Basic Aspects of Neurofibromatosis Type 1

#### **Chapter 6**

## Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1 by Induced-Neuronal (iN) Cells from Human Fibroblasts

*Noriaki Sagata, Yasunari Sakai and Takahiro A. Kato*

### **Abstract**

Direct conversion techniques, which generate induced-neuronal (iN) cells from human fibroblasts in less than two weeks, are expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. Here, we present unique gene expression and cell morphology profiles in iN cells derived from neurofibromatosis type 1 (NF1) patients. NF1 is a single-gene multifaceted disorder with relatively high co-occurrence of autism spectrum disorder (ASD). Adenylyl cyclase (AC) dysfunction is one of the candidate pathways in abnormal neuronal development in the brains of NF1 patients. In our study, microarray-based transcriptomic analysis of iN cells from healthy controls (males) and NF1 patients (males) revealed significantly different gene expression of 149 (110 were upregulated and 39 were downregulated). In iN cells derived from NF1 patients (NF1-iN cells), there was a change in the expression level of 90 genes with the addition of forskolin, an AC activator. Furthermore, treatment with forskolin dramatically changed the cell morphology, especially that of NF1-iN cells, from flatform to spherical-form. Current pilot data indicate the potential therapeutic effect of forskolin or AC activators on neuronal growth in NF1 patients. Further translational research is needed to validate the pilot findings for future drug development of ASD.

**Keywords:** adenylyl cyclases (ACs), autism spectrum disorder (ASD), forskolin, induced-neuronal (iN) cells, neurofibromatosis type 1 (NF1)

#### **1. Introduction**

Neurofibromatosis type 1 (NF1: also called von Recklinghausen disease) is a multifaceted disease with a variety of physical manifestations, including multiple café-au-lait spots, neurofibromas, Lisch nodules, scoliosis, and visual impairment [1–3]. NF1 patients also exhibit a variety of psychiatric symptoms such as mental retardation, epilepsy, and cognitive dysfunction/learning disabilities [4, 5]. Approximately half of NF1 patients exhibit impaired social information processing and disturbed social behavior [6–8]. In addition, about 30% of NF1 patients have comorbid autism spectrum disorder (ASD) [9–13]. These clinical reports suggest some kind of neurodevelopmental pathophysiology in the brains of NF1 patients.

NF1 is a monogenic disease, and the causative gene is the *NF1* gene, which encodes 'neurofibromin'. A recent mouse model study has shown that neurofibromin dysfunction increases the protein level of the anti-apoptotic protein BCL2 (B cell leukemia/lymphoma 2) in neural stem cells (NSCs) [14]. The strong association between neurofibromin and Ras-GTPase is widely known [15]. Some studies have shown that neurofibromin is involved not only in Ras-GTPase pathway, but also adenylyl cyclases (ACs) pathway of various cell types [16]. However, the detailed molecular basis for the ACs-mediated function of neurofibromin has not been elucidated. Interestingly, a recent study using the zebrafish model of NF1 has shown that the AC signaling pathway is involved in learning and the Ras-GTPase pathway is associated with memory (**Figure 1**) [17].

Few studies have shown whether such dysfunction is present in human living neuronal cells of NF1 patients. This is because it is difficult to directly analyze human brain cells containing neuronal cells. Very recently, a study has been published that reveals patient-common and mutation-dependent abnormalities using neural progenitor cells and cerebral organoids derived from induced pluripotent stem (iPS) cells in NF1 patients [18]. Regenerative medicine technologies using iPS cells from human tissues have been attracting attention to clarify the pathophysiology of brain disorders, including psychiatric disorders, at the cellular level [19, 20]. Direct conversion technologies without using iPS cells have also attracted attention as a useful translational research tool [21, 22]. The cells directly converted to neuronal cells are called "induced-neuronal (iN) cells" and were first developed from mouse fibroblasts transfected with the three transcriptional factors *Brn2*, *Ascl1*, and *Myt1l* (BAM factors) [23]. Human iN cells have been utilized in neuropsychiatric research, [24, 25] and several advantages have been reported that iN cells retain some of the aging-related physiological conditions that are lost in iPS cells [26, 27]. Using human-derived BAM factors, we succeeded in inducing iN cells from adult human fibroblasts in 2 weeks [28–32]. Briefly, in our protocol, the transfected fibroblasts were cultured in a medium containing 10 ng/mL FGF2, 1 mM valproic acid, 10 μM forskolin (optional), 0.8% N2 supplement, and 0.4% B27 supplement. And, we performed unbiased microarray analysis using SurePrint G3 Human Gene Expression Microarray 8 × 60 K v2 (Agilent Technologies) to investigate aberrant gene expression in NF1-iN cells. We reported the results of gene expression analysis of NF1 patients-derived iN cells (NF1-iN cells) [31]. Interestingly, forskolin, an

#### **Figure 1.**

*Schematic diagram of the pathways involved in neurofibromin. Two pathways involving neurofibromin have already been identified. One is the RAS pathway and the other is the AC pathway. Forskolin is an exogenous additive that activates the AC pathway.*

*Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1… DOI: http://dx.doi.org/10.5772/intechopen.98817*

activator of AC pathway, restored the aberrant gene expression in NF1-iN cells to the gene expression level of healthy controls-derived iN cells (HC-iN cells). Furthermore, prior to forskolin treatment, many HC-iN cells had neuron-like spherical-form cell morphology, whereas most NF1-iN cells were flat-form rather than spherical-form. After forskolin treatment, iN cells morphology changed rapidly and dramatically from flat-form to spherical-form, especially in NF1-iN cells. These pilot data show that forskolin or AC activators have possible therapeutic effects on neuronal growth in NF1 patients.

In this chapter, we present the results obtained to date on abnormalities in gene expression and cell morphology in iN cells derived from NF1 patients, and describe future prospects.

#### **2. Dysregulated gene expression in the neuronal cells of NF1 patients**

Direct conversion methods that generate human induced-neuronal (iN) cells from fibroblasts within two weeks are expected to discover unknown neuronal phenotypes in neuropsychiatric disorders. Here, we introduce unique gene expression profiles in iN cells of neurofibromatosis type 1 (NF1) patients, a single-gene multifaceted disorder with relatively high co-occurrence with autism spectrum disorder (ASD). The association between NF1 and adenylyl cyclases (ACs) activity has been reported in animal model studies, [16, 17] as far as we know, there are no experimental studies using human neuronal cells. To clarify how abnormalities in the ACs pathway affect the gene expression pattern of iN cells derived from NF1 patients (NF1-iN cells), a group treated with forskolin, an ACs activator, was included in the microarray analysis. First, an unbiased microarray analysis was performed to investigate aberrant gene expression in NF1-iN cells (6 male samples including 3 healthy controls (HC) and 3 NF1). Interestingly, in the iN cells, the expression of 149 genes was significantly different in NF1-iN cells compared to HC-iN cells (**Figure 2**). It is strongly suggested that these aberrant gene expressions in NF1 patients are shown only in iN cells and not in fibroblasts. In NF1-iN cells, the expression level of 90 genes was changed by the addition of the AC activator forskolin. Among the above149 genes (HC-iN cells vs. NF1-iN cells) and 90 genes (NF1-iN cells without forskolin vs. NF-iN cells with forskolin), 31 genes were overlapped (**Figure 2**). Interestingly, all of their expression levels in NF1-iN cells were rescued to HC level by the application of forskolin (**Figure 3**). These 31 genes may be strongly dysregulated via the AC pathway in neurons of NF1 patients (especially males).

To confirm the validity of the differences in expression of the 31 gene mentioned above, all samples on hand (3 male HC samples and 3 female NF1 samples) were added and reassessed by real-time PCR analysis. Unfortunately, when we validated these results with real-time PCR analysis using a total of six HC and six NF1 samples, including female samples, we could not reproduce most of the differences in the expression of the 31 genes. Recent epidemiological studies have shown that NF1 patients have a high comorbidity with ASD, and prevalence of ASD is about twice as high in males than females [12, 33]. Further investigation with larger samples may clarify our novel hypothesis about the tendency for neuronal pathologies to develop especially in male NF1 patients, and may lead to a better understanding of gender differences in ASD and other neuropsychiatric disorders.

#### **2.1** *MEX3D* **gene expression in the neuronal cells of NF1 patients**

Interestingly, in the real-time PCR analysis described above, only the gene expression of the *MEX3D* (Mex-3 RNA Binding Family Member D) was

#### **Figure 2.**

*Simplified schematic of microarray analysis. Circles show 6 samples groups: Healthy control fibroblast (HC-FB), NF1 patient fibroblast (NF1-FB), healthy control iN cells (HC-iN), NF1 patient iN cells (NF1-iN), healthy control iN cells with forskolin (HC-iN+FSK), and NF1 patient iN cells with forskolin (NF1-iN+FSK). Blue and orange double arrows indicate the number of aberrant genes between two groups (circles). A yellow double arrow indicates the number of overlapping genes between two blue double arrows. (modified from Sagata* et al. *2017).*

significantly downregulated in NF1-iN cells (p = 0.0040). MEX3D is a member of the RNA-binding protein family with homologous members: MEX3A, MEX3B, MEX3C, and MEX3D [34]. All members of the MEX3 family have two KH (K Homology) RNA-binding domains at the N-terminus, and a RING (Really Interesting New Gene) finger domain with ubiquitin E3 ligase activity at the C-terminus. A previous study has shown that MEX3D promotes the degradation of *BCL2* mRNA by interacting with its AU-rich elements (AREs) [35]. Therefore, we assessed the mRNA level of *BCL2* of the iN cells and found no difference between HC- and NF1-iN cells (p = 0.3134). AREs were initially reported to be present in the 3′-UTR (untranslated region) of the mRNAs of early response genes such as *FOS* (Fos Proto-Oncogene, AP-1 Transcription Factor Subunit), *MYC* (V-MYC Avian Myelocytomatosis Viral Oncogene Homolog), and *JUN* (Jun Proto-Oncogene, AP-1 Transcription Factor Subunit), which code for powerful transcriptional activators, and *CSF2* (Colony Stimulating Factor 2), *IL2* (Interleukin 2), *IL3*, and *IL6*, which code for growth factors and cytokines. These mRNAs are finely controlled in response to external stimuli and have a fast turnover [36, 37]. Therefore, we next assessed whether the reduction of *MEX3D* in NF1-iN cells was associated with expression levels of these mRNAs. The expression level of *FOS* mRNA in NF1-iN cells was significantly higher than that in HC-iN cells (p = 0.0428). Conversely, the mRNA expression level of *JUN* was significantly lower in NF1-iN cells (p = 0.0395). There were no significant differences in the expression levels of other genes (*MYC*, *CSF2*, *IL2*, *IL3*, and *IL6*). To the best our knowledge, there is no report showing a direct interaction between MEX3D and FOS or JUN in human neuronal cells.

To evaluate whether the reduction of *MEX3D* affects the expression levels of *FOS* and *JUN* mRNA in neuronal cells, we performed *Mex3d*-knockdown experiment using the Neuro2A cells, mouse neuroblastoma cell line. Knockdown of *Mex3d* with siRNA significantly increased the mRNA expression levels of both *Fos* and *Jun*

*Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1… DOI: http://dx.doi.org/10.5772/intechopen.98817*

**Figure 3.**

*Unique gene expression profile in iN cells from patients with NF1. Heatmap of the 31 genes that were revealed as aberrant in microarray analysis. \*limma adjusted p-value <0.05. Red indicates higher expression genes, and green indicates lower expression genes. (modified from Sagata* et al. *2017).*

in Neuro2A cells (p = 0.0002, 0.0360, respectively). This result suggests that there is a strong interaction between *MEX3D* (*Mex3d*) and *FOS*/*JUN* (*Fos*/*Jun*) not only in human neuronal cells but also in mouse cells. On the other hand, in Neuro2A cells, *Mex3d* knockdown did not change the *Nf1* mRNA expression level, and *Nf1* knockdown did not change the *Mex3d* mRNA expression level. The low expression level of *MEX3D* mRNA seen in human NF1-iN cells was not reproduced in the mouse neuronal cell line Neuro2A. This result suggests the importance of analyzing human cells in disease models.

#### **2.2** *BCL2* **gene expression in the neuronal cells of NF1 patients**

A previous study has shown that BCL2, an anti-apoptotic protein, is elevated in neuronal stem cells (NSCs) from *NF1* gene-disrupted mice [14]. To our knowledge, there are no data on BCL2 abnormalities in the mature neurons in *NF1*-disrupted mice or NF1 patients. Our data also showed that elevated *BCL2* mRNA was not observed in Day-14 mature iN cells. Therefore, we hypothesized that upregulation of *BCL2* by *NF1* gene-disruption could be a developmentally specific phenomenon in early-stage neuronal cells.

Treutlein *et al.* showed that the initial transcriptional response of iN cells generation occurs relatively homogeneously among fibroblasts, but during neuronal maturation of iN cells, a portion of the induced cells population takes on an alternative myogenic cell fate [38]. This should also imply that early-stage iN cells after transfection constitute a homogeneous population. In addition, although cell linage conversion and neuronal maturation are different events, we believe that early-stage iN cells may exhibit some characteristics of pre-mature neuronal cells in early developmental stage.

The morphology of Day-5 iN cells (early-stage iN cells) was not significantly different from that of fibroblasts. Surprisingly, forskolin transformed iN cells from a fibroblast-like shape to a long-branched neuron-like morphology even at Day 5. These Day-5 iN cells showed higher levels of *MAP2* (Microtubule Associated Protein 2: a pan-neuronal marker) than fibroblasts, even in the absence of forskolin (p < 0.0001). Day-14 iN cells showed higher expression level of *RBFOX3* (RNA Binding Protein, Fox-1 Homolog 3: a mature neuronal marker), while fibroblasts and Day-5 iN cells with/without forskolin showed no difference in *RBFOX3* expression. From these data, we speculate that Day-5 iN cells may exhibit some of the characteristics of pre-mature neuronal cells compared to Day-14 developed-stage iN cells. Interestingly, similar to the data of NSCs of *NF1*-disrupted mice, the expression level of *BCL2* mRNA in early-stage NF1-iN cells was significantly higher (p = 0.0002). These data partially support our hypothesis that high expression of BCL2 is observed only in early-stage neuronal cells in NF1 patients.

*BCL2* mRNA and protein are present at relatively high levels during the nervous system development and are reduced in the postnatal brain [39–41]. Abnormalities of apoptosis constitute the pathogenesis of neurodevelopmental disorders [42]. The majority of neurons are immature or premature at the stage of neurodevelopment, and apoptosis of immature / premature neurons needs to be highly controlled in order to form proper neural circuits. Therefore, in the brains of NF1 patients, BCL2 mediated neuronal apoptosis may be disturbed during neurodevelopment, thereby leading to the formation of abnormal neural circuits. Disruption of this pathway may be one of the pathogenic mechanisms underlying the development of ASD and other neurodevelopmental disorders in NF1 patients.

#### **2.3** *MAGEL2* **gene expression in the neuronal cells of NF1 patients**

Aberrant gene expression in NF1-iN cells has also been discovered from a completely different approach. Akamine *et al*. reported on a 45-year-old woman with NF1, epileptic encephalopathy of infantile onset, and severe developmental delay [32]. Whole genome sequencing confirmed *de novo* pathogenic mutations in *NF1* and *MAGEL2*, a gene associated with Schaaf-Yang syndrome. According to STRING (http://string-db.org/), a protein–protein interaction database, NF1 and MAGEL2 were predicted to be closely linked in this network through a common interacting protein. To test the possibility of a functional interaction between NF1 and MAGEL2, it was examined whether pathological mutations in *NF1* affect the neuronal expression of *MAGEL2*. Interestingly, NF1-iN cells had significantly lower expression of *MAGEL2* than HC-iN cells (54%, p = 0.0047) [32]. These data are the first to show that pathogenic mutations of *NF1* regulate the expression of other neurodevelopmental disease-associated genes. *De novo* mutations in multiple genes can cause severe developmental phenotypes due to their cumulative effects or synergistic interactions.

#### **3. Aberrant cell morphology of the neuronal cells of NF1 patients**

Adenylyl cyclase (AC) dysfunction is one of the candidate pathways in abnormal neuronal development in the brain of NF1 patients, but its dynamic abnormalities have not been observed. Therefore, we observed the dynamic effects of forskolin on iN cells. In HC-iN cells, most of cells were neuron-like spherical-form. On the other

*Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1… DOI: http://dx.doi.org/10.5772/intechopen.98817*

hand, in NF1-iN cells, most of the cells were thin and flat. Interestingly, after only 20 minutes of AC activation by forskolin treatment, most NF1-iN cells had a dense cell contour and their cell morphology changed dramatically to neuron-like spherical-form. This result suggests that most NF1-iN cells were unable to form neuronlike spherical-form cell morphology due to lack of AC ability. Counting the number of cells, NF1-iN cells had a significantly higher number of flat-form cells than HC-iN cells (**Figure 4**, p = 0.0164), and their cell morphology was significantly restored by forskolin treatment (**Figure 4**, p = 0.0059) [43]. In addition, forskolin appeared to promote neurite outgrowth in iN cells, so quantitative experiments and analysis with more samples should be conducted in the near future.

Forskolin activates intracellular ACs and increases intracellular cyclic adenosine monophosphate (cAMP) levels, and it has previously reported that forskolin regulates cytoskeletal formation in Y1 cells, a cell line derived from mouse adrenocortical tumors [44]. When intracellular cAMP levels increase, dephosphorylation of paxillin occurs at the cell edge, and paxillin moves from the focal adhesion to the cytoplasm [44]. Patients with NF1 have aberrant gene expression of neurofibromin that is known to regulate the activity of ACs and the intracellular cAMP levels [16]. Recently, we have shown that neurofibromin gene expression is also low in NF1-iN cells, [31] suggesting that intracellular cAMP levels are low in NF1-iN cells. As mentioned above, NF1-iN cells tend to have flat-form cell morphology compared to HC-iN cells, and these cell morphologies are restored by application of forskolin [43]. Thus, such morphological abnormalities may be attributed by abnormal cytoskeleton development due to decreased dephosphorylation levels of paxillin due to decreased activation of ACs and decreased intracellular cAMP levels in NF1-iN cells. Paxillin has been shown to be involved in neurite outgrowth in PC12 cells, a cell line derived from rat adrenal medulla pheochromocytoma [45]. Similarly, our findings suggest that forskolin alter the phosphorylation level of paxillin and activated neurite outgrowth.

Our pilot experiment showed that activation of ACs may normalize the development of neuronal cells in the brain of NF1 patients. We propose that administration of forskolin or forskolin-like AC activators into the brain during

#### **Figure 4.**

*The ratio of the number of neuronal-like spherical-form cells to the total number of cells. NF1-iN cells in the absence of forskolin had a significantly lower percentage of the spherical-form cells compared to HC-iN cells (p = 0.0164, two-way ANOVA/Tukey's test, n = 3 each group). In the presence of forskolin, the spherical-form cell morphology of NF1-iN cells was significantly higher (p = 0.0059, two-way ANOVA/Tukey's test, n = 3 each group) (modified from Sagata* et al. *2020).*

neurodevelopmental periods of NF1 patients may contribute to the prevention of neurodevelopmental disorders such as ASD and neuropsychiatric disorders in subsequent life.

#### **4. Conclusions**

In this chapter, we have presented unique gene expressions and cell morphology profiles in induced-neuronal (iN) cells of patients with neurofibromatosis type 1 (NF1), a single-gene multifaceted disorder with relatively high co-occurrence of autism spectrum disorder (ASD). Microarray analysis revealed that the expression of 149 genes was abnormal in the neuronal cells of NF1 male patients, and that the expression of 90 genes was altered in the presence of forskolin. Of these, 31 genes in particular were suggested to be normalized by improvement of the AC pathway. These abnormalities of gene expressions may be male-specific and may be related to gender differences in the development of ASD. Further cellular analysis, especially considering gender-specific neuronal dysregulation, should be performed to reveal unknown neurobiological roles of gender underlying the pathophysiology of ASD.

We also introduced that the effects of forskolin shows dramatic changes not only in gene expression but also in the cell morphology of neuronal cells in NF1 patients. We propose that research is needed to prevent the development of ASD and neuropsychiatric disorder later in life by administering forskolin and other AC activators, which are easily introduced in to the brain, to NF1 patients early in their developmental period.

Furthermore, we found that the expression of *FOS* and *BCL2* mRNA, which have anti-apoptotic effects in neuronal cells, were elevated in developed- and earlystage iN cells of NF1 patients, respectively. Therefore, neuronal apoptosis during neurodevelopmental period can be disturbed in NF1 patients.

Moreover, the findings presented here should be validated by additional analyses such as apoptosis analysis, protein level analysis and functional analysis of neurons. On the other hand, more detailed molecular mechanisms, especially the interactions between NF1, MEX3D, FOS, JUN, BCL2, and MAGEL2, will be the subject of future work. In addition, *in vitro* studies using mouse Neuro2A cells did not show some of interactions seen in the gene expression analysis of human NF1-iN cell (e.g., the interaction between *Nf1* and *Mex3d*), suggesting that these interactions may be unique to humans, highlighting the importance of studying human cellular models. Neuron studies derived from human iPS cell are expected to confirm the findings introduced here.

#### **Acknowledgements**

Our studies shown in this paper were partially supported by Grant-in-Aid for Scientific Research on (1) Innovative Areas "Will-Dynamics" of The Ministry of Education, Culture, Sports, Science, and Technology, Japan (JP16H06403 to T.A.K.), (2) The Japan Agency for Medical Research and Development (AMED) ("Syogaisya-Taisaku-Sogo-Kenkyu-Kaihatsu-Jigyo" JP19dk0307047 & JP19dk0307075, and "Yugo-no" JP19dm0107095 to T.A.K.), (3) KAKENHI - the Japan Society for the Promotion of Science ("Wakate A" JP26713039 and "Kiban A" JP18H04042 to T.A.K., and "Wakate B" JP26860932 & JP17K16386 to N.S.), (4) SENSHIN Medical Research Foundation (to T.A.K.), (5) Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.A.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

*Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1… DOI: http://dx.doi.org/10.5772/intechopen.98817*

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Noriaki Sagata1 , Yasunari Sakai<sup>2</sup> and Takahiro A. Kato1 \*

1 Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

2 Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

\*Address all correspondence to: kato.takahiro.015@m.kyushu-u.ac.jp

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Neurofibromatosis. Conference statement. National Institutes of Health Consensus Development Conference. Arch Neurol. 1988;45:575-8

[2] Ferner RE. Neurofibromatosis 1 and neurofibromatosis 2: a twenty first century perspective. Lancet Neurol. 2007;6:340-51. DOI: 10.1016/ S1474-4422(07)70075-3

[3] Jouhilahti EM, Peltonen S, Heape AM, Peltonen J. The pathoetiology of neurofibromatosis 1. Am J Pathol. 2011;178:1932-9. DOI: 10.1016/j.ajpath.2010.12.056

[4] Johnson NS, Saal HM, Lovell AM, Schorry EK. Social and emotional problems in children with neurofibromatosis type 1: evidence and proposed interventions. J Pediatr. 1999;134:767-72. DOI: 10.1016/ s0022-3476(99)70296-9

[5] Barton B, North K. Social skills of children with neurofibromatosis type 1. Dev Med Child Neurol. 2004;46:553- 63. DOI: 10.1017/s0012162204000921

[6] Noll RB, Reiter-Purtill J, Moore BD, Schorry EK, Lovell AM, Vannatta K, Gerhardt CA. Social, emotional, and behavioral functioning of children with NF1. Am J Med Genet A. 2007;143A: 2261-73. DOI: 10.1002/ajmg.a. 31923

[7] Lehtonen A, Howie E, Trump D, Huson SM. Behaviour in children with neurofibromatosis type 1: cognition, executive function, attention, emotion, and social competence. Dev Med Child Neurol. 2013;55:111-25. DOI: 10.1111/j.1469-8749.2012.04399.x

[8] Huijbregts SC, de Sonneville LM. Does cognitive impairment explain behavioral and social problems of children with neurofibromatosis type 1? Behav Genet. 2011;41:430-6. DOI: 10.1007/s10519-010-9430-5

[9] Morris SM, Acosta MT, Garg S, Green J, Huson S, Legius E, North KN, Payne JM, Plasschaert E, Frazier TW, Weiss LA, Zhang Y, Gutmann DH, Constantino JN. Disease Burden and Symptom Structure of Autism in Neurofibromatosis Type 1: A Study of the International NF1-ASD Consortium Team (INFACT). JAMA Psychiatry. 2016;73:1276-1284. DOI: 10.1001/ jamapsychiatry.2016.2600

[10] Plasschaert E, Descheemaeker MJ, Van Eylen L, Noens I, Steyaert J, Legius E. Prevalence of Autism Spectrum Disorder symptoms in children with neurofibromatosis type 1. Am J Med Genet B Neuropsychiatr Genet. 2015;168B:72-80. DOI: 10.1002/ ajmg.b.32280

[11] Garg S, Lehtonen A, Huson SM, Emsley R, Trump D, Evans DG, Green J. Autism and other psychiatric comorbidity in neurofibromatosis type 1: evidence from a population-based study. Dev Med Child Neurol. 2013;55:139-45. DOI: 10.1111/ dmcn.12043

[12] Garg S, Green J, Leadbitter K, Emsley R, Lehtonen A, Evans DG, Huson SM. Neurofibromatosis type 1 and autism spectrum disorder. Pediatrics. 2013;132:e1642-8. DOI: 10.1542/peds.2013-1868

[13] Walsh KS, Vélez JI, Kardel PG, Imas DM, Muenke M, Packer RJ, Castellanos FX, Acosta MT. Symptomatology of autism spectrum disorder in a population with neurofibromatosis type 1. Dev Med Child Neurol. 2013;55:131-8. DOI: 10.1111/dmcn.12038

[14] Dasgupta B, Gutmann DH. Neurofibromin regulates neural stem cell proliferation, survival, and astroglial differentiation in vitro and in *Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1… DOI: http://dx.doi.org/10.5772/intechopen.98817*

vivo. J Neurosci. 2005;25:5584-94. DOI: 10.1523/JNEUROSCI.4693-04. 2005

[15] Weiss B, Bollag G, Shannon K. Hyperactive Ras as a therapeutic target in neurofibromatosis type 1. Am J Med Genet. 1999;89:14-22.

[16] Tong J, Hannan F, Zhu Y, Bernards A, Zhong Y. Neurofibromin regulates G protein-stimulated adenylyl cyclase activity. Nat Neurosci. 2002;5:95-6. DOI: 10.1038/nn792

[17] Wolman MA, de Groh ED, McBride SM, Jongens TA, Granato M, Epstein JA. Modulation of cAMP and ras signaling pathways improves distinct behavioral deficits in a zebrafish model of neurofibromatosis type 1. Cell Rep. 2014;8:1265-70. DOI: 10.1016/j. celrep.2014.07.054

[18] Anastasaki C, Wegscheid ML, Hartigan K, Papke JB, Kopp ND, Chen J, Cobb O, Dougherty JD, Gutmann DH. Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations. Stem Cell Reports. 2020;14:541-550. DOI: 10.1016/j. stemcr.2020.03.007

[19] Ishii T, Ishikawa M, Fujimori K, Maeda T, Kushima I, Arioka Y, Mori D, Nakatake Y, Yamagata B, Nio S, Kato TA, Yang N, Wernig M, Kanba S, Mimura M, Ozaki N, Okano H. In Vitro Modeling of the Bipolar Disorder and Schizophrenia Using Patient-Derived Induced Pluripotent Stem Cells with Copy Number Variations of PCDH15 and RELN. eNeuro. 2019;6:ENEURO. 0403-18.2019. DOI: 10.1523/ENEURO. 0403-18.2019

[20] Wang M, Zhang L, Gage FH. Modeling neuropsychiatric disorders using human induced pluripotent stem cells. Protein Cell. 2020;11:45-59. DOI: 10.1007/s13238-019-0638-8

[21] Gamo NJ, Sawa A. Human stem cells and surrogate tissues for basic and translational study of mental disorders. Biol Psychiatry. 2014;75:918-9. DOI: 10.1016/j.biopsych.2014.03.025

[22] Liu YN, Lu SY, Yao J. Application of induced pluripotent stem cells to understand neurobiological basis of bipolar disorder and schizophrenia. Psychiatry Clin Neurosci. 2017;71:579- 599. DOI: 10.1111/pcn.12528

[23] Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010;463:1035- 41. DOI: 10.1038/nature08797

[24] Srivastava R, Faust T, Ramos A, Ishizuka K, Sawa A. Dynamic Changes of the Mitochondria in Psychiatric Illnesses: New Mechanistic Insights From Human Neuronal Models. Biol Psychiatry. 2018;83:751-760. DOI: 10.1016/j.biopsych.2018.01.007

[25] Yoshimizu T, Pan JQ, Mungenast AE, Madison JM, Su S, Ketterman J, Ongur D, McPhie D, Cohen B, Perlis R, Tsai LH. Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons. Mol Psychiatry. 2015;20:162-9. DOI: 10.1038/mp.2014.143

[26] Victor MB, Richner M, Olsen HE, Lee SW, Monteys AM, Ma C, Huh CJ, Zhang B, Davidson BL, Yang XW, Yoo AS. Striatal neurons directly converted from Huntington's disease patient fibroblasts recapitulate ageassociated disease phenotypes. Nat Neurosci. 2018;21:341-352. DOI: 10.1038/s41593-018-0075-7

[27] Tang BL. Patient-Derived iPSCs and iNs-Shedding New Light on the Cellular Etiology of Neurodegenerative Diseases. Cells. 2018;7:38. DOI: 10.3390/ cells7050038

[28] Kano S, Yuan M, Cardarelli RA, Maegawa G, Higurashi N, Gaval-Cruz M, Wilson AM, Tristan C, Kondo MA, Chen Y, Koga M, Obie C, Ishizuka K, Seshadri S, Srivastava R, Kato TA, Horiuchi Y, Sedlak TW, Lee Y, Rapoport JL, Hirose S, Okano H, Valle D, O'Donnell P, Sawa A, Kai M. Clinical utility of neuronal cells directly converted from fibroblasts of patients for neuropsychiatric disorders: studies of lysosomal storage diseases and channelopathy. Curr Mol Med. 2015;15:138-45. DOI: 10.2174/156652401 5666150303110300

[29] Passeri E, Wilson AM, Primerano A, Kondo MA, Sengupta S, Srivastava R, Koga M, Obie C, Zandi PP, Goes FS, Valle D, Rapoport JL, Sawa A, Kano S, Ishizuka K. Enhanced conversion of induced neuronal cells (iN cells) from human fibroblasts: Utility in uncovering cellular deficits in mental illnessassociated chromosomal abnormalities. Neurosci Res. 2015;101:57-61. DOI: 10.1016/j.neures.2015.07.011

[30] Passeri E, Jones-Brando L, Bordón C, Sengupta S, Wilson AM, Primerano A, Rapoport JL, Ishizuka K, Kano S, Yolken RH, Sawa A. Infection and characterization of Toxoplasma gondii in human induced neurons from patients with brain disorders and healthy controls. Microbes Infect. 2016;18:153-8. DOI: 10.1016/j. micinf.2015.09.023

[31] Sagata N, Kato TA, Kano SI, Ohgidani M, Shimokawa N, Sato-Kasai M, Hayakawa K, Kuwano N, Wilson AM, Ishizuka K, Kato S, Nakahara T, Nakahara-Kido M, Setoyama D, Sakai Y, Ohga S, Furue M, Sawa A, Kanba S. Dysregulated gene expressions of MEX3D, FOS and BCL2 in human induced-neuronal (iN) cells from NF1 patients: a pilot study. Sci Rep. 2017;7:13905. DOI: 10.1038/ s41598-017-14440-7

[32] Akamine S, Sagata N, Sakai Y, Kato TA, Nakahara T, Matsushita Y, Togao O, Hiwatashi A, Sanefuji M, Ishizaki Y, Torisu H, Saitsu H, Matsumoto N, Hara T, Sawa A, Kano S, Furue M, Kanba S, Shaw CA, Ohga S. Early-onset epileptic encephalopathy and severe developmental delay in an association with de novo double mutations in NF1 and MAGEL2. Epilepsia Open. 2017;3:81-85. DOI: 10.1002/epi4.12085

[33] Garg S, Heuvelman H, Huson S, Tobin H, Green J; Northern UK NF1 Research Network. Sex bias in autism spectrum disorder in neurofibromatosis type 1. J Neurodev Disord. 2016;8:26. DOI: 10.1186/s11689-016-9159-4

[34] Buchet-Poyau K, Courchet J, Le Hir H, Séraphin B, Scoazec JY, Duret L, Domon-Dell C, Freund JN, Billaud M. Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNAbinding proteins differentially localized to processing bodies. Nucleic Acids Res. 2007;35:1289-300. DOI: 10.1093/ nar/gkm016

[35] Donnini M, Lapucci A, Papucci L, Witort E, Jacquier A, Brewer G, Nicolin A, Capaccioli S, Schiavone N. Identification of TINO: a new evolutionarily conserved BCL-2 AU-rich element RNA-binding protein. J Biol Chem. 2004;279:20154-66. DOI: 10.1074/jbc.M314071200

[36] Stoecklin G, Stoeckle P, Lu M, Muehlemann O, Moroni C. Cellular mutants define a common mRNA degradation pathway targeting cytokine AU-rich elements. RNA. 2001;7:1578-88

[37] Chen CY, Shyu AB. Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol Cell Biol. 1994;14:8471-82. DOI: 10.1128/mcb.14.12.8471

[38] Treutlein B, Lee QY, Camp JG, Mall M, Koh W, Shariati SA, Sim S, *Clarifying the Pathophysiological Mechanisms of Neuronal Abnormalities of NF1… DOI: http://dx.doi.org/10.5772/intechopen.98817*

Neff NF, Skotheim JM, Wernig M, Quake SR. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature. 2016;534:391-5. DOI: 10.1038/ nature18323

[39] Abe-Dohmae S, Harada N, Yamada K, Tanaka R. Bcl-2 gene is highly expressed during neurogenesis in the central nervous system. Biochem Biophys Res Commun. 1993;191:915-21. DOI: 10.1006/bbrc.1993.1304

[40] Merry DE, Veis DJ, Hickey WF, Korsmeyer SJ. bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS. Development. 1994;120:301-11

[41] Akhtar RS, Ness JM, Roth KA. Bcl-2 family regulation of neuronal development and neurodegeneration. Biochim Biophys Acta. 2004;1644:189- 203. DOI: 10.1016/j.bbamcr.2003.10.013

[42] Margolis RL, Chuang DM, Post RM. Programmed cell death: implications for neuropsychiatric disorders. Biol Psychiatry. 1994;35:946-56. DOI: 10.1016/0006-3223(94)91241-6

[43] Sagata N, Kano SI, Ohgidani M, Inamine S, Sakai Y, Kato H, Masuda K, Nakahara T, Nakahara-Kido M, Ohga S, Furue M, Sawa A, Kanba S, Kato TA. Forskolin rapidly enhances neuron-like morphological change of directly induced-neuronal cells from neurofibromatosis type 1 patients. Neuropsychopharmacol Rep. 2020;40:396-400. DOI: 10.1002/ npr2.12144

[44] Han JD, Rubin CS. Regulation of cytoskeleton organization and paxillin dephosphorylation by cAMP. Studies on murine Y1 adrenal cells. J Biol Chem. 1996;271:29211-5. DOI: 10.1074/ jbc.271.46.29211

[45] Ishitani T, Ishitani S, Matsumoto K, Itoh M. Nemo-like kinase is involved in

NGF-induced neurite outgrowth via phosphorylating MAP1B and paxillin. J Neurochem. 2009;111:1104-18. DOI: 10.1111/j.1471-4159.2009.06400.x

#### **Chapter 7**

## Alternative Splicing of Neurofibromatosis Type 1 Exon 23a Modulates Ras/ERK Signaling and Learning Behaviors in Mice

*Karl Andreas Mader and Hua Lou*

#### **Abstract**

Neurofibromin is one of the few Ras-GTP activating proteins (Ras-GAPs) expressed in the brain. Disruption of its expression leads to the detrimental disease neurofibromatosis type 1 (NF1). Many studies have revealed the crucial role of *NF1* in developing and adult tissues. However, these studies have focused on the expression of the entire *NF1* gene and largely ignored the role of an alternative splicing event that controls the Ras-GAP function of neurofibromin. The focus of this chapter is *NF1* exon 23a. This exon is located in the GAP-related domain (GRD) of neurofibromin. Its expression level, indicated by the percentage of its inclusion in the *NF1* mRNA transcripts, has a profound effect on the Ras-GAP function of neurofibromin. In this chapter, we review the expression pattern of exon 23a and the molecular mechanisms that regulate its expression. We then discuss the role of its expression in Ras/ERK signaling and learning behaviors in mice. Lastly, we propose a few directions for future studies.

**Keywords:** NF1, alternative splicing, exon 23a, Ras-GAP, learning behaviors, mouse

#### **1. Introduction**

Neurofibromatosis type 1 (NF1) is a genetic disorder that affects approximately 1 in 2000–4000 individuals [1]. The disease hallmark includes tumors in the nervous system, most commonly, benign peripheral nerve tumors or neurofibromas, and café-au-lait macules [1]. In addition, many NF1 patients exhibit cognitive and behavioral problems, bone abnormalities and hypertension [1].

The underlying cause of the NF1 disease is the germline mutation in one of the two alleles of the *NF1* gene on chromosome 17q11.2. This gene, spanning more than 350 kilobases (kb) of genomic DNA, codes for neurofibromin, which is one of the major GTPase activating proteins (GAPs) that down regulates the activity of Ras [2–5]. Neurofibromin attenuates Ras signaling by converting it from its active GTPbound form to its inactive GDP-bound state via the GAP-related domain (GRD). Mutations in the *NF1* gene reduce/abolish the Ras-GAP function of neurofibromin, which leads to abnormally high cellular activity of the Ras signaling pathway [4, 5].

Proper expression of the GRD is critical to achieving the optimal Ras-GAP function of neurofibromin. In mammals, the *NF1* gene expression is highly regulated temporally and spatially. During embryonic development, *NF1* is expressed in many tissues.

#### **Figure 1.**

*A diagram showing that alternative splicing of the NF1/Nf1 exon 23a can give rise two isoforms of neurofibromin. Early* in vitro *experiments demonstrated that inclusion of exon 23a leads to reduced Ras-GAP activity of neurofibromin and thus increased active Ras activity. The two protein isoforms are depicted above and below and pre-mRNA in the middle. The diagram is not drawn to scale.*

However, in adults, its expression is highly enriched in the nervous system in neurons, oligodendrocytes and Schwann cells [6–8]. A recent study demonstrated that in both mouse and human brain, *NF1* expression is enriched in inhibitory neurons [9].

In addition to transcriptional regulation, the expression output of the *NF1* gene is subject to a post-transcriptional alternative splicing regulation that gives rise to two distinct NF1 transcripts: one with and the other without exon 23a (exon 31 in the current *NF1* nomenclature) [10]. Exon 23a is a 63 nucleotide in-frame cassette exon that, when translated, adds additional 21 amino acids in the neurofibromin protein [10]. The neurofibromin that does not contain the 21 amino acids is named type 1 isoform whereas the protein that contains the extra amino acids is named type 2 isoform (**Figure 1**) [10]. Because these additional amino acids are located in the GRD, it was immediately suspected when exon 23a was identified that inclusion of these amino acids would affect the Ras-GAP function of neurofibromin. As predicted, early *in vitro* analysis using truncated GRD expression plasmids with or without exon 23a demonstrated that the GRD polypeptide containing exon 23a showed up to 10 times lower Ras-GAP activity than that the GRD without exon 23a [10, 11]. For more than a decade after these initial studies, it remained unknown if expression of this alternative exon regulates the full-length neurofibromin protein in a similar fashion. Since 2002, our group has conducted extensive research to understand the biology of this alternative splicing event.

In this chapter, we will focus on exon 23a and discuss the following questions. What is the expression pattern of this exon? How is its expression regulated? How does its expression affect the Ras-GAP function of neurofibromin? How does its expression affect the signaling pathways downstream of Ras? How does disruption of its expression affect animal behaviors *in vivo*? Lastly, we will discuss the pressing remaining questions for future studies.

#### **2. The functional role of regulated expression of NF1 exon 23a**

#### **2.1 Expression of Nf1 exon 23a in mouse**

RT-PCR analysis indicated that the alternative inclusion of exon 23a is tightly regulated in tissue- and developmental stage-specific patterns. In adults, exon 23a

#### *Alternative Splicing of Neurofibromatosis Type 1 Exon 23a Modulates Ras/ERK Signaling… DOI: http://dx.doi.org/10.5772/intechopen.99678*

is predominantly skipped in the brain and testis leading to production of the type 1 NF1 isoform, while in other tissues, exon 23a is included to various extents leading to production of the type 2 isoform [6, 12, 13]. In adult mouse, exon 23a is included at 8% in the testis, 11% in the brain, 42% in the spleen, 58% in the heart, 62% in the liver, 78% in the kidney and 82% in the lung [14]. Within the brain, exon 23a is least included in hippocampus at 2–4% and slightly more included in the cortex at 10% [15]. In primary mouse cardiomyocytes, exon 23a is included at 70% [16].

During development, in the mouse brain, a switch from the isoform 2 to isoform 1 occurs during early embryonic development between day E10 and E11 [6, 13]. The biological significance of this switch has not been investigated.

#### **2.2 Molecular mechanisms regulating alternative splicing of exon 23a**

Most of our experiments were conducted using human *NF1* sequence and human, mouse or rat cells. All of the existing evidences indicate that this alternative splicing event is conserved in mammalian cells [17]. Consistent with the widely used nomenclature, the human gene is designated as *NF1* while the mouse gene is designated as *Nf1* throughout the chapter.

The differential splicing of exon 23a is under complex control by two distinct mechanisms (**Figure 2**). The first mechanism involves several regulatory RNA-binding proteins (RBPs) which promote either its skipping or inclusion (**Figure 2A**). Two families of RBPs which promote the skipping of exon 23a have been identified, Hu proteins, also known as ELAV-like proteins, and CUG-BP1 and ETR-3 like factors (CELF). Hu proteins bind to AU-rich regions of RNA both upstream and downstream of exon 23a while CELF proteins bind to UG-rich motifs upstream of exon 23a (**Figure 2A**) [18–20]. Mechanistically, upstream of exon 23a, Hu and CELF proteins function to block the splicing factor U2AF from binding to the 3′ splice site, while downstream of exon 23a, Hu proteins block splicing factors U1 and U6 snRNP complexes from binding to the 5′ splicing site [18, 20]. Two additional families of RBPs, TIA-1/TIAR and muscleblind-like (MBNL) proteins, on the other hand, promote the inclusion of exon 23a (**Figure 2A**). TIA-1/TIAR proteins, in direct competition for binding with Hu proteins, bind to the U-rich sequence downstream of exon 23a, promoting the U1 and U6 snRNP binding at the 5′ splice site and inclusion of the exon [18]. MBNL proteins binds to a sequence upstream of exon 23a to promote its inclusion (**Figure 2A**) [21].

The second mechanism involves epigenetic regulation of alternative splicing, at the chromatin level, through altering histone modifications and transcriptional elongation rate (**Figure 2B**). One of the models that explains the epigenetic regulation of splicing is the kinetics coupling model of transcription and splicing [22]. This model predicts that faster transcriptional elongation rate of RNAPII promotes skipping of an alternative exon, which is usually surrounded by suboptimal splicing signals, as in the case of NF1 exon 23a [23]. One of the factors regulating transcriptional elongation rate is the "openness" of chromatin modulated by histone acetylation [22]. The higher level of histone acetylation is correlated with more relaxed configuration of the chromatin, which allows RNAPII move faster during transcription. Exon 23a is subjected to this mode of regulation in two different ways as shown in **Figure 2B**. Studies using mouse primary cardiomyocytes where exon 23a is normally included at 70% demonstrated that an increase in Ca2+ by KCl-induced depolarization led to a significant reduction of inclusion to 10–15% through increasing histone acetylation on the body of the entire *Nf1* gene [16]. In neuronal cells, Hu proteins interact with HDAC2, a member of the histone deacetylase family, that reduces the deacetylation enzymatic activity of HDAC2 in a localized fashion [24]. In both cases, the transcriptional elongation rate is increased by histone hyperacetylation, leading to exon 23a skipping [16, 24].

#### **Figure 2.**

*Regulation of alternative splicing of* NF1/Nf1 *exon 23a. A. RBP-mediated regulatory mechanisms. TIA and MBNL proteins promote inclusion of exon 23a by recruiting splicing factors U1 and U6 to the 5′ splice site and U2AF to the 3′ splice site, respectively. Hu and CELF proteins promote skipping of exon 23a by preventing these splicing factors from binding. B. Epigenetic regulatory mechanisms. Both mechanisms involve increased histone acetylation, which leads to increased transcription elongation rate causing exon 23a to be skipped. The increased histone acetylation is triggered by either nuclear export of HDAC proteins (box 1) or decreased activity of HDAC2 (box 2). The up and down arrows indicate increase and decrease, respectively.*

#### **2.3 Role of exon 23a expression in cell signaling regulation**

In order to uncover the biological importance of exon 23a inclusion, our laboratory generated mutant embryonic stem (ES) cell lines through the classical gene-targeting knock-in approach [23]. We generated two contrasting mouse ES cell lines, one showing 100% exon 23a inclusion in the endogenously expressed *Nf1* gene, *Nf1*23aIN/23aIN, and the other 100% exon 23a skipping, *Nf1*23aΔ/23aΔ [23].

We then differentiated these ES cells into CNS-like neurons following an established protocol [25]. In this two week procedure, mouse ES cells were first grown in a non-adherent dish in the presence of retinoic acid to form cellular aggregates, which were then dissociated and plated on laminin-coated tissue culture plates in neuronal culture medium that support neural differentiation and maturation. This procedure was shown to produce pyramidal neurons with >90% homogeneity [25]. When the two mutant *Nf1* ES cell lines were differentiated into neuronal cells, they showed drastically different Ras signaling but similar cAMP activities [23]. Compared to wild type neurons (10%, exon 23a inclusion), the *Nf1*23aΔ/23aΔ neurons (0% exon 23a inclusion) exhibited a slightly lower level of Ras-GTP while the *Nf1*23aIN/23aIN neurons (100% exon 23a inclusion) exhibited at least three times more Ras-GTP [23]. These experiments establish that exon 23a expression affects the Ras-GAP function of the endogenously expressed neurofibromin. Interestingly,

#### *Alternative Splicing of Neurofibromatosis Type 1 Exon 23a Modulates Ras/ERK Signaling… DOI: http://dx.doi.org/10.5772/intechopen.99678*

*Nf1* exon 23a expression specifically affects the phospho-ERK1/2 level downstream of Ras but not the PI3K/Akt/mTOR pathway [23].

Using the *Nf1*23aIN/23aIN ES cells, we generated a mutant mouse line [14]. The *NF1*23aIN/+ mouse ES cells were from the 129 background. Chimeric 129:C57Bl/6 J mice were generated by blastocyst injection of the *Nf1*23aIN/+ ES cells and crossed with C57Bl/6 J mice. A founding *Nf1*23aIN/+ mouse was obtained. The mice were then crossed for 10 generations onto the C57Bl/6 J background. In the *Nf1*23aIN/23aIN mice, the *Nf1* gene only produces the isoform II neurofibromin where exon 23a is included in all cell types at 100% [14]. When the mouse brain proteins were analyzed, similar results were found as in the ES-derived neurons. While Ras-GTP level was barely detectable in the wild type mouse brain, it was significantly increased in the *Nf1*23aIN/23aIN brain [14]. The pERK1/2 is six times higher in the mutant than wild type brain while the PI3K/Akt/mTOR pathway was unaltered [14]. These findings support a model in which alternative splicing of exon 23a plays a crucial role in regulating the Ras–Raf–MEK–ERK signaling pathway *in vivo*.

#### **2.4 Role of exon 23a expression in mouse learning and memory behaviors**

To explore the link between exon 23a regulation of Ras and cognitive behaviors, a battery of learning and memory tests were conducted comparing the wildtype and mutant *Nf1*23aIN/23aIN mice. The results of these tests indicated clear impairments in learning and memory performance in the mutant mice [14].

To test short-term and long-term spatial memory, a T-maze and Morris water maze test were conducted, respectively. T-maze test is used to examine the short-term spatial memory. In this test, mice were placed in a T-shaped maze and allowed to explore the maze freely for 10 minutes while one of the arms was closed. Following the exploration, mice were returned to their home cage for 2 hours and then put back in the T-maze with all three arms open. Once put in the T-maze, mice were video recorded. The memory measurement was calculated as the time spent in the previously closed arm divided by the overall time spent in both arms, which was expected to be 50% by chance. The wild type mice were more likely to explore an unfamiliar lever arm than a familiar one, a preference indicative of an active short-term memory. The mutant *Nf1*23aIN/23aIN mice showed an impairment of this function and failed to display any preference between lever arms, with a selection rate around 50% [14].

Morris water maze test is used to examine the long-term spatial memory. In this test, mice were trained in a small water pool in a well-lit room replete of visual cues. A hidden escape platform was placed 0.5 cm beneath the water level in a particular location in the pool. Animals were tested for three trials per day over 4 days. For these trials, mice were placed in the water and allowed to swim for 60 seconds. If mice did not find the platform during the allotted time, they were guided toward it, and held for 15 seconds on the platform. Swim time and path length were recorded. Following the final session, the platform was removed for a probe trial to test for spatial strategy and retention. During the probe test, mice were allowed to swim for 60 seconds without the possibility of escape; the percentage of time spent in the quadrant where the platform was previously located was measured. In this test, the mutant *Nf1*23aIN/23aIN mice fell behind their wild type counterparts in their ability to find a hidden platform upon repeated exposure to the same conditions. Additionally, when the hidden platforms were removed, the mutant mice spent less time swimming in the region that the platform had been in previous trials [14].

The mutant *Nf1*23aIN/23aIN mice also exhibited impairment in the fear associative learning test in a fear conditioning experiment [14]. In this experiment, mice were placed in a cage and given a short electric shock after being given an audio cue.

After 24 hours, their freezing response times after being placed in the same cage and being played the audio cue were measured. The mutant mice showed increased freezing times over wild type mice after being placed back within the cage that shocks had been given [14]. When this testing was repeated over time, the mutant mice showed an inability to extinguish this conditioned response as compared with the wild type mice [14].

#### **3. Conclusions and future studies**

Our studies have demonstrated that *Nf1* exon 23a expression is tightly regulated and it plays a key role in controlling Ras signaling and learning behaviors in mice. In the brain, when exon 23a inclusion is increased, e.g., as shown in the mutant *Nf1*23aIN/23aIN mice, the Ras-GAP function of neurofibromin decreased, leading to an increase in pERK1/2 activity, which results in defects in learning and memory behaviors.

Given the role of Ras/ERK in many brain functions, it is reasonable to predict additional behavioral defects in the mutant *Nf1*23aIN/23aIN mice. For example, regulated Ras/ERK signaling is known to modulate circadian as well as depressive behaviors [26, 27]. Future experiments can be established to examine such behaviors in the mutant mice.

The behavioral defects observed in the mutant *Nf1*23aIN/23aIN mice is very interesting in light of a prior study by Costa and colleagues [28]. In this study, *Nf1* exon 23a was deleted from the *Nf1* gene in mice. These mice also suffered from learning and memory impairments. Specifically, similar long-term spatial memory defects were observed in these mice in the same Morris water maze test [28]. Without exon 23a, these mice should have very low Ras/ERK activity, opposite of our mutant mice. However, both mutant mice display the same learning defect. These results lead to a tantalizing hypothesis that both isoforms of neurofibromin are required for optimal brain functions. Even though isoform 1, the one without exon 23a, is the predominant isoform in the brain, its deletion is detrimental. Thus, it appears that the potential for this exon to be included is important for normal brain functions. Is it possible that the alternative splicing is regulated dynamically so under certain physiological conditions exon 23a is included significantly more in certain areas of the brain? Give the complex nature of this question, only exquisitely designed experiments will reveal the answer.

Lastly, the ratio of neurofibromin isoform 1 and isoform 2 in neuronal tissues in NF1 patients has never been examined. It will be interesting to study if the ratio changes in patients and if so, how does the change contributes to the disease development.

#### **Acknowledgements**

We thank the former members of the Lou laboratory, Victoria Fleming, Xuan Gao, Melissa Hinman, Hieu Nguyen, Alok Sharma, Hua-Lin Zhou and Hui Zhu, for their work discussed in this chapter. Karl Mader was supported by a Cystic Fibrosis Foundation pilot grant to Hua Lou as part of the CWRU RDP, DRUMM19R0.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Alternative Splicing of Neurofibromatosis Type 1 Exon 23a Modulates Ras/ERK Signaling… DOI: http://dx.doi.org/10.5772/intechopen.99678*

#### **Author details**

Karl Andreas Mader1 and Hua Lou1,2,3\*

1 Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, USA

2 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, USA

3 The Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, USA

\*Address all correspondence to: hua.lou@case.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Ly, K.I. and J.O. Blakeley, The diagnosis and Management of Neurofibromatosis Type 1. Med Clin North Am, 2019. 103(6): p. 1035-1054.

[2] Ballester, R., et al., The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell, 1990. 63(4): p. 851-859.

[3] Martin, G.A., et al., The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell, 1990. 63(4): p. 843-849.

[4] Xu, G.F., et al., The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell, 1990. 63(4): p. 835-841.

[5] DeClue, J.E., et al., Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell, 1992. 69(2): p. 265-273.

[6] Gutmann, D.H., J.L. Cole, and F.S. Collins, Expression of the neurofibromatosis type 1 (NF1) gene during mouse embryonic development. Prog Brain Res, 1995. 105: p. 327-335.

[7] Gutmann, D.H., et al., Expression of the neurofibromatosis 1 (NF1) isoforms in developing and adult rat tissues. Cell Growth Differ, 1995. 6(3): p. 315-323.

[8] Daston, M.M. and N. Ratner, Neurofibromin, a predominantly neuronal GTPase activating protein in the adult, is ubiquitously expressed during development. Dev Dyn, 1992. 195(3): p. 216-226.

[9] Ryu, H.H., et al., Enriched expression of NF1 in inhibitory neurons in both mouse and human brain. Mol Brain, 2019. 12(1): p. 60.

[10] Andersen, L.B., et al., A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity. Mol Cell Biol, 1993. 13(1): p. 487-495.

[11] Yunoue, S., et al., Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras. J Biol Chem, 2003. 278(29): p. 26958-26969.

[12] Baizer, L., et al., Regulated expression of the neurofibromin type I transcript in the developing chicken brain. J Neurochem, 1993. 61(6): p. 2054-2060.

[13] Huynh, D.P., T. Nechiporuk, and S.M. Pulst, Differential expression and tissue distribution of type I and type II neurofibromins during mouse fetal development. Dev Biol, 1994. 161(2): p. 538-551.

[14] Nguyen, H.T., et al., Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice. Hum Mol Genet, 2017. 26(19): p. 3797-3807.

[15] Guo, X., et al., Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay. J Vis Exp, 2018(138).

[16] Sharma, A., et al., Calciummediated histone modifications regulate alternative splicing in cardiomyocytes. Proc Natl Acad Sci U S A, 2014. 111(46): p. E4920-E4928.

[17] Barron, V.A. and H. Lou, Alternative splicing of the neurofibromatosis type I pre-mRNA. Biosci Rep, 2012. 32(2): p. 131-138.

*Alternative Splicing of Neurofibromatosis Type 1 Exon 23a Modulates Ras/ERK Signaling… DOI: http://dx.doi.org/10.5772/intechopen.99678*

[18] Zhu, H., et al., Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA. Mol Cell Biol, 2008. 28(4): p. 1240-1251.

[19] Hinman, M.N., et al., All three RNA recognition motifs and the hinge region of HuC play distinct roles in the regulation of alternative splicing. Nucleic Acids Res, 2013. 41(9): p. 5049-5061.

[20] Barron, V.A., et al., The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation. Nucleic Acids Res, 2010. 38(1): p. 253-264.

[21] Fleming, V.A., et al., Alternative splicing of the neurofibromatosis type 1 pre-mRNA is regulated by the muscleblind-like proteins and the CUG-BP and ELAV-like factors. BMC Mol Biol, 2012. 13: p. 35.

[22] Schor, I.E., L.I. Gomez Acuna, and A.R. Kornblihtt, Coupling between transcription and alternative splicing. Cancer Treat Res, 2013. 158: p. 1-24.

[23] Hinman, M.N., et al., Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signaling in neurons. Mol Cell Biol, 2014. 34(12): p. 2188-2197.

[24] Zhou, H.L., et al., Hu proteins regulate alternative splicing by inducing localized histone hyperacetylation in an RNA-dependent manner. Proc Natl Acad Sci U S A, 2011. 108(36): p. E627-E635.

[25] Bibel, M., et al., Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat Protoc, 2007. 2(5): p. 1034-1043.

[26] Serchov, T. and R. Heumann, Ras activity tunes the period and modulates the entrainment of the Suprachiasmatic clock. Front Neurol, 2017. 8: p. 264.

[27] Marsden, W.N., Synaptic plasticity in depression: Molecular, cellular and functional correlates. Prog Neuropsychopharmacol Biol Psychiatry, 2013. 43: p. 168-184.

[28] Costa, R.M., et al., Learning deficits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1. Nat Genet, 2001. 27(4): p. 399-405.

#### **Chapter 8**

## Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins

*Charoula Peta, Emmanouella Tsirimonaki, Constantinos Fedonidis, Xeni Koliou, Nikos Sakellaridis and Dimitra Mangoura*

#### **Abstract**

Neurofibromatosis type 1, NF-1, is a common monogenic (*NF1*) disease, characterized by highly variable clinical presentation and high predisposition for tumors, especially those of astrocytic origin (low- to high-grade gliomas). Unfortunately, very few genotype–phenotype correlations have been possible, and the numerous identified mutations do not offer help for prognosis and patient counselling. Whole gene deletion in animals does not successfully model the disease, as NF-1 cases caused by point mutations could be differentially affected by cell type-specific alternative splice variants of *NF1.* In this chapter, we will discuss the differential Microtubule-Associated-Protein (MAP) properties of NLS or ΔNLS neurofibromins, produced by the alternatively splicing of exon 51, which also contains a Nuclear Localization Sequence (NLS), in the assembly of the mitotic spindle and in faithful genome transmission. We will also commend on the major theme that emerges about NLS-containing tumor suppressors that function as mitotic MAPs.

**Keywords:** NLS and ΔNLS neurofibromins, astrocyte, glioblastoma, astral microtubules, spindle, Microtubule-Associated-Proteins, chromosome segregation

#### **1. Introduction**

Neurofibromatosis type 1 (NF-1) is a common, complex multisystem cancer predisposition syndrome, with a worldwide incidence at birth of 1: 2–3000 people [1] and a documented high mortality mostly due to malignancies [2]. NF-1 is caused by autosomal, dominantly inherited or de novo (50: 50 [3]) pathogenic mutations in the *NF1* gene, which encodes the large protein neurofibromin. The *NF1* gene was identified 30 years ago, yet with over 3000 different mutations identified thus far [4], only very few genotype–phenotype correlations have been postulated [5–7]. Affected individuals may present with a wide range of clinical manifestations, mostly from the Central Nervous System, CNS and the Peripheral Nervous System, PNS [1], as the *NF1* gene remains highly expressed there, whereas is downregulated in most other tissues in the adult. Thus, most NF-1 tumors are found in the CNS (gliomas) or the PNS (plexiform neurofibromas, malignant peripheral nerve sheath tumors, or the hallmark of the disease sub- and cutaneous neurofibromas), while there is increased risk for other cancers mostly of neural crest origin [8, 9].

In particular high-grade gliomas are more frequent in adults with NF-1, which have 5 times greater risk for glioblastoma (GBM) than the general population [10]. In addition, the great mutation rate of the *NF1* gene, which has also made its cloning impossible, is now recognized in ~20% of sporadic GBM [11]. In terms of specific treatments none are available for the cancers of NF-1 patients. Many drugs, like anti-angiogenic agents [12], have shown no responses, and MEK1/2 inhibitors have been approved only for plexiform neurofibromas. GBM prognosis remains dire (~2 years) even with the use of the highly cytotoxic temozolomide, while clinicians daily struggle with decisions for affected children. Unfortunately, gliomas frequently are resistant to temozolomide therapy and the candidate mechanism, for other tumors too, is the formation of tumor microtubes [13]. These recently recognized long, highly rich in actin, dynamic membrane protrusions of astrocytoma cells form a network for multicellular communication that promotes tumor growth and invasion of the brain. Therefore, understanding the cytoskeleton associations of neurofibromin is highly important in the effort to identify new therapeutic targets.

Confirmation of causative mutation with molecular diagnosis is a difficult task, as the large *NF1* gene of over 400 Kb and 57 exons has no mutational hot spots and one of the highest mutation rates in human genetic disorders, which explains the high incidence of *de novo* variants even within the same family. The complicated behavior of the gene is further highlighted from genetic manipulations of mice. When *Nf1* along with two more tumor suppressor genes (TSGs) were targeted with CRISPR/Cas in the forebrain of E13.5 mice, aggressive tumors resembling human GBMs were produced; however, whole-genome sequencing of the induced GBMs hinted to a very variable repair of CRISPR-induced double-strand breaks, potentially locus-specific [14]. Thus, mouse genetic NF-1 models have been marginally helpful in designing prognostic tests or therapies.

Even when timely, molecular diagnosis may only rarely offer help for prognosis or consultation [5, 7] and the challenge to correlate genotype–phenotype in this disease of uncontrolled cell growth and tumorigenesis remains largely unmet. It is our opinion that the impact of the various mutations will be best appreciated, once our currently limited knowledge on the functions of the distinct neurofibromin protein domains (**Figure 1**) will be expanded. As we will elaborate, these domains perform critical functions, evidently through inter- and intra-molecular interactions, most notably with tubulins, all of which are altered by cell type-specific, alternative

#### **Figure 1.**

*Major NF1 splicing events and neurofibromin domains with functional importance in the CNS. A, the two major alternative exons in the NF1 gene, namely 31 and 51, which produce* GRDI *or* GRDII *and*  NLS *or* ΔNLS *transcripts, respectively. B, Neurofibromin domains of known functional importance: CSRD, Cysteine/Serine-Rich domain; GRD, GAP-related domain; SEC14, Yeast Sec14p-like domain; PH, pleckstrin homology domain; CTD, C-Terminal domain and NLS, Nuclear localization sequence. Amino acid numbers for all putative isoforms are based on GRDI-NLS neurofibromin (Ensembl transcript NF1–201).*

#### *Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

splicing events and post-translational modifications, with mutations adding a supra level of complexity that must be met.

Indeed, the quest for genotype–phenotype correlation is complicated by developmental stage- and tissue- or cell type-specific alternative splicing events of the *NF1* gene, which is downregulated in most tissues in the adult except CNS and PNS tissues. While several alternative exons have been described, three are common in the CNS, namely 9a/9br, 31 (former 23), and 51 (former 43) [15–17]. Addition of the small alternative exon 9a/9br correlates well with neuronal differentiation and is downregulated in oligodendrogliomas [15, 18], but no specific function assignments have been made thus far. In contrast, inclusion of the other two, namely 31 and 51, does have important functional consequences, as will be discussed next.

Skipping of alternative exon 31, which corresponds to the center of the RasGAPrelated domain (GRD) of neurofibromin (**Figure 1A**) through which neurofibromin inactivates Ras, generates two variants accordingly named as GRD type I, GRDI and, if exon 31 is included, as GRD-type II, GRDII [16]. Exon 31 is mostly skipped in CNS neurons early on, whereas it is retained as the prominent transcript in astrocytes [19–21]. Due to the central role of Ras in many cellular functions and in carcinogenesis, GRDs have received high attention. Both GRDs are functional RasGAPs [22], when overexpressed in vitro [23] and as we showed in vivo [24], albeit GRDI is a much more potent RasGAP than GRDII.

Nevertheless, no significant rescue capacity of GRDs alone has been shown for NF-1 phenotypes, leading to the characterization of such phenotypes as Rasindependent by many researchers [25–27]. Along several such scientific efforts, the importance of other domains (**Figure 1B**) in the allosteric regulation of GRD has been established. Indeed, collective experimental evidence has postulated that neurofibromin domains may bind each other to form dimers [28], as well as, multiple proteins to coordinate Ras signalling ([25–27, 29, 30], reviewed in [31]). For example, in glioma cells overexpression of CSRD plus GRD -after phosphorylations by PKCε or α- binding to cortical F-actin increases and imposes a positive allosteric regulation on GRD and thus intense Ras deactivation, which is sufficient to switch the effect of EGF signalling from proliferation to differentiation [30]. While this mechanism was the first provided answer to the open question of how RasGAPs are recruited to the membrane, its clinical significance was directly postulated when large cohorts of NF-1 patients, heterozygous for nonsynonymous mutations of any of five successive amino acids in the CSRD, were found to have high, >50% predisposition to malignancies as compared to the general NF-1-affected population [5, 7]. SEC14, also reported to halt glioma cell invasion [32], is a domain that mediates binding to phospholipids [33, 34] and, as we showed, imposes, like CSRD, a positive allosteric regulation on GRD and accelerates Ras deactivation, potent enough to switch the activation of ERK from an analogous to a digital mode [24].

Exon 51 in the CTD contains the NLS, a sequence of basic amino acid clusters required for proteins of >45 kDa to dock onto the nuclear pore complex as a cargo for nuclear import. The necessary energy expenditure and the directionality of the import is provided by a gradient of RanGDP in the cytoplasm and RanGTP in the nucleus. A similar Ran gradient, generated around duplicated chromosomes during mitosis, allows the release of NLS-containing mitotic proteins that regulate spindle assembly and congression of chromosomes [35, 36]. *NF1* exon 51 may be also alternatively transcribed, producing *NLS* or *non NLS (ΔNLS)* transcripts and corresponding NLS or ΔNLS neurofibromin isoforms (**Figure 1**). We first identified in silico this bipartite NLS and documented experimentally that most neurofibromin molecules reside in the nucleus in neurons [37].

Later genetic analysis [17] revealed that in those fetal tissues, which will not retain high levels of *NF1* expression in the adult, *ΔNLS* transcripts are expressed in higher percentages. In contrast, in tissues that *NF1* remains high in the adult, fetal expression of *ΔNLS* is very low early on and increases with development. Typical examples for the former is the liver (*ΔNLS* constitutes 25% of total *NF1* expression in the fetus and only 15% in the adult) and for the latter is the brain, where the meek expression of 1% rises by 4-fold in the adult. Thus, there is an upregulation of *ΔNLS* transcripts in the tissue most implicated in NF-1 pathology, that is the CNS [17].

Moreover, we recently addressed the pressing question of developmental regulation of exon 51 skipping/inclusion in CNS cell types, using chick embryo or the early postnatal mouse or rat brains. We find that expression of *ΔNLS* is first detected only when neurons become postmitotic with its levels rising from negligent to 10% of total *NF1* in mature neurons; in astrocytes in culture, *ΔNLS* transcripts rise along with those for glial fibrillary acidic protein (GFAP) and reach a level of ~5% of total *NF1* [19]. Thus, our studies postulate that in both neurons and astrocytes as many as four variants and neurofibromin isoforms may be expressed (**Figure 1**), while expression of *ΔNLS* transcripts and ΔNLS neurofibromins correlates with neuronal and astrocytic differentiation and underline the necessity to study the individual functions of ΔNLS and NLS neurofibromins.

The importance of NLS inclusion was totally unexplored, till we documented a few years ago that neurofibromin controls the pivotal function of chromosome congression on the mitotic spindle [38] (**Figure 2**) and then proved that depletion of NLS neurofibromins deregulates spindle assembly and positioning, leading to aneuploidy and increased micronuclei formation [39]. More importantly, these studies established the function of neurofibromins as MAPs. All currently known impacts of this function will be presented in more detail in the next section.

In concluding this introduction, we believe that the importance of exploring novel yet fundamental questions on the functions of NLS neurofibromin isoforms is tantamount for understanding patient phenotypes and designing prognostic tools for NF-1 glioma growth and NF-1 mutation-specific therapies.

#### **Figure 2.**

*Neurofibromins regulate spindle configuration and chromosome congression. A, Mitotic spindles consist of three major types of microtubules (MTs): astral MTs that radiate from the centrosomes/poles, microtubule bundles (K-fibers) to link kinetochores to poles, and interpolar bundles to separate poles, elongate the spindle, and bridge K-fibers [40]. B, SF268 glioblastoma cells, transduced with mock or NF1*-*specific siRNAs, are stained for neurofibromin,* β*-tubulin, and chromatin, as indicated. In mock siRNA-cells, neurofibromin decorates astral MTs (asterisks) and both K-fibers and interpolar MTs (arrowheads), in a symmetric spindle with properly aligned chromosomes at its equator (white arrows). Depletion of neurofibromins (siNF1) causes irregularities in the spindle geometry and chromosome congression aberrations (yellow arrows) [38]. Images are the maximal intensity projection of 0.34* μ*m confocal plane stacks.*

Therefore, in this Chapter we will focus on novel insights on the MAP function of neurofibromins from our recent studies.

#### **2. Neurofibromins as MAPs and their role in mitosis**

Tubulins rapidly form highly dynamic noncovalent polymers, the microtubules, which execute essential functions for the constant yet ever-changing needs of all cells, such as function-coupled shapes, directed intracellular transport, migration, and, most importantly for the development of an organism, properly oriented cell divisions with accurate genomic transmission. For cell division, several types of MTs organize, elongate, and orient a bipolar spindle, through which chromosomes will position at the spindle equator for faithful sister chromatid separation and then segregation to the two daughter cells ([41–43] and **Figure 2A**).

Accordingly, multitudes of structurally different MAP proteins associate with MTs to regulate MT nucleation, polymerization, organization, bundling, and crosslinking in preparation for and completion of mitosis. The availability of mitotic MAPs is tightly regulated by coordinated transcription, as well as by cell cycle-dependent post-translational modifications, most often phosphorylations that control protein trafficking, homeostasis, and inter- or intramolecular interactions [44–46]. Aberrations in these processes may lead to aneuploidy and on to tumorigenesis, thus the ability of MAPs to alter MT dynamics is recognized for its prognostic value in cancer and as a target for cancer chemotherapies [47, 48].

Association of neurofibromin with cytosolic MTs was first established by confocal microscopy in fibroblasts and the molecule was proposed to act as a MAP, through a small segment (residues 815–834 in the CSRD) bearing in silico homology to MAPs Tau and MAP2 [49]. Since then, diverse experimental approaches, including co-immunoprecipitations, co-purifications, in vitro MT assembly, and affinity precipitations, have further documented this interaction with cytosolic [23, 24, 29, 37, 38, 50] and with mitotic MTs [38, 39].

Indeed, confocal image analysis of primary or tumor cells derived from the ectoderm or the neural crest and quadruply stained for β-tubulin, neurofibromin, F-actin and chromatin/chromosomes, shows that pools of endogenous neurofibromin colocalize with cytoplasmic MTs (e.g., rat astrocytes in **Figure 3A**, yellow arrows), as well as with F-actin, mainly at the cell cortex and lamellipodia ([37–39]; **Figure 3B**, yellow arrows). Interestingly, no association could be established with any intermediate filament in neural cells, as for example with the abundant astrocytic marker GFAP (**Figure 3C**), except for nuclear lamins [38].

In an earlier study with primary neurons, we found that neurofibromin, in addition to its colocalization with cytosolic MTs, localizes also in the nucleus and identified a bipartite NLS in the CTD (**Figure 1B**) as the probable mediator of nuclear entry [37]. Previously thought as an artifact in the skin epithelium, nuclear neurofibromin is detected with a variety of techniques, i.e., immunocytochemistry (e.g., white arrows in **Figure 3**), subcellular fractionations, or proteomics [28, 29, 37–39, 51–53] in all cells of an ectodermal origin.

We next provided evidence that the nuclear entry of neurofibromin is active, that is through interactions of its NLS with the Ran/importin system [38], as now shown in cancer breast cells [54]. Moreover, we established that a requirement for the cell-cycle regulated nuclear entry of neurofibromin is phosphorylation by Protein Kinase C (PKC) on Serine2808, a residue relatively close to the NLS [29, 38], which is retained in both isoforms. As neurofibromin expression patterns and nuclear regulation appeared to have all the attributes of a mitotic factor, in particular of a MAP, we next addressed the possibility in cells that regularly undergo mitosis.

#### **Figure 3.**

*Endogenous neurofibromins colocalize with both A. cytoplasmic MTs and B. F-actin, but not with C. GFAP. Rat primary astrocytes are stained for neurofibromin, chromatin (Hoechst 33258) and A,* β*-tubulin, B, F-actin (phalloidin) or C, GFAP. Images are the maximal intensity projection of 0.34* μ*m confocal planes; yellow arrows point to co-localization of neurofibromin with cytoplasmic MTs in A and actin in B; white arrows point to nuclear neurofibromin.*

Thus, we have postulated that neurofibromin primarily co-localizes with β-tubulin at all stages of spindle assembly from prophase to metaphase (e.g., **Figure 2B**) and then through the transformation of the spindle to a machinery for chromosome segregation and cell division, that's is through anaphase, telophase and cytokinesis [38, 39]. Again, neurofibromin's localization onto the spindle is apparent in all cells of ectodermal origin, with no exception. More relevant for gliomagenesis, endogenous neurofibromin in primary cortical or cerebellar astrocytes colocalizes with all three tubulin classes on microtubular structures, that is with α- and β-tubulin throughout mitosis and with γ-tubulin at the centrosomes at interphase and when duplicated for entrance to mitosis [19, 38, 39].

Experimentally, at least three neurofibromin domains have been previously identified to bind tubulins, namely GRD, SEC14, and CTD. Affinities to tubulin for the first two domains were explored for regulation of neurofibromin's RasGAP activity and the third for baiting neurofibromin associated proteins or for nuclear import studies. Thus, GRDI-tubulin interactions lead to a partial inhibition of its cytosolic GAP activity [23] and certain patient mutations in GRD impair the ability of neurofibromin to associate with MTs [55], while the competition of tubulin with H-Ras for binding to SEC14 that we found in COS cells may provide an explanation

*Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

and a mechanism for neurofibromin dissociation away from cytoplasmic microtubules [24]. As for CTD, it baits the plus-end MAP Collapsin response mediator protein 2 (CRMP2) [50], while we have shown high affinity of GFP-CTD(+NLS) to α-, β- and γ-tubulins [38].

The importance of neurofibromin as a mitotic MAP was first discovered, when we showed that siRNA-depletion of all transcripts and thus of all neurofibromin isoforms leads to severe errors in chromosome congression, with chromosomes remaining unattached or randomly away from the spindle equator even at full metaphasic spindle length (**Figure 2B**, yellow arrows and [38]). Typically, the unstable lateral interactions between kinetochores and microtubules, which dominate early prometaphase, lead to the reproducible arrangement of chromosomes in an equatorial ring, or torus-like distribution on the surface of the spindle [56]. This loss of the toroidal arrangement of chromosomes with neurofibromin depletion has to be the first evidence that neurofibromin may act to stabilize microtubule for chromosome congression. Consistent with this, using overexpressions of our human CTD construct but not of other domains, abnormal bypassing of mitotic arrest was rescued in the yeast, after the yeast homologs of neurofibromin Ira1 and 2 were deleted [25].

The importance of neurofibromin isoforms as major mitotic MAPs was next discovered, when we probed the individual effects of neurofibromin isoforms that differ in the sequence of the 41 amino acids encoded by exon 51, namely of ΔNLS- and NLS-neurofibromins, on mitotic spindle assembly and faithful genomic transmission [39]. These effects will be highlighted next.

#### **3. NLS and** Δ**NLS neurofibromins are different MAPS**

To further address the mechanism by which neurofibromin regulates chromosome congression and considering together that a. neurofibromin accumulates in the nucleus in a Ran-dependent manner at late S/G2 and resides on the spindle throughout mitosis [38], b. the major cellular target in NF-1 for abnormal proliferation and carcinogenesis is the astrocyte [10], and c. the higher expression of NLSover ΔNLS -*NF1* transcripts in astrocytes [19], we next evaluated separately the roles of ΔNLS- and NLS-isoforms in spindle assembly and chromosome segregation in an astrocytic cell context.

For these purposes, we have generated SF268 glioblastoma cell lines that stably express, under the control of doxycycline, shRNAs specifically designed to degrade either both GRDI- and GRDII-ΔNLS or both GRDI- and GRDII-NLS-*NF1* transcripts (referred to as NLS-cells and ΔNLS-cells, respectively). This reversible genetic modification has allowed us to pose the question of possible different functions of ΔNLS- and NLS-isoforms and dissect their roles in mitosis [39].

Confocal image analysis of cells immunostained for β-tubulin and neurofibromin and co-stained for filamentous actin, along quantitation of colocalization, postulates that ΔNLS neurofibromins are absent from the nucleus [39]. Moreover, in ΔNLScells association of neurofibromin with F-actin is significantly limited, especially in lamellipodia, whereas NLS-neurofibromins richly decorate them along other actin structures. Association with tubulin is not significantly reduced in ΔNLS-cells, yet microtubule organizing centers (MTOCs) are discerned with difficulty, because MTs organize a dense but non-radial network. To the contrary, NLS neurofibromin colocalization with β-tubulin is significantly enhanced, although β-tubulin intensity itself is not increased [39].

This different robustness of MTOC formation among ΔNLS-cells and the parental or NLS-cells is functionally validated with cell migration after wound (scratch) assays. In astrocytes, relocation of their major MTOC, the centrosome, between

the nucleus and the leading-edge during migration is well explained [57]. When positions of centrosomes and nuclei are observed in cells stained for γ-tubulin and Hoechst, respectively, confocal microscopy shows that, unlike parental and NLScells, it is readily apparent that centrosomes in ΔNLS-cells fail to position properly, remaining randomly oriented [39]. Hence, time-lapse video microscopy of cells during wound healing confirms that NLS-cells and the parental SF268 cells move with a directed, multicellular movement, while ΔNLS-cells, moving almost as fast, perform a palindromic motion and fail to repair the "scratch wound" (videos in [39]). Overall, this is the first time that neurofibromin is linked to astrocytic cell migration, and, at least the loss of NLS neurofibromins, to defective centrosome positioning and functional cell polarity [39].

Both types of neurofibromins retained colocalization with β-tubulin on the mitotic spindle, albeit colocalization levels with NLS-neurofibromin are, as also for cytosolic MTs, significantly raised (**Figure 4**, images, plots, and colocalization means; [39]). Considering together that NLS neurofibromins do not affect MT densities, whereas ΔNLS-neurofibromins inversely regulate MT densities both in the cytoplasm and the spindle [39], these data document differential MAP properties for ΔNLS-neurofibromins as compared to NLS-neurofibromins.

Examinations of colocalization with γ-tubulin on the duplicated centrosome, show a 25% decrease for NLS-neurofibromins, while ΔNLS neurofibromins show no statistical differences on this aspect. Yet, centrosomes in ΔNLS cells have larger volumes (1.8x), indicating that NLS neurofibromins may help form a more efficient mitotic centrosome, in terms of future spindle assembly [38, 39]. In human cells, the centrosome is the major MTOC for spindle MT assembly and duplicated centrosomes serve as poles to orient the spindle. More specifically, γ-tubulin and its several associated proteins form a large ring complex (γ-TuRC) that serves to

#### **Figure 4.**

*NLS- and* Δ*NLS-neurofibromins have different affinities for mitotic MTs. Naïve,* Δ*NLS-, and NLS- SF268 cells are stained for neurofibromin and* β*-tubulin. The first two columns contain single focal 0.34* μ*m planes of a confocal stack and the third column their mergings. Scatter plots show signal intensity in each plane (Volocity®) and numbers are the colocalization means±SE; arrow indicates the statistically significant difference in NLS- versus* Δ*NLS- or parental cells.*

*Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

nucleate highly dynamic MTs from the spindle, K-fibers from kinetochores, and interpolar bundles that elongate the spindle [43, 58, 59]. Because γ-TuRC is dispensable for this purpose on occasion [60], the role of MAP-dependent regulation in the nucleation to centrosomes, whether increasing [61] or inhibiting nucleation [62], has been highlighted.

The differential properties of the NLS and ΔNLS isoforms as MAPs on centrosome size are further highlighted by the effects on MT nucleation prior to nuclear envelope breakdown, when the relatively sparse microtubule formations of interphase cells transfigure into a bipolar spindle. Indeed, our studies have documented that the following parameters are greatly affected with depletion of NLS neurofibromins [39]:

#### **3.1 Astral MT formation and spindle positioning**

A most striking difference is the abnormal astral MT growth patterns with loss of NLS neurofibromins, as the average length of astral MTs in NLS-cells grows to 5.9 ± 0.33 μm over 4.0 ± 0.19 μm in parentals (p < 0.0001), while is robustly diminished in ΔNLS-cells (**Figures 4** and **5**, asterisks; [39]). Proper astral formation is required for spindle position and aberrations of astral MTs correlate well with spindle misorientation [41], and we too find that loss of astral MTs with loss of NLSneurofibromin leads to statistically differential positioning of the spindle by several degrees [39]. A number of diverse families of proteins impact the timely nucleation and maintenance of astral MTs, yet few may cause loss of astral MTs. Notably, functional ablation (phosphoablating mutants) of End-binding protein 2 (EB2), an MT plus-end MAP that binds to MT lattices in a phosphorylation-dependent manner

#### **Figure 5.**

*Spindle configuration and chromosome congression are regulated by neurofibromin isoforms. A, Immunofluorescence confocal images of cells at metaphase stained for* β*-tubulin and chromatin of SF268, NLS, and* Δ*NLS-SF268 cells. B, 3D reconstructions (IMARIS) of the same images for better viewing show that parental and NLS-cells have a rich array of astral MTs (asterisks) and properly congressed chromosomes (white arrows). In contrast,* Δ*NLS cells lack astral MTs (asterisks) and display abnormal chromosome congression (yellow arrows).*

during mitosis, leads to a marked delay in anaphase onset and abnormalities in chromosome congression [63]. Multifunction proteins ALIX and RACK1 acting through regulation of other MAPs [64] or motor proteins [65], are also essential for proper astral MT elongation, spindle orientation and chromosome segregation. Whether neurofibromins regulate in addition the actions of other MAPs remain to be investigated; it is clear, however, that NLS neurofibromins are essential for astral MTs formation.

#### **3.2 Spindle length**

Both parental and NLS-cells with fully developed metaphasic spindles have normally congressed chromosomes at the spindle equator (**Figure 5**, white arrows), albeit the latter have significantly shorter spindles (pole to pole distance *x* \_ = 8.91 ± 0.22 μm versus 10.8 ± 0.2 μm; p < 0.0001 [39]). In stark contrast, cells expressing only ΔNLS-neurofibromins have very poorly aligned chromosomes at the equator (**Figure 5**, yellow arrows), although their metaphasic spindle length is significantly longer (*x* \_ = 11.5 ± 0.15 μm; p < 0.01;). In over 50% of ΔNLS cells, the majority of chromosomes from a wide diffused ring and altogether lack the typical tight alignment seen at metaphase (**Figure 5**, yellow arrows).

#### **3.3 Spindle geometry**

When confocal z-planes of β-tubulin and Hoechst fluorescence signals are reconstructed in three-dimensions, the dramatically different spindle geometries, formed in the absence of NLS-neurofibromins, become readily apparent (**Figure 5B**). The anastral spindles of ΔNLS cells feature large hollows by the equator and chromosomes forming queues on some prominent thick K-fibers, while over half of the cells have unaligned chromosomes, or a 4-fold increase compared to control and NLS-cells [39]. In interpreting this geometry, we assume that thicker MT formations may result from upregulation of the augmin-mediated, local amplification of MTs, as augmin targets γ-TuRCs to nucleate preexisting MTs [66]; in parallel, bridging (**Figure 2A**) MTs [40] delay to develop, hence the spindle equator is almost devoid of tubulin signals. As these metaphasic patterns [39] strongly resemble those typically seen at prometaphase when unstable interactions of MTs dominate [56], the important role of neurofibromins as MAPs for mitosis is further highlighted.

#### **3.4 Duration of mitotic phases**

Abnormal positioning of the spindle often associates with altered times spent at mitotic stages. Quantification of the mitotic stage distribution for each cell type by flow cytometry validates this prediction, since loss of NLS-neurofibromin elicits an almost 50% increase in time spent at metaphase [39]. In contrast, NLS cells have significantly lower percentages in prophase and metaphase over parentals, which, combined with higher percentages in cytokinesis, reflected an overall acceleration through metaphase. Considered together, these results document for the first time that neurofibromin actively participates in the progression of mitosis. Moreover, these data further support the notion that NLS- and ΔNLS-neurofibromins may exert opposing effects during aster formation and spindle assembly, as, in parental cells, these two parameters appear to be the arithmetic sum of the results obtained with each isoform type [39].

#### **3.5 Chromosome segregation fidelity**

In parental (**Figures 2** and **5**, white arrows) and NLS-neurofibromin expressing cells (**Figure 5**, white arrows), chromosomes move in a coordinated manner towards the opposed poles and chromosome compaction is readily seen. In cells expressing only ΔNLS-neurofibromins, these parameters are again inversely regulated, namely, despite the prolonged time spent at metaphase, a significant >40% increase in cells with chromosome segregation errors mainly lagging chromosomes is documented (**Figure 5**, yellow arrows); similar delays in chromosome compaction in ΔNLS-neurofibromin cells are recorded in telophase [39]. The described effects on spindle assembly and chromosome segregation perturbations are readily traced in the high frequency of micronuclei, and a 5-fold increase in the numbers of cells with micronuclei within 10 mitotic cycles [39]. Micronuclei may facilitate rapid karyotype evolution, as their few chromosomes, unprotected from DNA damage, often undergo chromothripsis and chromoanasynthesis and then get incorporated into the genome of the host cell within just 1–2 mitoses [67].

Summarizing, these data establish for the first time that NLS- and ΔNLSneurofibromins actively participate in the formation of mitotic asters and spindles, and efficient, error-free chromosome congression, possibly by exerting opposing effects. The question then rises about the possible mechanisms that would explain their different interactions with tubulins and microtubules. Drawing from immunoprecipitations studies with various antibodies, whereby different amounts of endogenously expressed neurofibromin is recovered from ΔNLS- or NLS-cell lysates [39], we have to reasonably presume that inclusion, or not, of the 41 amino acids of exon 51 may alter the conformation of the molecule. Numerous examples exist when one to few residues change the functional properties of a protein by imposing changes on protein conformation and posttranslational modifications. Thus, an expected differential conformation of the ΔNLS or NLS neurofibromins would impact both its known intramolecular and intermolecular interactions.

In support of this argument the affinity of NLS- is higher for β- and lower for γ-tubulin when compared to ΔNLS-neurofibromins. Moreover, revisiting the question of MAP domains in the primary sequence of neurofibromin, we have identified, at higher percentages of similarity than the previously suggested [49], three other small Tau-like motifs [39], one of which corresponds to codons apposed to 50–51 or 50–52 exon junctions and could be affected by the inclusion or skipping of exon 51.

Our results show that the direction of the ΔNLS or NLS knockdown effects is most often opposite and suggest that the two functionally interact when both present. Whether this occurs through the formation of a dimer, if neurofibromins form dimers [28, 68] in eukaryotic cells at normal neurofibromin concentrations, is an intriguing question. Indeed, how the NLS and ΔNLS conformations may affect dimer formation is an interesting experimental goal.

Given the higher abundance of *NLS* transcripts irrespectively of GRD type that we observe in neurons and astrocytes [19], it is not possible to have only NLS-ΔNLS heterodimers. It is, however, possible to have homodimers only, or dimerization to be driven by properties that GRDI or GRDII attain. If any of the latter are entertained in eukaryotic cells, then an additional level of regulation is to be expected. At any rate, loss of the amino acids and the NLS encoded by exon 51 suffices to produce a different MAP. By the same token, the expression of two closely related isoforms yet with differential effects on MT structures further suggests that an extra layer of regulation on MT dynamics is thereby served by neurofibromins.

#### **4. Conclusions: NLS-containing, tumor suppressor MAPS**

A major theme that emerges from our studies and studies by others is that several MAPs have been described as tumor suppressors and correspondingly several proteins, identified as such, are found to function as mitotic MAPs. Another typical characteristic of such tumor suppressors is the presence of an NLS in their amino acid sequence, which regulates both their timely nuclear import in preparation of mitosis and their release during spindle assembly. All currently known tumor suppressor proteins with such attributes are listed in **Table 1**.

Perturbations of spindle assembly and chromosome segregation, when tumor suppressors that act as mitotic MAPs are lost or mutated, is a first step to aneuploidy. Given the usually compromised ability for DNA repair and the increased replication stress in these genetic backgrounds, the resulting aneuploidy may additionally feed chromosomal instability (CIN) and thus rapid evolution of karyotypes with clonal expansion advantages and tumorigenesis [104, 105]. Hence, the study of the regulation of NLS-containing tumor suppressors must receive high attention in the collective effort of understanding their mechanism of action and for developing better prognostic and possibly therapeutic approaches.


#### **Table 1.**

*Tumor Suppressors with a functional NLS and established roles as MAPs.*

*Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Nomenclature**


### **Author details**

Charoula Peta1,2, Emmanouella Tsirimonaki1,2, Constantinos Fedonidis1 , Xeni Koliou1 , Nikos Sakellaridis2 and Dimitra Mangoura1 \*

1 Basic Research Center, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

2 Pharmacology, Medical School, University of Thessaly, Larissa, Greece

\*Address all correspondence to: mangoura@bioacademy.gr

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Uusitalo E, Leppavirta J, Koffert A, Suominen S, Vahtera J, Vahlberg T, et al. Incidence and mortality of neurofibromatosis: a total population study in Finland. The Journal of investigative dermatology. 2015;135(3):904-6.

[2] Evans DG, O'Hara C, Wilding A, Ingham SL, Howard E, Dawson J, et al. Mortality in neurofibromatosis 1: in North West England: an assessment of actuarial survival in a region of the UK since 1989. European journal of human genetics : EJHG. 2011;19(11):1187-91.

[3] McKeever K, Shepherd CW, Crawford H, Morrison PJ. An epidemiological, clinical and genetic survey of neurofibromatosis type 1 in children under sixteen years of age. The Ulster medical journal. 2008;77(3):160-3.

[4] Stenson PD, Mort M, Ball EV, Chapman M, Evans K, Azevedo L, et al. The Human Gene Mutation Database (HGMD((R))): optimizing its use in a clinical diagnostic or research setting. Human genetics. 2020;139(10): 1197-207.

[5] Koczkowska M, Chen Y, Callens T, Gomes A, Sharp A, Johnson S, et al. Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844-848. American journal of human genetics. 2018;102(1):69-87.

[6] Upadhyaya M, Huson SM, Davies M, Thomas N, Chuzhanova N, Giovannini S, et al. An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation. American journal of human genetics. 2007;80(1):140-51.

[7] Xu M, Xiong H, Han Y, Li C, Mai S, Huang Z, et al. Identification of Mutation Regions on NF1 Responsible for High- and Low-Risk Development of Optic Pathway Glioma in Neurofibromatosis Type I. Frontiers in genetics. 2018;9:270.

[8] Holzel M, Huang S, Koster J, Ora I, Lakeman A, Caron H, et al. NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell. 2010;142(2):218-29.

[9] Opocher G, Conton P, Schiavi F, Macino B, Mantero F. Pheochromo cytoma in von Hippel-Lindau disease and neurofibromatosis type 1. Familial cancer. 2005;4(1):13-6.

[10] D'Angelo F, Ceccarelli M, Tala, Garofano L, Zhang J, Frattini V, et al. The molecular landscape of glioma in patients with Neurofibromatosis 1. Nature medicine. 2019;25(1):176-87.

[11] Cancer Genome Atlas Research N. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061-8.

[12] Gururangan S, Fangusaro J, Poussaint TY, McLendon RE, Onar-Thomas A, Wu S, et al. Efficacy of bevacizumab plus irinotecan in children with recurrent low-grade gliomas--a Pediatric Brain Tumor Consortium study. Neuro-oncology. 2014;16(2):310-7.

[13] Weil S, Osswald M, Solecki G, Grosch J, Jung E, Lemke D, et al. Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas. Neuro-oncology. 2017;19(10):1316-26.

[14] Zuckermann M, Hovestadt V, Knobbe-Thomsen CB, Zapatka M, Northcott PA, Schramm K, et al.

*Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nature communications. 2015;6:7391.

[15] Danglot G, Regnier V, Fauvet D, Vassal G, Kujas M, Bernheim A. Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5' part of the gene. Human molecular genetics. 1995;4(5):915-20.

[16] Suzuki Y, Suzuki H, Kayama T, Yoshimoto T, Shibahara S. Brain tumors predominantly express the neurofibromatosis type 1 gene transcripts containing the 63 base insert in the region coding for GTPase activating protein-related domain. Biochemical and biophysical research communications. 1991;181(3):955-61.

[17] Vandenbroucke I, Vandesompele J, De Paepe A, Messiaen L. Quantification of NF1 transcripts reveals novel highly expressed splice variants. FEBS letters. 2002;522(1-3):71-6.

[18] Gutmann DH, Zhang Y, Hirbe A. Developmental regulation of a neuronspecific neurofibromatosis 1 isoform. Annals of neurology. 1999;46(5): 777-82.

[19] Peta C, Tsirimonaki E, Fedonidis C, Koliou X, Mangoura D. Depletion of the developmentally-regulated, NLScontaining, major neurofibromin isoform imposes mitotic aberrations in mouse astrocytes. 2021.

[20] Karouzaki S, Theofilopoulos S, Mangoura D. Differential properties of neurofibromin GRD I and II in developing neurons. Journal of neurochemistry. 2011;118, Sup.1, p192.

[21] Barron VA, Lou H. Alternative splicing of the neurofibromatosis type I pre-mRNA. Bioscience reports. 2012;32(2):131-8.

[22] Andersen LB, Ballester R, Marchuk DA, Chang E, Gutmann DH, Saulino AM, et al. A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity. Molecular and cellular biology. 1993;13(1):487-95.

[23] Bollag G, McCormick F, Clark R. Characterization of full-length neurofibromin: tubulin inhibits Ras GAP activity. The EMBO journal. 1993;12(5):1923-7.

[24] Leondaritis G, Mangoura D, editors. Regulation of neurofibromin's GAPrelated domain RasGAP activity by a Sec14-homology domain-dependent allosteric switch. Trans 61th EEBMB; 2010.

[25] Luo G, Kim J, Song K. The C-terminal domains of human neurofibromin and its budding yeast homologs Ira1 and Ira2 regulate the metaphase to anaphase transition. Cell cycle. 2014;13(17):2780-9.

[26] Ozawa T, Araki N, Yunoue S, Tokuo H, Feng L, Patrakitkomjorn S, et al. The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2 cofilin pathway. The Journal of biological chemistry. 2005;280(47): 39524-33.

[27] Starinsky-Elbaz S, Faigenbloom L, Friedman E, Stein R, Kloog Y. The pre-GAP-related domain of neurofibromin regulates cell migration through the LIM kinase/cofilin pathway. Molecular and cellular neurosciences. 2009;42(4):278-87.

[28] Sherekar M, Han SW, Ghirlando R, Messing S, Drew M, Rabara D, et al. Biochemical and structural analyses reveal that the tumor suppressor

neurofibromin (NF1) forms a highaffinity dimer. The Journal of biological chemistry. 2020;295(4):1105-19.

[29] Leondaritis G, Petrikkos L, Mangoura D. Regulation of the Ras-GTPase activating protein neurofibromin by C-tail phosphorylation: implications for protein kinase C/Ras/extracellular signal-regulated kinase 1/2 pathway signaling and neuronal differentiation. Journal of neurochemistry. 2009;109(2):573-83.

[30] Mangoura D, Sun Y, Li C, Singh D, Gutmann DH, Flores A, et al. Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells. Oncogene. 2006;25(5):735-45.

[31] Scheffzek K, Shivalingaiah G. Ras-Specific GTPase-Activating Proteins-Structures, Mechanisms, and Interactions. Cold Spring Harbor perspectives in medicine. 2019;9(3).

[32] Fadhlullah SFB, Halim NBA, Yeo JYT, Ho RLY, Um P, Ang BT, et al. Pathogenic mutations in neurofibromin identifies a leucine-rich domain regulating glioma cell invasiveness. Oncogene. 2019;38(27):5367-80.

[33] D'Angelo I, Welti S, Bonneau F, Scheffzek K. A novel bipartite phospholipid-binding module in the neurofibromatosis type 1 protein. EMBO reports. 2006;7(2):174-9.

[34] Welti S, Fraterman S, D'Angelo I, Wilm M, Scheffzek K. The sec14 homology module of neurofibromin binds cellular glycerophospholipids: mass spectrometry and structure of a lipid complex. Journal of molecular biology. 2007;366(2):551-62.

[35] Kalab P, Pralle A, Isacoff EY, Heald R, Weis K. Analysis of a RanGTPregulated gradient in mitotic somatic cells. Nature. 2006;440(7084):697-701.

[36] Oh D, Yu CH, Needleman DJ. Spatial organization of the Ran pathway by microtubules in mitosis. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(31):8729-34.

[37] Li C, Cheng Y, Gutmann DA, Mangoura D. Differential localization of the neurofibromatosis 1 (NF1) gene product, neurofibromin, with the F-actin or microtubule cytoskeleton during differentiation of telencephalic neurons. Brain research Developmental brain research. 2001;130(2):231-48.

[38] Koliou X, Fedonidis C, Kalpachidou T, Mangoura D. Nuclear import mechanism of neurofibromin for localization on the spindle and function in chromosome congression. Journal of neurochemistry. 2016;136(1):78-91.

[39] Peta C, Tsirimonaki E, Samouil D, Georgiadou K, Mangoura D. Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation. Cells. 2020;9(11).

[40] Kajtez J, Solomatina A, Novak M, Polak B, Vukusic K, Rudiger J, et al. Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores. Nature communications. 2016;7:10298.

[41] di Pietro F, Echard A, Morin X. Regulation of mitotic spindle orientation: an integrated view. EMBO reports. 2016;17(8):1106-30.

[42] Maiato H, Gomes AM, Sousa F, Barisic M. Mechanisms of Chromosome Congression during Mitosis. Biology. 2017;6(1).

[43] Meraldi P. Centrosomes in spindle organization and chromosome segregation: a mechanistic view. Chromosome research : an international journal on the molecular, supramolecular and evolutionary

#### *Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

aspects of chromosome biology. 2016;24(1):19-34.

[44] Cuijpers SAG, Vertegaal ACO. Guiding Mitotic Progression by Crosstalk between Post-translational Modifications. Trends in biochemical sciences. 2018;43(4):251-68.

[45] He J, Zhang Z, Ouyang M, Yang F, Hao H, Lamb KL, et al. PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis. Nature communications. 2016;7:12355.

[46] Reed SI. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature reviews Molecular cell biology. 2003;4(11):855-64.

[47] Bhat KM, Setaluri V. Microtubuleassociated proteins as targets in cancer chemotherapy. Clinical cancer research : an official journal of the American Association for Cancer Research. 2007;13(10):2849-54.

[48] Schroeder C, Grell J, Hube-Magg C, Kluth M, Lang D, Simon R, et al. Aberrant expression of the microtubuleassociated protein tau is an independent prognostic feature in prostate cancer. BMC cancer. 2019;19(1):193.

[49] Gregory PE, Gutmann DH, Mitchell A, Park S, Boguski M, Jacks T, et al. Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules. Somatic cell and molecular genetics. 1993;19(3):265-74.

[50] Patrakitkomjorn S, Kobayashi D, Morikawa T, Wilson MM, Tsubota N, Irie A, et al. Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2. The Journal of biological chemistry. 2008;283(14):9399-413.

[51] Daub H, Olsen JV, Bairlein M, Gnad F, Oppermann FS, Korner R, et al. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. Molecular cell. 2008;31(3):438-48.

[52] Godin F, Villette S, Vallee B, Doudeau M, Morisset-Lopez S, Ardourel M, et al. A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line. Biochemical and biophysical research communications. 2012;418(4):689-94.

[53] Nousiainen M, Sillje HH, Sauer G, Nigg EA, Korner R. Phosphoproteome analysis of the human mitotic spindle. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(14):5391-6.

[54] Zheng ZY, Anurag M, Lei JT, Cao J, Singh P, Peng J, et al. Neurofibromin Is an Estrogen Receptor-alpha Transcriptional Co-repressor in Breast Cancer. Cancer cell. 2020;37(3): 387-402 e7.

[55] Xu H, Gutmann DH. Mutations in the GAP-related domain impair the ability of neurofibromin to associate with microtubules. Brain research. 1997;759(1):149-52.

[56] Magidson V, O'Connell CB, Loncarek J, Paul R, Mogilner A, Khodjakov A. The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly. Cell. 2011;146(4):555-67.

[57] Etienne-Manneville S, Hall A. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature. 2003;421(6924): 753-6.

[58] Maiato H, Rieder CL, Khodjakov A. Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis. The Journal of cell biology. 2004;167(5): 831-40.

[59] Meunier S, Vernos I. K-fibre minus ends are stabilized by a RanGTPdependent mechanism essential for functional spindle assembly. Nature cell biology. 2011;13(12):1406-14.

[60] Consolati T, Locke J, Roostalu J, Chen ZA, Gannon J, Asthana J, et al. Microtubule Nucleation Properties of Single Human gammaTuRCs Explained by Their Cryo-EM Structure. Developmental cell. 2020;53(5): 603-17 e8.

[61] Gard DL, Kirschner MW. A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. The Journal of cell biology. 1987;105(5):2203-15.

[62] Ringhoff DN, Cassimeris L. Stathmin regulates centrosomal nucleation of microtubules and tubulin dimer/polymer partitioning. Molecular biology of the cell. 2009;20(15):3451-8.

[63] Iimori M, Watanabe S, Kiyonari S, Matsuoka K, Sakasai R, Saeki H, et al. Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability. Nature communications. 2016;7:11117.

[64] Malerod L, Le Borgne R, Lie-Jensen A, Eikenes AH, Brech A, Liestol K, et al. Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics. The EMBO journal. 2018;37(13).

[65] Ai E, Poole DS, Skop AR. RACK-1 directs dynactin-dependent RAB-11 endosomal recycling during mitosis in *Caenorhabditis elegans*. Molecular biology of the cell. 2009;20(6):1629-38.

[66] Song JG, King MR, Zhang R, Kadzik RS, Thawani A, Petry S. Mechanism of how augmin directly targets the gamma-tubulin ring complex to microtubules. The Journal of cell biology. 2018;217(7):2417-28.

[67] Liu S, Kwon M, Mannino M, Yang N, Renda F, Khodjakov A, et al. Nuclear envelope assembly defects link mitotic errors to chromothripsis. Nature. 2018;561(7724):551-5.

[68] Lupton CJ, Bayly-Jones C, D'Andrea L, Huang C, Schittenhelm RB, Venugopal H, et al. The cryo-EM structure of the neurofibromin dimer reveals the molecular basis for von Recklinghausen disease. bioRxiv. 2021.

[69] Dikovskaya D, Khoudoli G, Newton IP, Chadha GS, Klotz D, Visvanathan A, et al. The adenomatous polyposis coli protein contributes to normal compaction of mitotic chromatin. PloS one. 2012;7(6):e38102.

[70] Green RA, Wollman R, Kaplan KB. APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Molecular biology of the cell. 2005;16(10):4609-22.

[71] Slep KC, Rogers SL, Elliott SL, Ohkura H, Kolodziej PA, Vale RD. Structural determinants for EB1 mediated recruitment of APC and spectraplakins to the microtubule plus end. The Journal of cell biology. 2005;168(4):587-98.

[72] Molina A, Velot L, Ghouinem L, Abdelkarim M, Bouchet BP, Luissint AC, et al. ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics. Cancer research. 2013;73(9):2905-15.

[73] Nehlig A, Seiler C, Steblyanko Y, Dingli F, Arras G, Loew D, et al. Reciprocal regulation of Aurora kinase A and ATIP3 in the control of metaphase spindle length. Cellular and molecular life sciences : CMLS. 2021;78(4):1765-79.

[74] Rodrigues-Ferreira S, Nehlig A, Moindjie H, Monchecourt C, Seiler C, *Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

Marangoni E, et al. Improving breast cancer sensitivity to paclitaxel by increasing aneuploidy. Proceedings of the National Academy of Sciences of the United States of America. 2019;116(47): 23691-7.

[75] Di Paolo A, Racca C, Calsou P, Larminat F. Loss of BRCA1 impairs centromeric cohesion and triggers chromosomal instability. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2014;28(12):5250-61.

[76] Joukov V, Groen AC, Prokhorova T, Gerson R, White E, Rodriguez A, et al. The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly. Cell. 2006;127(3): 539-52.

[77] Sung M, Giannakakou P. BRCA1 regulates microtubule dynamics and taxane-induced apoptotic cell signaling. Oncogene. 2014;33(11): 1418-28.

[78] Gao J, Huo L, Sun X, Liu M, Li D, Dong JT, et al. The tumor suppressor CYLD regulates microtubule dynamics and plays a role in cell migration. The Journal of biological chemistry. 2008;283(14):8802-9.

[79] Yang Y, Liu M, Li D, Ran J, Gao J, Suo S, et al. CYLD regulates spindle orientation by stabilizing astral microtubules and promoting dishevelled-NuMA-dynein/dynactin complex formation. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(6): 2158-63.

[80] Yu L, Shang ZF, Abdisalaam S, Lee KJ, Gupta A, Hsieh JT, et al. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochoremicrotubule attachments. Nucleic acids research. 2016;44(18):8842-54.

[81] Vitiello E, Ferreira JG, Maiato H, Balda MS, Matter K. The tumour suppressor DLC2 ensures mitotic fidelity by coordinating spindle positioning and cell-cell adhesion. Nature communications. 2014;5:5826.

[82] Etienne-Manneville S, Manneville JB, Nicholls S, Ferenczi MA, Hall A. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization. The Journal of cell biology. 2005;170(6):895-901.

[83] Ishii H, Vecchione A, Murakumo Y, Baldassarre G, Numata S, Trapasso F, et al. FEZ1/LZTS1 gene at 8p22 suppresses cancer cell growth and regulates mitosis. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(18):10374-9.

[84] Vecchione A, Baldassarre G, Ishii H, Nicoloso MS, Belletti B, Petrocca F, et al. Fez1/Lzts1 absence impairs Cdk1/ Cdc25C interaction during mitosis and predisposes mice to cancer development. Cancer cell. 2007;11(3): 275-89.

[85] Chaudhuri AR, Khan IA, Prasad V, Robinson AK, Luduena RF, Barnes LD. The tumor suppressor protein Fhit. A novel interaction with tubulin. The Journal of biological chemistry. 1999;274(34):24378-82.

[86] Vizeacoumar FJ, van Dyk N, F SV, Cheung V, Li J, Sydorskyy Y, et al. Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis. The Journal of cell biology. 2010;188(1):69-81.

[87] Cohen-Dvashi H, Ben-Chetrit N, Russell R, Carvalho S, Lauriola M, Nisani S, et al. Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression. EMBO molecular medicine. 2015;7(3): 299-314.

[88] den Bakker MA, Tascilar M, Riegman PH, Hekman AC, Boersma W, Janssen PJ, et al. Neurofibromatosis type 2 protein co-localizes with elements of the cytoskeleton. The American journal of pathology. 1995;147(5):1339-49.

[89] Smole Z, Thoma CR, Applegate KT, Duda M, Gutbrodt KL, Danuser G, et al. Tumor suppressor NF2/Merlin is a microtubule stabilizer. Cancer research. 2014;74(1):353-62.

[90] Yin F, Yu J, Zheng Y, Chen Q, Zhang N, Pan D. Spatial organization of Hippo signaling at the plasma membrane mediated by the tumor suppressor Merlin/NF2. Cell. 2013;154(6):1342-55.

[91] Contadini C, Monteonofrio L, Virdia I, Prodosmo A, Valente D, Chessa L, et al. p53 mitotic centrosome localization preserves centrosome integrity and works as sensor for the mitotic surveillance pathway. Cell death & disease. 2019;10(11):850.

[92] Giannakakou P, Sackett DL, Ward Y, Webster KR, Blagosklonny MV, Fojo T. p53 is associated with cellular microtubules and is transported to the nucleus by dynein. Nature cell biology. 2000;2(10):709-17.

[93] Morris VB, Brammall J, Noble J, Reddel R. p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study. Experimental cell research. 2000;256(1):122-30.

[94] van Ree JH, Nam HJ, Jeganathan KB, Kanakkanthara A, van Deursen JM. Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes. Nature cell biology. 2016;18(7):814-21.

[95] Dallol A, Hesson LB, Matallanas D, Cooper WN, O'Neill E, Maher ER, et al. RAN GTPase is a RASSF1A effector

involved in controlling microtubule organization. Current biology : CB. 2009;19(14):1227-32.

[96] Jeon HJ, Oh JS. RASSF1A Regulates Spindle Organization by Modulating Tubulin Acetylation via SIRT2 and HDAC6 in Mouse Oocytes. Frontiers in cell and developmental biology. 2020;8:601972.

[97] Rong R, Jin W, Zhang J, Sheikh MS, Huang Y. Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest. Oncogene. 2004;23(50): 8216-30.

[98] Coschi CH, Martens AL, Ritchie K, Francis SM, Chakrabarti S, Berube NG, et al. Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive. Genes & development. 2010;24(13):1351-63.

[99] Iovino F, Lentini L, Amato A, Di Leonardo A. RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts. Molecular cancer. 2006;5:38.

[100] Lyu J, Yang EJ, Zhang B, Wu C, Pardeshi L, Shi C, et al. Synthetic lethality of RB1 and aurora A is driven by stathmin-mediated disruption of microtubule dynamics. Nature communications. 2020;11(1):5105.

[101] Hell MP, Duda M, Weber TC, Moch H, Krek W. Tumor suppressor VHL functions in the control of mitotic fidelity. Cancer research. 2014;74(9): 2422-31.

[102] Thoma CR, Toso A, Gutbrodt KL, Reggi SP, Frew IJ, Schraml P, et al. VHL loss causes spindle misorientation and chromosome instability. Nature cell biology. 2009;11(8):994-1001.

[103] Shandilya J, Roberts SG. A role of WT1 in cell division and genomic stability. Cell cycle. 2015;14(9):1358-64. *Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins DOI: http://dx.doi.org/10.5772/intechopen.97574*

[104] Burrell RA, McClelland SE, Endesfelder D, Groth P, Weller MC, Shaikh N, et al. Replication stress links structural and numerical cancer chromosomal instability. Nature. 2013;494(7438):492-6.

[105] Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature. 2005;434(7035):907-13.

#### **Chapter 9**

## Metabolic Features of Neurofibromatosis Type 1-Associated Tumors

*Ionica Masgras and Andrea Rasola*

#### **Abstract**

Rewiring cellular metabolism is a key hallmark of cancer. Multiple evidences show that alterations in various metabolic circuits directly contribute to the tumorigenic process at different levels (e.g. cancer initiation, metastasis, resistance). However, the characterization of the metabolic profile of Neurofibromatosis type 1 (NF1)-related neoplastic cells has been only partially elucidated both in benign neurofibromas and in malignant peripheral nerve sheath tumors (MPNSTs). Here, we illustrate the state of the art on the knowledge of the metabolic features of tumors related to NF1 and discuss their potential implications for the development of novel therapeutic perspectives.

**Keywords:** NF1, metabolism, mitochondria, chaperones, sirtuins, MPNST, neurofibroma, glucose, glutamine, PET

#### **1. Introduction**

Neurofibromatosis type 1 (NF1) is a genetic multisystem disorder that predisposes to the onset of several tumor types and is characterized by a number of clinical manifestations encompassing café au lait macules in the skin, iris hamartomas (Lisch nodules), cognitive deficits, axillary or groin freckles, bone deformities, optic gliomas and Schwann cell neoplasms called neurofibromas. The presence of two or more of these clinical features is used as consensus diagnostic criteria for NF1 [1].

NF1 is inherited in an autosomal dominant way when inactivating mutations occur at the *NF1* locus that encodes for the Ras-GTPase activating protein (Ras-GAP) neurofibromin. Complete loss of neurofibromin activity, caused by second hit mutations, leads to hyperactivation of Ras signaling and tumor onset. The tumor type that hallmarks this genetic disease is neurofibroma, a benign neoplasm affecting peripheral nerves. Plexiform neurofibromas (PNs) involve perineural sheaths of nerve bundles and may occasionally transform into malignant peripheral nerve sheath tumors (MPNSTs), highly aggressive sarcomas endowed with a dismal prognosis. MPNSTs are currently untreatable, while only recently an inhibitor of MEK, a downstream effector of Ras signaling, has been approved for pediatric inoperable neurofibromas [2].

PN monitoring is critical for managing tumor progression and early malignancy diagnosis. To this purpose, imaging tools are extremely important in identifying suspicious lesions, and an increase in the avidity for the radioactive tracer

18F-fluorodeoxyglucose (FDG) during Positron Emission Tomography (PET) scans is a critical indication of malignant progression [3–5]. This increase in glucose uptake denotes that some neoplastic cells inside the PN mass are undergoing a metabolic rewiring. Glucose is used by various intracellular metabolic pathways for the overall energetic and anabolic needs of highly proliferative cells, as it provides them with several advantages, such as induction of nucleotide and amino acid biosynthetic pathways that stem from glycolysis intermediates, as well as enhancement of anti-oxidant defenses by boosting the pentose phosphate pathway [6]. Moreover, glycolysis induction is often accompanied by a repression in cellular respiration, *aka* oxidative phosphorylation (OXPHOS), making neoplastic cells less dependent on oxygen, as its availability can often be scarce in a growing neoplastic mass that is poorly vascularized [7]. The characterization of tumor metabolic features has gained increased attention in the attempt of identifying crucial regulators of metabolism that could be exploited as pharmacological targets.

#### **2. Metabolic features of NF1 patients**

Several indications suggest that dysregulation of Ras signaling in NF1 has metabolic effects. Indeed, metabolic alterations have been identified in NF1 patients at the systemic level (**Figure 1**). For instance, in fasting conditions they show a glucose level in the blood that is lower than in control people [8] and display an increased insulin sensitivity [9] that makes them less prone to diabetes mellitus development [10]. This could be caused by a general imbalance in the levels of several hormones, including lower levels of leptin and visfatin and higher adiponectin in NF1 patients with respect to control subjects. It remains to be explained the mechanistic connection between heterozygous loss of neurofibromin and these metabolic changes, confirmed in a large cohort of patients [11]. Moreover, NF1 individuals show reduced cerebral glucose metabolism, specifically in the thalamus [12]. Altogether, these observations put forward the hypothesis that neurofibromin haploinsufficiency may have systemic effects in overall glucose utilization. Thalamic glucose hypometabolism could be related to the neurological symptoms of NF1 (e.g. cognitive impairment). By using NF1 animal models it was also proposed that other dysmetabolic traits, such as disarrangements in neuronal usage of glutamate, γ-amino butyric acid (GABA) and dopamine, could be connected to the deficits in spatial learning, memory and attention observed in patients [13–15].

Changes in the levels of these neurotransmitters could affect the activity of several ion channels linked to the neurologic phenotype of NF1. For instance, augmented activity of voltage-gated sodium and calcium channels in sensory neurons dictates increased excitability and firing properties and underlies heightened pain sensations in NF1 patients [16]. In addition, changes in ion channel properties have repercussions on non-neuronal cells in NF1 and may participate in the overall alteration of ion homeostasis, as for Ca2+ signaling, which is altered in NF1 keratinocytes [17]. Ca2+ is a highly compartmentalized ion, and its mobilization has the capability of tuning a variety of cellular processes connected to mitochondrial metabolism and cell death pathways. Whether these Ca2+ alterations in neurofibromin haploinsufficient cells install adaptations that are relevant also in NF1-related tumors is an intriguing possibility.

At the muscular level, NF1 children may display reduced muscle function, which has been related to a role of neurofibromin in regulating fatty acid metabolism in this tissue [18]. Muscle specimens from limb-specific Nf1Prx1−/− conditional knockout mice show a 10-fold increase in muscle triglyceride content, upregulation in the activity of oxidative metabolism enzymes and increased expression of

*Metabolic Features of Neurofibromatosis Type 1-Associated Tumors DOI: http://dx.doi.org/10.5772/intechopen.98661*

#### **Figure 1.**

*The multisystem metabolic phenotype of NF1 disease. A NF1 patient is depicted with the most common metabolic-related features.*

fatty acid synthase and of the hormone leptin, whereas the expression of a number of fatty acid transporters is decreased. This genetic NF1 mouse models has shown that a lipid storage disease phenotype may underlie muscle weakness in NF1, thus displaying commonalities with the lipid storage myopathies (LSMs), which also present with progressive muscle weakness and muscle lipid accumulation, and may occasionally be treated with high dose L-carnitine supplementation [19]. Nf1 null muscle specimens are enriched in long chain fatty acid (LCFA) containing neutral lipids, such as cholesterol esters and triacyl glycerides, suggesting impaired LCFA metabolism [20]. Thus, Nf1Prx1−/− mice recapitulate the human NF1 myopathy and lipid storage excess inside muscle fibers, and a dietary intervention of reduced LCFAs and enrichment of medium-chain fatty acids with L-carnitine effectively rescues lipid accumulation and muscle weakness in knockout mice. These data link NF1 deficiency to fundamental shifts in muscle metabolism, and provide strong proof of principle that a dietary intervention can ameliorate muscle symptoms. On the same path, pharmacological intervention with the MEK inhibitor PD0325901 in pregnant mice is able to rescue body weight loss and lipid accumulation in the Nf1MyoD−/− progeny, suggesting a potential mechanism underlying the NF1-Ras-MAPK dependency of altered fatty acid metabolism [21]. Furthermore, a recent work has highlighted the requirement of neurofibromin for postnatal muscle growth and metabolic homeostasis [22].

In NF1 patients, skeletal problems including scoliosis, tibial pseudo-arthrosis and short stature are also common. Bone dysplasia is considered linked to mineralization defects and is a generalized metabolic bone disease [23]. Indeed, NF1 patients display a decreased bone mineral density, low levels of serum 25-hydroxy vitamin D3, increased osteoporosis and fracture risk [24]. Whether these systemic metabolic characteristics (*i.e*. increased glucose utilization and reduced fat depot mass) could affect the timing and type of tumor manifestations remains a puzzling issue. Neurofibroma onset and growth are accelerated by the heterozygous condition of the tumor microenvironment. Similarly, it could be envisioned that circulating factors determined by the peculiar metabolism of NF1 also participate in determining the extent of cancer predisposition. Moreover, given the sophisticated regulation and adaptability of human metabolism to external factors, the understanding of its potential involvement in NF1-related tumorigenesis may shed light on the patient-to-patient variability in the tumor burden of this disease.

**Take home message.** Altogether, these reports underline that NF1 has multisystem effects from the metabolic point of view. Recently, some of the metabolic and morphologic features of humans with NF1 have been fully recapitulated by Nf1 heterozygous mouse models of the disease [25].

#### **3. Metabolic adaptations of NF1-related tumors**

One of the most worrisome features of NF1 disease is the increased susceptibility of patients to several neoplasms. Beside the presence of neurofibromas, benign tumors that hallmark this disorder, gliomas, hematological neoplasms, breast cancer, pheochromocytomas, gastrointestinal tumors (GISTs) and MPNSTs may develop throughout lifetime. Following the loss of the tumor suppressor gene neurofibromin and the subsequent activation of the Ras pathway, several intracellular signaling cascades are rearranged and impact on cellular processes relevant to cancer progression (e.g. survival, growth, cell death, metabolism). Beside this network of deregulated pathways inside the tumor cell, a variety of inter-cellular signals are altered by neurofibromin haploinsufficiency. Neurofibromas show a highly heterotypic microenvironment composed mainly by mast cells, macrophages and fibroblasts, and neoplastic growth depends on the complex interplay between these cell types (**Figure 2**). For instance, the KIT growth factor is secreted by NF1 null Schwann cells and acts as a chemo-attractant for NF1 heterozygous mast cells. In turn, mast cells produce TGFβ, stimulating heterozygous fibroblasts to increase production of collagen and of other extracellular matrix (ECM) proteins. Mast cells also produce heparin, vascular endothelial growth factor (VEGF) and matrix metalloproteases (MMPs), which promote tumor vascularization and invasiveness. Aberrantly proliferating Schwann cells secrete colony-stimulating factor (CSF1), thereby recruiting macrophages that sustain tumor progression.

Apart from regulating survival and proliferation, some of these alterations in signal transduction can also directly affect cellular metabolism. Indeed, RAS signaling promotes oncogenic metabolism by coordinating numerous metabolic processes including lipid, nucleotide, and glycolytic pathways (**Figure 2**). Specifically, upregulation of the Ras pathway sustains a glycolytic and glutaminolytic metabolism by MYC induction, allowing cancer cells to preferentially use glucose and glutamine for anabolic purposes. This is accompanied by a decrease in OXPHOS that is characterized by blunted TCA cycle and reduced mitochondrial respiration. Ras downstream pathways, such as the mTOR signaling, also affect lipid and nucleotide synthesis for anabolic demands [26, 27].

#### *Metabolic Features of Neurofibromatosis Type 1-Associated Tumors DOI: http://dx.doi.org/10.5772/intechopen.98661*

#### **Figure 2.**

*Impact of neurofibromin loss on intra/intercellular signaling and metabolic pathways. Signaling cascades within Schwann cells and in the heterozygous microenvironment are highlighted in black. Metabolic pathways altered following neurofibromin loss are depicted in red.*

Several metabolic circuits converge on mitochondria, which are considered the powerhouse of the cell. They are in charge of energy supply and actively sustain biosynthetic pathways mandatory for cell replication. Moreover, mitochondria are involved in cell death signaling and contribute to oxidative stress regulation. Changes in several mitochondrial functions have been linked to the pro-neoplastic dysregulation of many fundamental biological processes, including a variety of bioenergetic circuities [28].

Tumor metabolism refers to a plethora of cancer features, spanning from the way neoplastic cells take up and utilize nutrients for growth and replication, to the diverse communication modes they establish with the neighboring cells. Altogether, these metabolic adaptations during cancer initiation and progression render aberrant cells capable of circumventing nutrient and oxygen shortage conditions that they may encounter, and often affect and constrain the behavior of the surrounding microenvironment [29].

So far, targeting strategies against cancer mainly rely on specifically blocking molecular signals that promote cell proliferation, hinder cell death, modulate the immune response or enhance angiogenesis and cell survival. However, most of these signaling pathways are either redundant or essential in healthy tissue making these types of target therapies challenging. A further strategy is to hit key metabolic transformations that occur in cancer cells, whereby the metabolic adaptations to hypoxic conditions seem to be specific for cancer cells, shared in many tumor types and required for neoplastic growth.

#### **3.1 Mitochondrial respiration**

Although the metabolic scenario of NF1 mutant cells is poorly defined, some bioenergetic alterations are starting to surface. For instance, respiratory complex II, *aka* succinate dehydrogenase (SDH), is a crucial metabolic enzyme at the crossroad between OXPHOS and Krebs cycle that is repressed in NF1-related tumor cells in an ERK-dependent manner following neurofibromin loss; this metabolic rewiring is compensated by an increased glycolytic pathway [30]. In detail, hyperactivation of the mitochondrial branch of Ras/ERK signaling causes phosphorylation of the mitochondrial chaperone TRAP1. Its consequent activation inhibits SDH enzymatic activity, triggering intracellular accumulation of the oncometabolite succinate that in turn stabilizes the pro-neoplastic transcription factor HIF1α. Importantly, genetic ablation of TRAP1 inhibits tumor growth [31]. Taken together, these data indicate that TRAP1 mediates a pseudo-hypoxic signaling, as it orchestrates a HIF1α-dependent program that is crucial in the neoplastic process and boosts tumor growth independently of environmental oxygen tension. Moreover, it was recently demonstrated that TRAP1 also executes the hypoxic response, as it is a transcriptional target of HIF1α induced in KRAS-dependent models of carcinogenesis, such as pancreatic adenocarcinoma, with a crucial role in handling the cell bioenergetic response to oxygen paucity [32]. In specific cases of familiar cancers (*i.e*. in the hereditary paraganglioma-phaeochromocytoma syndrome, HPGL/PCC), succinate increases following inactivating mutations of SDH subunits. In these settings, in addition to inducing HIF1α, high levels of succinate can impinge on cell epigenetics by inhibiting α-ketoglutarate dependent dioxygenases, such as histone and DNA demethylases, further contributing to neoplastic growth [33]. Therefore, it can be envisioned that similar complex changes in the epigenome landscape occur upon TRAP1-mediated SDH inhibition in NF1-related tumor cells.

As a consequence, pharmacological inhibition of TRAP1 has been proposed as an anti-neoplastic approach for MPNST and other tumor types. Recently, the identification of highly selective TRAP1 allosteric inhibitors has shown promising results, ablating *in vitro* tumorigenesis [34, 35]. Previous targeting of the HSP90 family of chaperones, to which TRAP1 belongs, has been pursued with the drug IPI-504 which, in combination with the mTOR inhibitor rapamycin, cooperates in the growth repression of NF1 mutant cancer cells [36]. Here, strong ER stress drastically represses cancer growth. Given the intense molecular crosstalk between ER and mitochondria and their coordinated regulation of Ca2+ homeostasis, it could be envisaged that the interplay between ER and mitochondria is crucial in the growth of NF1 deficient cells. Indeed, yeast synthetic lethality screens have identified Y100 as a molecule capable of interfering with mito-ER homeostasis, thus revealing crucial metabolic vulnerabilities of the yeast cells null for the homolog of NF1, called IRA2 [37].

Another report describes that neurofibromin-deficient cells display a decrease in the activity of NADH dehydrogenase, *aka* the first respiratory complex, with a consequent unbalance in NAD+ /NADH ratio [38]. This metabolic alteration negatively impacts on the activity of mitochondrial sirtuins, specifically SIRT3. SIRT3 reactivation through NAD+ precursor supply or genetic manipulation impairs tumorigenesis of neurofibromin-deficient cells and synergize with TRAP1 ablation in repressing MPNST growth in xenografts by preventing HIF1α stabilization. Furthermore, the repressed expression of several subunits of the NADH dehydrogenase respiratory complex I, one of the main ROS producers in mitochondria, renders neurofibromin-deficient cells more resistant to pro-oxidant drugs acting through complex I-mediated ROS increase.

**Take home message.** Altogether, these data indicate that NF1-related tumors display a pseudo-hypoxic signature that contributes to tumor proliferation and transition towards malignancy. Indeed, neurofibromin inactivation occurs in certain cancers through hypoxia-induced degradation, independently of *NF1* gene mutations [39]. These data suggest that the hypoxic response might affect the Ras/ERK signaling pathways downstream to neurofibromin loss and its genetic

inactivation installs a hypoxic-like response that may provide cells with an equipped and prompt response to any possible drop in oxygen availability.

#### **3.2 Glutamine metabolism**

As already shown for several cancers, NF1 null cells are highly sensitive to glutamine deprivation, and glutaminase (GLS) inhibitors such as BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3) or CB-839 have been proposed as antineoplastic agents in the context of NF1-associated neoplasms [40]. Glutamine is one of the most abundant intracellular amino acids and fuels several biosynthetic pathways by providing carbons to TCA cycle intermediates, glutathione, fatty acids, and nucleotides. Pharmacological GLS inhibition causes a shortage in multiple TCA cycle intermediates, among which α-ketoglutarate, succinate and fumarate.

Phase II Basket Trial of Glutaminase Inhibitor (BeGIN) CB-839 HCl in patients with metastatic or unresectable MPNST is ongoing (https://clinicaltrials.gov/ct2/ show/NCT03872427). Still, CB-839 resistance has been observed *in vitro,* whereby c-Myc induction takes place through epigenetic changes mediated by bromodomain-containing protein 4 (BRD4), which promotes transcription by recognizing acetylated lysines on histones. Indeed, CB-839 resistant cells are more sensitive to JQ1, a small molecule inhibitor of BRD4 [41]. Furthermore, glutamine dependency has been identified in lung adenocarcinomas where KRAS mutations coexist with Nf1 loss [42]. This work suggests that oncologic patient stratification for NF1 loss may uncover crucial targetable metabolic adaptations.

Similarly, the glutamine antagonist JHU395, a novel orally bioavailable prodrug designed to circulate in an inert form in plasma and to permeate and release the active drug within target tissues, is able to inhibit tumor growth in a murine flank MPNST model [43]. One of the major outcomes of JHU395 administration is the reduced usage of glutamine-dependent metabolites with a prominent effect on purine synthesis. Interestingly, glutamine utilization for anaplerotic purposes (*i.e.* supply of TCA intermediates such as glutamate, α-ketoglutarate and succinate) is not limited by JHU395. The different modes of action of drugs targeting glutamine metabolism indicate that multiple metabolic pathways in glutamine utilization might be critical for MPNST growth.

#### **3.3 Lipid metabolism**

During cancer growth, transformed cells experience nutrient and glucose shortage and must install metabolic adaptations to overcome these potentially harmful circumstances. Metabolic stress factors such as hypoxia and glucose deprivation increase expression of carnitine palmitoyltransferase 1C (CPT1C), member of a family of mitochondria-associated enzymes that regulate fatty acid metabolism. Its genetic ablation in a NF1 murine model delays tumor growth [44]. This finding exposes a susceptibility of NF1-related cancers to drugs targeting lipid metabolism when stressful conditions occur, as in the case of active chemotherapeutic regimens.

Lipid droplet accumulation has been reported in MPNSTs, which utilize both exogenous and endogenous lipids as a source of energy [45]. Indeed, either disruption of fatty acid oxidation and the use of the fatty acid synthase (FASN) inhibitors C75, orlistat and Irgasan reduce MPNST survival.

MPNSTs have been reported to secrete elevated levels of prostaglandin E2 (PGE2), an active lipid compound with hormone-like effects in animals [46]. It usually acts as an endocrine mediator of metabolic processes in homeostasis but also in inflammatory and neoplastic conditions. Remarkably, PGE2 receptor antagonists decreased the proliferation of MPNST cell lines. Prostaglandin administration has

also been linked to aberrant cAMP metabolism in MPNSTs that display two-fold increased cAMP levels compared to normal Schwann cells [47].

#### **3.4 Connections between genetic mutations and metabolic changes**

The HPGL/PCC syndrome, where loss-of-function mutations affect SDH and increase intracellular levels of the oncometabolite succinate, thus causing onset of pheochromocytoma and paraganglioma, is a proof-of-concept that metabolic changes can drive tumorigenesis. It is of note that NF1 patients can develop this kind of tumors in 5% of cases, whereas in non-NF1 related patients with HPGL/PCC history NF1 mutations have been reported in tumor cells [48]. This information, even though only correlative, is in accord with the observation that TRAP1 exerts a pro-neoplastic role in NF1 by inhibiting SDH, and suggests a possible overlapping path of metabolic adaptations existing between inactivation of NF1 and SDH components. Moreover, it must be highlighted that dysregulated signaling cascades can impinge on metabolic circuits, thus leading to neoplastic metabolic alterations either in the absence or in addition to specific mutations in metabolic enzymes.

Another interesting line of investigation links gene mutations to pro-neoplastic metabolic adaptations during neurofibroma growth. Indeed, it was reported that somatic mutations in mitochondrial DNA (mtDNA), which encodes 13 proteins of the OXPHOS machinery, are acquired and maintained by a high percentage of cutaneous and plexiform neurofibromas [49]. This suggests a possible positive selection in neoplastic cells for mutated mitochondrial genes, in keeping with observations that an aberrant mitochondrial respiration confers adaptive advantages to neurofibroma cells.

**Take home message.** Although the metabolic landscape of neurofibromindeficient cells and MPNST has been only partially investigated, uncovering the adaptations in the metabolic circuits of these tumor cells may shed light on novel targetable actors. Furthermore, despite the genetic variability in MPNSTs characterized by different acquired mutations (e.g. TP53, p16, PRC, SUZ12, *CDKN2A,* etc.), there is the possibility of conserved derangements in metabolic pathways that may render MPNST vulnerable to selective targeting.

**Perspectives.** Beside cell autonomous changes in metabolism of neurofibromindeficient tumor cells, their metabolic phenotype can be determined by alterations in intercellular communication within the tumor microenvironment. Recently it has been reported that fibroblast metabolic rewiring can promote growth of neural tumors [50]. Furthermore, beside the mitochondria to nucleus signaling mediated by succinate-dependent regulation of HIF1α and of epigenetic changes, this oncometabolite can exit tumor cells affecting immune cell responses. Thus, metabolic changes within neurofibromin null cells can affect the behavior of neighboring cells within the tumor microenvironment. Recent advances in immunotherapy approaches against MPNST growth have highlighted how these cancers might evade immune recognition and hijack immunological functions (e.g. tissue healing, angiogenesis, etc.) to their advantage. Whether the metabolic status of NF1-related tumors mediate the relationship between transformed cells and immune system is an exciting matter of investigation.

#### **4. Conclusions**

For a long time, pharmacological treatments suited for NF1-related neoplasms have been lacking. Only recently the first therapeutic approaches have been

#### *Metabolic Features of Neurofibromatosis Type 1-Associated Tumors DOI: http://dx.doi.org/10.5772/intechopen.98661*

translated from NF1 mouse models to patient bedside and further clinical trials are currently ongoing. Altogether, the major efforts in managing NF1-related neoplasms have been based on drugs targeting signaling transduction cascades such as RTK, RAS- RAF–MEK–ERK and PI3K-AKT–mTOR inhibitors. Selumetinib was the first drug approved in 2019 by the US FDA for pediatric NF1 patients with symptomatic and inoperable PN [51, 52] after a phase 2 clinical trial started a decade ago (https://clinicaltrials.gov/ct2/show/NCT01362803). Results indicate that 74% of patients display a partial response in terms of tumor volume shrinkage, and this is durable in 56% of patients. Albeit extremely positive, these results demand the urgent development of additional treatments. Previous attempts of targeting signaling cascades in neurofibroma microenvironment through imatinib mesylate administration, a dual SCF/cKIT inhibitor, have shown modest response rates limited only to small tumors [53] (https://clinicaltrials.gov/ct2/show/NCT01673009). Cabozantinib, an inhibitor of multiple tyrosine kinases among which c-Kit, vascular endothelial growth factor (VEGF) receptor (VEGFR)2, MET, RET, FMS-related RTK 3 (FLT3) and TAM family receptors (tyrosine kinases AXL, TYRO3 and MERTK) is now under study in a phase II trial against progressive or symptomatic, inoperable PN (https://clinicaltrials.gov/ct2/show/NCT02101736) as it has shown promising results in Nf1-mutant mice [54].

As for glioma, chemotherapy remains the first line treatment. More recently, epigenetic-based approaches in fighting MPNST growth have emerged [55] and drugs targeting the immune checkpoints are considered the emerging therapeutic option with ongoing clinical trials [56, 57] (https://clinicaltrials.gov/ct2/show/ NCT02691026).

In this scenario, beside the recently reviewed pharmacological options for MPNST treatment [58–60], targeting the metabolic features of NF1-related tumors constitutes an additional, promising therapeutic option. Although multiple metabolic routes have been shown to be affected in NF1 tumorigenesis, metabolic based anti-neoplastic approaches are limited in the field (BeGIN clinical trial) and others are at the preclinical stage (**Figure 3**). A recent report has resumed the idea of targeting the glycolytic pathway [61]; however, the drug employed, *i.e*. 3-bromopyruvate, has already been dismissed from past clinical trials for excessive and life-threatening toxicity.

As for MPNST, complete surgical excision with clear margins remains the only treatment in the case of a localized cancer. Given the lack of efficacy in targeting unique aspects of MPNST disease biology, some benefits could hopefully come from combinatorial therapeutic designs that consider and include innovative rational therapies, such as targeting bioenergetic circuities.

In this direction, despite the genetically heterogeneous phenotype of NF1-related malignancies, the annotation of conserved metabolic adaptations in the progression towards MPNST might open space for innovative therapeutic interventions [62].

**Perspectives.** Advancements in animal modeling of NF1-related neoplasms are meant to refine the understanding of PN tumorigenesis and put the basis for testing multi-targeted drug therapies and adaptive tumor response. For instance, atypical neurofibromas with an uncertain transforming potential have been recapitulated by *Cdkn2a* loss [63]. Uncovering the potential metabolic adaptations of the transitional stages of NF1 tumors from the benign to the malignant ones may equip clinicians with metabolic biomarkers to be monitored during NF1 patient surveillance.

Furthermore, the understanding of the metabolic interplay between cancer cells that have lost neurofibromin and other cell types present in the tumor microenvironment might uncover metabolic susceptibility of these cancers. For instance, MPNSTs display an increased number of macrophages with respect to PNs and

#### **Figure 3.**

*Treatment options against NF1-related neoplasms. Drugs under clinical (green) and preclinical (violet) evaluation against NF1-related tumors are reported.*

are highly glutamine-addicted. It is known that macrophages sense the lack of glutamine and install a synthetic pathway for glutamine supply based on glutamine synthetase induction. This metabolic rewiring characterizes the pro-tumorigenic polarization towards a tumor-associated macrophage phenotype. Given these tight and crucial metabolic crosstalks between tumor cells and the immunologic compartment, it can be envisioned that targeted therapies are accompanied by metabolic-based treatments hitting both neoplastic and environmental cells in order to overcome potential cancer resistance (e.g. CB-839 and JQ1, which combines metabolic and epigenetic treatments).

PET scans with labeled glucose uptake evaluation can provide an extremely useful tool for monitoring lesions at high potential for growth and at risk for malignant transformation; regular imaging is suggested especially in symptomatic neurofibromas [64]. We expect that metabolic tracking of additional nutrients such as glutamine could be employed in NF1 patients for the unraveling of metabolically active lesions. Imaging of labeled glutamine is currently under evaluations in cancer patients and has the potential of predicting cancer response to metabolic targeted therapies, thus helping the guidance of therapeutic decision-making [65, 66].

#### **Acknowledgements**

This work was supported by grants from University of Padova, Neurofibromatosis Therapeutic Acceleration Program and Associazione Italiana Ricerca Cancro (AIRC grant IG 2017/20749).

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Notes/thanks/other declarations**

Images were obtained with BioRender software (https://biorender.com).

#### **Author details**

Ionica Masgras1,2 and Andrea Rasola2 \*


\*Address all correspondence to: andrea.rasola@unipd.it

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Ferner RE, Huson SM, Thomas N, Moss C, Willshaw H, Evans DG, et al. Guidelines for the diagnosis and management of individuals with neurofibromatosis 1. J Med Genet 2007 Feb;44(2):81-88.

[2] Mukhopadhyay S, Maitra A, Choudhury S. Selumetinib: the first ever approved drug for neurofibromatosis-1 related inoperable plexiform neurofibroma. Curr Med Res Opin 2021 Mar 23:1-6.

[3] Ahlawat S, Blakeley JO, Langmead S, Belzberg AJ, Fayad LM. Current status and recommendations for imaging in neurofibromatosis type 1, neurofibromatosis type 2, and schwannomatosis. Skeletal Radiol 2020 Feb;49(2):199-219.

[4] Van Der Gucht A, Zehou O, Djelbani-Ahmed S, Valeyrie-Allanore L, Ortonne N, Brugières P, et al. Metabolic Tumour Burden Measured by 18F-FDG PET/CT Predicts Malignant Transformation in Patients with Neurofibromatosis Type-1. PLoS One 2016 Mar 17;11(3):e0151809.

[5] Urban T, Lim R, Merker VL, Muzikansky A, Harris GJ, Kassarjian A, et al. Anatomic and metabolic evaluation of peripheral nerve sheath tumors in patients with neurofibromatosis 1 using whole-body MRI and (18)F-FDG PET fusion. Clin Nucl Med 2014 May;39(5):e301-7.

[6] DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv 2016 May 27;2(5):e1600200.

[7] Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009 May 22;324(5930): 1029-1033.

[8] Martins AS, Jansen AK, Rodrigues LO, Matos CM, Souza ML, de Souza JF, et al. Lower fasting blood glucose in neurofibromatosis type 1. Endocr Connect 2016 Jan;5(1):28-33.

[9] Martins AS, Jansen AK, Rodrigues LOC, Matos CM, Souza MLR, Miranda DM, et al. Increased insulin sensitivity in individuals with neurofibromatosis type 1. Arch Endocrinol Metab 2018 Feb;62(1):41-46.

[10] Ozhan B, Ozguven AA, Ersoy B. Neurofibromatosis type 1 and diabetes mellitus: an unusual association. Case Rep Endocrinol 2013;2013:689107.

[11] Kallionpää RA, Peltonen S, Leppävirta J, Pöyhönen M, Auranen K, Järveläinen H, et al. Haploinsufficiency of the NF1 gene is associated with protection against diabetes. J Med Genet 2020 Jun 22.

[12] Apostolova I, Derlin T, Salamon J, Amthauer H, Granström S, Brenner W, et al. Cerebral glucose metabolism in adults with neurofibromatosis type 1. Brain Res 2015 Nov 2;1625:97-101.

[13] Costa RM, Federov NB, Kogan JH, Murphy GG, Stern J, Ohno M, et al. Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature 2002 Jan 31;415(6871): 526-530.

[14] Brown JA, Emnett RJ, White CR, Yuede CM, Conyers SB, O'Malley KL, et al. Reduced striatal dopamine underlies the attention system dysfunction in neurofibromatosis-1 mutant mice. Hum Mol Genet 2010 Nov 15;19(22): 4515-4528.

[15] Ryu HH, Lee YS. Cell type-specific roles of RAS-MAPK signaling in learning and memory: Implications in neurodevelopmental disorders.

*Metabolic Features of Neurofibromatosis Type 1-Associated Tumors DOI: http://dx.doi.org/10.5772/intechopen.98661*

Neurobiol Learn Mem 2016 Nov;135:13-21.

[16] Moutal A, Dustrude ET, Khanna R. Sensitization of Ion Channels Contributes to Central and Peripheral Dysfunction in Neurofibromatosis Type 1. Mol Neurobiol 2017 Jul;54(5): 3342-3349.

[17] Korkiamäki T, Ylä-Outinen H, Koivunen J, Karvonen SL, Peltonen J. Altered calcium-mediated cell signaling in keratinocytes cultured from patients with neurofibromatosis type 1. Am J Pathol 2002 Jun;160(6):1981-1990.

[18] Sullivan K, El-Hoss J, Quinlan KG, Deo N, Garton F, Seto JT, et al. NF1 is a critical regulator of muscle development and metabolism. Hum Mol Genet 2014 Mar 1;23(5):1250-1259.

[19] Summers MA, Quinlan KG, Payne JM, Little DG, North KN, Schindeler A. Skeletal muscle and motor deficits in Neurofibromatosis Type 1. J Musculoskelet Neuronal Interact 2015 Jun;15(2):161-170.

[20] Summers MA, Rupasinghe T, Vasiljevski ER, Evesson FJ, Mikulec K, Peacock L, et al. Dietary intervention rescues myopathy associated with neurofibromatosis type 1. Hum Mol Genet 2018 Feb 15;27(4):577-588.

[21] Summers MA, Vasiljevski ER, Mikulec K, Peacock L, Little DG, Schindeler A. Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse. Mol Genet Metab 2018 Apr;123(4):518-525.

[22] Wei X, Franke J, Ost M, Wardelmann K, Börno S, Timmermann B, et al. Cell autonomous requirement of neurofibromin (Nf1) for postnatal muscle hypertrophic growth and metabolic homeostasis. J Cachexia

Sarcopenia Muscle 2020 Dec;11(6): 1758-1778.

[23] Brunetti-Pierri N, Doty SB, Hicks J, Phan K, Mendoza-Londono R, Blazo M, et al. Generalized metabolic bone disease in Neurofibromatosis type I. Mol Genet Metab 2008 May;94(1): 105-111.

[24] Filopanti M, Verga U, Ulivieri FM, Giavoli C, Rodari G, Arosio M, et al. Trabecular Bone Score (TBS) and Bone Metabolism in Patients Affected with Type 1 Neurofibromatosis (NF1). Calcif Tissue Int 2019 Feb;104(2):207-213.

[25] Tritz R, Benson T, Harris V, Hudson FZ, Mintz J, Zhang H, et al. Nf1 heterozygous mice recapitulate the anthropometric and metabolic features of human neurofibromatosis type 1. Transl Res 2021 Feb;228:52-63.

[26] Park JH, Pyun WY, Park HW. Cancer Metabolism: Phenotype, Signaling and Therapeutic Targets. Cells 2020 Oct 16;9(10):2308. doi: 10.3390/ cells9102308.

[27] DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv 2016 May 27;2(5):e1600200.

[28] Cannino G, Ciscato F, Masgras I, Sánchez-Martín C, Rasola A. Metabolic Plasticity of Tumor Cell Mitochondria. Front Oncol 2018 Aug 24;8:333.

[29] Pavlova NN, Thompson CB. The Emerging Hallmarks of Cancer Metabolism. Cell Metab 2016 Jan 12;23(1):27-47.

[30] Masgras I, Ciscato F, Brunati AM, Tibaldi E, Indraccolo S, Curtarello M, et al. Absence of Neurofibromin Induces an Oncogenic Metabolic Switch via Mitochondrial ERK-Mediated Phosphorylation of the Chaperone TRAP1. Cell Rep 2017 Jan 17;18(3):659-672.

[31] Sciacovelli M, Guzzo G, Morello V, Frezza C, Zheng L, Nannini N, et al. The mitochondrial chaperone TRAP1 promotes neoplastic growth by inhibiting succinate dehydrogenase. Cell Metab 2013 Jun 4;17(6):988-999.

[32] Laquatra C, Sanchez-Martin C, Dinarello A, Cannino G, Minervini G, Moroni E, et al. HIF1α-dependent induction of the mitochondrial chaperone TRAP1 regulates bioenergetic adaptations to hypoxia. Cell Death Dis 2021 May 1;12(5):434-021-03716-6.

[33] Kaushik AK, DeBerardinis RJ. Applications of metabolomics to study cancer metabolism. Biochim Biophys Acta Rev Cancer 2018 Aug;1870(1):2-14.

[34] Sanchez-Martin C, Moroni E, Ferraro M, Laquatra C, Cannino G, Masgras I, et al. Rational Design of Allosteric and Selective Inhibitors of the Molecular Chaperone TRAP1. Cell Rep 2020 Apr 21;31(3):107531.

[35] Masgras I, Sanchez-Martin C, Colombo G, Rasola A. The Chaperone TRAP1 As a Modulator of the Mitochondrial Adaptations in Cancer Cells. Front Oncol 2017 Mar 29;7:58.

[36] De Raedt T, Walton Z, Yecies JL, Li D, Chen Y, Malone CF, et al. Exploiting cancer cell vulnerabilities to develop a combination therapy for ras-driven tumors. Cancer Cell 2011 Sep 13;20(3):400-413.

[37] Allaway RJ, Wood MD, Downey SL, Bouley SJ, Traphagen NA, Wells JD, et al. Exploiting mitochondrial and metabolic homeostasis as a vulnerability in NF1 deficient cells. Oncotarget 2017 Jul 18;9(22):15860-15875.

[38] Masgras I, Cannino G, Ciscato F, Sanchez-Martin C, Pizzi M, Menga A, et al. Tumor growth of neurofibromindeficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3. in press (2021)

[39] Green YS, Sargis T, Reichert EC, Rudasi E, Fuja D, Jonasch E, et al. Hypoxia-Associated Factor (HAF) Mediates Neurofibromin Ubiquitination and Degradation Leading to Ras-ERK Pathway Activation in Hypoxia. Mol Cancer Res 2019 May;17(5):1220-1232.

[40] Sheikh TN, Patwardhan PP, Cremers S, Schwartz GK. Targeted inhibition of glutaminase as a potential new approach for the treatment of NF1 associated soft tissue malignancies. Oncotarget 2017 Oct 6;8(55): 94054-94068.

[41] Sheikh TN, Lu C, Schwartz GK. Targeting compensatory metabolic pathways: Novel approaches to overcome resistance to glutaminase inhibition in NF1 driven malignant peripheral nerve sheath tumors. Proceedings of the American Association for Cancer Research Annual Meeting 2020 2020;80(16): Abstract nr 248.

[42] Wang X, Min S, Liu H, Wu N, Liu X, Wang T, et al. Nf1 loss promotes Kras-driven lung adenocarcinoma and results in Psat1-mediated glutamate dependence. EMBO Mol Med 2019 Jun;11(6):e9856. doi: 10.15252/ emmm.201809856.

[43] Lemberg KM, Zhao L, Wu Y, Veeravalli V, Alt J, Aguilar JMH, et al. The Novel Glutamine Antagonist Prodrug JHU395 Has Antitumor Activity in Malignant Peripheral Nerve Sheath Tumor. Mol Cancer Ther 2020 Feb;19(2):397-408.

[44] Sanchez-Macedo N, Feng J, Faubert B, Chang N, Elia A, Rushing EJ, et al. Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model. Cell Death Differ 2013 Apr;20(4): 659-668.

[45] Patel AV, Johansson G, Colbert MC, Dasgupta B, Ratner N. Fatty acid

*Metabolic Features of Neurofibromatosis Type 1-Associated Tumors DOI: http://dx.doi.org/10.5772/intechopen.98661*

synthase is a metabolic oncogene targetable in malignant peripheral nerve sheath tumors. Neuro Oncol 2015 Dec;17(12):1599-1608.

[46] Deadwyler GD, Dang I, Nelson J, Srikanth M, De Vries GH. Prostaglandin E(2) metabolism is activated in Schwann cell lines derived from human NF1 malignant peripheral nerve sheath tumors. Neuron Glia Biol 2004 May;1(2):149-155.

[47] Dang I, De Vries GH. Aberrant cAMP metabolism in NF1 malignant peripheral nerve sheath tumor cells. Neurochem Res 2011 Sep;36(9): 1697-1705.

[48] Dahia PL. Pheochromocytoma and paraganglioma pathogenesis: learning from genetic heterogeneity. Nat Rev Cancer 2014 Feb;14(2):108-119.

[49] Kurtz A, Lueth M, Kluwe L, Zhang T, Foster R, Mautner VF, et al. Somatic mitochondrial DNA mutations in neurofibromatosis type 1-associated tumors. Mol Cancer Res 2004 Aug;2(8):433-441.

[50] Wei CJ, Gu YH, Wang W, Ren JY, Cui XW, Lian X, et al. A narrative review of the role of fibroblasts in the growth and development of neurogenic tumors. Ann Transl Med 2020 Nov;8(21):1462-20-3218.

[51] Gross AM, Wolters PL, Dombi E, Baldwin A, Whitcomb P, Fisher MJ, et al. Selumetinib in Children with Inoperable Plexiform Neurofibromas. N Engl J Med 2020 Apr 9;382(15): 1430-1442.

[52] Dombi E, Baldwin A, Marcus LJ, Fisher MJ, Weiss B, Kim A, et al. Activity of Selumetinib in Neurofibromatosis Type 1-Related Plexiform Neurofibromas. N Engl J Med 2016 Dec 29;375(26):2550-2560.

[53] Robertson KA, Nalepa G, Yang FC, Bowers DC, Ho CY, Hutchins GD, et al. Imatinib mesylate for plexiform neurofibromas in patients with neurofibromatosis type 1: a phase 2 trial. Lancet Oncol 2012 Dec;13(12): 1218-1224.

[54] Fisher MJ, Shih CS, Rhodes SD, Armstrong AE, Wolters PL, Dombi E, et al. Cabozantinib for neurofibromatosis type 1-related plexiform neurofibromas: a phase 2 trial. Nat Med 2021 Jan;27(1): 165-173.

[55] Korfhage J, Lombard DB. Malignant Peripheral Nerve Sheath Tumors: From Epigenome to Bedside. Mol Cancer Res 2019 Jul;17(7):1417-1428.

[56] Farschtschi S, Kluwe L, Park SJ, Oh SJ, Mah N, Mautner VF, et al. Upregulated immuno-modulator PD-L1 in malignant peripheral nerve sheath tumors provides a potential biomarker and a therapeutic target. Cancer Immunol Immunother 2020 Jul;69(7):1307-1313.

[57] Wu LMN, Lu QR. Therapeutic targets for malignant peripheral nerve sheath tumors. Future Neurol 2019;14(1). doi: 10.2217/fnl-2018-0026

[58] Hassan A, Pestana RC, Parkes A. Systemic Options for Malignant Peripheral Nerve Sheath Tumors. Curr Treat Options Oncol 2021 Feb 27;22(4): 33-021-00830-7.

[59] Marjanska A, Galazka P, Wysocki M, Styczynski J. New Frontiers in Therapy of Peripheral Nerve Sheath Tumors in Patients With Neurofibromatosis Type 1: Latest Evidence and Clinical Implications. Anticancer Res 2020 Apr;40(4): 1817-1831.

[60] Foiadelli T, Naso M, Licari A, Orsini A, Magistrali M, Trabatti C, et al. Advanced pharmacological therapies for neurofibromatosis type 1-related tumors. Acta Biomed 2020 Jun 30;91(7-S):101-114.

[61] Linke C, Wösle M, Harder A. Anti-cancer agent 3-bromopyruvate reduces growth of MPNST and inhibits metabolic pathways in a representative in-vitro model. BMC Cancer 2020 Sep 18;20(1):896-020-07397-w.

[62] Lemberg KM, Wang J, Pratilas CA. From Genes to -Omics: The Evolving Molecular Landscape of Malignant Peripheral Nerve Sheath Tumor. Genes (Basel) 2020 Jun 24;11(6):691. doi: 10.3390/genes11060691.

[63] Chaney KE, Perrino MR, Kershner LJ, Patel AV, Wu J, Choi K, et al. Cdkn2a Loss in a Model of Neurofibroma Demonstrates Stepwise Tumor Progression to Atypical Neurofibroma and MPNST. Cancer Res 2020 Nov 1;80(21):4720-4730.

[64] Reinert CP, Schuhmann MU, Bender B, Gugel I, la Fougère C, Schäfer J, et al. Comprehensive anatomical and functional imaging in patients with type I neurofibromatosis using simultaneous FDG-PET/MRI. Eur J Nucl Med Mol Imaging 2019 Mar;46(3):776-787.

[65] Grkovski M, Goel R, Krebs S, Staton KD, Harding JJ, Mellinghoff IK, et al. Pharmacokinetic Assessment of (18)F-(2S,4R)-4-Fluoroglutamine in Patients with Cancer. J Nucl Med 2020 Mar;61(3):357-366.

[66] Faubert B, Solmonson A, DeBerardinis RJ. Metabolic reprogramming and cancer progression. Science 2020 Apr 10;368(6487):eaaw5473. doi: 10.1126/ science.aaw5473.

### *Edited by Juichiro Nakayama and Yuichi Yoshida*

Neurofibromatosis type 1 (NF1), also known as von Recklinghausen disease, is a major monogenic neurocutaneous disorder. The NF1 gene encodes the protein neurofibromin whose dysfunction promotes tumorigenesis in central and peripheral neuronal tissues. In addition to inducing the formation of cutaneous pigmented lesions or neurofibromas, NF1 affects multiple organ systems, resulting in neurological and psychiatric disorders, orthopedic conditions, and impaired endocrine functions. This book examines the fundamental, clinical, and basic aspects of NF1 over three sections and nine chapters. Topics addressed include bone lesions in children with NF1, diffuse neurofibromatous tissue, seizures in adults with NF1, Ras-GAP function of neurofibromin, endocrine disorders characteristic of NF1, and more.

Published in London, UK © 2022 IntechOpen © chiewr / iStock

Clinical and Basic Aspects of Neurofibromatosis Type 1

Clinical and Basic Aspects of

Neurofibromatosis Type 1

*Edited by Juichiro Nakayama and Yuichi Yoshida*