5. Pathophysiological function of SLC25A46

In the last 2 years, a series of experiments aiming at resolving the function of SLC25A46 and the pathogenesis of SLC25A46-associated diseases were conducted. Primary cultures of skin fibroblasts from HMSN6B patients (including PCH or Leigh syndrome patients) have been studied to investigate the pathophysiology of the diseases. Simultaneously, mutant SLC25A46 alleles or SLC25A46-targeted RNA-interference molecules were transfected into common cell lines (e.g., COS-7, HeLa, HEK293T, and HCT116) to study the consequences of the mutations or protein knock-down in a homogeneous genetic background. Finally, zebrafish, murine, and bovine models have been employed to examine the pathophysiological effects of SLC25A46 mutations in vivo (Table 2).

#### 5.1. Function of SLC25A46 in mitochondrial dynamics

In silico analysis led to the identification of a SLC25A46 homolog in yeast, namely Ugo1. The Ugo1 protein is a modified mitochondrial solute carrier expressed in the mitochondrial outer membrane that operates as a mitochondrial fusion factor and interacts physically with Mgm1 (homolog of human OPA1) and Fzo (homolog of human MFN2) [38]. A succession of studies in cells demonstrated consistently that, like Ugo1, MTCH1, and MTCH2, SLC25A46 also localizes to the mitochondrial outer membrane [17, 20, 22].

Animal

SLC25A46

SLC25A46

 proteins

 Age

Age of

Optic

Peripheral

Ataxia

Degeneration

Other features

dynamics

Mitochondrial

in

cerebellum

/brainstem

atrophy

neuropathy

of

death

onset

1 mo. Euthanasia

—

+

+

 +

Degenerative

Elongated

mitochondria

with abnormal

cristae.

lesions both in

gray matter

and white

matter;

demyelination

in certain peripheral

nerves.

around 2–

3 mo.

mutations

species

Bovine

c.376C>T

 p. R126C

Duchesne

et al. [43] Tg/ FVB/

Tg18: indel

p.

Val122Leu123delinsATIIYI;

2w

 3–4 w

—

+

Impaired

Elongated

mitochondria

growth, small

intestine,

with abnormal

cristae.

thymus, spleen

and liver,

severe

hypoglycemia;

low plasma

iron

concentrations

combined with

high ferritin.

Growth

Atypical

mitochondria

Neurodegenerative Diseases Associated with Mutations in *SLC25A46*

retardation,

severe thymic

in Purkinje

cells.

and splenic hypoplasia,

compromised

http://dx.doi.org/10.5772/intechopen.79992

Purkinje cell

dendritic

arborization

and reduced

synaptic

connectivity,

RGC

79

N mouse

12 bp;

Duchesne

Tg26:

p.Ala108fs\*159

del 75 bp

et al. [43]

atc/atc

c.283C>T

 p.Gln95fs\*

2 w 5 w

 +

 +

+

 +

> C57BL/6 J

mouse

Terzenidou

et al. [42]


mutation. As shown in Table 1, very severe SLC25A46-related disease has been linked to mutations that yield markedly reduced SLC25A46 levels, including homozygous or compound heterozygous nonsense mutations, (c.691C>T, p.Arg231\*; c.736A>T, p.Arg246\*; c.42C>G, p.Tyr14\*), a splice site variant (c.462+1G>A), and a micro-deletion (g.chr5: 110738771\_11074670del). In addition, Wan et al. and Janer et al. verified that the three missense mutations c.1022T>C (p.Leu341Pro), c.998C>T (p.Pro333Leu), and c.425C>T (p. Thr142Ile) destabilize the protein without nonsense-mediated mRNA decay, causing a marked loss of SLC25A46 function. Such protein-depriving mutations lead to severe clinical symptoms of PCH or Leigh syndrome. In contrast, missense mutations of SLC25A46 that are associated with normal or mildly reduced protein levels tend to result in a relatively mild phenotype. For instance, stable expression of the p.Gly249Asp mutant protein produces low-severity manifestations of optic atrophy spectrum disorder [17, 22] (Table 1). Thus, the more stable, functional, and abundant the mutant protein, the less severe the

In conclusion, the main molecular causes of SLC25A46-related neurological disease appear to be SLC25A46 loss of function or deficiency. The recessive inheritance pattern observed in all pathogenic SLC25A46 mutation-affected individuals and families thus far contrast with the dominant inheritance pattern observed with other mitochondrial dynamic genes, including OPA1, MFN1/2, and DRP1, for which haplo-insufficiency and dominant negative effects are

In the last 2 years, a series of experiments aiming at resolving the function of SLC25A46 and the pathogenesis of SLC25A46-associated diseases were conducted. Primary cultures of skin fibroblasts from HMSN6B patients (including PCH or Leigh syndrome patients) have been studied to investigate the pathophysiology of the diseases. Simultaneously, mutant SLC25A46 alleles or SLC25A46-targeted RNA-interference molecules were transfected into common cell lines (e.g., COS-7, HeLa, HEK293T, and HCT116) to study the consequences of the mutations or protein knock-down in a homogeneous genetic background. Finally, zebrafish, murine, and bovine models have been employed to examine the pathophysiolog-

In silico analysis led to the identification of a SLC25A46 homolog in yeast, namely Ugo1. The Ugo1 protein is a modified mitochondrial solute carrier expressed in the mitochondrial outer membrane that operates as a mitochondrial fusion factor and interacts physically with Mgm1 (homolog of human OPA1) and Fzo (homolog of human MFN2) [38]. A succession of studies in cells demonstrated consistently that, like Ugo1, MTCH1, and MTCH2, SLC25A46 also

clinical manifestations.

78 Recent Advances in Neurodegeneration

observed [8, 36, 37].

5. Pathophysiological function of SLC25A46

ical effects of SLC25A46 mutations in vivo (Table 2).

5.1. Function of SLC25A46 in mitochondrial dynamics

localizes to the mitochondrial outer membrane [17, 20, 22].

Neurodegenerative Diseases Associated with Mutations in *SLC25A46* http://dx.doi.org/10.5772/intechopen.79992 79


 mo represents month; 

Table 2. Clinical phenotypes associated with mutant SLC25A46 animal models.

Knock down of SLC25A46 in various cell lines by different research groups caused mitochondrial hyperfusion and abnormal cristae architecture visualized with fluorescent staining and electron microscopy [17, 20, 22]. In concordance, in an ultrastructural study of a SLC25A46 knock-out mouse model, we observed enlarged mitochondria with swollen cristae in Purkinje cell (PC) dendrites and sciatic nerves (Table 2) [39]. Hyperfused mitochondria consequent to SLC25A46 loss was unexpected because loss of Ugo1 function usually results in mitochondrial fission; however, it should be noted that strikingly similar cristae architecture abnormalities from loss of function are common to both genes [38, 40, 41]. Interestingly, in SLC25A46 mutant Purkinje cell bodies, ring-shaped or C-shaped mitochondria (a rarely reported morphology) were more commonly observed than hyperfused mitochondria [39, 42]. Furthermore, mitochondria were found to have an abnormal distribution and impaired movement within mutant Purkinje cells in a primary culture of mouse cerebellar cells [39]. These findings confirm that SLC25A46 plays an important role in the regulation of mitochondrial dynamics, including mitochondrial fusion/fission, distribution, and movement, as well as the maintenance of cristae architecture. Regarding the molecular actions of SLC25A46 in the balance of mitochondrial dynamics, recent research findings present three possible explanations: (1) SLC25A46 may act as an independent pro-fission factor; (2) SLC25A46 may serve as a regulator by interacting with mitochondrial fusion machinery, such as through an association with MFN1/2 oligomerization; and (3) SLC25A46 may regulate mitochondrial dynamics through its

Neurodegenerative Diseases Associated with Mutations in *SLC25A46*

http://dx.doi.org/10.5772/intechopen.79992

81

functions in lipid transfer between the endoplasmic reticulum (ER) and mitochondria.

nisms given the current conflicting results in the literature.

In an inter-institution collaborative exploratory study employing immunoprecipitation assays and mass spectrometry analysis, there was no evidence of SLC25A46 interacting with MFN2 or OPA1 in HEK293T cells, but rather SLC25A46 was observed forming a complex with mitofilin that was independent of MFN2 [17]. Furthermore, overexpression of wild-type SLC25A46 protein led to mitochondrial fragmentation and disruption of the mitochondrial network. Thus, SLC25A46 was proposed to act as a pro-fission factor [17]. In contrast, two subsequent studies using similar immunoprecipitation approaches in patient fibroblasts and two cell lines (HEK293T with stable wild-type SLC25A46 expression and LAN5 neuronal cells) showed SLC25A46 interactions with proteins involved in fission and fusion, including MFN1/ 2 and OPA1, as well as with components of the MICOS (mitochondrial contact site and cristae organizing system) complex (Figure 1) [18, 20]. Moreover, decreased expression of SLC25A46 resulted in increased stability and oligomerization of MFN1 and MFN2 in association with mitochondria, thus promoting mitochondrial hyperfusion [18]. In SLC25A46 knock-out mice, two independent mass spectrometry studies yielded opposite results regarding the interaction between SLC25A46 and common dynamic proteins [42, 43]. Hence, although it seems reasonable that SLC25A46 would have interaction relationships with MFN1/MFN2 and the MICOS complex similar to those of Ugo1, further studies are needed to resolve its molecular mecha-

MFN2 tethers the ER to the mitochondrial network, suggesting that the ER may have a physical relationship with the mitochondrial network [44]. SLC25A46 has also been shown to interact with all nine components of the endoplasmic reticulum membrane complex (EMC) [18, 20], an ER protein complex recently shown to be necessary for phospholipid transfer from the ER to mitochondria in yeast. Most mitochondrial phospholipid species were altered Knock down of SLC25A46 in various cell lines by different research groups caused mitochondrial hyperfusion and abnormal cristae architecture visualized with fluorescent staining and electron microscopy [17, 20, 22]. In concordance, in an ultrastructural study of a SLC25A46 knock-out mouse model, we observed enlarged mitochondria with swollen cristae in Purkinje cell (PC) dendrites and sciatic nerves (Table 2) [39]. Hyperfused mitochondria consequent to SLC25A46 loss was unexpected because loss of Ugo1 function usually results in mitochondrial fission; however, it should be noted that strikingly similar cristae architecture abnormalities from loss of function are common to both genes [38, 40, 41]. Interestingly, in SLC25A46 mutant Purkinje cell bodies, ring-shaped or C-shaped mitochondria (a rarely reported morphology) were more commonly observed than hyperfused mitochondria [39, 42]. Furthermore, mitochondria were found to have an abnormal distribution and impaired movement within mutant Purkinje cells in a primary culture of mouse cerebellar cells [39]. These findings confirm that SLC25A46 plays an important role in the regulation of mitochondrial dynamics, including mitochondrial fusion/fission, distribution, and movement, as well as the maintenance of cristae architecture. Regarding the molecular actions of SLC25A46 in the balance of mitochondrial dynamics, recent research findings present three possible explanations: (1) SLC25A46 may act as an independent pro-fission factor; (2) SLC25A46 may serve as a regulator by interacting with mitochondrial fusion machinery, such as through an association with MFN1/2 oligomerization; and (3) SLC25A46 may regulate mitochondrial dynamics through its functions in lipid transfer between the endoplasmic reticulum (ER) and mitochondria.

In an inter-institution collaborative exploratory study employing immunoprecipitation assays and mass spectrometry analysis, there was no evidence of SLC25A46 interacting with MFN2 or OPA1 in HEK293T cells, but rather SLC25A46 was observed forming a complex with mitofilin that was independent of MFN2 [17]. Furthermore, overexpression of wild-type SLC25A46 protein led to mitochondrial fragmentation and disruption of the mitochondrial network. Thus, SLC25A46 was proposed to act as a pro-fission factor [17]. In contrast, two subsequent studies using similar immunoprecipitation approaches in patient fibroblasts and two cell lines (HEK293T with stable wild-type SLC25A46 expression and LAN5 neuronal cells) showed SLC25A46 interactions with proteins involved in fission and fusion, including MFN1/ 2 and OPA1, as well as with components of the MICOS (mitochondrial contact site and cristae organizing system) complex (Figure 1) [18, 20]. Moreover, decreased expression of SLC25A46 resulted in increased stability and oligomerization of MFN1 and MFN2 in association with mitochondria, thus promoting mitochondrial hyperfusion [18]. In SLC25A46 knock-out mice, two independent mass spectrometry studies yielded opposite results regarding the interaction between SLC25A46 and common dynamic proteins [42, 43]. Hence, although it seems reasonable that SLC25A46 would have interaction relationships with MFN1/MFN2 and the MICOS complex similar to those of Ugo1, further studies are needed to resolve its molecular mechanisms given the current conflicting results in the literature.

MFN2 tethers the ER to the mitochondrial network, suggesting that the ER may have a physical relationship with the mitochondrial network [44]. SLC25A46 has also been shown to interact with all nine components of the endoplasmic reticulum membrane complex (EMC) [18, 20], an ER protein complex recently shown to be necessary for phospholipid transfer from the ER to mitochondria in yeast. Most mitochondrial phospholipid species were altered

Animal

SLC25A46

SLC25A46

 proteins

 Age

Age of

Optic

Peripheral

Ataxia

Degeneration

Other features

dynamics

Mitochondrial

in

cerebellum

/brainstem

aberrations,

improper

80 Recent Advances in Neurodegeneration

neuromuscular

junction.

Purkinje cell

Enlarged or

ring/C-shaped

mitochondria.

loss and dendritic

abnormalities,

degeneration

striatum,

corpus

callosum and

spinal cord;

axon

degeneration

and

demyelination.

 in

atrophy

neuropathy

of

death

onset

mutations

species

Slc/

c.992\_1037del

p.Leu331fs\*346

2 w 2–8 w

 +

 +

+

 +

B6D2

mouse Li

et al. [39] Note: mo represents Table 2. Clinical phenotypes

 associated with mutant SLC25A46

 animal models.

 month; w the week.

dramatically by the loss of SLC25A46, indicating that SLC25A46 provides direct coupling of lipid flux between the ER and mitochondria at outer-inner mitochondrial membrane contacts [20]. Meanwhile, endoplasmic reticulum chaperone BiP (a.k.a. 78 kDa glucose-regulated protein), which acts at the ER-mitochondria interface under stress conditions and is considered as a major regulator of the ER, was down-regulated in SLC25A46 knock-out mice [43]. These findings support the notion that the facilitation of lipid flux at ER contact sites may be a primary function of SLC25A46.

medial anterior horn at all levels from the cervical to the lumbar region. Rectus femoris muscle biopsies showed severely atrophied muscle fibers of all types, without type grouping [48].

Neurodegenerative Diseases Associated with Mutations in *SLC25A46*

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83

For now, most anatomical and histological analyses for SLC25A46-related diseases are based on animal models. Four SLC25A46 mutant mammalian models have been discovered or designed, including a calf model and 3 mouse models with different genetic backgrounds (Table 2). Turning calf syndrome, a neurodegenerative disease, is characterized during massive inbreeding in cattle. Genetic study determines that this disease resulted from a single substitution in the coding region of the SLC25A46 [43]. All affected calves manifested early onset of ataxia, especially of hind limbs, and paraparesis (2–6 weeks old). Despite symptomatic care, nervous symptoms progressed over the next months, leading to repetitive falls and ultimately resulting in permanent recumbency and inevitably euthanasia. Characteristic degenerative microscopic lesions were found in both gray matter (brainstem lateral vestibular nuclei and spinal cord thoracic nuclei) and white matter (dorsolateral and ventromedial funiculi of the spinal cord) regions of the central nervous system, as well as demyelination of some peripheral nerves [49]. Electron microscopy confirmed this neuropathy phenotype, revealing

Three SLC25A46 knock-out mouse models with different genetic backgrounds, including FVB/ N, C57BL/6J, and B6D2, were generated, respectively (Table 2). In spite of various mutation positions and sizes, three mouse lines displayed very similar phenotypes, including growth delay, progressive ataxia, optic atrophy, short life span, which recapitulated the pathological state in human. Further histopathologic studies have shown tissue- and cell-specific lesions in

Although macroscopic examination showed no overt abnormalities in the gross anatomy of mutant brain, histological staining revealed markedly reduced cerebellums, with Purkinje cells (PCs) that had stunted dendrites and were reduced in number. Degeneration (evidenced by Fluoro-Jade C dye) was selectively present in mutant PCs [39]. Examination via electron microscopy (EM) revealed that degenerated PC dendrites exhibited disorganized cytoskeleton, often containing remnants of mitochondria and other organelles. Numerous atypical mitochondria with cytoplasmic inclusions were found both in the soma and dendrites of PCs. In addition, a significant reduction in vGlut1 and vGlut2 immunoreactivity both in PCs and molecular layer indicated a paucity of glutamatergic synapses in mutant mice [42]. Apart from PCs, degenerative signals were also aggregated in the vestibular nucleus of brainstem, deep cerebellar nuclei, the striatum, the corpus callosum, and the spinal cord, but not in other parts of the brain [39]. The neurodegeneration was associated with astrogliosis and microgliosis in the cerebellum and spinal cord, indicating high levels of neuroinflammation [39, 42]. These observations suggest that although SLC25A46 mRNA is transcribed in most neural tissues, SLC25A46 may have a tissue- or cell-specific function. Interestingly, mice with conditional knock-out MFN2 or DRP1 mutations showed a similar phenomenon, with major changes in cerebellar Purkinje cells, whereas granule cells seemed to be spared [50]. It is not yet known why specific neurons are selectively affected by mitochondrial dysfunction. It could be that a large neuronal size makes particular neurons susceptible; Purkinje cells are one of the largest neuron types in the brain with long axons and extremely extensive dendritic branches. Their

discrete demyelinating lesions and some enlarged nodes of Ranvier.

both the central nervous system and peripheral nervous system (Table 2).

Studies implicating OPA1 and the MICOS complex in the maintenance of cristae architecture are compelling, but it is unclear how they may interact [45]. The observation that SLC25A46 interacts with OPA1 and MIC60, the major MICOS organizer, provides a molecular link that may integrate their functions in modulating cristae architecture [18, 20].

It now seems likely that SLC25A46 may possess multiple homeostatic functions in mitochondrial dynamics. Further studies are expected to reveal more refined details of the pathophysiological functions of SLC25A46, such as which domain interacts with dynamic proteins and which domain recognizes and communicates with the ER.
