Interventions: Medical and Non Medical

#### **Chapter 4**

## Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling Cerebellar Ataxias

*Roxana Deleanu*

### **Abstract**

The most affected cell types in cerebellar ataxias are the cerebellar neurons, which are not readily accessible for cellular and molecular investigation. Pluripotent stem cell (PSC) technology has emerged as an important tool for generating diverse types of neurons, which are used in order to better understand the human nervous system development and pathologies. In this chapter, the strategies for the differentiation of human PSCs toward cerebellar neurons are overviewed, followed by an outlook of their further optimization and diversification by implementing the knowledge from cerebellar development and new cell culture approaches. The optimization stategies are based on the recent progress made in defining the cell populations in mature and developing mouse and human cerebellum. The cellular phenotypes and organization in mouse and human cerebellum are briefly presented, followed by an overview of our current knowledge about their development, which includes pattering, proliferation, neurogenesis, gliogenesis, migration, connectivity and maturation. To date, however, relatively few studies have used induced PSCs (iPSCs) to model cerebellar ataxias and even fewer have looked directly to cerebellar neurons. The reported iPSC-derived *in vitro* models for cerebellar ataxias are reviewed, followed by an outlook of how to improve these models by generating and exporing the cerebellar neurons.

**Keywords:** cerebellar ataxias, iPSC-derived cellular models, cerebellar neurogenesis, Purkinje cells, cerebellar organoids

#### **1. Introduction**

Cerebellar ataxias constitute a very heterogeneous group of diseases in which the motor incoordination is caused by the dysfunction and degeneration of the cerebellar neurons. Although different causative genes or toxins have been identified and several pathological pathways have been investigated, the treatments for these conditions are still largely palliative. Therefore, it is an urgent need for disease-relevant cellular models for studying disease progression and screening for potential therapies.

The rapid development in the field of induced pluripotent stem cell (iPSC) technology offers the opportunity to combine the genetic authenticity of the

patient-derived cellular models with the disease-relevant cell types. Human iPSCs have been generated from a wide variety of easily accessible tissues, including skin and blood cells, using methods which nowadays are safer because they avoid the genomic integration of the viral vectors containing reprogramming factors. The potential of iPSCs to differentiate into any cell type of the body was previously explored by the studies with mouse and human embryonic stem cells (ESCs), which are blastocyst-derived pluripotent populations. Both iPSCs and ESCs may offer direct access to study the cells making the nervous system, but straightforth for disease models are the neurons differentiated from iPSCs, generated from patients with a variety of neurologic or neurodegenerative conditions [1, 2].

Although significant advances have been made, most of the protocols for the differentiation of human PSCs into neurons yield cellular populations which can only partially mirror the functional characteristics detected *in vivo*. In addition, most of the available neuronal characterization comes from the studies in rodents and we still know little about the phenotypes that the human neurons have in different stages of their development or degeneration. Nowadays, only few protocols generate efficiently specific neuronal classes, such as the midbrain dopaminergic neurons or the cortical neurons, while for the most neuronal types in the human brain, including the neurons of the cerebellum, the efficiency of the protocols is much lower and additional cell selection methods are required.

As it happened for the generation of other human neural or non-neural cells and especially for the generation of the cerebral cells (reviewed in [3, 4]), the improvements in the generation of cerebellar neurons will definitely come from a better knowledge of the human cerebellum and its developmental pathways.

The human adult cerebellum is the second largest brain part (after the cerebral cortex) and contains around 80 billion neurons (which represents four times more neurons than in the cerebral cortex) [5–8]. These neurons contribute to the complex cerebellar functions, including the control of movements for performing finetuning and coordination [9, 10], as well as of cognitive and emotional processes [11, 12]. The morphological and functional organization in the cerebellum, intensively investigated in rodents, is highly conserved across vertebrates [13]. Both human and mouse cerebella contain two lateral hemispheres connected by a region named vermis. The lateral hemispheres are subdivided into lobes and lobules and, together with vermis, covered by a uniformly pliated gray matter forming the cerebellar cortex. Cerebellar neurons have their cell bodies (somas) located in the cerebellar cortex and in the nuclei situated inside the white matter of each cerebellar hemisphere, called deep cerebellar nuclei (DCN). There are four distinctive DCN in mouse (dentate, fastigial, emboliform and globose), while the last two are fused as the interposed nucleus in human [10, 13].

The higher number of lobules in humans makes the cerebellar cortex more expanded relative to mice; in spite of the increase in size, both the volume of the cerebellum as a percentage of the total brain and the ratio of the number of neurons in the cerebellum to the cerebral cortex is remarkably constant across mammalian species, pointing to the concomitant increase of the cerebellum and the cerebral cortex in humans [6, 8, 14–17].

The morphological organization of the adult cerebellum is schematically presented in **Figure 1**. The neurons located in the cerebellar cortex form three laminar structures laying between the internal white matter and the external pia mater: the granular layer (GL, named also the inner GL), the Purkinje layer (PL) and the molecular layer (ML). The GL contains the densely packed granule cells, which are the most abundant cell type in cerebellum and in the whole brain, as well as few other cells, such as Golgi cells (with different subtypes, such as Lugano, globular and candelabrum) and unipolar brush cells. PL is a narrow middle zone

*Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

#### **Figure 1.**

*Cellular composition and organization in the adult cerebellum. The cerebellum contains, from exterior to interior, the cerebellar cortex with 3 layers, the molecular layer (ML), the Purkinje layer (PL) and the granular layer (GL), and the deep cerebellar nuclei (DCN) situated in the white matter (WM). Excitatory (red-orange) and inhibitory (green) neurons are located in the cortex (granule cells (GC), unipolar brush cells (UBC), Purkinje cells (PC), Golgi (G), basket (B) and stellate (S) cells) and in the DCN (E-DCN and I-DCN). GC and UBC receive external afferents via mossy fiber, while E-DCN via both mossy fibers and climbing fibers. PC receive external afferents via climbing fibers and internal afferents via parallel fibers, sending efferents to DCN. BG: Bergmann glia (gray).*

that contains the large cell bodies of the Purkinje cells, together with the cell bodies of a special type of glial cells named Bergmann glia. The ML contains mainly cell projections, but also a few entire neurons such as the basket cells located near the PL and stellate cells located near the pia mater.

In addition to the shape and location of their cell bodies, the cerebellar neurons are characterized by other intrinsic properties included in their neurochemical profiles (neurotransmitters, associated neuropeptides and receptors), electrophysiological profiles and, in the recent years, in high-throughput transcriptional fingerprints. Based on the neurotransmitters used for synaptic communication, cerebellar neurons are set into two *main classes*: excitatory neurons, which release glutamate, and inhibitory neurons, which release mainly γ-aminobutyric acid (GABA). Excitatory neurons are situated in the cerebellar cortex (granule and unipolar brush cells) and in the DCN. Inhibitory neurons are localized also in the cerebellar cortex (Purkinje cells, Golgi cells, basket and stellate cells) and in the DCN (**Figure 1**).

Regarding tissue architecture and connectivity, the cerebellar neurons are arranged as repeating units in a highly regular manner, relatively identical in all areas of the cerebellar cortex. Granule cells and excitatory neurons in DCN are projection neurons, while inhibitory neurons in the cortex (Golgi cells, stellate cells and basket cells) and DCN, and the unipolar brush cells are interneurons. Granule cells receive excitatory signals from neurons of the brainstem or spinal cord, mainly with a station in the middle or inferior cerebellar peduncle, *via* the mossy fiber afferents. The information from ~25 million mossy fibers is dispersed to ∼50 billion granule cells, but each dendrite apparently synapses with a single mossy fiber, in this way promoting combinatorial encoding and enhanced processing of sensory input to the cerebellum. Unipolar brush cells receive sensorimotor signals *via* mossy fibers, each cell forming a specialized giant synaptic junction with a single mossy fiber terminal. Their axons branch locally within the GL, where an intrinsic system superimposes on the canonical extrinsic mossy fiber system (reviewed in [15]).

The axons of granule cells project to the ML, where they form the parallel fibers, which intercept the dendrites of Purkinje cells at right angles. There are ~200 granule cells per Purkinje cell in mice, while in humans there are 3000 granule cells *per* Purkinje cell [8]. Purkinje cell bodies form a monolayer in the middle of PL, each neuron sending a monoplane-oriented expansive dendritic tree with thousands of little spines into the ML, while its axon projects towards and connects with one neuron in the DCN. In addition to the inputs from granule cells, each Purkinje cell receives excitatory signals from climbing fibers arising from the inferior olive neurons in the medulla (which receives sensory information from the cortex). Purkinje cells convey the results of the analysis of afferent information to the excitatory neurons in DCN, which form the main cerebellar output. Each excitatory neuron in DCN receives inputs from several Purkinje cells, but also inputs from the spinocerebellar tract *via* the mossy fibers and from the inferior olive *via* the climbing fibers. Excitatory neurons in DCN send projections back to the brainstem and to motor cortex *via* the thalamus [18, 19].

Remarkably, Purkinje cells can exhibit two distinct types of action potential, with simple and complex spikes. The simple spikes represent an autonomous pacemaker activity, with very little variability between spiking intervals, firing in absence of synaptic inputs. The simple spikes can be modulated by inputs from mossy fiber *via* the parralel fibers. Inhibitory interneurons in the ML, i.e. the stellate and basket cells, also influence circuit topography by making synapses with the dendritic tree and modulating the activity level of Purkinje cells. Additionally, Purkinje cells can evoke complex climbing fiber inputs. Integration of the inputs from climbing fibers and parralel fibers in Purkinje cells generates a unique form of heterosynaptic plasticity, that has been shown to underlie the motor learning [18, 20, 21]. In line with the recent multimodal characterization of the cerebral cortical neurons [22], a deeper investigation of the electrical profiles in human cerebellum is expected from the new Patch-seq techniques [23, 24].

A more extensive neuronal characterization was recently performed by high throughput sequencing, including single-cell sequencing for mouse and human cerebellar tissue [25, 26]. In spite of their quite regular morphology, the cerebellar neurons in each subclass appear as a heterogeneous population, different subsets being defined by several molecular cues, including co-neurotransmitters (e.g. glycine) and neuromodulators (e.g. calbindin, parvalbumin). Markers of some subclasses are related to the position in the cerebellar areas (reviewed in [27]). In addition, a comparative high throughput analysis of mouse versus human cerebellar cells using single cell-RNA sequencing showed that several genes are expressed in human but not in mouse Purkinje cells and confirmed at protein level the expression of novel and specific human Purkinje cell markers, in line with the data from the cerebral cortex [28, 29].

#### *Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

Recent progress in genetic technologies has significantly clarified how the cerebellar cells and their circuits are formed in model organisms, especially in mouse [30–33]. Remarkable advances were made not only in defining of the molecular phenotypes and the differentiation pathways for most of the neural progenitors, but also in understanding of how these synchronize for forming neuronal circuits. Purkinje cells have major roles also during development [34]. They orchestrate the long lasting neurogenesis of the granule cells, the most abundant local excitatory neurons, and the maturation of the local inhibitory neurons, which reciprocally respond by helping in their own maturation.

The human-specific morphological and functional attributes were intensively studied over the last two decades, including for the development of the cerebellum. Mouse mutants for different genes related to developmental diseases affecting the cerebellum in humans demonstrated a considerable evolutionary conservation of the molecular programs across species, but also revealed some human-specific differences. Recent investigations of the developing human cerebellum have emphasized some differences in the organization of the cerebellar progenitor pools. Other human specific differences have been outlined by the single-cell sequencing of different brain cells, including cells in the cerebellum. These high throughput results point out that we still have much to learn about the human cerebellar development, composition and functions.

To what extent can or could the cellular diversity in the adult human cerebellum, and, in the same time, the spatial precision in its organization *in vivo* be reproduced by the PSC-related differentiation protocols? Which would be a proper human model for cerebellum development and cerebellar diseases?

The reported strategies for the differentiation of human PSCs toward cerebellar neurons, especially toward Purkinje cells, are reviewed in this chaper, followed by an outlook of their further optimization and diversification by implementing the knowledge from cerebellar development and new cell culture approaches. This outlook incudes an overview of the recent progress made in defining the cell populations in developing mouse and human cerebellum, followed by our current knowledge about their development, which includes pattering, proliferation, neurogenesis, gliogenesis, migration, connectivity and maturation. This knowledge is also the basis for the establishment and optimization of the PSC-derived models for cerebellar ataxias. An overview of the reported *in vitro* patient-derived iPSC approaches for modeling cerebellar ataxias is presented, followed by an outlook of some challenges that remains to be overcome.

#### **2. Differentiation of pluripotent stem cells toward cerebellar neurons**

Over the past 20 years, human PSCs, including the ESCs and the iPSCs [35–38], have revolutionized the research on human development and diseases, particularly for the nervous system. Considerable progress has been made in converting human PSC into different types of neural progenitors, from which some continued to differentiate toward different classes of neurons, *in vitro* or after xenotransplantation.

Most of the reported human PSC-based protocols are an adaptation of the protocols that were previously developed for mouse ESCs, which reflect, to a various extent, different stages of neural differentiation in mouse embryo. On this line, the differentiation of the human PSCs is expected to reflect different stages of neural differentiation in human embryonic and fetal stages. Remarkably, recent data have demonstrated that several protocols starting from human PSCs produced authentic neurons and structured brain-like tissues, including the cerebral cortex,

the most complex structure in the human brain. However, many questions remain about the extent to which the relative simplistic *in vitro* settings could reproduce the high complexity of the adult brain structures, both in cell diversity and connectivity (reviewed in [3, 39]).

For the neurons making the human cerebellum, the progress of *in vitro* differentiation protocols was a lot slower comparing to other neuronal populations, such as the spinal cord motoneurons, midbrain dopaminergic neurons, and glutamatergic and GABAergic cortical neurons, between many others. The main reason is the complexity of the cerebellar development, which was only partially and only recently deciphered (overviewed in the next section), while the developmental mechanisms for the spinal cord, midbrain and cerebral cortex were much faster and deeper investigated [40–43].

Increasing understanding of cerebellar development has allowed the elaboration of several protocols in the last years, which made the production of some classes of cerebellar neurons possible, with increasing efficiencies. These protocols were implemented in 2D and 3D cell cultures, or in their combination. As for other brain regions, the differentiation protocols include "directed" steps, meaning controlled differentiation by using extrinsic manipulation approaches, but also steps in which the differentiation advances spontaneously. Most of the protocols use morphogens/ growth factors or small molecules with similar functions, which are sequentially administered to mimic the environment *in vivo*.

Two early studies implemented the mouse ESCs differentiation into cerebellar neurons, using different approaches [44, 45], which were followed by several protocols aimed to increase their efficiency. Su et al. [45] used non-adherent ESC cell clusters in serum-free medium supplemented with fibroblast growth factor 2 (FGF2) and insulin. The cellular spheroids, named serum-free embryoid bodies (SFEB, even though they contained mainly undifferentiated cells in this stage), gradually differentiated into more complex 3D cell aggregates containing a mixture of progenitor cells and neurons, which included some granule cell progenitors and few neurons expressing early Purkinje cell markers. Following the same conditions, Muguruma et al. [46] showed that the FGF2-treated neural progenitors presented a broad fate, but some cells organized in tissue-like structures resembling the cerebellum origin in the embryo. These 3D cell aggregates further formed brain organoids, which contained some areas organized as a primitive cerebellar tissue. When cyclopamine, a sonic hedgehog (SHH) antagonist, was added to block the spontaneous ventralization, the proportion of cerebellar cells was increased, including 35–42% Purkinje cell progenitors by day 11 of ESC differentiation. Additionally, this study introduced the selection of the cerebellar progenitor cells, addressing to a cell-surface marker expressed in this population (Kirrel2/Neph3). The selected cells survived and integrated into the mouse cerebellum following *in utero* transplantation at embryonic day (e) 15.5, but their surviving and differentiation into Purkinje cells *in vitro* was possible only in co-culture with dissociated mouse postnatal cerebellar cells [47]. Following the same protocol, Tao et al. [48] showed that the cerebellar organotypic slices prepared from mice at postnatal day (p) 6–8 supply an appropriate trophic environment for the differentiation and maturation of ESCderived Purkinje cells. Remarkably, after 28 days in co-culture, they showed the same characteristics as the neonatal Purkinje cells.

Salero and Hatten [44] succeeded in generating mouse ESC-derived granule cells at a relatively high efficiency by implementing a protocol in 2D culture based on step-related treatments with different morphogens. FGF8, WNT1 and retinoic acid (RA) were used in the first step, while bone morphogenic proteins (BMPs) were used in the next step to obtain the granule cell progenitors, which were next proliferated with SHH and Jagged1 and showed markers expressed in GL *in vivo*.

#### *Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

Again, for differentiation and maturation, granule cell progenitors were co-cultured with either postnatal mouse cerebellar neurons or glial-conditioned medium and the resulted neurons resembled the neonatal counterparts.

The pioneering studies of mouse ESC cerebellar differentiation were next translated to human PSCs and subsequently refined (**Table 1**). The protocol of Muguruma et al. [46] in 3D culture was applied to human ESC and iPSCs [49, 50, 52]. Human progenitor cells self-organized in polarized neuroepithelium containing around 10% KIRELL2+ cells after 20 days. Muguruma et al. [50] also refined this protocol and followed a long-term ESC differentiation in 3D culture, an approach which resembled the first generation of human brain organoids. They found that the dorsal hindbrain patterning is more efficient for human cells without cyclopamine. Sequential addition of FGF19 and stromal cell-derived factor 1 (SDF1) generated approximately 28% KIRREL2+ cells (representing the progenitors of the cerebellar inhibitory neurons) and 18% ATOH1+ cells (representing the progenitors of the cerebellar excitatory neurons) by day 35. As for the mouse protocol,


#### **Table 1.**

*Reported protocols for the differentiation of human PSCs toward cerebellar neurons.*

KIRREL2+ cells were subsequently selected by fluorescence activated cell sorting (FACS) and differentiated into Purkinje cells in co-culture with murine granule cell progenitors. The *in vitro* differentiation of the KIRREL2+ cells for 10 days generated ~45% Purkinje cell progenitors.

Other approaches aimed to increase the proportion of human ESC-derived cerebellar cells by applying the hindbrain patterning conditions tested for mouse ESCs [44]. Erceg et al. [53, 55] treated human ESCs aggregates with FGF8b and RA, followed by a manual selection of the neuroepithelial cells organized in polarized structures. This procedure yielded, after further differentiation, a heterogeneous population expressing markers of granule cells, Purkinje cells and glial cells. In a more directed differentiation approach, Sundberg et al. [54] used the WNT agonist CHIR99021, FGF8b and FGF2 for pattering the neuroepithelial cells resulted from the parallel neural induction of human ESCs with dual-SMAD inhibition [56]. The patterned progenitors gradually express the hindbrain, cerebellar and Purkinje cell progenitor markers, such as EN1/2, GBX2, PTF1a, KIRREL2 and SKOR2. Between days 24 and 48 of differentiation, markers of GABAergic phenotype and markers of immature Purkinje cells, such as PCP2, were detected. In order to enrich for the Purkinje cell population, instead of the previously used cell sorting for KIRELL2, Sundberg et al. [54] implemented the THY1+ cell selection, a method previously used to purify mouse Purkinje cells from primary cerebellar cultures [57]. The sorted THY1+ cells further matured into Purkinje cells expressing the early Purkinje cells marker PCP2 (or L7). The same team further optimized the directed differentiation protocol [28], by quantifying the effect of patterning molecules on directing the cerebellar cell phenotypes. They found that the combination of the GSK3 inhibitor CHIR99021 (1.5 μM) for 4 days with FGF8b (100 ng/ml) between days 5 and 12 of differentiation generated the highest proportion of Purkinje cell progenitors. From days 12 to 24, neural cell expressing the cerebellar marker KIRREL2 gave rise to increasing numbers of adjacently located cells expressing Purkinje cell markers. As early as day 35 of differentiation, subpopulations of iPSC-derived cells expressed markers of the primary cerebellar progenitor cells. The postmitotic Purkinje cell marker PCP2 was observed starting from day 18 onward. Flow cytometry analysis showed that ∼23% of cells expressed PCP2 at day 24 of differentiation. A changing element of this protocol was the selection of the immature human PSC-derived Purkinje cells in two steps, a negative selection by GD3 immunopanning and a positive selection by magnetic cell sorting (MACS) with NCAM antibodies [28].

As for the mouse cerebellar neurons, the conditions used for the *in vitro* maturation of the Purkinje cells and granule cells generated from human PSC were undefined, based on co-culture with different cerebellar tissue-derived populations (**Table 1**). The maturation into functional Purkinje neurons has so far been achieved in undefined conditions by co-culturing with either cerebellar granule cell precursors isolated from murine embryos [50–52], or with fetal or postnatal cerebellar organotypic slices [48, 49]. A protocol adapted from Muguruma et al. [50] eliminated the KIRREL2+ cell sorting and employed the differentiation of human cells in co-culture with e18.5 mouse cerebellar progenitors [52]. Again, markers of the cerebellar proliferative zones were detected at early times of differentiation and around 10% Calbindin+ Purkinje cells were detected from day 50 onward. Following long-term co-culture (up to 150 days), these neurons expressed the Purkinje cell markers L7, Calbindin, Aldolase C and LHX5 [50]. In the study of Sundberg et al. [54], the selected Purkinje cells were co-cultured with mouse cerebellar glia and then with mouse granule cells. With this methodology, human PSC-derived Purkinje cells formed synapses with mouse granule cells and had more differentiated morphologies. However, significant electrophysiological activity,

*Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

comparable with that of Purkinje cells *in vivo* of the iPSC-derived neurons, was observed only following co-culture with human fetal cerebellar slices [49].

#### **3. Strategies for the optimization of the human PSC-derived cerebellar cultures**

Even though the reported protocols have advanced in the generation of cerebellar neuron from human PSCs, they still need a lot of optimization in order to generate homogeneous population of cerebellar neurons in 2D cultures or cerebellar tissue-like aggregated in 3D cultures. Looking at the previous optimizated protocols for generating other neuronal populations, such as the midbrain neurons, the cortical neurons or the cortical organoids, it is relevant to follow again the steps which were gradually applied in order to achieve the efficiency and complexity they offer today (reviewed in [3, 4]). Following this aim, here the development principles of the cerebellar neurons are overviewed, from progenitor specification to neuronal assembles, followed by an outlook of how these principles could be applied for the optimization of the protocols generating cerebellar neurons from human PSCs.

During early embryo development, the human neural tube is formed by the folding of a sheet of neuroepithelium and is progressively closed and regionalized under the control of temporally and spatially coordinated gradients of morphogens secreted by organizer centers. At the end of the neurula stage, corresponding to embryonic day (E) 28, the neural tube is entirely closed and contains, from anterior to posterior, the three primary brain vesicles (forebrain, midbrain and hindbrain) and the spinal cord. Soon after the definition of the midbrain-hindbrain boundary (MHB), cerebellum starts to form at the most anterior and dorsal hindbrain territory. In humans, the cerebellar development is highly protracted, extending from E30 to the end of the second postnatal year. In mice, cerebellum almost completes over a period of around one month, starting from embryonic day (e) 9 and including the first three postnatal weeks (reviewed in [15, 58–60] (**Figure 2**). However, as for the whole brain, the mechanisms of cell differentiation and histogenesis in cerebellum are mainly conserved in mammals. While the development of the mouse cerebellum was intensively studied [15, 30, 32–34, 58, 61–65], the embryonic and fetal stages in human cerebellar development were only recently described in details [13, 16, 59, 60]. Notably, as for the other parts of the human brain, the embryonic and fetal stages of development are not available for cellular and functional studies, and their histological and clinical images represent only snapshots in time for one individual. Conversely, developmental time-course experiments in mice can be conducted on multiple mice of identical genotypes. These studies revealed that the ontogenesis of all neurons and glial cells in the nervous system, including the ones in the cerebellum, follows the same steps of (1) patterning and specification of the progenitor cells, (2) neurogenesis/gliogenesis and (3) migration, histogenesis, formation of the neuronal circuits and neuronal maturation (reviewed in [15, 27, 58, 61, 66, 67]). However, in contrast to other CNS areas, including the cerebral cortex, in which gliogenesis follows neurogenesis [68, 69], glia generation in cerebellum parallels or precedes the long-lasting generation of the granule cells and inhibitory neurons [15, 30, 32, 65, 68]. Even though the main developmental programs are conserved from mice to humans, some important specie-specific differences responsible for the expansion of the human cerebellum have been recently identified [59, 60]. In the following brief presentation, the main morphological, cellular and molecular events in mouse are complemented with the available information in human.

#### **Figure 2.**

*Timing and the aligned stages in mouse and human cerebellar development. Embryonic days in mouse (e) and human (E). GW-gestational weeks. NE- neuroepithelium (light blue). The cerebellar ventricular zone (VZ) (blue) is the origin of the inhibitory neurons and glial cells. Inhibitory neurons (green) are the Purkinje cells (PC), Golgi, basket and stellate cells, and the inhibitory neurons in the deep cerebellar nuclei (I-DCN). The rhombic lip (RL) (Lila) is the origin of the excitatory neurons in the cortex (Granule cells (GC) and unipolar brush cells (UBC) and in the DCN (E-DCN). Long-lasting progenitor stages for the GC progenitors (GCPviolet), and inhibitory interneuron and glial progenitors (INP and GP, blue). Long-lasting maturation of inhibitory neurons (light green) and of excitatory neurons (light pink-orange), and gliogenesis (gray) stages.*

#### **3.1 Patterning and specification of the cerebellar progenitor cells**

Several studies in mouse showed that all cerebellar neurons and glial cells originate from the hindbrain region corresponding to the dorsal (or alar) part (or plate) of the first rhombomere (r1) [30, 70]. The anterior limit of the cerebellum is defined by the MHB, named also isthmus, where an organizer center, named the isthmus organizer (IsO), forms early in development and has a major role in the anterior/posterior (A/P) patterning of the midbrain and hindbrain. IsO formation is preceded by a series of pattering events that start in the forming neural plate, where two transcription factors, Otx2 (Orthodenticle Homeobox 2) and Gbx2 (Gastrulation Brain Homeobox 2) define the primitive anterior and posterior domains, respectively [71]. They are further co-expressed in early IsO and then differentially express in the midbrain and hindbrain domains [72]. WNT signaling has a main role in the A/P patterning of the neural tube but also in IsO induction, showed by the loss of IsO in WNT1 homozygous mutants ([73]; reviewed in [74]). Shortly after the primary brain vesicles formation, Fibroblast Growth Factor 8 (FGF8) secreted by IsO patterns the adjacent territories [71, 75–80]. Additional A/P patterning by extra-neurally secreted retinoic acid (RA) defines the metencephalic and myelencephalic secondary hindbrain vesicles. The metencephalon expresses the homeobox gene *Hoxa1*, and formed the first hindbrain rhombomere (r1), where the FGF8 blocks the expression of other *Hox* genes. Next, the selective expression of negative regulators of the activated Ras–ERK pathway in r1 stops the local action of FGF8 [81]. In parallel with the A/P patterning, whole neural tube is patterned also dorsoventrally (D/V). The main ventralizing factor is Sonic Hedgehog (SHH), which by e9 is produced in the floor plate of the metencephalon [15, 74, 82, 83] and secreted into the neural tube's lumen, which at this level becomes the 4th ventricle. Consequently, the alar plate of the r1 territory

*Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

is patterned into the cerebellar domain (anlagen) (**Figure 3**), while anteriorly situated territory becomes the tectum domain, posteriorly, the r2 domain, and ventrally, the pons domain.

Between e9 and e12.5 and, the cerebellar neuroepithelium undergoes morphological changes: the midline remains as a single cell layer and forms the roof plate, while each lateral part forms two primary proliferative zones, known as the origins of the neural populations in the mouse cerebellum: the cerebellar ventricular zone (VZ) and rhombic lip (RL) (**Figures 2** and **3**) [30]. By e10, the roof plate becomes the second cerebellar organizer center and secretes factors belonging (TGF)-β family, such as the bone morphogenetic proteins (BMPs), the most important dorsalizing factors in the cerebellum, and gradually transforms into the choroid plexus epithelium (ChPe). By e12.5, ChPe additionally produces SHH. Genetic fate mapping proved that the morphogens secreted by IsO, roof plate and floor plate define the cerebellar domains which, in addition to the hindbrain restricted

#### **Figure 3.**

*Stages and distribution of cell populations in mouse early cerebellar development. Formation and differentiation of the cerebellar populations from embryonic day (e) 8 to e16, when all the neuronal populations or their long-lasting progenitors are formed. (A) Between e8 and 12, in the dorsal part of the first rhombomere (r1) of the hindbrain neural tube, the cerebellar ventricular zone (CVZ) (light blue) forms at e9–10, due to the dorsal FGF8 signal and ventral SHH signal, while the rhombic lip (RL) forms at after e10, being visible at the border between the CVZ and the roof plate (RP) (light Lila), due to the BMP signaling from the RP, which forms the choroid plexus epithelium (ChPe) (red). (B) Between e12 and 16, different progenitors arrive in the subventricular zone (SVZ) and mantle zone (MZ) of the neural tube. At e12–14, the Ptf1+ ventricular zone (VZ) of the CVZ primary domain contains the Olig2+ and the Gbx1+ subdomains, which generate the Purkinje cell progenitors (PCP) and the interneuron progenitors (INP, blue) domains, respectively, while the first postmitotic Purkinje cells (PC) already exit the SVZ. The VZ in RL contains Atoh1+ progenitors, which gradually form progenitors of the excitatory neurons in SVZ. They generate first the excitatory neurons for the deep cerebellar nuclei (E-DCN) and at later time points (e14–16), they start to generate the unipolar brush cells (UPC). The RL generates also the progenitors of the granule cells progenitors (GCP-violet), which migrate in waves in the MZ close to the pia mater (PM). In the CVZ, cells representing a subpopulation of the INP domain migrates in the MZ and join the E-DCN in a nuclear transitory zone (NTZ), where they start to differentiate into the inhibitory neurons of the DCN (I-DCN).*

expression of Gbx2, show the differential expression of two basic-helix–loop–helix (bHLH) transcription factors: Pancreatic transcription factor 1 (Ptf1) specifies the VZ domain and Atonal homolog 1 (Atoh1, also called Math1), specifies the RL progenitor domain [15, 58, 61, 84, 85].

Each cerebellar progenitor zone forms subdomains with their own spatial and temporal identities, which produce specific neuronal subtypes. VZ-derived progenitors give rise to all GABAergic neurons and glial cells of the cerebellum. VZ-derived neurogenesis starts at e10.5 and continues untill e17 in mouse. Before the neurogenesis starts (~e9), the VZ progenitor domain corresponds to the neuroepithelial cells localized in the VZ of the r1 neural tube (**Figure 3**). Most of the earliest Ptf1a + progenitors upregulate *Kirrel2/Neph3* and oligodendrocyte-specific bHLH gene *Olig2* expression [82, 86], while a small proportion in the early rostral VZ express homeodomain-containing transcription factor gene *Gsx1*. As the neural tube grows, the neuroepithelial cells gradually transform into radial glial progenitors and a subventricular zone appears evident in the VZ domain (SVZvz in **Figure 3**). Ptfa1+ and Olig2+ radial glial cells start to express *Lhx1*, *Lhx5* and *Skor2,* and become Purkinje cell progenitors, located in the SVZvz, which gradually express Neurogenin 1 and 2, start neurogenesis and migrate from the SVZvz. Ptfa1+ and Gbx1+ radial glial cells gradually commit to inhibitory interneuron and glial progenitors. The interneuron progenitors express *Lhx1, Lhx5* and *Pax2*. By e14.5, they become predominant in the SVZvz [87] and soon after start to migrate out of the SVZvz and form transient amplifying progenitor pools. Once all the neurons and transit amplifying progenitors exit the SVZvz, the remaining radial glial cells differentiate into Bergmann glia. The VZ-derived transit amplifying progenitors generate inhibitory interneurons, astrocytes and oligodendrocytes [58].

The neuroepithelium of the RL gives rise to all glutamatergic neurons in the cerebellum (**Figures 2** and **3**), but also to extracerebellar neurons such as the pontine neurons [66, 70]. RL Atoh1+ neuroepithelial cells situated between the roof plate and the VZ domain start their proliferation after the adjacent VZ progenitors (~e10). Also the RL neuroepithelial cells gradually acquire a radial glial phenotype and are patterned in subdomains, which express the paired box gene *Pax6* in combination with the zinc finger genes *Zic* and the homeobox gene *Meis*. First, *Pax6* and *Meis2* expressing progenitors commit to neurogenesis, when they gradually express *Tbr2* and *Tbr1* and generate the glutamatergic neurons in DCN. Later, the remaining RL progenitors co-expressing *Pax6, Meis1*, *Zic1/2* and *Barhl1* commit to granule cell progenitors, in parallel with the unipolar brush cell progenitors, which upregulate the *Tbr2* expression, downregulate *Pax6* expression and become unipolar brush cells [15, 88].

The cerebellar proliferative zones in human embryos have been only recently investigated. The human cerebellar VZ (gradually forming the SVZvz) undergoes massive expansion which covers the second month (E30–56), afterwards extinguishing its proliferative potential and remaining as a single cell layer. Conversely, the RL germinal zone remains small during the peak expansion of the VZ progenitors, but starts a significant expansion at around gestational week (GW) 11, when it forms the SVZRL, which persists long after birth [59, 60].

*GABAergic phenotypes.* Cerebellar inhibitory neurons, including Purkinje cells and interneurons (Golgi, stellate, basket and inhibitory neurons of the DCN) originate from different subdomains in cerebellar VZ (Ptf1a+), in different waves (**Figures 2** and **3**). Purkinje cell progenitors (expressing Skor2, Lhx1/5 and Corl2) gradually express Neurog1/2 and start neurogenesis, which in mouse is completed at e12.5. Once in the postmitotic stage, Purkinje cells start a short distance radial migration alongside the radial glial processes toward the mantle zone where they stack in a transient multilayered structure named the Purkinje cell plate and

#### *Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

gradually express markers such as Purkinje cell protein 2 (Pcp2, named also L7), Pcp4 and Calbindin 1 (Calb1) [15, 64]. In postnatal stages, due to extensive cerebellar expansion, multilayered Purkinje cells gradually form a monolayer while each neuron starts the development of its characteristic extensive and flattened dendritic arbor and the expression of mature markers synaptic markers [30, 58, 89].

In humans, all Purkinje cells are generated before the 8th GW, which places them among the earliest-born central neurons. They start to migrate at E44 outwards from the VZ along radial glial projections to the pial surface. A broad Purkinje cell multilayer extending in the mantle zone is evident between the GW 10 and 13 GW, while a monolayer distribution is achieved by GW 20–24 (**Figure 2**). Human Purkinje cells start to develop their characteristic extensive and flattened dendritic arbors and long axons in the early fetal stages, their final maturation being achieved postnatally, in a 6-fold longer period than in mice [59, 60, 90, 91].

Contrary to the Purkinje cells, which are postmitotic already into the cerebellar SVZvz, the Gbx1+ progenitors expressing the paired homeobox gene *Pax2* migrate in several waves from the SVZvz to the mantle zone, where they start to express the neurogenic genes *Neurog1* or *Ascl1* and differentiate into Pax2+ interneurons. In the first wave (from ~e10.5), the interneuron progenitors migrate to the rostral end of the cerebellar anlage in a Nuclear Transitory Zone (NTZ), which is transient zone for the DCN assembly [15]. After the progenitors settle near the already established excitatory neurons, they produce the inhibitory interneurons of the DCN. In later stages of development, NTZ is gradually organized into distinct DCN. In the second wave (from ~e13.5), the interneuron progenitors migrate to the Purkinje cell multilayer, continue their migration in the developing white matter and postnatally reach the developing granular layer where they generate postmitotic Golgi cells. At later stages, interneuron progenitors migrate radially in the white matter, continue to proliferate in a transit amplifying center and eventually generate the stellate and basket cells in the ML [78, 92]. In parallel with the late interneurons progenitors, the progenitors of astrocytes and oligodendrocytes continue to proliferate in the developing white matter (**Figure 2**).

Glutamatergic cerebellar neurons (excitatory neurons in DCN, granule cells and unipolar brush cells) originate from different subdomains of the RL, in different waves (**Figures 2** and **3**). The first cells leaving from the RL are the newborn excitatory neurons in DCN. Next, the granule cell progenitors migrate in waves out of the RL, where they continue the proliferation. In the first wave (e10.5–12.5), discrete subpopulations of rostrally situated Atoh1+ cells gradually upregulate *Pax6*, *Meis2*, *Lhx9 Tbr2* and *Tbr1* and become newborn glutamatergic neurons, which migrate rostrally and tangentially to the NTZ [15, 88, 93]. The allocation of a temporal framework of different DCN components is accompanied by a characterized sequence of transcriptional maturation that results in the first born neurons for the lateral nucleus (projecting to midbrain and thalamus), followed by neurons for the medial (fastigial) group.

The second wave covers middle to late embryonic stages, when Pax6+ granule cell progenitors leave the RL, migrate out toward the pial surface and undergo a prolonged expansion in a secondary germinal zone, or a second transit amplifying center, named the external granular layer (EGL) [64]. Granule cell progenitors retain the expression of *Atoh1* and migrate into the mantle zone where they express *Tbr2* and continue to proliferate to form the EGL [88]. During the early postnatal period, multiple mitogenic pathways expand the EGL. Peak EGL proliferation occurs around p7 and is complete by p15 (**Figure 2**). The main mitogen is the SHH, secreted by the underlying Purkinje cells [94], but also Jag1, a ligand the Notch2, acts locally in the EGL [95]. Exponential granule cell proliferation in the EGL drives cerebellar growth and foliation [96]. BMP4 and WNT3 secreted by the ChPe promote cell-cycle exit and neurogenesis [15]. The postmitotic granule cells downregulate *Atoh1* and upregulate *NeuroD1* [15]. Newborn granule cells migrate tangentially within the EGL and then exit the EGL migrating radially inwardly along Bergmann glial fibers, trailing a long T-shaped axon behind, interact with the flat, elaborate dendrites of Purkinje cells and form the parallel fibers in ML. Migrating granule cells settle below the developing PL to form the internal granule layer (IGL, corresponding to the adult GL), achieving the final laminar arrangement of the mature cerebellum, from where they extend dendrites to form synapses with mossy fiber afferent axons [15, 58].

Unipolar brush cell differentiation parallels the granule cell progenitor waves (**Figure 2**). Unipolar brush cells are born starting with e13.4, while continuing to p0–1. Progenitors of the unipolar brush cells express Wnt1 early in development (e10.5–13.5), but this expression is downregulated before they migrate from the RL. The newly generated neurons remain in the RL for an additional 1–2 days, after which they exit RL and migrate dorsally through the white matter to their final destination. Most unipolar brush cells reach the IGL by p10, several days before granule cell neurogenesis is complete. Their final maturation occurs between p2 and p28, which seems to coincide with the establishment of the first synaptic contacts with external mossy fibers [15, 27, 88].

#### **3.2 Coordinated formation of the cerebellar circuits**

The successful construction of the neuronal circuitry relies on the coordinated generation of functionally opposed neurons. Accordingly, the differentiation programs of cerebellar excitatory and inhibitory neurons are interdependent and defined as the coordinated integration of the VZ and RL-derived lineages in local circuits, in both the cortex and DCN. For the DCN, the cell fate of the excitatory neurons appears determined at the RL, in a temporal pattern, while the interneuron progenitors migrate, differentiate and integrate in the NTZ after receiving local signals from the excitatory neurons.

Purkinje cells have a remarkable capacity to regulate developmental events by sending SHH signals bi-directionally. Starting at e16.5 and continuing throughout adulthood, *SHH* expression in cerebellum is restricted to Purkinje cells and Bergmann glia [97]. Dendritic-derived SHH drives the granule progenitor cell proliferation, while axon-derived SHH disseminates to the neonatal white matter and contributes to the expansion of the VZ-derived progenitors for the late-born interneurons and glial cells during the postnatal period [98]. Additionally, Purkinje cells are critical for the terminal differentiation and morphogenesis of the interneurons in the ML, the basket and stellate cells. On the other side, signaling from differentiating granule cells influences the planarity and the elaborate branching pattern of the Purkinje cell dendritic tree, which occurs from p5 to p15 [99, 100]. Additionally, the dendritic differentiation of the interneurons in ML is sensitive to the granule cell-derived inputs, including BDNF signaling [15].

In the third trimester and postnatally, human cerebellum undergoes its major growth, primarily due to the prolonged expansion of the granule cell progenitors. By 10–11 GW, streams of cells which form the external GL (EGL) were observed along the pial surface connecting to the RL. Due to extensive EGL proliferation, human cerebellum increases 5 fold in size between GW 24–40 [90]. Differentiation and maturation of the human cerebellar neurons progress mainly as in the mouse, but there are some species-specific features. Foliation correlates with EGL proliferation and increases dramatically between GW 20–32, as the cerebellum rapidly increases in size and volume. The formation of the Purkinje cell monolayer coincides with the peak of EGL proliferation [89, 90]. The human cerebellar cortex still has a prominent EGL at birth. EGL gradually decreases in thickness as a result of

*Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

migration of granule cells into the internal GL. By the end of the second postnatal year, EGL is depleted while the thickness of the molecular layer and the length of the PL increase, concomitant with the increasing cerebellar volume [89, 90]. To date, there are few studies about the development of the human interneurons, both inhibitory and excitatory, which represent a minority comparing to the granule cells, but with a major role in the maturation of Purkinje cells and circuit formation [15, 34, 58, 91, 101].

In addition, the single-cell sequencing techniques have been applied for analyzing different stages of mouse cerebellar development [62, 102]. Carter et al. [62] performed single-cell RNA-sequencing and unbiased classification of around 40 thousand murine cerebellar cells from eight embryonic samples (at e10-e17) and 4 postnatal samples (at p0, p4, p7 and p10). Such approach allows for a more comprehensive detailing of the transcriptional and cellular heterogeneity among lineages of interest and can provide a valuable resource for answering further questions related to cerebellar development and diseases. In a similar study, Peng et al. [102] analyzed around 20 thousand cells from mouse postnatal cerebella and looked in addition to the dynamics of interneuron differentiation but also mitochondrial markers and ataxia risk genes. In a complementary approach, gene expression in the postnatal stages of mouse cerebellar development were analyzed by Buchholtz et al. [28] in Purkinje cell populations selected from mice expressing a *Egfp-Pcp2* reporter gene. Again, the dynamics of different pathways of mitochondrial and autophagy genes correlated with the developmental stages of Purkinje cells, which suggest their implication in several neurodevelopmental diseases.

#### **3.3 From development of the cerebellum to the optimization of the human PSC differentiation protocols**

There are several steps to be considered for the cerebellar protocols, which practically cover all the developmental stages: from neural induction and dorsal hindbrain patterning to the patterning and proliferation of the VZ-like and RL-like progenitors, to the neurogenesis of the selected progenitors, and lastly to the maturation of the neurons and the formation of the neuronal circuits. Are the previously used neural induction and early patterning conditions (in both 2D and 3D approaches) optimal for the generation of progenitors similar to the ones in the dorsal r1 in the neurula stage, which represent the origin of the neurons making the cerebellum? Are the previously used conditions optimal for the uniform generation of early VZ and RL progenitors? Which factors and what timing would be necessary for a uniform patterning towards VZ or RL subpopulations? Which conditions would be efficient to produce a uniform neurogenesis from different progenitors? What would the defined conditions for the neuronal maturation be? How can the neuronal maturation be faster? How can other neuronal subtypes, such as the interneurons in the cerebellar cortex and in the DCN, be generated uniformly and efficiently?

Some recent strategies were successful for the optimization of the protocols for the cerebral neurons and cerebral organoids. It remains to be checked whether these strategies can be extrapolated for the cerebellar cultures. Again, the solutions may come from the development principles. The main traiectories that could be followed from the human iPSC to the neuronal cell types contained in the cerebellum are outlooked in **Figure 4** and detailed in the following paragraphs.

*Improving neural induction and hindbrain patterning.* The first step for all the protocols regarding the neural differentiation of human PSCs implies the removing of the pluripotent cell proliferation factors, such as FGF2 and TGFβ. The additional use of several inhibitors such as BMP/Activin/TGFβ pathway inhibitors, alone (such as Noggin) or in combination (dual-SMAD inhibition by small molecules such as

#### **Figure 4.**

*In vitro trajectories from human induced pluripotent stem cells to cerebellar neurons by combining the differentiation protocols and the developmental principles. The differentiation conditions for some stages (meaning the combination of extrinsic factors, their concentration and time of action in the protocol) are previously established. However, for several steps, it remains to be established which treatments are necessary for patterning and proliferation of progenitor subpopulation in VZ and RL and in the secondary proliferation domains. Some factors which are known to act in the mouse cerebellar development could work also for the patterning and proliferation of human progenitor cells, but many question marks remain. These questions address both the treatments and the specific markers for subpopulation of progenitor cells and neurons.*

dorsomorphin or LDN and SB431542) [56], significantly increased the yield of neural induction in human PSCs cultured in serum-free medium, both in 2D and 3D systems [40]. Shortly after neural induction, human PAX6+ neuroepithelial cells acquire a primitive anterior identity, expressing *OTX2*, but no more caudal markers, like *EN1*, *GBX2*, or *HOX* genes [56, 103]. However, this anterior phenotype is transient and, depending on the presence of added or endogenously secreted morphogens such as WNTs, FGFs, and RA, neuroepithelial cells take on a definitive regional identity [41, 104–106].

Some previous protocols used FGF2 for amplifying the neuroepithelial population and showed that, although an anterior phenotype is kept for a few passages in the presence of FGF2, longer exposure gradually patterns human progenitors toward midbrain and hindbrain fates [105, 107, 108]. FGF2 was used by Muguruma et al. [50] for inducing a brought midbrain-hindbrain patterning, including the IsO-like cells, in 3D spontaneously differentiating human PSCs in serum-free medium, for a time approximating the MHB formation in human embryos. However, the reproducibility of this protocol is limited and the efficiency of the neural induction and pattering was not investigated, many cells in the 3D clusters could present a more anterior phenotype (and maybe non-neural phenotypes). Watson et al. [52] proposed the parallel neural induction and hindbrain patterning by using FGF2 in combination with the SMAD inhibitor SB431542 for around 20 days. Even though it showed an increased expression in hindbrain and cerebellar markers, yet the efficiency and the selectivity of this approach was not reported.

#### *Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

The implementation of WNT signaling was shown to increase the midbrain and hindbrain patterning and reduce the spontaneous forebrain patterning in human PSC-derived neural cultures [28, 41, 54, 109, 110]. In Kirkeby et al. [41] and Kirkeby et al. [110], neural induction with dual-SMAD inhibition and pattering were applied in parallel for 9 days. The GSK3 inhibitor CHIR99021 was used at 1–2 μM concentration for patterning the anterior r1 fate. Following this protocol with some modifications, Sundberg et al. [54] applied the neural induction and hindbrain patterning by WNT in the same time, for 12 days, with noggin and 1.7 μM CHIR99021, while in a following study coming from the same group [28], neural induction and patterning with CHIR99021 1.5 μM was applied for only 4 days. In both studies, FGF8b (100 ng/ml) was added from day 4 to day 12 of differentiation, while FGF2 applied at day 10–12 in Sundberg et al. [54] was excluded in the next protocol [28]. However, the resulted cell populations in both studies were not directly phenotyped, but after 16 or 32 days of differentiation, when they contained KIRREL2+ or THY1+ cells, respectively, which were selected by FACS. Further optimization for neural induction and hindbrain patterning requires a deeper investigation, including negative markers for forebrain, midbrain, hindbrain (excepting the r1), and ventral markers (especially for the r1). The dorsal r1 cells should concomitantly and uniformly express GBX2 and EN1/2. Obviously, reporter lines for different genes expressed solely in r1, such as HOXA1, would be very useful tools.

In addition, a study using human hindbrain tissue from embryos at GW 5–7 showed that the hindbrain neuroepithelial cells were stably expandable in FGF2 and EGF conditions, but the short treatment with FGF8 and WNT (for 1 passage) hugely increased the expression of GBX2, EN1 and EN2 [111]. A deeper investigation of the human embryonic dorsal hindbrain tissue could provide hints for the optimization of the human PSC differentiation protocol toward cerebellar cells. The human embryonic hindbrain neuroepithelial cells can be further patterned *in vitro* by BMPs (BMP6, BMP7 and GDF7) and WNT3A to RL progenitors (ATOH1), which generated granule cells after transplantation into the rat cerebellum [111]. Some additional hints are revealed by the pattering of the human embryonic hindbrain tissue. ATOH1 was not expressed if FGF8 was added together with BMPs or if FGF2 and EGF were maintained, FGF signaling appearing to counteract the BMP stimulation [111]. The same factors were applied for the RL patterning from human PSCs (reviewed in [40]). It appears clear that ATOH is not expressed by default, but only after BMP signaling, in spontaneous or directed differentiation approaches. Again, developing human PSC reporter lines for *ATOH* and a deeper phenotypic investigation, including negative markers such the ones express in vicinity of the RL, (e.g. in pons, tectum and neural crest), would be of great help. The same approach is necessary for the optimization of cerebellar VZ progenitors, which are favorized by FGFs and SHH treatments. It remains to be established which treatments with extrinsic factors (combination, concentration and time) are necessary for patterning and proliferation of progenitor subpopulation in VZ and RL, as well as out of them, in the secondary proliferation domain, as long-term proliferative populations (such as granule progenitor cells, interneuron progenitor cells and glial progenitor cells). Some factors known to act in the mouse cerebellar development could work also for human progenitor cells, but many question marks remain. These questions address both the treatments and the specific markers for subpopulation of progenitors and neurons (**Figure 4**).

*Increasing maturation of the cerebellar neurons in defined conditions.* One of the most consistent observations about human PSC-derived neurons is that they mature relatively slow and often incomplete (reviewed in [3]). An obvious reason is the time in culture: human PSC-derived Purkinje cells are usually kept in culture around 4 months, while they need over 2 years for maturation *in vivo*. An important challenge is the long-term culture and maturation of human PSC-derived cerebellar neurons without the presence of mouse cell/tissue co-cultures. Mature phenotypes of PSC-derived Purkinje cells and granule cells have so far only been demonstrated in co-culture or, more convincingly, by transplantation of differentiated cells into mouse cerebellum. While some of the *in vitro* and transplantation procedures demonstrated the potential of the PSC-derived neurons to mature into functional cerebellar neurons, they also highlighted the need to better understand the factors that promote their maturation. Significant variability in the efficiency to obtain functional Purkinje cells using different feeder cell sources was reported. For instance, feeder-free and co-culturing with rat granular progenitors failed to sustain Purkinje cell maturation and survival, while co-culture with rat cerebellar slices sustained Purkinje cells that nevertheless were devoided of any action potential or spontaneous post-synaptic currents. In contrast, co-culture with human fetal cerebellar slices resulted in electrophysiological active Purkinje cells [49], suggesting that human specific factors, as well as interactions with glial cells [112] are needed for proper maturation. The use of the co-culture system has limitations *per se*, feeder cells introducing inherent variability to the procedure [49]. A growing number of methods for reverse-engineering specific cellular micro-environments and the cells and molecules which constitute these [113] will definitely extend into the cerebellar field. It is likely that the combination of these technologies will help in elucidating key conditions for long-term survival and maturation of PSC-derived cerebellar neurons.

Another approach can come for the optimization of long-term cultures of cerebellar organoid, in line with the extensively investigated field of cerebral organoids [39]. As shown in different previous reports, functional synaptic connections are necessary for maturation and activity of the human PSC-derived neurons, which include glia and target neurons, all of these could be provided in the same cerebellar organoid.

Again, one limitation for most of the human PSC-derived neurons, as for the human neurons in general, is the lack of transcriptomic signatures, to rigorously identify specific types of neurons and to compare their development across species. A recent Metagene projection analysis of global gene expression patterns revealed that differentiating human PSC-derived Purkinje cells share classical and developmental gene expression signatures with developing mouse Purkinje cells. Remarkably, it revealed that the human PSC-derived Purkinje cells matured in co-culture for around two months are closest to late juvenile (p21) mouse Purkinje cells, suggesting that they are relatively mature. Gene expression profiling also identified human-specific genes in human PSC-derived Purkinje cells. Protein expression for one of these human-specific genes CD40LG, a tumor necrosis factor superfamily member, was confirmed in native human cerebellar tissue, arguing for the bona-fide nature of the human PSC-derived cerebellar neurons [28]. Obviously, the routine applications of the single-cell transcriptomics into the optimization steps of the human PSC-derived cerebellar differentiation protocols will hugely contribute to the progress in the field.

#### **4. iPSC-derived models for cerebellar ataxias**

The iPSC technology together with the cerebellar differentiation protocols offer the opportunity to indirectly generate and to directly study the most affected cells in patients with cerebellar ataxias, the cerebellar neurons. As schematically presented in **Figure 5**, somatic cells such as skin fibroblasts or white blood cells obtained from patients are reprogrammed into iPSCs, which can be theoretically differentiated

#### *Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

into any type of neurons. Ideally, the neuronal differentiation should address the most affected subpopulation in each disease, by following the existing protocols or optimized protocols in the desired direction (using development principles and combining efficient selection methods). Remarkably, for the inherited ataxias, the patient iPSC-derived neurons express the disease mutation in the authentic genetic background and cellular environment, which is not the case in the animal models.

The neuropathological events in hereditary cerebellar ataxias affect both cerebellar and extracerebellar territories. Nevertheless, degeneration and ultimate loss of cerebellar neurons is a neuropathological hallmark in cerebellar ataxias. The affected cerebellar neurons and the responsible genes for several cerebellar ataxias are presented in **Table 2**. Spinocerebellar ataxias (SCAs) are a family of over 40 currently described late-onset dominant diseases, manifesting clinically at middle age and gradually progressing with neurodegeneration in cerebellum and other CNS areas, [136–139] while in other genetic ataxias, such as the autosomal recessive Friedreich ataxia (FRDA) and ataxia-telangiectasia (AT), the disease manifests a lot earlier and, in addition to the nervous system, extraneural territories are affected [137, 138]. FRDA is considered a multi-systemic condition, including central and peripheral neuropathies, diabetes and cardiomyopathy [140, 141].

In cerebellum, SCA1, SCA3 and FRDA involve mainly the DCN, especially the dentate nucleus, but also extracerebellar territories such as the Clarke's column, which present with severe neuronal loss (reviewed in [142]). SCA2 predominantly affects the pontine nuclei, while the Purkinje cells and DCN seem to be secondarily affected. SCA31 is relatively restricted to the Purkinje cells. Although Purkinje cells are predominantly involved in SCA6, degeneration is evident also in the dentate nucleus and granule cells. Therefore, patients with SCA6 show more severe ataxia than those with SCA31. Several SCA subtypes have CAG repeat expansions in the coding region of different genes (http://www.scabase.eu/; [143–146]), resulting in PolyQ elongations in the respective proteins, the elongation size being correlated with the intensity of clinical manifestations. In other SCAs (SCA12, SCA31 and SCA36) or non-SCA monogenic ataxias, such as FRDA, the repeat expansion is intronic, but also in these diseases the cerebellar dysfunction is correlated with the elongation size [147].

Modeling these human genetic disorders in mice has reproduced to a certain extend the neuropathological aspects and has provided some insights into disease mechanisms. Many disease mechanisms that have been explored in mouse models are expected to be recapitulated in patient iPSC-derived neurons. However, some ataxias could not be modeled in mice using the same mutation as in the patients, suggestion that the human-specific environment is essential for the disease to

#### **Figure 5.**

*From ataxia patients to neuronal disease models. Somatic cells from patients with cerebellar ataxias are reprogrammed into induced pluripotent stem (iPS) cells, which can be genetically modified in order to correct the mutation. Patient and control/corrected iPS cells can be differentiated into neurons that are relevant for the cerebellar diseases, such as Purkinje cells. Additional stress or forced aging can be equally applied to the patient and control/corrected neurons or their progenitors, in order to amplify the phenotypic differences resulted from the ataxia's specific mutation.*


#### **Table 2.**

*Affected cerebellar neurons and iPSC-derived models for different ataxias.*

develop. Additional mechanistic understanding of the network of events produced by the mutation is crucial for the development of effective therapies, as none of the cerebellar ataxias is yet curable, treatable or preventable [143, 145, 147–149].

For modeling cerebellar ataxias, the iPSC-based models present three main advantages. First, most of cerebellar ataxias are monogenic diseases. Second, neurons bearing the mutation, which are not directly available from patients, can be generated *in vitro* from the patient iPSCs. Third, the human neurons generated *in vitro* seem to acquire a molecular profile close to the postnatal age in mouse, as in the previously mentioned Metagene analysis of key gene pathways, which showed that the human Purkinje cells generated *in vitro* have the closest molecular expression with the Purkinje cells in p21 mouse cerebellum [28]. As for many mouse models for cerebellar ataxias a disease phenotype was found close to this age, the *in vitro* generated human neurons are expected to behave similarly and to reveal the disease phenotype in early stage of maturation.

#### *Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

However, as presented in **Table 2**, relatively few studies have succeeded in generating iPSC-based models for cerebellar ataxias. An additional important question for the iPSC-based models is to what extend the mutated gene is expressed in the neurons generated *in vitro*. The most vulnerable and affected cells are neuronal subpopulations, most of them being located in cerebellum. From the reported iPSC-derived models, only a very few implemented the cerebellar differentiation protocols, including the pathways for generating the specific cerebellar cells affected in disease.

A handful of studies published to date addressed iPSC models of PolyQ SCAs (such as SCA1, 2, 3, 6, 7 and 12), non-PolyQ SCAs (such as SCA36 and 42), and other ataxias (such as FRDA and A-T). Most of the iPSC-based models used a generic differentiation towards the neural lineage, as opposed to the generation of specific neuronal subtypes, and very few characterized the neuronal phenotypes. The only reported iPSC-derived models addressing the cerebellar neurons were for SCA6 [51], SCA42 [127] and A-T [135].

For SCA1 and SCA12, only the generation of patient-derived iPSCs were until now reported [114, 115, 119, 125]. Several other SCA models have already addressed the neural phenotypes. SCA2 was modeled by Xia et al. [116] and by Chuang et al. [117] using patient iPSC-derived neural progenitors and central neurons. No cerebellar protocol has yet addressed SCA2, in which both Purkinje cells PCs and DCN neurons are affected. Whereas patient and control fibroblasts showed comparable levels of expression of the disease-causing protein Ataxin-2, its expression was decreased in patient iPSC-derived neural stem cells, which survived shorter in cell culture. Chuang et al. [117] reported that SCA2 neurons exhibited a glutamatedependent disease phenotype, which are suppressed by anti-glutamate drugs and a calcium stabilizer treatment.

One of the first studies using the generation of neurons from patient iPSCs addressed to SCA3, also called Machado-Joseph disease (MJD) [118]. In this model, neuronal excitation by glutamate promoted an increase in intracellular calcium concentration and proteolysis of Ataxin-3, triggering its aggregation—a hallmark of the disease in patients. This intraneuronal aggregation, (which was also found to depend on sodium and potassium channel function, as well as on ionotropic and voltage-gated calcium channel function), was abolished by calpain inhibition, pointing to a key role of this protease in Ataxin-3 cleavage. Furthermore, intracellular aggregations were not observed in patient iPSCs, fibroblasts or iPSC-derived glial cells, providing a clue for the neuron-specific phenotype observed in SCA3 patients. Hansen et al. [120] differentiated the SCA3 patient-derived iPSCs further into hindbrain neurons that expressed *GBX2* and *HOXA2*. They reported that glutamate loading or calcium increase by ionomycin did not induce Ataxin-3 accumulation in these hindbrain neurons. It remains to be investigated whether this discrepancy comes from a difference in cell types or in the applied protocols. In another study [121], SCA3 iPSCs differentiated into NeuN-positive (postmitotic) neurons showed accumulation of Ataxin-3 in the absence of stress. The activation of autophagy by rapamycin was effective for degradation of Ataxin-3, suggesting that autophagy could be a key for development of therapeutic treatments. Chuang et al. [117] reported that SCA3 iPSC-derived neurons again showed glutamate-dependent phenotypes, which were suppressed by anti-glutamate drugs. Ouyang et al. [122] applied gene editing techniques for the deletion of the expanded CAG in the *ATXN3* gene in SCA3 patient-derived iPSCs, which were further characterized. Such corrected iPSCs will be useful for SCA3 isogenic models. However, no further studies have addressed SCA3 iPSCderived cerebellar neurons and a directed protocol for the DCN neurons, the most affected in SCA3, is not yet available.

SCA6 is a very interesting case, first, by being one of the three diseases in which patient iPSC-derived cerebellar neurons were generated to date, and second, because of the bicistronic nature of the affected gene, *CACNA1A.* It encodes the α1A subunit of P/Q-type voltage-dependent calcium channel Cav2.1, and the α1ACT, with an identical sequence with the PolyQ bearing C-terminal segment of the longest isoform of α1A [150]. In addition, the gene is expressed mainly in neurons, contrary to the other ataxia-related genes, that are ubiquitous expressed. Utilizing the differentiation method for the cerebellar neurons [50], Ishida et al. [51] differentiated Purkinje cells from iPSCs derived from hetero- and homozygous SCA6 patients [51]. They found that SCA6-derived Purkinje cells exhibit decreased expression of α1ACT and its target molecules, TAF1 and BTG1. They further constructed a disease model in which SCA6 patient-derived Purkinje cells specifically degenerate by depletion of the thyroid hormone triiodothyronine (T3), which is necessary in late stages of maturation. Bavassano et al. [107] differentiated SCA6 patient-derived iPSCs into neurons expressing Cav2.1 and α1ACT, using the same differentiation and stress model as for the SCA3 [118]. The glutamate loading decreased the viability of SCA6 neurons, pointing toward a common pathway of stress response in PolyQ SCAs. In addition, SCA6 neurons showed differences in the expression of several genes previously reported to depend on the transcriptional regulation by the α1ACT, and showed no differences in the electric response of the Cav2.1 channel. Recent high-throughput investigations in the mouse and human cerebellum revealed complex functions of α1ACT [26] and further studies are expected to clarify the role of the mutated α1ACT in cerebellar neurons, especially in Purkinje cells.

For SCA7, in which cerebellar and retinal cells are degenerated [151], Luo et al. [123] reported the generation of iPSCs and neurons from a SCA7 patient, but did not characterize the neuronal phenotype and the disease phenotype. Ward et al. [124] generated SCA7 patient-derived iPSCs and their isogenic lines transduced with either normal or expanded ATXN7. They reported that SCA7 iPSC-derived neural progenitors exhibit altered metabolism and mitochondrial dysfunction.

SCA36 and SCA42 are non PolyQ autosomal dominant diseases, affecting the cerebellar neurons and other neurons. Matsuzono et al. [126] generated motor neurons from the patient-derived iPSCs and recapitulated an increase in RNA foci-positive cells that can be markedly suppressed by treatment of antisense oligonucleotide. SCA42 is caused by a mutation in *CACNA1G*, which encodes T-type voltage-dependent calcium channel Cav3.1 [127]. In addition to identifying the affected gene, [127] reported a model disease for which patient-derived iPSCs were differentiated into Purkinje cells. The SCA42-derived Purkinje cells would provide a useful tool for further phenotype analysis of the mutated CAV3.1, for which the investigation was till now limited to the HEK293 cell line.

For the FRDA, a pioneering work revealed that abnormal expansion of GAA repeats led to upregulation of the DNA mismatch repair protein MSH2 in FRDA patient-derived iPSCs [130]. They reported that the functional inhibition of *MSH2* by shRNA suppresses the repeat expansion. They further reported an inhibitor of histone deacetylase HDACi 109 increased the expression of *FXN* gene and Frataxin protein, pointing to the involvement of histone H3 lysine 9 in *FXN* expression. Polak et al. [131] also focused on epigenetic modifications in FRDA-derived iPSCs and performed drug evaluations. They found that an inhibitor of lysine-specific demethylation enzyme 1 (called Parnate or Tranylcypromine), and the HDAC inhibitor sodium butyrate have transient effects on decreasing the repeats and increasing *FXN* gene expression. Bird et al. [132] also reported a decrease in Frataxin expression in neurons differentiated from FRDA iPSCs, but could not

*Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

detect abnormality in mitochondrial functions. Hick et al. [133] reported decreased expression of *FXN* and Frataxin, a decrease in mitochondrial membrane potential and degeneration of mitochondria in FRDA IPSC-derived neurons. Eigentler et al. [128] showed a cell-specific decrease of frataxin in disease-vulnerable FRDA iPSC-derived peripheral neurons. Lai et al. [129] and Mazzara et al. [134] generated FRDA isogenic lines. Mazzara et al. [134] demonstrated that the entire intron 1 removal, and not solely the elongation, was necessary for the recovery of the *FXN* expression level in peripheral sensory neurons. Although several studies have provided insights into the pathogenesis of FRDA in cardiomyocytes and peripheral neurons, additional work is required to elucidate the role of Frataxin in other affected cell types, such as the neurons of the DCN.

For the A-T is caused by several mutations in the *ATM* gene [152], Nayler et al. [135] differentiated A-T patient-derived iPSCs into cerebellar neurons and performed RNA sequencing analysis with them. Remarkably, they found that the generated neurons acquired properties of the cerebellum at GW 22 and exhibited disrupted gene regulatory networks related to synaptic vesicle dynamics and oxidative stress.

#### **5. Strategies for optimizing the neuronal models of cerebellar ataxias**

Of particular interest in future research in the cerebellar ataxias is the comparison between affected and unaffected neuronal types, in order to identify particular characteristics that render specific neuronal populations vulnerable to a genetic insult which is ubiquitously presented. One of the most crucial needs is to establish a reliable and consistent disease phenotype in a relevant cell population, and those cell types to be generated in relatively large quantities *in vitro* [153].

Differentiation into specific and mature neurons that are the disease targets, such as Purkinje cells for several SCAs, or solely DCN neurons for some ataxias, or both of them for the most of SCAs (**Table 2**), will enable the construction of more reliable disease models [154]. However, the suitability of iPSC-derived neurons for modeling late-onset conditions remains controversial, particularly given the immature, fetal-like phenotypes of the neurons generated from these cells.

Remarkably, in contrast to the immature morphology observed for human PSCderived Purkinje cells, a recent bioinformatics analysis of their gene expression and developing showed that they most closely resembled late juvenile p21 mouse expression mouse Purkinje cells, when most of the cerebellar disease phenotypes in several animal models start to manifest. This finding suggests that the Purkinje cells are among the most mature human PSC-derived central neurons analyzed to date. This approach also underscores the utility of transcriptomic analysis for analyzing the maturation of human PSC-derived neurons and validates the use of hPSC-neurons for modeling cerebellar ataxias.

Still, it is possible that the disease phenotypes of adult-onset conditions, as the most of genetic SCAs are, may never be fully recapitulated under 2D cell culture conditions, even with directed protocols and optimized maturation. Generation of 3D cerebellar-like tissues as the cerebellar organoids may allow to increasing the neuronal maturation *in vitro*. The next generation or organoids or "assembloids", which will allow the proper combination of different cell types, including vascularization, can offer a good perspective but also limitations by increased heterogeneity. The multiomics approaches at single-cell level can definitely contribute to understand and quantify this heterogeneity and in the same time decipher the cell-type related disease phenotype.

Another way to model the late-onset diseases is the addition of neural stressors, such as reactive oxygen species, pro-inflammatory factors, and toxins or forced aging, as schematically presented in **Figure 5**. These approaches were already used for modeling several SCAs or other neurologic diseases [153, 155–157]. However, in an ideal situation, these stressors should only exacerbate the disease phenotype, which can be evident in a good model solely by the expression of the mutation in the disease-relevant cells. Another approach is to genetically manipulate the system for forcing the aging, such as by overexpression of progerin in neural progenitors. By this approach, the disease phenotype is expected to manifest *in vitro* in earlier stages of neuronal maturation [155, 156] (reviewed in [158]).

On the other side, recent evidence from cell and animal models indicates that abnormalities in early Purkinje cell development may contribute to the pathogenesis of the ataxias Purkinje cell developmental abnormalities are clearly evident in a wide range of ataxic mouse mutants, including models of the degenerative SCAs [26]. The observed Purkinje cell developmental defects commonly include impaired dendritic arborization, resulting in synaptic deficits affecting CF and PF connections and ultimately altering Purkinje cell physiology. Similar impairments in Purkinje cell dendritogenesis and synapse formation have been described in mouse models of SCA5, and in cell and mouse models of SCA14, SCA1, SCA3 and SCA5. Given the increasing evidence for Purkinje cell developmental abnormalities in cerebellar ataxias, it seems likely that iPSC-derived models, which are capable of recapitulating early developmental events *in vitro*, will be invaluable in unraveling the pathogenic complexities of these conditions. It will be important to better understand the underlying—likely common—molecular mechanisms, by which mutations in distinct genes cause abnormal Purkinje cell development and function [159]. These could offer attractive future therapeutic targets to alleviate motor dysfunction in cerebellar ataxia.

Another limitation in the field of modeling cerebellar ataxias is that most of the studies implemeted the production of iPSCs from a few patients. On one hand, addressing to larger patient cohorts may allow to identifying more accurate phenotypes. On the other hand, for investigating the pathological function of a mutation, the ideal situation is to compare the cells bearing the mutation with control cells with an identical genetic background. The rapid development of CRISPR/Cas9-mediated genome editing is likely to result in significant advances in the field, allowing the correction of disease-causing mutations into iPSCs, which can then be used to create paired isogenic lines to produce better disease models in which far less patient-derived cell lines will be necessary [160]. This was already performed even for the 'difficult to correct' elongations, like in SCA3, SCA7, it is expected in the near future to constitute 'the norm' for all iPSC-derived disease models.

The establishment of efficient, reproducible cellular models of cerebellar dysfunction and degeneration will be important not only in elucidating the molecular basis of these diseases, but also in the development of effective therapies. Establishment of special cell cultures, such as Purkinje cells from patients with cerebellar ataxia, provides opportunities to screen for drugs that may correct the observed disease phenotypes. These cell cultures can be combined with stressors capable of eliciting phenotypes in late-onset conditions and genotypic modifiers of disease progression and drug response. In addition, these cerebellar cell cultures may be used for toxicity screens, to assess the effects of novel compounds on relevant cell types, or for differentiation screens, to identify compounds capable of enhancing self-renewal, maturation or survival of specific cerebellar cells (**Figure 5**).

*Human Pluripotent Stem Cell-Derived Cerebellar Neurons: From Development to Modeling… DOI: http://dx.doi.org/10.5772/intechopen.96653*

#### **6. Final remarks**

Recent technologies for producing iPSCs from patients combined with the differentiation of PSCs into neural cells and the self-organizing 3D neural tissues have provided a new way to experimentally investigate the developmental and disease mechanisms of the human brain. While several challenges have hindered the generation of cerebellar neurons *in vitro,* starting from human PSCs, some important steps have been made. These protocols, combined with the patient-derived iPSCs, have been further applied for the investigation of several cerebellar diseases. In addition to the "classical" protocols aimed to generate specific types of neurons in two-dimensional (2D) cell cultures, recent progress has been made in culturing cells in three-dimensional (3D) structures, which may better reproduce the tissue organization and complexity *in vivo*, such as the PSC-derived brain organoids. Despite promising results, a number of issues remain to be addressed before the iPSC-based models to be widely adopted. Generation of the disease-relevant cerebellar cells and tissue *in vitro* remains a challenge, requiring a precise understanding of the complex molecular events during the development of each neuronal subtype, and an accurate set of markers by which to identify and characterize the generated cells. The 3D brain models in general and the 3D cerebellar models in particular still wait for improvements, including a better cellular characterization and an increased reliability, in order to contribute to better disease models.

However, human PSC-based models offer distinct advantages for the study of cerebellar ataxias. Cerebellar neuronal models are likely to provide valuable insights into the selective vulnerability of distinct neuronal subtypes, particularly the Purkinje cells. More directed and/or complex approaches will allow for the generation of accurate, disease-relevant models for the study of the molecular mechanisms underlying cerebellar ataxias, and the development of the long-awaited therapies.

#### **Acknowledgements**

This work was supported by Austrian Science Fund (FWF), Project P26886-B19, Austria.

#### **Author details**

Roxana Deleanu Institute for Neuroscience, Medical University of Innsbruck, Innsbruck, Austria

\*Address all correspondence to: irina-roxana.deleanu@i-med.ac.at

© 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.

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#### **Chapter 5**

## Rehabilitation for Spinocerebellar Ataxia

*Akiyoshi Matsugi, Kyota Bando, Yutaka Kikuchi, Yuki Kondo and Hideki Nakano*

#### **Abstract**

Rehabilitation is an important treatment for spinocerebellar ataxia (SCA). The lack of improvement in ataxia, deficit of motor learning, and unstable balance causes disability for activities of daily living and restricts participation in social activities, further resulting in a disturbance of the restoration of quality of life. This narrative review describes physical rehabilitation, including measurement of movement disorder, associated with ataxia and possible interventions. Several lines of evidence suggest that high-intensity individualized physical rehabilitation programs, especially for gait and balance training, improve motor function. Continuous exercise at home contributes to the maintenance of the gait and balance function. Moreover, videography and mechanical technology contribute to the evaluation of ataxia and motor learning ability, and assistive robotic systems may improve gait stability. Neuromodulation montages, such as repetitive transcranial magnetic stimulation and transcranial electrical stimulation, can enhance the effect of physical rehabilitation. Further research aimed at developing a more-effective physical rehabilitation for these patients is expected.

**Keywords:** spinocerebellar ataxia, rehabilitation, physical therapy, ataxia, assessment, gait training, balance training, motor learning, assistive technology, neuromodulation, noninvasive brain stimulation

#### **1. Introduction**

Spinocerebellar ataxia (SCA), which is included in spinocerebellar degeneration (SCD), is a genetically heterogeneous group of autosomal dominantly inherited progressive disorders [1]. Cerebellar atrophy is the most prominent clinical feature of this condition and is accompanied by spinal cord and sequential brain stem and basal ganglion damage. Therefore, coordinated movement of the eyes, head, trunk, and extremities is impaired. Therefore, the activities of daily living (ADL) and participation in social activities are limited, and the quality of life (QOL) is undisputedly impaired in these patients [2].

The effects of medication and surgery in this clinical setting depend on the cause of ataxia and the extent of neuronal damage [3, 4]; however, there is no rational effective treatment for SCA and it is difficult to slow the progression of the disease. Rehabilitation [5, 6], including physical therapy [7, 8], aimed at improving/maintaining motor function, ADL, and QOL [5] is an important intervention for patients with SCA. Here we provide a narrative review of physical rehabilitation for SCA.

#### **2. General features**

For the clinical diagnosis of cerebellar ataxia, specific blood studies and magnetic resonance imaging (MRI) have been performed [9]. Furthermore, genetic techniques improve the diagnosis of degenerative cerebellar ataxia [10]. Although the details of the findings of these genetic and blood studies are beyond the scope of this review of rehabilitation, cerebellar atrophy and cerebellar motor deficits are traditionally common observations in patients with degenerative cerebellar ataxia [9]. Furthermore, recently, the absence of motor cerebellar symptoms has also been recognized as being important for rehabilitation [11].

The cerebellum is the motor-control system in humans [12]. Clinically, the oculomotor deficit, speech deficits, ataxia in the trunk and extremities, balance disorder, and gait disturbance are the targets of rehabilitation in SCA [9, 13]. The possible underlying pathogenetic mechanisms include distorted timing, abnormal sensory acquisition, impaired sensory motor synchronization, impaired triggering of corticomotor excitability, and abnormal visuokinesthetic cerebro-cerebellar interactions [13].

Oculomotor deficits cause deoptimized vision. The vestibulo-ocular reflex and smooth pursuit [14] partially depend on motor prediction in static and dynamic movement and contribute to dynamic gazing [15]; moreover, the cerebellum contributes to the trainability of eye-head coordinated movements [16].

Abnormal excitability and modulation in the motor cortex and corticospinal tract causes a voluntary contraction deficit in [17, 18]. Cerebellar stimulation modulates the motor-evoked potential induced by transcranial magnetic stimulation (TMS) of the primary motor cortex [19–21]; however, this modulation is absent in patients with SCA [22, 23]. Furthermore, the cortical silent period, which reflects the excitability of the inhibitory GABAergic neural circuit in the primary motor cortex, is abnormal in these patients [24–29], and this cerebellar effect on the cortical silent period is characteristic of the healthy population [30]. Before muscle contraction for movement, the corticospinal excitability increases in healthy individuals; in contrast, this facilitation is insufficient in SCA [31]. In addition, in patients with SCA, muscle tones are decreased [11] and the spinal reflex excitability is facilitated by cerebellar stimulation [32–34]. The long latency spinal reflex, which is correlated with the cortical circuit, is disturbed in SCA [35]. Although this functional cerebellum-spine connection may contribute to the preparation for muscle contraction, there is insufficient evidence that these connections contribute to motor control in healthy and cerebellar ataxia populations.

In simple movements, such as extension of the elbow, coordinated activity of the biceps and triceps is needed. For ballistic elbow-extension movement practice, the triphasic muscle agonist and antagonist contraction patterns contribute to the smooth movement, but under/overshooting appears during the uncoordinated contraction pattern of patients with SCA [36, 37]. Furthermore, this contraction pattern may be obtained by temporal electrical stimulation in these individuals [37].

The cerebellar internal model contributes to predictable/online/offline motor control and motor learning/adaptation [38]. The symptoms associated with motor learning do not appear at the onset of the cerebral atrophy [39], because several brain areas, i.e., the prefrontal cortex, primary motor cortex, and basal ganglia, compensate for cerebellar function in early-stage SCA [5, 6, 39]. Recently, the motor learning deficit at the early stage of the disorder was reportedly detected using an adaptation task [40]. Therefore, the assessment of the capacity for motor learning may be important to strategize the interventions that are concretely described in the following sections.

Representative nonataxia symptoms include hyperreflexia, areflexia, extensor plantar, spasticity, paresis, muscle atrophy, fasciculations, myoclonus, rigidity, chorea/dyskinesia, dystonia, resting tremor, sensory symptoms, urinary dysfunction, cognitive impairment, and brain stem oculomotor signs [41]. The Inventory of NonAtaxia Symptoms (INAS) [41] is used to estimate these nonataxia symptoms. The appearance of these symptoms depends on the type of SCA [41].

#### **3. Assessment format**

We should conduct assessment to detect the degree of motor dysfunction and consider more effective intervention of physical rehabilitation. The first, the imaging technology such as MRI provides us with structural information about the atrophic areas of the brain associated with the disease. We described about neuroimaging technique in Section 3.1. The next, we can use some outcome measurement to estimate the motor dysfunction and verification in the physical rehabilitation. Then, we introduce the representable outcome measures for physical rehabilitation in SCA in Section 3.2. However, we had not established method to estimate the remaining of motor learning ability, which is one of the most important factors to predict the effect of physical rehabilitation. Therefore, we propose the possible assessment of motor learning ability in Section 3.3.

#### **3.1 Neuroimaging**

Neuroimaging is a technique that is used to visualize the structural and functional activities of the brain. MRI measurements, such as diffusion tensor imaging and surface-based morphometry, visualize the brain structures. Functional activity imaging is achieved using fMRI and NIRS, which are indicators of cerebral blood flow, and electroencephalogram (EEG) and magnetoencephalography, which are indicators of electrical activity. Positron emission tomography and single-photon emission computed tomography with nuclear tracers are also used in this setting. The application of neuroimaging in the rehabilitation of cerebellar disorders includes voxelbased lesion symptom mapping in patients with stroke, to investigate the recovery of upper arm reach [42] and walking ability [43] depending on the lesion site.

Although conventional MRI [44] is widely used for the neuroimaging of spinocerebellar degeneration, to obtain diagnostic findings, few studies have used neuroimaging as a guideline or outcome of rehabilitation. The lack of reports in this context hampers the quantification of cerebellar degeneration in SCA and its correlation with motor dysfunctions. In terms of measurement techniques, the cerebellum exhibits a much tighter folding compared with the cerebral cortex, with individual cortical sheets with a thickness of 1–2 mm and a sheet area of 1500–2000 cm2 , compared with a sheet area of 2200 cm2 with a thickness of 1.5–4 mm in the cerebral cortex. Therefore, the typical 2–4 mm3 spatial resolution of neuroimaging techniques is insufficient to capture local cerebellar changes. Patient factors include the difficulty in limiting the brain regions involved in movement disorders to the cerebellum, because the degenerative regions in SCD extend beyond this structure to multiple brain regions [45].

Among the neuroimaging modalities, the role of voxel-based morphometry (VBM) is notable in SCA rehabilitation. VBM is a statistical analysis of the entire brain in voxel units (1 mm3 ) that is used to identify the behavioral patterns and related brain morphological characteristics of patients [46]. Burciu et al. assessed the degree of cerebellar atrophy concerning motor and learning functions using VBM to evaluate brain structure changes after 2 weeks of balance training in patients with SCD; these authors reported the association between an increased volume of the dorsal premotor cortex and increased balance ability [47]. Matsgi et al. reported an association between VBM and neurophysiological markers in cerebellar brain inhibition (CBI), with atrophy of the dentate nucleus at VBM observed in cases of pure cerebellar ataxia that did not show CBI [48]. Bando et al. reported a correlation between adaptive learning ability and gray matter volume of the cerebellar IV-VII lobules and the supramarginal gyrus in a prismatic adaptation task in SCA [49]. Thus, VBM may be a biomarker to explain motor dysfunction in patients with SCA.

Conversely, VBM is not an ideal tool to show a causal relationship between brain structural changes and behavioral differences. As a solution to this problem, we can propose a combination of VBM and neurostimulation [50], as neurostimulation of the brain regions associated with the behavioral patterns obtained by VBM and the observation of behavioral changes before and after stimulation allow us to examine brain degeneration sites and behavior.

#### **3.2 Outcome measurement**

Gait disturbance is a major symptom of the cerebellar pathology in SCA [51]. The functional ambulation categories (FAC) is useful for the comprehensive assessment of walking ability; the FAC assesses gait for about 15 m and climbing stairs and classifies gait levels into 6 levels [52]. The FAC is also used in the exercise program created by Research Committee for Ataxia Disease (Research team under the jurisdiction of the Ministry of Health, Labour and Welfare in Japan, http:// ataxia.umin.ne.jp/rehabilitation/).

The quantitative assessment of cerebellar ataxia is very important in clinical practice. The International Cooperative Ataxia Rating Scale (ICARS) has been used as a quantitative assessment of ataxia symptoms. However, it has been noted that the test reliability of the eye movement items is low [53]. The Scale for Assessment and Rating of Ataxia (SARA) is an 8-item performance-based scale that yields a total score of 0–40 (most severe ataxia). The minimal detectable change (MDC) for individual score difference from the baseline to the 1-year follow-up in SARA was <3.5 (n = 171; SCA1, n = 43; SCA2, n = 61; SCA3, n = 37; and SCA6, n = 30; mean age, 50.9 ± 13.5 years; mean disease duration, 11.8 ± 5.6 years) [54]. SARA does not include an eye movement section. Schmahmann et al. noted the importance of assessing oculomotor abnormalities and developed the Brief Ataxia Rating Scale, a modification of ICARS [55]. Each SCA genotype exhibits specific symptoms [56]. Therefore, these assessments should be used differently for different symptoms. However, one feature that is consistent among these assessments is that the scoring range is large and does not allow the assessment of minute symptom changes. Honda et al. developed a system to measure the evaluation of SARA using a depth sensor [57]. Using this system, the degree of ataxia can be measured numerically. In addition, because the system is inexpensive, it can be installed at the patient's home, making it a useful tool for telemedicine.

The balance dysfunction in SCA has a significant impact on QOL [58]. The Berg Balance Scale and the Timed Up and Go test are widely used to assess balance dysfunction in SCA [59]. However, despite their widespread use, these assessments have not been examined for reliability and validity in SCA. Kondo et al. examined the test reliability of the Balance Evaluation Systems Test (BESTest) [60]. The BESTest is a multitask balance assessment tool that was developed to identify specific postural control problems (i.e., biomechanical constraints, stability limits, anticipatory postural adjustments, postural responses, sensory orientation, dynamic balance during gait, and cognitive effects) [61]. The MDC for an individual score difference from the baseline to the 4-week follow-up in

#### *Rehabilitation for Spinocerebellar Ataxia DOI: http://dx.doi.org/10.5772/intechopen.95999*

BESTest was <8.7 (n = 20; SCA3, n = 4; SCA6, n = 9; SCA31, n = 7; mean age, 63.7 ± 10.1 years; age at onset, 53.9 ± 10.5 years; baseline SARA, 9.9 ± 3.5) [61]. Many types of balance function measures have been reported. However, BESTest is the only scale that is considered to have absolute reliability in SCA.

Gait speed is often used as an outcome of intervention studies in SCA [62, 63]. However, some changes in the gait pattern (e.g., base of support and gait speed) most likely reflect cerebellar-unspecific, compensatory strategies, and a high spatiotemporal gait variability appears to be a distinctive feature of ataxic gait [58, 64]. The Gait Variability Index (GVI) is a measure of gait variability that has been examined regarding reliability and validity [65]. The MDC for an individual score difference from day 1 to day 2 in GVI was <8.6 (Friedreich's ataxia, n = 81; baseline ICARS, 70.4 ± 7.9) [65]. It has been suggested that gait instability in SCA are characterized by a stronger effect of balance-related impairments of cerebellar control during slow walking and a stronger effect of impaired intra-limb coordination during fast walking [58]. Therefore, in clinical practice, it is necessary to evaluate not only the optimal gait speed, but also slow walking and fast walking, to extract the characteristics of gait instability.

#### **3.3 Assessment of motor learning ability**

The cerebellum has the ability to compensate for tissue damage and loss of function. This is called the cerebellar reserve [6]. Mitoma et al. suggested that this is important for motor rehabilitation at a time when the cerebellar reserve is functioning [6]. Motor rehabilitation in the early stages may maintain and improve the cerebellar reserve [66, 67]. Therefore, it is important to assess this parameter.

Cerebellar ataxia is the main symptom of SCA. Ataxia symptoms may represent a compensation for predictive control using feedback control [6]. Predictive control requires a mechanism called internal model [38]. The internal model is constantly updated by motor learning [68]. In turn, motor learning is one of the most important functions of the cerebellum. Thus, a measure of motor learning ability may be useful as an assessment of the cerebellar reserve.

Prism adaptation (PA) is widely used as an assessment of motor learning ability in patients with SCA [40, 69]. The basic procedure of PA is shown in **Figure 1**. First, at the "baseline," the task is performed without a prism lens. Subsequently, the prism lens is introduced and the task is performed. In the initial phase, the lens is set off to either the left or right side of the target, but the error is corrected as the number of repetitions increases. This period is called the "initial error correction phase." Thereafter, a spatial realignment phase is performed under the prism lens. The purpose of this phase is to gather visuospatial information including the errors. Next, the prism is removed and an "after-effect phase" is performed. If the spatial information is being re-learned, errors are generated in the opposite direction to the initial error correction phase. Recently, Hashimoto et al. developed the Adaptability Index (AI), which is a composite index computed from several parameters measured PA (**Figure 2**). The clinical efficacy of the AI in discriminating patients with SCA from healthy individuals has been demonstrated [70]. Furthermore, Bando et al. found that a reduced AI was correlated with gray matter atrophy in the cerebellum in the SCA group [49]. In particular, the right lobule VI and the left Crus I showed the most robust correlation. These cerebellar regions are consistent with the correlates of PA detected in previous human and nonhuman primate studies [71, 72]. AI is considered as a motor learning index that reflects the cerebellar reserve (in this case, the degree of cerebellar atrophy).

PA can be implemented using a simple system. In addition, it takes only 20 min to complete a PA. Reaching tasks can be performed even in the period during which

#### **Figure 1.**

*Overview of prism adaptation. The ordinate shows the finger-touch error represented from the target to the touch point. Three phases are generally used: (1) absence of a prism lens (prism off), (2) presence of a prism lens (prism on), and (3) absence of a prism lens (prism off).*

#### **Figure 2.**

*Calculation of the adaptability index (AI). The AI is calculated as follows: AI = a × b × c, where "a" is the adaptation index defined as the probability of correct touches in the last 10 trials of the spatial realignment phase 1, "b" is the retention index defined as the probability of incorrect touches in the initial 5 trials of the after-effect phase, and "c" is the extinction index designated as the probability of correct touches in the last 10 trials of the spatial realignment phase 2.*

the patient is unable to walk, and the fact that the PA can be assessed continuously over a long period is an advantage. However, only cross-sectional studies have been conducted in previous reports [40, 49, 69, 70, 73, 74]. Future studies need to be designed to examine long-term changes and intervention effects.

#### **4. Rehabilitation**

The targets of rehabilitation in cerebellar ataxia are mainly disability in ADL, gait, and motor dysfunction. Therefore, GAS, FIM, 10-m walking test, TCA, SARA, ICARS, and BESTest are used as important outcomes in rehabilitation. The most important strategies of rehabilitation for cerebellar ataxia including SCA consists in balance training (see Section 4.3), gait training (see Section 4.2), and muscle strengthening training using a high-intensity program (see Section 4.1). Further, optional possible interventions are using assistive technology (see Section 4.4) and neuromodulation technique (see Section 4.5).

#### **4.1 Intensive and continuous training**

Rehabilitation methods for cerebellar ataxia have been reported [75]. The most important strategy is the increase in the intensity of physical training, such as balancing, gait, and strength [76]. Several systematic reviews [77–79] and narrative

#### *Rehabilitation for Spinocerebellar Ataxia DOI: http://dx.doi.org/10.5772/intechopen.95999*

reviews [3, 75, 80, 81] introduced and recommended intensive physical therapy for cerebellar ataxia in patients with SCA. Miyai et al. [62] reported that physical and occupational therapies of 2 h × 5 days +1 h × 2 days per week for 4 weeks were applied to inpatients and improved the SARA score and gait speed; however, the effect was carried over only up to 12 weeks after the training, and had disappeared at 24 weeks [62]. Conversely, Ilg et al. reported that intensive coordinative physiotherapy delivered over 4 weeks improved motor performance in degenerative cerebellar ataxia in a study with an intraindividual control design [63].

An outpatient rehabilitation program for 6 weeks applied to 19 participants with Friedreich's ataxia improved the motor domain item in the FIM score and Friedreich's Ataxia Impact Scale, but the posthome program could not maintain the effect [82]. Therefore, this finding indicates that continuous outpatient rehabilitation programs are important for maintaining the ADL in patients with Friedreich's ataxia. Additional large-scale studies are needed to investigate the long-term effect of outpatient rehabilitation programs and identify the characteristics of patients who respond to treatment. Therefore, the development of optimal individual programs is important to obtain the effect of training, regardless of the inpatient, outpatient, or home-self-training setting [83]. The semi-order program of the Research Committee for Ataxia Disease (Research team under the jurisdiction of the Ministry of Health, Labour and Welfare in Japan, http://ataxia.umin.ne.jp/ rehabilitation/) can be used for this purpose.

Subsequently, the continuity of the intensive training is an important factor, because degradation in physical function was reported. Therefore, approaches aimed at upkeeping these programs in a way that suits the patients are needed. For example, exergames contribute to the practice of exercise at home. In the future, tele-rehabilitation systems [84] should be tested for the improvement (or maintenance) of the function and continuity of exercise.

#### **4.2 Gait training**

Gait training has been reported to improve spatiotemporal gait parameters (cadence, step length/width, gait speed, etc.) [85–87], complex gait (Timed Up and Go test, Dynamic Gait Index) [85], independence (FAC) [86], ataxia (SARA) [88], and adaptive locomotor adjustments (ALA) [88]. Patients with SCA exhibit problems other than the gait disturbance itself, i.e., stiffening of the body in an attempt to avoid the occurrence of gait disturbances. Therefore, it is important to focus on gait disturbances and increasing the number of walking patterns when considering gait training in a person with SCA.

Disturbances of gait are the core features of SCA [89–92], thus leading to a risk of falling down [93]. Patients with cerebellar ataxia walk with a reduced walking speed and cadence, as well as reduced step length, stride length, and swing phase; increased walking base width, stride time, step time, stance phase, and double limb support phase; and increased variability of step length, stride length, and stride time [94]. These items are affected by both balance-related impairments and deficits related to limb control and intra-limb coordination [95]. We believe that balance training and coordination training are key to the improvement of gait disturbances. Regarding the details of balance training, please refer to the Section 4.3.

In addition, stiffening of the body leads to a decrease in the number of walking patterns; as a result, ALA deteriorates [96, 97]. ALA implies that obstacle avoidance is achieved by modifying basic walking patterns in response to obstacle properties, e.g., a sloping road, stepping over an obstacle, or dynamically changing the spaces created by pedestrians in a hallway. In persons with SCA, feelings of anxiety as a result of the frequent experience of falls, as well as deficits related to limb control

by ataxia, could negatively affect their ALA because of increased muscular cocontractions and reduced joint movements [98]. We will describe the approaches to improve ALA in the next paragraph.

The proposals for gait training are as follows: gait training without or with a treadmill. First, in gait training without a treadmill, we refer the reader to Section VI of the BESTest as gait adaptability training [61]. Section VI of the BESTest consists of a 7-item scale: (1) Gait Natural, (2) Change Speed, (3) Head Turns, (4) Pivot Turn, (5) Obstacles, (6) "Get Up & Go" Test, and (7) Cognitive Task "Get Up & Go" Test, aimed at evaluating the stability of the gait. These elements are important to improve ALA. As an example of gait training, persons with SCA are asked to walk while making an effort to change their walking speed according to therapist's instructions to engage is "fast (or slow)" walking as fast (or slow) as possible. If patients need assistance when walking, you might want to change the walking speed with the support of a therapist.

Second, gait training using a treadmill has advantages in that patients can practice a relatively large amount of gait training over a short period and the therapists can control the speed and incline easily. Gait training using a treadmill has been reported as a potentially promising tool for improving ALA in a person with SCA [88], as well as gait disturbances in a person with Parkinson's disease [99, 100]. It has been reported that variability was increased during slow and fast walking, but was normal during the preferred walking speed in a person with cerebellar ataxia [101]. Another study reported that, in ataxia, walking at the preferred speed minimizes the gait abnormalities, and the analysis of gait at a wide range of speeds is recommended [94]. For this reason, when using a treadmill in gait training, we suggest that walking be practiced at the speed at which the gait disturbance increases (i.e., slow or fast walking speed) for specific patients. When the fear of falling increases, the use of a harness is recommended, to provide a safe environment for gait without the fear of falling.

It is important to improve the balance ability and ALA during gait training in a person with SCA. Gait training is a relatively easy method; however, it is left to the therapist's discretion and experience. By changing the task itself or adjusting the difficulty level of the task, gait training may be able to overcome the limited walking patterns of these patients.

#### **4.3 Balance training**

All patients with SCA will develop balance difficulties during the course of the disease. Balance is essential for mobility, and is very important for QOL. Although there is no effective pharmacological treatment for decreasing the ataxia or slowing disease progression, physical therapy plays an important role in controlling ataxia and improving or maintaining function through training [76]. In general, the physical therapy programs for degenerative cerebellar ataxia are based on intensive static and dynamic balance and coordination training. There is some evidence that such therapeutic training programs alleviate the ataxic symptoms and improve functional activities in a person with cerebellar ataxia [63, 78, 102]. In these patients, the disease progressively damages the cerebellar structure that plays a crucial role in motor learning [103]; however, these studies have indicated that it is necessary for highly repetitive balance training for balance impairment in SCA. For this reason, highly repetitive balance training in patients with SCA should be the focus of future studies.

More concretely, balance training exercises in early stages of the disease, i.e., ambulation, include the following categories: (1) static balance training, (2) dynamic balance training, and (3) coordination training (**Figure 3**). In addition, combining a

#### *Rehabilitation for Spinocerebellar Ataxia DOI: http://dx.doi.org/10.5772/intechopen.95999*

#### **Figure 3.**

*National Center of Neurology and Psychiatry (NCNP) balance training program. This balance training program was devised through consultations with patients with SCA, medical doctors, and therapists at the NCNP in Japan. In the advanced stage of SCA, it is recommended to perform the programs indicated by an asterisk.*

dual task with balance training improves balance and reduces the number of falls in individuals with cerebellar ataxia [104].

Moreover, it is important to provide support for these approaches and make them a habit of exercising. For instance, if the patients with SCA have no habit of exercising, they should start with a small number of exercises (i.e., the minimum necessary) to get used to exercising, followed by the gradual increase in the number of exercises. If the patients with SCA have a habit of exercising, the therapist should teach them to adjust the exercise load (e.g., exercise more slowly and/or provide a small base of support). It is also important to adopt balance training that can be enjoyed, e.g., video games [105] and Tai Chi [106], as a means of continuing balance training.

In advanced stages of the disease (i.e., no ambulation), it is necessary to perform balance training under safe conditions (e.g., prone, supine, crawl, and sitting positions), to prevent the decrease in physical activity. Even in advanced stages, it has been reported that a person with degenerative ataxia may benefit from balance training [107]. In addition, it is necessary to focus on ADL and living infrastructure at this stage. If a patient with SCA requires assistance during transfer, engaging in repetitive transfer training with assistance and/or modification of the living infrastructure (e.g., installation of handrails) are necessary.

Focusing on highly repetitive balance training in patients with SCA might preserve the balance function. There is no scientific basis for the number of balance training exercises that are necessary to achieve this goal; however, we would like to recommend engaging in 30 repetitions at least per balance training session. Furthermore, the balance training must be designed to provide a significant challenge to the person's balance. If a person with SCA wants to preserve the balance function, they have to continue engaging in repetitive balance training, "use it or lose it." However, few studies have reported the effect of gait and balance training in persons with SCA.

Therefore, further studies are needed to clarify the clinical effectiveness of gait and/or balance training.

#### **4.4 Assistive technology**

In recent years, various technologies have been used in the assessment of and treatment based on rehabilitation, as well as to support daily life in patients with SCD. Curara, a wearable robotic system, assists both hip and knee movements and supports the wearer's rhythmic gait using a synchronization control based on a central pattern generator [108]. Gait support using the curara system has been reported to improve gait smoothness in patients with SCD [109]. In addition to these findings, a recent study addressed the effects of robotic gait training combined with noninvasive brain stimulation. This report showed that robot gait training using Lokomat-Pro in combination with cerebellar tDCS improved the functional scores on SARA, especially the scores on the subitems of gait, stance, sitting, and heel-shin slide compared with robot gait training alone [110]. Thus, hybrid training using robots and noninvasive brain stimulation will be applied to the rehabilitation treatment of patients with SCD in the future.

Accordingly, the use of walking aids is a complementary method for balance and gait impairment. In general, walking aids such as canes and walkers improve postural stability, but their improper use increases the risk of falling [111]. Because the manipulation of a cane requires coordinated upper limb movements [112], patients with SCD who have upper limb ataxia are likely to experience difficulty in using a cane. Conversely, because a walker does not require much coordinated movement of the upper limbs, technology-based walkers are being developed. Recently, a smart walker for mobility assistance and monitoring system aid, ASBGo, was developed and reported to improve gait parameters and postural stability in patients with SCA [113, 114]. In addition to technology, some studies on walking assistance using dogs and handkerchiefs have also been reported. Walking with a rehabilitation dog that has been specifically trained for goal-directed interventions or with an assistance dog that helps people with physical disability and mobility impairments has been reported to improve balance while walking in patients with SCD [115]. Furthermore, the handkerchief-guided gait, in which the patient with SCD walks along with the caregiver while maintaining light tension on a handkerchief by pulling lightly, has been shown to decrease body swaying and increase stride length and gait velocity during walking [116].

Moreover, technology is also being used as a tool to assess ataxia in patients with SCD living at home. Most of them represent attempts to evaluate SARA, which is a typical measure of ataxia, at home. In recent years, a technology aimed at objectively evaluating the speech, upper and lower limb, balance, and gait functions using wearable inertial sensors and a Kinect camera was developed, which makes it possible to discriminate between normal and abnormal functions and to detect ataxia at an early stage [117]. In addition, SaraHome has been developed to allow the remote evaluation of SARA items using Kinect and Leap Motion Controller [118]. Moreover, a spoon equipped with an inertial sensor, called Ataxia Instrumented Measurement-Spoon, has been developed, which allows the evaluation of upper limb function in ataxia while eating with a spoon [119–121]. Because SCD is an intractable neurological disease, it is difficult for many patients to leave their houses. Therefore, the contribution of technology to home-based rehabilitation is expected to increase in the future if a low-cost and easy method of assessing ataxia at home is established using the technologies and products of daily living described above.

#### *Rehabilitation for Spinocerebellar Ataxia DOI: http://dx.doi.org/10.5772/intechopen.95999*

Regarding the support of ADL, BMI studies have been reported. Patients with severe SCA often have difficulty in communicating because of language impairment. The application of BMI using event-related potentials and frequency bands of EEG is being investigated as a solution to this problem. The operational accuracy of BMI using P300 for event-related potentials was 82.9% in patients with SCA, which was similar to the accuracy observed in healthy subjects (83.2%) [122]. There are also reports of BMI manipulation in patients with SCD using the EEG frequency band associated with motor imagery [123]. BMI has a wide range of applications in diseases of the central nervous system, such as communication tools, transportation, and life support, and is expected to contribute to the QOL of patients with SCD.

#### **4.5 Neuromodulation**

Neuromodulation via noninvasive brain stimulation (NIBS) is a potential method for the treatment of cerebellar ataxia [19, 124]. A previous systematic review [125] reported the effectiveness of cerebellar neuromodulation using the TMS technique of transcranial direct current stimulation (tDCS). The SARA and ICARS scores in patients with SCA3, multiple system atrophy, and postlesion ataxia, as assessed using real cerebellar rTMS (1 Hz), were significantly lower than those detected in the sham stimulation group [125]. Furthermore, no harmful side effects were noted [125]. Cerebellar rTMS can modulate the plasticity of the vestibular reflex [16, 126]; therefore, cerebellar rTMS has potential for application in balance training to enhance vestibular contributions.

A single session of anodal cerebellar tDCS (2 mA, 20 min) significantly improved SARA, ICARS, 9-hole-peg test, and 8-m walking test scores [127]. Furthermore, combined anodal cerebellar tDCS and cathodal spinal DCS (5 days/week, 2 weeks) improved SARA score, ICARS score, 9-peg test, and 8-m walking time in patients with degenerative cerebellar ataxia [128]. There is insufficient evidence regarding whether simultaneous stimulation is more effective than single stimulation [129]; however, it is possible that this intervention method will produce improvements. Based on these findings, which were gleaned from small-sample studies, we suggest that a neuromodulation montage will improve the ataxia, balance, and gait ability. Therefore, we should perform further studies using a larger population.

#### **5. Conclusion**

Individualized physical rehabilitation programs for patients with SCA may improve/maintain their motor function, balance, gait ability, and ADL. In particular, the intensity and continuity of gait and balance training need to be considered to achieve effectiveness. Furthermore, several technologies, such as depth sensors, robotics, and NIBS, have contributed to the development of methods for the assessment and treatment of motor dysfunction in individuals with SCA. We should continue to study populations suffering from dysfunction caused by SCA.

#### **Acknowledgements**

This work was supported by Shijonawate Gakuen University and JSPS KAKENHI (Grant Number 20 K11298).

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Akiyoshi Matsugi1 \*, Kyota Bando2 , Yutaka Kikuchi3 , Yuki Kondo2 and Hideki Nakano4

1 Shijonawate Gakuen University, Daito, Osaka, Japan,

2 National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

3 Institute of Brain and Blood Vessels Mihara Memorial Hospital, Isesaki, Gunma, Japan

4 Kyoto Tachibana University, Kyoto-city, Kyoto, Japan

\*Address all correspondence to: a-matsugi@reha.shijonawate-gakuen.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.

*Rehabilitation for Spinocerebellar Ataxia DOI: http://dx.doi.org/10.5772/intechopen.95999*

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#### **Chapter 6**

## Living and Coping with Spinocerebellar Ataxia: Palliative Care Approach

*Caroline Bozzetto Ambrosi and Patricia Bozzetto Ambrosi*

#### **Abstract**

The discussion about the palliative care approach in spinocerebellar ataxia (SCA) has become extremely relevant. Mainly after considering that most progressive ataxias are incurable, there are few published studies on their palliative and end-of-life care. Although many patients with degenerative neurological diseases have a normal life expectancy, some forms of SCA (e.g., type 1, 2, 3, and 17) can progress rapidly, with a shorter life span. This chapter will discuss current guidelines and recommendations that have been drawn from the broader field of progressive neurological conditions. In addition, we also review aspects of strategic end-of-life care management, the involvement of the multidisciplinary team and the contribution of allied health professionals are essential for excellent patient support care in a palliative approach. More studies on your supportive care and end-of-life care to manage this serious illness to improve quality of life and reduce suffering, addressing complex medical symptoms, psychosocial issues, general well-being, and planning strategies for better living and coping are needed.

**Keywords:** spinocerebellar ataxia, palliative care, supportive care, end-of-life, wellness being, medical strategies

#### **1. Introduction**

*"We cannot avoid suffering but we can choose how to cope with it, find meaning in it, and move forward with renewed purpose." (Viktor E Frank)*

*"Our most cruel failure in how we treat the sick and the elderly is the failure to recognize that they have priorities beyond just being safe and living longer; that the chance to shape one's history is essential to sustaining the meaning of life; that we can reshape our institutions, our culture and our conversations in ways that transform the possibilities of the last chapters of everyone's life." (Atul Gawande)*

Spinocerebellar ataxia (SCA) is a term that refers to a group of inherited autosomal dominant ataxias characterized by degenerative changes in the part of the brain related to movement control (cerebellum) and in the spinal cord, its connections with progressive decline in functional capacity [1–3].

The significant improvement in the classification and correlation of the clinical profile of the different forms of cerebellar ataxias is due to genetic advances in medical diagnosis. Although several subtypes of SCAs have been identified,

phenotypically, cerebellar ataxia is a common feature of each type with individual differences regarding mutations in many different genes and the involvement of the cerebellum and its connections [3–10].

On the other hand, despite these advances in genetics, modifying therapies targeting specific genes or stem cells, there is no current definitive treatment able to stop the progression of most cases as in most degenerative neurological diseases. Strategic management of SCA to improve quality of life and reduce suffering, addressing complex medical symptoms, psychosocial issues, general well-being, and planning requires a broad and dedicated multidisciplinary involving palliative care approach. For patients with more complex needs help from a palliative care specialist team may be necessary [11–24].

In this chapter, it would be reviewed the main clinical aspects, the perspectives of therapeutic management, directed symptomatic management, support, and multidisciplinary team including the current guidelines regarding patients living and coping with SCA.

#### **2. Recognition and key clinical aspects**

Genetically and phenotypically, several subtypes of SCAs have been identified. Cerebellar ataxia is a feature of each type; other distinguishing features may suggest a specific type. However, more than ⅔ of patients with SCA have mutations at known loci that can be identified through genetic testing. In addition, among patients with apparently idiopathic sporadic cerebellar ataxia (no family history), an SCA mutation (types 1, 2, 3, 6, 7, 8, or 12; most often SCA-6) or Friedreich's ataxia can be identified in approximately ¼ of patients [1–4, 10, 25].

In most subtypes of SCAs (SCA 1, 2, 3, 6, 7, and 17) a genetic abnormality will be identified related to the expansion of CAG repeats in the region that encodes the polyglutamine tracts in protein products, like what is observed in Huntington's disease. Wild-type chromosomes with a stable CAG repeat have 6–34 repeat units; more than 36 repeats result in an unstable, expanded, disease-causing allele.

Clinically disorders associated with CAG repeat expansion share several relevant medical condition features [6, 7]:


#### **3. Perspectives of medical palliative care management**

The nihilistic view of the treatment of SCA, as it happens for long past years with many other neurodegenerative diseases, is no longer justified. In addition to rehabilitation therapies, there are specific complications to be looked for and treated. These interventions can significantly alleviate the problems of progressive ataxia and prevent potentially fatal complications. An enthusiastic, motivational, and well-informed medical approach in addition to follow-up by a multidisciplinary team can provide valuable support to a patient with SCA. When a patient approaches the end-of-life, specialized palliative care services should be involved to help to meet their specific needs as will be described in this chapter [20–24, 26–33].

#### **4. Symptomatic management**

A patient living with SCA, the care team services should be involved to help to meet their specific symptomatic needs. A variety of potential symptoms will need to be managed as treatment "strategies" are often derived from other neurological conditions with similar symptoms and work equally well.

The approach to treating spasticity and bladder symptoms, for example, is the same as for people with other neurological diseases [22, 23]. The assessment and management of these complications is best done by involving therapy specialists, and the work of the multidisciplinary team can improve patient care. Speech and language therapy are essential along the patient's journey, from monitoring the swallowing function in the initial stages and providing useful tips on how to avoid complications, to planning feeding by percutaneous gastrostomy [24].

The impact of cerebellar disease on cognition is not widely known, but it can have a significant impact on morbidity. These "remote" effects of cerebellar dysfunction can include frontal subcortical impairments affecting personality, behavior, and judgment.

Mental health complications (anxiety, depression) can exacerbate people's sense of isolation nowadays with Covid-19 pandemic and fear of the future. These symptoms often accompany sleep disturbances and fatigue but are barely recognized [26].

Management of cardiac complications is especially important in Friedreich's ataxia; it can be applied to SCA and patients need regular electrocardiographic checks and echocardiograms to detect the development of cardiomyopathy. Echocardiography may show concentric left ventricular hypertrophy (possibly in more than half of cases, especially early-onset ones).

As the disease progresses, hypertrophy regresses, resulting in a thin, dilated left ventricle. Cardiac enzymes may be asymptomatically elevated (in the absence of arrhythmia or acute coronary syndrome) and may help to have baseline values for future comparison. It is essential to involve an experienced cardiologist with knowledge in the treatment of neuromuscular disorders, initially to advise on medications to treat cardiomyopathy and heart failure, and later to manage arrhythmias and other complications related [27, 28].

#### **5. Multidisciplinary team work**

The multidisciplinary team is clearly important in evaluating and managing patients with SCA. Patients with SCA should be offered several times a year's reviews, ideally by a specialized team including a neurologist, advanced palliative care, nurse, and when it would be necessary, other members as social workers, psychiatrists, therapists, physiatrists.

Speech and linguistic therapy (for both communication and feeding), occupational therapy, and physiotherapy can each make important contributions at different stages of the patient's life. Patients require regular review to identify any new symptoms that may need treatment, and for patients to take advantage of advances in diagnosis and any newly available treatments [20–24].

These multidisciplinary interventions can significantly alleviate the problems of progressive ataxia and prevent potentially fatal complications. An enthusiastic and well-informed medical approach in addition to follow-up by a multidisciplinary team can provide valuable support to an SCA's patient.

In addition, those with no established cause for their ataxia can undergo a thorough and repeated review of the clinical features and investigation results, which sometimes leads to a clearer diagnosis. Patients and their families should be encouraged to contact patient support groups. When a family first receives the diagnosis of progressive ataxia, patients are usually not heard of the condition or come across other people with it. Support from patient organizations can, therefore, be particularly important at this stage. The possibility of meeting others in the same situation, receiving emotional support and information, and the opportunity to learn about research developments can all help [30, 33].

#### **6. Supportive care**

Given that most cases of SCA are difficult to manage, can progress rapidly and have a shortened life. Studies on their palliation and end-of-life care are needed. Most of the recommendations in guidelines at present are drawn from the broader field of other progressive neurological conditions. The supportive care comes alongside your current medical and neurology team to give an extra layer of support not only for patients but also for family [29–32].

Palliative care is for anyone living with a serious illness at any stage and can be offered at any facility wherever the patient is at the hospital, clinic outpatients, and at home [30]. To provide support to physical and psychological symptoms, social issues, community groups, talking about end-of-life worries, other issues (for example, copying distress) and spiritual concerns. Compared to usual care, it provides relief from suffering, works in quality of life, plans for decline, advances care planning, focuses on patient and family, and requires a consistent team approach and strategy. The members of neuro-palliative care are doctors who are going to review the history and physical examination, establishing goals of care, symptoms management and education, about disease and prognosis discussion which is very difficult related to SCA due to lack of key markers then normally is done about the point of care of each individual case.

The nursing team is going to make the medication review, identification of medical durable power of attorney, discussion of advanced care planning at the appropriate time besides for screening caregiver distress. The chaplain is going to address spiritual/existential concerns, exploring social and family issues, identifying, and discussing grief and screening for caregiver distress. The social worker is going to advice on financial and insurance issues. Providing resources for home health care, and logistical aspects of transition care.

#### **7. Planning care and final remarks**

As stated in our book and described in several chapters, the symptoms of ACS are much more than ataxia or movement disorders and include variability in

#### *Living and Coping with Spinocerebellar Ataxia: Palliative Care Approach DOI: http://dx.doi.org/10.5772/intechopen.104605*

cognitive complaints, mood disorders, fatigue, vision problems, problems eating, swallowing, neuropathy, cramps, muscle, heart, intestinal, and urinary problems among many others.

The psychiatrist Viktor Frankl identified three main sources for meaning in care and life [31]:


*In a position of utter desolation, when a man cannot express himself in positive action, when his only achievement may consist in enduring his sufferings in the right way—an honorable way—in such a position man can, through loving contemplation of the image he carries of his beloved, achieve fulfillment.*

*Love goes very far beyond the physical person of the beloved. It finds its deepest meaning in his spiritual being, his inner self.*

• In courage during difficult times. Suffering itself is meaningless, but our response to it gives it meaning.

Then understating an individual's value goals of care allows clinical to align the care with what is the most important to the patients with SCA and their families. Mainly addressing the value goals of care, for example, doing exploration about what was life before SCA? and other several important matters, asking questions about the quality of life and hoping to realize what is most important for the patient. Patients with intractable and/or distressing physical symptoms may benefit from referral for a specialist palliative care, which might also help those with complex social, psychological, or spiritual needs and plan of care.

The time for planning end-of-life care is when the clinician answers 'No' to the 'surprise question'—'Would you be surprised if this patient died in the next 12 months?'—as well there being generic and specific (for ataxia) indicators that the patients have reached the terminal phase of their illness. Management in this phase should be geared toward enabling a 'good death': being treated as an individual, with dignity and respect, without pain or other distressing symptoms, in familiar surroundings, and the company of close friends and family.

The plan of care will be a negotiation of goals of care and realistic medical options for management. Besides that, the unique psychosocial stressors such as changing roles in a relationship, loss of autonomy, financial strain, communication difficulties, social isolation (especially during Covid-19 pandemic), cosmetic effects, a social stigma that will require referral to an attorney, psychotherapy, support groups, ataxia specific programming in rehabilitation centers.

The spiritual distress includes grief, guilt, fear of cognitive decline, existential crisis, and death anxiety and needs to be addressed the caregiver distress as well as high levels of burden and depression. Establishing an advance care plan to ensure that patient wishes are known and planning the future associated with improvement of patient satisfaction, lower hospital admission rates, decreases significantly the psychological comorbidities and suffering for the family.

Having a diagnosis of SCA is very important to identify a care team to strategize how to bring back meaning to life, getting extra support at home, and community resources. Also, despite care holistic symptom management, long-term relationship with the care team, and establishing a plan for future advanced care planning [30, 32, 33].

In conclusion, the palliative care approach in patients living and coping with SCA should benefit the patient's life in many aspects, such as better quality of life, improved symptom burden, better life of patient and family, greater satisfaction with care, higher rates and quality of the advance plan of future and no adverse effects. In addition to that, further studies are needed as clinical priorities included to develop and implement models to integrate palliative care into neurology and to develop and implement informative quality measures to evaluate and compare palliative approaches in SCA through validated trials.

### **Conflict of interest**

The authors declare no conflict of interest or disclosures.

### **Author details**

Caroline Bozzetto Ambrosi1 and Patricia Bozzetto Ambrosi2 \*

1 Medical Doctor, Specialist in Palliative Care, United Kingdom

2 Medical Doctor, Alumni Sorbonne University, France

\*Address all correspondence to: patriciabozzettoadm@outlook.com

© 2022 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.

*Living and Coping with Spinocerebellar Ataxia: Palliative Care Approach DOI: http://dx.doi.org/10.5772/intechopen.104605*

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### *Edited by Patricia Bozzetto Ambrosi*

This book is about spinocerebellar ataxia (SCA), which is among the most challenging pathologies in the neurological landscape. It covers basic concepts, functional classification, and new approaches to medical and non-medical treatment including rehabilitation/palliative care approaches. The volume also describes a wide spectrum of generalities and particularities about various forms of clinical and genetic presentations of ACS that have life-threatening characteristics and long-standing presentation with tremendous variability in presentation and clinical severity. In addition, the book presents important aspects of cerebellar anatomy, nutrition impact, genetic subtypes, and functional classification of medical and non-medical interventions related to stem cells, rehabilitation, and palliative care.

Published in London, UK © 2022 IntechOpen © Koonsiri Boonnak / iStock

Spinocerebellar Ataxia - Concepts, Particularities and Generalities

Spinocerebellar Ataxia

Concepts, Particularities and Generalities

*Edited by Patricia Bozzetto Ambrosi*