Spinocerebellar Ataxia Type 1 Pathology Research Articles

Dynamic molecular network analysis of iPSC-Purkinje cells differentiation delineates roles of ISG15 in SCA1 at the earliest stage

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.

Therapeutic Strategies for Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder that affects one or two individuals per 100,000. The disease is caused by an extended CAG repeat in exon 8 of the ATXN1 gene and is characterized mostly by a profound loss of cerebellar Purkinje cells, leading to disturbances in coordination, balance, and gait. At present, no curative treatment is available for SCA1. However, increasing knowledge on the cellular and molecular mechanisms of SCA1 has led the way towards several therapeutic strategies that can potentially slow disease progression. SCA1 therapeutics can be classified as genetic, pharmacological, and cell replacement therapies. These different therapeutic strategies target either the (mutant) ATXN1 RNA or the ataxin-1 protein, pathways that play an important role in downstream SCA1 disease mechanisms or which help restore cells that are lost due to SCA1 pathology. In this review, we will provide a summary of the different therapeutic strategies that are currently being investigated for SCA1.

Indirect Negative Effect of Mutant Ataxin-1 on Short- and Long-Term Synaptic Plasticity in Mouse Models of Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is an intractable progressive neurodegenerative disease that leads to a range of movement and motor defects and is eventually lethal. Purkinje cells (PC) are typically the first to show signs of degeneration. SCA1 is caused by an expansion of the polyglutamine tract in the ATXN1 gene and the subsequent buildup of mutant Ataxin-1 protein. In addition to its toxicity, mutant Ataxin-1 protein interferes with gene expression and signal transduction in cells. Recently, it is evident that ATXN1 is not only expressed in neurons but also in glia, however, it is unclear the extent to which either contributes to the overall pathology of SCA1. There are various ways to model SCA1 in mice. Here, functional deficits at cerebellar synapses were investigated in two mouse models of SCA1 in which mutant ATXN1 is either nonspecifically expressed in all cell types of the cerebellum (SCA1 knock-in (KI)), or specifically in Bergmann glia with lentiviral vectors expressing mutant ATXN1 under the control of the astrocyte-specific GFAP promoter. We report impairment of motor performance in both SCA1 models. In both cases, prominent signs of astrocytosis were found using immunohistochemistry. Electrophysiological experiments revealed alteration of presynaptic plasticity at synapses between parallel fibers and PCs, and climbing fibers and PCs in SCA1 KI mice, which is not observed in animals expressing mutant ATXN1 solely in Bergmann glia. In contrast, short- and long-term synaptic plasticity was affected in both SCA1 KI mice and glia-targeted SCA1 mice. Thus, non-neuronal mechanisms may underlie some aspects of SCA1 pathology in the cerebellum. By combining the outcomes of our current work with our previous data from the B05 SCA1 model, we further our understanding of the mechanisms of SCA1.

Extracellular S100β Disrupts Bergman Glia Morphology and Synaptic Transmission in Cerebellar Purkinje Cells.

Astrogliosis is a pathological process that affects the density, morphology, and function of astrocytes. It is a common feature of brain trauma, autoimmune diseases, and neurodegeneration including spinocerebellar ataxia type 1 (SCA1), a poorly understood neurodegenerative disease. S100β is a Ca2+ binding protein. In SCA1, excessive excretion of S100β by reactive astrocytes and its uptake by Purkinje cells has been demonstrated previously. Under pathological conditions, excessive extracellular concentration of S100β stimulates the production of proinflammatory cytokines and induces apoptosis. We modeled astrogliosis by S100β injections into cerebellar cortex in mice. Injections of S100β led to significant changes in Bergmann glia (BG) cortical organization and affected their processes. S100β also changed morphology of the Purkinje cells (PCs), causing a significant reduction in the dendritic length. Moreover, the short-term synaptic plasticity and depolarization-induced suppression of synaptic transmission were disrupted after S100β injections. We speculate that these effects are the result of Ca2+-chelating properties of S100β protein. In summary, exogenous S100β induced astrogliosis in cerebellum could lead to neuronal dysfunction, which resembles a natural neurodegenerative process. We suggest that astrocytes play an essential role in SCA1 pathology, and that astrocytic S100β is an important contributor to this process.

PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein's stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.

Progressive impairment of cerebellar mGluR signalling and its therapeutic potential for cerebellar ataxia in spinocerebellar ataxia type 1 model mice.

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by a gene defect, leading to movement disorder such as cerebellar ataxia. It remains largely unknown which functional defect contributes to the cerebellar ataxic phenotype in SCA1. In this study, we report progressive dysfunction of metabotropic glutamate receptor (mGluR) signalling, which leads to smaller slow synaptic responses, reduced dendritic Ca2+ signals and impaired synaptic plasticity at cerebellar synapses, in the early disease stage of SCA1 model mice. We also show that enhancement of mGluR signalling by a clinically available drug, baclofen, leads to improvement of motor performance in SCA1 mice. SCA1 is an incurable disease with no effective treatment, and our results may provide mechanistic grounds for targeting mGluRs and a novel drug therapy with baclofen to treat SCA1 patients in the future. Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease that presents with cerebellar ataxia and motor learning defects. Previous studies have indicated that the pathology of SCA1, as well as other ataxic diseases, is related to signalling pathways mediated by the metabotropic glutamate receptor type 1 (mGluR1), which is indispensable for proper motor coordination and learning. However, the functional contribution of mGluR signalling to SCA1 pathology is unclear. In the present study, we show that SCA1 model mice develop a functional impairment of mGluR signalling which mediates slow synaptic responses, dendritic Ca2+ signals, and short- and long-term synaptic plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in a progressive manner from the early disease stage (5 postnatal weeks) prior to PC death. Notably, impairment of mGluR-mediated dendritic Ca2+ signals linearly correlated with a reduction of PC capacitance (cell surface area) in disease progression. Enhancement of mGluR signalling by baclofen, a clinically available GABAB receptor agonist, led to an improvement of motor performance in SCA1 mice and the improvement lasted ∼1 week after a single application of baclofen. Moreover, the restoration of motor performance in baclofen-treated SCA1 mice matched the functional recovery of mGluR-mediated slow synaptic currents and mGluR-dependent short- and long-term synaptic plasticity. These results suggest that impairment of synaptic mGluR cascades is one of the important contributing factors to cerebellar ataxia in early and middle stages of SCA1 pathology, and that modulation of mGluR signalling by baclofen or other clinical interventions may be therapeutic targets to treat SCA1.

HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice.

Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset.

Structural basis of the phosphorylation dependent complex formation of neurodegenerative disease protein Ataxin-1 and RBM17

Spinocerebellar Ataxia Type1 (SCA1) is a dominantly inherited neurodegenerative disease and belongs to polyglutamine expansion disorders. The polyglutamine expansion in Ataxin-1 (ATXN1) is responsible for SCA1 pathology. ATXN1 forms at least two distinct complexes with Capicua (CIC) or RNA-binding motif protein 17 (RBM17). The wild-type ATXN1 dominantly forms a complex with CIC and the polyglutamine expanded form of ATXN1 favors to form a complex with RBM17. The phosphorylation of Ser776 in ATXN1 is critical for SCA1 pathology and serves as a binding platform for RBM17. However, the molecular basis of the phospho-specific binging of ATXN1 to RBM17 is not delineated. Here, we present the modeled structure of RBM17 bound to the phosphorylated ATXN1 peptide. The structure reveals the phosphorylation specific interaction between ATXN1 and RBM17 through a salt-bridge network. Furthermore, the modeled structure and the interactions between RBM17 and ATXN1 were validated through mutagenesis study followed by Surface Plasmon Resonance binding experiments. This work delineates the molecular basis of the interaction between RBM17 and the phosphorylated form of ATXN1, which is critical for SCA1 pathology. Furthermore, the structure of RBM17 and pATXN1 peptide might be utilized to target RBM17–ATXN1 interaction to modulate SCA1 pathogenesis.

Systems biology analysis of Drosophila in vivo screen data elucidates core networks for DNA damage repair in SCA1

DNA damage repair is implicated in neurodegenerative diseases; however, the relative contributions of various DNA repair systems to the pathology of these diseases have not been investigated systematically. In this study, we performed a systematic in vivo screen of all available Drosophila melanogaster homolog DNA repair genes, and we tested the effect of their overexpression on lifespan and developmental viability in Spinocerebellar Ataxia Type 1 (SCA1) Drosophila models expressing human mutant Ataxin-1 (Atxn1). We identified genes previously unknown to be involved in CAG-/polyQ-related pathogenesis that function in multiple DNA damage repair systems. Beyond the significance of each repair system, systems biology analyses unraveled the core networks connecting positive genes in the gene screen that could contribute to SCA1 pathology. In particular, RpA1, which had the largest effect on lifespan in the SCA1 fly model, was located at the hub position linked to such core repair systems, including homologous recombination (HR). We revealed that Atxn1 actually interacted with RpA1 and its essential partners BRCA1/2. Furthermore, mutant but not normal Atxn1 impaired the dynamics of RpA1 in the nucleus after DNA damage. Uptake of BrdU by Purkinje cells was observed in mutant Atxn1 knockin mice, suggesting their abnormal entry to the S-phase. In addition, chemical and genetic inhibitions of Chk1 elongated lifespan and recovered eye degeneration. Collectively, we elucidated core networks for DNA damage repair in SCA1 that might include the aberrant usage of HR.

Polyglutamine Disease Toxicity Is Regulated by Nemo-like Kinase in Spinocerebellar Ataxia Type 1

Polyglutamine diseases are dominantly inherited neurodegenerative diseases caused by an expansion of a CAG trinucleotide repeat encoding a glutamine tract in the respective disease-causing proteins. Extensive studies have been performed to unravel disease pathogenesis and to develop therapeutics. Here, we report on several lines of evidence demonstrating that Nemo-like kinase (NLK) is a key molecule modulating disease toxicity in spinocerebellar ataxia type 1 (SCA1), a disease caused by a polyglutamine expansion in the protein ATAXIN1 (ATXN1). Specifically, we show that NLK, a serine/threonine kinase that interacts with ATXN1, modulates disease phenotypes of polyglutamine-expanded ATXN1 in a Drosophila model of SCA1. Importantly, the effect of NLK on SCA1 pathology is dependent upon NLK's enzymatic activity. Consistent with this, reduced Nlk expression suppresses the behavioral and neuropathological phenotypes in SCA1 knock-in mice. These data clearly indicate that either reducing NLK enzymatic activity or decreasing NLK expression levels can have beneficial effects against the toxicity induced by polyglutamine-expanded ATXN1.

D2R Agonist Bromocriptine Improves Neurodegeneration in Spinocerebellar Ataxia Type 1 Mouse Model (P05.021)

Objective: To explore the use of dopamine D2 receptor (D2R) agonist Bromocriptine (BRC) as a potential Spinocerebellar Ataxia Type 1 (SCA1) therapeutic. Background SCA1, a dominantly inherited neurodegenerative disease, results from an expanded CAG repeat mutation in the ataxin-1 gene which causes progressive ataxia, loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem. The prominent pathological features both in SCA1 patients and mouse model are remarkable PC dendritic atrophy and the development of S100B positive PC vacuoles. Mutant ataxin-1 aggregation and neurotoxicity is dependent on its phosphorylation status at S776. Previously, we have shown that D2R agonist BRC can reduce S776 phosphorylation and aggregation of the mutant ataxin-1 in cell culture models. Design/Methods: In this study, we treated SCA1 mice with 20mg/kg BRC every other day for 3 weeks. We explored the therapeutic potential of BRC at two age intervals corresponding to differential degenerative state of disease. After treatment, animals were analyzed for improvements in ataxic behavior on rotarod and bar tests and their brains processed for morphological and Western blot analysis. Results: A significant improvement was demonstrated on the bar test in SCA1 mice treated with BRC compared to vehicle treated mice. However, no significant changes were observed on the rotarod test. Immunohistochemical analysis showed improved PC morphology by increasing PC dendritic branch area and reducing S100B vacuole formation in SCA1 mice treated with BRC as compared to the vehicle treated animals. Furthermore, Western blot analysis revealed that BRC treated animals have increased levels of the calcium binding protein, CaB, a PC marker protein down regulated in SCA1, as compared to vehicle. Conclusions: Currently there is no treatment available for SCA1 patients. Our findings that BRC suppresses SCA1 phenotype suggest that BRC treatment may help reduce SCA1 pathology in human patients. Supported by: The Currier Spinocerebellar Degenerative Grant from the University of Mississippi Medical Center, Jackson, MS. Disclosure: Dr. Hearst has nothing to disclose. Dr. Lopez has nothing to disclose. Dr. Shao has nothing to disclose. Dr. Vig has nothing to disclose.

Disruption of metabotropic glutamate receptor signalling is a major defect at cerebellar parallel fibre–Purkinje cell synapses in staggerer mutant mice

Staggerer mutant mice have functional loss of a transcription factor, retinoid-related orphan receptor α (RORα), which is abundantly expressed in Purkinje cells (PCs) of the cerebellum.Homozygous staggerer (sg/sg)mice show cerebellar hypoplasia and congenital ataxia. Sg/sg mice serve as an important extreme mouse model of the hereditary spinocerebellar ataxia type 1 (SCA1), since it has been shown that RORα dysfunction is strongly correlated with SCA1 pathogenesis. However, synaptic abnormalities, especially at parallel fibre (PF)-PC synapses, in SCA1-related sg/sg mice have not been examined in detail electrophysiologically. In this study, we report that PFs can still establish functional synapses onto PCs in sg/sg mice in spite of reduction in the number of PF-PC synapses. Compared with PF-evoked EPSCs in the wild-type or heterozygotes, the success rate of the EPSC recordings in sg/sg was quite low (∼40%) and the EPSCs showed faster kinetics and slightly decreased paired pulse facilitation at short intervals. The prominent synaptic dysfunction is that sg/sg mice lack metabotropic glutamate receptor (mGluR)-mediated slow EPSCs completely. Neither intense PF stimulation nor an exogenously applied mGluR agonist, DHPG, could elicit mGluR-mediated responses.Western blot analysis in the sg/sg cerebellum revealed low-level expression of mGluR1 and TRPC3, both of which underlie mGluR-mediated slow currents in PCs. Immunohistochemical data demonstrated marked mislocalization of mGluR1 on sg/sg PCs.We found that mGluR-mediated retrograde suppression of PF-PC EPSCs by endocannabinoid is also impaired completely in sg/sg mice. These results suggest that disruption of mGluR signalling at PF-PC synapses is one of the major synaptic defects in sg/sg mice and may manifest itself in SCA1 pathology.

Duplication of Atxn1l suppresses SCA1 neuropathology by decreasing incorporation of polyglutamine-expanded ataxin-1 into native complexes

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine tract in ataxin-1 (ATXN1). SCA1 pathogenesis studies support a model in which the expanded glutamine tract causes toxicity by modulating the normal activities of ATXN1. To explore native interactions that modify the toxicity of ATXN1, we generated a targeted duplication of the mouse ataxin-1-like (Atxn1l, also known as Boat) locus, a highly conserved paralog of SCA1, and tested the role of this protein in SCA1 pathology. Using a knock-in mouse model of SCA1 that recapitulates the selective neurodegeneration seen in affected individuals, we found that elevated Atxn1l levels suppress neuropathology by displacing mutant Atxn1 from its native complex with Capicua (CIC). Our results provide genetic evidence that the selective neuropathology of SCA1 arises from modulation of a core functional activity of ATXN1, and they underscore the importance of studying the paralogs of genes mutated in neurodegenerative diseases to gain insight into mechanisms of pathogenesis.

Intranasal administration of IGF-I improves behavior and Purkinje cell pathology in SCA1 mice

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of polyglutamine repeat within ataxin-1 protein. Cerebellar Purkinje cells are the primary targets of SCA1 pathology. These cells synthesize insulin-like growth factor-I (IGF-I) and express its receptors during their entire life. The aim of present study was to determine if intranasally administered IGF-I to SCA1 transgenic mice suppresses toxic effects of ataxin-1. Two-week old SCA1 heterozygous animals were randomly divided into two treatment groups of IGF-I (30 and 60 μg IGF-I/animal) and a vehicle-treated control group. The wildtype animals served as normal controls. IGF-I or vehicle was administered at 48 h intervals for the total of 10 doses. Animals were then subjected to rotarod test, sacrificed, brains removed and processed for immunohistochemical and Western blot analysis. Radiolabeled IGF-I and bioactive TAT peptide accumulated in the brains of SCA1 mice following intranasal administration validating the use of intranasal route. SCA1 mice showed SCA1 pathology with impaired motor function and downregulation of calcium binding proteins as compared to wildtype mice. However, 30 and 60 μg IGF-I-treated animals showed improved performance on the rotarod as compared to vehicle-treated SCA1 mice with significant improvement ( p < 0.05) on day 3 in 60 μg IGF-I group. The immunohistochemical data further showed partial recovery in the expression of calbindin D28k and protein kinase C-γ in Purkinje cells in IGF-I-treated SCA1 animals. Our results indicate that suppression of ataxin-1-mediated adverse effects by intranasal IGF-I treatment may be of a therapeutic value to treat SCA1.

Coexistence of familial transthyretin amyloidosis ATTR Val30Met and spinocerebellar ataxia type 1 in a Japanese family – A follow-up autopsy report

Three brothers in a family with Val30Met transthyretin (TTR) amyloid polyneuropathy (FAP) in Iiyama, Japan were studied pathologically. In this family, affected members have been reported to show typical clinical features of FAP, and some have been documented to exhibit symptoms and signs of central nervous system (CNS) involvement consisting of cerebellar ataxia and pyramidal tract signs. After the original description, this family was regarded as a unique phenotype of this form of FAP; however, subsequent molecular genetic studies revealed that some patients and asymptomatic members in the family had Val30Met TTR and/or spinocerebellar ataxia type 1 (SCA1) gene mutations. In this study, pathological examination of two patients with both FAP and CNS symptoms showed (1) TTR-immunoreactive leptomeningeal and cerebrovascular amyloid deposition compatible with Val30Met TTR FAP, and (2) neuronal loss and gliosis mainly in the Purkinje cell layer, spinocerebellar system, olivo-ponto-cerebellar system, dentato-rubral system, gracile nuclei, cuneate nuclei, and various nuclei of cranial nerves, accompanied by anti-expanded polyglutamine tract antibody positive neuronal intranuclear inclusions, all of which were compatible with the pathological findings of SCA1. On the other hand, the remaining patient with FAP symptoms only showed the former pathological finding alone. The present study demonstrates, at the pathological level, that Val30Met TTR FAP and SCA1 coexist in the same family members, and that the CNS dysfunction seen in the patients in this family is ascribable to SCA1 pathology but not to CNS amyloidosis.