Purkinje-Enriched snRNA-seq in SCA7 Cerebellum Reveals Zebrin Identity Loss as a Central Feature of Polyglutamine Ataxias.

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion. SCA7 patients display a striking loss of Purkinje cell (PC) neurons with disease progression; however, PCs are rare, making them difficult to characterize. We developed a PC nuclei enrichment protocol and applied it to single-nucleus RNA-seq of a SCA7 knock-in mouse model. Our results unify prior observations into a central mechanism of cell identity loss, impacting both glia and PCs, driving accumulation of inhibitory synapses and altered PC spiking. Zebrin-II subtype dysregulation is the predominant signal in PCs, leading to complete loss of zebrin-II striping at motor symptom onset in SCA7 mice. We show this zebrin-II subtype degradation is shared across Polyglutamine Ataxia mouse models and SCA7 patients. It has been speculated that PC subtype organization is critical for cerebellar function, and our results suggest that a breakdown of zebrin-II parasagittal striping is pathological.

Purkinje cell ataxin-1 modulates climbing fiber synaptic input in developing and adult mouse cerebellum.

Previous studies indicate that while transgenic mice with ATXN1[30Q]-D776-induced disease share pathological features caused by ATXN1[82Q] having an expanded polyglutamine tract, they fail to manifest the age-related progressive neurodegeneration seen in spinocerebellar ataxia type 1. The shared features include morphological alterations in climbing fiber (CF) innervation of Purkinje cells (PCs). To further investigate the ability of ataxin-1 (ATXN1) to impact CF/PC innervation, this study used morphological and functional approaches to examine CF/PC innervation during postnatal development in ATXN1[30Q]-D776 and ATXN1[82Q] cerebella. Notably, ATXN1[30Q]-D776 induced morphological alterations consistent with the development of the innervation of PCs by CFs being compromised, including a reduction of CF translocation along the PC dendritic tree, and decreased pruning of CF terminals from the PC soma. As previously shown for ATXN1[82Q], ATXN1[30Q]-D776 must enter the nucleus of PCs to induce these alterations. Experiments using conditional ATXN1[30Q]-D776 mice demonstrate that both the levels and specific timing of mutant ATXN1 expression are critical for alteration of the CF-PC synapse. Together these observations suggest that ATXN1, expressed exclusively in PCs, alters expression of a gene(s) in the postsynaptic PC that are critical for its innervation by CFs. To investigate whether ATXN1[30Q]-D776 curbs the progressive disease in ATXN1[82Q]-S776 mice, we crossed ATXN1[30Q]-D776 and ATXN1[82Q]-S776 mice and found that double transgenic mice developed progressive PC atrophy. Thus, the results also show that to develop progressive cerebellar degeneration requires expressing ATXN1 with an expanded polyglutamine tract.

Abnormalities in the climbing fiber-Purkinje cell circuitry contribute to neuronal dysfunction in ATXN1[82Q] mice.

One fundamental unanswered question in the field of polyglutamine diseases concerns the pathophysiology of neuronal dysfunction. Is there dysfunction in a specific neuronal population or circuit initially that contributes the onset of behavioral abnormalities? This study used a systems-level approach to investigate the functional integrity of the excitatory cerebellar cortical circuitry in vivo from several transgenic ATXN1 mouse lines. We tested the hypotheses that there are functional climbing fiber (CF)-Purkinje cell (PC) and parallel fiber (PF)-PC circuit abnormalities using flavoprotein autofluorescence optical imaging and extracellular field potential recordings. In early-symptomatic and symptomatic animals expressing ATXN1[82Q], there is a marked reduction in PC responsiveness to CF activation. Immunostaining of vesicular glutamate transporter type 2 demonstrated a decrement in CF extension on PC dendrites in symptomatic ATXN1[82Q] mice. In contrast, responses to PF stimulation were relatively normal. Importantly, the deficits in CF-PC synaptic transmission required expression of pathogenic ataxin-1 (ATXN1[82Q]) and for its entrance into the nucleus of PCs. Loss of endogenous mouse Atxn1 had no discernible effects. Furthermore, the abnormalities in CF-PC synaptic transmission were ameliorated when mutant transgene expression was prevented during postnatal cerebellar development. The results demonstrate the preferential susceptibility of the CF-PC circuit to the effects of ATXN1[82Q]. Further, this deficit likely contributes to the abnormal motor phenotype of ATXN1[82Q] mice. For polyglutamine diseases generally, the findings support a model whereby specific neuronal circuits suffer insults that alter function before cell death.

Phosphorylation of ATXN1 at Ser776 in the cerebellum.

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative disorders caused by a mutant protein with an expanded polyglutamine tract. Phosphorylation of ataxin-1 (ATXN1) at serine 776 is implicated in SCA1 pathogenesis. Previous studies, utilizing transfected cell lines and a Drosophila photoreceptor model of SCA1, suggest that phosphorylating ATXN1 at S776 renders it less susceptible to degradation. This work also indicated that oncogene from AKR mouse thymoma (Akt) promotes the phosphorylation of ATXN1 at S776 and severity of neurodegeneration. Here, we examined the phosphorylation of ATXN1 at S776 in cerebellar Purkinje cells, a prominent site of pathology in SCA1. We found that while phosphorylation of S776 is associated with a stabilization of ATXN1 in Purkinje cells, inhibition of Akt either in vivo or in a cerebellar extract-based phosphorylation assay did not decrease the phosphorylation of ATXN1-S776. In contrast, immunodepletion and inhibition of cyclic AMP-dependent protein kinase decreased phosphorylation of ATXN1-S776. These results argue against Akt as the in vivo kinase that phosphorylates S776 of ATXN1 and suggest that cyclic AMP-dependent protein kinase is the active ATXN1-S776 kinase in the cerebellum.

ATAXIN-1 interacts with the repressor Capicua in its native complex to cause SCA1 neuropathology.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions.

Recovery from polyglutamine-induced neurodegeneration in conditional SCA1 transgenic mice.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant, polyglutamine-induced neurodegenerative disorder that results in loss of motor coordination caused primarily by a disruption of cerebellar Purkinje cell function. In this study, we developed a conditional SCA1 mouse model to examine whether stopping expression of mutant ataxin-1 alters the disease phenotype. After cessation of SCA1[82Q] transgene expression, mutant ataxin-1, including that in nuclear inclusions, was cleared rapidly from Purkinje cells. At an early stage of disease, Purkinje cell pathology and motor dysfunction were completely reversible. After halting SCA1 expression at later stages of disease, only a partial recovery was seen. Interestingly, restoration of the ability to perform a complex motor task, the accelerating Rotarod, correlated with localization of mGluR1alpha to the Purkinje cell-parallel fiber synapse. These results show that the progression of SCA1 pathogenesis is dependent on the continuous expression of mutant ataxin-1. Of note, even at a late stage of disease, Purkinje cells retain at least some ability to repair the damage caused by mutant ataxin-1.

Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice.

Polyglutamine-induced neurodegeneration in transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene is modulated by subcellular distribution of ataxin-1 and by components of the protein folding/degradation machinery. Since phosphorylation is a prominent mechanism by which these processes are regulated, we examined phosphorylation of ataxin-1 and found that serine 776 (S776) was phosphorylated. Residue 776 appeared to affect cellular deposition of ataxin-1[82Q] in that ataxin-1[82Q]-A776 failed to form nuclear inclusions in tissue culture cells. The importance of S776 for polyglutamine-induced pathogenesis was examined by generating ataxin-1[82Q]-A776 transgenic mice. These mice expressed ataxin-1[82Q]-A776 within Purkinje cell nuclei, yet the ability of ataxin-1[82Q]-A776 to induce disease was substantially reduced. These studies demonstrate that polyglutamine tract expansion and localization of ataxin-1 to the nucleus of Purkinje cells are not sufficient to induce disease. We suggest that S776 of ataxin-1 also has a critical role in SCA1 pathogenesis.