Antisense Oligonucleotide Silencing Reverses Abnormal Neurochemistry in Spinocerebellar Ataxia 3 Mice.

OBJECTIVE: Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia, and biomarkers are needed to noninvasively monitor disease progression and treatment response. Anti-ATXN3 antisense oligonucleotide (ASO) treatment has been shown to mitigate neuropathology and rescue motor phenotypes in SCA3 mice. Here, we investigated whether repeated ASO administration reverses brainstem and cerebellar neurochemical abnormalities by magnetic resonance spectroscopy (MRS). METHODS: Symptomatic SCA3 mice received intracerebroventricular treatment of ASO or vehicle and were compared to wild-type vehicle-treated littermates. To quantify neurochemical changes in treated mice, longitudinal 9.4T MRS of cerebellum and brainstem was performed. Acquired magnetic resonance (MR) group means were analyzed by 2-way analysis of variance mixed-effects sex-adjusted analysis with post hoc Sidak correlation for multiple comparisons. Pearson correlations were used to relate SCA3 pathology and behavior. RESULTS: MR spectra yielded 15 to 16 neurochemical concentrations in the cerebellum and brainstem. ASO treatment in SCA3 mice resulted in significant total choline rescue and partial reversals of taurine, glutamine, and total N-acetylaspartate across both regions. Some ASO-rescued neurochemicals correlated with reduction in diseased protein and nuclear ATXN3 accumulation. ASO-corrected motor activity correlated with total choline and total N-acetylaspartate levels early in disease. INTERPRETATION: SCA3 mouse cerebellar and brainstem neurochemical trends parallel those in patients with SCA3. Decreased total choline may reflect oligodendrocyte abnormalities, decreased total N-acetylaspartate highlights neuronal health disturbances, and high glutamine may indicate gliosis. ASO treatment fully or partially reversed select neurochemical abnormalities in SCA3 mice, indicating the potential for these measures to serve as noninvasive treatment biomarkers in future SCA3 gene silencing trials. ANN NEUROL 2023;94:658-671.

BDNF is altered in a brain-region specific manner and rescues deficits in Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset, dominantly inherited neurodegenerative disease caused by the expanded polyQ tract in the protein ATAXIN1 (ATXN1) and characterized by progressive motor and cognitive impairments. There are no disease-modifying treatments or cures for SCA1. Brain-derived neurotrophic factor (BDNF) plays important role in cerebellar physiology and has shown therapeutic potential for cerebellar pathology in the transgenic mouse model of SCA1, ATXN1[82Q] line that overexpress mutant ATXN1 under a cerebellar Purkinje-cell-specific promoter. Here we demonstrate decreased expression of brain derived neurotrophic factor (BDNF) in the cerebellum and medulla of patients with SCA1. Early stages of disease seem most amenable to therapy. Thus, we next quantified Bdnf expression in Atxn1154Q/2Q mice, a knock-in mouse model of SCA1, during the early symptomatic disease stage in four clinically relevant brain regions: cerebellum, medulla, hippocampus and motor cortex. We found that during the early stages of disease, Bdnf mRNA expression is reduced in the hippocampus and cerebellum, while it is increased in the cortex and brainstem. Importantly, we observed that pharmacological delivery of recombinant BDNF improved motor and cognitive performance, and mitigated pathology in the cerebellum and hippocampus of Atxn1154Q/2Q mice. Our findings demonstrate brain-region specific deficiency of BDNF in SCA1 and show that reversal of low BDNF levels offers the potential for meaningful treatment of motor and cognitive deficits in SCA1.

Cerebellar contribution to the cognitive alterations in SCA1: evidence from mouse models.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by abnormal expansion of glutamine (Q) encoding CAG repeats in the gene Ataxin-1 (ATXN1). Although motor and balance deficits are the core symptoms of SCA1, cognitive decline is also commonly observed in patients. While mutant ATXN1 is expressed throughout the brain, pathological findings reveal severe atrophy of cerebellar cortex in SCA1 patients. The cerebellum has recently been implicated in diverse cognitive functions, yet to what extent cerebellar neurodegeneration contributes to cognitive alterations in SCA1 remains poorly understood. Much of our understanding of the mechanisms underlying pathogenesis of motor symptoms in SCA1 comes from mouse models. Reasoning that mouse models could similarly offer important insights into the mechanisms of cognitive alterations in SCA1, we tested cognition in several mouse lines using Barnes maze and fear conditioning. We confirmed cognitive deficits in Atxn1154Q/2Q knock-in mice with brain-wide expression of mutant ATXN1 and in ATXN1 null mice. We found that shorter polyQ length and haploinsufficiency of ATXN1 do not cause significant cognitive deficits. Finally, ATXN1[82Q ] transgenic mice-with cerebellum limited expression of mutant ATXN1-demonstrated milder impairment in most aspects of cognition compared to Atxn1154Q/2Q mice, supporting the concept that cognitive deficits in SCA1 arise from a combination of cerebellar and extra-cerebellar dysfunctions.

Post-symptomatic Delivery of Brain-Derived Neurotrophic Factor (BDNF) Ameliorates Spinocerebellar Ataxia Type 1 (SCA1) Pathogenesis.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an abnormal expansion of CAG repeats in the Ataxin1 (ATXN1) gene. SCA1 is characterized by motor deficits, cerebellar neurodegeneration, and gliosis and gene expression changes. Expression of brain-derived neurotrophic factor (BDNF), growth factor important for the survival and function of cerebellar neurons, is decreased in ATXN1[82Q] mice, the Purkinje neuron specific transgenic mouse model of SCA1. As this decrease in BDNF expression may contribute to cerebellar neurodegeneration, we tested whether delivery of extrinsic human BDNF via osmotic ALZET pumps has a beneficial effect on disease severity in this mouse model of SCA1. Additionally, to test the effects of BDNF on established and progressing cerebellar pathogenesis and motor deficits, we delivered BDNF post-symptomatically. We have found that post-symptomatic delivery of extrinsic BDNF ameliorated motor deficits and cerebellar pathology (i.e., dendritic atrophy of Purkinje cells, and astrogliosis) indicating therapeutic potential of BDNF even after the onset of symptoms in SCA1. However, BDNF did not alter Purkinje cell gene expression changes indicating that certain aspects of disease pathogenesis cannot be ameliorated/slowed down with BDNF and that combinational therapies may be needed.

Polarization-sensitive optical coherence tomography reveals gray matter and white matter atrophy in SCA1 mouse models.

Spinocerebellar ataxia type 1 (SCA1) is a fatal inherited neurodegenerative disease. In this study, we demonstrate the label-free optical imaging methodology that can detect, with a high degree of sensitivity, discrete areas of degeneration in the cerebellum of the SCA1 mouse models. We used ATXN1[82Q] and ATXN1[30Q]-D776 mice in which the transgene is directed only to Purkinje cells. Molecular layer, granular layer, and white matter regions are analyzed using the intrinsic contrasts provided by polarization-sensitive optical coherence tomography. Cerebellar atrophy in SCA1 mice occurred both in gray matter and white matter. While gray matter atrophy is obvious, indications of white matter atrophy including different birefringence characteristics, and shortened and contorted branches are observed. Imaging results clearly show the loss or atrophy of myelinated axons in ATXN1[82Q] mice. The method provides unbiased contrasts that can facilitate the understanding of the pathological progression in neurodegenerative diseases and other neural disorders.

Assessing recovery from neurodegeneration in spinocerebellar ataxia 1: Comparison of in vivo magnetic resonance spectroscopy with motor testing, gene expression and histology.

Suppression of transgene expression in a conditional transgenic mouse model of spinocerebellar ataxia 1 (SCA1) reverses the Purkinje cell pathology and motor dysfunction that are hallmarks of SCA1. We previously showed that cerebellar neurochemical levels measured by magnetic resonance spectroscopy (MRS) correlate with progression of pathology and clinical status of patients and that abnormal neurochemical levels normalize upon suppression of transgene expression, indicating their potential as robust surrogate markers of treatment effects. Here we investigated the relative sensitivities of MRS, histology, transgene expression and motor behavioral testing to disease reversal in conditional SCA1 mice. Transgene expression was suppressed by doxycycline administration and treated and untreated mice were assessed by MRS at 9.4tesla before and after treatment and with an accelerating Rotarod, histology and quantitative polymerase chain reaction (qPCR) for ataxin-1 transgene expression following doxycycline treatment. The MRS-measured N-acetylaspartate-to-myo-inositol ratio (NAA/Ins) correlated significantly with the molecular layer (ML) thickness and transgene expression. NAA/Ins, ML thickness and transgene expression were highly significantly different between the treated vs. untreated groups (p<0.0001), while the Rotarod assessment showed a trend for treatment effect. MRS, qPCR and histology had high sensitivity/specificity to distinguish treated from untreated mice, all with areas under the curve (AUC)=0.97-0.98 in receiver operating characteristic (ROC) analyses, while Rotarod had significantly lower sensitivity and specificity (AUC=0.72). Therefore, MRS accurately reflects the extent of recovery from neurodegeneration with sensitivity similar to invasive measures, further validating its potential as a surrogate marker in pre-clinical and clinical treatment trials.

Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

Decreasing mutant ATXN1 nuclear localization improves a spectrum of SCA1-like phenotypes and brain region transcriptomic profiles.

Spinocerebellar ataxia type 1 (SCA1) is a dominant trinucleotide repeat neurodegenerative disease characterized by motor dysfunction, cognitive impairment, and premature death. Degeneration of cerebellar Purkinje cells is a frequent and prominent pathological feature of SCA1. We previously showed that transport of ATXN1 to Purkinje cell nuclei is required for pathology, where mutant ATXN1 alters transcription. To examine the role of ATXN1 nuclear localization broadly in SCA1-like disease pathogenesis, CRISPR-Cas9 was used to develop a mouse with an amino acid alteration (K772T) in the nuclear localization sequence of the expanded ATXN1 protein. Characterization of these mice indicates that proper nuclear localization of mutant ATXN1 contributes to many disease-like phenotypes including motor dysfunction, cognitive deficits, and premature lethality. RNA sequencing analysis of genes with expression corrected to WT levels in Atxn1175QK772T/2Q mice indicates that transcriptomic aspects of SCA1 pathogenesis differ between the cerebellum, brainstem, cerebral cortex, hippocampus, and striatum.

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model ( f-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin - Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

Spatial and Temporal Diversity of Astrocyte Phenotypes in Spinocerebellar Ataxia Type 1 Mice.

While astrocyte heterogeneity is an important feature of the healthy brain, less is understood about spatiotemporal heterogeneity of astrocytes in brain disease. Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by a CAG repeat expansion in the gene Ataxin1 (ATXN1). We characterized astrocytes across disease progression in the four clinically relevant brain regions, cerebellum, brainstem, hippocampus, and motor cortex, of Atxn1154Q/2Q mice, a knock-in mouse model of SCA1. We found brain region-specific changes in astrocyte density and GFAP expression and area, early in the disease and prior to neuronal loss. Expression of astrocytic core homeostatic genes was also altered in a brain region-specific manner and correlated with neuronal activity, indicating that astrocytes may compensate or exacerbate neuronal dysfunction. Late in disease, expression of astrocytic homeostatic genes was reduced in all four brain regions, indicating loss of astrocyte functions. We observed no obvious correlation between spatiotemporal changes in microglia and spatiotemporal astrocyte alterations, indicating a complex orchestration of glial phenotypes in disease. These results support spatiotemporal diversity of glial phenotypes as an important feature of the brain disease that may contribute to SCA1 pathogenesis in a brain region and disease stage-specific manner.

Antisense Oligonucleotide Therapeutic Approach for Suppression of Ataxin-1 Expression: A Safety Assessment.

Spinocerebellar ataxia type 1 (SCA1) is a lethal, autosomal dominant neurodegenerative disease caused by a polyglutamine expansion in the ATAXIN-1 (ATXN1) protein. Preclinical studies demonstrate the therapeutic efficacy of approaches that target and reduce Atxn1 expression in a non-allele-specific manner. However, studies using Atxn1-/- mice raise cautionary notes that therapeutic reductions of ATXN1 might lead to undesirable effects such as reduction in the activity of the tumor suppressor Capicua (CIC), activation of the protease ß-secretase 1 (BACE1) and subsequent increased amyloidogenic cleavage of the amyloid precursor protein (APP), or a reduction in hippocampal neuronal precursor cells that would impact hippocampal function. Here, we tested whether an antisense oligonucleotide (ASO)-mediated reduction of Atxn1 produced unwanted effects involving BACE1, CIC activity, or reduction in hippocampal neuronal precursor cells. Notably, no effects on BACE1, CIC tumor suppressor function, or number of hippocampal neuronal precursor cells were found in mice subjected to a chronic in vivo ASO-mediated reduction of Atxn1. These data provide further support for targeted reductions of ATXN1 as a therapeutic approach for SCA1.

Brain Derived Neurotrophic Factor (BDNF) Delays Onset of Pathogenesis in Transgenic Mouse Model of Spinocerebellar Ataxia Type 1 (SCA1).

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an abnormal expansion of CAG repeats in the Ataxin-1 (ATXN1) gene and characterized by motor deficits and cerebellar neurodegeneration. Even though mutant ATXN1 is expressed from an early age, disease onset usually occurs in patient's mid-thirties, indicating the presence of compensatory factors that limit the toxic effects of mutant ATXN1 early in disease. Brain derived neurotrophic factor (BDNF) is a growth factor known to be important for the survival and function of cerebellar neurons. Using gene expression analysis, we observed altered BDNF expression in the cerebella of Purkinje neuron specific transgenic mouse model of SCA1, ATXN1[82Q] mice, with increased expression during the early stage and decreased expression in the late stage of disease. We therefore investigated the potentially protective role of BDNF in early stage SCA1 through intraventricular delivery of BDNF via ALZET osmotic pumps. Extrinsic BDNF delivery delayed onset of motor deficits and Purkinje neuron pathology in ATXN1[82Q] mice supporting its use as a novel therapeutic for SCA1.

Antisense oligonucleotide-mediated ataxin-1 reduction prolongs survival in SCA1 mice and reveals disease-associated transcriptome profiles.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.