Biology subjects

Shakkottai, V. G.

Publications and source records attributed to Shakkottai, V. G..

6 recordsLinked to original sources

bioRxiv2023

Increased intrinsic membrane excitability is associated with hypertrophic olivary degeneration in spinocerebellar ataxia type 1

One of the characteristic areas of brainstem degeneration across multiple spinocerebellar ataxias (SCAs) is the inferior olive (IO), a medullary nucleus that plays a key role in motor learning. In addition to its vulnerability in SCAs, the IO is also susceptible to a distinct pathology known as hypertrophic olivary degeneration (HOD). Clinically, HOD has been exclusively observed after lesions in the brainstem disrupt inhibitory afferents to the IO. Here, for the first time, we describe HOD in another context: spinocerebellar ataxia type 1 (SCA1). Using the genetically-precise SCA1 knock-in mouse model (SCA1-KI; both sexes used), we assessed SCA1-associated changes in IO neuron structure and function. Concurrent with degeneration, we found that SCA1-KI IO neurons are hypertrophic, exhibiting early dendrite lengthening and later somatic expansion. Unlike in previous descriptions of HOD, we observed no clear loss of IO inhibitory innervation; nevertheless, patch-clamp recordings from brainstem slices reveal that SCA1-KI IO neurons are hyperexcitable. Rather than synaptic disinhibition, we identify increases in intrinsic membrane excitability as the more likely mechanism underlying this novel SCA1 phenotype. Specifically, transcriptome analysis indicates that SCA1-KI IO hyperexcitability is associated with a reduced medullary expression of ion channels responsible for spike afterhyperpolarization (AHP) in IO neurons - a result that has a functional consequence, as SCA1-KI IO neuron spikes exhibit a diminished AHP. These results reveal membrane excitability as a potential link between disparate causes of IO degeneration, suggesting that HOD can result from any cause, intrinsic or extrinsic, that increases excitability of the IO neuron membrane. Significance statementLittle is known about the factors that make inferior olive (IO) neurons susceptible to degeneration in the spinocerebellar ataxias (SCAs), a group of inherited neurodegenerative movement disorders. Another well-described form of IO degeneration, known as hypertrophic olivary degeneration (HOD), results from inhibitory denervation of the IO after brainstem injury. Here, we describe a novel finding of HOD in SCA1 without inhibitory denervation, in association with increased intrinsic membrane excitability and reduced potassium channel transcripts. This suggests that increased membrane excitability may be the underlying primary mechanism of HOD. Identifying hyperexcitability as the mechanistic driver of HOD would imply that reducing intrinsic IO excitability could be an effective strategy for treating diverse causes of both inherited and sporadic olivary degeneration.

neuroscience↗

bioRxiv2022

Neuronal activity regulates Matrin 3 levels and function in a calcium-dependent manner through calpain cleavage and calmodulin binding

RNA-binding protein (RBP) dysfunction is a fundamental hallmark of amyotrophic lateral sclerosis (ALS) and related neuromuscular disorders. Abnormal neuronal excitability is also a conserved feature in ALS patients and disease models, yet little is known about how activity-dependent processes regulate RBP levels and functions. Mutations in the gene encoding the RBP Matrin 3 (MATR3) cause familial disease, and MATR3 pathology has also been observed in sporadic ALS, suggesting a key role for MATR3 in disease pathogenesis. Here, we show that glutamatergic activity drives MATR3 degradation in a NMDAR-, Ca2+-, and calpain-dependent mechanism. The most common pathogenic MATR3 mutation renders it resistant to calpain degradation, suggesting a link between activity-dependent MATR3 regulation and disease. We also demonstrate that Ca2+ regulates MATR3 through a non-degradative process involving the binding of Ca2+/calmodulin (CaM) to MATR3 and inhibition of its RNA-binding ability. These findings indicate that neuronal activity impacts both the abundance and function of MATR3, and provide a foundation for further study of Ca2+-coupled regulation of RBPs implicated in ALS and related neurological diseases.

neuroscience↗

bioRxiv2022

Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies. SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.

neuroscience↗

bioRxiv2020

In vivo molecular signatures of cerebellar pathology in spinocerebellar ataxia type 3

BackgroundNo treatment exists for the most common dominantly inherited ataxia Machado-Joseph disease, or spinocerebellar ataxia type 3 (SCA3). Successful evaluation of candidate therapeutics will be facilitated by validated noninvasive biomarkers of aspects of disease pathology recapitulated by animal models. ObjectiveWe sought to identify shared neurochemical signatures in two mouse models of SCA3 that reflect aspects of the human disease pathology. MethodsCerebellar neurochemical concentrations in homozygous YACMJD84.2 (Q84/Q84) and hemizygous CMVMJD135 (Q135) mice were measured by magnetic resonance spectroscopy at 9.4 tesla. Motivated by the shared neurochemical abnormalities in the two models, we determined the levels of neurofilament medium (NFL, indicator of neuroaxonal integrity) and myelin basic protein (MBP, indicator of myelination) in cerebellar lysates from a subset of mice and from patients with SCA3. Finally, NFL and MBP levels were measured in cerebellar extracts of Q84/Q84 mice upon sustained silencing of the mutant ATXN3 gene from 6-8 weeks-of-age until death. ResultsBoth Q84/Q84 and Q135 mice displayed lower N-acetylaspartate than wild-type littermates, indicating neuroaxonal loss/dysfunction, and lower myo-inositol and total choline, indicating disturbances in phospholipid membrane metabolism and demyelination. Cerebellar NFL and MBP levels were accordingly lower in both models as well as in the cerebellar cortex of patients with SCA3 than controls. Furthermore, long-term sustained RNAi-mediated reduction of ATXN3 levels increased NFL and MBP in Q84/Q84 cerebella. ConclusionsN-acetylaspartate, myo-inositol and total choline levels in the cerebellum are candidate biomarkers of neuroaxonal and oligodendrocyte pathology in SCA3, which are reversible by reduction of mutant ATXN3 levels.

neuroscience↗

bioRxiv2019

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line.

Spinocerebellar ataxia type 3 (SCA3) is a fatal, late-onset neurodegenerative disorder characterized by selective neuropathology in the brainstem, cerebellum, spinal cord, and substantia nigra. Here, we characterize the first NIH-approved human embryonic stem cell (hESC) line derived from an embryo harboring the SCA3 mutation. Referred here as SCA3-hESC, this line is heterozygous for the mutant polyglutamine-encoding CAG repeat expansion in the ATXN3 gene within the pathogenic repeat range for SCA3. We observed relevant molecular hallmarks of the human disease at all differentiation stages from stem cells to cortical neurons, including robust ATXN3 aggregation and altered expression of key components of the protein quality control machinery. Finally, antisense oligonucleotide-mediated reduction of ATXN3 prevented the formation of p62-positive aggresomes in SCA3-hESCs. The SCA3-hESC line offers a unique and highly relevant human disease model that holds strong potential to advance understanding of SCA3 disease mechanisms and facilitate the evaluation of possible SCA3 therapies.\n\nHighlightsO_LIGenerated first NIH-approved SCA3 human embryonic stem cell line (SCA3-hESC)\nC_LIO_LISCA3-hESC exhibit robust ATXN3 aggregation pathology and form p62+ aggresomes\nC_LIO_LIAnti-ATXN3 antisense oligonucleotides rescue aggresome formation in SCA3-hESC\nC_LIO_LIDerived SCA3 neurons form aggregates and exhibit impaired protein quality control\nC_LI

neuroscience↗

bioRxiv2018

MTSS1/Src family kinase Dysregulation Underlies Multiple Inherited Ataxias

The genetically heterogeneous Spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of non-receptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-Metastasis (MTSS1) is a novel ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically-approved Src inhibitor corrects Purkinje basal firing, and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.\n\nSignificance StatementThe Src family of non-receptor tyrosine kinases (SFK) are essential for nervous system function, and may contribute to neurodegeneration. Spinocerebellar ataxias (SCAs) are neurodegenerative diseases where Purkinje neurons fire irregularly and degenerate leading to motor problems. We show that the SFK suppressor Missing-in-Metastasis (MTSS1) is a novel ataxia gene that links multiple SCAs. MTSS1 loss results in increased SFK activity, degenerating Purkinje neurons with low basal firing rates, and cell death. Surprisingly, mouse models for three different SCAs show elevated SFK activity, with SCA1 and SCA2 models displaying dramatically reduced MTSS1 protein levels. Treatment of each SCA model with SFK inhibitor corrects Purkinje basal firing, and delays ataxia progression in MTSS1 mutants. Our results identify a common link among disparate neurodegenerative diseases.

neuroscience↗