Biology subjects

Pulst, S. M.

Publications and source records attributed to Pulst, S. M..

15 recordsLinked to original sources

bioRxiv2026

Accumulation of FAM98B and tRNA-Splicing Ligase Complex Proteins in Neuronal Intranuclear Inclusions in Spinocerebellar ataxia type 4.

Background and Objectives: Spinocerebellar ataxia type 4 (SCA4) is caused by a GGC-repeat expansion in ZFHX3 encoding an expanded polyglycine tract causing p62- and ubiquitin-positive neuronal intranuclear inclusions (NIIs). In multiple polyglycine diseases NIIs sequester tRNA-splicing ligase complex (tRNA-LC) proteins, causing downstream functional defects linked to neurodegeneration. We assessed the localization of tRNA-LC proteins in SCA4 pontine neurons. Methods: Postmortem brain tissue from a genetically confirmed SCA4 patient was examined across multiple regions using immunofluorescence confocal microscopy for p62, ubiquitin and tRNA-LC proteins FAM98B, RTRAF, and RTCB. Results: In pontine neurons, FAM98B, RTRAF, and RTCB were incorporated into NII, accumulating in a distinct outer layer respective to a ubiquitin inner layer. This redistribution was accompanied by depletion of all three proteins from the soluble nucleoplasm. Discussion: Our findings link SCA4 to a tRNA-LC dysfunction phenotype shared by multiple GGC-repeat disorders, further supporting a unified pathological mechanism in polyglycine diseases.

neuroscience↗

bioRxiv2024

Viral vector-mediated SLC9A6 gene replacement reduces cerebellar dysfunction in the shaker rat model of Christianson syndrome

BackgroundChristianson syndrome (CS) is an x-linked recessive neurodevelopmental and neurodegenerative condition characterized by severe intellectual disability, cerebellar degeneration, ataxia, and epilepsy. Mutations to the SLC9A6 gene encoding NHE6 are responsible for CS, and we recently demonstrated that a mutation to the rat Slc9a6 gene causes a similar phenotype in the spontaneous shaker rat model, which exhibits cerebellar degeneration with motor dysfunction. In previous work, we used the PhP.eB-L7-Slc9a6-GFP adeno-associated viral (AAV) vector to demonstrate that gene replacement in Purkinje cells reduced the shaker motor and molecular phenotype. MethodsWe carried out a 20-week longitudinal study evaluating the impact of Purkinje cell-specific gene replacement on ataxia and tremor. Taking advantage of the high homology between human SLC9A6 and rat Slc9a6, we tested a more clinically relevant construct, AAV9-CAG-hSLC9A6 AAV vector in the shaker rat. In both experimental cohorts, we performed molecular studies to evaluate expression of NHE6 and key cerebellar markers. We then characterized the relationship between molecular markers and motor function, as well between tremor and ataxia. ResultsAdministration of either of PhP.eB-L7-Slc9a6-GFP or AAV9-CAG-hSLC9A6 AAV vectors led to significant improvement in the molecular and motor phenotypes. The abundance of each disease-relevant cerebellar proteins was significantly correlated to motor ataxia. Further, we found that the relationship between cerebellar ataxia and tremor devolved over time, with disease modifying therapy disrupting their temporal relationship. ConclusionsThese findings impact future SLC9A6-targeted gene therapy efforts for CS and strongly support gene replacement as a viable therapeutic strategy. Furthermore, tremor and ataxia phenotypes may arise from dissociable cerebellar mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/621435v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@368165org.highwire.dtl.DTLVardef@691b54org.highwire.dtl.DTLVardef@816174org.highwire.dtl.DTLVardef@2508fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

A novel naive Bayes approach to identifying grooming behaviors in the force-plate actometric platform

BackgroundSelf-grooming behavior in rodents serves as a valuable model for investigating stereotyped and perseverative responses. Most current grooming analyses primarily rely on video observation, which lacks standardization, efficiency, and quantitative information about force. To address these limitations, we developed an automated paradigm to analyze grooming using a force-plate actometer. New MethodGrooming behavior is quantified by calculating ratios of relevant movement power spectral bands. These ratios are then input into a naive Bayes classifier, trained with manual video observations. To validate the effectiveness of this method, we applied it to the behavioral analysis of the early-life striatal cholinergic interneuron depletion (CIN-d) mouse, a model of tic pathophysiology recently developed in our laboratory, which exhibits prolonged grooming responses to acute stressors. Behavioral monitoring was simultaneously conducted on the force-place actometer and by video recording. ResultsThe naive Bayes approach achieved 93.7% accurate classification and an area under the receiver operating characteristic curve of 0.894. We confirmed that male CIN-d mice displayed significantly longer grooming durations compared to controls. However, this elevation was not correlated with increases in grooming force. Notably, haloperidol, a benchmark therapy for tic disorders, reduced both grooming force and duration. Comparison with Existing MethodsIn contrast to observation-based approaches, our method affords rapid, unbiased, and automated assessment of grooming duration, frequency, and force. ConclusionsOur novel approach enables fast and accurate automated detection of grooming behaviors. This method holds promise for high-throughput assessments of grooming stereotypies in animal models of tic disorders and other psychiatric conditions.

neuroscience↗

bioRxiv2023

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.

genomics↗

bioRxiv2022

Targeting Staufen 1 with antisense oligonucleotides for treating ALS and SCA2

Staufen1 (STAU1) is a multifunctional RNA binding protein that controls mRNA degradation and subcellular localization. STAU1 interacts with the ATXN2 protein, that is polyglutamine expanded in spinocerebellar ataxia type 2 (SCA2). We previously showed that STAU1 is elevated and aggregated in cells from SCA2 patients, cells from amyotrophic lateral sclerosis (ALS) patients, and in SCA2 and ALS mouse models. We also found that reduction of STAU1 abundance in vivo by genetic interaction improved motor behavior in an SCA2 mouse model, normalized the levels of several SCA2-related proteins, and reduced aggregation of polyglutamine-expanded ATXN2. Here we developed antisense oligonucleotides (ASOs) lowering STAU1 expression toward developing a therapeutic that may be effective for treating SCA2 and ALS. We performed a screen of 118 20mer phosphorothioate 2-O-methoxyethyl (MOE) ASO gapmers targeting across the STAU1 mRNA coding region for lowering STAU1 expression in HEK-293 cells. ASO hits lowering STAU1 by >45 % were rescreened in SCA2 patient fibroblasts, and 10 of these were tested for lowering STAU1 abundance in vivo in a new BAC-STAU1 mouse model. This identified efficacious ASOs targeting human STAU1 in vivo that normalized autophagy marker proteins, including ASO-45 that also targets mouse Stau1. When delivered by intracerebroventricular (ICV) injection, ASO-45 normalized autophagy markers and abnormal mRNA abundances in cerebella of ATXN2-Q127 SCA2 mice, as well as ChAT, NeuN and cleaved caspase-3 in spinal cord of Thy1- TDP-43 transgenic mice. Targeting STAU1 may be an effective strategy for treating ALS and SCA2 as well as other disorders characterized by its overabundance.

neuroscience↗

bioRxiv2022

Slc9a6 mutation causes Purkinje cell loss and ataxia in the spontaneous shaker rat

BackgroundThe shaker rat carries a naturally occurring mutation leading to progressive ataxia characterized by Purkinje cell (PC) loss. We previously reported on fine-mapping the shaker locus to the long arm of the rat X chromosome. In this work, we sought to identify the mutated gene underlying the shaker phenotype and confirm its identity by functional complementation. MethodsWe fine-mapped the candidate region and analyzed cerebellar transcriptomes to identify deleterious variants. We generated an adeno-associated virus (AAV) targeting solute carrier family 9, member A6 (Slc9a6) expression to PCs using a mouse L7-6 (L7) promoter, as well as a control green fluorescent protein (GFP)-expressing virus. We administered AAVs prior to the onset of PC degeneration through intracerebroventricular injection and evaluated the molecular, cellular, and motor phenotypes. ResultsWe identified a XM_217630.9 (Slc9a6):c.[191_195delinsA] variant in the Slc9a6 gene that segregated with disease. This mutation is predicted to generate a truncated sodium-hydrogen exchanger 6 (NHE6) protein, p.(Ala64Glufs*23). Administration of AAV9-PHP.eB expressing rat Slc9a6 prior to symptom onset reduced the shaker motor, molecular, and cellular phenotypes. InterpretationSlc9a6 is mutated in shaker and also in human Christianson syndrome, an epileptic encephalopathy. AAV-based gene therapy may be a viable therapeutic strategy for Christianson syndrome, and the shaker rat model may aid in therapeutic development.

neuroscience↗

bioRxiv2021

MFN2 Influences Amyotrophic Lateral Sclerosis Pathology

ObjectiveTo better understand the pathology of amyotrophic lateral sclerosis, we used sequence data from patients seen at the University of Utah to identify novel disease-associated loci. We utilized both in vitro and in vivo studies to determine the biological effect of patient mutations in MFN2. MethodSequence data for a total of 140 patients were run through VAAST and Phevor to determine genes that were more burdened with rare, nonsynonymous variants compared to control longevity cohort. Variants identified in MFN2 were expressed in Mfn2 knockout cells to determine if mutant MFN2 could rescue mitochondrial morphology defects. We identified additional rare, nonsynonymous variants in MFN2 in ALSdb that were expressed in knockout mouse embryonic fibroblasts (MEFs). Membrane potential was measured to quantify mitochondrial health upon mutant MFN2 expression. mfn2 knockout zebrafish were used to examine movement compared to wildtype and protein aggregation in brain. ResultsMFN2 mutations identified in ALS patients from our University of Utah cohort and ALSdb were defective in rescuing morphological defects in Mfn2 knockout MEFs. Selected mutants showed decreased membrane potential compared to wildtype MFN2 expression. Zebrafish heterozygous and homozygous for loss of mfn2 showed increased TDP-43 levels in their hindbrain and cerebellum. ConclusionIn total, 21 rare, deleterious mutations in MFN2 were tested in Mfn2 knockout MEFs. Mutant MFN2 expression was not able to rescue the knockout phenotype, though at differing degrees of severity. Decreased membrane potential also argues for inhibited mitochondrial function. Increased TDP-43 levels in mutant zebrafish illustrates MFN2s function in ALS pathology. MFN2 variants influence ALS pathology and highlight the importance of mitochondria in neurodegeneration.

genetics↗

bioRxiv2021

RNA toxicity and perturbation of rRNA processing in spinocerebellar ataxia type 2

BACKGROUNDSpinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVEHere we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODSThe toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if rRNA processing is disrupted in SCA2 and Huntington disease (HD) human brain tissue. RESULTSexpATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin {beta}-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSIONThese findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications.

neuroscience↗

bioRxiv2020

Defining the impact of deep brain stimulation contact size and shape on neural selectivity

BackgroundUnderstanding neural selectivity is essential for optimizing medical applications of deep brain stimulation (DBS). We previously showed that modulation of the DBS waveform can induce changes in orientation-based selectivity, and that lengthening of DBS pulses or directional segmentation can reduce preferential selectivity for large axons. In this work, we sought to answer a simple, but important question: how do the size and shape of the contact influence neural selectivity? MethodsWe created multicompartment neuron models for several axon diameters and used finite element modeling with standard-sized cylindrical leads to determine the effects on changing contact size and shape on axon activation profiles and volumes of tissue activated. Contacts ranged in size from 0.04 to 16 mm2, compared with a standard size of 6 mm2. ResultsWe found that changes in contact size induce substantial changes in orientation-based selectivity in the context of a cylindrical lead, and rectangular shaping of the contact can alter this selectivity. Smaller contact sizes were more effective in constraining neural activation to small, nearby axons representative of grey matter. However, micro-scale contacts enable only limited spread of neural activation before exceeding standard charge density limitations; further, energetic efficiency is optimized by somewhat larger contacts. InterpretationsSmall-scale contacts are optimal for constraining stimulation in nearby grey matter and avoiding orientation-selective activation. However, given charge density limitations and energy inefficiency of micro-scale contacts, our results suggest that contacts about half the size of those on segmented clinical leads may optimize efficiency and charge density limitation avoidance.

neuroscience↗

bioRxiv2020

Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in Spinocerebellar ataxia type 1

Selective neuronal vulnerability in neurodegenerative disease is poorly understood. Using the ATXN1[82Q] model of spinocerebellar ataxia type 1 (SCA1), we explored the hypothesis that regional differences in Purkinje neuron degeneration could provide novel insights into selective vulnerability. ATXN1[82Q] Purkinje neurons from the anterior cerebellum were found to degenerate earlier than those from the nodular zone, and this early degeneration was associated with selective dysregulation of ion channel transcripts and altered Purkinje neuron spiking. Efforts to understand the basis for selective dysregulation of channel transcripts revealed modestly increased expression of the ATXN1 corepressor Capicua (Cic) in anterior cerebellar Purkinje neurons. Importantly, lentiviral overexpression of Cic in the nodular zone accelerated both aberrant Purkinje neuron spiking and neurodegeneration. These findings reinforce the central role for Cic in SCA1 cerebellar pathophysiology and suggest that only modest reductions in Cic are needed to have profound therapeutic impact in SCA1.

neuroscience↗

bioRxiv2019

Staufen 1 amplifies pro-apoptotic activation of the unfolded protein response.

Staufen-1 (STAU1) is an RNA binding protein that becomes highly overabundant in numerous neurodegenerative disease models, including those carrying mutations in presenilin1 (PSEN1), microtubule associated protein tau (MAPT), huntingtin (HTT), TAR DNA-binding protein-43 gene (TARDBP) or C9orf72. We previously reported that elevations in STAU1 determine autophagy defects. Additional functional consequences of STAU1 overabundance, however, have not been investigated. We studied the role of STAU1 in the chronic activation of the Unfolded Protein Response (UPR), a common feature among the neurodegenerative diseases where STAU1 is increased, and is directly associated with neuronal death. Here we report that STAU1 is a novel modulator of the UPR, and is required for apoptosis induced by activation of the PERK-CHOP pathway. STAU1 levels increased in response to multiple ER stressors and exogenous expression of STAU1 was sufficient to cause apoptosis through the PERK-CHOP pathway of the UPR. Cortical neurons and skin fibroblasts derived from Stau1-/- mice showed reduced UPR and apoptosis when challenged with thapsigargin. In fibroblasts from SCA2 patients or with ALS-causing TDP-43 and C9ORF72 mutations we found highly increased STAU1 and CHOP levels in basal conditions. STAU1 knockdown restored CHOP levels to normal. Taken together, these results show STAU1 overabundance reduces cellular resistance to ER stress and precipitates apoptosis.

neuroscience↗

bioRxiv2019

Staufen blocks autophagy in neurodegeneration

ObjectiveThe mechanistic target of rapamycin (mTOR) kinase is one of the master coordinators of cellular stress responses, regulating metabolism, autophagy, and apoptosis. We recently reported that Staufen1 (STAU1), a stress granule (SG) protein, was overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis, frontotemporal degeneration, Huntingtons, Alzheimers, and Parkinsons diseases as well as animal models, and patient tissues. STAU1 overabundance is associated with mTOR hyperactivation and links SG formation with autophagy. Our objective was to determine the mechanism of mTOR regulation by STAU1. MethodsWe determined STAU1 abundance with disease- and chemical-induced cellular stressors in patient cells and animal models. We also used RNA binding assays to contextualize STAU1 interaction with MTOR mRNA. ResultsSTAU1 and mTOR were overabundant in BAC-C9orf72, ATXN2Q127, and Thy1-TDP-43 transgenic mouse models. Reducing STAU1 levels in these mice normalized mTOR levels and activity and autophagy-related marker proteins. We also saw increased STAU1 levels in HEK293 cells transfected to express C9orf72-relevant dipeptide repeats (DPRs). Conversely, DPR accumulations were not observed in cells treated by STAU1 RNAi. Overexpression of STAU1 in HEK293 cells increased mTOR levels through direct MTOR mRNA interaction, activating downstream targets and impairing autophagic flux. Targeting mTOR by rapamycin or RNAi normalized STAU1 abundance in a SCA2 cellular model. InterpretationSTAU1 interaction with mTOR drives its hyperactivation and inhibits autophagic flux in multiple models of neurodegeneration. Staufen, therefore, constitutes a novel target to modulate mTOR activity, autophagy, and for the treatment of neurodegenerative diseases.

neuroscience↗

bioRxiv2019

Neural Selectivity, Efficiency, and Dose Equivalence in Deep Brain Stimulation through Pulse Width Tuning and Segmented Electrodes.

BackgroundAchieving deep brain stimulation (DBS) dose equivalence is challenging, especially with pulse width tuning and directional contacts. Further, the precise effects of pulse width tuning are unknown.\n\nMethodsWe created multicompartment neuron models for two axon diameters and used finite element modeling to determine extracellular influence from standard and segmented electrodes. We analyzed axon activation profiles and calculated volumes of tissue activated.\n\nResultsLong pulse widths focus the stimulation effect on small, nearby fibers, suppressing white matter tract activation (responsible for some DBS side effects) and improving battery utilization. Directional leads enable similar benefits to a greater degree. We derive equations for equivalent activation with pulse width tuning and segmented contacts.\n\nInterpretationsWe find agreement with classic studies and reinterpret recent articles concluding that short pulse widths focus the stimulation effect on small, nearby fibers, decrease side effects, and improve power consumption. Our field should reconsider shortened pulse widths.

neuroscience↗

bioRxiv2018

Loss of TP73 function contributes to amyotrophic lateral sclerosis pathogenesis

Much remains unknown about the genetics and pathophysiology underlying the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We analyzed exome sequences from a cohort of 87 sporadic ALS (SALS) patients and 324 healthy individuals. TP73, a homolog of the TP53 tumor suppressor gene, had five rare deleterious protein-coding variants; in a separate collection of >2,900 ALS patients we identified an additional 19 rare deleterious variants in TP73. An in vitro C2C12 myoblast growth assay confirmed that these variants impair or alter TP73 function. In vivo mutagenesis of zebrafish tp73 using CRISPR led to impaired motor neuron development and abnormal axonal morphology, concordant with ALS pathology. Together, these results demonstrate that TP73 is a risk factor for ALS, and identifies a novel dysfunctional cellular process in the pathogenesis of ALS.

genetics↗

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↗