Biology subjects

Pandey, U. B.

Publications and source records attributed to Pandey, U. B..

10 recordsLinked to original sources

bioRxiv2026

LAMA2-related muscular dystrophy alters Schwann cell calcium homeostasis and increases peripheral nerve inflammation

LAMA2-related muscular dystrophy (LAMA2-RD) affects skeletal muscle and peripheral nerves, yet the consequences of LAMA2 deficiency on peripheral nerve integrity beyond early postnatal development remain incompletely understood. We investigated sciatic nerve architecture and molecular alterations across postnatal life in hypomorphic Lama2dy2J/dy2J mice. LAMA2 localizes around the abaxonal surface of myelinating Schwann cells in control nerves. LAMA2-deficient nerves exhibited patchy regions lacking myelinated axons from early postnatal stages into adulthood, including expanded Remak bundles along with axons completely lacking normal Schwann cell ensheathment. In adult nerves, myelin infolding and reduced myelin circularity revealed abnormalities in the architecture of myelin that had formed. Bulk sciatic nerve RNA sequencing identified altered calcium-signaling and immune-related transcriptional signatures. Complementary Fura Red measurements in primary nerve-derived Schwann cell cultures demonstrated decreased intracellular calcium in LAMA2-deficient samples, while ATP-evoked responses were preserved. Older LAMA2-deficient nerves also exhibited increased CD45+CD11b+ myeloid-cell infiltration. Together, these observations extend the characterization of LAMA2-associated neuropathy into adulthood and identify calcium homeostasis and neuroimmune responses as candidate processes relevant to persistent peripheral nerve pathology.

neuroscience↗

bioRxiv2026

Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity

The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of {beta}-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce {beta}-sheet breaking proline residues into FUS LC with the goal of preventing cross-{beta}-driven aggregation without disrupting essential functions and phase separation. {beta}-sheet-deficient FUS variants maintain native-like global motions, disorder, and phase separation, but no longer show a liquid-to-solid transition (LST). Biochemical partitioning, cellular localization, and auto- and cross-regulatory functions of FUS all remain essentially unchanged. Conversely, FUS-induced neurodegeneration in several Drosophila models is drastically reduced. These findings suggest a strategy for mitigating disease-related toxicity through backbone structure modulation to prevent prion-like domain protein aggregation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/706410v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@d15f63org.highwire.dtl.DTLVardef@1cd6221org.highwire.dtl.DTLVardef@e58126org.highwire.dtl.DTLVardef@181ec67_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYThe RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation as part of its physiological function but can aberrantly aggregate into solid-like assemblies in amyotrophic lateral sclerosis and frontotemporal dementia. To dissect the role of {beta}-sheets in both function and pathological transition, we engineered {beta}-sheet-preventing FUS variants via targeted proline residue insertions in the prion-like disordered region. These variants retained native structure, motions, and phase behavior yet showed dramatically reduced aggregation, both as an isolated prion-like domain and in full-length FUS. Crucially, these variants maintained a panel of FUS cellular functions that depend on FUS condensation but prevented FUS toxicity in fly models of neurodegeneration. Our findings implicate {beta}-sheets as key drivers of FUS condensate maturation and neuronal toxicity, highlighting {beta}-sheet modulation as a therapeutic strategy against FUS-related neurodegeneration. HIGHLIGHTSO_LITargeted proline additions disrupt {beta}-sheet formation in FUS without altering native conformations, dynamics, or phase separation behavior C_LIO_LI{beta}-sheet-deficient FUS variants prevent aggregation and liquid-to-solid transitions while retaining key biological functions C_LIO_LIIn vivo models reveal attenuated toxicity of {beta}-sheet-deficient FUS in Drosophila C_LIO_LI{beta}-sheets are identified as central drivers of condensate maturation and neuronal death, offering a therapeutic entry point for modulating prion-like domain pathology C_LI

biochemistry↗

bioRxiv2023

Differential effects of MATR3 variants on its cryptic splicing repression function

MATR3 is an RNA-binding protein implicated in neurodegenerative and neurodevelopmental diseases. However, little is known regarding the role of MATR3 in cryptic splicing within the context of functional genes and how disease-associated variants impact this function. We show that loss of MATR3 leads to cryptic exon inclusion in many transcripts. We reveal that ALS-linked S85C pathogenic variant reduces MATR3 solubility but does not impair RNA binding. In parallel, we report a novel neurodevelopmental disease-associated M548T variant, located in the RRM2 domain, which reduces protein solubility and impairs RNA binding and cryptic splicing repression functions of MATR3. Altogether, our research identifies cryptic events within functional genes and demonstrates how disease-associated variants impact MATR3 cryptic splicing repression function.

molecular biology↗

bioRxiv2023

HuD (ELAVL4) gain-of-function impairs neuromuscular junctions and induces apoptosis in familial and sporadic amyotrophic lateral sclerosis models

Early defects at the neuromuscular junction (NMJ) are among the first hallmarks of the progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS). According to the "dying back" hypothesis, disruption of the NMJ not only precedes, but is also a trigger for the subsequent degeneration of the motoneuron in both sporadic and familial ALS, including ALS caused by the severe FUS pathogenic variant P525L. However, the mechanisms linking genetic and environmental factors to NMJ defects remain elusive. By taking advantage of co-cultures of motoneurons and skeletal muscle derived from human induced pluripotent stem cells (iPSCs), we show that the neural RNA binding protein HuD (ELAVL4) may underlie NMJ defects and apoptosis in FUS-ALS. HuD overexpression in motoneurons phenocopies the severe FUSP525L mutation, while HuD knockdown in FUSP525L co-cultures produces phenotypic rescue. We validated these findings in vivo in a Drosophila FUS-ALS model. Neuronal-restricted overexpression of the HuD-related gene, elav, produces per se a motor phenotype, while neuronal-restricted elav knockdown significantly rescues motor dysfunction caused by FUS. Finally, we show that HuD levels increase upon oxidative stress in human motoneurons and in sporadic ALS patients with an oxidative stress signature. On these bases, we propose HuD as an important player downstream of FUS mutation in familial ALS, with potential implications for sporadic ALS related to oxidative stress.

neuroscience↗

bioRxiv2023

C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is translated into dipeptide repeat proteins, among which poly-proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo. PR partitions to the nucleus when expressed in neurons and other cell types. Using drosophila and primary rat cortical neurons as model systems, we show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR accumulates in the nucleolus, a site of ribosome biogenesis that regulates the cell stress response. We examined the effect of nucleolar PR accumulation and its impact on nucleolar function and determined that PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels, either genetically or by increasing its degradation, also prevented PR-mediated neurotoxic phenotypes both in in-vitro and in-vivo models. We also investigated whether PR could cause the senescence phenotype in neurons but observed none. Instead, we found induction of apoptosis via caspase-3 activation. In summary, we uncovered the central role of nucleolar dysfunction upon PR expression in the context of C9-ALS/FTD.

neuroscience↗

bioRxiv2022

CLIP-Seq Analysis Enables the Design of Ribosomal RNA Bait Oligonucleotides That Protect Against C9orf72 ALS/FTD-Associated Poly-GR Pathophysiology

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind to low complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remains unclear. We used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits in cells and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal disfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

neuroscience↗

bioRxiv2022

Axon guidance pathways modulate neurotoxicity of ALS-associated UBQLN2

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

neuroscience↗

bioRxiv2022

Functional and structural deficiencies of Gemin5 variants associated with neurological disease

Dysfunction of RNA-binding proteins are often linked to a wide range of human disease in general, and particularly with neurological conditions. Gemin5 is a member of the survival of motor neuron (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 protein have been reported but the functional consequences of these variants remain elusive. Here we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the non-canonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization while the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, abolishing the cap-dependent and selective translation control of Gemin5, and establishing a functional difference with the wild type protein. Our study provides a molecular basis of disease associated with malfunction of Gemin5 protein.

molecular biology↗

bioRxiv2022

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

cell biology↗

bioRxiv2019

Nucleocytoplasmic Proteomic Analysis Uncovers ETF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by the C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift towards a more cytosolic proteome in C9-HRE cells. Amongst these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient iPSC-neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD-driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS/FTD.

neuroscience↗