Biology subjects

Pasinelli, P.

Publications and source records attributed to Pasinelli, P..

10 recordsLinked to original sources

bioRxiv2026

Engineered Chimeric Proteins Functionally Replace ALS-causing Endogenous FUS and TDP-43 to Improve Neuronal Health

Mislocalization and aggregation of the RNA-binding proteins (RBPs) FUS and TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), contributing to combined loss- and gain-of-function toxicity. Here, we present a protein engineering strategy that appends optimized anti-aggregation motifs to FUS and TDP-43, thereby enhancing their nuclear localization and solubility, and enabling functional replacement of disease-associated proteins. We show that these engineered chimeric RBPs reduce levels of aggregation-prone endogenous proteins through autoregulatory mechanisms. In addition, chimeric FUS and TDP-43 recapitulate key nuclear functions, including DNA repair and mRNA processing. Notably, chimeric FUS restores neurite outgrowth in diseased iPSC-derived neurons, while chimeric TDP-43 suppresses cryptic exon inclusion in patient-derived fibroblasts. Collectively, these results demonstrate that engineered chimeric RBPs can rebalance RBP homeostasis by simultaneously addressing protein aggregation and mislocalization, thus mitigating RBP-associated loss- and gain-of-function toxicity, highlighting a generalizable strategy for therapeutic protein replacement in neurodegenerative disease.

neuroscience↗

bioRxiv2026

A Human Neuronal Cell Model of Endogenous TDP-43 A315T Reveals Altered Protein Dynamics and Disease-Relevant Cellular Dysfunction

TAR DNA-binding protein 43 (TDP-43) aggregation is the defining pathological hallmark of nearly all cases of amyotrophic lateral sclerosis (ALS), yet physiologically relevant human models that faithfully recapitulate disease-associated TDP-43 proteinopathy and dysfunction remain limited. To cover this gap, we generated a novel human-based model of cortical neurons carrying the endogenous ALS-linked TDP-43 A315T mutation together with an in-frame Dendra2 fluorescent reporter, enabling temporal and spatial monitoring of the protein. Neurons expressing TDP-43 A315T exhibited progressive neurite degeneration, altered neuronal activity, and impaired mitochondrial respiration, recapitulating several ALS-associated phenotypes. Our model also displays autophagy-dependent accumulation of cytoplasmic aggregates of mutant TDP-43 without overt loss of nuclear function, maintaining normal processing of canonical cryptic exon targets. In contrast, experimental induction of TDP-43 nuclear exclusion readily triggered cryptic exon incorporation, demonstrating that the model faithfully reproduces loss-of-function phenotypes under stress conditions. In line with perturbed protein solubility, mutant TDP-43 neurons show increased stress granule (SG) formation at baseline and under oxidative stress condition. Finally, treatment with the RNA chaperone Clip34 significantly reduced TDP-43 aggregation under both basal and oxidative stress conditions as well as its localization to SGs. Taken together, these findings establish a physiologically relevant human model that separates early TDP-43 toxic gain-of-function from basal loss-of-function while providing a robust platform for investigating TDP-43 biology and accelerating therapeutic discovery in ALS.

neuroscience↗

bioRxiv2025

A CAR-T Cell-Based Strategy for Eliminating Pathogenic Microglia in ALS

Neurodegenerative diseases are defined by the propagation of neuroinflammation, driven in part by disease-associated microglia (DAM) that amplify inflammatory signaling and hasten neurodegeneration. Strategies to selectively eliminate DAM to attenuate disease progression remain elusive. Using existing datasets in combination with multiplexed immunofluorescence analysis of post-mortem ALS tissues, we identified the urokinase-type plasminogen activator receptor (uPAR) as a novel surface marker of DAM. uPAR protein is markedly elevated in IBA1/CD68 microglia within ALS-affected regions of both sporadic and familial cases, with negligible expression in unaffected areas or control tissues. These uPAR-high microglia drive neurite retraction in iPSC-derived neurons. To target these cells, we engineered 3rd-generation CAR-T cells expressing an anti-uPAR single-chain variable fragment, enabling specific recognition and elimination of uPAR-expressing microglia. Target specificity was assessed in human microglia C20 cells driven into a pathogenic state by poly(IC) or IFN{gamma} stimulation, which resulted in robust surface uPAR expression alongside phagocytic (CD68) and antigen-presenting (CD80) markers. uPAR-CAR-T cells induced antigen-dependent cytolysis of uPAR-high microglia, reducing their viability by over 80% while sparing resting microglia and neurons in a mixed culture system. These results position uPAR-directed CAR-T cells as a viable immunotherapeutic approach to selectively disrupt disease-amplifying microglial subsets and modify the trajectories of neuroinflammatory diseases. One Sentence SummaryCAR-T cells targeting uPAR selectively ablate pathogenic microglia while sparing neurons, enabling precision immunotherapy for ALS.

neuroscience↗

bioRxiv2025

Neuronal Activity-Dependent Gene Dysregulation in C9orf72 i3 Neuronal Models of ALS/FTD Pathogenesis

The GGGGCC nucleotide repeat expansion (NRE) mutation in the C9orf72 (C9) gene is the most common cause of ALS and FTD. Neuronal activity plays an essential role in shaping biological processes within both healthy and neurodegenerative disease scenarios. Here, we show that at baseline conditions, C9-NRE iPSC-cortical neurons display aberrations in several pathways, including synaptic signaling and transcriptional machinery, potentially priming diseased neurons for an altered response to neuronal stimulation. Indeed, exposure to two pathophysiologically relevant stimulation modes, prolonged membrane depolarization, or a blockade of K+ channels, followed by RNA sequencing, induces a temporally divergent activity-dependent transcriptome of C9-NRE cortical neurons compared to healthy controls. This study provides new insights into how neuronal activity influences the ALS/FTD-associated transcriptome, offering a dataset that enables further exploration of pathways necessary for conferring neuronal resilience or degeneration.

neuroscience↗

bioRxiv2024

Short RNA chaperones promote aggregation-resistant TDP-43 conformers to mitigate neurodegeneration

Aberrant aggregation of the prion-like, RNA-binding protein TDP-43 underlies several debilitating neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). Here, we define how short, specific RNAs antagonize TDP-43 aggregation. Short, specific RNAs engage and stabilize the TDP-43 RNA-recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. By mining sequence space, we uncover short RNAs with enhanced activity against TDP-43 and diverse disease-linked variants. The solubilizing activity of enhanced short RNA chaperones corrects aberrant TDP-43 phenotypes in optogenetic models and ALS patient-derived neurons. Remarkably, an enhanced short RNA chaperone mitigates TDP-43 proteinopathy and neurodegeneration in mice. Our studies reveal mechanisms of short RNA chaperones and pave the way for the development of short RNA therapeutics for fatal TDP-43 proteinopathies.

biochemistry↗

bioRxiv2023

Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice. We found that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also found that one of the arginine-rich DPRs (PR) can directly contribute to glucose metabolism and metabolic stress. These findings provide a mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and support a feedforward loop model that opens several opportunities for therapeutic intervention.

neuroscience↗

bioRxiv2023

EphrinB2 knockdown in spinal cord astrocytes preserves diaphragm innervation in a mutant SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

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

KapBeta2 is a modifier of the C9orf72-linked glycine-arginine dipeptide neurotoxicity

SummaryExpanded intronic G4C2 repeats in the C9orf72 gene cause several cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeats are translated through a non-AUG-dependent mechanism into five different dipeptides (DPRs), including poly-glycine-arginine (GR), which is aggregation-prone and eventually neurotoxic. Here, we report that Kap{beta}2 and GR interact, co-aggregating in primary neurons in-vitro and CNS tissue in-vivo. Importantly, this interaction improves the overall survival of neurons expressing GR. Downregulation of Kap {beta}2 is detrimental to the survival of neurons only if GR is expressed, whereas increased Kap {beta}2 levels mitigate GR-mediated neurotoxicity. notably, we did not find any changes in TDP-43 localization nor in the dynamic properties of the GR aggregates when Kap{beta}2 was over-expressed. These findings support the design of therapeutic strategies aimed at modulating Kap {beta}2 levels as a potential new avenue for contrasting neurodegeneration in C9orf72-ALS/FTD.

neuroscience↗

bioRxiv2021

Astrocytic expression of ALS-causative mutant FUS leads to TNFa-dependent neurodegeneration in vivo

Genetic mutations that cause Amyotrophic Lateral Sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein Fused in Sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-{kappa}B pathway and release of pro-inflammatory cytokine TNF. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that mtFUS expression causes TNF upregulation, motor function deficits, and spinal motor neuron loss. We further demonstrate a lack of phenotype in TNF knockout animals expressing mtFUS, and prevention of neurodegeneration in mtFUS-transduced animals through administration of TNF neutralizing antibodies. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS, establish that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo, and provide insights identifying FUS-ALS specific potential therapeutic targets.

neuroscience↗