Biology subjects

Paulson, H. L.

Publications and source records attributed to Paulson, H. L..

12 recordsLinked to original sources

bioRxiv2026

Ficd loss rescues motor impairments and reverses oligodendrocyte maturation deficits in a mouse model of spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is an inherited, fatal neurodegenerative disease caused by a pathological CAG repeat expansion in the ATXN3 gene, resulting in the selective degeneration of vulnerable neuronal populations. Recent work has identified impairments in oligodendrocyte maturation as a novel and robust feature of SCA3 pathogenesis. Oligodendrocytes synthesize myelin structural components through the endoplasmic reticulum (ER), rendering this organelle essential for white matter integrity. Despite this, the role of ER function in SCA3 remains unclear. In this study, we show that loss of FICD-mediated AMPylation, a post-translational modification regulating the ER-resident HSP70 chaperone, BiP, rescues motor impairments in a transgenic SCA3 mouse model. Ficd-/- SCA3 mice exhibit significantly reduced levels of nuclear ATXN3 in vulnerable brain regions, while Ficd+/+ littermates show an increased burden of AMPylated BiP in the spinal cord, identifying aberrant AMPylation as a novel contributor of SCA3 pathology. Using unbiased proteomics, we demonstrate that Ficd deletion mitigates the pathological decrease in myelin structural proteins and oligodendrocyte maturation factors, restoring levels of mature, myelinating oligodendrocytes. In parallel, we show that Ficd activates SREBP2-dependent cholesterol biosynthesis to support myelination. Taken as a whole, these findings posit ER homeostasis as a critical driver of oligodendrocyte pathology and identify FICD as a novel target for alleviating non-neuronal toxicity in SCA3.

molecular biology↗

bioRxiv2025

Repeat associated non-AUG translation as a common mechanism for the polyGln ataxias

Determining if repeat associated non-AUG (RAN) proteins contribute to the CAG polyGln-encoding spinocerebellar ataxias (CAG-SCAs) is critical for understanding mechanisms and developing therapies for these diseases. Immunohistochemistry using antibodies against polySer and polyLeu repeats and locus specific C-terminal regions show sense polySer (AGC frame) and antisense polyLeu (CUG frame) RAN proteins accumulate in affected grey and white-matter brain regions, throughout the cerebellum and pons, in SCA1, SCA2, SCA3, SCA6, and SCA7 autopsy brains. Cerebellar white matter regions with prominent polySer and polyLeu but minimal polyGln aggregates show demyelination, white matter loss, and activated microglia. In SCA3 mice, RAN proteins accumulate in an age-dependent manner. In neural cells, polySer and polyLeu RAN proteins are toxic and cause autophagic dysfunction. In cells, the FDA-approved drug metformin decreases RAN protein levels and reduces toxicity. Taken together, these data identify sense and antisense RAN proteins as a common molecular mechanism shared by the CAG-SCAs.

neuroscience↗

bioRxiv2024

Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

The human genome is replete with sequences derived from foreign elements including endogenous retrovirus-like proteins of unknown function. Here we show that UBQLN2, a ubiquitin-proteasome shuttle factor implicated in neurodegenerative diseases, is regulated by the linked actions of two retrovirus-like proteins, RTL8 and PEG10. RTL8 confers on UBQLN2 the ability to complex with and regulate PEG10. PEG10, a core component of stress granules, drives the recruitment of UBQLN2 to stress granules under various stress conditions, but can only do so when RTL8 is present. Changes in PEG10 levels further remodel the kinetics of stress granule disassembly and overall composition by incorporating select extracellular vesicle proteins. Within stress granules, PEG10 forms virus-like particles, underscoring the structural heterogeneity of this class of biomolecular condensates. Together, these results reveal an unexpected link between pathways of cellular proteostasis and endogenous retrovirus-like proteins.

neuroscience↗

bioRxiv2023

Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils

Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and -synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and -synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

bioengineering↗

bioRxiv2023

Quantitative flow cytometric selection of tau conformational nanobodies specific for pathological aggregates

1.Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimers and Parkinsons diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimers disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both transgenic tau mouse models and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.

bioengineering↗

bioRxiv2023

Regional and age-dependent changes in ubiquitination in cellular and mouse models of Spinocerebellar ataxia type 3

1Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the most common dominantly inherited ataxia. SCA3 is caused by a CAG repeat expansion in the ATXN3 gene that encodes an expanded tract of polyglutamine (polyQ) in the disease protein ataxin-3 (ATXN3). As a deubiquitinating enzyme, ATXN3 regulates numerous cellular processes including proteasome- and autophagy-mediated protein degradation. In SCA3 disease brain, polyQ-expanded ATXN3 accumulates with other cellular constituents, including ubiquitin (Ub)-modified proteins, in select areas like the cerebellum and the brainstem, but whether pathogenic ATXN3 affects the abundance of ubiquitinated species is unknown. Here, in mouse and cellular models of SCA3, we investigated whether elimination of murine Atxn3 or expression of wild-type or polyQ-expanded human ATXN3 alters soluble levels of overall ubiquitination, as well as K48-linked (K48-Ub) and K63-linked (K63-Ub) chains. Levels of ubiquitination were assessed in the cerebellum and brainstem of 7- and 47-week-old Atxn3 knockout and SCA3 transgenic mice, and also in relevant mouse and human cell lines. In older mice, we observed that wild-type ATXN3 impacts the cerebellar levels of K48-Ub proteins. In contrast, pathogenic ATXN3 leads to decreased brainstem abundance of K48-Ub species in younger mice and changes in both cerebellar and brainstem K63-Ub levels in an age-dependent manner: younger SCA3 mice have higher levels of K63-Ub while older mice have lower levels of K63-Ub compared to controls. Human SCA3 neuronal progenitor cells also show a relative increase in K63-Ub proteins upon autophagy inhibition. We conclude that wild-type and mutant ATXN3 differentially impact K48-Ub- and K63-Ub-modified proteins in the brain in a region- and age-dependent manner.

neuroscience↗

bioRxiv2022

Sleep alterations in a mouse model of Spinocerebellar ataxia type 3

BackgroundSpinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder showing progressive neuronal loss in several brain areas and a broad spectrum of motor and non-motor symptoms, including ataxia and altered sleep. While sleep disturbances are known to play pathophysiologic roles in other neurodegenerative disorders, their impact on SCA3 is unknown. ObjectivesUsing state-of-the art spectrographic measurements, we sought to quantitatively characterize sleep electroencephalography (EEG) in a SCA3 transgenic mouse model with confirmed disease phenotype. MethodsWe first measured motor phenotypes in 18-31-week-old homozygous and hemizygous SCA3 YACMJD84.2 mice and non-transgenic wild-type littermate mice during lights-on and lights-off periods. We next implanted electrodes to obtain 12-hour (zeitgeber time 0-12) EEG recordings for three consecutive days when the mice were 26-36 weeks old. We then analyzed EEG-based sleep structure data to quantify differences between homozygous, hemizygous, and wild-type mice. ResultsCompared to wild-type littermates, SCA3 homozygous mice display: i) increased duration of rapid-eye movement sleep (REM) and fragmentation in all sleep and wake states; ii) higher beta power oscillations during REM and non-REM (NREM); and iii) additional spectral power band alterations during REM and wake. ConclusionsOur data show that sleep architecture and EEG spectral power are dysregulated in homozygous SCA3 mice, indicating that common sleep-related etiologic factors may underlie mouse and human SCA3 phenotypes.

neuroscience↗

bioRxiv2021

GRASP55 regulates mutant huntingtin unconventional secretion and aggregation

Recent studies demonstrated that the Golgi stacking proteins, GRASPs, especially GRASP55, regulate Golgi-independent unconventional secretion, but the underlying mechanism remains unknown. Here, we used mutant huntingtin (Htt-Q74) as a model system to address this question. Our results demonstrate that Htt secretion is GRASP55- and autophagy-dependent, and is enhanced under stress conditions such as starvation and ER stress. Mechanistically, GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the ERGIC lumen. Moreover, GRASP55 level is upregulated by various stresses to facilitate unconventional secretion, while inhibition of Htt-Q74 secretion by GRASP55 knockout enhances Htt aggregation and toxicity. Lastly, comprehensive secretomic analysis identified novel cargoes secreted by the same unconventional pathway, such as TAGLN, PAICS and PRDX1. This study provides important information on the role of GRASP55 in unconventional protein secretion and Huntingtons disease progression.

cell biology↗

bioRxiv2020

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. We report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by the brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and levels of aggregation. This variability among ubiquilins along the continuum from liquid-like to solid likely informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in various neurodegenerative diseases.

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

Druggable genome screen identifies new regulators of the abundance and toxicity of ATXN3, the Spinocerebellar Ataxia Type 3 disease protein

BackgroundSpinocerebellar Ataxia type 3 (SCA3, also known as Machado-Joseph disease) is a neurodegenerative disorder caused by a CAG repeat expansion encoding an abnormally long polyglutamine (polyQ) tract in the disease protein, ataxin-3 (ATXN3). No preventive treatment is yet available for SCA3. Because SCA3 is likely caused by a toxic gain of ATXN3 function, a rational therapeutic strategy is to reduce mutant ATXN3 levels by targeting pathways that control its production or stability. Here, we sought to identify genes that modulate ATXN3 levels as potential therapeutic targets in this fatal disorder.\n\nMethodsWe screened a collection of siRNAs targeting 2742 druggable human genes using a cell-based assay based on luminescence readout of polyQ-expanded ATXN3. From 317 candidate genes identified in the primary screen, 100 genes were selected for validation. Among the 33 genes confirmed in secondary assays, 15 were validated in an independent cell model as modulators of pathogenic ATXN3 protein levels. Ten of these genes were then assessed in a Drosophila model of SCA3, and one was confirmed as a key modulator of physiological ATXN3 abundance in SCA3 neuronal progenitor cells.\n\nResultsAmong the 15 genes shown to modulate ATXN3 in mammalian cells, orthologs of CHD4, FBXL3, HR and MC3R regulate mutant ATXN3-mediated toxicity in fly eyes. Further mechanistic studies of one of these genes, FBXL3, encoding a F-box protein that is a component of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex, showed that it reduces levels of normal and pathogenic ATXN3 in SCA3 neuronal progenitor cells, primarily via a SCF complex-dependent manner. Bioinformatic analysis of the 15 genes revealed a potential molecular network with connections to tumor necrosis factor-/nuclear factor-kappa B (TNF/NF-kB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways.\n\nConclusionsWe identified 15 druggable genes with diverse functions to be suppressors or enhancers of pathogenic ATXN3 abundance. Among identified pathways highlighted by this screen, the FBXL3/SCF axis represents a novel molecular pathway that regulates physiological levels of ATXN3 protein.

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↗