Biology subjects

Brown, R. H.

Publications and source records attributed to Brown, R. H..

8 recordsLinked to original sources

bioRxiv2025

Dynamic changes in chromosome and nuclear architecture during maturation of normal and ALS C9orf72 motor neurons

We have investigated changes in chromosome conformation, nuclear organization, and transcription during differentiation and maturation of control and mutant motor neurons harboring hexanucleotide expansions in the C9orf72 gene that cause amyotrophic lateral sclerosis (ALS). Using an in vitro reprogramming, differentiation and neural maturation protocol, we obtained highly purified populations of post-mitotic motor neurons for both normal and diseased cells. As expected, as fibroblasts are reprogrammed into iPSCs, and as iPSCs differentiate into motor neurons, chromatin accessibility, chromosome conformation, and nuclear organization change along with large-scale alterations in transcriptional profiles. We find that the transcriptome changes extensively during the first three weeks of post-mitotic neuronal maturation, with thousands of genes changing expression, but then is relatively stable for the next three weeks. In contrast, chromosome conformation and nuclear organization continue to change over the entire 6-week maturation period: chromosome territoriality increases, long-range interactions along chromosomes decrease, compartmentalization strength increases, and centromeres and telomeres increasingly cluster. In motor neurons derived from ALS patients such changes in chromosome conformation were much reduced. Chromatin accessibility changes also showed delayed maturation. The transcriptome in these cells matured relatively normally but with notable changes in expression of genes involved in lipid, sterol and mitochondrial function. We conclude that neural maturation is associated with large scale post-mitotic changes in gene expression, chromosome conformation and nuclear organization, and that these processes are defective in motor neurons derived from ALS patients carrying C9orf72 hexanucleotide repeat expansions.

neuroscience↗

bioRxiv2024

RNAi-mediated silencing of SOD1 profoundly extends survival and functional outcomes in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo. With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo. Administered intraventricularly, di-siRNASOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders. One sentence summarySilencing SOD1 with chemically optimized divalent siRNA profoundly extends the lifespan of G93A mice and prevents neurodegenerative biochemical and behavioral phenotypes.

neuroscience↗

bioRxiv2023

Up-regulation of cholesterol synthesis pathways and limited neurodegeneration in a knock-in Sod1 mutant mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene (SOD1) are associated with [~]20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. To generate a model that more closely represents patient gene expression, we created a knock-in point mutation (G85R, a human ALS-causing mutation) in the endogenous mouse Sod1 gene, leading to mutant SOD1G85R protein expression. Heterozygous Sod1G85R mutant mice resemble wild type, whereas homozygous mutants have reduced body weight and lifespan, a mild neurodegenerative phenotype, and express very low mutant SOD1 protein levels with no detectable SOD1 activity. Homozygous mutants exhibit partial neuromuscular junction denervation at 3-4 months of age. Spinal cord motor neuron transcriptome analyses of homozygous Sod1G85R mice revealed up-regulation of cholesterol synthesis pathway genes compared to wild type. Transcriptome and phenotypic features of these mice are similar to Sod1 knock-out mice, suggesting the Sod1G85R phenotype is largely driven by loss of SOD1 function. By contrast, cholesterol synthesis genes are down-regulated in severely affected human TgSOD1G93A transgenic mice at 4 months. Our analyses implicate dysregulation of cholesterol or related lipid pathway genes in ALS pathogenesis. The Sod1G85R knock-in mouse is a useful ALS model to examine the importance of SOD1 activity in control of cholesterol homeostasis and motor neuron survival. SIGNIFICANCE STATEMENTAmyotrophic lateral sclerosis is a devastating disease involving the progressive loss of motor neurons and motor function for which there is currently no cure. Understanding biological mechanisms leading to motor neuron death is critical for developing new treatments. Using a new knock-in mutant mouse model carrying a Sod1 mutation that causes ALS in patients, and in the mouse, causes a limited neurodegenerative phenotype similar to Sod1 loss-of-function, we show that cholesterol synthesis pathway genes are up-regulated in mutant motor neurons, whereas the same genes are down-regulated in transgenic SOD1 mice with a severe phenotype. Our data implicate dysregulation of cholesterol or other related lipid genes in ALS pathogenesis and provide new insights that could contribute to strategies for disease intervention.

neuroscience↗

bioRxiv2022

CRISPR/Cas9-Mediated Excision of ALS/FTD-Causing Hexanucleotide Repeat Expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro

A hexanucleotide repeat expansion (HRE) consisting of GGGGCC24+ in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are fatal neurodegenerative diseases with no current approved treatments that significantly slow disease progression or extend life expectancy. Several hypotheses have emerged to explain how this HRE causes neuronal death, including C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. In the present study we used a CRISPR/Cas9 gene-editing approach to remove the HRE from the C9ORF72 genomic locus, designing guide RNAs (gRNAs) flanking the HRE, and delivered Cas9 and gRNAs using adeno-associated virus serotype 9 (AAV9) vectors. Here, we demonstrate successful excision of the HRE in C9ORF72 in primary cortical neurons and brains of three mouse models containing the C9ORF72 expanded HRE (ranging from 500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, the major hallmarks of C9-ALS/FTD, making this an extremely attractive therapeutic approach to these diseases.

molecular biology↗

bioRxiv2021

Low-level overexpression of wild type TDP-43 causes late-onset, progressive neurodegeneration and paralysis in mice

Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed [~]30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.

genetics↗

bioRxiv2021

Non-Coding Genetic Analysis Implicates Interleukin 18 Receptor Accessory Protein 3'UTR in Amyotrophic Lateral Sclerosis

The non-coding genome is substantially larger than the protein-coding genome but is largely unexplored by genetic association studies. Here, we performed region-based burden analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole-genomes and 70,403 non-ALS controls. We identified Interleukin-18 Receptor Accessory Protein (IL18RAP) 3'UTR variants significantly enriched in non-ALS genomes, replicated in an independent cohort, and associated with a five-fold reduced risk of developing ALS. Variant IL18RAP 3'UTR reduces mRNA stability and the binding of RNA-binding proteins. Variant IL18RAP 3'UTR further dampens neurotoxicity of human iPSC-derived C9orf72-ALS microglia that depends on NF-{kappa}B signaling. Therefore, the variant IL18RAP 3'UTR provides survival advantage for motor neurons co-cultured with C9-ALS microglia. The study reveals direct genetic evidence and therapeutic targets for neuro-inflammation, and emphasizes the importance of non-coding genetic association studies. One Sentence SummaryNon-coding genetic variants in IL-18 receptor 3UTR decrease ALS risk by modifying IL-18-NF-{kappa}B signaling in microglia.

neuroscience↗

bioRxiv2021

Quantifying and Mitigating Motor Phenotypes Induced by Antisense Oligonucleotides in the Central Nervous System

Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into the cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. The effect of sugar and phosphate modifications on these phenotypes has not previously been systematically studied. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogues, PS-DNA induces the strongest motor phenotype while 2-substituted RNA modifications improve the tolerability of PS-ASOs. This helps explain why gapmer ASOs have been more challenging to develop clinically relative to steric blocker ASOs, which have a reduced tendency to induce these effects. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces their efficacy or duration of effect. Reducing PS content improved the acute tolerability of ASOs in both mice and sheep. We show that this acute toxicity is not mediated by the major nucleic acid sensing innate immune pathways. Formulating ASOs with calcium ions before injecting into the CNS further improved their tolerability, but through a mechanism at least partially distinct from the reduction of PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry approaches that improve tolerability of this class of compounds.

pharmacology and toxicology↗

bioRxiv2018

Regional collapsing of rare variation implicates specific genic regions in ALS

Large-scale sequencing efforts in amyotrophic lateral sclerosis (ALS) have implicated novel genes using gene-based collapsing methods. However, pathogenic mutations may be concentrated in specific genic regions. To address this, we developed two collapsing strategies, one focuses rare variation collapsing on homology-based protein domains as the unit for collapsing and another gene-level approach that, unlike standard methods, leverages existing evidence of purifying selection against missense variation on said domains. The application of these two collapsing methods to 3,093 ALS cases and 8,186 controls of European ancestry, and also 3,239 cases and 11,808 controls of diversified populations, pinpoints risk regions of ALS genes including SOD1, NEK1, TARDBP and FUS. While not clearly implicating novel ALS genes, the new analyses not only pinpoint risk regions in known genes but also highlight candidate genes as well.

genetics↗