Biology subjects

Kalb, R. G.

Publications and source records attributed to Kalb, R. G..

4 recordsLinked to original sources

bioRxiv2025

The molecular mechanism of uptake and cell-to-cell transmission of arginine-containing dipeptide repeat proteins.

Micro-satellite repeat expansion of the 5 GGGGCC 3 sequence in the C9orf72 gene is the most common monogenic form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) translated from the mutant allele can be detected in postmortem brains of afflicted individuals. The arginine containing peptides, poly-PR and poly-GR, are particularly noxious to cells. Both have been shown to undergo cell-cell transmission, but the underlying mechanisms are not understood. We found rapid internalization and nucleolar localization of bath-applied hemagglutinin (HA) tagged poly-PR with twenty repeats (HA-PR20) in cell lines and neurons. Small molecule and RNAi approaches implicated a temperature-dependent, fluid phase endocytosis mechanism in HA-PR20 uptake. We sought to identify DPR-related cell surface uptake factors using a high-resolution proximity labeling technique developed in the MacMillan group, termed {micro}Map. DPR-iridium conjugates identified candidate cell-surface proteins which were interrogated in an RNAi screen. Focusing on our strongest candidate, chondroitin sulfate proteoglycan 4 (CSPG4), we showed that cellular uptake of HA-PR20 is blocked by inhibition of glycosaminoglycan chain synthesis (using drugs or RNAi) and knockdown or ablation of CSPG4 (using RNAi or CRISPR editing). Reduction of CSPG4 protected PR20-induced neuronal toxicity. We used a dual reporter system to interrogate in vitro neuron-to-neuron transmission of PR50 and found that PR50 synthesized by one neuron readily spread to neighboring neurons. Transmission was significantly reduced when CSPG4 was knocked down. These results suggest CSPG4 is an important factor in poly-PR internalization and transmission and therefore may be a therapeutic target to slow DPR transmission and disease progression. Significance StatementA GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common monogenic form of ALS/FTD. This expansion leads to dipeptide repeat protein (DPR) production through non-canonical translation of repeat-containing RNA. DPRs have been shown to transmit between cells, but how this occurs is not well understood. We identified the cell surface protein chondroitin sulfate proteoglycan 4 (CSPG4) as a mediator of the uptake and intercellular spread of toxic arginine-rich DPRs. Targeting CSPG4 may provide a strategy to block DPR transmission and slow disease progression.

neuroscience↗

bioRxiv2025

Cholinergic neuron circadian clock mediates RNA-binding protein function and contributes to ALS disease phenotypes

Circadian clocks are encoded by a transcription-translation feedback loop that aligns physiological processes with the solar cycle. Previous work linking the circadian clock to the regulation of RNA-binding proteins (RBPs) and alternative splicing provides a foundation for the vital examination of their mechanistic connections in the context of amyotrophic lateral sclerosis (ALS)--a fatal neurodegenerative disease commonly marked by disrupted RBP function. Here, we reveal that the spinal cord cholinergic neuron rhythmic transcriptome is enriched for genes associated with ALS and other neurodegenerative diseases. We show that there is time-of-day-dependent expression of ALS-linked RBP transcripts and rhythmic alternative splicing of genes involved in fundamental neuronal processes, such as microtubule cytoskeleton organization, intracellular trafficking, and synaptic function. We demonstrate clock-dependent expression of ALS-linked RBP Ataxin 2 in this neuronal subtype. Further, through in silico analysis of RNA sequencing data from sporadic ALS patients, we find that gene expression profiles altered in disease correspond with rhythmic gene networks. Finally, we report that clock disruption through cholinergic neuron-specific deletion of clock activator BMAL1 (i) increases lumbar spinal cord motor neuron loss and sciatic nerve axon degeneration and (ii) drives time-of-day-dependent alternative splicing of genes associated with RNA metabolism, including genes encoding ALS-linked RBPs (e.g., Matr3, Srsf7, and Ythdf2). Our results establish a role for the cholinergic neuron circadian clock in RNA metabolism and mediating neurodegeneration.

neuroscience↗

bioRxiv2021

The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of SPOP mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS. Significance statementThe G4C2 repeat expansion in the C9orf72 gene is a major cause of Fronto-Temporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins, PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both C. elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through the bromodomain protein BET-1 to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and druggable pathway that may represent a new strategy for treating these currently incurable diseases.

neuroscience↗

bioRxiv2020

Preconditioning of Caenorhabditis elegans to Anoxic Insult by Inactivation of Cholinergic, GABAergic, and Muscle Activity

For most metazoans, oxygen deprivation leads to cell dysfunction and if severe, death. Sublethal stress prior to a hypoxic or anoxic insult ("preconditioning") can protect cells from subsequent oxygen deprivation. The molecular mechanisms by which sublethal stress can buffer against a subsequent toxic insult and the role of the nervous system in the response are not well understood. We studied the role of neuronal activity preconditioning to oxygen deprivation in C. elegans. Animals expressing the histamine gated chloride channels (HisCl1) in select cell populations were used to temporally and spatially inactivate the nervous system or tissue prior to an anoxic insult. We find that inactivation of the nervous system for 3 hours prior to the insult confers resistance to a 48-hour anoxic insult in 4th-stage larval animals. Experiments show that this resistance can be attributed to loss of activity in cholinergic and GABAergic neurons as well as in body wall muscles. These observations indicate that the nervous system activity can mediate the organisms response to anoxia.

neuroscience↗