Biology subjects

Gitler, A. D.

Publications and source records attributed to Gitler, A. D..

29 records · Page 2Linked to original sources

bioRxiv2021

Poly(A)-binding protein is an ataxin-2 chaperone that emulsifies biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs) which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar that dictates the behavior of a few key scaffold proteins that make up these phases but how the partitioning of hundreds of other SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, a SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14aa sequence that acts as an ataxin-2 condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal a new molecular function for ancient poly(A)-binding proteins as emulsifiers of biomolecular condensate proteins. These findings may inspire novel approaches to therapeutically target aberrant phases in disease.

cell biology↗

bioRxiv2021

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

A hallmark pathological feature of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function.

neuroscience↗

bioRxiv2020

Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress.

Many organisms evolved strategies to survive and thrive under extreme desiccation. Plant seeds protect dehydrated embryos from a variety of stressors and can even lay dormant for millennia. While hydration is the key trigger that reactivates metabolism and kick-starts germination, the exact mechanism by which the embryo senses water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that the biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We also find intragenic, intraspecific, and interspecific natural variations in phase separation propensity of FLOE1 homologs. These findings demonstrate a physiological role of phase separation in a multicellular organism and have direct implications for plant ecology and agriculture, especially the design of drought resistant crops, in the face of climate change.

cell biology↗

bioRxiv2020

Single-cell transcriptomic analysis of the adult mouse spinal cord

The spinal cord is a fascinating structure responsible for coordinating all movement in vertebrates. Spinal motor neurons control the activity of virtually every organ and muscle throughout the body by transmitting signals that originate in the spinal cord. These neurons are remarkably heterogeneous in their activity and innervation targets. However, because motor neurons represent only a small fraction of cells within the spinal cord and are difficult to isolate, the full complement of motor neuron subtypes remains unknown. Here we comprehensively describe the molecular heterogeneity of motor neurons within the adult spinal cord. We profiled 43,890 single-nucleus transcriptomes using fluorescence-activated nuclei sorting to enrich for spinal motor neuron nuclei. These data reveal a transcriptional map of the adult mammalian spinal cord and the first unbiased characterization of all transcriptionally distinct autonomic and somatic spinal motor neuron subpopulations. We identify 16 sympathetic motor neuron subtypes that segregate spatially along the spinal cord. Many of these subtypes selectively express specific hormones and receptors, suggesting neuromodulatory signaling within the autonomic nervous system. We describe skeletal motor neuron heterogeneity in the adult spinal cord, revealing numerous novel markers that distinguish alpha and gamma motor neurons--cell populations that are specifically affected in neurodegenerative disease. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neurons. Collectively, these data provide a single-cell transcriptional atlas for investigating motor neuron diversity as well as the cellular and molecular basis of motor neuron function in health and disease.

neuroscience↗

bioRxiv2020

G-baToN: a versatile cell-cell interaction reporter system.

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various cell types, including diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher-order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

bioengineering↗

bioRxiv2019

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

Hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

neuroscience↗

bioRxiv2019

A memory of RPS25 loss drives resistance phenotypes

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Many cell types can nonetheless withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. However, the direct and indirect consequences from sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation.

molecular biology↗

bioRxiv2019

Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72

Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.

neuroscience↗

bioRxiv2019

LRRK2 modifies α-syn pathology and spread in mouse models and human neurons

Progressive aggregation of the protein alpha-synuclein (-syn) and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are key histopathological hallmarks of Parkinsons disease (PD). Accruing evidence suggests that -syn pathology can propagate through neuronal circuits in the brain, contributing to the progressive nature of the disease. Thus, it is therapeutically pertinent to identify modifiers of -syn transmission and aggregation as potential targets to slow down disease progression. A growing number of genetic mutations and risk factors have been identified in studies of both familial and sporadic forms of PD. However, how these genes affect -syn aggregation and pathological spread, and whether they can be targeted for therapeutic interventions, remains unclear. We performed a targeted genetic screen of risk genes associated with PD and parkinsonism for modifiers of -syn aggregation, using an -syn preformed-fibril (PFF) induction assay. We found that decreased expression of Lrrk2 and Gba modulated -syn aggregation in mouse primary neurons. Conversely, -syn aggregation increased in primary neurons from transgenic mice expressing the PD-linked LRRK2 G2019S mutation. In vivo, we investigated -syn spreading in a LRRK2 G2019S transgenic mouse model and observed acceleration of -syn aggregation and degeneration of dopaminergic neurons in the SNpc. Furthermore, LRRK2 G2019S transgenic mice displayed more pronounced degeneration-associated neuroinflammation and exacerbated behavioral deficits. To validate our findings in a human context, we established a human -syn transmission model using induced pluripotent stem cell (iPS)-derived neurons (iNs). Recombinant human -syn PFFs triggered aggregation of endogenous -syn in a time-dependent manner in human iNs. In isogenic lines of human iNs, the G2019S mutation enhanced -syn aggregation, whereas loss of LRRK2 decreased aggregation. Collectively, these findings establish a strong interaction between the PD risk gene LRRK2 and -syn transmission across mouse and human models. Since clinical trials of LRRK2 inhibitors in PD are currently underway, our findings raise the possibility that these may be effective in PD broadly, not just in the cases caused by LRRK2 mutations.

neuroscience↗

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↗

bioRxiv2017

CRISPR-Cas9 Screens In Human Cells And Primary Neurons Identify Modifiers Of C9orf72 Dipeptide Repeat Protein Toxicity

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from model systems. We also uncovered new potent modifiers of DPR toxicity whose gene products function in the endoplasmic reticulum (ER), proteasome, RNA processing pathways, and in chromatin modification. Since regulators of ER stress emerged prominently from the screens, we further investigated one such modifier, TMX2, which we identified as a modulator of the ER-stress signature elicited by C9orf72 DPRs in neurons. Together, this work identifies novel suppressors of DPR toxicity that represent potential therapeutic targets and demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases.\n\nOne Sentence SummaryGenome-wide CRISPR-Cas9 screens in human cells reveal mechanisms and targets for ALS-associated C9orf72 dipeptide repeat protein toxicity.

genetics↗