Biology subjects

Dawson, V. L.

Publications and source records attributed to Dawson, V. L..

26 records · Page 2Linked to original sources

bioRxiv2022

Poly ADP-Ribose Signaling is Dysregulated in Huntington Disease Patients

Huntington disease (HD) is a genetic neurodegenerative disease caused by CAG expansion in the Huntingtin (HTT) gene, translating to an expanded polyglutamine tract in the huntingtin (HTT) protein. Age at disease onset correlates to CAG repeat length but varies by decades between individuals with identical repeat lengths. Genome-wide association studies link HD modification to DNA repair and mitochondrial health pathways. Clinical studies show elevated DNA damage in HD, even at the premanifest stage. A major DNA repair node influencing neurodegenerative disease is the PARP pathway. Accumulation of poly ADP-ribose (PAR) has been implicated in Alzheimer and Parkinson diseases, as well as cerebellar ataxia. We report that HD mutation carriers have lower cerebrospinal fluid PAR levels than healthy controls, starting at the premanifest stage. Human HD iPSC-derived neurons and patient- derived fibroblasts have diminished PAR response in the context of elevated DNA damage. We have defined a PAR-binding motif in huntingtin, detected huntingtin complexed with PARylated proteins in human cells during stress, and localized huntingtin to mitotic chromosomes upon inhibition of PAR degradation. Direct huntingtin PAR binding was measured by fluorescence polarization and visualized by atomic force microscopy at the single molecule level. While wild type and mutant huntingtin did not differ in their PAR binding ability, purified wild type huntingtin protein increased in vitro PARP1 activity while mutant huntingtin did not. These results provide insight into an early molecular mechanism of HD, suggesting possible targets for the design of early preventive therapies. Significance statementA consensus on dysfunctional DNA repair has emerged in neurodegenerative disease research, with elevated poly ADP-ribose (PAR) signaling more recently implicated. In contrast, we have identified a deficient PAR response in Huntingtons disease (HD) patient spinal fluid samples and cells. This may be explained by the inability of huntingtin protein bearing the HD-causing mutation to stimulate production of PAR the way the wild type protein does. Since drugs that target PAR production and degradation have already been developed, these findings present an exciting avenue for therapeutic intervention for HD.

neuroscience↗

bioRxiv2022

Enhanced mTORC1 signaling and Protein Synthesis in Parkinson Disease Pathogenesis

Pathologic -syn destabilizes the TSC 1 and 2 complex leading to mTORC1 activation, enhanced protein translation and neurodegeneration in PD. AbstractPathological -synuclein (-syn) plays an important role in the pathogenesis of -synucleinopathies such as Parkinsons disease (PD). Disruption of protein homeostasis is thought be central to PD pathogenesis, however the molecular mechanism of this deregulation is poorly understood. Here we report that pathologic -syn binds to tuberous sclerosis protein (TSC) 2 and destabilizes the TSC1-TSC2 complex leading to activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and enhanced mRNA translation. Dopamine neuron loss, behavioral deficits and aberrant biochemical signaling in the -syn preformed fibril (PFF) and Drosophila -syn transgenic models of pathologic -syn induced degeneration were attenuated by genetic and pharmacologic inhibition of mTOR and protein translation. Our findings establish a potential molecular mechanism by which pathologic -syn activates mTORC1 leading to enhanced protein translation and concomitant neurodegeneration in PD.

neuroscience↗

bioRxiv2022

Molecular recording of calcium signaling via calcium-dependent protein proximity labeling

Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, calcium transients play a central role in regulating neurotransmitter release and synaptic plasticity. However, it is challenging to capture the molecular events associated with these localized and ephemeral calcium signals. Here we present an engineered biotin ligase that combines the power of genetically encoded calcium indicators with protein proximity labeling. The enzyme, Cal-ID, biotinylates nearby proteins within minutes in response to elevated local calcium levels. The biotinylated proteins can be identified via mass spectrometry and visualized using microscopy. In neurons, Cal-ID labeling is triggered by neuronal activity, leading to prominent protein biotinylation that enables transcription-independent activity mapping in the brain. Cal-ID produces a biochemical record of calcium signaling and neuronal activity with high spatial resolution and molecular specificity.

neuroscience↗

bioRxiv2022

Proteome-wide microarray-based screening of PAR-binding proteins

Poly(ADP-ribose) (PAR) plays a crucial role in intracellular signaling and scaffolding through covalent modification or non-covalent binding to target proteins. The non- covalent binding PARylome has not been extensively characterized. Here we performed a PAR-binding screen using a human protein microarray that covers most of the human proteome to characterize the non-covalent binding PARylome. A total of 356 PAR- binding proteins were identified. The PAR-binding PARylome suggests that PAR- binding regulates a variety of biological processes beyond well-characterized DNA damage signaling and DNA repair. Proteins that may be reprogrammed by PAR-binding include signaling molecules, transcription factors, nucleic acid binding proteins, calcium binding proteins, ligases, oxidoreductases, enzymes, transferases, hydrolases, and receptors. The global database of PAR-binding proteins that we established will be a valuable tool for further in-depth analysis of the role of PARylation in a wide range of biological contexts.

biochemistry↗

bioRxiv2021

Host regulator PARP1 contributes to sex differences and immune responses in a mouse model of tuberculosis

Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths annually that has a poorly understood male bias. Elucidating the basis of this male bias may enable precision medicine interventions for TB treatment and prevention. Here, we identify the master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as a driver of TB sex differences. We found that infection with M. tuberculosis (M. tb) triggers robust PARP activation in mouse lungs, suggesting that PARP1 activation is a fundamental host response to TB. Remarkably, PARP1 deletion abolished known sex differences in TB cytokine responses and blunted the early induction of TNF, IL-1{beta}, IFN{gamma}, MCP-1, and IL-6, particularly in male mice. In contrast, PARP1 was required for IL-10 induction in male or female mice. PARP1 deletion was protective against TB in female mice, resulting in significantly prolonged survival and reduced bacterial burden, but impaired TB containment in male mice. Our findings indicate that PARP1 contributes to TB sex differences via sexually divergent immune regulation and uniquely enhances early proinflammatory responses in males that prove beneficial for TB containment.

immunology↗

bioRxiv2020

Complement and coagulation cascades are potentially involved in dopaminergic neurodegeneration in alpha-synuclein-based mouse models of Parkinson's disease

Parkinsons disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and eventually, cognitive impairment. -Synuclein protein has been known to be the most culprit protein, but the underlying pathological mechanism still remains to be elucidated. As an effort to clarify the pathogenesis mechanism by -synuclein, various PD mouse models with -synuclein overexpression have been developed. However, the systemic analysis of protein abundance change by the overexpressed -synuclein in the whole proteome level has been still lacking. To address this issue, we established two sophisticated mouse models of PD by injecting -synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T -synuclein to discover overlapping pathways, which could be altered in the two different types of PD mouse model. For more accurate quantification of mouse brain proteome, stable isotope labeling with amino acid in mammal-based quantification was implemented. As a result, we have successfully identified a total of 8,355 proteins from both of the mouse models; [~]6,800 and [~]7,200 proteins from -synuclein PFF injected mice and human A53T -synuclein transgenic mice, respectively. From the pathway analysis of the differentially expressed proteins in common, the complement and coagulation cascade pathway were determined as the most enriched ones. This is the first study that highlights the significance of the complement and coagulation pathway in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.

neuroscience↗

bioRxiv2017

Experience-dependent translational state defined by cell type-specific ribosome profiling

Experience-dependent neuronal activity regulates the translation of mRNA, supporting memory formation. We have developed a new method termed translating ribosome affinity purification and ribosome profiling (TRiP) which allows us to determine cell type-specific ribosome occupancy of mRNA with nucleotide resolution. Using TRiP we show that a memory-inducing experience creates a distinct translational state in mouse CA1 pyramidal cells. The experience-dependent translation state is characterized by enhanced translation of protein-coding open reading frames (ORFs) including numerous components of the actin cytoskeleton and calcium/calmodulin binding proteins, and by decreased translation of a defined subset of genes containing upstream ORFs (uORFs). Using animals heterozygous for an unphosphorylatable allele of the eukaryotic translation initiation factor 2 (eIF2), we show that dephosphorylation of eIF2 contributes significantly to the experience-dependent translation state. These observations demonstrate that TRiP is a valuable methodology for studying physiologically relevant changes in translational state in genetically defined cell types.

genomics↗

bioRxiv2017

Towards the human cellular microRNAome

microRNAs are short RNAs that serve as master regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell autonomously and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data has caused considerable confusion and comprehensive cell-level data does not yet exist. Here we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent super-enhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 2,632 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder suggesting Drosha and Dicer-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to deconvolute variable cellular composition of adipose tissue samples highlighting one use of this cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species.

genomics↗