Biology subjects

Prasad, B. C.

Publications and source records attributed to Prasad, B. C..

5 recordsLinked to original sources

bioRxiv2026

Targeted quantification assays for DNA repair and handling proteins and interactions in Huntington's disease models

Huntingtons disease (HD) is a life-altering genetic neurodegenerative disorder, with cognitive, motor, and psycho-social effects that have consequential impacts on the individuals and their families. While current treatments improve disease symptoms, there are no FDA-approved therapies that prevent disease progression. Converging lines of evidence from human GWAS and mouse models point to DNA repair and handling (R/H) proteins as promising therapeutic targets due to their ability to modulate somatic expansion of the CAG repeat of HTT. The roles of DNA R/H HD modulator proteins are incompletely understood, in part, due to their relatively low cellular abundance and technical challenges in quantification. Here, we developed and validated targeted mass spectrometry assays quantifying DNA R/H proteins, spanning functions in mismatch repair, Fanconi anemia, and transcriptional regulation, using complementary workflows for timsTOF and Orbitrap platforms. We built species-specific experiment spectral libraries that outperformed in silico libraries for target detection. Applying this pipeline to an HTT-Q140 knock-in mouse HD model, we observed that DNA R/H protein abundances were largely unchanged in HD mice, while HTT and HAP40 showed increased nuclear association with disease progression. To facilitate translational research applications, we further developed a stable isotope dilution assay for absolute quantification of 11 human mismatch repair-associated proteins and generated an HTT knock-out human neuroblastoma cell line. Additionally, we used thermal proximity coaggregation profiling to characterize the endogenous interactomes of MMR proteins. We observed that HTT KO caused proteome down-regulation in selected DNA R/H proteins and reshaped the MMR protein interactome, with the most pronounced changes observed for MLH1 and PMS1 interactions. Overall, we established a validated, transferable assay for quantifying DNA R/H proteins in perturbation studies using human and mouse HD model systems and provide evidence that HTT influences the abundance and interaction landscape of proteins central to CAG repeat instability.

neuroscience↗

bioRxiv2026

Unscheduled DNA synthesis reveals a DNA repair hotspot and biomarker of somatic instability at the expanded-CAG repeat tract in the huntingtin gene

Somatic instability (SI) of expanded DNA repeats is a hallmark of repeat expansion disorder (REDs) and drives onset and progression in Huntingtons disease (HD) yet the absence of target engagement (TE) biomarkers for SI-modulating therapies represents a critical gap to clinical development. Here, we describe the development of the unscheduled repair synthesis assay (URSA)--combining 5-ethynyl-2-deoxyuridine (EdU) pulse-labeling with digital PCR or sequencing--and show that the CAG-expanded huntingtin (HTT) exon 1 allele is a highly active DNA repair hotspot in cells from people with HD (PwHD). Repair activity increases with repeat length, is allele-specific, and depends strongly on MSH3, a central driver of somatic expansion. URSA robustly quantifies MSH3 modulation in preclinical models within days compared to weeks or months required by conventional repeat-length measurements. Critically, substantial repair activity is detectable in peripheral blood mononuclear cells (PBMCs) from PwHD, where signal correlates with CAG length and improves predictive models of somatic expansion (SE) beyond age and CAG length alone. Unlike repeat-length changes, which require years to accumulate in blood, URSA signal is measurable within days. These findings establish DNA repair activity at the mutant HTT locus as a mechanistically grounded pharmacodynamic biomarker, enabling TE monitoring on a clinically actionable timescale, and with broad applicability to REDs and other diseases where modulation of the DNA damage response is therapeutically targeted.

neuroscience↗

bioRxiv2024

A FAN1 point mutation associated with accelerated Huntington's disease progression alters its PCNA-mediated assembly on DNA

FAN1 is an endo- and exo-nuclease involved in DNA and interstrand crosslink repair. Genome-wide association studies of people with Huntingtons disease revealed a strong association between the FAN1 R507H mutation and early disease onset, however the underlying mechanism(s) remains unclear. FAN1 has previously been implicated in modulating triplet repeat expansion in a PCNA dependent manner. To examine the role of PCNA on FAN1 activation, we solved the cryo-EM structures of a PCNA-FAN1-DNA complex. Our findings reveal that the FAN1 R507 residue directly interacts with PCNA D232. Biophysical interaction studies demonstrated that FAN1 enhances the binding affinity of PCNA for DNA, a synergistic effect disrupted in mutants carrying the R507H mutation. In contrast, PCNA does not affect the affinity of FAN1 for DNA but does modulate FAN1 activity upon ternary complex formation. The weakened and functionally altered FAN1 R507H-PCNA-DNA complex may partly impair the FAN1-mediated repair of CAG extrahelical extrusions, providing a potential explanation for the mutations role in accelerating disease progression.

biophysics↗

bioRxiv2024

Msh3 and Pms1 Set Neuronal CAG-repeat Migration Rate to Drive Selective Striatal and Cortical Pathogenesis in HD Mice

Modifiers of Huntingtons disease (HD) include mismatch repair (MMR) genes; however, their underlying disease-altering mechanisms remain unresolved. Knockout (KO) alleles for 9 HD GWAS modifiers/MMR genes were crossed to the Q140 Huntingtin (mHtt) knock-in mice to probe such mechanisms. Four KO mice strongly (Msh3 and Pms1) or moderately (Msh2 and Mlh1) rescue a triad of adult-onset, striatal medium-spiny-neuron (MSN)-selective phenotypes: somatic Htt DNA CAG-repeat expansion, transcriptionopathy, and mHtt protein aggregation. Comparatively, Q140 cortex also exhibits an analogous, but later-onset, pathogenic triad that is Msh3-dependent. Remarkably, Q140/homozygous Msh3-KO lacks visible mHtt aggregates in the brain, even at advanced ages (20-months). Moreover, Msh3-deficiency prevents striatal synaptic marker loss, astrogliosis, and locomotor impairment in HD mice. Purified Q140 MSN nuclei exhibit highly linear age-dependent mHtt DNA repeat expansion (i.e. repeat migration), with modal-CAG increasing at +8.8 repeats/month (R2=0.98). This linear rate is reduced to 2.3 and 0.3 repeats/month in Q140 with Msh3 heterozygous and homozygous alleles, respectively. Our study defines somatic Htt CAG-repeat thresholds below which there are no detectable mHtt nuclear or neuropil aggregates. Mild transcriptionopathy can still occur in Q140 mice with stabilized Htt 140-CAG repeats, but the majority of transcriptomic changes are due to somatic repeat expansion. Our analysis reveals 479 genes with expression levels highly correlated with modal-CAG length in MSNs. Thus, our study mechanistically connects HD GWAS genes to selective neuronal vulnerability in HD, in which Msh3 and Pms1 set the linear rate of neuronal mHtt CAG-repeat migration to drive repeat-length dependent pathogenesis; and provides a preclinical platform for targeting these genes for HD suppression across brain regions. One Sentence SummaryMsh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.

neuroscience↗

bioRxiv2023

Conformational dynamics and DNA recognition by human MutSbeta

Human and mouse genetic studies have demonstrated a role for DNA mismatch repair (MMR) molecular machines in modulating the rate of somatic expansion of the huntingtin (HTT) CAG repeats, and onset and progression of Huntingtons Disease (HD). MutS{beta}, a key component of the MMR pathway, is a heterodimeric protein of MSH2 and MSH3 that recognizes and initiates the repair of extrahelical DNA extrusions. Loss-of-function of mouse Msh3 and reduced-expression alleles of human MSH3 lead to slower rates of somatic expansion and delayed disease onset in humans, signifying MSH3 as a promising therapeutic target for HD. Here we report biochemical and cryo-electron microscopy analyses of human MutS{beta}, demonstrating MutS{beta} undergoes conformational changes induced by nucleotide and DNA binding. We present multiple conformations of MutS{beta} including the DNA-free MutS{beta} compatible with homoduplex DNA binding, two distinct structures of MutS{beta} bound to (CAG)2 DNA, a sliding clamp form and a DNA-unbound, ATP-bound conformation. Along with evidence for novel conformational states adopted by MutS{beta} to initiate the MMR cascade, these structures provide a foundation for structure-guided drug discovery.

biophysics↗