Biology subjects

Ramaswamy, P.

Publications and source records attributed to Ramaswamy, P..

4 recordsLinked to original sources

bioRxiv2026

Stimulating microglial neuroinflammation with amyloid-beta oligomers compatible with solid-state NMR structural characterization

Soluble amyloid-{beta} (A{beta}) oligomers are considered the primary neurotoxic species in Alzheimer's disease, yet the relationship between oligomer structure and biological activity remains poorly understood. Oligomer sample preparations amenable to high-resolution structural characterization have not been tested in cell-based assays, mostly because detergents used to drive oligomer formation are toxic to cells. Here, we describe a detergent-free protocol in which size-exclusion chromatography-isolated A{beta}42 monomers spontaneously oligomerize at 37 {degrees}C in a concentration- and temperature-dependent manner. Biophysical characterization shows a {beta}-sheet structure by circular dichroism, a self-associating population above 100 kDa by silver-stained SDS-PAGE, and bead-like strings by negative-stain TEM resembling the previously characterized SDS-induced 150 kDa oligomer. Solid-state NMR spectroscopy of selectively 13C/15N-labeled 37 {degrees}C oligomers yields well-resolved spectra with {beta}-strand chemical shifts, and two-dimensional 13C-13C DARR and CHHC experiments provide initial inter-residue distance constraints that reveal both similarities to and differences from the 150 kDa oligomer. Treatment of mouse microglial cells (MMCs) with 50 nM 37 {degrees}C oligomers elicited a robust pro-inflammatory cytokine response that exceeded that of monomeric A{beta}42, as demonstrated by Luminex multiplex profiling and principal component analysis. hiPSC-derived microglia (iMGs) treated with 1 {micro}M oligomers also separated from vehicle, establishing activity in a human cellular model. Monomeric A{beta}42 was inactive in both systems, indicating that inflammatory activity depends on the assembly state of A{beta}42 rather than on the presence of peptide alone. These results establish a detergent-free A{beta}42 oligomer preparation that is simultaneously compatible with solid-state NMR structural characterization and biologically active in neuroinflammation assays, providing a platform for studying microglial immune responses to oligomers.

neuroscience↗

bioRxiv2025

Aβ-42 sidechain deamidation at Q15 and N27 modulate protein aggregation and microglial responses via altered cytokine production and CD68 expression

The progressive aggregation of amyloid beta (A{beta}) monomers into oligomers is a critical factor in Alzheimers disease (AD) pathogenesis. Although mutated forms of A{beta} have been shown to display altered aggregation dynamics, the specific effects of deamidated A{beta} on microglial function remain understudied. Our research group previously found that the deamidated variant A{beta}-42-N27D modified A{beta} aggregation, reduced neurotoxicity, and reduced microglial reactivity, but the impact of A{beta}-42 side chain deamidation in general on such parameters remained unclear. Here, we expanded on our prior work by investigating how two site-specific A{beta}-42 mutations (Q15E & N27D), where neutral amide side chains are replaced with negatively charged carboxylic acids, affect aggregation and microglial immune response using a mouse microglial cell line. Size exclusion chromatography revealed that A{beta}-42-Q15E and A{beta}-42-N27D exhibit distinct aggregation profiles compared to A{beta}-42 wild type (WT). Multiplexed analysis of 8 cytokines secreted into the culture medium revealed that A{beta}-42-Q15E and A{beta}-42-N27D decrease the expression of inflammatory cytokines such as IL-6, IP-10, and MIP-1 relative to A{beta}-42-WT. Immunocytochemistry revealed that A{beta}-42-Q15E and A{beta}-42-N27D decrease CD68 expression relative to A{beta}-42-WT. These findings demonstrate that deamidation significantly alters A{beta}-42 aggregation and microglial activation, suggesting structural modifications to A{beta}-42 modulate inflammatory signaling in AD. This work provides a foundation for future studies on A{beta}-42 post-translational modifications as potential therapeutic targets in AD.S

neuroscience↗

bioRxiv2024

Headache-specific Hyperexcitation Sensitises and Habituates on different Time Scales: An Event Related Potential study of Pattern-Glare

Cortical hyperexcitability is a key pathophysiological feature in several neurological disorders, including migraine, epilepsy, tinnitus, and Alzheimers disease. We examined the temporal characteristics of Evoked Related Potentials (ERPs) in a healthy population using the Pattern Glare Test, a diagnostic tool used to assess patients with sensitivity to cortical hyperexcitability. During the experiment, participants recorded state measures with this study focussing on susceptibility to migraine. We investigated two timeframes: habituation over the course of the experiment and sensitization over the course of stimulus presentation. We found evidence of hyperexcitability in the visual cortex, for the clinically aggravating stimuli (medium). Participants who reported a higher state measure exhibited a higher degree of habituation and sensitization, which was dependent on susceptibility to migraine. These findings suggest that the same experimental paradigm and analysis should be performed on a clinically diagnosed population.

neuroscience↗

bioRxiv2023

Identification of novel oncogenic transcriptional targets of mutant p53 in Esophageal Squamous Cell Carcinoma

Missense mutations in the DNA binding domain of p53 are observed frequently in Esophageal Squamous Cell Carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common hotspot p53 mutations while rarer mutations are poorly characterized. We had previously identified SMARCD1 as an oncogenic transcriptional target of rare non-hotspot p53 mutants detected from squamous cell carcinoma of the oral tongue (SCCOT). We now report the characterization of non-hotspot p53 mutations from ESCC. In-vitro tumorigenic assays performed following ectopic-expression of non-hotspot mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harbouring mutant p53. Of these, ARF6, C1QBP and TRIM23 were studied further due to their previously reported pro-oncogenic roles. Reverse transcription quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumor samples revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild type and several mutant p53 forms followed by RT-qPCR, Chromatin affinity-purification and Promoter-luciferase assays indicated the exclusive recruitment of p53 mutants - P190T and P278L, to the target genes leading to activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effect of the genes was confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of rare non-hotspot mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor specific p53 mutations.

cancer biology↗