Biology subjects

Mehla, J.

Publications and source records attributed to Mehla, J..

9 recordsLinked to original sources

bioRxiv2025

Post-translational modifications distinguish amyloid-β isoform patterns extracted from vascular deposits and parenchymal plaques

Deposition of amyloid-{beta} (A{beta}) aggregates is a core pathological hallmark of both cerebral amyloid angiopathy (CAA) and extracellular parenchymal plaques in Alzheimers disease (AD). While both disease processes share progressive, decades-long deposition of fibrillar A{beta} peptide, they differ in isoform composition. We hypothesized that post-translational modifications (PTMs) on A{beta} would also differ between CAA and parenchymal plaques. Using Lys-N enzymatic digestion followed by quantitative mass spectrometry, we profiled A{beta} isoforms and N-terminus PTMs (aspartic acid isomerization and pyroglutamate formation) across CAA severity and compared them to parenchymal plaque A{beta} in AD. Moderate to severe CAA primarily featured intact N-terminus (A{beta}1-x) ([~]95%) with minimal N-truncated species (A{beta}2-x, A{beta}3pGlu-x, A{beta}4-x), whereas parenchymal plaques displayed diverse N-terminus truncations and PTMs. Increasing CAA severity correlated with a shift from longer, hydrophobic C-terminal isoforms (A{beta}41, A{beta}42, A{beta}43) to shorter, less hydrophobic C-terminal isoforms (A{beta}37, A{beta}38, A{beta}39, A{beta}40). Importantly, moderate and severe CAA displayed minimal isomerization of Asp-1 and Asp-7 residues, which correlated significantly (r > 0.9) with shorter C-terminal isoforms (A{beta}37, A{beta}38, A{beta}39, A{beta}40). These patterns suggest distinct A{beta} aggregation mechanisms in CAA versus parenchymal plaques. We propose that the intact N-terminus found in CAA with limited Asp isomerization is due to its inclusion within the protofibril structure (less disordered and inaccessible to PTMs), unlike the parenchymal plaques, where the N-terminus is more disordered and accessible to PTMs. These biochemical differences may reflect distinct protofibril architectures with potential implications for biomarker development for early CAA detection and therapeutic targeting of vascular and parenchymal A{beta}.

neuroscience↗

bioRxiv2023

Predicting Permeation of Compounds across the Outer Membrane of P. aeruginosa Using Molecular Descriptors: Advantages and Limitations

The ability of Gram-negative pathogens to adapt and protect themselves against antibiotics is a growing threat to public health. The low permeability of the outer membrane (OM) in combination with effective multidrug efflux pumps, constitute the two main antibiotic resistance mechanisms. Though much efforts have been devoted to discover new antibiotics that can bypass these defense mechanisms, no new antibiotic classes have been introduced into clinics in the last 35 years. Models that identify specific descriptors of molecular properties and predict the likelihood that a given compound is capable of successfully permeate the OM and inhibit bacterial growth while avoiding efflux could facilitate the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors of 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. While part of these descriptors are computed using traditional approaches based on the physicochemical properties intrinsic to the compounds, ensemble docking and all-atom molecular dynamics (MD) simulations are used to derive additional bacterium-specific mechanistic properties. Descriptors of compound permeation across the OM were calculated using all-atom MD simulations of the compounds in different subregions of the OM model. Descriptors of interactions with efflux pumps were calculated from ensemble docking of compounds targeting specific binding pockets of MexB, the major efflux transporter of P. aeruginosa. Using these descriptors and the measured antibacterial inhibitory concentrations of compounds, we design and implement a statistical protocol to identify a subset of the molecular properties that are predictive of whether a given compound is a strong or weak permeator across the Gram-negative OM. Our results indicate that 88.4% of the compounds that show measurable antibacterial activity, follow very consistent rules of permeation, which highlight the critical role that the interaction between the compound and the OM have at predicting permeation. The remaining 11.6% of the compounds, although less predictive, are characterized by distinctive structural markers that can be used to minimize classification errors. An implementation of the permeation rules and the structural markers uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Taken together, our analysis sheds new light on the key molecular properties that drug candidates should have in order to be effective at OM permeation/inhibition of P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens.

biophysics↗

bioRxiv2023

Molecular determinants of avoidance and inhibition of Pseudomonas aeruginosa MexB efflux pump

Transporters of the Resistance-Nodulation-cell Division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. Previous analyses of antibacterial and biochemical activities showed that these compounds vary broadly in their efficiency as inhibitors or substrates of MexAB and can be categorized into different functional classes. Here, we performed multi-copy molecular dynamics simulations, machine learning analyses and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of the various classes. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket, and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. ImportanceMultidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning, computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.

microbiology↗

bioRxiv2022

Gradual cerebral hypoperfusion in a knock-in mouse model of Alzheimer's disease triggers cortical network dysfunctions

Alzheimers disease (AD) is characterized neuropathologically by amyloid-{beta} (A{beta}) plaques and neurofibrillary tangles. Vascular pathology caused by chronic cerebral hypoperfusion (HP) is hypothesised to exacerbate AD pathology and has emerged as an increasing cause of age-related cognitive impairment. In this study we examined the effects of gradual cerebral HP on cognitive dysfunction, A{beta} pathology, microgliosis, and cortical network dynamics in C57BL/6J mice and a single App knock-in mouse model of AD (AppNL-G-F). We performed unilateral common carotid artery gradual occlusion (UCAgO) in two-month-old mice using an ameroid constrictor. At 4 months of age, animals were tested in a behavioral battery consisting of tests of spatial learning and memory (Morris water task), recognition memory (novel object recognition task), and motor coordination (balance beam). Following behavioural testing, in vivo mesoscale wide-field voltage imaging was done to assess cortical functional connectivity and sensory-evoked cortical activity, and brains were harvested for pathology characterization using immunohistochemistry. We found that UCAgO reduced cerebral blood flow (CBF) in the occluded hemisphere (OH), however, subtle behavioural deficits were observed due to HP. A dissociative effect of HP was observed in resting-state functional connectivity analysis, where HP led to hyper-connectivity in C57 mice and hypo-connectivity in App mice. Interestingly, sensory stimulation of limbs contralateral to OH revealed hyper-cortical activations in the non-occluded hemisphere of C57 HP mice, however, hypo-cortical activations were observed in App HP mice. Furthermore, we found that the UCAgO increased cortical and hippocampal microgliosis in both hemispheres of C57 and App mice, a bilateral increase in A{beta} deposition was only observed in App mice. These results suggest that gradual cerebral HP leads to cortical network alterations in AD, which is partly mediated via activation of microglia.

neuroscience↗

bioRxiv2021

YhcB (DUF1043), a novel cell division protein conserved across gamma-proteobacteria

YhcB, an uncharacterized protein conserved across gamma-proteobacteria, is composed predominantly of a single Domain of Unknown Function (DUF 1043) with an N-terminal transmembrane -helix. Here, we show that E. coli YhcB is a conditionally essential protein that interacts with the proteins of the cell divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). We found 7 interactions of YhcB that are conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we identified several point mutations that abolished interactions of YhcB with FtsI and RodZ. The yhcB knock-out strain does not grow at 45{degrees}C and is hypersensitive to cell-wall acting antibiotics even in stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septa development. The 2.8 [A] crystal structure for the cytosolic domain from Haemophilus ducreyi YhcB shows a unique tetrameric -helical coiled-coil structure that combines parallel and anti-parallel coiled-coil intersubunit interactions. This structure is likely to organize interprotein oligomeric interactions on the inner surface of the cytoplasmic membrane, possibly involved in regulation of cell division and/or envelope biogenesis/integrity in proteobacteria. In summary, YhcB is a conserved and conditionally essential protein that is predicted to play a role in cell division and consequently or in addition affects envelope biogenesis. ImportanceOnly 0.8 % of the protein annotations in the UniProt are based on experimental evidence and thus, functional characterization of unknown proteins remains a rate-limiting step in molecular biology. Herein, the functional properties of YhcB (DUF1043) were investigated using an integrated approach combining X-ray crystallography with genetics and molecular biology. YhcB is a conserved protein that appears to be needed for the transition from exponential to stationary growth and is involved in cell division and/or envelope biogenesis/integrity. This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

microbiology↗

bioRxiv2020

Repeated multi-domain, but not single-domain, cognitive training prevents cognitive decline and amyloid pathology found in the APPNL-G-F mouse model of Alzheimer disease

Education, occupation, and an active lifestyle, comprising enhanced social, physical, and mental components are associated with improved cognitive functions in aged people and may prevent/ or delay the progression of various neurodegenerative diseases including Alzheimers disease (AD). To investigate this protective effect, APPNL-G-F/NL-G-F mice at 3 months of age were exposed to repeated, single- or multi-domain cognitive training. Cognitive training was given at the age of 3, 6, 9 & 12 months of age. Single-domain cognitive training was limited to a spatial navigation task. Multi-domain cognitive training consisted of a spatial navigation task, object recognition, and fear conditioning. At the age of 12 months, behavioral tests were completed for cognitive training groups and control group. After completion of behavioral testing, mice were sacrificed, and their brains were assessed for pathology. AppNL-G-F mice given multi-domain cognitive training compared to APPNL-G-F control group showed an improvement in cognitive functions, reductions in amyloid load and microgliosis, and a preservation of cholinergic function. There were mild reductions in microglosis in the brain of APPNL-G-F mice with singledomain cognitive training. These findings provide causal evidence for the potential of certain forms of cognitive training to mitigate the cognitive deficits in Alzheimer disease.

animal behavior and cognition↗

bioRxiv2020

Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology

More than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in the mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates transition to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi of an amyloid precursor protein knock-in mouse model and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimers disease (AD). This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity.

genomics↗

bioRxiv2017

A variant of the Morris water task for assessing learning and memory processes in mice

The Morris water task (MWT) is commonly used to assess rodent spatial learning and memory. Our goal was to develop a 3-phase variant of the hidden goal water task to assess old and new spatial memories acquired in the same context using various measures of spatial learning in C57BL/6 mice. In the first phase, mice were pre-trained to an initially hidden location. The second phase consisted of a massed training session to a new location in the same apparatus and context. The final phase consisted of a competition test between the original and new platform locations. AppNL-G-F/NL-G-F mice, a novel transgenic mouse model for Alzheimers disease (AD), were also used as an independent variable to validate this 3-phase variant of MWT. The results of the present study showed that C57 mice acquired and retained both the old and new location representations; however, AppNL-G-F/NL-G-F mice retained a recently acquired spatial memory but did not remember the old location acquired in the same apparatus and context. The results showed that C57 mice can show precise place learning and memory with the right amount of training and acquire and retain multiple spatial memory locations in the same environment whereas this ability was impaired in AppNL-G-F/NL-G-F mice. In the visible platform test, however, all groups of mice showed normal sensorimotor ability and motivation. These findings indicate that this new version of the MWT provides a robust way for assessment of old and new memories in mice. This paradigm could also be exploited to assess manipulations of neural circuits implicated in learning and memory processes as well as for research investigating human brain diseases.

neuroscience↗

bioRxiv2017

Gradual Cerebral Hypoperfusion Impair Fear Conditioning and Object Recognition Learning and Memory in Mice: Potential Roles of Neurodegeneration and Cholinergic Dysfunction

In the present study, male C57BL/6J mice were subjected to gradual cerebral hypoperfusion by implanting an ameroid constrictor (AC) on the left common carotid artery (CCA) and a stenosis on the right CCA. In the sham group, all surgical procedures were kept the same except no AC was implanted and stenosis was not performed. One month following the surgical procedures, fear conditioning and object recognition tests were conducted to evaluate learning and memory functions and motor functions were assessed using a balance beam test. At the experimental endpoint, mice were perfused and brains were collected for immunostaining and histology. Learning and memory as well as motor functions were significantly impaired in the hypoperfusion group. The immunoreactivity to choline acetyltransferase was decreased in dorsal striatum and basal forebrain of the hypoperfusion group indicating that cholinergic tone in these brain regions was compromised. In addition, an increased number of Fluoro-Jade positive neurons was also found in cerebral cortex, dorsal striatum and hippocampus indicating neurodegeneration in these brain regions. Based on this pattern of data, we argued that this mouse model would be a useful tool to investigate the therapeutic interventions for the treatment of vascular dementia. Additionally, this model could be employed to exploit the effect of microvascular occlusions on cognitive impairment in the absence and presence of Alzheimer pathology.

neuroscience↗