A gene signature for Alzheimer’s disease using RNAi in C. elegans

Alzheimer’s disease (AD) is a complex multifactorial disorder that is responsible for the large majority of the 50 million cases of dementia worldwide. This disease is still incurable, a situation caused at least in part by the fact that its genetics are incompletely known. In our laboratory, we have developed a novel computational approach—Network-based Transcriptome-Wide Association Studies (nTWAS)—that seeks to identify the genes associated with AD by comparing gene expression patterns across tissues in the brain. nTWAS acts as an in silico pre-screen by providing a list of gene candidates, thus enabling us to pursue investigations into each gene candidate with significantly more depth. To that end, we use RNA interference in an AD model in the nematode worm Caenorhabditis elegans to validate the results of this pre-screen. C. elegans is a well-established research tool in biological and biochemical research for its ease of culture, small size, short generation time, and relative simplicity. Furthermore, the worm’s facility for genetic manipulation and remarkably similar cellular characteristics to those of humans have allowed for numerous advances in the study of cancer, neurodegeneration, and ageing. Our approach takes advantage of an automated worm tracking platform, developed in our laboratory, that can simultaneously track hundreds of worms and make precise measurements of their motility, defects of which has been shown to correlate with neurological and muscular toxicity. While standard approaches typically only take data on dozens of worms, the vastly increased population size of our approach greatly improves the statistical power of our screen. We have leveraged these improvements in screening methods to associate the differences in distributions of these parameters with phenotypic changes across various siRNA conditions. Through both motility screening and validation by imaging, we identified ckr-2, skr-21, and Y92H12A.2 as modulators of amyloid beta aggregation. While skr-21 and Y92H12A.2 are both components of the ubiquitin-proteasome system, ckr-2 is an ortholog of a neuronal cholecystokinin receptor which has been suggested to be a biomarker of AD but for which no mechanism is known. The results of this work thus contribute to extending our understanding of the gene signature of AD

Study of serum lactate dehydrogenase level and seasonal variation in preeclampsia and eclampsia with its obstetric outcome

Background: Preeclampsia and eclampsia complicate 6–8% of all pregnancies and lead to various maternal and fetal complications. LDH is an intracellular enzyme and its level is increased in this women due to cellular death. So, serum LDH levels can be used to assess the severity of disease, to improve the maternal and fetal outcome. Studies in several countries have shown higher incidence of the disease in the winter season. This study is being conducted to correlate serum LDH levels and seasonal variation in preeclampsia and eclampsia.Methods: It is a retrospective observational study. Data for 102 cases were collected from the parturition register and patient discharge record from January to December 2016. All singleton pregnant women who came to R L Jalappa Hospital with severe preeclampsia and eclampsia were included in the study.Results: Total of 102 patients were studied. Incidence of the disease was most commonly seen in younger age group, which was statistically significant (p=0.020). Even though most of the cases presented in winter (39), there was no statistically significant association between seasonal variation in occurrence of the disease and serum LDH levels. LDH raised to >800IU/L in the cases was seen more in the younger age group.Conclusions: This study only showed that preeclampsia and eclampsia occurred most commonly in younger women. This study did not show any variation in serum LDH levels in patients presenting in different seasons

Evaluation of vitamin D levels in term pregnancy and its obstetric outcome in Indian women

Background: Vitamin D deficiency is currently a global pandemic affecting all age groups. Vitamin D is considered a fundamental hormone in calcium homeostasis and bone health. Risk of vitamin D deficiency increases during pregnancy due to increased maternal and fetal demands and altered vitamin D metabolism. Recently, maternal vitamin D deficiency has been linked to adverse pregnancy outcomes, including preeclampsia, gestational diabetes, fetal growth restriction and preterm birth. Adequate vitamin D status appears to be relevant to health at all ages, and even in prenatal life.Methods: This is a cross sectional, observational study conducted in the department of obstetrics and gynaecology at R. L. Jalappa Hospital. A total number of 160 subjects were included. 5 ml of venous blood was collected and was centrifuged at 3000 rpm and stored at - 80°C till analysis. Analysis of 25-hydroxy Vitamin D was done using ELISA.Results: Majority of the subjects were vitamin D deficient (81.87%) and 12.5% were vitamin D insufficient and only 5.63% were vitamin D sufficient. The prevalence of vitamin D deficiency was more among primigravidas (85.6%) and was associated with higher rates of caesarean section (92.4%). High prevalence of vitamin D deficiency was seen in lower middle socioeconomic class (62.5%). Maternal vitamin D deficiency was associated low birth weight of neonates (100%).Conclusions: In this study it was concluded that majority of subjects were vitamin D deficient and belonged to lower middle socioeconomic class. Majority of this subjects who underwent caesarean section were vitamin D deficient. Vitamin D deficiency was associated only with low birth weight of neonates and no other adverse obstetric outcome

Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation.

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.This work was supported by the UK BBSRC and the Wellcome Trust (CMD, TPJK, MV), the Frances and Augustus Newman Foundation (TPJK), Magdalene College, Cambridge (AKB) , St John’s College, Cambridge (TCTM), the Cambridge Home and EU Scholarship Scheme (GM), Elan Pharmaceuticals (CMD, TPJK, MV, CG) and the Leverhulme Trust (AKB).This is the accepted manuscript. The final version is available from NPG at http://www.nature.com/nchembio/journal/v11/n3/abs/nchembio.1750.htm

Repair or destruction: an intimate liaison between ubiquitin ligases and molecular chaperones in proteostasis

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability [1, 2]. Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin/proteasome-system (UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite the large number of structurally unrelated substrates, ubiquitin conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 ubiquitin ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this Review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discusse

DNA-Liposome Hybrid Carriers for Triggered Cargo Release

The design of simple and versatile synthetic routes to accomplish triggered-release properties in carriers is of particular interest for drug delivery purposes. In this context, the programmability and adaptability of DNA nanoarchitectures in combination with liposomes have great potential to render biocompatible hybrid carriers for triggered cargo release. We present an approach to form a DNA mesh on large unilamellar liposomes incorporating a stimuli-responsive DNA building block. Upon incubation with a single-stranded DNA trigger sequence, a hairpin closes, and the DNA building block is allowed to self-contract. We demonstrate the actuation of this building block by single-molecule Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching, and fluorescence quenching measurements. By triggering this process, we demonstrate the elevated release of the dye calcein from the DNA-liposome hybrid carriers. Interestingly, the incubation of the doxorubicin-laden active hybrid carrier with HEK293T cells suggests increased cytotoxicity relative to a control carrier without the triggered-release mechanism. In the future, the trigger could be provided by peritumoral nucleic acid sequences and lead to site-selective release of encapsulated chemotherapeutics. © 2022 American Chemical Society. All rights reserved

Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

A metastable subproteome underlies inclusion formation in muscle proteinopathies

Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue

Supersaturated proteins are enriched at synapses and underlie cell and tissue vulnerability in Alzheimer's disease.

Neurodegenerative disorders progress across the brain in characteristic spatio-temporal patterns. A better understanding of the factors underlying the specific cell and tissue vulnerability responsible for such patterns could help identify the molecular origins of these conditions. To investigate these factors, based on the observation that neurodegenerative disorders are closely associated with the presence of aberrant protein deposits, we made the hypothesis that the vulnerability of cells and tissues is associated to the overall levels of supersaturated proteins, which are those most metastable against aggregation. By analyzing single-cell transcriptomic and subcellular proteomics data on healthy brains of ages much younger than those typical of disease onset, we found that the most supersaturated proteins are enriched in cells and tissues that succumb first to neurodegeneration. Then, by focusing the analysis on a metastable subproteome specific to Alzheimer's disease, we show that it is possible to recapitulate the pattern of disease progression using data from healthy brains. We found that this metastable subproteome is significantly enriched for synaptic processes and mitochondrial energy metabolism, thus rendering the synaptic environment dangerous for aggregation. The present identification of protein supersaturation as a signature of cell and tissue vulnerability in neurodegenerative disorders could facilitate the search for effective treatments by providing clearer points of intervention