Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis.

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.

Up-Regulation of the Long Noncoding RNA X-Inactive-Specific Transcript and the Sex Bias in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a sex-biased disease. Increased expression and activity of the long-noncoding RNA X-inactive-specific transcript (Xist), essential for X-chromosome inactivation and dosage compensation of X-linked genes, may explain the sex bias of PAH. The present studies used a murine model of plexiform PAH, the intersectin-1s (ITSN) heterozygous knockout (KOITSN+/-) mouse transduced with an ITSN fragment (EHITSN) possessing endothelial cell proliferative activity, in conjunction with molecular, cell biology, biochemical, morphologic, and functional approaches. The data demonstrate significant sex-centered differences with regard to EHITSN-induced alterations in pulmonary artery remodeling, lung hemodynamics, and p38/ETS domain containing protein/c-Fos signaling, altogether leading to a more severe female lung PAH phenotype. Moreover, the long-noncoding RNA-Xist is up-regulated in the lungs of female EHITSN-KOITSN+/- mice compared with that in female wild-type mice, leading to sex-specific modulation of the X-linked gene ETS domain containing protein and its target, two molecular events also characteristic to female human PAH lung. More importantly, cyclin A1 expression in the S and G2/M phases of the cell cycle of synchronized pulmonary artery endothelial cells of female PAH patients is greater versus controls, suggesting functional hyperproliferation. Thus, Xist up-regulation leading to female pulmonary artery endothelial cell sexual dimorphic behavior may provide a better understanding of the origin of sex bias in PAH. Notably, the EHITSN-KOITSN+/- mouse is a unique experimental animal model of PAH that recapitulates most of the sexually dimorphic characteristics of human disease.

Trends and Geographic Patterns in Drug and Synthetic Opioid Overdose Deaths - United States, 2013-2019.

Deaths involving synthetic opioids other than methadone (synthetic opioids), which largely consist of illicitly manufactured fentanyl; psychostimulants with abuse potential (e.g., methamphetamine); and cocaine have increased in recent years, particularly since 2013 (1,2). In 2019, a total of 70,630 drug overdose deaths occurred, corresponding to an age-adjusted rate of 21.6 per 100,000 population and a 4.3% increase from the 2018 rate (20.7) (3). CDC analyzed trends in age-adjusted overdose death rates involving synthetic opioids, psychostimulants, cocaine, heroin, and prescription opioids during 2013-2019, as well as geographic patterns in synthetic opioid- and psychostimulant-involved deaths during 2018-2019. From 2013 to 2019, the synthetic opioid-involved death rate increased 1,040%, from 1.0 to 11.4 per 100,000 age-adjusted (3,105 to 36,359). The psychostimulant-involved death rate increased 317%, from 1.2 (3,627) in 2013 to 5.0 (16,167) in 2019. In the presence of synthetic opioid coinvolvement, death rates for prescription opioids, heroin, psychostimulants, and cocaine increased. In the absence of synthetic opioid coinvolvement, death rates increased only for psychostimulants and cocaine. From 2018 to 2019, the largest relative increase in the synthetic opioid-involved death rate occurred in the West (67.9%), and the largest relative increase in the psychostimulant-involved death rate occurred in the Northeast (43.8%); these increases represent important changes in the geographic distribution of drug overdose deaths. Evidence-based prevention and response strategies including substance use disorder treatment and overdose prevention and response efforts focused on polysubstance use must be adapted to address the evolving drug overdose epidemic.

Early modulation of the gut microbiome by female sex hormones alters amyloid pathology and microglial function.

It is well-established that women are disproportionately affected by Alzheimer's disease. The mechanisms underlying this sex-specific disparity are not fully understood, but several factors that are often associated-including interactions of sex hormones, genetic factors, and the gut microbiome-likely contribute to the disease's etiology. Here, we have examined the role of sex hormones and the gut microbiome in mediating Aß amyloidosis and neuroinflammation in APPPS1-21 mice. We report that postnatal gut microbiome perturbation in female APPPS1-21 mice leads to an elevation in levels of circulating estradiol. Early stage ovariectomy (OVX) leads to a reduction of plasma estradiol that is correlated with a significant alteration of gut microbiome composition and reduction in Aß pathology. On the other hand, supplementation of OVX-treated animals with estradiol restores Aß burden and influences gut microbiome composition. The reduction of Aß pathology with OVX is paralleled by diminished levels of plaque-associated microglia that acquire a neurodegenerative phenotype (MGnD-type) while estradiol supplementation of OVX-treated animals leads to a restoration of activated microglia around plaques. In summary, our investigation elucidates the complex interplay between sex-specific hormonal modulations, gut microbiome dynamics, metabolic perturbations, and microglial functionality in the pathogenesis of Alzheimer's disease.

Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner.

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aß deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aß amyloidosis in the 5XFAD mouse model that were treated at a point when Aß burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aß amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aß burden was detectable upto 12 weeks of age when Aß burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aß burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-ß deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aß deposition or when given after Aß deposition is already at higher levels.

Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis.

Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.

Chromatin reprogramming and bone regeneration in vitro and in vivo via the microtopography-induced constriction of cell nuclei.

Topographical cues on cells can, through contact guidance, alter cellular plasticity and accelerate the regeneration of cultured tissue. Here we show how changes in the nuclear and cellular morphologies of human mesenchymal stromal cells induced by micropillar patterns via contact guidance influence the conformation of the cells' chromatin and their osteogenic differentiation in vitro and in vivo. The micropillars impacted nuclear architecture, lamin A/C multimerization and 3D chromatin conformation, and the ensuing transcriptional reprogramming enhanced the cells' responsiveness to osteogenic differentiation factors and decreased their plasticity and off-target differentiation. In mice with critical-size cranial defects, implants with micropillar patterns inducing nuclear constriction altered the cells' chromatin conformation and enhanced bone regeneration without the need for exogenous signalling molecules. Our findings suggest that medical device topographies could be designed to facilitate bone regeneration via chromatin reprogramming.

Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer.

Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer. SIGNIFICANCE: This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.

A tumor suppressor role for EZH2 in diffuse midline glioma pathogenesis.

Pediatric high-grade gliomas, specifically diffuse midline gliomas, account for only 20% of clinical cases but are 100% fatal. A majority of the DMG cases are characterized by the signature K27M mutation in histone H3. The H3K27M mutation opposes the function of enhancer of zeste homolog 2 (EZH2), the methyltransferase enzyme of the polycomb repressor complex 2. However, the role of EZH2 in DMG pathogenesis is unclear. In this study, we demonstrate a tumor suppressor function for EZH2 using Ezh2 loss- and gain-of-function studies in H3WT DMG mouse models. Genetic ablation of Ezh2 increased cell proliferation and tumor grade while expression of an Ezh2 gain-of-function mutation significantly reduced tumor incidence and increased tumor latency. Transcriptomic analysis revealed that Ezh2 deletion upregulates an inflammatory response with upregulation of immunoproteasome genes such as Psmb8, Psmb9, and Psmb10. Ezh2 gain-of-function resulted in enrichment of the oxidative phosphorylation/mitochondrial metabolic pathway namely the isocitrate dehydrogenase Idh1/2/3 genes. Pharmacological inhibition of EZH2 augmented neural progenitor cell proliferation, supporting the tumor suppressive role of EZH2. In vivo 7-day treatment of H3K27M DMG tumor bearing mice with an EZH2 inhibitor, Tazemetostat, did not alter proliferation or significantly impact survival. Together our results suggest that EZH2 has a tumor suppressor function in DMG and warrants caution in clinical translation of EZH2 inhibitors to treat patients with DMG.

Gut microbiota-driven brain Aß amyloidosis in mice requires microglia.

We previously demonstrated that lifelong antibiotic (ABX) perturbations of the gut microbiome in male APPPS1-21 mice lead to reductions in amyloid ß (Aß) plaque pathology and altered phenotypes of plaque-associated microglia. Here, we show that a short, 7-d treatment of preweaned male mice with high-dose ABX is associated with reductions of Aß amyloidosis, plaque-localized microglia morphologies, and Aß-associated degenerative changes at 9 wk of age in male mice only. More importantly, fecal microbiota transplantation (FMT) from transgenic (Tg) or WT male donors into ABX-treated male mice completely restored Aß amyloidosis, plaque-localized microglia morphologies, and Aß-associated degenerative changes. Transcriptomic studies revealed significant differences between vehicle versus ABX-treated male mice and FMT from Tg mice into ABX-treated mice largely restored the transcriptome profiles to that of the Tg donor animals. Finally, colony-stimulating factor 1 receptor (CSF1R) inhibitor-mediated depletion of microglia in ABX-treated male mice failed to reduce cerebral Aß amyloidosis. Thus, microglia play a critical role in driving gut microbiome-mediated alterations of cerebral Aß deposition.

Machine learning-assisted elucidation of CD81-CD44 interactions in promoting cancer stemness and extracellular vesicle integrity.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Selective progesterone receptor blockade prevents BRCA1-associated mouse mammary tumors through modulation of epithelial and stromal genes.

Pharmacological approaches to breast cancer risk-reduction for BRCA1 mutation carriers would provide an alternative to mastectomy. BRCA1-deficiency dysregulates progesterone signaling, promoting tumorigenesis. Selective progesterone receptor (PR) modulators (SPRMs) are therefore candidate prevention agents. However, their efficacy varies in different BRCA1-deficient mouse models. We examined chemopreventive efficacy of telapristone acetate (TPA), ulipristal acetate (UPA) and mifepristone (MFP) in mice with a conditional knockout of the Brca1 C-terminal domain. The SPRMs displayed a spectrum of efficacy: UPA was most effective, TPA less, and MFP ineffective. Compared to no-treatment controls, UPA reduced tumorigenesis (p = 0.04), and increased tumor latency (p = 0.03). In benign mammary glands, UPA decreased Ki67 (p < 0.001) and increased PR expression (p < 0.0001). RNA sequencing analysis revealed distinct gene expression in response to UPA and MFP. UPA downregulated glycolysis and extracellular matrix-inflammation genes (Fn1, Ptgs2, Tgfb2, Tgfb3) whereas MFP downregulated claudin genes and upregulated amino acid metabolism and inflammation genes. The anti-glucocorticoid effects of MFP appeared not to be tumor-protective, while altering estrogen receptor signaling and NF-kB activation. Our study points to an important role of epithelial PR and its paracrine action on the microenvironment in BRCA1-deficient mammary tumorigenesis, and prevention.

Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population.

Purpose: Single-cell RNA-sequencing (scRNA-seq) was used to interrogate the relatively rare stem (SC) and early transit amplifying (TA) cell populations in limbal/corneal epithelia from wild-type and autophagy-compromised mice. Methods: We conducted scRNA-seq on ocular anterior segmental tissue from wild-type and beclin 1-deficient (beclin1+/-) mice, using a 10X Gemomics pipeline. Cell populations were distinguished by t-distributed stochastic neighbor embedding. Seurat analysis was conducted to compare gene expression profiles between these two groups of mice. Differential protein expression patterns were validated by immunofluorescence staining and immunoblotting. Results: Unbiased clustering detected 10 distinct populations: three clusters of mesenchymal and seven clusters of epithelial cells, based on their unique molecular signatures. A discrete group of mesenchymal cells expressed genes associated with corneal stromal SCs. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. Thioredoxin-interacting protein and PDZ-binding kinase (PBK) were identified as novel regulators of stem/early TA cell quiescence. PBK arrested corneal epithelial cells in G2/M phase of the cell cycle. Beclin1+/- mice displayed a decrease in proliferation-associated (Ki67, Lrig1) and stress-response (H2ax) genes. The most increased gene in beclin1+/- mice was transcription factor ATF3, which negatively regulates limbal epithelial cell proliferation. Conclusions: Establishment of a comprehensive atlas of genes expressed by stromal and epithelial cells from limbus and cornea forms the foundation for unraveling regulatory networks among these distinct tissues. Similarly, scRNA-seq profiling of the anterior segmental epithelia from wild-type and autophagy-deficient mice provides new insights into how autophagy influences proliferation in these tissues.

A dynamic thermoregulatory material inspired by squid skin.

Effective thermal management is critical for the operation of many modern technologies, such as electronic circuits, smart clothing, and building environment control systems. By leveraging the static infrared-reflecting design of the space blanket and drawing inspiration from the dynamic color-changing ability of squid skin, we have developed a composite material with tunable thermoregulatory properties. Our material demonstrates an on/off switching ratio of ~25 for the transmittance, regulates a heat flux of ~36 W/m2 with an estimated mechanical power input of ~3 W/m2, and features a dynamic environmental setpoint temperature window of ~8 °C. Moreover, the composite can manage one fourth of the metabolic heat flux expected for a sedentary individual and can also modulate localized changes in a wearer's body temperature by nearly 10-fold. Due to such functionality and associated figures of merit, our material may substantially reduce building energy consumption upon widespread deployment and adoption.

An introduction to color-changing systems from the cephalopod protein reflectin.

Cephalopods possess unrivaled camouflage and signaling abilities that are enabled by their sophisticated skin, wherein multiple layers contain chromatophore pigment cells (as part of larger chromatophore organs) and different types of reflective cells called iridocytes and leucophores. The optical functionality of these cells (and thus cephalopod skin) critically relies upon subcellular structures partially composed of unusual structural proteins known as reflectins. Herein, we highlight studies that have investigated reflectins as materials within the context of color-changing coatings. We in turn discuss these proteins' multi-faceted properties, associated challenges, and future potential. Through our presentation of selected case studies, we hope to stimulate additional dialogue and spur further research on photonic technologies based on and inspired by reflectins.

Cephalopod-Derived Biopolymers for Ionic and Protonic Transistors.

Cephalopods (e.g., squid, octopuses, and cuttlefish) have long fascinated scientists and the general public alike due to their complex behavioral characteristics and remarkable camouflage abilities. As such, these animals are explored as model systems in neuroscience and represent a well-known commercial resource. Herein, selected literature examples related to the electrical properties of cephalopod-derived biopolymers (eumelanins, chitosans, and reflectins) and to the use of these materials in voltage-gated devices (i.e., transistors) are highlighted. Moreover, some potential future directions and challenges in this area are described, with the aim of inspiring additional research effort on ionic and protonic transistors from cephalopod-derived biopolymers.

Mutation-based structural modification and dynamics study of amyloid beta peptide (1-42): An in-silico-based analysis to cognize the mechanism of aggregation.

Alzheimer's disease is the prevalent cause of premature senility, a progressive mental disorder due to degeneration in brain and deposition of amyloid ß peptide (1-42, a misfolded protein) in the form of aggregation that prevails for a prolonged time and obstructs every aspect of life. One of the primary hallmarks of the neuropathological disease is the accretion of amyloid ß peptide in the brain that leads to Alzheimer's disease, but the mechanism is still a mystery. Several investigations have shown that mutations at specific positions have a significant impact in stability of the peptide as predicted from aggregation profiles. Here in our study, we have analyzed the mutations by substituting residues at position A22G, E22G, E22K, E22Q, D23N, L34V and molecular dynamics have been performed to check the deviation in stability and conformation of the peptide. The results validated that the mutations at specific positions lead to instability and the proline substitution at E22P and L34P stalled the aggregation of the peptide.