Hsa-miR-4520-2-3p: A Potential Modulator of COVID-related ACE2

ACE2 is a transmembrane receptor located in cells in various tissues around the body. Its normal role is the conversion of Angiotensin II to Angiotensin 1-7 leading to vasodilation and a subsequent reduction in blood pressure via the renin-angiotensin-aldosterone system. ACE2 also plays a pivotal role in the infection of COVID-19 as it determines entry of virus into human cells. SARS-CoV-2 uses one of its four structural proteins, the spike (S) glycoprotein, to bind to the ACE2 receptor. This entry into the cell begins the process of infection and spread of the disease; because of the abundance of ACE2 throughout the body, viruses are able to enter a number of different organs. This multi-cell-entry via ACE2 is why COVID-19 affects multiple organ systems in the human body. Currently, therapies targeting ACE2 expression are limited. MicroRNA (miRNA)s are short non-coding RNA that downregulate the expression of a variety of proteins, and therefore offer a mechanism by which to inhibit the expression of ACE2. MiRNAs can potentially downregulate ACE2 protein synthesis by binding to the mRNA of ACE2 at a seed sequence. This binding results in mRNA degradation or inhibition of translation. Thus, our goal was to identify miRNA that may potentially target and downregulate ACE2. TargetScan, DIANA-MicroT, and PicTar are three algorithms that predict miRNAs that can bind to the mRNA of ACE2 and downregulate its expression. Together, the prediction tools resulted in 57 shared human miRNAs that target ACE2. We then utilized self-defined parameters to narrow down our list and identified hsa-miR-4520-2-3p as our predicted miRNA to target ACE2. This miRNA will be experimentally verified in the future. Supervisor: Prof Nipun Chopra, Ph

The microrna-mediated regulation of proteins implicated in the pathogenesis of Alzheimer's Disease

Indiana University-Purdue University Indianapolis (IUPUI)Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the post-mortem deposition of amyloid-beta (Aβ) containing neuritic plaques and tau-loaded tangles. According to the amyloid hypothesis, the generation of Aβ via the cleavage of Aβ precursor protein (APP) by β-APP site-cleaving enzyme 1 (BACE1) is a causative step in the development of AD. Therefore, targeting the production and/or clearance of Aβ peptide (by Aβ-degrading enzymes such as Neprilysin) would help understand the disorder as well as serves as therapeutic potential to treat the disorder. MicroRNA are small, noncoding RNA capable of modulating protein expression by primarily targeting their 3’UTR. Therefore, identifying miRNA which target APP, BACE1 and Neprilysin (NEP) would elucidate the complicated regulatory mechanisms involved in protein turnover and provide novel drug targets. We identified miR-20b as a modulator of APP and soluble Aβ. We also identified the target site for miR-20b’s binding on the APP 3’UTR. Further, miR-20b exerts influence on neuronal morphology, likely due to its APP reduction. We also identified miR-298 as a dual regulator of APP and BACE1 and confirmed miR-298’s targeting of both 3’UTRs. We also showed that miR-298 overexpression reduced levels of both soluble Aβ40 and Aβ42 peptides. Additionally, we identified two SNPs in proximity to the MIR298 gene, which are associated with AD-related biomarkers. Based on these results, we showed miR-298 targets a specific isoform of tau by putatively binding a non-canonical target site on the MAPT 3’UTR. Finally, the insertion of the NEP 3’UTR into a reporter vector increases reporter expression; suggesting regulatory elements targeting the 3’UTR. We subsequently identified miR-216 as reducing NEP 3’UTR-mediated luciferase activity. We also measured levels of NEP protein in various mammalian tissue – such as rodent and human fetal tissue, and subsequently showed measurable Aβ levels in correlation with NEP expression. Therefore, herein, we have identified miRNA involved in the regulation of proteins implicated in the pathogenesis of AD

MicroRNA-298 reduces levels of human amyloid-β precursor protein (APP), β-site APP-converting enzyme 1 (BACE1) and specific tau protein moieties

Alzheimer’s disease (AD) is the most common age-related form of dementia, associated with deposition of intracellular neuronal tangles consisting primarily of hyperphosphorylated microtubule-associated protein tau (p-tau) and extracellular plaques primarily comprising amyloid- β (Aβ) peptide. The p-tau tangle unit is a posttranslational modification of normal tau protein. Aβ is a neurotoxic peptide excised from the amyloid-β precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. MicroRNAs (miRNAs) are short, single-stranded RNAs that modulate protein expression as part of the RNA-induced silencing complex (RISC). We identified miR-298 as a repressor of APP, BACE1, and the two primary forms of Aβ (Aβ40 and Aβ42) in a primary human cell culture model. Further, we discovered a novel effect of miR-298 on posttranslational levels of two specific tau moieties. Notably, miR-298 significantly reduced levels of ~55 and 50 kDa forms of the tau protein without significant alterations of total tau or other forms. In vivo overexpression of human miR-298 resulted in nonsignificant reduction of APP, BACE1, and tau in mice. Moreover, we identified two miR-298 SNPs associated with higher cerebrospinal fluid (CSF) p-tau and lower CSF Aβ42 levels in a cohort of human AD patients. Finally, levels of miR-298 varied in postmortem human temporal lobe between AD patients and age-matched non-AD controls. Our results suggest that miR-298 may be a suitable target for AD therapy

Inhibiting S100β and Troubleshooting Cell Growth

S100B is a protein that is upregulated in neuronal injury. The upregulation of S100B has been observed to start multiple cascades that result in neuronal death. The inhibition of S100B would be a promising treatment for neuronal injury. In our research, we attempt to do exactly so

Novel Nuclear Factor-KappaB Targeting Peptide Suppresses β-Amyloid Induced Inflammatory and Apoptotic Responses in Neuronal Cells

In the central nervous system (CNS), activation of the transcription factor nuclear factor-kappa B (NF-κβ) is associated with both neuronal survival and increased vulnerability to apoptosis. The mechanisms underlying these dichotomous effects are attributed to the composition of NF-κΒ dimers. In Alzheimer’s disease (AD), β-amyloid (Aβ) and other aggregates upregulate activation of p65:p50 dimers in CNS cells and enhance transactivation of pathological mediators that cause neuroinflammation and neurodegeneration. Hence selective targeting of activated p65 is an attractive therapeutic strategy for AD. Here we report the design, structural and functional characterization of peptide analogs of a p65 interacting protein, the glucocorticoid induced leucine zipper (GILZ). By virtue of binding the transactivation domain of p65 exposed after release from the inhibitory IκΒ proteins in activated cells, the GILZ analogs can act as highly selective inhibitors of activated p65 with minimal potential for off-target effects

Identifying an appropriate in vitro model to study the effects of microRNA-4705 on S100β expression

Traumatic brain injury (TBI) is caused by external force to the head. In addition to the primary injury sustained by the impact, TBI can trigger a chronic inflammatory response that causes further tissue damage and neuronal death. Following TBI, glial cells of the central nervous system secrete elevated levels of the S100βprotein. S100βcan activate and upregulate the Receptor for Advanced Glycation End Products (RAGE). The RAGE pathway activates NFκB, a transcription factor involved in production of pro-inflammatory cytokines, resulting in infiltration of immune cells from the peripheral blood. Unchecked, the inflammatory response is thought to cause excessive tissue damage and neuronal death. It is hypothesized that downregulation of S100βfollowing TBI may ameliorate damage caused by chronic neuroinflammation. Our lab aims to downregulate S100βusing microRNA. Previously, our lab used bioinformatics to identify a candidate microRNA, miR-4705, that might target S100β. We aim to investigate whether miR-4705 can downregulate S100βin human SK-MEL-28 cells. Thus far, we have gathered evidence that SK-MEL-28 cells express S100β (shown in Fig. 1). However, we have not been able to demonstrate that S100βsiRNA downregulates S100β, suggesting that the transfection was unsuccessful. These preliminary experiments have served to test the validity of our in vitro model for studying S100β expression. Our next steps will be to repeat our siRNA experiments and to begin testing the effect of miR-4705 on S100β expression in SK-MEL-28 cells

Effect of a Hexylamine Derivative on Cancer Cell Viability

2-[(p-Chlorophenyl)hydroxymethyl]-1-[(methylamino)hexyl]cyclohexanol is a drug produced by hexyl amine and an epoxide through an aldol epoxidation reaction. Motifs of β-amino alcohols and nonpolar R groups in organic compounds have been found to have cytotoxic properties. Past studies in Dr. Hansen’s lab has shown that this hexylamine derivative has similar LC50 values to other antitumor agents. They also found that the drug was cytotoxic to HL-60 cancer cells. No other cell lines have been tested with this drug. Our study investigates the effect of our compound on varying cell lines to further determine its anticancer properties. Mouse NIH/3T3, Human HEK293, and Human SK-MEL-28 cell lines were cultured and plated into 96-well plates. Varying concentrations of the hexylamine derivative were administered and incubated for 48 hours. MTT assays detected the levels of cell viability. Results showed a significant decrease in HEK293 cells at a 30 μM concentration of our drug. The mouse and cancer cell lines did not produce significant results after statistical ANOVA tests. Future directions include further validation of the current results as well as research on the mechanisms by which this drug causes decreased cell viability. This research includes LDH and wound healing assays in addition to determining which proteins are down- and up-regulated in the process. Our study has found that the compound reduces human embryonic cell viability but does not significantly affect mouse cells or human melanoma cells. Further research is required to determine the methods of the drug and its potential in tumor treatment

Lithium alters expression of RNAs in a type-specific manner in differentiated human neuroblastoma neuronal cultures, including specific genes involved in Alzheimer's disease.

Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple nervous system disorders, including Alzheimer's disease (AD). While perturbation of RNA levels by Li has been previously reported, its effects on the whole transcriptome has been given little attention. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels. We cultured and differentiated human neuroblastoma (SK-N-SH) cells to neuronal cells with all-trans retinoic acid (ATRA). We exposed cultures for one week to lithium chloride or distilled water, extracted total RNA, depleted ribosomal RNA and performed whole-transcriptome RT-sequencing. We analyzed results by RNA length and type. We further analyzed expression and protein interaction networks between selected Li-altered protein-coding RNAs and common AD-associated gene products. Lithium changed expression of RNAs in both non-specific (inverse to sequence length) and specific (according to RNA type) fashions. The non-coding small nucleolar RNAs (snoRNAs) were subject to the greatest length-adjusted Li influence. When RNA length effects were taken into account, microRNAs as a group were significantly less likely to have had levels altered by Li treatment. Notably, several Li-influenced protein-coding RNAs were co-expressed or produced proteins that interacted with several common AD-associated genes and proteins. Lithium's modification of RNA levels depends on both RNA length and type. Li activity on snoRNA levels may pertain to bipolar disorders while Li modification of protein coding RNAs may be relevant to AD

Regulation of Proteins Implicated in Alzheimer’s Disease by MicroRNAs

poster abstractAlzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the deposition of Amyloid-Beta (Aβ) peptide in the brain. This toxic peptide is generated by the sequential cleavage of Amyloid Precursor Protein (APP) by Beta-site APP-cleaving enzyme-1 (BACE-1) and γ-secretase. The disorder is also characterized by the perturbation of calcium homeostasis in neurons. MicroRNAs are short, single-stranded RNAs that are able to influence protein expression by targeting the 3’ Untranslated region (UTR) or 5’ UTR of mRNAs. Previous work in our laboratory has shown that miR-101, miR-153 and miR-346 can regulate APP whereas miR-339-5p can lower BACE1 expression. Here, we aim to reduce APP, BACE1 and Aβ levels, in vitro, by the addition of microRNAs that target the 3’ UTR of APP and BACE1. We show that in a human astrocytoma-glioblastoma (U373) cell line, the expression of BACE1 protein is significantly reduced compared to the mock condition upon transfecting miR-298, miR-328 and miR-144. miR-298 also reduces Aβ levels in these cells. Similarly, in HeLa cells, we show that miR-520c, miR-20b and miR-144 produce a reduction in APP expression compared to both mock and a negative control microRNA mimic. Additionally, we observed that knocking down APP using siRNA, but not knocking down BACE1, lowers basal intracellular calcium levels as well as changes the kinetics of Potassium Chloride (KCl)-induced intracellular calcium influx in a human fetal brain (HFB) culture, when compared to control. miR-346 increases basal calcium levels, but does not affect KCl-induced calcium transients in our HFB culture. Taken together, these results show that miRNAs can influence both the protein expression as well as calcium homeostasis in different human cell culture models. By reducing levels of proteins implicated in AD pathology and by reversing calcium dysregulation, our results will benefit AD research and generate possibilities for novel therapeutics

Repeated electromagnetic field stimulation lowers amyloid-β peptide levels in primary human mixed brain tissue cultures

Late Onset Alzheimer’s Disease is the most common cause of dementia, characterized by extracellular deposition of plaques primarily of amyloid-β (Aβ) peptide and tangles primarily of hyperphosphorylated tau protein. We present data to suggest a noninvasive strategy to decrease potentially toxic Aβ levels, using repeated electromagnetic field stimulation (REMFS) in primary human brain (PHB) cultures. We examined effects of REMFS on Aβ levels (Aβ40 and Aβ42, that are 40 or 42 amino acid residues in length, respectively) in PHB cultures at different frequencies, powers, and specific absorption rates (SAR). PHB cultures at day in vitro 7 (DIV7) treated with 64 MHz, and 1 hour daily for 14 days (DIV 21) had significantly reduced levels of secreted Aβ40 (p = 001) and Aβ42 (p = 0.029) peptides, compared to untreated cultures. PHB cultures (DIV7) treated at 64 MHz, for 1 or 2 hour during 14 days also produced significantly lower Aβ levels. PHB cultures (DIV28) treated with 64 MHz 1 hour/day during 4 or 8 days produced a similar significant reduction in Aβ40 levels. 0.4 W/kg was the minimum SAR required to produce a biological effect. Exposure did not result in cellular toxicity nor significant changes in secreted Aβ precursor protein-α (sAPPα) levels, suggesting the decrease in Aβ did not likely result from redirection toward the α-secretase pathway. EMF frequency and power used in our work is utilized in human magnetic resonance imaging (MRI, thus suggesting REMFS can be further developed in clinical settings to modulate Aβ deposition