Garrison Institute on Aging

MicroRNAs (miRNAs) are involved in growth, development, and occurrence and progression of many diseases. MiRNA mediated post-transcriptional regulation is poorly understood in vascular biology and pathology. The purpose of our study is to determine circulatory miRNAs as early detectable peripheral biomarkers in patients with ischemic stroke (IS). MiRNAs expression levels were measured in IS serum samples and healthy controls using Illumina deep sequencing analysis and identified differentially expressed miRNAs. Differentially expressed miRNAs were further validated using SYBR-green based quantitative real-time PCR assay (qRT-PCR) in postmortem IS brains, lymphoblastiod IS cell lines, OGD/R treated human and mouse neuroblastoma cells, and mouse models of hypoxia and ischemia (HI) induced stroke. A total of 4,656 moRNAs were differentially expressed in IS serum samples relative to healthy controls. Out of 4,656 miRNAs, 272 were found to be significantly deregulated in IS patients. Interestingly, we found several novel and previously unreported miRNAs in IS patients relative to healthy controls. Further analysis revealed that some candidate miRNAs and its target genes were involved in the revealed that some candidate miRNAs and its target genes were involved in the regulation of the stroke. To the best of our knowledge, this is the first study identified potential novel candidate miRNAs in IS serum samples from the residents of rural West Texas. MiRNAs identified in the present study could potentially be used as a biomarker and the development of novel therapeutic approaches for stroke. Further studies are necessary to better understand miRNAs-regulated stroke cellular changes.

Authors: Vijayan, Kumar, Yin, Zafer, Chanana, Cengiz, Reddy PH

The purpose of our study was to determine the toxic effects of hippocampal mutant APP and amyloid beta (AB) in human mutant APP (mAPP) cDNA transfected with primary mouse hippocampal neurons (HTT22). Hippocampal tissues are the best source of studying learning and memory functions in patients with Alzheimer's disease (AD) and healthy controls. However, investigating immortalized hippocampal neurons that express AD proteins provide an excellent opportunity for drug testing. Using quantitative RT-PCR, immunoblotting & immunofluorescence, and transmission electron microscopy, we assessed mRNA and protein levels of synaptic, sutophagy, mitophagy, mitochondrial dynamics, diogenesis, dendritic, dendritic protein MAP2, an assessed mitochondrial number and length in mAPP-HT22 cells that express Swedich/Indiana mutations. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid perovidation, cytochrome c oxidase activity and mitochondrial ATP. Increased levels of mRNA and protein levels of mitochondrial fission genes, Drp1 and Fis1 and decreased levels fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1a, NRF1, NRF2 & TFAM). autophagy (ATG5 & LC3BI, LC3BII), mitophagy (PINK1 & TERT, BCL2 & BNIPBL), synaptic (Synaptophysin & PSD95) and dendritic (MAP2) genes were found in mAPP-HT22 cells relative to WT-HT22 cells. Cell survival was significantly reduced mAPP-HT22 cells. GTPase-Dp1 enzymatic activity was increased in mAPP-HT22 cells. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in mAPP-HT22 cells. These findings suggest that hippocampal accumulation of mutant APP and AB is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins & reduced dendritic spines and mitochondrial structural and functional changes in mutant APP hippocampal cels. These observations strongly suggest that accumulation of mAPP and AB causes mitochondrial, synaptic and autophagy/mitophagy abnormalities in hippocampal neurons, leading to neuronal dysfuntion.

Authors: Reddy PH, Yin, Manczah, Kumar, Jangampalli Adi, Vijayan, Reddy AP

A non-invasive and early-detectable peripheral biomarker is urgently needed for Alzheimer's disease (AD). The present study is a step forward to verify the biomarker Properties of human mocroRNA-455-3p (Hsa-miR-455-3p) in AD patients. Our previous findings on mild cognitive impaired subjects, AD patients and AD cells and mouse models unveiled the miR-455-3p as a potential peripheral biomarker for AD. In the current study, we verified the differential expression of miR-455-3p in post-mortem AD brains obtained from NIH NeuroBioBank, and fibroblasts and B-lympho-cytes from both familial and sporadic AD patients from Coriell Cell Repository of National Institutes on Aging. Total RNA was extracted from the fibroblasts, B-lympho-cytes and AD postmortem brains, and expression of miR-455-3p was measured by real-time reverse-transcriptase RT-PCR. Our real-time RT-PCR analysis showed a significant (P=0.0002) upregulation of miR-455-3p expression in AD postmortem brains compared to healthy control samples. Expression of miR-455-3p was also upregulated in the fibroblasts from AD patients, however a significant difference in miR-455-3p level was observed in the cell from sporadic AD patients (P=0.014) compared to healthy controls. Similarly, in B-lymphocytes, miR-455-3p level was also higher (P=0.044) especially in sporadic AD cases compared to controls. Receiver operating characteristic (ROC) curve analysis indicated the significant area under ROC curve (AUROC) value of miR-455-3p in AD postmortem brain (AUROC=0.792; P=0.001) and AD fibroblasts cells (AUROC=0.861;P=0.03), whereas in B-lymphocytes AUROC value of miR-455-3p was not significant. Further, in-silico analysis for miRNA targets predictions showed the binding capacity of miR-455-3p with several AD associated key genes such as APP, NGF, USP25, PDRG1, SMAD4, UBQLN1, SMAD2, TP73, VAMP2, HSPBAP1, and NRXN1. Hence, these observations further revealed that miR-455-3p is a potential biomarker for AD and its possible therapeutic target for AD. 

Authors: Kumar, Reddy PH

The purpose of our study was to determine the toxic effects of hippocampal mutant APP and amyloid beta (AB) in 12 month old APP transgenic mice. Using rotarod and Morris water maze tests, immunoblotting and immunoflourescence, Golgi-cox staining and transmission electron microscopy, we assessed cognitive behavior, protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2 and quantified dendritic spines and mitochondrial number and length in 12 month-old APP mice that express Swedish mutation. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid perocidation, cytochrome c oxidase activity and mitochondrial ATP. Morris water maze and rotarod test revealed that hippocampal learning and memory and motor learning and coordination were impaired in APP mice relative to wild-type (WT) mice. Increased levels of mitochondrial fission proteins, Drp1 and Fis1 and decreased levels of fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1a, NRF1, NRF2 and TFAM), autophagy )ATG5 and LC3BI, LC3BII), mitophagy (Pink1 and TERT), synaptic (Synaptophysin and PSD95) and dendritic (MAP2) proteins were found in 12 month-old APP mice relative to age-matched non-transgenic WT mice. Golgi-cox staining analysis revealed that dendritic spines are significantly reduced. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in APP mice. These findings suggest that hippocampal accumulation of mutant APP and AB is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins and reduced dendritic spines and hippocampal-based learning and memory impairments and mitochondrial structural and functional changes in 12 month-old APP mice.

Authors: Manczak, Kandimalla, Yin, Reddy PH

The purpose of our study was to determine the synergistic protective effects of mitochondria-targeted antioxidant SS31 and mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Using biochemical methods, we assessed mitochondrial function by measuring the levels of hydrogen peroxide, lipid peroxidation, cyto-chrome c oxidase activity, mitochondrial ATP, and GTPase Drp1 enzymatic activity in mutant ABPP cells. Using biochemical methods, we also measured cell survival and apoptotic cell death. Amyloid-B (AB) levels were measured using sandwich ELISA, and using real-time quantitative RT-PCR, we assessed mtDNA (mtDNA) copy number in relation to nuclear DNA (nDNA) in all groups of cells, We found significantly reduced levels of AB40 and AB42 in mutant ABPP cells treated with SS31, Mdici1, and SS31+Mdivi1, and the reduction of AB42 levels were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. The levels of mtDNA copy number and cell survival were significantly increased in SS31, Mdivi1, and SS31+Mdivi1 treated mutant ABPP cells; however, the increased levels of mtDNA copy number and cell survival were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. Mitochondrial dysfunctions is significantly reduced in SS31, Mdivi1, and SS31+Mdivi1 treated mutant ABPP cells; however, the reduction is much higher in cells treated with both SS31+Mdvi1. Similarly, GTPase Drp1 activity is reduced in all treatments, but reduced much higher in SS31+Mdivi1 treat cells. These observations strongly suggest that combined treatment of SS31+Mdivi1 is effective than individual treatments of SS31 and Mdivi1. Therefore, we propose that combined treatment of SS31+Mdivi1 is a better therapeutic strategy for AD. Ours is the first study to investigate combined treatment of mitochondria-targeted antioxidant SS31 and mitochondrial division inhibitor 1 in AD neurons.

Authors: Reddy PH, Manczak, Yin, Reddy AP

The purpose of our article is to assess the current understanding of Indian spice, curcumin, against amyloid-B (AB)-induced toxicity in Alzheimer's disease (AD) pathogenesis. Natural products, such as ginger, curcumin, and gingko biloba have been used a diets and dietary supplements to treat human disesases, including cancer, cardiovascular, respiratory, infectious, diabetes, obesity, metabolic syndromes and neurological disorders. Products derived from plants are known to have protective effects, including anti-infammatory, antioxidant, anti-arthritis, pro-healing, and boosting memory cognitive functions. In the last decade, several groups have designed and synthesized curcumin and its derivatives and extensively tested using cell and mouse models of AD. Recent research of AM and curcumin has revealed that curcumin prevents AB in humans. Recent research has also reported that curcumin ameliorates cognitive decline and improves synaptic functions in mouse models of AD. Further, recent groups have initiated studies on elderly individuals and patients with AD and the outcome of these studies is currently being assessed. This article highlights the beneficial effects of curcumin on AD. This article also critically assesses the current limitations of curcumin's bioavailabitlity and urgent need for new formulations to increase its brain levels to treat patients with AD. 

Authors; Reddy PH, Manczak, Yin, Grady, Mitchell, Tonk, Kuruva, Bhatti, Kandimalla, Vijayan, Kumar, Wang, Pradeepkiran, Ogunmokun, Thamarai, Quesada, Boles, Reddy AP

The purpose of our study was to understand the toxic effects of hippocampal phosphorylated tau in tau mice. Using rotarod and Morris water maze (MWM) tests, immunoblotting and immunoflourescence, Golgi-Cox staining and transmission electron microscopy, we assessed cognitive behavior, measured protein levels of mitochondrial dynamics, MAP2, total and phosphorylated tau, and quantified dendritic spines and mitochondrial number and length in 12 month-old tau mice with P301L mutation. Mitochondrial function was assessed by measuring the levels of H202, lipid perovidation, cytochrome oxidase activity and mitochondrial ATP. MWM and rotarod tests revealed that hoppocampal learning and memory and motor learning and coordination were impaired in tau mice relative to wild-type (WT) mice. Increased levels of mitochondrial fission proteins, Mfn1, Mfn2 and Pop1 were found in 12 month-old tau mice relative to age matched WT mice, indicating that the presence of abnormal mitochondrial dynamics in tau mice. Decreased levels of dendritic proteins were found in tau mice relative to WT mice. Mitochondrial functions was defective. Golgi-Cox staining analysis revealed that dendritic spines are significantly reduced. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in tau mice. These findings suggest that hippo-campal accumulation of phosphorylated tau is responsible for abnormal mitochondrial dynamics and reducing dendritic protein MAP2 and dendritic spines and hippocampal based learning and memory impairments, and mitochondrial structural and functional changes in tau mice. Based on these observations, we propose that reduced hippocampal phosphorylated tau is an important therapeutic strategy for AD and other tauopathies.

Authors: Kandimalla, Manczak, Yin, Wang, Reddy PH