Protective effects of a small-molecule inhibitor DDQ against tau-induced toxicities in a transgenic tau mouse model of Alzheimer's disease.

The purpose of our study is to determine DDQ (diethyl (3,4-dihydroxyphenethylamino) (quinolin-4-yl) methylphosphonate)-a newly discovered molecule that has been shown to protect against phosphorylated tau (p-tau) in Alzheimer's disease (AD) pathogenesis. We used a well-studied tau (P301L) transgenic mouse model to achieve our goal. We administered DDQ into 12-month-old Tau mice, at 20 mg/kg body weight intraperitoneally two times per week for 2 months. We also assessed DDQ levels in the blood, skeletal muscle and brain using biochemical and molecular techniques. We investigated the mRNA and protein levels of mitochondrial dynamics, biogenesis, synaptic, p-tau and longevity genes sirtuins in DDQ-treated tau mice using real-time quantitative PCR (q-RT-PCR), immunoblotting and immunofluorescence techniques. Our extensive pharmacodynamics investigations revealed that skeletal muscle had the greatest peak levels of DDQ, followed by serum and brain. Interestingly, DDQ-treated tau mice had higher levels of mitochondrial fusion, biogenesis, synaptic genes and sirtuins than DDQ-untreated tau mice. In addition, DDQ-treated tau mice had lower levels of mitochondrial fission and p-tau than untreated tau mice. The current findings, combined with our prior findings, firmly show that DDQ possesses anti-aging, anti-amyloid-beta and anti-p-tau properties, making it a promising molecule for reducing age-related, amyloid-beta and p-tau-induced synaptic and mitochondrial toxicities in AD.

Reduced VDAC1, Maintained Mitochondrial Dynamics and Enhanced Mitochondrial Biogenesis in a Transgenic Tau Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most common forms of neurodegeneration, defined by reduced cognitive function, which is caused by the gradual death of neurons in the brain. Recent studies have shown an age-dependent rise in the levels of voltage-dependent anion channel 1 (VDAC1) in AD. In addition, we discovered an aberrant interaction between VDAC1 and P-TAU in the brains of AD patients, which led to abnormalities in the structural and functional integrity of the mitochondria. The purpose of our study is to understand the protective effects of reduced VDAC1 against impaired mitochondrial dynamics and defective mitochondrial biogenesis in transgenic TAU mice. Recently, we crossed heterozygote VDAC1 knockout (VDAC1+/-) mice with transgenic TAU mice to obtain double-mutant VDAC1+/-/TAU mice. Our goal was to evaluate whether a partial decrease in VDAC1 lessens the amount of mitochondrial toxicity in transgenic Tau (P301L) mice. We found that mitochondrial fission proteins were significantly reduced, and mitochondrial fusion and biogenesis proteins were increased in double-mutant mice compared to TAU mice. On the basis of these discoveries, the current work may have significance for the development of reduced-VDAC1-based treatments for individuals suffering from AD as well as other tauopathies.

Behavioral Evidence for a Tau and HIV-gp120 Interaction.

Despite successful virologic control with combination antiretroviral therapy (cART), about half of people living with the human immunodeficiency virus-1 (HIV) develop an HIV-associated neurocognitive disorder (HAND). It is estimated that 50% of individuals who are HIV-positive in the United States are aged 50 years or older. Therefore, a new challenge looms as individuals living with HIV increase in age. There is concern that Alzheimer's disease (AD) may become prevalent with an earlier onset of cognitive decline in people living with HIV (PLWH). Clinical data studies reported the presence of AD biomarkers in PLWH. However, the functional significance of the interaction between HIV or HIV viral proteins and AD biomarkers is still not well studied. The main goal of the present study is to address this knowledge gap by determining if the HIV envelope glycoprotein 120 (HIV-gp120) can affect the cognitive functions in the Tau mouse AD model. Male Tau and age-matched, wild-type (WT) control mice were treated intracerebroventricularly (ICV) with HIV-gp120. The animals were evaluated for cognitive function using a Y-maze. We found that HIV-gp120 altered cognitive function in Tau mice. Notably, HIV-gp120 was able to promote a cognitive decline in transgenic Tau (P301L) mice compared to the control (HIV-gp120 and WT). We provide the first in vivo evidence of a cognitive interaction between an HIV viral protein and Tau mice.

Rlip76: An Unexplored Player in Neurodegeneration and Alzheimer's Disease?

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common cause of dementia in older people. AD is associated with the loss of synapses, oxidative stress, mitochondrial structural and functional abnormalities, microRNA deregulation, inflammatory responses, neuronal loss, accumulation of amyloid-beta (Aß) and phosphorylated tau (p-tau). AD occurs in two forms: early onset, familial AD and late-onset, sporadic AD. Causal factors are still unknown for a vast majority of AD patients. Genetic polymorphisms are proposed to contribute to late-onset AD via age-dependent increases in oxidative stress and mitochondrial abnormalities. Recent research from our lab revealed that reduced levels of Rlip76 induce oxidative stress, mitochondrial dysfunction and synaptic damage, leading to molecular and behavioral phenotypes resembling late-onset AD. Rlip76 is a multifunctional 76 kDa protein encoded by the RALBP1 gene, located on chromosome 18. Rlip is a stress-protective ATPase of the mercapturic acid pathway that couples clathrin-dependent endocytosis with the efflux of glutathione-electrophile conjugates. Rlip is evolutionarily highly conserved across species and is ubiquitously expressed in all tissues, including AD-affected brain regions, the cerebral cortex and hippocampus, where highly active neuronal metabolisms render the cells highly susceptible to intracellular oxidative damage. In the current article, we summarize molecular and cellular features of Rlip and how depleted Rlip may exacerbate oxidative stress, mitochondrial dysfunction and synaptic damage in AD. We also discuss the possible role of Rlip in aspects of learning and memory via axonal growth, dendritic remodeling, and receptor regulation. We conclude with a discussion of the potential for the contribution of genetic polymorphisms in Rlip to AD progression and the potential for Rlip-based therapies.

HLA-DRB1 genes and the expression dynamics of HLA CIITA determine the susceptibility to T2DM.

Type 2 diabetes mellitus (T2DM) is a disease with polygenic inheritance. The expression of major histocompatibility complex class II genes are regulated by several trans-activators. We have studied the expression of HLA-DRB1, RFX, CIITA-P1, PIV transactivators, immunophenotyping of cells, SNPs in CIITA-168 (A/G) and IFN-γ + 874 (T/A) in T2DM patients and controls (n = 201 each). We observed increased frequencies of DRB1*03, DRB1*04 and DRB1*07 and decreased frequencies of DRB1*10, DRB1*14, and DRB1*15 alleles among patients. Significant up-regulations of HLA-DRB1 genes were observed in patients (p < 0.0001). Down-regulated expressions were documented in DRB1*03-homo (p < 0.002) and DRB1*04-homo (p < 0.009) patients. No significant differences were observed for CIITA-P1 expression except DRB1*04-pooled (p < 0.0113). The CIITA-PIV was up-regulated in overall (p < 0.0001), DRB1*03-pooled (p < 0.0006), DRB1*03-hetero (p < 0.0006) and DRB1*03-homo (p < 0.001) T2DM patients. However, significant down-regulations were documented for DRB1*04-pooled (p < 0.040), DRB1*04-hetero (p < 0.060), and DRB1*04-homo (p < 0.027) combinations. Further, significant down-regulations of RFX5 were observed in overall (p < 0.0006), DRB1*04-pooled (p < 0.0022), and DRB1*04-hetero (p < 0.0004) combinations. Immunophenotyping studies revealed significant increase of CD45+ CD14-, CD19+, CD14- and CD8 cells and elevated level of expression of IFN-γ (p < 0.0001) in patients. A significant increase of TT (p < 3.35 × 10-6) and decrease of TA (p < 4.57 × 10-4) genotypes of IFN-γ + 874 (T/A) and an increase of GG (p < 0.001) and decrease of AG (p < 8.24 × 10-5) genotypes of CIITA-168 A/G SNPs were observed. The combinatorial analysis revealed susceptible associations for DRB1*03 + AA, *03 + AG, *03 + GG and *04 + GG and protective associations for DRB1*10 + AG, *10 + GG, *15 + AG, and *14 + GG combinations. Thus, the present study corroborated the effect of differential expressions of promoters of risk alleles in the pathogenesis of T2DM.

Identification of novel circulatory microRNA signatures linked to patients with ischemic stroke.

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 this 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 (qRT-PCR) assay in postmortem IS brains, lymphoblastoid IS cell lines, oxygen and glucose deprivation/reoxygenation -treated human and mouse neuroblastoma cells, and mouse models of hypoxia and ischemia (HI)-induced stroke. A total of 4656 miRNAs were differentially expressed in IS serum samples relative to healthy controls. Out of 4656 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 analyses 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 this 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.

Mutant APP and amyloid beta-induced defective autophagy, mitophagy, mitochondrial structural and functional changes and synaptic damage in hippocampal neurons from Alzheimer's disease.

The purpose of our study was to determine the toxic effects of hippocampal mutant APP (mAPP) and amyloid beta (Aß) in human mAPP complementary DNA (cDNA) transfected with primary mouse hippocampal neurons (HT22). 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 reverse transcriptase-polymerase chain reaction, immunoblotting & immunofluorescence and transmission electron microscopy, we assessed messenger RNA (mRNA) and protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2 and assessed mitochondrial number and length in mAPP-HT22 cells that express Swedish/Indiana mutations. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial adenosine triphosphate. Increased levels of mRNA and protein levels of mitochondrial fission genes, Drp1 and Fis1 and decreased levels fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, 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-Drp1 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 mAPP and Aß 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 mAPP hippocampal cells. These observations strongly suggest that accumulation of mAPP and Aß causes mitochondrial, synaptic and autophagy/mitophagy abnormalities in hippocampal neurons, leading to neuronal dysfunction.

MicroRNA-455-3p as a potential peripheral biomarker for Alzheimer's disease.

The purpose of our study was to identify microRNAs (miRNAs) as early detectable peripheral biomarkers in Alzheimer's disease (AD). To achieve our objective, we assessed miRNAs in serum samples from AD patients and Mild cognitive impairment (MCI) subjects relative to healthy controls. We used Affymetrix microarray analysis and validated differentially expressed miRNAs using qRT-PCR. We further validated miRNA data using AD postmortem brains, amyloid precursor protein transgenic mice and AD cell lines. We identified a gradual upregulation of four miRNAs: miR-455-3p, miR-4668-5p, miR-3613-3p and miR-4674. A fifth miRNA, mir-6722, was down-regulated in persons with AD and mild cognitive impairment compared with controls. Validation analysis by qRT-PCR showed significant upregulation of only miR-455-3p (P = 0.007) and miR-4668-5p (P = 0.016) in AD patients compared with healthy controls. Furthermore, qRT-PCR analysis of the AD postmortem brains with different Braak stages also showed upregulation of miR-455-3p (P = 0.016). However, receiver operating characteristic curves (ROC) curve analysis revealed a significant area under curve (AUC) value only for miR-455-3p in the serum (AUROC = 0.79; P = 0.015) and brains (AUROC = 0.86; P = 0.016) of AD patients. Expression analysis of amyloid precursor protein transgenic mice also revealed high level of mmu-miR-455-3p (P = 0.004) in the cerebral cortex (AD-affected) region of brain and low in the non-affected area, i.e. cerebellum. Furthermore, human and mouse neuroblastoma cells treated with the amyloid-ß(1-42) peptide also showed a similarly higher expression of miR-455-3p. Functional analysis of differentially expressed miRNAs via the miR-path indicated that miR-455-3p was associated in the regulation of several biological pathways. Genes associated with these pathways were found to have a crucial role in AD pathogenesis. An increase in miR-455-3p expression found in AD patients and Aß pathologies unveiled its biomarker characteristics and a precise role in AD pathogenesis.

Diversity and association of HLA/KIR receptors with type 2 diabetes in South India.

The present study was undertaken to delineate the association(s) of KIR-HLA combination in South Indian Type 2 diabetes mellitus (T2DM) patients. The T2DM patients (n = 343) and healthy controls (n = 309) were genotyped for KIR/HLA ligands by PCR-SSP method. The increased frequency of activatory KIR (aKIR) 2DS2 (OR = 1.91; p < 2.91 × 10-4 ) was observed in patients suggesting a susceptible association. The frequencies of iKIR 2DL2 (OR = 0.38; p < 1.55 × 10-5 ) and aKIRs 2DS1 (OR = 0.60; p < 0.001) and 3DS1 (OR = 0.52; p < 5.83 × 10-5 ) were decreased in patients suggesting protective associations. The C1/C2 combinatorial analysis has revealed an increased frequency of C1+ /C2- in T2DM patients (OR = 1.62; p < 0.014). The KIR "AB" genotype (OR = 2.41; p < 3.87 × 10-5 ) was observed to be higher in patients. However, the "BB" genotype (OR = 0.32; p < 4.71 × 10-7 ) was increased in controls. The KIR motifs, "Tel-B/B" (OR = 1.84; p < 0.007), were observed higher among patients. However, the frequency of "Tel-A/B" motif genotype was decreased in patients (OR = 0.56; p < 3.13 × 10-4 ). The iKIR/HLA combinations such as 2DL2/3 +C1 and 3DL2+A3/A11 were increased in patients (OR = 3.90; p < 7.5 × 10-5 ) suggesting susceptible associations. On the contrary, the aKIR+HLA combinations such as 2DS2+C1, 2DS1+C2 and 3DS1+Bw4 were less frequent in patients (OR = 0.32; p < 4.2 × 10-4 ) suggesting protective associations. Thus, the present study clearly establishes the positive and negative associations of different KIR-HLA receptor combinations with T2DM in South India.

Peripheral biomarkers of stroke: Focus on circulatory microRNAs.

Stroke is the second leading cause of death in the world. Stroke occurs when blood flow stops, and that stoppage results in reduced oxygen supply to neurons in the brain. The occurrence of stroke increases with age, but anyone at any age can suffer from stroke. Recent research has implicated multiple cellular changes in stroke patients, including oxidative stress and mitochondrial dysfunction, inflammatory responses, and changes in mRNA and proteins. Recent research has also revealed that stroke is associated with modifiable and non-modifiable risk factors. Stroke can be controlled by modifiable risk factors, including diet, cardiovascular, hypertension, smoking, diabetes, obesity, metabolic syndrome, depression and traumatic brain injury. Stroke is the major risk factor for vascular dementia (VaD) and Alzheimer's disease (AD). The purpose of this article is to review the latest developments in research efforts directed at identifying 1) latest developments in identifying biomarkers in peripheral and central nervous system tissues, 2) changes in microRNAs (miRNAs) in patients with stroke, 3) miRNA profile and function in animal brain, and 4) protein biomarkers in ischemic stroke. This article also reviews research investigating circulatory miRNAs as peripheral biomarkers of stroke.

A critical evaluation of neuroprotective and neurodegenerative MicroRNAs in Alzheimer's disease.

Currently, 5.4 million Americans suffer from AD, and these numbers are expected to increase up to 16 million by 2050. Despite tremendous research efforts, we still do not have drugs or agents that can delay, or prevent AD and its progression, and we still do not have early detectable biomarkers for AD. Multiple cellular changes have been implicated in AD, including synaptic damage, mitochondrial damage, production and accumulation of Aß and phosphorylated tau, inflammatory response, deficits in neurotransmitters, deregulation of the cell cycle, and hormonal imbalance. Research into AD has revealed that miRNAs are involved in each of these cellular changes and interfere with gene regulation and translation. Recent discoveries in molecular biology have also revealed that microRNAs play a major role in post-translational regulation of gene expression. The purpose of this article is to review research that has assessed neuroprotective and neurodegenerative characteristics of microRNAs in brain samples from AD transgenic mouse models and patients with AD.

WT1 and NPHS2 gene mutation analysis and clinical management of steroid-resistant nephrotic syndrome.

Nephrotic syndrome (NS) is a kidney disease predominantly present in children with idiopathic condition; final stage of the disease progresses into end-stage renal disease. Generally, NS is treated using standard steroid therapy, however; most of the children are steroid sensitive and about 15-20% are non-responders (SRNS). Non-responsiveness of these children would be a risk with the possibility of mutational changes in podocyte genes (NPHS1, NPHS2, WT1, PLCE1). The mutation in podocyte genes is associated with SRNS. NPHS1, NPHS2, and WT1 genes are identified/directly linked to SRNS. The present study is a surveillance on the mutation analysis of WT1 (exons 8 and 9) and NPHS2 (exons 1-8) gene in SRNS followed by clinical management. In the present study, we analyzed these two genes in a total of 117 SRNS (73 boys and 44 girls) children. A total of five mutations were detected in six children. First, WT1 mutation was detected at 9th intron-IVS 9 + 4C > T position in one SRNS female patient. This WT1 mutation was identified in a girl having Frasier Syndrome (FS) with focal segmental glomerulosclerosis and a complete sex reversal found through molecular and karyological screening. In NPHS2, missense mutations of P20L (in two children), P316S, and p.R229Q, and a frame shift mutation of 42delG were detected. Thus, applying molecular investigation helped us to decide on treatment plan of SRNS patients, mainly to avoid unnecessary immunosuppressive treatment.

Association of HLA-DR/DQ alleles and haplotypes with nephrotic syndrome.

BACKGROUND: Nephrotic syndrome (NS) is a debilitating renal problem in children resulting from an interaction between environmental and genetic factors including human leukocyte antigen genes (HLA). The aim of this work was to study the probable link between HLA alleles/haplotypes and NS in south India. METHODS: HLA DRB1*/DQB1* alleles were genotyped in 183 NS (76 steroid sensitive-SSNS; 107 steroid resistant-SRNS) and paediatric healthy controls (PHCs; n = 91) using polymerase chain reaction-sequence specific primers (PCR-SSP). HLA-A/-B genotyping was performed for patients (n = 70) positive for DRB1*07-DQB1*02 haplotype to identify four locus extended haplotype. RESULTS: The following alleles and haplotypes were strongly associated with NS (P < 0.05 as significant): DRB1*07 (SSNS, P < 7.98 × 10(-6) ; SRNS, P < 0.008), DQB1*02 (SSNS, P < 3.99 × 10(-6) ; SRNS, P < 0.002), DRB1*07-DQB1*02 (SSNS, P < 1.32 × 10(-4) ; SRNS, P < 0.010), DRB1*07-DQB1*0301,0304 (DQ7) (SSNS, P < 0.001) and DRB1*03-DQB1*02 (SRNS, P < 0.048). Protective associations were observed for alleles DRB1*10 (SRNS, P < 0.013), DQB1*05 (SSNS, P < 4.34 × 10(-6) ; SRNS, P < 0.01), DQB1*06 (SSNS, P < 0.003), and haplotypes DRB1*10-DQB1*06 (SSNS, P < 0.046; SRNS, P < 0.032) and DRB1*15-DQB1*05 (SSNS, P < 0.018). HLA-A/-B typing of 70 NS cases with two locus haplotype DRB1*07-DQB1*02 (70/183; 38.25%) revealed the presence of an extended haplotype 'A*03-B*07-DRB1*07-DQB1*02' (n = 35; 50%). CONCLUSION: Our study revealed strong susceptible association of DRB1*07 with SRNS and DQB1*02 with SSNS. A gender predominant protective association was observed for DRB1*10 with SRNS females; DQB1*05 with SSNS and SRNS males. Further, the study documented the presence of an extended haplotype and pleiotropic action of DRB1*/DQB1* alleles in immune-mediated aetiology of NS in south India.

Unveiling the Role of Novel miRNA PC-5P-12969 in Alleviating Alzheimer's Disease.

Background: The intricate and complex molecular mechanisms that underlie the progression of Alzheimer's disease (AD) have prompted a concerted and vigorous research endeavor aimed at uncovering potential avenues for therapeutic intervention. Objective: This study aims to elucidate the role of miRNA PC-5P-12969 in the pathogenesis of AD. Methods: We assessed the differential expression of miRNA PC-5P-12969 in postmortem AD brains, AD animal and cell models using real-time reverse-transcriptase RT-PCR, we also checked the gene and protein expression of GSK3α and APP. Results: Our investigation revealed a notable upregulation of miRNA PC-5P-12969 in postmortem brains of AD patients, in transgenic mouse models of AD, and in mutant APP overexpressing-HT22 cells. Additionally, our findings indicate that overexpression of miRNA PC-5P-12969 exerts a protective effect on cell survival, while concurrently mitigating apoptotic cell death. Further-more, we established a robust and specific interaction between miRNA PC-5P-12969 and GSK3α. Our luciferase reporter assays provided confirmation of the binding between miRNA PC-5P-12969 and the 3'-UTR of the GSK3α gene. Manipulation of miRNA PC-5P-12969 levels in cellular models of AD yielded noteworthy alterations in the gene and protein expression levels of both GSK3α and APP. Remarkably, the manipulation of miRNA PC-5P-12969 levels yielded significant enhancements in mitochondrial respiration and ATP production, concurrently with a reduction in mitochondrial fragmentation, thus unveiling a potential regulatory role of miRNA PC-5P-12969 in these vital cellular processes. Conclusions: In summary, this study sheds light on the crucial role of miRNA PC-5P-12969 and its direct interaction with GSK3α in the context of AD.

A Comprehensive Review of Membrane Transporters and MicroRNA Regulation in Alzheimer's Disease.

Alzheimer's disease (AD) is a distressing neurodegenerative condition characterized by the accumulation of amyloid-beta (Aß) plaques and tau tangles within the brain. The interconnectedness between membrane transporters (SLCs) and microRNAs (miRNAs) in AD pathogenesis has gained increasing attention. This review explores the localization, substrates, and functions of SLC transporters in the brain, emphasizing the roles of transporters for glutamate, glucose, nucleosides, and other essential compounds. The examination delves into the significance of SLCs in AD, their potential for drug development, and the intricate realm of miRNAs, encompassing their transcription, processing, functions, and regulation. MiRNAs have emerged as significant players in AD, including those associated with mitochondria and synapses. Furthermore, this review discusses the intriguing nexus of miRNAs targeting SLC transporters and their potential as therapeutic targets in AD. Finally, the review underscores the interaction between SLC transporters and miRNA regulation within the context of Alzheimer's disease, underscoring the need for further research in this area. This comprehensive review aims to shed light on the complex mechanisms underlying the causation of AD and provides insights into potential therapeutic approaches.

The role of RLIP76 in oxidative stress and mitochondrial dysfunction: Evidence based on autopsy brains from Alzheimer's disease patients.

Several converging lines of evidence from our group support a potential role of RLIP76 (AKA Rlip) in neurodegenerative disorders, including Alzheimer's Disease (AD). However, the role of Rlip in Alzheimer's and other neurodegenerative diseases is not well understood. The purpose of the present study is to determine the role of Rlip in the brains of AD patients and control subjects. To achieve our goals, we used frozen tissues and formalin-fixed paraffin-embedded postmortem brains from AD patients of different Braak stages and age-matched control subjects. Our immunohistology and immunoblotting blotting analysis revealed that expression of Rlip protein gradually and significantly decreased (p = 0.0001) with AD progression, being lowest in Braak stage IV-V. Rlip was colocalized with Amyloid beta (Aß) and phosphorylated tau (p-Tau) as observed by IHC staining and co-immunoprecipitation studies. Lipid peroxidation (4-HNE generation) and H2O2 production were significantly higher (p = 0.004 and 0.0001 respectively) in AD patients compared to controls, and this was accompanied by lower ATP production in AD (p = 0.0009). Oxidative DNA damage was measured by 8-Hydroxyguanosine (8-OHdG) in tissue lysates by ELISA and COMET assay. AD 8-OHdG levels were significantly higher (p = 0.0001) compared to controls. COMET assay was performed in brain cells, isolated from frozen postmortem samples. The control samples showed minimal DNA in comets representing few DNA strand breaks (<20 %), (score-0-1). However, the AD group showed an average of 50 % to 65 % of DNA in comet tails (score-4-5) indicating numerous DNA strand breaks. The difference between the two groups was significant (p = 0.001), as analyzed by Open Comet by ImageJ. Elevated DNA damage was further examined by western blot analysis for phosphorylated histone variant H2AX (γH2AX). Induction of γH2AX was very significant (p < 0.0001) and confirmed the presence of double-strand breaks in DNA. Overall, our results indicate an important role for Rlip in maintaining neuronal health and homeostasis by suppressing cellular oxidative stress and DNA damage. Based on our findings, we cautiously conclude that Rlip is a promising therapeutic target for Alzheimer's disease.

A Novel Role for BRIP1/FANCJ in Neuronal Cells Health and in Resolving Oxidative Stress-Induced DNA Lesions.

BACKGROUND: DNA damage accumulation and mitochondrial abnormalities are elevated in neurons during aging and may contribute to neurodegenerative pathologic conditions such as Alzheimer's disease. BRCA1 interacting protein 1 or BRIP1 is a 5' to 3' DNA helicase that catalyzes many abnormal DNA structures during DNA replication, gene transcription, and recombination, and contribute to genomic integrity. OBJECTIVE: BRIP1 functions were reasonably well studied in DNA repair; however, there is limited data on its role and regulation during aging and neurodegenerative diseases. METHODS: We used immunohistochemistry, western blot, and qRT-PCR assays to analyze the expression of BRIP1. Immunofluorescence studies were performed to study the formation of R-loops, reactive oxygen species (ROS) generation, and mitochondrial morphology. Flow cytometry and transmission electron microscopy were used to evaluate mitochondrial ROS and mitochondrial structures, respectively. Oxygen consumption rate was measured using Seahorse, and the Presto Blue™ assays were used to evaluate cell viability. RESULTS: Our results demonstrate the expression of BRIP1 in mouse and human brain tissues and in neuronal cell lines. BRIP1 levels were elevated in the hippocampal regions of the brains, specifically in the dentate gyrus. BRIP1 downregulation in neuronal cells caused increased R-loop formation basally and in response to H2O2 treatment. Furthermore, BRIP1 deficient cells exhibited elevated levels of excitotoxicity induced by L-Glutamic acid exposure as evidenced by (mitochondrial) ROS levels, deteriorated mitochondrial health, and cell death compared to BRIP1 proficient neuronal cells. CONCLUSION: Overall, our results indicate an important role for BRIP1 in maintaining neuronal cell health and homeostasis by suppressing cellular oxidative stress.

A partial reduction of VDAC1 enhances mitophagy, autophagy, synaptic activities in a transgenic Tau mouse model.

Alzheimer's disease (AD) is the most common cause of mental dementia in the aged population. AD is characterized by the progressive decline of memory and multiple cognitive functions, and changes in behavior and personality. Recent research has revealed age-dependent increased levels of VDAC1 in postmortem AD brains and cerebral cortices of APP, APPxPS1, and 3xAD.Tg mice. Further, we found abnormal interaction between VDAC1 and P-Tau in the AD brains, leading to mitochondrial structural and functional defects. Our current study aimed to understand the impact of a partial reduction of voltage-dependent anion channel 1 (VDAC1) protein on mitophagy/autophagy, mitochondrial and synaptic activities, and behavior changes in transgenic TAU mice in Alzheimer's disease. To determine if a partial reduction of VDAC1 reduces mitochondrial and synaptic toxicities in transgenic Tau (P301L) mice, we crossed heterozygote VDAC1 knockout (VDAC1+/- ) mice with TAU mice and generated double mutant (VDAC1+/- /TAU) mice. We assessed phenotypic behavior, protein levels of mitophagy, autophagy, synaptic, other key proteins, mitochondrial morphology, and dendritic spines in TAU mice relative to double mutant mice. Partial reduction of VDAC1 rescued the TAU-induced behavioral impairments such as motor coordination and exploratory behavioral changes, and learning and spatial memory impairments in VDAC1+/- /TAU mice. Protein levels of mitophagy, autophagy, and synaptic proteins were significantly increased in double mutant mice compared with TAU mice. In addition, dendritic spines were significantly increased; the mitochondrial number was significantly reduced, and mitochondrial length was increased in double mutant mice. Based on these observations, we conclude that reduced VDAC1 is beneficial in symptomatic-transgenic TAU mice.

Early Cellular, Molecular, Morphological and Behavioral Changes in the Humanized Amyloid-Beta-Knock-In Mouse Model of Late-Onset Alzheimer's Disease.

The purpose of our study is to investigate early cellular, molecular, morphological and behavioral changes in humanized amyloid-beta-knock-in (hAbKI) mice. Using seven-month-old homozygous hAbKI mice, we studied behavioral phenotype parameters, including spatial learning and memory (Morris Water Maze), locomotor activity (open field), working memory (Y-maze) and motor coordination (rotarod); mRNA abundance, protein levels, soluble amyloid-beta 40 and 42 levels and regional immunoreactivities of key markers of mitochondrial dynamics, mitochondrial biogenesis, synaptic health, mitophagy and autophagy; mitochondrial function and using transmission electron microscopy & Golgi-Cox staining, we assessed mitochondrial morphology and dendritic spines. Our extensive behavioral analysis revealed that seven-month-old hAbKI mice showed impairments in motor coordination, reduced locomotor and exploration activities, impairments in working memory and spatial learning and memory. Our mRNA and protein analyses revealed the increased expression of mitochondrial-fission genes and reduced expression of mitochondrial-fusion, mitochondrial-biogenesis, synaptic, autophagy and mitophagy genes in seven-month-old hAbKI mice. An immunofluorescence analysis revealed altered immunoreactivities and agreed with the immunoblot results. Transmission-electron-microscopy data revealed increased mitochondrial fragmentation and reduced mitochondrial length in both hippocampal and cortical tissues of seven-month-old hAbKI mice and mitochondrial function defective. A Golgi-Cox-staining analysis revealed reduced dendritic spines in both cerebral cortices and hippocampi of hAbKI mice. Soluble amyloid-beta (1-40 and 1-42) were detected in three-month-old hAbKI mice and progressively increased in seven-month-old mice. These observations suggest that the human amyloid-beta peptide is sufficient to cause behavioral, mitochondrial, synaptic and ultrastructural changes in seven-month-old hAbKI mice. Our study findings also suggest that hAbKI mice might serve as a model for preclinical studies of preventive therapies.

A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer's Disease.

In the current study, for the first time, we study mitophagy enhancer urolithin A and a combination of urolithin A+green tea extract EGCG against human Aß peptide-induced mitochondrial and synaptic, dendritic, inflammatory toxicities and behavioral changes in humanized homozygous amyloid beta knockin (hAbKI) mice of late-onset Alzheimer's disease (AD). Our findings reveal significantly increased positive effects of urolithin A and a combination treatment of urolithin A+EGCG in hAbKI mice for phenotypic behavioral changes including motor coordination, locomotion/exploratory activity, spatial learning and working memory. mRNA and protein levels of mitochondrial fusion, synaptic, mitophagy and autophagy genes were upregulated, and mitochondrial fission genes are downregulated in urolithin A and combine treatment in hAbKI mice; however, the effect is stronger in combined treatment. Immunofluorescence analysis of hippocampal brain sections shows similar findings of mRNA and protein levels. Mitochondrial dysfunction is significantly reduced in both treatment groups, but a stronger reduction is observed in combined treatment. Dendritic spines and lengths are significantly increased in both treatment groups, but the effect is stronger in combined treatment. The fragmented number of mitochondria is reduced, and mitochondrial length is increased, and mitophagosomal formations are increased in both the groups, but the effect is stronger in the combined treatment. The levels of amyloid beta (Aß) 40 and Aß42 are reduced in both treatments, however, the reduction is higher for combined treatment. These observations suggest that urolithin A is protective against human Aß peptide-induced toxicities; however, combined treatment of urolithin A+EGCG is effective and stronger, indicating that combined therapy is promising to treat late-onset AD patients.