Project description:Background: Dementia is one of the most common diseases in elderly people and hundreds of thousand new cases per year of Alzheimer’s disease (AD) are estimated. While the recent decade has seen significant advances in the development of novel biomarkers to identify dementias at their early stage, a great effort has been recently made to identify biomarkers able to improve differential diagnosis. However, only few potential candidates, mainly detectable in cerebrospinal fluid (CSF), have been described so far. Methods: We searched for miRNAs regulating MAPT translation. We employed a capture technology able to find the miRNAs directly bound to the MAPT transcript in cell lines. Afterwards, we evaluated the levels of these miRNAs in plasma samples from FTD (n = 42) and AD patients (n = 33) and relative healthy controls (HCs) (n = 42) by using qRT-PCR. Results: Firstly, we found all miRNAs that interact with the MAPT transcript. Ten miRNAs have been selected to verify their effect on Tau levels increasing or reducing miRNA levels by using cell transfections with plasmids expressing the miRNAs genes or LNA antagomiRs. Following the results obtained, miR-92a-3p, miR-320a and miR-320b were selected to analyse their levels in plasma samples of patients with FTD and AD respect to HCs. The analysis showed that the miR-92a-1-3p was under-expressed in both AD and FTD compared to HCs. Moreover, miR-320a was upregulated in FTD vs. AD patients, particularly in men when we stratified by sex. Respect to HC, the only difference is showed in men with AD who have reduced levels of this miRNA. Instead, miR-320b is up-regulated in both dementias, but only patients with FTD maintain this trend in both genders. Conclusions: Our results seem to identify miR-92a-3p and miR-320a as possible good biomarkers to discriminate AD from HC, while miR-320b to discriminate FTD from HC, particularly in males. Combining three miRNAs improves the accuracy only in females, particularly for differential diagnosis (FTD vs. AD) and to distinguish FTD from HC.

Project description:Background Circular RNAs (circRNAs) are highly stable regulatory molecules, often accumulated in the mammalian brain and thought to serve as “memory molecules” that govern the long process of aging. Mounting evidence demonstrated circRNA dysregulation in postmortem cortical regions of Alzheimer’s disease (AD) patients. However, whether and how circRNA dysregulation underlies AD progression remains unexplored. Methods Poly(A)-tailing/RNase R digestion was coupled with our recently published computational algorithm CARP to identify genome-wide dysregulation of circRNAs and their downstream pathways in the 5xFAD mouse cerebral cortex between 5- and 7-month of age, a critical window that marks the transition from reversible to irreversible pathogenic progression. Irreversibly dysregulated circRNAs and pathways in 5xFAD cortex were systematically compared with a large human AD cohort study. A top ranked circRNA conserved and commonly affected in AD patients and 5xFAD mice was depleted in cultured cells to confirm its causal roles in AD-relevant molecular and cellular changes. Results We discovered genome-wide circRNA alterations specifically in the 5xFAD cortex associated with AD progression and found numerous circRNAs also consistently affected in AD patient postmortem cortical areas. Among such circRNAs, circGigyf2 showed the highest net reduction starting from 7-month of age, which is highly conserved and negatively correlated with human AD severity. Mechanistically, we found multiple AD-affected splicing factors that are essential for circGigyf2 biogenesis. Functionally, we identified and experimentally validated the conserved roles of circGigyf2 in sponging AD-relevant miRNAs and AD-associated RNA binding proteins (RBPs) including the cleavage and polyadenylation factor 6 (CPSF6). CircGigyf2 downregulation in AD exert silencing activities of its sponged miRNAs and aberrantly enhanced the polyadenylation efficiency of many CPSF6 targets. These post-transcriptionally tuned mRNAs by circGigyf2 were enriched in apoptotic pathways. Furthermore, circGigyf2 depletion in a mouse immortalized neuronal cell line dysregulated circGigyf2-miRNA and circGigyf2-CPSF6 axes and potentiated neuronal responses to apoptotic insults, which strongly support the causative role of circGigyf2 deficiency in AD disease progression. Conclusions Together, our results unveiled pathological alterations of the brain circRNA landscape associated with irreversible disease stage in an AD mouse model and identified novel molecular mechanisms underlying dysregulation of conserved circRNA pathways that contribute to AD pathogenesis.

Project description:Background: Molecular adaptations in the striatum mediated by dopamine (DA) denervation or Levodopa (L-dopa) treatment have been implicated with the motor deficits found in Parkinsonâs disease (PD). Alterations in glutamatergic neurotransmission and anti-oxidant mechanisms are reported to play important roles in mediating these changes. However, the mechanisms mediating the molecular adaptations in the striatum are not well understood. In recent years, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of gene expression with fundamental roles in numerous biological processes. miRNAs are known to influence the development and maintenance of striatal neurons. To determine the contribution of miRNAs in molecular adaptations found in PD, we screened the expression of 800 miRNAs in postmortem striatum samples obtained from PD and neurologically normal controls. We detected the expression of approximately 250 miRNAs in postmortem human putamen samples collected from patients with PD and healthy controls. There was an abundance of a subset of 17 miRNAs (10 up- and 7 down-regulated) differing substantially between PD and the control, suggesting that miRNAs are altered in the striatum during the course of PD. Computational analysis show that many of these miRNAs targets pro-inflammatory factors in the striatum. Therefore, we sought to detrmine the expression of experimentally validated pro-inflammatory transcripts in PD striatum. Methods: Using a digital gene expression platform to quantify 134 gene transcripts, we compared human postmortem putamen tissues from patients with PD and neurologically normal controls. Results: We identified deregulated expression of 10 gene transcripts (4 up- and 6 down-regulated mRNAs) in postmortem human putamen samples collected fromPD patients compared to controls. The expression of these genes negative correlates with the expression of many of the differentially epxressed miRNAs. Moreover, many of these genes are predicted targets of the differentially expressed miRNAs. Conclusions: We identified deregulated mRNAs most likely associated with anti-oxidant mechanims in PD striatum. This approach may provide insights into pathogenesis of the disease. Human postmortem putamen tissues from 12 patients with PD symptoms and 12 neurologically normal controls were used in the study. Samples were obtained from the Human Brain and Spinal Fluid Resource Center, Los Angeles, CA through NIH NeuroBioBank, and stored at -80Â°C until RNA isolation. Total RNA was extracted using the mirVana RNA isolation kit, following the manufacturerâs instructions (Ambion). Using 225ng total RNA, mRNA levels were assayed by direct digital detection using Nanostring nCounter assay kits (Nanostring Technologies).

Project description:Alzheimer’s Disease (AD) is the most common cause of dementia in the elderly population without a cure or early diagnostics currently available. Despite the demonstrated ability of extracellular vesicles (EVs) to spread tau and Aβ pathology in AD models and their potential utility as a diagnostic and treatment-monitoring tool, our knowledge of human brain EV subpopulations, their molecular composition and roles in disease progression is very limited. Genome-wide association studies linked multiple AD genetic risk factors to microglia-specific pathways suggesting a potential role of microglia-derived EVs in AD progression. Here we are presenting a method for isolation of microglial CD11b-positive small EVs from cryopreserved human brain tissue and multi-omics analysis of the EVs from the parietal cortex of 4 late-stage AD (Braak V-VI) and 3 age-matched normal/low pathology (NL) cases. We identified a total of 1000 proteins by quantitative proteomics, analyzed 594 individual lipid species by targeted lipidomics, and 105 miRNAs using a NanoString miRNA expression panel. We found significant reduction in abundance of homeostatic microglia markers, P2RY12 and TMEM119, and increased levels of disease associated microglia markers, FTH1 and TREM2, in microglial EVs from AD brain compared to NL cases. Protein tau abundance was also significantly higher in AD brain-derived microglial EVs. These changes were accompanied by upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free-cholesterol were elevated in microglial EVs from AD brain. Lipidomic analysis also revealed a pro-inflammatory lipid profile, endolysosomal dysfunction and significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids of different classes, suggesting a potential defect in acyl-chain remodeling. Several immune pathways and senescence were among the top pathways controlled by 4 miRNAs significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may accompany endolysosomal impairment and release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. These data validate a method of isolation of small cell-type specific EVs from human brain tissue, suggests new potential EV-associated markers and disease-related pathways, and provides a framework for future large-scale multi-omics study.

Project description:Background: Molecular adaptations in the striatum mediated by dopamine (DA) denervation or Levodopa (L-dopa) treatment have been implicated with the motor deficits found in Parkinson’s disease (PD). Alterations in glutamatergic neurotransmission and anti-oxidant mechanisms are reported to play important roles in mediating these changes. However, the mechanisms mediating the molecular adaptations in the striatum are not well understood. In recent years, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of gene expression with fundamental roles in numerous biological processes. miRNAs are known to influence the development and maintenance of striatal neurons. To determine the contribution of miRNAs in molecular adaptations found in PD, we screened the expression of 800 miRNAs in postmortem striatum samples obtained from PD and neurologically normal controls. We detected the expression of approximately 250 miRNAs in postmortem human putamen samples collected from patients with PD and healthy controls. There was an abundance of a subset of 17 miRNAs (10 up- and 7 down-regulated) differing substantially between PD and the control, suggesting that miRNAs are altered in the striatum during the course of PD. Computational analysis show that many of these miRNAs targets pro-inflammatory factors in the striatum. Therefore, we sought to detrmine the expression of experimentally validated pro-inflammatory transcripts in PD striatum. Methods: Using a digital gene expression platform to quantify 134 gene transcripts, we compared human postmortem putamen tissues from patients with PD and neurologically normal controls. Results: We identified deregulated expression of 10 gene transcripts (4 up- and 6 down-regulated mRNAs) in postmortem human putamen samples collected fromPD patients compared to controls. The expression of these genes negative correlates with the expression of many of the differentially epxressed miRNAs. Moreover, many of these genes are predicted targets of the differentially expressed miRNAs. Conclusions: We identified deregulated mRNAs most likely associated with anti-oxidant mechanims in PD striatum. This approach may provide insights into pathogenesis of the disease.

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Synapse-centered novel omics targets in Alzheimer’s disease

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Integrated multi-omics analyses of synaptosomes revealed synapse-associated novel targets in Alzheimer's disease.

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