Transcriptomics and Proteomics Approach for the Identification of Altered Blood microRNAs and Plasma Proteins in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective destruction of dopaminergic neurons (DA-nergic). Clinically, PD is diagnosed based on developing signs and symptoms. A neurological and physical examination and sometimes medical and family history also help in the diagnosis of PD. However, most of these features are visible when more than 80% of the dopaminergic neurons have degenerated. An understanding of the selective degeneration process at the cellular and molecular level and the development of new biomarkers are required for effective PD management. Several studies have been carried out using a selected set of miRNAs/ mRNAs and proteins to develop biomarkers of PD; however, an unbiased and combined miRNA-protein profiling study was required to identify the markers of progressive and selected degeneration of dopaminergic neurons in PD patients. In the present study, we have carried out global protein profiling through LC-MS/MS and miRNA profiling by using a "brain-specific" miRNA array panel of 112 miRNAs in PD patients and healthy controls to find the unprejudiced group of proteins and miRNAs that are deregulating in PD. In the whole blood samples of PD patients compared to healthy controls, the expression of 23 miRNAs and 289 proteins was significantly increased, whereas the expression of 4 miRNAs and 132 proteins was considerably downregulated. Network analysis, functional enrichment, annotation, and analysis of miRNA-protein interactions were also performed as part of the bioinformatics investigation of the discovered miRNAs and proteins revealing several pathways that lead to PD development and pathogenesis. Based on the analysis of miRNA and protein profiling, we have identified four miRNAs (hsa-miR-186-5p, miR-29b, miR-139 & has-miR-150-5p) and four proteins (YWHAZ, PSMA4, HYOU1, & SERPINA1), which can be targeted for the development of new biomarkers of PD. In vitro studies have identified the role of miR-186-5p in regulating the levels of the YWHAZ/YWHAB & CALM2 gene, which has shown maximum downregulation in PD patients and is known for its role in neuroprotection from apoptotic cell death & calcium regulation. In conclusion, our research has identified a group of miRNA-proteins that can be developed as PD biomarkers; however, future studies on the release of these miRNAs and proteins in extracellular vesicles circulating in the blood of PD patients can further validate these as specific biomarkers of PD.

Neuronal melatonin type 1 receptor overexpression promotes M2 microglia polarization in cerebral ischemia/reperfusion-induced injury.

Microglial activation is readily detected following cerebral ischemia/reperfusion-induced injury. Activated microglia polarize into either classic pro-inflammatory M1 or protective M2 microglia following ischemia/reperfusion-induced injury. Melatonin is protective immediately after ischemia/reperfusion-induced brain injury. However, the ability of melatonin to affect longer-term recovery from ischemic/reperfusion-induced injury as well as its ability to modulate microglia/macrophage polarization are unknown. The goal of this study is to understand the impact of melatonin on mice 14 days after injury, as well as to understand how melatonin affects microglial polarization of neuronal MT1 activation following cerebral ischemia/reperfusion. We utilized NSEMT1-GFP transgenic mice which overexpress MT1 (melatonin type 1 receptor) in neurons. Melatonin-treated or vehicle treated wild type and NSEMT1-GFP mice underwent middle cerebral artery occlusion (MCAO)/reperfusion and followed for 14 days. Neuronal MT1 overexpression significantly reduced infarct volumes, improved motor function, and ameliorated weight loss. Additionally, melatonin treatment reduced infarct volume in NSEMT1-GFP mice as compared to untreated wild type, melatonin treated wild type, and untreated NSEMT1-GFP mice. Melatonin improved neurological function and prevented weight loss in NSEMT1-GFP mice compared with melatonin treated wild type mice. Finally, melatonin treatment in combination with MT1 overexpression reduced the numbers of Iba1+/CD16+ M1 microglia and increased the numbers of Iba1+/ CD206+ M2 microglia after ischemic injury. In conclusion, neuronal MT1 mediates melatonin-induced long-term recovery after cerebral ischemia, at least in part, by shifting microglial polarization toward the neuroprotective M2 phenotype.

Neurodevelopmental disorders and neurotoxicity: MicroRNA in focus.

Controlled protein synthesis in different cell types is a prerequisite for the proper development and functioning of organs. Alterations in proteostasis either due to neurotoxicants (biological, chemical, or genetic) or aging promotes neuronal degeneration and result in the development of neurological disorders. Gene expression is controlled at the transcriptional, post-transcriptional, and translational levels to maintain the proteostasis of the cells. The complexity of gene regulatory networks increases during nervous system development and regulates the formation of neuronal and glial cells from a common progenitor cell. Complex features of brain development, brain maturation, and brain function are achieved by overlapping molecular networks of gene regulatory circuits. Gene expression alterations or dysregulation by either genetic, biological, or environmental insults during brain development results in neurodevelopmental disorders. Discovery of microRNAs (miRNAs) has opened a new window and provides an opportunity to understand molecular mechanisms of complex gene regulatory networks. Here, we review all the important and recent findings available so far in the literature, which deals with the regulation and role of miRNAs in neurodevelopmental disorders and neurotoxicity.

Two hit mitochondrial-driven model of synapse loss in neurodegeneration.

In healthy neurons, a mitochondrial membrane potential gradient exists whereby membrane potential is highest in the soma and decreases with distance from the nucleus. Correspondingly, distal mitochondria have more oxidative damage and slower protein import than somal mitochondria. Due to these differences, distal mitochondria have an intrinsic first stressor that somal mitochondria do not have, resulting in synaptic mitochondrial vulnerability. A second stressor may result from mutant protein expression, situational stress, or aging, exacerbating vulnerable mitochondria activating stress responses. Under these conditions, distal mitochondria release cytochrome c and mitochondrial DNA, leading to compartmentalized sub-lethal caspase-3 activation and cytokine production. In this two-hit mitochondrial-driven synaptic loss model, synapse vulnerability during neurodegeneration is explained as a superposition of pre-existing lower synaptic mitochondrial membrane potential (hit one) with additional mitochondrial stress (hit two). This two-hit mechanism occurs in synaptic mitochondria, activating signaling pathways leading to synaptic degeneration, as a potential preamble to neuronal death.

Coordinated Action of miR-146a and Parkin Gene Regulate Rotenone-induced Neurodegeneration.

Mitochondrial dysfunction is a common cause in pathophysiology of different neurodegenerative diseases. Elimination of dysfunctional and damaged mitochondria is a key requirement for maintaining homeostasis and bioenergetics of degenerating neurons. Using global microRNA (miRNA) profiling in a systemic rotenone model of Parkinson's disease, we have identified miR-146a as upmost-regulated miRNA, which is known as inflammation regulatory miRNA. Here, we report the role of activated nuclear factor kappa beta (NF-kß) in miR-146a-mediated downregulation of Parkin protein, which inhibits clearance of damaged mitochondria and induces neurodegeneration. Our studies have shown that 4-week rotenone exposure (2.5 mg/kg b.wt) induced oxidative imbalance-mediated NF-kß activation in 1-year-old rat's brain. Activated NF-kß binds in promoter region of miR-146a gene and induces its transcription, which downregulates levels of Parkin protein. Decreased amount of Parkin protein results in accumulation of damaged and dysfunctional mitochondria, which further promotes the generation of reactive oxygen species in degenerating neurons. In conclusion, our studies have identified direct role of NF-kß-mediated upregulation of miR-146a in regulating mitophagy through inhibition of the Parkin gene.

Melatonin inhibits cytosolic mitochondrial DNA-induced neuroinflammatory signaling in accelerated aging and neurodegeneration.

Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis, resulting in mitochondrial DNA (mtDNA) release and activation of cytosolic DNA-mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential, with higher mtDNA release in brain and primary cerebro-cortical neurons of melatonin-deficient aralkylamine N-acetyltransferase (AANAT) knockout mice. Cytosolic mtDNA activated the cGAS/STING/IRF3 pathway, stimulating inflammatory cytokine generation. We found that Huntington's disease mice had increased mtDNA release, cGAS activation, and inflammation, all inhibited by exogenous melatonin. Thus, we demonstrated that cytosolic mtDNA activated the inflammatory response in aging and neurodegeneration, a process modulated by melatonin. Furthermore, our data suggest that AANAT knockout mice are a model of accelerated aging.

Correction to: Plasma Fibrinogen Is a Natural Deterrent to Amyloid ß-Induced Platelet Activation and Neuronal Toxicity.

Following publication of the original article [1], the author reported an error in Figure 1. The correct version of Figure 1 is as follows.

Correction to: Expression of miR-145 and Its Target Proteins Are Regulated by miR-29b in Differentiated Neurons.

The original version of this article unfortunately contained an error. In Figure 8, the image under section a) NTC, and b) NTC + PFTα are copied by mistake.

MiR-34 and MiR-200: Regulator of Cell Fate Plasticity and Neural Development.

Studies from last two decades have established microRNAs (miRNAs) as the most influential regulator of gene expression, especially at the post-transcriptional stage. The family of small RNA molecules including miRNAs is highly conserved and expressed throughout the multicellular organism. MiRNAs regulate gene expression by binding to 3' UTR of protein-coding mRNAs and initiating either decay or movement of mRNAs to stress granules. Tissues or cells, which go through cell fate transformation like stem cells, brain cells, iPSCs, or cancer cells show very dynamic expression profile of miRNAs. Inability to pass the developmental stages of Dicer (miRNA maturation enzyme) knockout animals has confirmed that expression of mature and functional miRNAs is essential for proper development of different organs and tissues. Studies from our laboratory and elsewhere have demonstrated the role of miR-200 and miR-34 families in neural development and have shown higher expression of both families in mature and differentiated neurons. In present review, we have provided a general overview of miRNAs and focused on the role of miR-34 and miR-200, two miRNA families, which have the capability to change the phenotype and fate of a cell in different tissues and situations.

Expression of miR-145 and Its Target Proteins Are Regulated by miR-29b in Differentiated Neurons.

MicroRNAs (miRNAs) are emerging as the most potential regulator of neuronal development. Recent studies from our lab and elsewhere have demonstrated a direct role of miRNAs in regulating neuronal differentiation and synaptogenesis. MicroRNA-145, a miRNA identified to regulate pluripotency of stem cells, downregulates the protein levels of reprogramming transcription factors (RTFs) like OCT4, SOX2, and KLF4 (cell, 137,647-658,2009). Studies have shown that miR-145 is multifunctional and crucial for fate determination of neurons. In our recently published study, we have identified a set of miRNAs including miR-145 and miR-29b families differentially expressed in SH-SY5Y cells exposed sequentially with retinoic acid + brain-derived neurotrophic factor (RA+BDNF) for differentiation into mature neurons (Mol Neurobiol (2016) doi: https://doi.org/10.1007/s12035-016-0042-9 ). In the present study, we have identified the role of miR-29b in upregulation of miR-145, which is upregulated after exposure of RA+BDNF in a P53-dependent manner. In differentiating SH-SY5Y cells, expression of miR-29b downregulates expression of P85α, a P53 inhibitor, which results in upregulation of miR-145 and downregulation of RTF proteins. Ectopic expression of miR-145 and miR-29b in amounts equivalent to their endogenous expression has induced G1 phase cell cycle arrest. In conclusion, our studies have identified miR-29b as an upstream regulator of miR-145 and targets its RTF genes during differentiation of SH-SY5Y cells.

Regulation of miR-34 Family in Neuronal Development.

Differentiation of neural stem cells (NSC's) to mature and functional neurons requires coordinated expression of mRNA, microRNAs (miRNAs) and regulatory proteins. Our earlier unbiased miRNA profiling studies have identified miR-200, miR-34 and miR-221/222 as maximally up-regulated miRNA families in differentiating PC12 cells and demonstrated the capability of miR-200 family in inducing neuronal differentiation (J. Neurochem, 2015, 133, 640-652). In present study, we have investigated role of miR-34 family in neuronal differentiation and identified P53 as mediator of nerve growth factor (NGF) induced miR-34a expression in differentiating PC12 cells. Our studies have shown that NGF induced miR-34a, arrests proliferating PC12 cells to G1 phase, which is pre-requisite for neuronal differentiation. Our studies have also shown that increased expression of miR-34a controls the P53 level in differentiated PC12 cells in feedback inhibition manner, which probably prevents differentiated cells from P53 induced apoptosis. Expression profiling of miR-34 family in different neuronal, non-neuronal and developing cells have identified differentiated and aged brain cells as richest source of miR-34, which also indicates that higher expression of miR-34 family helps in maintaining the mature neurons in non-proliferative stage. In conclusion, our studies have shown that miR-34 is brain enriched miRNA family, which up-regulates with neuronal maturation and brain ageing and co-operative regulation of P53 and miR-34a helps in neuronal differentiation by arresting cells in G1 phase.

Differentiation Induces Dramatic Changes in miRNA Profile, Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence.

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.

Plasma Fibrinogen Is a Natural Deterrent to Amyloid Beta-Induced Platelet Activation.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, characterized by extensive loss of neurons, and deposition of amyloid beta (Aß) in the form of extracellular plaques. Aß is considered to have critical role in synaptic loss and neuronal death underlying cognitive decline. Platelets contribute to 95% of circulating amyloid-precursor protein that releases Aß into circulation. We have recently demonstrated that, Aß active fragment containing amino acid sequence 25-35 (Aß25-35) is highly thrombogenic in nature, and elicits strong aggregation of washed human platelets in RhoA-dependent manner. In the present study we evaluated the influence of fibrinogen on Aß-induced platelet activation. Intriguingly, Aß failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aß25-35, which could be reversed by increasing doses of Aß. Fibrinogen also attenuated Aß-induced platelet responses like secretion, clot retraction, rise in cytosolic Ca+2 and reactive oxygen species (ROS). Fibrinogen prevented intracellular accumulation of full length amyloid beta peptide (Aß42) in platelets as well as neuronal cells. We conclude that fibrinogen serves as a physiological check against the adverse effects of Aß by preventing its interaction with cells.

Critical role of the miR-200 family in regulating differentiation and proliferation of neurons.

The generation of differentiated and functional neurons is a complex process, which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200, miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells, induction of miR-200 family was observed in differentiating neural stem cells, demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However, inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells, an inverse relationship was observed between expression of reprogramming transcription factors (SOX2, KLF4, NANOG, OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover, we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion, our results indicate that increased expression of the miR-200 family promotes neuronal differentiation, while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4.

Regulatory triangle of neurodegeneration, adult neurogenesis and microRNAs.

MicroRNAs (miRNAs) have emerged as a new class of RNA molecules which are short in length, less in number but play bigger role in regulation of cellular events. miRNAs keep cellular homeostasis in tight control by fine tuning expression of protein coding genes at post-transcriptional level. Neurogenesis and neurodegeneration are two complex processes which are regulated by dynamic expression of regulatory proteins like transcription factors and signaling proteins. Evidences are accumulating that expression of miRNAs play major role in fate determination of neuronal cells undergoing neurogenesis or neurodegeneration. Neurodegeneration either induced by genetic factors or environmental chemicals results in development of neurodegenerative disorders like Parkinson's or Alzheimer's. With increasing acceptance of adult neurogenesis, it seems possible that inducing neurogenesis in adult brain can help in fighting with neurodegenerative disorders. Regulatory RNA molecules, like miRNAs are presenting them as potential therapeutic targets for inducing neurogenesis and controlling neurodegeneration. In the current review, we are exploring the link between neurodegeneration and adult neurogenesis regulation by focusing on miRNAs.