Basic Science of Neuroinflammation and Involvement of the Inflammatory Response in Disorders of the Nervous System.

Neuroinflammation is a key immune response observed in many neurologic diseases. Although an appropriate immune response can be beneficial, aberrant activation of this response recruits excessive proinflammatory cells to cause damage. Because the central nervous system is separated from the periphery by the blood-brain barrier (BBB) that creates an immune-privileged site, it has its own unique immune cells and immune response. Moreover, neuroinflammation can compromise the BBB causing an influx of peripheral immune cells and factors. Recent advances have brought a deeper understanding of neuroinflammation that can be leveraged to develop more potent therapies and improve patient selection.

PAD2 dysregulation and aberrant protein citrullination feature prominently in reactive astrogliosis and myelin protein aggregation in sporadic ALS.

Alteration in protein citrullination (PC), a common posttranslational modification (PTM), contributes to pathogenesis in various inflammatory disorders. We previously reported that PC and protein arginine deiminase 2 (PAD2), the predominant enzyme isoform that catalyzes this PTM in the central nervous system (CNS), are altered in mouse models of amyotrophic lateral sclerosis (ALS). We now demonstrate that PAD2 expression and PC are altered in human postmortem ALS spinal cord and motor cortex compared to controls, increasing in astrocytes while trending lower in neurons. Furthermore, PC is enriched in protein aggregates that contain the myelin proteins PLP and MBP in ALS. These results confirm our findings in ALS mouse models and suggest that altered PAD2 and PC contribute to neurodegeneration in ALS.

The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models.

Increased protein citrullination (PC) and dysregulated protein arginine deiminase (PAD) activity have been observed in several neurodegenerative diseases. PC is a posttranslational modification catalyzed by the PADs. PC converts peptidyl-arginine to peptidyl-citrulline, thereby reducing the positive charges and altering structure and function of proteins. Of the five PADs, PAD2 is the dominant isoform in the central nervous system (CNS). Abnormal PC and PAD dysregulation are associated with numerous pathological conditions, including inflammatory diseases and neurodegeneration. Animal model studies have shown therapeutic efficacy from inhibition of PADs, thus suggesting a role of PC in pathogenesis. To determine whether PC contribute to amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis, and eventual death, we investigated alterations of PC and PAD2 in two different transgenic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively. PC and PAD2 expression are altered dynamically in the spinal cord during disease progression in both models. PC and PAD2 increase progressively in astrocytes with the development of reactive astrogliosis, while decreasing in neurons. Importantly, in the spinal cord white matter, PC accumulates in protein aggregates that contain the myelin proteins PLP and MBP. PC also accumulates progressively in insoluble protein fractions during disease progression. Finally, increased PC and PAD2 expression spatially correlate with areas of the CNS with the most severe motor neuron degeneration. These results suggest that altered PC is an integral part of the neurodegenerative process and potential biomarkers for disease progression in ALS. Moreover, increased PC may contribute to disease-associated processes such as myelin protein aggregation, myelin degeneration, and astrogliosis.

Bioinformatic Investigation of Micro RNA-802 Target Genes, Protein Networks, and Its Potential Prognostic Value in Breast Cancer.

Background: An increasing number of studies have suggested that unveiling the molecular network of miRNAs may provide novel therapeutic targets or biomarkers. In this study, we investigated the probable molecular functions that are related to microRNA-802 (miR-802) and evaluated its prognostic value in breast cancer utilizing bioinformatics tools. Methods: PPI network, pathway enrichment and transcription factor analysis were applied to obtain hub genes among overlapping genes of four miRNA target prediction databases. Prognosis value assessments and expression analysis of hub genes using bioinformatics tools, as well as their literature validation were performed. Results: Our results showed a significant correlation of the miR-802 overexpression with poor patient survival rate (BC, p=2.7e-5). We determined 247 target genes significant for GO and KEGG terms. Analysis of TFs by TRUST showed that RUNX3, FOXO3, and E2F1 are possible TFs that regulate the miR-802 expression and target genes network. According to our analysis; 21 genes might have an important function in miR-802 molecular processes and regulatory networks. The result shows that among these 21 genes, 8 genes (CASC3, ITGA4, AGO3, TARDBP, MED13L, SF1, SNRPE and CRNKL1) are positively correlated with patient survival. Therefore these genes could be considered and experimentally evaluated as a prognostic biomarker for breast cancer. Conclusion: The comprehensive bioinformatics study on miR-802 target genes provided insight into miR-802 mediated pathways and processes. Furthermore, representing candidate target genes by prognostic values indicates the potential clinical application of miR-802 in breast cancer.

Low-level overexpression of wild type TDP-43 causes late-onset, progressive neurodegeneration and paralysis in mice.

Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ~30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.

Preclinical Evaluation of miR-15/107 Family Members as Multifactorial Drug Targets for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial, fatal neurodegenerative disorder characterized by the abnormal accumulation of Aß and Tau deposits in the brain. There is no cure for AD, and failure at different clinical trials emphasizes the need for new treatments. In recent years, significant progress has been made toward the development of miRNA-based therapeutics for human disorders. This study was designed to evaluate the efficiency and potential safety of miRNA replacement therapy in AD, using miR-15/107 paralogues as candidate drug targets. We identified miR-16 as a potent inhibitor of amyloid precursor protein (APP) and BACE1 expression, Aß peptide production, and Tau phosphorylation in cells. Brain delivery of miR-16 mimics in mice resulted in a reduction of AD-related genes APP, BACE1, and Tau in a region-dependent manner. We further identified Nicastrin, a γ-secretase component involved in Aß generation, as a target of miR-16. Proteomics analysis identified a number of additional putative miR-16 targets in vivo, including α-Synuclein and Transferrin receptor 1. Top-ranking biological networks associated with miR-16 delivery included AD and oxidative stress. Collectively, our data suggest that miR-16 is a good candidate for future drug development by targeting simultaneously endogenous regulators of AD biomarkers (i.e., Aß and Tau), inflammation, and oxidative stress.

miR-132/212 deficiency impairs tau metabolism and promotes pathological aggregation in vivo.

Alzheimer's disease (AD) and related tauopathies comprise a large group of neurodegenerative diseases associated with the pathological aggregation of tau protein. While much effort has focused on understanding the function of tau, little is known about the endogenous mechanisms regulating tau metabolism in vivo and how these contribute to disease. Previously, we have shown that the microRNA (miRNA) cluster miR-132/212 is downregulated in tauopathies such as AD. Here, we report that miR-132/212 deficiency in mice leads to increased tau expression, phosphorylation and aggregation. Using reporter assays and cell-based studies, we demonstrate that miR-132 directly targets tau mRNA to regulate its expression. We identified GSK-3ß and PP2B as effectors of abnormal tau phosphorylation in vivo. Deletion of miR-132/212 induced tau aggregation in mice expressing endogenous or human mutant tau, an effect associated with autophagy dysfunction. Conversely, treatment of AD mice with miR-132 mimics restored in part memory function and tau metabolism. Finally, miR-132 and miR-212 levels correlated with insoluble tau and cognitive impairment in humans. These findings support a role for miR-132/212 in the regulation of tau pathology in mice and humans and provide new alternatives for therapeutic development.

Experimental verification of a conserved intronic microRNA located in the human TrkC gene with a cell type-dependent apoptotic function.

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75(NTR) receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.

Inhibitory effect of hsa-miR-590-5p on cardiosphere-derived stem cells differentiation through downregulation of TGFB signaling.

The cardiac cells generation via stem cells differentiation is a promising approach to restore the myocardial infarction. Promoted by our primary bioinformatics analysis as well as some previously published data on potential role of hsa-miR-590-3p in cardiogenesis, we have tried to decipher the role of miR-590-5p during the course of differentiation of cardiosphere-derived cells (CDCs). The differentiation induction of CDCs by TGFB1 was confirmed by real-time PCR, ICC, and flow cytometry. The expression pattern of hsa-miR-590-5p and some related genes were examined during the differentiation process. In order to study the role of miR-590-5p in cardiac differentiation, the effect of miR-590 overexpression in CDCs was studied. Evaluating the expression patterns of miR-590 and its potential targets (TGFBRs) during the course of differentiation, demonstrated a significant downregulation of miR-590 and an upregulation of TGFBR2, following the treatment of CDCs with TGFB1. Therefore, we proposed a model in which TGFB1 exerts its differentiation induction via downregulation of miR-590, and hence the higher transcriptional expression level of TGFBR2. In accordance with our proposed model, transfection of CDCs by a pLenti-III-hsa-mir-590-GFP expression vector before or after the first TGFB1 treatment caused a significant alteration in the expression levels of TGFBRs. Moreover, our data revealed that overexpression of miR-590 before TGFB1 induction was able to attenuate the CDCs differentiation probably via the reduction of TGFBR2 expression level. Altogether, our data suggest an inhibitory role of miR-590 during the cardiac differentiation of CDCs which its suppression might elevate the rate of differentiation.

Experimental verification of a predicted intronic microRNA in human NGFR gene with a potential pro-apoptotic function.

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells.