Disrupted phase behavior of FUS underlies poly-PR-induced DNA damage in amyotrophic lateral sclerosis.

GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of the chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Among the five dipeptide repeat proteins translated from G4C2 HRE, arginine-rich poly-PR (proline:arginine) is extremely toxic. However, the molecular mechanism responsible for poly-PR-induced cell toxicity remains incompletely understood. Here, we found that poly-PR overexpression triggers severe DNA damage in cultured cells, primary cortical neurons, and the motor cortex of a poly-PR transgenic mouse model. Interestingly, we identified a linkage between poly-PR and RNA-binding protein fused in sarcoma (FUS), another ALS-related gene product associated with DNA repair. Poly-PR interacts with FUS both in vitro and in vivo, phase separates with FUS in a poly-PR concentration-dependent manner, and impairs the fluidity of FUS droplets in vitro and in cells. Moreover, poly-PR impedes the recruitment of FUS and its downstream protein XRCC1 to DNA damage foci after microirradiation. Importantly, overexpression of FUS significantly decreased the level of DNA damage and dramatically reduced poly-PR-induced cell death. Our data suggest the severe DNA damage caused by poly-PR and highlight the interconnection between poly-PR and FUS, enlightening the potential therapeutic role of FUS in alleviating poly-PR-induced cell toxicity.

Bioinformatics and network-based screening and discovery of potential molecular targets and small molecular drugs for breast cancer.

Accurate identification of molecular targets of disease plays an important role in diagnosis, prognosis, and therapies. Breast cancer (BC) is one of the most common malignant cancers in women worldwide. Thus, the objective of this study was to accurately identify a set of molecular targets and small molecular drugs that might be effective for BC diagnosis, prognosis, and therapies, by using existing bioinformatics and network-based approaches. Nine gene expression profiles (GSE54002, GSE29431, GSE124646, GSE42568, GSE45827, GSE10810, GSE65216, GSE36295, and GSE109169) collected from the Gene Expression Omnibus (GEO) database were used for bioinformatics analysis in this study. Two packages, LIMMA and clusterProfiler, in R were used to identify overlapping differential expressed genes (oDEGs) and significant GO and KEGG enrichment terms. We constructed a PPI (protein-protein interaction) network through the STRING database and identified eight key genes (KGs) EGFR, FN1, EZH2, MET, CDK1, AURKA, TOP2A, and BIRC5 by using six topological measures, betweenness, closeness, eccentricity, degree, MCC, and MNC, in the Analyze Network tool in Cytoscape. Three online databases GSCALite, Network Analyst, and GEPIA were used to analyze drug enrichment, regulatory interaction networks, and gene expression levels of KGs. We checked the prognostic power of KGs through the prediction model using the popular machine learning algorithm support vector machine (SVM). We suggested four TFs (TP63, MYC, SOX2, and KDM5B) and four miRNAs (hsa-mir-16-5p, hsa-mir-34a-5p, hsa-mir-1-3p, and hsa-mir-23b-3p) as key transcriptional and posttranscriptional regulators of KGs. Finally, we proposed 16 candidate repurposing drugs YM201636, masitinib, SB590885, GSK1070916, GSK2126458, ZSTK474, dasatinib, fedratinib, dabrafenib, methotrexate, trametinib, tubastatin A, BIX02189, CP466722, afatinib, and belinostat for BC through molecular docking analysis. Using BC cell lines, we validated that masitinib inhibits the mTOR signaling pathway and induces apoptotic cell death. Therefore, the proposed results might play an effective role in the treatment of BC patients.

Autophagy and Polyglutamine Disease.

Polyglutamine (polyQ) disease is a type of fatal neurodegenerative disease caused by an expansion of CAG repeats in a specific gene, resulting in a protein with an abnormal polyQ fragment. The age of onset and the degree of pathological deterioration are related to the length of the polyQ fragment. At least 9 kinds of polyglutamine diseases have been discovered, including Huntington disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), spinobulbar muscular atrophy (SBMA) and six spinocerebellar ataxia (SCA) such as SCA1, 2, 3, 6, 7 and 17 subtypes (Table 9.1). Previous studies suggest that autophagy plays a major role in the quality control of disease proteins in polyQ diseases. In this chapter, we majorly focused on three representative polyQ diseases, including spinocerebellar Ataxia type 3 (SCA3), spinocerebellar ataxia type 7 (SCA7) and Huntington's disease (HD). The relationship of the ubiquitin-proteasome system and autophagy involved in disease protein accumulation were summarized.

DJ-1 regulates tyrosine hydroxylase expression through CaMKKß/CaMKIV/CREB1 pathway in vitro and in vivo.

Lack of dopamine production and neurodegeneration of dopaminergic neurons in the substantia nigra are considered as the major characteristics of Parkinson's disease, a prevalent movement disorder worldwide. DJ-1 mutation leading to loss of its protein functions is a genetic factor of PD. In this study, our results illustrated that DJ-1 can directly interact with Ca2+ /calmodulin-dependent protein kinase kinase ß (CaMKKß) and modifies the cAMP-responsive element binding protein 1 (CREB1) activity, thus regulates tyrosine hydroxylase (TH) expression. In Dj-1 knockout mouse substantia nigra, the levels of TH and the phosphorylation of CREB1 Ser133 are significantly decreased. Moreover, Dj-1 deficiency suppresses the phosphorylation of CaMKIV (Thr196/200) and CREB1 (Ser133), subsequently inhibits TH expression in vitro. Furthermore, Knockdown of Creb1 abolishes the effects of DJ-1 on TH regulation. Our data reveal a novel pathway in which DJ-1 regulates CaMKKß/CaMKIV/CREB1 activities to facilitate TH expression.

Poly-PR in C9ORF72-Related Amyotrophic Lateral Sclerosis/Frontotemporal Dementia Causes Neurotoxicity by Clathrin-Dependent Endocytosis.

GGGGCC repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9ORF72 produce five dipeptide repeat (DPR) proteins by an unconventional repeat-associated non-ATG (RAN) translation. Within the five DPR proteins, poly-PR and poly-GR that contain arginine are more toxic than the other DPRs (poly-GA, poly-GP, and poly-PA). Here, we demonstrated that poly-PR peptides transferred into cells by endocytosis in a clathrin-dependent manner, leading to endoplasmic reticulum stress and cell death. In SH-SY5Y cells and primary cortical neurons, poly-PR activated JUN amino-terminal kinase (JNK) and increased the levels of p53 and Bax. The uptake of poly-PR peptides by cells was significantly inhibited by knockdown of clathrin or by chlorpromazine, an inhibitor that blocks clathrin-mediated endocytosis. Inhibition of clathrin-dependent endocytosis by chlorpromazine significantly blocked the transfer of poly-PR peptides into cells, and attenuated poly-PR-induced JNK activation and cell death. Our data revealed that the uptake of poly-PR undergoes clathrin-dependent endocytosis and blockade of this process prevents the toxic effects of synthetic poly-PR peptides.

Pharmacological activation of REV-ERBα represses LPS-induced microglial activation through the NF-κB pathway.

REV-ERBα, the NR1D1 (nuclear receptor subfamily 1, group D, member 1) gene product, is a dominant transcriptional silencer that represses the expression of genes involved in numerous physiological functions, including circadian rhythm, inflammation, and metabolism, and plays a crucial role in maintaining immune functions. Microglia-mediated neuroinflammation is tightly associated with various neurodegenerative diseases and psychiatric disorders. However, the role of REV-ERBα in neuroinflammation is largely unclear. In this study, we investigated whether and how pharmacological activation of REV-ERBα affected lipopolysaccharide (LPS)-induced neuroinflammation in mouse microglia in vitro and in vivo. In BV2 cells or primary mouse cultured microglia, application of REV-ERBα agonist GSK4112 or SR9011 dose-dependently suppressed LPS-induced microglial activation through the nuclear factor kappa B (NF-κB) pathway. In BV2 cells, pretreatment with GSK4112 inhibited LPS-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα) kinase (IκK), thus restraining the phosphorylation and degradation of IκBα, and blocked the nuclear translocation of p65, a NF-κB subunit, thereby suppressing the expression and secretion of the proinflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor α (TNFα). Moreover, REV-ERBα agonist-induced inhibition on neuroinflammation protected neurons from microglial activation-induced damage, which were also demonstrated in mice with their ventral midbrain microinjected with GSK4112, and then stimulated with LPS. Our results reveal that enhanced REV-ERBα activity suppresses microglial activation through the NF-κB pathway in the central nervous system.

Tyrosine hydroxylase down-regulation after loss of Abelson helper integration site 1 (AHI1) promotes depression via the circadian clock pathway in mice.

Abelson helper integration site 1 (AHI1) is associated with several neuropsychiatric and brain developmental disorders, such as schizophrenia, depression, autism, and Joubert syndrome. Ahi1 deficiency in mice leads to behaviors typical of depression. However, the mechanisms by which AHI1 regulates behavior remain to be elucidated. Here, we found that down-regulation of expression of the rate-limiting enzyme in dopamine biosynthesis, tyrosine hydroxylase (TH), in the midbrains of Ahi1-knockout (KO) mice is responsible for Ahi1-deficiency-mediated depressive symptoms. We also found that Rev-Erbα, a TH transcriptional repressor and circadian regulator, is up-regulated in the Ahi1-KO mouse midbrains and Ahi1-knockdown Neuro-2a cells. Moreover, brain and muscle Arnt-like protein 1 (BMAL1), the Rev-Erbα transcriptional regulator, is also increased in the Ahi1-KO mouse midbrains and Ahi1-knockdown cells. Our results further revealed that AHI1 decreases BMAL1/Rev-Erbα expression by interacting with and repressing retinoic acid receptor-related orphan receptor α, a nuclear receptor and transcriptional regulator of circadian genes. Of note, Bmal1 deficiency reversed the reduction in TH expression induced by Ahi1 deficiency. Moreover, microinfusion of the Rev-Erbα inhibitor SR8278 into the ventral midbrain of Ahi1-KO mice significantly increased TH expression in the ventral tegmental area and improved their depressive symptoms. These findings provide a mechanistic explanation for a link between AHI1-related behaviors and the circadian clock pathway, indicating an involvement of circadian regulatory proteins in AHI1-regulated mood and behavior.

TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration.