Polycystin-1 loss of function increases susceptibility to atrial fibrillation through impaired DNA damage response.

BACKGROUND: The increasing prevalence of atrial fibrillation (AF) and chronic kidney diseases highlights the need for a deeper comprehension of the molecular mechanisms linking them. Mutations in PKD1, the gene encoding Polycystin-1 (PKD1 or PC1), account for 85% of autosomal dominant polycystic kidney disease (ADPKD) cases. This disease often includes cardiac complications such as AF. In cardiomyocytes, PC1 deletion reduces hypertrophic response to pressure overload but promotes baseline ventricular dysfunction, while deletion in fibroblasts ameliorates post-myocardial infarction fibrosis. Despite its known cardiac impact, the role of PC1 in atrial cardiomyocytes and arrhythmias is less understood. Here, we sought to investigate the role of PC1 in AF. METHODS: We used intracardiac programmed stimulation and optical mapping to evaluate AF inducibility in two mouse models, Pkd1 R3277C, which recapitulates human ADPKD progression, and cardiomyocyte-specific Pkd1 deletion, and their respective controls. Isolated adult mouse atrial cardiomyocytes, human iPSC-derived atrial cardiomyocytes (hiPSC-aCM), and HL-1 cells served as in vitro cellular models. Molecular mechanisms were evaluated using optical mapping and molecular and biochemical approaches. RESULTS: Loss-of-function PC1 mutations significantly increased AF susceptibility in vivo and facilitated local reentry in ex vivo left atrial appendages. Comprehensive in vitro experiments supported a direct effect of PC1 in atrial cardiomyocytes. PC1-deficient monolayers exhibited increased arrhythmic events, escalating into reentrant spiral waves post-tachypacing. Transcriptomics analysis revealed PC1-dependent regulation of DNA repair, with PC1 deficiency leading to increased DNA damage under stress. PARP1 inhibitors or nicotinamide riboside, which counteract DNA damage-related metabolic consequences, reduced in vitro arrhythmias PC1-deficient monolayers. Overexpression of the C-terminus of PC1 had the opposite effects in DNA repair genes, suggesting its regulatory effects in atrial cardiomyocytes through retinoblastoma/E2F. Analyses of human atrial tissue from non-ADPKD patients showed reduced levels of mature PC1, suggesting a broader relevance of impaired PC1 in AF. CONCLUSIONS: Impaired PC1 increases in vivo AF inducibility under programmed electrical stimulation and promotes in vitro arrhythmias in hiPSC-aCM and HL-1 cells. Our findings indicate that PC1 protects against DNA damage to reduce AF susceptibility.

Regulation of DNA damage response by RNA/DNA-binding proteins: Implications for neurological disorders and aging.

RNA-binding proteins (RBPs) are evolutionarily conserved across most forms of life, with an estimated 1500 RBPs in humans. Traditionally associated with post-transcriptional gene regulation, RBPs contribute to nearly every known aspect of RNA biology, including RNA splicing, transport, and decay. In recent years, an increasing subset of RBPs have been recognized for their DNA binding properties and involvement in DNA transactions. We refer to these RBPs with well-characterized DNA binding activity as RNA/DNA binding proteins (RDBPs), many of which are linked to neurological diseases. RDBPs are associated with both nuclear and mitochondrial DNA repair. Furthermore, the presence of intrinsically disordered domains in RDBPs appears to be critical for regulating their diverse interactions and plays a key role in controlling protein aggregation, which is implicated in neurodegeneration. In this review, we discuss the emerging roles of common RDBPs from the heterogeneous nuclear ribonucleoprotein (hnRNP) family, such as TAR DNA binding protein-43 (TDP43) and fused in sarcoma (FUS) in controlling DNA damage response (DDR). We also explore the implications of RDBP pathology in aging and neurodegenerative diseases and provide a prospective on the therapeutic potential of targeting RDBP pathology mediated DDR defects for motor neuron diseases and aging.

TDP43 interacts with MLH1 and MSH6 proteins in a DNA damage-inducible manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.

TDP43 Interacts with MLH1 and MSH6 Proteins in A DNA Damage-Inducible Manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

FUS unveiled in mitochondrial DNA repair and targeted ligase-1 expression rescues repair-defects in FUS-linked motor neuron disease.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.

lncRNA Sequencing Reveals Neurodegeneration-Associated FUS Mutations Alter Transcriptional Landscape of iPS Cells That Persists in Motor Neurons.

Fused-in sarcoma (FUS) gene mutations have been implicated in amyotrophic lateral sclerosis (ALS). This study aimed to investigate the impact of FUS mutations (R521H and P525L) on the transcriptome of induced pluripotent stem cells (iPSCs) and iPSC-derived motor neurons (iMNs). Using RNA sequencing (RNA Seq), we characterized differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELs) and subsequently predicted lncRNA-mRNA target pairs (TAR pairs). Our results show that FUS mutations significantly altered the expression profiles of mRNAs and lncRNAs in iPSCs. Using this large dataset, we identified and verified six key differentially regulated TAR pairs in iPSCs that were also altered in iMNs. These target transcripts included: GPR149, NR4A, LMO3, SLC15A4, ZNF404, and CRACD. These findings indicated that selected mutant FUS-induced transcriptional alterations persist from iPSCs into differentiated iMNs. Functional enrichment analyses of DEGs indicated pathways associated with neuronal development and carcinogenesis as likely altered by these FUS mutations. Furthermore, ingenuity pathway analysis (IPA) and GO network analysis of lncRNA-targeted mRNAs indicated associations between RNA metabolism, lncRNA regulation, and DNA damage repair. Our findings provide insights into potential molecular mechanisms underlying the pathophysiology of ALS-associated FUS mutations and suggest potential therapeutic targets for the treatment of ALS.

FUS Unveiled in Mitochondrial DNA Repair and Targeted Ligase-1 Expression Rescues Repair-Defects in FUS-Linked Neurodegeneration.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated neurodegeneration.

lncRNA Sequencing Reveals Neurodegeneration-associated FUS Mutations Alter Transcriptional Landscape of iPS Cells That Persists In Motor Neurons.

Fused-in Sarcoma (FUS) gene mutations have been implicated in amyotrophic lateral sclerosis (ALS). This study aimed to investigate the impact of FUS mutations (R521H and P525L) on the transcriptome of induced pluripotent stem cells (iPSCs) and iPSC-derived motor neurons (iMNs). Using RNA sequencing (RNA Seq), we characterized differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs), and subsequently predicted lncRNA-mRNA target pairs (TAR pairs). Our results show that FUS mutations significantly altered expression profiles of mRNAs and lncRNAs in iPSCs. We identified key differentially regulated TAR pairs, including LMO3, TMEM132D, ERMN, GPR149, CRACD, and ZNF404 in mutant FUS iPSCs. We performed reverse transcription PCR (RT-PCR) validation in iPSCs and iMNs. Validation confirmed RNA-Seq findings and suggested that mutant FUS-induced transcriptional alterations persisted from iPSCs into differentiated iMNs. Functional enrichment analyses of DEGs indicated pathways associated with neuronal development and carcinogenesis that were likely altered by FUS mutations. Ingenuity Pathway Analysis (IPA) and GO network analysis of lncRNA-targeted mRNAs indicated associations related to RNA metabolism, lncRNA regulation, and DNA damage repair. Our findings provide insights into the molecular mechanisms underlying the pathophysiology of ALS-associated FUS mutations and suggest potential therapeutic targets for the treatment of ALS.