Human DNA polymerase η promotes RNA-templated error-free repair of DNA double-strand breaks.

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.

Mobility Enhancement in CVD-Grown Monolayer MoS2 Via Patterned Substrate-Induced Nonuniform Straining.

The extraordinary mechanical properties of two-dimensional transition-metal dichalcogenides make them ideal candidates for investigating strain-induced control of various physical properties. Here we explore the role of nonuniform strain in modulating optical, electronic, and transport properties of semiconducting, chemical vapor deposited monolayer MoS2, on periodically nanostructured substrates. A combination of spatially resolved spectroscopic and electronic properties explore and quantify the differential strain distribution and carrier density on a monolayer, as it conformally drapes over the periodic nanostructures. The observed accumulation in electron density at the strained regions is supported by theoretical calculations which form the likely basis for the ensuing ×60 increase in field effect mobility in strained samples. Though spatially nonuniform, the pattern-induced strain is shown to be readily controlled by changing the periodicity of the nanostructures thus providing a robust yet useful macroscopic control on strain and mobility in these systems.

Two-Dimensional Silver-Chalcogenolate-Based Cluster-Assembled Material: A p-type Semiconductor.

We have synthesized and characterized a new two-dimensional honeycomb architecture resembling a single-layer of atomically precise silver cluster-assembled material (CAM), [Ag12(StBu)6(CF3COO)6(4,4'-azopyridine)3] (Ag12-azo-bpy). The interlayer noncovalent van der Waals interactions within the single-crystals were successfully disrupted, leading to the creation of this unique structure. The optimized Ag12-azo-bpy CAM demonstrates a valence band that is localized on the Ag12 cluster node situated near the Fermi energy level. This localization induces electron injection from the linker to the cluster node, facilitating efficient charge transportation along the plane. Exploiting this single-layer structure as a distinctive platform for p-type channel material, it was employed in a field-effect transistor configuration. Remarkably, the transistor exhibits a high hole mobility of 1.215 cm2 V-1 s-1 and an impressive ON/OFF current ratio of ∼4500 at room-temperature.

Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.

Deficiency in classical nonhomologous end-joining-mediated repair of transcribed genes is linked to SCA3 pathogenesis.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.

Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects.

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

DNA Double-Strand Breaks as Pathogenic Lesions in Neurological Disorders.

The damage and repair of DNA is a continuous process required to maintain genomic integrity. DNA double-strand breaks (DSBs) are the most lethal type of DNA damage and require timely repair by dedicated machinery. DSB repair is uniquely important to nondividing, post-mitotic cells of the central nervous system (CNS). These long-lived cells must rely on the intact genome for a lifetime while maintaining high metabolic activity. When these mechanisms fail, the loss of certain neuronal populations upset delicate neural networks required for higher cognition and disrupt vital motor functions. Mammalian cells engage with several different strategies to recognize and repair chromosomal DSBs based on the cellular context and cell cycle phase, including homologous recombination (HR)/homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the classic non-homologous end-joining (NHEJ). In addition to these repair pathways, a growing body of evidence has emphasized the importance of DNA damage response (DDR) signaling, and the involvement of heterogeneous nuclear ribonucleoprotein (hnRNP) family proteins in the repair of neuronal DSBs, many of which are linked to age-associated neurological disorders. In this review, we describe contemporary research characterizing the mechanistic roles of these non-canonical proteins in neuronal DSB repair, as well as their contributions to the etiopathogenesis of selected common neurological diseases.

Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis.

Dominant mutations in the RNA/DNA-binding protein TDP-43 have been linked to amyotrophic lateral sclerosis (ALS). Here, we screened genomic DNA extracted from spinal cord specimens of sporadic ALS patients for mutations in the TARDBP gene and identified a patient specimen with previously reported Q331K mutation. The patient spinal cord tissue with Q331K mutation showed accumulation of higher levels of DNA strand breaks and the DNA double-strand break (DSB) marker γH2AX, compared to age-matched controls, suggesting a role of the Q331K mutation in genome-damage accumulation. Using conditional SH-SY5Y lines ectopically expressing wild-type (WT) or Q331K-mutant TDP-43, we confirmed the increased cytosolic sequestration of the poly-ubiquitinated and aggregated form of mutant TDP-43, which correlated with increased genomic DNA strand breaks, activation of the DNA damage response factors phospho-ataxia-telangiectasia mutated (ATM), phospho-53BP1, γH2AX and neuronal apoptosis. We recently reported the involvement of WT TDP-43 in non-homologous end joining (NHEJ)-mediated DSB repair, where it acts as a scaffold for the recruitment of XRCC4-DNA ligase 4 complex. Here, the mutant TDP-43, due to its reduced interaction and enhanced cytosolic mislocalization, prevented the nuclear translocation of XRCC4-DNA ligase 4. Consistently, the mutant cells showed significantly reduced DNA strand break sealing activity and were sensitized to DNA-damaging drugs. In addition, the mutant cells showed elevated levels of reactive oxygen species, suggesting both dominant negative and loss-of-function effects of the mutation. Together, our study uncovered an association of sporadic Q331K mutation with persistent genome damage accumulation due to both damage induction and repair defects.

Enhancement of Photoacoustic Signal Strength with Continuous Wave Optical Pre-Illumination: A Non-Invasive Technique.

Use of portable and affordable pulse light sources (light emitting diodes (LED) and laser diodes) for tissue illumination offers an opportunity to accelerate the clinical translation of photoacoustic imaging (PAI) technology. However, imaging depth in this case is limited because of low output (optical) power of these light sources. In this work, we developed a noninvasive technique for enhancing strength (amplitude) of photoacoustic (PA) signal. This is a photothermal-based technique in which a continuous wave (CW) optical beam, in addition to short-pulse ~ nsec laser beam, is employed to irradiate and, thus, raise the temperature of sample material selectively over a pre-specified region of interest (we call the process as pre-illumination). The increase in temperature, in turn enhances the PA-signal strength. Experiments were conducted in methylene blue, which is one of the commonly used contrast agents in laboratory research studies, to validate change in temperature and subsequent enhancement of PA-signal strength for the following cases: (1) concentration or optical absorption coefficient of sample, (2) optical power of CW-optical beam, and (3) time duration of pre-illumination. A theoretical hypothesis, being validated by numerical simulation, is presented. To validate the proposed technique for clinical and/or pre-clinical applications (diagnosis and treatments of cancer, pressure ulcers, and minimally invasive procedures including vascular access and fetal surgery), experiments were conducted in tissue-mimicking Agar phantom and ex-vivo animal tissue (chicken breast). Results demonstrate that pre-illumination significantly enhances PA-signal strength (up to ~70% (methylene blue), ~48% (Agar phantom), and ~40% (chicken tissue)). The proposed technique addresses one of the primary challenges in the clinical translation of LED-based PAI systems (more specifically, to obtain a detectable PA-signal from deep-seated tissue targets).

Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A.

Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.

The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD.

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.

MAIN CONCLUSION: Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Syn vip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Syn vip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (syn bar) gene. Marker gene excision was achieved in hybrids with up to 41.18 % recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII + cre-syn bar), were established in subsequent generation through genetic segregation.

Transgenic expression of an unedited mitochondrial orfB gene product from wild abortive (WA) cytoplasm of rice (Oryza sativa L.) generates male sterility in fertile rice lines.

MAIN CONCLUSION: Over-expression of the unedited mitochondrial orfB gene product generates male sterility in fertile indica rice lines in a dose-dependent manner. Cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration are widespread developmental features in plant reproductive systems. In self-pollinated crop plants, these processes often provide useful tools to exploit hybrid vigour. The wild abortive CMS has been employed in the majority of the "three-line" hybrid rice production since 1970s. In the present study, we provide experimental evidence for a positive functional relationship between the 1.1-kb unedited orfB gene transcript, and its translated product in the mitochondria with male sterility. The generation of the 1.1-kb unedited orfB gene transcripts increased during flowering, resulting in low ATP synthase activity in sterile plants. Following insertion of the unedited orfB gene into the genome of male-fertile plants, the plants became male sterile in a dose-dependent manner with concomitant reduction of ATPase activity of F1F0-ATP synthase (complex V). Fertility of the transgenic lines and normal activity of ATP synthase were restored by down-regulation of the unedited orfB gene expression through RNAi-mediated silencing. The genetic elements deciphered in this study could further be tested for their use in hybrid rice development.

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.

Mitochondria-Targeted Oligomeric α-Synuclein Induces TOM40 Degradation and Mitochondrial Dysfunction in Parkinson's Disease and Parkinsonism-Dementia of Guam.

Mitochondrial dysfunction is a central aspect of Parkinson's disease (PD) pathology, yet the underlying mechanisms are not fully understood. This study investigates the link between α-Synuclein (α-Syn) pathology and the loss of translocase of the outer mitochondrial membrane 40 (TOM40), unraveling its implications for mitochondrial dysfunctions in neurons. We discovered that TOM40 protein depletion occurs in the brains of patients with Guam Parkinsonism Dementia (Guam PD) and cultured neurons expressing α-Syn proteinopathy, notably, without corresponding changes in TOM40 mRNA levels. Cultured neurons expressing α-Syn mutants, with or without a mitochondria-targeting signal (MTS) underscore the role of α-Syn's mitochondrial localization in inducing TOM40 degradation. Parkinson's Disease related etiological factors, such as 6-hydroxy dopamine or ROS/metal ions stress, which promote α-Syn oligomerization, exacerbate TOM40 depletion in PD patient-derived cells with SNCA gene triplication. Although α-Syn interacts with both TOM40 and TOM20 in the outer mitochondrial membrane, degradation is selective for TOM40, which occurs via the ubiquitin-proteasome system (UPS) pathway. Our comprehensive analyses using Seahorse technology, mitochondrial DNA sequencing, and damage assessments, demonstrate that mutant α-Syn-induced TOM40 loss results in mitochondrial dysfunction, characterized by reduced membrane potential, accumulation of mtDNA damage, deletion/insertion mutations, and altered oxygen consumption rates. Notably, ectopic supplementation of TOM40 or reducing pathological forms of α-Syn using ADP-ribosylation inhibitors ameliorate these mitochondrial defects, suggesting potential therapeutic avenues. In conclusion, our findings provide crucial mechanistic insights into how α-Syn accumulation leads to TOM40 degradation and mitochondrial dysfunction, offering insights for targeted interventions to alleviate mitochondrial defects in PD.

Endogenous TDP-43 mislocalization in a novel knock-in mouse model reveals DNA repair impairment, inflammation, and neuronal senescence.

TDP-43 mislocalization and aggregation are key pathological features of motor neuron diseases (MND) including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, transgenic hTDP-43 WT or ΔNLS-overexpression animal models mainly capture late-stages TDP-43 proteinopathy, and do not provide a complete understanding of early motor neuron-specific pathology during pre-symptomatic phases. We have now addressed this shortcoming by generating a new endogenous knock-in (KI) mouse model using a combination of CRISPR/Cas9 and FLEX Cre-switch strategy for the conditional expression of a mislocalized Tdp-43ΔNLS variant of mouse Tdp-43. This variant is either expressed conditionally in whole mice or specifically in the motor neurons. The mice exhibit loss of nuclear Tdp-43 concomitant with its cytosolic accumulation and aggregation in targeted cells, leading to increased DNA double-strand breaks (DSBs), signs of inflammation and DNA damage-associated cellular senescence. Notably, unlike WT Tdp43 which functionally interacts with Xrcc4 and DNA Ligase 4, the key DSB repair proteins in the non-homologous end-joining (NHEJ) pathway, the Tdp-43ΔNLS mutant sequesters them into cytosolic aggregates, exacerbating neuronal damage in mice brain. The mutant mice also exhibit myogenic degeneration in limb muscles and distinct motor deficits, consistent with the characteristics of MND. Our findings reveal progressive degenerative mechanisms in motor neurons expressing endogenous Tdp-43ΔNLS mutant, independent of TDP-43 overexpression or other confounding etiological factors. Thus, this unique Tdp-43 KI mouse model, which displays key molecular and phenotypic features of Tdp-43 proteinopathy, offers a significant opportunity to further characterize the early-stage progression of MND and also opens avenues for developing DNA repair-targeted approaches for treating TDP-43 pathology-linked neurodegenerative diseases.

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.

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.