The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis

The pathology of the RNA-/DNA-binding protein TDP-43, first implicated a decade ago in the motor neuron disease amyotrophic lateral sclerosis (ALS), has been subsequently linked to a wide spectrum of neurodegenerative diseases, including frontotemporal dementia (FTD), Alzheimer’s disease (AD), and related dementia-associated disorders. ALS, also known as Lou Gehrig’s disease, is a progressive, degenerative motor neuron disorder, characterized by a diverse etiopathology. TDP-43 pathology, mediated by a combination of several mutations in the TARDBP gene and stress factors, has been linked to more than 97% of ALS patients. We recently identified, for the first time, the critical involvement of TDP-43 in neuronal genome maintenance and the repair of DNA double-strand breaks (DSBs). Our studies showed that TDP-43 regulates the DNA break-sealing activities of the XRCC4-DNA Ligase 4 (LIG4) complex in DSB repair, suggesting that loss of genomic integrity in TDP-43-associated neurodegeneration may be amenable to a DNA repair-based intervention. In this chapter, we discuss the broader aspects of TDP-43 toxicity-induced pathomechanisms, including the emerging role of TDP-43 in neuronal DSB repair and its synergistic genotoxic effects with other neurodegeneration-associated etiologies that contribute significantly to neuronal dysfunction. We also discuss potential future perspectives and underscore how unraveling the molecular basis and implications of TDP-43-induced genome instability in ALS could guide the development of neuroprotective therapies

Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New Paradigm and Its Potential as Therapeutic Target

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disorder, characterized by a diverse etiopathology. While ALS is predominantly sporadic, mutations in one or more of a dozen risk factors have been linked to approximately 10% of familial ALS patients. The multifunctional RNA/DNA-binding protein fused in sarcoma (FUS) is one such protein whose autosomal dominant missense mutations were identified in a subset of familial and sporadic ALS patients. Initial studies linked FUS with both RNA-related and genome maintenance functions, yet the mechanisms and potential implications to neurodegeneration were not completely understood. We recently identified a novel function of FUS in repairing single-strand break (SSB) in the genome. FUS directly interacts and recruits XRCC1/DNA Ligase IIIα (LigIII) to DNA oxidative damage sites in a PARP1 activity-dependent manner, which facilitates optimal oxidative genome damage repair. Besides, FUS regulates DNA strand break sealing by enhancing ligation activity of LigIII. The mutation of FUS induces accumulation of oxidative DNA damage as well as DNA repair deficiency in ALS patients. The novel findings provide insights into a previously undescribed mechanism of DNA repair defect in FUS-associated neurodegeneration, and raise the pentientials of developing neuroprotective therapies by targeting DNA break ligating defects

Regulation of Oxidized Base Repair in Human Chromatin by Posttranslational Modification

Base excision repair (BER) is the major pathway for the repair of oxidized bases and apurinic/apyrimidinic (abasic; AP) sites produced by reaction with reactive oxygen/nitrogen species (ROS/RNS). These metabolites are generated spontaneously by endogenous cellular processes and also by environmental agents. Because most of these lesions are promutagenic, linked to diverse disease-associated somatic mutations, as well as heritable single nucleotide polymorphisms (SNPs) in the normal human population, their prompt repair is warranted. Impairment of repair leading to mutation, a hallmark of cancer, underscores the essentiality of BER for maintaining genome integrity in humans and other mammals. In mammals, repair of oxidized bases and other BER substrates is initiated by DNA glycosylases (DGs), which excise the damaged bases and cleave the DNA strands at the resulting AP sites, followed by sequential end processing, gap-filling DNA synthesis, and ligation. In vitro BER performed with naked DNA substrates has been extensively studied, which delineates its basic mechanistic steps and subpathways. However, recent interest is directed to unraveling BER in cell chromatin, including its regulation via posttranslational modifications (PTMs), which occurs possibly in concert with nucleosome remodeling. Emerging reports on various PTMs of BER enzymes indicate that the PTMs, while dispensable for the enzymatic activity, regulate overall repair by modulating interactions with other repair proteins and chromatin factors, assembly of BER complexes, as well as turnover of the proteins, and may ultimately dictate the cellular phenotype. Here, we discuss recent advances in the BER field by reviewing the PTMs and how they regulate BER in chromatin

Revisiting Metal Toxicity in Neurodegenerative Diseases and Stroke: Therapeutic Potential

Excessive accumulation of pro-oxidant metals, observed in affected brain regions, has consistently been implicated as a contributor to the brain pathology including neurodegenerative diseases and acute injuries such as stroke. Furthermore, the potential interactions between metal toxicity and other commonly associated etiological factors, such as misfolding/aggregation of amyloidogenic proteins or genomic damage, are poorly understood. Decades of research provide compelling evidence implicating metal overload in neurological diseases and stroke. However, the utility of metal toxicity as a therapeutic target is controversial, possibly due to a lack of comprehensive understanding of metal dyshomeostasismediated neuronal pathology. In this article, we discuss the current understanding of metal toxicity and the challenges associated with metal-targeted therapies.Excessive accumulation of pro-oxidant metals, observed in affected brain regions, has consistently been implicated as a contributor to the brain pathology including neurodegenerative diseases and acute injuries such as stroke. Furthermore, the potential interactions between metal toxicity and other commonly associated etiological factors, such as misfolding/aggregation of amyloidogenic proteins or genomic damage, are poorly understood. Decades of research provide compelling evidence implicating metal overload in neurological diseases and stroke. However, the utility of metal toxicity as a therapeutic target is controversial, possibly due to a lack of comprehensive understanding of metal dyshomeostasismediated neuronal pathology. In this article, we discuss the current understanding of metal toxicity and the challenges associated with metal-targeted therapies

New Perspectives on Oxidized Genome Damage and Repair Inhibition by Pro-Oxidant Metals in Neurological Diseases

The primary cause(s) of neuronal death in most cases of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease, are still unknown. However, the association of certain etiological factors, e.g., oxidative stress, protein misfolding/aggregation, redox metal accumulation and various types of damage to the genome, to pathological changes in the affected brain region(s) have been consistently observed. While redox metal toxicity received major attention in the last decade, its potential as a therapeutic target is still at a cross-roads, mostly because of the lack of mechanistic understanding of metal dyshomeostasis in affected neurons. Furthermore, previous studies have established the role of metals in causing genome damage, both directly and via the generation of reactive oxygen species (ROS), but little was known about their impact on genome repair. Our recent studies demonstrated that excess levels of iron and copper observed in neurodegenerative disease-affected brain neurons could not only induce genome damage in neurons, but also affect their repair by oxidatively inhibiting NEIL DNA glycosylases, which initiate the repair of oxidized DNA bases. The inhibitory effect was reversed by a combination of metal chelators and reducing agents, which underscore the need for elucidating the molecular basis for the neuronal toxicity of metals in order to develop effective therapeutic approaches. In this review, we have focused on the oxidative genome damage repair pathway as a potential target for reducing pro-oxidant metal toxicity in neurological diseases.The primary cause(s) of neuronal death in most cases of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease, are still unknown. However, the association of certain etiological factors, e.g., oxidative stress, protein misfolding/aggregation, redox metal accumulation and various types of damage to the genome, to pathological changes in the affected brain region(s) have been consistently observed. While redox metal toxicity received major attention in the last decade, its potential as a therapeutic target is still at a cross-roads, mostly because of the lack of mechanistic understanding of metal dyshomeostasis in affected neurons. Furthermore, previous studies have established the role of metals in causing genome damage, both directly and via the generation of reactive oxygen species (ROS), but little was known about their impact on genome repair. Our recent studies demonstrated that excess levels of iron and copper observed in neurodegenerative disease-affected brain neurons could not only induce genome damage in neurons, but also affect their repair by oxidatively inhibiting NEIL DNA glycosylases, which initiate the repair of oxidized DNA bases. The inhibitory effect was reversed by a combination of metal chelators and reducing agents, which underscore the need for elucidating the molecular basis for the neuronal toxicity of metals in order to develop effective therapeutic approaches. In this review, we have focused on the oxidative genome damage repair pathway as a potential target for reducing pro-oxidant metal toxicity in neurological diseases

An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease

Altered expression of α-synuclein is linked to Parkinson’s disease (PD). A major challenge to explore how the increased α-synuclein affect neurotoxicity is the lack of a suitable human neuronal cell model that mimics this scenario. Its expression in neural precursors affects their differentiation process, in addition to the neuronal adaptability and variability in maintaining a constant level of expression across passages. Here, we describe an SH-SY5Y line harboring Tet-ON SNCA cDNA cassette that allows for induction of controlled α-synuclein expression after neuronal differentiation, which can be an important tool for PD research.Altered expression of α-synuclein is linked to Parkinson’s disease (PD). A major challenge to explore how the increased α-synuclein affect neurotoxicity is the lack of a suitable human neuronal cell model that mimics this scenario. Its expression in neural precursors affects their differentiation process, in addition to the neuronal adaptability and variability in maintaining a constant level of expression across passages. Here, we describe an SH-SY5Y line harboring Tet-ON SNCA cDNA cassette that allows for induction of controlled α-synuclein expression after neuronal differentiation, which can be an important tool for PD research

Carotenoids as Novel Therapeutic Molecules Against Neurodegenerative Disorders: Chemistry and Molecular Docking Analysis

Alzheimer’s disease (AD) is the most devastating neurodegenerative disorder that affects the aging population worldwide. Endogenous and exogenous factors are involved in triggering this complex and multifactorial disease, whose hallmark is Amyloid-β (Aβ), formed by cleavage of amyloid precursor protein by β- and γ-secretase. While there is no definitive cure for AD to date, many neuroprotective natural products, such as polyphenol and carotenoid compounds, have shown promising preventive activity, as well as helping in slowing down disease progression. In this article, we focus on the chemistry as well as structure of carotenoid compounds and their neuroprotective activity against Aβ aggregation using molecular docking analysis. In addition to examining the most prevalent anti-amyloidogenic carotenoid lutein, we studied cryptocapsin, astaxanthin, fucoxanthin, and the apocarotenoid bixin. Our computational structure-based drug design analysis and molecular docking simulation revealed important interactions between carotenoids and Aβ via hydrogen bonding and van der Waals interactions, and shows that carotenoids are powerful anti-amyloidogenic molecules with a potential role in preventing AD, especially since most of them can cross the blood-brain barrier and are considered nutraceutical compounds. Our studies thus illuminate mechanistic insights on how carotenoids inhibit Aβ aggregation. The potential role of carotenoids as novel therapeutic molecules in treating AD and other neurodegenerative disorders are discussed.Alzheimer’s disease (AD) is the most devastating neurodegenerative disorder that affects the aging population worldwide. Endogenous and exogenous factors are involved in triggering this complex and multifactorial disease, whose hallmark is Amyloid-β (Aβ), formed by cleavage of amyloid precursor protein by β- and γ-secretase. While there is no definitive cure for AD to date, many neuroprotective natural products, such as polyphenol and carotenoid compounds, have shown promising preventive activity, as well as helping in slowing down disease progression. In this article, we focus on the chemistry as well as structure of carotenoid compounds and their neuroprotective activity against Aβ aggregation using molecular docking analysis. In addition to examining the most prevalent anti-amyloidogenic carotenoid lutein, we studied cryptocapsin, astaxanthin, fucoxanthin, and the apocarotenoid bixin. Our computational structure-based drug design analysis and molecular docking simulation revealed important interactions between carotenoids and Aβ via hydrogen bonding and van der Waals interactions, and shows that carotenoids are powerful anti-amyloidogenic molecules with a potential role in preventing AD, especially since most of them can cross the blood-brain barrier and are considered nutraceutical compounds. Our studies thus illuminate mechanistic insights on how carotenoids inhibit Aβ aggregation. The potential role of carotenoids as novel therapeutic molecules in treating AD and other neurodegenerative disorders are discussed

The C-terminal Domain (CTD) of Human DNA GlycosylaseNEIL1 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

Chromatin-bound oxidized α-synuclein causes strand breaks in neuronal genomes in in vitro models of Parkinson's Disease

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD