DNA Damage In Neurons Research Articles

Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM.

Short Peptides Protect Fibroblast-Derived Induced Neurons from Age-Related Changes

Neurons become more vulnerable to stress factors with age, which leads to increased oxidative DNA damage, decreased activity of mitochondria and lysosomes, increased levels of p16, decreased LaminB1 proteins, and the depletion of the dendritic tree. These changes are exacerbated in vulnerable neuronal populations during the development of neurodegenerative diseases. Glu-Asp-Arg (EDR) and Lys-Glu-Asp (KED), and Ala-Glu-Asp-Gly (AEDG) peptides have previously demonstrated neuroprotective effects in various models of Alzheimer’s disease. In this study, we investigated the influence of EDR, KED, and AEDG peptides on the aging of fibroblast-derived induced neurons. We used a new in vitro cellular model of human neuronal aging based on the transdifferentiation of aged dermal fibroblasts from elderly donors into induced cortical neurons. All peptides promote the arborization of the dendritic tree, increasing both the number of primary processes and the total length of dendrites. Tripeptides have no effect on the activity of mitochondria and lysosomes and the level of p16 protein in induced neurons. EDR peptide reduces oxidative DNA damage in induced neurons derived from elderly donor fibroblasts. Short peptides partially protect induced neurons from age-related changes and stimulate dendritogenesis in neurons. They can be recommended for use as neuroprotective agents.

SimplySmart_v1, a new tool for the analysis of DNA damage optimized in primary neuronal cultures

BackgroundThe increased interest in research on DNA damage in neurodegeneration has created a need for the development of tools dedicated to the analysis of DNA damage in neurons. Double-stranded breaks (DSBs) are among the most detrimental types of DNA damage and have become a subject of intensive research. DSBs result in DNA damage foci, which are detectable with the marker γH2AX. Manual counting of DNA damage foci is challenging and biased, and there is a lack of open-source programs optimized specifically in neurons. Thus, we developed a new, fully automated application, SimplySmart_v1, for DNA damage quantification and optimized its performance specifically in primary neurons cultured in vitro.ResultsCompared with control neurons, SimplySmart_v1 accurately identifies the induction of DNA damage with etoposide in primary neurons. It also accurately quantifies DNA damage in the desired fraction of cells and processes a batch of images within a few seconds. SimplySmart_v1 was also capable of quantifying DNA damage effectively regardless of the cell type (neuron or NSC-34). The comparative analysis of SimplySmart_v1 with other open-source tools, such as Fiji, CellProfiler and a focinator, revealed that SimplySmart_v1 is the most ‘user-friendly’ and the quickest tool among others and provides highly accurate results free of variability between measurements. In the context of neurodegenerative research, SimplySmart_v1 revealed an increase in DNA damage in primary neurons expressing abnormal TAR DNA/RNA binding protein (TDP-43).ConclusionsThese findings showed that SimplySmart_v1 is a new and effective tool for research on DNA damage and can successfully replace other available software.

Multiple lines of evidence for disruption of nuclear lamina and nucleoporins in FUS amyotrophic lateral sclerosis.

Advanced pathological and genetic approaches have revealed that mutations in fused in sarcoma/translated in liposarcoma (FUS/TLS), which is pivotal for DNA repair, alternative splicing, translation and RNA transport, cause familial amyotrophic lateral sclerosis (ALS). The generation of suitable animal models for ALS is essential for understanding its pathogenesis and developing therapies. Therefore, we used CRISPR-Cas9 to generate FUS-ALS mutation in the non-classical nuclear localization signal (NLS), H517D (mouse position: H509D) and genome-edited mice. Fus WT/H509D mice showed progressive motor impairment (accelerating rotarod and DigiGait system) with age, which was associated with the loss of motor neurons and disruption of the nuclear lamina and nucleoporins and DNA damage in spinal cord motor neurons. We confirmed the validity of our model by showing that nuclear lamina and nucleoporin disruption were observed in lower motor neurons differentiated from patient-derived human induced pluripotent stem cells (hiPSC-LMNs) with FUS-H517D and in the post-mortem spinal cord of patients with ALS. RNA sequence analysis revealed that most nuclear lamina and nucleoporin-linking genes were significantly decreased in FUS-H517D hiPSC-LMNs. This evidence suggests that disruption of the nuclear lamina and nucleoporins is crucial for ALS pathomechanisms. Combined with patient-derived hiPSC-LMNs and autopsy samples, this mouse model might provide a more reliable understanding of ALS pathogenesis and might aid in the development of therapeutic strategies.

An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging neurons.

Genomic stability is critical for cellular function, however, in the central nervous system highly metabolically active differentiated neurons are challenged to maintain their genome over the organismal lifespan without replication. DNA damage in neurons increases with chronological age and accelerates in neurodegenerative disorders, resulting in cellular and systemic dysregulation. Distinct DNA damage response strategies have evolved with a host of polymerases. The Y-family translesion synthesis (TLS) polymerases are well known for bypassing and repairing damaged DNA in dividing cells. However, their expression, dynamics, and role if any, in enduring postmitotic differentiated neurons of the brain are completely unknown. We show through systematic longitudinal studies for the first time that DNA polymerase kappa (POLK), a member of the Y-family polymerases, is highly expressed in neurons. With chronological age, there is a progressive and significant reduction of nuclear POLK with a concomitant accumulation in the cytoplasm that is predictive of brain tissue age. The reduction of nuclear POLK in old brains is congruent with an increase in DNA damage markers. The nuclear POLK colocalizes with damaged sites and DNA repair proteins. The cytoplasmic POLK accumulates with stress granules and endo/lysosomal markers. Nuclear POLK expression is significantly higher in GABAergic interneurons compared to excitatory pyramidal neurons and lowest in non-neurons, possibly reflective of the inherent biological differences such as firing rates and neuronal activity. Interneurons associated with microglia have significantly higher levels of cytoplasmic POLK in old age. Finally, we show that neuronal activity itself can lead to an increase in nuclear POLK levels and a reduction of the cytoplasmic fraction. Our findings open a new avenue in understanding how different classes of postmitotic neurons deploy TLS polymerase(s) to maintain their genomic integrity over time, which will help design strategies for longevity, healthspan, and prevention of neurodegeneration.

Early oxidative stress and DNA damage in Aβ-burdened hippocampal neurons in an Alzheimer’s-like transgenic rat model

Oxidative stress is a key contributor to AD pathology. However, the earliest role of pre-plaque neuronal oxidative stress, remains elusive. Using laser microdissected hippocampal neurons extracted from McGill-R-Thy1-APP transgenic rats we found that intraneuronal amyloid beta (iAβ)-burdened neurons had increased expression of genes related to oxidative stress and DNA damage responses including Ercc2, Fancc, Sod2, Gsr, and Idh1. DNA damage was further evidenced by increased neuronal levels of XPD (Ercc2) and γH2AX foci, indicative of DNA double stranded breaks (DSBs), and by increased expression of Ercc6, Rad51, and Fen1, and decreased Sirt6 in hippocampal homogenates. We also found increased expression of synaptic plasticity genes (Grin2b (NR2B), CamkIIα, Bdnf, c-fos, and Homer1A) and increased protein levels of TOP2β. Our findings indicate that early accumulation of iAβ, prior to Aβ plaques, is accompanied by incipient oxidative stress and DSBs that may arise directly from oxidative stress or from maladaptive synaptic plasticity.

The effect of crocin on movement disorders and oxidative DNA damage in Parkinson's disease: Insights from a randomized controlled trial

BackgroundParkinson's disease (PD), the second most prevalent neurological disorder in the elderly, manifests with distinctive movement disorders, including bradykinesia, resting tremor, and stiffness. With a progressive course, current treatment strategies primarily target symptomatic relief. Crocin is a chemical compound isolated from the dry stigma of Crocus sativus, and has demonstrated neuroprotective properties. ObjectivesThis study explores the impact of crocin on movement disorders and neuronal oxidative DNA damage in PD patients. MethodConducted as a randomized, blinded, and controlled trial, this research focused on patients aged 30 to 80 with idiopathic PD. Using the second and third parts of the movement disorder society-unified PD rating scale (MDS-UPDRS), aspects of daily life activity and movement disorders were assessed before and after an 8-week intervention. Patients in the crocin groups received capsules containing 30 mg of crocin twice daily. Additionally, the 8-hydroxy-2-deoxydiguanosine (8-OHdG) to urinary creatinine ratio (8-OHdG/uCr) was measured to evaluate neuronal oxidative DNA damage. ResultsOut of the initially evaluated 164 patients, 30 were randomly assigned to each group, with 53 subjects completing the study. Within-group analysis revealed a significant improvement in the second and third parts of MDS-UPDRS after 8 weeks of crocin intervention (P < 0.05). However, the 8-OHdG/uCr did not show significant changes. The well-tolerated daily dose of 60 mg of crocin demonstrated minimal side effects. ConclusionThis study establishes the efficacy of crocin in enhancing daily life activities and mitigating movement disorders, suggesting its potential as a supplementary intervention alongside conventional PD medications.

Mono- and Biallelic Inactivation of Huntingtin Gene in Patient-Specific Induced Pluripotent Stem Cells Reveal HTT Roles in Striatal Development and Neuronal Functions.

Mutations in the Huntingtin (HTT) gene cause Huntington's disease (HD), a neurodegenerative disorder. As a scaffold protein, HTT is involved in numerous cellular functions, but its normal and pathogenic functions during human forebrain development are poorly understood. To investigate the developmental component of HD, with a specific emphasis on understanding the functions of wild-type and mutant HTT alleles during forebrain neuron development in individuals carrying HD mutations. We used CRISPR/Cas9 gene-editing technology to disrupt the ATG region of the HTT gene via non-homologous end joining to produce mono- or biallelic HTT knock-out human induced pluripotent stem cell (iPSC) clones. We showed that the loss of wild-type, mutant, or both HTT isoforms does not affect the pluripotency of iPSCs or their transition into neural cells. However, we observed that HTT loss causes division impairments in forebrain neuro-epithelial cells and alters maturation of striatal projection neurons (SPNs) particularly in the acquisition of DARPP32 expression, a key functional marker of SPNs. Finally, young post-mitotic neurons derived from HTT-/- human iPSCs display cellular dysfunctions observed in adult HD neurons. We described a novel collection of isogenic clones with mono- and biallelic HTT inactivation that complement existing HD-hiPSC isogenic series to explore HTT functions and test therapeutic strategies in particular HTT-lowering drugs. Characterizing neural and neuronal derivatives from human iPSCs of this collection, we show evidence that HTT loss or mutation has impacts on neuro-epithelial and striatal neurons maturation, and on basal DNA damage and BDNF axonal transport in post-mitotic neurons.

Abstract 3222: Acute and long-term responses to treatment with the HDAC inhibitor quisinostat in preclinical models of glioblastoma

Abstract Introduction: High expression of histone deacetylases (HDACs), particularly HDAC1, is strongly associated with poor prognosis for glioblastoma (GBM) patients. Quisinostat is a second-generation, class I-II selective HDAC inhibitor with high specificity for HDAC1 and potent anti-tumor activity. Here, we evaluated GBM PDX tissues and glioma stem cells (GSCs) for cellular, transcriptomic and phenotypic changes after acute and long-term treatment with quisinostat. Methods: Multiple GBM PDX models and PDX-derived GSCs were treated with quisinostat, radiation, or a combination of quisinostat and radiation. Tissue and cells were processed for RNA-seq, qRT-PCR, and immunofluorescence assays to examine alterations in neuronal signaling, cell fate, DNA damage, and markers of stemness and differentiation. Results: Bulk RNA sequencing of tumor tissue revealed that chronic co-treatment with quisinostat and radiation led to increased cellular expression of neuronal signaling and neuronal development pathways compared to the untreated control group. Increase in expression of neuronal signaling genes was also observed upon in vitro treatment of GSCs with quisinostat. Immunocytochemistry and immunofluorescence staining confirmed changes in neuronal signaling and neuronal cell fate proteins after quisinostat treatment. Conclusions: Quisinostat treatment induces a shift in tumor cells towards a neuronal-like cell fate. Considering that GSCs have been shown to establish synaptic junctions with healthy neurons to evoke enhanced excitatory currents within glioma cells and contribute to treatment resistance, further investigation of this shift upon HDAC inhibition is crucial. Ongoing studies are aimed at evaluating the significance of these cellular and molecular changes resulting from quisinostat treatment and whether the quisinostat-induced cell fate changes can be exploited for future combination therapy for GBM. Citation Format: Keely Orndorff, James McNamara, Mariya Stavnichuk, Costanza Lo Cascio, Connor White, Ernesto Luna Melendez, Gozde Uzunalli, Nader Sanai, Shwetal Mehta. Acute and long-term responses to treatment with the HDAC inhibitor quisinostat in preclinical models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3222.

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI).Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence.In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death.Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Relationships of brain cholesterol and cholesterol biosynthetic enzymes to Alzheimer’s pathology and dementia in the CFAS population-derived neuropathology cohort

Altered cholesterol metabolism is implicated in brain ageing and Alzheimer’s disease. We examined whether key genes regulating cholesterol metabolism and levels of brain cholesterol are altered in dementia and Alzheimer’s disease neuropathological change (ADNC). Temporal cortex (n = 99) was obtained from the Cognitive Function and Ageing Study. Expression of the cholesterol biosynthesis rate-limiting enzyme HMG-CoA reductase (HMGCR) and its regulator, SREBP2, were detected using immunohistochemistry. Expression of HMGCR, SREBP2, CYP46A1 and ABCA1 were quantified by qPCR in samples enriched for astrocyte and neuronal RNA following laser-capture microdissection. Total cortical cholesterol was measured using the Amplex Red assay. HMGCR and SREBP2 proteins were predominantly expressed in pyramidal neurones, and in glia. Neuronal HMGCR did not vary with ADNC, oxidative stress, neuroinflammation or dementia status. Expression of HMGCR neuronal mRNA decreased with ADNC (p = 0.022) and increased with neuronal DNA damage (p = 0.049), whilst SREBP2 increased with ADNC (p = 0.005). High or moderate tertiles for cholesterol levels were associated with increased dementia risk (OR 1.44, 1.58). APOE ε4 allele was not associated with cortical cholesterol levels. ADNC is associated with gene expression changes that may impair cholesterol biosynthesis in neurones but not astrocytes, whilst levels of cortical cholesterol show a weak relationship to dementia status.

Hexavalent Chromium Induces Neurotoxicity by Triggering Mitochondrial Dysfunction and ROS-Mediated Signals.

Hexavalent chromium (Cr (VI)), one of the most detrimental pollutants, has been ubiquitously present in the environment and causes serious toxicity to humans, such as hepatotoxicity, nephrotoxicity, pulmonary toxicity, and cardiotoxicity. However, Cr (VI)-induced neurotoxicity in primary neuron level has not been well explored yet. Herein, potassium dichromate (K2Cr2O7) was employed to examine the neurotoxicity of Cr (VI) in rat primary hippocampal neurons. MTT test was used to examine the neural viability. Mitochondrial dysfunction was assessed by the JC-1 probe and Mito-Tracker probe. DCFH-DA and Mito-SOX Red were utilized to evaluate the oxidative status. Bcl-2 family and MAPKs expression were investigated using Western blotting. The results demonstrated that Cr (VI) treatment dose- and time-dependently inhibited neural viability. Mechanism investigation found that Cr (VI) treatment causes mitochondrial dysfunction by affecting Bcl-2 family expression. Moreover, Cr (VI) treatment also induces intracellular reactive oxygen species (ROS) generation, DNA damage, and MAPKs activation in neurons. However, inhibition of ROS by glutathione (GSH) effectually balanced Bcl-2 family expression, attenuated DNA damage and the MAPKs activation, and eventually improved neural viability neurons. Collectively, these above results above suggest that Cr (VI) causes significant neurotoxicity by triggering mitochondrial dysfunction, ROS-mediated oxidative damage and MAKPs activation.

Chemotherapy-induced neuronal DNA damage: an intriguing toolbox to elucidate DNA repair mechanisms in the brain

Chemotherapy-induced neuronal DNA damage: an intriguing toolbox to elucidate DNA repair mechanisms in the brain

Protective Effects of Heat-Killed Ruminococcus albus against β-Amyloid-Induced Apoptosis on SH-SY5Y Cells.

A high level of β-amyloid (Aβ) in the brains of patients with Alzheimer's disease (AD) generates reactive oxygen species that induce neuronal death and DNA damage. The interaction between the gut microbiota and brain health has attracted attention in recent years. Heat-killed Ruminococcus albus (hkRA) reportedly protects neurons against damage induced by oxidative stress. However, whether hkRA can inhibit Aβ-induced apoptosis and thus alleviate AD remains unclear. Hence, we aimed to evaluate the protective effects of hkRA against Aβ-induced apoptosis on the human neuroblastoma SH-SY5Y cell. HkRA treatment (108 cells/ml) significantly decreased the Aβ-induced cytotoxicity and DNA damage in the SH-SY5Y cells. It also showed a significant increase of the bax/bcl-2 ratio in the Aβ-treated SH-SY5Y cells. Moreover, hkRA treatment stimulated the expression of antioxidation-related genes HO-1, Nrf2, and PKC-δ and increased the expression of brain-derived neurotrophic factor (BDNF). Meanwhile, it significantly decreased the activity of caspase-3 and protein expression of cleaved caspase-3 in the Aβ-treated SH-SY5Y cells. Additionally, the protein levels of mitochondrial and cytosolic cytochrome c increased and decreased, respectively, in the cells. These results suggest that hkRA protects human neuroblastoma cells from Aβ-induced apoptosis and oxidative stress. Thus, hkRA may be developed into a health-promoting paraprobiotic (the inactivated microbial cells of probiotics) for patients with AD.

Loss of TDP-43 function contributes to genomic instability in amyotrophic lateral sclerosis.

A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the cytoplasmic mislocalization and aggregation of the DNA/RNA-binding protein TDP-43, but how loss of nuclear TDP-43 function contributes to ALS and FTD pathogenesis remains largely unknown. Here, using large-scale RNAi screening, we identify TARDBP, which encodes TDP-43, as a gene whose loss-of-function results in elevated DNA mutation rate and genomic instability. Consistent with this finding, we observe increased DNA damage in induced pluripotent stem cells (iPSCs) and iPSC-derived post-mitotic neurons generated from ALS patients harboring TARDBP mutations. We find that the increase in DNA damage in ALS iPSC-derived neurons is due to defects in two major pathways for DNA double-strand break repair: non-homologous end joining and homologous recombination. Cells with defects in DNA repair are sensitive to DNA damaging agents and, accordingly, we find that ALS iPSC-derived neurons show a marked reduction in survival following treatment with a DNA damaging agent. Importantly, we find that increased DNA damage is also observed in neurons with nuclear TDP-43 depletion from ALS/FTD patient brain tissues. Collectively, our results demonstrate that ALS neurons with loss of nuclear TDP-43 function have elevated levels of DNA damage and contribute to the idea that genomic instability is a defining pathological feature of ALS/FTD patients with TDP-43 pathology.

Oxidative DNA Damage-induced PARP-1-mediated Autophagic Flux Disruption Contributes to Bupivacaine-induced Neurotoxicity During Pregnancy.

Severe neurologic complications after spinal anesthesia are rare but highly distressing, especially in pregnant women. Bupivacaine is widely used in spinal anesthesia, but its neurotoxic effects have gained attention. Furthermore, the etiology of bupivacaine-mediated neurotoxicity in obstetric patients remains unclear. Female C57BL/6 mice were intrathecally injected with 0.75% bupivacaine on the 18th day of pregnancy. We used immunohistochemistry to examine DNA damage after bupivacaine treatment in pregnant mice and measured γ-H2AX (Ser139) and 8-OHdG in the spinal cord. A PARP-1 inhibitor (PJ34) and autophagy inhibitor (3-MA) were administered with bupivacaine in pregnant mice. Parp-1flox/flox mice were crossed with Nes-Cre transgenic mice to obtain neuronal conditional knockdown mice. Then, LC3B and P62 staining were performed to evaluate autophagic flux in the spinal cords of pregnant wild-type (WT) and Parp-1-/- mice. We performed transmission electron microscopy (TEM) to evaluate autophagosomes. The present study showed that oxidative stress-mediated DNA damage and neuronal injury were increased after bupivacaine treatment in the spinal cords of pregnant mice. Moreover, PARP-1 was significantly activated, and autophagic flux was disrupted. Further studies revealed that PARP-1 knockdown and autophagy inhibitors could alleviate bupivacaine-mediated neurotoxicity in pregnant mice. Bupivacaine may cause neuronal DNA damage and PARP-1 activation in pregnant mice. PARP-1 further obstructed autophagic flux and ultimately led to neurotoxicity.

Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin

Noncanonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-strand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells.

Lewy body disease as a potential negative outcome modifier of glioblastoma treatment: a case report

BackgroundElderly patients with glioblastoma are particularly susceptible to the adverse effects of ionizing radiation to the brain. This population also has an increasing prevalence of dementia in the successive seventh, eighth and nineth decade of life, and dementia with Lewy bodies is characterized by pathologic α-synucleins, proteins that take part in neuronal DNA damage repair.Case presentationWe report a 77-year-old man, with a history of coronary artery disease and mild cognitive impairment, who experienced subacute behavioral changes over 3 months with wording-finding difficulty, memory loss, confusion, perseveration, and irritable mood. Neuroimaging studies disclosed a 2.5 × 2.4 × 2.7 cm cystic enhancing mass with central necrosis in the left temporal lobe of the brain. Gross total resection of the tumor revealed IDH-1 wild-type glioblastoma. After treatment with radiation and temozolomide chemotherapy, his cognitive status deteriorated rapidly, and he died from unexpected sudden death 2 months after radiation. Autopsy of his brain revealed (i) tumor cells with atypical nuclei and small lymphocytes, (ii) neuronal cytoplasmic inclusions and Lewy bodies that were positive for α-synuclein in the midbrain, pons, amygdala, putamen and globus pallidus, and (iii) no amyloid plaques and only rare neurofibrillary tangles near the hippocampi.ConclusionsThis patient most likely had pre-clinical limbic subtype of dementia with Lewy bodies prior to his diagnosis of glioblastoma. The radiation and temozolomide that was used to treat his tumor may have accelerated neuronal damage due to induction of DNA breakage when his brain was already compromised by pathologic α-synucleins. α-Synucleinopathy could be a negative outcome modifier in glioblastoma patients.

MDM2 upregulation induces mitophagy deficiency via Mic60 ubiquitination in fetal microglial inflammation and consequently neuronal DNA damage caused by exposure to ZnO-NPs during pregnancy

During pregnancy, the human body is quite vulnerable to external stimuli. Zinc oxide nanoparticles (ZnO-NPs) are widely used in daily life, and they enter the human body via environmental or biomedical exposure, thus having potential risks. Although accumulating studies have demonstrated the toxic effects of ZnO-NPs, few studies have addressed the effect of prenatal ZnO-NP exposure on fetal brain tissue development. Here, we systematically studied ZnO-NP-induced fetal brain damage and the underlying mechanism. Using in vivo and in vitro assays, we found that ZnO-NPs could cross the underdeveloped bloodbrain barrier and enter fetal brain tissue, where they could be endocytosed by microglia. ZnO-NP exposure impaired mitochondrial function and induced autophagosome overaccumulation by downregulation of Mic60, thus inducing microglial inflammation. Mechanistically, ZnO-NPs increased Mic60 ubiquitination by activating MDM2, resulting in imbalanced mitochondrial homeostasis. Inhibition of Mic60 ubiquitination by MDM2 silencing significantly attenuated the mitochondrial damage induced by ZnO-NPs, thereby preventing autophagosome overaccumulation and reducing ZnO-NP-mediated inflammation and neuronal DNA damage. Our results demonstrate that ZnO-NPs are likely to disrupt mitochondrial homeostasis, inducing abnormal autophagic flux and microglial inflammation and secondary neuronal damage in the fetus. We hope the information provided in our study will improve the understanding of the effects of prenatal ZnO-NP exposure on fetal brain tissue development and draw more attention to the daily use of and therapeutic exposure to ZnO-NPs among pregnant women.

The Tardigrade damage suppressor protein Dsup promotes DNA damage in neurons

Tardigrades are microscopic invertebrates, which are capable of withstanding extreme environmental conditions, including high levels of radiation. A Tardigrade protein, Dsup (Damage Suppressor), protects the Tardigrade's DNA during harsh environmental stress and X-rays. When expressed in cancer cells, Dsup protects DNA from single- and double-strand breaks (DSBs) induced by radiation, increases survival of irradiated cells, and protects DNA from reactive oxygen species. These unusual properties of Dsup suggested that understanding how the protein functions may help in the design of small molecules that could protect humans during radiotherapy or space travel. Here, we investigated if Dsup is protective in cortical neurons cultured from rat embryos. We discovered that, in cortical neurons, the codon-optimized Dsup localizes to the nucleus and, surprisingly, promotes neurotoxicity, leading to neurodegeneration. Unexpectedly, we found that Dsup expression results in the formation of DNA DSBs in cultured neurons. With electron microscopy, we discovered that Dsup promotes chromatin condensation. Unlike Dsup's protective properties in cancerous cells, in neurons, Dsup promotes neurotoxicity, induces DNA damage, and rearranges chromatin. Neurons are sensitive to Dsup, and Dsup is a doubtful surrogate for DNA protection in neuronal cells.