DNA Damage In Neurons Research Articles

Repair of oxidative DNA damage, cell-cycle regulation and neuronal death may influence the clinical manifestation of Alzheimer's disease.

Alzheimer’s disease (AD) is characterized by progressive cognitive decline associated with a featured neuropathology (neuritic plaques and neurofibrillary tangles). Several studies have implicated oxidative damage to DNA, DNA repair, and altered cell-cycle regulation in addition to cell death in AD post-mitotic neurons. However, there is a lack of studies that systematically assess those biological processes in patients with AD neuropathology but with no evidence of cognitive impairment. We evaluated markers of oxidative DNA damage (8-OHdG, H2AX), DNA repair (p53, BRCA1, PTEN), and cell-cycle (Cdk1, Cdk4, Cdk5, Cyclin B1, Cyclin D1, p27Kip1, phospho-Rb and E2F1) through immunohistochemistry and cell death through TUNEL in autopsy hippocampal tissue samples arrayed in a tissue microarray (TMA) composed of three groups: I) “clinical-pathological AD” (CP-AD) - subjects with neuropathological AD (Braak≥IV and CERAD = B or C) and clinical dementia (CDR≥2, IQCODE>3.8); II) “pathological AD” (P-AD) - subjects with neuropathological AD (Braak≥IV and CERAD = B or C) and without cognitive impairment (CDR 0, IQCODE<3.2); and III) “normal aging” (N) - subjects without neuropathological AD (Braak≤II and CERAD 0 or A) and with normal cognitive function (CDR 0, IQCODE<3.2). Our results show that high levels of oxidative DNA damage are present in all groups. However, significant reductions in DNA repair and cell-cycle inhibition markers and increases in cell-cycle progression and cell death markers in subjects with CP-AD were detected when compared to both P-AD and N groups, whereas there were no significant differences in the studied markers between P-AD individuals and N subjects. This study indicates that, even in the setting of pathological AD, healthy cognition may be associated with a preserved repair to DNA damage, cell-cycle regulation, and cell death in post-mitotic neurons.

Apomorphine attenuates ethanol-induced neurodegeneration in the adult rat cortex

Apomorphine, therapeutically used for Parkinson's disease, is a dopamine D1/D2 receptor agonist that has been determined to be a potent antioxidant and to prevent the reaction of free radicals in the brain. Alcohol is a neurotoxic agent that induces neurodegeneration possibly through the generation of free radicals. In this study, we investigated the antioxidant potential of apomorphine upon ethanol-induced neurodegeneration in the cortex of adult rats. Ethanol-induced apoptotic neurodegeneration was measured via the suppression of Bcl-2, the induction of Bax, the release of cytochrome C and the activation of caspase-9 and caspase-3. Moreover, ethanol-induced elevated levels of cleaved PARP-1 indicated exaggerated neuronal DNA damage. Our results demonstrated the neuroprotective effect of apomorphine by reversing the ethanol-induced apoptotic trend as observed by the increased expression of Bcl-2, down regulation of Bax, inhibition of mitochondrial cytochrome C release and inhibition of activated caspase-9 and caspase-3. Moreover, apomorphine treatment further decreased the expression of cleaved PARP-1 to reveal a reduction in ethanol-induced neuronal damage. Immunohistochemical analysis and Nissl staining also revealed neuroprotective effect of apomorphine after ethanol-induced neuronal cell death. In this study, our results indicated that apomorphine at doses of 1 and 5mg/kg has neuroprotective effects for ethanol-induced neuronal damage. Finally, we can conclude that apomorphine has effective therapeutic potential to protect the brain against ethanol-induced neurotoxicity.

Cocaine induces DNA damage in distinct brain areas of female rats under different hormonal conditions

We evaluated levels of neuronal DNA damage after acute or repeated cocaine treatment in different brain areas of female rats after ovariectomy or sham surgery. Rats in the control and acute groups were given saline i.p., whereas in the repeated group were given 15 mg/kg, i.p., cocaine for 8 days. After a 10 day washout period, the control group was given saline i.p., whereas rats in the acute and repeated groups were given a challenge dose of 15 mg/kg, i.p., cocaine. After behavioural assessment, rats were killed and the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected for the Comet assay. Acute cocaine exposure induced DNA damage in all brain areas. This effect persisted after repeated administration, except in the hypothalamus, where repeated treatment did not cause increased DNA damage. Sexual hormones exhibited a neuroprotective effect, decreasing cocaine-induced DNA damage in cycling rats in all brain areas.

17β-Estradiol increases expression of the oxidative stress response and DNA repair protein apurinic endonuclease (Ape1) in the cerebral cortex of female mice following hypoxia

While it is well established that 17β-estradiol (E2) protects the rodent brain from ischemia-induced damage, it has been unclear how this neuroprotective effect is mediated. Interestingly, convincing evidence has also demonstrated that maintaining or increasing the expression of the oxidative stress response and DNA repair protein apurinic endonuclease 1 (Ape1) is instrumental in reducing ischemia-induced damage in the brain. Since E2 increases expression of the oxidative stress response proteins Cu/Zn superoxide dismutase and thioredoxin in the brain, we hypothesized that E2 may also increase Ape1 expression and that this E2-induced expression of Ape1 may help to mediate the neuroprotective effects of E2 in the brain. To test this hypothesis, we utilized three model systems including primary cortical neurons, brain slice cultures, and whole animals. Although estrogen receptor α and Ape1 were expressed in primary cortical neurons, E2 did not alter Ape1 expression in these cells. However, immunofluorescent staining and quantitative Western blot analysis demonstrated that estrogen receptor α and Ape1 were expressed in the nuclei of cortical neurons in brain slice cultures and that E2 increased Ape1 expression in the cerebral cortex of these cultures. Furthermore, Ape1 expression was increased and oxidative DNA damage was decreased in the cerebral cortices of ovariectomized female C57Bl/6J mice that had been treated with E2 and exposed to hypoxia. Taken together, our studies demonstrate that the neuronal microenvironment may be required for increased Ape1 expression and that E2 enhances expression of Ape1 and reduces oxidative DNA damage, which may in turn help to reduce ischemia-induced damage in the cerebral cortex and mediate the neuroprotective effects of E2.

Genistein attenuates ischemic oxidative damage and behavioral deficits via eNOS/Nrf2/HO-1 signaling

Global cerebral ischemia, such as occurs following cardiac arrest, can lead to oxidative stress, hippocampal neuronal cell death, and cognitive defects. The current study examined the potential beneficial effect and underlying mechanisms of post-treatment with the naturally occurring isoflavonic phytoestrogen, genistein, which has been implicated to attenuate oxidative stress. Genistein (1 mg kg(-1)) was administered i.v. 5 min after reperfusion in rats subjected to four-vessel global cerebral ischemia (GCI). The results revealed that genistein exerted significant neuroprotection of hippocampal CA1 neurons following GCI, as evidenced by an increase in NeuN-positive neurons and the decrease in TUNEL-positive neurons. Furthermore, genistein treatment also resulted in significantly improved spatial learning and memory as compared to vehicle control animals. The beneficial effects of genistein appear to be mediated by an increase of phosphorylation/activation of eNOS, with subsequent activation of the antioxidant/detoxification Nrf2/Keap1 transcription system. Along these lines, genistein increased keap1 S-nitrosylation, with a corresponding nuclear accumulation and enhanced DNA binding activity of Nrf2. Genistein also enhanced levels of the Nrf2 downstream antioxidant protein, heme oxygenase (HO)-1, as compared to vehicle control groups. In accordance with its induction of Nrf2 activation, genistein exerted a robust attenuation of oxidative DNA damage and lipid peroxidative damage in hippocampal CA1 neurons after GCI, as measured by immunofluorescence staining of the oxidative stress markers, 8-hydroxy-2-deoxyguanosine (8-OHdG) and 4-Hydroxynonenal (4-HNE). Interestingly, the aforementioned effects of genistein were abolished by pretreatment with L-NAME, an inhibitor of eNOS activation. In conclusion, the results of the study demonstrate that low dose genistein can exert significant antioxidant, neuroprotective, and cognitive-enhancing effects in the hippocampal CA1 region following GCI. Mechanistically, the beneficial effects of genistein appear to be mediated by enhanced eNOS phosphorylation/activation and nitric oxide (NO)-mediated thiol modification of Keap1, with subsequent upregulation of the Nrf2/HO-1 antioxidative signaling pathway and a resultant attenuation of oxidative stress.

The role of ATM and DNA damage in neurons: Upstream and downstream connections

ATM (ataxia-telangiectasia mutated) is a large protein kinase whose best-known function is as a participant in the process of DNA damage repair, specifically lesions that result in double strand breaks. In the cells of the nervous system, however, the symptoms of children with ataxia-telangiectasia and the phenotypes of mice with engineered mutations in their ATM gene argue for a broader range of protein functions. ATM is now appreciated to play a role in vesicle dynamics as well as in the maintenance of the epigenetic code of histone modifications. Finally, the decline of ATM levels with age suggest that late onset neurodegenerative diseases may owe part of their pathogenesis to deficits in ATM signaling. Evidence from the location of HDAC4 in the hippocampal pyramidal cells of the Alzheimer's disease brain supports this hypothesis. These multiple functions of the ATM protein are in keeping with the complex multi-system nature of the symptoms of ataxia-telangiectasia and encourage us to look beyond DNA damage for the full understanding of the disease and its consequences.

Lewy body pathology is associated with mitochondrial DNA damage in Parkinson's disease

Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson's disease (PD) and Alzheimer's disease (AD), but its relation to protein aggregation is unclear. PD is characterized by synuclein aggregation (i.e., Lewy body [LB] formation). In AD, the abnormal accumulation of tau protein forms neurofibrillary tangles. In this study, we laser-dissected LB-positive and -negative neurons from the substantia nigra of postmortem PD brains, and tau-positive and -negative hippocampal neurons from AD brains. We quantified mitochondrial DNA deletions in relation to the cellular phenotype and in comparison with age-matched controls. Deletion levels were highest in LB-positive neurons of PD brains (40.5 ± 16.8%), followed by LB-negative neurons of PD cases (31.8 ± 14.4%) and control subjects (25.6 ± 17.5%; analysis of variance p < 0.005). In hippocampal neurons, deletion levels were 25%–30%, independent of disease status and neurofibrillary tangles. The presented findings imply increased mitochondrial DNA damage in LB-positive midbrain neurons, but do not support a direct causative link of respiratory chain dysfunction and protein aggregation.

Cadmium Induced Cell Apoptosis, DNA Damage, Decreased DNA Repair Capacity, and Genomic Instability during Malignant Transformation of Human Bronchial Epithelial Cells

Cadmium and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood. Our study was designed to elucidate the mechanisms of DNA damage in cadmium-induced malignant transformation of human bronchial epithelial cells. We analyzed cell cycle, apoptosis, DNA damage, gene expression, genomic instability, and the sequence of exons in DNA repair genes in several kinds of cells. These cells consisted of untreated control cells, cells in the fifth, 15th, and 35th passage of cadmium-treated cells, and tumorigenic cells from nude mice using flow cytometry, Hoechst 33258 staining, comet assay, quantitative real-time polymerase chain reaction (PCR), Western blot analysis, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis. We observed a progressive increase in cell population of the G0/G1 phase of the cell cycle and the rate of apoptosis, DNA damage, and cadmium-induced apoptotic morphological changes in cerebral cortical neurons during malignant transformation. Gene expression analysis revealed increased expression of cell proliferation (PCNA), cell cycle (CyclinD1), pro-apoptotic activity (Bax), and DNA damage of the checkpoint genes ATM, ATR, Chk1, Chk2, Cdc25A. Decreased expression of the anti-apoptotic gene Bcl-2 and the DNA repair genes hMSH2, hMLH1, ERCC1, ERCC2, and hOGG1 was observed. RAPD-PCR revealed genomic instability in cadmium-exposed cells, and sequence analysis showed mutation of exons in hMSH2, ERCC1, XRCC1, and hOGG1 in tumorigenic cells. This study suggests that Cadmium can increase cell apoptosis and DNA damage, decrease DNA repair capacity, and cause mutations, and genomic instability leading to malignant transformation. This process could be a viable mechanism for cadmium-induced cancers.

Intranuclear matrix metalloproteinases promote DNA damage and apoptosis induced by oxygen–glucose deprivation in neurons

Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood–brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen–glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons during acute stroke injury.

Deregulation of BRCA1 Leads to Impaired Spatiotemporal Dynamics of γ-H2AX and DNA Damage Responses in Huntington’s Disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of mid-life onset characterized by involuntary movements and progressive cognitive decline caused by a CAG repeat expansion in exon 1 of the Huntingtin (Htt) gene. Neuronal DNA damage is one of the major features of neurodegeneration in HD, but it is not known how it arises or relates to the triplet repeat expansion mutation in the Htt gene. Herein, we found that imbalanced levels of non-phosphorylated and phosphorylated BRCA1 contribute to the DNA damage response in HD. Notably, nuclear foci of γ-H2AX, the molecular component that recruits various DNA damage repair factors to damage sites including BRCA1, were deregulated when DNA was damaged in HD cell lines. BRCA1 specifically interacted with γ-H2AX via the BRCT domain, and this association was reduced in HD. BRCA1 overexpression restored γ-H2AX level in the nucleus of HD cells, while BRCA1 knockdown reduced the spatiotemporal propagation of γ-H2AX foci to the nucleoplasm. The deregulation of BRCA1 correlated with an abnormal nuclear distribution of γ-H2AX in striatal neurons of HD transgenic (R6/2) mice and BRCA1(+/-) mice. Our data indicate that BRCA1 is required for the efficient focal recruitment of γ-H2AX to the sites of neuronal DNA damage. Taken together, our results show that BRCA1 directly modulates the spatiotemporal dynamics of γ-H2AX upon genotoxic stress and serves as a molecular maker for neuronal DNA damage response in HD.

Apolipoprotein epsilon 3 alleles are associated with indicators of neuronal resilience

BackgroundEpilepsy is associated with precocious development of Alzheimer-type neuropathological changes, including appearance of senile plaques, neuronal loss and glial activation. As inheritance of APOE ε4 allele(s) is reported to favor this outcome, we sought to investigate neuronal and glial responses that differ according to APOE genotype. With an eye toward defining ways in which APOE ε3 alleles may foster neuronal well-being in epilepsy and/or APOE ε4 alleles exacerbate neuronal decline, neuronal and glial characteristics were studied in temporal lobectomy specimens from epilepsy patients of either APOE ε4,4 or APOE ε3,3 genotype.MethodsTissue and/or cellular expressions of interleukin-1 alpha (IL-1α), apolipoprotein E (ApoE), amyloid β (Aβ) precursor protein (βAPP), synaptophysin, phosphorylated tau, and Aβ were determined in frozen and paraffin-embedded tissues from 52 APOE ε3,3 and 7 APOE ε4,4 (0.25 to 71 years) epilepsy patients, and 5 neurologically normal patients using Western blot, RT-PCR, and fluorescence immunohistochemistry.ResultsTissue levels of IL-1α were elevated in patients of both APOE ε3,3 and APOE ε4,4 genotypes, and this elevation was apparent as an increase in the number of activated microglia per neuron (APOE ε3,3 vs APOE ε4,4 = 3.7 ± 1.2 vs 1.5 ± 0.4; P < 0.05). This, together with increases in βAPP and ApoE, was associated with apparent neuronal sparing in that APOE ε4,4 genotype was associated with smaller neuron size (APOE ε4,4 vs APOE ε3,3 = 173 ± 27 vs 356 ± 45; P ≤ 0.01) and greater DNA damage (APOE ε4,4 vs APOE ε3,3 = 67 ± 10 vs 39 ± 2; P = 0.01). 3) Aβ plaques were noted at early ages in our epilepsy patients, regardless of APOE genotype (APOE ε4,4 age 10; APOE ε3,3 age 17).ConclusionsOur findings of neuronal and glial events, which correlate with lesser neuronal DNA damage and larger, more robust neurons in epilepsy patients of APOE ε3,3 genotype compared to APOE ε4,4 genotype carriers, are consistent with the idea that the APOE ε3,3 genotype better protects neurons subjected to the hyperexcitability of epilepsy and thus confers less risk of AD (Alzheimer's disease).Please see related article: http://www.biomedcentral.com/1741-7015/10/36

Ginkgo biloba protects against intermittent hypoxia-induced memory deficits and hippocampal DNA damage in rats

The aim of the present study was to explore the potential protective effect of Ginkgo biloba extract (EGb 761) on intermittent hypoxia (IH)-induced memory deficits and oxidative stress in rats. MethodsThe passive avoidance reflex (PAR) test was employed to assess the effect of concurrent EGb 761 treatment in different dose levels on the memory deficits that were induced by concurrent long-term exposure to IH (21 days). The levels of hippocampal malondialdehyde (MDA), nitric oxide (NO), and intracellular glutathione (GSH) and the activity of glutathione peroxidase (GSH-Px) were estimated. In addition, serum and hippocampal 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were assessed to study the effect of EGb 761 on hippocampal oxidative DNA damage induced by IH. ResultsExposure to long-term IH in rats induced marked memory impairment that was indicated by a significant decrease in the retention latency in the PAR test. This effect was accompanied by a significant increase in hippocampal oxidative stress and DNA damage. EGb 761 that was administered in either 50- or 100-mg/kg doses per day reversed IH-induced memory deficits, an effect that was accompanied by a significant decrease in hippocampal MDA and NO levels. The antioxidant defence (GSH and GSH-Px) that was depressed by IH was significantly reactivated by EGb 761. Furthermore, serum and hippocampal levels of 8-OHdG that were elevated by IH were significantly reduced. ConclusionsEGb 761 can protect against IH-induced memory impairment, oxidative stress and neuronal DNA damage, possibly through multiple mechanisms involving its potential anti-oxidative effect.

Oxidative DNA Damage in Neurons: Implication of Ku in Neuronal Homeostasis and Survival

Oxidative DNA damage is produced by reactive oxygen species (ROS) which are generated by exogenous and endogenous sources and continuously challenge the cell. One of the most severe DNA lesions is the double-strand break (DSB), which is mainly repaired by nonhomologous end joining (NHEJ) pathway in mammals. NHEJ directly joins the broken ends, without using the homologous template. Ku70/86 heterodimer, also known as Ku, is the first component of NHEJ as it directly binds DNA and recruits other NHEJ factors to promote the repair of the broken ends. Neurons are particularly metabolically active, displaying high rates of transcription and translation, which are associated with high metabolic and mitochondrial activity as well as oxygen consumption. In such a way, excessive oxygen radicals can be generated and constantly attack DNA, thereby producing several lesions. This condition, together with defective DNA repair systems, can lead to a high accumulation of DNA damage resulting in neurodegenerative processes and defects in neurodevelopment. In light of recent findings, in this paper, we will discuss the possible implication of Ku in neurodevelopment and in mediating the DNA repair dysfunction observed in certain neurodegenerations.

Complex Regulation of p73 Isoforms after Alteration of Amyloid Precursor Polypeptide (APP) Function and DNA Damage in Neurons

Genetic ablations of p73 have shown its implication in the development of the nervous system. However, the relative contribution of ΔNp73 and TAp73 isoforms in neuronal functions is still unclear. In this study, we have analyzed the expression of these isoforms during neuronal death induced by alteration of the amyloid-β precursor protein function or cisplatin. We observed a concomitant up-regulation of a TAp73 isoform and a down-regulation of a ΔNp73 isoform. The shift in favor of the pro-apoptotic isoform correlated with an induction of the p53/p73 target genes such as Noxa. At a functional level, we showed that TAp73 induced neuronal death and that ΔNp73 has a neuroprotective role toward amyloid-β precursor protein alteration or cisplatin. We investigated the mechanisms of p73 expression and found that the TAp73 expression was regulated at the promoter level. In contrast, regulation of ΔNp73 protein levels was regulated by phosphorylation at residue 86 and multiple proteases. Thus, this study indicates that tight transcriptional and post-translational mechanisms regulate the p73 isoform ratios that play an important role in neuronal survival.

Striatal Dysfunctions Associated with Mitochondrial DNA Damage in Dopaminergic Neurons in a Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is one of the most common progressive neurodegenerative disorders, characterized by resting tremor, rigidity, bradykinesia, and postural instability. These symptoms are associated with massive loss of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) causing an estimated 70-80% depletion of dopamine (DA) in the striatum, where their projections are located. Although the etiology of PD is unknown, mitochondrial dysfunctions have been associated with the disease pathophysiology. We used a mouse model expressing a mitochondria-targeted restriction enzyme, PstI or mito-PstI, to damage mitochondrial DNA (mtDNA) in dopaminergic neurons. The expression of mito-PstI induces double-strand breaks in the mtDNA, leading to an oxidative phosphorylation deficiency, mostly due to mtDNA depletion. Taking advantage of a dopamine transporter (DAT) promoter-driven tetracycline transactivator protein (tTA), we expressed mito-PstI exclusively in dopaminergic neurons, creating a novel PD transgenic mouse model (PD-mito-PstI mouse). These mice recapitulate most of the major features of PD: they have a motor phenotype that is reversible with l-DOPA treatment, a progressive neurodegeneration of the SN dopaminergic population, and striatal DA depletion. Our results also showed that behavioral phenotypes in PD-mito-PstI mice were associated with striatal dysfunctions preceding SN loss of tyrosine hydroxylase-positive neurons and that other neurotransmitter systems [noradrenaline (NE) and serotonin (5-HT)] were increased after the disruption of DA neurons, potentially as a compensatory mechanism. This transgenic mouse model provides a novel model to study the role of mitochondrial defects in the axonal projections of the striatum in the pathophysiology of PD.

Differential function of NBS1 and ATR in neurogenesis

MRN (MRE11/RAD50/NBS) helps to activate ATM in response to DNA double strand breaks (DSBs) and also facilitates ATR activation by catalyzing the formation and extension of DNA single strand breaks (SSBs). Mutations of NBS1 and ATR cause human genomic instability syndrome NBS and ATR-Seckel, respectively, both of which feature neurodevelopmental defects. Whether these two DNA damage response components interact to prevent neuropathology is largely unknown. Here we show that a deletion of Nbs1 or Atr in the mouse central nervous system (CNS) results in neurodevelopmental defects characterized by reduced proliferation and increased apoptosis in embryonic brains. In contrast to Nbs1, deletion of Atr alone and both Nbs1 and Atr in the CNS causes early postnatal lethality, indicating a wider function of Atr. Importantly, deletion of Nbs1 and Atr together results in dramatic proliferation defects in neuroprogenitors. Whereas most apoptosis in the Nbs1-deleted cortex is restricted to the highly proliferating progenitors, Atr knockout induces apoptosis in both proliferating and non-proliferating neural cells. Consistently, an inducible deletion of Atr or Nbs1–Atr, but not of Nbs1, triggers a p53-independent cell death pathway in differentiated neurons, albeit elevated DNA damage in Nbs1 null neurons. Altogether, we identify a distinct function of Nbs1 and Atr in neurogenesis, namely a specific function of Nbs1 in proliferating neuroprogenitors and of Atr in both proliferating and non-dividing cells.

Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy

OBJECTIVEErythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.RESEARCH DESIGN AND METHODSAfter 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1–30 μg/kg pHBSP or control peptide).RESULTSpHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01–0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.CONCLUSIONSTreatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Modeling the impact of mitochondrial DNA damage in forebrain neurons and beyond

We have generated an inducible transgenic mouse model, which expresses a mutated version of UNG1 (mutUNG1) that removes thymine, in addition to uracil from mitochondrial DNA. The abasic-sites (AP-sites) generated by removal of thymine or uracil are a threat to genomic integrity, and are particularly harmful in mitochondria due to inhibition of mitochondrial DNA polymerase. MutUNG1, accompanied by a luciferase reporter-gene, is controlled by the Tet-on system. Transgene expression is spatially regulated by the forebrain specific CaMKIIα-promoter, and temporally by the addition of doxycycline. Mice harboring this transgene develop compromised mitochondrial dynamics, neurodegeneration and impaired behavior.

Neuroprotection by Minocycline Caused by Direct and Specific Scavenging of Peroxynitrite

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.