Preferential Involvement of BRCA1/BARD1, Not Tip60/Fe65, in DNA Double-Strand Break Repair in Presenilin-1 P117L Alzheimer Models.

Recently, we showed that DNA double-strand breaks (DSBs) are increased by the Aß 42-amyloid peptide and decreased by all-trans retinoic acid (RA) in SH-SY5Y cells and C57BL/6J mice. The present work was aimed at investigating DSBs in cells and murine models of Alzheimer's disease carrying the preseniline-1 (PS1) P117L mutation. We observed that DSBs could hardly decrease following RA treatment in the mutated cells compared to the wild-type cells. The activation of the amyloidogenic pathway is proposed in the former case as Aß 42- and RA-dependent DSBs changes were reproduced by an α-secretase and a γ-secretase inhibitions, respectively. Unexpectedly, the PS1 P117L cells showed lower DSB levels than the controls. As the DSB repair proteins Tip60 and Fe65 were less expressed in the mutated cell nuclei, they do not appear to contribute to this difference. On the contrary, full-length BRCA1 and BARD1 proteins were significantly increased in the chromatin compartment of the mutated cells, suggesting that they decrease DSBs in the pathological situation. These Western blot data were corroborated by in situ proximity ligation assays: the numbers of BRCA1-BARD1, not of Fe65-Tip60 heterodimers, were increased only in the mutated cell nuclei. RA also enhanced the expression of BARD1 and of the 90 kDa BRCA1 isoform. The increased BRCA1 expression in the mutated cells can be related to the enhanced difficulty to inhibit this pathway by BRCA1 siRNA in these cells. Overall, our study suggests that at earlier stages of the disease, similarly to PS1 P117L cells, a compensatory mechanism exists that decreases DSB levels via an activation of the BRCA1/BARD1 pathway. This supports the importance of this pathway in neuroprotection against Alzheimer's disease.

Neuroprotection against Amyloid-ß-Induced DNA Double-Strand Breaks Is Mediated by Multiple Retinoic Acid-Dependent Pathways.

In this study, we have investigated the role of all-trans-retinoic acid (RA) as a neuroprotective agent against Aß 1-42-induced DNA double-strand breaks (DSBs) in neuronal SH-SY5Y and astrocytic DI TNC1 cell lines and in murine brain tissues, by single-cell gel electrophoresis. We showed that RA does not only repair Aß 1-42-induced DSBs, as already known, but also prevents their occurrence. This effect is independent of that of other antioxidants studied, such as vitamin C, and appears to be mediated, at least in part, by changes in expression, not of the RARα, but of the PPARß/δ and of antiamyloidogenic proteins, such as ADAM10, implying a decreased production of endogenous Aß. Whereas Aß 1-42 needs transcription and translation for DSB production, RA protects against Aß 1-42-induced DSBs at the posttranslational level through both the RARα/ß/γ and PPARß/δ receptors as demonstrated by using specific antagonists. Furthermore, it could be shown by a proximity ligation assay that the PPARß/δ-RXR interactions, not the RARα/ß/γ-RXR interactions, increased in the cells when a 10 min RA treatment was followed by a 20 min Aß 1-42 treatment. Thus, the PPARß/δ receptor, known for its antiapoptotic function, might for these short-time treatments play a role in neuroprotection via PPARß/δ-RXR heterodimerization and possibly expression of antiamyloidogenic genes. Overall, this study shows that RA can not only repair Aß 1-42-induced DSBs but also prevent them via the RARα/ß/γ and PPARß/δ receptors. It suggests that the RA-dependent pathways belong to an anti-DSB Adaptative Gene Expression (DSB-AGE) system that can be targeted by prevention strategies to preserve memory in Alzheimer's disease and aging.

The Vitamin A Derivative All-Trans Retinoic Acid Repairs Amyloid-ß-Induced Double-Strand Breaks in Neural Cells and in the Murine Neocortex.

The amyloid-ß peptide or Aß is the key player in the amyloid-cascade hypothesis of Alzheimer's disease. Aß appears to trigger cell death but also production of double-strand breaks (DSBs) in aging and Alzheimer's disease. All-trans retinoic acid (RA), a derivative of vitamin A, was already known for its neuroprotective effects against the amyloid cascade. It diminishes, for instance, the production of Aß peptides and their oligomerisation. In the present work we investigated the possible implication of RA receptor (RAR) in repair of Aß-induced DSBs. We demonstrated that RA, as well as RAR agonist Am80, but not AGN 193109 antagonist, repair Aß-induced DSBs in SH-SY5Y cells and an astrocytic cell line as well as in the murine cortical tissue of young and aged mice. The nonhomologous end joining pathway and the Ataxia Telangiectasia Mutated kinase were shown to be involved in RA-mediated DSBs repair in the SH-SY5Y cells. Our data suggest that RA, besides increasing cell viability in the cortex of young and even of aged mice, might also result in targeted DNA repair of genes important for cell or synaptic maintenance. This phenomenon would remain functional up to a point when Aß increase and RA decrease probably lead to a pathological state.

Comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aß peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aß(1-42), okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aß and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aß and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Maladaptive exploratory behavior and neuropathology of the PS-1 P117L Alzheimer transgenic mice.

Patients with the early-onset Alzheimer's disease P117L mutation in the presenilin-1 gene (PS-1) present pathological hallmarks in the hippocampus, the frontal cortex and the basal ganglia. In the present work we determined by immunohistochemistry which brain regions were injured in the transgenic PS-1 P117L mice, in comparison to their littermates, the B6D2 mice. Furthermore, as these regions are involved in novelty detection, we investigated the behavior of these mice in tests for object and place novelty recognition. Limited numbers of senile plaques and neurofibrillary tangles were detected in aged PS-1 P117L mice in the CA1 only, indicating that the disease is restrained to an initial neuropathological stage. Western blots showed a change in PSD-95 expression (p=0.03), not in NR2A subunit, NR2B subunit and synaptophysin expressions in the frontal cortex, suggesting specific synaptic alterations. The behavioral tests repeatedly revealed, despite a non-significant preference for object or place novelty, maladaptive exploratory behavior of the PS-1 P117L mice in novel environmental conditions, not due to locomotor problems. These mice, unlike the B6D2 mice, were less inhibited to visit the center of the cages (p=0.01) and they continued to move excessively in the presence of a displaced object (p=0.021). Overall, the PS-1 P117L mice appear to be in an initial Alzheimer's disease-like neuropathological stage, and they showed a lack of reaction toward novel environmental conditions.

Postsynaptic density protein PSD-95 expression in Alzheimer's disease and okadaic acid induced neuritic retraction.

In order to understand how plasticity is related to neurodegeneration, we studied synaptic proteins with quantitative immunohistochemistry in the entorhinal cortex from Alzheimer patients and age-matched controls. We observed a significant decrease in presynaptic synaptophysin and an increase in postsynaptic density protein PSD-95, positively correlated with beta amyloid and phosphorylated Tau proteins in Alzheimer cases. Furthermore, Alzheimer-like neuritic retraction was generated in okadaic acid (OA) treated SH-SY5Y neuroblastoma cells with no decrease in PSD-95 expression. However, in a SH-SY5Y clone with decreased expression of transcription regulator LMO4 (as observed in Alzheimer's disease) and increased neuritic length, PSD-95 expression was enhanced but did not change with OA treatment. Therefore, increased PSD-95 immunoreactivity in the entorhinal cortex might result from compensatory mechanisms, as in the SH-SY5Y clone, whereas increased Alzheimer-like Tau phosphorylation is not related to PSD-95 expression, as suggested by the OA-treated cell models.

Transcription regulator LMO4 interferes with neuritogenesis in human SH-SY5Y neuroblastoma cells.

LMO4 is a transcription regulator interacting with proteins involved, among else, in tumorigenesis. Its function in the nervous system, and particularly in the adult nervous system, has however still to be elucidated. We decided to modify its expression in a neuronal model, human SH-SY5Y neuroblastoma cells, by permanent transfection of sense or anti-sense Lmo4 cDNAs. Generated clones overexpressing the Lmo4 transcript in sense orientation tended to aggregate. They showed significantly reduced average number of neurites per cell and average neuritic length per cell. The opposite was observed with clones overexpressing the anti-sense Lmo4 transcript. Furthermore, selected clones were subjected to 72 h long-term treatments with retinoic acid and phorbol ester (TPA), two biochemicals known to stimulate differentiation of non-transfected SH-SY5Y cells and other neuroblastoma cells. Neuritogenesis occurred after retinoic acid stimulation in all cases. The inhibitory effect of sense Lmo4 RNA overexpression on neuritic outgrowth was indeed prevented. The protein kinase C activator TPA could not induce neuritogenesis in SH-SY5Y cells overexpressing sense Lmo4 RNA. Thus, sense Lmo4 RNA overexpression, not Lmo4 endogenous transcription, overrides the stimulatory effect of TPA upon neuritic outgrowth. We also showed that Lmo4-dependent neuritic retraction and outgrowth correspond to altered phosphorylation of cytoskeletal proteins. Overall, Lmo4 RNA overexpression interferes with neuritic outgrowth, whereas anti-sense Lmo4 RNA expression favors neuritogenesis in SH-SY5Y cells. Consequently, changes in Lmo4 RNA expression levels might alter the rate of neuritic outgrowth in the developing and adult nervous system.