Manganese-induced neurotoxicity in cerebellar granule neurons due to perturbation of cell network pathways with potential implications for neurodegenerative disorders.

Manganese (Mn) is essential for living organisms, playing an important role in nervous system function. Nevertheless, chronic and/or acute exposure to this metal, especially during early life stages, can lead to neurotoxicity and dementia by unclear mechanisms. Thus, based on previous works of our group with yeast and zebrafish, we hypothesized that the mechanisms mediating manganese-induced neurotoxicity can be associated with the alteration of protein metabolism. These mechanisms may also depend on the chemical speciation of manganese. Therefore, the current study aimed at investigating the mechanisms mediating the toxic effects of manganese in primary cultures of cerebellar granule neurons (CGNs). By exposing cultured CGNs to different chemical species of manganese ([[2-[(dithiocarboxy)amino]ethyl]carbamodithioato]](2-)-kS,kS']manganese, named maneb (MB), and [[1,2-ethanediylbis[carbamodithioato]](2-)]manganese mixture with [[1,2-ethanediylbis[carbamodithioato]](2-)]zinc, named mancozeb (MZ), and manganese chloride (MnCl2)), and using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, we observed that both MB and MZ induced similar cytotoxicity (LC50∼ 7-9 µM), which was higher than that of MnCl2 (LC50∼ 27 µM). Subsequently, we applied systems biology approaches, including metallomics, proteomics, gene expression and bioinformatics, and revealed that independent of chemical speciation, for non-cytotoxic concentrations (0.3-3 µM), Mn-induced neurotoxicity in CGNs is associated with metal dyshomeostasis and impaired protein metabolism. In this way, we verified that MB induced more post-translational alterations than MnCl2, which can be a plausible explanation for cytotoxic differences between both chemical species. The metabolism of proteins is one of the most energy consuming cellular processes and its impairment appears to be a key event of some cellular stress processes reported separately in other studies such as cell cycle arrest, energy impairment, cell signaling, excitotoxicity, immune response, potential protein accumulation and apoptosis. Interestingly, we verified that Mn-induced neurotoxicity shares pathways associated with the development of Alzheimer's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, and Parkinson's disease. This has been observed in baker's yeast and zebrafish suggesting that the mode of action of Mn may be evolutionarily conserved.

Pink1 regulates FKBP5 interaction with AKT/PHLPP and protects neurons from neurotoxin stress induced by MPP.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

DJ-1 modulates the unfolded protein response and cell death via upregulation of ATF4 following ER stress.

The unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress is a feature of many neurodegenerative diseases including Alzheimer's disease, Huntington's disease and Parkinson's disease (PD). Although the vast majority of PD is sporadic, mutations in a number of genes including PARK7 which encodes the protein DJ-1 have been linked to early-onset, familial PD. In this regard, both PD of sporadic and genetic origins exhibit markers of ER stress-induced UPR. However, the relationship between pathogenic mutations in PARK7 and ER stress-induced UPR in PD pathogenesis remains unclear. In most contexts, DJ-1 has been shown to protect against neuronal injury. However, we find that DJ-1 deficiency ameliorates death in the context of acute ER stress in vitro and in vivo. DJ-1 loss decreases protein and transcript levels of ATF4, a transcription factor critical to the ER response and reduces the levels of CHOP and BiP, its downstream effectors. The converse is observed with DJ-1 over-expression. Importantly, we find that over-expression of wild-type and PD-associated mutant form of PARK7L166P, enhances ER stress-induced neuronal death by regulating ATF4 transcription and translation. Our results demonstrate a previously unreported role for wild-type and mutant DJ-1 in the regulation of UPR and provides a potential link to PD pathogenesis.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.

Regulation of ischemic neuronal death by E2F4-p130 protein complexes.

Inappropriate activation of cell cycle proteins, in particular cyclin D/Cdk4, is implicated in neuronal death induced by various pathologic stresses, including DNA damage and ischemia. Key targets of Cdk4 in proliferating cells include members of the E2F transcription factors, which mediate the expression of cell cycle proteins as well as death-inducing genes. However, the presence of multiple E2F family members complicates our understanding of their role in death. We focused on whether E2F4, an E2F member believed to exhibit crucial control over the maintenance of a differentiated state of neurons, may be critical in ischemic neuronal death. We observed that, in contrast to E2F1 and E2F3, which sensitize to death, E2F4 plays a crucial protective role in neuronal death evoked by DNA damage, hypoxia, and global ischemic insult both in vitro and in vivo. E2F4 occupies promoter regions of proapoptotic factors, such as B-Myb, under basal conditions. Following stress exposure, E2F4-p130 complexes are lost rapidly along with the presence of E2F4 at E2F-containing B-Myb promoter sites. In contrast, the presence of E2F1 at B-Myb sites increases with stress. Furthermore, B-Myb and C-Myb expression increases with ischemic insult. Taken together, we propose a model by which E2F4 plays a protective role in neurons from ischemic insult by forming repressive complexes that prevent prodeath factors such as Myb from being expressed.

Delayed combinatorial treatment with flavopiridol and minocycline provides longer term protection for neuronal soma but not dendrites following global ischemia.

We previously reported that delayed administration of the general cyclin-dependent kinase inhibitor flavopiridol following global ischemia provided transient neuroprotection and improved behavioral performance. However, it failed to provide longer term protection. In the present study, we investigate the ability of delayed flavopiridol in combination with delayed minocycline, another neuroprotectant to provide sustained protection following global ischemia. We report that a delayed combinatorial treatment of flavopiridol and minocycline provides synergistic protection both 2 and 10 weeks following ischemia. However, protected neurons in the hippocampal CA1 are synaptically impaired as assessed by electrophysio logical field potential recordings. This is likely because of the presence of degenerated processes in the CA1 even with combinatorial therapy. This indicates that while we have addressed one important pre-clinical parameter by dramatically improving long-term neuronal survival with delayed combinatorial therapy, the issue of synaptic preservation of protected neurons still exists. These results also highlight the important observation that protection does not always lead to proper function.