Mutations in several genes, including Parkin, PTEN-induced kinase 1 (Pink1) and DJ-1, are associated with rare inherited forms of Parkinson's disease (PD). Despite recent attention on the function of these genes, the interplay between DJ-1, Pink1 and Parkin in PD pathogenesis remains unclear. In particular, whether these genes regulate mitochondrial control pathways in neurons is highly controversial. Here we report that Pink1-dependent Parkin translocation does occur in mouse cortical neurons in response to a variety of mitochondrial damaging agents. This translocation only occurs in the absence of antioxidants in the neuronal culturing medium, implicating a key role of reactive oxygen species (ROS) in this response. Consistent with these observations, ROS blockers also prevent Parkin recruitment in mouse embryonic fibroblasts. Loss of DJ-1, a gene linked to ROS management, results in increased stress-induced Parkin recruitment and increased mitophagy. Expression of wild-type DJ-1, but not a cysteine-106 mutant associated with defective ROS response, rescues this accelerated Parkin recruitment. Interestingly, DJ-1 levels increase at mitochondria following oxidative damage in both fibroblasts and neurons, and this process also depends on Parkin and possibly Pink1. These results not only highlight the presence of a Parkin/Pink1-mediated pathway of mitochondrial quality control (MQC) in neurons, they also delineate a complex reciprocal relationship between DJ-1 and the Pink1/Parkin pathway of MQC.
Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H 2 O 2 in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.neurodegeneration | Parkinson disease | reactive oxygen species
Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.
Progranulin (PGRN) has recently emerged as a key player in a subset of frontotemporal dementias (FTD). Numerous mutations in the progranulin gene have been identified in patients with familial or sporadic frontotemporal lobar degeneration (FTLD). In order to understand the molecular mechanisms by which PGRN deficiency leads to FTLD, we examined activity of PGRN in mouse cortical and hippocampal neurons and in human neuroblastoma SH-SY5Y cells. Treatment of mouse neurons with PGRN protein resulted in an increase in neurite outgrowth, supporting the role of PGRN as a neurotrophic factor. PGRN treatment stimulated phosphorylation of glycogen synthase kinase-3 beta (GSK-3β) in cultured neurons. Knockdown of PGRN in SH-SY5Y cells impaired retinoic acid induced differentiation and reduced the level of phosphorylated GSK-3β. PGRN knockdown cells were also more sensitized to staurosporine-induced apoptosis. These results reveal an important role of PGRN in neurite outgrowth and involvement of GSK-3β in mediating PGRN activity. Identification of GSK-3β activation as a downstream event for PGRN signaling provides a mechanistic explanation for PGRN activity in the nervous system. Our work also suggest that loss of axonal growth stimulation during neural injury repair or deficits in axonal repair may contribute to neuronal damage or axonal loss in FTLD associated with PGRN mutations. Finally, our study suggests that modulating GSK-3β or similar signaling events may provide therapeutic benefits for FTLD cases associated with PGRN mutations.
Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer's disease (AD). We here combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain (RC), but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the RC. To validate these predictions, we utilized fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, nonintuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity.
Emerging evidence has demonstrated a growing genetic component in Parkinson disease (PD). For instance, loss-of-function mutations in PINK1 or PARKIN can cause autosomal recessive PD. Recently, PINK1 and PARKIN have been implicated in the same signaling pathway to regulate mitochondrial clearance through recruitment of PARKIN by stabilization of PINK1 on the outer membrane of depolarized mitochondria. The precise mechanisms that govern this process remain enigmatic. In this study, we identify Bcl2-associated athanogene 2 (BAG2) as a factor that promotes mitophagy. BAG2 inhibits PINK1 degradation by blocking the ubiquitination pathway. Stabilization of PINK1 by BAG2 triggers PARKIN-mediated mitophagy and protects neurons against 1-methyl-4-phenylpyridinium-induced oxidative stress in an in vitro cell model of PD. Collectively, our findings support the notion that BAG2 is an upstream regulator of the PINK1/PARKIN signaling pathway. Parkinson disease (PD)2 is the second most common neurodegenerative disorder, characterized by selective loss of the pigmented dopaminergic neurons of the substantia nigra pars compacta (1). Although most PD cases are sporadic in nature (2), mutations in several genes have been linked to familial forms of PD (reviewed in Ref.3). Indeed, these familial genes serve as important vehicles to study the potential mechanisms of pathogenesis in PD. In this regard, increasing evidence suggests that mitochondrial dysfunction may play a critical role in both the inherited and sporadic forms of PD, although the precise role of mitochondrial dysfunction in PD is unclear (4). Recently, two familial recessive PD genes, PTEN-induced putative kinase 1 (PINK1), a mitochondrially localized serine/threonine kinase gene, and PARKIN, an E3 ubiquitin ligase gene, have been identified as acting along similar pathways in regulating mitochondrial quality control in mammalian systems (5-8). These findings are supported by genetic studies in Drosophila models of PD that show that PARKIN and PINK1 function in a common pathway, with PARKIN being a downstream player of PINK1 (9 -11). A third recessive PD gene, DJ-1, associated with the regulation of oxidative stress, also regulates PINK1/PARKIN-mediated control of mitochondrial health (12).PINK1 is normally shuttled to the inner mitochondrial membrane where it is processed by multiple proteases (13). The endogenous role of PINK1 at this site is unknown. However, PINK1-deficient cells display altered calcium homeostasis at the mitochondria as well as altered mitochondrial function (14). Processed PINK1 at the inner mitochondrial membrane has also been proposed to be shuttled to the cytosol, where it is degraded by the proteasome (15). However, under conditions of mitochondrial stress such as treatment with the mitochondrial uncoupler carbonyl cyanide p-chlorophenylhydrazone (CCCP) or with the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP ϩ ), PINK1 can also regulate mitophagy in a PARKIN-dependent fashion (12, 16 -19). The framework by which this mitochondrial ...
Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.
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/.
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