Loss of PTEN-Induced Kinase 1 Regulates Oncogenic Ras-Driven Tumor Growth By Inhibiting Mitochondrial Fission.

Mitochondrial metabolism and dynamics (fission and fusion) critically regulate cell survival and proliferation, and abnormalities in these pathways are implicated in both neurodegenerative disorders and cancer. Mitochondrial fission is necessary for the growth of mutant Ras-dependent tumors. Here, we investigated whether loss of PTEN-induced kinase 1 (PINK1) - a mitochondrial kinase linked to recessive familial Parkinsonism - affects the growth of oncogenic Ras-induced tumor growth in vitro and in vivo. We show that RasG12D-transformed embryonic fibroblasts (MEFs) from PINK1-deficient mice display reduced growth in soft agar and in nude mice, as well as increased necrosis and decreased cell cycle progression, compared to RasG12D-transformed MEFs derived from wildtype mice. PINK1 re-expression (overexpression) at least partially rescues these phenotypes. Neither PINK1 deletion nor PINK1 overexpression altered Ras expression levels. Intriguingly, PINK1-deficient Ras-transformed MEFs exhibited elongated mitochondria and altered DRP1 phosphorylation, a key event in regulating mitochondrial fission. Inhibition of DRP1 diminished PINK1-regulated mitochondria morphological changes and tumor growth suggesting that PINK1 deficiency primarily inhibits Ras-driven tumor growth through disturbances in mitochondrial fission and associated cell necrosis and cell cycle defects. Moreover, we substantiate the requirement of PINK1 for optimal growth of Ras-transformed cells by showing that human HCT116 colon carcinoma cells (carrying an endogenous RasG13D mutation) with CRISPR/Cas9-introduced PINK1 gene deletions also show reduced mitochondrial fission and decreased growth. Our results support the importance of mitochondrial function and dynamics in regulating the growth of Ras-dependent tumor cells and provide insight into possible mechanisms underlying the lower incidence of cancers in Parkinson's disease and other neurodegenerative disorders.

Mitochondrial and Autophagic Regulation of Adult Neurogenesis in the Healthy and Diseased Brain.

Adult neurogenesis is a highly regulated process during which new neurons are generated from neural stem cells in two discrete regions of the adult brain: the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus. Defects of adult hippocampal neurogenesis have been linked to cognitive decline and dysfunction during natural aging and in neurodegenerative diseases, as well as psychological stress-induced mood disorders. Understanding the mechanisms and pathways that regulate adult neurogenesis is crucial to improving preventative measures and therapies for these conditions. Accumulating evidence shows that mitochondria directly regulate various steps and phases of adult neurogenesis. This review summarizes recent findings on how mitochondrial metabolism, dynamics, and reactive oxygen species control several aspects of adult neural stem cell function and their differentiation to newborn neurons. It also discusses the importance of autophagy for adult neurogenesis, and how mitochondrial and autophagic dysfunction may contribute to cognitive defects and stress-induced mood disorders by compromising adult neurogenesis. Finally, I suggest possible ways to target mitochondrial function as a strategy for stem cell-based interventions and treatments for cognitive and mood disorders.

Proteasome inhibition promotes mono-ubiquitination and nuclear translocation of mature (52 kDa) PINK1.

Mutations of PTEN-induced kinase 1 (PINK1) cause recessive familial Parkinson's disease. Cells lacking PINK1 display mitochondrial deficits and increased sensitivity to oxidative and proteasomal stress. It has been shown that the 52-kDa (mature) form of PINK1 in the cytoplasm mitigates proteasomal stress-induced cell death by enhancing aggresomes formation and autophagy. Here we newly demonstrate that proteasome dysfunction triggers mono-ubiquitination and nuclear translocation of mature PINK1. Enhancing PINK1 mono-ubiquitination by two different means increased nuclear accumulation of PINK1 independent of proteasome inhibition. Moreover, we show that PINK1 harbors a hitherto unknown nuclear export sequence (NES) in its C-terminus. Blocking CRM1-dependent nuclear export with leptomycin B augmented PINK1 levels in the nucleus of MG132-treated cells but not in normal cells. Overall, these results show that proteasomal stress-induced mono-ubiquitination of PINK1 mediates PINK1 nuclear translocation, while PINK1 is excluded from the nucleus of healthy cells via its NES. Therefore, mature PINK1 may have a nuclear function in cells under proteasomal stress.

PINK1 deficiency is associated with increased deficits of adult hippocampal neurogenesis and lowers the threshold for stress-induced depression in mice.

Parkinson's disease (PD) is characterized by motor impairments and several non-motor features, including frequent depression and anxiety. Stress-induced deficits of adult hippocampal neurogenesis (AHN) have been linked with abnormal affective behavior in animals. It has been speculated that AHN defects may contribute to affective symptoms in PD, but this hypothesis remains insufficiently tested in animal models. Mice that lack the PD-linked kinase PINK1 show impaired differentiation of adult-born neurons in the hippocampus. Here, we examined the relationship between AHN deficits and affective behavior in PINK1-/- mice under basal (no stress) conditions and after exposure to chronic stress. PINK1 loss and corticosterone negatively and jointly affected AHN, leading to lower numbers of neural stem cells and newborn neurons in the dentate gyrus of corticosterone-treated PINK1-/- mice. Despite increased basal AHN deficits, PINK1-deficient mice showed normal affective behavior. However, lack of PINK1 sensitized mice to corticosterone-induced behavioral despair in the tail suspension test at a dose where wildtype mice were unaffected. Moreover, after two weeks of chronic restraint stress male PINK1-/- mice displayed increased immobility in the forced swim test, and protein expression of the glucocorticoid receptor in the hippocampus was reduced. Thus, while impaired AHN as such is insufficient to cause affective dysfunction in this PD model, PINK1 deficiency may lower the threshold for chronic stress-induced depression in PD. Finally, PINK1-deficient mice displayed reduced basal voluntary wheel running but normal rotarod performance, a finding whose mechanisms remain to be determined.

Lack of PINK1 alters glia innate immune responses and enhances inflammation-induced, nitric oxide-mediated neuron death.

Neuroinflammation is involved in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. We show that lack of PINK1- a mitochondrial kinase linked to recessive familial PD - leads to glia type-specific abnormalities of innate immunity. PINK1 loss enhances LPS/IFN-γ stimulated pro-inflammatory phenotypes of mixed astrocytes/microglia (increased iNOS, nitric oxide and COX-2, reduced IL-10) and pure astrocytes (increased iNOS, nitric oxide, TNF-α and IL-1ß), while attenuating expression of both pro-inflammatory (TNF-α, IL-1ß) and anti-inflammatory (IL-10) cytokines in microglia. These abnormalities are associated with increased inflammation-induced NF-κB signaling in astrocytes, and cause enhanced death of neurons co-cultured with inflamed PINK1 -/- mixed glia and neuroblastoma cells exposed to conditioned medium from LPS/IFN-γ treated PINK1 -/- mixed glia. Neuroblastoma cell death is prevented with an iNOS inhibitor, implicating increased nitric oxide production as the cause for enhanced death. Finally, we show for the first time that lack of a recessive PD gene (PINK1) increases α-Synuclein-induced nitric oxide production in all glia types (mixed glia, astrocytes and microglia). Our results describe a novel pathogenic mechanism in recessive PD, where PINK1 deficiency may increase neuron death via exacerbation of inflammatory stimuli-induced nitric oxide production and abnormal innate immune responses in glia cells.

Loss of PINK1 leads to metabolic deficits in adult neural stem cells and impedes differentiation of newborn neurons in the mouse hippocampus.

Emerging evidence suggests that mitochondrial dynamics regulates adult hippocampal neurogenesis (AHN). Although abnormal AHN has been linked to depression, anxiety, and cognitive dysfunction, which are features of neurodegenerative conditions, including Parkinson's disease (PD), the impact of mitochondrial deficits on AHN have not been explored previously in a model of neurodegeneration. Here, we used PTEN-induced kinase 1-deficient (PINK1-/- ) mice that lacked a mitochondrial kinase mutated in recessive familial PD. We show that mitochondrial defects, elevated glycolysis, and increased apoptosis are associated with impaired but not abrogated differentiation of PINK1-deficient neural stem cells (NSCs) in culture. In the dentate gyrus of PINK1-/- mice, newly generated doublecortin-positive neurons show aberrant dendritic morphology, and their maturation is compromised compared with wild-type mice. In addition, in vivo labeling of NSCs with 5-ethynyl-2'-deoxyuridine shows that proliferating NSC numbers are normal, but the differentiation of NSCs to doublecortin-positive neuroblasts and mature NeuN+ neurons is impeded in PINK1-/- mice. Finally, we demonstrate that home cage activity and corticosterone levels of PINK1-/- mice are normal, thereby excluding reduced physical activity and increased stress as causes of neurogenesis defects. Our results reveal a new and important relationship between mitochondrial dysfunction and impaired AHN in a genetic PD model. Targeting mitochondrial function and metabolism to increase AHN may hold promise for the treatment of affective disorders and the mitigation of related symptoms in PD and other neurodegenerative conditions.-Agnihotri, S. K., Shen, R., Li, J., Gao, X., Büeler, H. Loss of PINK1 leads to metabolic deficits in adult neural stem cells and impedes differentiation of newborn neurons in the mouse hippocampus.

Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease.

Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70, heat-shock-related 70 kDa protein 2; IDHc, cytoplasmic isocitrate dehydrogenase [NADP+]; MAPK1, mitogen-activated protein kinase 1; MEK1, MAP kinase kinase 1; MDHc, cytoplasmic malate dehydrogenase; NFM, neurofilament medium polypeptide; NSF, N-ethylmaleimide-sensitive fusion protein; PHB, prohibitin; PINK1, PTEN-induced putative kinase 1; PPIaseA, peptidyl-prolyl cis-trans isomerase A; PSA2, proteasome subunit alpha type-2; TK, transketolase; VDAC-2, voltage-dependent anion-selective channel protein 2.

The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system.

Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (<5 kb) making the co-expression of two genes (e.g., from two promoters) difficult. An internal ribosomal entry site has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot-and-mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.

Unaltered striatal dopamine release levels in young Parkin knockout, Pink1 knockout, DJ-1 knockout and LRRK2 R1441G transgenic mice.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative brain diseases; it is accompanied by extensive loss of dopamine (DA) neurons of the substantia nigra that project to the putamen, leading to impaired motor functions. Several genes have been associated with hereditary forms of the disease and transgenic mice have been developed by a number of groups to produce animal models of PD and to explore the basic functions of these genes. Surprisingly, most of the various mouse lines generated such as Parkin KO, Pink1 KO, DJ-1 KO and LRRK2 transgenic have been reported to lack degeneration of nigral DA neuron, one of the hallmarks of PD. However, modest impairments of motor behavior have been reported, suggesting the possibility that the models recapitulate at least some of the early stages of PD, including early dysfunction of DA axon terminals. To further evaluate this possibility, here we provide for the first time a systematic comparison of DA release in four different mouse lines, examined at a young age range, prior to potential age-dependent compensations. Using fast scan cyclic voltammetry in striatal sections prepared from young, 6-8 weeks old mice, we examined sub-second DA overflow evoked by single pulses and action potential trains. Unexpectedly, none of the models displayed any dysfunction of DA overflow or reuptake. These results, compatible with the lack of DA neuron loss in these models, suggest that molecular dysfunctions caused by the absence or mutation of these individual genes are not sufficient to perturb the function and survival of mouse DA neurons.

Mitochondrial and cytosolic roles of PINK1 shape induced regulatory T-cell development and function.

Mutations in PTEN-induced kinase 1 (PINK1), a serine/threonine kinase linked to familial early-onset Parkinsonism, compromise mitochondrial integrity and metabolism and impair AKT signaling. As the activation of a naïve T cell requires an AKT-dependent reorganization of a cell's metabolic machinery, we sought to determine if PINK1-deficient T cells lack the ability to undergo activation and differentiation. We show that CD4(+) T cells from PINK1 knockout mice fail to properly phosphorylate AKT upon activation, resulting in reduced expression of the IL-2 receptor subunit CD25. Following, deficient IL-2 signaling mutes the activation-induced increase in respiratory capacity and mitochondrial membrane potential. Under polarization conditions favoring the development of induced regulatory T cells, PINK1(-/-) T cells exhibit a reduced ability to suppress bystander T-cell proliferation despite normal FoxP3 expression kinetics. Our results describe a critical role for PINK1 in integrating extracellular signals with metabolic state during T-cell fate determination, and may have implications for the understanding of altered T-cell populations and immunity during the progression of active Parkinson's disease or other immunopathologies.

Metabolic stress modulates Alzheimer's ß-secretase gene transcription via SIRT1-PPARγ-PGC-1 in neurons.

Classic cardio-metabolic risk factors such as hypertension, stroke, diabetes, and hypercholesterolemia all increase the risk of Alzheimer's disease. We found increased transcription of ß-secretase/BACE1, the rate-limiting enzyme for Aß generation, in eNOS-deficient mouse brains and after feeding mice a high-fat, high-cholesterol diet. Up- or downregulation of PGC-1α reciprocally regulated BACE1 in vitro and in vivo. Modest fasting in mice reduced BACE1 transcription in the brains, which was accompanied by elevated PGC-1 expression and activity. Moreover, the suppressive effect of PGC-1 was dependent on activated PPARγ, likely via SIRT1-mediated deacetylation in a ligand-independent manner. The BACE1 promoter contains multiple PPAR-RXR sites, and direct interactions among SIRT1-PPARγ-PGC-1 at these sites were enhanced with fasting. The interference on the BACE1 gene identified here represents a unique noncanonical mechanism of PPARγ-PGC-1 in transcriptional repression in neurons in response to metabolic signals that may involve recruitment of corepressor NCoR.

Shared and cell type-specific mitochondrial defects and metabolic adaptations in primary cells from PINK1-deficient mice.

BACKGROUND: Mutations in PTEN-induced kinase 1 (PINK1) cause early-onset recessive parkinsonism. PINK1 and Parkin regulate mitochondrial quality control. However, PINK1 ablation in Drosophila and cultured mammalian cell lines affected mitochondrial function/dynamics in opposite ways, confounding the elucidation of the role of PINK1 in these processes. OBJECTIVE: We recently generated PINK1-deficient (PINK1-/-) mice and reasoned that primary cells from these mice provide a more physiological substrate to study the role of PINK1 in mammals and to investigate metabolic adaptations and neuron-specific vulnerability in PINK1 deficiency. METHODS AND RESULTS: Using real-time measurement of oxygen consumption and extracellular acidification, we show that basal mitochondrial respiration is increased, while maximum respiration and spare respiratory capacity are decreased in PINK1-/- mouse embryonic fibroblasts (MEF), as is the membrane potential. In addition, a Warburg-like effect in PINK1-/- MEF promotes survival that is abrogated by inhibition of glycolysis. Expression of uncoupling protein-2 is decreased in PINK1-/- MEF and the striatum of PINK1-/- mice, possibly increasing the sensitivity to oxidative stress. Mitochondria accumulate in large foci in PINK1-/- MEF, indicative of abnormal mitochondrial dynamics and/or transport. Like in PINK1-/- Drosophila, enlarged/swollen mitochondria accumulate in three different cell types from PINK1-/- mice (MEF, primary cortical neurons and embryonic stem cells). However, mitochondrial enlargement is greatest and most prominent in primary cortical neurons that also develop cristae fragmentation and disintegration. CONCLUSION: Our results reveal mechanisms of PINK1-related parkinsonism, show that the function of PINK1 is conserved between Drosophila and mammals when studied in primary cells, and demonstrate that the same PINK1 mutation can affect mitochondrial morphology/degeneration in a cell type-specific manner, suggesting that tissue-/cell-specific metabolic capacity and adaptations determine phenotypes and cellular vulnerability in PINK1-/- mice and cells.

PINK1 enhances insulin-like growth factor-1-dependent Akt signaling and protection against apoptosis.

Mutations in the PARK6 gene coding for PTEN-induced kinase 1 (PINK1) cause recessive early-onset Parkinsonism. Although PINK1 and Parkin promote the degradation of depolarized mitochondria in cultured cells, little is known about changes in signaling pathways that may additionally contribute to dopamine neuron loss in recessive Parkinsonism. Accumulating evidence implicates impaired Akt cell survival signaling in sporadic and familial PD (PD). IGF-1/Akt signaling inhibits dopamine neuron loss in several animal models of PD and both IGF-1 and insulin are neuroprotective in various settings. Here, we tested whether PINK1 is required for insulin-like growth factor 1 (IGF-1) and insulin dependent phosphorylation of Akt and the regulation of downstream Akt target proteins. Our results show that embryonic fibroblasts from PINK1-deficient mice display significantly reduced Akt phosphorylation in response to both IGF-1 and insulin. Moreover, phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) and nuclear exclusion of FoxO1 are decreased in IGF-1 treated PINK1-deficient cells. In addition, phosphorylation of ribosomal protein S6 is reduced indicating decreased activity of mitochondrial target of rapamycin (mTOR) in IGF-1 treated PINK1(-/-) cells. Importantly, the protection afforded by IGF-1 against staurosporine-induced metabolic dysfunction and apoptosis is abrogated in PINK1-deficient cells. Moreover, IGF-1-induced Akt phosphorylation is impaired in primary cortical neurons from PINK1-deficient mice. Inhibition of cellular Ser/Thr phosphatases did not increase the amount of phosphorylated Akt in PINK1(-/-) cells, suggesting that components upstream of Akt phosphorylation are compromised in PINK1-deficient cells. Our studies show that PINK1 is required for optimal IGF-1 and insulin dependent Akt signal transduction, and raise the possibility that impaired IGF-1/Akt signaling is involved in PINK1-related Parkinsonism by increasing the vulnerability of dopaminergic neurons to stress-induced cell death.

Increased mitochondrial calcium sensitivity and abnormal expression of innate immunity genes precede dopaminergic defects in Pink1-deficient mice.

BACKGROUND: PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca²+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson's disease (PD) display altered activity in the nigrostriatal system of Pink1⁻/⁻ mice. METHODS AND FINDINGS: Purified brain mitochondria of Pink1⁻/⁻ mice showed impaired Ca²+ storage capacity, resulting in increased Ca²+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1⁻/⁻ mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1⁻/⁻ mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1⁻/⁻ mice had increased levels of IL-1ß, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-ß (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1⁻/⁻ mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting. CONCLUSIONS: Increased mitochondrial Ca²+ sensitivity and JNK activity are early defects in Pink1⁻/⁻ mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses. While some differentially expressed genes may mitigate neurodegeneration, increased LPS-induced brain cytokine expression and impaired cytokine-induced NF-κB activation may predispose neurons of Pink1⁻/⁻ mice to inflammation and injury-induced cell death.

Extended lifespan of Drosophila parkin mutants through sequestration of redox-active metals and enhancement of anti-oxidative pathways.

The mechanisms underlying neuron death in Parkinson's disease are unknown, but both genetic defects and environmental factors are implicated in its pathogenesis. Mutations in the parkin gene lead to autosomal recessive juvenile Parkinsonism (AR-JP). Here we report that compared to control flies, Drosophila lacking parkin show significantly reduced lifespan but no difference in dopamine neuron numbers when raised on food supplemented with environmental pesticides or mitochondrial toxins. Moreover, chelation of redox-active metals, anti-oxidants and overexpression of superoxide dismutase 1 all significantly reversed the reduced longevity of parkin-deficient flies. Finally, parkin deficiency exacerbated the rough eye phenotype of Drosophila caused by overexpression of the copper importer B (Ctr1B). Taken together, our results demonstrate an important function of parkin in the protection against redox-active metals and pesticides implicated in the etiology of Parkinson's disease. They also corroborate that oxidative stress, perhaps as a consequence of mitochondrial dysfunction, is a major determinant of morbidity in parkin mutant flies.

Mitochondrial dynamics, cell death and the pathogenesis of Parkinson's disease.

The structure and function of the mitochondrial network is regulated by mitochondrial biogenesis, fission, fusion, transport and degradation. A well-maintained balance of these processes (mitochondrial dynamics) is essential for neuronal signaling, plasticity and transmitter release. Core proteins of the mitochondrial dynamics machinery play important roles in the regulation of apoptosis, and mutations or abnormal expression of these factors are associated with inherited and age-dependent neurodegenerative disorders. In Parkinson's disease (PD), oxidative stress and mitochondrial dysfunction underlie the development of neuropathology. The recessive Parkinsonism-linked genes PTEN-induced kinase 1 (PINK1) and Parkin maintain mitochondrial integrity by regulating diverse aspects of mitochondrial function, including membrane potential, calcium homeostasis, cristae structure, respiratory activity, and mtDNA integrity. In addition, Parkin is crucial for autophagy-dependent clearance of dysfunctional mitochondria. In the absence of PINK1 or Parkin, cells often develop fragmented mitochondria. Whereas excessive fission may cause apoptosis, coordinated induction of fission and autophagy is believed to facilitate the removal of damaged mitochondria through mitophagy, and has been observed in some types of cells. Compensatory mechanisms may also occur in mice lacking PINK1 that, in contrast to cells and Drosophila, have only mild mitochondrial dysfunction and lack dopaminergic neuron loss. A better understanding of the relationship between the specific changes in mitochondrial dynamics/turnover and cell death will be instrumental to identify potentially neuroprotective pathways steering PINK1-deficient cells towards survival. Such pathways may be manipulated in the future by specific drugs to treat PD and perhaps other neurodegenerative disorders characterized by abnormal mitochondrial function and dynamics.

Differential sensitivity of the inner ear sensory cell populations to forced cell cycle re-entry and p53 induction.

Previous studies have shown that the maintenance of post-mitotic state is critical for the life-long survival of the inner ear mechanosensory cells, the hair cells. A general concept is that differentiated, post-mitotic cells rapidly die following cell cycle re-entry. Here we have compared the response of postnatal cochlear (auditory) and utricular (balance) hair cells to forced cell cycle reactivation and p53 up-regulation. Forced S-phase entry was triggered through the human papillomavirus-16 E7 oncogene misexpression in explant cultures. It induced DNA damage and p53 induction in cochlear outer hair cells and these cells were rapidly lost, before entry into mitosis. The death was attenuated by p53 inactivation. In contrast, despite DNA damage and p53 induction, utricular hair cells showed longer term survival and a proportion of them progressed into mitosis. Consistently, pharmacological elevation of p53 levels by nutlin-3a led to a death-prone phenotype of cochlear outer hair cells, while other hair cell populations were death-resistant. These data have important clinical implications as they show the importance of p53 in sensory cells that are essential in hearing function.

Impaired mitochondrial dynamics and function in the pathogenesis of Parkinson's disease.

Parkinson's disease (PD), the most frequent movement disorder, is caused by the progressive loss of the dopamine neurons within the substantia nigra pars compacta (SNc) and the associated deficiency of the neurotransmitter dopamine in the striatum. Most cases of PD occur sporadically with unknown cause, but mutations in several genes have been linked to genetic forms of PD (alpha-synuclein, Parkin, DJ-1, PINK1, and LRRK2). These genes have provided exciting new avenues to study PD pathogenesis and the mechanisms underlying the selective dopaminergic neuron death in PD. Epidemiological studies in humans, as well as molecular studies in toxin-induced and genetic animal models of PD show that mitochondrial dysfunction is a defect occurring early in the pathogenesis of both sporadic and familial PD. Mitochondrial dynamics (fission, fusion, migration) is important for neurotransmission, synaptic maintenance and neuronal survival. Recent studies have shown that PINK1 and Parkin play crucial roles in the regulation of mitochondrial dynamics and function. Mutations in DJ-1 and Parkin render animals more susceptible to oxidative stress and mitochondrial toxins implicated in sporadic PD, lending support to the hypothesis that some PD cases may be caused by gene-environmental factor interactions. A small proportion of alpha-synuclein is imported into mitochondria, where it accumulates in the brains of PD patients and may impair respiratory complex I activity. Accumulation of clonal, somatic mitochondrial DNA deletions has been observed in the substantia nigra during aging and in PD, suggesting that mitochondrial DNA mutations in some instances may pre-dispose to dopamine neuron death by impairing respiration. Besides compromising cellular energy production, mitochondrial dysfunction is associated with the generation of oxidative stress, and dysfunctional mitochondria more readily mediate the induction of apoptosis, especially in the face of cellular stress. Collectively, the studies examined and summarized here reveal an important causal role for mitochondrial dysfunction in PD pathogenesis, and suggest that drugs and genetic approaches with the ability to modulate mitochondrial dynamics, function and biogenesis may have important clinical applications in the future treatment of PD.

Bidirectional changes in water-maze learning following recombinant adenovirus-associated viral vector (rAAV)-mediated brain-derived neurotrophic factor expression in the rat hippocampus.

Alterations in hippocampal brain-derived neurotrophic factor (BDNF) expression have been implicated in the pathogenesis of emotional and cognitive dysfunction. Here, we induced BDNF overexpression in the rat hippocampus using recombinant adenovirus-associated viral (rAAV) vectors, and studied its long-term (2 months postinduction) effects on anxiety-related behaviour, exploration in the open field, and spatial learning in the water maze. Although the treatment successfully led to substantial elevation of hippocampal BDNF levels, its effect on spatial learning was bidirectional: a subset of rAAV-induced BDNF-overexpressing rats performed well above control level, whereas the rest were clearly impaired. This behavioural distinction corresponded to two markedly different levels of BDNF overexpression. The increase in dorsal hippocampal BDNF content achieved in the 'water-maze-impaired' subgroup was twice that attained in the 'water-maze-improved' rats. Although neither subgroup of rAAV-induced BDNF-overexpressing rats differed from controls in the open field, the 'water-maze-impaired' subgroup also showed a significant anxiolytic effect. Our results suggest that hippocampal BDNF elevation significantly affects cognitive and emotional behaviours, but the direction and magnitude of the effects critically depend on the precise levels of overexpression. This factor must be taken into account in future studies examining the functional consequences of hippocampal BDNF overexpression.

DJ-1 and Parkin modulate dopamine-dependent behavior and inhibit MPTP-induced nigral dopamine neuron loss in mice.

Parkin-deficient animals exhibit mitochondrial degeneration and increased oxidative stress vulnerability, and both mice and flies lacking DJ-1 are hypersensitive to environmental toxins associated with Parkinson's disease (PD). We used recombinant adeno-associated virus (AAV) gene transfer to study the influence of DJ-1 and Parkin on the dopaminergic system of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, a model for sporadic PD. After MPTP lesioning, significantly more dopamine neurons survived in the virus-injected substantia nigra of the AAV-DJ-1 and AAV-Parkin mice when compared with AAV-enhanced green fluorescent protein injected controls. Protection at the neuronal level was supported by increased amphetamine-induced contralateral turning behavior. Normal mice expressing DJ-1 showed apomorphine-induced ipsilateral turning, suggesting a hyporesponsiveness of striatal dopamine D1 receptors in the DJ-1-expressing hemisphere. MPTP drastically reduced dopamine to 19% of normal levels and neither DJ-1 nor Parkin protected against MPTP-induced catecholamine loss under these conditions. Our results show that Parkin and DJ-1 inhibit dopamine neuron death and enhance amphetamine-induced dopaminergic function in a mouse model of idiopathic PD. However, DJ-1 overexpression also reduced postsynaptic dopamine receptor responses in normal mice. These results warrant further exploration of DJ-1 and Parkin gene therapy for PD, although a better understanding of their effects on behavior and dopamine neurotransmission is required before these proteins can be safely used.