Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson''s disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechanism of recruitment and the ubiquitination target are unclear. Here we show in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. We hypothesize that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy.     

Impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice

Parkinson's disease (PD) is characterized by the selective vulnerability of the nigrostriatal dopaminergic circuit. Recently, loss-of-function mutations in the PTEN-induced kinase 1 (PINK1) gene have been linked to early-onset PD. How PINK1 deficiency causes dopaminergic dysfunction and degeneration in PD patients is unknown. Here, we investigate the physiological role of PINK1 in the nigrostriatal dopaminergic circuit through the generation and multidisciplinary analysis of PINK1(-/-) mutant mice. We found that numbers of dopaminergic neurons and levels of striatal dopamine (DA) and DA receptors are unchanged in PINK1(-/-) mice. Amperometric recordings, however, revealed decreases in evoked DA release in striatal slices and reductions in the quantal size and release frequency of catecholamine in dissociated chromaffin cells. Intracellular recordings of striatal medium spiny neurons, the major dopaminergic target, showed specific impairments of corticostriatal long-term potentiation and long-term depression in PINK1(-/-) mice. Consistent with a decrease in evoked DA release, these striatal plasticity impairments could be rescued by either DA receptor agonists or agents that increase DA release, such as amphetamine or l-dopa. These results reveal a critical role for PINK1 in DA release and striatal synaptic plasticity in the nigrostriatal circuit and suggest that altered dopaminergic physiology may be a pathogenic precursor to nigrostriatal degeneration.     

The kinase domain of mitochondrial PINK1 faces the cytoplasm

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD.     

Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability

Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser/Thr protein kinase targeted to mitochondria. In this study, we have investigated the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. We chose to examine two point mutations: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. We were able to confirm kinase activity and produce artificial "kinase-dead" mutants that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. We also examined the localization of PINK1 in transfected mammalian cells by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria.     

DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function

Mutations or deletions in PARKIN/PARK2, PINK1/PARK6, and DJ-1/PARK7 lead to autosomal recessive parkinsonism. In Drosophila, deletions in parkin and pink1 result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship between DJ-1 and mitochondria, however, remains unclear. We now report that Drosophila and mouse mutants in DJ-1 show compromised mitochondrial function with age. Flies deleted for DJ-1 manifest similar defects as pink1 and parkin mutants: male sterility, shortened lifespan, and reduced climbing ability. We further found poorly coupled mitochondria in vitro and reduced ATP levels in fly and mouse DJ-1 mutants. Surprisingly, up-regulation of DJ-1 can ameliorate pink1, but not parkin, mutants in Drosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest that DJ-1 is important for proper mitochondrial function and acts downstream of, or in parallel to, pink1. These findings link DJ-1, pink1, and parkin to mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism.     

PINK1-dependent recruitment of Parkin to mitochondria in mitophagy

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson''s disease. Upon a loss of mitochondrial membrane potential (ΔΨ_m) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨ_m relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨ_m. We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson''s disease.     

Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin MPTP

PTEN-induced putative kinase 1 (Pink1) is a recently identified gene linked to a recessive form of familial Parkinson's disease (PD). The kinase contains a mitochondrial localization sequence and is reported to reside, at least in part, in mitochondria. However, neither the manner by which the loss of Pink1 contributes to dopamine neuron loss nor its impact on mitochondrial function and relevance to death is clear. Here, we report that depletion of Pink1 by RNAi increased neuronal toxicity induced by MPP(+). Moreover, wild-type Pink1, but not the G309D mutant linked to familial PD or an engineered kinase-dead mutant K219M, protects neurons against MPTP both in vitro and in vivo. Intriguingly, a mutant that contains a deletion of the putative mitochondrial-targeting motif was targeted to the cytoplasm but still provided protection against 1-methyl-4-phenylpyridine (MPP(+))/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. In addition, we also show that endogenous Pink1 is localized to cytosolic as well as mitochondrial fractions. Thus, our findings indicate that Pink1 plays a functional role in the survival of neurons and that cytoplasmic targets, in addition to its other actions in the mitochondria, may be important for this protective effect.     

From the Cover: PNAS Plus: Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism

Prions are proteins that adopt alternative conformations that become self-propagating; the PrP^Sc prion causes the rare human disorder Creutzfeldt–Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T–YFP) and TgM83^+/− mice expressing α-synuclein (A53T). The TgM83^+/− mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83^+/+ mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83^+/− mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T–YFP in cultured cells, whereas none of six Parkinson’s disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83^+/+ mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.Looking back almost 50 y ago, kuru was the first human prion disease to be transmitted to an experimental animal (1). Subsequently, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker disease, and fatal familial insomnia were transmitted to nonhuman primates or transgenic (Tg) mice; all of these disorders were eventually found to be caused by PrP^Sc prions that were initially discovered in hamsters with experimental scrapie. Attempts to transmit other neurodegenerative diseases, including Alzheimer’s and Parkinson’s, to monkeys were disappointing; none of the animals developed signs of neurological dysfunction, and none showed recognizable neuropathological changes at autopsy (2).In 1960, Milton Shy and Glenn Drager described two male patients suffering from orthostatic hypotension, additional forms of autonomic insufficiency, and a movement disorder resembling Parkinson’s disease (PD). They also found an additional 40 cases of idiopathic hypotension in the literature, which shared many features with their patients. Nine years later, Graham and Oppenheimer suggested that Shy–Drager syndrome should be combined with striatonigral degeneration and olivopontocerebellar atrophy and that these three entities should be called multiple system atrophy (MSA) (3). They presciently argued that all three disorders were likely caused by a similar neurodegenerative process. Two decades passed before support for this hypothesis began to emerge when the brains of 11 MSA patients were reported to contain silver-positive accumulations or glial cytoplasmic inclusions (GCIs) primarily in oligodendrocytes (4). The nature of these GCIs remained elusive for another decade until three groups reported that GCIs exhibited positive immunostaining for α-synuclein (5–7). The discovery that MSA is a synucleinopathy followed a study reported 1 y earlier showing that Lewy bodies in PD contain α-synuclein by immunostaining (8). Such investigations were prompted by molecular genetic studies showing genetic linkage between the A53T point mutation in α-synuclein and inherited PD (9).MSA is a sporadic, adult-onset, progressive neurodegenerative disorder with an annual incidence of ∼3 per 100,000 individuals over the age of 50 (10, 11). The duration of MSA is generally 5–10 y and is substantially shorter than most cases of PD, which leads to death in 10–20 y. MSA has been subdivided based on the predominance of Parkinson’s symptoms (MSA-P) or cerebellar dysfunction (MSA-C) (12).The unanticipated results of an earlier study in 2013 showed that two cases of MSA transmitted CNS dysfunction to transgenic (TgM83^+/−) mice expressing mutant human α-synuclein*A53T protein (13). In that initial report, brain homogenates prepared from two cases of MSA were intracerebrally (IC) injected into TgM83^+/− mice, which resulted in progressive CNS dysfunction after ∼120 d. The brains of the Tg mice exhibited extensive phosphorylated α-synuclein deposits in the cytoplasm and axons of neurons.To determine whether the transmissions of two MSA cases were anomalous, we inoculated TgM83^+/− mice with another dozen cases from three different countries: the United Kingdom, Australia, and the United States. We report here that homogenates prepared from each of the additional 12 cases produced an experimental synucleinopathy in all of the IC inoculated TgM83^+/− mice with incubation times of ∼120 d. The mice developed intraneuronal deposits of aggregated, phosphorylated α-synuclein in their brainstems and some other CNS regions. Using multiple brain regions from some of the MSA cases, a total of 19 homogenates from 14 MSA cases produced CNS dysfunction in TgM83^+/− mice and infected human embryonic kidney (HEK) cells expressing α-syn140*A53T–YFP, resulting in cytoplasmic aggregates of the fusion protein that were measured by fluorescence microscopy (14). From these transmission studies in both TgM83^+/− mice and cultured cells, we conclude that MSA is a transmissible human neurodegenerative disease caused by α-synuclein prions.     

Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways,accumulation of α-synuclein,and apoptotic cell death in aged mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson''s disease. LRRK2 is a large protein containing a small GTPase domain and a kinase domain, but its physiological role is unknown. To identify the normal function of LRRK2 in vivo, we generated two independent lines of germ-line deletion mice. The dopaminergic system of LRRK2^−/− mice appears normal, and numbers of dopaminergic neurons and levels of striatal dopamine are unchanged. However, LRRK2^−/− kidneys, which suffer the greatest loss of LRRK compared with other organs, develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins at 20 months of age. The autophagy–lysosomal pathway is also impaired in the absence of LRRK2, as indicated by accumulation of lipofuscin granules as well as altered levels of LC3-II and p62. Furthermore, loss of LRRK2 dramatically increases apoptotic cell death, inflammatory responses, and oxidative damage. Collectively, our findings show that LRRK2 plays an essential and unexpected role in the regulation of protein homeostasis during aging, and suggest that LRRK2 mutations may cause Parkinson''s disease and cell death via impairment of protein degradation pathways, leading to α-synuclein accumulation and aggregation over time.     

Effects of a growth hormone-releasing hormone antagonist on telomerase activity, oxidative stress, longevity, and aging in mice

Both deficiency and excess of growth hormone (GH) are associated with increased mortality and morbidity. GH replacement in otherwise healthy subjects leads to complications, whereas individuals with isolated GH deficiency such as Laron dwarfs show increased life span. Here, we determined the effects of treatment with the GH-releasing hormone (GHRH) receptor antagonist MZ-5-156 on aging in SAMP8 mice, a strain that develops with aging cognitive deficits and has a shortened life expectancy. Starting at age 10 mo, mice received daily s.c. injections of 10 μg/mouse of MZ-5-156. Mice treated for 4 mo with MZ-5-156 showed increased telomerase activity, improvement in some measures of oxidative stress in brain, and improved pole balance, but no change in muscle strength. MZ-5-156 improved cognition after 2 mo and 4 mo, but not after 7 mo of treatment (ages 12, 14 mo, and 17 mo, respectively). Mean life expectancy increased by 8 wk with no increase in maximal life span, and tumor incidence decreased from 10 to 1.7%. These results show that treatment with a GHRH antagonist has positive effects on some aspects of aging, including an increase in telomerase activity.     

Cyclin-dependent kinase 5 regulates dopaminergic and glutamatergic transmission in the striatum

Dopaminergic and glutamatergic neurotransmissions in the striatum play an essential role in motor- and reward-related behaviors. Dysfunction of these neurotransmitter systems has been found in Parkinson's disease, schizophrenia, and drug addiction. Cyclin-dependent kinase 5 (CDK5) negatively regulates postsynaptic signaling of dopamine in the striatum. This kinase also reduces the behavioral effects of cocaine. Here we demonstrate that, in addition to a postsynaptic role, CDK5 negatively regulates dopamine release in the striatum. Inhibitors of CDK5 increase evoked dopamine release in a way that is additive to that of cocaine. This presynaptic action of CDK5 also regulates glutamatergic transmission. Indeed, inhibition of CDK5 increases the activity and phosphorylation of N-methyl-d-aspartate receptors, and these effects are reduced by a dopamine D1 receptor antagonist. Using mice with a point mutation of the CDK5 site of the postsynaptic protein DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, molecular mass of 32 kDa), in the absence or in the presence of a dopamine D1 receptor antagonist, we provide evidence that CDK5 inhibitors potentiate dopaminergic transmission at both presynaptic and postsynaptic locations. These findings, together with the known ability of CDK5 inhibitors to prevent degeneration of dopaminergic neurons, suggest that this class of compounds could potentially be used as a novel treatment for disorders associated with dopamine deficiency, such as Parkinson's disease.     

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.     

Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein alpha-synuclein (alphaS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, alphaS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric alphaS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of alphaS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.     

Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds

Recent experimental evidence suggests that transcellular propagation of fibrillar protein aggregates drives the progression of neurodegenerative diseases in a prion-like manner. This phenomenon is now well described in cell and animal models and involves the release of protein aggregates into the extracellular space. Free aggregates then enter neighboring cells to seed further fibrillization. The mechanism by which aggregated extracellular proteins such as tau and α-synuclein bind and enter cells to trigger intracellular fibril formation is unknown. Prior work indicates that prion protein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface to transmit pathologic processes. Here, we find that tau fibril uptake also occurs via HSPG binding. This is blocked in cultured cells and primary neurons by heparin, chlorate, heparinase, and genetic knockdown of a key HSPG synthetic enzyme, Ext1. Interference with tau binding to HSPGs prevents recombinant tau fibrils from inducing intracellular aggregation and blocks transcellular aggregate propagation. In vivo, a heparin mimetic, F6, blocks neuronal uptake of stereotactically injected tau fibrils. Finally, uptake and seeding by α-synuclein fibrils, but not huntingtin fibrils, occurs by the same mechanism as tau. This work suggests a unifying mechanism of cell uptake and propagation for tauopathy and synucleinopathy.Alzheimer’s disease (AD), frontotemporal dementia, and other tauopathies feature conversion of soluble, native tau protein into filamentous aggregates. In AD, tau pathology and its associated neural atrophy do not distribute randomly throughout the brain, but progress in association with neural networks (1–4), implying a role for connectivity and the transcellular movement of a pathological agent (1, 2, 4, 5). Prior studies by our laboratory and others have demonstrated that internalized tau aggregates can trigger fibrillization of native tau protein (6–11). We have previously observed that tau aggregates propagate the misfolded state among cells in culture via release of fibrils into the extracellular space. These aggregates trigger further fibrillization by direct protein–protein contact with native tau in the recipient cells (12). Thus, fibrillar tau appears to spread pathologic processes by mechanisms fundamentally similar to prion pathogenesis. Although the phenomenology is now well described, the basic mechanisms that mediate transcellular propagation of tau aggregation remain unknown, including the mechanism of aggregate uptake to seed intracellular fibrillization. Infectious prion protein is known to bind heparan sulfate proteoglycans (HSPGs) on the cell surface, a requirement for propagation of the pathological conformation (13, 14). This study elucidates a mechanism whereby tau aggregates bind HSPGs to stimulate cell uptake via macropinocytosis and seed further aggregation. Further, we find that HSPGs also mediate uptake and seeding of α-synuclein fibrils, but not huntingtin fibrils, consistent with a unifying mechanism for two major classes of neurodegenerative disease.     

The role of working memory and attentional disengagement on inhibitory control: effects of aging and Alzheimer's disease

Patients with Alzheimer''s disease have an impairment of inhibitory control for reasons that are currently unclear. Using an eye-tracking task (the gap-overlap paradigm), we examined whether the uncorrected errors relate to the task of attentional disengagement in preparation for action. Alternatively, the difficulty in correcting for errors may be caused by the working memory representation of the task. A major aim of this study was to distinguish between the effects of healthy aging and neurodegenerative disease on the voluntary control of saccadic eye movements. Using the antisaccade task (AST) and pro-saccade task (PST) with the ‘gap’ and ‘overlap’ procedures, we obtained detailed eye-tracking measures in patients, with 18 patients with probable Alzheimer''s disease, 25 patients with Parkinson''s disease and 17 healthy young and 18 old participants. Uncorrected errors in the AST were selectively increased in Alzheimer''s disease, but not in Parkinson''s disease compared to the control groups. These uncorrected errors were strongly correlated with spatial working memory. There was an increase in the saccade reaction times to targets that were presented simultaneously with the fixation stimulus, compared to the removal of fixation. This ‘gap’ effect (i.e. overlap–gap) saccade reaction time was elevated in the older groups compared to young group, which yielded a strong effect of aging and no specific effect of neurodegenerative disease. Healthy aging, rather than neurodegenerative disease, accounted for the increase in the saccade reaction times to the target that are presented simultaneously with a fixation stimulus. These results suggest that the impairment of inhibitory control in the AST may provide a convenient and putative mark of working memory dysfunction in Alzheimer''s disease.     

Tyrosine kinase nerve growth factor receptor switches from prosurvival to proapoptotic activity via Abeta-mediated phosphorylation

The present study shows that increased Abeta production in hippocampal neurons, due to a failure of NGF signal, induces an unexpected phosphorylation of tyrosine kinase receptor A (TrkA), followed by activation of the phospholipase C γ (PLCγ) pathway and neuronal death. Such phosphorylation seems causally connected with 2 kinases known be involved in amyloidogenesis, Src and CDK5, and associated with α and γ secretase–mediated p75 processing. Pharmacologic inhibition of TrkA phosphorylation and partial silencing of TrkA and/or p75 receptors prevent PLCγ activation and protect neurons from death. Concomitantly with these events, TrkA, p75, Abeta peptides, and PS1 protein coimmunoprecipitate, suggesting their direct interplay in the subsequent onset of apoptotic death. Together, these findings depict a cellular mechanism whereby the same cellular transducing system may invert its intracellular message from trophic and antiapoptotic to a death signaling, which could also have relevance in the onset of Alzheimer''s disease.     

Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.     

Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells

Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber''s hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.