Jordi Magrané

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 Parkinsons disease. Upon a loss of mitochondrial membrane potential (ΔΨm) 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 ΔΨm relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨm. 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 Parkinsons disease.

Heat Shock Protein 70 Participates in the Neuroprotective Response to Intracellularly Expressed β-Amyloid in Neurons

Intracellular β-amyloid 42 (Aβ42) accumulation is increasingly recognized as an early event in the pathogenesis of Alzheimers disease (AD). We have developed a doxycycline-inducible adenoviral-based system that directs intracellular Aβ42 expression and accumulation into the endoplasmic reticulum of primary neuronal cultures in a regulated manner. Aβ42 exhibited a perinuclear distribution in cell bodies and an association with vesicular compartments. Virally expressed intracellular Aβ42 was toxic to neuronal cultures 24 hr after induction in a dose-dependent manner. Aβ42 expression prompted the rapid induction of stress-inducible Hsp70 protein in neurons, and virally mediated Hsp70 overexpression rescued neurons from the toxic effects of intracellular Aβ accumulation. Together, these results implicate the cellular stress response as a possible modulator of Aβ-induced toxicity in neuronal cultures.

α-Synuclein is localized to mitochondria-associated ER membranes.

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by motor neuron degeneration. Mutations in Cu,Zn-superoxide dismutase (SOD1) are responsible for 20% of familial ALS cases via a toxic gain of function. In mutant SOD1 transgenic mice, mitochondria of spinal motor neurons develop abnormal morphology, bioenergetic defects and degeneration, which are presumably implicated in disease pathogenesis. SOD1 is mostly a cytosolic protein, but a substantial portion is associated with organelles, including mitochondria, where it localizes predominantly in the intermembrane space (IMS). However, whether mitochondrial mutant SOD1 contributes to disease pathogenesis remains to be elucidated. We have generated NSC34 motor neuronal cell lines expressing wild-type or mutant SOD1 containing a cleavable IMS targeting signal to directly investigate the pathogenic role of mutant SOD1 in mitochondria. We show that mitochondrially-targeted SOD1 localizes to the IMS, where it is enzymatically active. We prove that mutant IMS-targeted SOD1 causes neuronal toxicity under metabolic and oxidative stress conditions. Furthermore, we demonstrate for the first time neurite mitochondrial fragmentation and impaired mitochondrial dynamics in motor neurons expressing IMS mutant SOD1. These defects are associated with impaired maintenance of neuritic processes. Our findings demonstrate that mutant SOD1 localized in the IMS is sufficient to determine mitochondrial abnormalities and neuronal toxicity, and contributes to ALS pathogenesis.

Internalized antibodies to the Aβ domain of APP reduce neuronal Aβ and protect against synaptic alterations

Immunotherapy against β-amyloid peptide (Aβ) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Aβ immunization reduces Aβ plaque pathology and improves cognitive function. However, the biological mechanisms by which Aβ antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Aβ antibodies decreases levels of intracellular Aβ. Antibody-mediated reduction in cellular Aβ appears to require that the antibody binds to the extracellular Aβ domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Aβ antibodies protects against synaptic alterations that occur in APP mutant neurons.

PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinsons disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation.

Abnormal Mitochondrial Transport and Morphology are Common Pathological Denominators in SOD1 and TDP43 ALS Mouse Models

Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.

Intraneuronal β-Amyloid Expression Downregulates the Akt Survival Pathway and Blunts the Stress Response

Early events in Alzheimers disease (AD) pathogenesis implicate the accumulation of β-amyloid (Aβ) peptide inside neurons in vulnerable brain regions. However, little is known about the consequences of intraneuronal Aβ on signaling mechanisms. Here, we demonstrate, using an inducible viral vector system to drive intracellular expression of Aβ42 peptide in primary neuronal cultures, that this accumulation results in the inhibition of the Akt survival signaling pathway. Induction of intraneuronal Aβ42 expression leads to a sequential decrease in levels of phospho-Akt, increase in activation of glycogen synthase kinase-3β, and apoptosis. Downregulation of Akt also paralleled intracellular Aβ accumulation in vivo in the Tg2576 AD mouse model. Overexpression of constitutively active Akt reversed the toxic effects of Aβ through a mechanism involving the induction of heat shock proteins (Hsps). We used a small-interfering RNA approach to explore the possibility of a link between Akt activity and Hsp70 expression and concluded that neuroprotection by Akt could be mediated through downstream induction of Hsp70 expression. These results suggest that the early dysfunction associated with intraneuronal Aβ accumulation in AD involve the associated impairments of Akt signaling and suppression of the stress response.

Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons

Mutations in Cu,Zn superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS), a rapidly fatal motor neuron disease. Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild-type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons, in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild-type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS.

SOD1 targeted to the mitochondrial intermembrane space prevents motor neuropathy in the Sod1 knockout mouse

Motor axon degeneration is a critical but poorly understood event leading to weakness and muscle atrophy in motor neuron diseases. Here, we investigated oxidative stress-mediated axonal degeneration in mice lacking the antioxidant enzyme, Cu,Zn superoxide dismutase (SOD1). We demonstrate a progressive motor axonopathy in these mice and show that Sod1(-/-) primary motor neurons extend short axons in vitro with reduced mitochondrial density. Sod1(-/-) neurons also show oxidation of mitochondrial--but not cytosolic--thioredoxin, suggesting that loss of SOD1 causes preferential oxidative stress in mitochondria, a primary source of superoxide in cells. SOD1 is widely regarded as the cytosolic isoform of superoxide dismutase, but is also found in the mitochondrial intermembrane space. The functional significance of SOD1 in the intermembrane space is unknown. We used a transgenic approach to express SOD1 exclusively in the intermembrane space and found that mitochondrial SOD1 is sufficient to prevent biochemical and morphological defects in the Sod1(-/-) model, and to rescue the motor phenotype of these mice when followed to 12 months of age. These results suggest that SOD1 in the mitochondrial intermembrane space is fundamental for motor axon maintenance, and implicate oxidative damage initiated at mitochondrial sites in the pathogenesis of motor axon degeneration.