Alison Wood-Kaczmar

PINK1-Associated Parkinson's Disease Is Caused by Neuronal Vulnerability to Calcium-Induced Cell Death

Summary Mutations in PINK1 cause autosomal recessive Parkinsons disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na+/Ca2+ exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.

PINK1 Is Necessary for Long Term Survival and Mitochondrial Function in Human Dopaminergic Neurons

Parkinsons disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinsons disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.

Glycogen synthase kinase-3beta phosphorylation of MAP1B at Ser1260 and Thr1265 is spatially restricted to growing axons.

Recent experiments show that the microtubule-associated protein (MAP) 1B is a major phosphorylation substrate for the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) in differentiating neurons. GSK-3β phosphorylation of MAP1B appears to act as a molecular switch regulating the control that MAP1B exerts on microtubule dynamics in growing axons and growth cones. Maintaining a population of dynamically unstable microtubules in growth cones is important for axon growth and growth cone pathfinding. We have mapped two GSK-3β phosphorylation sites on mouse MAP1B to Ser1260 and Thr1265 using site-directed point mutagenesis of recombinant MAP1B proteins, in vitro kinase assays and phospho-specific antibodies. We raised phospho-specific polyclonal antibodies to these two sites and used them to show that MAP1B is phosphorylated by GSK-3β at Ser1260 and Thr1265 in vivo. We also showed that in the developing nervous system of rat embryos, the expression of GSK-3β phosphorylated MAP1B is spatially restricted to growing axons, in a gradient that is highest distally, despite the expression of MAP1B and GSK-3β throughout the entire neuron. This suggests that there is a mechanism that spatially regulates the GSK-3β phosphorylation of MAP1B in differentiating neurons. Heterologous cell transfection experiments with full-length MAP1B, in which either phosphorylation site was separately mutated to a valine or, in a double mutant, in which both sites were mutated, showed that these GSK-3β phosphorylation sites contribute to the regulation of microtubule dynamics by MAP1B.

What Have PINK1 and HtrA2 Genes Told Us about the Role of Mitochondria in Parkinson's Disease?

Parkinsons disease (PD) is a common, disabling, neurodegenerative disease. Our knowledge of the molecular events leading to PD is being greatly enhanced by the study of relatively rare familial form of the disease. Nevertheless, the pathways leading from the genetic mutations to nigral cell degeneration and the other features in PD remain poorly understood. The identification of PINK1, a mitochondrial putative protein kinase, has helped understand the pathophysiology of mitochondria and their potential role in PD. Mutations in PINK1 are associated with the PARK6 autosomal recessive, early‐onset, PD‐susceptibility locus. Point mutations in another mitochondrial protein, HtrA2, are a susceptibility factor for PD (PARK13 locus). We report here the results of investigations into the interactors and pathways of these two mitochondrial molecules (PINK1 and HtrA2) in a range of models and human PD tissue.

The Role of the Mitochondrial NCX in the Mechanism of Neurodegeneration in Parkinson’s Disease

Mitochondrial Na(+)/Ca(2+) exchange (NCXmito) is critical for neuronal Ca(2+) homeostasis and prevention of cell death from excessive mitochondrial Ca(2+) (m[Ca(2+)]) accumulation. The mitochondrial kinase PINK1 appears to regulate the mCa(2+) efflux from dopaminergic (DAergic) neurons, possibly by directly regulating NCXmito. However, the precise molecular identity of NCXmito is unknown and has been the subject of great controversy. Here we propose that the previously characterised plasmalemmal NCX isoforms (NCX2, NCX3) contribute to mitochondrial Na(+)/Ca(2+) exchange in human DAergic neurons and may act downstream of PINK1 in the prevention of neurodegeneration by m[Ca(2+)] accumulation. Firstly, we definitively show the existence of a mitochondrial pool of endogenous plasmalemmal NCX isoforms in human DAergic neurons and cell lines using immunolocalisation and fluorescence-assisted organelle sorting (FAOS). Secondly, we demonstrate reduced mitochondrial Ca(2+) efflux occurs following inhibition of NCX2 or NCX3 (but not NCX1) using siRNA or antibody blocking. This study has potentially revealed a new molecular target in Parkinsons disease pathology which ultimately may open up new avenues for future therapeutic intervention.

FAN1 modifies Huntington’s disease progression by stabilising the expanded HTT CAG repeat

Abstract Huntington’s disease (HD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the huntingtin (HTT) gene. CAG repeat length explains around half of the variation in age at onset (AAO) but genetic variation elsewhere in the genome accounts for a significant proportion of the remainder. Genome-wide association studies have identified a bidirectional signal on chromosome 15, likely underlain by FANCD2- and FANCI-associated nuclease 1 (FAN1), a nuclease involved in DNA interstrand cross link repair. Here we show that increased FAN1 expression is significantly associated with delayed AAO and slower progression of HD, suggesting FAN1 is protective in the context of an expanded HTT CAG repeat. FAN1 overexpression in human cells reduces CAG repeat expansion in exogenously expressed mutant HTT exon 1, and in patient-derived stem cells and differentiated medium spiny neurons, FAN1 knockdown increases CAG repeat expansion. The stabilizing effects are FAN1 concentration and CAG repeat length-dependent. We show that FAN1 binds to the expanded HTT CAG repeat DNA and its nuclease activity is not required for protection against CAG repeat expansion. These data shed new mechanistic insights into how the genetic modifiers of HD act to alter disease progression and show that FAN1 affects somatic expansion of the CAG repeat through a nuclease-independent mechanism. This provides new avenues for therapeutic interventions in HD and potentially other triplet repeat disorders.

B10 Inclusion formation in mutant HTT exon 1 expressing human neuronal cells

Background Neuronal inclusion formation is a pathognomonic feature of Huntington’s disease (HD). Recent evidence has suggested that these inclusion bodies (IBs) may comprise a heterogeneous population of huntingtin (HTT) protein species, a subset of which may lead to impaired cell viability and ultimately cell death. Aim To investigate the formation of inclusions in human neurons derived from neural stem cells (NSCs) (ReNcell VM) stably transduced to over-express HTT exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71 Q and 129Q). Methods High-content screening of neurons stained with anti-HTT antibody S830 was carried out using a Perkin Elmer Opera LX confocal microscope. Subsequent quantitative image analysis was carried out using ImageJ and R. Individual cells were analysed in more detail using super resolution imaging (N-SIM Super Resolution System). The effect of mHTT exon 1 on neuronal viability was assessed using a variety of biochemical techniques (LDH assay, MTT and Alamar Blue). Results Inclusion bodies form in a small proportion of (1%) of neurons, and are predominantly intra-nuclear, though a lower proportion of cells display smaller perinuclear inclusions. The formation of nuclear inclusions increases in a polyQ- length dependent manner and over time. Super resolution microscopy of individual cells has demonstrated this to be an all-or-nothing phenomenon. A diffuse cytosolic form of mHTT also accumulates in the cells over time. There is no overt effect on basal viability in these mHTT exon 1 expressing neurons. Conclusions We have characterised the spatio-temporal profile of mHTT inclusion formation in mHTT expressing NSCs and neurons, and have demonstrated poly-Q and time-dependent accumulation of IBs. A deeper understanding of relationship between the different forms of mHTT and neuronal vulnerability is essential for the design of targeted therapeutics.

B27 Abnormal bioenergetics in inclusion-containing mutant HTT exon 1 primary human neurons

Background Mitochondrial dysfunction is a known component of HD pathogenesis, but the precise mechanisms and temporal order of events linked to mHTT inclusion formation and neurodegeneration are unclear. For example, observations from clinical samples and in vitro models implicate respiratory impairment and enhanced glycolysis in HD cells. Aim To identify early signs of mitochondrial dysfunction and perturbed bioenergetics in a human HD neuronal model, and better understand the interaction between the proteotoxic environment of mHTT-expressing cells and their metabolic capacity. Methods We are using a human neural stem cell (NSC) line (ReNcell VM) that over-expresses huntingtin (HTT) exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71Q and 129Q), and can be rapidly differentiated into mixed populations of neurons and glia in various culture formats. The pathogenic HTT exon 1 lines develop both intranuclear and cytoplasmic mHTT-containing inclusions, as well as accumulating a diffuse aggregated form of mHTT in a time and poly Q-dependent manner. We have correlated with this measures of oxygen consumption rate and extracellular acidification rates using an XF96 Seahorse Bioanalyser, mitochondrial volume and basal ΔΨm by live confocal imaging and potential impairment individual respiratory chain complexes using in vitro assays. Results Despite no overt reduction in cell viability in the mHTT exon 1 expressing lines we have uncovered sub-pathological alterations to mitochondrial function. We report differences in key respiratory parameters (e.g. basal and maximal respiration) in line with a significant decrease in basal mitochondrial ΔΨm in mHTT exon 1 cells. Here, we will present our findings correlating the inclusion phenotypes and mitochondrial dysfunction in the presence of mHTT exon 1. Conclusions We have established a robust and disease-relevant in vitro cellular model of HD that has revealed potentially important early changes to neuronal bioenergetics and mitochondrial function. Using this system we aim to not only identify novel pathways in disease pathogenesis that may be amenable to therapeutic targeting, but also to simultaneously develop a novel in vitro cellular platform for high-throughput compound screening.