Welcome to the AD/PD™ 2021 Interactive Program
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- Live Session | 
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FOLLOWING THE LIVE DISCUSSION, THE RECORDING WILL BE AVAILABLE IN THE ON-DEMAND SECTION OF THE AUDITORIUM.
A REDOX FUNCTION FOR PARKIN EXPLAINS ITS SELECTIVE NEUROPROTECTION IN ADULT HUMAN BRAIN
Abstract
Abstract Body
Loss of parkin causes early onset recessive Parkinson disease. We hypothesized that parkin confers thiol-dependent antioxidant activity as a redox molecule, thus protecting against radicals such as hydrogen peroxide (H2O2) and electrophilic dopamine metabolites. We found that in human brain, including normal midbrain, parkin is oxidized and transitions into insolubility between the ages of 28 and 42 years. This correlates with rising H2O2 levels and is specific to human brain; this transition does not occur in human spinal cord, skeletal muscle, or rodent brain. Using human and mouse tissues, cellular models and recombinant proteins, we found that parkin fulfils criteria for a redox molecule: One, it interacts with redox stressors via its thiol groups. Two, parkin’s structure and solubility change upon oxidation at select residues, including at Cys95 and Cys253 in human brain. Three, through its oxidation parkin effects redox change by lowering ROS levels and augmenting melanin formation in a Cys95-dependent manner. Further, in human midbrain parkin is physically associated with neuromelanin. We also demonstrate that parkin expression alters glutathione (GSH) metabolism. In human and murine parkin-deficient brain, we detected increased H2O2 and carbonyl content as evidence of chronically elevated oxidative stress; we also found compensatory elevation in reduced GSH and the GSH:GSSG ratio. Taken together, we present an expanded concept for parkin functions in that its oxidation is linked to redox balance and sequestration of dopamine radicals in human brain. We propose that these non-E3-mediated redox effects contribute to dopamine neuron health in ageing human midbrain.
WNT AND NFAT SIGNALLING CHANGES IN LRRK2 PARKINSON’S DISEASE MODELS
Abstract
Aims
LRRK2 mediated Wnt and Ca2+ cell signalling changes were reported previously. These include Ca2+-dependent immune responses and autophagy. In addition, LRRK2 protective and pathogenic genetic PD variants affect canonical Wnt signalling in opposite directions showing a clear correlation between Wnt signalling activity and neurodegenerative disease susceptibility. As most evidence to date was based on in vitro experiments, this project is aimed to obtain a better understanding of LRRK2 signalling function and dysfunction in vivo.
Methods
We observed Wnt and NFAT signalling activities in different brain regions of wild type (WT), LRRK2 knock-out (KO), and G2019S knock-in (KI) mouse models. TCF/LEF-GFP lentiviral biosensors were transduced at P0 and signalling activity distribution in the brain investigated using immunohistochemistry. This data was complemented by measuring mRNA and protein expression changes, and signalling activity under basal and stimulated conditions in primary neuronal cultures from different brain regions.
Results
We observed Wnt signalling pathway activation in different brain regions. Localised differences in Wnt pathway mediators were also confirmed using mRNA and protein expression analysis. Wnt signalling activity differed between investigated brain regions and cell types and was dependent on LRRK2 genotype and sex.
Conclusions
LRRK2 KO and LRRK2 G2019S KI alter gene and protein expression of Wnt and NFAT signalling cascades. The observed signalling dysregulation was most prominent in the cortex and striatum, and also suggested changes in glia. Further investigation into detailed regulatory effects of LRRK2 mediated Wnt signalling activities under physiological and pathological conditions is ongoing.
LOSS OF FRAGILE X MENTAL RETARDATION PROTEIN (FMRP) PRECEDES LEWY PATHOLOGY IN PARKINSON’S DISEASE (PD).
Abstract
Aims
Previous research implicated alterations of neuronal excitability in DA neuron cell death. Because the Fragile X Mental Retardation Protein (FMRP) has been shown to control a large number of genes related to neuronal excitability and synaptic function, we here investigated the role of FMRP in PD.
Methods
We combined biochemical, electrophysiological and imaging techniques to examine the effect of a-syn on FMRP in vitro and in vivo. In addition, we investigated the abundance of FMRP in SNc DA neurons of post-mortem human brain tissue from PD patients and from subjects that had incidental Lewy body disease (iLBD).
Results
We found FMRP to be decreased in cultured DA neurons and in the mouse brain in response to a-syn overexpression. Likewise, FMRP was lost in SNc DA neurons of PD patients and in neurons from iLBD cases. Similar to Fragile X Syndrome (FXS) neurons, a-syn-overexpressing cells had an increase in membrane N-type calcium channels, enhanced N-type-mediated calcium currents, an increased phosphorylation of the protein translation initiation machinery (p-ERK1/2, p-eIF4E and p-S6) and, as a consequence, an increased overall protein synthesis. The loss of FMRP appeared to have a protective effect in SNc DA neurons, because FMRP knockout mice were resistant to the effect of a-syn on striatal dopamine release.
Conclusions
Our results reveal a new and previously unrecognized role of FMRP in PD. Our data thus suggest the loss of FMRP to be an early pathogenic event that precedes Lewy pathology in PD and therefore support the examination of FMRP-regulated genes in PD onset and progression.
BRAIN REGION SPECIFIC PATHOLOGY SPREAD IN SPORADIC PARKINSON’S DISEASE AND DEMENTIA WITH LEWY BODIES IS GOVERNED BY Α-SYNUCLEIN CONFORMATIONS.
Abstract
Aims
The α-synuclein (αSYN) protein is encoded by the SNCA gene and is thought to be a driver in the pathogenic mechanism of the two neurodegenerative diseases Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). What underlies the differences in region specific dysfunction in both diseases is still unclear. The αSYN protein exists in different physiological conformations: the cytosolic, active but susceptible (aggregation-prone) monomer (αSYNCS), and higher-order, cytosolic protective (aggregations-resistant) multimers (αSYNCP). It has been shown that SNCA mutations associated with familial PD destabilize the equilibrium of αSYNCS/ αSYNCP and that relative increase of the aggregation-prone monomer is causal for the neurodegeneration in model systems.
Methods
Here, we characterized the balance and levels of αSYNCS and αSYNCP in post-mortem native human brain tissue from sporadic PD and DLB patients in different brain regions according to the spreading theory of αSYN and classical Braak staging system.
Results
We show that the equilibrium of αSYNCS/ αSYNCP is destabilized in sporadic PD and DLB patients resulting in brain region-specific alterations according to the spread of Lewy body pathology, implying the αSYNCS/ αSYNCP equilibrium as an determining factor of temporal and spatial disease progression. Moreover, demented PD and DLB patients exhibited a specific decrease of the αSYNCS/ αSYNCP equilibrium in neocortical regions, implying that the region specific decrease is important for the dementia component of synculeinopathies.
Conclusions
Thus, our data suggest an association of αSYNCS/ αSYNCP destabilization in sporadic synucleinopathies related to the spreading of αSYN pathology and subsequently different (clinical) manifestations of neurodegeneration.
ALPHA-SYNUCLEIN INDUCES COFILIN PATHOLOGY: IMPLICATIONS FOR PARKINSON’S DISEASE DEMENTIA
Abstract
Aims
Besides the typical motor symptoms, dementia is recognized as a major complication in Parkinson’s disease (PD). PD-dementia appears to be associated with the accumulation of alpha-synuclein (aSyn) in the hippocampus. Cofilin-actin rods are neuropathology-related structures which have been implicated in synaptic dysfunction and cognitive impairment mainly in Alzheimer’s disease (AD). Here, we investigated whether hippocampal cofilin pathology impacts on cognitive dysfunction and dementia in PD.
Methods
Cofilin pathology was studied in vitro by the overexpression of aSyn or exogenous addition of aSyn pre-formed fribils (PFFs), in primary cultures of hippocampal neurons and in cultured hippocampal slices. Additionally, rod formation was evaluated in a PD mouse model overexpressing human aSyn in neurons (Thy1-aSyn mice) and presenting cognitive dysfunction, and in post mortem hippocampal sections from PD patients with cognitive impairment and dementia.
Results
Our in vitro results showed that elevated levels of aSyn induce cofilin-actin rods in hippocampal neurons, impacting on dendritic spine number and morphology, through a mechanism involving PrPC and NADPH oxidase pathways. Importantly cofilin pathology was validated in the hippocampus of Thy1-aSyn mice, at the same age where cognitive impairment is observed, and in hippocampal sections from PD patients with dementia.
Conclusions
This work proposes the innovative hypothesis that hippocampal cofilin pathology underlies synaptic impairment and cognitive dysfunction in PD. Currently, we are further exploring in vivo the consequences of cofilin pathology, and of its modulation using rod inhibitors, on synaptic dysfunction and cognitive impairment using the Thy1-aSyn mice. This will contribute to establish cofilin-actin rods as a novel therapeutic target for dementia in PD.
PROLONGED STABILIZATION OF WILDTYPE AND E46K-AMPLIFIED MUTANT ALPHA-SYNUCLEIN TETRAMERS PREVENTS AGGREGATE FORMATION, SECONDARY PD CYTOPATHOLOGY AND MOTOR PHENOTYPES
Abstract
Aims
Converging evidence suggests pathologically active forms of α-synuclein (αS) in Parkinson’s disease (PD) brain reflect progressive accumulation of self-aggregating αS monomers. The variability of phenotypes by genetic mutations or sex in PD cases have begun to challenge the assumption that an uniform monomeric αS level underlies neuronal dysfunction. We recently showed that female sex hormones delay progressive PD-like phenotypes of certain tg mice by normalizing the αS tetramer-to-monomer (T:M) ratios. Stabilizing physiological αS tetramers by several distinct methods represents a novel therapeutic approach to lower pathologic αS monomer levels, but the consequences of a prolonged upregulation of the αS T:M ratio in vivo have yet not been studied.
Methods
We systematically analyzed mice expressing WT hu αS or fPD E46K-like “3K” mutant αS and undergoing prolonged treatment with αS tetramer-stabilizing female hormones or lipid modulators (inhibitors of stearoyl CoA desaturase (SCD)). We used biochemistry, immunohistology and behavioral assessment. We analyzed the covariability of sex and/or compared the treated mice to mice with genetic downregulation of SCD (SCD heterozygous KO mice), providing target validation, respectively.
Results
Elevating αS T:M ratios in the 3K mutant and in WT hu αS mice by treating with female sex-hormones or with SCD inhibitors improved lysosomal cytopathology, slowed or prevented accumulation of PK-resistant, lipid-rich αS aggregates, and rescued the neurite deficits and resultant PD-like motor phenotypes.
Conclusions
Our findings demonstrate that chronic increase of αS T:M ratios represents an efficacious therapeutic approach to ameliorating PD-type pathology in vivo.
INTERPLAY BETWEEN SYNUCLEINOPATHY PROPAGATION AND NEUROINFLAMMATION
Abstract
Abstract Body
The clinical progression of neurodegenerative diseases correlates with the spread of proteinopathy in the brain. The dominant theory for how proteinopathy spreads is templated seeding, a self-perpetuating process in which aggregated proteins act as templates that convert nearby soluble proteins into their aggregated forms. However, some experimental observations do not support this theory1-4, suggesting that other fundamental mechanisms might be involved either on their own or in concert with templated seeding. Here, we propose that inflammation is fundamental to proteinopathy spread. In vitro, a sequence variant of a-synuclein (V40G) was much less capable of fibril formation than wild-type α-synuclein (WT-syn) and, when mixed with WT-syn, interfered with its fibrillation. Yet when V40G was injected intracerebrally into mice, it induced aggregate spreading even more effectively than WT-syn. Transcriptome analysis and immunohistochemistry revealed that the aggregate spreading was preceded by sustained microgliosis and inflammatory responses, which were more robust with V40G than with WT-syn. Oral administration of an anti-inflammatory agent (aspirin) suppressed aggregate spreading, inflammation, and behavioral deficits in the V40G-injected mice. Furthermore, exposure of cells in vitro to inflammatory cytokines increased the cell-to-cell propagation of α-synuclein. These results suggest that the inflammatory microenvironment is the major driver of the spread of synucleinopathy in the brain, thereby playing an important role in disease progression.
MONITORING ALPHA-SYNUCLEIN AGGREGATION IN MAMMALIAN CELLS AND MICE BY IN-CELL NMR AND PROTEOMICS
Abstract
Aims
1) characterize at atomic level the conformational changes that aSyn undergo in mammalian cells with inactive HSP90B1 and HSC70; 2) study the proteomic changes triggered by accumulation of cell-to-cell transmitted aSyn in the rodent brain.
Methods
1) alpha-Synuclein (aSyn) was analyzed by in-cell NMR in mammalian cells depleted of chaperones. Its amide fingerprint was monitored by heteronuclear (1H-15N HSQC/HMQC) multidimensional NMR that highlighted the structural transitions that aSyn undergo upon HSP90B1&HSC70 loss-of-function. 2) A variant of aSyn with increased cell-to-cell propagation was expressed in neurons of non-transgenic mice by virus-mediated gene transfer. Likewise, aSyn pre-formed fibrils (pffs) were administrated by stereotaxic surgery. A shotgun mass spectrometry-coupled to label free quantification approach was used to quantify the proteome of micro-dissected brains. Differentially expressed proteins were subjected to bioinformatics analyses to identify the cellular responses triggered by the two aSyn.
Results
1) In cells aSyn transiently interacts with the chaperones HSP90 and HSC70 through its N- and C-termini and Y39. aSyn re-localizes into mitochondria upon chaperones inhibition. The mitochondria interaction leads to aSyn aggregation and formation of inclusions. 2) Cell-to-cell transmitted aSyn accumulated in striatal and nigral neurons within Lewy body-like inclusions. Animals with burden of aSyn displayed, in addition to Lewy body-like pathology, neurodegeneration, inflammation, and motor deficits. Certain pathways were affected by cell-to-cell transmitted aSyn.
Conclusions
The disordered nature of aSyn is maintained by its transient interactions with HSP90 and HSC70. The PD-like pathology induced by cell-to-cell transmitted and pffs aSyn is characterized by a neuronal adaptive response involving key biological processes.