Welcome to the AD/PD™ 2022 Interactive Program

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Displaying One Session

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114

EPIGENETIC AND EPITRANSCRIPTOMIC REGULATION OF GENE EXPRESSION IN MODELS OF PARKINSON'S DISEASE

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
02:45 PM - 03:00 PM

Abstract

Abstract Body

Synaptic dysfunction is an early alteration in multiple neurodegenerative disorders including Parkinson’s disease and other synucleinopathies, disorders characterised by the accumulation of α-synuclein (aSyn) in pathological protein inclusions. aSyn is known as a pre-synaptic protein involved in synaptic vesicle trafficking, and SNARE complex formation at the nerve terminals. In pathological conditions, it is associated with alterations of synaptic function. Interestingly, aSyn also occurs in the nucleus where it induces epigenetic changes. RNA-mediated processes contribute to synaptic remodelling by RNA translocation to the synaptic compartment. This is particularly relevant for microRNAs (miRNAs) that can regulate mRNA expression by complementary binding. We are investigating aSyn-mediated epigenetic and epitranscriptomic alterations to uncover the molecular mechanisms underlying alterations in synaptic processes that may contribute to synapse degeneration.

We performed small RNA-Sequencing of the midbrain of 6-month-old transgenic mice expressing A30P mutant αsyn, present in familial forms of PD. Gene ontology (GO) functional annotation and pathway analysis of differentially expressed genes and miRNAs revealed several deregulated biological processes linked with the synaptic compartment.

Our data support the emerging role of specific microRNAs, and RNA modifications, as key regulators of gene expression alterations associated with aSyn. Ultimately, the understanding of the epigenetic and epitranscriptomic alterations in synucleinopathies may lead to the identification of targets for therapeutic intervention and for the development of novel biomarkers.

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ON THE STRUCTURAL BIOLOGY OF A-SYNUCLEIN IN CELLS

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
03:00 PM - 03:15 PM

Abstract

Aims

From a structural perspective A-synuclein is a chameleon-like molecule. It is an intrinsically disordered protein (IDP), but not quite, that undergoes environment dependent phase transition into liquid droplets and amyloid fibrils into distinct environment-dictated polymorphs, can bind transiently membranes and amongst others chaperones such as HSP70/90.

Methods

Structures and protein and ligand interactions have been studied at (near)atomic resolution using (in cell) NMR, cryo EM (in collaboration with H. Stahlberg, EPFL), and MS (in collaboration with P. Picotti, ETH) in part with in cell and in part in vitro samples.

Results

In concert with the transient interaction with chaperones, A-Synuclein’s residual structure of the monomer between its N-terminal and C-terminal segment is interfering with aggregation. If the chaperone interaction is perturbed A-synuclein is dislocated to mitochondria where it interacts transiently with a diverse set of proteins unfolding its IDP-like state further opening an avenue towards aggregation, while also influencing the ATP homeostasis by its interactions with other proteins.

Furthermore, in a proof of concept study the stabilization of the monomeric state by small molecule binders, the design of a A-synuclein fibril specific small molecule towards a PET tracer as well as inhibitors of A-synuclein aggregation and A-synuclein mitochondria protein interactions following a structure-based strategy are discussed.

B.M. Burmann et al. Nature 577:127-132 (2020)

R. Guerrero-Ferreira Elife 8:e48907 (2019)

P. Kumari et al. Proc. Natl. Acad. Sci 118: e2012171118 (2021)

S. Ray et al. Nat Chem. 12: 705-716 (2020)

Conclusions

The structural landscape of A-synuclein enables a detailed mechanistic understanding of the process of action associated with PD.

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LYSOSOMES ARE A HUB FOR SEEDING NEW ALPHA-SYNUCLEIN AGGREGATES AND PROMOTE THEIR INTERCELLULAR SPREADING THROUGH TUNNELING NANOTUBES

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
03:15 PM - 03:30 PM

Abstract

Aims

The accumulation of alpha-synuclein aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson’s disease. Alpha-synuclein aggregates propagate in a “prion-like” manner and can be transferred inside lysosomes to recipient cells through tunneling nanotubes (TNTs), which are actin-based, thin cellular protrusions connecting remote cells. However, how lysosomes contribute to the spreading of alpha-synuclein aggregates is unknown.

Methods

We performed super-resolution and electron microscopy, immunocytochemistry, and gene overexpression/downregulation experiments to study the role of lysosomes in the propagation of alpha-synuclein pathology in neuronal cells.

Results

We found that alpha-synuclein fibrils affect the morphology of lysosomes and impair their function. In addition, we demonstrated that alpha-synuclein fibrils induce peripheral redistribution of lysosomes, likely mediated by TFEB, increasing the efficiency of fibrils’ transfer to neighboring cells. We also showed that lysosomal membrane permeabilization allows the seeding of soluble alpha-synuclein in cells that have taken up alpha-synuclein fibrils from the culture medium and, more importantly, in healthy cells in co-culture following the lysosome-mediated transfer of the fibrils. Moreover, we demonstrated that seeding occurs mainly at lysosomes in both donor and acceptor cells after the uptake of alpha-synuclein fibrils from the medium and following their transfer. Finally, by using a heterotypic co-culture system, we showed that donor cells bearing alpha-synuclein fibrils transfer damaged lysosomes to acceptor cells, while also receiving healthy lysosomes from them.

Conclusions

Collectively, our findings indicate that lysosomes damaged by alpha-synuclein fibrils become a hub for seeding new aggregates and function as a Trojan horse facilitating the dissemination of misfolded aggregates through TNTs.

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ULTRASTRUCTURE OF ALPHA-SYNUCLEIN PATHOLOGY PROVIDES NEW INSIGHTS INTO MULTIPLE SYSTEM ATROPHY

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
03:30 PM - 03:45 PM

Abstract

Aims

Multiple System Atrophy (MSA) is characterized by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs). Despite many efforts, it remains unclear which mechanisms lead to their assembly. Thus, in order to increase our understanding of the disease, we investigated the ultrastructure of aSyn immunopositive pathology in the postmortem brain of four MSA donors.

Methods

Brain tissue (substantia nigra and putamen) from clinically diagnosed and pathologically confirmed MSA-P donors with postmortem delays of under 6 hours was processed for correlative light and electron microscopy (CLEM).

Results

We localized 123 GCIs, four microglial cytoplasmic aSyn aggregates and one Lewy body-like NCI. We found that GCIs are composed of a meshwork of fuzzy filaments that colocalized with lysosomes, autophagosomes and peroxisomes. Microglial aSyn aggregates varied in terms of ultrastructures, with some resembling GCIs, while others showed amorphous proteinaceous material without visible filaments. Lysosomes and autophagosomes were also found to colocalize with these aggregates. In contrast to GCIs and the NCIs that have previously been described in MSA literature, we found that the Lewy body-like NCI consisted of a highly dense accumulation of membrane fragments and vesicles, without visible filaments.

Conclusions

We can confirm that GCIs are filamentous and further show that they are composed of various organelles. We also demonstrate the unique ultrastructure of microglial aSyn aggregates and a Lewy body-like NCI for the first time using CLEM in tissue of MSA patients. Together, our findings may ultimately aid in confirming suitable MSA models in the future.

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A ROLE FOR EPIGENETIC MECHANISMS IN THE LEWY BODY DEMENTIAS

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
03:45 PM - 04:00 PM

Abstract

Aims

The Lewy body diseases, Dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and Parkinson’s disease dementia (PDD) are all neurodegenerative diseases classified by the accumulation of alpha-synuclein in neurons, forming Lewy bodies (LB). We hypothesise that these LBs cause epigenetic changes within neurons and surrounding cells and that these changes can be used to distinguish the different diseases from one another.

Methods

Bulk tissue from the cingulate gyrus and prefrontal cortex was run on the Illumina Infinium Methylation EPIC array generating a quantitative measure of DNA methylation for over 850,000 CpG sites (n=~100/disease group). Linear regression and pathway analyses were then used to identify loci that are significantly different or specific to each disease.

Results

Study groups have been sourced consisting of cases with PD, PDD and DLB based on LB deposition and clinical symptom staging. Control cases have been selected for matched age and levels of concomitant AD pathology. We have identified significant changes in methylation associated with both phenotype and neuropathology alongside the cellular pathways these changes correspond with.

Conclusions

We have collated a well powered study cohort to interrogate the epigenetic basis of neuropathological progression and clinical staging of LB disease, controlling for levels of concomitant AD pathology. We have completed bulk methylation analysis for two disease relevant brain regions and identified both phenotypic and neuropathologic changes within these regions. Processing of samples for fluorescence activated nuclei sorting has begun (n=15/group) to assess the cell type specificity of the methylation changes.

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STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF BRAIN DERIVED Α-SYNUCLEIN FIBRILS FROM PD AND MSA

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
04:00 PM - 04:15 PM

Abstract

Aims

Parkinson’s disease (PD) and multiple system atrophy (MSA) are characterized by the accumulation of filamentous α-synuclein in the brain. However, limited studies have scrutinized the brain derived α-synuclein fibrils and examined how these differ from the fibrils generated with seeding amplification assay like real-time quaking induced conversion (RT-QuIC). Here, we characterize the structural and biochemical properties of the human α-synuclein fibrils. Also, we compare the properties between the human and RT-QuIC amplified fibrils.

Methods

We extracted α-synuclein fibrils from the PD and MSA brains and in parallel generated RT-QuIC amplified fibrils. The morphology of the fibrils was observed using transmission electron microscopy (TEM) and immunogold TEM with antibodies targeting different regions of α-synuclein. Also, biochemical characterization was performed using slot-blot with the same set of antibodies. These observations were further examined with the clinical and neuropathological information.

Results

PD α-synuclein fibrils displayed two structurally distinct TEM morphologies, which were ‘straight’ and ‘twisted’. On the other hand, MSA α-synuclein fibrils were only composed of ‘straight’ fibrils. Furthermore, immunogold antibodies showed different labelling patterns with the α-synuclein fibrils, which provided structural information about the exposed and embedded regions of the fibrils. Also using the slot-blot, we were able to identify different α-synuclein species in the PD and MSA cases.

Conclusions

PD and MSA α-synuclein fibrils showed distinct structural and biochemical characteristics. This finding might provide a further insight about the mechanism on how the α-synuclein strains associate with different synucleinopathies and the causal link to the clinical and neuropathological variability, especially in PD.

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PRE-RECORDED: INTERPLAY BETWEEN NEUROINFLAMMATION AND AGGREGATE PROPAGATION

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
04:15 PM - 04:30 PM

Abstract

Abstract Body

Cell-to-cell propagation of α-synuclein has been thought to be the underlying mechanism of progression of Parkinson’s disease. Recent evidence suggests that inflammation plays an important role in propagation of protein aggregates. However, the mechanism by which inflammation regulates aggregate propagation remains unknown. Here, we show that one of the pro-inflammatory cytokines, TNFα, promotes α-synuclein propagation through activating neuronal senescence. In an in vitro culture, factors secreted from activated microglia promotes cell-to-cell propagation of α-synuclein. Production of the propagation stimulator depended on AP-1 transcription factor. AP-1 target inflammatory factors were screened for propagation stimulating activity. We have identified both stimulatory and inhibitory factors, among which TNFα showed the most robust stimulatory activity. Transcriptome analysis in neurons exposed to TNFα showed that TNFα triggered cellular senescence, as well as immune responses and apoptotic processes. Experimentally, neurons showed senescent phenotypes upon exposure to TNFα. Interestingly, secretion of α-synuclein was increased in senescent neurons through the senescence associated secretory phenotype (SASP). Using vacuolin, an inhibitor of lysosomal exocytosis, and RNAi against rab27a, we demonstrated that the SASP was mediated by lysosomal exocytosis. Combined light and electron microscopy confirmed that propagating α-synuclein aggregates were present in electron-dense lysosome-like compartments. TNFα promoted the SASP through stimulating lysosomal exocytosis, thereby increasing the secretion of α-synuclein. Collectively, these results suggest that TNFα is one of the major inflammatory factors that drive cell-to-cell propagation of α-synuclein through stimulating SASP-mediated secretion of α-synuclein.

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PRE-RECORDED: APPLYING DR. HORNYKIEWICZ’S LEGACY: STUDYING THE HUMAN BRAIN WILL REVEAL SECRETS (AND VARIANTS) OF PARKINSON’S

Session Type
SYMPOSIUM
Date
Thu, 17.03.2022
Session Time
02:45 PM - 04:45 PM
Room
ONSITE: 114
Lecture Time
04:30 PM - 04:45 PM

Abstract

Abstract Body

The twin discoveries by Dr. Oleh Hornykiecz of nigrostriatal dopamine deficiency in Parkinson disease (PD) and the amelioration of symptoms by L-DOPA administration represent milestone events in the history of medicine. What did Dr. Hornykiewicz wish to pass on to younger scientists? One of his frequently communicated conviction was that a disease of the human brain should be studied in human brain. Here, a former student demonstrates two examples of applying that legacy to research on the functions of two PD-linked genes: PRKN and SNCA. By studying >100 human brains, we discovered a novel function for parkin as an antioxidant. Parkin neutralizes reactive oxygen species and sequesters reactive electrophilic species (including dopamine quinone) through the function of the primate-specific residue C95. The sequestration effect may also contribute to neuromelanin formation [Tokarew et al., 2021]. In parallel work on olfactory epithelia studied in autopsy material and murine skulls, we discovered high expression levels of alpha-synuclein in olfactory sensory neurons, including the formation of proteinase K-resistant oligomers in their axons. These observations led to the identification of two functions of alpha-synuclein, namely in mammalian olfaction and antimicrobial defence against select RNA viruses [Tomlinson et al., 2017]. Ongoing research efforts focus on further validation of parkin's antioxidant role during dopamine metabolism and the characterization of alpha-synuclein misprocessing in olfactory neurons downstream of microbial encounters in the nasal cavity. These are aimed at better differentiating the pathogenesis of recessive PD versus late-onset, hyposmia-associated PD. The advice of Dr. Hornykiewicz to prioritize studies of the human brain in PD research continues to guide our field.

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