Work Plan
Work package I: Protein misfolding
1 The role of protein aggregation in neurodegeneration
2 The role of protein degradation in neurodegeneration
Work package II: Cellular stress
3 The role of stress in neurodegeneration
4 Mechanisms of neuronal dysfunctional/death in neurodegeneration
1. Study on the role of alternative proteases and proteolytic systems in Huntington's disease.
Main applicant Nico Dantuma (P4) in collaboration with Patrik Brundin (P1).
WP 1.1, 1.2
There is a large body of evidence in favour of a role of the ubiquitin/proteasome system (UPS) in neurodegeneration. The UPS is typically unable to degrade aggregated proteins such as found in neurodegenerative disorders. We have developed cellular and animal models for functional analysis of the UPS. These models are based on the constitutive expression of green fluorescent protein (GFP) based reporter substrates that accumulate under conditions of dysfunctional UPS. Studies from our lab suggest that various stress conditions can compromise the functionality of the UPS. We hypothesize that neurons can induce alternative proteolytic systems which compensate the UPS in degradation of misfolded proteins and relieve the burden from the UPS. This can explain why impairment is observed in some but not all neurodegenerative disorders. In this project we will 1) compare the effect of acute versus chronic exposure of mutHtt in vivo using UPS reporter mice and 2) characterize in vitro and in vivo changes in the proteolytic machinery of cells that are induced by the presence of mutant huntingtin.
2. Assessing the role of novel Alzheimer's disease-associated protein UBQLN1 in protein aggregation and ubiquitin-proteasome system
Main applicant Hilkka Soininen/Mikko Hiltunen, (P7), collaborator Nico Dantuma (P4)
WP 1.1, 1.2
The current project has a multidisciplinary approach, in which expertise in cell imaging/microscopy, molecular/cell biology and biochemistry will be employed to investigate the functional role of the AD-associated protein UBQLN1 in protein aggregation and UPS under proteasomal inhibition and endoplasmic reticulum (ER) stress. Since proteasomal dysfunction and UPR have been shown to play a role in AD pathogenesis and since we have previously demonstrated that UPS-related UBQLN1 protein is genetically and functionally associated with AD, it is important to assess whether UBQLN1 affects protein aggregation when proteasomal degradation is compromised. Our findings may thus provide novel insights on the pathogenesis of AD and other neurodegenerative diseases. In addition, we are interested in whether UBQLN1 affects ER-associated degradation (ERAD) of proteins under ER stress.
3. The role of cytoplasmic dopamine in alpha-synuclein-related toxicity
Main applicant Deniz Kirik (P2), collaborator Poul Henning Jensen (P8)
WP 1.1, 2.3
Ageing and dopamine neuronal phenotype are fundamental risk factors with respect to the neuropathology associated with Parkinson's disease (PD). Although the precise mechanisms underlying the selective vulnerability of midbrain dopamine (DA) neurons is unknown, given these risk factors, it has been suggested that age-related impairments in the capacity of these cells to handle cytoplasmic dopamine may be a significant/triggering factor. Furthermore, there is now mounting evidence suggesting that toxicity of alpha-synuclein in dopaminergic neurons is mediated by direct interaction between cytoplasmic DA and alpha-synuclein. This interaction leads to formation of abnormal alpha-synuclein species, which may remain in the cell as soluble non-physiological forms (possibly oligomeres) and initiate a process leading to cellular dysfunction and neurodegeneration. In this project, we propose a carefully designed set of experiments to directly test the hypothesis that impairments in the vesicular uptake of DA leads to elevated DA levels (and its metabolic or oxidization end-products) thereby perpetuating a pathogenic interaction between alpha-synuclein and DA. Although the DA toxicity hypothesis has already been widely speculated upon within the field, direct evidence to support this view has not yet been presented in an in vivo setting. Thus, the objective of the current proposal is to prove or dispute the hypothesis that the interaction between DA and alpha-synuclein can facilitate neuronal dysfunction and neurodegeneration.
4. Role of oxidative stress and DNA damage in CAGn stability and neurodegeneration of Huntington's disease
Main applicant: Arne Klungland,(P11) in collaboration with Patrik Brundin (P1).
WP 2.3
Genomic instability at repeated sequences is a landmark for numerous diseases. These include sporadic and hereditary cancers as well as several neurodegenerative diseases linked to expanding trinucleotide sequences. Oxidative DNA damage has for long been associated with ageing and neurological disease; although mechanistic connections of oxidation to these phenotypes have remained elusive. In a collaborative study, we show that the age-dependent somatic mutation associated with Huntington's disease (HD) occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine (8-oxoG) DNA glycosylase. Somatic, age-dependent, triplet expansions present a molecular link between oxidative damage and toxicity in post-mitotic neurons through error-prone repair of single-strand breaks. This project aim to further describe the link between somatic CAGn instability and DNA repair, using HD transgenic mice combined with OGG1 and FEN1 deficiency. Thus, this project aims at addressing the role of oxidative stress in neurodegeneration and mechanisms of neuronal dysfunction.
5. Mutant huntingtin as a cure for Parkinson's disease
Main applicant Patrik Brundin (P1) in collaboration with Karina Fog, Lundbeck A/S (P9).
WP 2.4
Novel therapies that can protect neurons against degeneration are greatly needed. Typically, experimental strategies have targeted a single cellular pathway known to mediate death in neurons, but have then failed clinically. The failures of neuroprotective therapies indicate that one has to rethink this classical approach and seek novel means of inhibiting cell death. We hypothesise that the neuroprotective effect exerted by N-terminal fragments of mutant huntingtin can be utilized to protect against neurological disease. We think that it is not directly due to the poly-Q region, but instead the poly-Q stretch influences the tertiary structure of a 50 amino acid-long sequence located immediately after the poly-Q stretch and thereby alters interactions with other protein partners. We will test this hypothesis and explore the signalling pathways involved in mediating the neuroprotective effect.
6. Road blockade versus motor failure? Axonal defects in models of Parkinson's and Huntington's diseases
Main applicants: Patrik Brundin and Jia-Yi Li (P1) in collaboration with Poul Henning Jensen
(P8) and Deniz Kirik (P2)
WP 2.4
Neurons are highly polarized cells with long dendrites and axons. Most proteins/peptides required in axons and synapses are transported over long distances after being synthesized within the cell body. Alterations in protein transport due to defects in the transport machinery may lead to perturbed synaptic activity and impaired trophic signaling and then lead to neurodegeneration. Defects in axonal transport occur in neurodegenerative diseases including Parkinson's and Huntington's diseases (PD and HD). The exact nature of the defects, how they are initiated, and their temporal relationship to disease progression are poorly understood. Defects in axonal trafficking contribute to PD and HD initiation and progression, leading to deficits in energy metabolism due to disrupted mitochondrial trafficking, and to lack of synaptic vesicle precursors and axonal membranes at synapses. The project aims to focus on 1) determining whether and how transport in axons is impaired and 2) determining how the early components relate to vulnerable axonal trafficking in PD and HD so as to help to identify possible targets for therapeutic intervention.
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