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The impact of MHCII expression on α-synuclein toxicity in the rat brain

Tontanahal, Ashmita (2016) MOBT01 20161
Degree Projects in Molecular Biology
Abstract
Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by a progressive loss of dopaminergic neurons (DNs) in substantia nigra pars compact and accumulation of aggregated forms of the protein α-synuclein known as Lewy Bodies (LBs). LBs are present in surviving DNs and other regions of the central nervous system (CNS) in PD patients. PD patients also display local CNS inflammation, with T cell infiltration, gliosis and major histocompatibility complex class II (MHCII) expression. In previous studies, it was shown that the Vra4 locus regulates MHCII expression in rats and Vra4 locus was fine-mapped to the MHCII transactivator gene (MHC2ta). Polymorphism in the promotor region of MHC2ta was... (More)
Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by a progressive loss of dopaminergic neurons (DNs) in substantia nigra pars compact and accumulation of aggregated forms of the protein α-synuclein known as Lewy Bodies (LBs). LBs are present in surviving DNs and other regions of the central nervous system (CNS) in PD patients. PD patients also display local CNS inflammation, with T cell infiltration, gliosis and major histocompatibility complex class II (MHCII) expression. In previous studies, it was shown that the Vra4 locus regulates MHCII expression in rats and Vra4 locus was fine-mapped to the MHCII transactivator gene (MHC2ta). Polymorphism in the promotor region of MHC2ta was subsequently shown to modulate susceptibility to experimental neuroinflammation and, in humans, to be associated with inflammatory diseases. This project evaluates the effects of the Vra4 locus/MHCII on α-syn toxicity and dopaminergic neurodegeneration. For this, we have overexpressed human α-syn in two different strains of rats, Dark Agouti (DA) and DA-Vra4 congenic, expressing different levels of MHCII due to different MHC2ta alleles. The DA-Vra4 congenic strain is identical to the DA parental strain except for having Piebald Virol Glaxo alleles in the Vra4 locus on chromosome 10. This confers lower MHCII expression levels compared to the DA parental rats. In order to study the effects on toxicity, a recombinant adeno-associated virus (rAAV) carrying the human a-syn gene was injected to the SN, to determine the effects on behaviour, a rAAV was injected into the SN, in combination with an injection in the striatum of preformed fibrils (PFFs) synthesized from recombinant human α-syn. The study revealed a significant loss in Tyrosine hydroxylase (TH) positive fibres in the dorsal striatum which correlated to the loss of DNs in the SN. Preliminary data also showed that the administration of α-syn in the SN and PFFs in the striatum gave rise to ~50 % reduction in relative use of contralateral paw in the behavioural experiments. (Less)
Popular Abstract
Parkinson’s disease (PD) is the second most common neurodegenerative disorder. This means that the brain slowly loses its ability to produce a neurotransmitter dopamine, which is involved in motor control pathways. PD is characterized by a progressive loss of dopaminergic neurons (DNs) in substantia nigra (SN) and accumulation of aggregated forms of α-synuclein (α-syn) into structures known as Lewy Bodies. α-syn is an abundant human brain protein, localised in the presynaptic terminals. These are distal terminals of the neurons which are specialized for neurotransmitter release.

The loss of DNs in SN leads to loss of motor control due to lack of dopamine synthesis. Thus giving rise to motor symptoms such as tremor (shaking), impaired... (More)
Parkinson’s disease (PD) is the second most common neurodegenerative disorder. This means that the brain slowly loses its ability to produce a neurotransmitter dopamine, which is involved in motor control pathways. PD is characterized by a progressive loss of dopaminergic neurons (DNs) in substantia nigra (SN) and accumulation of aggregated forms of α-synuclein (α-syn) into structures known as Lewy Bodies. α-syn is an abundant human brain protein, localised in the presynaptic terminals. These are distal terminals of the neurons which are specialized for neurotransmitter release.

The loss of DNs in SN leads to loss of motor control due to lack of dopamine synthesis. Thus giving rise to motor symptoms such as tremor (shaking), impaired balance (postural stability), bradykinesia (slow motion) and muscle rigidity (stiffness). While PD patients also develop non-motor symptoms such as fatigue (loss of energy), dementia (loss of memory), constipation (difficulty in emptying bowel), dysphagia (swallowing difficulties), depression (mood disorder), sleeping problems to name a few. PD is also thought to be coupled with an inflammatory response as patients display local CNS inflammation, T cell infiltration, gliosis (a response to CNS damage resulting in nonspecific reactive change of local cells) and expression of the inflammatory protein major histocompatibility complex class II (MHCII) expression. In previous studies, it was shown that the Vra4 locus regulates MHCII expression in rats and Vra4 was fine-mapped to the MHCII transactivator gene. This gene was subsequently shown to modulate susceptibility to experimental neuroinflammation and, in humans, it is associated with inflammatory diseases. In this project we wanted to study the effects of differential expression of MHCII molecules on α-syn toxicity in the rat brain. The experimental animals employed in this study, were the parental Dark Agouti (DA) and a congenic DA-Vra4 rat strains. The experimental animals are identical and only differ in the Vra4 locus on chromosome 10, which in DA-Vra4 rats is exchanged from the original DNA sequence found in DA to that of the obtained from Piebald Virol Glaxo (PVG) strain. This confers lower MHCII expression in DA-Vra4compared to the DA parental rats. The experiments were designed in such a way that the effect of MHCII expression on neurodegeneration (loss of neurons) and behavioural impairment could be analysed.

In the study dealing with the effects of MHCII expression on neurodegeneration, the rats received unilateral (single site) injections of adeno-associated recombinant virus (rAAV) coupled with human α-syn or green fluorescent protein (GFP) in the SN. The experimental animals were observed and sacrificed 12 weeks after intanigral injection and samples were used to determine the loss of dopaminergic cell bodies in the SN and the loss of dopaminergic fibers from these cells in the striatum.

To study the effects of MHCII expression on behavioural impairment received an intranigral injection of rAAV coupled α-syn or GFP and an intrastriatal injection of bovine serum albumin (BSA) or pre formed α-syn fibrils (PFFs) two weeks later. PFFs were used to determine the impact of MHCII on the spread of α-syn toxicity and neuropathology. The behaviour of the rats was assessed at different time-points (0, 3 and 6 weeks) during the course of the experiment and the rats were sacrificed 6 weeks after the first surgery.

The experiments revealed a stronger denervation (loss of dopaminergic fibres in the striatum) in DA-Vra4 rats in comparison to DA rats after overexpression of α-syn in SN. A significant loss of behaviour was not observed over time in animals treated with a combination of α-syn overexpression and injection of α-syn fibrils. We expect to see an exaggerated response, due to an increased α-syn aggregation as result of a seeding affect induced by PFFs. In the future experiments, we aim at studying the effects of PFFs administration on denervation and neurodegeneration

Advisor: Maria Swanberg and Itzia Jimenez
Master’s Degree Project in Molecular Biology and Medical Biology, 60 credits, 2016
Department of Biology, Lund University
Advisors Unit/Department: Neurodegeneration and Inflammation genetics, Department of Experimental Medical Sciences, BioMedical Center, Lund University (Less)
Please use this url to cite or link to this publication:
author
Tontanahal, Ashmita
supervisor
organization
course
MOBT01 20161
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8891693
date added to LUP
2016-09-14 11:17:21
date last changed
2016-09-14 11:17:21
@misc{8891693,
  abstract     = {Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by a progressive loss of dopaminergic neurons (DNs) in substantia nigra pars compact and accumulation of aggregated forms of the protein α-synuclein known as Lewy Bodies (LBs). LBs are present in surviving DNs and other regions of the central nervous system (CNS) in PD patients. PD patients also display local CNS inflammation, with T cell infiltration, gliosis and major histocompatibility complex class II (MHCII) expression. In previous studies, it was shown that the Vra4 locus regulates MHCII expression in rats and Vra4 locus was fine-mapped to the MHCII transactivator gene (MHC2ta). Polymorphism in the promotor region of MHC2ta was subsequently shown to modulate susceptibility to experimental neuroinflammation and, in humans, to be associated with inflammatory diseases. This project evaluates the effects of the Vra4 locus/MHCII on α-syn toxicity and dopaminergic neurodegeneration. For this, we have overexpressed human α-syn in two different strains of rats, Dark Agouti (DA) and DA-Vra4 congenic, expressing different levels of MHCII due to different MHC2ta alleles. The DA-Vra4 congenic strain is identical to the DA parental strain except for having Piebald Virol Glaxo alleles in the Vra4 locus on chromosome 10. This confers lower MHCII expression levels compared to the DA parental rats. In order to study the effects on toxicity, a recombinant adeno-associated virus (rAAV) carrying the human a-syn gene was injected to the SN, to determine the effects on behaviour, a rAAV was injected into the SN, in combination with an injection in the striatum of preformed fibrils (PFFs) synthesized from recombinant human α-syn. The study revealed a significant loss in Tyrosine hydroxylase (TH) positive fibres in the dorsal striatum which correlated to the loss of DNs in the SN. Preliminary data also showed that the administration of α-syn in the SN and PFFs in the striatum gave rise to ~50 % reduction in relative use of contralateral paw in the behavioural experiments.},
  author       = {Tontanahal, Ashmita},
  language     = {eng},
  note         = {Student Paper},
  title        = {The impact of MHCII expression on α-synuclein toxicity in the rat brain},
  year         = {2016},
}