Final Report Summary - MODIFYPD (Effects of post-translational modifications of alpha synuclein on dopaminergic neurodegeneration in a novel viral vector mediated in vivo model of Parkinson's disease)
Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting about 4 million people worldwide, a number expected to double by 2030. It has a significant impact on health resource utilisation by placing a high economic burden on the society. The economic costs of PD include both direct health care costs for drugs, hospitalisation and physician services and indirect costs for lost worker productivity. In addition, it significantly reduces health-related quality of life in patients. Importantly, its incidence increases steadily with age and seems to become higher in the future when considering the demographic development of the society.
PD is characterised by the selective loss of a neuronal population, the Substantia nigra (SN), which is located in the ventral midbrain. One of the main functions of this group of cells is to facilitate movements via the release of the neurotransmitter dopamine. When these dopaminergic neurons die, the typical symptoms of PD occur. These motor features include the so-called resting tremor, which are small muscle contractions and relaxations recognised as 'shaking', slowness of movements and stiffness.
Today, the exact mechanism by which the dopamine containing cells in the brain degenerate, is unknown. More importantly, there is no treatment option available, which can prevent or delay disease progression. In order to develop treatments that can stop the disease, mechanisms underlying the disease process need to be understood. There is evidence that a specific protein in the brain, named alpha-synuclein (alpha-syn), plays an important role in PD since this protein was found to accumulate in so-called Lewy bodies inside the cells affected from the disease process. More specifically, a pathogenic interaction between alpha-syn and dopamine as well as specific post-translational modifications of alpha-syn were suggested to contribute to the selective death of nigral neurons. The fellow's work focused on investigating the contribution of increased cytoplasmic dopamine levels to alpha-syn-induced neurodegeneration, as well as the consequences of increased Ser 129 phosphorylation of alpha-syn.
Previous studies indicate that a pathogenic interaction between alpha-syn and dopamine or its metabolites results in the accumulation of soluble toxic alpha-syn species, which appear to be the pathogenic species triggering the selective death of dopaminergic neurons. Although the dopamine toxicity hypothesis has been widely discussed within the scientific community, it has not yet been proven in an in vivo setting, which was one of the objectives of the fellowship. Towards testing the hypothesis, we employed the VMAT2 hypomorph mouse model, which is characterised by about 5 % expression of the vesicular monoamine transporter (VMAT) 2 resulting in elevated cytosolic dopamine levels. We used recombinant adeno-associated viral vectors (rAAV) to mediate robust and specific overexpression of human alpha-syn in the SN of these mice.
Eight weeks after injection, alpha-syn overexpression in the wild-type and heterozygous mice led to a 30-35 % decrease in the number of Dopamine transporter (DAT)-immunoreactive neurons in the injected SN compared to the intact side of the same animal. Importantly, in the homozygous VMAT2 hypomorph mice, nigral cell loss was significantly higher. As a next step, we analysed whether inhibition of dopamine synthesis could reverse alpha-syn-induced toxicity. For this purpose, we injected rAAV vectors encoding for both alpha-syn and a short hairpin Ribonucleic acid (RNA) to knock down tyrosine hydroxylase (shTH), which is the rate-limiting enzyme for dopamine synthesis. Indeed, co-expression of alpha-syn with shTH reduced the toxicity of alpha-syn in the homozygous hypomorph group and no significant difference was any longer present between the genotypes. This finding supported the hypothesis that impairments in vesicularisation of dopamine, which in turn would be expected to cause increased cytoplasmic breakdown, increased the vulnerability of these cells to alpha-syn mediated neurodegeneration.
The second aim of the project was to establish a causal link between neuropathology in PD and increased Ser 129 alpha-syn phosphorylation. The single phosphorylation of alpha-syn at Ser 129 was found to preferentially and consistently accumulate in Lewy bodies and thus can be hypothesised to trigger the formation of Lewy bodies. Previously, phosphorylation of alpha-syn at Ser 129 was difficult to study because kinases specifically and efficiently phosphorylating alpha-syn at Ser 129 were unknown. Recently, polo-like kinase (PLK) 2 and 3 were shown to efficiently phosphorylate alpha-syn at this residue in vitro and were therefore used in this in vivo study.
We overexpressed PLK2 and 3 in the rat SN by means of rAAV vectors, which lead to enhanced levels of alpha-syn specifically phosphorylated at Ser 129, whereas injection of kinase dead mutants of PLK2 and PLK3 as control were not capable of phosphorylating alpha-syn. Stereological estimation of TH-positive cell numbers in the SN as indicator of dopaminergic cell death revealed no differences between the active and mutant kinases suggesting that the single post-translational phosphorylation of endogenous alpha-syn at Ser 129 does not trigger neurodegeneration, at least in its own right. However, we looked beyond the neurotoxic properties and found alterations in dopamine handling in animals with elevated P(S129)-alpha-syn levels. Based on this finding, we decided to extend our project and study the physiological role of phosphorylated alpha-syn.
Taken together, we demonstrated that impaired dopamine handling contributes to and enhances alpha-syn mediated neurodegeneration. However, although elevated dopamine levels increased the susceptibility to alpha-syn toxicity in vivo, it is not the main contributing factor under 'normal' conditions, where dopamine levels are in the physiological range. In this case, different mechanisms most likely induce alpha-syn cell death. Amongst others, post-translational modifications of alpha-syn, in particular phosphorylation at Ser 129, was suggested to be critical for cell death in PD. We investigated this correlation and found that increased P(S129)-alpha-syn levels do not induce neurodegeneration per se. However, we discovered that enhanced levels of phosphorylated alpha-syn lead to alterations in dopamine handling, which might indirectly or in conjunction with other triggers contribute to alpha-syn pathology.
The studies performed during this fellowship significantly contributed to our understanding of the mechanisms underlying the selective neurodegeneration observed in PD. We uncovered that two of the factors, which were assumed to be critical in modulating cell death, are not directly involved in alpha-syn-induced toxicity in vivo.
PD is characterised by the selective loss of a neuronal population, the Substantia nigra (SN), which is located in the ventral midbrain. One of the main functions of this group of cells is to facilitate movements via the release of the neurotransmitter dopamine. When these dopaminergic neurons die, the typical symptoms of PD occur. These motor features include the so-called resting tremor, which are small muscle contractions and relaxations recognised as 'shaking', slowness of movements and stiffness.
Today, the exact mechanism by which the dopamine containing cells in the brain degenerate, is unknown. More importantly, there is no treatment option available, which can prevent or delay disease progression. In order to develop treatments that can stop the disease, mechanisms underlying the disease process need to be understood. There is evidence that a specific protein in the brain, named alpha-synuclein (alpha-syn), plays an important role in PD since this protein was found to accumulate in so-called Lewy bodies inside the cells affected from the disease process. More specifically, a pathogenic interaction between alpha-syn and dopamine as well as specific post-translational modifications of alpha-syn were suggested to contribute to the selective death of nigral neurons. The fellow's work focused on investigating the contribution of increased cytoplasmic dopamine levels to alpha-syn-induced neurodegeneration, as well as the consequences of increased Ser 129 phosphorylation of alpha-syn.
Previous studies indicate that a pathogenic interaction between alpha-syn and dopamine or its metabolites results in the accumulation of soluble toxic alpha-syn species, which appear to be the pathogenic species triggering the selective death of dopaminergic neurons. Although the dopamine toxicity hypothesis has been widely discussed within the scientific community, it has not yet been proven in an in vivo setting, which was one of the objectives of the fellowship. Towards testing the hypothesis, we employed the VMAT2 hypomorph mouse model, which is characterised by about 5 % expression of the vesicular monoamine transporter (VMAT) 2 resulting in elevated cytosolic dopamine levels. We used recombinant adeno-associated viral vectors (rAAV) to mediate robust and specific overexpression of human alpha-syn in the SN of these mice.
Eight weeks after injection, alpha-syn overexpression in the wild-type and heterozygous mice led to a 30-35 % decrease in the number of Dopamine transporter (DAT)-immunoreactive neurons in the injected SN compared to the intact side of the same animal. Importantly, in the homozygous VMAT2 hypomorph mice, nigral cell loss was significantly higher. As a next step, we analysed whether inhibition of dopamine synthesis could reverse alpha-syn-induced toxicity. For this purpose, we injected rAAV vectors encoding for both alpha-syn and a short hairpin Ribonucleic acid (RNA) to knock down tyrosine hydroxylase (shTH), which is the rate-limiting enzyme for dopamine synthesis. Indeed, co-expression of alpha-syn with shTH reduced the toxicity of alpha-syn in the homozygous hypomorph group and no significant difference was any longer present between the genotypes. This finding supported the hypothesis that impairments in vesicularisation of dopamine, which in turn would be expected to cause increased cytoplasmic breakdown, increased the vulnerability of these cells to alpha-syn mediated neurodegeneration.
The second aim of the project was to establish a causal link between neuropathology in PD and increased Ser 129 alpha-syn phosphorylation. The single phosphorylation of alpha-syn at Ser 129 was found to preferentially and consistently accumulate in Lewy bodies and thus can be hypothesised to trigger the formation of Lewy bodies. Previously, phosphorylation of alpha-syn at Ser 129 was difficult to study because kinases specifically and efficiently phosphorylating alpha-syn at Ser 129 were unknown. Recently, polo-like kinase (PLK) 2 and 3 were shown to efficiently phosphorylate alpha-syn at this residue in vitro and were therefore used in this in vivo study.
We overexpressed PLK2 and 3 in the rat SN by means of rAAV vectors, which lead to enhanced levels of alpha-syn specifically phosphorylated at Ser 129, whereas injection of kinase dead mutants of PLK2 and PLK3 as control were not capable of phosphorylating alpha-syn. Stereological estimation of TH-positive cell numbers in the SN as indicator of dopaminergic cell death revealed no differences between the active and mutant kinases suggesting that the single post-translational phosphorylation of endogenous alpha-syn at Ser 129 does not trigger neurodegeneration, at least in its own right. However, we looked beyond the neurotoxic properties and found alterations in dopamine handling in animals with elevated P(S129)-alpha-syn levels. Based on this finding, we decided to extend our project and study the physiological role of phosphorylated alpha-syn.
Taken together, we demonstrated that impaired dopamine handling contributes to and enhances alpha-syn mediated neurodegeneration. However, although elevated dopamine levels increased the susceptibility to alpha-syn toxicity in vivo, it is not the main contributing factor under 'normal' conditions, where dopamine levels are in the physiological range. In this case, different mechanisms most likely induce alpha-syn cell death. Amongst others, post-translational modifications of alpha-syn, in particular phosphorylation at Ser 129, was suggested to be critical for cell death in PD. We investigated this correlation and found that increased P(S129)-alpha-syn levels do not induce neurodegeneration per se. However, we discovered that enhanced levels of phosphorylated alpha-syn lead to alterations in dopamine handling, which might indirectly or in conjunction with other triggers contribute to alpha-syn pathology.
The studies performed during this fellowship significantly contributed to our understanding of the mechanisms underlying the selective neurodegeneration observed in PD. We uncovered that two of the factors, which were assumed to be critical in modulating cell death, are not directly involved in alpha-syn-induced toxicity in vivo.