- Email: [email protected]
2.305 The mechanism of alpha-synuclein fibril formation studied with high resolution Atomic Force Microscopy
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this study was to assess tau-protein, beta-amyloid (1-42) and cystatin C CSF levels in PD patients and in the control group (CG) and to compare CSF levels between these two groups and correlate to the both PD duration and severity of motor impairment. Method: Neurodegenerative markers in CSF were assessed in 32 patients with PD (23 males, 9 females, aged 37−73, mean 59.9±11.96 years), and CG of 20 patients (11 males, 9 females, aged 35−75, mean 51.2±10.74 years). Hoehn–Yahr scale was used for the assessment of the severity of motor impairment (H&Y). Kruskal-Wallis test and ANOVA were used when assessing statistical significance of the results. Results: The following statistically significant differences in the CSF were found: higher tau-protein levels in PD patients versus CG (p = 0.05), higher tau-protein levels (p = 0.03) and tau-proteinbeta-amlyoid (1-42) ratio (p = 0.04) in PD patients with duration less than 2 years vs. PD with duration more than 2 years. No significant correlation was found between the tau-protein CSF levels and the H&Y of PD. No difference in levels of beta-amyloid (1-42) and cystatin C in CSF was found in the CG and PD patients groups. Conclusion: Tau-protein CSF levels were higher in the group of PD patients with disease duration less than 2 years. It can be assumed that the maximum of neurodegenerative changes in PD is happening in the first two years after the disease onset.
2.302 Pattern of alpha-synuclein and phosphorylated tau pathology in the olfactory bulb, brainstem and limbic regions in aged individuals W. van de Berg1° , S. Zweekhorst, P. Voorn, H. Groenewegen, P. Hoogland, A.M. Rozemuller 1 Amsterdam, Netherlands Objective: To test the hypothesis that pathology in the olfactory bulb is related to pathology in the brainstem and limbic regions in non-demented individuals above the age of 45 years. Method: Brain tissue from aged, neurologically unimpaired individuals (n = 76), Parkinson (PD: n = 12) and Alzheimer (AD: n = 13) patients were obtained from the Netherlands Brain Bank. Paraffin sections of the olfactory bulb, brainstem and limbic regions were cut and stained for alpha-synuclein and hyperphosphorylated tau using immunohistochemistry. Lewy body pathology and tangle pathology was scored semi-quantitatively on sections collected throughout the regions. Braak staging was evaluated for all cases. Results: Twenty percent (17/76) of the aged individuals had alphasynuclein pathology and 87% (31/34) had tau pathology in the olfactory bulb. All cases with alpha-synuclein pathology in the dorsal motor nucleus of the vagal nerve exhibited alpha-synuclein pathology in the olfactory bulb. Few aged individuals had alpha-synuclein and tau pathology in the olfactory bulb, amygdala and entorhinal cortex without involvement of brainstem regions (8/17). In almost all aged individuals, the substantia nigra pars compacta was intact and did not reveal any Lewy bodies or neurites. All PD cases revealed alpha-synuclein pathology in the olfactory bulb, brainstem and limbic regions (12/12). All AD cases revealed tau pathology in olfactory bulb and limbic regions. Few AD cases revealed alpha-synuclein pathology in brainstem regions (4/13). Conclusion: These data (1) largely confirm Braak’s staging of alphasynuclein pathology in the brainstem and olfactory bulb; (2) suggest that olfactory and limbic alpha-synuclein pathology can co-occur without brainstem pathology; (3) illustrate the high incidence of alpha-synuclein and tau pathology in the olfactory bulb of aged individuals. Non-random samplings might introduce a selection bias. Therefore our numbers may not reflect true population estimates.
2.303 Lipoxidized, nitrated and phosphorylated alphasynuclein in amygdala-predominant Lewy body disease A. Mart´ınez1° 1 Spain
Objective: Dementia with Lewy bodies (DLB) is a degenerative disease manifested by dementia and parkinsonism, and characterised by the widespread distribution in the brainstem, amygdala, diencephalon and cerebral cortex of Lewy bodies and aberrant neurites composed of protein aggregates, mainly alpha-synuclein. Method: DLB is usually accompanied by Alzheimer’s disease pathology including beta-amyloid plaques and neurofibrillary tangles. In addition, many Alzheimer’s disease cases showed a predominance of Lewy bodies in the amygdala whereas other regions are minimally affected. Such cases have been considered an atypical form of alpha-synucleinopathy (AD with amygdala-predominant Lewy body disease). Results: The present study compares morphological and biochemical characteristics of alpha-synuclein in the amygdala in DLB and AD with amygdala-predominant LBD. Biochemical studies have shown abnormal solubility and aggregation of alpha-synuclein, alpha-synuclein phosphorylation, nitration/oxidation and lipoxidation in DLB and AD with amygdalapredominant LBD. Immunofluorescence and confocal microscopy disclosed similar morphology of Lewy bodies and neurites in both conditions, although large numbers of alpha-synuclein immunoreactive neurites were more common in AD with amygdala-predominant LBD than in DLB. Conclusion: These findings show that the amygdala is vulnerable to similar alpha-synuclein post-traslational modifications in LBD and amygdalapredominant LBD. 2.304 Neuropathology of conditional alpha-synuclein transgenic mouse models of Parkinson’s disease S. Nuber1° , E. Petrasch-Parwez, B. Winner, J. Winkler, S. von H¨orsten, P. Teismann, J.B. Schulz, M. Neumann, M. Fendt, F.N. Gellerich, O. Riess 1 Tuebingen, Germany Alpha-synuclein has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson’s disease (PD). PD is based on progressive neuropathological alterations leading to motor abnormalities that are frequently predated by olfactory dysfunction and often accompanied by cognitive decline in later stages of the disease. Whether the neurodegenerative process might be halted or even reversed is presently unknown. In order to explore whether alpha-synuclein induces these alterations we generated conditional mouse models by using the tet-regulatable system. Mice expressing high levels of human wildtype alpha-synuclein in several brain regions developed nigral and hippocampal neuropathology, including reduced neurogenesis and neurodegeneration, leading to progressive motor decline and impaired long-term memory. Turning off transgene expression in aged mice halted progression of motor symptoms but did not reverse the symptoms. Mice expressing the mutated (A30P) alpha-synuclein limited to the olfactory bulb showed a reduction of monoamines in this region. In a conducted TMT-smell test these mice also revealed an impaired anxiety reaction and an increased exploratory behavior; the latter might be a depressive core symptom. Our data suggest that approaches targeting a-syn induced pathological pathways might be of benefit in early disease stages. 2.305 The mechanism of alpha-synuclein fibril formation studied with high resolution Atomic Force Microscopy I. Segers-Nolten1° , G. Veldhuis, K. van der Werf, M. van Raaij, V. Subramaniam 1 Enschede, Netherlands Objective: In the brains of Parkinson’s patients high concentrations of fibrillar aggregates of alpha-synuclein protein are found. Although a
Poster Presentations: Alpha-synuclein and Lewy Bodies relation between the aggregation of alpha-synuclein and development of the disease has been proposed, the underlying aggregation mechanism is not yet known. We have employed high resolution atomic force microscopy (AFM) to get more insight in the fibril assembly mechanism. Detailed knowledge on the mechanism of fibril assembly, possibly a process fundamental to many protein misfolding diseases, is important in view of the development of means for therapeutic intervention. Method: We have performed tapping mode AFM height and phase imaging in air on fibrils formed in vitro by the recombinantly produced wild-type alpha-synuclein protein, and by the familial disease-related A30P, E46K and A53T variants. The height images were analyzed quantitatively for fibril height, modulation depth and periodicity. From phase images additional morphological information was obtained. To study the mechanical properties of fibrils we applied force during scanning in contact mode. Results: The fibril heights measured for wild-type, A30P, E46K and A53T alpha-synuclein were respectively: 7.5, 8.7, 9.8 and 10.4 nm. The E46K mutant displayed a more distinct and smaller periodicity than the other variants. The modulation depth for all mutants was very similar, but was smaller for wild-type protein commensurate with the lower fibril height. The obtained structural data are compatible with a twisted hierarchical assembly model. The detailed morphology observed in phase images, however, indicates that fibrils may also be formed through the association of fibril segments. The application of force resulted in characteristic deformation of protein fibrils with a periodicity corresponding to the modulation observed in tapping mode. Conclusion: Our observations suggest that alpha-synuclein fibril assembly may not exclusively occur according to the hierarchical assembly model, but multiple modes of fibril assembly may play a role, including segmented assembly.
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2.307 p25a induces alpha-synuclein dependent cell death in a cellular model for neurodegenerative disorders C. Petersen1° C, Denmark
1 Aarhus
Objective: The neurodegenerative disorders, Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) belong to the group of a-synucleinopathies. Their unifying hallmark is the presence of intracellular inclusions containing aggregates of a-synuclein (Asyn). The inclusions comprise neuronal Lewy bodies in PD and DLB and glial cytoplasmic inclusions in MSA. The mechanims underlying Asyn aggregation are not clear but are likely to involve an altered expression of pro-aggregatory factors. We have identified the brain-specific protein, p25a, as an accelerator of Asyn aggregation suggesting a role for p25a in the neurodegenerative process. The cellular expression of p25a is abnormal in degenerative neurons and oligodendrocytes in PD and MSA where it co-localizes with Asyn in Lewy bodies and glial cytoplasmic inclusions. Method: Here we have investigated the role of p25a on cellular toxicity in an oligodendrocyte cell line stably expressing human wild-type Asyn. Results: These cells displays cellular degeneration when transiently transfected with p25a as demonstrated by shrinkage of the cytosol, perinuclear localization of the microtubule, and nuclear condensation. Cell death is partially blocked by anti-aggregatory compounds. Furthermore, the p25a mediated cell death is significantly reduced by certain caspase inhibitors. Conclusion: Our results indicate that the p25a-mediated Asyndependent cell death is caused by the proaggregatory activity of p25a. Furthermore, a specific caspase cascade seems to be activated in the apoptotic process. Based on our results, we suggest that p25a contributes to the cytotoxity underlying several a-synucleinopathies.
2.306 A yeast-based screening system for mouse brain proteins that modify the toxicity of alpha-synuclein S. de Graeve1° , S. Marinelli, P. Van Dijck, J. Thevelein, F. Stolz Belgium
2.308 Characterization of the expression of alpha-synuclein and suppressor/enhancer proteins in Saccharomyces cerevisiae
Objective: a-Synuclein (a-SYN) is the major building block of the filaments that form the proteinacious inclusions that are characteristic for many disorders, including Lewy bodies and Lewy neurites in Parkinson’s disease (PD). Recently, it has been shown that, when expressed in the yeast Saccharomyces cerevisiae, a-SYN causes a growth defect, especially in strains where a non-essential gene involved in vesicle-mediated transport was deleted. Our objective was to develop a yeast-based screening system to discover mouse brain cDNA’s that counteract this toxic effect of a-SYN. This system was then used to identify novel interaction partners of a-SYN. Method: We have cloned the human WT a-SYN gene behind the galactose-inducible promoter of the yeast GAL1 gene and integrated two copies into the genome of the cog6D yeast mutant strain, which caused a severe growth defect on galactose-containing medium. After transformation with a mouse brain cDNA-library, the cells were plated out on galactosecontaining medium to check for cells that contain possible inhibitors of a-SYN toxicity. From the growing yeast colonies cDNA plasmids were isolated and retransformed into the yeast strain to confirm their potential to suppress the a-SYN toxicity effect. Results: We have identified two mouse brain proteins that suppress toxicity of a-SYN in the yeast system. In addition we have isolated a protein that surprisingly enhanced the toxicity of a-SYN, while it had no effect on growth of the strain without a-SYN. At present we are performing coimmunoprecipitation experiments to address whether these proteins work through a direct interaction with the a-SYN protein or in an indirect way. Conclusion: Yeast cells can be used as a tool to screen for brain proteins that modify the toxic effect of the PD-related protein a-SYN. Our method has led to the identification of two suppressors and one enhancer of a-SYN toxicity in yeast.
S. Marinelli1° , S. de Graeve, J. Vandamme, Y. Engelborghs, P. Van Dijck, J. Thevelein, F. Stolz 1 Heverlee, Belgium
1 Leuven,
Objective: Parkinson’s disease (PD) is a common neuronal disorder that is hallmarked by the loss of dopaminergic neurons and the presence of Lewy Body inclusions, which contain alpha-synuclein (aSYN) as a major component. A yeast model was established to screen for proteins that can modify the toxic phenotype of aSYN expression. Two suppressors (S1, S2) and one enhancer protein (E1) were identified and confirmed. The goal of this study was to characterize their effect on aSYN localization and on aSYN induced accumulation of lipid droplets in aSYN sensitive yeast mutants. Method: A fusion construct of aSYN wild type and green fluorescent protein (GFP) was synthesized and transformed into several yeast strains (BY4742, cog6D). The localization of aSYN-GFP was studied in the absence and in the presence of the confirmed suppressor/enhancer proteins. In addition cells co-expressing aSYN and a suppressor/enhancer protein were stained with Nile Red and a confocal microscopy experiment was performed to study the effect of expression on the accumulation of lipid droplets in several strains. Results: Three stages of aSYN localization were observed depending in time: plasma membrane localization, an intermediate stage and an aggregated stage. Expression of the suppressor proteins delays the aggregation process in yeast. Co-expression of suppressor S1 and aSYN or of enhancer E1 and aSYN causes a high increase of lipid droplets in comparison with cells that only express aSYN. In contrast cells co-expressing suppressor S2 and aSYN accumulate less lipid droplets. Conclusion: Expression of a suppressor protein (S1 or S2) causes a delay in the aSYN aggregation process. Suppressor S1 and S2 are able
Tuesday, 11 December 2007
this study was to assess tau-protein, beta-amyloid (1-42) and cystatin C CSF levels in PD patients and in the control group (CG) and to compare CSF levels between these two groups and correlate to the both PD duration and severity of motor impairment. Method: Neurodegenerative markers in CSF were assessed in 32 patients with PD (23 males, 9 females, aged 37−73, mean 59.9±11.96 years), and CG of 20 patients (11 males, 9 females, aged 35−75, mean 51.2±10.74 years). Hoehn–Yahr scale was used for the assessment of the severity of motor impairment (H&Y). Kruskal-Wallis test and ANOVA were used when assessing statistical significance of the results. Results: The following statistically significant differences in the CSF were found: higher tau-protein levels in PD patients versus CG (p = 0.05), higher tau-protein levels (p = 0.03) and tau-proteinbeta-amlyoid (1-42) ratio (p = 0.04) in PD patients with duration less than 2 years vs. PD with duration more than 2 years. No significant correlation was found between the tau-protein CSF levels and the H&Y of PD. No difference in levels of beta-amyloid (1-42) and cystatin C in CSF was found in the CG and PD patients groups. Conclusion: Tau-protein CSF levels were higher in the group of PD patients with disease duration less than 2 years. It can be assumed that the maximum of neurodegenerative changes in PD is happening in the first two years after the disease onset.
2.302 Pattern of alpha-synuclein and phosphorylated tau pathology in the olfactory bulb, brainstem and limbic regions in aged individuals W. van de Berg1° , S. Zweekhorst, P. Voorn, H. Groenewegen, P. Hoogland, A.M. Rozemuller 1 Amsterdam, Netherlands Objective: To test the hypothesis that pathology in the olfactory bulb is related to pathology in the brainstem and limbic regions in non-demented individuals above the age of 45 years. Method: Brain tissue from aged, neurologically unimpaired individuals (n = 76), Parkinson (PD: n = 12) and Alzheimer (AD: n = 13) patients were obtained from the Netherlands Brain Bank. Paraffin sections of the olfactory bulb, brainstem and limbic regions were cut and stained for alpha-synuclein and hyperphosphorylated tau using immunohistochemistry. Lewy body pathology and tangle pathology was scored semi-quantitatively on sections collected throughout the regions. Braak staging was evaluated for all cases. Results: Twenty percent (17/76) of the aged individuals had alphasynuclein pathology and 87% (31/34) had tau pathology in the olfactory bulb. All cases with alpha-synuclein pathology in the dorsal motor nucleus of the vagal nerve exhibited alpha-synuclein pathology in the olfactory bulb. Few aged individuals had alpha-synuclein and tau pathology in the olfactory bulb, amygdala and entorhinal cortex without involvement of brainstem regions (8/17). In almost all aged individuals, the substantia nigra pars compacta was intact and did not reveal any Lewy bodies or neurites. All PD cases revealed alpha-synuclein pathology in the olfactory bulb, brainstem and limbic regions (12/12). All AD cases revealed tau pathology in olfactory bulb and limbic regions. Few AD cases revealed alpha-synuclein pathology in brainstem regions (4/13). Conclusion: These data (1) largely confirm Braak’s staging of alphasynuclein pathology in the brainstem and olfactory bulb; (2) suggest that olfactory and limbic alpha-synuclein pathology can co-occur without brainstem pathology; (3) illustrate the high incidence of alpha-synuclein and tau pathology in the olfactory bulb of aged individuals. Non-random samplings might introduce a selection bias. Therefore our numbers may not reflect true population estimates.
2.303 Lipoxidized, nitrated and phosphorylated alphasynuclein in amygdala-predominant Lewy body disease A. Mart´ınez1° 1 Spain
Objective: Dementia with Lewy bodies (DLB) is a degenerative disease manifested by dementia and parkinsonism, and characterised by the widespread distribution in the brainstem, amygdala, diencephalon and cerebral cortex of Lewy bodies and aberrant neurites composed of protein aggregates, mainly alpha-synuclein. Method: DLB is usually accompanied by Alzheimer’s disease pathology including beta-amyloid plaques and neurofibrillary tangles. In addition, many Alzheimer’s disease cases showed a predominance of Lewy bodies in the amygdala whereas other regions are minimally affected. Such cases have been considered an atypical form of alpha-synucleinopathy (AD with amygdala-predominant Lewy body disease). Results: The present study compares morphological and biochemical characteristics of alpha-synuclein in the amygdala in DLB and AD with amygdala-predominant LBD. Biochemical studies have shown abnormal solubility and aggregation of alpha-synuclein, alpha-synuclein phosphorylation, nitration/oxidation and lipoxidation in DLB and AD with amygdalapredominant LBD. Immunofluorescence and confocal microscopy disclosed similar morphology of Lewy bodies and neurites in both conditions, although large numbers of alpha-synuclein immunoreactive neurites were more common in AD with amygdala-predominant LBD than in DLB. Conclusion: These findings show that the amygdala is vulnerable to similar alpha-synuclein post-traslational modifications in LBD and amygdalapredominant LBD. 2.304 Neuropathology of conditional alpha-synuclein transgenic mouse models of Parkinson’s disease S. Nuber1° , E. Petrasch-Parwez, B. Winner, J. Winkler, S. von H¨orsten, P. Teismann, J.B. Schulz, M. Neumann, M. Fendt, F.N. Gellerich, O. Riess 1 Tuebingen, Germany Alpha-synuclein has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson’s disease (PD). PD is based on progressive neuropathological alterations leading to motor abnormalities that are frequently predated by olfactory dysfunction and often accompanied by cognitive decline in later stages of the disease. Whether the neurodegenerative process might be halted or even reversed is presently unknown. In order to explore whether alpha-synuclein induces these alterations we generated conditional mouse models by using the tet-regulatable system. Mice expressing high levels of human wildtype alpha-synuclein in several brain regions developed nigral and hippocampal neuropathology, including reduced neurogenesis and neurodegeneration, leading to progressive motor decline and impaired long-term memory. Turning off transgene expression in aged mice halted progression of motor symptoms but did not reverse the symptoms. Mice expressing the mutated (A30P) alpha-synuclein limited to the olfactory bulb showed a reduction of monoamines in this region. In a conducted TMT-smell test these mice also revealed an impaired anxiety reaction and an increased exploratory behavior; the latter might be a depressive core symptom. Our data suggest that approaches targeting a-syn induced pathological pathways might be of benefit in early disease stages. 2.305 The mechanism of alpha-synuclein fibril formation studied with high resolution Atomic Force Microscopy I. Segers-Nolten1° , G. Veldhuis, K. van der Werf, M. van Raaij, V. Subramaniam 1 Enschede, Netherlands Objective: In the brains of Parkinson’s patients high concentrations of fibrillar aggregates of alpha-synuclein protein are found. Although a
Poster Presentations: Alpha-synuclein and Lewy Bodies relation between the aggregation of alpha-synuclein and development of the disease has been proposed, the underlying aggregation mechanism is not yet known. We have employed high resolution atomic force microscopy (AFM) to get more insight in the fibril assembly mechanism. Detailed knowledge on the mechanism of fibril assembly, possibly a process fundamental to many protein misfolding diseases, is important in view of the development of means for therapeutic intervention. Method: We have performed tapping mode AFM height and phase imaging in air on fibrils formed in vitro by the recombinantly produced wild-type alpha-synuclein protein, and by the familial disease-related A30P, E46K and A53T variants. The height images were analyzed quantitatively for fibril height, modulation depth and periodicity. From phase images additional morphological information was obtained. To study the mechanical properties of fibrils we applied force during scanning in contact mode. Results: The fibril heights measured for wild-type, A30P, E46K and A53T alpha-synuclein were respectively: 7.5, 8.7, 9.8 and 10.4 nm. The E46K mutant displayed a more distinct and smaller periodicity than the other variants. The modulation depth for all mutants was very similar, but was smaller for wild-type protein commensurate with the lower fibril height. The obtained structural data are compatible with a twisted hierarchical assembly model. The detailed morphology observed in phase images, however, indicates that fibrils may also be formed through the association of fibril segments. The application of force resulted in characteristic deformation of protein fibrils with a periodicity corresponding to the modulation observed in tapping mode. Conclusion: Our observations suggest that alpha-synuclein fibril assembly may not exclusively occur according to the hierarchical assembly model, but multiple modes of fibril assembly may play a role, including segmented assembly.
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2.307 p25a induces alpha-synuclein dependent cell death in a cellular model for neurodegenerative disorders C. Petersen1° C, Denmark
1 Aarhus
Objective: The neurodegenerative disorders, Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) belong to the group of a-synucleinopathies. Their unifying hallmark is the presence of intracellular inclusions containing aggregates of a-synuclein (Asyn). The inclusions comprise neuronal Lewy bodies in PD and DLB and glial cytoplasmic inclusions in MSA. The mechanims underlying Asyn aggregation are not clear but are likely to involve an altered expression of pro-aggregatory factors. We have identified the brain-specific protein, p25a, as an accelerator of Asyn aggregation suggesting a role for p25a in the neurodegenerative process. The cellular expression of p25a is abnormal in degenerative neurons and oligodendrocytes in PD and MSA where it co-localizes with Asyn in Lewy bodies and glial cytoplasmic inclusions. Method: Here we have investigated the role of p25a on cellular toxicity in an oligodendrocyte cell line stably expressing human wild-type Asyn. Results: These cells displays cellular degeneration when transiently transfected with p25a as demonstrated by shrinkage of the cytosol, perinuclear localization of the microtubule, and nuclear condensation. Cell death is partially blocked by anti-aggregatory compounds. Furthermore, the p25a mediated cell death is significantly reduced by certain caspase inhibitors. Conclusion: Our results indicate that the p25a-mediated Asyndependent cell death is caused by the proaggregatory activity of p25a. Furthermore, a specific caspase cascade seems to be activated in the apoptotic process. Based on our results, we suggest that p25a contributes to the cytotoxity underlying several a-synucleinopathies.
2.306 A yeast-based screening system for mouse brain proteins that modify the toxicity of alpha-synuclein S. de Graeve1° , S. Marinelli, P. Van Dijck, J. Thevelein, F. Stolz Belgium
2.308 Characterization of the expression of alpha-synuclein and suppressor/enhancer proteins in Saccharomyces cerevisiae
Objective: a-Synuclein (a-SYN) is the major building block of the filaments that form the proteinacious inclusions that are characteristic for many disorders, including Lewy bodies and Lewy neurites in Parkinson’s disease (PD). Recently, it has been shown that, when expressed in the yeast Saccharomyces cerevisiae, a-SYN causes a growth defect, especially in strains where a non-essential gene involved in vesicle-mediated transport was deleted. Our objective was to develop a yeast-based screening system to discover mouse brain cDNA’s that counteract this toxic effect of a-SYN. This system was then used to identify novel interaction partners of a-SYN. Method: We have cloned the human WT a-SYN gene behind the galactose-inducible promoter of the yeast GAL1 gene and integrated two copies into the genome of the cog6D yeast mutant strain, which caused a severe growth defect on galactose-containing medium. After transformation with a mouse brain cDNA-library, the cells were plated out on galactosecontaining medium to check for cells that contain possible inhibitors of a-SYN toxicity. From the growing yeast colonies cDNA plasmids were isolated and retransformed into the yeast strain to confirm their potential to suppress the a-SYN toxicity effect. Results: We have identified two mouse brain proteins that suppress toxicity of a-SYN in the yeast system. In addition we have isolated a protein that surprisingly enhanced the toxicity of a-SYN, while it had no effect on growth of the strain without a-SYN. At present we are performing coimmunoprecipitation experiments to address whether these proteins work through a direct interaction with the a-SYN protein or in an indirect way. Conclusion: Yeast cells can be used as a tool to screen for brain proteins that modify the toxic effect of the PD-related protein a-SYN. Our method has led to the identification of two suppressors and one enhancer of a-SYN toxicity in yeast.
S. Marinelli1° , S. de Graeve, J. Vandamme, Y. Engelborghs, P. Van Dijck, J. Thevelein, F. Stolz 1 Heverlee, Belgium
1 Leuven,
Objective: Parkinson’s disease (PD) is a common neuronal disorder that is hallmarked by the loss of dopaminergic neurons and the presence of Lewy Body inclusions, which contain alpha-synuclein (aSYN) as a major component. A yeast model was established to screen for proteins that can modify the toxic phenotype of aSYN expression. Two suppressors (S1, S2) and one enhancer protein (E1) were identified and confirmed. The goal of this study was to characterize their effect on aSYN localization and on aSYN induced accumulation of lipid droplets in aSYN sensitive yeast mutants. Method: A fusion construct of aSYN wild type and green fluorescent protein (GFP) was synthesized and transformed into several yeast strains (BY4742, cog6D). The localization of aSYN-GFP was studied in the absence and in the presence of the confirmed suppressor/enhancer proteins. In addition cells co-expressing aSYN and a suppressor/enhancer protein were stained with Nile Red and a confocal microscopy experiment was performed to study the effect of expression on the accumulation of lipid droplets in several strains. Results: Three stages of aSYN localization were observed depending in time: plasma membrane localization, an intermediate stage and an aggregated stage. Expression of the suppressor proteins delays the aggregation process in yeast. Co-expression of suppressor S1 and aSYN or of enhancer E1 and aSYN causes a high increase of lipid droplets in comparison with cells that only express aSYN. In contrast cells co-expressing suppressor S2 and aSYN accumulate less lipid droplets. Conclusion: Expression of a suppressor protein (S1 or S2) causes a delay in the aSYN aggregation process. Suppressor S1 and S2 are able