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2.321 Do chronic non-specific brain insults predispose to incidental Lewy Body Disease? An exploratory study
Poster Presentations: Animal models, Neurodegeneration and Neuroprotection no significant differences could be detected between both hemispheres. In both nuclei there was a difference between the expression level of both transporters between the non-lesioned hemisphere of the hemi-Parkinson rat and a control animal. As for the reuptake activity, we measured in both nuclei of control animals increases in extracellular glutamate concentrations of approximately 1000% and 250% when using respectively L-PDC and DHK. The increase in extracellular glutamate concentration reflects the reuptake activity for glutamate and will be compared between hemiParkinson and control rats. Conclusion: Our data are indicative for complex changes at the level of the glial glutamate transporters as a function of time after lesioning, in the 6-OHDA rat model. Further experiments will reveal whether these changes are the result of or the reason for neurodegeneration observed during Parkinson’s disease.
2.321 Do chronic non-specific brain insults predispose to incidental Lewy Body Disease? An exploratory study R. Frigerio1° , G. Glass, K. Klos, N. Schneider, K. Josephs, D. Dickson, H. Apaydin, J. Parisi, B. Boeve, J.E. Ahlskog 1 Rochester, USA Objective: Incidental Lewy Body Disease (ILBD) is 10-times more common than PD. It may represent preclinical PD, aborted PD, or a non-specific finding. Various systemic conditions that are common in seniors may accelerate brain aging or cause neuropathologic changes. Here we considered whether chronic brain insults related to general medical conditions might non-specifically predispose to ILBD. Method: We employed the Tissue Registry of the Rochester Epidemiology Project to identify subjects ages 60 and older at death during the time period 1990–2004. We excluded subjects with medical records documentation of parkinsonism, tremor, dementia, or other neurodegenerative diseases; and required multiple physician evaluations within the last five years of life. Formalin-fixed, paraffin-embedded brain tissue blocks were stained for H&E and alpha-synuclein. Blinded to clinical history, the blocks were examined for Lewy bodies and neurites in selected brain regions. Blinded to pathologic findings, the medical records were abstracted regarding the presence or absence of factors that may be markers of chronic brain insults. The factors we considered included: history of terminal illness, diabetes mellitus, stroke, coronary artery disease, peripheral vascular disease, hypertension, congestive heart failure, COPD, excessive alcohol intake, and systemic chemotherapy. Results: Of 93 subjects included, 12 had ILBD and 81 did not. The ages at death, gender distributions, and frequencies of medical contacts were similar in both groups. None of the general medical conditions that we considered were significantly associated with ILBD. Conclusion: ILBD was not associated with any of these general medical conditions that are common in aging. This suggests that Lewy bodies may be specific to the PD neuropathologic process, rather than a non-specific consequence of age-related chronic brain insults. However, we acknowledge that our sample size was small and that we only considered surrogate markers of chronic brain insults. Further exploration of our hypothesis may therefore deserve consideration.
2.322 Demonstration of the formation of Lewy bodies in parkinsonism from disrupted neurotransmitter-storage organelles and their interaction with alpha-synuclein M. Chrysanthou1° , P. Spiliopoulos, S. Havaki, I. Kloukina, M.R. Issidorides Greece
1 Athens,
Objective: Our previous histochemical and ultrastructural studies since 1978 (Issidorides, Mytilineou, Whetsell and Yahr, 1978) have identified, in human monoamine neurons abundant, spherical inclusions – termed protein bodies (pb) – that store the neurotransmitter (Issidorides, Havaki, Arvanitis, Chrysanthou-Piterou, 2004). In Parkinsonism (PD), pb are disrupted or
S127
missing. However, some of their major constituents, i.e. basic proteins and sphingomyelin are located in Lewy bodies (LB). Our objective is to study by what process pb give rise to LB, and whether ubiquitin and a-synuclein, the two components that are consistently demonstrated in LB, are operational. Method: We used postmortem substantia nigra and locus coeruleus tissues from 20 PD patients and 10 controls. We applied histochemical techniques and antibodies against ubiquitin and a-synuclein, for light and electron microscopy to study their immunolocalization pattern in normal and altered pb in PD. Results: Histochemistry for basic proteins and sphingomyelin showed a continuum for these substances in normal pb, in disrupted or coalesced pb and in LB. With the application of ubiquitin antibody no immunoreactivity (UBQ-IR) was evident in pb of control tissues. In contrast, UBQ-IR in PD tissue, localized not only in spherical, mature LB, but, also, in irregular compact formations, as well as in circular formations, surrounding “clear areas” in the cytoplasm, presumably enclosing constituents of damaged pb, as shown by aniline blue staining in light microscopy. The a-synuclein antibody, similarly, did not bind to pb of control tissues. In PD tissue, it localized in multiple irregular aggregates, amidst and on the neuromelanin granules, in light and electron microscopy. Conclusion: In PD, free basic proteins and sphingomyelin of disrupted pb in the cytoplasm induce production of UBQ and cause aggregation of a-synuclein due to its well known interaction with polycations and membrane lipids. These results signify defective sequestration of dopamine due to this absence of normal neurotransmitter storage sites.
Animal models, Neurodegeneration and Neuroprotection 2.401 Silencing plus over expression of selected genes as a novel model of sporadic Parkinson’s disease T. Fishman1° , M.B.H. Youdim, S. Mandel Israel
1 Haifa,
Objective: Our objective is to develop a model of sporadic PD by selectively silencing genes identified in our transcriptomic and protein profiling of human SNpc from sporadic PD brains, while enforcing the expression of others. Method: The mouse substantia nigra-derived cell-line SN4741 was employed as a model to study the involvement of the identified proteins intimately connected to the neurotoxic cascade (e.g. SKP1A, HSC-70 and ALDH1A) and the response to cell injury induced by the dopaminergic neurotoxin MPP+, serum deprivation and H2 O2 . SN4741 cells were infected with small-hairpin RNA (shRNA)-encoding lentiviruses targeting the selected transcripts or with non-target shRNA control. The percentage of silencing was evaluated using real-time PCR and Western blot analysis. Parameters of cell survival were assessed. Alterations in cell cycle were analyzed by flow cytometry. Results: MPP+ and H2 O2 induced a time and dose-dependent alteration in the protein expression of the oxygen and iron-regulated proteins transferrin receptor and prolyl hydroxylase (PHD2/EGLN1) and decreases in SKP1A, HSC-70 and ALDH1A. ShRNA-mediated silencing of Skp1A, a component of the SCF ubiquitin (E3) ligase complex, showed a delay in completion of the cell cycle and an increased susceptibility to MPP+ and serum starvation. Conclusion: The approach to develop an “ultimate model” of PD by silencing or over expressing single genes that were identified as mutated in the familial cases has not met with the expected success. We have reproduced to a significant extent the gene and protein alterations initially described in our studies with SNpc from Parkinsonian subjects, in a dopaminergic cell line from mice SNpc. The successful results obtained with the present study will help us to develop an experimental mice model of sporadic PD based on viral-mediated RNAi delivery which will be of help for developing novel neuroprotective drugs.
2.321 Do chronic non-specific brain insults predispose to incidental Lewy Body Disease? An exploratory study R. Frigerio1° , G. Glass, K. Klos, N. Schneider, K. Josephs, D. Dickson, H. Apaydin, J. Parisi, B. Boeve, J.E. Ahlskog 1 Rochester, USA Objective: Incidental Lewy Body Disease (ILBD) is 10-times more common than PD. It may represent preclinical PD, aborted PD, or a non-specific finding. Various systemic conditions that are common in seniors may accelerate brain aging or cause neuropathologic changes. Here we considered whether chronic brain insults related to general medical conditions might non-specifically predispose to ILBD. Method: We employed the Tissue Registry of the Rochester Epidemiology Project to identify subjects ages 60 and older at death during the time period 1990–2004. We excluded subjects with medical records documentation of parkinsonism, tremor, dementia, or other neurodegenerative diseases; and required multiple physician evaluations within the last five years of life. Formalin-fixed, paraffin-embedded brain tissue blocks were stained for H&E and alpha-synuclein. Blinded to clinical history, the blocks were examined for Lewy bodies and neurites in selected brain regions. Blinded to pathologic findings, the medical records were abstracted regarding the presence or absence of factors that may be markers of chronic brain insults. The factors we considered included: history of terminal illness, diabetes mellitus, stroke, coronary artery disease, peripheral vascular disease, hypertension, congestive heart failure, COPD, excessive alcohol intake, and systemic chemotherapy. Results: Of 93 subjects included, 12 had ILBD and 81 did not. The ages at death, gender distributions, and frequencies of medical contacts were similar in both groups. None of the general medical conditions that we considered were significantly associated with ILBD. Conclusion: ILBD was not associated with any of these general medical conditions that are common in aging. This suggests that Lewy bodies may be specific to the PD neuropathologic process, rather than a non-specific consequence of age-related chronic brain insults. However, we acknowledge that our sample size was small and that we only considered surrogate markers of chronic brain insults. Further exploration of our hypothesis may therefore deserve consideration.
2.322 Demonstration of the formation of Lewy bodies in parkinsonism from disrupted neurotransmitter-storage organelles and their interaction with alpha-synuclein M. Chrysanthou1° , P. Spiliopoulos, S. Havaki, I. Kloukina, M.R. Issidorides Greece
1 Athens,
Objective: Our previous histochemical and ultrastructural studies since 1978 (Issidorides, Mytilineou, Whetsell and Yahr, 1978) have identified, in human monoamine neurons abundant, spherical inclusions – termed protein bodies (pb) – that store the neurotransmitter (Issidorides, Havaki, Arvanitis, Chrysanthou-Piterou, 2004). In Parkinsonism (PD), pb are disrupted or
S127
missing. However, some of their major constituents, i.e. basic proteins and sphingomyelin are located in Lewy bodies (LB). Our objective is to study by what process pb give rise to LB, and whether ubiquitin and a-synuclein, the two components that are consistently demonstrated in LB, are operational. Method: We used postmortem substantia nigra and locus coeruleus tissues from 20 PD patients and 10 controls. We applied histochemical techniques and antibodies against ubiquitin and a-synuclein, for light and electron microscopy to study their immunolocalization pattern in normal and altered pb in PD. Results: Histochemistry for basic proteins and sphingomyelin showed a continuum for these substances in normal pb, in disrupted or coalesced pb and in LB. With the application of ubiquitin antibody no immunoreactivity (UBQ-IR) was evident in pb of control tissues. In contrast, UBQ-IR in PD tissue, localized not only in spherical, mature LB, but, also, in irregular compact formations, as well as in circular formations, surrounding “clear areas” in the cytoplasm, presumably enclosing constituents of damaged pb, as shown by aniline blue staining in light microscopy. The a-synuclein antibody, similarly, did not bind to pb of control tissues. In PD tissue, it localized in multiple irregular aggregates, amidst and on the neuromelanin granules, in light and electron microscopy. Conclusion: In PD, free basic proteins and sphingomyelin of disrupted pb in the cytoplasm induce production of UBQ and cause aggregation of a-synuclein due to its well known interaction with polycations and membrane lipids. These results signify defective sequestration of dopamine due to this absence of normal neurotransmitter storage sites.
Animal models, Neurodegeneration and Neuroprotection 2.401 Silencing plus over expression of selected genes as a novel model of sporadic Parkinson’s disease T. Fishman1° , M.B.H. Youdim, S. Mandel Israel
1 Haifa,
Objective: Our objective is to develop a model of sporadic PD by selectively silencing genes identified in our transcriptomic and protein profiling of human SNpc from sporadic PD brains, while enforcing the expression of others. Method: The mouse substantia nigra-derived cell-line SN4741 was employed as a model to study the involvement of the identified proteins intimately connected to the neurotoxic cascade (e.g. SKP1A, HSC-70 and ALDH1A) and the response to cell injury induced by the dopaminergic neurotoxin MPP+, serum deprivation and H2 O2 . SN4741 cells were infected with small-hairpin RNA (shRNA)-encoding lentiviruses targeting the selected transcripts or with non-target shRNA control. The percentage of silencing was evaluated using real-time PCR and Western blot analysis. Parameters of cell survival were assessed. Alterations in cell cycle were analyzed by flow cytometry. Results: MPP+ and H2 O2 induced a time and dose-dependent alteration in the protein expression of the oxygen and iron-regulated proteins transferrin receptor and prolyl hydroxylase (PHD2/EGLN1) and decreases in SKP1A, HSC-70 and ALDH1A. ShRNA-mediated silencing of Skp1A, a component of the SCF ubiquitin (E3) ligase complex, showed a delay in completion of the cell cycle and an increased susceptibility to MPP+ and serum starvation. Conclusion: The approach to develop an “ultimate model” of PD by silencing or over expressing single genes that were identified as mutated in the familial cases has not met with the expected success. We have reproduced to a significant extent the gene and protein alterations initially described in our studies with SNpc from Parkinsonian subjects, in a dopaminergic cell line from mice SNpc. The successful results obtained with the present study will help us to develop an experimental mice model of sporadic PD based on viral-mediated RNAi delivery which will be of help for developing novel neuroprotective drugs.