- Email: [email protected]
DISTINCT PATTERNS OF H2A.X PHOSPHORYLATION IN NEURONS: POTENTIAL ROLES IN HEALTH AND NEURODEGENERATIVE DISEASE
P990 P3-144
Poster Presentations: Tuesday, July 18, 2017 DISTINCT PATTERNS OF H2A.X PHOSPHORYLATION IN NEURONS: POTENTIAL ROLES IN HEALTH AND NEURODEGENERATIVE DISEASE
Mark D. Evans1,2, Lennart Mucke1,2, 1Gladstone Institute of Neurological Disease, San Francisco, CA, USA; 2University of California San Francisco, San Francisco, CA, USA. Contact e-mail: [email protected] Background: Genomic integrity is crucial to neuronal function, but
becomes severely disrupted in neurodegenerative diseases. Central to maintaining genomic integrity is the DNA damage response, which acts to recruit proteins necessary for the repair of damaged DNA. Phosphorylation of histone H2A.X at serine 139 (gH2A.X), an early event in the DNA damage response, is required for the retention of multiple repair factors at DNA double-strand breaks (DSBs), including 53BP1. However, gH2A.X also occurs at sites of DNA single-strand breaks and chromatin remodeling, and can be induced during cellular stress and apoptosis. Thus, gH2A.X appears to play a role in the cellular response to a variety of insults, including DSBs. In neurons, gH2A.X levels are elevated by increased circuit activity, but the mechanisms underlying this response and its function(s) remain unclear. Methods: We analyzed gH2A.X and 53BP1 immunoreactivity at baseline and after neuronal activity manipulations in primary neuronal cultures and in brain sections from wildtype and human amyloid precursor protein (hAPP) transgenic mice. Results: We show that neuronal gH2A.X immunoreactivity exists in two major patterns: (1) focal, likely representing DSBs, especially when colocalized with 53BP1, and (2) nucleus-wide, which is unlikely to represent DSBs. Pharmacological stimulation of primary neuronal cultures led to striking activity-dependent increases in nucleus-wide gH2A.X, but not 53BP1. This response was reversed 6 h after cultures were returned to baseline conditions. Pharmacologically induced epilepsy in wildtype mice also increased nucleus-wide gH2A.X in neuronal populations throughout the hippocampus in vivo. Conclusions: These data suggest that, independent of DSBs, neuronal gH2A.X formation is involved in activity-induced chromatin remodeling and, possibly, changes in gene expression. Ongoing investigations use mass spectrometry-based approaches to assess the abundance of multiple DNA repair factors in postmortem tissues from patients with Alzheimer’s disease and controls, as well as genetic modulations of such factors in experimental models.
Background: Platelets are considered a good model system to study a number of elements associated to neuronal pathways as they share biochemical similarities. Platelets represent the major source of amyloid-b (Ab) in blood contributing to the Ab accumulation in the brain parenchyma and vasculature1. Ab accumulation in brain is one of the main neuropathological hallmarks in Alzheimer’s disease (AD). AD-related alterations in the brain have been linked to the peripheral blood platelets2,3. These include membrane fluidity changes, abnormal cytoplasmic calcium fluxes or increased oxidative stress levels. Therefore, platelets can be considered a peripheral available model to study metabolic mechanisms, occurring in AD. In this study we examined platelet proteins expression in AD patients compared to healthy aged-matched controls to investigate peripheral molecular alterations in AD. Methods: Platelet samples from elderly non-demented subjects, mild cognitive impairment (MCI), probable mild AD and probable moderate-severe AD patients were collected and analysed. Minimental Status Examination (MMSE) and ApoE genotype were determined in all cases. A twodimensional difference gel electrophoresis (2D DIGE) discovery phase and a western blot validation analysis were performed. Results: Differences between patients and healthy controls were found in four proteins: Talin, Vinculin, Moesin and C3b, which are known to be involved in cytoskeletal regulation including focal adhesions, inflammation and immune functions. Talin was found to be increased in mild AD and moderate-severe AD groups versus control, while the other three were found to be decreased in AD patients versus controls. 2D DIGE and Western Blot analysis showed the same pattern of variation. Conclusions: These findings confirm and extend previous reports concerning the presence of human platelet morphological alterations and dysfunction in AD. The identification of differences in protein platelet expression strength the idea of AD as a systemic disease. References: 1. Chen M, Inestrosa NC, Ross GS, Fernandez HL. Platelets are the primary source of amyloid b-peptide in human blood. Biochem Biophys Res Commun. 1995;213(1):96-103. http://dx.doi.org/10. 1006/bbrc.1995.2103. 2. Veitinger M, Varga B, Guterres SB, Zellner M. Platelets, a reliable source for peripheral Alzheimer’s disease biomarkers? Acta Neuropathol Commun. 2014;2:65. http:// dx.doi.org/10.1186/2051-5960-2-65. 3. Catricala S, Torti M, Ricevuti G. Alzheimer disease and platelets: how’s that relevant. Immun Ageing. 2012;9:20. http://dx.doi.org/10.1186/1742-4933-9-20.
P3-146
P3-145
Niraj M. Shanbhag1,2, Mark D. Evans1,2, Lennart Mucke1,2, 1 Gladstone Institute of Neurological Disease, San Francisco, CA, USA; 2 University of California San Francisco, San Francisco, CA, USA. Contact e-mail: [email protected]
DIFFERENTIAL PROTEIN EXPRESSION IN PLATELETS FROM SUBJECTS WITH MILD COGNITIVE IMPAIRMENT AND ALZHEIMER’S DISEASE 1,2
1
THE ROLE OF BRCA1 IN ALZHEIMER’S DISEASE
1
Marta Gonz alez S anchez , Teresa Dıaz , Consuelo Pascual , Sara Llamas Velasco1,2, Alejandro Herrero San Martın1,2, David Perez Martınez1,2, Alberto Villarejo Galende1,2, Eva Carro1,2, Fernando Bartolome1,2, 1Research Institute Hospital, Madrid, Spain; 2 Biomedical Research Networking Center in Neurodegenerative Diseases (CIBERNED), Madrid, Spain. Contact e-mail: [email protected]
Background: The accumulation of DNA damage is emerging as a driving force behind normal aging as well as neuronal degeneration. As a testament to this, mutations in DNA repair proteins underlie diseases that can manifest neurodegenerative phenotypes, such as ataxia telangiectasia and xeroderma pigmentosum. It is therefore possible that acquired deficits in DNA repair could contribute to late onset neurodegenerative disorders such as
Poster Presentations: Tuesday, July 18, 2017 DISTINCT PATTERNS OF H2A.X PHOSPHORYLATION IN NEURONS: POTENTIAL ROLES IN HEALTH AND NEURODEGENERATIVE DISEASE
Mark D. Evans1,2, Lennart Mucke1,2, 1Gladstone Institute of Neurological Disease, San Francisco, CA, USA; 2University of California San Francisco, San Francisco, CA, USA. Contact e-mail: [email protected] Background: Genomic integrity is crucial to neuronal function, but
becomes severely disrupted in neurodegenerative diseases. Central to maintaining genomic integrity is the DNA damage response, which acts to recruit proteins necessary for the repair of damaged DNA. Phosphorylation of histone H2A.X at serine 139 (gH2A.X), an early event in the DNA damage response, is required for the retention of multiple repair factors at DNA double-strand breaks (DSBs), including 53BP1. However, gH2A.X also occurs at sites of DNA single-strand breaks and chromatin remodeling, and can be induced during cellular stress and apoptosis. Thus, gH2A.X appears to play a role in the cellular response to a variety of insults, including DSBs. In neurons, gH2A.X levels are elevated by increased circuit activity, but the mechanisms underlying this response and its function(s) remain unclear. Methods: We analyzed gH2A.X and 53BP1 immunoreactivity at baseline and after neuronal activity manipulations in primary neuronal cultures and in brain sections from wildtype and human amyloid precursor protein (hAPP) transgenic mice. Results: We show that neuronal gH2A.X immunoreactivity exists in two major patterns: (1) focal, likely representing DSBs, especially when colocalized with 53BP1, and (2) nucleus-wide, which is unlikely to represent DSBs. Pharmacological stimulation of primary neuronal cultures led to striking activity-dependent increases in nucleus-wide gH2A.X, but not 53BP1. This response was reversed 6 h after cultures were returned to baseline conditions. Pharmacologically induced epilepsy in wildtype mice also increased nucleus-wide gH2A.X in neuronal populations throughout the hippocampus in vivo. Conclusions: These data suggest that, independent of DSBs, neuronal gH2A.X formation is involved in activity-induced chromatin remodeling and, possibly, changes in gene expression. Ongoing investigations use mass spectrometry-based approaches to assess the abundance of multiple DNA repair factors in postmortem tissues from patients with Alzheimer’s disease and controls, as well as genetic modulations of such factors in experimental models.
Background: Platelets are considered a good model system to study a number of elements associated to neuronal pathways as they share biochemical similarities. Platelets represent the major source of amyloid-b (Ab) in blood contributing to the Ab accumulation in the brain parenchyma and vasculature1. Ab accumulation in brain is one of the main neuropathological hallmarks in Alzheimer’s disease (AD). AD-related alterations in the brain have been linked to the peripheral blood platelets2,3. These include membrane fluidity changes, abnormal cytoplasmic calcium fluxes or increased oxidative stress levels. Therefore, platelets can be considered a peripheral available model to study metabolic mechanisms, occurring in AD. In this study we examined platelet proteins expression in AD patients compared to healthy aged-matched controls to investigate peripheral molecular alterations in AD. Methods: Platelet samples from elderly non-demented subjects, mild cognitive impairment (MCI), probable mild AD and probable moderate-severe AD patients were collected and analysed. Minimental Status Examination (MMSE) and ApoE genotype were determined in all cases. A twodimensional difference gel electrophoresis (2D DIGE) discovery phase and a western blot validation analysis were performed. Results: Differences between patients and healthy controls were found in four proteins: Talin, Vinculin, Moesin and C3b, which are known to be involved in cytoskeletal regulation including focal adhesions, inflammation and immune functions. Talin was found to be increased in mild AD and moderate-severe AD groups versus control, while the other three were found to be decreased in AD patients versus controls. 2D DIGE and Western Blot analysis showed the same pattern of variation. Conclusions: These findings confirm and extend previous reports concerning the presence of human platelet morphological alterations and dysfunction in AD. The identification of differences in protein platelet expression strength the idea of AD as a systemic disease. References: 1. Chen M, Inestrosa NC, Ross GS, Fernandez HL. Platelets are the primary source of amyloid b-peptide in human blood. Biochem Biophys Res Commun. 1995;213(1):96-103. http://dx.doi.org/10. 1006/bbrc.1995.2103. 2. Veitinger M, Varga B, Guterres SB, Zellner M. Platelets, a reliable source for peripheral Alzheimer’s disease biomarkers? Acta Neuropathol Commun. 2014;2:65. http:// dx.doi.org/10.1186/2051-5960-2-65. 3. Catricala S, Torti M, Ricevuti G. Alzheimer disease and platelets: how’s that relevant. Immun Ageing. 2012;9:20. http://dx.doi.org/10.1186/1742-4933-9-20.
P3-146
P3-145
Niraj M. Shanbhag1,2, Mark D. Evans1,2, Lennart Mucke1,2, 1 Gladstone Institute of Neurological Disease, San Francisco, CA, USA; 2 University of California San Francisco, San Francisco, CA, USA. Contact e-mail: [email protected]
DIFFERENTIAL PROTEIN EXPRESSION IN PLATELETS FROM SUBJECTS WITH MILD COGNITIVE IMPAIRMENT AND ALZHEIMER’S DISEASE 1,2
1
THE ROLE OF BRCA1 IN ALZHEIMER’S DISEASE
1
Marta Gonz alez S anchez , Teresa Dıaz , Consuelo Pascual , Sara Llamas Velasco1,2, Alejandro Herrero San Martın1,2, David Perez Martınez1,2, Alberto Villarejo Galende1,2, Eva Carro1,2, Fernando Bartolome1,2, 1Research Institute Hospital, Madrid, Spain; 2 Biomedical Research Networking Center in Neurodegenerative Diseases (CIBERNED), Madrid, Spain. Contact e-mail: [email protected]
Background: The accumulation of DNA damage is emerging as a driving force behind normal aging as well as neuronal degeneration. As a testament to this, mutations in DNA repair proteins underlie diseases that can manifest neurodegenerative phenotypes, such as ataxia telangiectasia and xeroderma pigmentosum. It is therefore possible that acquired deficits in DNA repair could contribute to late onset neurodegenerative disorders such as