- Email: [email protected]
ASTROCYTE RESPONSE TO HYPERHOMOCYSTEINEMIA (HHCY)
P318 P1-197
Poster Presentations: Sunday, July 16, 2017 CHARACTERISING THE CHOLINERGIC PHENOTYPES IN TRANSGENIC MODELS FOR ALZHEIMER’S DISEASE AND FRONTOTEMPORAL LOBAR DEGENERATION BY CHAT, P75NTR, P-TRKA AND NGF PROTEINS STAINING
Adrianna Wysocka1, Maciej Zadrozny1, James Hyatt2, Marta Steczkowska1, Ewelina Palasz1, Radoslaw Folcik1, Grazyna Niewiadomska1, 1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Keele University, Staffordshire, United Kingdom. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is associated with severe loss of cholinergic markers in the brain, and such loss may be due to the toxic interaction of tau with muscarinic receptors. So far, significance of tau signaling through muscarinic receptor in solely in vivo tauopathic models remains uncertain. Methods: In this study, we evaluated cholinergic neurons morphology in two tauopathic mouse models: Line 1 (L1), with mild AD-like tauopathy and Line 66 (L66) with severe frontotemporal lobar degeneration-like tauopathy (FTLD), and wild-type mice as controls (Melis et al. 2015). Analysis was performed in 3-(young), 6-(aging) and 9month-old (aged) mice. Immunohistochemical staining against cholinergic markers ChAT, p75NTR, p-TrkA and NGF, as well as ELISA for NGF in cortex and hippocampus were performed. Results: We found that ChAT and p75NTR immunostainings were decreased in the basal forebrain cholinergic neurons only in L1. Decreased number of immunopositive cells and atrophic changes in cholinergic neurons morphology of L1 mice were observed in 3-mo-old animals before tauopathic symptoms developed. Alterations of the normal cholinergic phenotype were observed in striatal interneurons, as well as in projective neurons of medial septum, horizontal and vertical limbs of diagonal band and in magnocellular basal nucleus. Changes in cholinergic neurons number and morphology were not observed in L66. NGF level reduction was the largest in line L1, what corresponds with the changes in intensity of stainings against p-TrkA and p75NTR in most structures by the age of 3 months. Conclusions: Morphological changes in cholinergic system in line L1 correspond to those observed in AD. In opposite, the lack of changes in cholinergic system in line L66 is compatible with the data that there is no cholinergic deficit in FTLD patients and in animal models of the disease. Phenotype impairment of cholinergic neurons may be related to the type of pathological forms of tau protein - short oligomers or low aggregated forms such as PHF in L1, in contrast to the highly aggregated strands of NFT- type in L66, forming in brain due to the use of different tau constructs during transgenisation. Sponsored by NCN UMO-2014/15/B/NZ4/05041 grant and WisTa Laboratories Ltd.
P1-198
ASTROCYTE RESPONSE TO HYPERHOMOCYSTEINEMIA (HHCY)
Brittani R. Price1, Tiffany L. Sudduth1, Erica M. Weekman1, Donna M. Wilcock2, Abigail Woolums1, 1University of Kentucky, Lexington, KY, USA; 2Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA. Contact e-mail: [email protected] Background: Homocysteine (Hcy) is a non-protein forming amino acid involved in the production of both methionine and cysteine.
Elevated Hcy can result from mutations in methylenetetrahydrofolate reductase (MTHFR) or cystathionine beta synthase (CBS) genes, as well as low levels of vitamins B6, B9, and B12. Elevated Hcy is termed hyperhomocysteinemia (HHcy). HHcy is a risk factor for vascular cognitive impairment and dementia (VCID), as well as Alzheimer’s disease. The mechanism by which HHcy promotes VCID or AD remains unknown. Astrocytes play a crucial role in both potassium buffering and neurovascular coupling. The astrocytic end-feet essentially ensheath intraparenchymal blood vessels in the brain and express a variety of channels and markers indicative of their specialized functions. Channels enriched at the astrocytic end-feet include the aquaporin 4 water channel (AQP4), the inward rectifying potassium channel Kir4.1 and the calcium-dependent potassium channel BK. These channels are anchored at the astrocytic end-foot by dystrophin-1 (Dp71), as well as other anchoring proteins. Methods: To determine the cell specific effects of Hcy, C8D1A astrocytes were treated with 50mM of Hcy for 24, 48, 72 and 96 hours. Gene expression for several astrocyte specific markers was then analyzed. To determine the in vivo effects of HHcy, wildtype mice were subject to a HHcy-inducing diet for a period of 6, 10 or 14 weeks. Tissue was then histologically examined for the astrocytic end-foot markers AQP4, Kir4.1, BK and dystrophin-1 (Dp71). Results: Astrocytes treated with homocysteine exhibit an increased GFAP expression, suggesting homocysteine to be an inflammatory stimulus for astrocytes. While GFAP is increased, AQP4, Kir4.1, and BK expression levels were decreased. Our HHcy model displays significant astrocyctic end-foot disruptions. While AQP4 becomes dislocalized from the end-feet, Kir4.1 and BK protein expression decreases. In addition, the anchoring protein Dp71, also decreases. We examined mice subjected to the HHcy-inducing diet for 6, 10, and 14 weeks and find that end-disruption becomes progressively more severe as the mice continue the diet. This corresponds with progressively worse cognitive performance in the radial-arm water maze. Conclusions: HHcy results in the disruption of the astrocytic end-foot connection, which we hypothesize contributes to the cognitive decline observed in our HHcy mouse model.
P1-199
PROTECTIVE PROPERTY OF P3-ALCb AGAINST A NEUROTOXICITY INDUCED BY Ab OLIGOMER
Haruka Saitoh1, Chiori Omori2, Ayano Kimura1, Masaaki Waragai3, Masahiro Maeda4, Saori Hata1, Toshiharu Suzuki1 and Hokkaido, 1Hokkaido University, Sapporo, Japan; 2University of Tokyo, Kashiwa, Japan; 3Higashimatsudo Hospital, Chiba, Japan; 4IBL Co, Ltd., Fujioka, Japan. Contact e-mail: [email protected] Background: Neuronal p3-Alcb peptide is generated from the precursor protein Alcadeinb (Alcb) by a cleavage of a- and g-secretases. Alcb generates the C-terminal variant peptides including a major p3-Alcb37, as does APP. It is understood that Ab42 level tends to decrease in AD patients in cerebrospinal fluid (CSF) by deposition of Ab aggregate in brain compared to age-matched non-demented subjects. Although, p3-Alcb levels are comparable to those of Ab levels, the trend of non-aggregatable p3-Alcb levels in CSF of AD subjects has not been analyzed. Moreover, function of p3-Alcb remains unclear. In this study, we analyzed the change
Poster Presentations: Sunday, July 16, 2017 CHARACTERISING THE CHOLINERGIC PHENOTYPES IN TRANSGENIC MODELS FOR ALZHEIMER’S DISEASE AND FRONTOTEMPORAL LOBAR DEGENERATION BY CHAT, P75NTR, P-TRKA AND NGF PROTEINS STAINING
Adrianna Wysocka1, Maciej Zadrozny1, James Hyatt2, Marta Steczkowska1, Ewelina Palasz1, Radoslaw Folcik1, Grazyna Niewiadomska1, 1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Keele University, Staffordshire, United Kingdom. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is associated with severe loss of cholinergic markers in the brain, and such loss may be due to the toxic interaction of tau with muscarinic receptors. So far, significance of tau signaling through muscarinic receptor in solely in vivo tauopathic models remains uncertain. Methods: In this study, we evaluated cholinergic neurons morphology in two tauopathic mouse models: Line 1 (L1), with mild AD-like tauopathy and Line 66 (L66) with severe frontotemporal lobar degeneration-like tauopathy (FTLD), and wild-type mice as controls (Melis et al. 2015). Analysis was performed in 3-(young), 6-(aging) and 9month-old (aged) mice. Immunohistochemical staining against cholinergic markers ChAT, p75NTR, p-TrkA and NGF, as well as ELISA for NGF in cortex and hippocampus were performed. Results: We found that ChAT and p75NTR immunostainings were decreased in the basal forebrain cholinergic neurons only in L1. Decreased number of immunopositive cells and atrophic changes in cholinergic neurons morphology of L1 mice were observed in 3-mo-old animals before tauopathic symptoms developed. Alterations of the normal cholinergic phenotype were observed in striatal interneurons, as well as in projective neurons of medial septum, horizontal and vertical limbs of diagonal band and in magnocellular basal nucleus. Changes in cholinergic neurons number and morphology were not observed in L66. NGF level reduction was the largest in line L1, what corresponds with the changes in intensity of stainings against p-TrkA and p75NTR in most structures by the age of 3 months. Conclusions: Morphological changes in cholinergic system in line L1 correspond to those observed in AD. In opposite, the lack of changes in cholinergic system in line L66 is compatible with the data that there is no cholinergic deficit in FTLD patients and in animal models of the disease. Phenotype impairment of cholinergic neurons may be related to the type of pathological forms of tau protein - short oligomers or low aggregated forms such as PHF in L1, in contrast to the highly aggregated strands of NFT- type in L66, forming in brain due to the use of different tau constructs during transgenisation. Sponsored by NCN UMO-2014/15/B/NZ4/05041 grant and WisTa Laboratories Ltd.
P1-198
ASTROCYTE RESPONSE TO HYPERHOMOCYSTEINEMIA (HHCY)
Brittani R. Price1, Tiffany L. Sudduth1, Erica M. Weekman1, Donna M. Wilcock2, Abigail Woolums1, 1University of Kentucky, Lexington, KY, USA; 2Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA. Contact e-mail: [email protected] Background: Homocysteine (Hcy) is a non-protein forming amino acid involved in the production of both methionine and cysteine.
Elevated Hcy can result from mutations in methylenetetrahydrofolate reductase (MTHFR) or cystathionine beta synthase (CBS) genes, as well as low levels of vitamins B6, B9, and B12. Elevated Hcy is termed hyperhomocysteinemia (HHcy). HHcy is a risk factor for vascular cognitive impairment and dementia (VCID), as well as Alzheimer’s disease. The mechanism by which HHcy promotes VCID or AD remains unknown. Astrocytes play a crucial role in both potassium buffering and neurovascular coupling. The astrocytic end-feet essentially ensheath intraparenchymal blood vessels in the brain and express a variety of channels and markers indicative of their specialized functions. Channels enriched at the astrocytic end-feet include the aquaporin 4 water channel (AQP4), the inward rectifying potassium channel Kir4.1 and the calcium-dependent potassium channel BK. These channels are anchored at the astrocytic end-foot by dystrophin-1 (Dp71), as well as other anchoring proteins. Methods: To determine the cell specific effects of Hcy, C8D1A astrocytes were treated with 50mM of Hcy for 24, 48, 72 and 96 hours. Gene expression for several astrocyte specific markers was then analyzed. To determine the in vivo effects of HHcy, wildtype mice were subject to a HHcy-inducing diet for a period of 6, 10 or 14 weeks. Tissue was then histologically examined for the astrocytic end-foot markers AQP4, Kir4.1, BK and dystrophin-1 (Dp71). Results: Astrocytes treated with homocysteine exhibit an increased GFAP expression, suggesting homocysteine to be an inflammatory stimulus for astrocytes. While GFAP is increased, AQP4, Kir4.1, and BK expression levels were decreased. Our HHcy model displays significant astrocyctic end-foot disruptions. While AQP4 becomes dislocalized from the end-feet, Kir4.1 and BK protein expression decreases. In addition, the anchoring protein Dp71, also decreases. We examined mice subjected to the HHcy-inducing diet for 6, 10, and 14 weeks and find that end-disruption becomes progressively more severe as the mice continue the diet. This corresponds with progressively worse cognitive performance in the radial-arm water maze. Conclusions: HHcy results in the disruption of the astrocytic end-foot connection, which we hypothesize contributes to the cognitive decline observed in our HHcy mouse model.
P1-199
PROTECTIVE PROPERTY OF P3-ALCb AGAINST A NEUROTOXICITY INDUCED BY Ab OLIGOMER
Haruka Saitoh1, Chiori Omori2, Ayano Kimura1, Masaaki Waragai3, Masahiro Maeda4, Saori Hata1, Toshiharu Suzuki1 and Hokkaido, 1Hokkaido University, Sapporo, Japan; 2University of Tokyo, Kashiwa, Japan; 3Higashimatsudo Hospital, Chiba, Japan; 4IBL Co, Ltd., Fujioka, Japan. Contact e-mail: [email protected] Background: Neuronal p3-Alcb peptide is generated from the precursor protein Alcadeinb (Alcb) by a cleavage of a- and g-secretases. Alcb generates the C-terminal variant peptides including a major p3-Alcb37, as does APP. It is understood that Ab42 level tends to decrease in AD patients in cerebrospinal fluid (CSF) by deposition of Ab aggregate in brain compared to age-matched non-demented subjects. Although, p3-Alcb levels are comparable to those of Ab levels, the trend of non-aggregatable p3-Alcb levels in CSF of AD subjects has not been analyzed. Moreover, function of p3-Alcb remains unclear. In this study, we analyzed the change