- Email: [email protected]
THE CANDIDATE THERAPEUTIC TARGET IN A RAT MODEL OF DEPRESSION
Poster Presentations: Monday, July 25, 2016 P2-123
THE CANDIDATE THERAPEUTIC TARGET IN A RAT MODEL OF DEPRESSION
Yan Shi, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: After chronic stress, rats’ spontaneous activity weakened in the open-field experiments, the immobility time was significantly longer in forced swimming test, and the percentage of sucrose consumption reduced. Chronic stress-induced depressive rats showed spatial learning and memory deficit with decreasing of neurons and dendritic spines in hippocampus. Methods: After seven days of water maze learning and training, spatial learning improved depressive symptoms. However, the rats without learning showed no significant change. The study was performed on hippocampus of the learning and non-learning depression rats. Using TMT labeling and HPLC fractionation followed by high-resolution LC-MS/MS analysis, quantitative global proteome analysis was performed. Results: In total, more than 3000 proteins were quantified and 34 proteins were found to be differentially expressed. The learning group was associated with increased expression of Calcium/Calmodulin-Dependent Protein Kinase II Delta (CAMK2D) which currently investigated phosphorylates HDAC5 and increases the activity of the MEF2 transcription factor, resulted in produces rapid antidepressant-like effect in animal assays for depression. Besides, our results indicate that increased expression of PSAP, GLB1, VDBP, TSPAN2 within the hippocampal regions could be part of a depression improvement mechanism. Conclusions: Chronic stress-induced depressive rats showed decreasing of neurons and dendritic spines in hippocampus. Spatial learning improved depressive symptoms and these studies have shown the key role of hippocampus in depression.
P2-124
BRAIN ISOPRENOIDS AND ALZHEIMER’S DISEASE
Sandra Pelleieux1, Youla Tsantrizos2, Louise Theroux2, Doris Dea3, Judes Poirier2, 1Dougals Mental Health University Institute, Montreal, QC, Canada; 2McGill University, Montreal, QC, Canada; 3McGill University, Montreal, QC, Canada. Contact e-mail: [email protected] Background: The mevalonate pathway has been described to play a
key role in Alzheimer’s disease (AD). Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are non-sterol isoprenoid intermediates from mevalonate. They lead to protein prenylation which plays a crucial role in controlling cell signalling and proliferation. hFPP and hGGPP synthases lead to prenylation of small GTPase protein RhoA-cdc42, that have been shown to be associated with the accumulation of phospho-tau protein in the brain. It is hypothesise that these intermediates are implicated in tau dysregulation and the progression of AD. A better understanding of the role of hFPPs and hGGPPs in the pathophysiology of Alzheimer’s disease may prove to be essential to assess the function of the isoprenoids pathway as a potential target in prevention of AD. Methods: In the first part of this work, we measured cortical mRNA levels of hFPPs and hGGPPs (RT-qPCR) and, GGPP and FPP concentrations by Liquid chromatography-tandem mass spectrometry (LC/MS/MS) in control and AD brain tissues obtained from the Douglas-Bell Brain Bank (Montreal, Canada). In the second part, hFPPs and hGGPPs inhibitors derived from the nitrogen-containing bisphosphonates (N-BP) were tested on human SH-SY5Y neurons and their effect on the accumulation of tau and Phospho-tau was measured as a function of time and concentrations. Results: In autopsied frontal cortex, we found a significant positive correlation
P659
between mRNA levels (p<0.001, n¼124), FFP and GGPP (p<0.05, n¼116) and disease status. The levels of mRNA for hFFPs and hGGPPs also correlated with the amount of phospho-tau protein levels (p<0.05, n¼38) and neurofibrillary tangles density (p<0.006, n¼40) in frontal cortex. We also found that high levels of hFPPS and hGGPPS mRNA prevalence is significantly associated with earlier age of onset in AD (p<0.05, n¼64). Finally, preliminary data indicates that our novel families of inhibitors can decrease phospho-tau levels in SH-SY5Y neurons without any detectable toxicity. Conclusions: Together, these results suggest that accumulation of phospho-tau in the AD brain is related to increase levels of neuronal isoprenoids; a phenomenon that could be alleviated by the administration of specific and potent bi-phosphonate derivatives. supported by the J.L. Levesques Foundation, Canadian Institute of Health Research and FQRNT.
P2-125
ANALYSIS OF WILD TYPE AND R47H MUTANT TREM2 EXPRESSION IN ALZHEIMER’S DISEASE BRAINS
Daniel Sevlever, Mariet Younkin, Li Ma, Sakae Nobutaka, Gina Bisceglio, Dennis W. Dickson, Nilufer Ertekin-Taner, Steven G. Younkin, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Genetic analyses showed that the triggering receptor
expressed in myeloid cells 2 (TREM2) R47H variant increases the risk for AD. In the brain TREM2 is predominantly expressed in microglia. The question of whether the R47H mutation affects expression or function of the receptor remains unanswered. In this study we quantitated mRNA and analyzed protein profiles of WT and R47H TREM2 in human brains to determine if the mutation affects expression or processing of TREM2. Methods: There are at least three TREM2 transcripts that are expressed in human brain including one encoding for a soluble form of the receptor. qRT-PCR was performed using 2 sets of primers one that detects all TREM2 mRNA isoforms and one specific for the alternative spliced isoform (TREM2alt) that encodes for the extracellular domain. All analysis were carried out in R software using linear regression adjusting for Age, Sex, APOEε4dose, qRT-PCR plate, RNA integrity number, and expression levels of AIF1 (as a microglia marker). For TREM2 protein quantitation and N-glycosylation processing, RIPA brain extracts were analyzed by Western blot before and after Endo H and PNGAse F treatments. Results: We identified significantly increased levels of TREM2 transcripts in the temporal cortex of AD subjects when compared with age matched controls (p¼8.8E-03); TREM2alt was likewise higher in AD cases, but did not reach significance (p¼6.9E-02). Among AD subjects (N¼41), we observed suggestively higher TREM2 levels in carriers of the R47H risk allele (N¼18, p¼8.5E-02). Expression levels of TREM2 and TREM2alt were highly correlated in both sets of samples assessed (r2 > 0.72). At the protein level, SDS-gel profiles that included the carboxy-terminal fragment of WT and R47H samples were undistinguishable. Furthermore, TREM2 from either WT or R47H brains was equally susceptible to Endo H and PNGase F treatments. Conclusions: 1) The levels of all TREM2 transcripts are increased in AD. 2) In R47H carriers higher levels of TREM2 transcripts were found, but it failed to reach significance. 3) R47H mutation does not affect processing of TREM2 protein. Our data support previous findings that suggest that the R47H variant affects TREM2 function by altering binding properties of the receptor.
THE CANDIDATE THERAPEUTIC TARGET IN A RAT MODEL OF DEPRESSION
Yan Shi, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: After chronic stress, rats’ spontaneous activity weakened in the open-field experiments, the immobility time was significantly longer in forced swimming test, and the percentage of sucrose consumption reduced. Chronic stress-induced depressive rats showed spatial learning and memory deficit with decreasing of neurons and dendritic spines in hippocampus. Methods: After seven days of water maze learning and training, spatial learning improved depressive symptoms. However, the rats without learning showed no significant change. The study was performed on hippocampus of the learning and non-learning depression rats. Using TMT labeling and HPLC fractionation followed by high-resolution LC-MS/MS analysis, quantitative global proteome analysis was performed. Results: In total, more than 3000 proteins were quantified and 34 proteins were found to be differentially expressed. The learning group was associated with increased expression of Calcium/Calmodulin-Dependent Protein Kinase II Delta (CAMK2D) which currently investigated phosphorylates HDAC5 and increases the activity of the MEF2 transcription factor, resulted in produces rapid antidepressant-like effect in animal assays for depression. Besides, our results indicate that increased expression of PSAP, GLB1, VDBP, TSPAN2 within the hippocampal regions could be part of a depression improvement mechanism. Conclusions: Chronic stress-induced depressive rats showed decreasing of neurons and dendritic spines in hippocampus. Spatial learning improved depressive symptoms and these studies have shown the key role of hippocampus in depression.
P2-124
BRAIN ISOPRENOIDS AND ALZHEIMER’S DISEASE
Sandra Pelleieux1, Youla Tsantrizos2, Louise Theroux2, Doris Dea3, Judes Poirier2, 1Dougals Mental Health University Institute, Montreal, QC, Canada; 2McGill University, Montreal, QC, Canada; 3McGill University, Montreal, QC, Canada. Contact e-mail: [email protected] Background: The mevalonate pathway has been described to play a
key role in Alzheimer’s disease (AD). Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are non-sterol isoprenoid intermediates from mevalonate. They lead to protein prenylation which plays a crucial role in controlling cell signalling and proliferation. hFPP and hGGPP synthases lead to prenylation of small GTPase protein RhoA-cdc42, that have been shown to be associated with the accumulation of phospho-tau protein in the brain. It is hypothesise that these intermediates are implicated in tau dysregulation and the progression of AD. A better understanding of the role of hFPPs and hGGPPs in the pathophysiology of Alzheimer’s disease may prove to be essential to assess the function of the isoprenoids pathway as a potential target in prevention of AD. Methods: In the first part of this work, we measured cortical mRNA levels of hFPPs and hGGPPs (RT-qPCR) and, GGPP and FPP concentrations by Liquid chromatography-tandem mass spectrometry (LC/MS/MS) in control and AD brain tissues obtained from the Douglas-Bell Brain Bank (Montreal, Canada). In the second part, hFPPs and hGGPPs inhibitors derived from the nitrogen-containing bisphosphonates (N-BP) were tested on human SH-SY5Y neurons and their effect on the accumulation of tau and Phospho-tau was measured as a function of time and concentrations. Results: In autopsied frontal cortex, we found a significant positive correlation
P659
between mRNA levels (p<0.001, n¼124), FFP and GGPP (p<0.05, n¼116) and disease status. The levels of mRNA for hFFPs and hGGPPs also correlated with the amount of phospho-tau protein levels (p<0.05, n¼38) and neurofibrillary tangles density (p<0.006, n¼40) in frontal cortex. We also found that high levels of hFPPS and hGGPPS mRNA prevalence is significantly associated with earlier age of onset in AD (p<0.05, n¼64). Finally, preliminary data indicates that our novel families of inhibitors can decrease phospho-tau levels in SH-SY5Y neurons without any detectable toxicity. Conclusions: Together, these results suggest that accumulation of phospho-tau in the AD brain is related to increase levels of neuronal isoprenoids; a phenomenon that could be alleviated by the administration of specific and potent bi-phosphonate derivatives. supported by the J.L. Levesques Foundation, Canadian Institute of Health Research and FQRNT.
P2-125
ANALYSIS OF WILD TYPE AND R47H MUTANT TREM2 EXPRESSION IN ALZHEIMER’S DISEASE BRAINS
Daniel Sevlever, Mariet Younkin, Li Ma, Sakae Nobutaka, Gina Bisceglio, Dennis W. Dickson, Nilufer Ertekin-Taner, Steven G. Younkin, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Genetic analyses showed that the triggering receptor
expressed in myeloid cells 2 (TREM2) R47H variant increases the risk for AD. In the brain TREM2 is predominantly expressed in microglia. The question of whether the R47H mutation affects expression or function of the receptor remains unanswered. In this study we quantitated mRNA and analyzed protein profiles of WT and R47H TREM2 in human brains to determine if the mutation affects expression or processing of TREM2. Methods: There are at least three TREM2 transcripts that are expressed in human brain including one encoding for a soluble form of the receptor. qRT-PCR was performed using 2 sets of primers one that detects all TREM2 mRNA isoforms and one specific for the alternative spliced isoform (TREM2alt) that encodes for the extracellular domain. All analysis were carried out in R software using linear regression adjusting for Age, Sex, APOEε4dose, qRT-PCR plate, RNA integrity number, and expression levels of AIF1 (as a microglia marker). For TREM2 protein quantitation and N-glycosylation processing, RIPA brain extracts were analyzed by Western blot before and after Endo H and PNGAse F treatments. Results: We identified significantly increased levels of TREM2 transcripts in the temporal cortex of AD subjects when compared with age matched controls (p¼8.8E-03); TREM2alt was likewise higher in AD cases, but did not reach significance (p¼6.9E-02). Among AD subjects (N¼41), we observed suggestively higher TREM2 levels in carriers of the R47H risk allele (N¼18, p¼8.5E-02). Expression levels of TREM2 and TREM2alt were highly correlated in both sets of samples assessed (r2 > 0.72). At the protein level, SDS-gel profiles that included the carboxy-terminal fragment of WT and R47H samples were undistinguishable. Furthermore, TREM2 from either WT or R47H brains was equally susceptible to Endo H and PNGase F treatments. Conclusions: 1) The levels of all TREM2 transcripts are increased in AD. 2) In R47H carriers higher levels of TREM2 transcripts were found, but it failed to reach significance. 3) R47H mutation does not affect processing of TREM2 protein. Our data support previous findings that suggest that the R47H variant affects TREM2 function by altering binding properties of the receptor.