- Email: [email protected]
MICRORNA-140 MEDIATED REGULATION OF ADAM10
Poster Presentations: Sunday, July 16, 2017 Background: The levels of NGF and BDNF neurotrophins are
compromised in Alzheimer’s disease; however, the causes and progressive evolution of these changes are not completely understood. Methods: We used the McGill-R-Thy1-APP rat model of Alzheimer’s disease-like pathology, which progressively accumulate intraneuronal Ab peptide, exhibit amyloid plaques and cognitive deficits to explore the evolution of NGF and BDNF deregulation and the impact on cholinergic synapses during the progression of Ab pathology. Animals belonged to one of three groups: young, pre-plaque, transgenic rats (3-6 months), middle-aged (13-15 months) and old (18-21 months) post-plaque McGill-R-Thy1-APP rats. Age-matched wild type rats, used as controls, were non-transgenic littermates (hAPP negative). Results: Neurochemical analyses revealed a differential dysregulation of the neurotrophins NGF and BDNF in the cortices of McGill-R-Thy1-APP transgenic rats. While BDNF mRNA levels were significantly reduced at very early stages of the amyloid pathology (3-6 months), before amyloid plaques appeared, there were no changes in NGF mRNA expression even at advanced stages. Surprisingly, the protein levels of the NGF precursor, proNGF, were increased despite the normal expression of NGF mRNA. Alterations in the NGF metabolic pathway affecting proNGF maturation, explain the upregulation of proNGF. The alterations in proNGF proteases and regulators were detected in middle-aged (13-15 months) and old (18-21 months) homozygous McGill APP transgenic rats, when extensive intracellular Ab and extracellular amyloid plaques are present. In parallel, intraneuronal Ab leads to significant reductions in BDNF mRNA expression, which further correlate with learning and memory deficits. The reduction in BDNF levels precedes the NGF dysmetabolism and the decline in VAChT-immunoreactive boutons, which occurs at the postplaque stage. Conclusions: The reduction in BDNF mRNA occurs at very early stages of amyloid pathology, before plaques appeared and correlates with cognitive deficits, consistent with human studies (Peng et al., 2005b, Buchman et al., 2016). A marked dysregulation of the NGF metabolic pathway take place at advanced stages with extracellular amyloid plaques, similar to that previously found in the brains of Alzheimer’s disease patients (Bruno et al, 2009a; Bruno et al, 2009b) and in Down syndrome individuals with dementia (Iulita et al, 2014c).
P1-210
SEX DIFFERENCES IN METABOLIC AND NEUROLOGICAL OUTCOMES IN HUMANIZED APOE-ε4 KNOCK-IN RAT MODEL
Aarti Mishra1, Fei Yin1, Zisu Mao1, Roberta Diaz Brinton1,2, 1University of Southern California, Los Angeles, CA, USA; 2University of Arizona, Tucson, AZ, USA. Contact e-mail: [email protected] Background: Women APOE-ε4 carriers are at increased risk of
developing Alzheimer’s disease (AD) than men. Women APOE-ε4 carriers are susceptible to accelerated aging and undergo faster rates of cognitive decline during Mild Cognitive Impairment (MCI). Using a novel rat model with humanized APOE-ε4 gene knock-in, we conducted a longitudinal study spanning 9 months (between age 6-15 months) to characterize effect of sex and APOE-ε4 genotype during the aging process
P323
by evaluating metabolic and neurological outcomes. Methods: We used Sprague Dawley rats with humanized APOE-ε4 knockin from Horizon Discovery. For longitudinal analyses, we conducted micro PET-CT (Positron Emmision TomorgraphyComputed Tomography) to measure glucose uptake and tail vein blood collection to estimate blood based metabolic markersketone bodies, triglycerides, insulin, lipidomics and inflammatory markers. Longitudinal analyses included comparisons between APOE-ε4 male, female rats and Wild type (WT) male, female rats, across the following aging windows: 7-8 months (m), 9-10 m, 12-13 m and 15-16 m. Reproductive cyclicity in females was established by collection of vaginal lavages in APOE-ε4 and WT rats at 6-7 m, 9-10 m and 12-13 m. Results: Longitudinal follow-up revealed that in comparison to WT rats, APOE-ε4 rats had significantly elevated plasma triglyceride levels across all aging windows. Male APOE-ε4 rats had significantly higher triglyceride, ketone body and insulin levels across all aging comparisons. In APOE-ε4 females, age related decline in insulin levels while increase in ketone body levels was evident. Glucose uptake, as determined by micro PET-CT, was significantly higher in APOE-ε4 males than females at 9-10 m and 12-13 m. On undergoing the perimenopausal transition, APOE-ε4 females experienced a significant decline in glucose uptake. Conclusions: This longitudinal study helps in identifying aging windows in APOEε4 carriers, which are predictive of metabolic and neurological changes. Female APOE-ε4 rats demonstrate a state of bioenergetic deficit after the perimenopausal transition. Coincident increase in ketone body levels may reflect a metabolic state, which is shifting to a ketogenic system from a glucogenic system, and may be related to an increase in white matter catabolism. Grant support: National Institute on Aging (NIA) grants R01AG032236 and P01AG026572 to RDB, Paul Slavic Trust and Alzheimer’s association.
P1-211
MICRORNA-140 MEDIATED REGULATION OF ADAM10
Rumana Akhter1, Yvonne Shao1, McKenzie Shaw1, Shane Formica2, Maria Khrestian1, James B. Leverenz3, Lynn M. Bekris1, 1Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA; 2Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA; 3Lou Ruvo Center for Brain Health, Cleveland Clinic, Cleveland, OH, USA. Contact e-mail: bekrisl@ ccf.org Background: Recent reports in Alzheimer’s Disease (AD) research
suggests that alterations in microRNA (miRNA) expression is associated with disease pathology. Our previous studies show that genetic variation in ADAM10 (A Disintegrin and Metalloproteinase 10) domain which encodes the major a-secretase responsible for cleaving Amyloid Precursor Protein (APP) is associated with: higher CSF sAPP a levels in cognitively normal controls compared with AD patients along with higher ADAM10 protein levels in subjects with low plaque scores compared with those with high plaque scores, and higher promoter activity for promoter-only reporter constructs compared with promoter 3’ untranslated region (3’UTR) constructs in the human neuroblastoma SHSY5Y cell line. These findings suggest that ADAM10expression is modulated according to an extended regulatory region that includes the 3’UTR. Methods: In this study we
P324
Poster Presentations: Sunday, July 16, 2017
have investigated the role of trans-acting factors; transcription factor SOX2 and miRNA-140, in ADAM10 gene regulation. The entire sequence of ADAM10 gene is analyzed by computational bioinformatics and screened for probable trans-actors with high regulatory importance. Results: Our study shows that miRNA-140-5p has enhanced expression in AD post-mortem brain hippocampus using high throughput micro-arrays and qRT-PCR. Interestingly we have also seen that miRNA-140 seed sequence is present on both ADAM10 and SOX2 3’UTR and thus reporter constructs containing regulatory elements were transfected into human cell lines along with miRNA-140 mimics and inhibitors to evaluate their interaction by dual luciferase reporter assays. The specific interaction of miRNA-140 with both ADAM10 and its transcription factor SOX2 signifies high regulatory importance of this miRNA in controlling ADAM10 expression. Conclusions: Thus our proposed investigation unravels the mechanisms underlying ADAM10 downregulation by miR-140 that exacerbates Alzheimer’s disease related neurotoxic effects. Our findings could reflect a strong basis for future research aimed at understanding the potential contribution of trans-acting factors as a biomarker or modulator of AD pathophysiology.
P1-212
CEREBROSPINAL FLUID LEVELS OF ANGIOTENSIN-CONVERTING ENZYME ARE ASSOCIATED WITH AMYLOID-b IN ALZHEIMER’S DISEASE
Natalia P. Rocha1,2, Andre ASF. Toledo2, Laiane TS. Corgosinho2, Leonardo Cruz de Souza2, Elisa de Paula Franc¸a Resende2, Nayara FT. Braz2, Ana C. Simoes e Silva2, Paulo Caramelli2, Antonio L. Teixeira2,3, 1The University of Texas Health Science Center at Houston, Houston, TX, USA; 2Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3The University of Texas Health Science Center at Houston, Houston, TX, USA. Contact e-mail: [email protected] Background: Although the renin-angiotensin system (RAS) is traditionally regarded as a system that maintains blood pressure and fluid homeostasis, a range of evidence points to its role in different cerebral functions. For instance, all RAS components are found in the central nervous system. Experimental and epidemiological studies indicate that both angiotensin receptor type 1 (AT1) antagonists and angiotensin converting enzyme
(ACE) inhibitors may have beneficial effects for Alzheimer’s disease (AD). In the current study, we aimed to determine cerebrospinal (CSF) levels of RAS components in patients with AD and controls. We also investigated potential associations
Table 1 Clinical characteristics and cerebrospinal fluid levels of angiotensin-converting enzyme (ACE), amyloid-b, phosphorylated Tau (pTau) and Tau protein (hTau) in Alzheimer’s disease (AD) patients and controls.
Age in years, mean 6 SD (median) Female sex, N (%) Educational level MMSE, mean 6 SD (median) Ab40 (pg/mL), mean 6 SD (median) Ab42 (pg/mL), mean 6 SD (median) hTau (pg/mL), mean 6 SD (median) pTau (pg/mL), mean 6 SD (median) ACE (pg/mL), mean 6 SD (median) ACE2 (pg/mL), mean 6 SD (median) IATI, mean 6 SD (median) hTau/Ab42, mean 6 SD (median) pTau/Ab42, mean 6 SD (median)
Controls (n ¼ 10)
AD (n ¼ 18)
P value
69.0069.45 (65.50) 5 (50%) 3.4461.81 (3.00) 25.4463.13 (27.00) 1208.846732.42 (914.61) 1183.256248.30 (1275.70) 275.27676.44 (263.16) 52.77615.36 (48.41) 53.5564.01 (53.81) 12.4565.56 (53.81) 12.4565.56 (11.75) 0.2460.06 (0.24) 0.0560.01 (0.05)
66.6968.75 (67.50) 9 (50%) 8.9265.06 (8.00) 20.1567.82 (23.00) 638.046508.93 (549.78) 513.856225.22 (504.99) 1096.056957.24 (809.84) 106.73649.09 (104.67) 49.6965.01 (49.55) 13.9064.92 (13.67) 0.4260.17 (0.45) 2.1261.67 (1.53) 0.2160.07 (0.19)
0.531a 1.000b 0.003c 0.071c 0.020a <0.0001c <0.0001c 0.002a 0.046a 0.477a <0.0001a <0.0001c <0.0001c
MMSE ¼ mini-mental state examination, IATI ¼ Innotest Amyloid Tau Index a Student’s t test; bFisher’s exact test; cMann-Whitney test. Significant values are highlighted in bold.
compromised in Alzheimer’s disease; however, the causes and progressive evolution of these changes are not completely understood. Methods: We used the McGill-R-Thy1-APP rat model of Alzheimer’s disease-like pathology, which progressively accumulate intraneuronal Ab peptide, exhibit amyloid plaques and cognitive deficits to explore the evolution of NGF and BDNF deregulation and the impact on cholinergic synapses during the progression of Ab pathology. Animals belonged to one of three groups: young, pre-plaque, transgenic rats (3-6 months), middle-aged (13-15 months) and old (18-21 months) post-plaque McGill-R-Thy1-APP rats. Age-matched wild type rats, used as controls, were non-transgenic littermates (hAPP negative). Results: Neurochemical analyses revealed a differential dysregulation of the neurotrophins NGF and BDNF in the cortices of McGill-R-Thy1-APP transgenic rats. While BDNF mRNA levels were significantly reduced at very early stages of the amyloid pathology (3-6 months), before amyloid plaques appeared, there were no changes in NGF mRNA expression even at advanced stages. Surprisingly, the protein levels of the NGF precursor, proNGF, were increased despite the normal expression of NGF mRNA. Alterations in the NGF metabolic pathway affecting proNGF maturation, explain the upregulation of proNGF. The alterations in proNGF proteases and regulators were detected in middle-aged (13-15 months) and old (18-21 months) homozygous McGill APP transgenic rats, when extensive intracellular Ab and extracellular amyloid plaques are present. In parallel, intraneuronal Ab leads to significant reductions in BDNF mRNA expression, which further correlate with learning and memory deficits. The reduction in BDNF levels precedes the NGF dysmetabolism and the decline in VAChT-immunoreactive boutons, which occurs at the postplaque stage. Conclusions: The reduction in BDNF mRNA occurs at very early stages of amyloid pathology, before plaques appeared and correlates with cognitive deficits, consistent with human studies (Peng et al., 2005b, Buchman et al., 2016). A marked dysregulation of the NGF metabolic pathway take place at advanced stages with extracellular amyloid plaques, similar to that previously found in the brains of Alzheimer’s disease patients (Bruno et al, 2009a; Bruno et al, 2009b) and in Down syndrome individuals with dementia (Iulita et al, 2014c).
P1-210
SEX DIFFERENCES IN METABOLIC AND NEUROLOGICAL OUTCOMES IN HUMANIZED APOE-ε4 KNOCK-IN RAT MODEL
Aarti Mishra1, Fei Yin1, Zisu Mao1, Roberta Diaz Brinton1,2, 1University of Southern California, Los Angeles, CA, USA; 2University of Arizona, Tucson, AZ, USA. Contact e-mail: [email protected] Background: Women APOE-ε4 carriers are at increased risk of
developing Alzheimer’s disease (AD) than men. Women APOE-ε4 carriers are susceptible to accelerated aging and undergo faster rates of cognitive decline during Mild Cognitive Impairment (MCI). Using a novel rat model with humanized APOE-ε4 gene knock-in, we conducted a longitudinal study spanning 9 months (between age 6-15 months) to characterize effect of sex and APOE-ε4 genotype during the aging process
P323
by evaluating metabolic and neurological outcomes. Methods: We used Sprague Dawley rats with humanized APOE-ε4 knockin from Horizon Discovery. For longitudinal analyses, we conducted micro PET-CT (Positron Emmision TomorgraphyComputed Tomography) to measure glucose uptake and tail vein blood collection to estimate blood based metabolic markersketone bodies, triglycerides, insulin, lipidomics and inflammatory markers. Longitudinal analyses included comparisons between APOE-ε4 male, female rats and Wild type (WT) male, female rats, across the following aging windows: 7-8 months (m), 9-10 m, 12-13 m and 15-16 m. Reproductive cyclicity in females was established by collection of vaginal lavages in APOE-ε4 and WT rats at 6-7 m, 9-10 m and 12-13 m. Results: Longitudinal follow-up revealed that in comparison to WT rats, APOE-ε4 rats had significantly elevated plasma triglyceride levels across all aging windows. Male APOE-ε4 rats had significantly higher triglyceride, ketone body and insulin levels across all aging comparisons. In APOE-ε4 females, age related decline in insulin levels while increase in ketone body levels was evident. Glucose uptake, as determined by micro PET-CT, was significantly higher in APOE-ε4 males than females at 9-10 m and 12-13 m. On undergoing the perimenopausal transition, APOE-ε4 females experienced a significant decline in glucose uptake. Conclusions: This longitudinal study helps in identifying aging windows in APOEε4 carriers, which are predictive of metabolic and neurological changes. Female APOE-ε4 rats demonstrate a state of bioenergetic deficit after the perimenopausal transition. Coincident increase in ketone body levels may reflect a metabolic state, which is shifting to a ketogenic system from a glucogenic system, and may be related to an increase in white matter catabolism. Grant support: National Institute on Aging (NIA) grants R01AG032236 and P01AG026572 to RDB, Paul Slavic Trust and Alzheimer’s association.
P1-211
MICRORNA-140 MEDIATED REGULATION OF ADAM10
Rumana Akhter1, Yvonne Shao1, McKenzie Shaw1, Shane Formica2, Maria Khrestian1, James B. Leverenz3, Lynn M. Bekris1, 1Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA; 2Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA; 3Lou Ruvo Center for Brain Health, Cleveland Clinic, Cleveland, OH, USA. Contact e-mail: bekrisl@ ccf.org Background: Recent reports in Alzheimer’s Disease (AD) research
suggests that alterations in microRNA (miRNA) expression is associated with disease pathology. Our previous studies show that genetic variation in ADAM10 (A Disintegrin and Metalloproteinase 10) domain which encodes the major a-secretase responsible for cleaving Amyloid Precursor Protein (APP) is associated with: higher CSF sAPP a levels in cognitively normal controls compared with AD patients along with higher ADAM10 protein levels in subjects with low plaque scores compared with those with high plaque scores, and higher promoter activity for promoter-only reporter constructs compared with promoter 3’ untranslated region (3’UTR) constructs in the human neuroblastoma SHSY5Y cell line. These findings suggest that ADAM10expression is modulated according to an extended regulatory region that includes the 3’UTR. Methods: In this study we
P324
Poster Presentations: Sunday, July 16, 2017
have investigated the role of trans-acting factors; transcription factor SOX2 and miRNA-140, in ADAM10 gene regulation. The entire sequence of ADAM10 gene is analyzed by computational bioinformatics and screened for probable trans-actors with high regulatory importance. Results: Our study shows that miRNA-140-5p has enhanced expression in AD post-mortem brain hippocampus using high throughput micro-arrays and qRT-PCR. Interestingly we have also seen that miRNA-140 seed sequence is present on both ADAM10 and SOX2 3’UTR and thus reporter constructs containing regulatory elements were transfected into human cell lines along with miRNA-140 mimics and inhibitors to evaluate their interaction by dual luciferase reporter assays. The specific interaction of miRNA-140 with both ADAM10 and its transcription factor SOX2 signifies high regulatory importance of this miRNA in controlling ADAM10 expression. Conclusions: Thus our proposed investigation unravels the mechanisms underlying ADAM10 downregulation by miR-140 that exacerbates Alzheimer’s disease related neurotoxic effects. Our findings could reflect a strong basis for future research aimed at understanding the potential contribution of trans-acting factors as a biomarker or modulator of AD pathophysiology.
P1-212
CEREBROSPINAL FLUID LEVELS OF ANGIOTENSIN-CONVERTING ENZYME ARE ASSOCIATED WITH AMYLOID-b IN ALZHEIMER’S DISEASE
Natalia P. Rocha1,2, Andre ASF. Toledo2, Laiane TS. Corgosinho2, Leonardo Cruz de Souza2, Elisa de Paula Franc¸a Resende2, Nayara FT. Braz2, Ana C. Simoes e Silva2, Paulo Caramelli2, Antonio L. Teixeira2,3, 1The University of Texas Health Science Center at Houston, Houston, TX, USA; 2Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3The University of Texas Health Science Center at Houston, Houston, TX, USA. Contact e-mail: [email protected] Background: Although the renin-angiotensin system (RAS) is traditionally regarded as a system that maintains blood pressure and fluid homeostasis, a range of evidence points to its role in different cerebral functions. For instance, all RAS components are found in the central nervous system. Experimental and epidemiological studies indicate that both angiotensin receptor type 1 (AT1) antagonists and angiotensin converting enzyme
(ACE) inhibitors may have beneficial effects for Alzheimer’s disease (AD). In the current study, we aimed to determine cerebrospinal (CSF) levels of RAS components in patients with AD and controls. We also investigated potential associations
Table 1 Clinical characteristics and cerebrospinal fluid levels of angiotensin-converting enzyme (ACE), amyloid-b, phosphorylated Tau (pTau) and Tau protein (hTau) in Alzheimer’s disease (AD) patients and controls.
Age in years, mean 6 SD (median) Female sex, N (%) Educational level MMSE, mean 6 SD (median) Ab40 (pg/mL), mean 6 SD (median) Ab42 (pg/mL), mean 6 SD (median) hTau (pg/mL), mean 6 SD (median) pTau (pg/mL), mean 6 SD (median) ACE (pg/mL), mean 6 SD (median) ACE2 (pg/mL), mean 6 SD (median) IATI, mean 6 SD (median) hTau/Ab42, mean 6 SD (median) pTau/Ab42, mean 6 SD (median)
Controls (n ¼ 10)
AD (n ¼ 18)
P value
69.0069.45 (65.50) 5 (50%) 3.4461.81 (3.00) 25.4463.13 (27.00) 1208.846732.42 (914.61) 1183.256248.30 (1275.70) 275.27676.44 (263.16) 52.77615.36 (48.41) 53.5564.01 (53.81) 12.4565.56 (53.81) 12.4565.56 (11.75) 0.2460.06 (0.24) 0.0560.01 (0.05)
66.6968.75 (67.50) 9 (50%) 8.9265.06 (8.00) 20.1567.82 (23.00) 638.046508.93 (549.78) 513.856225.22 (504.99) 1096.056957.24 (809.84) 106.73649.09 (104.67) 49.6965.01 (49.55) 13.9064.92 (13.67) 0.4260.17 (0.45) 2.1261.67 (1.53) 0.2160.07 (0.19)
0.531a 1.000b 0.003c 0.071c 0.020a <0.0001c <0.0001c 0.002a 0.046a 0.477a <0.0001a <0.0001c <0.0001c
MMSE ¼ mini-mental state examination, IATI ¼ Innotest Amyloid Tau Index a Student’s t test; bFisher’s exact test; cMann-Whitney test. Significant values are highlighted in bold.