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Protecting against the outcome of taupathy: NAP – from concept to the clinic
Abstracts / Neuropeptides 40 (2006) 417–445
The melanin-concentrating hormone-1 receptor (MCHR1) may present a promising novel mechanism that modulates food consumption, stress responses and affective states. The role of MCHR1 in neuroendocrine, behavioral and neurochemical stress and anxiety-related responses was examined by monitoring the effects of melanin-concentrating hormone (MCH) and the selective MCHR1 antagonist, GW3430, in inbred C57Bl/6NTac and MCHR1-knockout (KO) and wild-type (WT) mice. Intracerebroventricular injection of MCH increased plasma ACTH and corticosterone, and produced anxiety-related responses in the elevated plus maze. The selective MCHR1 antagonist, GW3430, blocked the neuroendocrine and behavioral effects of MCH and produced anxiolytic-like effects by itself in distinct, validated animal models of anxiety. Moreover, MCHR1KO mice had an anxiolytic-like phenotype in behavioral models of anxiety, and GW3430 had anxiolytic-like effects in WT, but not MCHR1-KO mice. In addition, predator-odor stress evoked acetylcholine release within the prefrontal cortex of WT mice and MCHR1-KO mice. GW3430 reduced the increase seen in the WT but not the MCHR1-KO. In feeding studies, GW3430 selectively decreased food intake in ob/ob mice. We show that MCH elicits anxiety-like responses and that the effects of a selective MCHR1 antagonist and the phenotype of MCHR1-KO mice are consistent with anxiolytic-like action. Distinct behavioral, physiological and neurochemical stress and anxiety-related responses were selectively modulated by the MCHR1, and these actions may involve limbic cortical regulation of stress and anxiety processing centers. In addition, a specific MCHR1 antagonist decreased food intake in ob/ob mice. These results are consistent with a role for MCHR1 antagonists for the treatment of psychiatric disorders and obesity. doi:10.1016/j.npep.2006.09.015
OBESITY IN THE CARBOXYPEPTIDASE E KNOCKOUT MOUSE N.X. Cawley, M. Sridhar, T. Yanik, Y.P. Loh; Section on Cellular Neurobiology, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, United States Carboxypeptidase E (CPE) is an exopeptidase responsible for the removal of carboxy-terminally extended arginines and lysines from peptide hormone and neuropeptide intermediates in the regulated secretory pathway. The absence of CPE would therefore be expected to have many endocrine, neuro-endocrine and central defects. The CPE knockout (KO) mouse
429
has shown several such defects including diabetes, infertility and obesity in addition to developmental defects and deficits in learning and memory. The obesity phenotype affected both male and female mice, however; the females were affected more severely as their rate of weight gain exceeded those of the males. Weight gain, which was due primarily to fat accumulation, appears to be a consequence of several factors. The animals were less active, had a lower basal metabolic rate as measured by indirect calorimetry, had a lower respiratory quotient and were hyperphagic. The control of eating behavior is complex, involving communications between the endocrine system and higher brain functions mediated by the hypothalamus. We analyzed a number of neuropeptides in the hypothalamus to try to understand if and where any breakdown in the signaling of this pathway could account for the hyperphagic behavior. Gene array and quantitative PCR analyses revealed that levels of POMC expression are similar in both WT and KO animals, indicating that the metabolism of POMC (the precursor to alpha-MSH, an anorectic peptide) was normal. Indeed, Western blot analysis of hypothalamic extracts revealed similar levels of POMC between WT and KO mice. This is surprising because POMC expression is normally upregulated by leptin, which in the KO mice is significantly elevated (4-fold) compared to WT mice. This apparent disconnection between leptin and POMC expression may be mediated by low levels of the leptin receptor; however, expression of the leptin receptor in the hypothalamus was significantly upregulated and suggests a possible defect in leptin receptor signaling. doi:10.1016/j.npep.2006.09.016
ADNP, NAP & ADNF-9 – DEVELOPMENTAL DISORDERS AND ALZHEIMER’S DISEASE Chair: Illana Gozes, Israel, Douglas E. Brenneman, USA PROTECTING AGAINST THE OUTCOME OF TAUPATHY: NAP – FROM CONCEPT TO THE CLINIC I. Gozes a,1, Natalia Shiryaev a, Yan Jouroukhim a, Inna Vulih a, Albert Pinhasov a, Hanna Rosenmann b; a Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; b Hadassah Medical School, The Hebrew University, Jerusalem, Israel
1
I. Gozes holds the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and serves as the Chief Scientific Officer of Allon Therapeutics Inc.
430
Abstracts / Neuropeptides 40 (2006) 417–445
NAP is an eight amino acid peptide derived from activity-dependent neuroprotective protein (ADNP). Complete ADNP deficiency results in neural tube closure defects and embryonic lethality [Brain Res. Dev. Brain Res. 2003, Aug 12;144(1):83–90]. Partial ADNP deficiency (±heterozygote) is associated with increased tau phosphorylation which may be mediated by glycogen synthase kinase-3b. ADNP may interact with tubulin and NAP enhances microtubule assembly (J. Neurochem., in press) thereby protecting neurons in vitro and in vivo. As microtubule breakdown and tau hyperphosphorylation are the main pathological characteristics of AD we chose to evaluate NAP activity in a taupathy model. Tau transgenic mice that express the human double mutant tau protein [P301S;K257T] regulated by the original tau promoter provide an authentic animal model for tauopathy and AD (Rosenmann et al., 2004 Neural Plasticity, 12, 51). Daily treatment (5 days a week) of both wild type and tau transgenic male mice with intranasal NAP for 4.5 months resulted in improvement in short-term memory at 6.5 months of age (n P 8/ group). Probe test assessing spatial memory showed a statistically significant difference between NAP-treated and vehicle-treated tau transgenic mice (P < 0.05). When exposed to an open field for 3 min NAP treatment significantly reduced the number of fecal boli and liquid excretion points (representing an anxiety measure) in the tau transgenic male mice (P < 0.01). In the elevated plus maze, the frequency of entrance to the open arms was significantly higher in the tau transgenic NAP-treated mice as compared to vehicletreated tau transgenics. Furthermore, the relative amount of time spent in the closed arms was significantly lower in the NAP-treated mice. At 6.5 month there were no motor deficits in the tau transgenic male mice as measured in the staircase test and the Morris water maze. Motor coordination measured in the rotarod test indicated that the tau transgenic male mice exhibited reduced coordination at 9 month of age. Treatment with NAP partially ameliorated this deficit. Taken together, our results show that chronic treatment with NAP significantly reduced anxiety and enhanced cognitive performance in the tau transgenic mice. NAP is currently entering phase II clinical trials by Allon Therapeutics Inc. (Gozes I, Morimoto BH, Tiong J, Fox A, Sutherland K, Dangoor D, Holser-Cochav M, Vered K, Newton P, Aisen PS, Matsuoka Y, van Dyck CH, Thal L. CNS Drug Rev. 2005;11(4):353–368)’’. Support: NICHD, NIA, BSF, ISF, ISOA, Gildor Chair, Dr. Diana and Zelman Elton (Elbaum) Laboratory, Allon Therapeutics Inc. doi:10.1016/j.npep.2006.09.017
REVEALING THE ROLE OF ACTIVITY DEPENDENT NEUROPROTECTIVE PROTEIN DURING EMBRYOGENESIS S. Mandel a, I. Gozes a,b,1; a Human Molecular Genetics and Biochemistry, Israel; b Sackler Med. Sch., Tel Aviv University, Israel Activity dependent neuroprotective protein (ADNP) is a VIP responsive protein associated with its neuroprotective cascade (J. Neurochem., 1999, 72, 1283). ADNP contains an eight amino acid peptide (NAP) that was shown to possess neuroprotective properties probably through interaction with tubulin (J. Biol. Chem. 2004; 279(27):28531–28538; J. Alzheimer’s Dis. 2004; 6: S37– 41; J. Neurosci. in press). ADNP has been implicated in cell protective activities in vitro and is an essential protein for embryogenesis and brain formation (Brain Res. 2003; 144:83). ADNP null embryos die at day 9–9.5 of gestation, a time by which a series of major developmental events take place. The current study set out to reveal the specific pathways through which ADNP operates during embryonic development. A 22690 probe-Affymetrix gene array was used in order to compare gene expression patterns of ADNP knock out embryos to normal and heterozygous littermates. Data were analyzed using the Affymetrix web tools and the novel expender and prima software (Elkon et al. Genome Res. 2003, 13, 773). Data analysis enabled the partial deduction of a presumed mechanism by which ADNP deficiency may cause lethality as follows. The lack of ADNP expression resulted in a dramatic down-regulation of a number of essential genes associated with neurogenesis such as neurogenin1, neuroD1 and galanin as well as genes that are associated with heart development such as Myosin light chain 2. On the other hand, we showed a dramatic upregulation of genes associated with the following functional groups: lipid transport and metabolism, lytic vacuole and coagulation all associated with extra embryonic activity a tissue that was separated from the embryos. Most of the dramatically upregulated genes were not expected to be expressed in the embryonic body at this developmental stage. Results implicate ADNP as a repressor of a set of extra embryonic endoderm associated genes in the embryonic developing body, thus enabling proper embryogenesis. doi:10.1016/j.npep.2006.09.018
1
I. Gozes holds the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and serves as the Chief Scientific Officer of Allon Therapeutics Inc. that develops NAP (AL-108 & AL208) for clinical use in Alzheimer’s disease and mild cognitive impairment. Supported, in part, by ISF, BSF and Allon Therapeutics.
The melanin-concentrating hormone-1 receptor (MCHR1) may present a promising novel mechanism that modulates food consumption, stress responses and affective states. The role of MCHR1 in neuroendocrine, behavioral and neurochemical stress and anxiety-related responses was examined by monitoring the effects of melanin-concentrating hormone (MCH) and the selective MCHR1 antagonist, GW3430, in inbred C57Bl/6NTac and MCHR1-knockout (KO) and wild-type (WT) mice. Intracerebroventricular injection of MCH increased plasma ACTH and corticosterone, and produced anxiety-related responses in the elevated plus maze. The selective MCHR1 antagonist, GW3430, blocked the neuroendocrine and behavioral effects of MCH and produced anxiolytic-like effects by itself in distinct, validated animal models of anxiety. Moreover, MCHR1KO mice had an anxiolytic-like phenotype in behavioral models of anxiety, and GW3430 had anxiolytic-like effects in WT, but not MCHR1-KO mice. In addition, predator-odor stress evoked acetylcholine release within the prefrontal cortex of WT mice and MCHR1-KO mice. GW3430 reduced the increase seen in the WT but not the MCHR1-KO. In feeding studies, GW3430 selectively decreased food intake in ob/ob mice. We show that MCH elicits anxiety-like responses and that the effects of a selective MCHR1 antagonist and the phenotype of MCHR1-KO mice are consistent with anxiolytic-like action. Distinct behavioral, physiological and neurochemical stress and anxiety-related responses were selectively modulated by the MCHR1, and these actions may involve limbic cortical regulation of stress and anxiety processing centers. In addition, a specific MCHR1 antagonist decreased food intake in ob/ob mice. These results are consistent with a role for MCHR1 antagonists for the treatment of psychiatric disorders and obesity. doi:10.1016/j.npep.2006.09.015
OBESITY IN THE CARBOXYPEPTIDASE E KNOCKOUT MOUSE N.X. Cawley, M. Sridhar, T. Yanik, Y.P. Loh; Section on Cellular Neurobiology, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, United States Carboxypeptidase E (CPE) is an exopeptidase responsible for the removal of carboxy-terminally extended arginines and lysines from peptide hormone and neuropeptide intermediates in the regulated secretory pathway. The absence of CPE would therefore be expected to have many endocrine, neuro-endocrine and central defects. The CPE knockout (KO) mouse
429
has shown several such defects including diabetes, infertility and obesity in addition to developmental defects and deficits in learning and memory. The obesity phenotype affected both male and female mice, however; the females were affected more severely as their rate of weight gain exceeded those of the males. Weight gain, which was due primarily to fat accumulation, appears to be a consequence of several factors. The animals were less active, had a lower basal metabolic rate as measured by indirect calorimetry, had a lower respiratory quotient and were hyperphagic. The control of eating behavior is complex, involving communications between the endocrine system and higher brain functions mediated by the hypothalamus. We analyzed a number of neuropeptides in the hypothalamus to try to understand if and where any breakdown in the signaling of this pathway could account for the hyperphagic behavior. Gene array and quantitative PCR analyses revealed that levels of POMC expression are similar in both WT and KO animals, indicating that the metabolism of POMC (the precursor to alpha-MSH, an anorectic peptide) was normal. Indeed, Western blot analysis of hypothalamic extracts revealed similar levels of POMC between WT and KO mice. This is surprising because POMC expression is normally upregulated by leptin, which in the KO mice is significantly elevated (4-fold) compared to WT mice. This apparent disconnection between leptin and POMC expression may be mediated by low levels of the leptin receptor; however, expression of the leptin receptor in the hypothalamus was significantly upregulated and suggests a possible defect in leptin receptor signaling. doi:10.1016/j.npep.2006.09.016
ADNP, NAP & ADNF-9 – DEVELOPMENTAL DISORDERS AND ALZHEIMER’S DISEASE Chair: Illana Gozes, Israel, Douglas E. Brenneman, USA PROTECTING AGAINST THE OUTCOME OF TAUPATHY: NAP – FROM CONCEPT TO THE CLINIC I. Gozes a,1, Natalia Shiryaev a, Yan Jouroukhim a, Inna Vulih a, Albert Pinhasov a, Hanna Rosenmann b; a Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; b Hadassah Medical School, The Hebrew University, Jerusalem, Israel
1
I. Gozes holds the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and serves as the Chief Scientific Officer of Allon Therapeutics Inc.
430
Abstracts / Neuropeptides 40 (2006) 417–445
NAP is an eight amino acid peptide derived from activity-dependent neuroprotective protein (ADNP). Complete ADNP deficiency results in neural tube closure defects and embryonic lethality [Brain Res. Dev. Brain Res. 2003, Aug 12;144(1):83–90]. Partial ADNP deficiency (±heterozygote) is associated with increased tau phosphorylation which may be mediated by glycogen synthase kinase-3b. ADNP may interact with tubulin and NAP enhances microtubule assembly (J. Neurochem., in press) thereby protecting neurons in vitro and in vivo. As microtubule breakdown and tau hyperphosphorylation are the main pathological characteristics of AD we chose to evaluate NAP activity in a taupathy model. Tau transgenic mice that express the human double mutant tau protein [P301S;K257T] regulated by the original tau promoter provide an authentic animal model for tauopathy and AD (Rosenmann et al., 2004 Neural Plasticity, 12, 51). Daily treatment (5 days a week) of both wild type and tau transgenic male mice with intranasal NAP for 4.5 months resulted in improvement in short-term memory at 6.5 months of age (n P 8/ group). Probe test assessing spatial memory showed a statistically significant difference between NAP-treated and vehicle-treated tau transgenic mice (P < 0.05). When exposed to an open field for 3 min NAP treatment significantly reduced the number of fecal boli and liquid excretion points (representing an anxiety measure) in the tau transgenic male mice (P < 0.01). In the elevated plus maze, the frequency of entrance to the open arms was significantly higher in the tau transgenic NAP-treated mice as compared to vehicletreated tau transgenics. Furthermore, the relative amount of time spent in the closed arms was significantly lower in the NAP-treated mice. At 6.5 month there were no motor deficits in the tau transgenic male mice as measured in the staircase test and the Morris water maze. Motor coordination measured in the rotarod test indicated that the tau transgenic male mice exhibited reduced coordination at 9 month of age. Treatment with NAP partially ameliorated this deficit. Taken together, our results show that chronic treatment with NAP significantly reduced anxiety and enhanced cognitive performance in the tau transgenic mice. NAP is currently entering phase II clinical trials by Allon Therapeutics Inc. (Gozes I, Morimoto BH, Tiong J, Fox A, Sutherland K, Dangoor D, Holser-Cochav M, Vered K, Newton P, Aisen PS, Matsuoka Y, van Dyck CH, Thal L. CNS Drug Rev. 2005;11(4):353–368)’’. Support: NICHD, NIA, BSF, ISF, ISOA, Gildor Chair, Dr. Diana and Zelman Elton (Elbaum) Laboratory, Allon Therapeutics Inc. doi:10.1016/j.npep.2006.09.017
REVEALING THE ROLE OF ACTIVITY DEPENDENT NEUROPROTECTIVE PROTEIN DURING EMBRYOGENESIS S. Mandel a, I. Gozes a,b,1; a Human Molecular Genetics and Biochemistry, Israel; b Sackler Med. Sch., Tel Aviv University, Israel Activity dependent neuroprotective protein (ADNP) is a VIP responsive protein associated with its neuroprotective cascade (J. Neurochem., 1999, 72, 1283). ADNP contains an eight amino acid peptide (NAP) that was shown to possess neuroprotective properties probably through interaction with tubulin (J. Biol. Chem. 2004; 279(27):28531–28538; J. Alzheimer’s Dis. 2004; 6: S37– 41; J. Neurosci. in press). ADNP has been implicated in cell protective activities in vitro and is an essential protein for embryogenesis and brain formation (Brain Res. 2003; 144:83). ADNP null embryos die at day 9–9.5 of gestation, a time by which a series of major developmental events take place. The current study set out to reveal the specific pathways through which ADNP operates during embryonic development. A 22690 probe-Affymetrix gene array was used in order to compare gene expression patterns of ADNP knock out embryos to normal and heterozygous littermates. Data were analyzed using the Affymetrix web tools and the novel expender and prima software (Elkon et al. Genome Res. 2003, 13, 773). Data analysis enabled the partial deduction of a presumed mechanism by which ADNP deficiency may cause lethality as follows. The lack of ADNP expression resulted in a dramatic down-regulation of a number of essential genes associated with neurogenesis such as neurogenin1, neuroD1 and galanin as well as genes that are associated with heart development such as Myosin light chain 2. On the other hand, we showed a dramatic upregulation of genes associated with the following functional groups: lipid transport and metabolism, lytic vacuole and coagulation all associated with extra embryonic activity a tissue that was separated from the embryos. Most of the dramatically upregulated genes were not expected to be expressed in the embryonic body at this developmental stage. Results implicate ADNP as a repressor of a set of extra embryonic endoderm associated genes in the embryonic developing body, thus enabling proper embryogenesis. doi:10.1016/j.npep.2006.09.018
1
I. Gozes holds the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and serves as the Chief Scientific Officer of Allon Therapeutics Inc. that develops NAP (AL-108 & AL208) for clinical use in Alzheimer’s disease and mild cognitive impairment. Supported, in part, by ISF, BSF and Allon Therapeutics.