- Email: [email protected]
Pathophysiological role of proteolytic cleavage of neuroligins
P144
Oral Sessions: O1-09: Biomarkers: Clinical Implications
mechanism known as the unfolded protein response (UPR). When unfolded proteins accumulate in the endoplasmic reticulum (ER), UPR aims to restore cellular homeostasis. However, if stress is prolonged, it induces cell death and, thus, neurodegeneration. We investigated the order of events and the impact of UPR activation in Alzheimer’s disease (AD) model systems. Specifically, we developed an in vitro model system of human neuroblastoma cells (SK-N-SH) that overexpress wild type (WT) amyloid-b precursor protein (APP) or familial AD (FAD)-associated mutants, Swedish (S) and Swedish/Indiana (S/I) forms. Methods: We ’artificially’ induced ER stress in differentiated cells using tunicamycin at eight time points and assessed their viability. Subsequently, Real time PCR (RT-PCR) was used to analyse the gene expression levels of eight UPR markers downstream of all three receptors (IRE1, PERK, and ATF6). This allowed us to examine which ER stress pathways are activated and when in the three model systems. Results: Our data demonstrate that all cells activate stress post tunicamycin treatment. However, only the WT cells recover. More specifically, WT cells show stress activation in terms of ATF4 at three hours. ATF4 is part of the PERK pathway and functions by promoting translational attenuation, which is essential for cell survival. In mutant cells this activation occurs at four hours which we hypothesise that is too late for the UPR mechanism to promote cell survival and the cells are possibly already fated to apoptosis. We also have evidence that the S mutation is more toxic than the S/I or WT APP overexpression. S cells exhibit higher levels of stress as seen from the mRNA levels of their respective markers and appear to be more susceptible to apoptosis. Conclusions: Overall, there is strong evidence that UPR is involved in the progression of AD. The data support the hypothesis that an initial activation of the UPR in AD neurons might work as neuroprotective to restore homeostasis while its sustained activation could induce neurodegeneration. Future studies need to address the therapeutic opportunities of this pathway for the treatment of AD.
sults: To assess the in vivo function of zebrafish Bace1 (zBace1) we generated zBace1 knock out fish by zinc finger nuclease mediated genome editing. bace1 mutants (bace1 -/-) are hypomyelinated in the PNS while the CNS is not affected. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1 -/-. Double homozygous bace1 -/-; bace2 -/- fish do not enhance the single mutant phenotypes indicating non-redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile. To understand the function of BACE1 in myelination we investigated proteolytic processing of Neuregulin 1 (NRG1) type III and demonstrate that the ectodomain can be cleaved by three different sheddases, namely ADAM10, ADAM17, and BACE1. Surprisingly, we not only found cleavage by ADAM10, ADAM17 and BACE1 C-terminal to the epidermal growth factor (EGF)-like domain, which is believed to play a pivotal role in signaling, but also additional cleavage sites for ADAM17 and BACE1 N-terminal to that domain. Proteolytic processing N- and C-terminal of the EGF-like domain results in the secretion of this domain from NRG1 type III. The soluble EGF-like domain is functionally active and stimulates ErbB receptor phosphorylation in tissue culture assays. Moreover, the soluble EGF-like domain is capable to rescue hypomyelination in bace1 -/- zebrafish suggesting that NRG1 type III dependent myelination is not only controlled by membrane retained NRG1 type III but also in a paracrine manner via proteolytic liberation of the EGF-like domain. Conclusions: The identification of a specific bace2 -/associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo. Moreover, our findings not only demonstrate a pivotal function of BACE1 in myelination, but also show that this function is restricted to the PNS.
O1-08-06
PATHOPHYSIOLOGICAL ROLE OF PROTEOLYTIC CLEAVAGE OF NEUROLIGINS
Taisuke Tomita, Kunimichi Suzuki, Azusa Shiohara, Satoko Osawa, Takeshi Iwatsubo, University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected]
O1-08-05
DISTINCT FUNCTIONS OF BACE1 AND BACE2 IN NEUREGULIN PROCESSING AND MELANOCYTE MIGRATION
Christian Haass1, Frauke van Bebber2, Alexander Hruscha2, Michael Willem3, Bettina Schmid2, 1Adolf-Butenandt Institute & German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; 2 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; 3Adolf-Butenandt Institute, Munich, Germany. Contact e-mail: [email protected] Background: Beta-side APP cleaving enzyme BACE1 mediates the initial cleavage required for the release of Amyloid beta-peptide. Functionally, BACE1 is involved in the regulation of myelination. The function of the BACE1 homologue BACE2 is unknown. Methods: Standard methods of cell biology, biochemistry and zebrafish genetics were used. Re-
Background: Neuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to cognate presynaptic ligands. Recent genetic studies revealed that point mutation or copy number variation in NLG genes are linked to autism as well as schizophrenia. Moreover, normalization of NLG1 protein levels restores the social behavior deficits in autism model mice, suggesting that the regulatory mechanism of NLG protein level is an important drug target for neuropsychiatric diseases. Methods: Expression and proteolytic processing of NLG proteins were analyzed in cell lines, primary neurons and mouse brains. Results: We report that NLG proteins undergo ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG and a membrane-tethered C-terminal fragment (CTF). Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. In contrast, ADAM10 was not involved in the NLG2 shedding. NLG-CTF was subsequently cleaved by gamma-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Conclusions: Our data suggest that neuronal activity-dependent proteolytic processing determines the expression level of NLG proteins in vivo and negatively regulate the remodeling of synapses. ORAL SESSIONS: O1-09 BIOMARKERS: CLINICAL IMPLICATIONS O1-09-01
DIAGNOSTIC IMPACT OF CSF BIOMARKERS FOR ALZHEIMER’S DISEASE IN A MEMORY CLINIC SETTING
Flora Duits1, Niels Prins2, Evelien Lemstra2, Yolande Pijnenburg2, Femke Bouwman2, Charlotte Teunissen2, Marinus A. Blankenstein2,
Oral Sessions: O1-09: Biomarkers: Clinical Implications
mechanism known as the unfolded protein response (UPR). When unfolded proteins accumulate in the endoplasmic reticulum (ER), UPR aims to restore cellular homeostasis. However, if stress is prolonged, it induces cell death and, thus, neurodegeneration. We investigated the order of events and the impact of UPR activation in Alzheimer’s disease (AD) model systems. Specifically, we developed an in vitro model system of human neuroblastoma cells (SK-N-SH) that overexpress wild type (WT) amyloid-b precursor protein (APP) or familial AD (FAD)-associated mutants, Swedish (S) and Swedish/Indiana (S/I) forms. Methods: We ’artificially’ induced ER stress in differentiated cells using tunicamycin at eight time points and assessed their viability. Subsequently, Real time PCR (RT-PCR) was used to analyse the gene expression levels of eight UPR markers downstream of all three receptors (IRE1, PERK, and ATF6). This allowed us to examine which ER stress pathways are activated and when in the three model systems. Results: Our data demonstrate that all cells activate stress post tunicamycin treatment. However, only the WT cells recover. More specifically, WT cells show stress activation in terms of ATF4 at three hours. ATF4 is part of the PERK pathway and functions by promoting translational attenuation, which is essential for cell survival. In mutant cells this activation occurs at four hours which we hypothesise that is too late for the UPR mechanism to promote cell survival and the cells are possibly already fated to apoptosis. We also have evidence that the S mutation is more toxic than the S/I or WT APP overexpression. S cells exhibit higher levels of stress as seen from the mRNA levels of their respective markers and appear to be more susceptible to apoptosis. Conclusions: Overall, there is strong evidence that UPR is involved in the progression of AD. The data support the hypothesis that an initial activation of the UPR in AD neurons might work as neuroprotective to restore homeostasis while its sustained activation could induce neurodegeneration. Future studies need to address the therapeutic opportunities of this pathway for the treatment of AD.
sults: To assess the in vivo function of zebrafish Bace1 (zBace1) we generated zBace1 knock out fish by zinc finger nuclease mediated genome editing. bace1 mutants (bace1 -/-) are hypomyelinated in the PNS while the CNS is not affected. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1 -/-. Double homozygous bace1 -/-; bace2 -/- fish do not enhance the single mutant phenotypes indicating non-redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile. To understand the function of BACE1 in myelination we investigated proteolytic processing of Neuregulin 1 (NRG1) type III and demonstrate that the ectodomain can be cleaved by three different sheddases, namely ADAM10, ADAM17, and BACE1. Surprisingly, we not only found cleavage by ADAM10, ADAM17 and BACE1 C-terminal to the epidermal growth factor (EGF)-like domain, which is believed to play a pivotal role in signaling, but also additional cleavage sites for ADAM17 and BACE1 N-terminal to that domain. Proteolytic processing N- and C-terminal of the EGF-like domain results in the secretion of this domain from NRG1 type III. The soluble EGF-like domain is functionally active and stimulates ErbB receptor phosphorylation in tissue culture assays. Moreover, the soluble EGF-like domain is capable to rescue hypomyelination in bace1 -/- zebrafish suggesting that NRG1 type III dependent myelination is not only controlled by membrane retained NRG1 type III but also in a paracrine manner via proteolytic liberation of the EGF-like domain. Conclusions: The identification of a specific bace2 -/associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo. Moreover, our findings not only demonstrate a pivotal function of BACE1 in myelination, but also show that this function is restricted to the PNS.
O1-08-06
PATHOPHYSIOLOGICAL ROLE OF PROTEOLYTIC CLEAVAGE OF NEUROLIGINS
Taisuke Tomita, Kunimichi Suzuki, Azusa Shiohara, Satoko Osawa, Takeshi Iwatsubo, University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected]
O1-08-05
DISTINCT FUNCTIONS OF BACE1 AND BACE2 IN NEUREGULIN PROCESSING AND MELANOCYTE MIGRATION
Christian Haass1, Frauke van Bebber2, Alexander Hruscha2, Michael Willem3, Bettina Schmid2, 1Adolf-Butenandt Institute & German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; 2 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; 3Adolf-Butenandt Institute, Munich, Germany. Contact e-mail: [email protected] Background: Beta-side APP cleaving enzyme BACE1 mediates the initial cleavage required for the release of Amyloid beta-peptide. Functionally, BACE1 is involved in the regulation of myelination. The function of the BACE1 homologue BACE2 is unknown. Methods: Standard methods of cell biology, biochemistry and zebrafish genetics were used. Re-
Background: Neuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to cognate presynaptic ligands. Recent genetic studies revealed that point mutation or copy number variation in NLG genes are linked to autism as well as schizophrenia. Moreover, normalization of NLG1 protein levels restores the social behavior deficits in autism model mice, suggesting that the regulatory mechanism of NLG protein level is an important drug target for neuropsychiatric diseases. Methods: Expression and proteolytic processing of NLG proteins were analyzed in cell lines, primary neurons and mouse brains. Results: We report that NLG proteins undergo ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG and a membrane-tethered C-terminal fragment (CTF). Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. In contrast, ADAM10 was not involved in the NLG2 shedding. NLG-CTF was subsequently cleaved by gamma-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Conclusions: Our data suggest that neuronal activity-dependent proteolytic processing determines the expression level of NLG proteins in vivo and negatively regulate the remodeling of synapses. ORAL SESSIONS: O1-09 BIOMARKERS: CLINICAL IMPLICATIONS O1-09-01
DIAGNOSTIC IMPACT OF CSF BIOMARKERS FOR ALZHEIMER’S DISEASE IN A MEMORY CLINIC SETTING
Flora Duits1, Niels Prins2, Evelien Lemstra2, Yolande Pijnenburg2, Femke Bouwman2, Charlotte Teunissen2, Marinus A. Blankenstein2,