N-Methyl-D-Aspartate Receptor Link to the MAP Kinase Pathway in Cortical and Hippocampal Neurons and Microglia Is Dependent on Calcium Sensors and Is Blocked by α-Synuclein, Tau, and Phospho-Tau in Non-transgenic and Transgenic APPSw,Ind Mice

N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APPSw,Ind transgenic Alzheimer’s disease model. Interestingly, a very marked increase in NMDAR–NCS1 complexes was identified in neurons and a marked increase of both NMDAR–NCS1 and NMDAR–CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor–calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APPSw,Ind mouse embryos/pups

Expression of Melatonin and Dopamine D Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D receptors (DRs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by DR and either MT (MTR) or MT (MTR) melatonin receptors. Heteromerization led to the blockade of DR-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of DR-MTR and DR-MTR was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2

Molecular mechanisms regulated by the transcriptional coactivator CRTC1 in synaptic plasticity

La remodelació sinàptica depenent d’activitat o plasticitat sinàptica és considerada la base cel·lular de diferents processos fisiològics cerebrals com l’aprenentatge i la memòria. Un proteoma equilibrat és essencial per a una transmissió sinàptica eficient i la seva desregulació és característica de malalties neurodegeneratives com la malaltia d’Alzheimer. El factor sinaptonuclear coactivador transcripcional regulat per CREB 1 (CRTC1) connecta l’activació de receptors glutamatèrgics amb programes gènics dependents de CREB, contribuint així al desenvolupament, supervivència i plasticitat neuronal. No obstant això, els mecanismes molecular dependents de CRTC1 que regulen el proteoma sinàptic en condicions fisiològiques i patològiques es desconeixen. Les hipòtesis d’aquesta tesi doctoral són: (1) que CRTC1 modula la plasticitat sinàptica localment en la sinapsi regulant el seu proteoma, i distalment en el nucli modulant l’expressió de gens de neuroplasticitat; i (2) que la desregulació de CRTC1 està associada a canvis neuropatològics en tauopaties. Els objectius d’aquesta tesi doctoral són investigar els mecanismes moleculars sinàptics dependents de CRTC1 implicats en plasticitat sinàptica, i estudiar la relació de CRTC1 i presenilines (PS) en la presència de tau patològica en sinapsis. Els nostres resultats indiquen que CRTC1 és essencial per a la memòria associativa a llarg termini i que modula la neurotransmissió mediada pels receptors N-metil-D-aspartat (NMDARs; GluN). CRTC1 regula la composició, la fosforilació de la subunitat GluN1 dependent de la proteïna quinasa C (PKC) i la localització sinàptica dels receptors NMDA. A més, mitjançant l’ús d’un mutant de CRTC1 constitutivament citosòlic, que alberga la triple mutació S64/151/245A, he demostrat que la fosforilació i localització sinàptica de GluN1 és independent de l’activitat nuclear de CRTC1. Així mateix, CRTC1 contribueix al manteniment del proteoma neuronal promovent la localització dendrítica de mRNAs i la seva traducció local a proteïnes. Els anàlisis bioquímics de sinaptosomes purificats del model murí knockout de PS1 (PS1 cKO);Tau mostren un increment de tau patològica i disminució de CRTC1 en sinapsi. L’estudi de l’autofàgia en el model murí PS1 cKO;Tau i en mostres humanes indica que PS1 regula negativament la inducció de l’autofàgia i manté l’eliminació dels autolisosomes, i que la seva pèrdua probablement contribueix a l’acumulació i agregació de tau patològica. En conclusió, els nostres resultats indiquen que el CRTC1 sinàptic modula localment la neurotransmissió mediada pels receptors NMDA i que un augment en l’acumulació patològica de tau a causa de la pèrdua de funció de PS1 condueix a la desregulació de CRTC1 i a la patologia sinàptica.La remodelación sináptica dependiente de actividad o plasticidad sináptica es considerada la base celular de diferentes procesos fisiológicos cerebrales, incluidos el aprendizaje y la memoria. Un proteoma equilibrado es esencial para una transmisión sináptica eficiente y su desregulación es característica de enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA). El factor sinaptonuclear coactivador transcripcional regulado por CREB 1 (CRTC1) conecta la activación de receptores glutamatérgicos con programas génicos dependientes de CREB, contribuyendo así al desarrollo, la supervivencia y la plasticidad neuronal. Sin embargo, los mecanismos moleculares dependientes de CRTC1 que regulan el proteoma sináptico en condiciones fisiológicas y patológicas se desconocen. Las hipótesis de esta tesis doctoral son: (1) que CRTC1 media la plasticidad sináptica localmente en la sinapsis regulando su proteoma, y distalmente en el núcleo modulando la expresión de genes de neuroplasticidad; y (2) que la desregulación de CRTC1 está asociada a cambios neuropatológicos en tauopatías. Los objetivos de esta tesis doctoral son investigar los mecanismos moleculares sinápticos dependientes de CRTC1 implicados en plasticidad sináptica, y estudiar la relación de CRTC1 y presenilinas (PS) en la presencia de tau patológica en sinapsis. Nuestros resultados indican que CRTC1 es esencial para la memoria asociativa a largo plazo y que modula la neurotransmisión mediada por los receptores N-metil-D-aspartato (NMDARs; GluN). CRTC1 regula la composición, la fosforilación de la subunidad GluN1 dependiente de la proteína quinasa C (PKC) y la localización sináptica de los receptores NMDA. Además, mediante el uso de un mutante de CRTC1 constitutivamente citosólico, que alberga la triple mutación S64/151/245A, he demostrado que la fosforilación y localización sináptica de GluN1 es independiente de la actividad nuclear de CRTC1. Asimismo, CRTC1 contribuye al mantenimiento del proteoma neuronal promoviendo la localización dendrítica de mRNAs y su traducción local a proteínas. Los análisis bioquímicos de sinaptosomas purificados del modelo murino knockout de PS1 (PS1 cKO);Tau muestran un incremento de tau patológica y disminución de CRTC1 en sinapsis. El estudio de la autofagia en el modelo murino PS1 cKO;Tau y en muestras humanas indica que PS1 regula negativamente la inducción de la autofagia y mantiene la eliminación de los autolisosomas, y que su pérdida probablemente contribuya a la acumulación y agregación de tau patológica. En conclusión, nuestros resultados indican que el CRTC1 sináptico modula localmente la neurotransmisión mediada por los receptores NMDA y que un aumento en la acumulación patológica de tau debido a la pérdida de función de PS1 conduce a la desregulación de CRTC1 y a la patología sináptica.Activity-dependent remodeling of synapses or synaptic plasticity is considered the cellular basis of several brain physiological processes, including learning and memory. A balanced proteome is essential for efficient synaptic transmission and its deregulation is a hallmark of neurodegenerative diseases such as Alzheimer’s disease (AD). The synaptonuclear factor CREB-regulated transcription coactivator-1 (CRTC1) links glutamate receptor activation to CREB-dependent gene programs at the nucleus, contributing to neuronal development, survival, and plasticity. However, the CRTC1-dependent molecular mechanisms regulating synaptic proteome in physiological and pathological conditions remain poorly understood. The hypotheses of this doctoral thesis are: (1) that CRTC1 mediates synaptic plasticity locally at synapses by regulating its proteome, and distally at the nucleus by modulating the expression of neuroplasticity genes; and (2) that CRTC1 deregulation is associated with neuropathological changes in tauopathy dementias. The objectives of this doctoral thesis are to investigate the synaptic CRTC1-dependent molecular mechanisms involved in synaptic plasticity, and to study the link of CRTC1 and presenilin (PS) in pathological tau at synapses. Our results show that CRTC1 is essential for long-term associative memory and modulates N-methyl-D-aspartate receptors (NMDARs; GluN)-mediated neurotransmission. CRTC1 regulates NMDARs subunit composition, protein kinase C (PKC)-dependent GluN1 phosphorylation and synaptic localization. Remarkably, by using a constitutively cytosolic CRTC1 mutant harboring triple S64/151/245A mutation, I demonstrate that GluN1 phosphorylation and synaptic localization are independent of CRTC1 nuclear activity. In addition, CRTC1 contributes to maintain neuronal proteome by promoting mRNA transport and protein synthesis at dendritic compartments. Biochemical analyses of purified synaptosomes from PS1 conditional knockout (PS1 cKO);Tau mouse model reveal increased pathological tau and reduced CRTC1 at synapses. Analysis of autophagy in PS1 cKO;Tau mouse model and human samples show that PS1 negatively regulates autophagy induction and maintains autolysosomes clearance, and its loss likely contributes to accumulation and aggregation of pathological tau. In conclusion, our findings indicate that synaptic CRTC1 locally modulates NMDAR-mediated neurotransmission and that increased pathological tau accumulation due to PS1 loss of function leads to CRTC1 deregulation and synapse pathology.Universitat Autònoma de Barcelona. Programa de Doctorat en Neurocièncie

Molecular mechanisms regulated by the transcriptional coactivator CRTC1 in synaptic plasticity

La remodelació sinàptica depenent d'activitat o plasticitat sinàptica és considerada la base cel·lular de diferents processos fisiològics cerebrals com l'aprenentatge i la memòria. Un proteoma equilibrat és essencial per a una transmissió sinàptica eficient i la seva desregulació és característica de malalties neurodegeneratives com la malaltia d'Alzheimer. El factor sinaptonuclear coactivador transcripcional regulat per CREB 1 (CRTC1) connecta l'activació de receptors glutamatèrgics amb programes gènics dependents de CREB, contribuint així al desenvolupament, supervivència i plasticitat neuronal. No obstant això, els mecanismes molecular dependents de CRTC1 que regulen el proteoma sinàptic en condicions fisiològiques i patològiques es desconeixen. Les hipòtesis d'aquesta tesi doctoral són: (1) que CRTC1 modula la plasticitat sinàptica localment en la sinapsi regulant el seu proteoma, i distalment en el nucli modulant l'expressió de gens de neuroplasticitat; i (2) que la desregulació de CRTC1 està associada a canvis neuropatològics en tauopaties. Els objectius d'aquesta tesi doctoral són investigar els mecanismes moleculars sinàptics dependents de CRTC1 implicats en plasticitat sinàptica, i estudiar la relació de CRTC1 i presenilines (PS) en la presència de tau patològica en sinapsis. Els nostres resultats indiquen que CRTC1 és essencial per a la memòria associativa a llarg termini i que modula la neurotransmissió mediada pels receptors N-metil-D-aspartat (NMDARs; GluN). CRTC1 regula la composició, la fosforilació de la subunitat GluN1 dependent de la proteïna quinasa C (PKC) i la localització sinàptica dels receptors NMDA. A més, mitjançant l'ús d'un mutant de CRTC1 constitutivament citosòlic, que alberga la triple mutació S64/151/245A, he demostrat que la fosforilació i localització sinàptica de GluN1 és independent de l'activitat nuclear de CRTC1. Així mateix, CRTC1 contribueix al manteniment del proteoma neuronal promovent la localització dendrítica de mRNAs i la seva traducció local a proteïnes. Els anàlisis bioquímics de sinaptosomes purificats del model murí knockout de PS1 (PS1 cKO);Tau mostren un increment de tau patològica i disminució de CRTC1 en sinapsi. L'estudi de l'autofàgia en el model murí PS1 cKO;Tau i en mostres humanes indica que PS1 regula negativament la inducció de l'autofàgia i manté l'eliminació dels autolisosomes, i que la seva pèrdua probablement contribueix a l'acumulació i agregació de tau patològica. En conclusió, els nostres resultats indiquen que el CRTC1 sinàptic modula localment la neurotransmissió mediada pels receptors NMDA i que un augment en l'acumulació patològica de tau a causa de la pèrdua de funció de PS1 condueix a la desregulació de CRTC1 i a la patologia sinàptica.La remodelación sináptica dependiente de actividad o plasticidad sináptica es considerada la base celular de diferentes procesos fisiológicos cerebrales, incluidos el aprendizaje y la memoria. Un proteoma equilibrado es esencial para una transmisión sináptica eficiente y su desregulación es característica de enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA). El factor sinaptonuclear coactivador transcripcional regulado por CREB 1 (CRTC1) conecta la activación de receptores glutamatérgicos con programas génicos dependientes de CREB, contribuyendo así al desarrollo, la supervivencia y la plasticidad neuronal. Sin embargo, los mecanismos moleculares dependientes de CRTC1 que regulan el proteoma sináptico en condiciones fisiológicas y patológicas se desconocen. Las hipótesis de esta tesis doctoral son: (1) que CRTC1 media la plasticidad sináptica localmente en la sinapsis regulando su proteoma, y distalmente en el núcleo modulando la expresión de genes de neuroplasticidad; y (2) que la desregulación de CRTC1 está asociada a cambios neuropatológicos en tauopatías. Los objetivos de esta tesis doctoral son investigar los mecanismos moleculares sinápticos dependientes de CRTC1 implicados en plasticidad sináptica, y estudiar la relación de CRTC1 y presenilinas (PS) en la presencia de tau patológica en sinapsis. Nuestros resultados indican que CRTC1 es esencial para la memoria asociativa a largo plazo y que modula la neurotransmisión mediada por los receptores N-metil-D-aspartato (NMDARs; GluN). CRTC1 regula la composición, la fosforilación de la subunidad GluN1 dependiente de la proteína quinasa C (PKC) y la localización sináptica de los receptores NMDA. Además, mediante el uso de un mutante de CRTC1 constitutivamente citosólico, que alberga la triple mutación S64/151/245A, he demostrado que la fosforilación y localización sináptica de GluN1 es independiente de la actividad nuclear de CRTC1. Asimismo, CRTC1 contribuye al mantenimiento del proteoma neuronal promoviendo la localización dendrítica de mRNAs y su traducción local a proteínas. Los análisis bioquímicos de sinaptosomas purificados del modelo murino knockout de PS1 (PS1 cKO);Tau muestran un incremento de tau patológica y disminución de CRTC1 en sinapsis. El estudio de la autofagia en el modelo murino PS1 cKO;Tau y en muestras humanas indica que PS1 regula negativamente la inducción de la autofagia y mantiene la eliminación de los autolisosomas, y que su pérdida probablemente contribuya a la acumulación y agregación de tau patológica. En conclusión, nuestros resultados indican que el CRTC1 sináptico modula localmente la neurotransmisión mediada por los receptores NMDA y que un aumento en la acumulación patológica de tau debido a la pérdida de función de PS1 conduce a la desregulación de CRTC1 y a la patología sináptica.Activity-dependent remodeling of synapses or synaptic plasticity is considered the cellular basis of several brain physiological processes, including learning and memory. A balanced proteome is essential for efficient synaptic transmission and its deregulation is a hallmark of neurodegenerative diseases such as Alzheimer's disease (AD). The synaptonuclear factor CREB-regulated transcription coactivator-1 (CRTC1) links glutamate receptor activation to CREB-dependent gene programs at the nucleus, contributing to neuronal development, survival, and plasticity. However, the CRTC1-dependent molecular mechanisms regulating synaptic proteome in physiological and pathological conditions remain poorly understood. The hypotheses of this doctoral thesis are: (1) that CRTC1 mediates synaptic plasticity locally at synapses by regulating its proteome, and distally at the nucleus by modulating the expression of neuroplasticity genes; and (2) that CRTC1 deregulation is associated with neuropathological changes in tauopathy dementias. The objectives of this doctoral thesis are to investigate the synaptic CRTC1-dependent molecular mechanisms involved in synaptic plasticity, and to study the link of CRTC1 and presenilin (PS) in pathological tau at synapses. Our results show that CRTC1 is essential for long-term associative memory and modulates N-methyl-D-aspartate receptors (NMDARs; GluN)-mediated neurotransmission. CRTC1 regulates NMDARs subunit composition, protein kinase C (PKC)-dependent GluN1 phosphorylation and synaptic localization. Remarkably, by using a constitutively cytosolic CRTC1 mutant harboring triple S64/151/245A mutation, I demonstrate that GluN1 phosphorylation and synaptic localization are independent of CRTC1 nuclear activity. In addition, CRTC1 contributes to maintain neuronal proteome by promoting mRNA transport and protein synthesis at dendritic compartments. Biochemical analyses of purified synaptosomes from PS1 conditional knockout (PS1 cKO);Tau mouse model reveal increased pathological tau and reduced CRTC1 at synapses. Analysis of autophagy in PS1 cKO;Tau mouse model and human samples show that PS1 negatively regulates autophagy induction and maintains autolysosomes clearance, and its loss likely contributes to accumulation and aggregation of pathological tau. In conclusion, our findings indicate that synaptic CRTC1 locally modulates NMDAR-mediated neurotransmission and that increased pathological tau accumulation due to PS1 loss of function leads to CRTC1 deregulation and synapse pathology

N-Methyl-D-Aspartate Receptor Link to the MAP Kinase Pathway in Cortical and Hippocampal Neurons and Microglia Is Dependent on Calcium Sensors and Is Blocked by α-Synuclein, Tau, and Phospho-Tau in Non-transgenic and Transgenic APPSw,Ind Mice

N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APPSw,Ind transgenic Alzheimer's disease model. Interestingly, a very marked increase in NMDAR-NCS1 complexes was identified in neurons and a marked increase of both NMDAR-NCS1 and NMDAR-CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor-calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APPSw,Ind mouse embryos/pups

Expression of Melatonin and Dopamine D Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D receptors (DRs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by DR and either MT (MTR) or MT (MTR) melatonin receptors. Heteromerization led to the blockade of DR-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of DR-MTR and DR-MTR was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2