Role of the p75 neurotrophin receptor (p75NTR) in the basal forebrain cholinergic neurons (BFCNs)

225 págs, figuras y tablas[EN] The basal forebrain cholinergic neurons (BFCNs) are implicated in high cognitive processes such attention and memory as they project to regions like the cortex and the hippocampus. These neurons express high amounts of the p75 neurotrophin receptor (p75NTR) during their whole life, nevertheless, the role that the receptor plays in them is not fully understood. During the experimental work of this thesis, the role that p75NTR plays during ageing and the role of the proteolytic processing of p75NTR have been studied. The first objective was achieved using a mouse model of accelerated senescence, SAMP8. This mouse model was crossed with the knock-out of p75NTR, obtaining the SAMP8-p75NTR-/- mouse. The results showed how at young ages (2 months) there is an increase in the number of BFCNs in SAMP8-p75NTR-/- compared to SAMP8-p75NTR+/+ mice. However, in adult mice (6 months) and aged mice (10 months) the number of cholinergic neurons between SAMP8-p75NTR+/+ and SAMP8-p75NTR-/- are the same, indicating a cholinergic neuronal loss. In addition, the same result has been reported in the C57BL/6 background. The number of cholinergic neurons were quantified at postnatal day 16 and 2, 10 and 24 months. The results showed an increase in the number of BFCNs at 16 days old in C57BL/6-p75NTR-/- mice compared to C57BL/6-p75NTR+/+ animals. Nevertheless, the levels were reduce at 2, 10 and 24 months in C57BL/6-p75NTR-/- mice. The reduction in the number of cholinergic neurons is accompanied by a reduction in the levels of specific cholinergic markers such as: ChAT, acetylcholine and Acly in SAMP8-p75NTR-/- mice at 6 months. The results showed that neuronal cholinergic death is not due to a reduction in the oxidative stress response, since the SAMP8-p75NTR+/+ and the SAMP8-p75NTR-/- have the same oxidative response at 6 months of age. Nonetheless, SAMP8-p75NTR-/- mice present different levels in key enzymes of the cholesterol synthesis and endocytic pathway. Indicating that in the SAMP8-p75NTR-/- mice there might be a deregulation in the homeostasis of cholesterol explaining why cholinergic neurons degenerate. In addition, the cholinergic neuronal loss has a spatial memory effect as the SAMP8-p75NTR-/- mice perform worst the Y-maze test at 6 months compared to 2 months. The p75NTR is proteolytically cleaved by the α-secretase generating the C-terminal domain, which is further cleaved by the γ-secretase. To study the effect of the p75NTR cleavage, primary cultures of cholinergic neurons were used and the γ-secretase was chemically inhibited. The addition of CE (a γ-secretase inhibitor) to the cholinergic neurons specifically increases their complexity and length. Furthermore, it was seen that this effect is p75NTR-dependent as in p75NTR-/- cultures the effect disappears. In addition, we have proved that the increase in complexity is through the inhibition of the RhoA protein, a cytoskeleton regulatory protein. These results were confirmed in organotypic brain slices, as the addition of CE causes the same effect, an increase in the complexity of cholinergic neurons. Furthermore, in mouse models in which the γ-secretase is genetically inhibited, there is an increase in acetylcholinesterase fibres in the cortex and hippocampus, places of innervation of cholinergic neurons. This is an indication of increased complexity in a in vivo situation. All together the results of this thesis have amplified the knowledge of the p75NTR in the basal forebrain cholinergic neurons. Demonstrating a cholinergic dependence of the receptor during adulthood and ageing, probably to a cholesterol homeostasis deregulation. Moreover, the accumulation of p75NTR-CTF, increases cholinergic complexity, indicating another role of p75NTR in the cholinergic neurons regulating their morphology.[ES] Las neuronas colinérgicas del prosencéfalo basal están implicadas en procesos cognitivos superiores como la atención y la memoria dado que proyectan en regiones como el hipocampo y la corteza. Estas neuronas expresan altos niveles del receptor de neurotrofinas p75 (p75NTR) durante toda su vida. Sin embargo, el papel que juega en la fisiología de estas neuronas no esta del todo claro. En el trabajo experimental de esta tesis hemos estudiado el papel de p75NTR durante el envejecimiento y el papel del corte de p75NTR en neuronas maduras. El primer objetivo se estudio usando una cepa de senescencia acelerada: SAMP8. Este modelo fue cruzado con el knock-out de p75NTR. Los resultados muestran cómo a edades jóvenes (2 meses) el SAMP8-p75NTR-/- tiene un mayor número de neuronas colinérgicas comparado con SAMP8-p75NTR+/+. No obstante, a edades adultas (6 meses) y viejas (10 meses) el número de neuronas disminuye hasta los niveles de SAMP8-p75NTR+/+. Además, este mismo resultado fue comprobado en ratones C57BL/6-p75NTR-/-. El número de neuronas colinérgicas se comparó a día postnatal 16 y a 2, 10 y 24 meses. Así como en los ratones SAMP8-p75NTR-/-, los ratones C57BL/6-p75NTR-/- presentan un aumento en el número de neuronas colinérgicas a día postnatal 16 y una posterior reducción a 2, 10 y 24 meses. La reducción en el número de neuronas colinérgicas va acompañado de una reducción en los niveles de marcadores específicos de neuronas colinérgicas como: ChAT, acetilcolina y Acly en los ratones SAMP8-p75NTR-/- de 6 meses. Los resultados muestran cómo la muerte colinérgica no es debida a una disminución a la respuesta al estrés oxidativo, ya que los ratones SAMP8-p75NTR+/+ y los ratones SAMP8-p75NTR-/- tienen la misma respuesta a 6 meses. En cambio, los ratones SAMP8-p75NTR-/- sí que presentan unos niveles distintos en enzimas claves para la síntesis de colesterol. Estos resultados sugieren que los ratones SAMP8-p75NTR-/- podrían tener una desregulación en la homeostasis y síntesis de colesterol y por eso las neuronas colinérgicas degeneran y acaban muriendo. Además se comprobó que la perdida neuronal tiene un efecto en la memoria espacial ya que los ratones SAMP8-p75NTR-/- ejecutan peor el test-Y a los 6 meses de edad. p75NTR es proteolíticamente procesado por una actividad α-secretasa y posteriormente por la γ-secretasa. Para estudiar el efecto del corte de p75NTR se usaron cultivos primarios de neuronas colinérgicas en los cuales la γ-secretasa se inhibió. Al añadir CE (el inhibidor de la γ-secretasa) se vio un aumento específico en la complejidad y longitud de las neuronas colinérgicas. Además se ha comprobado que este efecto es dependiente de p75NTR ya que en cultivos p75NTR-/- el efecto desaparece. Además hemos comprobado cómo el aumento de complejidad es realizado a través de la proteína RhoA, una proteína reguladora de citoesqueleto. Estos resultados fueron comprobados en rodajas de cultivos organotípicos y la adición del CE causa del mismo modo un aumento en la complejidad de las neuronas colinérgicas. Además, en ratones donde la γ-secretasa esta inhibida genéticamente, hay un aumento de fibras acetilcolinesterasa en la corteza cerebral y en el hipocampo, sitios de inervación de las neuronas colinérgicas. Esto nos indica un aumento en la complejidad en una situación in vivo. En conclusión esta tesis ha ampliado el conocimiento de p75NTR en las neuronas colinérgicas del prosencéfalo basal. Demostrando una dependencia colinérgica del receptor durante la edad adulta y el envejecimiento, probablemente debido a una desregulación en la homeostasis del colesterol. Además, la acumulación de p75NTR-CTF, incrementa la complejidad colinérgica, indicando otro papel de p75NTR en las neuronas colinérgicas modulando su morfología.La realización de este trabajo de tesis doctoral ha sido posible gracias al Proyecto de Investigación: Plan Nacional I+D SAF2017-84096-R del Ministerio de Economía y Competitividad.Peer reviewe

Role of the p75 neurotrophin receptor (p75NTR) in the basal forebrain cholinergic neurons (BFCNs)

Las neuronas colinérgicas del prosencéfalo basal están implicadas en procesos cognitivos superiores como la atención y la memoria dado que proyectan en regiones como el hipocampo y la corteza. Estas neuronas expresan altos niveles del receptor de neurotrofinas p75 (p75NTR) durante toda su vida. Sin embargo, el papel que juega en la fisiología de estas neuronas no esta del todo claro. En el trabajo experimental de esta tesis hemos estudiado el papel de p75NTR durante el envejecimiento y el papel del corte de p75NTR en neuronas maduras. El primer objetivo se estudio usando una cepa de senescencia acelerada: SAMP8. Este modelo fue cruzado con el knock-out de p75NTR. Los resultados muestran cómo a edades jóvenes (2 meses) el SAMP8-p75NTR-/- tiene un mayor número de neuronas colinérgicas comparado con SAMP8-p75NTR+/+. No obstante, a edades adultas (6 meses) y viejas (10 meses) el número de neuronas disminuye hasta los niveles de SAMP8-p75NTR+/+. Además, este mismo resultado fue comprobado en ratones C57BL/6-p75NTR-/-. El número de neuronas colinérgicas se comparó a día postnatal 16 y a 2, 10 y 24 meses. Así como en los ratones SAMP8-p75NTR-/-, los ratones C57BL/6-p75NTR-/- presentan un aumento en el número de neuronas colinérgicas a día postnatal 16 y una posterior reducción a 2, 10 y 24 meses. La reducción en el número de neuronas colinérgicas va acompañado de una reducción en los niveles de marcadores específicos de neuronas colinérgicas como: ChAT, acetilcolina y Acly en los ratones SAMP8-p75NTR-/- de 6 meses. Los resultados muestran cómo la muerte colinérgica no es debida a una disminución a la respuesta al estrés oxidativo, ya que los ratones SAMP8-p75NTR+/+ y los ratones SAMP8-p75NTR-/- tienen la misma respuesta a 6 meses. En cambio, los ratones SAMP8-p75NTR-/- sí que presentan unos niveles distintos en enzimas claves para la síntesis de colesterol. Estos resultados sugieren que los ratones SAMP8-p75NTR-/- podrían tener una desregulación en la homeostasis y síntesis de colesterol y por eso las neuronas colinérgicas degeneran y acaban muriendo. Además se comprobó que la perdida neuronal tiene un efecto en la memoria espacial ya que los ratones SAMP8-p75NTR-/- ejecutan peor el test-Y a los 6 meses de edad. p75NTR es proteolíticamente procesado por una actividad α-secretasa y posteriormente por la γ-secretasa. Para estudiar el efecto del corte de p75NTR se usaron cultivos primarios de neuronas colinérgicas en los cuales la γ-secretasa se inhibió. Al añadir CE (el inhibidor de la γ-secretasa) se vio un aumento específico en la complejidad y longitud de las neuronas colinérgicas. Además se ha comprobado que este efecto es dependiente de p75NTR ya que en cultivos p75NTR-/- el efecto desaparece. Además hemos comprobado cómo el aumento de complejidad es realizado a través de la proteína RhoA, una proteína reguladora de citoesqueleto. Estos resultados fueron comprobados en rodajas de cultivos organotípicos y la adición del CE causa del mismo modo un aumento en la complejidad de las neuronas colinérgicas. Además, en ratones donde la γ-secretasa esta inhibida genéticamente, hay un aumento de fibras acetilcolinesterasa en la corteza cerebral y en el hipocampo, sitios de inervación de las neuronas colinérgicas. Esto nos indica un aumento en la complejidad en una situación in vivo. En conclusión esta tesis ha ampliado el conocimiento de p75NTR en las neuronas colinérgicas del prosencéfalo basal. Demostrando una dependencia colinérgica del receptor durante la edad adulta y el envejecimiento, probablemente debido a una desregulación en la homeostasis del colesterol. Además, la acumulación de p75NTR-CTF, incrementa la complejidad colinérgica, indicando otro papel de p75NTR en las neuronas colinérgicas modulando su morfología.The basal forebrain cholinergic neurons (BFCNs) are implicated in high cognitive processes such attention and memory as they project to regions like the cortex and the hippocampus. These neurons express high amounts of the p75 neurotrophin receptor (p75NTR) during their whole life, nevertheless, the role that the receptor plays in them is not fully understood. During the experimental work of this thesis, the role that p75NTR plays during ageing and the role of the proteolytic processing of p75NTR have been studied. The first objective was achieved using a mouse model of accelerated senescence, SAMP8. This mouse model was crossed with the knock-out of p75NTR, obtaining the SAMP8-p75NTR-/- mouse. The results showed how at young ages (2 months) there is an increase in the number of BFCNs in SAMP8-p75NTR-/- compared to SAMP8-p75NTR+/+ mice. However, in adult mice (6 months) and aged mice (10 months) the number of cholinergic neurons between SAMP8-p75NTR+/+ and SAMP8-p75NTR-/- are the same, indicating a cholinergic neuronal loss. In addition, the same result has been reported in the C57BL/6 background. The number of cholinergic neurons were quantified at postnatal day 16 and 2, 10 and 24 months. The results showed an increase in the number of BFCNs at 16 days old in C57BL/6-p75NTR-/- mice compared to C57BL/6-p75NTR+/+ animals. Nevertheless, the levels were reduce at 2, 10 and 24 months in C57BL/6-p75NTR-/- mice. The reduction in the number of cholinergic neurons is accompanied by a reduction in the levels of specific cholinergic markers such as: ChAT, acetylcholine and Acly in SAMP8-p75NTR-/- mice at 6 months. The results showed that neuronal cholinergic death is not due to a reduction in the oxidative stress response, since the SAMP8-p75NTR+/+ and the SAMP8-p75NTR-/- have the same oxidative response at 6 months of age. Nonetheless, SAMP8-p75NTR-/- mice present different levels in key enzymes of the cholesterol synthesis and endocytic pathway. Indicating that in the SAMP8-p75NTR-/- mice there might be a deregulation in the homeostasis of cholesterol explaining why cholinergic neurons degenerate. In addition, the cholinergic neuronal loss has a spatial memory effect as the SAMP8-p75NTR-/- mice perform worst the Y-maze test at 6 months compared to 2 months. The p75NTR is proteolytically cleaved by the α-secretase generating the C-terminal domain, which is further cleaved by the γ-secretase. To study the effect of the p75NTR cleavage, primary cultures of cholinergic neurons were used and the γ-secretase was chemically inhibited. The addition of CE (a γ-secretase inhibitor) to the cholinergic neurons specifically increases their complexity and length. Furthermore, it was seen that this effect is p75NTR-dependent as in p75NTR-/- cultures the effect disappears. In addition, we have proved that the increase in complexity is through the inhibition of the RhoA protein, a cytoskeleton regulatory protein. These results were confirmed in organotypic brain slices, as the addition of CE causes the same effect, an increase in the complexity of cholinergic neurons. Furthermore, in mouse models in which the γ-secretase is genetically inhibited, there is an increase in acetylcholinesterase fibres in the cortex and hippocampus, places of innervation of cholinergic neurons. This is an indication of increased complexity in a in vivo situation. All together the results of this thesis have amplified the knowledge of the p75NTR in the basal forebrain cholinergic neurons. Demonstrating a cholinergic dependence of the receptor during adulthood and ageing, probably to a cholesterol homeostasis deregulation. Moreover, the accumulation of p75NTR-CTF, increases cholinergic complexity, indicating another role of p75NTR in the cholinergic neurons regulating their morphology

TrkA-mediated endocytosis of p75-CTF prevents cholinergic neuron death upon γ-secretase inhibition

20 páginas, 8 figurasγ-secretase inhibitors (GSI) were developed to reduce the generation of Aβ peptide to find new Alzheimer's disease treatments. Clinical trials on Alzheimer's disease patients, however, showed several side effects that worsened the cognitive symptoms of the treated patients. The observed side effects were partially attributed to Notch signaling. However, the effect on other γ-secretase substrates, such as the p75 neurotrophin receptor (p75NTR) has not been studied in detail. p75NTR is highly expressed in the basal forebrain cholinergic neurons (BFCNs) during all life. Here, we show that GSI treatment induces the oligomerization of p75CTF leading to the cell death of BFCNs, and that this event is dependent on TrkA activity. The oligomerization of p75CTF requires an intact cholesterol recognition sequence (CRAC) and the constitutive binding of TRAF6, which activates the JNK and p38 pathways. Remarkably, TrkA rescues from cell death by a mechanism involving the endocytosis of p75CTF. These results suggest that the inhibition of γ-secretase activity in aged patients, where the expression of TrkA in the BFCNs is already reduced, could accelerate cholinergic dysfunction and promote neurodegeneration.This study was supported by the Spanish Minister of Economy and Competitiveness grant SAF2017-84096-R and by the Generalitat Valenciana 2018-55 to M Vilar. I García-Carpio was supported by an Formación de Personal Investigador (FPI) pre-doctoral fellowship (BFU2013/42746-P) and a mobility grant (EEBB-I-15-10278) from the Spanish Minister of Economy and Competitiveness. This work was funded by the Stichting Alzheimer Onderzoek (S16013) and the Fonds voor Wetenschappelijk Onderzoek or Flanders Research Foundation (FWO) research project (G0B2519N) to L Chavez-GutiérrezPeer reviewe

Cholinergic neurodegeneration and cholesterol metabolism dysregulation by constitutive p75NTR signaling in the p75exonIII-KO mice

16 páginas, 7 figurasDegeneration of basal forebrain cholinergic neurons (BFCNs) is a hallmark of Alzheimer's disease (AD). However, few mouse models of AD recapitulate the neurodegeneration of the cholinergic system. The p75 neurotrophin receptor, p75NTR, has been associated with the degeneration of BFCNs in AD. The senescence-accelerated mouse prone number 8 (SAMP8) is a well-accepted model of accelerated and pathological aging. To gain a better understanding of the role of p75NTR in the basal forebrain during aging, we generated a new mouse line, the SAMP8-p75exonIII-/-. Deletion of p75NTR in the SAMP8 background induces an increase in the number of BFCNs at birth, followed by a rapid decline during aging compared to the C57/BL6 background. This decrease in the number of BFCNs correlates with a worsening in the Y-maze memory test at 6 months in the SAMP8-p75exonIII-/-. We found that SAMP8-p75exonIII-/- and C57/BL6-p75exonIII-/- mice expressed constitutively a short isoform of p75NTR that correlates with an upregulation of the protein levels of SREBP2 and its targets, HMGCR and LDLR, in the BF of both SAMP8-p75exonIII-/- and C57/BL6-p75exonIII-/- mice. As the neurodegeneration of the cholinergic system and the dysregulation of cholesterol metabolism are implicated in AD, we postulate that the generated SAMP8-p75exonIII-/- mouse strain might constitute a good model to study long-term cholinergic neurodegeneration in the CNS. In addition, our results support the role of p75NTR signaling in cholesterol biosynthesis regulation.MV acknowledges the Spanish Ministry of Science and Innovation (grants SAG2017/84096-R and PID2021-127600NB-I00) and the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global). AB-M has a INVESTIGO contract from the Generalitat Valenciana (INVEST/2022/456). MV and HM to Generalitat Valenciana (PROMETEO 2018/055 and AICO23 CIAICO/2022/074) and HM to Spanish Ministry of Science and Innovation (grant PID2019-111225RB-I00).Peer reviewe