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Entre el silenci dels records: recerca d’una memòria perduda. Nous enfocaments terapèutics en la malaltia de l’Alzheimer
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[cat] En l’actualitat, la malaltia de l’Alzheimer és la causa més comuna de demència a nivell mundial. Aquesta malaltia és un trastorn neurodegeneratiu progressiu que es caracteritza per l’acumulació de plaques d’Aβ extracel·lulars i de cabdells neurofibril·lars de la proteïna p-Tau que van associats a un context de neuroinflamació que acaba degenerant en la disfunció sinàptica i la mort neuronal. La presència d’agregats d’Aβ augmenta els nivells de glutamat extracel·lulars, ja que s’alteren els processos de captació i reciclatge d’aquest neurotransmissor, induint una disfunció sinàptica i neurotoxicitat. Conseqüentment, els pacients pateixen una pèrdua progressiva de la memòria i l’aparició de dèficits cognitius i alteracions de la conducta. No es coneix l’etiologia de la malaltia de l’Alzheimer, però se sap que hi participen factors genètics, ambientals i d’estil de vida, tot i que l’edat avançada és el causant més important. A més, encara no se’n coneix la cura, però existeixen tractaments que busquen alleujar els símptomes i alentir la progressió de la malaltia.
En el context de la malaltia d’Alzheimer, els composts cannabinoides presenten certs efectes beneficiosos, ja que presenten efectes antiinflamatoris i neuroprotectors. Concretament, la senyalització retrògrada dels composts endocannabinoides ajuda a disminuir l’excitotoxicitat a través del CB1R localitzat pre-sinàpticament. Amb l’objectiu de potenciar els efectes beneficiosos dels composts cannabinoides i disminuir els efectes adversos, hem volgut estudiar les vies d'entrada d’aquests compostos al centre ortostèric del CB1R. D’aquesta manera, aportem informació clau per a la síntesi de nous compostos amb una major activitat terapèutica dirigits al CB1R. Entre els efectes secundaris de l’activació del CB1R trobem efectes psicoactius i d’addicció associats al consum de cànnabis. En aquesta tesis doctoral hem volgut descriure el paper regulador del CB1R en el procés d’addició a les drogues d’abús, emprant models de rosegador amb auto-administració de cocaïna. Els resultats obtinguts demostren que la sobreactivació de les neurones de l’hipocamp a través de la deleció dirigida de CB1R, incrementa la recerca de recompensa i el grau de recaiguda. Per contra, la inhibició de les neurones dopaminèrgiques de l’àrea tegmental ventral (VTA) mitjançant la deleció CB1R en neurones gabaèrgiques del ventral pàl·lidum (VP) redueix la recerca de recompensa. Aquests resultats demostren la importància del paper regulador de CB1R en el sistema de recompensa i la necessitat d’especificar la funció d’aquest receptor en diferents àrees del cervell implicades en l’addicció.
Un dels primers signes de la malaltia d’Alzheimer és l’augment dels nivells de glutamat extracel·lulars que hiperactiva el receptor del N-metil-D-aspartat (NMDAR) induint efectes citotòxics. Un dels tractaments actuals de la MA és l’administració de la memantina, un antagonista del NMDAR que s’empra en els estadis més avançats de la malaltia. En l’Alzheimer, s’ha observat que l’Aβ activa específicament els NMDAR-GluN2B extrasinàptics, fet que agreuja la disfunció sinàptica i la neurodegeneració de la malaltia. En canvi, la presència d’Aβ promou la internalització i inhibició del canal NMDAR-GluN2A localitzat sinàpticament que estan associats a la supervivència neuronal i a la plasticitat sinàptica. La memantina bloqueja tant el NMDAR-GluN2A com el NMDAR-GluN2B. Per aquest motiu, en aquesta tesis vam decidir cercar proteïnes
capaces de regular de manera diferencial els receptors de glutamat que contenien la subunitat NR2A i NR2B. Hem descobert que l’activació del CB1R modula de forma diferencial el canal NMDAR-GluN2A i NMDA-GluN2B a través de la formació de complexes heteromèrics. Potencia els efectes neuroprotectors del NMDA-GluN2A i reduint la toxicitat induïda pel NMDA-GluN2B. A més, l’agonista selectiu de CB1R, ACEA, disminueix el transport axonal de p-Tau, especialment a les neurones que expressa NMDA-GluN2A i que són KO per NMDA-GluN2B. Els resultats obtinguts destaquen el potencial del CB1R com a diana terapèutica en la malaltia de l’Alzheimer, ja que actua com a modulador del canal de NMDAR, afavorint els efectes neuroprotectors i reduint els efectes citotòxics.
S’ha descrit que en la malaltia d’Alzheimer, en un context de neuroinflamació pronunciada, l’expressió del CB2R a les cèl·lules microglials augmenta. A més, l’activació del CB2R promou la conversió del fenotip microglial proinflamatori (M1) al anti-inflamatori (M2), induint efectes neuroprotectors. D’altra banda, un dels símptomes més comuns de la MA és la desregulació del cicle son-vigília. Precisament, aquests trastorns del son alteren la correcta eliminació de residus tòxics que s’acumulen al líquid cefalorraquidi durant l’estat de vigília, com els agregats d’Aβ i de p-Tau, tot accelerant la progressió de la malaltia. El sistema orexinèrgic es troba involucrat en el control del cicle son-vigília. De fet, la senyalització orexinèrgica excessiva contribueix a les interrupcions del son, accelerant la deposició d’Aβ. En aquesta línia, l’evidència d’estudis realitzats en models animals i humans in vivo suggereix que la reducció del to orexigènic mitjançant antagonistes duals dels receptors orexinèrgics (DORA) millora l’alteració del son i el deteriorament cognitiu, prevenint, així, el procés neurodegeneratiu de la MA.
En aquesta tesi doctoral analitzem de forma exhaustiva els rols dels sistemes endocannabinoide i orexinèrgic en la MA. De forma interessant, hem descobert que ambdós sistemes es regulen entre ells en la MA. Concretament, hem descrit que el receptor de cannabinoides CB1 forma complexes heteromèrics amb el receptor d’orexina OX1 i que la seva expressió augmenta en neurones d’escorça tractades amb agregats del pèptid Aβ. A més, en neurones d’escorça de ratolins adults 5XFAD, model d’Alzheimer, hem observat que l’activació del receptor OX1 bloqueja l’efecte neuroprotector cannabinoide.
En l’Alzheimer, el CB2R es troba sobreexpressat en condicions d’inflamació i la seva activació desencadena una resposta neuroprotectora. Per aquest motiu, en aquest estudi també hem analitzat el paper regulador del receptor OX1 sobre el potencial terapèutic del CB2R. Hem descrit la formació de complexes CB2R-OX1R i un augment de l’expressió d’aquests heteròmers en condicions de la MA. De manera interessant hem descrit que el bloqueig del receptor OX1 potencia els efectes neuroprotectors cannabinoides en la micròglia de ratolins d’Alzheimer APPSw/Ind. Convertint als antagonistes d’OX1 en nous potencials compostos per combatre la malaltia.
El sistema orexinèrgic està constituït per dos receptor d’orexina: l’OX1 i l’ OX2. L’OX2 està menys estudiat, però sembla tenir un rol important en la regulació de l’activitat d’OX1R, presentant uns efectes neutres o fins i tot protectors en malalties neurodegeneratives que cursen amb l’alteració del son. En aquesta tesi doctoral, hem demostrat que en les
cèl·lules microglials en repòs els receptors CB2 i OX2 es bloquegen entre si. Mentre que en condicions de neuroinflamació actuen de forma sinèrgica per combatre aquesta simptomatologia, induint el fenotip microglial antiinflamatori (M2) i disminuint els nivells de ROS i mort cel·lular. Per tant, el complex de receptors CB2-OX2 esdevé una nova diana terapèutica amb gran potencial per combatre la MA.
Un dels efectes que dona més potencial al sistema endocannabinoide i a la recerca de nous compostos cannabinoides és la seva capacitat per pal·liar el dolor. Actualment, però, un dels sistemes més estudiants pel seus efectes analgèsics és el sistema opioide. El sistema opioide es troba involucrat en regular el dolor, no obstant, presenta importants efectes adversos i un fort component additiu. Per aquest motiu, en aquesta tesi doctoral hem intentat cercar proteïnes amb capacitat de regular aquest sistema, per tal de potenciar els efectes analgèsics i disminuir els efectes adversos. De forma interessant, hem pogut demostrar que el receptor orfe GPR88 modula la internalització del δOR a través de la formació del complex δOR-GPR88. Aquest fet demostra i explica l’acumulació de δOR als compartiments intracel·lulars i perquè els agonistes de δOR no actuen de forma més eficaç i no han aconseguit superar els assajos clínics.
[eng] Currently, Alzheimer’s disease is the most common cause of dementia worldwide. This disease is a progressive neurodegenerative disorder characterized by the accumulation of extracellular Aβ plaques and neurofibrillary tangles of the p-Tau protein, associated with a context of neuroinflammation that ultimately leads to synaptic dysfunction and neuronal death. The presence of Aβ aggregates increases extracellular glutamate levels, as the uptake and recycling processes of this neurotransmitter are impaired, inducing synaptic dysfunction and neurotoxicity. Consequently, patients suffer a progressive loss of memory, the onset of cognitive deficits, and behavioral alterations. The etiology of Alzheimer’s disease remains unknown, but it is known that genetic, environmental, and lifestyle factors play a role, with advanced age being the most important risk factor. Moreover, although there is no cure, treatments exist that aim to relieve symptoms and slow the progression of the disease. In the context of Alzheimer’s disease, cannabinoid compounds show beneficial effects due to their anti-inflammatory and neuroprotective properties. Specifically, retrograde signalling of endocannabinoids helps to decrease excitotoxicity through the presynaptic CB1R. With the aim of enhancing the beneficial effects of cannabinoid compounds and reducing adverse effects, we studied the entry pathways of these compounds into the orthosteric site of CB1R. In this way, we provide key information for the synthesis of new compounds with greater therapeutic activity targeting CB1R. Among the side effects of CB1R activation are psychoactive and addictive effects associated with cannabis consumption. In this doctoral thesis, we sought to describe the regulatory role of CB1R in drug addiction processes, using rodent models of cocaine self-administration. The results obtained demonstrate that overactivation of hippocampal neurons through targeted CB1R deletion increases reward-seeking and relapse. In contrast, inhibition of dopaminergic neurons in the ventral tegmental area (VTA) via CB1R deletion in GABAergic neurons of the ventral pallidum (VP) reduces reward-seeking. These results highlight the regulatory role of CB1R in the reward system and the need to specify its function in different brain areas involved in addiction. The first signs of Alzheimer’s disease is the increase in extracellular glutamate levels, which hyperactivates the N-methyl-D-aspartate receptor (NMDAR), inducing cytotoxic effects. One of the current treatments for AD is the administration of memantine, an NMDAR antagonist used in the more advanced stages of the disease. In Alzheimer’s, Aβ has been observed to specifically activate extrasynaptic NMDAR-GluN2B, worsening synaptic dysfunction and neurodegeneration. In contrast, Aβ promotes the internalization and inhibition of synaptic NMDAR-GluN2A channels, which are associated with neuronal survival and synaptic plasticity. Memantine blocks both NMDAR-GluN2A and NMDAR-GluN2B. For this reason, in this thesis we decided to search for proteins capable of differentially regulating glutamate receptors containing the NR2A and NR2B subunits. We discovered that CB1R activation differentially modulates NMDAR-GluN2A and NMDAR-GluN2B channels through the formation of heteromeric complexes. It enhances the neuroprotective effects of NMDAR-GluN2A and reduces the toxicity induced by NMDAR-GluN2B. In addition, the selective CB1R agonist ACEA decreases axonal transport of p-Tau, especially in neurons expressing NMDAR- GluN2A and lacking NMDAR-GluN2B. The results obtained highlight the potential of CB1R as a therapeutic target in Alzheimer’s disease, as it acts as a modulator of NMDAR channels, favouring neuroprotective effects and reducing cytotoxicity. It has been described that in Alzheimer’s disease, under conditions of pronounced neuroinflammation, the expression of CB2R in microglial cells increases. Furthermore, CB2R activation promotes the conversion of the pro-inflammatory (M1) microglial phenotype to the anti-inflammatory (M2) phenotype, inducing neuroprotective effects. On the other hand, one of the most common symptoms of AD is dysregulation of the sleep-wake cycle. These sleep disturbances impair the proper clearance of toxic waste that accumulates in cerebrospinal fluid during wakefulness, such as Aβ and p-Tau aggregates, thereby accelerating disease progression. The orexinergic system is involved in the regulation of the sleep-wake cycle. Indeed, excessive orexinergic signalling contributes to sleep interruptions, accelerating Aβ deposition. In this regard, evidence from studies conducted in animal models and in humans in vivo suggests that reducing orexinergic tone through dual orexin receptor antagonists (DORAs) improves sleep disturbances and cognitive decline, thus preventing the neurodegenerative process of AD. In this doctoral thesis, we analysed in detail the roles of the endocannabinoid and orexinergic systems in AD. Interestingly, we discovered that both systems regulate each other in AD. Specifically, we described that the cannabinoid receptor CB1 forms heteromeric complexes with the orexin receptor OX1, and that their expression increases in cortical neurons treated with Aβ aggregates. Moreover, in cortical neurons of adult 5XFAD mice, an Alzheimer’s model, we observed that OX1 receptor activation blocks the cannabinoid neuroprotective effect. In Alzheimer’s, CB2R is overexpressed under inflammatory conditions, and its activation triggers a neuroprotective response. For this reason, in this study we also analysed the regulatory role of the OX1 receptor on the therapeutic potential of CB2R. We described the formation of CB2R-OX1R complexes and an increase in their expression under AD conditions. Interestingly, we found that OX1 receptor blockade enhances cannabinoid neuroprotective effects in the microglia of APPSw/Ind Alzheimer’s mice, making OX1 antagonists’ potential new compounds to combat the disease. The orexinergic system consists of two orexin receptors: OX1 and OX2. OX2 is less studied, but it seems to play an important role in regulating OX1R activity, presenting neutral or even protective effects in neurodegenerative diseases associated with sleep disruption. In this doctoral thesis, we demonstrated that in resting microglial cells, CB2 and OX2 receptors antagonize each other. However, under neuroinflammatory conditions, they act synergistically to counteract this pathology, inducing the anti-inflammatory (M2) microglial phenotype and decreasing ROS levels and cell death. Therefore, the CB2-OX2 receptor complex emerges as a new therapeutic target with great potential to combat AD. One of the features that makes the endocannabinoid system particularly promising in the search for new cannabinoid compounds is its ability to alleviate pain. Currently, however, one of the most studied systems for its analgesic effects is the opioid system. The opioid system is involved in pain regulation, but it presents significant adverse effects and a strong addictive component. For this reason, in this doctoral thesis we sought proteins capable of regulating this system, to enhance analgesic effects while reducing adverse effects. Interestingly, we were able to demonstrate that the orphan receptor GPR88 modulates δOR internalization through the formation of the δOR-GPR88 complex. This finding explains the accumulation of δOR in intracellular compartments and why δOR agonists do not act more effectively and have failed to succeed in clinical trials.
[eng] Currently, Alzheimer’s disease is the most common cause of dementia worldwide. This disease is a progressive neurodegenerative disorder characterized by the accumulation of extracellular Aβ plaques and neurofibrillary tangles of the p-Tau protein, associated with a context of neuroinflammation that ultimately leads to synaptic dysfunction and neuronal death. The presence of Aβ aggregates increases extracellular glutamate levels, as the uptake and recycling processes of this neurotransmitter are impaired, inducing synaptic dysfunction and neurotoxicity. Consequently, patients suffer a progressive loss of memory, the onset of cognitive deficits, and behavioral alterations. The etiology of Alzheimer’s disease remains unknown, but it is known that genetic, environmental, and lifestyle factors play a role, with advanced age being the most important risk factor. Moreover, although there is no cure, treatments exist that aim to relieve symptoms and slow the progression of the disease. In the context of Alzheimer’s disease, cannabinoid compounds show beneficial effects due to their anti-inflammatory and neuroprotective properties. Specifically, retrograde signalling of endocannabinoids helps to decrease excitotoxicity through the presynaptic CB1R. With the aim of enhancing the beneficial effects of cannabinoid compounds and reducing adverse effects, we studied the entry pathways of these compounds into the orthosteric site of CB1R. In this way, we provide key information for the synthesis of new compounds with greater therapeutic activity targeting CB1R. Among the side effects of CB1R activation are psychoactive and addictive effects associated with cannabis consumption. In this doctoral thesis, we sought to describe the regulatory role of CB1R in drug addiction processes, using rodent models of cocaine self-administration. The results obtained demonstrate that overactivation of hippocampal neurons through targeted CB1R deletion increases reward-seeking and relapse. In contrast, inhibition of dopaminergic neurons in the ventral tegmental area (VTA) via CB1R deletion in GABAergic neurons of the ventral pallidum (VP) reduces reward-seeking. These results highlight the regulatory role of CB1R in the reward system and the need to specify its function in different brain areas involved in addiction. The first signs of Alzheimer’s disease is the increase in extracellular glutamate levels, which hyperactivates the N-methyl-D-aspartate receptor (NMDAR), inducing cytotoxic effects. One of the current treatments for AD is the administration of memantine, an NMDAR antagonist used in the more advanced stages of the disease. In Alzheimer’s, Aβ has been observed to specifically activate extrasynaptic NMDAR-GluN2B, worsening synaptic dysfunction and neurodegeneration. In contrast, Aβ promotes the internalization and inhibition of synaptic NMDAR-GluN2A channels, which are associated with neuronal survival and synaptic plasticity. Memantine blocks both NMDAR-GluN2A and NMDAR-GluN2B. For this reason, in this thesis we decided to search for proteins capable of differentially regulating glutamate receptors containing the NR2A and NR2B subunits. We discovered that CB1R activation differentially modulates NMDAR-GluN2A and NMDAR-GluN2B channels through the formation of heteromeric complexes. It enhances the neuroprotective effects of NMDAR-GluN2A and reduces the toxicity induced by NMDAR-GluN2B. In addition, the selective CB1R agonist ACEA decreases axonal transport of p-Tau, especially in neurons expressing NMDAR- GluN2A and lacking NMDAR-GluN2B. The results obtained highlight the potential of CB1R as a therapeutic target in Alzheimer’s disease, as it acts as a modulator of NMDAR channels, favouring neuroprotective effects and reducing cytotoxicity. It has been described that in Alzheimer’s disease, under conditions of pronounced neuroinflammation, the expression of CB2R in microglial cells increases. Furthermore, CB2R activation promotes the conversion of the pro-inflammatory (M1) microglial phenotype to the anti-inflammatory (M2) phenotype, inducing neuroprotective effects. On the other hand, one of the most common symptoms of AD is dysregulation of the sleep-wake cycle. These sleep disturbances impair the proper clearance of toxic waste that accumulates in cerebrospinal fluid during wakefulness, such as Aβ and p-Tau aggregates, thereby accelerating disease progression. The orexinergic system is involved in the regulation of the sleep-wake cycle. Indeed, excessive orexinergic signalling contributes to sleep interruptions, accelerating Aβ deposition. In this regard, evidence from studies conducted in animal models and in humans in vivo suggests that reducing orexinergic tone through dual orexin receptor antagonists (DORAs) improves sleep disturbances and cognitive decline, thus preventing the neurodegenerative process of AD. In this doctoral thesis, we analysed in detail the roles of the endocannabinoid and orexinergic systems in AD. Interestingly, we discovered that both systems regulate each other in AD. Specifically, we described that the cannabinoid receptor CB1 forms heteromeric complexes with the orexin receptor OX1, and that their expression increases in cortical neurons treated with Aβ aggregates. Moreover, in cortical neurons of adult 5XFAD mice, an Alzheimer’s model, we observed that OX1 receptor activation blocks the cannabinoid neuroprotective effect. In Alzheimer’s, CB2R is overexpressed under inflammatory conditions, and its activation triggers a neuroprotective response. For this reason, in this study we also analysed the regulatory role of the OX1 receptor on the therapeutic potential of CB2R. We described the formation of CB2R-OX1R complexes and an increase in their expression under AD conditions. Interestingly, we found that OX1 receptor blockade enhances cannabinoid neuroprotective effects in the microglia of APPSw/Ind Alzheimer’s mice, making OX1 antagonists’ potential new compounds to combat the disease. The orexinergic system consists of two orexin receptors: OX1 and OX2. OX2 is less studied, but it seems to play an important role in regulating OX1R activity, presenting neutral or even protective effects in neurodegenerative diseases associated with sleep disruption. In this doctoral thesis, we demonstrated that in resting microglial cells, CB2 and OX2 receptors antagonize each other. However, under neuroinflammatory conditions, they act synergistically to counteract this pathology, inducing the anti-inflammatory (M2) microglial phenotype and decreasing ROS levels and cell death. Therefore, the CB2-OX2 receptor complex emerges as a new therapeutic target with great potential to combat AD. One of the features that makes the endocannabinoid system particularly promising in the search for new cannabinoid compounds is its ability to alleviate pain. Currently, however, one of the most studied systems for its analgesic effects is the opioid system. The opioid system is involved in pain regulation, but it presents significant adverse effects and a strong addictive component. For this reason, in this doctoral thesis we sought proteins capable of regulating this system, to enhance analgesic effects while reducing adverse effects. Interestingly, we were able to demonstrate that the orphan receptor GPR88 modulates δOR internalization through the formation of the δOR-GPR88 complex. This finding explains the accumulation of δOR in intracellular compartments and why δOR agonists do not act more effectively and have failed to succeed in clinical trials.
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Citació
REBASSA PALOU, Joan Biel. Entre el silenci dels records: recerca d’una memòria perduda. Nous enfocaments terapèutics en la malaltia de l’Alzheimer. [consulted: 25 of May of 2026]. Available at: https://hdl.handle.net/2445/229339