Dual role of CDK5 on cognitive deficits and striatal vulnerability in Huntington’s disease

Abstract

Huntington’s disease (HD) is a neurodegenerative disorder caused by an autosomic mutation on the Huntingtin (HTT) coding gene. HD is mainly characterized by the appearance of motor symptoms or choreas, which are associated to the selective degeneration of striatal neurons, and by the presence of cognitive disturbances, which are attributed to alterations in corticostriatal connectivity and to hippocampal dysfunction. For this reason, finding targets involved both on striatal vulnerability and cognitive disturbances, might result in therapeutic strategies able to act simultaneously on HD’s motor and cognitive symptoms. In this Thesis we have focused on Cyclin-dependent kinase 5 (Cdk5) as one of these putative targets. Cdk5 acts mainly in the central nervous system, where its activator p35 is expressed, and it plays a major role on synaptic plasticity regulation. In addition, altered Cdk5 activity has been described in several neurodegenerative disorders, including HD, where Cdk5 deregulation has been associated to increased striatal vulnerability to excitotoxicity. Moreover, alteration of Cdk5 activity and/or subcellular distribution has also been linked to neuronal cell cycle re-entry, which has been proposed as a possible mechanism leading to neuronal dysfunction and eventual death in several neurodegenerative conditions. Therefore, on one hand, we aimed to study Cdk5 involvement in cognitive deficits and synaptic plasticity alterations in HD. To this end, we generated a new double mutant mice model which expresses one copy of mutant HTT (mHTT) (knock-in or KI), and is conditionally heterozygous for Cdk5 (Cdk5+/). We described that double mutant mice (KI:Cdk5+/-) presented restored corticostriatal and hippocampal cognitive function when compared to their KI littermates. We also observed that preserved corticostriatal function correlated with recovery of corticostriatal NR2B surface levels, which were reduced in KI mice. Moreover, recovery of NR2B surface levels was associated to normalization of NR2B total levels and of the pSrc/pNR2B pathway in the cortex of KI:Cdk5+/- mice. On the other hand, preserved hippocampal cognitive function correlated with recovery of CA1 dendritic spine density, as well as, with increased Rac1 activity in KI:Cdk5+/- mice. Restoration of dendritic spine density was also observed in layer V cortical neurons, in a Rac1-independent manner. Finally, we described that KI mice showed reduced physiological p35 plasma membrane levels in the cortex, which was recovered in KI:Cdk5+/- mice, correlating with preferential alteration of Cdk5 substrates phosphorylation levels in this brain region. In sum, our results demonstrate Cdk5 complex and brain region-specific involvement in cognitive deficits appearance and in synaptic alterations in HD. On the other hand, we also assessed whether Cdk5 deregulation might cause cell cycle re-entry of striatal neurons in HD. Cdk5 forms a nuclear complex with p27 and E2F1 in differentiated neurons, thus preventing E2F1 from binding to its coactivator DP1 and from activating transcription of cell cycle progression genes. For this reason, we analysed nuclear levels of Cdk5 and p27, and we observed that KI mice showed reduced Cdk5 and p27 nuclear levels, which could induce neuronal cell cycle re-entry. In agreement, we also observed increased levels of CyclinD1 in the striatum of KI mice since early symptomatic stages, and increased Cdk4 levels at late disease stages. Finally, we observed that NMDA treatment of striatal primary cultures caused a general reduction of cell cycle proteins neuronal expression, and importantly, it altered their subcellular distribution, reducing nuclear localization of the cell cycle inhibitor p27 and inducing nuclear presence of cell cycle progression proteins, E2F1 and Cdk4. Our results also suggested that presence of mHTT might further potentiate NMDA-induced subcellular distribution alteration of cell cycle proteins. Therefore, we suggest that reduction of Cdk5 nuclear levels might induce cell cycle re-entry of striatal neurons, a process which could be favoured by alterations in NMDA receptors activation, present in HD.

La malaltia de Huntington (MH) és un desordre neurodegeneratiu causat per una mutació al gen que codifica per la proteïna Huntingtina (HTT), i que consisteix principalment en l’aparició de dèficits motors, associats a la degeneració selectiva de l’estriat; i en l’aparició de dèficits cognitius, associats a una alteració en la connectivitat corticoestriatal i a una disfunció hipocampal. En aquesta Tesi, hem analitzat la implicació de la cinasa Cdk5, per una banda, en l’aparició dels dèficits cognitius; i per l’altre banda, en la reentrada neuronal al cicle cel·lular com a un possible mecanisme de susceptibilitat a la vulnerabilitat estriatal en la MH. Els nostres resultats han mostrat que la reducció genètica de Cdk5 en un model murí de la MH (KI), prevé l’aparició dels dèficits cognitius corticoestriatal i hipocampals. Aquesta millora cognitiva està associada a la recuperació dels nivells de membrana de NR2B a nivell corticoestriatal, i a la restauració de la densitat d’espines dendrítiques a l’hipocamp i a l’escorça, indicant una implicació de Cdk5, complexa i específica de regió cerebral, en les alteracions sinàptiques i l’aparició dels dèficits cognitius en la MH. D’altre banda, hem observat que els nivells nuclears de Cdk5 estan disminuïts a l’estriat dels ratolins KI, cosa que podria alterar la seva funció com a inhibidor de la progressió del cicle cel·lular en neurones diferenciades. En concordança amb aquesta hipòtesi, diferents proteïnes del cicle cel·lular presenten una alteració en els seus nivells proteics, tant en ratolins KI, com en mostres de pacients humans. A més, l’activació dels receptors NMDA en neurones estriatals porta a una alteració de la distribució subcel·lular de les proteïnes del cicle cel·lular prèviament analitzades, un efecte que podria ser potenciat per la presència de la HTT mutada. En conclusió, els resultats d’aquesta Tesi, mostren la complexa implicació de Cdk5 en l’aparició dels dèficits cognitius en la MH, i suggereixen que l’alteració de la localització nuclear de Cdk5 podria portar a la desregulació de diferents proteïnes del cicle cel·lular, un mecanisme que es podria veure afavorit per alteracions en l’activació dels receptors NMDA, presents en la MH.