Search

Ki67 positive cells are increased in anti-aggregant slices at DIV30. OHSCs were cultured until DIV30. The slices were then fixed and immunostained with NeuN antibody for neurons and Ki67, a marker of proliferating cells. Note that with advanced age the level of proliferation is higher in anti-aggregant TauRDÎ KPP slices (top row), compared with controls (bottom). (PDF 113 kb)

Total Tau and GSK3ß levels in the aged anti-aggregant mice. Hippocampal tissue lysates from 16month old control and anti-aggregant animals were subjected to western blot analysis for total Tau, active GSK3ß (phosphorylated at Y216) and total GSK3ß. There was no significant change, though there was a significant 50% increase in the total Tau levels. Results show the mean ±SEM of 6-7 animals per condition and represent the ratio between the analyzed protein and actin levels. Quantification was obtained by densitometry. (PDF 81 kb)

Rate of proliferation and neuronal differentiation is higher in anti-aggregant TauRDΔKPP slices at DIV30. The rate of proliferation (as assessed by BrdU) is compared to the rate of neuronal differentiation. OHSCs from the controls, anti-aggregant TauRDΔKPP and pro-aggregant TauRDΔK pups were cultured until DIV30. BrdU was applied to the culture media from DIV15 until DIV30. Later the slices were fixed with 4% formaldehyde and immunostained for BrdU (for proliferating cells) and NeuN (for neurons). (A) Graph representing the number of proliferating cells (BrdU, empty bars) vs. the number of cells differentiated into neurons (BrdU+NeuN, hatched bars) in the controls (grey) and anti-aggregant TauRDΔKPP slices (pink) at DIV30. In the anti-aggregant TauRDΔKPP slices the number of BrdU positive cells increased by 90% in CA1, 70% in CA3 and 100% in DG (compare empty grey and pink bars 1 to 3, 5 to 7 and 9 to11). Almost 75% of the proliferating cells get differentiated into mature neurons both in the DG and the CA3 region and 50% of the cells in the CA1 differentiate into neurons (bars 3 to 4, 7 to 8 and 11 to 12). Data was analyzed by Student's t test. *p

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the exon-1 of the huntingtin (htt) gene. The presence of mutant htt (mhtt) results in multiple physiopathological changes, including protein aggregation, transcriptional deregulation, decreased trophic support, alteration in signaling pathways and excitotoxicity. Indeed, the presence of mhtt induces changes in the activities/levels of different kinases, phosphatases and transcription factors that can impact on cell survival. Many studies have provided evidence that transcription may be a major target of mhtt, as gene dysregulation occurs before the onset of symptoms. The greatest number of downregulated genes in HD has led to test the ability of a large number of compounds to restore gene transcription in mouse models of HD. On the other hand, mhtt engenders multiple cellular dysfunctions including an increase of pathological glutamate-mediated excitotoxicity. For that reason, targeting the excess of glutamate has been the goal for many promising drugs leading to clinical trials. Although advances in developing effective therapies are evident, currently, there is no known cure for HD and existing symptomatic treatments are limited.

[cat] Huntington’s disease is a progressive neurodegenerative disorder caused by the expansion of a CAG tract in the exon-1 of the huntingtin gene. Mutant huntingtin induces a large amount of toxic effects that trigger cell dysfunction and consequently, behavioral alterations such as motor dysfunction, cognitive decline and psychological disturbances. However, before the onset of symptoms individuals are healthy. Thus, it is plausible that compensatory mechanisms may be activated to regulate a … [+] Resum: La malaltia de Huntington és un trastorn neurodegeneratiu progressiu caracteritzat per la presencia d’alteracions motores i dèficits cognitius. Aquesta malaltia està causada per l’expansió anòmala del triplet CAG en l’exó 1 del gen que codifica per la proteïna huntingtina. La huntingtina mutada indueix gran quantitat d’efectes tòxics que desencadenen la disfunció cel•lular i, en conseqüència, les alteracions en la conducta característiques d’aquesta malaltia. Tot i així, abans de l’aparició del símptomes, els individus són sans. Per tant, és plausible pensar que es produeix una activació de mecanismes compensatoris per a regular l’equilibri entre la mort i la supervivència cel•lular. En aquesta tesis, ens hem centrat en dos objectius principals: (1) l’estudi dels mecanismes compensatoris activats en la presencia de la huntingtina mutada per tal de millorar la supervivència cel•lular i (2) la identificació de dianes moleculars per a reduir els dèficits motors i cognitius, així com per a millorar la plasticitat sinàptica en models murins de la malaltia de Huntington. Específicament, els nostres resultats han ajudat a entendre el paper de la proteïna cinasa p90Rsk i del factor de transcripció Elk-1 en la susceptibilitat de les neurones estriatals a la mort induïda per la huntingtina mutada. Per altre banda, mostrem per primera vegada el potencial terapèutic dels receptors de la prostaglandina E2, EP1 i Ep2, per al tractament dels símptomes clínics y histopatològics en la malaltia de Huntington., [eng]Huntington’s disease is a progressive neurodegenerative disorder caused by the expansion of a CAG tract in the exon-1 of the huntingtin gene. Mutant huntingtin induces a large amount of toxic effects that trigger cell dysfunction and consequently, behavioral alterations such as motor dysfunction, cognitive decline and psychological disturbances. However, before the onset of symptoms individuals are healthy. Thus, it is plausible that compensatory mechanisms may be activated to regulate a balance between cell death and survival. This compensatory mechanism might modulate the progression of Huntington’s disease. Understanding altered mechanisms due to mutant huntingtin expression in order to find new therapeutic targets to reduce neuronal dysfunction/death in Huntington’s disease must be a priority. It is believed that there is a balance between positive signaling for cell survives or dies. Many of the pathways involved in these processes are controlled at the level of phosphorylation and transcription. In this thesis we have studied mechanisms of transcription and protein activation, which are considered one of the main causes that trigger to mutant huntingtin-induced neuronal dysfunction. We have identified two targets, the protein kinase p90Rsk and the transcription factor Elk-1, that are activated during the progression of Huntington’s disease in order to protect striatal cells against mutant huntingtin-induced cell death. These two proteins are tightly related to neuronal survival and transcription regulation. p90Rsk is a kinase that phosphorylates substrates in the cytoplasm inhibiting their pro-apoptotic activity and phosphorylate transcription factors in the nucleus promoting their pro-survival activity. Likewise, Elk-1 promotes the transcription of many immediate early genes related to synaptic plasticity and neuronal survival. We observed an up-regulation of these to proteins during the progression of Huntington’s disease in different mouse models. This up-regulation was necessary to protect striatal cells from mutant-huntingtin. The prevention of cell death is an important point for neurodegenerative diseases; even so, it is unlikely that cellular machinery works well until the death of the cells. In many neurodegenerative diseases, such as Huntington’s disease, neuronal and synaptic dysfunction precedes cell death and occurs long before, or sometimes in absence of cell death. Recent studies suggest that targeting early pathophysiological disturbances in models of Huntignton’s disease can reverse neuronal dysfunction and delay progression to neurodegeneration. In this thesis, we also point out for the first time the modulation of Prostaglandin E2 (PGE2) EP receptors, namely EP1 and EP2, as a therapeutic targets in Huntington’s disease. The activation of EP1 receptor produces an increase in intracellular levels of calcium, while EP2 receptor activation increases cAMP; therefore, the activation of different EP receptors can have opposite effects. Whereas the blockade of EP2-EP4 receptors can aggravate neurodegeneration, antagonizing EP1 receptor has neuroprotective effects. We observed that pharmacological inhibition of EP1 receptor improves motor coordination and reduces memory decline in R6/1mice model of Huntington’s disease. Moreover, EP1 antagonism increases the expression of specific synaptic markers in the striatum and the hippocampus of these mice and also improves the long-term potentiation in the hippocampus. Finally, EP1 receptor antagonism reduces the presence of striatal and hippocampal mutant huntingtin nuclear aggregates in the striatum and the hippocampus of treated-R6/1 mice. Contrary to EP1 receptor, EP2 receptor plays a neuroprotective role in Huntington’s disease. We observe that EP2 receptor activation improves long-term memory in R6/1 mice. Moreover, EP2 activator increases dendritic branching in a BDNF-dependent manner, increases the protein levels of BDNF and the total number of PSD-95+ spines in the hippocampus of these mice. In conclusion, we propose the modulation of PGE2 receptors as a new therapeutic strategy in Huntington’s disease.

Background The 90-kDa ribosomal S6 kinase (Rsk) family is involved in cell survival. Rsk activation is regulated by sequential phosphorylations controlled by extracellular signal-regulated kinase (ERK) 1/2 and 3-phosphoinositide-dependent protein kinase 1 (PDK1). Altered ERK1/2 and PDK1 phosphorylation have been described in Huntington's disease (HD), characterized by the expression of mutant huntingtin (mhtt) and striatal degeneration. However, the role of Rsk in this neurodegenerative disease remains unknown. Here, we analyzed the protein levels, activity and role of Rsk in in vivo and in vitro HD models. Results We observed increased protein levels of Rsk1 and Rsk2 in the striatum of HdhQ111/Q111 and R6/1 mice, STHdhQ111/Q111 cells and striatal cells transfected with full-length mhtt. Analysis of the phosphorylation of Rsk in Hdh mice and STHdh cells showed reduced levels of phospho Ser-380 (dependent on ERK1/2), whereas phosphorylation at Ser-221 (dependent on PDK1) was increased. Moreover, we found that elevated Rsk activity in STHdhQ111/Q111 cells was mainly due to PDK1 activity, as assessed by transfection with Rsk mutant constructs. The increase of Rsk in STHdhQ111/Q111 cells occurred in the cytosol and in the nucleus, which results in enhanced phosphorylation of both cytosolic and nuclear Rsk targets. Finally, pharmacological inhibition of Rsk, knock-down and overexpression experiments indicated that Rsk activity exerts a protective effect against mhtt-induced cell death in STHdhQ7/Q7 cells transfected with mhtt. Conclusion The increase of Rsk levels and activity would act as a compensatory mechanism with capacity to prevent mhtt-mediated cell death. We propose Rsk as a good target for neuroprotective therapies in HD.