Increased levels of rictor prevent mutant huntingtin-induced neuronal degeneration

Rictor associates with mTOR to form the mTORC2 complex, which activity regulates neuronal function and survival. Neurodegenerative diseases are characterized by the presence of neuronal dysfunction and cell death in specific brain regions such as for example Huntington's disease (HD), which is characterized by the loss of striatal projection neurons leading to motor dysfunction. Although HD is caused by the expression of mutant huntingtin, cell death occurs gradually suggesting that neurons have the capability to activate compensatory mechanisms to deal with neuronal dysfunction and later cell death. Here, we analyzed whether mTORC2 activity could be altered by the presence of mutant huntingtin. We observed that Rictor levels are specifically increased in the striatum of HD mouse models and in the putamen of HD patients. Rictor-mTOR interaction and the phosphorylation levels of Akt, one of the targets of the mTORC2 complex, were increased in the striatum of the R6/1 mouse model of HD suggesting increased mTORC2 signaling. Interestingly, acute downregulation of Rictor in striatal cells in vitro reduced mTORC2 activity, as shown by reduced levels of phospho-Akt, and increased mutant huntingtin-induced cell death. Accordingly, overexpression of Rictor increased mTORC2 activity counteracting cell death. Furthermore, normalization of endogenous Rictor levels in the striatum of R6/1 mouse worsened motor symptoms suggesting an induction of neuronal dysfunction. In conclusion, our results suggest that increased Rictor striatal levels could counteract neuronal dysfunction induced by mutant huntingtin

Striatal-Enriched Protein Tyrosine Phosphatase Expression and Activity in Huntington's Disease: A STEP in the Resistance to Excitotoxicity

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity

RTP801 is involved in mutant huntingtin-induced cell death

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease

Impacte ambiental dels iots en els ports de Sitges : anàlisi comparativa

Els ports esportius a nivell català han augmentat considerablement a causa de la creixent afició a la navegació i els esports nàutics. L'increment de la demanda d'amarratges al port i la conseqüent concentració d'iots, ha implicat la necessitat d'un estudi dels seus impactes potencials. El Port Garraf té un impacte més reduït degut a que prioritzen les embarcacions de vela, en canvi pel que fa al Port d'Aiguadolç i Port Ginesta la proporció d'embarcacions a motor és superior, per tant tindran un consum més elevat de combustible. Pel que fa a residus especials, concretament olis, els tres ports tenen un impacte sever per la difícil recuperació del medi en cas d'accident, encara que hi ha una bona gestió en els ports. Per acabar el Port d'Aiguadolç és el que té un consum d'aigua superior.Los puertos deportivos a nivel catalán han aumentado considerablemente a causa de la creciente afición de la navegación y los deportes náuticos. El incremento de la demanda de amarres en el puerto y la consecuente concentración de yates, ha implicado la necesidad de un estudio de los posibles impactos potenciales. El Port Garraf tiene un impacto más reducido debido a que se priorizan las embarcaciones de vela, en cambio, en el Port d'Aiguadolç y el Port Ginesta, la proporción de embarcaciones a motor es superior, por lo tanto, tendrán un consumo más elevado de combustible. Por lo que se refiere a los residuos especiales, concretamente aceites, los tres puertos tienen un impacto severo por la difícil recuperación del medio en caso de accidente, aunque haya una buena gestión en los puertos. Para acabar, el Port d'Aiguadolç es el que tiene un consumo de agua superior.The marinas has increased considerably in Catalonia because of the growing hobby of sailing and water sports. The increase in the demand of moorings in the marinas and the consequent concentration of yachts, has involved the need of a study of the potential environmental impacts. The Port of Garraf has a smaller environmental impact because it prioritises sailboats; however, the proportion of motor boats is greater in the Port of Ginesta and in the Port of Aiguadolç. That is why they both have a higher consumption of fuel. Regarding the hazardous waste, particularly oils, the three ports have a severe impact because of the difficult recovery of the environment in case of accident, although there is a good management in the ports. To conclude, the Port of Aiguadolç is the most consumptive of water. According to the comparison that has been made of the different flows, it can be concluded that the Port of Ginesta is the most sustainable. Thanks to the good management of the port and its user awareness. Although there is more surface and more boats, this port is the one which has the lower potential of impacts

Characterization of the mechanisms underlying alterations in macroautophagy and survival signalling in Huntington’s disease

[cat] La malaltia de Huntington és un trastorn neurodegeneratiu progressiu causat per una expansió de repeticions del triplet CAG (més de 37) en l’exó 1 del gen de la huntingtina que genera una proteïna aberrant. Aquesta proteïna modificada és tòxica i provoca una pèrdua selectiva de neurones GABAèrgiques de projecció en el nucli estriat, tot i que també s’han detectat alteracions i degeneració en altres àrees de l’encèfal, generant una simptomatologia complexa que engloba alteracions motores, cognitives i emocionals. Un marcador de la patologia és la formació d’agregats proteics, principalment compostos per fragments N-terminals de la huntingtina mutada que es generen per l’acció de proteases. Un dels processos que condueix a la mort selectiva de les neurones és l’activació de l’apoptosi. L’equilibri entre vies pro-apoptòtiques i vies de supervivència no només és el que regula el destí de la cèl•lula, sinó que també podria participar en la regulació de l’aparició dels primers símptomes de la patologia en un individu afectat. En aquesta Tesi es descriuen tres possibles mecanismes cel•lulars activats en un model murí de malaltia de Huntington, que podrien participar en compensar la toxicitat que genera la huntingtina mutada i així retardar-ne la patologia: (1) la sobreactivació de l’autofàgia selectiva a estadis inicials de la patologia, important per la degradació de la huntingtina mutada; (2) la sobreactivació de la via de senyalització mTOR-AKT, que participa en mecanismes de supervivència cel•lular; i (3) la inhibició de la via de senyalització de la PKCδ, que quan es troba activada genera apoptosis cel•lular en cèl•lules que expressen la huntingtina mutada. Potenciar des d’edats primerenques qualsevol d’aquests tres mecanismes, doncs, podria ser una bona estratègia terapèutica per a la malaltia de Huntington.[eng] Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG expansion (more than 37 repeats) in the exon 1 of the huntingtin gene, which results in the synthesis of a mutant protein that is toxic for some neuronal types. The striatum is the most affected brain region, although other brain areas, such as the cerebral cortex or hippocampus are also affected. Aggregates are a pathological hallmark of the disease, which are mainly composed by N-terminal mutant huntingtin fragments and are present within the cells in both cytoplasm and nucleus. Apoptotic activation is one of the mechansims by which neurodegeneration occurs. Thus, whether a neuron lives or dies in pathological condition is the result of a complex balance between anti- and pro-apoptotic signals, which is crucial to determine cell fate. Moreover, this balance might strongly regulate the onset of the disease. Here, we have characterized three putative compensatory pro-survival processes that are altered in Huntington’s disease and could be involved in delaying the pathology progression. First, we have studied selective autophagy, a mechanism that degrades toxic mutant huntingtin species. To this end, we analyzed protein levels and intracellular localization of p62 and NBR1 (two selective autophagy receptors that specifically recognize cell components that need to be removed by means of autophagy) in the R6/1 mouse model of HD along the progression of the disease.,. We have observed that at early stages of the disease, p62 and NBR1 protein levels are reduced, indicating increased autophagic activity that could play a role in degrading mutant huntingtin. However, at later stages of the disease protein levels of both proteins have a different pattern depending on the cerebral region analyzed. In the cortex protein levels of both proteins are still reduced, indicating that selective autophagy is overactivated until later stages of the disease. However, in the striatum and in the hippocampus they accumulate due to distinct factors. p62 is sequestered by nuclear mutant huntingtin aggregates, while NBR1 is still in the cytoplasm, thus still taking part in the autophagy process. Its accumulatiion could be due to an inefficient selective autophagy that could get worst with age. We have also studied whether different intracellular signalling pathways, involved in cell survival or apoptosis, are altered by mutant huntingtin expression. We have observed that the mTOR signalling pathway, through the mTORC2 but not mTORC1 complex, is over-activated in the striatum of R6/1 mice, and we think that this overactivation could play a role in the previous reported increased phosphorylation of the pro-survival kinase AKT in the same HD mouse model. An increase in the activity of the mTORC2 complex, could be due to an increase in Rictor protein levels that have been found specifically in the striatum of R6/1 mice and in the putamen of HD patients. Finally, we also analyzed the PKC signalling pathway, since PKCs regulate different processes important for neuronal survival and plasticity. We have observed that protein levels of different PKC isoforms, PKCα, PKCβII and PKCδ, are reduced in the striatum, cortex and hippocampus of R6/1 mice. The most important reduction was observed for the proapototic PKC isoform PKCδ, which started already at early stages of the disease. This suggests that neurons could try to block this signalling pathway in order to reduce mutant huntingtin toxicity. We have obsereved that striatal cells that express mutant huntingtin, but not wild-type huntingtin, are more vulnerable to undergo apoptosis when they overexpress PKCδ. The present Thesis describes the up-regulation of several compensatory prosurvival mechanisms in HD to counteract the mutant huntingtin-induced toxicity. The potentiation of such prosurvival mechanisms could be a good therapeutical approach in HD

Increased 90-kDa ribosomal S6 kinase (Rsk) activity is protective against mutant huntingtin toxicity

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

Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics

The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aβ1–40 levels could be observed in mice injected with anti-TfR/BACE1 bispecific antibodies when compared to vehicle-injected mice. In summary, we found two nanobodies that could bind both human and cynomolgus TfR with the potential to be used clinically to increase the brain permeability of therapeutic biologicals

Increased 90-kDa ribosomal S6 kinase (Rsk) activity is protective against mutant huntingtin toxicity

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 phosphoinositidedependent 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 H

Impact of diversity of antibiotic use on the development of antimicrobial resistance

Objectives: To evaluate the impact of different antibiotic strategies on acquisition of resistant microorganisms. Methods: A prospective study was conducted over a 44 month period in a single ICU. Four empirical antibiotic strategies for ventilator-associated pneumonia (VAP) were sequentially implemented. Over the initial 10 months, patient-specific antibiotic therapy was prescribed; then, 4 month periods of prioritization or restriction rotation cycles of various antimicrobial agents were implemented for a total of 24 months; and, finally, during the last 10 months (mixing period) the first-line antibiotic for VAP was changed following a pre-established schedule to ensure maximum heterogeneity. Antibiotic consumption was closely monitored every month, and antimicrobial resistance patterns were regularly assessed. Antimicrobial heterogeneity was estimated using a modified Peterson index (AHI) measuring the ratios for the five most used antibiotics. Colonization by targeted microorganisms and susceptibility patterns were compared with the patient-specific period. Results: Higher diversity of antibiotic prescription was obtained during patient-specific therapy (AHI = 0.93) or mixing periods (AHI = 0.95) than during prioritization (AHI = 0.70) or restriction periods (AHI = 0.68). High homogeneity was associated with increases in carbapenem-resistant Acinetobacter baumannii (CR-Ab) [relative risk (RR) 15.5; 95%CI 5.5–42.8], extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae (RR 4.2; 95%CI 1.9–9.3) and Enterococcus faecalis (RR 1.7; 95%CI 1.1–2.9). During the restriction period, incidence of ESBL-producing Enterobacteriaceae and E. faecalis returned to patient-specific rates but CR-Ab remained higher. Conclusions: Antibiotic prescription patterns balancing the use of different antimicrobials should be promoted to reduce the selection pressure that aids the development of resistance