Prion-mediated neurodegeneration is associated with early impairment of the ubiquitin-proteasome system.

Prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of misfolded prion protein (PrP(Sc)) in the brain. The critical relationship between aberrant protein misfolding and neurotoxicity currently remains unclear. The accumulation of aggregation-prone proteins has been linked to impairment of the ubiquitin-proteasome system (UPS) in a variety of neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington's diseases. As the principal route for protein degradation in mammalian cells, this could have profound detrimental effects on neuronal function and survival. Here, we determine the temporal onset of UPS dysfunction in prion-infected Ub(G76V)-GFP reporter mice, which express a ubiquitin fusion proteasome substrate to measure in vivo UPS activity. We show that the onset of UPS dysfunction correlates closely with PrP(Sc) deposition, preceding earliest behavioural deficits and neuronal loss. UPS impairment was accompanied by accumulation of polyubiquitinated substrates and found to affect both neuronal and astrocytic cell populations. In prion-infected CAD5 cells, we demonstrate that activation of the UPS by the small molecule inhibitor IU1 is sufficient to induce clearance of polyubiquitinated substrates and reduce misfolded PrP(Sc) load. Taken together, these results identify the UPS as a possible early mediator of prion pathogenesis and promising target for development of future therapeutics.

CB1 Cannabinoid Receptor-Dependent Activation of mTORC1/Pax6 Signaling Drives Tbr2 Expression and Basal Progenitor Expansion in the Developing Mouse Cortex.

The CB1 cannabinoid receptor regulates cortical progenitor proliferation during embryonic development, but the molecular mechanism of this action remains unknown. Here, we report that CB1-deficient mouse embryos show premature cell cycle exit, decreased Pax6- and Tbr2-positive cell number, and reduced mammalian target of rapamycin complex 1 (mTORC1) activation in the ventricular and subventricular cortical zones. Pharmacological stimulation of the CB1 receptor in cortical slices and progenitor cell cultures activated the mTORC1 pathway and increased the number of Pax6- and Tbr2-expressing cells. Likewise, acute CB1 knockdown in utero reduced mTORC1 activation and cannabinoid-induced Tbr2-positive cell generation. Luciferase reporter and chromatin immunoprecipitation assays revealed that the CB1 receptor drives Tbr2 expression downstream of Pax6 induction in an mTORC1-dependent manner. Altogether, our results demonstrate that the CB1 receptor tunes dorsal telencephalic progenitor proliferation by sustaining the transcriptional activity of the Pax6-Tbr2 axis via the mTORC1 pathway, and suggest that alterations of CB1 receptor signaling, by producing the missexpression of progenitor identity determinants may contribute to neurodevelopmental alterations.

CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling.

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.

The anxiolytic effect of cannabidiol on chronically stressed mice depends on hippocampal neurogenesis: involvement of the endocannabinoid system.

Cannabidiol (CBD), the main non-psychotomimetic component of the plant Cannabis sativa, exerts therapeutically promising effects on human mental health such as inhibition of psychosis, anxiety and depression. However, the mechanistic bases of CBD action are unclear. Here we investigate the potential involvement of hippocampal neurogenesis in the anxiolytic effect of CBD in mice subjected to 14 d chronic unpredictable stress (CUS). Repeated administration of CBD (30 mg/kg i.p., 2 h after each daily stressor) increased hippocampal progenitor proliferation and neurogenesis in wild-type mice. Ganciclovir administration to GFAP-thymidine kinase (GFAP-TK) transgenic mice, which express thymidine kinase in adult neural progenitor cells, abrogated CBD-induced hippocampal neurogenesis. CBD administration prevented the anxiogenic effect of CUS in wild type but not in GFAP-TK mice as evidenced in the novelty suppressed feeding test and the elevated plus maze. This anxiolytic effect of CBD involved the participation of the CB1 cannabinoid receptor, as CBD administration increased hippocampal anandamide levels and administration of the CB1-selective antagonist AM251 prevented CBD actions. Studies conducted with hippocampal progenitor cells in culture showed that CBD promotes progenitor proliferation and cell cycle progression and mimics the proliferative effect of CB1 and CB2 cannabinoid receptor activation. Moreover, antagonists of these two receptors or endocannabinoid depletion by fatty acid amide hydrolase overexpression prevented CBD-induced cell proliferation. These findings support that the anxiolytic effect of chronic CBD administration in stressed mice depends on its proneurogenic action in the adult hippocampus by facilitating endocannabinoid-mediated signalling.

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.

Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment.

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.

Acute polyglutamine expression in inducible mouse model unravels ubiquitin/proteasome system impairment and permanent recovery attributable to aggregate formation.

The presence of intracellular ubiquitylated inclusions in neurodegenerative disorders and the role of the ubiquitin/proteasome system (UPS) in degrading abnormal hazardous proteins have given rise to the hypothesis that UPS-impairment underlies neurodegenerative processes. However, this remains controversial for polyglutamine disorders such as Huntington disease (HD). Whereas studies in cellular models have provided evidence in favor of UPS-impairment attributable to expression of the N-terminal fragment of mutant huntingtin (N-mutHtt), similar studies on mouse models failed to do so. Furthermore, we have recently shown that the increase in polyubiquitin conjugates reported in the brain of N-mutHtt mice occurs in the absence of a general UPS-impairment. In the present study we aim to clarify the potential of N-mutHtt to impair UPS function in vivo as well as the mechanisms by which neurons may adapt after prolonged exposure to N-mutHtt in genetic models. By combining UPS reporter mice with an inducible mouse model of HD, we demonstrate for the first time polyglutamine-induced global UPS-impairment in vivo. UPS-impairment occurred transiently after acute N-mutHtt expression and restoration correlated with appearance of inclusion bodies (IBs). Consistently, UPS recovery did not take place when IB formation was prevented through administration of N-mutHtt aggregation-inhibitors in both cellular and animal models. Finally, no UPS-impairment was detected in old mice constitutively expressing N-mutHtt despite the age-associated decrease in brain proteasome activity. Therefore, our data reconcile previous contradictory reports by showing that N-mutHtt can indeed impair UPS function in vivo and that N-mutHtt aggregation leads to long lasting restoration of UPS function.

Boys-Specific Text-Comprehension Enhancement With Dual Visual-Auditory Text Presentation Among 12-14 Years-Old Students.

Quality of language comprehension determines performance in all kinds of activities including academics. Processing of words initially develops as auditory, and gradually extends to visual as children learn to read. School failure is highly related to listening and reading comprehension problems. In this study we analyzed sex-differences in comprehension of texts in Spanish (standardized reading test PROLEC-R) in three modalities (visual, auditory, and both simultaneously: dual-modality) presented to 12-14-years old students, native in Spanish. We controlled relevant cognitive variables such as attention (d2), phonological and semantic fluency (FAS) and speed of processing (WISC subtest Coding). Girls' comprehension was similar in the three modalities of presentation, however boys were importantly benefited by dual-modality as compared to boys exposed only to visual or auditory text presentation. With respect to the relation of text comprehension and school performance, students with low grades in Spanish showed low auditory comprehension. Interestingly, visual and dual modalities preserved comprehension levels in these low skilled students. Our results suggest that the use of visual-text support during auditory language presentation could be beneficial for low school performance students, especially boys, and encourage future research to evaluate the implementation in classes of the rapidly developing technology of simultaneous speech transcription, that could be, in addition, beneficial to non-native students, especially those recently incorporated into school or newly arrived in a country from abroad.

Contribution of Altered Endocannabinoid System to Overactive mTORC1 Signaling in Focal Cortical Dysplasia.

Alterations of the PI3K/Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway are causally involved in a subset of malformations of cortical development (MCDs) ranging from focal cortical dysplasia (FCD) to hemimegalencephaly and megalencephaly. These MCDs represent a frequent cause of refractory pediatric epilepsy. The endocannabinoid system -especially cannabinoid CB1 receptor- exerts a neurodevelopmental regulatory role at least in part via activation of mTORC1 signaling. Therefore, we sought to characterize the possible contribution of endocannabinoid system signaling to FCD. Confocal microscopy characterization of the CB1 receptor expression and mTORC1 activation was conducted in FCD Type II resection samples. FCD samples were subjected to single nucleotide polymorphism screening for endocannabinoid system elements, as well as CB1 receptor gene sequencing. Cannabinoid CB1 receptor levels were increased in FCD with overactive mTORC1 signaling. CB1 receptors were enriched in phospho-S6-positive cells including balloon cells (BCs) that co-express aberrant markers of undifferentiated cells and dysplastic neurons. Pharmacological regulation of CB1 receptors and the mTORC1 pathway was performed in fresh FCD-derived organotypic cultures. HU-210-evoked activation of CB1 receptors was unable to further activate mTORC1 signaling, whereas CB1 receptor blockade with rimonabant attenuated mTORC1 overactivation. Alterations of the endocannabinoid system may thus contribute to FCD pathological features, and blockade of cannabinoid signaling might be a new therapeutic intervention in FCD.

Ubiquitin-proteasome system involvement in Huntington's disease.

Huntington's disease (HD) is a genetic autosomal dominant neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin (htt) gene. This triplet expansion encodes a polyglutamine stretch (polyQ) in the N-terminus of the high molecular weight (348-kDa) and ubiquitously expressed protein htt. Normal individuals have between 6 and 35 CAG triplets, while expansions longer than 40 repeats lead to HD. The onset and severity of the disease depend on the length of the polyQ tract: the longer the polyglutamine stretch (polyQ) is, the earlier the disease begins and the more severe the symptoms are. One of the main histopathological hallmarks of HD is the presence of intraneuronal proteinaceous inclusion bodies, whose prominent and invariant feature is the presence of ubiquitin (Ub); therefore, they can be detected with anti-ubiquitin and anti-proteasome antibodies. This, together with the observation that mutations in components of the ubiquitin-proteasome system (UPS) give rise to some neurodegenerative diseases, suggests that UPS impairment may be causative of HD. Even though the link between disrupted Ub homeostasis and protein aggregation to HD is undisputed, the functional significance of these correlations and their mechanistic implications remains unresolved. Moreover, there is no consistent evidence documenting an accompanying decrease in levels of free Ub or disruption of Ub pool dynamics in neurodegenerative disease or models thus suggesting that the Ub-conjugate accumulation may be benign and just underlie lesion in 26S function. In this chapter we will elaborate on the different studies that have been performed using different experimental approaches, in order to shed light to this matter.

Protein oxidation in Huntington disease affects energy production and vitamin B6 metabolism.

Huntington disease (HD) is an inherited neurodegenerative disorder that initially affects the striatum and progressively the cortex. Oxidative stress in HD has been described as important to disease progression. In this study, protein carbonylation, used as a marker of protein oxidation, was analyzed in human brain striatum. A comparison of HD samples to matched controls identified 13 carbonylated proteins, including enzymes involved in the glycolytic pathway and mitochondrial proteins related to ATP production. Oxidation of the mitochondrial enzymes resulted in decreased catalytic activity, in good agreement with the energy deficiency observed in HD. We also found carbonylation of pyridoxal kinase and antiquitin 1, both involved in the metabolism of pyridoxal 5-phosphate, the active form of vitamin B6. The Tet/HD94 conditional mouse model allowed us to demonstrate that increased carbonylation in striatum is dependent on mutant huntingtin expression. As in humans, pyridoxal kinase showed decreased levels and was highly carbonylated in the gene-on mice; these modifications were reverted in the gene-off mice. We hypothesize that both pyridoxal kinase and antiquitin 1 oxidation could result in decreased pyridoxal 5-phosphate availability necessary as a cofactor in transaminations, synthesis of glutathione, and synthesis of GABA and dopamine, two neurotransmitters that play a key role in HD pathology.