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Background Extracellular vesicles (EVs) play a crucial role in intercellular communication, participating in the paracrine trophic support or in the propagation of toxic molecules, including proteins. RTP801 is a stress-regulated protein, whose levels are elevated during neurodegeneration and induce neuron death. However, whether RTP801 toxicity is transferred trans-neuronally via EVs remains unknown. Methods We overexpressed or silenced RTP801 protein in cultured cortical neurons and isolated the neural-derived EVs (RTP801-EVs, or shRTP801-EVs respectively). We characterized their protein content by mass spectrometry (MS) and western blotting (WB). RTP801-EVs toxicity was assessed by treating cultured neurons with these EVs and quantifying apoptotic neuron death and branching. We also tested shRTP801-EVs functionality in the pathologic in vitro model of 6-Hydroxydopamine (6-OHDA) by biochemical analyses. Results Expression of RTP801 increased the number of EVs released by neurons. Moreover, RTP801 led to a distinct proteomic signature of neural-derived EVs, containing more pro-apoptotic markers. Hence, we observed that RTP801 toxicity was transferred to neurons via EVs, activating apoptosis and impairing neuron morphology complexity. In contrast, EVs derived from neurons where RTP801 was silenced were able to increase the arborization of recipient neurons. We also showed that 6-OHDA neurotoxin elevated protein levels of RTP801 in both cell lysates and EVs. Furthermore, neural-derived EVs induced phosphorylation of Ser473-Akt and Ser235/236-RPS6 in recipient neurons, and this effect was lost when EVs were derived from neurons treated with 6-OHDA. Interestingly, EVs derived from neurons where RTP801 was silenced prior to exposing them to 6-OHDA maintained Akt and RPS6 transactivation in recipient neurons. Conclusion Taken together, these results suggest that RTP801 toxicity can be spread via EVs and therefore, it could contribute to the progression of neurodegenerative diseases in which RTP801 is involved.

The purpose of this study was to investigate whether differential phosphorylation states of blood markers can identify patients with LRRK2 Parkinson's disease (PD). We assessed phospho(P)-Ser-935-LRRK2 and P-Ser-473-AKT levels in peripheral blood cells from patients with G2019S LRRK2-associated PD (L2PD, n = 31), G2019S LRRK2 non-manifesting carriers (L2NMC, n = 26), idiopathic PD (iPD, n = 25), and controls (n = 40, total n = 122). We found no differences at P-Ser-935-LRRK2 between groups but detected a specific increase of P-Ser-473-AKT levels in all G2019S carriers, either L2PD or L2NMC, absent in iPD. Although insensitive to LRRK2 inhibition, our study identifies P-Ser-473-AKT as an endogenous candidate biomarker for peripheral inflammation in G2019S carriers using accessible blood cells. ANN NEUROL 2022;92:888-894.

Niemann-Pick type C disease (NP-C) is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NP-C disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NP-C disease is common. Recently, we demonstrated that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NP-C disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurological and hepatic functions, as well as liver histology and ultrastructure. Strikingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype but further deteriorated their compromised hepatic functions and reduced lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice do not reverse neurological and motor dysfunction and it further worsens overall liver function, exacerbating hepatic failure in NP-C disease.

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene. Striatal projection neurons are mainly affected, leading to motor symptoms, but molecular mechanisms involved in their vulnerability are not fully characterized. Here, we show that eIF4E binding protein (4E-BP), a protein that inhibits translation, is inactivated in Huntington's disease striatum by increased phosphorylation. Accordingly, we detected aberrant de novo protein synthesis. Proteomic characterization indicates that translation specifically affects sets of proteins as we observed upregulation of ribosomal and oxidative phosphorylation proteins and downregulation of proteins related to neuronal structure and function. Interestingly, treatment with the translation inhibitor 4EGI-1 prevented R6/1 mice motor deficits, although corticostriatal long-term depression was not markedly changed in behaving animals. At the molecular level, injection of 4EGI-1 normalized protein synthesis and ribosomal content in R6/1 mouse striatum. In conclusion, our results indicate that dysregulation of protein synthesis is involved in mutant huntingtin-induced striatal neuron dysfunction.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an expansion of a CAG repeat in the huntingtin (htt) gene, which results in an aberrant form of the protein (mhtt). This leads to motor and cognitive deficits associated with corticostriatal and hippocampal alterations. The levels of STriatal-Enriched protein tyrosine Phosphatase (STEP), a neural-specific tyrosine phosphatase that opposes the development of synaptic strengthening, are decreased in the striatum of HD patients and also in R6/1 mice, thereby contributing to the resistance to excitotoxicity described in this HD mouse model. Here, we aimed to analyze whether STEP inactivation plays a role in the pathophysiology of HD by investigating its effect on motor and cognitive impairment in the R6/1 mouse model of HD. We found that genetic deletion of STEP delayed the onset of motor dysfunction and prevented the appearance of cognitive deficits in R6/1 mice. This phenotype was accompanied by an increase in pERK1/2 levels, a delay in the decrease of striatal DARPP-32 levels and a reduction in the size of mhtt aggregates, both in the striatum and CA1 hippocampal region. We also found that acute pharmacological inhibition of STEP with TC-2153 improved cognitive function in R6/1 mice. In conclusion, our results show that deletion of STEP has a beneficial effect on motor coordination and cognition in a mouse model of HD suggesting that STEP inhibition could be a good therapeutic strategy in HD patients.

Huntington’s disease (HD) is an inherited neurodegenerative disorder with onset of characteristic motor symptoms at midlife, preceded by subtle cognitive and behavioral disturbances. Transcriptional dysregulation emerges early in the disease course and is considered central to HD pathogenesis. Using wild-type and HD knock-in mouse striatal cell lines we observed a HD genotype-dependent reduction in the protein levels of transcription factor 4 (TCF4), a member of the basic helix-loop-helix family with critical roles in brain development and function. We characterized mouse Tcf4 gene structure and expression of alternative mRNAs and protein isoforms in cell-based models of HD, and in four different brain regions of male transgenic HD mice (R6/1) from young to mature adulthood. The largest decrease in the levels of TCF4 at mRNA and specific protein isoforms were detected in the R6/1 mouse hippocampus. Translating this finding to human disease, we found reduced expression of long TCF4 isoforms in the post-mortem hippocampal CA1 area and in the cerebral cortex of HD patients. Additionally, TCF4 protein isoforms showed differential synergism with the proneural transcription factor ASCL1 in activating reporter gene transcription in hippocampal and cortical cultured neurons. Induction of neuronal activity increased these synergistic effects in hippocampal but not in cortical neurons, suggesting brain region-dependent differences in TCF4 functions. Collectively, this study demonstrates isoform-specific changes in TCF4 expression in HD that could contribute to the progressive impairment of transcriptional regulation and neuronal function in this disease. Significance Statement Historically, HD has been considered a neurodegenerative disease. However, research of the last decade has revealed disrupted neurogenesis and cognitive dysfunction preceding pathological neuronal cell death, suggesting that HD is also a neurodevelopmental disease. One of the major molecular mechanisms of HD is dysregulation of transcription. Studying transcription factors with functions in neurogenesis and neural plasticity is of interest for their potential participation in the cognitive impairment in HD etiology. Here we show reduced expression of the transcription factor TCF4, previously linked with neurodevelopmental and neuropsychiatric diseases, in hippocampus and cerebral cortex of R6/1 mouse and HD patients. Our results shed light on the potential neurodevelopmental aspect of HD, and could be applicable for developing alleviating therapies for HD.

Lamins are crucial proteins for nuclear functionality. Here, we provide new evidence showing that increased lamin B1 levels contribute to the pathophysiology of Huntington’s disease (HD), a CAG repeat‐associated neurodegenerative disorder. Through fluorescence‐activated nuclear suspension imaging, we show that nucleus from striatal medium‐sized spiny and CA1 hippocampal neurons display increased lamin B1 levels, in correlation with altered nuclear morphology and nucleocytoplasmic transport disruption. Moreover, ChIP‐sequencing analysis shows an alteration of lamin‐associated chromatin domains in hippocampal nuclei, accompanied by changes in chromatin accessibility and transcriptional dysregulation. Supporting lamin B1 alterations as a causal role in mutant huntingtin‐mediated neurodegeneration, pharmacological normalization of lamin B1 levels in the hippocampus of the R6/1 mouse model of HD by betulinic acid administration restored nuclear homeostasis and prevented motor and cognitive dysfunction. Collectively, our work points increased lamin B1 levels as a new pathogenic mechanism in HD and provides a novel target for its intervention., The study shows that increased lamin B1 levels contribute to altered nuclear function of specific neurons in Huntington's disease (HD) brain. Results highlight this alteration as a new pathogenic mechanism for HD and provide a novel target for HD intervention.

RTP801/REDD1 is a stress responsive protein that mediates mutant huntingtin (mhtt) toxicity in cellular models and is up regulated in Huntington’s disease (HD) patients’ putamen. Here, we investigated whether RTP801 is involved in motor impairment in HD by affecting striatal synaptic plasticity.Ectopic mhtt was over expressed in cultured rat primary neurons. The protein levels of RTP801 were assessed in homogenates and crude synaptic fractions from human postmortem HD brains and mouse models of HD. Striatal RTP801 expression was knocked down with adeno-associated viral particles containing a shRNA in the R6/1 mouse model of HD and motor learning was then tested.Ectopic mhtt elevated RTP801 in synapses of cultured neurons. RTP801 was also up regulated in striatal synapses from HD patients and mouse models. Knocking down RTP801 in the R6/1 mouse striatum prevented motor learning impairment. RTP801 silencing normalized the Ser473 Akt hyperphosphorylation by downregulating Rictor and it induced synaptic elevation of calcium permeable GluA1 subunit and TrkB receptor levels, suggesting an enhancement in synaptic plasticity.These results indicate that mhtt-induced RTP801 mediates motor dysfunction in a HD murine model, revealing a potential role in the human disease. These findings open a new therapeutic framework focused on the RTP801/Akt/mTOR axis.

Lamins are crucial proteins for nuclear functionality. Here, we provide new evidence showing an involvement of increased lamin B1 levels in the pathophysiology of Huntington’s disease (HD), a CAG repeat-associated neurodegenerative disorder. Through fluorescence-activated nuclear suspension imaging we demonstrate that nucleus from striatal medium-sized spiny and CA1 hippocampal neurons display increased lamin B1 levels, in correlation with altered nuclear morphology and nucleocytoplasmic transport disruption. Moreover, ChIP-sequencing analysis shows an alteration of lamin-associated chromatin domains in hippocampal nuclei, which could contribute to transcriptional alterations we determined by RNA sequencing. Supporting lamin B1 alterations as a causal role in mutant-huntingtin mediated neurodegeneration, pharmacological normalization of lamin B1 levels by betulinic acid administration in the R6/1 mouse model of HD restored nuclear homeostasis and prevented motor and cognitive dysfunction. Collectively, our work point out increased lamin B1 levels as a new pathogenic mechanism in HD and provides a novel target for its intervention.

Striatal-enriched protein tyrosine phosphatase (STEP) modulates key signaling molecules involved in synaptic plasticity and neuronal function. It is postulated that STEP opposes the development of long-term potentiation (LTP) and that it exerts a restraint on long-term memory (LTM). Here, we examined whether STEP61 levels are regulated during hippocampal LTP and after training in hippocampal-dependent tasks. We found that after inducing LTP by high frequency stimulation or theta-burst stimulation STEP61 levels were significantly reduced, with a concomitant increase of STEP33 levels, a product of calpain cleavage. Importantly, inhibition of STEP with TC-2153 improved LTP in hippocampal slices. Moreover, we observed that after training in the passive avoidance and the T-maze spontaneous alternation task, hippocampal STEP61 levels were significantly reduced, but STEP33 levels were unchanged. Yet, hippocampal BDNF content and TrkB levels were increased in trained mice, and it is known that BDNF promotes STEP degradation through the proteasome. Accordingly, hippocampal pTrkBTyr816, pPLCγTyr783, and protein ubiquitination levels were increased in T-SAT trained mice. Remarkably, injection of the TrkB antagonist ANA-12 (2 mg/Kg, but not 0.5 mg/Kg) elicited LTM deficits and promoted STEP61 accumulation in the hippocampus. Also, STEP knockout mice outperformed wild-type animals in an age- and test-dependent manner. Summarizing, STEP61 undergoes proteolytic degradation in conditions leading to synaptic strengthening and memory formation, thus highlighting its role as a molecular constrain, which is removed to enable the activation of pathways important for plasticity processes., This work was supported by the Ministerio de Economia y Competitividad, Spain (SAF2016-08573-R to E. PérezNavarro and BFU2014-57929-P, cofinanced by FEDER, to E.D. Martín).

Introduction: Huntington’s disease (HD), an autosomal dominant neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin gene, has long been characterized by the presence of motor symptoms due to the loss of striatal projection neurons. Cognitive dysfunction and neuropsychiatric symptoms are also present and they occur in the absence of cell death in most mouse models, pointing to neuronal dysfunction and abnormal synaptic plasticity as causative mechanisms.Areas covered: Here, we focus on those common mechanisms altered by the presence of mutant huntingtin affecting corticostriatal and hippocampal function as therapeutic targets that could prove beneficial to ameliorate both cognitive and motor function in HD. Specifically, we discuss the importance of reestablishing the balance in (1) synaptic/extrasynaptic N-methyl-D-aspartate receptor signaling, (2) mitochondrial dynamics/trafficking, (3) TrkB/p75NTR signaling, and (4) transcriptional activity.Expert opinion: Mutant hu...

RTP801/REDD1 is a stress-regulated protein whose levels are increased in several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s diseases (HD). RTP801 downregulation ameliorates behavioral abnormalities in several mouse models of these disorders. In HD, RTP801 mediates mutant huntingtin (mhtt) toxicity in in vitro models and its levels are increased in human iPSCs, human postmortem putamen samples and in striatal synaptosomes from mouse models of the disease. Here, we investigated the role of RTP801 in the hippocampal pathophysiology of HD. We found that RTP801 levels are increased in the hippocampus of HD patients in correlation with gliosis markers. Although RTP801 expression is not altered in the hippocampus of the R6/1 mouse model of HD, neuronal RTP801 silencing in the dorsal hippocampus with shRNA-containing AAV particles ameliorates cognitive alterations. This recovery is associated with a partial rescue of synaptic markers and with a reduction of inflammatory events, especially microgliosis. Altogether, our results indicate that RTP801 could be a marker of hippocampal neuroinflammation in HD patients and a promising therapeutic target of the disease.

Background Chelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells. Methods In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line. Results We found that 10 μM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular Ca2+ levels that mediates calpain activation. In addition, we found that chelerythrine induces ERK1/2- and calpain-independent caspase-3 activation that can be prevented by the Ca2+ chelator BAPTA-AM. Conclusions This is the first report showing that chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells, which has consequences for the interpretation of studies using this compound. General significance Chelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works.

No abstract available.

One of the main focuses in Huntington's disease (HD) research, as well as in most neurodegenerative diseases, is the development of new therapeutic strategies, as currently there is no treatment to delay or prevent the progression of the disease. Neuronal dysfunction and neuronal death in HD are caused by a combination of interrelated pathogenic processes that lead to motor, cognitive and psychiatric symptoms. Understanding how mutant huntingtin impacts on a plethora of cellular functions could help to identify new molecular targets. Although HD has been classically classified as a neurodegenerative disease affecting voluntary movement, lately cognitive dysfunction is receiving increased attention as it is very invalidating for patients. Thus, an ambitious goal in HD research is to find altered molecular mechanisms that contribute to cognitive decline. In this review, we have focused on those findings related to corticostriatal and hippocampal cognitive dysfunction in HD, as well as on the underlying molecular mechanisms, which constitute potential therapeutic targets. These include alterations in synaptic plasticity, transcriptional machinery and neurotrophic and neurotransmitter signaling.

Huntington's disease (HD) is a polyglutamine disorder caused by a CAG expansion in the Huntingtin (HTT) gene exon 1. This expansion encodes a mutant protein whose abnormal function is traditionally associated with HD pathogenesis; however, recent evidence has also linked HD pathogenesis to RNA stable hairpins formed by the mutant HTT expansion. Here, we have shown that a locked nucleic acid-modified antisense oligonucleotide complementary to the CAG repeat (LNA-CTG) preferentially binds to mutant HTT without affecting HTT mRNA or protein levels. LNA-CTGs produced rapid and sustained improvement of motor deficits in an R6/2 mouse HD model that was paralleled by persistent binding of LNA-CTG to the expanded HTT exon 1 transgene. Motor improvement was accompanied by a pronounced recovery in the levels of several striatal neuronal markers severely impaired in R6/2 mice. Furthermore, in R6/2 mice, LNA-CTG blocked several pathogenic mechanisms caused by expanded CAG RNA, including small RNA toxicity and decreased Rn45s expression levels. These results suggest that LNA-CTGs promote neuroprotection by blocking the detrimental activity of CAG repeats within HTT mRNA. The present data emphasize the relevance of expanded CAG RNA to HD pathogenesis, indicate that inhibition of HTT expression is not required to reverse motor deficits, and further suggest a therapeutic potential for LNA-CTG in polyglutamine disorders. This work was supported by the Spanish government through the Plan Nacional de I+D+I and cofunded by grants from the Instituto de Salud Carlos III (ISCIII) – Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER) (project PI11/02036, to EM, and PI13/01250, to EPN); the Spanish Ministerio de Economía y Competitividad (MINECO) (SAF2008-00357 and SAF2013-49108-R, to XE, and SAF2014-60551-R: iRPaD, to EM); and the Generalitat de Catalunya, Departament Economia i Coneixement, Secretaria Universitats i Recerca (AGAUR 2014 SGR-1138, to XE).

Caffeine has cognitive-enhancing properties with effects on learning and memory, concentration, arousal and mood. These effects imply changes at circuital and synaptic level, but the mechanism by which caffeine modifies synaptic plasticity remains elusive. Here we report that caffeine, at concentrations representing moderate to high levels of consumption in humans, induces an NMDA receptor-independent form of LTP (CAFLTP) in the CA1 region of the hippocampus by promoting calcium-dependent secretion of BDNF, which subsequently activates TrkB-mediated signaling required for the expression of CAFLTP. Our data include the novel observation that insulin receptor substrate 2 (IRS2) is phosphorylated during induction of CAFLTP, a process that requires cytosolic free Ca2+. Consistent with the involvement of IRS2 signals in caffeine-mediated synaptic plasticity, phosphorylation of Akt (Ser473) in response to LTP induction is defective in Irs2−/− mice, demonstrating that these plasticity changes are associated with downstream targets of the phosphoinositide 3-kinase (PI3K) pathway. These findings indicate that TrkB-IRS2 signals are essential for activation of PI3K during the induction of LTP by caffeine.

Striatal-enriched protein tyrosine phosphatase (STEP) modulates key signaling molecules involved in synaptic plasticity and neuronal function. It is postulated that STEP opposes the development of long-term potentiation (LTP) and that it exerts a restraint on long-term memory (LTM). Here, we examined whether STEP

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.

Positive allosteric modulators of cc-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are small molecules that decrease deactivation of AMPARs via an allosteric site. These molecules keep the receptor in an active state. Interestingly, this type of modulator has been proposed for treating cognitive decline in ageing, dementias, and Alzheimer's disease (AD). S 47445 (8-cyclopropyl-3[2-(3-fluorophenyflethy1]-7,8-dihydro-3H-[1,3]oxazino[6,5-g][1,2,3]benzotriazine-4,9-dione) is a novel AMPAR positive allosteric modulator (AMPA-PAM). Here, the mechanisms by which S 47445 could improve synaptic strength and connectivity were studied and compared between young and old mice. A single oral administration of S 47445 at 10 mg/kg significantly increased long-term potentiation (LTP) in CA3-CA1 hippocampal synapses in alert young mice in comparison to control mice. Moreover, chronic treatment with S 47445 at 10 mg/kg in old alert animals significantly counteracted the deficit of LTP due to age. Accordingly, chronic treatment with S 47445 at 10 mg/kg seems to preserve synaptic cytoarchitecture in old mice as compared with young control mice. It was shown that the significant decreases in number and size of pre-synaptic buttons stained for VGlutl, and post-synaptic dendritic spines stained for spinophilin, observed in old mice were significantly prevented after chronic treatment with 10 mg/kg of S 47445. Altogether, by its different effects on LTP, VGlutl-positive particles, and spinophilin, S 47445 is able to modulate both the structure and function of hippocampal excitatory synapses known to be involved in learning and memory processes. These results open a new window for the treatment of specific age-dependent cognitive decline and dementias such as AD. (C) 2017 The Authors. Published by Elsevier Ltd.

Recent results indicate that STriatal-Enriched protein tyrosine Phosphatase (STEP) levels are regulated by brain-derived neurotrophic factor (BDNF), whose expression changes during postnatal development and aging. Here, we studied STEP ontogeny in mouse brain and changes in STEP with age with emphasis on the possible regulation by BDNF. We found that STEP expression increased during the first weeks of life, reaching adult levels by 2-3 weeks of age in the striatum and cortex, and by postnatal day (P) 7 in the hippocampus. STEP protein levels were unaffected in BDNF+/- mice, but were significantly reduced in the striatum and cortex, but not in the hippocampus, of BDNF-/- mice at P7 and P14. In adult wild-type mice there were no changes in cortical and hippocampal STEP61 levels with age. Conversely, striatal STEP levels were reduced from 12 months of age, correlating with higher ubiquitination and increased BDNF content and signaling. Lower STEP levels in older mice were paralleled by increased phosphorylation of its substrates. Since altered STEP levels are involved in cellular malfunctioning events, its reduction in the striatum with increasing age should encourage future studies of how this imbalance might participate in the aging process.

Dopamine replacement with l-DOPA is the most effective therapy in Parkinson's disease. However, with chronic treatment, half of the patients develop an abnormal motor response including dyskinesias. The specific molecular mechanisms underlying dyskinesias are not fully understood. In this study, we used a well-characterized animal model to first establish the molecular differences between rats that did and did not develop dyskinesias. We then investigated the molecular substrates implicated in the anti-dyskinetic effect of buspirone, a 5HT1A partial agonist. Striatal protein expression profile of dyskinetic animals revealed increased levels of the dopamine receptor (DR)D3, ΔFosB and phospho (p)CREB, as well as an over-activation of the DRD1 signalling pathway, reflected by elevated ratios of phosphorylated DARPP32 and ERK2. Buspirone reduced the abnormal involuntary motor response in dyskinetic rats in a dose-dependent fashion. Buspirone (4 mg/kg) dramatically reduced the presence and severity of dyskinesias (by 83%) and normalized DARPP32 and ERK2 phosphorylation ratios, while the increases in DRD3, ΔFosB and pCREB observed in dyskinetic rats were not modified. Pharmacological experiments combining buspirone with 5HT1A and DRD3 antagonists confirmed that normalization of both pDARPP32 and pERK2 is required, but not sufficient, for blocking dyskinesias. The correlation between pDARPP32 ratio and dyskinesias was significant but not strong, pointing to the involvement of convergent factors and signalling pathways. Our results suggest that in dyskinetic rats DRD3 striatal over-expression could be instrumental in the activation of DRD1-downstream signalling and demonstrate that the anti-dyskinetic effect of buspirone in this model is correlated with DRD1 pathway normalization.

A balance between cell survival and apoptosis is crucial to avoid neurodegeneration. Here, we analyzed whether the pro-apoptotic protein PKCδ, and the pro-survival PKCα and βII, were dysregulated in the brain of R6/1 mouse model of Huntington's disease (HD). Protein levels of the three PKCs examined were reduced in all the brain regions analyzed being PKCδ the most affected isoform. Interestingly, PKCδ protein levels were also decreased in the striatum and cortex of R6/2 and Hdh(Q111/Q111) mice, and in the putamen of HD patients. Nuclear PKCδ induces apoptosis, but we detected reduced PKCδ in both cytoplasmic and nuclear enriched fractions from R6/1 mouse striatum, cortex and hippocampus. In addition, we show that phosphorylation and ubiquitination of PKCδ are increased in 30-week-old R6/1 mouse brain. All together these results suggest a pro-survival role of reduced PKCδ levels in response to mutant huntingtin-induced toxicity. In fact, we show that over-expression of PKCδ increases mutant huntingtin-induced cell death in vitro, whereas over-expression of a PKCδ dominant negative form or silencing of endogenous PKCδ partially blocks mutant huntingtin-induced cell death. Finally, we show that the analysis of lamin B protein levels could be a good marker of PKCδ activity, but it is not involved in PKCδ-mediated cell death in mutant huntingtin-expressing cells. In conclusion, our results suggest that neurons increase the degradation of PKCδ as a compensatory pro-survival mechanism in response to mutant huntingtin-induced toxicity that can help to understand why cell death appears late in the disease.

Huntington's disease (HD) causes motor disturbances, preceded by cognitive impairment, in patients and mouse models. We showed that increased hippocampal cAMP-dependent protein kinase (PKA) signaling disrupts recognition and spatial memories in R6 HD mouse models. However, unchanged levels of hippocampal phosphorylated (p) cAMP-responsive element-binding protein (CREB) suggested unaltered nuclear PKA activity in R6 mice. Here, we extend this finding by showing that nuclear pPKA catalytic subunit (Thr197) and pPKA substrate levels were unaltered in the hippocampus of R6/1 mice. Phosphodiesterases (PDEs) play an important role in the regulation of PKA activity. PDE10A, a cAMP/cGMP dual-substrate PDE, was reported to be restricted to the nuclear region in nonstriatal neurons. Using cell fractionation we confirmed that PDE10A was enriched in nuclear fractions, both in wild-type and R6/1 mice hippocampus, without differences in its levels or intracellular distribution between genotypes. We next investigated whether inhibition of PDE10 with papaverine could improve cognitive function in HD mice. Papaverine treatment improved spatial and object recognition memories in R6/1 mice, and significantly increased pGluA1 and pCREB levels in R6/1 mice hippocampus. Papaverine likely acted through the activation of the PKA pathway as the phosphorylation level of distinct cGMP-dependent kinase (cGK) substrates was not modified in either genotype. Moreover, hippocampal cAMP, but not cGMP, levels were increased after acute papaverine injection. Our results show that inhibition of PDE10 improves cognition in R6 mice, at least in part through increased GluA1 and CREB phosphorylation. Thus, PDE10 might be a good therapeutic target to improve cognitive impairment in HD.

Huntington's disease is characterized by the formation of protein aggregates, which can be degraded by macroautophagy. Here, we studied protein levels and intracellular distribution of p62 and NBR1, two macroautophagy cargo receptors, during disease progression. In R6/1 mice, p62 and NBR1 protein levels were decreased in all brain regions analyzed early in the disease, whereas at late stages they accumulated in the striatum and hippocampus, but not in the cortex. The accumulation of p62, but not NBR1, occurred in neuronal nuclei, where it co-localized with mutant huntingtin inclusions, both in R6/1 and Huntington's disease patients. Moreover, exportin-1 was selectively decreased in old R6/1 mice brain, and could worsen p62 nuclear accumulation. In conclusion, p62 interacts with mutant huntingtin and is retained in the nucleus along the progression of the disease, mostly in striatal and hippocampal neurons. Thus, cytoplasmic NBR1 might be important to maintain basal levels of selective macroautophagy in these neurons.

Chelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells.In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line.We found that 10μM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular CaThis is the first report showing that chelerythrine promotes CaChelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works.

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.

Huntington's disease (HD) patients and mouse models show learning and memory impairment even before the onset of motor symptoms. However, the molecular events involved in this cognitive decline are still poorly understood. Here, using three different paradigms, the novel object recognition test, the T-maze spontaneous alternation task and the Morris water maze, we detected severe cognitive deficits in the R6/1 mouse model of HD before the onset of motor symptoms. When we examined the putative molecular pathways involved in these alterations, we observed hippocampal cAMP-dependent protein kinase (PKA) hyper-activation in naïve R6/1 mice compared with wild-type (WT) mice, whereas extracellular signal-regulated kinase 1/2 and calcineurin activities were not modified. Increased PKA activity resulted in hyper-phosphorylation of its substrates N-methyl-D-aspartate receptor subunit 1, Ras-guanine nucleotide releasing factor-1 and striatal-enriched protein tyrosine phosphatase, but not cAMP-responsive element binding protein or the microtubule-associated protein tau. In correlation with the over-activation of the PKA pathway, we found a down-regulation of the protein levels of some phosphodiesterase (PDE) 4 family members. Similar molecular changes were found in the hippocampus of R6/2 mice and HD patients. Furthermore, chronic treatment of WT mice with the PDE4 inhibitor rolipram up-regulated PKA activity, and induced learning and memory deficits similar to those seen in R6 mice, but had no effect on R6/1 mice cognitive impairment. Importantly, hippocampal PKA inhibition by infusion of Rp-cAMPS restored long-term memory in R6/2 mice. Thus, our results suggest that occlusion of PKA-dependent processes is one of the molecular mechanisms underlying cognitive decline in R6 animals.

6 páginas, 4 figuras.-- El pdf del artículo es el manuscrito de autor.-- et al., Caveolins (CAVs) are essential components of caveolae, plasma membrane invaginations with reduced fluidity, reflecting cholesterol accumulation [1]. CAV proteins bind cholesterol, and CAV's ability to move between cellular compartments helps control intracellular cholesterol fluxes [ [1], [2] and [3]]. In humans, CAV1 mutations result in lipodystrophy, cell transformation, and cancer [ [4], [5], [6] and [7]]. CAV1 gene-disrupted mice exhibit cardiovascular diseases, diabetes, cancer, atherosclerosis, and pulmonary fibrosis [ [8] and [9]]. The mechanism or mechanisms underlying these disparate effects are unknown, but our past work suggested that CAV1 deficiency might alter metabolism: CAV1−/− mice exhibit impaired liver regeneration unless supplemented with glucose, suggesting systemic inefficiencies requiring additional metabolic intermediates [10]. Establishing a functional link between CAV1 and metabolism would provide a unifying theme to explain these myriad pathologies [11]. Here we demonstrate that impaired proliferation and low survival with glucose restriction is a shortcoming of CAV1-deficient cells caused by impaired mitochondrial function. Without CAV1, free cholesterol accumulates in mitochondrial membranes, increasing membrane condensation and reducing efficiency of the respiratory chain and intrinsic antioxidant defense. Upon activation of oxidative phosphorylation, this promotes accumulation of reactive oxygen species, resulting in cell death. We confirm that this mitochondrial dysfunction predisposes CAV1-deficient animals to mitochondrial-related diseases such as steatohepatitis and neurodegeneration., A.P. is supported by grants BFU2008-00345, CSD2009-00016, and Marató de TV3, M.M. is supported by grant PI10/02114, A.C. is supported by grant SAF2010-15760, F.T. is supported by grant BFU2006-15474, C.G.-R. is supported by grants SAF2008-02199 and Mutua Madrileña, and C.E. is supported by grants BFU2009-10335 and CSD2009-00016 from Ministerio de Ciencia e Innovación. C.E. is also supported by grant PI040236/Marató TV3, S.P.G. is supported by grant GM64624/NIH, E.P. is supported by grant PI071183, and J.C.F.-C. is supported by grants SAF2009-11417, HI2007-0244/MCI, P50-AA-11999/NIAAA/NIH, and Marató de TV3.

Sustained activation of AMPA and kainate receptors in rat oligodendrocytes induces cytosolic calcium overload, mitochondrial depolarization, and an increase of reactive oxygen species, resulting in cell death. Here, we provide evidence that Bax, a proapoptotic member of the Bcl-2 protein family, is involved in excitotoxic apoptotic death of oligodendrocytes and that calpain mediates Bax activation. Cultured Bax−/−oligodendrocytes, obtained from the optic nerve of Bax knock-out mice, were resistant to AMPA and kainate receptor-mediated insults. In turn, both mitochondrial calcium uptake and mitochondrial alterations after excitotoxic insults were diminished in Bax-null oligodendrocytes. Moreover, pretreatment with furosemide, a blocker of Bax translocation to mitochondria, significantly protected rat and mouse oligodendrocytes from AMPA- and kainate-induced damage; in contrast, bongkrekic acid, a blocker of the mitochondrial permeability transition pore, had no effect. Finally, we analyzed the participation of calpain, which cleaves Bax and is activated by AMPA and kainate, in oligodendrocyte death. Pretreatment with 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606), a broad cell-permeable calpain inhibitor, and two additional calpain inhibitors diminished Bax activation, inhibited its translocation to mitochondria, and attenuated all apoptotic events resulting from excitotoxic insults to rat oligodendrocytes. Together, these results indicate that Bax and calpain are essential intermediaries of the mitochondria-dependent death pathway, triggered by AMPA and kainate receptor activation in oligodendrocytes.

Calcineurin is a serine/threonine phosphatase involved in the regulation of glutamate receptors signaling. Here, we analyzed whether the regulation of calcineurin protein levels and activity modulates the susceptibility of striatal neurons to excitotoxicity in R6/1 and R6/1:BDNF+/− mouse models of Huntington's disease. We show that calcineurin inhibition in wild-type mice drastically reduced quinolinic acid-induced striatal cell death. Moreover, calcineurin A and B were differentially regulated during disease progression with a specific reduction of calcineurin A protein levels and calcineurin activity at the onset of the disease in R6/1:BDNF+/− mice. Analysis of the conditional mouse model Tet/HD94 showed that mutant huntingtin specifically controls calcineurin A protein levels. Finally, calcineurin activation induced by intrastriatal quinolinic acid injection in R6/1 mouse was lower than in wild-type mice. Therefore, reduction of calcineurin activity by alteration of calcineurin A expression participates in the pathophysiology of Huntington's disease and contributes to the excitotoxic resistance observed in exon-1 mouse models.

Bone morphogenetic proteins are members of the transforming growth factor-β superfamily that have multiple functions in the developing nervous system. One of them, bone morphogenetic protein-2 (BMP-2), promotes the differentiation of cultured striatal neurones, enhancing dendrite growth and calbindin-positive phenotype. Bone morphogenetic proteins have been implicated in cooperative interactions with other neurotrophic factors. Here we examined whether the effects of BMP-2 on cultured striatal neurones are mediated or enhanced by other neurotrophic factors. BMP-2 had a cooperative effect with low doses of brain-derived neurotrophic factor or neurotrophin-3 (but not with other neurotrophic factors such as glial cell line-derived neurotrophic factor, neurturin or transforming growth factor-β2) on the number of calbindin-positive striatal neurones. Moreover, BMP-2 induced phosphorylated Trk immunoreactivity in cultured striatal neurones, suggesting that neurotrophins are involved in BMP-2 neurotrophic effects. The addition of TrkB-IgG or antibodies against brain-derived neurotrophic factor abolished the effects of BMP-2 on the number and degree of differentiation of calbindin-positive striatal neurones. Indeed, BMP-2 treatment increased brain-derived neurotrophic factor protein levels in treated cultures media and BDNF immunocytochemistry revealed that this neurotrophin was produced by neuronal cells. Taken together, these results indicate that brain-derived neurotrophic factor mediates the effects of BMP-2 on striatal neurones.

Excitotoxicity has been proposed as one of the mechanisms involved in the specific loss of striatal neurons that occurs in Huntington's disease. Here, we studied the role of calcineurin in the vulnerability of striatal neurons expressing mutant huntingtin to excitotoxicity. To this end, we induced excitotoxicity by adding NMDA to a striatal precursor cell line expressing full-length wild-type (STHdh(Q7/Q7)) or mutant (STHdh(Q111/Q111)) huntingtin. We observed that cell death appeared earlier in STHdh(Q111/Q111) cells than in STHdh(Q7/Q7) cells. Interestingly, these former cells expressed higher levels of calcineurin A that resulted in a greater increase of its activity after NMDA receptor stimulation. Moreover, transfection of full-length mutant huntingtin in different striatal-derived cells (STHdh(Q7/Q7), M213 and primary cultures) increased calcineurin A protein levels. To determine whether high levels of calcineurin A might account for the earlier activation of cell death in mutant huntingtin knock-in cells, wild-type cells were transfected with calcineurin A. Calcineurin A-transfected STHdh(Q7/Q7) cells displayed a significant increase in cell death compared with that recorded in green fluorescent protein-transfected cells after NMDA treatment. Notably, addition of the calcineurin inhibitor FK-506 produced a more robust reduction in cell death in mutant huntingtin knock-in cells than it did in wild-type cells. These results suggest that high levels of calcineurin A could account for the increased vulnerability of striatal cells expressing mutant huntingtin to excitotoxicity.

The striatum is one of the brain areas most vulnerable to excitotoxicity, a lesion that can be prevented by neurotrophins. In the present study, intrastriatal injection of the N-methyl-d-aspartate receptor (NMDAR) agonist quinolinate (QUIN) was performed in mice heterozygous for neurotrophin-3 (NT3 +/-) or brain-derived neurotrophic factor (BDNF +/-) to analyze the role of endogenous neurotrophins on the regulation of striatal neurons susceptibility to excitotoxic injury. QUIN injection induced a decrease in dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) protein levels that was higher in NT-3 +/- than in BDNF+/- or wild type animals. This enhanced susceptibility was specific for enkephalin- and tachykinin-positive projection neurons, and also for parvalbumin-positive interneurons. However the excitotoxic damage in large interneurons was not modified in NT-3 +/- mice compared with wild type animals. This effect can be related to the regulation of NMDARs by endogenous NT-3. Thus, our results show that there is an age-dependent regulation of NMDAR subunits NR1 and NR2A, but not NR2B, in NT-3 +/- mice. The deficit of endogenous NT-3 induced a decrease in NR1 and NR2A subunits at postnatal day (P) 0 and P3 mice respectively, whereas an upregulation was observed in 12 week old NT-3 +/- mice. This differential effect was also observed after administration of exogenous NT-3. In primary striatal cultures, NT-3 treatment induced an enhancement in NR2A, but not NR2B, protein levels. However, intrastriatal grafting of NT-3 secreting-cells in adult wild type mice produced a down-regulation of NR2A subunit. In conclusion, NT-3 regulates the expression of NMDAR subunits modifying striatal neuronal properties that confers the differential vulnerability to excitotoxicity in projection neurons and interneurons in the striatum.