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Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IRdeficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1¿ and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling., We are thankful to M.Garcia and R. Cañadas for technical support. This work was funded by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) (Instituto de Salud CarlosIII, Spain) to I.T.A., A.G., and T.I.; an Inter-CIBER project (PIE14/00061) to I.T.A.that forms part of the projects PID2019-104376RB-I00 (I.T.A.) and RTI2018-094887-B-I00 (M.N.) funded by MCIN/AEI/10.13039/501100011033; a grant from Junta de Andalucia Consejería de Economía y Conocimiento (P18-RT-2233 to A.G.) cofinanced by Programa Operativo FEDER 2014–2020; a grant from Instituto de Salud Carlos III Spain (cofinanced by FEDER funds from the European Union; PI21/00915 to A.G.); Grant PID2020-115218RB-I00 to T.I. funded by Ministerio de Ciencia e Innovación/Agencia Española de Investigación (MCIN/AEI/10.13039/501100011033); and a grant from Comunidad de Madrid through the European Social Fund (ESF)–financed programme Neurometabolismo-Comunidad de Madrid (NEUROMETAB-CM) (B2017/BMD-3700 to I.T.A.and T.I.). M.N. was also supported by the Spanish Ministry of Science and Innovation (Ramón y Cajal RYC-2016-20414). J.P.-U. was contracted by CIBERNED.

Huntington’s disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophy–linked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamine–responsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD., This work was supported by CIBERNED-ISCIII collaborative grants 2013/09-2 to J.J.L. and 2015-2/06 and 2018/06-5 to J.J.L. and T.I., grants SAF2015-65371-R (MINECO/AEI/FEDER, UE), and grants from the Spanish Ministry of Science, Innovation and Universities (MINECO/MICINN): RTI2018-096322-B-I00 (MCIU/AEI/FEDER, UE) to J.J.L., SAF2017-88885-R (MINECO/AEI/FEDER, UE) to T.I., BFU2014-54122-P (MINECO/AEI/FEDER, UE) to R.M., and ISCIII-FEDER (PI17/00199; PI20/00625) to P.N. This work was also supported by the European Union FEDER funds, Fundación Botín–Banco Santander through its Santander Universities Global Division and Fundación Ramón Areces. A.P. and S.P. were recipients of FPI fellowships from MICINN/MINECO. A.P. was also hired through CIBERNED. J.P.-U. was contracted by SAF2017-88885-R (MINECO/AEI/FEDER, UE). D.M.-G. was supported by the “Río Hortega” programme (CM18/00142) from ISCIII-FEDER. A.E. was a recipient of Juan de la Cierva contracts from MICINN/MINECO.

Background Although promising gene-silencing therapies are being tested for Huntington’s disease (HD), no disease-modifying treatments are available. Thus, study of molecular mechanisms underneath Htt-mutation must continue to identify easily druggable targets. Cytoplasmic polyadenylation element binding proteins 1–4 (CPEB1–4) are RNA-binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Alteration of CPEB-dependent transcriptome polyadenylation has been associated to diseases like cancer, autism and epilepsy. Aims Analyze CPEBs and polyadenylation in HD. Identify easily druggable targets among genes mis-expressed due to altered CPEB-dependent polyadenylation, to assay them in HD mice. Methods a) Western blot and immunostaining of CPEBs in brains of HD patients and mouse models. b) Genome-wide poly(A)-tail analysis through poly(U) chromatography+gene chip. c) status of CPEB targets and related metabolites by western blot and HPLC. d) radiological, neuropathological and behavioural analysis of HD mice receiving target-related treatment. Results There is a CPEB1/4 imbalance in HD striatum with concomitant altered transcriptome polyadenylation affecting many neurodegeneration-linked genes like PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS and HTT. Among top deadenylated genes was SLC19A3 (ThTr2 thiamine transporter) whose mutation causes biotin+thiamine responsive basal ganglia disease (BTBGD). Decreased ThTr2 in HD and HD mice led us to discover that HD is in part a BTBG-like thiamine deficiency. Remarkably, high dose biotin+thiamine treatment prevented the thiamine deficiency of HD mice and attenuated their radiological, neuropathological and motor phenotypes. Conclusions This study unveils altered polyadenylation as a new molecular mechanism in neurodegeneration uncovering HD as a thiamine deficiency and, therefore, an easy to implement therapy.

© The Author(s) 2019., Excitotoxic neuronal death induced by high concentrations of glutamate is a pathological event common to multiple acute or chronic neurodegenerative diseases. Excitotoxicity is mediated through overactivation of the N-Methyl-D-aspartate type of ionotropic glutamate receptors (NMDARs). Physiological stimulation of NMDARs triggers their endocytosis from the neuronal surface, inducing synaptic activity and survival. However almost nothing is known about the internalization of overactivated NMDARs and their interacting proteins, and how this endocytic process is connected with neuronal death has been poorly explored. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a component of NMDAR complexes essential for neuronal viability by the control of ERK activation. Here we have investigated Kidins220 endocytosis induced by NMDAR overstimulation and the participation of this internalization step in the molecular mechanisms of excitotoxicity. We show that excitotoxicity induces Kidins220 and GluN1 traffic to the Golgi apparatus (GA) before Kidins220 is degraded by the protease calpain. We also find that excitotoxicity triggers an early activation of Rap1-GTPase followed by its inactivation. Kidins220 excitotoxic endocytosis and subsequent calpain-mediated downregulation governs this late inactivation of Rap1 that is associated to decreases in ERK activity preceding neuronal death. Furthermore, we identify the molecular mechanisms involved in the excitotoxic shutoff of Kidins220/Rap1/ERK prosurvival cascade that depends on calpain processing of Rap1-activation complexes. Our data fit in a model where Kidins220 targeting to the GA during early excitotoxicity would facilitate Rap1 activation and subsequent stimulation of ERK. At later times, activation of Golgi-associated calpain, would promote the degradation of GA-targeted Kidins220 and two additional components of the specific Rap1 activation complex, PDZ-GEF1, and S-SCAM. In this way, late excitotoxicity would turn off Rap1/ERK cascade and compromise neuronal survival., This work was funded by grants SAF2014-52737-P and SAF2017-88885-R to T.I., BFU2016-75973-R to M.D.-G and BFU2015-63769-R to R.L. (Spanish Ministry ofScience, Innovation and Universities—MICIU, Spain). It was also supported by grant SBPLY/17/180501/000229 from Junta de Comunidades de Castilla-La Mancha to R.L. Additional funding to T.I. was received by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED, Instituto de Salud Carlos III, Spain) as well as by grant S2017/BMD3700 (NEUROMETAB-CM) from Comunidad de Madrid co-financed with the Structural Funds of the European Union (FEDER). A.G.-M. was funded by contracts from CIBERNED. Contracts to A.S.-G and J.P.U were funded by grants S2017/BMD-3700 (NEUROMETAB-CM) and SAF2017-88885-R, respectively. The cost of this publication has been paid in part by FEDER funds.

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-¿B/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-¿B/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders., This work was supported by grants SAF2014-52737-P to T.I., SAF2013-45258-P to M.R. C., BFU2016-77885-P to F.H., SAF2014-54070-JIN to A.M. from Ministerio de Economía, Industria y Competitividad (Spain). It was also funded by P2010/BMD-2332 (Neurodegmodels) from Comunidad de Madrid to T.I., F.H. and J.A.) and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Instituto de Salud Carlos III, Spain) to T.I., F.H., J.A., and I.F.). J.P.-U. is a recipient of a predoctoral contract from SAF2014-52737-P; L.G.-G. was funded by a contract from CIBERNED; A.G.-M. and A.S.S. were funded by contracts from CIBERNED cooperative projects 2013/07 and CIBERNED 2015-2/06, respectively. A.M., A.D.P. is a recipient of a Juan de la Cierva formación fellowship (Ministerio de Economía, Industria y Competitividad, Spain) associated to CIBERNED. J.J.-A. was funded by a predoctoral contract from CSIC (JAEPredoc program) and by CIBERNED. The cost of this publication has been paid in part by FEDER (European Funds for Regional Development) funds.

Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes., This work was funded by MINECO (Spain) grants SAF2010-60051 and SAF2013-40710-R, and by CIBERNED. E.H.-G. was partially funded by a fellowship from ColFuturo and CIBERNED. T.I. is funded by MINECO (SAF2014-52737-P). T.I. and I.T.A. are funded by CIBERNED and Comunidad de Madrid, Spain (P2010/BMD-2331-Neurodegmodels).

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration., This work was supported by the Ministerio de Economía y Competitividad [SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R-C.]; Comunidad de Madrid [S2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas – CIBERNED, Instituto de Salud Carlos III, to T.I. Postdoctoral fellows L.S-R. and L.G-G. have been funded by research contracts from CIBERNED; Clara Aicart-Ramos is a recipient of a FPU predoctoral fellowship from Ministerio de Economía y Competitividad.