GSEA of mouse and human mitochondriomes reveals fatty acid oxidation in astrocytes

The prevalent view in neuroenergetics is that glucose is the main brain fuel, with neurons being mostly oxidative and astrocytes glycolytic. Evidence supporting that astrocyte mitochondria are functional has been overlooked. Here we sought to determine what is unique about astrocyte mitochondria by performing unbiased statistical comparisons of the mitochondriome in astrocytes and neurons. Using MitoCarta, a compendium of mitochondrial proteins, together with transcriptomes of mouse neurons and astrocytes, we generated cell-specific databases of nuclear genes encoding for mitochondrion proteins, ranked according to relative expression. Standard and in-house Gene Set Enrichment Analyses (GSEA) of five mouse transcriptomes revealed that genes encoding for enzymes involved in fatty acid oxidation (FAO) and amino acid catabolism are consistently more expressed in astrocytes than in neurons. FAO and oxidative-metabolism-related genes are also up-regulated in human cortical astrocytesversus the whole cortex, and in adult astrocytes versus fetal astrocytes. We thus present the first evidence of FAO in human astrocytes. Further, as shown in vitro, FAO coexists with glycolysis in astrocytes and is inhibited by glutamate. Altogether, these analyses provide arguments against the glucose-centered view of energy metabolism in astrocytes and reveal mitochondria as specialized organelles in these cells

CREB Regulates Distinct Adaptive Transcriptional Programs in Astrocytes and Neurons

The cyclic AMP response element binding protein (CREB) is a primary hub of a activity-driven genetic programs in neurons controlling plasticity, neurogenesis and survival. By contrast, the gene networks coordinated by CREB in astrocytes are Unknown despite the fact that the astrocytic CREB is a also activity-driven and neuroprotective. Herein we identified the transcriptional programs regulated by CREB in astrocytes as compared to neurons using, as study materials, transcriptome databases of astrocyte exposed to weII-known activators of CREB-dependent transcription as well as publidy available transcriptomes of neuronal cultures. Functional CREB signatures were extracted from the transcriptomes using Gene Ontology, adult-brain gene lists generated by Translating Ribosome Affinity Purification (TRAP) and CREB-target gene repositories. We found minimal overlap between CREB signatures in astrocytes and neurons. In astrocytes, the top triad of functions regulated by CREB consists of'Gene expression', 'Mitochondria', and 'Signa Iling', while in neurons it is 'Neurotransmission', 'Signalling' and 'Gene expression', the latter being represented by different genes from those in astrocytes. The newly gene rated data bases Will provide a tool to explore novel means whereby CREB impinges on brain functions requiring adaptive, long-lasting changes by coordinating transcriptionaI cascades in astrocytes

Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease

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

Neurodevelopmental alterations in X-linked adrenoleukodystrophy

La adrenoleucodistrofia ligada al cromosoma X (X-ALD, de sus siglas en inglés) es una enfermedad rara causada por mutaciones en el transportador peroxisomal ABCD1, presentando dos formas de manifestación clínica de neurodegeneración: demielinización aguda y letal en el cerebro de niños con la forma cerebral de X-ALD (CCALD), y degeneración crónica de los tractos de la médula espinal y neuropatía periférica en adultos con adrenomielopatía (AMN). Las alteraciones psiquiátricas son un signo temprano de CCALD y coexisten con la neurodegeneración periférica de la médula espinal en AMN, poniendo de manifiesto la patología cerebral. Teniendo en cuenta que la X-ALD es una condición genética quisimos determinar si las alteraciones psiquiátricas observadas pueden ser debidas, al menos en parte, a la formación aberrante de circuitos cerebrales durante el desarrollo y no a la neurodegeneración. Con este objetivo, usamos una aproximación de arriba hacia abajo, partiendo de datos de transcriptomas de pacientes, donde hemos buscado vías de neurodesarrollo desreguladas, hasta modelos murinos de X-ALD in vitro basados en el silenciamiento de los genes Abcd1 y Abcd2 para diseccionar la compartimentalización entre neuronas y astrocitos. Se presentan cuatro resultados principales. Primero, existen vías de neurodesarrollo desreguladas en CCALD, AMN y en los cultivos neuronales deficientes en ABCD, asociado con alteración en los procesos de neuritogénesis, espinogénesis, y axonogénesis. Segundo, un crecimiento aberrante de las espinas sinápticas es en parte debido a alteraciones en la vía canoníca de señalización Wnt puesto que la espinogénesis se recupera parcialmente mediante la activación de la vía de señalización Wnt tras la inhibición farmacológica de GSK-3. Tercero, la síntesis de colesterol y la localización del mismo se encuentran cambiadas en los astrocitos deficientes en ABCD. Cuarto, el silenciamiento de los transportadores ABCD causa alteraciones metabólicas en astrocitos incluyendo disminución de la oxidación de ácidos grasos, incremento del ratio NAD+/NADH, depleción de ATP, y disminución de la cantidad total de glutatión sugiriendo el deterioro conjunto de la defensa antioxidante y la bioenergética. Quinto, los astrocitos X-ALD presentan una señalización de calcio mediada por agonistas alterada y estrés del retículo endoplamático. Concluimos que i) la X-ALD presenta un componente de neurodesarrollo que puede explicar los síntomas psiquiátricos, y quizá contribuir a la progresión de la forma CCALD, y a la conversión de la AMN en una forma cerebral de la enfermedad, y ii) la deregulacion metabólica y de la excitabilidad en astrocitos apoya la disfunción global de los astrocitos que puede poner en peligro el papel computacional y homeostático de los astrocitos en los circuitos cerebrales.X-linked adrenoleukodystrophy (X-ALD) is a rare disease caused by mutations in the peroxisomal ABCD1 transporter, with two major clinical manifestations of neurodegeneration: acute and lethal brain demyelination in the child cerebral X-ALD (CCALD), and chronic degeneration of spinal-cord tracts and peripheral neuropathy in the adult adrenomyeloneuropathy (AMN). Psychiatric alterations are an early sign of CCALD and coexist with spinal-cord and peripheral neurodegeneration in AMN, revealing concomitant brain pathology. Since X-ALD is a genetic condition, we sought to determine whether psychiatric alterations could be due, at least in part, to abnormal formation of brain circuits during brain development and not to neurodegeneration. To this end, we used a top-down approach, moving from patient transcriptome data, where we searched for dysregulated neurodevelopmental pathways, to in vitro murine models of X-ALD based on Abcd1/Abcd2 silencing to dissect out astrocyte versus neurons compartmentalization. There are four major findings. First, developmental pathways are dysregulated in CCALD, CAMN and in ABCD-null neuronal cultures, associated with altered neuritogenesis, spinogenesis, and axonogenesis. Second, aberrant spine growth is in part due to alterations in canonical Wnt signalling alteration since the spinogenesis is partially rescued by activation of WNT pathways by pharmacological GSK-3 inhibition. Third, cholesterol synthesis and localization is changed in ABCD-null astrocytes. Four, silencing of ABCD transporters causes metabolic alterations in astrocytes including fatty acid oxidation impairment, increase of the ratio NAD+/NADH, ATP depletion, decreases in total glutathione, suggesting joint impairment of antioxidant defense and bioenergetics. Fifth, X-ALD astrocytes present altered agonist-induced calcium signaling and ER stress. We conclude that i) X-ALD has a neurodevelopmental component that may account for psychiatric symptoms, and perhaps contribute to the progression of CCALD, and to the conversion of AMN into a cerebral condition, and ii) metabolic and excitability dysregulation in astrocytes support global astrocytic dysfunction that may jeopardize computational and homeostatic role of astrocytes in neural circuits

Neurodevelopmental alterations in X-linked adrenoleukodystrophy

Departament responsable de la tesi: Departament de Bioquímica i Biologia Molecular.La adrenoleucodistrofia ligada al cromosoma X (X-ALD, de sus siglas en inglés) es una enfermedad rara causada por mutaciones en el transportador peroxisomal ABCD1, presentando dos formas de manifestación clínica de neurodegeneración: demielinización aguda y letal en el cerebro de niños con la forma cerebral de X-ALD (CCALD), y degeneración crónica de los tractos de la médula espinal y neuropatía periférica en adultos con adrenomielopatía (AMN). Las alteraciones psiquiátricas son un signo temprano de CCALD y coexisten con la neurodegeneración periférica de la médula espinal en AMN, poniendo de manifiesto la patología cerebral. Teniendo en cuenta que la X-ALD es una condición genética quisimos determinar si las alteraciones psiquiátricas observadas pueden ser debidas, al menos en parte, a la formación aberrante de circuitos cerebrales durante el desarrollo y no a la neurodegeneración. Con este objetivo, usamos una aproximación de arriba hacia abajo, partiendo de datos de transcriptomas de pacientes, donde hemos buscado vías de neurodesarrollo desreguladas, hasta modelos murinos de X-ALD in vitro basados en el silenciamiento de los genes Abcd1 y Abcd2 para diseccionar la compartimentalización entre neuronas y astrocitos. Se presentan cuatro resultados principales. Primero, existen vías de neurodesarrollo desreguladas en CCALD, AMN y en los cultivos neuronales deficientes en ABCD, asociado con alteración en los procesos de neuritogénesis, espinogénesis, y axonogénesis. Segundo, un crecimiento aberrante de las espinas sinápticas es en parte debido a alteraciones en la vía canoníca de señalización Wnt puesto que la espinogénesis se recupera parcialmente mediante la activación de la vía de señalización Wnt tras la inhibición farmacológica de GSK-3. Tercero, la síntesis de colesterol y la localización del mismo se encuentran cambiadas en los astrocitos deficientes en ABCD. Cuarto, el silenciamiento de los transportadores ABCD causa alteraciones metabólicas en astrocitos incluyendo disminución de la oxidación de ácidos grasos, incremento del ratio NAD+/NADH, depleción de ATP, y disminución de la cantidad total de glutatión sugiriendo el deterioro conjunto de la defensa antioxidante y la bioenergética. Quinto, los astrocitos X-ALD presentan una señalización de calcio mediada por agonistas alterada y estrés del retículo endoplamático. Concluimos que i) la X-ALD presenta un componente de neurodesarrollo que puede explicar los síntomas psiquiátricos, y quizá contribuir a la progresión de la forma CCALD, y a la conversión de la AMN en una forma cerebral de la enfermedad, y ii) la deregulacion metabólica y de la excitabilidad en astrocitos apoya la disfunción global de los astrocitos que puede poner en peligro el papel computacional y homeostático de los astrocitos en los circuitos cerebrales.X-linked adrenoleukodystrophy (X-ALD) is a rare disease caused by mutations in the peroxisomal ABCD1 transporter, with two major clinical manifestations of neurodegeneration: acute and lethal brain demyelination in the child cerebral X-ALD (CCALD), and chronic degeneration of spinal-cord tracts and peripheral neuropathy in the adult adrenomyeloneuropathy (AMN). Psychiatric alterations are an early sign of CCALD and coexist with spinal-cord and peripheral neurodegeneration in AMN, revealing concomitant brain pathology. Since X-ALD is a genetic condition, we sought to determine whether psychiatric alterations could be due, at least in part, to abnormal formation of brain circuits during brain development and not to neurodegeneration. To this end, we used a top-down approach, moving from patient transcriptome data, where we searched for dysregulated neurodevelopmental pathways, to in vitro murine models of X-ALD based on Abcd1/Abcd2 silencing to dissect out astrocyte versus neurons compartmentalization. There are four major findings. First, developmental pathways are dysregulated in CCALD, CAMN and in ABCD-null neuronal cultures, associated with altered neuritogenesis, spinogenesis, and axonogenesis. Second, aberrant spine growth is in part due to alterations in canonical Wnt signalling alteration since the spinogenesis is partially rescued by activation of WNT pathways by pharmacological GSK-3 inhibition. Third, cholesterol synthesis and localization is changed in ABCD-null astrocytes. Four, silencing of ABCD transporters causes metabolic alterations in astrocytes including fatty acid oxidation impairment, increase of the ratio NAD+/NADH, ATP depletion, decreases in total glutathione, suggesting joint impairment of antioxidant defense and bioenergetics. Fifth, X-ALD astrocytes present altered agonist-induced calcium signaling and ER stress. We conclude that i) X-ALD has a neurodevelopmental component that may account for psychiatric symptoms, and perhaps contribute to the progression of CCALD, and to the conversion of AMN into a cerebral condition, and ii) metabolic and excitability dysregulation in astrocytes support global astrocytic dysfunction that may jeopardize computational and homeostatic role of astrocytes in neural circuits

CREB Regulates Distinct Adaptive Transcriptional Programs in Astrocytes and Neurons

The cyclic AMP response element binding protein (CREB) is a primary hub of a activity-driven genetic programs in neurons controlling plasticity, neurogenesis and survival. By contrast, the gene networks coordinated by CREB in astrocytes are Unknown despite the fact that the astrocytic CREB is a also activity-driven and neuroprotective. Herein we identified the transcriptional programs regulated by CREB in astrocytes as compared to neurons using, as study materials, transcriptome databases of astrocyte exposed to weII-known activators of CREB-dependent transcription as well as publidy available transcriptomes of neuronal cultures. Functional CREB signatures were extracted from the transcriptomes using Gene Ontology, adult-brain gene lists generated by Translating Ribosome Affinity Purification (TRAP) and CREB-target gene repositories. We found minimal overlap between CREB signatures in astrocytes and neurons. In astrocytes, the top triad of functions regulated by CREB consists of'Gene expression', 'Mitochondria', and 'Signa Iling', while in neurons it is 'Neurotransmission', 'Signalling' and 'Gene expression', the latter being represented by different genes from those in astrocytes. The newly gene rated data bases Will provide a tool to explore novel means whereby CREB impinges on brain functions requiring adaptive, long-lasting changes by coordinating transcriptionaI cascades in astrocytes