Development and functional study of a mice model of hyperactivity obtained by dopaminergic neonatal lesion

La dopamina (DA) es un neurotransrnisor que participa en diversas funciones del sistema nervioso central de los mamíferos, como la regulación del movimiento y los procesos cognitivos. Existen varias patologías humanas asociadas a la disfunción dopaminérgica, entre las cuales se postula el déficit de atención e hiperactividad (ADHD), un desorden que afecta del 3 al 5 % de la población infantil mundial. Las lesiones neonatales con 6-hidroxidopamina en la rata se utilizan como modelo experimental de ADHD, porque inducen aspectos comportamentales presentes en el síndrome humano. En este trabajo desarrollamos este modelo en el ratón, cuya ventaja reside en que permite estudiar los genes potencialmente involucrados utilizando ratones modificados genéticamente. Los ratones lesionados neonatalmente con 6-OHDA evidenciaron hiperactividad, respuesta hipolocomotora paradójica frente a la administración de psicoestimulantes, déficits en la inhibición comportamental y en la coordinación motora. Los cambios neuroquírnicos detectados incluyen una disminución del contenido de DA del 80 % en el cuerpo estriado y del 35 % en la corteza prefrontal. A partir de los estudios genéticos que asocian al receptor dopaminérgico D4 (D4R) con el ADHD, estudiamos el papel fimcional del D4R en este modelo. Para esto, realizamos lesiones neonatales con 6-OHDA en ratones carentes del D4R (Drdf-/-). A pesar de mostrar las mismas alteraciones neuroquimicas que sus hermanos de genotipo normal, los ratones Drd4 -/- no desarrollaron hiperactividad ni déficit en la inhibición comportamental, demostrando que la expresión de tales fenotipos depende de la estimulación del D4R. Dado que las lesiones neonatales con 6-OHDA inducen aumento en el contenido de 5-HT estriatal, también analizamos la participación de la 5-HT en este modelo. La reducción de 5-HT normalizó la actividad locomotora de los ratones lesionados pero no previno la respuesta paradójica a la anfetamina. En conjunto estos resultados demuestran que tanto el D4R como la elevada S-HT estriatal son esenciales para la manifestación de las características más relevantes de este modelo murino.Dopamine (DA) is a neurotransmitter involved in several functions of mammalian brain, like motor control, cognition and emotion. Several human disorders are related to DA dysfunction; among them is attention deficit/hyperactivity disorder (ADHD), a syndrome that affects 3-5 % of school-aged population. Neonatal lesions of dopaminergic pathways with 6-hydroxydopamine (6-OHDA) in rats has been widely used as a model of ADHD. In this work we adapted this model into the mouse, to test the participation of candidate genes in knockout mice. Neonatally 6-OHDA-lesioned mice exhibited hyperactivity, paradoxical hypolocomotor response to psychostimulants, poor behavioral inhibition and deficit in motor coordination. The most salient neurochemical changes included 70-80 % reduction of striatal DA contents and 35 % reduction in the prefrontal cortex. Since genetic studies in humans have associated ADHD with the dopamine D4 receptor (D4R) we aimed to determine whether the D4R plays a role in the behavioral phenotypes of our model, performing neonatal 6-OHDA lesions in mice lacking D4Rs (Drd4-/-). Although striatal DA contents were equally reduced than in their wild-type siblings, Drd4-/- mice did not develop hyperactivity and showed normal behavioral inhibition, demonstrating that the D4R is essential for the expression of these phenotypes. Because neonatal DA depletion leads to an increase in serotonin (S-HT) striatal content we also studied the functional role of S-HT in our model. The reduction of striatal S-HT contents in 6-OHDA neonatally-lesioned mice reversed their hyperactivity to normal locomotor scores, but it did not prevent the paradoxical response to amphetamine. Together, our results from a combination of genetic and pharmacological approaches demonstrate that D4R and elevated S-HT are both essential for the expression of some relevant features present in this mouse model.Fil:Avale, María Elena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Partial ablation of postsynaptic dopamine D2 receptors in the central nucleus of the amygdala increases risk avoidance in exploratory tasks

The central nucleus of the amygdala (CeA) is involved in the expression of fear and has been implicated in several anxiety disorders. This structure is densely innervated by DAergic projections that impinge on amygdalar neurons expressing various dopamine (DA) receptor subtypes, including D2 receptors (D2Rs). Although various pharmacological approaches have assessed the role of D2Rs in the CeA, the actual participation of postsynaptic D2Rs in the CeA to defensive behaviors remains unclear. Here, we investigated the distribution of D2Rs in the CeA and their role in modifying neuronal activity and fear related behaviors in mice. First, using the mouse reporter strain D2R-EGFP, we verified that D2Rs are present both in neurons of the CeA and in A10 dorsocaudal (A10dc) DAergic neurons that innervate the CeA. Moreover, we showed that pharmacological stimulation of D2Rs increases the activity of protein kinase C (PKC)δ cells present in the CeA, a type of neuron previously associated with reduced defensive behaviors. Finally, using a molecular genetics approach that discriminates postsynaptic D2Rs from presynaptic D2 autoreceptors, we demonstrated that mice carrying targeted deletions of postsynaptic D2Rs in the CeA display increased risk avoidance in exploratory tasks. Together, our results indicate that postsynaptic D2Rs in the CeA attenuate behavioral reactions to potential environmental threats

Trans-splicing correction of tau isoform imbalance in a mouse model of tau mis-splicing

Abnormal metabolism of the tau protein is central to the pathogenesis of a number of dementias, including Alzheimer's disease. Aberrant alternative splicing of exon 10 in the tau pre-mRNA resulting in an imbalance of tau isoforms is one of the molecular causes of the inherited tauopathy, FTDP-17. We showed previously in heterologous systems that exon 10 inclusion in tau mRNA could be modulated by spliceosome-mediated RNA trans-splicing (SMaRT). Here, we evaluated the potential of trans-splicing RNA reprogramming to correct tau mis-splicing in differentiated neurons in a mouse model of tau mis-splicing, the htau transgenic mouse line, expressing the human MAPT gene in a null mouse Mapt background. Trans-splicing molecules designed to increase exon 10 inclusion were delivered to neurons using lentiviral vectors. We demonstrate reprogramming of tau transcripts at the RNA level after transduction of cultured neurons or after direct delivery and long-term expression of viral vectors into the brain of htau mice in vivo. Tau RNA trans-splicing resulted in an increase in exon 10 inclusion in the mature tau mRNA. Importantly, we also show that the trans-spliced product is translated into a full-length chimeric tau protein. These results validate the potential of SMaRT to correct tau mis-splicing and provide a framework for its therapeutic application to neurodegenerative conditions linked to aberrant RNA processing.Fil: Avale, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Institute of Psychiatry. King’s College London. Centre for Neurodegeneration Research. Department of Clinical Neuroscience; Reino UnidoFil: Rodrigez Martin, Teresa. Institute of Psychiatry. King’s College London. Centre for Neurodegeneration Research. Department of Clinical Neuroscience; Reino UnidoFil: Gallo, Jean-Marc. Institute of Psychiatry. King’s College London. Centre for Neurodegeneration Research. Department of Clinical Neuroscience; Reino Unid

Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting

The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity

Decrease of a Current Mediated by Kv1.3 Channels Causes Striatal Cholinergic Interneuron Hyperexcitability in Experimental Parkinsonism

The mechanism underlying a hypercholinergic state in Parkinson’s disease (PD) remains uncertain. Here, we show that disruption of the Kv1 channel-mediated function causes hyperexcitability of striatal cholinergic interneurons in a mouse model of PD. Specifically, our data reveal that Kv1 channels containing Kv1.3 subunits contribute significantly to the orphan potassium current known as IsAHP in striatal cholinergic interneurons. Typically, this Kv1 current provides negative feedback to depolarization that limits burst firing and slows the tonic activity of cholinergic interneurons. However, such inhibitory control of cholinergic interneuron excitability by Kv1.3-mediated current is markedly diminished in the parkinsonian striatum, suggesting that targeting Kv1.3 subunits and their regulatory pathways may have therapeutic potential in PD therapy. These studies reveal unexpected roles of Kv1.3 subunit-containing channels in the regulation of firing patterns of striatal cholinergic interneurons, which were thought to be largely dependent on KCa channels

Modulation of Tau Isoforms Imbalance Precludes Tau Pathology and Cognitive Decline in a Mouse Model of Tauopathy

Summary: The microtubule-associated protein tau regulates myriad neuronal functions, such as microtubule dynamics, axonal transport and neurite outgrowth. Tauopathies are neurodegenerative disorders characterized by the abnormal metabolism of tau, which accumulates as insoluble neuronal deposits. The adult human brain contains equal amounts of tau isoforms with three (3R) or four (4R) repeats of microtubule-binding domains, derived from the alternative splicing of exon 10 (E10) in the tau transcript. Several tauopathies are associated with imbalances of tau isoforms, due to splicing deficits. Here, we used a trans-splicing strategy to shift the inclusion of E10 in a mouse model of tauopathy that produces abnormal excess of 3R tau. Modulating the 3R/4R ratio in the prefrontal cortex led to a significant reduction of pathological tau accumulation concomitant with improvement of neuronal firing and reduction of cognitive impairments. Our results suggest promising potential for the use of RNA reprogramming in human neurodegenerative diseases. : Tau isoform imbalances in humans lead to neurological disorders. Espíndola et al. show that in vivo reprogramming of tau mRNA by trans-splicing in adult transgenic mice corrects tau isoform imbalance, yielding reduced pathological markers and preventing the loss of key functions such as neuronal activity and cognitive performance. Keywords: dementia, tauopathy, gene therapy, neurodegeneration, MAPT, alternative splicin

Detection of prodromal early phenotypes and potential therapeutic window in a model of tauopathy

Tau is a microtubule-associated protein predominantly expressed in neurons, which participates in microtubule polymerization and axonal transport. The alternative splicing of exon 10 (E10) in the Tau transcript produces protein isoforms with three (3R) or four (4R) microtubule binding repeats, which are expressed in equal amounts in the normal adult human brain. Here aimed to characterize early phenotypes of htau mice, at 3, 6 and 12 months old, to establish the time course of the progression state of tau pathology and identify the brain nuclei involved in these phenotypes. We performed behavioral tests to identify cognitive deficits, anxiety phenotypes, motor impulsivity and loss of behavioral inhibition. In addition, we assessed electrophysiological neuronal activity during the time course of pathological phenotypes, as well as molecular and histological markers. Finally, using an RNA trans-splicing strategy to modulate E10 inclusion we demonstrate that local shifting of 3R to 4R tau into the striatum of htau mice improved some of the htau phenotypes. Together, our results suggest that tau isoforms imbalance could develop early phenotypes that can be identified to generate elaborate strategies to restore the isoform balance.Fil: Muñiz, Javier Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Facal, Carolina Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Pereyra, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Falasco, German. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Urrutia, Leandro. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Páez Paz, Indiana de María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ferrario, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; ArgentinaFil: Damianich, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Avale, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaSAN2022 MeetingCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Investigaciones en Neurociencia