Normal spatial learning and improved spatial working memory in mice (mus musculus) lacking dopamine d4 receptors

Dopamine terminals in the hippocampus and prefrontal cortex modulate cognitive processes such as spatial learning and working memory. Because dopamine D4 receptors are expressed in these brain areas we have analyzed mutant mice lacking this receptor subtype (Drd4-/-). Wild-type and Drd4-/- mice were challenged in two spatial learning paradigms: the Morris water maze and an alternation T-maze. Drd4-/- mice showed normal place learning ability to find a hidden platform based on spatial extra-maze cues. In addition, Drd4-/- mice were able to find a new platform location with the same learning plasticity as wild type-mice. Spatial working memory assessed on a T maze showed that Drd4-/- mice were more efficient than wild-type mice in acquiring the maximum plateau of correct alternation scores. These results provide further evidence that the functional consequence of lacking D4 receptors is more evident in behaviors dependent on the integrity of the prefrontal cortex.Fil: Falzone, Tomas Luis. 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"; ArgentinaFil: Gelman, Diego Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Dopaminergic innervation at the central nucleus of the amygdala reveals distinct topographically segregated regions

The central nucleus of the amygdala (CeA) is involved in the expression of fear and anxiety disorders. Anatomically, it is divided into medial (CeM), lateral (CeL), and capsular (CeC) divisions. The CeA is densely innervated by dopaminergic projections that originate in the ventral periaqueductal gray/dorsal raphe (vPAG/DR) and the ventral tegmental area (VTA). However, whether dopamine (DA) exerts a homogenous control over the CeA or differentially regulates the various CeA subdivisions is still unknown. Here, we performed a neuroanatomical analysis of the mouse CeA and found that DAergic innervations from the PAG/DR and VTA constitute distinct, non-overlapping, pathways differing also in the relative expression of the dopamine transporter. By quantifying the distribution of DAergic fibers and the origin of DA inputs we identified two distinct regions in the CeL: a frontal region innervated by the VTA and vPAG/DR, a caudal region innervated only by the vPAG/DR, and three distinct regions in the CeC: fronto-dorsal innervated only by the VTA, fronto-ventral with sparse DAergic innervation, and a caudal region with low innervation from the vPAG/DR. In addition, we found that each region displays a distinct pattern of c-Fos activation following the administration of various DAeric drugs such as cocaine, SKF 38,393, quinpirole or haloperidol. In summary, we revealed unique properties of the DAergic pathways innervating the CeA, distinguishing six topographically segregated and functionally distinct regions. This unanticipated level of heterogeneity calls for more precise neuroanatomical specificity in future functional studies of the CeA.Fil: Casey, Eric. 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"; ArgentinaFil: Kravitz, Alexxai. Washington University in St. Louis; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Evaluation of RNA trans-splicing as a therapeutic strategy for spinocerebellar ataxia type 1

Background and objective Spinocerebellar ataxia type 1 (SCA1) is caused by an expanded polyglutamine (polyQ) tract in the protein ataxin-1 encoded by the ATXN1 gene. The exact pathogenic mechanism is not understood but phosphorylation of ataxin-1 at S776 is critical for the stabilisation and neurotoxicity of polyQ-expanded ataxin-1. Our objective is to evaluate the therapeutic potential of preventing pathogenic phosphorylation of ataxin-1 using an RNA reprogramming technology. Methods Spliceosome-mediated RNA trans-splicing (SMaRT) creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and an exogenously delivered pre-trans-splicing molecule (PTM). We constructed and tested, in-vitro, several PTMs designed to substitute S776 or S752 (the mouse homologue for S776) for alanine. PTMs were constructed in pcDNA3.1 and used to generate lentiviral constructs containing a GFP expression cassette. Trans-splicing in transfected, or transduced cells was analysed by RT-PCR and sequencing. Endogenous and ataxin-1 minigene transcripts were analysed, minigenes were constructed using the pSPL3 exon trapping vector. Results Human (SH-SY5Y) and mouse (N2a) cell lines were transfected with PTMs with and without minigenes. SMaRT successfully edited, in-vitro, mouse and human ATXN1 transcripts to substitute S752 or S776 for alanine, with efficiencies of approximately 30% for endogenous human transcripts. We additionally observed trans-splicing of endogenous ataxin-1 in cultured primary cortical neurons from wild-type mice. The most efficient PTM design hybridises with the 3’ end of intron 8, upstream of the branch point. Discussion and conclusion SCA1 is an excellent prototypic system to demonstrate that a SCA-causing protein can be converted into a non-toxic form by SMaRT. SMaRT can theoretically repair any mutation downstream of the PTM binding site and is particularly suited for dominant gain of function mutations characteristic of SCAs. Our work demonstrates the potential of SMaRT to prevent a pathogenic phosphorylation event and provides proof-of-concept for in-vivo pre-clinical development

SMaRT modulation of tau isoforms rescues cognitive and motor impairments in a preclinical model of tauopathy

Tau is a microtubule-associated protein predominantly expressed in neurons, which participates in microtubule polymerization and axonal transport. Abnormal tau metabolism leads to neurodegenerative diseases named tauopathies, such as Alzheimer’s disease and frontotemporal dementia. The alternative splicing of exon 10 (E10) in the primary transcript produces tau protein isoforms with three (3R) or four (4R) microtubule binding repeats, which are found in equal amounts in the normal adult human brain. Several tauopathies are associated with abnormal E10 alternative splicing, leading to an imbalance between 3R and 4R isoforms, which underlies disease. Correction of such imbalance represents a potential disease-modifying therapy for those tauopathies. We have previously optimized a trans-splicing RNA reprogramming strategy to modulate the 3R:4R tau content in a mouse model of tauopathy related to tau mis-splicing (htau mice), and showed that local modulation of E10 inclusion in the prefrontal cortex prevents cognitive decline, neuronal firing impairments and hyperphosphorylated tau accumulation. Furthermore, local shifting of 3R–4R tau into the striatum of htau mice prevented motor coordination deficits. However, a major bottleneck of our previous work is that local splicing regulation was performed in young mice, before the onset of pathological phenotypes. Here we tested whether regulation of tau E10 splicing could rescue tau pathology phenotypes in htau mice, after the onset of cognitive and motor impairments, comparable to early stages of human tauopathies. To determine phenotypic time course and affected brain nuclei, we assessed htau mice using behavioural tests and microPET FDG imaging over time, similarly to diagnosis methods used in patients. Based on these analyses, we performed local delivery of pre-trans splicing molecules to regulate E10 inclusion either into the medial prefrontal cortex (mPFC) or the striatum at 6-month-old once behavioral phenotypes and metabolic changes were detected. Tau isoforms modulation into the mPFC restored cognitive performance in mice that previously showed mild to severe memory impairment while motor coordination deficit was rescued after striatal injection of trans-splicing molecules. Our data suggest that tau regulation could recover pathological phenotypes early after phenotypic onset, raising promising perspectives for the use of RNA based therapies in tauopathies related to MAPT abnormal splicing.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: Urrutia, Leandro. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Clerici Delville, Ramiro. 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: Ferrario, Juan Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Falasco, Germán Alfredo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Avale, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Fyn knock-down prevents levodopa-induced dyskinesia in a mouse model of parkinson’s disease

Dopamine replacement by levodopa (L-DOPA) is the most widely used therapy for Parkinson’s disease (PD), however patients often develop side effects, known as L-DOPA-induced dyskinesia (LID), that usually need therapeutic intervention. There are no suitable therapeutic options for LID, except for the use of the NMDA receptor (NMDA-R) antagonist amantadine, which has limited efficacy. The NMDA-R is indeed the most plausible target to manage LID in PD and recently the kinase Fyn, one of its key regulators, became a new putative molecular target involved in LID. The aim of this work was to reduce Fyn expression to alleviate LID in a mouse model of PD. We performed intrastriatal delivery of a designed micro-RNA against Fyn (miRNA-Fyn) in 6-OHDA-lesioned mice treated with L-DOPA. The miRNA-Fyn was delivered either before or after L-DOPA exposure to assess its ability to prevent or revert dyskinesia. Preadministration of miRNA-Fyn reduced LID with a concomitant reduction of FosB-DFosB protein levels, a marker of LID, as well as decreased phosphorylation of the NR2B-NMDA subunit, which is a main target of Fyn. On the other hand, post-L-DOPA delivery of miRNA-Fyn was less effective to revert already established dyskinesia, suggest-ing that early blocking of Fyn activity might be a more efficient therapeutic approach. Together, our results provide proof of concept about Fyn as a plausible therapeutic target to manage LID, and validate RNA si-lencing as a potential approach to locally reduce striatal Fyn, rising new perspectives for RNA therapy interventions in PD.Fil: Bordone, Melina Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Damianich, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bernardi, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Eidelman, Tomas. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sanz Blasco, Sara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Gershanik, Oscar Samuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Avale, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ferrario, Juan Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

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

Reward-seeking and discrimination deficits displayed by hypodopaminergic mice are prevented in mice lacking dopamine D4 receptors

The dopamine D4 receptor (D4R) is predominantly expressed in the prefrontal cortex, a brain area that integrates motor, rewarding, and cognitive information. Because participation of D4Rs in executive learning is largely unknown, we challenged D4R knockout mice (Drd4 -/- ) and their wild-type (WT) littermates, neonatally treated with 6-hydroxydopamine (6-OHDA; icv) or vehicle in two operant learning paradigms. A continuous reinforcement task, in which one food-pellet was delivered after every lever press, showed that 6-OHDA-treated mice (hypodopaminergic) WT mice pressed the reinforcing lever at much lower rates than normodopaminergic WT mice. In contrast, Drd4 -/- mice displayed increased lever pressing rates, regardless of their dopamine content. In another study, mice were trained to solve an operant two-choice task in which a first showing lever was coupled to the delivery of one food pellet only after a second lever emerged. Interval between presentation of both levers was initially 12 s and progressively shortened to 6, 2, and finally 0.5 s. Normodopaminergic WT mice obtained a pellet reward in more than 75% of the trials at 12, 6, and 2 s, whereas hypodopaminergic WT mice were severely impaired to select the reward-paired lever. Absence of D4Rs was not detrimental in this task. Moreover, hypodopaminergic Drd4 -/- mice were as efficient as their normodopaminergic Drd4 -/- siblings in selecting the reward-paired lever. In summary, hypodopaminergic mice exhibit severe impairments to retrieve rewards in two operant positive reinforcement tasks, but these deleterious effects are totally prevented in the absence of functional D4Rs.Fil: Nemirovsky, Sergio Ivan. 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"; ArgentinaFil: Brunner, Daniela. PsychoGenics; Estados UnidosFil: Rubinstein, Marcelo. 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. Departamento de Fisiología, Biología Molecular y Celular; Argentin