Non-hyperpolarizing GABA B receptor activation regulates neuronal migration and neurite growth and specification by cAMP/LKB1

γ-Aminobutyric acid is the principal inhibitory neurotransmitter in adults, acting through ionotropic chloride-permeable GABAA receptors (GABAARs), and metabotropic GABABRs coupled to calcium or potassium channels, and cyclic AMP signalling. During early development, γ-aminobutyric acid is the main neurotransmitter and is not hyperpolarizing, as GABAAR activation is depolarizing while GABABRs lack coupling to potassium channels. Despite extensive knowledge on GABAARs as key factors in neuronal development, the role of GABABRs remains unclear. Here we address GABABR function during rat cortical development by in utero knockdown (short interfering RNA) of GABABR in pyramidal-neuron progenitors. GABABR short interfering RNA impairs neuronal migration and axon/dendrite morphological maturation by disrupting cyclic AMP signalling. Furthermore, GABABR activation reduces cyclic AMP-dependent phosphorylation of LKB1, a kinase involved in neuronal polarization, and rescues LKB1 overexpression-induced defects in cortical development. Thus, non-hyperpolarizing activation of GABABRs during development promotes neuronal migration and morphological maturation by cyclic AMP/LKB1 signalling

Polarized Expression of p75NTR Specifies Axons during Development and Adult Neurogenesis

VIDEO ABSTRACT: Newly generated neurons initiate polarizing signals that specify a single axon and multiple dendrites, a process critical for patterning neuronal circuits in vivo. Here, we report that the pan-neurotrophin receptor p75(NTR) is a polarity regulator that localizes asymmetrically in differentiating neurons in response to neurotrophins and is required for specification of the future axon. In cultured hippocampal neurons, local exposure to neurotrophins causes early accumulation of p75(NTR) into one undifferentiated neurite to specify axon fate. Moreover, knockout or knockdown of p75(NTR) results in failure to initiate an axon in newborn neurons upon cell-cycle exit in vitro and in the developing cortex, as well as during adult hippocampal neurogenesis in vivo. Hence, p75(NTR) governs neuronal polarity, determining pattern and assembly of neuronal circuits in adult hippocampus and cortical development

53 a neuro specific gene therapy approach to treat cognitive impairment in down syndrome by rna interference

Down syndrome (DS) is a genetic disorder caused by the presence of a third copy of chromosome 21. DS affects multiple organs, resulting in characteristic facial features, muscular hypotonia, heart defects, brain development impairment, and varying degrees of intellectual disability. Trisomic mouse models of DS reproduce the main cognitive disabilities of the human syndrome. In particular, DS mice show structural and functional synaptic impairment as well as learning and memory deficits, largely determined by altered GABAergic transmission through chloride-permeable GABAa receptors (GABAaR). In particular, we have recently found that intracellular chloride accumulation shifts GABAAR-mediated signaling from inhibitory to excitatory in the adult brain of the Ts65Dn mouse model of DS. Accordingly, intracellular chloride accumulation was paralleled by increased expression of the chloride importer NKCC1 (Na-K-Cl cotransporter) in the brains of both trisomic mice and DS patients.Our findings on NKCC1 as a pivotal molecular target for the rescue of cognitive deficits in DS opens the possibility of a gene therapy approach to treat the disease. Here, to normalize NKCC1 expression and rescue synaptic dysfunctions as well as cognitive deficits in Ts65Dn mice we have developed and characterized a knock-down approach to normalize NKCC1 activity. Reducing the expression of the chloride importer NKCC1 by RNA interference restored GABAAR-mediated inhibition and also rescued the structural dendritic deficits found in trisomic neurons in vitro. Most importantly, focal administration of an AAV expressing a silencing RNA under the transcriptional control of a neuron-specific promoter in the hippocampus of Ts65Dn animals mediated NKCC1 knockdown in vivo and rescued behavioral performance on different learning and memory tests at levels undistinguishable from those of WT mice.Our findings demonstrate that NKCC1 overexpression drives excitatory GABAAR signaling in trisomic cells, leading to structural neuronal abnormalities and behavioral impairments in DS mice. Moreover, our study identifies a new gene therapy target for treatments aimed at rescuing cognitive disabilities in individuals with DS

In vivo methods for acute modulation of gene expression in the central nervous system

Accurate and timely expression of specific genes guarantees the healthy development and function of the brain. Indeed, variations in the correct amount or timing of gene expression lead to improper development and/or pathological conditions. Almost forty years after the first successful gene transfection in in vitro cell cultures, it is currently possible to regulate gene expression in an area-specific manner at any step of central nervous system development and in adulthood in experimental animals in vivo, even overcoming the very poor accessibility of the brain. Here, we will review the diverse approaches for acute gene transfer in vivo, highlighting their advantages and disadvantages with respect to the efficiency and specificity of transfection as well as to brain accessibility. In particular, we will present well-established chemical, physical and virus-based approaches suitable for different animal models, pointing out their current and future possible applications in basic and translational research as well as in gene therapy

Indagini istologiche, immunoistochimiche e immunobiochimiche in capre clinicamente sane provenienti da un focolaio di scrapie

Nel corso del 2003, nell’ambito del piano di sorveglianza passiva sulle Encefalopatie Spongiformi Trasmissibili (TSEs), per la prima volta in Sardegna, è stata diagnosticata istologicamente ed immunoistochimicamente la scrapie in una capra. Il soggetto proveniva da un gregge nel quale da 2 anni si manifestava una patologia polimorfa caratterizzata talvolta da sintomi neurologici e da una mortalità intorno al 20%. Sulla base della legge attualmente in vigore in Italia si è proceduto all’abbattimento di tutte le capre presenti in allevamento (n = 93). Durante l’abbattimento venivano prelevati da ciascun capo obex, linfonodi retrofaringei e tonsille. L’esame istologico dell’obex non evidenziava quadri lesivi di tipo spongiforme mentre l’esame immunoistochimico del tessuto nervoso e linfatico evidenziava la presenza di cinque soggetti nello stato pre-clinico della malattia

Quali-quantitative evaluation of ileal peyer's patches innervation in scrapie-free or scrapie-affected sarda breed ovines

Although Peyer's patches (PPs) and the enteric nervous system (ENS) play a key role in early sheep scrapie pathogenesis, little is known on the kinetics of ENS plexuses colonization. This study was aimed at quali-quantitatively evaluating ileal PP innervation in 29 Sarda breed ovines (12 scrapie-free, 2 months-old lambs, 4 ARQ/ARQ, 4 ARR/ARQ and 4 ARR/ARR, respectively; 12 scrapie-free, 2-4 years-old sheep, 3 ARQ/ARQ, 7 ARR/ARQ and 2 ARR/ARR, respectively; 5 ARQ/ARQ scrapie-affected sheep)

Neurotrophic-mimetic strategy to rescue synaptic plasticity and cognitive functions in a mouse model of Down syndrome

Down syndrome (DS) or trisomy 21 is the most frequent genetic cause of intellectual disability in children and adults. Although numerous studies have shown that cognitive impairment possibly arises from dysfunction of the hippocampal circuit, there has been little progress in defining effective treatments. Previous studies have shown that impaired synaptic plasticity of mature hippocampal neurons and decreased hippocampal adult neurogenesis are main determinants in reducing cognitive functions in DS animal models. Currently, most preclinical therapeutic approaches in DS mice have focused on rescuing either one or the other of these impairments. Here, we have found that the expression of Brain-Derived Neurotrophic Factor (BDNF) is decreased in the brains of individuals with DS. Interestingly, a large body of literature indicates that BDNF signaling modulates both synaptic plasticity, and adult neurogenesis. Therefore, we propose here to promote BDNF/TrkB signaling using a BDNF-mimetic drug with the twofold aim of rescuing synaptic plasticity and increase adult neurogenesis toward the rescue of cognitive functions in the Ts65Dn mouse model of DS. Our results indicate that indeed promoting BDNF/TrkB signaling rescued hippocampal synaptic plasticity, increased hippocampal adult neurogenesis and restored cognitive performances in different behavioral tasks in Ts65Dn mice. The molecular mechanisms of impaired BDNF/TrkB signaling in trisomic mice are currently under investigation. Overall, our experiments show in a reliable animal model of DS the efficacy of a novel and multifaceted therapeutic approach with good potential to be translated into clinical practice

The three-electrode device: A new frontier for the in utero electroporation

The understanding of brain function requires the development of new methods to perturb and track distinct neuronal populations in the developing and adult central nervous system. Over the past ten years, in utero electroporation (IUE) has arisen as an extremely powerful tool to transfect and manipulate neuronal precursor cells of the parietal-cortex and their progeny in vivo. Although this technique has tremendous potentialities in targeting numerous brain areas, the results obtained so far have been generally hindered by low reliability of transfection in some regions and by the physical impossibility to reach other regions. Here, we present an innovative IUE configuration, which allows highly reliable transfection at various brain locations, including regions and cell types never targeted before. Our device, based on the usage of three independent electrodes upon an easy and highly reliable re-orientation of the electrode’s positions and polarities, allows consistent expression of genes of interest in an array of brain areas including the hippocampus, the visual and motor cortices, and the cerebellum. Moreover, depending on the developmental stage of the embryos, it is possible to target distinct neuronal cell types, which may be particularly relevant in the cerebellum. The importance of such a tool in comparison to other methods arises in those particular applications where tissues and circuits integrity are essential points, and in those where traditional electroporation configuration is the limiting step of the experimental approach

Hypofractionated radiotherapy for macroscopic canine soft tissue sarcoma: A retrospective study of 50 cas-es treated with a 5x6 gy protocol with or without metronomic chemotherapy

Wide surgical resection or a marginal/incomplete resection followed by full-course radiation therapy is the current standard of care for canine soft tissue sarcoma. The purpose of this retrospective, descriptive, bi-institutional study was to determine the effectiveness and toxicity of a hypofractionated 5 × 6 Gy protocol on macroscopic canine soft tissue sarcoma in terms of progression-free interval (PFI) and overall survival (OS), and to identify prognostic factors for patient outcome. Dogs with macroscopic soft tissue sarcoma irradiated with 5 × 6 Gy were eligible for the study. Progression-free interval and OS were compared with respect to different tumor and patient characteristics by the Kaplan-Meier method and multivariable Cox regression analysis. Fifty dogs with macroscopic disease were included. All dogs received the same radiation therapy protocol; part of the group (n = 20) received postradiation metronomic chemotherapy. Median PFI for all cases was 419 days (95% confidence interval (CI): 287-551) and median OS was 513 days (95% CI: 368-658). Dogs with tumors on the limbs had significantly longer PFI and OS, compared with head or trunk. Increasing tumor burden decreased OS. The addition of metronomic chemotherapy yielded a significantly longer OS (757 days (95% CI: 570-944) compared with dogs that did not receive systemic treatment (286 days (95% CI: 0-518), (P = 0.023)), but did not influence progression-free interval. Toxicity was low throughout all treatments. The 5 × 6 Gy radiation therapy protocol was well tolerated and provided long PFI and OS in dogs with macroscopic soft tissue sarcoma

High-performance and site-directed in utero electroporation by a triple-electrode probe

In utero electroporation is a powerful tool to transfect and manipulate neural-precursor cells of the rodent parietal cortex and their progeny in vivo. Although this technique can potentially target numerous brain areas, reliability of transfection in some brain regions is low or physical access is limited. Here we present a new in utero electroporation configuration based on the use of three electrodes, the relative position and polarities of which can be adjusted. The technique allows easy access and exceedingly reliable monolateral or bilateral transfection at brain locations that could only be sporadically targeted before. By improvement in the efficiency of the electrical field distribution, demonstrated here by a mathematical simulation, the multi-electrode configuration also extends the developmental timeframe for reliable in utero electroporation, allowing for the first time specific transfection of Purkinje cells in the rat cerebellum