Andrea Contestabile

Communication breaks-Down: From neurodevelopment defects to cognitive disabilities in Down syndrome

Down syndrome (DS) is the leading cause of genetically-defined intellectual disability and congenital birth defects. Despite being one of the first genetic diseases identified, only recently, thanks to the phenotypic analysis of DS mouse genetic models, we have begun to understand how trisomy may impact cognitive function. Cognitive disabilities in DS appear to result mainly from two pathological processes: neurogenesis impairment and Alzheimer-like degeneration. In DS brain, suboptimal network architecture and altered synaptic communication arising from neurodevelopmental impairment are key determinants of cognitive defects. Hypocellularity and hypoplasia start at early developmental stages and likely depend upon impaired proliferation of neuronal precursors, resulting in reduction of numbers of neurons and synaptic contacts. The impairment of neuronal precursor proliferation extends to adult neurogenesis and may affect learning and memory. Neurodegenerative mechanisms also contribute to DS cognitive impairment. Early onset Alzheimer disease occurs with extremely high incidence in DS patients and is causally-related to overexpression of beta-amyloid precursor protein (betaAPP), which is one of the triplicated genes in DS. In this review, we will survey the available findings on neurodevelopmental and neurodegenerative changes occurring in DS throughout life. Moreover, we will discuss the potential mechanisms by which defects in neurogenesis and neurodegenerative processes lead to altered formation of neural circuits and impair cognitive function, in connection with findings on pharmacological treatments of potential benefit for DS.

Lithium rescues synaptic plasticity and memory in Down syndrome mice

Down syndrome (DS) patients exhibit abnormalities of hippocampal-dependent explicit memory, a feature that is replicated in relevant mouse models of the disease. Adult hippocampal neurogenesis, which is impaired in DS and other neuropsychiatric diseases, plays a key role in hippocampal circuit plasticity and has been implicated in learning and memory. However, it remains unknown whether increasing adult neurogenesis improves hippocampal plasticity and behavioral performance in the multifactorial context of DS. We report that, in the Ts65Dn mouse model of DS, chronic administration of lithium, a clinically used mood stabilizer, promoted the proliferation of neuronal precursor cells through the pharmacological activation of the Wnt/β-catenin pathway and restored adult neurogenesis in the hippocampal dentate gyrus (DG) to physiological levels. The restoration of adult neurogenesis completely rescued the synaptic plasticity of newborn neurons in the DG and led to the full recovery of behavioral performance in fear conditioning, object location, and novel object recognition tests. These findings indicate that reestablishing a functional population of hippocampal newborn neurons in adult DS mice rescues hippocampal plasticity and memory and implicate adult neurogenesis as a promising therapeutic target to alleviate cognitive deficits in DS patients.

Reversing excitatory GABA A R signaling restores synaptic plasticity and memory in a mouse model of Down syndrome

Down syndrome (DS) is the most frequent genetic cause of intellectual disability, and altered GABAergic transmission through Cl−-permeable GABAA receptors (GABAARs) contributes considerably to learning and memory deficits in DS mouse models. However, the efficacy of GABAergic transmission has never been directly assessed in DS. Here GABAAR signaling was found to be excitatory rather than inhibitory, and the reversal potential for GABAAR-driven Cl− currents (ECl) was shifted toward more positive potentials in the hippocampi of adult DS mice. Accordingly, hippocampal expression of the cation Cl− cotransporter NKCC1 was increased in both trisomic mice and individuals with DS. Notably, NKCC1 inhibition by the FDA-approved drug bumetanide restored ECl, synaptic plasticity and hippocampus-dependent memory in adult DS mice. Our findings demonstrate that GABA is excitatory in adult DS mice and identify a new therapeutic approach for the potential rescue of cognitive disabilities in individuals with DS.

Cortical cultures coupled to micro-electrode arrays: a novel approach to perform in vitro excitotoxicity testing.

In vitro neuronal cultures exhibit spontaneous electrophysiological activity that can be modulated by chemical stimulation and can be monitored over time by using Micro-Electrode Arrays (MEAs), devices composed by a glass substrate and metal electrodes. Dissociated networks respond to transmitters, their blockers and many other pharmacological substances, including neurotoxic compounds. In this paper we present results related to the effects, both acute (i.e. 1 hour after the treatment) and chronic (3 days after the treatment), of increasing glutamatergic transmission induced by the application of rising concentrations of glutamate and its agonists (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid - AMPA, N-methyl-D-aspartate - NMDA and AMPA together with cyclothiazide - CTZ). Increase of available glutamate was obtained in two ways: 1) by direct application of exogenous glutamate and 2) by inhibiting the clearance of the endogenously released glutamate through DL-threo-β-benzyloxyaspartate (TBOA). Our findings show that fine modulations (i.e. low concentrations of drug) of the excitatory synaptic transmission are reflected in the electrophysiological activation of the network, while intervention leading to excessive direct stimulation of glutamatergic pathways (i.e. medium and high concentrations of drug) results in the abolishment of the electrophysiological activity and eventually cell death. The results obtained by means of the MEA recordings have been compared to the analysis of cell viability to confirm the excitotoxic effect of the applied drug. In conclusion, our study demonstrates that MEA-coupled cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission and might provide sensitive endpoints to detect acute and chronic neurotoxic effects of chemicals and drugs for predictive toxicity testing.

Dominant β-catenin mutations cause intellectual disability with recognizable syndromic features.

The recent identification of multiple dominant mutations in the gene encoding β-catenin in both humans and mice has enabled exploration of the molecular and cellular basis of β-catenin function in cognitive impairment. In humans, β-catenin mutations that cause a spectrum of neurodevelopmental disorders have been identified. We identified de novo β-catenin mutations in patients with intellectual disability, carefully characterized their phenotypes, and were able to define a recognizable intellectual disability syndrome. In parallel, characterization of a chemically mutagenized mouse line that displays features similar to those of human patients with β-catenin mutations enabled us to investigate the consequences of β-catenin dysfunction through development and into adulthood. The mouse mutant, designated batface (Bfc), carries a Thr653Lys substitution in the C-terminal armadillo repeat of β-catenin and displayed a reduced affinity for membrane-associated cadherins. In association with this decreased cadherin interaction, we found that the mutation results in decreased intrahemispheric connections, with deficits in dendritic branching, long-term potentiation, and cognitive function. Our study provides in vivo evidence that dominant mutations in β-catenin underlie losses in its adhesion-related functions, which leads to severe consequences, including intellectual disability, childhood hypotonia, progressive spasticity of lower limbs, and abnormal craniofacial features in adults.

REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage‐gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element‐1 Silencing Transcription Factor/Neuron‐Restrictive Silencer Factor (REST/NRSF). We showed that 4‐aminopyridine‐induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage‐gated Na+ channels, thereby decreasing the neuronal Na+ current density. This mechanism plays an important role in the downregulation of the firing activity at the single‐cell level, re‐establishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.

Non-hyperpolarizing GABAB 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.

Localization of focal adhesion kinase isoforms in cells of the central nervous system

Focal adhesion kinase (FAK) is a non‐receptor tyrosine kinase which in non‐neuronal cells is localized to focal adhesions, where it participates to adhesion‐dependent intracellular signalling. FAK is highly expressed in the central nervous system both during development and in the adult. FAK+, a splice isoform of FAK selectively enriched in neurons, contains a three‐amino acid insertion in the carboxy‐terminal sequence responsible for the localization of FAK to focal adhesions. Enhanced green fluorescent protein‐tagged constructs were used to study the targeting of FAK and FAK+ in neuronal and non‐neuronal cells of the central nervous system. In transfected non‐neuronal cells, both fusion proteins colocalized with vinculin in focal contacts. When expressed in hippocampal neurons in culture, both chimeras were locally concentrated in the growth cone, where they overlapped with F‐actin enrichments but not with vinculin. In the growth cone of living neurons, the FAK+ chimera showed a dynamic relocalization to membrane ruffles and to the tips of the membrane protrusions induced by cytochalasin D treatment, indicating a dependence of FAK distribution on F‐actin organization. Since virtually identical patterns of distribution were found for FAK and FAK+ chimeras, it follows that the additional insertion in FAK+ is not responsible for the localization of the kinase. Finally, we showed that the carboxy‐terminal domain of both FAK and FAK+ is sufficient to mediate the localization of the proteins to focal adhesions in non‐neuronal cells and to maintain their correct intracellular targeting in neurons.

The polyphenols resveratrol and epigallocatechin-3-gallate restore the severe impairment of mitochondria in hippocampal progenitor cells from a Down syndrome mouse model.

Mitochondrial dysfunctions critically impair nervous system development and are potentially involved in the pathogenesis of various neurodevelopmental disorders, including Down syndrome (DS), the most common genetic cause of intellectual disability. Previous studies from our group demonstrated impaired mitochondrial activity in peripheral cells from DS subjects and the efficacy of epigallocatechin-3-gallate (EGCG) - a natural polyphenol major component of green tea - to counteract the mitochondrial energy deficit. In this study, to gain insight into the possible role of mitochondria in DS intellectual disability, mitochondrial functions were analyzed in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice, a widely used model of DS which recapitulates many major brain structural and functional phenotypes of the syndrome, including impaired hippocampal neurogenesis. We found that, during NPC proliferation, mitochondrial bioenergetics and mitochondrial biogenic program were strongly compromised in Ts65Dn cells, but not associated with free radical accumulation. These data point to a central role of mitochondrial dysfunction as an inherent feature of DS and not as a consequence of cell oxidative stress. Further, we disclose that, besides EGCG, also the natural polyphenol resveratrol, which displays a neuroprotective action in various human diseases but never tested in DS, restores oxidative phosphorylation efficiency and mitochondrial biogenesis, and improves proliferation of NPCs. These effects were associated with the activation of PGC-1α/Sirt1/AMPK axis by both polyphenols. This research paves the way for using nutraceuticals as a potential therapeutic tool in preventing or managing some energy deficit-associated DS clinical manifestations.

The GABAergic Hypothesis for Cognitive Disabilities in Down Syndrome

Down syndrome (DS) is a genetic disorder caused by the presence of a third copy of chromosome 21. DS affects multiple organs, but it invariably results in altered brain development and diverse degrees of intellectual disability. A large body of evidence has shown that synaptic deficits and memory impairment are largely determined by altered GABAergic signaling in trisomic mouse models of DS. These alterations arise during brain development while extending into adulthood, and include genesis of GABAergic neurons, variation of the inhibitory drive and modifications in the control of neural-network excitability. Accordingly, different pharmacological interventions targeting GABAergic signaling have proven promising preclinical approaches to rescue cognitive impairment in DS mouse models. In this review, we will discuss recent data regarding the complex scenario of GABAergic dysfunctions in the trisomic brain of DS mice and patients, and we will evaluate the state of current clinical research targeting GABAergic signaling in individuals with DS.