Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type–specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.

A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function. Genetic analysis indicates that 20% to 30% of the total open reading frame encodes for integral membrane proteins (1). Although less abundant than cytosolic proteins, membrane-passing proteins contribute to the regulation of all major cell processes and signaling pathways. In particular, membrane proteins play an important role in the establishment of functional neuronal circuitries during development. This process initially entails the growth, guidance, and stabilization of neuronal processes (axons and dendrites) in a timely, ordered manner involving cell surface molecules that sense the extracellular surroundings and activate signaling cascades (2).Then, specialized cell-to-cell connections, the synapses, are formed. These connections allow information to flow from one neuron to another and relay the precise juxtaposition and interactions between the pre-and postsynaptic membrane proteins to support their final functional establishment. Several families of synaptic transmembrane or membrane proteins, such as semaphorin, neuroligin, neurexin, and the immunoglobulin superfamily (IgSF), 1 are implicated in neurite formation and synapse establishment (3). However, the picture of membrane proteins expressed in neurons is still far from being completely resolved, and it is expected that many other key molecules are awaiting identification (4). Thus, uncovering the nature of the dynamic multiprotein complexes expressed at the plasma membrane will possibly strongly support the understanding of the mechanism controlling structural and functional neuron development. Here, we describe a biochemical approach to isolate and quantify proteins exposed at the extracellular side of the plasma membrane. Our assay utilized affinity purification on streptavidin resin of biotinylated membrane proteins extracted from a crude synaptosomal preparation. We combined this cell surface biotinylation assay with MS/MS analysis and label-free quantification to investigate protein patterns characterizing immature and m...

A variety of pathophysiological mechanisms are implicated in Huntington's disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from presymptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.

Long-term memory formation (LTM) is a process accompanied by energy-demanding structural changes at synapses and increased spine density. Concomitant increases in both spine volume and postsynaptic density (PSD) surface area have been suggested but never quantified in vivo by clear-cut experimental evidence. Using novel object recognition in mice as a learning task followed by 3D electron microscopy analysis, we demonstrate that LTM induced all aforementioned synaptic changes, together with an increase in the size of astrocytic glycogen granules, which are a source of lactate for neurons. The selective inhibition of glycogen metabolism in astrocytes impaired learning, affecting all the related synaptic changes. Intrahippocampal administration of l-lactate rescued the behavioral phenotype, along with spine density within 24 hours. Spine dynamics in hippocampal organotypic slices undergoing theta burst-induced long-term potentiation was similarly affected by inhibition of glycogen metabolism and rescued by l-lactate. These results suggest that learning primes astrocytic energy stores and signaling to sustain synaptic plasticity via l-lactate.

Intellectual disability affects 2–3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1–GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1–GluA2 binding restored synaptic function in Tm4sf2−/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2−/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.

No abstract

Myosin IXa (Myo9a) is a motor protein that is highly expressed in the brain. However, the role of Myo9a in neurons remains unknown. Here, we investigated Myo9a function in hippocampal synapses. In rat hippocampal neurons, Myo9a localizes to the postsynaptic density (PSD) and binds the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA2 subunit. Myo9a+/- mice displayed a thicker PSD and increased levels of PSD95 and surface AMPAR expression. Furthermore, synaptic transmission, long-term potentiation (LTP) and cognitive functions were impaired in Myo9a+/- mice. Together, these results support a key role for Myo9a in controlling the molecular structure and function of hippocampal synapses.