GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3Ј-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14 -29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of ϳ3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

It is widely thought that Alzheimer's disease (AD) begins as a malfunction of synapses, eventually leading to cognitive impairment and dementia. Homeostatic synaptic scaling is a mechanism that could be crucial at the onset of AD but has not been examined experimentally. In this process, the synaptic strength of a neuron is modified so that the overall excitability of the cell is maintained. Here, we investigate whether synaptic scaling mediated by L-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) contributes to pathology in double knockin (2؋KI) mice carrying human mutations in the genes for amyloid precursor protein and presenilin-1. By using whole-cell recordings, we show that 2؋KI mice exhibit age-related downscaling of AMPARmediated evoked currents and spontaneous, miniature currents. Electron microscopic analysis further corroborates the synaptic AMPAR decrease. Additionally, 2؋KI mice show age-related deficits in bidirectional plasticity (long-term potentiation and longterm depression) and memory flexibility. These results suggest that AMPARs are important synaptic targets for AD and provide evidence that cognitive impairment may involve downscaling of postsynaptic AMPAR function.amyloid precursor protein ͉ glutamate ͉ presenilin E xtensive work on Alzheimer's disease (AD) has led to the hypothesis that the memory failure exhibited by patients in the early stages of AD results from synaptic disruption (1-3), without frank neuronal loss, which is caused by toxicity of the 42-aa variant of the amyloid ␤ protein (A␤ 42 ). Work in AD models (4) shows that A␤ 42 impairs synaptic plasticity in brain regions, such as the hippocampus, that are recognized early targets for AD. Transgenic (Tg) mice with familial AD mutations display disruptions of long-term potentiation (LTP), an electrophysiological correlate of memory encoding (5). The LTP impairment occurs before deposition of A␤ plaques (4, 6), making it a sensitive marker for early AD dysfunction. Notably, the late phase of LTP (called expression) is highly susceptible to disruption (6-9). LTP expression relies on alterations of L-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors (AMPARs), including phosphorylation by kinases and recruitment of AMPARs to the synaptic membrane (10). Conversely, AMPAR removal is thought to mediate the activitydependent decrease of excitatory transmission (11), which is elicited by the paradigm of long-term depression (LTD) (12). Importantly, LTD has been scarcely studied in AD models (4, 13), particularly Tg-AD mice.Recently, AMPARs have been implicated in a slower form of synaptic plasticity, termed homeostatic synaptic scaling, in which the total synaptic strength of a neuron is modified to regulate its excitability (14). Synaptic scaling involves, among other factors, the postsynaptic insertion and removal of AMPARs and changes in the turnover rate of functional receptors (14-16). Adjustments by synaptic scaling operate in vivo (17) and seem critical for regulating synaptic strength during lear...

The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin-neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1␤ are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1␤ with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.

BackgroundHyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies.MethodsGenetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A “click” chemistry labeling method is developed for the detection of O-GlcNAcylated tau.ResultsSubstantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity.ConclusionThe present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer’s disease and other neurodegenerative tauopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0181-0) contains supplementary material, which is available to authorized users.