Dvorzhak, A. | Search bioRxiv

bioRxiv2020

C-terminal-dependent control of EAAT2 signaling, corticostriatal synaptic glutamate clearance and spontaneous motor activity in mice with hypokinesia

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. Despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have remained unclear. This limits the options for pharmacological treatment of motor disorders associated with glutamate excitotoxicity. Therefore, using the Q175 mouse model of Huntingtons disease (HD), we explored the effects of structural changes in the astrocytic excitatory amino acid transporter type 2 (EAAT2). We report that expression of a C-terminal-modified variant of EAAT2 can alleviate the symptoms of hypokinesia in mice with already advanced HD. At a cellular level, this beneficial outcome correlated with faster synaptic glutamate clearance, higher astrocytic glutamate uptake and larger amounts of native EAAT2 protein. Proteomics data indicate a partial reversal of HD-induced changes in the EAAT2 interactor spectrum. Thus, astrocytic glutamate transport remains a target for therapeutic intervention.

neuroscience↗

bioRxiv2020

Increased and synchronous recruitment of release sites underlies hippocampal mossy fiber presynaptic potentiation

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of long-term potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed two-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Moreover, time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between immunofluorescence signals from calcium channels and release sites. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements likely enable long-term increase in synaptic strength.

neuroscience↗

bioRxiv2018

Glutamate uptake and release at single adult corticostriatal synapses of normal and Huntington mice

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGluu. We report findings from individual corticostriatal synapses in acute slices prepared from mice aged >1 year. Contrasting patterns of short-term plasticity and a size criterion identified 2 classes of terminals, presumably corresponding to the previously defined IT and PT synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGluu single synapse study in Q175 mice, a model of Huntingtons disease (HD). It was found that in HD the time constant of perisynaptic [Glu] decay (TauD, as indicator of uptake) and the peak iGluu amplitude (as indicator of release) were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nM) mimicked the TauD changes in homozygotes (HOM). Considering the largest TauD values encountered in WT, about 40% of PT terminals tested in Q175 heterozygotes (HET) can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGluu. Finally, EAAT2 immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention. SIGNIFICANCE STATEMENTAlterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor [Glu] at individual corticostriatal synapses we can now quantify the time constant of perisynaptic [Glu] decay (as indicator of uptake) and the maximal [Glu] elevation next to the active zone (as indicator of Glu release). The results provide a positive answer to the hitherto unresolved question whether neurodegeneration (e.g. Huntingtons disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

neuroscience↗