Independent functions of hsp90 in neurotransmitter release and in the continuous synaptic cycling of AMPA receptors

Abstract

The delivery of neurotransmitter receptors into synapses is essential for synaptic function and plasticity. In particular, AMPA-type glutamate receptors (AMPA receptors) reach excitatory synapses according to two distinct routes: a regulated pathway, which operates transiently during synaptic plasticity, and a constitutive pathway, which maintains synaptic function under conditions of basal transmission. However, the specific mechanisms that distinguish these two trafficking pathways are essentially unknown. Here, we evaluate the role of the molecular chaperone hsp90 (heat shock protein 90) in excitatory synaptic transmission in the hippocampus. On one hand, we found that hsp90 is necessary for the efficient neurotransmitter release at the presynaptic terminal. In addition, we identified hsp90 as a critical component of the cellular machinery that delivers AMPA receptors into the postsynaptic membrane. Using the hsp90-specific inhibitors radicicol and geldanamycin, we show that hsp90 is required for the constitutive trafficking of AMPA receptors into synapses during their continuous cycling between synaptic and nonsynaptic sites. In contrast, hsp90 function is not required for either the surface delivery of AMPA receptors into the nonsynaptic plasma membrane or for the acute, regulated delivery of AMPA receptors into synapses during plasticity induction (long-term potentiation). The synaptic cycling of AMPA receptors was also blocked by an hsp90-binding tetratricopeptide repeat (TPR) domain, suggesting that the role of hsp90 in AMPA receptor trafficking is mediated by a TPR domain-containing protein. These results demonstrate new roles for hsp90 in synaptic function by controlling neurotransmitter release and, independently, by mediating the continuous cycling of synaptic AMPA receptors.

Figure 5.

hsp90 is necessary for the constitutive cycling of GluR2 receptors but not for the stability of GluR1 receptors at synapses. Average rectification values (AMPA-mediated response at –60 mV/AMPA-mediated response at +40 mV) for CA1 neurons in the absence (Control) and presence of radicicol (Rad), as indicated, with or without expression of the rectifying GluR2 (R586Q) (A) or GluR1 plus constitutively active αCaMKII (B). Insets, Sample trace of evoked AMPA receptor-mediated synaptic responses recorded at –60 and +40 mV from control, GluR2 (R586Q)-infected cells or GluR1-tCaM-transfected cells in the absence or presence of radicicol, as indicated. Calibration: 20 pA, 20 msec.

Figure 1.

Radicicol, an inhibitor of hsp90, diminishes AMPA and NMDA receptor-mediated responses. A, AMPA and NMDA receptor-mediated evoked synaptic responses were recorded in CA1 neurons of the hippocampus during stimulation of the Schaffer collateral pathway. Radicicol (20 μm; Rad) or the equivalent amount of the radicicol vehicle (0.1% DMSO) was added to the perfusion system at the time indicated with an arrow. AMPA and NMDA receptor-mediated currents were recorded in separate experiments, at –60 and +40 mV, respectively. Values are presented as mean ± SEM. Insets, Sample trace of evoked AMPA or NMDA receptor-mediated synaptic responses (as indicated) before (thin line) and 15 min after (thick line) the addition of radicicol or DMSO. Calibration: 20 pA, 20 msec. B, Average series resistance for the recordings shown in A. In addition, we confirmed that radicicol application does not alter membrane input resistance (control, 182 ± 13 MΩ; plus radicicol, 194 ± 15 MΩ).

Figure 2.

Inhibition of hsp90 enhances paired-pulse facilitation (PPF). Hippocampal slices were perfused with 20 μm radicicol (Rad) for at least 30 min, and then paired-pulse facilitation was monitored at different interstimulus intervals (50, 100, 200, and 400 msec). Paired-pulse facilitation is expressed as the ratio between the amplitude of the second response versus the amplitude of the first response. Note that paired-pulse facilitation was significantly enhanced in slices perfused with radicicol at interstimulus intervals of 50, 100, and 200 msec. At 400 msec, there was no significant facilitation either with or without radicicol. Insets, Sample trace of evoked AMPA receptor-mediated synaptic responses with an interstimulus interval of 100 msec. Calibration: 20 pA, 50 msec.

Figure 3.

Inhibition of hsp90 decreases AMPA/NMDA ratio. Hippocampal slices were perfused with 20 μm radicicol (Rad), 20 μm geldanamycin (Geld), or its vehicle (0.1% DMSO) for at least 30 min, and then AMPA and NMDA responses were recorded from individual cells. Control slices were maintained in regular perfusion solution. Both radicicol and geldanamycin significantly decreased AMPA/NMDA ratio (p is the probability measured by Student's t test comparing AMPA/NMDA ratio in the presence of the vehicle and in the presence of the drug). Values are presented as mean ± SEM.

Figure 4.

Surface cross-linking of AMPA receptors in hippocampal slices exposed to radicicol. Top, Western blot analysis of the fraction of AMPA receptor GluR1 subunit cross-linked on the cell surface with the membrane-impermeant cross-linker BS³. Slices were treated with radicicol or DMSO, as indicated, for 30 min. –BS³ indicates control slices not cross-linked. Each lane in the Western blot is the result of pooling together extracts from four slices treated in parallel. Bottom, Quantification by densitometric scanning of six independent experiments as the one shown on top. Quantification of the intracellular fraction was calculated as described in Materials and Methods.

Figure 6.

hsp90 and NSF act on the same pool of cycling AMPA receptors. A, Whole-cell recordings of AMPA receptor-mediated responses in the presence of the pep2M/G10 peptide in the internal solution. Recordings were performed on naive slices or on slices pretreated with radicicol (Rad) for at least 30 min, as indicated. B, Average series resistance from the recordings shown in A.

Figure 7.

Overexpression of an hsp90-binding TPR domain decreases AMPA receptor-mediated transmission. Left, Average AMPA receptor-mediated current amplitude from infected neurons coexpressing TPR domain and GFP (A) or TPR (R101A) mutant and GFP (B) and control neighboring cells not expressing the recombinant protein (Uninf); n represents the number of pathways from cell pairs. Right, Average AMPA/NMDA ratios for uninfected and infected cells (n represents the number of pathways).

Figure 8.

Blocking hsp90 postsynaptically does not impair LTP. Organotypic slice cultures were infected with virus expressing either TPR or TPR (R101A). Whole-cell recordings were established from neurons expressing the desired proteins or uninfected cells, and LTP was induced by pairing, as described previously (Hayashi et al., 2000). Experiments were done blind with respect to which construct was expressed. Pairing significantly increased AMPA receptor-mediated responses in control, TPR-expressing, and TPR (R101A)-expressing neurons. No significant difference in the amount of potentiation was observed among the three groups at any time point (p = 0.95). Inset, Sample trace of evoked AMPA receptor-mediated synaptic responses before pairing (thin line) and 30 min after pairing (thick line). Calibration: 20 pA, 40 msec.