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The small GTPase Rac1 promotes actin polymerization and plays a critical and increasingly appreciated role in the development and plasticity of glutamatergic synapses. Growing evidence suggests that disruption of the Rac1 signaling pathway at glutamatergic synapses contributes to Autism Spectrum Disorder/intellectual disability (ASD/ID)-related behaviors seen in animal models of ASD/ID. Rac1 has also been proposed as a strong candidate of convergence for many factors implicated in the development of ASD/ID. However, the effects of ASD/ID-related mutations in Rac1 itself have not been explored in neurons. Here, we investigate a recently reported de novo missense mutation in Rac1 found in an individual with severe ID. Our modeling predicts that this mutation will strongly inhibit Rac1 activation by occluding Rac1’s GTP binding pocket.
Indeed, we find that this de novo mutation prevents Rac1 function and results in a selective reduction in synaptic AMPA receptor function. Furthermore, this mutation prevents the induction of long-term potentiation (LTP), the cellular mechanism underlying learning and memory formation. Together, our findings strongly suggest that this mutation contributes to the development of ID in this individual. This research demonstrates the importance of Rac1 in synaptic function and plasticity and contributes to a growing body of evidence pointing to dysregulation of actin polymerization at glutamatergic synapses as a contributing factor to ASD/ID.
A de novo missense mutation in the P-loop region of Rac1 in an individual with severe intellectual disability (ID) is predicted to prevent Rac1 activation. (Top) Rac1 protein regions are indicated, starting with the N terminus: P-loop region, switch I and switch II. Location of a de novo ID-related missense mutation and control missense mutations are shown.
(Bottom) The predicted effect of the severe ID-related mutation is shown. Rac1’s ribbon structure is shown in orange (Protein Data Bank code 3TH5). Rac1’s GTP binding pocket is shown as a transparent white surface.
The tyrosine at position 18 observed in an individual with severe ID is labeled and shown in stick representation and colored magenta. A GTP analog (GMP-PNP) is shown in stick representation with carbon atoms colored pale yellow. Organotypic Hippocampal Slices 400 μm organotypic hippocampal slice cultures were prepared from P6 to P9 Sprague-Dawley rat pups as described previously (Stoppini et al., ). Culture media was exchanged every other day.
Sparse biolistic transfections of organotypic slice cultures were carried out on DIV4 or DIV12 as described (Schnell et al., ). Construct expression was confirmed by GFP and mCherry co-transfection. Paired whole-cell recordings from transfected neurons and non-transfected control neurons were performed on DIV7 or DIV15 slices. During recording, all slices were maintained in room temperature artificial cerebrospinal fluid (aCSF) saturated with 95% O 2/5% CO 2. ACSF contained 119 mM NaCl, 2.5 mM KCl, 1 mM NaH 2PO 4, 26.2 mM NaHCO 3, 11 mM glucose, 4 mM CaCl 2, 4 mM MgSO 4, supplemented with 5 μM 2-chloroadenosine to dampen epileptiform activity and 0.1 mM picrotoxin to block GABA(A) receptors. The internal whole-cell recording solution contained: 135 mM CsMeSO 4, 8 mM NaCl, 10 mM HEPES, 0.3 mM EGTA, 5 mM QX-314, 4 mM Mg-ATP, and 0.3 mM Na-GTP.
The internal solution was pH buffered at 7.3–7.4 and osmolarity was adjusted to 290–295 mOsm. Whole-cell recordings were carried out as described (Sadybekov et al., ). Synaptic responses were evoked by stimulating with a monopolar glass electrode filled with aCSF in stratum radiatum of CA1. AMPA receptor-mediated currents were measured at −70 mV. NMDA receptor-mediated currents were recorded at +40 mV, temporally isolated from AMPAR currents by measuring amplitudes 250 ms following the stimulus.
In most cases AMPAR and NMDAR mediated currents were recorded from the same neuron by changing the membrane potential. In the scatterplot, each open circle represents one paired recording, with the y-axis value for transfected neuron eEPSC amplitude and the x-axis value for control neuron eEPSC amplitude.
When eEPSC amplitudes in control neurons are higher than that of transfected neurons, data points fall below the diagonal line. When eEPSC amplitudes in control neurons are lower than that of transfected neurons, data points fall above the diagonal line. Paired-pulse ratio was recorded by delivering two stimuli at intervals of 20 ms, 40 ms, 70 ms and 100 ms and dividing the peak response of stimulus two by the peak response of stimulus one.
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