Disrupted-in-Schizophrenia 1 (DISC1) regulates spines of the glutamate synapse via Rac1

Abstract

Synaptic spines are dynamic structures that regulate neuronal responsiveness and plasticity. We examined the role of the schizophrenia risk factor DISC1 in the maintenance of spine morphology and function. We found that DISC1 anchored Kalirin-7 (Kal-7), regulating access of Kal-7 to Rac1 and controlling the duration and intensity of Rac1 activation in response to NMDA receptor activation in both cortical cultures and rat brain in vivo. These results explain why Rac1 and its activator (Kal-7) serve as important mediators of spine enlargement and why constitutive Rac1 activation decreases spine size. This mechanism likely underlies disturbances in glutamatergic neurotransmission that have been frequently reported in schizophrenia that can lead to alteration of dendritic spines with consequential major pathological changes in brain function. Furthermore, the concept of a signalosome involving disease-associated factors, such as DISC1 and glutamate, may well contribute to the multifactorial and polygenetic characteristics of schizophrenia.

Figure 1. Short-term knockdown of DISC1 elicits spine enlargement in rat primary cortical neurons

(a) Spine changes in mature neurons by short-term knockdown (2 days) of DISC1 with use of two independent RNAi against DISC1. Scale bar, 50 μm. (b) Enhanced surface expression of GluR1 on the spine, Arrowheads, GluR1 clustering on spines. (c) Increase in the frequency of miniature excitatory postsynaptic currents (mEPSC). Left, representative mEPSC traces; right, mEPSC amplitude and frequency. Bar, s.e.m. *P < 0.05, #P < 0.001.

Figure 2. Protein interaction of DISC1/Kal-7 regulates spine morphology in rat primary cortical neurons

(a) Endogenous interactions of DISC1 with Kal-7 and PSD95 (red asterisks) by co-immunoprecipitation (IP) from primary cortical neurons and rat cerebral cortex. DISC1 did not bind Tiam1 norβPIX. Strong interactions of DISC1/Kal-7 and DISC1/PSD95 are observed in the synaptosomal fractions (double asterisks). Full-length blots are presented in Supplementary Fig. 17. (b) Spine shrinkage and reduced spine density by overexpression (for 2 days) of full length DISC1 (DISC-FL), but not by DISC1 lacking Kal-7 interaction (DISC1-ΔKal-7). Both DISC1-FL and DISC1-ΔKal-7 were localized in the dendritic spine (arrowheads). (c) Normalization of DISC1 knockdown-induced spine enlargement by DISC1-FL, but not by DISC1-ΔKal-7. (d) Increase in the frequency of mEPSC was normalized by the overexpression of DISC1-FL^R, but not by that of DISC1-ΔKal-7^R. Left, representative mEPSC traces; right, mEPSC amplitude and frequency. Bar, s.e.m. *P < 0.05, #P < 0.001.

Figure 3. Augmentation of Kal-7/PSD95 protein binding by DISC1 in rat primary cortical neurons

(a) Increased Kal-7/PSD95 protein binding by overexpression of DISC1. Left, immunofluorescent cell staining indicating that majority of neurons are infected with Sindbis virus expressing DISC1-FL-HA or DISC1-ΔKal-7-HA. Middle and right, increased binding of Kal-7 and PSD95 by overexpression of DISC1-FL (asterisk), but not DISC1-ΔKal-7. Scale bar, 20 μm. (b) Decrease in Kal-7/PSD95 binding upon lentivirus-based DISC1 knockdown. *P < 0.05, #P < 0.001. Full-length blots are presented in Supplementary Fig. 17.

Figure 4. Protein interaction of DISC1/Kal-7/PSD95 influenced by activation of the NMDA-type glutamate receptor

(a) Decrease in interactions among DISC1, Kal-7, and PSD95 (asterisks) three min after electroconvulsive treatment (ECT) in rat brains in vivo. Full-length blots are presented in Supplementary Fig. 18. (b) Decrease in interactions of DISC1/Kal-7 and DISC1/PSD95 (asterisk) as well as activation of Rac1 (input, asterisk) after selective activation of the NMDA receptor by APV withdrawal (WD). *P < 0.05. Full-length blots are presented in Supplementary Fig. 18.

Figure 5. Regulation of Rac1 activity via “signalosome” of DISC1/Kal-7

(a) Inhibition of Rac1 activity (level of GTP-Rac1) by DISC1-FL (red asterisk), but not DISC1-ΔKal-7, and augmentation of the activity by DISC1 knockdown (green asterisk) in primary cortical neurons. Augmented Rac1 activity is also monitored by phosphorylation of Pak1. Full-length blots are presented in Supplementary Fig. 18. (b) Decreased Rac1/Kal-7 binding by DISC1-FL (red asterisk), not by DISC1-ΔKal-7. (c) Key role of Rac1 in DISC1-regulated spine enlargement, as evident by its blockade by expression of Rac1-DN (dominant-negative). Scale bar, 20 μm. Bar, s.e.m. Significant effects of Rac1-DN compared to mock are shown as *P < 0.05, †P < 0.01, #P < 0.001. Full-length blots are presented in Supplementary Fig. 18.

Figure 6. Long-term disturbance of DISC expression leads to spine shrinkage in rat primary cortical neurons

(a) Short- or long-term effect of Rac1 [WT (wild-type), CA (constitutively-active), or DN] on spine morphology in mature neurons. Bars, -fold changes of spine size by introduction with Rac1-CA or -DN relative to that with Rac1-WT. (b) Short- or long-term effect of DISC1 knockdown (in green) or overexpression (in red) in mature neurons. (c) Reduced spine size by knockdown of DISC1 for 6 days in primary cortical culture. (d) Decrease in mEPSC by long-term DISC1 knockdown. (e) Spine deterioration (decreased spine size) by long-term knockdown of DISC1 was normalized by overexpression of DISC1-FL, but not by that of DISC1-ΔKal-7. *P < 0.05, #P < 0.001. (f) Spine deterioration (decreased mEPSC frequency) by long-term knockdown of DISC1 was normalized by overexpression of DISC1-FL, but not by that of DISC1-ΔKal-7. Bar, s.e.m. *P < 0.05. Scale bar, 10 μm.

Figure 7. Long-term suppression of DISC1 leads to spine shrinkage in slices and brains in vivo

(a) Effect of long-term DISC1 knockdown on spine morphology in rat organotypic cortical culture. Representative image of culture with DISC1 RNAi (green). Scale bar, 5 mm. (b) Long-term effect of DISC1 knockdown on spine size in the medial prefrontal cortex. Bar, s.e.m. #P < 0.001.