Pathogenic disruption of DISC1-serine racemase binding elicits schizophrenia-like behavior via D-serine depletion

Abstract

Perturbation of Disrupted-In-Schizophrenia-1 (DISC1) and D-serine/NMDA receptor hypofunction have both been implicated in the pathophysiology of schizophrenia and other psychiatric disorders. In the present study, we demonstrate that these two pathways intersect with behavioral consequences. DISC1 binds to and stabilizes serine racemase (SR), the enzyme that generates D-serine, an endogenous co-agonist of the NMDA receptor. Mutant DISC1 fails to bind to SR, facilitating ubiquitination and degradation of SR and a decrease in D-serine production. To elucidate DISC1-SR interactions in vivo, we generated a mouse model of selective and inducible expression of mutant DISC1 in astrocytes, the main source of D-serine in the brain. Expression of mutant DISC1 downregulates endogenous DISC1 and decreases protein but not mRNA levels of SR, resulting in diminished production of D-serine. In contrast, mutant DISC1 does not alter levels of ALDH1L1, connexins, GLT-1 or binding partners of DISC1 and SR, LIS1 or PICK1. Adult male and female mice with lifelong expression of mutant DISC1 exhibit behavioral abnormalities consistent with hypofunction of NMDA neurotransmission. Specifically, mutant mice display greater responses to an NMDA antagonist, MK-801, in open field and pre-pulse inhibition of the acoustic startle tests and are significantly more sensitive to the ameliorative effects of D-serine. These findings support a model wherein mutant DISC1 leads to SR degradation via dominant negative effects, resulting in D-serine deficiency that diminishes NMDA neurotransmission thus linking DISC1 and NMDA pathophysiological mechanisms in mental illness.

Figure 1. SR interacts with DISC1 and co-localizes in primary astrocytes

[a] Co-immunoprecipitation of overexpressed DISC1 and SR in HEK-293 cells. Note that full-length DISC1 (100 kDa) binds to SR, but not the truncated mutant DISC1 (64 kDa). mSR, mouse SR; hSR, human SR. Cell lysates containing 500 ug of protein were used for co-immunoprecipitation. [b] Co-immunoprecipitation of DISC1 and SR in HT22 cells. [c] Endogenous DISC1 binds to SR in mouse primary astrocytes. Cell lysates containing 700 ug of protein were used for co-immunoprecipitation. [d] DISC1 immobilized to HA affinity beads binds to SR in mouse brain lysates. pCMV-HA is used as a control. [e] Subcellular fractionation of primary astrocytes indicates that DISC1 is present in both the nuclear (N) and cytosolic (C) fraction while SR is exclusively in the cytosolic (C) fraction. C, cytosol; M, membrane; N, nucleus; CK, cytoskeleton. [f] Immunofluorescent staining of DISC1 and SR in the mouse primary astrocytes, scale bar - 10 µm.

Figure 2. Mutant DISC1 depletes SR

[a] SR protein is depleted when co-expressed with mutant DISC1 in HEK-293, HT-22 and N2A cell lines compared to full-length DISC1. Quantification of mouse [b] and human SR protein [c] when co-expressed with different forms of DISC1 in HEK-293 cells. mSR, mouse SR; hSR, human SR. Data are representative of 4 independent experiments, *p<0.05, ***p<0.0005. [d] Mutant DISC1 decreases D-serine production. L-serine (10 mM) was added to the cell culture media of HEK-293 cells transfected with different constructs and 24 h later, D-serine in the media was measured by the spectrophotometric assay. *p<0.05, **p<0.005.

Figure 3. SR level is decreased in primary astrocytes and brain tissue from the GFAP-ΔC hDISC1 mice

[a] Both DISC1 and SR were expressed in astrocytes in 6-week-old mouse brain. DISC1 and SR immunofluorescence was carried out with glial marker GFAP in the mouse forebrain sections, scale bar - 20 µm; [b] ΔC-hDISC1 is expressed in the astrocytes of the mutant mouse but not in the neurons or in the control mouse; [c] Immunofluorescence showing that mutant DISC1 is expressed in astrocytes of the mouse hippocampus, scale bar - 10 µm; [d] Expression time course of ΔC-hDISC1 in the mutant mouse forebrain, with the highest expression level around E18. PC, positive control, brain tissue from a mouse that expresses mutant DISC1 under the CAMKII promoter; [e] Expression of SR, ΔC-hDISC1, endogenous mouse DISC1 and an astrocytic marker, ALDH1L1, in samples from primary astrocytes. Below, quantitative evaluation of expression of these markers, n=7–10 per group, *p<0.05, ** p<0.005 vs. controls, Student’s t-test. The exposure time of the ΔC-hDISC1 and endogenous mouse DISC1 blots is the same. [f] Expression of SR, ΔC-hDISC1, endogenous mouse DISC1 and an astrocytic marker, ALDH1L1, in samples from forebrain area of newborn mice. Below, quantitative evaluation of expression of these markers, n=7–9 per group, *p<0.05 vs. controls, Student’s t-test. [g] D-serine level in the culture media of primary astrocytes. L-serine was added to the culture media to a final concentration of 4 mM and D-serine level was measured 48 h later. Control, n=6; mutant, n=7, *** p<0.0005; [h] D-serine level (expressed as [D-serine]/[total serine]) in the brain of P0, P7, P21 and P90 mice determined by HPLC. Each bar represents ~4–6 mice. In the P0 mouse cortex, mutant has ~20% decrease in D-serine level compared to the control, while in the P21 mouse cortex and hippocampus, there is a small but significant increase, *p<0.05, ** p<0.005, NS, not significant. CTX - cortex. [i] SR mRNA level in primary astrocytes is unaltered as measured by quantitative Real Time-PCR. Control, n=7; mutant, n=6.

Figure 4. Mutant DISC1 depletes SR protein by increasing its ubiquitination level

[a] Cycloheximide chase assay. HEK-293 cells were treated with 70 mg/ml cycloheximide 24–36 h after transfection and SR level was quantified. Protein levels were expressed as percentage of the level at time 0. The half-life of SR is significantly shorter when co-expressed with mutant DISC1. [b,c] In vivo ubiquitination assay of mouse [b] and human SR [c] in HEK-293 cells. The levels of mouse and human SR ubiquitination are higher when co-expressed with mutant DISC1 as quantified in [d] and [e], respectively. Primary astrocytes [f] were treated with the proteasome inhibitor MG-132 (30 µM for 10 h), and relative SR protein levels are quantified below the blots. The value of SR level in primary astrocytes from control mice before treatment has been normalized to 1.0.

Figure 5. DISC1 mutant mice exhibit greater responses to an NMDA antagonist, MK-801, and D-serine treatment

[a] Locomotor activity in open field of male mice before and after treatment with MK-801 (0.3 mg/kg, i.p.) followed by saline or D-serine injections (2.7 g/kg, i.p.). Compared to control male mice, mutant male mice display significantly greater activity in response to MK-801. Compared to control male mice, mutant mice also exhibit significantly greater sensitivity to the ameliorative effects of D-serine. Numbers of mice in each group are indicated on the graph. [b] Locomotor activity in open field of female mice before and after treatments with MK-801 (0.3 mg/kg, i.p.) followed by saline or D-serine injections (2.7 g/kg, i.p.). Compared to control female mice, mutant female mice exhibit significantly higher locomotor activity in response to MK-801. Compared to control female mice, mutant female mice show significantly greater sensitivity to the ameliorative effects of D-serine. Numbers of mice in each group are indicated on the graph. [c] PPI of the acoustic startle of male mice before and after treatment with MK-801 (0.3 mg/kg, i.p.) followed by saline or D-serine injections (2.7 g/kg, i.p.). p4–20 are the intensities of pre-pulses above the background noise level (70dB). Compared to control male mice, mutant male mice display greater MK-801 induced impairment in PPI at P4 and P8. Compared to control male mice, mutant mice also exhibit significantly greater sensitivity to the ameliorative effects of D-serine at P4. Numbers of mice in each group are indicated on the graph. [d] PPI of the acoustic startle of female mice before and after treatment with MK-801 (0.3 mg/kg, i.p.) followed by saline or D-serine injections (2.7 g/kg, i.p.). Numbers of mice in each group are indicated on the graph.