Impaired NMDA receptor transmission alters striatal synapses and DISC1 protein in an age-dependent manner - PubMed (original) (raw)

Impaired NMDA receptor transmission alters striatal synapses and DISC1 protein in an age-dependent manner

Amy J Ramsey et al. Proc Natl Acad Sci U S A. 2011.

Abstract

NMDA receptors are key regulators of synaptic plasticity, and their hypofunction is thought to contribute to the pathophysiology of CNS disorders. Furthermore, NMDA receptors participate in the formation, maintenance, and elimination of synapses. The consequences of NMDA receptor hypofunction on synapse biology were explored in a genetic mouse model, in which the levels of NMDA receptors are reduced to 10% of normal levels (i.e., NR1-knockdown mice). In these mice, synapse number is reduced in an age-dependent manner; reductions are observed at the postpubertal age of 6 wk, but normal at 2 wk of age. Efforts to uncover the biochemical underpinnings of this phenomenon reveal synapse-specific reductions in 14-3-3ε protein and in Disrupted in Schizophrenia-1 (DISC1), two schizophrenia susceptibility factors that have been implicated in the regulation of spine density. Subchronic administration of MK-801, an NMDA receptor antagonist, produces similar synaptic reductions in both spine density and DISC1, indicating that synaptic levels of DISC1 are regulated by NMDA receptor function. The synaptic reduction of DISC1 and 14-3-3ε is developmentally correlated with the age-dependent decrease in striatal spine density.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

NR1-KD mice show an age-dependent reduction in spine density of MSNs. Representative photomicrographs of dendritic shaft and spines taken from WT or NR1-KD mice at P14 (A and B) and P40 (C and D). Spine images were taken from acute slices by using patch-clamp cell labeling with Alexa Fluor 594 and two-photon laser scanning microscopy. (E) Quantification of spine density: combined counts of mushroom, thin, and stubby spines per 100 μm of dendrite (n = 6 neurons from three animals for each genotype; P > 0.05, two-tailed t test). (F) Quantification of spine density: combined counts of mushroom, thin, and stubby spines per 100 μm of dendrite (n = 8 neurons from three animals for each genotype; *P < 0.05, two-tailed t test). (G) Quantification of spine density in F for each of the three classes of spine morphology: mushroom shaped (Mush), stubby (Stub), and thin.

Fig. 2.

Fig. 2.

NR1-KD mice have a synapse-specific depletion of 14–3-3ε and DISC1 proteins. (A) Western blot of total striatal extracts (25 μg) and (B) striatal synaptic plasma membrane fractions (15 μg protein). Monoclonal antibody to 14–3-3ε and polyclonal antibody to DISC1 (Invitrogen antibody, I) detects a 29-kDa isoform of 14–3-3ε and 100-kDa and 70-kDa isoforms of DISC1. (C) 14–3-3ε and DISC1 levels normalized to GAPDH demonstrate a synapse-specific decrease in 14–3-3ε, and for DISC1 a modest decrease in total extract, but substantial depletion of DISC1 in synaptic extracts (n = 6 for each genotype; *P < 0.05, two-tailed t test). (D) Representative photomicrographs of DISC1 labeling by postembedding immunogold transmission EM. DISC1 immunoreactivity is detected in pre- and postsynaptic membranes of asymmetric synapses of the striatum (circled synapse with arrows), and in mitochondria (uncircled arrows). (E) Quantification of DISC1 immunogold labeling on asymmetric synapses or within mitochondria (n = 3 animals for each genotype; *P < 0.05, two-tailed t test). (F) Western blot of total (25 μg) and synaptic (15 μg) striatal protein extracts to detect relative levels of PDE4B, LIS1, and NDEL1 from WT and NR1KD mice. (G) Levels normalized to GAPDH indicate no change in PDE4B or NDEL1, a modest decrease in total LIS1 levels, and an increase in synaptic LIS1 (n = 6 for each genotype; *P < 0.05, two-tailed t test).

Fig. 3.

Fig. 3.

Subchronic treatment with MK-801 reduces synapse number and DISC1 protein in adult striatum. (A) Representative photomicrographs of dendritic shaft and spines from MNs of saline solution- and MK801-treated mice (0.2 mg/kg/h, 7 d). (Scale bar: 10 μm.) (B) Quantification of spine density per 100 μm of dendrite from mice treated with saline solution or 0.1 or 0.2 mg/kg/h MK801 (n = 6 neurons from three animals for each treatment; P = 0.051 two-tailed t test). (C) Total striatal protein extracts (25 μg) and synaptic plasma membrane extracts (15 μg) taken from mice treated with saline solution or a 14-d infusion of MK-801 delivered by osmotic minipump (0.2 mg/kg/h); Western blot detects 14–3-3ε and DISC1 (Santa Cruz, S). (D) Relative levels of DISC1 and 14–3-3ε normalized to GAPDH levels (n = 6 for each treatment group; *P < 0.05 and **P < 0.01, two-tailed t test).

Fig. 4.

Fig. 4.

Synaptic decreases in DISC1 are less substantial in juvenile NR1-KD mice. (A) Western blot of total protein extracts (25 μg) from WT and NR1-KD mice at P14, blotted for NR1 C-terminus, DISC1 (Santa Cruz, S), and 14–3-3ε. (B) Western blot of synaptic plasma membrane striatal extracts (15 μg) from the same experimental groups. (C) Relative levels of proteins normalized to GAPDH levels (n = 6 for each genotype; *P < 0.05, two-tailed t test).

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