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. 2014 Mar-Jun;28(1-2):98-111.
doi: 10.3109/01677063.2014.892486. Epub 2014 Apr 22.

Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95

Jingping Zhang  1 , Taixiang Saur, Angela N Duke, Seth G N Grant, Donna M Platt, James K Rowlett, Ole Isacson, Wei-Dong Yao
Affiliations

Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95

Jingping Zhang et al. J Neurogenet. 2014 Mar-Jun.

Abstract

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.

Keywords: Huntington; dopamine; glutamate; striatum.

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Figures

Figure 1
Figure 1
Reduced locomotor activity and impaired motor coordination in PSD-95 deficient mice. (a) Time courses of horizontal activity per 5 min of WT, PSD-95+/-, and PSD-95-/- mice when placed in a novel environment, an open field activity chamber. (b) Summary of total horizontal and vertical activity of WT, PSD-95+/- and PSD-95-/- mice in response to a novel environment. **, p < 0.01, one-way ANOVA with post-hoc Dunnett's test vs. WT. (c) Total horizontal activity of WT, PSD-95+/-, and PSD-95-/-mice during a 5-day test period. Mice were placed in the same locomotor activity monitor on test days for 60 min. Horizontal and vertical activity was measured as distance traveled in blocks of 5 min. *, p < 0.05, one-way ANOVA with post-hoc Dunnett's test vs. corresponding WT controls. (d) Summary of beam walking data. Inset, a WT mouse performing the beam walking test. *, p < 0.05 versus WT, Student's t test. Numbers in parentheses designate numbers of mice used.
Figure 2
Figure 2
Progressive limb-clasping symptoms in PSD-95 deficient mice. (a) Examples of hind-limb, all-limb and full body clasping in PSD-95-/- mice compared to a WT control. (b) Clasping duration in a 15-s interval. **, p < 0.01; ***, p≪0.001, Student's t-tests. A clasping event is defined by the retraction of limbs into the body and toward the midline. (c) Developmental progression of clasping phenotype. **, p < 0.01; ***, p≪0.001, one-way ANOVA followed by post hoc Newman–Keuls tests. Mice were observed during a 15 s tail suspension test to monitor the progression of the clasping phenotype between 4 and 20 weeks of age. Numbers in parentheses designate numbers of mice used.
Figure 3
Figure 3
Loss of striatal MSNs in aged symptomatic PSD-95 deficient mice. (a) Sectioning scheme for immunohistochemistry and stereological analysis. Serial coronal sections (240 μm apart) from Tubes #2 and #8 were used in DARPP-32 staining and subsequent cell counting. (b, c) DARPP-32 staining of a WT and a PSD-95-/- mouse striatal section, viewed at 2.5× (b) and 40× (c). Arrows indicate ventricles. (d-f) Summary of total DARPP-32-positive cells (d), cell density (e), and striatal volume (f) in 3-month- and >12-month-old WT and PSD-95-/- symptomatic mice. Cell counting was made at 40×, using Stereo Investigator 7. n = 8 mice each group, *, p < 0.05; **, p < 0.01 vs. WT, two-way ANOVA with Bonferroni post-hoc tests.
Figure 4
Figure 4
Increased striatal NMDA/AMPA ratio in PSD-95-ΔGK mice. (a) Representative EPSCs recorded from WT and PSD-95-/- MSNs at holding potentials of -60 (to record AMPA-EPSCs) and +40 mV (to record both AMPA-EPSCs and NMDAR-EPSCs). (b) Summary of mean NMDA/AMPA ratios, defined as the amplitude of the NMDAR component 80 ms after stimulation at -40 mV divided by the peak AMPAR component at -60 mV. Numbers in parentheses indicate numbers of cells analyzed. *, p < 0.05 vs. WT; Student's t tests.
Figure 5
Figure 5
Increased striatal NMDAR activity in PSD-95-ΔGK mice. (a) Representative AMPAR-mediated mEPSCs recorded from WT and mutant MSNs. (b,c) Summaries of mean AMPA-mEPSC amplitudes (b) and frequencies (c). (d) Representative mEPSCs in the absence (to record total mEPSCs mediated by both NMDA and AMPA receptors) followed by the presence (to record AMPA-mEPSCs) of Mg2+. (e) Summary of charge transfer mediated through NMDA-mEPSCs. The NMDAR component of mEPSCs was derived by subtracting the average AMPA-mEPSC from the average total mEPSC, and the area under the resultant NMDA-mEPSC was measured. Numbers in parentheses indicate numbers of cells analyzed. *, p < 0.05 vs. WT, Student's t tests.
Figure 6
Figure 6
Enhanced SKF81297 modulation of synaptic NMDAR currents in PSD-95-ΔGK mice. (a) Representative NMDA-EPSCs recorded from WT and mutant PFC pyramidal neurons before and (30-40 min) after bath application of SKF81297 (1 μM). (b) Summary of SKF81297 effect on NMDA-EPSCs in WT and PSD-95-/- mice. NMDA-EPSCs were pharmacologically isolated in Mg2+-free ACSF containing CNQX (20 μM), glycine (1 μM), and picrotoxin (100 μM). Neurons were voltage clamped at −60 mV. Following a 10-min baseline recording, a brief (5-10 min) application of SKF81297 elicited a long-lasting potentiation of NMDA-EPSCs in WT neurons, which was further enhanced in PSD-95-ΔGK neurons. Numbers in parentheses indicate numbers of cells analyzed.
Figure 7
Figure 7
Exacerbating effect of cocaine exposure on limb-clasping behavior. (a) Repeated cocaine administration (7 days, 20 mg/kg, i.p.) induced limb clasping in asymptomatic PSD-95-/- mice, but not in WT mice. The same WT and mutant mice before and after cocaine treatments are shown. (b) Development and recovery time course of cocaine-elicited limb clasping behavior. Clasping score designates clasping duration in a 15-s interval. Numbers in parentheses indicate numbers of mice analyzed. Age = 7 months.
Figure 8
Figure 8
Proposed model by which PSD-95 may regulate neuronal integrity by regulating NMDAR function, DA D1 signaling, and DA-glutamate interaction at synapses. In dendritic spines PSD-95 regulates D1 trafficking and inhibits D1 signaling, and dampens the positive coupling between D1 and NMDAR. Deletion of PSD-95 in mutant mice produces enhanced D1 signaling, measured as increased cAMP production, which in turn further enhances NMDAR activity. The concomitant overactivation of both D1 and NMDAR make the neurons more susceptible to NMDAR-mediated excitotoxicity, causing cell death and the neurological phenotypes observed in mutant mice.
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