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The Full-length version of this article is also available, published online February 9, 2005 as doi:10.1096/fj.04-1854fje.
Published as doi: 10.1096/fj.04-1854fje.
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(The FASEB Journal. 2005;19:583-585.)
© 2005 FASEB

Subcellular redistribution of the synapse-associated proteins PSD-95 and SAP97 in animal models of Parkinson’s disease and L-DOPA-induced dyskinesia

J. E. Nash*,{dagger},{ddagger},1, T. H. Johnston*, G. L. Collingridge{ddagger}, C. C. Garner{ddagger} and J. M. Brotchie*

* Toronto Western Research Institute, Toronto Western Hospital, Toronto, Ontario, Canada;
{dagger} Department of Psychiatry and Behavioural Sciences, Stanford University, Palo Alto, California, USA; and
{ddagger} MRC Centre for Synaptic Plasticity, Department of Anatomy, School of Medical Sciences, University of Bristol, Bristol, UK

1Correspondence: Toronto Western Research Institute, Toronto Western Hospital, 399 Bathurst St., MC 11-419, Toronto, ON, M5T 2S8, Canada. E-mail:jnash{at}uhnres.utoronto.ca

SPECIFIC AIMS

Abnormalities in neurotransmission at many receptors within the striatum have been identified as contributing factors in Parkinson’s disease (PD) and the major side effect in its treatment, L-DOPA-induced dyskinesia (LID). We hypothesized that processes fundamental to the regulation of synaptic transmission may underlie these disorders.

Synaptic proteins, including receptors, are trafficked, anchored, and clustered by PDZ domain-containing proteins, two of the most well characterized being PSD-95 and SAP97. To determine whether SAP97 and PSD-95 are dysfunctional and to raise the possibility they may be responsible for changes in synaptic plasticity in PD and LID, we assessed the expression and subcellular distribution of PSD-95 and SAP97 in the striatum in rat models of PD and LID.

PRINCIPAL FINDINGS

1. In parkinsonism, total and synaptic levels of striatal PSD-95 and SAP97 are decreased whereas vesicular levels are increased
We performed SDS-PAGE electrophoresis, followed by Western blot analysis using antibodies against SAP97 and PSD-95 on tissue prepared from the striatum of the 6-hyroxydopamine (6-OHDA) -lesioned rat model of PD and compared it to levels in the unoperated side of sham-operated rats by densitometric analysis. Striatal dopamine depletion > 95% was demonstrated by HPLC for dopamine, DOPAC, and HVA and by levels of tyrosine hydroxylase. In total striatal membranes, levels of PSD-95 and SAP97 in the dopamine-depleted striatum of 6-OHDA-lesioned rats were decreased by 25.6 ± 9.9% (P<0.05) and 19.0 ± 5.0% (P<0.001), respectively, of vehicle-treated, sham-operated levels Fig. 1 ). To ensure that equal volumes of protein had been loaded onto the gel in each lane, levels of the endogenous housekeeping proteins ß-tubulin and glutathione transferase (GST) were measured. There was no significant difference in tubulin or GST levels between the treatment groups (Fig. 1) .



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  		Figure 1. Total and subcellular distribution of SAP97 and PSD-95 levels in striatum in sham-operated and 6-OHDA-lesioned rats after L-DOPA or vehicle treatment. A, C, E)Western blots of striatal membranes (i.e., P100 pellet from hypotonically lysed striatal homogenates) probed with antibodies against SAP97, PSD-95, tyrosine hydroxylase (TH), ß-tubulin (ß-tub), or GST. A) Total striatal levels; C) heavy membrane fractions (layers 6 and 7 containing synaptic membranes); E) light membrane fractions (layers 4 and 5 of the flotation assay). B, D, F)Mean optical density values of the blots (n=7). Mean optical density value in sham-operated animals after vehicle treatment on the operated side was taken at 100% (control) and compared with other treatment groups. Comparison with *sham-operated, vehicle-treated, same side; asame treatment group, other side; c6-OHDA-lesioned, L-DOPA treated, same side. One symbol: P< 0.05; two symbols: P< 0.01; three symbols: P< 0.001.

To determine the subcellular location of the remaining SAP97 and PSD-95, we used a flotation assay. Individual striata were hypotonically lysed and centrifuged to yield the supernatant (S100) and pellet (P100) fraction. The P100 fraction (which contains all membrane-associated proteins but no cytosolic components) was adjusted to 2M sucrose and loaded as a layer of a discontinuous sucrose gradient underneath layers of 1.2, 0.8, and 0.3M sucrose. After centrifugation fractions were assayed for levels of SAP97 and PSD-95 by SDS-PAGE followed by Western blot analysis. Proteins that float between the 1.2 and 0.8M gradient contain predominantly proteins associated with vesicles; proteins that remain at the bottom of the tube, heavy membrane fractions, contain predominantly synaptic membranes. When the P100 fraction was treated with the membrane permeabilizing agent, Triton X100, SAP97, and PSD-95 were found only at the bottom of the gradient, indicating that the ability of these proteins to float and be present on vesicles is dependent on membrane integrity.

There was a redistribution PSD-95 and SAP97 from the synaptic membrane to the vesicular component in 6-OHDA-lesioned rats vs. control. SAP97 and PSD-95 levels in the synaptic membrane (heavy membrane) fraction showed a pattern of change similar to that found in crude striatal membranes. In the 6-OHDA-lesioned striatum, in the synaptic membrane-containing fraction, PSD-95 and SAP97 levels were reduced to 20.3 ± 14.2% and 17.0 ± 5.0% of vehicle-treated, sham-operated levels on the same side (P<0.05 and P<0.001 for PSD-95 and SAP97, respectively). In light flotation fractions there was a significant increase in SAP97 and PSD-95 levels in 6-OHDA-lesioned rats compared with control. On the operated side of vehicle-treated 6-OHDA-lesioned rats, PSD-95 and SAP97 were increased by 37.4 ± 11.3% and 33.6 ± 6.7%, respectively, compared with vehicle-treated, sham-operated rats (P<0.01 for PSD-95, P>0.05 for SAP97).

To determine whether changes in the rate of protein synthesis of PSD-95 and SAP97 were responsible for changes in striatal levels of these proteins, in situ hybridization was used with 33P-labeled SAP97 and PSD-95 mRNA probes in coronal sections of rat brain at striatal levels in 6-OHDA-lesioned rats, followed by densitometric analysis of autoradiographs. In unilaterally 6-OHDA-lesioned animals (lesion confirmed by HPLC), PSD-95 mRNA levels were significantly decreased on the operated side in vehicle-treated 6-OHDA lesioned rats compared with vehicle-treated sham-operated animals (90±2.9% of vehicle-treated, sham-operated animals, P<0.01). There was no significant difference between SAP97 mRNA levels on the operated side in vehicle-treated 6-OHDA lesioned rats vs. vehicle-treated sham-operated animals.

Thus, in a well-validated animal model of PD, striatal levels of PSD-95 and SAP97 are decreased. The remaining protein undergoes redistribution such that it is preferentially found in vesicular rather than synaptic membrane fractions.

2. In L-DOPA-induced dyskinesia, total and synaptic levels of striatal PSD-95 and SAP97 are increased whereas vesicular levels are decreased
After 21 days of L-DOPA-methyl ester (6.5 mg/kg i.p. bid) administration, 6-OHDA-lesioned animals exhibited a marked rotational response contraversive to the 6-OHDA-induced lesion (758±114 rotations/2 h) whereas vehicle-treated 6-OHDA-lesioned rats showed mild ipsiversive rotations (–15.3±2.5 rotations/2 h) (P<0.001). In the L-DOPA-treated group, the rotational response on day 21 was significantly higher than that on days 1, 7, and 14. On no day was response to vehicle significantly higher than that of vehicle treatment on day 1. This rotational behavior represents a rodent correlate of LID.

Total striatal membrane levels of PSD-95 and SAP97 on the operated side of L-DOPA-treated 6-OHDA-lesioned rats were significantly increased over the same side of vehicle-treated, sham-operated rats (to 367.4±43.2% and 159.9±7.0% of vehicle-treated sham-operated rats, respectively, P<0.001 for both) (Fig. 1) .

In the synaptic membrane enriched fraction, PSD-95 and SAP7 levels in L-DOPA-treated 6-OHDA-lesioned rats were significantly higher than vehicle-treated, sham-operated levels, being 239.3 ± 6.3% and 155.9 ± 7.0%, respectively (P<0.001 for both).

Although total striatal levels of both proteins were elevated after L-DOPA treatment in 6-OHDA-lesioned rats, there was a dramatic decrease in both proteins in vesicular fractions of the same animals (to 7.0±5.0% and 13.9±6.3% for PSD-95 and SAP97 of vehicle treated sham-operated levels, respectively, P<0.001 for both).

After long-term administration of L-DOPA, striatal PSD-95 mRNA was significantly higher on the operated side of 6-OHDA-lesioned animals than on the operated side of vehicle-treated sham-operated animals, 114.8 ± 2.8% (P<0.001) and vehicle-treated 6-OHDA-lesioned animals by 126.3 ± 3.0% (P<0.001). There was no significant difference in striatal PSD-95 expression on the unoperated side of L-DOPA-treated, sham-operated rats vs. the same side in 6-OHDA-lesioned animals.

After L-DOPA treatment, striatal SAP97 expression was significantly higher on the operated side of 6-OHDA-lesioned animals than on the operated side of vehicle-treated sham-operated animals, 136 ± 3.1% (P<0.001), and vehicle-treated 6-OHDA-lesioned animals, 117.1 ± 4.4% (P<0.05).

CONCLUSIONS AND SIGNIFICANCE

We show that in a rat model of Parkinson’s disease, total levels of SAP97 and PSD-95 are dramatically decreased and are increased above normal levels in a rodent model of dyskinesia. In parkinsonism, there is a relative redistribution of both proteins from the synapse to vesicles. The reverse is true in LID.

SAP97 and PSD-95 are crucial for the formation of macromolecular assemblies within the synapse and in vesicular compartments. By multiple protein-protein binding domains, they link receptors with proteins regulating targeting, trafficking, and downstream signaling; they are key players in the mechanisms of synaptic plasticity (Fig. 2 ). These findings support the notion that redistribution of SAP97 and PSD-95 may be responsible for the multitude of changes in receptor-mediated signaling documented in parkinsonism and LID. Since alterations in trafficking and modulation of receptor function underlie synaptic plasticity, alteration of PSD-95 and SAP97 at the synaptic membrane or in vesicles may explain the abnormalities in synaptic plasticity observed in animal models of PD and LID. Current treatments for PD and LID are not optimal. Novel therapeutic approaches typically target only one abnormal receptor system. Manipulation of abnormalities in SAP function may focus on the root of the problems underlying these disorders and allow restoration of function of several receptor systems, which may prove more beneficial in treating neurological disorders than present therapies.



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  		Figure 2. Functional implications of subcellular redistribution of PSD-95 and SAP97 in PD and LID. A) PSD-95 and SAP97 play critical roles in coordinating macromolecular assemblies at 1) the synaptic membrane, where they regulate interactions between receptors and signaling molecules and control the stability of localization of receptors at the synapse by regulating insertion/exocytosis and internalization/endocytosis, and 2) in vesicular compartments, where they regulate the transport of receptors between endoplasmic reticulum, Golgi, and the synaptic membrane. The complexity of their interactions might define which proteins are delivered with that receptor, and thus with which proteins it might form important interactions at the synapse. B) In parkinsonism, reduction of synaptic SAP97 and PSD-95 may lead to inappropriate combinations of receptors and other proteins, perhaps leading to enhanced NMDA receptor activity in parkinsonism by virtue of abnormal phosphorylation (due to inappropriate association with kinases and phosphatases), abnormal cellular localization of receptors (ref), or abnormal association with other signaling molecules (e.g., adenosine A2a receptors). C) In LID, reduction in vesicular PSD-95 and SAP97 may lead to abnormalities in delivery and trafficking of receptors. Inappropriate association of receptors with trafficking molecules may lead to abnormal delivery or delivery with inappropriate partners. Such processes may lead to changes in signaling in multiple transmitter systems and to abnormal interactions between such systems and their downstream signaling molecules.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1854fje;




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