Cortico-striatal Synaptic Defects And OCD-like Behaviors In SAPAP3 Mutant Mice

To reveal the molecular mechanism of synaptic function is critical for better understanding of how information is communicated between cells in the nervous system. The molecular basis of post-synaptic assembly and function is not well understood, although as many as several hundreds proteins have been identified in the post-synaptic density (PSD), few of these proteins have known functions. A current prevalent model regarding postsynaptic development is that a group of key scaffolding proteins orchestrate the assembly of macromolecular complex at the postsynaptic membrane by providing multiple protein docking sites as well as connections to the actin cytoskeleton. The goal of this dissertation was to investigate the in vivo physiological functions of the SAPAPs (SAP90/PSD95 associated protein), one family of PSD proteins, as one step toward defining a molecular understanding of post-synaptic assembly and functions. We applied combined methods of genetics, biochemistry, electrophysiology and behavior to determine the SAPAP3 in synaptic development and function, and its implication in Obsessive-compulsive disorder (OCD)-like behavior.To examine in vivo the functions of SAPAPs, a mouse null for the SAPAP3 protein was successfully generated which was confirmed by tail PCR and southern blotting and western blotting. The SAPAP3 Knock-Out (SAPAP3 KO) mice initially appeared grossly normal and were fertile. However, at 4-8 monthes of age, SAPAP3 KO mice developed facial lesions with 100% penetrance. These lesions were likely self-inflicted and may indicate a behavior defect. By decoding the behaviors recorded by continuously videotaped for a 24-hour period in home cage, SAPAP3 KO mice showed dramatically increased grooming bouts and durations compared to wild type littermates. Importantly, the post-lesion mutant mice (with facial lesions) and pre-lesion ones (not yet to develop lesions) showed a similar level of increased grooming behavior. These data demonstrate that SAPAP3 KO mice have pathological levels of grooming behavior. To test whether SAPAP3 KO mice also increased anxiety, we performed the open field test, elevated zero maze test, and dark to light emergence test. In all these three anxiety behavior tests, SAPAP3 KO mice manifested excess anxiety levels.Both the over-grooming behavior and anxiety behaviors are reminiscent of the symptoms of human OCD spectrum disorders. Since Selective serotonin reuptake inhibitors (SSRI) are widely used to treat OCD patients and are effective to varying degrees in about 50% of OCD patients, we had tried to treat SAPAP3 KO mice with fluoxetine for 6 days. And we found fluoxetine significantly reduced both the excessive grooming and anxiety-like behaviors in SAPAP3 KO mice.Obsessive-compulsive disorder (OCD) is an anxiety disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striatal-thalamo-cortical circuitry is implicated in OCD, though the underlying pathogenic mechanisms are largely unknown. The fact that SAPAP3 is the only member of SAPAP3 family highly expressed in the striatum and SAPAP3 KO mouse is the only mutant mouse showing OCD-like behavior strongly suggests that there might be striatal synaptic defects in SAPAP3 KO mice. So we performed field recordings from acute striatal brain slices. We found the maximal total field responses were reduced but the NMDA-mediated field responses were elevated in the SAPAP3 KO mice. These findings implicate a role for SAPAP3 in determining synaptic activity of both AMPA- and NMDA-type glutamate receptors, but in opposite directions. Further we investigated the postsynaptic protein assembly in SAPAP3 KO mice by using biochemistry methods. Data showed the protein level of NR1 from the PSD fractions was significantly increased in the striatums of KO mice. Interestingly, we also found that the level of NR2B in the PSD, the "juvenile" subunit, was significantly increased whereas NR2A, the "adult" subunit, was significantly reduced. These data suggest that this developmental switch either does not happen or may not be maintained in SAPAP3 KO mice. Combined with electrophysiology data, these results raised an intriguing possibility that SAPAP3 plays an important role in the functional maturation of cortica-striatal synapses.Our findings of altered cortico-striatal synaptic function and structure in SAPAP3 mutant mice in conjunction with the pre-existing evidence for the role of striatal circuitry in the pathogenesis of OC-spectrum disorders led us to ask whether loss of SAPAP3 in the striatam was critical to the behavioral phenotypes of SAPAP3 KO mice. To address this question, we investigated whether selective expression of SAPAP3 in the striatum was sufficient to rescue the defects of phenotypes of SAPAP3 KO mice. We injected bilaterally lentiviral vectors which express GFP-SAPAP3 fusion protein into the striatum of SAPAP3 mice at multiple locations. At 4-6 months after injection, behavioral analyses were conducted to assess grooming, facial lesions, and anxiety-like behaviors. By continuous videotaping, we found excessive grooming was markedly reduced in SAPAP3 KO mice injected with GFP-SAPAP3 lentivirus. Facial lesions were also greatly alleviated in these mice. In addition, SAPAP3 KO mice injected with GFP-SAPAP3 lentivirus also showed reduced anxiety-like behavior in the dark-light emergence test. Taken together, these data show that selective expression of GFP-SAPAP3 in the striatum is sufficient to alleviate the manifestations of the OCD-like behavior in SAPAP3 KO mice. We then investigated the cortico-striatal field excitatory postsynaptic potentials (fEPSP) recordings of acute brain slices from GFP-SAPAP3 lentivirus or GFP lentivirus (as control) injected SAPAP3 KO mice, and found striatal fEPSP defects were completely rescued in GFP-SAPAP3 lentivirus-injected SAPAP3-/- mice, These data further support the idea that SAPAP3 plays a critical role at cortico-striatal synapses and indicate the importance of cortico-striatal circuitry in OCD-like behaviors, and demonstrated the determinative role of SAPAP3 in the pathogenesis of OCD-like behavior of SAPAP3 mutant mice.Since our data strongly suggest the dysfunction of cortico-striatal-thalamic -cortical circuitry mediated by SAPAP3 may play a key role in the pathogenesis of OCD-like behavior, we tried to directly test this hypothesis by generating Bac transgenic mice in which the expression of SAPAP3 is inducible by Cre recombinase. When combined with spatially restricted or temporally controllable Cre-expressing mouse lines, these SAPAP3 transgenic mice will allow us to determine the neural circuits and developmental stages involved in behavioral in SAPAP3 KO mice. First, we generated 6 founder lines of SAPAP3 transgenic mice in which LoxP-flanked STOP cassette was placed in front of the SAPAP3 cDNA, also an HA tag was put to the N-terminal of the SAPAP3 protein to facilitate detection of the transgenic SAPAP3 gene product. By crossing these 6 SAPAP3 BAC transgenic mice (named HA-SAPAP3^STOP mice) to a line of germline Cre mouse, we were able to detect the expression of the HA-SAPAP3 transgene by anti-HA staining. We found that the expression pattern of HA-SAPAP3 transgene is very comparable with that of endogenous SAPSP3. So these HA-SAPAP3^STOP mice would be very useful tools for further research.In conclusions, our findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and illustrate the importance of cortico-striatal circuitry in OCD-like behaviors.