Understanding cognitive deficits in neurodevelopmental disorders using a candidate

In Chapter 2, we validated Pcdh10 as a key molecule that regulates synaptic structure in the amygdala, and found that loss of one allele of Pchd10 led to deficits in social behavior and amygdala-dependent learning. Previously, genome-wide association studies of families with high incidence of ASD identified a novel copy number variation (CNV) affecting a region near multiple genes encoding members of the cadherin and protocadherin superfamilies, including Pcdh10 (Morrow et al. 2008). Because the CNV is more than 500kb downstream of the nearest protocadherin locus (Pcdh10) and did not include promoter or coding regions of the proposed candidate genes, it was unclear whether the deletion was likely to alter expression levels at these loci. Additionally,

Pcdh10 is a novel ASD candidate gene with limited genetic and molecular studies to

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pathophysiology of ASD, functional studies are needed to demonstrate a role for PCDH10 in endophenotypes associated with the disorder.

Because many neurodevelopmental disorders have complicated etiology likely involving multiple genetic and environmental factors, identification and validation of candidate genes and molecular pathways is critical to gain an understanding of central

mechanisms. Studies of PCDH10 function in the brain are limited, but functional studies link it to the Fragile X mental retardation protein FMRP (Tsai et al. 2012). Pcdh10 is a regulatory target of FMRP linked to regulation of post-synaptic density stability and synapse number, key neuronal processes disrupted in Fragile X disorder (FX) (Tsai et al. 2012). In Chapter 2, we used genetically modified mice to show that loss of a single copy of Pcdh10 resulted in cognitive deficits and social withdrawal behaviors, as well as alterations in structural and functional properties of synaptic connections that are

remarkably similar to phenotypes observed in mice lacking FMRP expression. These studies provide further evidence that Pcdh10 regulates structural and functional

properties of synapses, consistent with it being a functional target of FMRP that supports consolidation of emotional memory in the amygdala.

PCDH10 is a newly identified functional target of FMRP, and it is unknown whether it plays a role in the pathophysiology of FX. Knockdown experiments in primary cultured neurons from wild-type mice support a role for PCDH10 in dendritic spine elimination that is disrupted by aberrant translation elongation factor EF1α in Fmr1 KO mice, leading to increased spine density (Tsai et al. 2012). In Pcdh10+/- mice, increased spine density

and reduced gamma synchrony in amygdala neurons mirror structural and functional changes in Fmr1 KO cortical neurons, hinting at a common mechanism, yet important mechanistic differences are predicted in the two mouse lines. Fmr1 KO mice exhibit

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increased mGluR5 activity and overactive translation machinery- key functional changes proposed to underlie changes in spine density and synaptic plasticity (Tsai et al. 2012, Iliff et al. 2012). In contrast, PCDH10 has yet to be linked to either translational

regulation or mGluR5 activity. We could start to address these possibilities by

measuring protein translation rates in Pcdh10+/- tissue using a protein synthesis reporter

system such as puromycin labeling. Additionally, western blot experiments measuring activation of signaling molecules downstream of type I mGluRs such as PLC (see Fig.

3.7) would indicate whether activity of mGluR5 was altered in Pcdh10+/- tissue. Further

dissection of synaptic mechanisms underlying synaptic phenotypes in Pcdh10+/- and

Fmr1 KO neurons is a critical next step in understanding molecular pathways underlying

changes in spine density in Fmr1 KO mice as well as the role of PCDH10 in spine elimination.

In Chapter 2, we also showed that social deficits in juvenile Pcdh10+/- males could be

rescued by treatment with the NMDAR partial agonist d-cycloserine (dCS).

Experimental reduction of NMDAR subunit NR1 levels yields deficits in social approach and fear learning, as well as impaired gamma synchrony in mice (Dzirasa et al. 2009, Halene et al., 2009). This array of phenotypes is highly similar to behavioral and electrophysiological changes in Pcdh10+/- mice, suggesting that reduced expression of

NR1 could underlie the social deficts and therapeutic response we observed. To test this hypothesis, we are currently measuring synaptic levels of NMDAR subunits in

subcellular fractions isolated from amygdala tissue of Pcdh10+/- mice. Preliminary

results suggest that levels of NR1 protein are reduced in the post-synaptic density of Pcdh10+/- mice (A. Bannerjee, unpublished results).

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PCDH10 facilitates degradation of PSD95, a synaptic structural protein involved in clustering NMDARs and AMPARs at the PSD (Tsai et al. 2012, Yan et al. 2014);

however, the reduction in NR1 protein levels suggested by our preliminary studies is not entirely consistent with the current model of PCDH10 function. In the Huber model (Tsai et al. 2012), knockdown of PCDH10 levels in cultured primary cortical neurons leads to stabilization of PSD95 and spine preservation. Increased synaptic PSD95 is associated with increased spine density and increased AMPAR-mediated synaptic transmission (El- Husseini et al. 2000, Beique and Andrade 2002). In our mouse, reduced PCDH10 is associated with increased spine density but the dCS rescue and NR1 protein levels suggest a mechanism involving NDMAR hypofunction. We did not observe a change in amplitude of field responses in the BLA (Fig. 2.10C); suggesting that reduced NMDAR function may be accompanied by increased activation of other receptor types, including AMPARs. To unravel this mechanism, more specific dissection of NMDAR/AMPAR function is needed. Whole cell patch clamp recordings designed to distinguish NMDAR activity from AMPAR activity will provide a critical functional readout of the relative activity of these receptor types, and indicate whether one or both currents are altered in Pcdh10+/- neurons. Additionally, Western blots for synaptic and total levels of PSD95 in

Pcdh10+/- amygdala tissue will indicate whether reduction of PCDH10 in our mouse

affects levels of PSD95. Because the current model of PCDH10 function is based off of experiments conducted in cultured cortical neurons harvested from neonatal mice, important molecular differences between tissues and developmental stages explain the different patterns of electrophysiological and protein changes we observe in 30d old mice. In rodents, expression of NMDAR subunits and functional properties of the resulting channels are highly dynamic during the first post-natal week (Monyer et al. 1994). Certain experiments in our study were conducted in juvenile mice (social testing,

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protein levels, electrophysiology), while other experiments were conducted in adult animals (cognitive tasks, spine counts). Additional studies of PSD composition, dendritic spine morphology, and electrophysiological properties of Pcdh10+/- neurons from mice of

different ages will provide important insight into the function of PCDH10 and its regulation of structural and functional properties of synapses across development.

A major theme from Chapter 2 is that exploration of molecular pathways containing existing ASD-associated proteins is not only critical for understanding pathological processes, but is also a strategy for discovering novel candidates. In a molecular pathway that is a critical mechanism underlying ASD phenotypes, major regulatory and enzymatic factors should be investigated as potential risk genes by merit of their

essential role in the pathway. In the FMRP-PCDH10 pathway, modification of PSD95 by the ubiquitin ligase murine double minute 2 (MDM2) is a critical step upstream of its degradation. An oncogene best studied for its role in promoting degradation of the tumor suppressor p53, MDM2 is only beginning to be explored in the context of neuronal function and cognition (Tsai et al. 2012, Richmany et al. 2013). Existing

pharmacological tools for inhibiting MDM2-mediated degradation of p53 could potentially be used to experimentally stabilize PSD95 (Rew et al. 2012). Future studies of MDM2 and its role in synaptic structure and memory formation will provide insight into this novel mechanism for regulating a major post-synaptic structural protein.

5.2 Gene silencing is a critical regulatory mechanism during memory formation.