Schizophrenia Risk Proteins ZNF804A and NT5C2 Interact at Synapses

The zinc finger protein 804A (ZNF804A) and the 5′-nucleotidase cytosolic II (NT5C2) genes have been identified as robust susceptibility genes in large-scale genome-wide association studies of schizophrenia. The ZNF804A and NT5C2 proteins are highly expressed in developing and mature cortical neurons. ZNF804A has been implicated in regulating the development of neuronal morphology; it localises to synapses and is required for activity-dependent modifications of dendritic spines. NT5C2 has been shown to regulate 5′ adenosine monophosphate-activated protein kinase activity and implicated in influencing protein synthesis in neural progenitor cells. But despite these findings, a better understanding of the role these proteins play in regulating neuronal function is needed. A recent yeast two-hybrid screen has identified ZNF804A and NT5C2 as potential interacting proteins, but whether this occurs in situ; and moreover, in cortical neurons, is unknown. Here we show that ZNF804A and Nt5C2 colocalise and interact in hEK293T cells. Furthermore, their rodent homolouges, ZFP804A and NT5C2, specifically colocalise at synapses and form a protein complex in cortical neurons. Knockdown of Zfp804A or Nt5c2 resulted in a significant decrease in synaptic expression of both proteins, suggesting that both proteins are required for the synaptic targeting of each other. Taken together, these data indicate that ZNF804A/ZFP804A and NT5C2 interact together in cortical neurons and indicate that these GWAS risk factors may function as a complex to regulate neuronal function.

The ZNF804A protein has been found to be expressed in the human cerebral 66 cortex, particularly in pyramidal neurons (Tao et al., 2014). ZNF804A has also been 67 found to localise to putative synapses, as demonstrated by co-localisation with the 68 synaptic proteins PSD-95 and GluN1 (Deans et al., 2017). Similarly, ZFP804A (rodent 69 homologue) was found to be enriched in synaptic fractions, and to localise to dendritic 70 spines, where the majority of excitatory synapses occur in the mammalian brain 71 (Deans et al., 2017). Consistent with a role for the encoded zinc finger protein at 72 synapses, knockdown or knockout of Zfp804A resulted in a reduction of dendritic spine density (Deans et al., 2017;Huang et al., 2020), whereas an increase in spine density 74 was observed when the full length or shorter disease-associated isoform of the protein 75 was overexpressed in cortical neurons (Dong et al., 2021;Zhou et al., 2020). 76 Moreover, activity-dependent remodelling of dendritic spines was impaired in cortical 77 neurons with reduced ZNF804A expression levels (Deans et al., 2017). Together, 78 these data indicate a role for ZNF804A at synapses. 79 The NT5C2 protein also appears to be enriched in cortical neurons (Duarte et . Whereas it appears that NT5C2 has a function in the developing and adult 88 brain, the precise underlying neurological mechanisms are yet to be established.

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A recent interactome study using a yeast two-hybrid assay has indicated that 90 ZNF804A interacts with multiple proteins, including NT5C2 (Zhou et al., 2018 ZNF804A and NT5C2 form a protein complex in heterologous cells. 107 As a yest-two hybrid assays has revealed that ZNF804A and NT5C2 are  Figure 1C). This was confirmed by a line graph construction, whereby 119 the fluorescence intensity of both proteins was measured ( Figure 1Di & ii), and the 120 overlap between the intensities provided evidence of co-localisation.

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To validate these findings and further determine whether ZNF804A and NT5C2 122 formed a protein complex, we performed a co-immunoprecipitation using hEK293T 123 cells ectopically expressing both proteins. Cells co-transfected with HA-ZNF804A and 124 Myc-NT5C2 were immunoprecipitated with an anti-Myc antibody to isolate NT5C2 and 125 its interacting partners ( Figure 1E). As expected, Myc-NT5C2 was readily detected in 126 the immunoprecipitation (IP) sample. In addition, HA-ZNF804A was also present in 127 the IP sample indicating that ZNF804A and NT5C2 were part of a protein complex 128 ( Figure 1E). Taken together, these data indicate that ZNF804A and NT5C2 co-localise 129 in the cell cytoplasm, near the membrane, and that they form a protein complex in 130 hEK293T cells.   Considering that ZNF804A and NT5C2 form a protein complex in Hek293T 167 cells, and that ZFP804A and NT5C2 localise to the same subcellular compartments in 168 cortical neurons, we sought to explore whether these proteins were part of a protein complex in rat cortical neurons, in situ. First, we examined whether both proteins co-

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To demonstrate this colocalization represented a potential interaction between 184 the two proteins, we performed a co-immunoprecipitation assay for ZFP804A from 185 cortical neuron lysates. Consistent with our data from heterologous cells, 186 immunoblotting revealed that NT5C2 was present in lysates immunoprecipitated for 187 ZFP804A, but not for rabbit IgG ( Figure 3D). These data indicate that ZFP804A and 188 NT5C2 colocalize near synaptic regions in cortical neurons, and that they are part of 189 a protein complex.  p=0.026, n=3)) ( Figure 5B+C). Similar to the ZFP804A knockdown, no effect on 235 ZFP804A puncta was observed in dendrites when Nt5c2 was knocked down. These 236 data suggest a bidirectional signalling interaction occurs between NT5C2 and 237 ZFP804A which operate in concert at synapses but not in dendrites.

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In this study we demonstrate that exogenous ZNF804A and NT5C2 form a          525 The authors declare that they have no conflict of interest.