Review coordinated by Life Science Editors Foundation

Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation

Potential Conflicts of Interest: None

Punch line: Rare risk variants associated with schizophrenia converge on the cAMP/PKA pathway.

Why is this interesting? The cAMP/PKA pathway could be a mechanism & therapeutic target for neuropsychiatric disorders arising from different mutations.

Background: * About 1-4% of people will develop psychosis or schizophrenia. * Schizophrenia is a highly heritable disease. * Genetic loci associated with schizophrenia can be common variants, which typically have small effects on risk, or rare variants, which can have large effects. * Rare, protein-truncating variants substantially increase the risk for mental illnesses like schizophrenia. * Disease-associated genes have diverse functions (e.g.): 1. RNA binding (RBM12) 2. transcriptional regulation (SP4, RB1CC1, SETD1A) 3. splicing (SRRM2) 4. signaling (AKAP11) 5. ion transport (CACNA1G, GRIN2A, GRIA3) 6. neuronal migration and growth (TRIO) 7. nuclear transport (XPO7) 8. ubiquitin ligation (CUL1, HERC1) ** What are the pathological mechanisms?* * A genetic screen identified the risk gene RBM12 as a novel repressor of GPCR/cAMP signaling (Semesta et al., PLOS Genetics, 2020). * Dysregulation of GPCR activity in the brain contributes to the pathophysiology of several neurological and neuropsychiatric disorders. * cAMP is a critical second messenger that mediates all important aspects of neuronal function, including development, excitability, and plasticity.

Results: * Use knockout HEK293 cells to verify that RBM12 is novel repressor of the GPCR/cAMP pathway that extends to multiple GPCRs coupled to the stimulatory G protein (e.g. dopamine 1 receptor, beta-2 adrenergic receptor). * Show RBM12 also represses this pathway in iPSC-derived neurons. RBM12 knockdown yielded hyperactive upregulation of NR4A1 and FOS mRNAs, two known CREB-dependent immediate early genes induced by neuronal activity. * RBM12 loss leads to increased PKA activity and supraphysiological CREB-dependent transcriptional responses. * RBM12 loss increased expression of the endogenous β2-AR transcriptional target mRNAs, PCK1 and FOS. * RBM12 loss increased CREB transcriptional reporter expression in response to a panel of endogenous or synthetic β2-AR agonists.<br /> * Transcriptional responses are orchestrated from endosomal β2-ARs in wild-type cells but from both plasma membrane and endosomal β2-ARs in RBM12 knockout cells. * Their results suggest that cAMP production and transcriptional signaling are independently subject to RBM12 regulation. * The neuropsychiatric disease-linked mutations fail to rescue GPCR-dependent hyperactivation in cells depleted of RBM12. * Defined β2-AR-dependent transcriptional targets in “wild-type” and RBM12 knockdown neurons by differential expression analysis between each respective basal and isoproterenol conditions. 669 unique β2-AR-dependent transcriptional targets across the two cell lines. * Discerned β2-AR-dependent targets that were exclusive to wild-type or RBM12 knockdown only (qualitatively distinct targets) versus targets that are in wt and RBM12 kd but upregulated to different extents (quantitatively distinct targets). * 21 wild-type- and 115 RBM12 knockdown-specific target genes. Factors involved in synaptic plasticity and schizophrenia such as JUN, ARC (encoding the activity-regulated cytoskeleton-associated protein), BDNF, and NRXN3 (encoding the cell adhesion molecule neurexin-3-alpha) were induced by GPCR signaling only in RBM12 knockdown neurons, while GRIA2 (encoding the AMPA receptor) and CBLN2 (encoding cerebellin 2 precursor) were upregulated upon GPCR signaling only in wt neurons. * the remaining 533 genes were induced in both wt & RBM12-depeleted, with a trend toward RBM12-dependent hyperactivation. * loss of RBM12 leads to aberrant expression of ADCY, PDE, and PRKACA, suggesting this mechanism underlies the hyperactive GPCR/cAMP/PKA signaling phenotypes.

Discussion: * Dysregulation of GPCR signaling could contribute to the neuronal pathologies stemming from loss of RBM12. * RBM12 function is required for normal cAMP production downstream of many Gαs-coupled receptors with established roles in the nervous system consistent with dysregulation of cAMP/PKA pathway. Specifically, the entire repertoire of targets, many of which orchestrate processes essential for neuronal differentiation, gene reprogramming, and memory and learning, shows a trend towards hyperactivation in RBM12 depleted neurons. * Over 100 genes are induced in response to receptor stimulation only in the knockdown (e.g. ARC and BDNF, with crucial roles in synaptic function, plasticity, and learning. * RBM12 could act through other mechanisms, given that RBM12 knockdown neurons also affects the expression of genes involved in neuron differentiation, synapse organization, and neurogenesis. * A study on post-mortem brains of patients with bipolar affective disorder demonstrated elevated levels of the PKAcat subunit Cα in temporal and frontal cortices compared to matched normal brains. * A different report on patient-derived platelet cells found that the catalytic subunit of cAMP-dependent protein kinase was significantly upregulated in untreated depressed and manic patients with bipolar disorder compared with untreated euthymic patients with bipolar disorder and healthy subjects. * Mutations in the schizophrenia risk gene histone methyltransferase SET domain-containing protein 1 A (SETD1A) also led to transcriptional and signaling signatures supporting hyperactivation of the cAMP pathway through upregulation of adenylyl cyclases and downregulation of PDEs. This in turn resulted in increased dendritic branching and length and altered network activity in human iPSC-derived glutamatergic neurons.

Beautiful follow up to their PLOS Genetics paper, and compelling pathological mechanism in combination with the recent SETD1A Cell Reports paper.

Future work: * Does loss of RBM12 increase dendritic branching and length and alter network activity in human iPSC-derived glutamatergic neurons (e.g. does it phenocopy loss of SETD1A). * Does pharmacologically targeting the cAMP pathway rescue the phenotypes caused by loss of RBM12? * If RBM12 is ubiquitously expressed, why is the disease neuronal? What is the relevant GPCR/neuronal mechanism? * How does RBM12 affect the abundance of the transcripts encoding the GPCR/cAMP effectors? * Do mutations in any of these other rare risk genes converge on GPCR? (transcriptional regulation (SP4, RB1CC1), splicing (SRRM2). signaling (AKAP11). ion transport (CACNA1G, GRIN2A, GRIA3), neuronal migration and growth (TRIO), nuclear transport (XPO7). ubiquitin ligation (CUL1, HERC1))