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Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077.

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

Regulation of gene expression is important for the development and function of the nervous system. However, the transcriptional programs altered in psychiatric diseases are not completely characterized. Human gene association studies and analysis of mutant mice suggest that the transcription factor specificity protein 4 (SP4) may be implicated in the pathophysiology of psychiatric diseases. We hypothesized that SP4 levels may be altered in the brain of bipolar disorder (BD) subjects and regulated by neuronal activity and drug treatment. We analyzed messenger RNA (mRNA) and protein levels of SP4 and SP1 in the postmortem prefrontal cortex and cerebellum of BD subjects (n = 10) and controls (n = 10). We also examined regulation of SP4 mRNA and protein levels by neuronal activity and lithium in rat cerebellar granule neurons. We report a reduction of SP4 and SP1 proteins, but not mRNA levels, in the cerebellum of BD subjects. SP4 protein and mRNA levels were also reduced in the prefrontal cortex. Moreover, we found in rat cerebellar granule neurons that under non-depolarizing conditions SP4, but not SP1, was polyubiquitinated and degraded by the proteasome while lithium stabilized SP4 protein. Our study provides the first evidence of altered SP4 protein in the cerebellum and prefrontal cortex in BD subjects supporting a possible role of transcription factor SP4 in the pathogenesis of the disease. In addition, our finding that SP4 stability is regulated by depolarization and lithium provides a pathway through which neuronal activity and lithium could control gene expression suggesting that normalization of SP4 levels could contribute to treatment of affective disorders.

The remarkable capacity of neurons to reorganize their structure, function, and connections in an activity-dependent manner is supported by an extensive network of molecules and effectors. Among those, Activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1) is considered one of the central, most versatile, players. Arc is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation, while also oligomerizing to form capsids that can be released and can move independent of the synapse. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are regulated both temporally and spatially in neurons. To answer to this question, the regulation of Arc expression must be monitored at various levels. This thesis analyzes how Arc can be transcriptionally and post-translationally regulated and how this confers neurobiological specificity. Being an immediate early gene, transcription of Arc needs to act rapidly in response to a neuronal stimulus, such as BDNF. The first aim of this thesis provides a model to describe how the Arc promoter can overcome topological constraints to facilitate rapid and persistent transcription, up to 6 hours post-BDNF exposure. Using chromatin analysis and biochemical techniques we have mapped regions of the Arc promoter that undergo double stranded DNA breaks to facilitate rapid transcription, and that BDNF-induced Arc non-coding RNA elements interact with DNA repair proteins such as Brd4 to effectively end transcription. The second aim of this thesis aims to determine how phosphorylation can spatiotemporally dictate the function of Arc. Mass spectrometry and sequence prediction strategies were used to map novel Arc phosphorylation sites. This approach led us to recognize S67 and T278 as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons and been implicated as a risk factor for psychiatric disorders. Characterization of TNIK-dependant phosphorylation at these two sites can strongly influence Arc's subcellular distribution and self-assembly as capsids. The multifaceted nature of Arc’s neurobiology ranges from being a key component of long-term memory consolidation to participating in viral capsid intracellular communication. This thesis provides insight into Arc’s moonlighting capabilities both at the transcription and post-translational levels.

Calcium (Ca2+) is a pivotal signaling molecule in non-excitable and excitable cells. In neurons, it regulates processes from transcription to excitability. Though they have specialized Ca2+ channels that influx massive amounts of this cation during activity, these channels are closed during rest. So, to regulate smaller oscillations, neurons use store-operated Ca2+ entry (SOCE). This mechanism utilizes the ER Ca2+ sensor STIM and the Ca2+ channel ORAI for influx after Ca2+ stores in the ER are depleted. This influx is known to regulate transcription, differentiation, survivability, and excitability, among other processes. Since the various roles SOCE contributes to are cell type-specific, we wanted to assess its impact on transcription and differentiation with a model seldom used for SOCE-specific research: human-derived neural progenitor cells (NPCs). SOCE is known to modulate activity of transcription factors such as NFAT and Sp4. We sought to test whether CREB, a crucial transcription factor in neurogenesis, could be modulated by SOCE. We found that CREB was phosphorylated as soon as five minutes after SOCE activation, though not by one of its canonical pathways. We also found that SOCE activation contributed broadly to transcription as its activation upregulated several immediate early genes. If SOCE indeed influences neurogenesis in this way, dysregulation in the brain could contribute to neurodevelopmental disease. Though Ca2+ signaling is known to be dysregulated in bipolar disorder (BD), few studies have explored potential SOCE dysregulation in cells derived from these patients. We uncovered an attenuation in SOCE-specific Ca2+ influx in BD-derived NPCs (BD-NPCs) that was dependent on differentiation. From this we explored neurodevelopmental deficits in these cells, which included a unique transcriptome, upregulated microRNAs, reduced rate of proliferation, increased migration, longer neurite outgrowth, and abnormal subventricular structures after generating BD organoids. This all suggests that BD pathophysiology may begin long before symptoms of the disease present in early adulthood. Continuing to study the impact SOCE has on NPCs and how dysregulated neurodevelopmental processes in BD-NPCs contribute to disease could uncover new cell signaling pathways and potential therapeutics for the disease.

Estrogens have been implicated extensively in learning, memory, dendritic spinogenesis, and synaptic plasticity. The molecular pathways underlying estrogens’ rapid, nongenomic actions are not well understood, however protein translation of dendritically-localized mRNA transcripts may be a possible mechanism of action. The Activity-regulated cytoskeleton-association protein (Arc) has emerged as a strong candidate for the mediation of these effects. Here, we present findings from mouse in vivo and primary cortical cell culture models demonstrating Arc’s dynamic regulation following stimulation by 17beta-estradiol (E2) within 15- to 40-minutes of treatment. Further, we show that E2 induces a differential localization of dendritic Arc dependent on the length of time that follows E2 treatment. In sum, these data suggest a novel role for Arc acting within a mechanism of E2-facilitated neuronal actions that is dose-, time-, and dendritic region-dependent, advocating for a multi-mechanism approach to studying Arc’s involvement in estrogenic short-term memory facilitation within the mouse brain.