Abstract Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

TAR DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS) are RNA binding proteins (RBPs) primarily located in the nucleus, and involved in numerous aspects of RNA metabolism. Both proteins can be found to be depleted from the nucleus and accumulated in cytoplasmic inclusions in two major neurodegenerative conditions, amyotrophic lateral sclerosis and frontotemporal dementia. Recent evidences suggest that, in addition to their nuclear functions, both TDP-43 and FUS are involved in multiple processes in the cytoplasm, including mRNA stability and transport, translation, the stress response, mitochondrial and autophagy regulation. Here, we review the most recent advances in understanding their functions in the cytoplasm and how these are affected in disease.

Medications that target the noradrenergic system are important therapeutics for depression and anxiety disorders. More recently, clinical studies have shown that the α2-noradrenergic receptor (α2AR) agonist guanfacine can decrease stress-induced smoking relapse during acute abstinence, suggesting that targeting the noradrenergic system may aid in smoking cessation through effects on stress pathways in the brain. Acetylcholine (ACh), like the nicotine in tobacco, acts at nicotinic acetylcholine receptors (nAChRs) to regulate behaviors related to anxiety and depression. We therefore investigated interactions between guanfacine and ACh signaling in tests of anxiolytic and antidepressant efficacy in female and male C57BL/6J mice, focusing on the amygdala as a potential site of noradrenergic/cholinergic interaction. The antidepressant-like effects of guanfacine were blocked by shRNA-mediated knockdown of α2AR in amygdala. Knockdown of the high-affinity β2 nAChR subunit in amygdala also prevented antidepressant-like effects of guanfacine, suggesting that these behavioral effects require ACh signaling through β2-containing nAChRs in this brain area. Ablation of NE terminals prevented the anxiolytic- and antidepressant-like effects of the nicotinic partial agonist cytisine, whereas administration of the cholinesterase antagonist physostigmine induced a depression-like phenotype that was not altered by knocking down α2AR in the amygdala. These studies suggest that ACh and NE have opposing actions on behaviors related to anxiety and depression and that cholinergic signaling through β2-containing nAChRs and noradrenergic signaling through α2a receptors in neurons of the amygdala are critical for regulation of these behaviors.

Rationale The α2a-noradrenergic agonist guanfacine decreases stress-induced smoking in female, but not male, human smokers. It is not known whether these effects are due to effects on mood regulation and/or result from nicotinic-cholinergic interactions. Objectives To determine whether there are sex-differences in the effect of guanfacine in tests of anxiolytic- and antidepressant-efficacy in mice at baseline and in a hypercholinergic model of depression induced by the acetylcholinesterase inhibitor physostigmine. Methods The effects of guanfacine were measured in the light/dark box, tail suspension and the forced swim test in female and male C57BL/6J mice. In parallel, electrophysiological properties were evaluated in prefrontal cortex, a critical brain region involved in stress responses. c-fos immunoreactivity was measured in other brain regions known to regulate mood. Results Despite a baseline sex difference in behavior in the forced swim test (female mice were more immobile), guanfacine had similar, dose-dependent, antidepressant-like effects in mice of both sexes (optimal dose: 0.15 mg/kg). An antidepressant-like effect of guanfacine was also observed following pre-treatment with physostigmine. A sex difference in the paired-pulse ratio in PFC (male: 1.4; female, 2.1) was observed at baseline that was normalized by guanfacine. Other brain regions areas involved in cholinergic control of depression-like behaviors, including basolateral amygdala and lateral septum, showed sex-specific changes in c-fos expression. Conclusions Guanfacine has a robust antidepressant-like effect and can reverse a depression-like state induced by increased ACh signaling. These data suggest that different brain areas are recruited in female and male mice, despite similar behavioral responses to guanfacine.

Nicotinic acetylcholine receptor (nAChR) blockers potentiate the effects of selective serotonin reuptake inhibitors (SSRIs) in some treatment-resistant patients; however, it is not known whether these effects are independent, or whether the two neurotransmitter systems act synergistically. We first determined that the SSRI fluoxetine and the nicotinic partial agonist cytisine have synergistic effects in a mouse model of antidepressant efficacy, whereas serotonin depletion blocked the effects of cytisine. Using a pharmacological approach, we found that the 5-HT1A agonist 8-OH-DPAT also potentiated the antidepressant-like effects of cytisine, suggesting that this subtype might mediate the interaction between the serotonergic and cholinergic systems. The 5-HT1A receptors are located both presynaptically and postsynaptically. We therefore knocked down 5-HT1A receptors in either the dorsal raphe (presynaptic autoreceptors) or the hippocampus (a brain area with high expression of 5-HT1A heteroreceptors sensitive to cholinergic effects on affective behaviors). Knockdown of 5-HT1A receptors in hippocampus, but not dorsal raphe, significantly decreased the antidepressant-like effect of cytisine. This study suggests that serotonin signaling through postsynaptic 5-HT1A receptors in the hippocampus is critical for the antidepressantlike effects of a cholinergic drug and begins to elucidate the molecular mechanisms underlying interactions between the serotonergic and cholinergic systems related to mood disorders.

Acetylcholine (ACh) levels are elevated in actively depressed subjects. Conversely, antagonism of either nicotinic or muscarinic ACh receptors can have antidepressant effects in humans and decrease stress-relevant behaviors in rodents. Consistent with a role for ACh in mediating maladaptive responses to stress, brain ACh levels increase in response to stressful challenges, whereas systemically blocking acetylcholinesterase (AChE, the primary ACh degradative enzyme) elicits depression-like symptoms in human subjects, and selectively blocking AChE in the hippocampus increases relevant behaviors in rodents. We used an ACh sensor to characterize stress-evoked ACh release, then used chemogenetic, optogenetic and pharmacological approaches to determine whether cholinergic inputs from the medial septum/diagonal bands of Broca (MSDBB) or ChAT-positive neurons intrinsic to the hippocampus mediate stress-relevant behaviors in mice. Chemogenetic inhibition or activation of MSDBB cholinergic neurons did not result in significant behavioral effects, while inhibition attenuated the behavioral effects of physostigmine. In contrast, optogenetic stimulation of septohippocampal terminals or selective chemogenetic activation of ChAT-positive inputs to hippocampus increased stress-related behaviors. Finally, stimulation of sparse ChAT-positive hippocampal neurons increased stress-related behaviors in one ChAT-Cre line, which were attenuated by local infusion of cholinergic antagonists. These studies suggest that ACh signaling results in maladaptive behavioral responses to stress if the balance of signaling is shifted toward increased hippocampal engagement.

In a recent investigation Deshaies and colleagues identify HNRNP A1-7B, an isoform of the RNA binding protein (RBP) HNRNP A1, as present in post mortem amyotrophic lateral sclerosis (ALS) spinal motor neuron inclusions (Deshaies et al., 2018). HNRNP A1 is an important player in ALS, as mutations in its low complexity domain (LCD) can be causative for the disease (Kim et al., 2013). Intriguingly, the isoform described by Deshaies et al.includes exon 7B, which produces an extension of the LCD by 52 amino acids, pointing to a link between this alternative splicing event and neuronal protein aggregation. The authors further suggest that loss of RBP TDP-43 can influence this splicing event and result in an increase of the aggregation-prone 7B isoform. TDP-43 is abnormally mislocalised in >95% of ALS cases and when mutated can cause ALS (Harrison and Shorter, 2017). TDP-43 binds RNA, preferentially at UG repeats, and is involved in alternative splicing (Buratti and Baralle, 2001). Importantly, its activity is extremely dosage sensitive, making physiological expression an essential condition for studying the effect of TDP-43 mutations on splicing. Recently, we and others have published an allelic series of novel physiological TDP-43 mouse mutant models and shown that C-terminal mutations induce RNA splicing gain of function (Fratta et al., 2018; White et al., 2018). In order to investigate the link between TDP-43, its mutations and Hnrnpa1 exon 7B splicing, we utilised recently published

Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease in which the loss of motor neurons induces progressive weakness and death from respiratory failure, typically only 3-5 years after onset.FUS has been established to have a role in numerous aspects of RNA processing, including splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterised, as most disease models have been based on FUS overexpression, which in itself alters its RNA processing functions. To overcome this, we and others have recently created knock-in models, and have generated high depth RNA-sequencing data on FUS mutants in parallel to FUS knockout. We combined three independent datasets with a joint modelling approach, allowing us to compare the mutation-induced changes to genuine loss of function. We find that FUS ALS-mutations induce a widespread loss of function on expression and splicing, with a preferential effect on RNA binding proteins. Mutant FUS induces intron retention changes through RNA binding, and we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS regulation has been linked to disease, and intriguingly, we find FUS autoregulation to be altered not only by FUS mutations, but also in other genetic forms of ALS, including those caused by TDP-43,