In this study, we established the causal effects of MCH signaling pathways on cilia length. We first showed that MCH treatment causes cilia shortening in the striatal and cortical brain slice cultures. We then demonstrated that the stimulation of the MCH system through direct agonist activation of the MCHR1 or via optogenetic and chemogenic excitation of MCH neurons causes a significant decrease in cilia length. In contrast, the inactivation of the MCH system through pharmacological blockade of MCHR1 or genetic manipulation (MCHR1 germline deletion or MCH neurons' conditional ablation), causes a significant increase in cilia length.
MCHR1 is widely distributed throughout the brain, with high density in the CPu, NAc, CA1 of the CA1, and the PFC (Hervieu et al., 2000; Saito et al., 2001). Therefore, we focused on these regions in examining the effects of MCH system activation and inactivation on cilia length. Unexpectedly, we found that cilia lengths differ among brain regions and different mouse strains.
The various manipulation approaches allowed us to 1) recapitulate conditions used previously to substantiate MCH physiological functions; 2) differentiate between the effects of MCH neurons versus MCHR1 manipulation; 3) distinguish the effects of acute versus chronic activation/inactivation of MCH system; and 4) determine the effects of early-life versus adult stage MCH system manipulations.
The role of MCH in regulating a wide variety of physiological functions such as feeding, obesity, reward, and sleep has been established using pharmacological and genetic manipulations (Chung et al., 2009; Chung et al., 2011; Garcia-Fuster et al., 2012; Hu et al., 2017; Shimazaki et al., 2006). For example, central chronic infusions of MCH had long been shown to induce mild obesity in wild-type mice (Gomori et al., 2003). In contrast, subchronic administration of MCHR1 antagonists and MCHR1/MCH genetic deletion are known to produce anti-obesity effects in mice (Gehlert et al., 2009; Marsh et al., 2002). The ciliopathy Bardet-Biedl Syndrome (BBS) is characterized by obesity, and mutations affecting the mouse orthologs of BBS-associated genes disrupt the localization of MCHR1 (Berbari et al., 2008b; Zhang et al., 2011). Thus, failure of MCHR1 to reach the cilium could potentially be associated with failure in the MCHR1 signaling pathway in the cilia, leading to obesity in BBS (Engle et al., 2021; Tsang et al., 2018).
Given that MCH neurons synthesize and release other neurotransmitters/neuropeptides, including GABA, NEI, and NGE (Nahon et al., 1989; Parkes and Vale, 1992), it is important to distinguish the selective role of MCHR1 from that of MCH neurons in regulating cilia length. Hence, MCHR1 pharmacological activation and blockade by MCH central infusion and GW803430 treatment respectively and MCHR1 germline deletion allowed for exploring the selective role of the MCHR1. In parallel, optogenetic and DREADD stimulation of the MCH neurons and IDTR- dependent ablation of MCH neurons allowed for exploring the role of MCH neurons. Optogenetics and DREADD technologies have also been previously used to explore MCH neurons' role in sleep, reward, and feeding (Blanco-Centurion et al., 2016; Dilsiz et al., 2020; Naganuma et al., 2018).
Previous reports demonstrated distinctive effects of acute versus chronic activations of MCH systems on body weight (Shearman et al., 2003). Therefore we examined the effects of acute (optogenetic and chemogenetic stimulation) and chronic activations (MCH icv administration for 7 days) of the MCH system on cilia length. We previously showed that developmental and adulthood inactivation of the MCH system in mice leads to distinct behavioral deficits. For example, germline MCHR1 deletion but not adult IDTR- dependent ablation of MCH neurons caused olfactory impairment and social deficits (Vawter et al., 2020a). Therefore, we examined the effects of germline MCHR1 deletion and adulthood conditional MCH ablation on cilia length.
Strikingly, we found that the different MCH system manipulations produced consistent patterns of alterations in cilia length. The activation of MCH system consistently shortened cilia length, while the inactivation of MCH system lengthened cilia length. The findings that both acute and chronic activation of MCH system shortened cilia length and that both early life and adulthood inactivation of the MCH system lengthened cilia substantiate the dynamic nature of the cilia system and suggest that cilia's morphology undergoes rapid changes in response to its environment. Most importantly, our findings provide the first evidence for the direct and moment-to-moment regulation of the brain cilia structure by the MCH system.
Unexpectedly, unilateral optogenetic stimulation of MCH neurons in the lateral hypothalamus caused bilateral shortening of cilia length in the PFC, CA1, CPu, and NAc. This finding may suggest that MCH neurons project ipsilaterally and contralaterally to these brain regions. Another explanation might be that MCH activation may produce ipsilateral modifications of other neurotransmitter systems, which may result in contralateral shortening of cilia length. It is well established that MCH neurons innervate serotonergic neurons in the raphe nucleus and regulate the activity of these neurons (Devera et al., 2015; Urbanavicius et al., 2016). Indeed, specific MCH-regulated functions are mediated through the serotonin system (Lopez Hill et al., 2013; Urbanavicius et al., 2014). Thus, the activation of MCH neurons may regulate cilia indirectly through its interaction with the serotonin system. Interestingly, MCHR1 and 5-HT6 are among the very few GPCRs that preferentially localize to primary cilia (Bansal et al., 2019), and the inhibition of 5-HT6 receptors is known to shorten cilia length (Brodsky et al., 2017; Hu et al., 2017).
Defects in the assembly such as cilia shortening have been shown to cause a range of severe diseases and developmental disorders called ciliopathies, which are associated with neurological deficits such as abnormal cortical formation and cognitive deficits (Hartwig et al., 2018; Reiter and Leroux, 2017; Valente et al., 2014). We have recently shown that brain cilia genes were differentially expressed in major psychiatric disorders, including schizophrenia, autism spectrum disorder, depression, and bipolar disorder (Alhassen et al., 2021). On the other hand, evidence from our own work and others strongly support an essential role for the MCH system, particularly MCHR1, in the pathophysiology of schizophrenia. We showed that MCHR1 mRNA is decreased in the PFC of patients with schizophrenia (Vawter et al., 2020a). We also demonstrated that genetic manipulation of the MCH system via the deletion of MCHR1 and the conditional ablation of MCH neurons resulted in behavioral abnormalities mimicking schizophrenia-like phenotypes, including repetitive behavior, social impairment, impaired sensorimotor gating, and disrupted cognitive functions (Vawter et al., 2020b). We also showed that MCHR1 germline deletion causes in mice alterations in depressive-like behavior that is sex-dependent (Vawter et al., 2020a). These same genetic models were used in our current study to examine the consequences of this genetic manipulation on cilia length. In both IDTR + pMchCre + and MCHR1KO animals, we found a significant increase in cilia in all regions, including the PFC, CA1, CPu, and NAc. Our previous and current data may point to the role of ciliary MCHR1 in the pathopgysiology of psychiatric disorders such as schizophrenia and depression. Together, our previous and current studies point at the cilia elongation as a possible mechanism through which MCH system dysregulation causes social and cognitive deficits in mice and is associated with psychiatric disorders in humans (Alhassen et al., 2021; Vawter et al., 2020a). Our results also prompt the question on whether selective targeting of cilia MCHR1 might offer a novel approach to treat psychiatric disorders. Indeed, studies have demonstrated that some pharmacological agents can alter cilia length. For example, lithium, a mood stabilizer that is used in treatment of bipolar disorder and acute mania, increases MCHR1-positive cilia length in several cell types, including neuronal cells the dorsal striatum and NAc (Miyoshi et al., 2009).
In conclusion, our present study has established the causal regulatory effects of the MCH system signaling on cilia length. The findings of this study are significant because (1) they show for the first time in vivo that primary cilia length can be regulated by the MCH signaling, proving the link between GPCRs and cilia length regulation, and (2) they implicate ciliary MCHR1 as a potential therapeutic target for the treatment of pathological conditions characterized by impaired cilia function.