The melanin-concentrating hormone (MCH) system, composed of the hypothalamic neuropeptide MCH and its receptor MCHR1, is a critical regulator of several functions, including energy homeostasis, food intake, sleep, stress, mood, aggression, reward, and cognition. The MCH system is expressed primarily in the lateral hypothalamus and zona incerta and projects throughout the central nervous system. MCHR1 is widely distributed in several brain regions, particularly in the frontal cortex, amygdala, nucleus accumbens, and hippocampus, suggesting that MCH may modulate social, emotional, and cognitive functions. MCHR1 is a G protein-coupled receptor that is located in the primary neuronal cilia.Primary cilia are small microtubule backbones, hair-like structures that protrude from the plasma membrane of almost every cell, including neurons. They serve as sensory organelles that detect and transduce extracellular signals, such as mechanical and chemical stimuli, into intracellular signals that regulate cell signaling pathways and gene expression. Ciliopathies are a group of inherited disorders caused by defects in cilia structure or function, and many of the genes implicated in these disorders have been linked to neurological deficits, including cognitive
impairments. Despite the evidence suggesting that cilia dysfunction may play a role in psychiatric disorders, such as schizophrenia, autism spectrum disorder, bipolar disorder, and major depressive disorder, the specific mechanisms underlying this association remain poorly understood. Alongside, almost all brain cells have cilia that are made of microtubules that play critical roles in brain functions. They are essential for brain formation and maturation during neurodevelopmental stages, and in the adult brain, they act as signaling hubs that receive and transduce various signals, regulating cell-to-cell communications. Cilia are intricate and adaptable sub-cellular systems that work in a coordinated way to perform their structural and functional roles. These roles involve sensing environmental stimuli that follow circadian rhythms, which suggests that genes that encode the components of cilia might also have circadian patterns of expression.
G-protein-coupled receptors (GPCRs) are crucial to the neurobiology of psychiatric disorders, as they mediate the effects of most neurotransmitters implicated in these disorders and are the primary targets of psychotropic drugs. However, their precise role in the development and progression of psychiatric disorders remains poorly understood. The MCH system is a critical player in several physiological and behavioral functions, and the MCHR1 receptor's distribution in neuronal primary cilia suggests that MCH may regulate these functions by modulating cellular signaling pathways. Further research is necessary to understand the exact mechanisms by which MCH exerts its action and how modulation of the MCH system could be utilized for therapeutic purposes.
This thesis investigates the dysregulation of cilia genes in psychiatric disorders, with a focus on circadian patterns, age-related changes, and region-dependent functions. Additionally, the thesis examines the involvement of brain primary cilia in MCH signaling and its role in the manifestation of behavioral deficits related to social and cognitive impairments in animal models with time-dependent ciliary MCHR1 deletion. Finally, we begin to explore the potential of MCH fragment analogues as treatments for ciliopathies or psychiatric disorders.
To begin we identified patterns of cilia gene dysregulation in psychiatric disorders by analyzing differentially expressed genes from publicly available databases. We found that a significant portion of brain-expressed cilia genes were differentially expressed in these disorders, indicating that cilia signaling pathways may be involved in their pathophysiology. Additionally, we revealed that genes encoding proteins of almost all sub-cilia structural and functional compartments were dysregulated in these disorders, suggesting that cilia dysfunctions may be involved in various aspects of disease pathology. We also found that genes encoding for certain cilia proteins were differentially expressed across multiple psychiatric disorders, indicating that cilia signaling may be a common pathway in their pathophysiology. Overall, this study represents the first step towards understanding the role that cilia components play in the pathophysiological processes of major psychiatric disorders. By shedding light on the role of cilia signaling in these disorders, this study may lead to the development of novel therapeutic targets for these disorders.
Disruptions to the cilia-circadian rhythm connection have been linked to various diseases and disorders, such as obesity, diabetes, and sleep disorders, highlighting the crucial role of cilia in maintaining proper circadian rhythm and overall physiological function. By analyzing the gene expression atlas of primates using computational techniques, we found that approximately 73% of cilia transcripts showed circadian rhythmicity in at least one of the 22 brain regions studied. Furthermore, cilia transcriptomes in 12 brain regions were enriched with circadian oscillating transcripts compared to the rest of the transcriptome. Notably, cilia circadian transcripts shared between the basal ganglia nuclei and prefrontal cortex peaked in a sequential pattern similar to the order of activation of the basal ganglia-cortical circuitry, which is essential for movement
coordination. These findings suggest that the spatiotemporal orchestration of cilia genes expression might play a critical role in the normal physiology of the basal ganglia-cortical circuit and motor control.
It is unknown if MCH system activation in vivo causally regulates cilia length, which is highly dynamic in morphology and length. To investigate this, we used different experimental models and methodologies, including organotypic brain slice cultures from rat prefrontal cortex (PFC) and caudate-putamen (CPu), in vivo pharmacological approaches, germline and conditional genetic deletion of MCHR1 and MCH, optogenetic, and chemogenetic methods. Our results revealed that activation of the MCH system through MCHR1 agonism or optogenetic and chemogenetic excitation of MCH-neurons caused cilia shortening, while MCH signaling inactivation via MCHR1 antagonism or genetic manipulation resulted in cilia lengthening. Our findings indicate that the MCH system plays a significant role in ciliary signaling and highlight MCHR1 located at primary cilia as a potential therapeutic target for pathological conditions associated with abnormal primary cilia function and modification of its length.
Next, we wanted to understand cilia’s role in higher-order brain functions as it remains largely unknown. Acting as a hub that senses and transduces environmental stimuli to generate appropriate cellular responses, cilia-rich brain structures, such as the striatum, receive and integrate various types of information to drive appropriate motor responses. In this study, we employed loxP/Cre technology to remove cilia from the dorsal striatum of male mice and observed the behavioral outcomes. Our results suggest a critical role for striatal cilia in the acquisition and brief storage of information, specifically in learning new motor skills, but not in the consolidation of long-term information or the maintenance of learned motor skills. Moreover, the deficits observed in the behavior of mice without striatal cilia were clustered around the clinical manifestations of neuro-psychiatric disorders that involve striatal functions and timing perception. Therefore, striatal cilia may act as regulators of the timing functions of the basal ganglia-cortical circuit by maintaining accurate timing perception.
MCHR1's role in primary cilia is not yet fully understood, but has been implicated in regulating a range of physiological processes, such as appetite and energy balance, as well as behaviors related to reward, motivation, and mood. To better understand the role of ciliary MCHR1 in social and cognitive deficits, we utilized an inducible knockout model. Our results revealed that late deletion of ciliary MCHR1 does not significantly affect sociability but leads to an increase in hyperactivity and deficits in cognition and sensorimotor gating. On the other hand, early deletion of ciliary MCHR1 leads to deficits in both social and cognitive function, as well as sensorimotor gating deficits. Additionally, we quantified the amount of ciliary and non-ciliary MCHR1 that localizes to primary cilia to better understand the role they play in these deficits. Our findings suggest that the MCH system's disruption interferes with neurodevelopmental processes, which could contribute to the pathogenesis of schizophrenia.
Lastly, we began to design MCH analogues with improved binding affinity for MCH receptors to potentially develop new therapies for these conditions. We used various in vitro binding techniques to analyze the affinity of the MCH analogues for MCH receptors. In the in vivo experiments, we injected MCH and MCH analogues intracerebroventricularly in mice to study their effects on feeding behavior, energy homeostasis, and cilia length. We discovered an MCH fragment analogue with a reduced number of amino acids and molecular weight that showed potential to bind in vivo. This MCH fragment analogue had a potency comparable to the full MCH peptide and caused cilia shortening in the adult mouse brain and was reversed when administered with an MCHR1 antagonist. We also found that when administered i.c.v similarly to MCH, the mice gained weight. When given simultaneously with an antagonist, it resulted in weight loss. This suggests that MCH fragment analogues could potentially be used as potential treatments for conditions associated with abnormal MCH signaling, such as ciliopathies or psychiatric disorders. In conclusion, our study provides new insights into the design of MCH analogues with improved binding affinity for MCH receptors. We believe that these findings will contribute to the development of new therapeutic approaches for conditions associated with MCH signaling abnormalities. By providing new insights into the underlying mechanisms of schizophrenia and other neurological disorders, the studies in this thesis may pave the way for the development of novel therapeutic targets for the treatment of these conditions.
