Dynamic Changes of Brain Cilia Transcriptomes across the Human Lifespan

Almost all brain cells contain primary cilia, antennae-like microtubule sensory organelles, on their surface, which play critical roles in brain functions. During neurodevelopmental stages, cilia are essential for brain formation and maturation. In the adult brain, cilia play vital roles as signaling hubs that receive and transduce various signals and regulate cell-to-cell communications. These distinct roles suggest that cilia functions, and probably structures, change throughout the human lifespan. To further understand the age-dependent changes in cilia roles, we identified and analyzed age-dependent patterns of expression of cilia’s structural and functional components across the human lifespan. We acquired cilia transcriptomic data for 16 brain regions from the BrainSpan Atlas and analyzed the age-dependent expression patterns using a linear regression model by calculating the regression coefficient. We found that 67% of cilia transcripts were differentially expressed genes with age (DEGAs) in at least one brain region. The age-dependent expression was region-specific, with the highest and lowest numbers of DEGAs expressed in the ventrolateral prefrontal cortex and hippocampus, respectively. The majority of cilia DEGAs displayed upregulation with age in most of the brain regions. The transcripts encoding cilia basal body components formed the majority of cilia DEGAs, and adjacent cerebral cortices exhibited large overlapping pairs of cilia DEGAs. Most remarkably, specific α/β-tubulin subunits (TUBA1A, TUBB2A, and TUBB2B) and SNAP-25 exhibited the highest rates of downregulation and upregulation, respectively, across age in almost all brain regions. α/β-tubulins and SNAP-25 expressions are known to be dysregulated in age-related neurodevelopmental and neurodegenerative disorders. Our results support a role for the high dynamics of cilia structural and functional components across the lifespan in the normal physiology of brain circuits. Furthermore, they suggest a crucial role for cilia signaling in the pathophysiological mechanisms of age-related psychiatric/neurological disorders

Regulation of Brain Primary Cilia Length by MCH Signaling: Evidence from Pharmacological, Genetic, Optogenetic, and Chemogenic Manipulations

The melanin-concentrating hormone (MCH) system is involved in numerous functions, including energy homeostasis, food intake, sleep, stress, mood, aggression, reward, maternal behavior, social behavior, and cognition. In rodents, MCH acts on MCHR1, a G protein-coupled receptor, which is widely expressed in the brain and abundantly localized to neuronal primary cilia. Cilia act as cells’ antennas and play crucial roles in cell signaling to detect and transduce external stimuli to regulate cell differentiation and migration. Cilia are highly dynamic in terms of their length and morphology; however, it is not known if cilia length is causally regulated by MCH system activation in vivo. In the current work, we examined the effects of activation and inactivation of MCH system on cilia lengths by using different experimental models and methodologies, including organotypic brain slice cultures from rat prefrontal cortex (PFC) and caudate–putamen (CPu), in vivo pharmacological (MCHR1 agonist and antagonist GW803430), germline and conditional genetic deletion of MCHR1 and MCH, optogenetic, and chemogenetic (designer receptors exclusively activated by designer drugs (DREADD)) approaches. We found that stimulation of MCH system either directly through MCHR1 activation or indirectly through optogenetic and chemogenetic-mediated excitation of MCH-neuron, caused cilia shortening, detected by the quantification of the presence of ADCY3 protein, a known primary cilia marker. In contrast, inactivation of MCH signaling through pharmacological MCHR1 blockade or through genetic manipulations — germline deletion of MCHR1 and conditional ablation of MCH neurons — induced cilia lengthening. Our study is the first to uncover the causal effects of the MCH system in the regulation of the length of brain neuronal primary cilia. These findings place MCH system at a unique position in the ciliary signaling in physiological and pathological conditions and implicate MCHR1 present at primary cilia as a potential therapeutic target for the treatment of pathological conditions characterized by impaired primary cilia function associated with the modification of its length

Patterns of Cilia Gene Dysregulations in Major Psychiatric Disorders

Primary cilia function as cells\u27 antennas to detect and transduce external stimuli and play crucial roles in cell signaling and communication. The vast majority of cilia genes that are causally linked with ciliopathies are also associated with neurological deficits, such as cognitive impairments. Yet, the roles of cilia dysfunctions in the pathogenesis of psychiatric disorders have not been studied. Our aim is to identify patterns of cilia gene dysregulation in the four major psychiatric disorders: schizophrenia (SCZ), autism spectrum disorder (ASD), bipolar disorder (BP), and major depressive disorder (MDD). For this purpose, we acquired differentially expressed genes (DEGs) from the largest and most recent publicly available databases. We found that 42%, 24%, 17%, and 15% of brain-expressed cilia genes were significantly differentially expressed in SCZ, ASD, BP, and MDD, respectively. Several genes exhibited cross-disorder overlap, suggesting that typical cilia signaling pathways\u27 dysfunctions determine susceptibility to more than one psychiatric disorder or may partially underlie their pathophysiology. Our study revealed that genes encoding proteins of almost all sub-cilia structural and functional compartments were dysregulated in the four psychiatric disorders. Strikingly, the genes of 75% of cilia GPCRs and 50% of the transition zone proteins were differentially expressed in SCZ. The present study is the first to draw associations between cilia and major psychiatric disorders, and is the first step toward understanding the role that cilia components play in their pathophysiological processes, which may lead to novel therapeutic targets for these disorders

Exploring the relationship of primary cilia and psychiatric disorders to further define the role of ciliary MCHR1 in social & cognitive functions

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

Cilia in the Striatum Mediate Timing-Dependent Functions

Almost all brain cells contain cilia, antennae-like microtubule-based organelles. Yet, the significance of cilia, once considered vestigial organelles, in the higher-order brain functions is unknown. Cilia act as a hub that senses and transduces environmental sensory stimuli to generate an appropriate cellular response. Similarly, the striatum, a brain structure enriched in cilia, functions as a hub that receives and integrates various types of environmental information to drive appropriate motor response. To understand cilia's role in the striatum functions, we used loxP/Cre technology to ablate cilia from the dorsal striatum of male mice and monitored the behavioral consequences. Our results revealed an essential role for striatal cilia in the acquisition and brief storage of information, including learning new motor skills, but not in long-term consolidation of information or maintaining habitual/learned motor skills. A fundamental aspect of all disrupted functions was the "time perception/judgment deficit." Furthermore, the observed behavioral deficits form a cluster pertaining to clinical manifestations overlapping across psychiatric disorders that involve the striatum functions and are known to exhibit timing deficits. Thus, striatal cilia may act as a calibrator of the timing functions of the basal ganglia-cortical circuit by maintaining proper timing perception. Our findings suggest that dysfunctional cilia may contribute to the pathophysiology of neuro-psychiatric disorders, as related to deficits in timing perception