Cognitive-Affective Functions of the Cerebellum.

The cerebellum, traditionally associated with motor coordination and balance, also plays a crucial role in various aspects of higher-order function and dysfunction. Emerging research has shed light on the cerebellum's broader contributions to cognitive, emotional, and reward processes. The cerebellum's influence on autonomic function further highlights its significance in regulating motivational and emotional states. Perturbations in cerebellar development and function have been implicated in various neurodevelopmental disorders, including autism spectrum disorder and attention deficit hyperactivity disorder. An increasing appreciation for neuropsychiatric symptoms that arise from cerebellar dysfunction underscores the importance of elucidating the circuit mechanisms that underlie complex interactions between the cerebellum and other brain regions for a comprehensive understanding of complex behavior. By briefly discussing new advances in mapping cerebellar function in affective, cognitive, autonomic, and social processing and reviewing the role of the cerebellum in neuropathology beyond the motor domain, this Mini-Symposium review aims to provide a broad perspective of cerebellar intersections with the limbic brain in health and disease.

Social memory deficit caused by dysregulation of the cerebellar vermis.

Social recognition memory (SRM) is a key determinant of social interactions. While the cerebellum emerges as an important region for social behavior, how cerebellar activity affects social functions remains unclear. We selectively increased the excitability of molecular layer interneurons (MLIs) to suppress Purkinje cell firing in the mouse cerebellar vermis. Chemogenetic perturbation of MLIs impaired SRM without affecting sociability, anxiety levels, motor coordination or object recognition. Optogenetic interference of MLIs during distinct phases of a social recognition test revealed the cerebellar engagement in the retrieval, but not encoding, of social information. c-Fos mapping after the social recognition test showed that cerebellar manipulation decreased brain-wide interregional correlations and altered network structure from medial prefrontal cortex and hippocampus-centered to amygdala-centered modules. Anatomical tracing demonstrated hierarchical projections from the central cerebellum to the social brain network integrating amygdalar connections. Our findings suggest that the cerebellum organizes the neural matrix necessary for SRM.

Limited expression of Nrf2 in neurons across the central nervous system.

Nrf2, encoded by the gene Nfe2l2, is a broadly expressed transcription factor that regulates gene expression in response to reactive oxygen species (ROS) and oxidative stress. It is commonly referred to as a ubiquitous pathway, but this generalization overlooks work indicating that Nrf2 is essentially unexpressed in some neuronal populations. To explore whether this pattern extends throughout the central nervous system (CNS), we quantified Nfe2l2 expression and chromatin accessibility at the Nfe2l2 locus across multiple single cell datasets. In both the mouse and human CNS, Nfe2l2 was repressed in almost all mature neurons, but highly expressed in non-neuronal support cells, and this pattern was robust across multiple human CNS diseases. A subset of key Nrf2 target genes, like Slc7a11, also remained low in neurons. Thus, these data suggest that while most cells express Nfe2l2, with activity determined by ROS levels, neurons actively avoid Nrf2 activity by keeping Nfe2l2 expression low.

Limited Expression of Nrf2 in Neurons Across the Central Nervous System.

Nrf2 is a broadly expressed transcription factor that regulates gene expression in response to reactive oxygen species (ROS) and oxidative stress. It is commonly referred to as a ubiquitous pathway, but this generalization overlooks work indicating that Nrf2 is essentially unexpressed in some neuronal populations. To explore whether this pattern extends throughout the central nervous system (CNS), we quantified Nrf2 expression and chromatin accessibility at the Nrf2 locus across multiple single cell datasets. In both the mouse and human CNS, Nrf2 was repressed in almost all mature neurons, but highly expressed in non-neuronal support cells, and this pattern was robust across multiple human CNS diseases. A subset of key Nrf2 target genes, like Slc7a11 , also remained low in neurons. Thus, these data suggest that while most cells express Nrf2, with activity determined by ROS levels, neurons actively avoid Nrf2 activity by keeping Nrf2 expression low.

Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition.

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes (Ntng2, Mfrp, Nr4a2, Thbs1, Atxn1, and Atxn3) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD.

Functional Convergence of Motor and Social Processes in Lobule IV/V of the Mouse Cerebellum.

Topographic organization of the cerebellum is largely segregated into the anterior and posterior lobes that represent its "motor" and "non-motor" functions, respectively. Although patients with damage to the anterior cerebellum often exhibit motor deficits, it remains unclear whether and how such an injury affects cognitive and social behaviors. To address this, we perturbed the activity of major anterior lobule IV/V in mice by either neurotoxic lesion or chemogenetic excitation of Purkinje cells in the cerebellar cortex. We found that both of the manipulations impaired motor coordination, but not general locomotion or anxiety-related behavior. The lesioned animals showed memory deficits in object recognition and social-associative recognition tests, which were confounded by a lack of exploration. Chemogenetic excitation of Purkinje cells disrupted the animals' social approach in a less-preferred context and social memory, without affecting their overall exploration and object-based memory. In a free social interaction test, the two groups exhibited less interaction with a stranger conspecific. Subsequent c-Fos imaging indicated that decreased neuronal activities in the medial prefrontal cortex, hippocampal dentate gyrus, parahippocampal cortices, and basolateral amygdala, as well as disorganized modular structures of the brain networks might underlie the reduced social interaction. These findings suggest that the anterior cerebellum plays an intricate role in processing motor, cognitive, and social functions.

Targeting inhibitory cerebellar circuitry to alleviate behavioral deficits in a mouse model for studying idiopathic autism.

Autism spectrum disorder (ASD) encompasses wide-ranging neuropsychiatric symptoms with unclear etiology. Although the cerebellum is a key region implicated in ASD, it remains elusive how the cerebellar circuitry is altered and whether the cerebellum can serve as a therapeutic target to rectify the phenotype of idiopathic ASD with polygenic abnormalities. Using a syndromic ASD model, e.g., Black and Tan BRachyury T+Itpr3tf/J (BTBR) mice, we revealed that increased excitability of presynaptic interneurons (INs) and decreased intrinsic excitability of postsynaptic Purkinje neurons (PNs) resulted in low PN firing rates in the cerebellum. Knowing that downregulation of Kv1.2 potassium channel in the IN nerve terminals likely augmented their excitability and GABA release, we applied a positive Kv1.2 modulator to mitigate the presynaptic over-inhibition and social impairment of BTBR mice. Selective restoration of the PN activity by a new chemogenetic approach alleviated core ASD-like behaviors of the BTBR strain. These findings highlight complex mechanisms converging onto the cerebellar dysfunction in the phenotypic model and provide effective strategies for potential therapies of ASD.

The eIF2α Kinase GCN2 Modulates Period and Rhythmicity of the Circadian Clock by Translational Control of Atf4.

The integrated stress response (ISR) is activated in response to diverse stress stimuli to maintain homeostasis in neurons. Central to this process is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Here, we report a critical role for ISR in regulating the mammalian circadian clock. The eIF2α kinase GCN2 rhythmically phosphorylates eIF2α in the suprachiasmatic circadian clock. Increased eIF2α phosphorylation shortens the circadian period in both fibroblasts and mice, whereas reduced eIF2α phosphorylation lengthens the circadian period and impairs circadian rhythmicity in animals. Mechanistically, phosphorylation of eIF2α promotes mRNA translation of Atf4. ATF4 binding motifs are identified in multiple clock genes, including Per2, Per3, Cry1, Cry2, and Clock. ATF4 binds to the TTGCAGCA motif in the Per2 promoter and activates its transcription. Together, these results demonstrate a significant role for ISR in circadian physiology and provide a potential link between dysregulated ISR and circadian dysfunction in brain diseases.

mTOR signaling in VIP neurons regulates circadian clock synchrony and olfaction.

Mammalian/mechanistic target of rapamycin (mTOR) signaling controls cell growth, proliferation, and metabolism in dividing cells. Less is known regarding its function in postmitotic neurons in the adult brain. Here we created a conditional mTOR knockout mouse model to address this question. Using the Cre-LoxP system, the mTOR gene was specifically knocked out in cells expressing Vip (vasoactive intestinal peptide), which represent a major population of interneurons widely distributed in the neocortex, suprachiasmatic nucleus (SCN), olfactory bulb (OB), and other brain regions. Using a combination of biochemical, behavioral, and imaging approaches, we found that mice lacking mTOR in VIP neurons displayed erratic circadian behavior and weakened synchronization among cells in the SCN, the master circadian pacemaker in mammals. Furthermore, we have discovered a critical role for mTOR signaling in mediating olfaction. Odor stimulated mTOR activation in the OB, anterior olfactory nucleus, as well as piriform cortex. Odor-evoked c-Fos responses along the olfactory pathway were abolished in mice lacking mTOR in VIP neurons, which is consistent with reduced olfactory sensitivity in these animals. Together, these results demonstrate that mTOR is a key regulator of SCN circadian clock synchrony and olfaction.

Histone Lysine Demethylases of JMJD2 or KDM4 Family are Important Epigenetic Regulators in Reward Circuitry in the Etiopathology of Depression.

Major depressive disorder (MDD) is debilitating mental illness and is one of the leading contributors to global burden of disease, but unfortunately newer and better drugs are not forthcoming. The reason is lack of complete understanding of molecular mechanisms underlying the development of this disorder. Recent research shows dysregulation in epigenetic regulatory mechanisms, particularly the transcriptionally repressive di- and tri-methylation of histone 3 lysine 9 (H3K9me2/me3) in nucleus accumbens (NAc), a critical region of the reward pathway involved in the development of anhedonia, the hallmark of depression. However, the role of histone lysine demethylases, which can remove methylation from H3K9, in particular Jumonji domain containing demethylases 2 or Jmjd2 family, has not been studied. Using social defeat stress-induced mouse model of depression, this study uncovered that transcripts of most of the Jmjd2 members were unchanged after 5 days of defeat during the onset of depression, but were downregulated after 10 days of defeat in full-blown depression. Blocking the Jumonji domain containing demethylases by chronic administration of inhibitors dimethyloxalylglycine (DMOG) and ML324 resulted in depression-like phenotype even in absence of stress exposure, which was associated with an increase in transcriptionally repressive epigenetic marks H3K9me2/me3 in NAc, causing altered neuroplastic changes as reported in NAc in depression models. Thus, we report for the first time that Jmjd2 class demethylases are critical epigenetic regulators involved in etiopathology of depression and related disorders and activation of these demethylases can be a good strategy in the treatment of MDD and related psychiatric disorders.

Epigenetic regulatory mechanisms in stress-induced behavior.

Stress response is considered to have adaptive value for organisms faced with stressful condition. Chronic stress however adversely affects the physiology and may lead to neuropsychiatric disorders. Repeated stressful events in animal models have been shown to cause long-lasting changes in neural circuitries at molecular, cellular, and physiological level, leading to disorders of mood as well as cognition. Molecular studies in recent years have implicated diverse epigenetic mechanisms, including histone modifications, DNA methylation, and noncoding RNAs, that underlie dysregulation of genes in the affected neural circuitries in chronic stress-induced pathophysiology. A review of the myriad epigenetic regulatory mechanisms associated with neural and behavioral responses in animal models of stress-induced neuropsychiatric disorders is presented here. The review also deals with clinical evidence of the epigenetic dysregulation of genes in psychiatric disorders where chronic stress appears to underlie the etiopathology.