Myeloid deficiency of the intrinsic clock protein BMAL1 accelerates cognitive aging by disrupting microglial synaptic pruning.

Aging is associated with loss of circadian immune responses and circadian gene transcription in peripheral macrophages. Microglia, the resident macrophages of the brain, also show diurnal rhythmicity in regulating local immune responses and synaptic remodeling. To investigate the interaction between aging and microglial circadian rhythmicity, we examined mice deficient in the core clock transcription factor, BMAL1. Aging Cd11bcre;Bmallox/lox mice demonstrated accelerated cognitive decline in association with suppressed hippocampal long-term potentiation and increases in immature dendritic spines. C1q deposition at synapses and synaptic engulfment were significantly decreased in aging Bmal1-deficient microglia, suggesting that BMAL1 plays a role in regulating synaptic pruning in aging. In addition to accelerated age-associated hippocampal deficits, Cd11bcre;Bmallox/lox mice also showed deficits in the sleep-wake cycle with increased wakefulness across light and dark phases. These results highlight an essential role of microglial BMAL1 in maintenance of synapse homeostasis in the aging brain.

Neurexin-2: An inhibitory neurexin that restricts excitatory synapse formation in the hippocampus.

Neurexins are widely thought to promote synapse formation and to organize synapse properties. Here we found that in contrast to neurexin-1 and neurexin-3, neurexin-2 unexpectedly restricts synapse formation. In the hippocampus, constitutive or neuron-specific deletions of neurexin-2 nearly doubled the strength of excitatory CA3➔CA1 region synaptic connections and markedly increased their release probability. No effect on inhibitory synapses was detected. Stochastic optical reconstruction microscopy (STORM) superresolution microscopy revealed that the neuron-specific neurexin-2 deletion elevated the density of excitatory CA1 region synapses nearly twofold. Moreover, hippocampal neurexin-2 deletions also increased synaptic connectivity in the CA1 region when induced in mature mice and impaired the cognitive flexibility of spatial memory. Thus, neurexin-2 controls the dynamics of hippocampal synaptic circuits by repressing synapse assembly throughout life, a restrictive function that markedly differs from that of neurexin-1 and neurexin-3 and of other synaptic adhesion molecules, suggesting that neurexins evolutionarily diverged into opposing pro- and antisynaptogenic organizers.

GluD1 is a signal transduction device disguised as an ionotropic receptor.

Ionotropic glutamate delta receptors 1 (GluD1) and 2 (GluD2) exhibit the molecular architecture of postsynaptic ionotropic glutamate receptors, but assemble into trans-synaptic adhesion complexes by binding to secreted cerebellins that in turn interact with presynaptic neurexins1-4. It is unclear whether neurexin-cerebellin-GluD1/2 assemblies serve an adhesive synapse-formation function or mediate trans-synaptic signalling. Here we show in hippocampal synapses, that binding of presynaptic neurexin-cerebellin complexes to postsynaptic GluD1 controls glutamate receptor activity without affecting synapse numbers. Specifically, neurexin-1-cerebellin-2 and neurexin-3-cerebellin-2 complexes differentially regulate NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors by activating distinct postsynaptic GluD1 effector signals. Of note, minimal GluD1 and GluD2 constructs containing only their N-terminal cerebellin-binding and C-terminal cytoplasmic domains, joined by an unrelated transmembrane region, fully control the levels of NMDA and AMPA receptors. The distinct signalling specificity of presynaptic neurexin-1 and neurexin-35,6 is encoded by their alternatively spliced splice site 4 sequences, whereas the regulatory functions of postsynaptic GluD1 are mediated by conserved cytoplasmic sequence motifs spanning 5-13 residues. Thus, GluDs are signalling molecules that regulate NMDA and AMPA receptors by an unexpected transduction mechanism that bypasses their ionotropic receptor architecture and directly converts extracellular neurexin-cerebellin signals into postsynaptic receptor responses.

Cerebellin-2 regulates a serotonergic dorsal raphe circuit that controls compulsive behaviors.

Cerebellin-1 (Cbln1) and cerebellin-2 (Cbln2) are secreted glycoproteins that are expressed in distinct subsets of neurons throughout the brain. Cbln1 and Cbln2 simultaneously bind to presynaptic neurexins and postsynaptic GluD1 and GluD2, thereby forming trans-synaptic adhesion complexes. Genetic associations link cerebellins, neurexins and GluD's to neuropsychiatric disorders involving compulsive behaviors, such as Tourette syndrome, attention-deficit hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD). Extensive evidence implicates dysfunction of serotonergic signaling in these neuropsychiatric disorders. Here, we report that constitutive Cbln2 KO mice, but not Cbln1 KO mice, display robust compulsive behaviors, including stereotypic pattern running, marble burying, explosive jumping, and excessive nest building, and exhibit decreased brain serotonin levels. Strikingly, treatment of Cbln2 KO mice with the serotonin precursor 5-hydroxytryptophan or the serotonin reuptake-inhibitor fluoxetine alleviated compulsive behaviors. Conditional deletion of Cbln2 both from dorsal raphe neurons and from presynaptic neurons synapsing onto dorsal raphe neurons reproduced the compulsive behaviors of Cbln2 KO mice. Finally, injection of recombinant Cbln2 protein into the dorsal raphe of Cbln2 KO mice largely reversed their compulsive behaviors. Taken together, our results show that Cbln2 controls compulsive behaviors by regulating serotonergic circuits in the dorsal raphe.

Hypocretin (Orexin) Replacement Therapies.

Twenty-two years after their discovery, the hypocretins (Hcrts), also known as orexins, are two of the most studied peptidergic systems, involved in myriad physiological systems that range from sleep, arousal, motivation, homeostatic regulation, fear, anxiety and learning. A causal relationship between activity of Hcrt and arousal stability was established shortly after their discovery and have led to the development of a new class of drugs to treat insomnia. In this review we discuss the many faces of the Hcrt system and examine recent findings that implicate decreased Hcrt function in the pathogenesis of a number of neuropsychiatric conditions. We also discuss future therapeutic strategies to replace or enhance Hcrt function as a treatment option for these neuropsychiatric conditions.

Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs.

Cerebellins are important neurexin ligands that remain incompletely understood. Two critical questions in particular remain unanswered: do different cerebellins perform distinct functions, and do these functions act in the initial establishment of synapses or in rendering nascent synapses capable of normal synaptic transmission? Here we show that in mice, Cbln2 and Cbln4 are expressed in the medial habenula (MHb) nucleus in different types of neurons that project to distinct target neurons in the interpeduncular nucleus. Conditional genetic deletion of Cbln2 in the MHb impaired synaptic transmission at Cbln2+ synapses in the interpeduncular neurons within 3 wk, but decreased synapse numbers only after 3 mo, suggesting a functional, but not a structural, requirement for Cbln2 in synapses formed by Cbln2-expressing neurons. In contrast, genetic deletions of Cbln4 in the MHb had no major effect on synaptic transmission or synapse numbers in interpeduncular target neurons. Nevertheless, MHb ablation of both Cbln2 and Cbln4 significantly impaired behavioral responses in mice, but affected different types of behaviors. Specifically, Cbln2 MHb deletions decreased spatial learning, as measured in the water T-maze, whereas Cbln4 MHb deletions increased anxiety levels, as monitored in the open field test and elevated plus maze. Thus, Cbln2 and Cbln4 are expressed in distinct MHb neurons that contribute to different behaviors.

Genetic Ablation of All Cerebellins Reveals Synapse Organizer Functions in Multiple Regions Throughout the Brain.

Cerebellins are synaptic organizer molecules that bind to presynaptic neurexins and postsynaptic receptors. They are well studied in the cerebellum, but three of the four cerebellins (Cbln1, Cbln2, and Cbln4) are also broadly expressed outside of the cerebellum, suggesting that they perform general functions throughout the brain. Here, we generated male and female constitutive single (KO), double KO (dKO), and triple KO (tKO) mice of Cbln1, Cbln2, and Cbln4. We found that all constitutive cerebellin-deficient mice were viable and fertile, suggesting that cerebellins are not essential for survival. Cbln1/2 dKO mice exhibited salience-induced seizures that were aggravated in Cbln1/2/4 tKO mice, suggesting that all cerebellins contribute to brain function. As described previously, Cbln1 KO mice displayed major motor impairments that were aggravated by additional KO of Cbln2. Strikingly, the Cbln1/2 dKO did not cause alterations in synapse density in the hippocampus of young adult (1- and 2-month-old) mice, but produced a selective ∼50% decrease in hippocampal synapse density in the stratum lacunosum moleculare of the CA1 region and in the dentate gyrus of aging, 6-month-old mice. A similar decrease in excitatory synapse density was observed in the striatum and retrosplenial cortex. Behaviorally, the Cbln1 KO produced dramatic changes in motor behaviors that were partly aggravated by additional deletion of Cbln2 and/or Cbln4. Our results show that cerebellins are not essential for survival and do not contribute to initial synapse formation, but perform multiple functions throughout the brain; as a consequence, their ablation results in a delayed loss of synapses and in behavioral impairments.SIGNIFICANCE STATEMENT Cerebellins (Cbln1-4) are trans-synaptic cell adhesion molecules. In the cerebellum, Cbln1 functions as a bidirectional organizer of parallel fiber-Purkinje cell synapses by binding to presynaptic neurexins and postsynaptic GluRδ2. Little is known about the function of cerebellins outside of the cerebellum; therefore, the present study used single, double, and triple constitutive KO mice of Cbln1, Cbln2, and Cbln4 to analyze the overall function of cerebellins. We show that cerebellins act as important synaptic organizers in specific subsets of neurons and likely contribute to many different brain functions. We also show that cerebellins are not initially required for synapse formation, but rather for specification and long-term synapse maintenance and demonstrate that all cerebellins, not just Cbln1, contribute to brain function.

Cerebellins are differentially expressed in selective subsets of neurons throughout the brain.

Cerebellins are secreted hexameric proteins that form tripartite complexes with the presynaptic cell-adhesion molecules neurexins or 'deleted-in-colorectal-cancer', and the postsynaptic glutamate-receptor-related proteins GluD1 and GluD2. These tripartite complexes are thought to regulate synapses. However, cerebellins are expressed in multiple isoforms whose relative distributions and overall functions are not understood. Three of the four cerebellins, Cbln1, Cbln2, and Cbln4, autonomously assemble into homohexamers, whereas the Cbln3 requires Cbln1 for assembly and secretion. Here, we show that Cbln1, Cbln2, and Cbln4 are abundantly expressed in nearly all brain regions, but exhibit strikingly different expression patterns and developmental dynamics. Using newly generated knockin reporter mice for Cbln2 and Cbln4, we find that Cbln2 and Cbln4 are not universally expressed in all neurons, but only in specific subsets of neurons. For example, Cbln2 and Cbln4 are broadly expressed in largely non-overlapping subpopulations of excitatory cortical neurons, but only sparse expression was observed in excitatory hippocampal neurons of the CA1- or CA3-region. Similarly, Cbln2 and Cbln4 are selectively expressed, respectively, in inhibitory interneurons and excitatory mitral projection neurons of the main olfactory bulb; here, these two classes of neurons form dendrodendritic reciprocal synapses with each other. A few brain regions, such as the nucleus of the lateral olfactory tract, exhibit astoundingly high Cbln2 expression levels. Viewed together, our data show that cerebellins are abundantly expressed in relatively small subsets of neurons, suggesting specific roles restricted to subsets of synapses.

Knockout of G protein ß5 impairs brain development and causes multiple neurologic abnormalities in mice.

Gß5 is a divergent member of the signal-transducing G protein ß subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gß isoforms, Gß5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling proteins that contain G protein-γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gß5 is an essential stabilizer of such regulator of G protein signaling proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gß5 in the brain outside the visual system. We show here that mice lacking Gß5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe-walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gß5-deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gß5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression.