Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Protocadherin-αC2 is required for diffuse projections of serotonergic axons.

Serotonergic axons extend diffuse projections throughout various brain areas, and serotonergic system disruption causes neuropsychiatric diseases. Loss of the cytoplasmic region of protocadherin-α (Pcdh-α) family proteins, products of the diverse clustered Pcdh genes, causes unbalanced distributions (densification and sparsification) of serotonergic axons in various target regions. However, which Pcdh-α member(s) are responsible for the phenotype is unknown. Here we demonstrated that Pcdh-αC2 (αC2), a Pcdh-α isoform, was highly expressed in serotonergic neurons, and was required for normal diffusion in single-axon-level analyses of serotonergic axons. The loss of αC2 from serotonergic neurons, but not from their target brain regions, led to unbalanced distributions of serotonergic axons. Our results suggest that αC2 expressed in serotonergic neurons is required for serotonergic axon diffusion in various brain areas. The αC2 extracellular domain displays homophilic binding activity, suggesting that its homophilic interaction between serotonergic axons regulates axonal density via αC2's cytoplasmic domain.

Spinal RacGAP α-Chimaerin Is Required to Establish the Midline Barrier for Proper Corticospinal Axon Guidance.

In the developing CNS, the midline barrier, which comprises guidance molecule-expressing midline glial somata and processes, plays a pivotal role in midline axon guidance. Accumulating evidence has revealed the molecular mechanisms by which the midline barrier ensures proper midline guidance for axons. In contrast, the mechanisms for establishing the midline barrier remain obscure. Here, we report that Rac-specific GTPase-activating protein (RacGAP) α-chimaerin is required for both axonal repulsion at and establishment of the midline barrier in the spinal cord. We generated cortex-specific and spinal-cord-specific α-chimaerin gene (Chn1) knock-out mice (Cx-Chn1KO and Sp-Chn1KO mice, respectively) and found that both showed aberrant corticospinal tract (CST) axon midline crossing in the spinal cord. Strikingly, Sp-Chn1KO mice had breaks (holes) in the ephrinB3(+) spinal midline barrier and EphA4(+) CST axons aberrantly crossed the midline through these holes. During normal embryonic development, EphA4(+) spinal cells are located in juxta-midline areas but are excluded from the midline. In contrast, in Chn1KO embryos, several EphA4(+) cells were aberrantly relocated into the midline and the midline barrier was broken around these cells. Similarly, the spinal cord midline of Epha4KO mice was invaded by juxta-midline EphA4 cells (i.e., Epha4 promoter-active cells) during the embryonic stage and holes were formed in the midline barrier. Juxta-midline EphA4 cells in the spinal cord expressed α-chimaerin. We propose that spinal α-chimaerin aids in establishing an intact spinal midline barrier by mediating juxta-midline EphA4(+) cell repulsion, thus preventing these cells from breaking into the ephrinB3(+) midline barrier.SIGNIFICANCE STATEMENT The midline barrier plays a critical role in midline axon guidance, which is fundamental to the formation of neural circuits that are responsible for proper left-right coordination of the body. Studies have revealed some of the mechanisms underlying how the midline barrier navigates axons. In contrast, the establishment of the midline barrier during embryonic development remains unclear. In this study, we determined that α-chimaerin is required for the formation of an intact midline barrier. Spinal-cord-specific α-chimaerin knock-out mice had spinal midline barriers with numerous breaks (holes), through which corticospinal axons aberrantly crossed the midline. We propose that α-chimaerin protects the midline barrier by mediating cell-repulsive signaling in juxta-midline cells, which prevents these cells from invading the midline.

Total expression and dual gene-regulatory mechanisms maintained in deletions and duplications of the Pcdha cluster.

The clustered protocadherin-alpha (Pcdha) genes, which are expressed in the vertebrate brain, encode diverse membrane proteins whose functions are involved in axonal projection and in learning and memory. The Pcdha cluster consists of 14 tandemly arranged genes (Pcdha1-Pcdha12, Pcdhac1, and Pcdhac2, from 5' to 3'). Each first exon (the variable exons) is transcribed from its own promoter, and spliced to the constant exons, which are common to all the Pcdha genes. Cerebellar Purkinje cells show dual expression patterns for Pcdha. In individual Purkinje cells, different sets of the 5' genes in the cluster, Pcdha1-12, are randomly expressed, whereas both 3' genes, Pcdhac1 and Pcdhac2, are expressed constitutively. To elucidate the relationship between the genomic structure of the Pcdha cluster and their expression in Purkinje cells, we deleted or duplicated multiple variable exons and analyzed the expression of Pcdha genes in the mouse brain. In all mutant mice, transcript levels of the constant exons and the dual expression patterns were maintained. In the deletion mutants, the missing genes were flexibly compensated by the remaining variable exons. On the other hand, in duplication mutants, the levels of the duplicated genes were trimmed. These results indicate that the Pcdha genes are comprehensively regulated as a cluster unit, and that the regulators that randomly and constitutively drive Pcdha gene expression are intact in the deleted or duplicated mutant alleles. These dual regulatory mechanisms may play important roles in the diversity and fundamental functions of neurons.

Protocadherin-alpha family is required for serotonergic projections to appropriately innervate target brain areas.

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain, where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles, we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons, which are common to the gene cluster. In the first week after birth, the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type, but by 3 weeks of age, when the serotonergic axonal termini complete their arborizations, the distribution of the projections was abnormal. In some target regions, notably the globus pallidus and substantia nigra, the normally even distribution of serotonin axonal terminals was, in the mutants, dense at the periphery of each region, but sparse in the center. In the stratum lacunosum-molecular of the hippocampus, the mutants showed denser serotonergic innervation than in wild-type, and in the dentate gyrus of the hippocampus and the caudate-putamen, the innervation was sparser. Together, the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections.

Down-regulation of protocadherin-alpha A isoforms in mice changes contextual fear conditioning and spatial working memory.

Diverse protocadherins (Pcdhs), which are encoded as a large cluster (composed of alpha, beta and gamma clusters) in the genome, are localized to axons and synapses. The Pcdhs have been proposed to contribute to the generation of sophisticated neural networks and to regulate brain function. To address the molecular roles of Pcdhs in regulating individual behavior, here we generated knockdown mice of Pcdh-alpha proteins and examined their behavioral abnormalities. There are two alternative splicing variants of the Pcdh-alpha constant region, Pcdh-alpha A and B isoforms, with different cytoplasmic tails. Pcdh-alpha(DeltaBneo/DeltaBneo) mice, in which the Pcdh-alpha B splicing variant was absent and the Pcdh-alpha A isoforms were down-regulated to approximately 20% of the wild-type level, exhibited enhanced contextual fear conditioning and disparities in an eight-arm radial maze. Similar abnormalities were found in Pcdh-alpha(DeltaAneo/DeltaAneo) mice, which lacked 57 amino acids of the Pcdh-alpha A cytoplasmic tail. These learning abnormalities were, however, not seen in Pcdh-alpha(DeltaB/DeltaB) mice [in which the neomycin-resistance (neo) gene cassette was removed from the Pcdh-alpha(DeltaBneo/DeltaBneo) alleles], in which the expression level of the Pcdh-alpha A isoforms was recovered, although the Pcdh-alpha B isoforms were still completely missing in the brain. In addition, the amount of 5-hydroxytryptamine increased in the hippocampus of the hypomorphic Pcdh-alpha A mutant mice but not in recovery Pcdh-alpha(DeltaB/DeltaB). These results suggested that the level of Pcdh-alpha A isoforms in the brain has an important role in regulating learning and memory functions and the amount of 5-hydroxytryptamine in the hippocampus.

The protocadherin-alpha family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse.

Olfactory sensory neurons (OSNs) that express the same odorant receptor project their axons to specific glomeruli in the main olfactory bulb. Protocadherin-alpha (Pcdha) proteins, diverse cadherin-related molecules that are encoded as a gene cluster, are highly concentrated in OSN axons and olfactory glomeruli. Here, we describe Pcdha mutant mice, in which the constant region of the Pcdha gene cluster has been deleted by gene targeting. The mutant mice show abnormal sorting of OSN axons into glomeruli. There are multiple, small, extraneous glomeruli for the odorant receptors M71 and MOR23. These abnormal patterns of M71 and MOR23 glomeruli persist until adulthood. Many M71 glomeruli, but apparently not MOR23 glomeruli, are heterogeneous in axonal innervation. Thus, Pcdha molecules are involved in coalescence of OSN axons into OR-specific glomeruli of the olfactory bulb.