Changes in cytokine and cytokine receptor levels during postnatal development of the human dorsolateral prefrontal cortex.

In addition to their traditional roles in immune cell communication, cytokines regulate brain development. Cytokines are known to influence neural cell generation, differentiation, maturation, and survival. However, most work on the role of cytokines in brain development investigates rodents or focuses on prenatal events. Here, we investigate how mRNA and protein levels of key cytokines and cytokine receptors change during postnatal development of the human prefrontal cortex. We find that most cytokine transcripts investigated (IL1B, IL18, IL6, TNF, IL13) are lowest at birth and increase between 1.5 and 5 years old. After 5 years old, transcriptional patterns proceeded in one of two directions: decreased expression in teens and young adults (IL1B, p = 0.002; and IL18, p = 0.004) or increased mean expression with maturation, particularly in teenagers (IL6, p = 0.004; TNF, p = 0.002; IL13, p < 0.001). In contrast, cytokine proteins tended to remain elevated after peaking significantly around 3 years of age (IL1B, p = 0.012; IL18, p = 0.026; IL6, p = 0.039; TNF, p < 0.001), with TNF protein being highest in teenagers. An mRNA-only analysis of cytokine receptor transcripts found that early developmental increases in cytokines were paralleled by increases in their ligand-binding receptor subunits, such as IL1R1 (p = 0.033) and IL6R (p < 0.001) transcripts. In contrast, cytokine receptor-associated signaling subunits, IL1RAP and IL6ST, did not change significantly between age groups. Of the two TNF receptors, the 'pro-death' TNFRSF1A and 'pro-survival' TNFRSF1B, only TNFRSF1B was significantly changed (p = 0.028), increasing first in toddlers and again in young adults. Finally, the cytokine inhibitor, IL13, was elevated first in toddlers (p = 0.006) and again in young adults (p = 0.053). While the mean expression of interleukin-1 receptor antagonist (IL1RN) was highest in toddlers, this increase was not statistically significant. The fluctuations in cytokine expression reported here support a role for increases in specific cytokines at two different stages of human cortical development. The first is during the toddler/preschool period (IL1B, IL18, and IL13), and the other occurs at adolescence/young adult maturation (IL6, TNF and IL13).

Reduced adult neurogenesis is associated with increased macrophages in the subependymal zone in schizophrenia.

Neural stem cells in the human subependymal zone (SEZ) generate neuronal progenitor cells that can differentiate and integrate as inhibitory interneurons into cortical and subcortical brain regions; yet the extent of adult neurogenesis remains unexplored in schizophrenia and bipolar disorder. We verified the existence of neurogenesis across the lifespan by chartering transcriptional alterations (2 days-103 years, n = 70) and identifying cells indicative of different stages of neurogenesis in the human SEZ. Expression of most neural stem and neuronal progenitor cell markers decreased during the first postnatal years and remained stable from childhood into ageing. We next discovered reduced neural stem and neuronal progenitor cell marker expression in the adult SEZ in schizophrenia and bipolar disorder compared to controls (n = 29-32 per group). RNA sequencing identified increased expression of the macrophage marker CD163 as the most significant molecular change in schizophrenia. CD163+ macrophages, which were localised along blood vessels and in the parenchyma within 10 µm of neural stem and progenitor cells, had increased density in schizophrenia but not in bipolar disorder. Macrophage marker expression negatively correlated with neuronal progenitor marker expression in schizophrenia but not in controls or bipolar disorder. Reduced neurogenesis and increased macrophage marker expression were also associated with polygenic risk for schizophrenia. Our results support that the human SEZ retains the capacity to generate neuronal progenitor cells throughout life, although this capacity is limited in schizophrenia and bipolar disorder. The increase in macrophages in schizophrenia but not in bipolar disorder indicates that immune cells may impair neurogenesis in the adult SEZ in a disease-specific manner.

Deletion of POMT2 in Zebrafish Causes Degeneration of Photoreceptors.

Mutations in the extracellular matrix protein eyes shut homolog (EYS) are a common cause of retinitis pigmentosa, a blinding disease characterized by photoreceptor degeneration. EYS binds to matriglycan, a carbohydrate modification on O-mannosyl glycan substitutions of the cell-surface glycoprotein α-dystroglycan. Patients with mutations in enzymes required for the biosynthesis of matriglycan exhibit syndromic retinal atrophy, along with brain malformations and congenital muscular dystrophy. Protein O-mannosyltransferase 2 (POMT2) is an enzyme required for the synthesis of O-mannosyl glycans. To evaluate the roles of O-mannosyl glycans in photoreceptor health, we generated protein O-mannosyltransferase 2 (pomt2) mutant zebrafish by CRISPR. pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to α-dystroglycan. Mutant zebrafish presented with hydrocephalus and hypoplasia of the cerebellum, as well as muscular dystrophy. EYS protein was enriched near photoreceptor connecting cilia in the wild-type, but its presence and proper localization was significantly reduced in mutant animals. The mutant retina exhibited mis-localization of opsins and increased apoptosis in both rod and cone photoreceptors. Immunofluorescence intensity of G protein subunit alpha transducin 2 (GNAT2) antibody (a general cone marker) and 1D4 antibody (a long double cone marker) in mutant retinas did not differ from wild-type retinas at 1-month post fertilization, but was reduced at 6 months post fertilization, indicating significant cone degeneration. These data suggest that POMT2-mediated O-mannosyl glycosylation is required for EYS protein localization to the connecting cilium region and photoreceptor survival.

Interactions between C8orf37 and FAM161A, Two Ciliary Proteins Essential for Photoreceptor Survival.

Mutations in C8orf37 cause Bardet-Biedl syndrome (BBS), retinitis pigmentosa (RP), and cone-rod dystrophy (CRD), all manifest in photoreceptor degeneration. Little is known about which proteins C8orf37 interacts with to contribute to photoreceptor survival. To determine the proteins that potentially interact with C8orf37, we carried out a yeast two-hybrid (Y2H) screen using C8orf37 as a bait. FAM161A, a microtubule-binding protein localized at the photoreceptor cilium required for photoreceptor survival, was identified as one of the preys. Double immunofluorescence staining and proximity ligation assay (PLA) of marmoset retinal sections showed that C8orf37 was enriched and was co-localized with FAM161A at the ciliary base of photoreceptors. Epitope-tagged C8orf37 and FAM161A, expressed in HEK293 cells, were also found to be co-localized by double immunofluorescence staining and PLA. Furthermore, interaction domain mapping assays identified that the N-terminal region of C8orf37 and amino acid residues 341-517 within the PFAM UPF0564 domain of FAM161A were critical for C8orf37-FAM161A interaction. These data suggest that the two photoreceptor survival proteins, C8orf37 and FAM161A, interact with each other which may contribute to photoreceptor health.

Trajectory of change in brain complement factors from neonatal to young adult humans.

Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F = 2.41 -3.325, p = .01-0.05). We found that C4 protein was higher at 1-5 years (H = 16.378, p = .012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F = 4.186, p = .003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F > 4.4, p < .01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5 years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.

A schizophrenia subgroup with elevated inflammation displays reduced microglia, increased peripheral immune cell and altered neurogenesis marker gene expression in the subependymal zone.

Inflammation regulates neurogenesis, and the brains of patients with schizophrenia and bipolar disorder have reduced expression of neurogenesis markers in the subependymal zone (SEZ), the birthplace of inhibitory interneurons. Inflammation is associated with cortical interneuron deficits, but the relationship between inflammation and reduced neurogenesis in schizophrenia and bipolar disorder remains unexplored. Therefore, we investigated inflammation in the SEZ by defining those with low and high levels of inflammation using cluster analysis of IL6, IL6R, IL1R1 and SERPINA3 gene expression in 32 controls, 32 schizophrenia and 29 bipolar disorder cases. We then determined whether mRNAs for markers of glia, immune cells and neurogenesis varied with inflammation. A significantly greater proportion of schizophrenia (37%) and bipolar disorder cases (32%) were in high inflammation subgroups compared to controls (10%, p < 0.05). Across the high inflammation subgroups of psychiatric disorders, mRNAs of markers for phagocytic microglia were reduced (P2RY12, P2RY13), while mRNAs of markers for perivascular macrophages (CD163), pro-inflammatory macrophages (CD64), monocytes (CD14), natural killer cells (FCGR3A) and adhesion molecules (ICAM1) were increased. Specific to high inflammation schizophrenia, quiescent stem cell marker mRNA (GFAPD) was reduced, whereas neuronal progenitor (ASCL1) and immature neuron marker mRNAs (DCX) were decreased compared to low inflammation control and schizophrenia subgroups. Thus, a heightened state of inflammation may dampen microglial response and recruit peripheral immune cells in psychiatric disorders. The findings elucidate differential neurogenic responses to inflammation within psychiatric disorders and highlight that inflammation may impair neuronal differentiation in the SEZ in schizophrenia.

Eyes shut homolog (EYS) interacts with matriglycan of O-mannosyl glycans whose deficiency results in EYS mislocalization and degeneration of photoreceptors.

Mutations in eyes shut homolog (EYS), a secreted extracellular matrix protein containing multiple laminin globular (LG) domains, and in protein O-mannose ß1, 2-N-acetylglucosaminyl transferase 1 (POMGnT1), an enzyme involved in O-mannosyl glycosylation, cause retinitis pigmentosa (RP), RP25 and RP76, respectively. How EYS and POMGnT1 regulate photoreceptor survival is poorly understood. Since some LG domain-containing proteins function by binding to the matriglycan moiety of O-mannosyl glycans, we hypothesized that EYS interacted with matriglycans as well. To test this hypothesis, we performed EYS Far-Western blotting assay and generated pomgnt1 mutant zebrafish. The results showed that EYS bound to matriglycans. Pomgnt1 mutation in zebrafish resulted in a loss of matriglycan, retention of synaptotagmin-1-positive EYS secretory vesicles within the outer nuclear layer, and diminished EYS protein near the connecting cilia. Photoreceptor density in 2-month old pomgnt1 mutant retina was similar to the wild-type animals but was significantly reduced at 6-months. These results indicate that EYS protein localization to the connecting cilia requires interaction with the matriglycan and that O-mannosyl glycosylation is required for photoreceptor survival in zebrafish. This study identified a novel interaction between EYS and matriglycan demonstrating that RP25 and RP76 are mechanistically linked in that O-mannosyl glycosylation controls targeting of EYS protein.