Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase.

The formation of primary cilia is a highly choreographed process that can be disrupted in developing neurons by overexpressing neuromodulatory G-protein-coupled receptors GPCRs or by blocking intraflagellar transport. Here, we examined the effects of overexpressing the ciliary GPCRs, 5HT6 and SSTR3, on cilia structure and the differentiation of neocortical neurons. Neuronal overexpression of 5HT6 and SSTR3 was achieved by electroporating mouse embryo cortex in utero with vectors encoding these receptors. We found that overexpression of ciliary GPCRs in cortical neurons, especially 5HT6, induced the formation of long (>30 µm) and often forked cilia. These changes were associated with increased levels of intraflagellar transport proteins and accelerated ciliogenesis in neonatal neocortex, the induction of which required Kif3a, an anterograde motor critical for cilia protein trafficking and growth. GPCR overexpression also altered the complement of signaling molecules within the cilia. We found that SSTR3 and type III adenylyl cyclase (ACIII), proteins normally enriched in neuronal cilia, were rarely detected in 5HT6-elongated cilia. Intriguingly, the changes in cilia structure were accompanied by changes in neuronal morphology. Specifically, disruption of normal ciliogenesis in developing neocortical neurons, either by overexpressing cilia GPCRs or a dominant-negative form of Kif3a, significantly impaired dendrite outgrowth. Remarkably, coexpression of ACIII with 5HT6 restored ACIII to cilia, normalized cilia structure, and restored dendrite outgrowth, effects that were not observed in neurons coexpressing ACIII and dominant-negative form of Kif3a. Collectively, our data suggest the formation of neuronal dendrites in developing neocortex requires structurally normal cilia enriched with ACIII.

Detection of primary cilia in human glioblastoma.

Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies.

A model of neocortical area patterning in the lissencephalic mouse may hold for larger gyrencephalic brains.

In the mouse, two telencephalic signaling centers orchestrate embryonic patterning of the cerebral cortex. From the rostral patterning center in the telencephalon, the Fibroblast Growth Factor, FGF8, disperses as a morphogen to establish the rostral to caudal axis of the neocortical area map. FGF8 coordinates with Wnt3a from the cortical hem to regulate graded expression of transcription factors that position neocortical areas, and control hippocampal development. Whether similar signaling centers pattern the much larger cortices of carnivore and primate species, however, is unclear. The limited dispersion range of FGF8 and Wnt3a is inconsistent with patterning larger cortical primordia. Yet the implication that different mechanisms organize cortex in different mammals flies in the face of the tenet that developmental patterning mechanisms are conserved across vertebrate species. In the present study, both signaling centers were identified in the ferret telencephalon, as were expression gradients of the patterning transcription factor genes regulated by FGF8 and Wnt3a. Notably, at the stage corresponding to the peak period of FGF8 signaling in the mouse neocortical primordium (NP), the NP was the same size in ferret and mouse, which would allow morphogen patterning of the ferret NP. Subsequently, the size of ferret neocortex shot past that of the mouse. Images from online databases further suggest that NP growth in humans, too, is slowed in early cortical development. We propose that if early growth in larger brains is held back, mechanisms that pattern the neocortical area map in the mouse could be conserved across mammalian species.

Type 3 Adenylyl Cyclase and Somatostatin Receptor 3 Expression Persists in Aged Rat Neocortical and Hippocampal Neuronal Cilia.

The primary cilia of forebrain neurons assemble around birth and become enriched with neuromodulatory receptors. Our understanding of the permanence of these structures and their associated signaling pathways in the aging brain is poor, but they are worthy of investigation because disruptions in neuronal cilia signaling have been implicated in changes in learning and memory, depression-like symptoms, and sleep anomalies. Here, we asked whether neurons in aged forebrain retain primary cilia and whether the staining characteristics of aged cilia for type 3 adenylyl cyclase (ACIII), somatostatin receptor 3 (SSTR3), and pericentrin resemble those of cilia in younger forebrain. To test this, we analyzed immunostained sections of forebrain tissues taken from young and aged male Fischer 344 (F344) and F344 × Brown Norway (F344 × BN) rats. Analyses of ACIII and SSTR3 in young and aged cortices of both strains of rats revealed that the staining patterns in the neocortex and hippocampus were comparable. Virtually every NeuN positive cell examined possessed an ACIII positive cilium. The lengths of ACIII positive cilia in neocortex were similar between young and aged for both strains, whereas in F344 × BN hippocampus, the cilia lengths increased with age in CA1 and CA3, but not in dentate gyrus (DG). Additionally, the percentages of ACIII positive cilia that were also SSTR3 positive did not differ between young and aged tissues in either strain. We also found that pericentrin, a protein that localizes to the basal bodies of neuronal cilia and functions in primary cilia assembly, persisted in aged cortical neurons of both rat strains. Collectively, our data show that neurons in aged rat forebrain possess primary cilia and that these cilia, like those present in younger brain, continue to localize ACIII, SSTR3, and pericentrin. Further studies will be required to determine if the function and signaling pathways regulated by cilia are similar in aged compared to young brain.

Influences of primary cilia on cortical morphogenesis and neuronal subtype maturation.

Recognition that virtually every neuronal progenitor cell and neuron in the cerebral cortex is ciliated has triggered intense interest in neuronal cilia function. Here, we review recent studies that suggest the primary cilia of cortical progenitor cells are required for establishing and maintaining the organization within pools of proliferative cells. In addition, signaling via primary cilia differentially influence the migration and differentiation of excitatory and inhibitory neurons in the developing cortex. Specifically, the primary cilia of excitatory neurons appear to play a significant role in regulating the post-migratory differentiation of these neurons whereas cilia of inhibitory neurons appear to be required for the proper migration and positioning of those cells in cortex. Given the recently discovered functions of cilia in proliferation, neuronal migration, and differentiation, it is likely that further studies of cilia signaling will improve our understanding of how these basic developmental processes are regulated and may provide insight into how mutations in specific cilia genes linked to ciliopathies lead to the many neurological deficits associated with these diseases.

Development and distribution of neuronal cilia in mouse neocortex.

Neuronal primary cilia are not generally recognized, but they are considered to extend from most, if not all, neurons in the neocortex. However, when and how cilia develop in neurons are not known. This study used immunohistochemistry for adenylyl cyclase III (ACIII), a marker of primary cilia, and electron microscopic analysis to describe the development and maturation of cilia in mouse neocortical neurons. Our results indicate that ciliogenesis is initiated in late fetal stages after neuroblast migration, when the mother centriole docks with the plasma membrane, becomes a basal body, and grows a cilia bud that we call a procilium. This procilium consists of a membranous protrusion extending from the basal body but lacking axonemal structure and remains undifferentiated until development of the axoneme and cilia elongation starts at about postnatal day 4. Neuronal cilia elongation and final cilia length depend on layer position, and the process extends for a long time, lasting 8-12 weeks. We show that, in addition to pyramidal neurons, inhibitory interneurons also grow cilia of comparable length, suggesting that cilia are indeed present in all neocortical neuron subtypes. Furthermore, the study of mice with defective ciliogenesis suggested that failed elongation of cilia is not essential for proper neuronal migration and laminar organization or establishment of neuronal polarity. Thus, the function of this organelle in neocortical neurons remains elusive.