Neuronal primary cilia regulate pyramidal cell positioning to the deep and superficial sublayers in the hippocampal CA1

It is well-recognized that primary cilia regulate embryonic neurodevelopment, but little is known about their roles in postnatal neurodevelopment. The striatum pyramidal (SP) of hippocampal CA1 consists of superficial and deep sublayers, however, it is not well understood how early- and late-born pyramidal neurons position to two sublayers postnatally. Here we show that neuronal primary cilia emerge after CA1 pyramidal cells have reached SP, but before final neuronal positioning. The axonemes of primary cilia of early-born neurons point to the stratum oriens (SO), whereas late-born neuronal cilia orient toward the stratum radiatum (SR), reflecting an inside-out lamination pattern. Neuronal primary cilia in SP undergo marked changes in morphology and orientation from postnatal day 5 (P5) to P14, concurrent with pyramidal cell positioning to the deep and superficial sublayers and with neuronal maturation. Transgenic overexpression of Arl13B, a protein regulating ciliogenesis, not only elongates primary cilia and promotes earlier cilia protrusion, but also affects centriole positioning and cilia orientation in SP. The centrioles of late-born neurons migrate excessively to cluster at SP bottom before primary cilia protrusion and a reverse movement back to the main SP. Similarly, this pull-back movement of centriole/cilia is also identified on late-born cortical pyramidal neurons, although early- and late-born cortical neurons display the same cilia orientation. Together, this study provides the first evidence demonstrating that late-born pyramidal neurons exhibit a reverse movement for cell positioning, and primary cilia regulate pyramidal neuronal positioning to the deep and superficial sublayers in the hippocampus.

An N-terminal Fusion Allele to Study Melanin Concentrating Hormone Receptor 1

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (Mchr1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

HTR6 and SSTR3 targeting to primary cilia

Primary cilia are hair-like projections of the cell membrane supported by an inner microtubule scaffold, the axoneme, which polymerizes out of a membrane-docked centriole at the ciliary base. By working as specialized signaling compartments, primary cilia provide an optimal environment for many G protein-coupled receptors (GPCRs) and their effectors to efficiently transmit their signals to the rest of the cell. For this to occur, however, all necessary receptors and signal transducers must first accumulate at the ciliary membrane. Serotonin receptor 6 (HTR6) and Somatostatin receptor 3 (SSTR3) are two GPCRs whose signaling in brain neuronal cilia affects cognition and is implicated in psychiatric, neurodegenerative, and oncologic diseases. Over a decade ago, the third intracellular loops (IC3s) of HTR6 and SSTR3 were shown to contain ciliary localization sequences (CLSs) that, when grafted onto non-ciliary GPCRs, could drive their ciliary accumulation. Nevertheless, these CLSs were dispensable for ciliary targeting of HTR6 and SSTR3, suggesting the presence of additional CLSs, which we have recently identified in their C-terminal tails. Herein, we review the discovery and mapping of these CLSs, as well as the state of the art regarding how these CLSs may orchestrate ciliary accumulation of these GPCRs by controlling when and where they interact with the ciliary entry and exit machinery via adaptors such as TULP3, RABL2 and the BBSome.

Artificial Intelligence Approaches to Assessing Primary Cilia

ABSTRACTCilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize neuronal cilia in images from both in vivo and in vitro samples. After using our trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and colocalization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.SUMMARYThe use of Artificial Intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

Differentiation of ciliated human midbrain-derived LUHMES neurons

ABSTRACTMany human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing timecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

Differentiation of ciliated human midbrain-derived LUHMES neurons

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing.LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain.In LUHMES, neuronal cilia biology can be investigated along a complete timeline: from proliferation through differentiation to mature neurons.Summary StatementWith LUHMES, a ciliated human neuronal cell model, the underlying “neurobiology” of cilia and ciliopathies can be investigated along a complete time line: from proliferation through differentiation to mature neurons.

Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

Ciliary microtubules are subject to post-translational modifications that act as a “Tubulin Code” to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

Kinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.