Ulk4 Deficiency Impairs Motile Cilia and CSF Flow
Material below summarizes the article Ulk4 Is Essential for Ciliogenesis and CSF Flow, published on July 20, 2016, in JNeurosci and authored by Min Liu, Zhenlong Guan, Qin Shen, Pierce Lalor, Una Fitzgerald, Timothy O'Brien, Peter Dockery, and Sanbing Shen.
Tiny hair-like subcellular structures called motile cilia project from the ependymal surface to ventricular cavities in the brain. Their synchronized beating drives directional flow of the cerebrospinal fluid (CSF). Defects in ciliary development and/or function, termed ciliopathies, impair CSF flow, leading to hydrocephalus.
In fact, ciliopathies are now recognized as an emerging class of devastating disorders with pleiotropic symptoms. These include Joubert syndrome, Bardet-Biedl syndrome, Meckel-Gruber syndrome, Oral–facial–digital syndrome type 1, Nephronophthisis, dyslexia, and schizophrenia, many of which are associated with hydrocephalus.
We recently demonstrated that the Unc51-like-kinase 4 (ULK4) is a rare risk factor for schizophrenia, autism, and bipolar disorder. We investigated the neuroanatomy of a mutant mouse, Ulk4 tm1a /tm1a. The mouse was created by an insertion of IRES-LacZ-PA reporter into the intron 6 of the Ulk4 loci, to express the lacZ reporter and to knockout the Ulk4 gene.
We compared homozygous newborn mice with wild-type littermate controls and observed a mild but significant dilation of the lateral ventricles (LV) and third ventricle (3V) in mutants. Further histological analyses of mouse brains at postnatal day 12 revealed a typical hydrocephalus phenotype, with 17.5-fold enlargement of the LV and approximately five-fold dilation of the 3V. However, the morphology of subcommissural organ (SCO), the sizes of aqueduct and the fourth ventricle (4V) were not significantly altered.
These data suggest the Ulk4 hydrocephalus is congenital, the blockage is at the aqueduct level, and the phenotype gets severer during early postnatal development, coinciding with the genesis that drive CSF flow.
To examine CSF flow in Ulk4 mutants, we injected Evans Blue dye into P12 LV. The dye can bind to serum albumin in the CSF with a high affinity and be circulated with the CSF. In WT mice, a well-restrained distribution of the dye was detected throughout the brain cavities 20 minutes after injection, from the LV, 3V, SCO, aqueduct, 4V to the spinal canal. However, in the mutants, there was little evidence of directional CSF flow. Diffused dye was observed in the LV and 3V, and almost nothing reached to the SCO, aqueduct, 4V, or spinal canal.
Therefore, the functional analyses consistently demonstrate an impaired CSF circulation in the Ulk4 mutants.
Directional flow of the CSF from the anterior to posterior brain requires coordinated beating of ependymal cilia. Is Ulk4 gene expressed in ependymal cells?
We took advantage of the IRES-lacZ knock-in and carried out X-gal staining. While weak β-gal activity was detected in the choroid plexus where the CSF was produced, the most intense X-gal staining was identified in the ependymal layer of the brain ventricles, which were associated with motile cilia. Remarkably, LacZ was abundantly expressed in the lateral wall of the aqueduct with motile cilia, but not on the roof of the aqueduct which was occupied by non-ciliated cells. Thus, Ulk4 are highly expressed in multi-ciliated cells in brain cavities.
Motile cilia are largely developed and matured in the first two postnatal weeks in mice. What do mutant cilia look like?
To answer this question, we stained P12 brain with an antibody against acetylated α-tubulin, a ciliary marker, and observed highly organized cilium bundles on the WT ependymal wall, but less dense and disorganized cilia in Ulk4tm1a/tm1a cells.
To further confirm this, we undertook scanning electron microscopy and showed that arrays of densely populated ciliary bundles in wild-type animals were orientated towards the same direction, an indication of coordinated directional beating. However, in the homozygous mutants, the number of cilium bundles was dramatically reduced, and the mutant cilia were highly disorganized and randomly scattered on the ependymal surface, implying of dysfunctional cilia with no coordinated beating.
While non-motile primary cilia possess nine pairs of peripheral microtubular doublets (“9+0”), motile cilia also have a central pair of microtubular doublet (“9+2”). We deployed transmission electron microscopy and examined the ultra-structure of motile cilia. Whereas a typical feature of “9+2” axonemal structure was consistently present in the wild-type mice, this was replaced with a mixture of “9+2”, “9+0”, “8+4” and “8+0” ultrastructure in the Ulk4 mutants.
During ciliogenesis, basal bodies are originated from amplified centrioles. On its way to cell surface, centriole forms a complex with cytoplasmic membrane vesicles and then fuses with the plasma membrane. The axonemal microtubules will then extend from the basal body and project to the ventricular cavity. The position of the basal body determines cilium orientation. In WT ependymal cells, clustered basal bodies were aligned in parallel and formed the root for coordinated beating of multi-cilia, which were fully developed, matured, and oriented in the same direction in individual ependymal cells. However, in Ulk4tm1a/tm1a mice, the basal bodes were dys-arrayed, and the ependymal cilia were markedly fewer.
All together, these data demonstrate fewer, disorganized, structurally fault and functionally impaired ependymal cilia in Ulk4tm1a/tm1a mice.
How can this happen? Ulk4 is not a transcriptional factor, but belongs to a relatively novel Ser/Thr kinase family, and nothing is known about its mechanism.
We next carried out whole genome RNA sequencing and obtained quantitative reads of 19,652 genes from cortical RNA. Bioinformatics analyses identified 1,824 significantly up-regulated genes and 1,005 down-regulated genes in Ulk4tm1a/tm1a cortex, which could be a mixture of the first and secondary targets of Ulk4. Pathway analyses revealed that Foxj1 gene was specifically dysregulated, together with a series of other molecules involved in centriole amplification, basal body alignment, assembly of microtubular doublets and cilia beating.
Thus, we propose Ulk4 as a scaffold protein and a key player during ciliogenesis, which modulates expression of the master ciliogenesis regulator Foxj1 and other ciliogeneic factors, thereby regulating ciliary development, axonemal structure, coordinated beating, and CSF flow. Deficiency of the Ulk4 leads to malformation and dysfunction of cilia and causes hydrocephalus. ULK4 may therefore become a drug target for ciliopathy-related disorders.
Ulk4 Is Essential for Ciliogenesis and CSF Flow. Liu M, Guan Z, Shen Q, Lalor P, Fitzgerald U, O'Brien T, Dockery P, Shen S. JNeuroSci Jul 20; 36(29):7589-600. DOI: 10.1523/JNEUROSCI.0621-16.2016.