Roles of a novel serine/threonine protein kinase Ulk4 in mouse brain development and functions
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SUMMARY UNC-51-like kinase 4 (ULK4) is a novel gene encoding a potential serine/threonine kinase (STK), and little is known about its functions. Limited studies have been carried out so far, and most of the current 12 publications are genetic studies, which suggest that ULK4 may be involved in hypertension, myeloma and neurodevelopmental disorders. We were the first to show that ULK4 gene can be a rare risk factor for neurodevelopmental/ neuropsychiatric disorders including schizophrenia, autism, bipolar disorder and depression (Lang, Pu et al. 2014). Increased copy number variations (CNVs) were detected in patients of schizophrenia and autism, and single nucleotide polymorphisms (SNP) of the ULK4 were also found to be associated with the major depression (Lang, Pu et al. 2014). Functional analyses of the ULK4 gene was carried out in vitro by a knockdown approach in neuroblastoma cells, which showed that depleted ULK4 expression disrupted the composition of microtubules, and compromised neuritogenesis and cell motility. ULK4 also modulated multiple signaling pathways including ERK, p38, PKC, and JNK, which were implicated in neurodevelopmental disorders and involved in stress response. Ulk4 expression was regulated by developmental cues, and there was a developmental switch in Ulk4 isoform expression during mouse brain development and neuronal maturation. I contributed to the publication (Lang, Pu et al. 2014) by providing evidence that targeted deletion was shown to compromise the integrity of the corpus callosum in newborn mice, a phenotype which is commonly associated with ciliopathies and neurodevelopmental disorders. We have additional unpublished data that ULK4 is deleted in a small proportion of patients with heterogeneous clinical features including developmental delay, language delay and severe intellectual disability. In a recent comparative study, we characterized the Ulk4 gene expression during Xenopus development, and found co-expression of the Ulk4 mRNA with Sox3 (a neural stem/progenitor cell marker) and Blbp (a radial glial marker) in the ventricular regions of the forebrain (Dominguez, Schlosser et al. 2015). However, in both mouse and human adult brains, ULK4 was co-expressed with GAD67+ GABAergic neurons in cerebral cortex and hippocampus (Lang, Pu et al. 2014), highlighting its potential function of ULK4 gene in excitation/inhibition balance. The aim of this PhD project is to investigate the roles of Ulk4 in brain development and function. I have carried out a systematic study in the Ulk4-/- mice and littermate controls, and the research outcomes of this PhD project are summarized in four Results Chapters, which demonstrate that (1) ULK4 gene is crucial for cilia development and flow of cerebrospinal fluid (CSF), (2) ULK4 is essential for white matter integrity and myelination, (3) ULK4 regulates cell cycle and neurogenesis, and (4) ULK4 regulates excitation and inhibition balance and anxiety-related behavior. Firstly in Chapter 3, I described that (1) Ulk4 is predominantly expressed in mouse ventricular system, (2) Ulk4-/- ependymal cells display reduced and disorganized cilia with abnormal microtubular ultrastructure, (3) CSF flow is functionally impaired in the Ulk4-/- mice, (4) Ulk4 regulates Foxj1, a master regulator of ciliogenesis and a range of other cilia components and ciliogenesis genes. This provides the subcellular and molecular mechanisms and networks how disruption of Ulk4 can lead to non-communicating hydrocephalus phenotype. I also proposed that Ulk4 may act as a scaffold protein, in this aspect, regulating different processes of ciliogenesis and coordinating cilia formation and ciliary beating, as Ulk4 lacks evolutionally conserved critical amino acid in the N-terminus (see Chapter3) as a kinase. In Chapter 4, I followed up the early observation that the integrity of the corpus callosum was compromised in Ulk4-/- newborn mice (Lang, Pu et al. 2014), and presented the evidence that (1) Ulk4 is involved in white matter integrity in postnatal mice, (2) oligodendrocyte production and myelination are significantly reduced in Ulk4-/- mice, and (3) the Ulk4-/- mice exhibit an increased neuro-inflammation which includes activation of astrocytes and microglia (see Chapter 4). In Chapter 5, I started with an initial observation that in the Ulk4-/- newborn mice the subventricular zone (SVZ), where adult neural stem cells reside, was substantially smaller. I followed up this initial observation and presented evidence that, (1) Ulk4 is expressed in a cell cycle–dependent manner in vivo and in vitro, with a peak of expression in the G2 and M phases of the cell cycle; (2) Ulk4 deficiency reduces middle neurogenesis and compromises the generation of layer II-IV pyramidal neurons, which are commonly implicated in neurodevelopmental disorders including schizophrenia; (3) In the absence of the Ulk4, the neural stem cell pool is dramatically reduced in the SVZ of newborn mice, which is the starting point of adult neurogenesis and neuronal renewal, and this will have a wide implication in both neurodevelopmental and neurodegenerative disorders. In addition, I identified a cluster of genes, which are involved in cell cycle regulation and neural stem cell proliferation, but dysregulated in the Ulk4-/- mice. This suggests that Ulk4 regulates neurogenesis through the Wnt signaling (see Chapter 4). In order to determine how Ulk4 deletion may impact on behavior, we performed a series of behavioral tests on the Ulk4+/- mice, in collaboration with Dr Michelle Roche in the NUI Galway and Dr Steve Clapcote in the University of Leeds. In Chapter 6, I described that Ulk4+/- mice display an anxiety-related phenotype. I also provided the evidence that a reduction in GABAergic neurons in amygdala and hippocampus may partially account for the mechanisms of the anxiety-related behavior in the Ulk4+/- mice. In addition, I presented the negative data that the immobility of depressive tests was not altered in forced swimming and tail suspension tests. In summary, I demonstrated, through this PhD project, that Ulk4 is critical for proper brain development and function. Deletion, mutation and/or polymorphism of the Ulk4 gene may have an implication in a wide range of neurodevelopmental and neuropsychiatric disorders.
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