Association of Unc-51-like Kinase 4 (ULK4) with the reactivity of the extended reward system in response to conditioned stimuli.

OBJECTIVES: ULK4 is an established candidate gene for mental disorders and antipsychotic treatment response. We investigated the association of functional genetic variation at the ULK4 locus with the human extended dopaminergic reward system using fMRI during the performance of a well-established reward paradigm. METHODS: Two hundred and thirty-four patients were included in this study. Association of genetic variation in the ULK4 gene with reward system functioning were determined using the Desire-Reason-Dilemma (DRD) paradigm which allows to assess brain activation in response to conditioned reward stimuli. RESULTS: Variant prioritisation revealed the strongest functional signatures for the ULK4 variant rs17215589, coding for amino acid exchange Ala715Thr. For rs17215589 minor allele carriers, we detected increased activation responses to conditioned reward stimuli in the ventral tegmental area, nucleus accumbens and several cortical brain regions of the extended reward system. CONCLUSIONS: Our findings provide further evidence in humans that genetic variation in ULK4 may increase the vulnerability to mental disorders, by modulating the extended reward system function. Future studies are needed to confirm the modulation of the extended reward system by ULK4 and to specify the role of this mechanism in the pathogenesis of psychiatric disorders.

Ulk4 promotes Shh signaling by regulating Stk36 ciliary localization and Gli2 phosphorylation.

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism has remained poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and with Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.

The putative protein kinase Stk36 is essential for ciliogenesis and CSF flow by associating with Ulk4.

Motile cilia lining on the ependymal cells are crucial for cerebrospinal fluid (CSF) flow and its dysfunction is often associated with hydrocephalus. Unc51-like-kinase 4 (Ulk4) was previously linked to CSF flow and motile ciliogenesis in mice, as the hypomorph mutant of Ulk4 (Ulk4tm1a/tm1a ) developed hydrocephalic phenotype resulted from defective ciliogenesis and disturbed ciliary motility, while the underling mechanism is largely obscure. Here, we report that serine/threonine kinase 36 (STK36), a paralog of ULK4, directly interacts with ULK4 and this was demonstrated by yeast two-hybrid (Y2H) in yeast and coimmunoprecipitation (co-IP) assays in HEK293T cells, respectively. The interaction region was confined to their respective N-terminal kinase domain. The hypomorph mutant of Stk36 (Stk36tmE4-/- ) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. Stk36tmE4-/- also disturbed the expression of Foxj1 transcription factor and a range of other ciliogenesis-related genes. All these morphological changes, motile cilia defects and transcriptional dysregulation in the Stk36tmE4-/- are practically copied from that in Ulk4tm1a/tm1a mice. Taken together, we conclude that both Stk36 and Ulk4 are crucial for CSF flow, they cooperate by direct binding with their kinase domain to regulate the Foxj1 transcription factor pathways for ciliogenesis and cilia function, not limited to CSF flow. The underlying molecular mechanism probably conserved in evolution and could be extended to other metazoans.

Association of genetic variants in ULK4 with the age of first onset of type B aortic dissection.

Background: The association between autophagy, structural alterations of the aortic wall, and endothelial dysfunction in humans has yet to be fully elucidated. The family of ULK (UNC51-like) enzymes plays critical roles in autophagy and development. This study aimed to evaluate the association between ULK gene family members and patient age of first type B aortic dissection (TBAD) onset. Methods: The genotype data in a TBAD cohort from China and the related summary-level datasets were analyzed. We applied the sequence kernel association test (SKAT) to test the association between single-nucleotide polymorphisms (SNPs) and age of first onset of TBAD controlling for gender, hypertension, and renal function. Next, we performed a 2-sample Mendelian randomization (MR) to explore the potential causal relationship between ULK4 and early onset of TBAD at the level of gene expression coupled with DNA methylation with genetic variants as instrumental variables. Results: A total of 159 TBAD patients with 1,180,097 SNPs were included. Concerning the association between the ULK gene family and the age of first onset of the TBAD, only ULK4 was found to be significant according to SKAT analysis (q-FDR = 0.0088). From 2-sample MR, the high level of ULK4 gene expression was related to a later age of first onset of TBAD (ß = 4.58, p = 0.0214). Conclusion: This is the first study of the ULK gene family in TBAD, regarding the association with the first onset age. We demonstrated that the ULK4 gene is associated with the time of onset of TBAD based on both the SKAT and 2-sample MR analyses.

ULK4 in Neurodevelopmental and Neuropsychiatric Disorders.

The gene Unc51-like kinase 4 (ULK4) belongs to the Unc-51-like serine/threonine kinase family and is assumed to encode a pseudokinase with unclear function. Recently, emerging evidence has suggested that ULK4 may be etiologically involved in a spectrum of neuropsychiatric disorders including schizophrenia, but the underlying mechanism remains unaddressed. Here, we summarize the key findings of the structure and function of the ULK4 protein to provide comprehensive insights to better understand ULK4-related neurodevelopmental and neuropsychiatric disorders and to aid in the development of a ULK4-based therapeutic strategy.

Schizophrenia predisposition gene Unc-51-like kinase 4 for the improvement of cerebral ischemia/reperfusion injury.

BACKGROUND: Cerebral ischemia/reperfusion injury (CIRI) has complex pathogenesis, and inhibiting apoptosis and supporting neural progenitor proliferation are extremely beneficial strategies for treating CIRI. Unc-51-like kinase 4 (ULK4), a susceptibility gene for schizophrenia, promotes neural progenitors proliferation. The phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in CIRI via inhibition of apoptosis. Therefore, the relationship among ULK4, the PI3K pathway, and apoptosis in the context of CIRI has attracted our great interest. METHODS AND RESULTS: Primary cortical neurons were subjected to oxygen-glucose deprivation/reperfusion (OGD/R), and rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Transfection of the ULK4-overexpression lentivirus was performed alone or in combination with PI3K inhibitor treatment. Here, we revealed that ULK4 was poorly expressed in the cortex in MCAO/R rats and OGD/R-treated primary cortical neurons, ULK4 overexpression inhibited apoptosis, and reduced neurological deficit scores, cerebral infarct volume, and histopathological damage. Moreover, ULK4 overexpression increased PI3K expression and the p-protein kinase B/AKT and p-glycogen synthase kinase 3 beta (GSK3ß)/GSK3ß ratios, and inhibited apoptosis, while a PI3K inhibitor reversed the effects of ULK4 overexpression on CIRI. CONCLUSIONS: ULK4 protects against CIRI, and the underlying mechanism involves PI3K pathway activation which in turn inhibits apoptosis.

Ulk4, a Newly Discovered Susceptibility Gene for Schizophrenia, Regulates Corticogenesis in Mice.

Schizophrenia (SCZ) is a chronic and severe mental disease that affects around 1% of the population. The precise etiology of SCZ still remains largely unknown, and no conclusive mechanisms are firmly established. Recent advances in epidemiological and clinical investigation support an overwhelmingly strong neurodevelopmental origin for SCZ. Here, we demonstrated that Unc-51-like kinase 4 (Ulk4), a novel risk factor for major mental disorders including schizophrenia, is involved in the corticogenesis. Deletion of Ulk4 in mice led to significantly thinner layers of II-III, and V in the cerebral cortex, which was confirmed in conditional Ulk4 deletion mice achieved by Cre-loxp strategy. This abnormality might be caused by decreased intermediate neural progenitors and increased apoptosis. Thus, our data suggest that Ulk4 manipulates the behaviors of neural progenitors during brain development and, when functionally defective, leads to the reduction of specific cortical layers. This anomaly may increase predisposition to a range of neurodevelopmental disorders, including SCZ.

Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4.

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the co-factor is required for structural stability of ULK4. Here, we present a high-resolution structure of a ULK4-ATPγS complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop, which stabilizes the C helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin-associated proteins and several active kinases suggesting interesting regulatory roles for ULK4.

Intragenic Microdeletion of ULK4 and Partial Microduplication of BRWD3 in Siblings with Neuropsychiatric Features and Obesity.

ULK4 and BRWD3 deletions have been identified in patients with developmental/language delay and intellectual disability. Both genes play pivotal roles in brain development. In particular, ULK4 encodes serine/threonine kinases that are critical for the development and function of the nervous system, while BRWD3 plays a crucial role in ubiquitination, as part of the ubiquitin/proteasome system. We report on 2 brothers, aged 7.6 and 20 years, presenting with cognitive impairment, epilepsy, autistic features, hearing loss, and obesity. Array-CGH analysis demonstrated 2 rare CNVs in both siblings: a paternally inherited microdeletion of ∼145 kb at 3p22.1, disrupting the ULK4 gene, and a maternally inherited microduplication of ∼117 kb at Xq21.1 including only the BRWD3 gene. As already described for other recurrent syndromes with variable phenotype, these findings are challenging in genetic counseling because of an evident variable penetrance. We discuss the possible correlations between the clinical phenotype of our patients and the function of the genes involved in these microrearrangements.

Ulk4 deficiency leads to hypomyelination in mice.

Brain nerve fibers are insulated by myelin which is produced by oligodendrocytes. Defects in myelination are increasingly recognized as a common pathology underlying neuropsychiatric and neurodevelopmental disorders, which are associated with deletions of the Unc-51-like kinase 4 (ULK4) gene. Key transcription factors have been identified for oligodendrogenesis, but little is known about their associated regulators. Here we report that Ulk4 acts as a key regulator of myelination. Myelination is reduced by half in the Ulk4tm1a/tm1a hypomorph brain, whereas expression of axonal marker genes Tubb3, Nefh, Nefl and Nefm remains unaltered. Transcriptome analyses reveal that 8 (Gfap, Mbp, Mobp, Plp1, Slc1a2, Ttr, Cnp, Scd2) of the 10 most significantly altered genes in the Ulk4tm1a/tm1a brain are myelination-related. Ulk4 is co-expressed in Olig2+ (pan-oligodendrocyte marker) and CC1+ (mature myelinated oligodendrocyte marker) cells during postnatal development. Major oligodendrogeneic transcription factors, including Olig2, Olig1, Myrf, Sox10, Sox8, Sox6, Sox17, Nkx2-2, Nkx6-2 and Carhsp1, are significantly downregulated in the mutants. mRNA transcripts enriched in oligodendrocyte progenitor cells (OPCs), the newly formed oligodendrocytes (NFOs) and myelinating oligodendrocytes (MOs), are significantly attenuated. Expression of stage-specific oligodendrocyte factors including Cspg4, Sox17, Nfasc, Enpp6, Sirt2, Cnp, Plp1, Mbp, Ugt8, Mag and Mog are markedly decreased. Indirect effects of axon caliber and neuroinflammation may also contribute to the hypomyelination, as Ulk4 mutants display smaller axons and increased neuroinflammation. This is the first evidence demonstrating that ULK4 is a crucial regulator of myelination, and ULK4 may therefore become a novel therapeutic target for hypomyelination diseases.

Multiple roles of Ulk4 in neurogenesis and brain function.

Neurogenesis is essential for proper brain formation and function, and abnormal neural proliferation is an underlying neuropathology of many brain disorders. Recent advances on adult neurogenesis demonstrate that neural stem cells (NSCs) at the subventricular zone (SVZ) are largely derived during mid-embryonic neurogenesis from a subset of cells, which slow down in their pace of cell division,1 become quiescent cells and can be reactivated in need.2 The NSCs at birth constitute the stem cell pool for both postnatal oligodendrogenesis3 and adult neurogenesis.1,2 However, little is known about factors that control the size of NSC pool. The article published in Stem Cells on Jun 14, 2016 by Liu and colleagues described a member of the Unc-51-like serine/threonine kinase family, Ulk4, which plays a critical role in regulating the NSC pool size.4 Authors presented evidence of cell cycle-dependent Ulk4 expression in vitro and in vivo, and reduced NSC pool in targetedly disrupted Ulk4 newborn mice, with disturbed pathways of cell cycle regulation and WNT signaling (Fig. 1), suggesting that ULK4 may be associated with neurodevelopmental, neuropsychiatric as well as neurodegenerative diseases.

Ulk4 Is Essential for Ciliogenesis and CSF Flow.

UNLABELLED: Ciliopathies are an emerging class of devastating disorders with pleiotropic symptoms affecting both the central and peripheral systems and commonly associated with hydrocephalus. Even though ciliary components and three master transcriptional regulators have been identified, little is known about the signaling molecules involved. We previously identified a novel gene, Unc51-like-kinase 4 (ULK4), as a risk factor of neurodevelopmental disorders. Here we took multidisciplinary approaches and uncovered essential roles of Ulk4 in ciliogenesis. We show that Ulk4 is predominantly expressed in the ventricular system, and Ulk4(tm1a/tm1a) ependymal cells display reduced/disorganized cilia with abnormal axonemes. Ulk4(tm1a/tm1a) mice exhibit dysfunctional subcommissural organs, obstructive aqueducts, and impaired CSF flow. Mechanistically, we performed whole-genome RNA sequencing and discovered that Ulk4 regulates the Foxj1 pathway specifically and an array of other ciliogenesis molecules. This is the first evidence demonstrating that ULK4 plays a vital role in ciliogenesis and that deficiency of ULK4 can cause hydrocephalus and ciliopathy-related disorders. SIGNIFICANCE STATEMENT: Ciliopathies are an emerging class of devastating disorders with pleiotropic symptoms affecting both the central and peripheral systems. Ciliopathies are commonly associated with hydrocephalus, and Unc51-like-kinase 4 (Ulk4) has been identified as one of 12 genes causing hydrocephalus in mutants. Here we uncover an essential role of Ulk4 in ciliogenesis. Ulk4 is predominantly expressed in the ventricles, and mutant ependymal cells display reduced/disorganized/nonfunctional motile cilia with abnormal axonemes and impaired CSF flow. Ulk4 modulates expression of the master regulator of ciliogenesis, Foxj1, and other ciliogenesis molecules. This is the first report demonstrating a vital role of Ulk4 in ciliogenesis. ULK4 deficiency may be implicated in human hydrocephalus and other ciliopathy-related disorders.

Ulk4 Regulates Neural Stem Cell Pool.

The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4(tm1a/tm1a) mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4(tm1a/tm1a) mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016;34:2318-2331.

Long noncoding RNAs in hepatitis B virus-related hepatocellular carcinoma.

AIM: To study the expression of long noncoding RNAs (lncRNAs) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: The lncRNA profiles between HBV-related HCC tissues and corresponding normal liver tissues were generated using microarray analysis. Datasets were analyzed using multiple algorithms to depict alterations in gene expression on the basis of gene ontology (GO), pathway analysis, and lncRNA levels. RESULTS: The microarray revealed that 1772 lncRNAs and 2508 mRNAs were differently expressed. The pathway analysis demonstrated that the cell cycle, cytokine-cytokine receptor interaction, chemokine signaling pathway, and phosphoinositide 3-kinase-protein kinase B signaling pathway may play important roles in HCC. Several GO terms, such as cell cycle, DNA replication, immune response, and signal transduction, were enriched in gene lists, suggesting a potential correlation with HBV-related HCC. The upregulated large intergenic noncoding RNA ULK4P2 was physically combined with enhancer of zeste homolog 2. Therefore, the lncRNAs may participate in regulating HBV-related HCC. CONCLUSION: lncRNAs play important roles in HCC, future studies should verify whether large intergenic noncoding ULK4P2 functions by combining with enhancer of zeste homolog 2 in HCC.

Expression of a novel serine/threonine kinase gene, Ulk4, in neural progenitors during Xenopus laevis forebrain development.

We have analyzed the expression pattern of a novel serine/threonine kinase gene Ulk4 during forebrain development in Xenopus laevis. To this aim, we firstly cloned a Ulk4 cDNA fragment from X.laevis and generated a RNA probe that was used for its detection by in situ hybridization. Throughout development xUlk4 expression was detected along the ventricular (vz) and subventricular zones (svz) of all forebrain regions, with the exception of the vz of the striatum. In the adult, xUlk4 was also mainly located in the vz, with some xUlk4 expressing cells reaching the svz/mantle zone (mz). This xUlk4 expression was especially remarkable in forebrain regions involving the homeostatic control of the brain such as the preoptic region, the hypothalamic territory and some neurosecretory circumventricular organs (CVOs). We further combined in situ hybridization for xUlk4 with immunohistochemistry for the neural progenitor cell marker SOX3, the radial glial marker brain lipid-binding protein (BLBP), neuronal markers MAP2 and doublecortin (DCX) and the specific neuronal marker tyrosine hydroxylase (TH). xUlk4 was co-expressed with the neural stem/progenitor cell marker SOX3 in the vz of all the forebrain regions throughout development and in the adult, and this co-expression was also especially evident in the svz of the hypothalamic region. xUlk4 was also expressed in the radial glia along the whole brain. We have also found minor expression of xUlk4 in some DCX- or MAP2-positive cells but not in TH-positive neurons. These findings suggest that Ulk4 may play roles in neural stem/progenitor cells during neurogenesis both in development and in the adulthood, in migrating cells as well as in cells committed to neuronal fate in Xenopus. Moreover, the results obtained in this study argue for an involvement of Ulk4 in the control of the neuroendocrine homeostatic functions in the brain.

Recurrent deletions of ULK4 in schizophrenia: a gene crucial for neuritogenesis and neuronal motility.

Although many pathogenic copy number variations (CNVs) are associated with neuropsychiatric diseases, few of them have been functionally characterised. Here we report multiple schizophrenia cases with CNV abnormalities specific to unc-51-like kinase 4 (ULK4), a serine/threonine kinase gene. Deletions spanning exons 21-34 of ULK4 were present in 4 out of 3391 schizophrenia patients from the International Schizophrenia Consortium, but absent in 3181 controls. Deletions removing exons 33 and 34 of the large splice variant of ULK4 also were enriched in Icelandic schizophrenia and bipolar patients compared with 98,022 controls (P = 0.0007 for schizophrenia plus bipolar disorder). Combining the two cohorts gives a P-value less than 0.0001 for schizophrenia, or for schizophrenia plus bipolar disorder. The expression of ULK4 is neuron-specific and developmentally regulated. ULK4 modulates multiple signalling pathways that include ERK, p38, PKC and JNK, which are involved in stress responses and implicated in schizophrenia. Knockdown of ULK4 disrupts the composition of microtubules and compromises neuritogenesis and cell motility. Targeted Ulk4 deletion causes corpus callosum agenesis in mice. Our findings indicate that ULK4 is a rare susceptibility gene for schizophrenia.