Here we report that neuron-specific abolition of either UFL1 or UFBP1, two components of the UFM1-specific E3 ligase, led to significant loss of neurons in cortex and hippocampal CA1 regions and caused elevated inflammatory response. Furthermore, we found that heterozygous mice with loss of one UFBP1 wild-type allele exhibited abnormal EEGs that indicate occurrence of spontaneous seizure-like events. Our study have first demonstrated that the UFM1 E3 ligase is essential for normal function of murine central nervous system.
Laboratory studies of various mouse genetic models have demonstrated the indispensable role of the UFM1 system in embryogenesis, development and normal physiology of multiple tissues and organs . Its importance is further supported by identification of multiple UFMylation gene variants in genetic diseases such as skeletal dysplasia and neurological disorders. Recent human genetic studies indicate that the neural system appears to be more susceptible to perturbation of the UFMylation pathway than other systems. Most disease-causing mutations of human UFM1, UBA5 (E1) and UFC1 (E2) genes were found in pediatric patients with neurological anomalies, ranging from dystonia, ataxia, abnormal EEGs, epileptic seizure to microcephaly and global developmental delay [15–25]. These abnormalities were manifested in various syndromes, such as hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC), severe early-onset encephalopathy with progressive microcephaly, severe infantile-onset encephalopathy, and autosomal recessive cerebellar ataxia (ARCA) [15–25]. Biochemically, mutations identified in UBA5 and UFC1 genes compromise their enzymatic activity, thereby resulting in attenuation of the UFMylation pathway [15, 24]. Interestingly, a pathogenic variant of UFSP2, a tentative de-UFMylase, was recently identified in pediatric patient with a severe syndrome of neurodevelopmental disability and epilepsy . Although the impact of the mutations in these genes on UFMylation of specific targets remains to be further evaluated, it is reasonable to postulate that a well-balanced UFMylation activity is essential for normal development and function of neurons, and disruption of this balance (either insufficient or over-accumulated UFMylated products) may lead to neurological disorders with varying degree of severity.
Although the critical role of UBA5 (UFM1-specific E1) and UFC1 (UFM1-specific E2) in the nervous system has been strongly supported by both clinical data and animal studies, the involvement of UFM1-specific E3 ligase in brain development remains largely unknown. Using neuron-specific knockout mouse models in the current study, we have demonstrated that UFL1/UFBP1 complex, the only known UFM1-specific E3 ligase identified so far, is indeed important for normal function of murine brain. As originally reported, the Cre recombinase activity in Camk2a-Cre line T29-1 is predominantly expressed in the CA1 pyramidal cell layer in the hippocampus during the third and fourth postnatal week, and then spreads to the forebrain and other regions in later postnatal stages ( and https://www.jax.org/strain/005359). Consistent with the distribution of Cre activity in adult brain, Cre-mediated knockout of either UFL1 or UFBP1 mainly affects the neurons in the cortex and CA1 region of hippocampus. More TUNEL positive cells are present in KO mice indicating the essential role of UFL1 and UFBP1 in neuronal survival (Fig. 3). The underlying cause of neuronal death remains to be further elucidated. The UFMylation pathway has been implicated in cell processes such as autophagy, protein translation, ribosome-associated protein quality control (RQC) and Unfolded Protein Response (UPR) . Impairment of one or multiple of these pathways may lead to disruption of neuronal proteostasis, thereby resulting in elevated cell death. Further mechanistic investigation is needed to identify key UFM1 substrates and elucidate functional impact of UFMylation of these targets in neuronal survival and cellular processes.
In addition to neuronal survival, UFL1 or UFBP1 may be also involved in normal neuronal function. This notion is supported by our finding of seizure-like phenotype manifested by UFBP1 heterozygous mice. Although we cannot exclude the contribution of non-neuronal effect of UFBP1 deletion to this phenotype at this stage, this finding raises an intriguing possibility that partial loss of UFMylation function may play a broader role in the pathophysiology of neurological disorders than our current view, which is mainly focused on early-onset pediatric encephalopathy. Interestingly, GWAS study has identified UFBP1 (also known as DDRGK1) as a new risk locus in Parkinson’s disease , while UFL1 expression is decreased in schizophrenia patients . Thus, further investigation of the UFM1 system in the neural system may unravel a novel insight into the pathogenic mechanism of neurological disorders as well as new therapeutic targets for these diseases.