TurboID Proximity Labeling of a Protocadherin Protein to Characterize Interacting Protein Complex

The study of the neuron has always been a fundamental aspect when it came to studying mental illnesses such as autism and depression. The protein protocadherin-9 (PCDH9) is an important transmembrane protein in the development of the neuron synapse. Hence, research on its protein interactome is key to understanding its functionality and specific properties. A newly discovered biotin ligase, TurboID, is a proximity labeler that is designed to be able to label and observe transmembrane proteins, something that previous methods struggled with. The TurboID method is verified in HEK293T cells and primary cultured mouse cortical neurons. Results have proven the validity of the TurboID method in observing PCDH9-interacting proteins.

Association of TRMT2B gene variants with juvenile amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons,and it demonstrates high clinical heterogeneity and complex genetic architecture.A variation within TRMT2B(c.1356G>T;p.K452N)was identified to be associated with ALS in a family comprising two patients with juvenile ALS(JALS).Two missense variations and one splicing variation were identified in 10 patients with ALS in a cohort with 910 patients with ALS,and three more variants were identified in a public ALS database including 3317 patients with ALS.A decreased number of mitochondria,swollen mitochondria,lower expression of ND1,decreased mitochondrial complex I activities,lower mitochondrial aerobic respiration,and a high level of ROS were observed functionally in patient-originated lymphoblastoid cell lines and TRMT2B interfering HEK293 cells.Further,TRMT2B variations overexpression cells also displayed decreased ND1.In conclusion,a novel JALS-associated gene called TRMT2B was identified,thus broadening the clinical and genetic spectrum of ALS.

PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton,modulating emotional behavior

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function.Synaptic abnormalities,such as defects in the density and morphology of postsynaptic dendritic spines,underlie the pathology of various neuropsychiatric disorders.Protocadherin 17(PCDH17)is associated with major mood disorders,including bipolar disorder and depression.However,the molecular mechanisms by which PCDH17 regulates spine number,morphology,and behavior remain elusive.In this study,we found that PCDH17 functions at postsynaptic sites,restricting the number and size of dendritic spines in excitatory neurons.Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety-and depression-like behaviors in mice.Mechanistically,PCDH17 interacts with actin-relevant proteins and regulates actin filament(F-actin)organization.Specifically,PCDH17 binds to ROCK2,increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3(Ser3).Inhibition of ROCK2 activity with belumosudil(KD025)ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression,suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development.Hence,these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior,providing pathological insights into the neurobiological basis of mood disorders.

Current and future therapies for abnormal early embryogenesis with assisted reproductive technology

Each stage of embryonic development,including normal gamete maturation,fertilization,zygotic genome activation,and cleavage,is crucial for human reproduction.Early embryo arrest is a common phenomenon.It is estimated that about 40%–70%of human embryos are arrested at early developmental stages.However,the exact mechanism remains largely uncertain.Embryos can be investigated in vitro by way of the development of in vitro fertilization/intracytoplasmic sperm injection.In addition to iatrogenic factors related to abnormal oocyte/embryo development,multiple gene mutations have been found to be involved in such phenotypes.Based on the knowledge of known etiological factors,several therapies are proposed to improve clinical outcomes.Here,we shed light on current and potential therapies for treating these conditions through reviewing articles and combining with our clinical and research experience.

Association of variants in the KIF1A gene with amyotrophic lateral sclerosis

Background:Amyotrophic lateral sclerosis(ALS)is a devastating progressive neurodegenerative disease that affects neurons in the central nervous system and the spinal cord.As in many other neurodegenerative disorders,the genetic risk factors and pathogenesis of ALS involve dysregulation of cytoskeleton and neuronal transport.Notably,sen-sory and motor neuron diseases such as hereditary sensory and autonomic neuropathy type 2(HSAN2)and spastic paraplegia 30(SPG30)share several causative genes with ALS,as well as having common clinical phenotypes.KIF1A encodes a kinesin 3 motor that transports presynaptic vesicle precursors(SVPs)and dense core vesicles and has been reported as a causative gene for HSAN2 and SPG30.Methods:Here,we analyzed whole-exome sequencing data from 941 patients with ALS to investigate the genetic association of KIF1A with ALS.Results:We identified rare damage variants(RDVs)in the KIF1A gene associated with ALS and delineated the clini-cal characteristics of ALS patients with KIF1A RDVs.Clinically,these patients tended to exhibit sensory disturbance.Interestingly,the majority of these variants are located at the C-terminal cargo-binding region of the KIF1A protein.Functional examination revealed that the ALS-associated KIF1A variants located in the C-terminal region preferentially enhanced the binding of SVPs containing RAB3A,VAMP2,and synaptophysin.Expression of several disease-related KIF1A mutants in cultured mouse cortical neurons led to enhanced colocalization of RAB3A or VAMP2 with the KIF1A motor.Conclusions:Our study highlighted the importance of KIF1A motor-mediated transport in the pathogenesis of ALS,indicating KIF1A as an important player in the oligogenic scenario of ALS.

Transcriptional pausing induced by ionizing radiation enables the acquisition of radioresistance in nasopharyngeal carcinoma

Lesions on the DNA template can impact transcription via distinct regulatory pathways.Ionizing radiation(IR)as the mainstay modality for many malignancies elicits most of the cytotoxicity by inducing a variety of DNA damages in the genome.How the IR treatment alters the transcription cycle and whether it contributes to the development of radioresistance remain poorly understood.Here,we report an increase in the paused RNA polymerase II(RNAPII),as indicated by the phosphorylation at serine 5 residue of its C-terminal domain,in recurrent nasopharyngeal carcinoma(NPC)patient samples after IR treatment and cultured NPC cells developing IR resistance.Reducing the pool of paused RNAPII by either inhibiting TFIIH-associated CDK7 or stimulating the positive transcription elongation factor b,a CDK9-CycT1 heterodimer,attenuates IR resistance of NPC cells.Interestingly,the poly(ADP-ribosyl)ation of CycT1,which disrupts its phase separation,is elevated in the IR-resistant cells.Mutation of the major poly(ADP-ribosyl)ation sites of CycT1 decreases RNAPII pausing and restores IR sensitivity.Genome-wide chromatin immunoprecipitation followed by sequencing analyses reveal that several genes involved in radiation response and cell cycle control are subject to the regulation imposed by the paused RNAPII.Particularly,we identify the NIMA-related kinase NEK7 under such regulation as a new radioresistancefactor,whose downregulation results in the increased chromosome instability,enabling the development of IR resistance.Overall,our results highlight a novel link between the alteration in the transcription cycle and the acquisition of IR resistance,opening up new opportunities to increase the efficacy of radiotherapy and thwart radioresistance in NpC.

Rif1 interacts with non-canonical polycomb repressive complex PRC1.6 to regulate mouse embryonic stem cells fate potential

Mouse embryonic stem cells(mESCs)cycle in and out of a transient 2-cell(2C)-like totipotent state,driven by a com-plex genetic circuit involves both the coding and repetitive sections of the genome.While a vast array of regulators,including the multi-functional protein Rif1,has been reported to influence the switch of fate potential,how they act in concert to achieve this cellular plasticity remains elusive.Here,by modularizing the known totipotency regulatory factors,we identify an unprecedented functional connection between Rif1 and the non-canonical polycomb repres-sive complex PRC1.6.Downregulation of the expression of either Rif1 or PRC1.6 subunits imposes similar impacts on the transcriptome of mESCs.The LacO-LacI induced ectopic colocalization assay detects a specific interaction between Rif1 and Pcgf6,bolstering the intactness of the PRC1.6 complex.Chromatin immunoprecipitation followed by sequencing(ChIP-seq)analysis further reveals that Rif1 is required for the accurate targeting of Pcgf6 to a group of genomic loci encompassing many genes involved in the regulation of the 2C-like state.Depletion of Rif1 or Pcgf6 not only activates 2C genes such as Zscan4 and Zfp352,but also derepresses a group of the endogenous retroviral element MERVL,a key marker for totipotency.Collectively,our findings discover that Rif1 can serve as a novel auxiliary component in the PRC1.6 complex to restrain the genetic circuit underlying totipotent fate potential,shedding new mechanistic insights into its function in regulating the cellular plasticity of embryonic stem cells.

Rewiring the transcriptional circuitries in cancer by endogenous retroviruses

With sequence similarities to exogenous retroviruses,the proviral DNA elements named endogenous retroviruses(ERVs)make up∼8% of human genome,threatening genomic stability meanwhile nurturing regulatory innovations.Diverse host epigenetic mechanisms are enlisted to limit the ERVs’activity.However,in certain physiological and pathophysiological processes,distinct ERVs become abnormally activated,rewiring and perplexing the host regulons at different levels(Cosby et al.,2019).