Ablation of the 14‐3‐3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex

14‐3‐3 proteins are ubiquitously‐expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14‐3‐3epsilon and 14‐3‐3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14‐3‐3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14‐3‐3gamma‐deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14‐3‐3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time‐lapse live imaging of brain slices revealed that the ablation of the 14‐3‐3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14‐3‐3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14‐3‐3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14‐3‐3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600–614, 2016     

Fatty acid binding protein 7 and n‐3 poly unsaturated fatty acid supply in early rat brain development

Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin‐dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid‐rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 287–297, 2016     

CiRS‐7 targeting miR‐7 modulates the progression of non‐small cell lung cancer in a manner dependent on NF‐κB signalling

The purpose of this study was to figure out the effect of ciRS‐7/miR‐7/NF‐κB axis on the development of non‐small cell lung cancer (NSCLC). In response, the expressions of ciRS‐7, miR‐7 and NF‐κB subunit (ie RELA) within NSCLC tissues and cell lines were determined with real‐time polymerase chain reaction (RT‐PCR) and Western blot. Moreover, the NSCLC cells were transfected with pcDNA3‐ciRS‐7‐ir, pcDNA3‐ciRS‐7, miR‐NC and miR‐7 mimic. Furthermore, the targeted relationships between ciRS‐7 and miR‐7, as well as between miR‐7 and RELA, were confirmed by luciferase reporter assay. The proliferation, migration and apoptosis of NSCLC cells were, successively, measured using CCK‐8 assay, wound‐healing assay and flow cytometry test. Consequently, ciRS‐7, miR‐7, histopathological grade, lymph node metastasis and histopathological stage could independently predict the prognosis of patients with NSCLC (all P < .05). Moreover, remarkably up‐regulated ciRS‐7 and RELA expressions, as along with down‐regulated miR‐7 expressions, were found within NSCLC tissues and cells in comparison with normal ones (P < .05). Besides, overexpressed ciRS‐7 and underexpressed miR‐7 were correlated with increased proliferation, migration and invasion, yet reduced apoptosis rate of NSCLC cells (P < .05). More than that, ciRS‐7 specifically targeted miR‐7 to reduce its expressions (P < .05). Ultimately, the NSCLC cells within miR‐7 + RELA group were observed with superior proliferative, migratory and invasive capabilities than those within miR‐7 group (P < .05), and RELA expression was also significantly modified by both ciRS‐7 and miR‐7 (P < .05). In conclusion, the ciRS‐7/miR‐7/NF‐kB axis could exert pronounced impacts on the proliferation, migration, invasion and apoptosis of NSCLC cells.     

Imbalanced genomic imprinting in brain development: an evolutionary basis for the aetiology of autism

We describe a new hypothesis for the development of autism, that it is driven by imbalances in brain development involving enhanced effects of paternally expressed imprinted genes, deficits of effects from maternally expressed genes, or both. This hypothesis is supported by: (1) the strong genomic-imprinting component to the genetic and developmental mechanisms of autism, Angelman syndrome, Rett syndrome and Turner syndrome; (2) the core behavioural features of autism, such as self-focused behaviour, altered social interactions and language, and enhanced spatial and mechanistic cognition and abilities, and (3) the degree to which relevant brain functions and structures are altered in autism and related disorders. The imprinted brain theory of autism has important implications for understanding the genetic, epigenetic, neurological and cognitive bases of autism, as ultimately due to imbalances in the outcomes of intragenomic conflict between effects of maternally vs. paternally expressed genes.     

Evidence for an angiotensin‐(1‐7) neuropeptidase expressed in the brain medulla and CSF of sheep

Angiotensin‐(1‐7) [Ang‐(1‐7)] is an alternative product of the brain renin‐angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang‐(1‐7) to the inactive metabolite product Ang‐(1‐4) in CSF of adult sheep. This study purified the peptidase 1445‐fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o‐phenanthroline and EDTA, as well as the mercury compound p‐chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin‐converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV‐390 was a potent inhibitor of Ang‐(1‐7) hydrolysis (Ki = 0.8 nM). Kinetic studies using ¹²⁵I‐labeled Ang‐(1‐7), Ang II, and Ang I revealed comparable apparent K_m values (2.6, 2.8, and 4.3 μM, respectively), but a higher apparent V_max for Ang‐(1‐7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang‐(1‐7) to Ang‐(1‐4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin‐13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang‐(1‐7) within the brain.

     

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

LIN28B is an RNA‐binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let‐7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let‐7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first‐trimester trophoblast cell line, ACH‐3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral‐based vectors. LIN28B knockdown in ACH‐3P cells resulted in significantly decreased levels of AR and increased levels of Let‐7 miRNAs. Moreover, treatment of ACH‐3P cells with Let‐7c mimic, but not Let‐7e or Let‐7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin‐induced syncytialization and Let‐7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW‐1 and a significant decrease in AR in ACH‐3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let‐7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.     

Male‐specific alteration in excitatory post‐synaptic development and social interaction in pre‐natal valproic acid exposure model of autism spectrum disorder

Autism spectrum disorder (ASD) is a pervasive developmental disorder characterized by three main behavioral symptoms including social deficits, impaired communication, and stereotyped and repetitive behaviors. ASD prevalence shows gender bias to male. Prenatal exposure to valproic acid (VPA), a drug used in epilepsy and bipolar disorder, induces autistic symptoms in both human and rodents. As we reported previously, prenatally VPA‐exposed animals at E12 showed impairment in social behavior without any overt reproductive toxicity. Social interactions were not significantly different between male and female rats in control condition. However, VPA‐exposed male offspring showed significantly impaired social interaction while female offspring showed only marginal deficits in social interaction. Similar male inclination was observed in hyperactivity behavior induced by VPA. In addition to the ASD‐like behavioral phenotype, prenatally VPA‐exposed rat offspring shows crooked tail phenotype, which was not different between male and female groups. Both male and female rat showed reduced GABAergic neuronal marker GAD and increased glutamatergic neuronal marker vGluT1 expression. Interestingly, despite of the similar increased expression of vGluT1, post‐synaptic marker proteins such as PSD‐95 and α‐CAMKII expression was significantly elevated only in male offspring. Electron microscopy showed increased number of post‐synapse in male but not in female at 4 weeks of age. These results might suggest that the altered glutamatergic neuronal differentiation leads to deranged post‐synaptic maturation only in male offspring prenatally exposed to VPA. Consistent with the increased post‐synaptic compartment, VPA‐exposed male rats showed higher sensitivity to electric shock than VPA‐exposed female rats. These results suggest that prenatally VPA‐exposed rats show the male preponderance of ASD‐like behaviors including defective social interaction similar to human autistic patients, which might be caused by ectopic increase in glutamatergic synapses in male rats.     

Immunoregulatory protein B7‐H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells

B7‐H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7‐H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small‐molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK‐162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API‐2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7‐H3 monoclonal antibody, while the opposite was seen in B7‐H3‐overexpressing cells. Further, combining B7‐H3 inhibition with small‐molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF^V600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7‐H3 may be a novel alternative to improve current therapy of metastatic melanoma.     

Synthesis of a novel fluorescent probe based on 7‐nitrobenzo‐2‐oxa‐1,3‐diazole skeleton for the rapid determination of vitamin B12 in pharmaceuticals

A new fluorescent probe, 4‐N,N‐di(2‐hydroxyethyl)imino‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (HINBD) was synthesized in a single step with reasonably good yield. The water‐soluble HINBD emits strongly in the visible region (λ_ex = 479 nm, λ_em = 545 nm) and is stable over a wide range of pH values. It was found that vitamin B₁₂ (VB₁₂) had the ability to quench the fluorescence of HINBD, and the quenched fluorescence intensity was proportional to the concentration of VB₁₂. A method for VB₁₂ determination based on the quenching fluorescence of HINBD was thus established. Interference effects of various substances, including sugars, vitamins, amino acids, inorganic cations and some organic substances have been studied. Under optimal conditions, the linear range is 0.0–2.4 × 10^–5 mol/L. The determination limit is 8.3 × 10^–8 mol/L. The method was applied to measure VB₁₂ in pharmaceutical preparations with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

DKWSLLL,a Versatile DXXXLL‐Type Signal with Distinct Roles in the Cu+‐Regulated Trafficking of ATP7B

In the liver, the P‐type ATPase and membrane pump ATP7B plays a crucial role in Cu⁺ donation to cuproenzymes and in the elimination of excess Cu⁺. ATP7B is endowed with a COOH‐cytoplasmic (DE)XXXLL‐type traffic signal. We find that accessory (Lys ?3, Trp ?2, Ser ?1 and Leu +2) and canonical (D ?4, Leu 0 and Leu +1) residues confer the DKWSLLL signal with the versatility required for the Cu⁺‐regulated cycling of ATP7B between the trans‐Golgi network (TGN) and the plasma membrane (PM). The separate mutation of these residues caused a disruption of the signal, resulting in different ATP7B distribution phenotypes. These phenotypes indicate the key roles of specific residues at separate steps of ATP7B trafficking, including sorting at the TGN, transport from the TGN to the PM and its endocytosis, and recycling to the TGN and PM. The distinct roles of ATP7B in the TGN and PM and the variety of phenotypes caused by the mutation of the canonical and accessory residues of the DKWSLLL signal can explain the separate or joined presentation of Wilson's cuprotoxicosis and the dysfunction of the cuproenzymes that accept Cu⁺ at the TGN.