Faculty Bibliography

Neferine, an alkaloid isolated from Lotus seeds, displays multiple pharmacological effects that counter cancer, oxidants, and arrhythmia. It was initially identified as a strong inducer for macroautophagy in cancer cells by suppressing AMPK/mTOR signaling. In this study, we found that autophagy signaling was inhibited in the condition of neferine treatment. Exposure to neferine resulted in the accumulation of LC3-II and an associated adaptor protein, p62/SQSTM1. Knockdown of ATG5 failed to reduce the accumulation of LC3-II induced by neferine. The electron microscopy (EM) images showed that neferine induce accumulation of multi-vesicle bodies (MVB) and failure of lysosome maturation. Moreover, exposure to neferine reduced maturation of cathepsin D and impaired the degradation of autophagic and phagocytic cargos. Rather than stimulate autophagic flux, the data indicate that neferine impaired lysosomes to block degradation within phagolysosomes.

The ability to understand in great details, at the molecular level, the process of myelination in the peripheral nervous system (PNS) is, in no minor part, due to the availability of an in vitro culture model of PNS myelination. This culture system is based on the ability to prepare large population of highly purified Schwann cells and dorsal root ganglia neurons that, once co-cultured, can be driven to form in vitro well-defined myelinated axon units. In this chapter, we present our detailed protocols to establish these cell cultures that are derived from modifications of procedures developed 35-40 years ago.

The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafishnanogmutants. Whereas zygoticnanogmutants survive to adulthood, maternal-zygotic (MZnanog) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZnanogembryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation.

AIM/OBJECTIVE:Schizophrenia (SZ) and bipolar disorder (BD) have been known to share genetic and environmental risk factors, and complex gene-environmental interactions may contribute to their pathophysiology. In contrast to high genetic overlap between SZ and BD, as revealed by genome-wide association studies, the extent of epigenetic overlap remains largely unknown. In the present study, we explored whether SZ and BD share epigenetic risk factors in the same manner as they share genetic components. METHODS:We performed DNA methylation analyses of the CpG sites in the top five candidate regions (FAM63B, ARHGAP26, CTAGE11P, TBC1D22A, and intergenic region [IR] on chromosome 16) reported in a previous methylome-wide association study (MWAS) of SZ, using whole blood samples from subjects with BD and controls. RESULTS:Among the five candidate regions, the CpG sites in FAM63B and IR on chromosome 16 were significantly hypomethylated in the samples from subjects with BD as well as those from subjects with SZ. On the other hand, the CpG sites in TBC1D22A were hypermethylated in the samples from subjects with BD, in contrast to hypomethylation in the samples from subjects with SZ. CONCLUSION/CONCLUSIONS:Hypomethylation of FAM63B and IR on chromosome 16 could be common epigenetic risk factors for SZ and BD. Further comprehensive epigenetic studies for BD, such as MWAS, will uncover the extent of similarity and uniqueness of epigenetic alterations.

Lentiviral transduction is a gene delivery method that provides numerous advantages over direct transfection and traditional retroviral or adenoviral delivery methods. It facilitates for the transduction of primary cells inherently difficult to transfect, delivers constructs of interest to nondividing as well as dividing cells, and permits the long-term expression of sizable DNA inserts (e.g., <7 kb). The study of peripheral nerve myelination at the molecular level has long benefited from the Schwann cells/dorsal root ganglia (DRG) neurons myelinating co-culture system. As this culture system takes about a month to develop and perform experiments with, lentiviral-delivered constructs can be used to manipulate gene expression in Schwann cells and DRG neurons, primary cells that are otherwise resilient to direct transfection. Here we present our protocol for lentiviral production and purification and subsequent infection of large numbers of Schwann cells and/or DRG neurons for the molecular study of peripheral nerve myelination in vitro.

BACKGROUND:Oxytocin is a peptide hormone that influences the integration of social cognition with behavior and affect regulation. Oxytocin also prominently directs the transition of neuronal GABA neurotransmission from excitatory to inhibitory after birth. The oxytocin receptor (OXTR) is linked to schizophrenia, a heterogeneous syndrome. Relationships of OXTR polymorphisms with specific clinical features could aid in evaluating any role of oxytocin in the pathogenesis of schizophrenia. METHOD/METHODS:Schizophrenia cases with rare missense coding OXTR single nucleotide variants (SNVs) were identified from a well-characterized sample of cases and controls who were assessed for symptoms, cognition and early life trauma. RESULTS:Five of 48 cases showed rare OXTR variants. Compared to the other cases they had less severe negative symptoms (deficits in emotional expression and motivation) and less severe general psychopathology scores (depression and anxiety). They demonstrated lower nonverbal (performance) than verbal intelligence due to deficient perceptual organization and slow processing speed. They also reported greater early trauma exposure (physical and sexual abuse and emotional trauma). CONCLUSION/CONCLUSIONS:Cases carrying rare OXTR SNVs had less negative and affective symptoms than other cases, but similar psychotic symptoms, along with specific cognitive deficits. The clinical characterization of these cases occurred in association with environmental exposure to early trauma, especially sexual abuse, which may have influenced the expression of schizophrenia in subjects harboring specific SNVs in the OXTR.

Battlefield blast exposure related to improvised explosive devices (IEDs) has become the most common cause of traumatic brain injury (TBI) in the recent conflicts in Iraq and Afghanistan. Mental health problems are common after TBI. A striking feature in the most recent veterans has been the frequency with which mild TBI (mTBI) and posttraumatic stress disorder (PTSD) have appeared together, in contrast to the classical situations in which the presence of mTBI has excluded the diagnosis of PTSD. However, treatment of PTSD-related symptoms that follow blast injury has become a significant problem. BCI-838 (MGS0210) is a Group II metabotropic glutamate receptor (mGluR2/3) antagonist prodrug, and its active metabolite BCI-632 (MGS0039) has proneurogenic, procognitive, and antidepressant activities in animal models. In humans, BCI-838 is currently in clinical trials for refractory depression and suicidality. The aim of the current study was to determine whether BCI-838 could modify the anxiety response and reverse PTSD-related behaviors in rats exposed to a series of low-level blast exposures designed to mimic a human mTBI or subclinical blast exposure. BCI-838 treatment reversed PTSD-related behavioral traits improving anxiety and fear-related behaviors as well as long-term recognition memory. Treatment with BCI-838 also increased neurogenesis in the dentate gyrus (DG) of blast-exposed rats. The safety profile of BCI-838 together with the therapeutic activities reported here, make BCI-838 a promising drug for the treatment of former battlefield Warfighters suffering from PTSD-related symptoms following blast-induced mTBI.