Progresses in the establishment, evaluation, and application of in vitro blood-brain barrier models.

The blood-brain barrier (BBB) is a barrier between the circulatory system and the central nervous system (CNS), contributing to CNS protection and maintaining the brain homeostasis. Establishment of in vitro BBB models that are closer to the microenvironment of the human brain is helpful for evaluating the potential and efficiency of a drug penetrating BBB and thus the clinical application value of the drug. The in vitro BBB models not only provide great convenience for screening new drugs that can access to CNS but also help people to have a deeper study on the mechanism of substances entering and leaving the brain, which makes people have greater opportunities in the treatment of CNS diseases. Up to now, although much effort has been paid to the researches on the in vitro BBB models and many progresses have been achieved, no unified method has been described for establishing a BBB model and there is much work to do and many challenges to be faced with in the future. This review summarizes the research progresses in the establishment, evaluation, and application of in vitro BBB models.

Effects of latroeggtoxin-VI on dopamine and α-synuclein in PC12 cells and the implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by death of dopaminergic neurons leading to dopamine deficiency, excessive α-synuclein facilitating Lewy body formation, etc. Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin discovered from the eggs of spider L. tredecimguttatus, was previously found to promote the synthesis and release of PC12 cells, showing a great potential as a drug candidate for PD. However, the relevant mechanisms have not been understood completely. The present study explored the mechanism underlying the effects of LETX-VI on dopamine and α-synuclein of PC12 cells and the implications for PD. RESULTS: After PC12 cells were treated with LETX-VI, the level of dopamine was significantly increased in a dose-dependent way within a certain range of concentrations. Further mechanism analysis showed that LETX-VI upregulated the expression of tyrosine hydroxylase (TH) and L-dopa decarboxylase to enhance the biosynthesis of dopamine, and downregulated that of monoamine oxidase B to reduce the degradation of dopamine. At the same time, LETX-VI promoted the transport and release of dopamine through modulating the abundance and/or posttranslational modification of vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT). While the level of dopamine was increased by LETX-VI treatment, α-synuclein content was reduced by the spider toxin. α-Synuclein overexpression significantly decreased the dopamine level and LETX-VI efficiently alleviated the inhibitory action of excessive α-synuclein on dopamine. In the MPTP-induced mouse model of PD, application of LETX-VI ameliorated parkinsonian behaviors of the mice, and reduced the magnitude of MPTP-induced α-synuclein upregulation and TH downregulation. In addition, LETX-VI displayed neuroprotective effects by inhibiting MPTP-induced decrease in the numbers of TH-positive and Nissl-stained neurons in mouse brain tissues. CONCLUSIONS: All the results demonstrate that LETX-VI promotes the synthesis and release of dopamine in PC12 cells via multiple mechanisms including preventing abnormal α-synuclein accumulation, showing implications in the prevention and treatment of PD.

Analysis of differentially expressed genes discovers Latroeggtoxin VI-induced changes and SYNJ1 as a main target in PC12 cells.

BACKGROUND: Previous preliminary work found that Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin from the eggs of spider Latrodectus tredecimguttatus, could promote the synthesis and release of dopamine in PC12 cells. However, the underlying mechanisms have not been fully clear. Here, the effects of LETX-VI on the gene expression profile and dopamine in PC12 cells were analyzed with the differential transcriptome-based strategies. RESULTS: After treatment of PC12 cells with LETX-VI for 24 h, a total of 356 differentially expressed transcripts were identified. Of them 165 were up-regulated and 191 down-regulated. Relevant GO analysis indicated that LETX-VI modulated the expression of certain genes and thereby affected multiple biological processes in PC12 cells, including protein metabolism, nucleic acid metabolism, substance transport, signaling, neurotransmitter metabolism and release. When western blot analysis was employed to confirm the abundance levels of synaptojanin 1 and synuclein alpha interacting protein, the representatives of highly up- and down-regulated transcript-encoded proteins that are closely related with dopamine respectively, it was found that the level of synaptojanin 1 in the PC12 cells treated with LETX-VI was increased, whereas that of synuclein alpha interacting protein was not obviously altered, suggesting that synaptojanin 1 may be much more involved in the effects of LETX-VI on dopamine. After synaptojanin 1 level was knocked down using siRNA, the levels of both total and released dopamine were significantly decreased, indicating that synaptojanin 1 is a protein positively modulating the synthesis and secretion of dopamine. When the PC12 cells with knocked down synaptojanin 1 were treated by LETX-VI, the adverse effects of synaptojanin 1 knockdown on dopamine were attenuated, confirming that LETX-VI promotes the synthesis and secretion of dopamine at least partially by enhancing the expression of the gene SYNJ1 encoding synaptojanin 1. CONCLUSIONS: This work demonstrates that LETX-VI exerts multiple regulatory effects on the cellular processes in PC12 cells by altering the gene expression profile. LETX-VI modulates the expression of the genes closely related to the synthesis, transport and release of neurotransmitters especially dopamine in PC12 cells, with the gene SYNJ1 encoding synaptojanin 1 as a main target.

Insights into the mediation of Ca2+ signaling in the promoting effects of LETX-VI on the synthesis and release of dopamine.

Latroeggtoxin-VI (LETX-VI) is an active protein and was previously demonstrated to have effects on the synthesis and release of dopamine. Hererin, the involvement of Ca2+ signaling in the effects of LETX-VI on dopamine was systematically investigated, using PC12 cells as a neuron model. LETX-VI was shown to promote dopamine release from PC12 cells both in the presence and absence of extracellular Ca2+; however the presence of extracellular Ca2+ was favorable for enhancing the promoting effects of LETX-VI on dopamine, because LETX-VI facilitated the influx of extracellular Ca2+ through the L-type calcium channels in plasma membrane (PM) to increase cytosolic Ca2+ concentration. LETX-VI was able to penetrate the PM of PC12 cells to act on the Ca2+ channel proteins IP3Rs and RyRs in the endoplasm reticulum (ER) membrane, opening the Ca2+ channels and promoting the release of ER Ca2+ to elevate cytosolic Ca2+ level. With the help of intracellular Ca2+ chelator BAPTA, the elevated cytosolic Ca2+ level was proven to play crucial role for the enhanced promoting effects of LETX-VI on dopamine. Taken together, LETX-VI is able to open the Ca2+ channels in both PM and ER membrane simultaneously to facilitate extracellular Ca2+ influx and ER Ca2+ release, and thus increases the cytosolic Ca2+ concentration to enhance the promoting effects on the synthesis and release of dopamine.

Latroeggtoxin-VI protects nerve cells and prevents depression by inhibiting NF-κB signaling pathway activation and excessive inflammation.

Depression has a high incidence and seriously endangers human health. Accumulated evidence indicates that targeting neuroinflammation is a potential avenue for neuroprotection and thus depression prevention. Herein, the effects of latroeggtoxin-VI (LETX-VI), a bioactive protein from the eggs of spider Latrodectus tredecimguttatus, on lipopolysaccharide (LPS)-induced inflammation and depression were systematically investigated using RAW264.7 macrophages and depression mouse model. Pretreatment with LETX-VI suppressed LPS-evoked NF-κB signaling pathway activation, inhibited LPS-induced over-production of NO, iNOS, IL-6 and TNF-α; at the same time LETX-VI mitigated the inhibitory effect of LPS on the expression of anti-inflammatory factors such as Arg-1, thereby suppressing oxidative stress and excessive inflammation. Culture of PC12 cells with the conditioned medium of RAW264.7 cells pretreated with LETX-VI demonstrated the neuroprotective effect of LETX-VI due to its anti-inflammation effect. In the LPS-induced depression mouse model, pretreatment with LETX-VI improved the LPS-induced depression-like behaviors, inhibited the activation of microglia and astrocytes, prevented the down-regulation of Nurr1 expression and alleviated the LPS-caused adverse changes in the brain tissues. Taken together, these in vitro and in vivo findings provide powerful insights into the anti-inflammation-based neuroprotective and antidepressant mechanisms of LETX-VI, which is helpful to deeply reveal the biological effects and potential applications of LETX-VI.

Rab3 and synaptotagmin proteins in the regulation of vesicle fusion and neurotransmitter release.

Ca2+-triggered neurotransmitter release involves complex regulatory mechanisms, including a series of protein-protein interactions. Three proteins, synaptobrevin (VAMP), synaptosomal-associated protein of 25kDa (SNAP-25) and syntaxin, constitute the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex that plays key roles in controlling vesicle fusion and exocytosis. Many other proteins participate in the regulation of the processes via direct and/or indirect interaction with the SNARE complex. Although much effort has been made, the regulatory mechanism for exocytosis is still not completely clear. Accumulated evidence indicates that the small GTPase Rab3 and synaptotagmin proteins play important regulatory roles during vesicle fusion and neurotransmitter release. This review outlines our present understanding of the two regulatory proteins, with the focus on the interaction of Rab3 with synaptotagmin in the regulatory process.