Lycium barbarum is used both as a food additive and as a medicinal herb in many countries, and L. barbarum polysaccharides (LBPs), a major cell component, are reported to have a wide range of beneficial effects including neuroprotection, anti-aging and anticancer properties, and immune modulation. The effects of LBPs on neuronal function, neurogenesis, and drug-induced learning and memory deficits have not been assessed. We report the therapeutic effects of LBPs on learning and memory and neurogenesis in scopolamine (SCO)-treated rats. LBPs were administered via gastric perfusion for 2 weeks before the onset of subcutaneous SCO treatment for a further 4 weeks. As expected, SCO impaired performance in novel object and object location recognition tasks, and Morris water maze. However, dual SCO- and LBP-treated rats spent significantly more time exploring the novel object or location in the recognition tasks and had significant shorter escape latency in the water maze. SCO administration led to a decrease in Ki67- or DCX-immunoreactive cells in the dentate gyrus and damage of dendritic development of the new neurons; LBP prevented these SCO-induced reductions in cell proliferation and neuroblast differentiation. LBP also protected SCO-induced loss of neuronal processes in DCX-immunoreactive neurons. Biochemical investigation indicated that LBP decreased the SCO-induced oxidative stress in hippocampus and reversed the ratio Bax/Bcl-2 that exhibited increase after SCO treatment. However, decrease of BDNF and increase of AChE induced by SCO showed no response to LBP administration. These results suggest that LBPs can prevent SCO-induced cognitive and memory deficits and reductions in cell proliferation and neuroblast differentiation. Suppression of oxidative stress and apoptosis may be involved in the above effects of LBPs that may be a promising candidate to restore memory functions and neurogenesis.

Our previous studies indicated that transcription factor Brn-4 is upregulated in the surgically denervated hippocampus in vivo, promoting neuronal differentiation of hippocampal neural stem cells (NSCs) in vitro. The molecules mediating Brn-4 upregulation in the denervated hippocampus remain unknown. In this study we examined the levels of insulin-like growth factor-1 (IGF-1) in hippocampus following denervation. Surgical denervation led to a significant increase in IGF-1 expression in vivo. We also report that IGF-1 treatment on NSCs in vitro led to a marked acceleration of Brn-4 expression and cell differentiation down neuronal pathways. The promotion effects were blocked by PI3K-specific inhibitor (LY294002), but not MAPK inhibitor (PD98059); levels of phospho-Akt were increased by IGF-1 treatment. In addition, inhibition of IGF-1 receptor (AG1024) and mTOR (rapamycin) both attenuated the increased expression of Brn-4 induced by IGF-1. Together, the results demonstrated that upregulation of IGF-1 induced by hippocampal denervation injury leads to activation of the PI3K/Akt signaling pathway, which in turn gives rise to upregulation of the Brn-4 and subsequent stem cell differentiation down neuronal pathways.

Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs.

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