SummaryZika virus (ZIKV) infection causes microcephaly in a subset of infants born to infected pregnant mothers. It is unknown whether human individual differences contribute to differential susceptibility of ZIKV-related neuropathology. Here, we use an Asian-lineage ZIKV strain, isolated from the 2015 Mexican outbreak (Mex1-7), to infect primary human neural stem cells (hNSCs) originally derived from three individual fetal brains. All three strains of hNSCs exhibited similar rates of Mex1-7 infection and reduced proliferation. However, Mex1-7 decreased neuronal differentiation in only two of the three stem cell strains. Correspondingly, ZIKA-mediated transcriptome alterations were similar in these two strains but significantly different from that of the third strain with no ZIKV-induced neuronal reduction. This study thus confirms that an Asian-lineage ZIKV strain infects primary hNSCs and demonstrates a cell-strain-dependent response of hNSCs to ZIKV infection.

Fibroblast growth factor (FGF) and epidermal growth factor (EGF) are critical for the development of the nervous system. We previously discovered that FGF2 and EGF had opposite effects on motor neuron differentiation from human fetal neural stem cells (hNSCs), but the underlying mechanisms remain unclear. Here, we show that FGF2 and EGF differentially affect the temporal patterns of Akt and glycogen synthase kinase 3 beta (GSK3β) activation. High levels of phosphatidylinositol 3-kinase (PI3K)/Akt activation accompanied with GSK3β inactivation result in reduction of the motor neuron transcription factor HB9. Inhibition of PI3K/Akt by chemical inhibitors or RNA interference or overexpression of a constitutively active form of GSK3β enhances HB9 expression. Consequently, PI3K inhibition increases hNSCs differentiation into HB9+/microtubule-associated protein 2 (MAP2)+ motor neurons in vitro. More importantly, blocking PI3K not only enhances motor neuron differentiation from hNSCs grafted into the ventral horn of adult rat spinal cords, but also permits ectopic generation of motor neurons in the dorsal horn by overriding environmental influences. Our data suggest that FGF2 and EGF affect the motor neuron fate decision in hNSCs differently through a fine tuning of the PI3K/AKT/GSK3β pathway, and that manipulation of this pathway can enhance motor neuron generation.

Neural stem cells (NSCs) promote recovery from brain trauma, but neuronal replacement is unlikely the sole underlying mechanism. We hypothesize that grafted NSCs enhance neural repair at least partially through modulating the host immune response after traumatic brain injury (TBI). C57BL/6 mice were intracerebrally injected with primed human NSCs (hNSCs) or vehicle 24 h after a severe controlled cortical impact injury. Six days after transplantation, brain tissues were collected for Western blot and immunohistochemical analyses. Observations included indicators of microglia/macrophage activation, M1 and M2 phenotypes, axonal injury detected by amyloid precursor protein (APP), lesion size, and the fate of grafted hNSCs. Animals receiving hNSC transplantation did not show significant decreases of brain lesion volumes compared to transplantation procedures with vehicle alone, but did show significantly reduced injury-dependent accumulation of APP. Furthermore, intracerebral transplantation of hNSCs reduced microglial activation as shown by a diminished intensity of Iba1 immunostaining and a transition of microglia/macrophages toward the M2 anti-inflammatory phenotype. The latter was represented by an increase in the brain M2/M1 ratio and increases of M2 microglial proteins. These phenotypic switches were accompanied by the increased expression of anti-inflammatory interleukin-4 receptor a and decreased proinflammatory interferon-g receptor b. Finally, grafted hNSCs mainly differentiated into neurons and were phagocytized by either M1 or M2 microglia/macrophages. Thus, intracerebral transplantation of primed hNSCs efficiently leads host microglia/macrophages toward an anti-inflammatory phenotype that presumably contributes to stem cell-mediated neuroprotective effects after severe TBI in mice.

Transplantation of neural stem cells (NSCs) improves functional outcomes following traumatic brain injury (TBI). Previously we demonstrated that human NSCs (hNSCs) via releasing glial cell line-derived neurotrophic factor (GDNF), preserved cognitive function in rats following parasagittal fluid percussion. However, the underlying mechanisms remain elusive. In this study, we report that NSC grafts significantly reduce TBI-induced axonal injury in the fimbria and other brain regions by blocking abnormal accumulation of amyloid precursor protein (APP). A preliminary mass spectrometry proteomics study revealed the opposite effects of TBI and NSCs on many of the cytoskeletal proteins in the CA3 region of the hippocampus, including α-smooth muscle actin (α-SMA), the main stress fiber component. Further, Western blot and immunostaining studies confirmed that TBI significantly increased the expression of α-SMA in hippocampal neurons, whereas NSC grafts counteracted the effect of TBI. In an in vitro model, rapid stretch injury significantly shortened lengths of axons and dendrites, increased the expression of both APP and α-SMA, and induced actin aggregation, effects offset by GDNF treatment. These GDNF protective effects were reversed by a GDNF-neutralizing antibody or a specific calcineurin inhibitor, and were mimicked by a specific Rho inhibitor. In summary, we demonstrate for the first time that hNSC grafts and treatment with GDNF acutely reduce traumatic axonal injury and promote neurite outgrowth. Possible mechanisms underlying GDNF-mediated neurite protection include balancing the activity of calcineurin, whereas GDNF-induced neurite outgrowth may result from the reduction of the abnormal α-SMA expression and actin aggregation via blocking Rho signals. Our study also suggests the necessity of further exploring the roles of α-SMA in the central nervous system (CNS), which may lead to a new avenue to facilitate recovery after TBI and other injuries.

Alcohol intoxication results in neuronal apoptosis, neurodegeneration and manifest with impaired balance, loss of muscle coordination and behavioral changes. One of the early events of alcohol intoxication is mitochondrial (Mt) dysfunction and disruption of intracellular redox homeostasis. The mechanisms by which alcohol causes Mt dysfunction, disrupts cellular redox homeostasis and triggers neurodegeneration remains to be further investigated. Proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) plays critical roles in regulating Mt biogenesis and respiration, cellular antioxidant defense mechanism, and maintenance of neuronal integrity and function. In this study, we sought to investigate whether alcohol causes Mt dysfunction and triggers neurodegeneration by suppressing PGC-1α expression. We report that ethanol suppresses PGC-1α expression, and impairs mitochondrial function and enhances cellular toxicity in cultured neuronal cell line and also in human fetal brain neural stem cell-derived primary neurons. Moreover, we report that cells over-expressing exogenous PGC-1α or treated with Rolipram, a selective phosphodiesterase-4 inhibitor, ameliorate alcohol-induced cellular toxicity. Further analysis show that ethanol decreases steady-state intracellular cAMP levels, and thus depletes phosphorylation of cAMP-response element binding protein (p-CREB), the key transcription factor that regulates transcription of PGC-1α gene. Accordingly, we found PGC-1α promoter activity and transcription was dramatically repressed in neuronal cells when exposed to ethanol, suggesting that ethanol blunts cAMP→CREB signaling pathway to interfere with the transcription of PGC-1α. Ethanol-mediated decrease in PGC-1α activity results in the disruption of Mt respiration and function and higher cellular toxicity. This study might lead to potential therapeutic intervention to ameliorate alcohol-induced apoptosis and/or neurodegeneration by targeting PGC-1α.