Our data show that: 1) TLR2 is expressed in the developing mouse brain and in cultured embryonic NPC. 2) The presence of TLR2 does not influence NPC proliferation both in vitro
and in vivo
, whereas its activation inhibits NPC proliferation in vitro
and decreases cell proliferation in vivo
. 3) Activation of TLR2 with bacterial ligands in utero
results in ventricular dysplasia. The lack of a difference in the proliferation rate of NPC from WT and TLR2KO mice, and the similar ventricular size and gross morphology of brains in WT and TLR2KO embryos, suggests that TLR2 signaling does not play a critical role in embryonic brain development under normal conditions. However, we cannot rule out more subtle effects of endogenous TLR2 signaling on the development of cortical cytoarchitecture, or on the behavior of NPC in the adult brain. In a study of adult hippocampal NPC, TLR2 deficiency did not alter the proliferation of NPC, but did affect their phenotypic fate by increasing the formation of astrocytes at the expense of neurons (Rolls et al. 2007
). In contrast, in the present study the absence of TLR2 did not alter the proportion of astrocytes, oligodendrocytes and neurons that differentiate from embryonic NPC. This apparent discrepancy may result from differences in NPC-specific developmental transcriptional changes at embryonic and adult stages, or alternatively from differences between NPC niches in the developing and adult brain. TLR2 activation in differentiating embryonic NPC derived from either E12 or E15 developmental stages does not affect differentiation proportions to neurons or astrocytes, limiting the effects of TLR2 activation in NPC to proliferation inhibition only.
Very little data exist regarding the expression and functions of TLRs in the developing nervous system. TLR8 is abundant in embryonic brain as early as E12, however this expression is limited to sympathetic ganglia and postmitotic migrating cortical neurons and axons and is not present in the proliferating ventricular zone (Ma et al. 2006
). In contrast, NPC express TLRs 2, 3 and 4 from early in CNS development (Lathia et al. 2008
). TLR3 is transiently increased from early brain development (E12.5) and begins to diminish at E17.5 (Lathia et al. 2008
). In contrast, TLR4 maintains relatively constant expression levels throughout neuronal development and into postnatal stages (Lathia et al. 2008
). Our data indicate that TLR2 mRNA is expressed at E11 and into postnatal ages in vivo
as well as in SOX2-expressing NPC in vitro
. Notably, while both mRNA and protein levels for TLR2 increase during embryogeneis and into postnatal ages, mRNA for MyD88 increases while its protein levels remain constant during embryogenesis and early postnatal stages. This could be due to reduced stability of the mRNA for MyD88, which requires higher levels of the mRNA to maintain high protein expression levels. The increased expression of TLR2 during cortical development was contrasted by the decreased expression of IRF5, a transcription factor that is activated by TLR2, suggesting a developmental role of IRF5 that may be independent of TLR2. Further, we show that TLR2 and its heterodimer partner, TLR6, but not TLR1, are expressed in proliferating cells in the SVZ. The presence of TLR1 mRNA but not protein levels could imply lack of expression, but could also be the result of differences between in vivo
and in vitro
growth conditions. We further show that TLR2 is indeed functional and can be activated in response to both exogenous and endogenous TLR2-specific stimuli which results in Akt phosphorylation and activation of the transcription factor NF-κB, in a manner similar to that shown in immune cells. While in earlier developmental stages, the percentage of proliferating cells is greater than in later developmental stages, the changes discussed above observed in embryonic cortical tissue reflect total mRNA and protein levels not only in SVZ proliferating cells, but also in neurons, glia and to a lower extent possibly other cell types.
Emerging findings suggest that TLRs, whether activated by endogenous or exogenous ligands, often act as negative regulators of NPC proliferation and differentiation. TLR4 activation restricts retinal progenitor cell proliferation in early postnatal eye development (Shechter et al. 2008
). TLR8 is expressed in the cortex as early as E12, where its activation restricts neurite outgrowth and induces apoptosis in differentiated neurons (Ma et al. 2006
). Similarly, activation of TLR3 inhibits neurite outgrowth and causes growth cone collapse (Cameron et al. 2007
). TLR3 was recently reported to negatively regulate NPC proliferation during early stages of neuronal development and that TLR3 activation with PolyI:C decreases NPC proliferation (Lathia et al. 2008
). The absence of functional TLR3 during embryogenesis promotes proliferation of NPC suggesting that TLR3 may be activated by an endogenous ligand. In contrast, we found that elimination of TLR2 neither affects the proliferative capacity of embryonic NPC, nor alters the fate of cells during differentiation in vitro
. However, similar to TLR3 activation, activation of TLR2/6 or TLR2/1 heterodimers with either FSL1 or Pam3
respectively, or with an endogenous ligand for TLR2, LMW-HA, inhibits NPC proliferation in vitro
. Therefore, while TLR2 itself may not participate in restricting NPC proliferation, its activation by either exogenous or endogenous ligands diminishes the proliferative capacity of NPC.
Activation of TLR2 with FSL1, Pam3CSK4 or with LMW-HA inhibited neurosphere formation in vitro and caused ventricular dysplasia when injected into the ventricles of E15 mouse embryos as indicated by a significant increase in ventricle size, decrease in the proliferative area, increase in cell density in the proliferative area, an increase in the number of PH3+ cells and a decrease in the number of BrdU+ cells in the SVZ. This suggests that TLR2 activation interferes with the cell cycle of NPC, prevents cells from incorporating BrdU and prevents them from exiting M-phase of the cell cycle, thereby causing telencephalic dysplasia. The mechanism for these effects could involve both direct actions on the NPC and indirect effects. Indirect effects could result from an increase in cell density followed by cellular stress leading to deleterious counter effects, or also ventricular edema induced by TLR2 signaling in ependymal cells could cause ventricular volume increase, which culminates in decreased SVZ volume. Interestingly, there were no significant changes in all the tested parameters following in utero injection between the ipsilateral and contralateral embryonic hemispheres. This could be due to either a rapid dispersion of Pam3CSK4 throughout the embryonic CNS or as suggested above, due to an extrinsic effect such as a released factor(s) that culminate in telencephalic dysplasia.
Despite the high degree of specificity of TLR2/1 and TLR2/6 heterodimers to their ligands, we did not observe any differences in the effects conferred by the two ligands. Equimolar doses of both TLR2/6 and TLR2/1 activation induced comparable rates of neurosphere formation inhibition. TLR2 stimulation results in NF-κB activation in macrophages (Schwandner et al. 1999
), AP-1 in gastric cells (Chang et al. 2005
) and Nrf-2 in tracheal smooth muscle cells (Lee et al. 2008
). Inhibition of NPC proliferation by both ligands was correlated with NF-κB promoter activity but not AP-1 or Nrf-2. While FSL1 administration resulted in greater NF-κB promoter activity compared to Pam3
, FSL1 was not more potent than Pam3
in inhibiting NPC proliferation, suggesting that the increase evoked by Pam3
was sufficient to maximally inhibit NPC proliferation. In addition, the basal activity of AP-1 was not affected by either TLR2 ligands, and no promoter activity of Nrf-2 was observed in NPC even in response to sulforaphane, an Nrf-2 activator (Ellis 2007
). Moreover, co-administration of Pam3
and FSL1 resulted in a significant but mild additive inhibitory effect, which likely results from mutual downstream signaling induced by the two ligands.
Our findings are of interest in the context of alterations in brain development caused by infectious agents, ischemia and inflammatory states. TLR2 activation has long been known to occur in immune cells during bacterial infections. Bacterial infection is common during pregnancy, including bacterial vaginosis, and can result in preterm birth and spontaneous abortion (Leitich et al. 2003
, Sorensen et al. 2008
). Prenatal infection can also have profound lasting effects on brain development. For example, maternal bacterial infection is correlated with increased risk of schizophrenia (Sorensen et al. 2008
). Maternal infection with lipopolysaccharide (LPS), a TLR4 agonist, is associated with altered hippocampal morphology and decreased synaptic transmission (Golan et al. 2005
, Lowe et al. 2008
). LPS administration results in cerebral death and sensitizes the developing chick brain to hypoxic damage and death (Wang et al. 2008
). Further, prenatal LPS infection selectively reduces baseline numbers of dopaminergic neurons in the substantia nigra, thereby increasing the vulnerability of the animals to a Parkinsonian toxin (6-hydroxydopamine) later in life (Ling et al. 2004
). Our findings suggest that in utero
exposure of the brain to infectious agents that produce TLR2 ligands or endogenous activation of TLR2 such as those used in the present study may diminish the proliferative capacity of embryonic NPC and possibly lead to lasting abnormalities of neuronal plasticity and behavior. One particularly interesting long-term effect is whether TLR2-mediated telencephalic dysplasia affects the rostral migratory stream of NPC to the olfactory bulb, and whether this culminates in behavioral abnormalities in the adult mouse.