6.1.1Working model for CRS and RUBV induced neurological diseases
The overarching goal of this dissertation was to elucidate the mechanism underlying the develop- ment of neurological abnormalities in CRS. Since the primary time in gestation when RUBV exhibits pro- found teratogenicity (i.e, the first trimester) coincides with organogenesis in the developing fetus, the fa- vored hypothesis is that alterations induced in infected fetal stem/progenitor cells ultimately affect organ development. The attractiveness of this hypothesis increased, especially in CNS complications, consider- ing the recently developed “schizophrenia hypothesis” which stated that schizophrenia results from a dis- ruption in programmed maturation of the brain in prenatal and early neonatal life [109]. Meanwhile, RUBV specific antibody can be detected in the CNS of infants with CRS for a prolonged time after birth and in PRP with onset of disease in the second-decade of life in individuals with CRS, indicating the in- fectious process continues within in brain even after birth and that virus persistence may play an im- portant role for the pathogenesis of RUBV-associated neurological diseases. Our model is that hNPCs are the major cell type that RUBV impacts in the developing of CNS. The cells support RUBV replication and persistence and therefore it serves as a reservoir for long-term viral residence in brain RUBV limits hNPCs cell doubling potential and deactivates the genetic program of cell death in infected cells, albeit in a small number of cells. Overall, however, this explains the general features of CRS-induced disease, namely reduced cell number and aberrations, but not to the extent that fetal death occurs, Since hNPCs still function in adult brain, it is highly possible that the depletion of hNPCs over the years would signifi- cantly hamper the local brain tissue repair and regeneration ability and thus contribute partially to the later onset neurological diseases, such as autism or schizophrenia. Our model is also proposed in conjunction with the studies in this dissertation, which found that viral persistency may play an important role for the disease development: the non-productive replication of RUBV in hNPCs may serve as a mechanism to
avoid local cytokine/inflammatory responses in brain at early embryonic development. However, with the progressive maturity of adaptive immune response and increased humoral competency, virus can be de- tected and therefore trigger massive immune responses that contribute to CRS late-onset diseases. The neuronal and glial cell damages could possibly be triggered by viral mediated demyelination (molecular mimicry between viral envelope protein and MOG) and cytokine mediated local necrosis. Our results may complement the clonal hypothesis by Simons (REF), which posits that viral persistence in congenital ru- bella could be explained by the effect of many interacting factors: the intracellular location of RUBV may avoid the activity of specific antibodies; the impossibility of activating the genetic program of cell death by at least part of the infected cells during embryogenesis; the slower growth rate and limited doubling potential observed in clones of infected cells.
6.1.2hESC derived neural progenitors as a model to study viral teratogenesis
Numerous attempts have been made to establish a model of RUBV and other teratogenic viruses both in vitro and in vivo. Overall, definitive conclusions cannot be drawn for mechanisms of teratogenesis of RUBV since none of the previous studies was performed in a human lineage that mimics early stages of fetal development. hNPCs derived from hESCs presumably mimic the in utero progression from the pluripotent cells of the inner cell mass to differentiated neuronal phenotype. Thus, the first advantage of using such cells to study RUBV and other viral teratogens is that they well reflect the most important de- velopmental events during first trimester of pregnancy the time period in which the fetus is most vulnera- ble to the malformations. Indeed, in our study, the results of RUBV infection of hNPCs are consistent with earlier in vitro and in vivo CRS studies in brain suggesting that hNPCs provide a reliable model for RUBV-induced teratogenesis research. Another advantage we can obviously point out after all these ex- periments is that: hESC derived hNPCs have uniform performance over proliferation and differentiation, However, this is hard to achieve in hNPCs isolated from abortuses; the performance of the cells largely depend on the gestational age of fetus and genetic background. Finally, the diverse cell responses of hNPCs to different virus infections (SINV and RUBV in our study and VZV by Goldstein) supports that
hESC-derived progenitor cultures have potential broad application for detailed pathogenesis studies with RUBV and other clinically important teratogenic viruses.
Taken together, our work demonstrates the utility of hESCs derived culture systems as a platform to study the interactions between embryonic development, virus infection and pathogenesis in CNS. Fu- ture work should focus on broadening the genetic background of the cells, which can be achieved by em- ploying other hESC lines and reprogramming cells of interested individuals (see NIH hESCs cell line reg- istry for a complete list of hESC lines eligible for NIH funding). Also, additional work has to be done by stem cell biologists to further explore the multipotency of those progenitor cells such as to characterize specific lineages that can be derived from progenitor cells. Thus, moving to more efficient protocols for complete terminal differentiation of hNPCs would be a great step-forward in the field.
6.1.3Significance
The work described in this dissertation adds to the expanding breath of knowledge concerning the CNS disorders associated with RUBV infection. It is important to continue this work, as important paral- lels could be drawn between RUBV and other teratogenic/ neurotropic viruses in the pathogenesis of var- ious brain anomalies. In comparison and contrast with another neurotropic virus, SINV, our study also illustrated how multiple factors come together to influence the final outcome of an infection in same type of cells therefore the neuropathological manifestations. It is intriguing that RUBV, although inducinng a wide spectrum of neurological disease throughout life of congenitally infected individuals is not particu- larly neurovirulent to the brain. As similarly seen in VZV, this represents a category of neuropathologen- esis associated with teratogenic viruses that requires further exploration. RUBV infected hNPCs could further serves as a model of chronic viral infections in perinatal/postnatal period which via disruptions of NPCs growth and neurogenesis, which might lead to the onset of neurovahavioral abnormalities.