New neurons and astrocytes in the adult brain. Studies on adult rat neural progenitors in vivo and in vitro with special reference to the effects of IGF-I and CNTF regarding proliferation and lineage determination

Abstract

The hippocampal dentate gyrus in several species, including man, continues to produce new neurons and glial cells in adulthood. The generation of new neurons and glial cells in the adult hippocampus is under strict regulation. Two major events of significance for cell genesis from progenitor cells are proliferation and lineage determination. In the present thesis, the importance of two endogenous proteins, namely ciliary neurotrophic factor (CNTF) and insulin-like growth factor I (IGF-I), in adult hippocampal progenitor cell regulation was investigated concerning proliferation and lineage determination. Recent studies suggest that CNTF may play a role in reactive gliosis, a hallmark of most CNS disorders. The CNTF receptor a-subunit (CNTFRa) was expressed in fibroblast growth factor-2 (FGF-2) expanded adult-derived rat hippocampal neural progenitors (AHPs). Binding of CNTF to CNTFRa resulted in activation of the Janus kinase - signal transducer and activator of transcription (Jak - STAT) pathway. It was demonstrated that CNTF is an instructive signal for astroglial type 2 cell fate in AHPs, and by electroporation of antisense oligonucleotides against STAT3 it was shown that the astroglial lineage determination is specifically mediated via activation of STAT3. However, inhibition of the mitogen-activated protein kinase (MAPK) signalling pathway did not block CNTF-induced gliogenesis in progenitor cells. These experiments demonstrated that microelectroporation is a novel powerful method for introducing agents into single cells in complex cellular networks. In vivo we investigated the effect of the peripheral administration of IGF-I on cellular proliferation in the adult rat dentate subgranular proliferative zone, and on the subsequent migration and differentiation of progenitor cells within the granule cell layer (GCL). Using bromodeoxyuridine (BrdU) labelling, we found a significant increase of BrdU-immunoreactive progenitors in the GCL after 6 days of peripheral IGF-I administration. BrdU-positive cells also increased significantly in animals treated with IGF-I for 20 days. Furthermore, the fraction of newly generated neurons in the GCL increased, as evaluated by the co-localisation of neuronal markers and BrdU after 20 days of IGF-I treatment. Thus, our results show that peripheral infusion of IGF-I increases progenitor cell proliferation and induces neurogenesis in progeny of adult neural progenitor cells. This corresponds to a 78 + 17% (P < 0.001) increase in the number of new neurons in IGF-I-treated animals compared to controls. In vitro we found that the IGF-I receptor, IGF binding protein-2 and -4 (IGFBP-2 and IGFBP-4) are expressed in AHPs. IGF-I-treated cultures showed a dose-dependent increase in DNA synthesis and number of cells, well separated from the effects mediated by insulin, demonstrating a proliferative effect of IGF-I. The proliferative effect of IGF-I was additive to the proliferative effect of FGF-2. We also demonstrated by means of inhibitors and dominant negative constructs that the MAPK signalling pathway was required for IGF-I-stimulated proliferation in AHPs. Furthermore, an analysis of the phenotypic distribution showed that the fraction of newly generated neurons increased significantly. These findings demonstrate the importance of extracellular signals and indicate that proliferation and cell fate determination are regulated by complex interactions between AHPs and extracellular cues like the CNTF and IGF-I systems. As peripheral IGF-I has been shown to mediate an exercise-induced increase in hippocampal neurogenesis which in turn has been shown to improve memory in rodents, these results suggest that cognitive functions may be affected by peripheral IGF-I. Increased information about the regulation of progenitor cell proliferation and cell fate determination provides new insight into neurogenesis and its possible therapeutic potential.