In 1912, Dr. Ezra Allen was the first to make the observation that “after the dividing cells have disappeared from all other areas of the brain they are still to be found in the limited zone along the lateral ventricles of the cerebrum”. It took over 80 years for the scientific community to accept adult neurogenesis as a real phenomenon. For this historical reason, adult neurogenesis is still a relatively new field and researchers are still defining the basic components of adult neurogenic systems. There are two main neurogenic regions in the adult brain: the subgranular zone of the hippocampus and the subventricular zone (SVZ) lining the lateral ventricles. The SVZ is the largest neurogenic niche and present throughout a variety of species, including mice and humans.
The SVZ is comprised of five main cell types. Ependymal cells separate the brain parenchyma from the lateral ventricle. B1 astrocytes sit atop this layer, in prime position to receive cues from the cerebral spinal fluid and blood. They also receive trophic support from non-neurogenic B2 astrocytes scattered throughout the SVZ. B1 astrocytes are the quiescent neural stem cells in the adult brain. They asymmetrically divide to give rise to transit amplifying progenitor cells (C cells). C cells, in turn, produce neuroblasts. In most species, neuroblasts exit the SVZ and travel through the rostral migratory stream to the olfactory bulb (OB), where they differentiate to become functional interneurons. Although the cellular progression within the SVZ is well defined, cell type specific marker expression is not. Chapter 2 aims to clarify marker expression within the adult rodent SVZ. The most important finding from this chapter is that marker expression is not static. Instead, proliferative cells gain and lose marker expression depending upon their place in the lineage progression.
Chapter 2 also introduces Glial fibrillary acidic protein (GFAP). GFAP is expressed by both B1 and B2 astrocytes within the SVZ. GFAP is an intermediate filament protein commonly found in astrocytes. Intermediate filament proteins are integral components of the cytoskeleton. Here, intermediate filaments function as signaling platforms for diverse processes including proliferation, motility, morphology, and stress responses. GFAP has several isoforms including GFAPa and GFAPd. The canonical isoform GFAPa is the most highly expressed GFAP isoform throughout the central nervous system. GFAPd is an alternatively spliced isoform characterized by the inclusion of the alternative exon, exon 7+. Thereby, GFAPd differs from GFAPa in its C terminal tail.
GFAPd has raised some considerable interest in the scientific community, as GFAPd is a preferential marker for radial glia in the developing brain and B1 astrocytes in the adult SVZ in the human brain. GFAPd is also expressed sparsely in other human brain regions such as the hippocampus, glia limitans, and around the third ventricle. That being said, adult human GFAPd+ SVZ stem cells can self-renew and are multipotent. Human GFAPd also interacts with presenilin, a crucial component of the notch pathway. Recent data, again, demonstrates the importance of GFAPd to stem cell biology. GFAPd directly interacts with notch, having direct effects on neurosphere formation. Combined, this evidence shows that within the human SVZ, GFAPd is a neural stem cell marker and an active component human neural stem cell biology.
The main questions of this thesis are: (1) Is GFAPd also a neural stem cell marker in mouse as it is in human? (2) Is GFAP important for adult neurogenesis in the mouse SVZ? and (3) What tools can be employed to specifically modulate GFAP isoform expression within the adult mouse SVZ?
(1) Is GFAPd also a neural stem cell marker in mouse as it is in human?
Though much is known about GFAPd in the human, there was little known about it in mouse. Therefore, GFAPd expression was characterized in the mouse throughout development, in primary astrocyte and neural stem cell cultures (Chapter 3), as well as in adulthood (Chapter 4). GFAPd expression in mouse is strikingly different to what is seen in human. To begin with, GFAPd expression does not coincide with GFAP expression as it does in human. GFAPd is first seen on protein level at embryonic day 18 (E18), whereas GFAP can be detected at E12. Moreover, GFAPd is expressed by a heterogeneous population of cells that includes radial glia and non-neurogenic astrocytes, such as those in the supragranular and fimbrial bundles of the hippocampus. This observation indicates that GFAPd is present in all mouse astrocytes, regardless of their neurogenic potential. In vitro data further supports this finding because GFAPd is highly expressed by both primary mouse astrocytes and neural stem cells. Moreover, both astrocytes and neural stem cells have a comparable Gfapd/Gfapa transcript ratio. An interesting finding in Chapter 3 is that Gfapd transcripts are observed much earlier (E12) than GFAPd protein (E18). Although this could be due to the lack of sensitivity of the antibody, it is most probably attributed to a translational uncoupling. Such an uncoupling has been proposed before in GFAP during mouse and rat development. It is currently unknown whether this phenomenon is also seen during human development or whether the Gfap gene has evolved in such a way to eliminate this uncoupling. Once Gfapd transcripts can be reliably detected, the Gfapd/Gfapa transcript ratio does not change throughout development.
Chapter 4 shows that the Gfapd/Gfapa transcript ratio also remains stable throughout neurogenic and non-neurogenic brain regions in the adult mouse, despite the regional differences in Gfap expression. Moreover, GFAPd is expressed by virtually all cells that express GFAPa. This is in stark contrast to the adult human situation, where GFAPd marks specific populations of cells. Another difference between human and mouse Gfap expression is that mice do not express all the Gfap isoforms that humans do. There was no evidence for GfapΔexon6, GfapΔ135, and GfapΔ164 expression under normal (wildtype) or pathological (Alzheimer) conditions. Interestingly at the transcript level, reactive astrocytes in the Alzheimer brain proportionally upregulated all alternatively spliced Gfap isoforms, but upregulated Gfapa to a seemingly higher degree. Thus emphasizing the importance of the canonical isoform Gfapa in reactive gliosis. Within the human brain, GFAP isoforms mark distinct subpopulations of astrocytes. However, within the mouse brain Gfap isoforms tend to be expressed in similar ratios throughout all brain regions. Together Chapter 3 and Chapter 4 show that GFAPd is broadly expressed throughout mouse development and adulthood. Therefore, GFAPd is not a specific neural stem cell marker in the mouse brain.
(2) Is GFAP important for adult neurogenesis in the mouse SVZ?
GFAP is highly expressed by both B1 and B2 astrocytes within the SVZ. Therefore, it was asked whether GFAP, itself, or individual GFAP isoforms were important for adult neurogenesis. Chapter 5 characterizes adult neurogenesis within the GFAP knockout (-/-) mouse. Interestingly, the absence of GFAP does not affect the architecture or proliferation potential of the SVZ. However, the loss of GFAP leads to an altered environmental milieu characterized by less glial derived neurotropic factor (Gdnf) and neural adhesion cell molecule (Ncam), among other transcript changes. Typically, these changes should give rise to an altered neurogenic phenotype. However, B1 astrocytes and C Cells actively adapt to the altered environment by, for example, upregulating receptors such as the epidermal growth factor receptor (Egfr). B1 astrocyte and C cells also quickly adapt in vitro, as GFAP-/- adult neurospheres show a reduced proliferation potential only at passage 1. These B1 astrocyte and C cell adaptations, therefore, presumably restore a “normal” proliferation profile within the GFAP-/- SVZ. The GFAP-/- OB also displays an altered environmental milieu. Here, there is an upregulation of the pro-survival factor S100b. This environment theoretically fosters the increased number of neuroblasts that reach and incorporate into the OB. Together data from the GFAP-/- mouse indicates that GFAP is not a crucial functional component to B1 astrocytes. Instead, GFAP plays more of a background role in non-neurogenic astrocytes - assisting them in the production of balanced trophic support.
Chapter 7 and Chapter 8 aimed to decipher whether GFAPa or GFAPd have individual roles within the adult mouse SVZ. Two different RNA interference (RNAi) approaches were used in Chapter 7 to specifically downregulate Gfapd. shRNA successfully downregulated Gfapd transcripts after 3 weeks. However, this downregulation did not affect the SVZ neurogenic niche and was transient. It is possible that the lack of phenotype seen in this study was a result of insufficient Gfapd downregulation and possible off-target effects. In Chapter 8 the opposite approach was used; here, GFAPa or GFAPd were reintroduced into the adult GFAP-/- SVZ. Again, this isoform modulation did not affect the SVZ niche. As GFAPa or GFAPd were successfully re-expressed within the SVZ, this observation possibly indicates that a proper intermediate filament network is required in order for GFAP isoforms to subtly exert an effect. Combined, data from these three chapters indicate that GFAP is not strictly necessary for the process of adult neurogenesis, but it does help to subtly regulate the environmental milieu. Therefore, GFAP is an understudy in adult mouse SVZ neurogenesis.
(3) What tools can be employed to specifically modulate GFAP isoform expression within the adult mouse SVZ?
In vivo modulation of specific cell populations is a powerful tool to uncover how a protein of interest affects cellular processes. Chapter 6 reviews a variety of methods including electroporation, Cre/LoxP technology, and viral vectors in regards to their ability to specifically modulate B1 astrocytes in vivo. Ultimately, it was decided that viral vectors such as lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) had the highest potential for modulating GFAP isoform expression within the adult mouse SVZ. Chapter 7 compares the tropism of AAV4, AAV5, and LV pseudotyped with a vesicular stomatis virus envelope (VSV-G) within the SVZ. AAV4 showed the most limited tropism – transducing only ependymal cells and the choroid plexus. Based upon this observation, AAV4 is not suitable for B1 astrocyte modulation. Both AAV5 and LV VSV-G transduced B1 astrocytes, but not specifically. This lack of specificity resulted in off-target effects when using two different RNAi constructs. Chapter 8 reintroduced GFAPa or GFAPd into the GFAP-/- SVZ using LV VSV-G. Again, the broad tropism of LV VSV-G led to GFAPa and GFAPd expression in astrocytes along with other cell types, impeding data interpretation. Together, these chapters clearly show the vital importance of specific targeting.
The end of Chapter 10 revisits the methodology surrounding cell type specific targeting. There are a variety of ways to limit transgene expression in distinct cell populations. In regards to B1 astrocytes, LVs can be pseudotyped with envelopes that have favorable astrocyte tropism. Promoters, such as gfa2, can also be used to guide transgene expression. Furthermore, newer technologies, such as Split-Cre, hold an even more refined in vivo targeting potential. It is likely that a combination of these methods will result in B1 astrocyte specific targeting. Nonetheless, more time and effort must be placed in the development of a transgene delivery system that is B1 astrocyte specific.
GFAPd displays a differential expression pattern in the human and mouse brain, whereas, GFAPa has a similar pattern in both species. GFAPd is not a specific neural stem cell marker in the developing and adult mouse brain. GFAP isoforms are highly expressed within the adult mouse SVZ. But despite this strong expression, GFAP, itself, is not a crucial component in adult mouse neural stem cells. Instead, within the mouse brain, GFAP dwells in the background and is most likely involved in the subtle regulation of the environmental milieu. More specific targeting tools need to be used in order understand the exact subtleties that GFAP elicits within the mouse brain. This refinement might help uncover why GFAP isoforms have acquired species-specific profiles. Chapter 10 delves deeper into how this species difference could have emerged throughout evolution and discusses the validity of mouse models for the study of adult neurogenesis. In conclusion, although GFAP is an understudy in adult neurogenesis, its subtle regulation and intricate ties to astrocytes make it a star.