Dr. Soshi Kanemoto (Postdoctoral Fellow)
Professor Paul Fraser and Professor Peter St. George-Hyslop
Repair of CNS Damaged by Neurodegenerative Disease
Introduction
Until a few years ago, conventional wisdom had led to the belief that the population of nerve cells in the adult brain was fixed, and that new nerve cells could not be generated in adulthood. However, this now appears to be inaccurate. Small numbers of new neurons can be found in selected regions of the adult brain and in the hippocampus in particular. The hippocampus is a brain structure critically involved in some aspects of memory and learning. This same brain region is also heavily impacted by the pathology of Alzheimer’s disease, and is one of the earliest regions affected by AD.
These new observations have led us to ask the question as to whether the presence of neurotoxic amyloid beta peptide influences the appearance of new neurons inside the hippocampus. This question has two important implications. First, while it is clear that amyloid-beta (Abeta) peptide kills established neurons in the adult brain, it is unclear whether this accelerated loss is compounded by an inhibition of the normal replacement of nerve cells through the process of adult neurogenesis. If Abeta causes both accelerated loss of existing neurons and reduced replacement by new neurons, there may need to be therapeutic strategies designed not only to protect existing neurons, but also to protect new neurons in the cortex as well. The second reason for the importance of this question relates to future attempts to repair brain injured by AD.
The process of inserting new neurons into the cortex requires a series of steps that include the birth of new nerve cells in deep brain structures, the migration of those cells to the cortex, the differentiation of those cells into functional adult nerve cells, and finally, their incorporation into functional neural circuits. The knowledge of which step/steps are affected by the presence of neurotoxic Abeta will shape the type of strategies that can be applied to facilitate brain repair in the future. Thus, defects in the migration from deep brain structures might be circumvented through surgically harvesting the precursors from deep brain structures, followed by simple autologous re-implantation of precursor cells into the desired brain regions. However, defects in the functional integration of new neurons into memory circuits will require additional work to understand the nature of the signals that are required for this integration process. That knowledge can then be applied to overcome any Abeta-mediated blockade of the relevant signaling process.
The work that will be developed in the next several years in this research program will be designed, therefore, to ask the question as to whether Abeta causes changes in:
- Rates of adult neurogenesis in deep brain structures;
- The rates of migration of these precursors to the hippocampus;
- The differentiation of these precursors into adult nerve cells in the hippocampus upon arrival; and
- The integration of these new neurons into memory circuits associated with learning spatial tasks.
These studies will be conducted in a robust model of human AD (TgCRND8 mice) and in non-transgenic littermate controls. While TgCRND8 mice do not have large amounts of neuronal death, they do have high levels of cerebral Abeta as well as functional and morphological evidence of neuronal injury (Figure 1). The absence of major amounts of cell death and gross anatomical changes in this model is important because migratory failure could also occur if large numbers of cells were lost from their normal anatomical locations, thereby disrupting the anatomical landmarks necessary for new neurons to navigate to their correct locations.
Methods
Two groups of animals at different stages of development were investigated to determine the effects of amyloid pathology on neurogenesis. Transgenics at 5-weeks and 12-weeks of age were injected with bromodeoxyuridine (BrdU) intraperitoneally twice daily for 5 days. BrdU is a synthetic nucleotide analog of thymidine which is incorporated into dividing and proliferating neurons as well as glia cells. Both groups of animals were then sampled at intervals of 1, 2, 4, 6 or 8 weeks from the starting point of each BrdU injection.
Brain sections were analyzed by immunohistochemsitry using a BrdU-specific antibody. BrdU-positive cells in the dentate gyrus and other regions of the brain were counted using confocal microscopy. To identify the cell types undergoing proliferation, sections were also counterstained with markers for neurons and astroglial. As markers for neuron, astroglia and neural progenitor cells, NeuN, s100beta and GFAP are used, respectively.
Results
Proliferation in the dentate gyrus is increased in the younger transgenic mice.
Two groups of animals, 5 and 12 weeks of age, were specifically selected as these time points represent significant differences in the amyloid pathology which is observed. The younger TgCRND transgenics have a high level of soluble Abeta that is found in an aggregated state but these have not been deposited as amyloid plaques. In the older mice, the Abeta has aggregated to a much greater extent and this results in the formation of insoluble amyloid fibrils which accumulates within the hippocampus and other affected regions associated with Alzheimer’s disease. The investigation of these two groups will therefore allow us to dissect out the relative contributions of the different amyloid species.
Both groups were initially examined for their total BrdU labeling which corresponds to the overall number of dividing cells. We found that the number of BrdU-labeled cells in the dentate gyrus of TgCRND8 mouse may have been moderately increased of the 5-week-old group compared with the
wild type mice (Figure 2). However, this does not represent a significant difference which suggests that there was no differences in the total number of proliferating cells in the TgCRND8 or control groups. This was confirmed in 12-week-old group where the number of BrdU-labeled cells in the dentate gyrus was virtually identical in both the TgCRND8 and wild type mice (Figure 3). These findings indicate that Abeta oligomers and amyloid plaques may not globally decrease the levels of all dividing cells. But these are composed of several different subgroups that include mature neurons and astrocytes as well as yet to be differentiated neural progenitors.
Decreased Neural Progenitor Cells in Amyloid Transgenic Mice
The different subgroups of dividing cells are divided into three basic categories: mature neuronal cells which are destined to develop into new neurons; mature astroglial cells that are enroute to becoming mature glia; and a population of neural progenitor cells (NPCs) which are the group of cells destined to become new neurons. By utilizing immunohistochemical approaches it is possible to identify each of these proliferating cell groups in the brain. All three cell lines are BrdU-positive and the cells are distinguished by secondary markers where mature neuronal lines are also NeuN-positive, mature astroglial lines are S100beta-positive while neural progenitors are labeled with the glia acid fibrillary protein (GFAP). Using antibodies specific for BrdU and the marker proteins it is possible to visualize and count the number of cells for each of these three groups (Figure 4).
TgCRND8 and wild-type control mice were injected with BrdU at 5 weeks of age and one group of animals was examined one week post-injection (6 weeks old) and another following eight weeks (13 weeks old) of BrdU labeling. Immunohistochemistry of the 6 week old mice indicated that the relative number of proliferating astroglial cells was unchanged which was determined by both BrdU and S100beta positive staining (Figure 5). In contrast, the number of Brdu-NeuN positive cells was decreased by approximately 35-40% in the TgCRND8 mice as compared to controls. The loss of neural progenitors in the amyloid transgenic animals was even more pronounced as shown by the number of BrdU-GFAP positive cells which was decreased by ~70%.
Mice labeled with BrdU for eight weeks (13 weeks of age total) displayed a relatively similar pattern. The number of astroglial cells (BrdU-S100beta) were unchanged in both wild-type and TgCRND8 amyloid mice (Figure 6; top). Unlike the one week labeling group, the mature neuronal population was not significantly reduced in the TgCRND8 mice to the extent seen in the younger animals (Figure 6; middle). However, the neural progenitor cells identified by BrdU-GFAP double labeling were consistently decreased, in this case, by approximately 80%. Cumulatively, these finding indicate that amyloid aggregates possibly both the soluble oligomers and the deposited plaques significantly reduce the number of neuronal progenitor population.
The older animals injected with BrdU at 12 weeks of age are currently being examined by similar immuno-histochemical techniques to determine if a comparable and specific decrease in GFAP-positive neural progenitors cells is observed. At this age the TgCRND8 animals will have begun to deposit classical amyloid plaques and the information from this aspect of the investigation will provide critical information on the roles of amyloid fibrils as opposed to diffusible Abeta oligomeric aggregates.
Next Phase
Neurogenesis and Cognitive Changes in Abeta-Immunized mice
Our group has previously demonstrated that vaccination of TgCRND8 mice with synthetic Abeta peptides results in a substantial decrease in the number and size of the amyloid plaques in vivo. This was also correlated with an improvement in cognitive ability of the mice consistent with a direct correlation of Abeta with the functional impairment of neuronal transmission. Abeta vaccines have since been investigated in clinical trials as a potential therapeutic strategy for Alzheimer disease. Given the reduced levels of neuronal progenitors we have observed in the amyloid transgenics, we will using a similar Abeta immunization strategy to determine if the number of BrdU-GFAP and BrdU-NeuN positive cells can be rescued by the elimination of the amyloid aggregates.
This will be achieved by active immunization of TgCRND8 and wild-type mice using synthetic Abeta (residues 1-40) as compared to a non-specific negative control peptide. The density of amyloid plaques and diffuse Abeta-positive deposits will be identified by immunocytochemistry using an amyloid-beta specific antibody and correlated with the location of BrdU-positive neurons. Spatial learning in TgCRND8 animals will be determined using the Morris Water Maze and compared to non-transgenic littermate controls. This technique involves a learning process based on unique cues that are linked to a submerged platform in the water maze. Animals learning and memory assessments are based on their ability to find and remember the platform location. The combination of these data on pathology, nerve cell proliferation and memory will indicate if AD-related amyloid alters neurogenesis and the ability to regenerate neuronal networks.
Neurosphere Isolation and Neural Progenitor Cell Proliferation Assay
The data obtained in vivo indicates that Abeta aggregates are detrimental to the survival and proliferation of the neural progenitor cells. This implicates amyloid as a primary factor in the process and to investigate this further and confirm the correlation and in vitro analyses will be conducted.
To isolate neural progenitor cells (NPCs), the subventricular zones of dentate gyrus from TgCRND8 and control mice will be isolated and cells will be dissociated. Culturing under specific conditions to induce neurosphere formation will be utilized which provides the source of NPCs. The proliferation of the total NPC population will by analyzed to determine if there are any instrinsic differences between the transgenic and control mice. More importantly, to confirm the role of amyloid in the specific reduction of BrdU-GFAP cells, cultured NPCs will be treated with amyloid aggregates produced by synthetic Abeta peptides. NPCs will then be labeled with BrdU and examined by immunohistochemistry using NeuN, S100beta and GFAP to quantify the levels of the different cell populations. If a similar and specific reduction of the GFAP-positive cells is observed under these in vitro conditions then these findings will provide compelling evidence on the adverse effect of Abeta on CNS regeneration in Alzheimer’s disease.
