CROSS INTERACTION BETWEEN B-AMYLOID PROTEIN AND ADULT NEUROGENESIS DURING NEURODEGENERATION: TOWARDS THE IDENTIFICATION OF NEW THERAPEUTICAL STRATEGIES FOR ALZHEIMER'S DISEASE

Abstract

Adult neurogenesis is a multi-step process, which consists in the continuous generation of new neurons in the mammalian brain throughout adulthood. This process occurs in two brain regions of mammals, also called neurogenic niches, which are the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus (DG-HP). The role of adult neurogenesis in neurodegenerative disorders, such as Alzheimer’s disease (AD), is still under debate. Since the latter stages of AD are characterized by a massive loss of neurons, possible role of adult neurogenesis in the pathogenesis of this disease has been investigated. Indeed, the complete understanding of the role of neurogenesis in the pathology would exploit this physiological process as a possible therapy for Alzheimer’s disease. Alzheimer’s disease (AD) animal models are still useful to study dynamic alteration of adult neurogenesis due to the fact that similar studies are no possible in humans. Conflicting observations have been reported regarding the level of neurogenesis in animal models of Alzheimer’s disease. Furthermore, very little is known about differences in neurogenesis at the early stages of AD, before the onset of amyloid-plaque formation, one of the typical hallmarks of pathology progression. Indeed, there are increasing evidences that adult neurogenesis is under the influence of key molecules underlying Alzheimer’s disease, such as the - amyloid (A) peptide. During disease progression, A peptides assemble into various aggregation forms, ranging from dimers and oligomers to fibrils in amyloid plaques. However, the magnitude of amyloid plaque deposition in the brain correlates poorly with cognitive decline, and emerging evidence suggests that A oligomers may be the major causes in this regard. However, it is still not clear how the pathophysiological environment in the AD brain, and in particular the different A species, affect neural stem cell (NSC) biology and thus adult neurogenesis. Previous studies obtained controversial results, reporting that extracellular A either decreases or increases proliferation, prevents or induces neurogenesis of NSCs. These discrepancies are likely due to the different animal and cellular models used and, more important, the different A species. Furthermore, none of these studies explored the role of intracellular A generation and oligomerization, which is one of the earliest event in AD pathogenesis. The aim of this thesis is to define how neurogenesis is regulated in Alzheimer’s disease and if alterations in adult neurogenesis represent an early event in AD pathogenesis. I investigated neurogenesis in Tg2576 transgenic mice at an age (1-2 months) that is considered pre-symptomatic, in terms of Aβ accumulation and neurodegeneration. Moreover, since in these mice there is an overproduction Aβ oligomers (AβOs) in the nervous system, I investigated if naturally occurring Aβ oligomers, which represent the most neurotoxic species in AD, modulate NSCs biology, in view of possible cross-interaction between impaired neurogenesis and the amyloidogenic processing pathway. To better investigate this aim SVZ-and DG-derived aNSCs have been analyzed in vitro for their proliferation and differentiation potential in relationship with the biochemical profile of the different Aβ species. The ultimate goal of this research is to gain new insights into the molecular mechanisms that control adult neurogenesis in AD neurodegeneration and to develop new strategies to restore normal neurogenesis specifically in those brain regions where it is impaired. By analyzing the proliferative and differentiative features of resident or SVZ-derived adult neural stem cells (aNSCs) I found that Tg2576 aNSCs proliferate significantly less, with respect to their control counterparts. Tg2576 neurospheres are enriched in DCX+ neuroblasts, which failed to terminally differentiate, as demonstrated by the lower degree of maturation in terms of neurites arborization, and give rise to less GFAP+ astrocytes, with an aberrant morphology. These defects have been confirmed also in vivo, where a defective olfactory bulbs (OBs) neurogenesis is observed, as demonstrated by the reduced number of newborn interneurons NueN+ and CalR+ in Tg2576 OBs. I demonstrated that reduced proliferation and the differentiation impairment of Tg2576 progenitors in vitro are caused by endogenous oligomeric Aβ: indeed, both defects are rescued by the expression of a conformation-specific intrabody, which selectively interferes with the early intracellular generation of Aβ oligomers. Noteworthy, the AβO-selective intrabody interference restores the pathological microtubule hyperstabilization of newly formed neurons, which is tau mediated. Finally, lentiviral-mediated in vivo expression of the intrabody in Tg2576 SVZ rescues the olfactory bulbs neurogenesis, and restores SVZ-derived aNSCs proliferation and differentiation. Early alterations in adult neurogenesis were also present in the other neurogenic niche of Tg2576 mice, the dentate gyrus of the hippocampus (DG-HP). Similarly to the SVZ-derived progenitors, hippocampal progenitors displayed high amount of intracellular Aβ oligomers and undergo neuronal and astrocytic differentiation impairment. Both defects were rescued by the AβOs intracellular targeting with an inducible scFvA13-KDEL intrabody. Comparable results were obtained in human neural progenitors (NPs) that stably expressed the ScFvA13 intrabody. Moreover, in order to analyze also the role of extracellular AβOs accumulation, that leads to Aβ plaques formation at the last stage of AD, Tg2576 aNSCs have been treated with the recombinant nanobody. In this way, I demonstrated that also extracellular AβOs played an important role in controlling both proliferation and differentiation of aNSCs. In fact, the extracellular administration of ScFvA13 nanobody to Tg2576 aNSCs significantly rescues their proliferative impairment as well as their neurogenic defects, in a dose dependent manner. Strikingly, I demonstrated the efficacy of ScFvA13, both as intrabody and nanobody, in restoring AβOs-dependent proliferative and differentiative impairment also in human-AD NPs. Altogether, my results demonstrate that impaired neurogenesis is an early event, occurring at a presymptomatic age prior to overt neurodegeneration and is caused by endogenous AβOs accumulation in the neurogenic niches. Notably, the intra- or extracellular interception of AβOs by scFvA13 antibody in aNSCs reestablishes proper neuronal and glial differentiation. This important finding demonstrates the potential efficacy of targeting AbOs in aNSCs as a therapeutic strategy to restrain neuronal degeneration in AD and validates the scFvA13 antibody as a new therapeutic tool, able to restore a functional neurogenesis.