(I-346) Direct Reprogramming-based Vasculogenic and Neurogenic Cell Therapies Drive Memory Improvement and Reduced Neuropathological Burden in a Mouse Model of Alzheimer’s Disease.

Introduction:: Alzheimer’s Disease (AD) is characterized by the presence of two core neuropathological lesions in the brain, amyloid-beta plaques, and neurofibrillary tangles. During the disease development these core lesions are accompanied by activation of resident and peripheral immune cells that lead to chronic inflammatory processes and a subsequent loss of synaptic connections and neurons. All these pathological processes ultimately result in a progressive and profound cognitive and executive decline of the AD patients. In addition to the main neuropathological mechanisms underlying AD, recent studies indicate that even before the emergence of amyloid-beta plaques, and neurofibrillary tangles, AD patients present significant cerebrovascular impairments, including reduced Cerebral Blood Flow (CBF) and Blood-Brain-Barrier (BBB) dysfunction. These observations suggest that impaired cerebrovascular function plays a key role in the onset and/or progression of AD and that therapeutic interventions aiming to reestablish the CBF and BBB function may attenuate AD development. Cell-based therapies constitute promising strategies to address vascular and neurogenic deficiencies in the brain. However, the reliance on primary cells or stem cell sources together with the use of viral vectors to manipulate cell plasticity, drastically reduce the translational potential of cell therapies due to substantial risks of uncontrolled differentiation, tumorigenesis, genetic abnormalities, and immunogenicity.

Materials and Methods:: To implement safe and efficient cell therapies in AD, we used electroporation, to deliver 3 pro-vasculogenic (Etv2, Foxc2, Fli1) and 3 pro-neurogenic (Ascl1, Brn2, Myt1l) reprogramming factors to reprogram mouse primary embryonic fibroblasts (pMEFs) into induced endothelial cells (iECs) induced neurons and (iN) respectively. To evaluate their therapeutic potential, pMEFs labeled with 5-bromo-2’-deoxyuridine (BrdU) and transfected with Etv2, Foxc2, Fli1(EFF) or an empty plasmid were delivered with 3 intracranial injections (8x10⁵ cells/injection) into the lateral ventricles (LV) of females from the triple transgenic murine model of AD (3xTg-AD) or Wild-Type Controls (n=10 per group, 40 total). Two weeks after the last injection (26 weeks-old mice) spatial memory was analyzed with the Barnes Maze, and recognitivn memory was evaluated by Novel Object Recognition (NOR) assay. Subsequently, brain tissue was processed for immunostaining and biochemical analysis. Similarly, pMEFs labeled with BrdU and transfected with Ascl1, Brn2, Myt1l (ABM), ABM+EFF, or an empty plasmid were injected into the hippocampus (1x10⁵ cells/injection) of 3xTg-AD or Wild-Type Controls females (n=10 per group, 40 total). 4 weeks after the injection (51 weeks old mice) spatial memory was analyzed with the Barnes Maze and recognition memory by NOR. One week later, brain tissue was processed for immunostaining and biochemical analysis.


Results, Conclusions, and Discussions:: Our preliminary results indicate that pMEFs pre-programmed into iECs and delivered to the LVs induce and increase in global CBF (Figure 1A) a meaningful reduction of the spatial memory deficits observed in 3xTg-AD (Figure 1B). In addition, injected cells can survive for at least 4 weeks inside the brain, migrate to the brain vasculature and induce and increase in the total vascular area in the cortex of the 3xTg-AD mice (Figure 1D-E). Finally, injection of EFF transfected cells seems to induce the activation of resident microglia cells together with a reduction in amyloid-beta load (Figure 1F). On the other hand, pMEFs pre-programmed into iNs can survive for at least 6 weeks and express markers of mature neurons inside the brain parenchyma (Figure 1G) and induce a reduction of amyloid-beta levels in the hippocampus of 3xTg-AD mice (Figure 1H). Finally, co-injection of ABM and EFF transfected cells in the hippocampus leads to a reduction of NOR deficits (Figure 1I). Together our results indicate that the development of electroporation-based cell therapies by direct reprogramming constitute a promising approach to treat AD and AD-related dementias. Our approach overcome many of the limitations of current therapies, as it does not rely on progenitor cells, and does not require viral vectors and/or labor-intensive ex vivo pre-processing to induce lineage conversions. More importantly, our innovative cell-based therapy has the potential to reduce cognitive deficits and activate cellular and tissue-level mechanism to reduce the levels and aggregation of the amyloid-beta peptide observed in AD.


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