Nanotechnology-driven Direct Cell Reprogramming Therapies Induce Memory Improvement and Reduction of Neuropathological Burden in a Mouse Model of Alzheimer’s Disease

Introduction: Alzheimer’s Disease (AD) is characterized by a gradual loss of cognitive and executive functions. This correlates with the presence of two core neuropathological lesions amyloid-beta plaques, and neurofibrillary tangles, and also correlates with chronic inflammation, progressive reduction of synapses, and neuronal loss. Recent studies indicate that before the formation of the neuropathological lesions and the loss of synapses and neurons, AD patients present reduced cerebral blood flow (CBF) and Blood-Brain-Barrier (BBB) dysfunction, suggesting that impaired cerebrovascular function plays a key role in the onset of AD and that therapeutic interventions aiming to reestablish the CBF and BBB function may attenuate AD development. Our lab has pioneered the use of nanotechnology-mediated cell therapies against vascular disorders, implementing methods with exceptional translational potential. In this work, we show that cell-based therapies constitute promising strategies to address neurovascular deficiencies that drive AD development

Materials and

Methods: To implement cell therapies in AD, we used electroporation, to deliver 3 pro-vasculogenic reprogramming factors Etv2, Foxc2, and Fli1(EFF) to reprogram mouse primary embryonic fibroblasts (pMEFs) into induced endothelial cells (iECs). To evaluate their therapeutic potential, pMEFs labeled with 5-bromo-2’-deoxyuridine (BrdU) and transfected with 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 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. Subsequently, brain tissue was processed for immunostaining, transcriptomics, and biochemical analysis

Results, Conclusions, and Discussions: Our preliminary results indicate transfection with the EFF factors leads to an angiogenic transcriptional program that correlates with an Endothelial cell phenotype (Figure 1A,B). Furthermore, pMEFs pre-programmed into iECs and delivered to the LVs induce an increase in global CBF (Figure 1C,D). More importantly, multiple injections of the iECs pre-programmed cells correlate with a significant reduction of the spatial memory deficits in the Barnes Maze observed in 3xTg-AD (Figure 1E-J), specifically a reduction in the number of errors (Figure 1G), distance (Figure 1H), and the mean distance from scape hole (Figure 1I), and increasing the time around the target area (Figure 1J). In addition, injected cells labeled with BrdU can survive for at least 4 weeks inside the brain, migrate to the brain vasculature (Figure 1K), and induce an increase in the total vascular area in the cortex of the 3xTg-AD mice (Figure 1L,M). Injection of EFF transfected also seems to correlate with a reduction in cortical amyloid-beta load (Figure 1N,O). Finally, transcriptome analysis identified potential gene expression programs that may explain the effects of EFF-transfected cells in the control and AD brains (Figure 1P-S). Of particular importance, we identified genes involved in fatty acid oxidation such as the Peroxisome proliferator-activated receptor alpha (PPAR-α) (Figure 1S). This gene has documented effects in the reduction of amyloidogenic processing of the amyloid precursor protein, tau phosphorylation, and neuronal inflammation. Together our results indicate that the development of electroporation-based cell therapies by direct reprogramming constitutes 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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