Modeling Mixed Vascular and Alzheimer's Dementia Using Focal Subcortical Ischemic Stroke in Human ApoE4-TR:5XFAD Transgenic Mice.

Subcortical white matter ischemic lesions are increasingly recognized to have pathologic overlap in individuals with Alzheimer's disease (AD). The interaction of white matter ischemic lesions with amyloid pathology seen in AD is poorly characterized. We designed a novel mouse model of subcortical white matter ischemic stroke and AD that can inform our understanding of the cellular and molecular mechanisms of mixed vascular and AD dementia. Subcortical white matter ischemic stroke underlying forelimb motor cortex was induced by local stereotactic injection of an irreversible eNOS inhibitor. Subcortical white matter ischemic stroke or sham procedures were performed on human ApoE4-targeted-replacement (TR):5XFAD mice at 8 weeks of age. Behavioral tests were done at 7, 10, 15, and 20 weeks. A subset of animals underwent 18FDG-PET/CT. At 20 weeks of age, brain tissue was examined for amyloid plaque accumulation and cellular changes. Compared with sham E4-TR:5XFAD mice, those with an early subcortical ischemic stroke showed a significant reduction in amyloid plaque burden in the region of cortex overlying the subcortical stroke. Cognitive performance was improved in E4-TR:5XFAD mice with stroke compared with sham E4-TR:5XFAD animals. Iba-1+ microglial cells in the region of cortex overlying the subcortical stroke were increased in number and morphologic complexity compared with sham E4-TR:5XFAD mice, suggesting that amyloid clearance may be promoted by an interaction between activated microglia and cortical neurons in response to subcortical stroke. This novel approach to modeling mixed vascular and AD dementia provides a valuable tool for dissecting the molecular interactions between these two common pathologies.

Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation.

Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer's disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation.

Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression.

Oligonucleotide drugs are experiencing greater success in the clinic, encouraging the initiation of new projects. Resources are insufficient to develop every potentially important project, and persuasive experimental data using cell lines close to disease target tissue is needed to prioritize candidates. Friedreich's ataxia (FRDA) is a devastating and currently incurable disease caused by insufficient expression of the enzyme frataxin (FXN). We have previously shown that synthetic nucleic acids can activate FXN expression in human patient-derived fibroblast cells. We chose to further test these compounds in induced pluripotent stem cell-derived neuronal progenitor cells (iPSC-NPCs). Here we describe methods to deliver oligonucleotides and duplex RNAs into iPSC-NPCs using electroporation. Activation of FXN expression is potent, easily reproducible, and potencies parallel those determined using patient-derived fibroblast cells. A duplex RNA and several antisense oligonucleotides (ASOs) with different combinations of 2'-methoxyethyl (2'-MOE), 2'-fluoro (2'-F), and constrained ethyl (cEt) were active, providing multiple starting points for further development and highlighting improved potency as an important goal for preclinical development. Our data support the conclusion that ASO-mediated activation of FXN is a feasible approach for treating FRDA and that electroporation is a robust method for introducing ASOs to modulate gene expressions in neuronal cells.