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.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

CD4(+)CD25(hi)Foxp3(+) Cells Exacerbate Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a fatal lung disease with a median survival of 2 to 5 years. A decade of studies has downplayed inflammation contributing to its pathogenesis. However, these studies preceded the discovery of regulatory T cells (Tregs) and all of their functions. On the basis of human studies demonstrating Tregs can decrease graft-versus-host disease and vasculitides, there is consideration of their use to treat idiopathic pulmonary fibrosis. We hypothesized that Treg therapy would attenuate the fibroplasia involved in a preclinical murine model of pulmonary fibrosis. IL-2 complex was used in vivo to expand CD4(+)CD25(hi)Foxp3(+) cells in the lung during intratracheal bleomycin challenge; however, this unexpectedly led to an increase in lung fibrosis. More important, this increase in fibrosis was a lymphocyte-dependent process. We corroborated these results using a CD4(+)CD25(hi)Foxp3(+) cellular-based therapy. Mechanistically, we demonstrated that CD4(+)CD25(hi)Foxp3(+) cells undergo alterations during bleomycin challenge and the IL-2 complex had no effect on profibrotic (eg, transforming growth factor-ß) or type 17 immune response cytokines; however, there was a marked down-regulation of the type 1 and augmentation of the type 2 immune response cytokines from the lungs. Collectively, our animal studies show that a specific lung injury can induce Treg alterations, which can augment pulmonary fibrosis.

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Profiling of volatile impurities in tetramethylenedisulfotetramine (TETS) for synthetic-route determination.

This study investigated the feasibility of using volatile impurities from the rodenticide tetramethylenedisulfotetramine (TETS) for the discrimination of TETS produced by three synthetic routes. Each route was used to make one batch of TETS by reacting sulfamide with one of three formaldehyde analogs in the presence of either trifluroacetic acid (TFA) or hydrochloric acid. Ten impurities useful for differentiating the three TETS batches were discovered and tentatively identified by headspace solid-phase microextraction comprehensive two-dimensional gas chromatography-mass spectrometry (HS-SPME/GC×CG-MS). Of the ten identified impurities, the alkyl trifluoroacetate and alkyl chloride impurities distinguished TETS routes based on their use of either TFA or HCl as catalyst. On the other hand, four 6-carbon ketone impurities appeared to be batch specific rather than route specific and hence potentially useful for sample matching. Interestingly, 1,3,5-trioxane was not found in the TETS batch where it was used as a reactant, but instead was found in the two batches that did not have 1,3,5-trioxane as the reactant. In brief, the limited work discussed in this paper supports: (1) the feasibility of sampling and detecting volatile organic impurities from a solid chemical-threat agent, (2) the probable forensic benefit of catalysts acting as reactants in side reactions, (3) the uniqueness of a synthetic batch's impurity profile for potential sample matching, and (4) the possibility that some impurities, such as formaldehyde analogs, are not forensically helpful and may lead to an incorrect estimate about the synthetic route if not supported by sound chemical knowledge.