Cerebral hypoperfusion exacerbates traumatic brain injury in male but not female mice.

Mild-moderate traumatic brain injuries (TBIs) are prevalent, and while many individuals recover, there is evidence that a significant number experience long-term health impacts, including increased vulnerability to neurodegenerative diseases. These effects are influenced by other risk factors, such as cardiovascular disease. Our study tested the hypothesis that a pre-injury reduction in cerebral blood flow (CBF), mimicking cardiovascular disease, worsens TBI recovery. We induced bilateral carotid artery stenosis (BCAS) and a mild-moderate closed-head TBI in male and female mice, either alone or in combination, and analyzed CBF, spatial learning, memory, axonal damage, and gene expression. Findings showed that BCAS and TBI independently caused a ~10% decrease in CBF. Mice subjected to both BCAS and TBI experienced more significant CBF reductions, notably affecting spatial learning and memory, particularly in males. Additionally, male mice showed increased axonal damage with both BCAS and TBI compared to either condition alone. Females exhibited spatial memory deficits due to BCAS, but these were not worsened by subsequent TBI. Gene expression analysis in male mice highlighted that TBI and BCAS individually altered neuronal and glial profiles. However, the combination of BCAS and TBI resulted in markedly different transcriptional patterns. Our results suggest that mild cerebrovascular impairments, serving as a stand-in for preexisting cardiovascular conditions, can significantly worsen TBI outcomes in males. This highlights the potential for mild comorbidities to modify TBI outcomes and increase the risk of secondary diseases.

Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes.

The bottleneck in drug discovery for central nervous system diseases is the absence of effective systemic drug delivery technology for delivering therapeutic drugs into the brain. Despite the advances in the technology used in drug discovery, such as Adeno-Associated Virus (AAV) vectors, the development of drugs for central nervous system diseases remains challenging. In this manuscript, we describe, for the first time, the development of a workflow to generate a novel brain-targeted drug delivery system that involves the generation of genetically engineered exosomes by first selecting various functional AAV capsid-specific peptides (collectively called CAPs) known to be involved in brain-targeted high-expression gene delivery, and then expressing the CAPs in frame with lysosome-associated membrane glycoprotein (Lamp2b) followed by expressing CAP-Lamp2b fusion protein on the surface of mesenchymal stem cell-derived exosomes, thus generating CAP-exosomes. Intravenous injection of green fluorescent protein (GFP) gene-loaded CAP-exosomes in mice transferred the GFP gene throughout the CNS as measured by monitoring brain sections for GFP expression with confocal microscopy. GFP gene transfer efficiency was at least 20-fold greater than that of the control Lamp2b-exosomes, and GFP gene transduction to mouse liver was low.

Mild traumatic brain injury induces pericyte detachment independent of stroke vulnerability.

Mild traumatic brain injury (mTBI) is an independent risk factor for ischemic stroke and can result in poorer outcomes- an effect presumed to involve the cerebral vasculature. Here we tested the hypothesis that mTBI-induced pericyte detachment from the cerebrovascular endothelium is responsible for worsened stroke outcomes. We performed a mild closed-head injury and/or treated C57/bl6 mice with imatinib mesylate, a tyrosine kinase inhibitor that induces pericyte detachment. The time course of pericyte detachment was assessed 7, 14, and 28 days post injury (DPI). To test the role of pericytes in TBI-induced exacerbation of ischemic stroke outcomes, we induced mTBI and/or treated mice with imatinib for one week prior to transient middle cerebral artery occlusion. We found that injury promoted pericyte detachment from the vasculature commensurate with the levels of detachment seen in imatinib-only treated animals, and that the detachment persisted for at least 14DPI, but recovered to sham levels by 28DPI. Further, mTBI, but not imatinib-induced pericyte detachment, increased infarct volume. Thus, we conclude that the transient detachment of pericytes caused by mTBI may not be sufficient to exacerbate subsequent ischemic stroke damage. These data have important implications for understanding cerebrovascular dysfunction following mTBI and potential mechanisms of increased risk for future ischemic strokes.

Microvascular degeneration occurs before plaque onset and progresses with age in 3xTg AD mice.

Heart disease and vascular disease positively correlate with the incidence of Alzheimer's disease (AD). Although there is ostensible involvement of dysfunctional cerebrovasculature in AD pathophysiology, the characterization of the specific changes and development of vascular injury during AD remains unclear. In the present study, we established a time-course for the structural changes and degeneration of the angioarchitecture in AD. We used cerebrovascular corrosion cast and µCT imaging to evaluate the geometry, topology, and complexity of the angioarchitecture in the brain of wild type and 3xTg AD mice. We hypothesized that changes to the microvasculature occur early during the disease, and these early identifiable aberrations would be more prominent in the brain subregions implicated in the cognitive decline of AD. Whole-brain analysis of the angioarchitecture indicated early morphological abnormalities and degeneration of microvascular networks in 3xTg AD mice. Our analysis of the hippocampus and cortical subregions revealed microvascular degeneration with onset and progression that was subregion dependent.

IL-1ß Antibody Protects Brain from Neuropathology of Hypoperfusion.

Chronic brain hypoperfusion is the primary cause of vascular dementia and has been implicated in the development of white matter disease and lacunar infarcts. Cerebral hypoperfusion leads to a chronic state of brain inflammation with immune cell activation and production of pro-inflammatory cytokines, including IL-1ß. In the present study, we induced chronic, progressive brain hypoperfusion in mice using ameroid constrictor, arterial stenosis (ACAS) surgery and tested the efficacy of an IL-1ß antibody on the resulting brain damage. We observed that ACAS surgery causes a reduction in cerebral blood flow (CBF) of about 30% and grey and white matter damage in and around the hippocampus. The IL-1ß antibody treatment did not significantly affect CBF but largely eliminated grey matter damage and reduced white matter damage caused by ACAS surgery. Over the course of hypoperfusion/injury, grip strength, coordination, and memory-related behavior were not significantly affected by ACAS surgery or antibody treatment. We conclude that antibody neutralization of IL-1ß is protective from the brain damage caused by chronic, progressive brain hypoperfusion.

MiR-34a Interacts with Cytochrome c and Shapes Stroke Outcomes.

Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC) in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a-/- mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assay in vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.

MiR-34a and stroke: Assessment of non-modifiable biological risk factors in cerebral ischemia.

Aging of the nervous system, and the occurrence of age-related brain diseases such as stroke, are associated with changes to a variety of cellular processes controlled by many distinct genes. MicroRNAs (miRNAs), short non-coding functional RNAs that can induce translational repression or site-specific cleavage of numerous target mRNAs, have recently emerged as important regulators of cellular senescence, aging, and the response to neurological insult. Here, we focused on the assessment of the role of miR-34a in stroke. We noted increases in miR-34a expression in the blood of stroke patients as well as in blood and brain of mice subjected to experimental stroke. Our methodical genetic manipulation of miR-34a expression substantially impacted stroke-associated preclinical outcomes and we have in vitro evidence that these changes may be driven at least in part by disruptions to blood brain barrier integrity and mitochondrial oxidative phosphorylation in endothelial cells. Finally, aging, independent of brain injury, appears to be associated with shifts in circulating miRNA profiles. Taken together, these data support a role for miRNAs, and specifically miR-34a, in brain aging and the physiological response to age-related neurological insult, and lay the groundwork for future investigation of this novel therapeutic target.

Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.

Coenzyme A (CoA) is a cofactor that is central to energy metabolism and CoA synthesis is controlled by the enzyme pantothenate kinase (PanK). A transgenic mouse strain expressing human PANK2 was derived to determine the physiological impact of PANK overexpression and elevated CoA levels. The Tg(PANK2) mice expressed high levels of the transgene in skeletal muscle and heart; however, CoA was substantially elevated only in skeletal muscle, possibly associated with the comparatively low endogenous levels of acetyl-CoA, a potent feedback inhibitor of PANK2. Tg(PANK2) mice were smaller, had less skeletal muscle mass and displayed significantly impaired exercise tolerance and grip strength. Skeletal myofibers were characterized by centralized nuclei and aberrant mitochondria. Both the content of fully assembled complex I of the electron transport chain and ATP levels were reduced, while markers of oxidative stress were elevated in Tg(PANK2) skeletal muscle. These abnormalities were not detected in the Tg(PANK2) heart muscle, with the exception of spotty loss of cristae organization in the mitochondria. The data demonstrate that excessively high CoA may be detrimental to skeletal muscle function.

DOCK2 is critical for CD8(+) TCR(-) graft facilitating cells to enhance engraftment of hematopoietic stem and progenitor cells.

CD8(+) TCR(-) graft facilitating cells (FCs) enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic and syngeneic recipients. The mechanisms by which FCs promote HSC engraftment and tolerance induction have not been fully elucidated. Here, we provide data to support a critical role for dedicator of cytokinesis 2 (DOCK2) in multiple aspects of FCs function. DOCK2(-/-) FCs exhibit compromised facilitative function in vivo as evidenced by the loss of engraftment-enhancing capability for c-Kit(+) Sca-1(+) lineage(-) (KSL) cells, and compromised ability to promote KSL cell homing and lodgment in hematopoietic niche. Deletion of DOCK2 abrogates the ability of FCs to induce differentiation of naïve CD4(+) CD25(-) T cells into FoxP3(+) regulatory T cells and interleukin-10-producing type 1 regulatory T cells in vitro. Moreover, DOCK2(-/-) FCs are unable to promote survival of KSL cells when cocultured with KSL cells. DOCK2(-/-) FCs also exhibit compromised migration to stroma-derived factor-1 in vitro and impaired homing to the bone marrow in vivo. In conclusion, our results demonstrate that DOCK2 is critical for FCs to maintain its immunomodulatory function and exert its trophic effects on KSL cells. These findings may have direct clinical relevance to promote HSC engraftment for treatment of autoimmunity, hemoglobinopathies, and to induce transplantation tolerance.

A clinically feasible approach to induce delayed tolerance in recipients of prior kidney or vascularized composite allotransplants.

BACKGROUND: Mixed chimerism induces donor-specific tolerance to kidney and vascularized composite allotransplants (VCA). However, simultaneous kidney or VCA and bone marrow transplantation (BMT) is problematic because of the combined risk and time required for conditioning. Here, we developed a delayed tolerance induction strategy with mixed chimerism through BMT in prior kidney or VCA recipients. METHODS: Wistar Furth rats that received kidney transplantation (KTx) or VCA from allogeneic August-Copenhagen Irish donors were maintained on immunosuppression (IS) for 8 weeks. These recipients were then conditioned with anti-αß-T-cell receptor and anti-CD8 monoclonal antibodies, total body irradiation, cyclosporine A and mycophenolate mofetil (12 doses), and antilymphocyte serum (one dose); and transplanted with T-cell-depleted donor marrow. All IS was discontinued on day 11 after BMT. RESULTS: Cyclosporine A monotherapy prevented acute rejection of KTx or VCA. However, all allografts were rejected after IS withdrawal in KTx or VCA recipients who were conditioned but did not receive BMT. After delayed BMT, mixed chimerism was initially achieved in all KTx or VCA recipients with 200-, 300-, and 400-cGy total body irradiation. Long-term tolerance to KTx or VCA was achieved in most of these recipients with total IS withdrawal. The tolerance achieved with delayed BMT was donor specific as confirmed by acceptance of donor skin and rejection of third-party skin graft. CONCLUSIONS: IS-free donor-specific tolerance can be successfully induced with delayed BMT to previous recipients of kidney transplantation or VCA. These findings have significant clinical implications for transplant recipients who receive an organ from either a living donor or a deceased donor with frozen bone marrow cells available.

Regulation of integrin endocytic recycling and chemotactic cell migration by syntaxin 6 and VAMP3 interaction.

Integrins are the primary receptors of cells adhering to the extracellular matrix, and play key roles in various cellular processes including migration, proliferation and survival. The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. The present study examines the function of syntaxin 6 (STX6), a t-SNARE located in the trans-Golgi network, in integrin trafficking. STX6 is overexpressed in many types of human cancer. We show that depletion of STX6 inhibits chemotactic cell migration and the delivery of the laminin receptor α3ß1 integrin to the cell surface, whereas STX6 overexpression stimulates chemotactic cell migration, integrin delivery, and integrin-initiated activation of focal adhesion kinase. These data indicate that STX6 plays a rate-limiting role in cell migration and integrin trafficking. In STX6-depleted cells, α3ß1 integrin is accumulated in recycling endosomes that contain the v-SNARE VAMP3. Importantly, we show that STX6 and VAMP3 form a v-/t-SNARE complex, VAMP3 is required in α3ß1 integrin delivery to the cell surface, and endocytosed α3ß1 integrin traffics to both VAMP3 and STX6 compartments. Collectively, our data suggest a new integrin trafficking pathway in which endocytosed integrins are transported from VAMP3-containing recycling endosomes to STX6-containing trans-Golgi network before being recycled to the plasma membrane.

Syntaxins 3 and 4 mediate vesicular trafficking of α5ß1 and α3ß1 integrins and cancer cell migration.

Integrins, a family of heterodimeric receptors for cell adhesion to the extracellular matrix (ECM), play key roles in cell migration, cancer progression and metastasis. As transmembrane proteins, integrins are transported in vesicles and delivered to the cell surface by vesicular trafficking. The final step for integrin delivery, i.e., fusion of integrin-containing vesicles with the plasma membrane, is poorly understood at the molecular level. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxins 1, 2, 3 and 4 are present at the plasma membrane to drive vesicle fusion. In this study, we examined the roles of syntaxins 1, 2, 3 and 4 in vesicular trafficking of α5ß1 and α3ß1 integrins. We showed that syntaxins 2, 3 and 4 were expressed in HeLa cervical adenocarcinoma cells and PANC-1 pancreatic adenocarcinoma cells. In migrating HeLa and PANC-1 cells, syntaxins 2, 3 and 4 co-localized with the lipid raft constituent GM1 ganglioside at the leading edge. siRNA knockdown (KD) of syntaxins 3 and 4, but not of syntaxin 2, in HeLa cells reduced cell surface expression of α5ß1 and α3ß1 integrins and accumulated the integrins in cytoplasmic vesicles, indicating that syntaxins 3 and 4 mediate vesicular trafficking of α5ß1 and α3ß1 integrins to the cell surface. In addition, KD of syntaxins 3 and 4 inhibited cell adhesion to fibronectin, suppressed chemotactic cell migration and triggered apoptosis. Collectively, these data suggest that syntaxins 3- and 4-dependent integrin trafficking is important in cancer cell migration and survival, and may be a valuable target for cancer therapy.

Fusogenic pairings of vesicle-associated membrane proteins (VAMPs) and plasma membrane t-SNAREs--VAMP5 as the exception.

BACKGROUND: Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of ß-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30-50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction. CONCLUSIONS/SIGNIFICANCE: These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.