Lung infection by Pseudomonas aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

BACKGROUND: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. METHODS: Lung infection in mice was induced by instilling Pseudomonas aeruginosa (PA) intratracheally. We determined bacterial colonization in tissue, microvascular leakage, expression of cytokines and leukocyte infiltration into the brain. RESULTS: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 h and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + CD45+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) and adherens junction (AJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. CONCLUSIONS: Lung bacterial infection is associated with BBB disruption and behavioral changes, which are mediated by systemic cytokine release.

Improving Gene Therapy for Angelman Syndrome with Secreted Human UBE3A.

The rare genetic neurodevelopmental disease Angelman syndrome (AS) is caused by the loss of function of UBE3A, a ubiquitin ligase. The disease results in a lifetime of severe symptoms, including intellectual disability and motor impairments for which there are no effective treatments. One avenue of treatment for AS is the use of gene therapy to reintroduce a functional copy of the UBE3A gene. Our group had previously shown that recombinant adeno-associated virus (rAAV) expressing mouse Ube3a could rescue deficits in a mouse model of AS. Here, we expand on this work and show that this approach could be successfully replicated in a second AS model using the human UBE3A gene. Furthermore, we address the challenge of limited vector distribution in the brain by developing a novel modified form of UBE3A. This modified protein, termed STUB, was designed with a secretion signal and a cell-penetrating peptide. This allowed transduced cells to act as factories for the production of UBE3A protein that could be taken up by neighboring non-transduced cells, thus increasing the number of neurons receiving the therapeutic protein. Combining this construct with intracerebroventricular injections to maximize rAAV distribution within the brain, we demonstrate that this novel approach improves the recovery of behavioral and electrophysiological deficits in the AS rat model. More importantly, a comparison of rAAV-STUB to a rAAV expressing the normal human UBE3A gene showed that STUB was a more effective therapeutic. These data suggest that rAAV-STUB is a new potential approach for the treatment of AS.

CCL2 Overexpression in the Brain Promotes Glial Activation and Accelerates Tau Pathology in a Mouse Model of Tauopathy.

Innate immune activation is a major contributor to Alzheimer's Disease (AD) pathophysiology, although the mechanisms involved are poorly understood. Chemokine C-C motif ligand (CCL) 2 is produced by neurons and glial cells and is upregulated in the AD brain. Transgene expression of CCL2 in mouse models of amyloidosis produces microglia-induced amyloid ß oligomerization, a strong indication of the role of these activation pathways in the amyloidogenic processes of AD. We have previously shown that CCL2 polarizes microglia in wild type mice. However, how CCL2 signaling contributes to tau pathogenesis remains unknown. To address this question, CCL2 was delivered via recombinant adeno-associated virus serotype 9 into both cortex and hippocampus of a mouse model with tau pathology (rTg4510). We report that CCL2 overexpression aggravated tau pathology in rTg4510 as shown by the increase in Gallyas stained neurofibrillary tangles as well as phosphorylated tau-positive inclusions. In addition, biochemical analysis showed a reduction in the levels of detergent-soluble tau species followed by increase in the insoluble fraction, indicating a shift toward larger tau aggregates. Indeed, increased levels of high molecular weight species of phosphorylated tau were found in the mice injected with CCL2. We also report that worsening of tau pathology following CCL2 overexpression was accompanied by a distinct inflammatory response. We report an increase in leukocyte common antigen (CD45) and Cluster of differentiation 68 (CD68) expression in the brain of rTg4510 mice without altering the expression levels of a cell-surface protein Transmembrane Protein 119 (Tmem119) and ionized calcium-binding adaptor molecule 1 (Iba-1) in resident microglia. Furthermore, the analysis of cytokines in brain extract showed a significant increase in interleukin (IL)-6 and CCL3, while CCL5 levels were decreased in CCL2 mice. No changes were observed in IL-1α, IL-1ß, TNF-α. IL-4, Vascular endothelial growth factor-VEGF, IL-13 and CCL11. Taken together our data report for the first time that overexpression of CCL2 promotes the increase of pathogenic tau species and is associated with glial neuroinflammatory changes that are deleterious. We propose that these events may contribute to the pathogenesis of Alzheimer's disease and other tauopathies.

Disruption of normal circadian clock function in a mouse model of tauopathy.

Disruption of normal circadian rhythm physiology is associated with neurodegenerative disease, which can lead to symptoms such as altered sleep cycles. In Alzheimer's disease (AD), circadian dysfunction has been attributed to ß-amyloidosis. However, it is unclear whether tauopathy, another AD-associated neuropathology, can disrupt the circadian clock. We have evaluated the status of the circadian clock in a mouse model of tauopathy (Tg4510). Tg4510 mice display a long free-running period at an age when tauopathy is present, and show evidence of tauopathy in the suprachiasmatic nucleus (SCN) of the hypothalamus - the site of the master circadian clock. Additionally, cyclic expression of the core clock protein PER2 is disrupted in the hypothalamus of Tg4510 mice. Finally, disruption of the cyclic expression of PER2 and BMAL1, another core circadian clock protein, is evident in the Tg4510 hippocampus. These results demonstrate that tauopathy disrupts normal circadian clock function both at the behavioral and molecular levels, which may be attributed to the tauopathy-induced neuropathology in the SCN. Furthermore, these results establish the Tg4510 mouse line as a model to study how tauopathy disrupts normal circadian rhythm biology.

Calorie restriction does not restore brain mitochondrial function in P301L tau mice, but it does decrease mitochondrial F0F1-ATPase activity.

Calorie restriction (CR) has been shown to increase lifespan and delay aging phenotypes in many diverse eukaryotic species. In mouse models of Alzheimer's disease (AD), CR has been shown to decrease amyloid-beta and hyperphosphorylated tau levels and preserve cognitive function. Overexpression of human mutant tau protein has been shown to induce deficits in mitochondrial electron transport chain complex I activity. Therefore, experiments were performed to determine the effects of 4-month CR on brain mitochondrial function in Tg4510 mice, which express human P301L tau. Expression of mutant tau led to decreased ADP-stimulated respiratory rates, but not uncoupler-stimulated respiratory rates. The membrane potential was also slightly higher in mitochondria from the P301L tau mice. As shown previously, tau expression decreased mitochondrial complex I activity. The decreased complex I activity, decreased ADP-stimulated respiratory rate, and increased mitochondrial membrane potential occurring in mitochondria from Tg4510 mice were not restored by CR. However, the CR diet did result in a genotype independent decrease in mitochondrial F0F1-ATPase activity. This decrease in F0F1-ATPase activity was not due to lowered levels of the alpha or beta subunits of F0F1-ATPase. The possible mechanisms through which CR reduces the F0F1-ATPase activity in brain mitochondria are discussed.

Intraventricular human immunoglobulin distributes extensively but fails to modify amyloid in a mouse model of amyloid deposition.

Intravenous immunoglobulin infusions into Alzheimer patients have been found to provide cognitive benefit over a period of 6 mo in open label studies. One suggestion has been that these preparations contain small amounts of antibodies directed against monomeric and oligomeric Aß which underlie their effectiveness in patients. To test this hypothesis, we infused Gammagard, a version of intravenous immunoglobulin (IVIG), into the lateral ventricle of amyloid precursor protein (APP) transgenic mice with pre-existing amyloid deposits. Mice were infused over 4 weeks, and tested behaviorally for the last 2 weeks of treatment. Brains were analyzed for histopathology. We found widespread distribution of human-immunoglobulin G (h-IgG) staining in the mouse forebrain, including cerebral cortices and hippocampus. Some cortical neurons appeared to concentrate the h-IgG, but we did not detect evidence of amyloid plaque labeling by h-IgG. The IVIG-treated mice had no change in phenotype compared to saline-infused animals with respect to activity, learning and memory, or amyloid deposition. APP mice infused with an anti-Aß monoclonal antibody did show some reduction in amyloid deposits. These data do not support the argument that anti-Aß antibodies in IVIG preparations are responsible for cognitive benefits seen with these preparations.