Seiko Ikezu

Depletion of microglia and inhibition of exosome synthesis halt tau propagation

Accumulation of pathological tau protein is a major hallmark of Alzheimers disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target.

miR-155 Is Essential for Inflammation-Induced Hippocampal Neurogenic Dysfunction.

Peripheral and CNS inflammation leads to aberrations in developmental and postnatal neurogenesis, yet little is known about the mechanism linking inflammation to neurogenic abnormalities. Specific miRs regulate peripheral and CNS inflammatory responses. miR-155 is the most significantly upregulated miR in primary murine microglia stimulated with lipopolysaccharide (LPS), a proinflammatory Toll-Like Receptor 4 ligand. Here, we demonstrate that miR-155 is essential for robust IL6 gene induction in microglia under LPS stimulation in vitro. LPS-stimulated microglia enhance astrogliogenesis of cocultured neural stem cells (NSCs), whereas blockade of IL6 or genetic ablation of microglial miR-155 restores neural differentiation. miR-155 knock-out mice show reversal of LPS-induced neurogenic deficits and microglial activation in vivo. Moreover, mice with transgenic elevated expression of miR-155 in nestin-positive neural and hematopoietic stem cells, including microglia, show increased cell proliferation and ectopically localized doublecortin-positive immature neurons and radial glia-like cells in the hippocampal dentate gyrus (DG) granular cell layer. Microglia have proliferative and neurogenic effects on NSCs, which are significantly altered by microglial miR-155 overexpression. In addition, miR-155 elevation leads to increased microglial numbers and amoeboid morphology in the DG. Our study demonstrates that miR-155 is essential for inflammation-induced neurogenic deficits via microglial activation and induction of IL6 and is sufficient for disrupting normal hippocampal development.

Tau-tubulin kinase

Tau-tubulin kinase (TTBK) belongs to casein kinase superfamily and phosphorylates microtubule-associated protein tau and tubulin. TTBK has two isoforms, TTBK1 and TTBK2, which contain highly homologous catalytic domains but their non-catalytic domains are distinctly different. TTBK1 is expressed specifically in the central nervous system and is involved in phosphorylation and aggregation of tau. TTBK2 is ubiquitously expressed in multiple tissues and genetically linked to spinocerebellar ataxia type 11. TTBK1 directly phosphorylates tau protein, especially at Ser422, and also activates cycline-dependent kinase 5 in a unique mechanism. TTBK1 protein expression is significantly elevated in Alzheimer’s disease (AD) brains, and genetic variations of the TTBK1 gene are associated with late-onset Alzheimer’s disease in two cohorts of Chinese and Spanish populations. TTBK1 transgenic mice harboring the entire 55-kilobase genomic sequence of human TTBK1 show progression of tau accumulation, neuroinflammation, and neurodegeneration when crossed with tau mutant mice. Our recent study shows that there is a striking switch in mononuclear phagocyte and activation phenotypes in the anterior horn of the spinal cord from alternatively activated (M2-skewed) microglia in P301L tau mutant mice to pro-inflammatory (M1-skewed) infiltrating peripheral monocytes by crossing the tau mice with TTBK1 transgenic mice. TTBK1 is responsible for mediating M1-activated microglia-induced neurotoxicity, and its overexpression induces axonal degeneration in vitro. These studies suggest that TTBK1 is an important molecule for the inflammatory axonal degeneration, which may be relevant to the pathobiology of tauopathy including AD.

Accelerated Neurodegeneration and Neuroinflammation in Transgenic Mice Expressing P301L Tau Mutant and Tau-Tubulin Kinase 1

Tau-tubulin kinase-1 (TTBK1) is a central nervous system (CNS)-specific protein kinase implicated in the pathological phosphorylation of tau. TTBK1-transgenic mice show enhanced neuroinflammation in the CNS. Double-transgenic mice expressing TTBK1 and frontotemporal dementia with parkinsonism-17-linked P301L (JNPL3) tau mutant (TTBK1/JNPL3) show increased accumulation of oligomeric tau protein in the CNS and enhanced loss of motor neurons in the ventral horn of the lumbar spinal cord. To determine the role of TTBK1-induced neuroinflammation in tauopathy-related neuropathogenesis, age-matched TTBK1/JNPL3, JNPL3, TTBK1, and non-transgenic littermates were systematically characterized. There was a striking switch in the activation phenotype and population of mononuclear phagocytes (resident microglia and infiltrating macrophages) in the affected spinal cord region: JNPL3 mice showed accumulation of alternatively activated microglia, whereas TTBK1 and TTBK1/JNPL3 mice showed accumulation of classically activated infiltrating peripheral monocytes. In addition, expression of chemokine ligand 2, a chemokine important for the recruitment of peripheral monocytes, was enhanced in TTBK1 and TTBK1/JNPL3 but not in other groups in the spinal cord. Furthermore, primary cultured mouse motor neurons showed axonal degeneration after transient expression of the TTBK1 gene or treatment with conditioned media derived from lipopolysaccharide-stimulated microglia; this was partially blocked by silencing of the endogenous TTBK1 gene in neurons. These data suggest that TTBK1 accelerates motor neuron neurodegeneration by recruiting proinflammatory monocytes and enhancing sensitivity to neurotoxicity in inflammatory conditions.

A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimers disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.

EXOSOMES CONTAINING SPECIFIC TAU OLIGOMER FORMATIONS ACCELERATE PATHOLOGICAL TAU PHOSPHORYLATION IN C57BL/6 MICE

P1-025 EXOSOMES CONTAINING SPECIFIC TAU OLIGOMER FORMATIONS ACCELERATE PATHOLOGICALTAUPHOSPHORYLATION IN C57BL/6 MICE Zhi Ruan, Annina M. DeLeo, Asuka Yoshii-Kitahara, Seiko Ikezu, Rakez Kayed, Santhi Golantla, Howard E. Gendelman, Tsuneya Ikezu, Boston University School of Medicine, Boston, MA, USA; University of Texas Medical Branch, Galveston, TX, USA; University of Nebraska Medical Center, Omaha, NE, USA. Contact e-mail: zhiruan@bu.edu

COMPREHENSIVE CHARACTERIZATION OF HUMAN ALZHEIMER’S DISEASE BRAIN-DERIVED EXOSOMES

cell types, is amenable to repeated analysis, and can be readily manipulated. Long-term organotypic hippocampal slice cultures (OHSCs) from neonatal amyloid mice offer an excellent compromise between in vivo and primary culture studies, largely retaining the cellular composition and neuronal architecture of the in vivo hippocampus, but with the in vitro advantages of accessibility to live imaging, sampling and intervention. Methods:OHSCs are made using tissue from P6-P9 TgCRND8 mice and maintained on membrane inserts for up to 2 months. Pathology development was closely monitored using AbELISA, western blotting for synaptic proteins, and immunofluorescence for Ab, neurons and glia. The mechanisms behind presynaptic disruption were probed via pharmacological manipulation of Ab production and genetic knockout of endogenousmouse tau.Results:Ab1-40 and Ab1-42 rise progressively in transgenic slice culture medium and stabilise when regular feeding balances continued production. In contrast, intra-axonal Ab continues to accumulate in close correlation with a specific decline in presynaptic proteins. There are no fibrillar plaques under these conditions indicating that plaques are not needed for synaptic disruption. Beta secretase inhibitor treatment completely abolished the accumulation of Ab1-42 in the medium but surprisingly this did not rescue synaptophysin levels, evenwhen continually present from before synaptophysin declines. This raises the question of whether BACE1-independent APP products underlie some synaptic defects and provides an effective experimental system to test this. Interestingly, genetic deletion of tau does rescue the loss of synaptophysin, opening lines of investigation for potential therapeutics. Conclusions: Organotypic brain slices from TgCRND8 mice represent an important new system for understanding mechanisms of presynaptic disruption induced by the presence of mutant huAPP. The pathology observed in these cultures will allow for rapid assessment of disease modifying compounds in a system amenable to manipulation and observation. Future work will involve examination of the mechanisms resulting in synaptophysin depletion, particularly in relation to the involvement of tau and alternative APP processing products.