Tomomi Kiyota

Phosphorylation of Claudin-5 and Occludin by Rho Kinase in Brain Endothelial Cells

Critical to the proper maintenance of blood-brain-barrier (BBB) integrity are the endothelial tight junctions (TJs). Posttranslational modifications of essential endothelial TJ proteins, occludin and claudin-5, contribute and possibly disrupt BBB integrity. Our previous work has shown that Rho kinase (RhoK) activation mediates occludin and claudin-5 phosphorylation resulting in diminished barrier tightness and enhanced monocyte migration across BBB in the setting of human immunodeficiency virus-1 encephalitis (HIVE). To determine whether RhoK can directly phosphorylate TJ proteins, we examined phosphorylation of cytoplasmic domains of recombinant claudin-5 and occludin by RhoK. We found that RhoK predominately phosphorylated two sites on occludin (T382 and S507) and one site on claudin-5 (T207). Specific anti-phosphopeptide antibodies were developed for these sites, allowing the detection of phosphorylated occludin at T382 and S507, and claudin-5 at T207 from full-length recombinant occludin and claudin-5 transiently expressed in COS-7 cells and mouse brain microvascular endothelial cells. Finally, these phosphospecific antibodies demonstrated enhanced staining of brain endothelial cells in the mouse model for HIVE and human HIVE brains featuring mononuclear cell infiltration across disrupted BBB. Our results demonstrated the direct phosphorylation of occludin and claudin-5 by RhoK at specific sites, which was increased in encephalitic brain tissue. These antibodies could be useful reagents for monitoring BBB dysfunction in vivo.

CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer’s disease-like pathogenesis in APP+PS1 bigenic mice

Cytokines play an emerging role as neurotransmitters, neuromodulators, and neurohormones in the brain. This paradigm shift in cytokine function offers a new framework to understand their roles in ameliorating neurodegenerative disorders, such as Alzheimers disease (AD). Molecular adjuvant therapy of AD animal models with glatiramer acetate induces antiinflammatory responses and therapeutic effects. Although these effects are potentially mediated through anti‐inflammatory cytokine signaling, the exact molecular identities and pathways are poorly understood. Here, we show that virus‐mediated expression of the mouse interleukin (IL)‐4 gene in β‐amyloid precursor protein + presenilin‐1 (APP+PS1) bigenic mice attenuates AD pathogenesis. Introduction of an adeno‐associated viral (AAV) vector encoding IL‐4 into the hippocampus resulted in sustained expression of IL‐4, reduced astro/microgliosis, amyloid‐β peptide (Aβ) oligomerization and deposition, and enhanced neurogenesis. Moreover, increased levels of IL‐4 improved spatial learning, promoted phosphorylation of N‐methyl‐D‐aspartate receptor subunit 2B at Tyr 1472, and enhanced its cell surface retention both in vivo and in vitro. Our data suggest that neuronal anti‐inflammatory cytokine signaling may be a potential alternative target for non‐Aβ‐mediated treatment of AD.—Kiyota, T., Okuyama, S., Swan, R. J., Jacobsen, M. T., Gendelman, H. E., Ikezu, T. CNS expression of anti‐inflammatory cytokine interleukin‐4 attenuates Alzheimers disease‐like pathogenesis in APP+PS1 bigenic mice. FASEB J. FASEB J. 24, 3093–3102 (2010). www.fasebj.org

AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimers disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

FGF2 gene transfer restores hippocampal functions in mouse models of Alzheimer's disease and has therapeutic implications for neurocognitive disorders

The adult hippocampus plays a central role in memory formation, synaptic plasticity, and neurogenesis. The subgranular zone of the dentate gyrus contains neural progenitor cells with self-renewal and multilineage potency. Transgene expression of familial Alzheimers disease-linked mutants of β-amyloid precursor protein (APP) and presenilin-1 leads to a significant inhibition of neurogenesis, which is potentially linked to age-dependent memory loss. To investigate the effect of neurogenesis on cognitive function in a relevant disease model, FGF2 gene is delivered bilaterally to the hippocampi of APP+presenilin-1 bigenic mice via an adenoassociated virus serotype 2/1 hybrid (AAV2/1-FGF2). Animals injected with AAV2/1-FGF2 at a pre- or postsymptomatic stage show significantly improved spatial learning in the radial arm water maze test. A neuropathological investigation demonstrates that AAV2/1-FGF2 injection enhances the number of doublecortin, BrdU/NeuN, and c-fos–positive cells in the dentate gyrus, and the clearance of fibrillar amyloid-β peptide (Aβ) in the hippocampus. AAV2/1-FGF2 injection also enhances long-term potentiation in another APP mouse model (J20) compared with control AAV2/1-GFP–injected littermates. An in vitro study confirmed the enhanced neurogenesis of mouse neural stem cells by direct AAV2/1-FGF2 infection in an Aβ oligomer-sensitive manner. Further, FGF2 enhances Aβ phagocytosis in primary cultured microglia, and reduces Aβ production from primary cultured neurons after AAV2/1-FGF2 infection. Thus, our data indicate that virus-mediated FGF2 gene delivery has potential as an alternative therapy of Alzheimers disease and possibly other neurocognitive disorders.

CCL2 Accelerates Microglia-Mediated Aβ Oligomer Formation and Progression of Neurocognitive Dysfunction

Background The linkages between neuroinflammation and Alzheimers disease (AD) pathogenesis are well established. What is not, however, is how specific immune pathways and proteins affect the disease. To this end, we previously demonstrated that transgenic over-expression of CCL2 enhanced microgliosis and induced diffuse amyloid plaque deposition in Tg2576 mice. This rodent model of AD expresses a Swedish β-amyloid (Aβ) precursor protein mutant. Methodology/Principal Findings We now report that CCL2 transgene expression accelerates deficits in spatial and working memory and hippocampal synaptic transmission in β-amyloid precursor protein (APP) mice as early as 2–3 months of age. This is followed by increased numbers of microglia that are seen surrounding Aβ oligomers. CCL2 does not suppress Aβ degradation. Rather, CCL2 and tumor necrosis factor-α directly facilitated Aβ uptake, intracellular Aβ oligomerization, and protein secretion. Conclusions/Significance We posit that CCL2 facilitates Aβ oligomer formation in microglia and propose that such events accelerate memory dysfunction by affecting Aβ seeding in the brain.

Cytokine-Mediated Inhibition of Fibrillar Amyloid-β Peptide Degradation by Human Mononuclear Phagocytes

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-β peptides (Aβ) in brain. Although Aβ uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Aβ degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Aβ using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer 125I-Aβ40 and Aβ42. Initial uptake of fibrillar Aβ40 and Aβ42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Aβ less than 0.5% and saturation of degradation within 24 h. IFN-γ increased the intracellular retention of fibrillar Aβ40 and Aβ42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-β1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Aβ degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-γ and TNF-α directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Aβ degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Aβ degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.

AAV1/2-mediated CNS Gene Delivery of Dominant-negative CCL2 Mutant Suppresses Gliosis, β-amyloidosis, and Learning Impairment of APP/PS1 Mice

Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimers disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.Accumulation of aggregated amyloid-β (Aβ) peptide was studied as an initial step for Alzheimers disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, β-amyloidosis, including suppression of both fibrillar and oligomer Aβ accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.

CCL2 affects β-amyloidosis and progressive neurocognitive dysfunction in a mouse model of Alzheimer's disease.

Neuroinflammation affects the pathobiology of Alzheimers disease (AD). Notably, β-amyloid (Aβ) deposition induces microglial activation and the subsequent production of proinflammatory neurotoxic factors. In maintaining brain homeostasis, microglial plasticity also enables phenotypic transition between toxic and trophic activation states. One important control for such cell activation is through the CC-chemokine ligand 2 (CCL2) and its receptor, the CC-chemokine receptor 2. Both affect microglia and peripheral macrophage immune responses and for the latter, cell ingress across the blood-brain barrier. However, how CCL2-CC-chemokine receptor 2 signaling contributes to AD pathogenesis is not well understood. To this end, we now report that CCL2 deficiency influences behavioral abnormalities and disease progression in Aβ precursor protein/presenilin-1 double-transgenic mice. Here, increased cortical and hippocampal Aβ deposition is coincident with the formulation of Aβ oligomers. Deficits in peripheral Aβ clearance and in scavenger, neuroprogenitor, and microglial cell functions are linked to deficient Aβ uptake. All serve to accelerate memory dysfunction. Taken together, these data support a role of CCL2 in innate immune functions relevant to AD pathogenesis.

Phenolic Bis-styrylbenzenes as β-Amyloid Binding Ligands and Free Radical Scavengers

Starting from bisphenolic bis-styrylbenzene DF-9 (4), β-amyloid (Aβ) binding affinity and specificity for phenolic bis-styrylbenzenes, monostyrylbenzenes, and alkyne controls were determined by fluorescence titration with β-amyloid peptide Aβ(1-40) and a fluorescence assay using APP/PS1 transgenic mouse brain sections. Bis-styrylbenzene SAR is derived largely from work on symmetrical compounds. This study is the first to describe Aβ binding data for bis-styrylbenzenes unsymmetrical in the outer rings. With one exception, binding affinity and specificity were decreased by adding and/or changing the substitution pattern of phenol functional groups, changing the orientation about the central phenyl ring, replacing the alkene with alkyne bonds, or eliminating the central phenyl ring. The only compound with an Aβ binding affinity and specificity comparable to 4 was its 3-hydroxy regioisomer 8. Like 4, 8 crossed the blood-brain barrier and bound to Aβ plaques in vivo. By use of a DPPH assay, phenol functional groups with para orientations seem to be a necessary, but insufficient, criterion for good free radical scavenging properties in these compounds.

A model of nitric oxide induced α-synuclein misfolding in Parkinson's disease

Inducible nitric oxide synthase (iNOS) upregulation and consequent NO formation are well-recognized neuroinflammatory responses associated with Parkinsons disease (PD). These contribute to nitrosative protein modifications affecting neuronal injury and cell death. Indeed, a pathobiologic signature for PD is Lewy body formation containing misfolded and aggregated forms of alpha-synuclein (α-syn). Moreover, nitration of α-syn promotes protein aggregation in disease. To model such pathological events, we constructed controllable iNOS and bicistronic α-syn-IRES-tTA adeno-associated virus (AAV) expression vectors. Transduction of iNOS and α-syn AAV constructs led to nitration of α-syn in neurons and overexpression of iNOS promoted protein aggregation. We posit that this AAV system mimics critical protein misfolding events associated with the pathogenesis of PD.