Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-␤ peptide (A␤) accumulation is poorly understood. Thus, we generated a novel Swedish ␤-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-␥ receptor type I was knocked out (APP/GRKO). IFN-␥ signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated A␤ induced IFN-␥ production from co-culture of astrocytes and microglia, and IFN-␥ elicited tumor necrosis factor (TNF)-␣ secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-␥ and TNF-␣ enhanced A␤ production from APP-expressing astrocytes and cortical neurons. TNF-␣ directly stimulated ␤-site APP-cleaving enzyme (BACE1) expression and enhanced ␤-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-␥ and TNF-␣ activation of WT microglia suppressed A␤ degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-␥ and TNF-␣ enhance A␤ deposition through BACE1 expression and suppression of A␤ clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer's disease pathogenesis. Accumulating evidence supports the idea that neuroinflammation plays a significant role in the neuropathogenesis of Alzheimer's disease (AD).1,2 Amyloid-␤ peptide (A␤) aggregation and accumulation, a principal part of AD neuropathology, is linked directly to disease progression 3 and is regulated and directly affected by innate immune responses. 4 -6 Indeed, A␤ modulates microglial inflammatory responses and abilities and speed at which microglia digest and clear this protein from brain underlines disease severity. 7A␤ is processed from the ␤-amyloid precursor protein (APP). This is accomplished by processing enzymes (secretases), which include the ␤-site APP-cleaving enzyme (BACE1, a ␤-secretase) 8 as well as the ␥-secretase complexes of presenilin (PS)-1, aph-1, pen-2, and nicastrin. 9 Mutant forms of PS-1, PS-2, and APP genes are transmitted as autosomal dominants in early onset familial AD (FAD) and are linked to A␤ aggregation and deposition.10 Transgenic mice expressing Swedish FAD APP mutant (Tg2576) 11 mimic pathobiological features of human disease including neural dysfunction, amyloid deposition, and neuroinflammation.12-14 Each disease component affects one another. Indeed, for neuroinflammation, chronic expression of monocyte chemotactic protein-1/CCL2, a major mononuclear phagocyte chemoattractant, recruits monocytes and macrophages into the brain and enhances diffuse plaque formation in APP/CCL2 bigenic mice. 15 Moreover, proinflammatory cytokines, such as interferon (IFN)-␥, interleukin (IL)-1␤, transforming growth factor (TGF)-1␤, and tumor necrosis factor (TNF...

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 Alzheimer's disease (AD). Molecular adjuvant therapy of AD animal models with glatiramer acetate induces anti-inflammatory 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 beta-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-beta peptide (Abeta) 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-Abeta-mediated treatment of AD.

Critical to the proper maintenance of blood-brainbarrier (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). The blood-brain-barrier (BBB) is composed of specialized nonfenestrated brain microvascular endothelial cells (BMVECs) connected by tight junctions (TJs) in an impermeable monolayer devoid of transcellular pores.1 TJs are composed of claudins and occludin (OCC, integral membrane proteins) and intracellular proteins, zonula occludens (ZO-1 to ZO-3).2 OCC (65-kDa protein) is highly expressed in BMVECs, and it is consistently found along the cell borders of brain endothelium.3,4 OCC is composed of four transmembranous domains with the carboxyl and amino terminals oriented to the cytoplasm and two extracellular loops (44 amino acids and 45 amino acids) spanning the intercellular cleft.5 OCC content is much lower in endothelial cells outside of the central nervous system 6,7 suggesting its active role in BBB function. The phosphorylation state of OCC regulates its association with the cell membrane and barrier permeability, and multiple phosphorylation sites have been identified on OCC serine and threonine residues.8 -10 The cytoplasmic C-terminal domain provides the connection of OCC with the cytoskeleton via accessory proteins, ZO-1 and ZO-2. 11Up to 24 claudins (20-to 24-kDa proteins) sharing the high sequence homology in the first and fourth transmembranous domains and extracellular loops have been identified in mammals.12 Contiguous staining for claudins is found along endothelial cell borders in and outside the central nervous system. BMVECs express predominantly claudin-3 and -5 (CLD5).3,13 The homophilic and heterophilic interactions between the extracellular loops of clauSupported in part by the National Institutes of Health (research grants P01 NS043985, R01 AA015913, and R01 MH65151 to Y.P.; R01 MH072539 to T.I.; and National Center for Research Resources P20RR15635 to T.I. and R.L.C.).M.Y. and S.H.R. contributed equally in this study.

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 pro-inflammatory 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 Alzheimer’s 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 APP+PS1 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, while 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.

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 Alzheimer's 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 Alzheimer's disease and possibly other neurocognitive disorders.