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...

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.

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Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.

The cholinergic pedunculopontine tegmental nucleus (PPTN) is one of the major ascending arousal systems in the brain stem and is linked to motor, limbic, and sensory systems. Based on previous studies, we hypothesized that PPTN would be related to the integrative control of movement, reinforcement, and performance of tasks in behaving animals. To investigate how PPTN contributes to the behavioral control, we analyzed the activity of PPTN neurons during visually guided saccade tasks in three monkeys in relation to saccade preparation, execution, reward, and performance of the task. During visually guided saccades, we observed saccade-related burst (26/70) and pause neurons (19/70), indicating that a subset of PPTN neurons are related to both saccade execution and fixation. Burst neurons exhibited greater selectivity for saccade direction than pause neurons. The preferred directions for both burst and pause neurons were not aligned with either horizontal or vertical axes, nor biased strongly in either the ipsilateral or the contralateral direction. The spatial representation of the saccade-related activity of PPTN neurons is different from other brain stem saccade systems and may therefore relay saccade-related activity from different areas. Increasing discharges were observed around reward onset in a subset of neurons (22/70). These neurons responded to the freely delivered rewards within ~140 ms. However, during the saccade task, the latencies of the responses around reward onset ranged between 100 ms before and 200 ms after the reward onset. These results suggest that the activity observed after appropriate saccade during the task may include response associated with reward. We found that the reaction time to the appearance of the fixation point (FP) was longer when the animal tended to fail in the ensuring task. This reaction time to FP appearance (RTFP) served as an index of motivation. The RTFP could be predicted by the neuronal activity of a subset of PPTN neurons (13/70) that varied their activity levels with task performance, discharging at a higher rate in successful versus error trials. A combination of responses related to saccade execution, reward delivery, and task performance was observed in PPTN neurons. We conclude from the multimodality of responses in PPTN neurons that PPTN may serve as an integrative interface between the various signals required for performing purposive behaviors.