We have previously developed and characterized isolated microglia and astrocyte cultures from rapid (<4 h) brain autopsies of Alzheimer's disease (AD) and nondemented elderly control (ND) patients. In the present study, we evaluate the inflammatory repertoire of AD and ND microglia cultured from white matter (corpus callosum) and gray matter (superior frontal gyrus) with respect to three major proinflammatory cytokines, three chemokines, a classical pathway complement component, a scavenger cell growth factor, and a reactive nitrogen intermediate. Significant, dose-dependent increases in the production of pro-interleukin-1beta (pro-IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1alpha (MIP-1alpha), IL-8, and macrophage colony-stimulating factor (M-CSF) were observed after exposure to pre-aggregated amyloid beta peptide (1-42) (Abeta1-42). Across constitutive and Abeta-stimulated conditions, secretion of complement component C1q, a reactive nitrogen intermediate, and M-CSF was significantly higher in AD compared with ND microglia. Taken together with previous in situ hybridization findings, these results demonstrate unequivocally that elderly human microglia provide a brain endogenous source for a wide range of inflammatory mediators.

The aspartyl protease ␤-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid ␤ (A␤) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of A␤-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(؊/؊) mice demonstrated that BACE1 is absolutely required for both A␤ generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for A␤ generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in A␤ levels in young mice, it nonetheless resulted in a dramatic reduction in A␤ plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain A␤ levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant A␤ levels lead to dramatic inhibition of A␤-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.Alzheimer disease is the major cause of dementia in elderly people and is characterized by progressive cognitive decline. There is no cure, current treatments offer only temporary relief, and death invariably ensues. Substantial evidence suggests that the amyloid ␤ peptide (A␤) 6 is the cause of Alzheimer disease (AD)-associated neuropathology (1). A␤ is derived by sequential proteolysis of the amyloid precursor protein (APP) through ␤-and ␥-secretase activities and is widely deposited in amyloid plaques in the brains of individuals with AD (2, 3). Therefore, inhibiting the action of one or both of these enzymatic activities may provide inaugural disease-modifying therapies for AD.The aspartyl protease BACE1 is the primary ␤-secretase (4 -6) and is the sole ␤-secretase in mice, since its genetic ablation fully abolishes A␤ generation (7-9). Early reports indicated that BACE1 knock-out animals are healthy and fertile, with no histological pathologies, suggesting that inhibition of BACE1 for therapeutic intervention in AD would have no mechanism related toxicities (7, 9, 10). In contrast, recent reports of partially penetrant lethality and cognitive deficits in BACE1 knock-out animals do suggest potential liabilities of complete BACE1 inhibition (11, 12). As the initiating enzyme in the generation of A␤, BACE1 is a key drug target and would be predicted to abrogate pathologies associated with any form of A␤. To avoid potential side effects re...

To gain a molecular understanding of neuronal responses to amyloid-β peptide (Aβ), we have analyzed the effects of Aβ treatment on neuronal gene expressionin vitroby quantitative reverse transcription-PCR andin situhybridization. Treatment of cultured rat cortical neurons with Aβ1–40results in a widespread apoptotic neuronal death. Associated with death is an induction of several members of the immediate early gene family. Specifically, we (1) report the time-dependent and robust induction ofc-jun,junB,c-fos, andfosB, as well astransin, which is induced by c-Jun/c-Fos heterodimers and encodes an extracellular matrix protease; these gene inductions appear to be selective because other Jun and Fos family members, i.e.,junDandfra-1, are induced only marginally; (2) show that thec-juninduction is widespread, whereasc-fosexpression is restricted to a subset of neurons, typically those with condensed chromatin, which is a hallmark of apoptosis; (3) correlate gene induction and neuronal death by showing that each has a similar dose–response to Aβ; and (4) demonstrate that both cell death and immediate early gene induction are dependent on Aβ aggregation state. This overall gene expression pattern during this “physiologically inappropriate” apoptotic stimulus is markedly similar to the pattern we previously identified after a “physiologically appropriate” stimulus, i.e., the NGF deprivation-induced death of sympathetic neurons. Hence, the parallels identified here further our understanding of the genetic alterations that may lead neurons to apoptosis in response to markedly different insults.

Triple-transgenic mice (3xTgAD) overexpressing Swedish-mutated ␤-amyloid precursor protein (␤APP swe ), P310L-Tau (Tau P301L ), and physiological levels of M146V-presenilin-1 (PS1 M146V ) display extracellular amyloid-␤ peptides (A␤) deposits and Tau tangles. More disputed is the observation that these mice accumulate intraneuronal A␤ that has been linked to synaptic dysfunction and cognitive deficits. Here, we provide immunohistological, genetic, and pharmacological evidences for early, age-dependent, and hippocampusspecific accumulation of the ␤-secretase-derived ␤APP fragment C99 that is observed from 3 months of age and enhanced by pharmacological blockade of ␥-secretase. Notably, intracellular A␤ is only detectable several months later and appears, as is the case of C99, in enlarged cathepsin B-positive structures, while extracellular A␤ deposits are detected ϳ12 months of age and beyond. Early C99 production occurs mainly in the CA1/subicular interchange area of the hippocampus corresponding to the first region exhibiting plaques and tangles in old mice. Furthermore, the comparison of 3xTgAD mice with double-transgenic mice bearing the ␤APP swe and Tau P301L mutations but expressing endogenous PS1 (2xTgAD) demonstrate that C99 accumulation is not accounted for by a loss of function triggered by PS1 mutation that would have prevented C99 secondary cleavage by ␥-secretase. Together, our work identifies C99 as the earliest ␤APP catabolite and main contributor to the intracellular ␤APP-related immunoreactivity in 3xTgAD mice, suggesting its implication as an initiator of the neurodegenerative process and cognitive alterations taking place in this mouse model.

Several neurological diseases, includingThe importance of ␣-synuclein to the pathogenesis of Parkinson disease (PD) 4 and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1)(2)(3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4 -6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7-9), which increase levels of ␣-synuclein with no alteration in sequence, also cause PD or DLB.␣-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11,12). Phosphorylation at tyrosine residues has been observed by some investigators (13,14) but not by others (10 -12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress ␣-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of ␣-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate ␣-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an ␣-synuclein kinase. The ability of PLK2 to directly phosphorylate ␣-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates ␣-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brai...

␣ 1 -Antichymotrypsin (ACT) is an acute phase protein expressed in the brain which specifically colocalizes with amyloid-␤ during Alzheimer's disease. We analyzed ACT synthesis in cultured human cortical astrocytes in response to various cytokines and growth factors. Oncostatin M (OSM) and interleukin (IL)-1␤ were potent stimulators of ACT mRNA expression, whereas tumor necrosis factor-␣ had modest activity, and IL-6 and leukemia inhibitory factor (LIF) were ineffective. The finding that OSM, but not LIF or IL-6, stimulated ACT expression suggests that human astrocytes express a "specific" OSM receptor, but not IL-6 or LIF receptors. However, cotreatment of human astrocytes with soluble IL-6 receptor (sIL-6R)⅐IL-6 complex did result in potent stimulation of ACT expression. When the human ACT gene was cloned, two elements binding STAT1 and STAT3 (signal transducer and activator of transcription) in response to OSM or IL-6⅐sIL-6R complexes could be identified and characterized. Taken together, these findings indicate that OSM or IL-6⅐sIL-6 complexes may regulate ACT expression in human astrocytes and thus directly or indirectly contribute to the pathogenesis of Alzheimer's disease.1 is one of the major positive human acute phase proteins produced by the liver and secreted into blood plasma (1, 2). The expression of this proteinase inhibitor in hepatic cells is enhanced by interleukin (IL)-6 and glucocorticoids and to a lesser extent by IL-1 (3, 4). Although ACT is also found in the brain, the plasma-derived inhibitor is separated from this origin by a tight blood-brain barrier consisting of endothelial cells. For this reason it is believed that astrocytes are the likely source of ACT produced within the central nervous system (5). Significantly, ACT has been identified as one of the amyloid-associated proteins found in the brains of patients with Alzheimer's disease (6, 7). The pathological feature of this disease is cerebral degeneration with neuronal cell death and deposition of abnormal proteins in the form of amyloid plaques and neurofibrillary tangles. Because the expression of ACT is enhanced dramatically in affected brain regions in Alzheimer's disease, a state of cerebral "acute phase" in response to neuronal degradation has been proposed. IL-1 and IL-6, which are produced by cells of CNS, were suggested to induce ACT expression in astrocytes (8). Indeed, the induction of ACT expression by IL-1 has been shown in human astrocyte cultures (9); however, regulation by IL-6 has not been confirmed.To understand the control of ACT expression in the brain we used human astrocyte cultures and analyzed the pattern of its synthesis after stimulation with a variety of factors including IL-1 and cytokines of the IL-6 family. We have also cloned the 5Ј-flanking region of the ACT gene and performed analysis of its transcriptional activity. Our results suggest that at least one cytokine, oncostatin M (OSM), may play an important role in up-regulating ACT expression in astrocytes, whereas IL-6 requires the presence of solu...

Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson’s disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.

Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3–P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower βamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS.