To date, there is no reasonable explanation as to why plaques and tangles simultaneously accumulate in Alzheimer's disease (AD). We demonstrate here by Western blotting and ELISA that a stable complex can form between tau and amyloid-␤ protein (A␤). This complex enhances tau phosphorylation by GSK3␤, but the phosphorylation then promotes dissociation of the complex. We have localized the sites of this interaction by using peptide membrane arrays. A␤ binds to multiple tau peptides, especially those in exons 7 and 9. This binding is sharply reduced or abolished by phosphorylation of specific serine and threonine residues. Conversely, tau binds to multiple A␤ peptides in the mid to C-terminal regions of A␤. This binding is also significantly decreased by GSK3␤ phosphorylation of tau. We used surface plasmon resonance to determine the binding affinity of A␤ for tau and found it to be in the low nanomolar range and almost 1,000-fold higher than tau for itself. In soluble extracts from AD and control brain tissue, we detected A␤ bound to tau in ELISAs. We also found by double immunostaining of AD brain tissue that phosphorylated tau and A␤ form separate insoluble complexes within the same neurons and their processes. We hypothesize that in AD, an initial step in the pathogenesis may be the intracellular binding of soluble A␤ to soluble nonphosphorylated tau, thus promoting tau phosphorylation and A␤ nucleation. Blocking the sites where A␤ initially binds to tau might arrest the simultaneous formation of plaques and tangles in AD.immunochemistry ͉ neurofibrillary tangles ͉ senile plaques ͉ surface plasmon resonance S o far, no reasonable interpretation has been advanced to explain the simultaneous appearance of senile plaques and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). Senile plaques develop extracellularly with the main component being aggregated amyloid-␤ protein (A␤), whereas NFTs develop intracellularly with the main component being aggregated forms of phosphorylated tau. The two aggregation processes appear to occur independently, because NFTs develop in neuronal cell bodies and senile plaques develop around their nerve endings. This observation has led to two schools of thought regarding AD causation. The tau hypothesis holds that the disease is driven by tangles resulting from an excess of tau phosphorylation or a deficiency of its dephosphorylation (1). The hypothesis does not explain plaques. The amyloid hypothesis holds that the disease is driven by excess production of A␤ (2, 3). This hypothesis does not explain NFTs. Nevertheless, the amyloid cascade hypothesis is dominant because mutations in amyloid precursor protein (APP), which enhance A␤ production, cause autosomal dominant AD but not other types of dementia (2). On the other hand, mutations in tau that promote tau aggregation produce autosomal dominant frontotemporal dementia but not AD (4, 5). As a result, the presumption is that excess A␤ is a major cause and an upstream event of AD tangle formation, but exactly how an A␤ excess ...

alpha-Synuclein, a gene whose mutations, duplication, and triplication has been linked to autosomal dominant familial Parkinson's disease (fPD), appears to play a central role in the pathogenesis of sporadic PD (sPD) as well. Enhancement of neurodegeneration induced by mutant alpha-synuclein has been attributed to date largely to faster formation of alpha-synuclein aggregates in neurons. Recently, we reported that microglial activation enhances wild type (WT) alpha-synuclein-elicited dopaminergic neurodegeneration. In the present study, using a primary mesencephalic culture system, we tested whether mutated alpha-synuclein could activate microglia more powerfully than WT alpha-synuclein, thereby contributing to the accelerated neurodegeneration observed in fPD. The results showed that alpha-synuclein with the A30P or A53T mutations caused greater microglial activation than WT alpha-synuclein. Furthermore, the extent of microglial activation paralleled the degree of dopaminergic neurotoxicity induced by WT and mutant alpha-synuclein. Mutant alpha-synuclein also induced greater production of reactive oxygen species than WT alpha-synuclein by NADPH oxidase (PHOX), and PHOX activation was linked to direct activation of macrophage antigen-1 (Mac-1) receptor, rather than alpha-synuclein internalization via scavenger receptors. These results have, for the first time, demonstrated that microglia are also critical in enhanced neurotoxicity induced by mutant alpha-synuclein.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal-dominant late-onset Parkinson disease. We report that by reverse transcriptase-polymerase chain reaction, the mRNA of LRRK2 is expressed in soluble extracts of human brain, liver, and heart and in cultured human astrocytes, microglia, and oligodendroglia as well as in human neuroblastoma cell lines. We find by Western blotting using a polyclonal antibody of the leucine-rich repeat kinase 2 protein (Lrrk2) specific for C-terminal residues 2,511-2,527 that an apparent full-length protein and several of its fractions are expressed in soluble extracts of normal human brain. By immunocytochemistry, the antibody recognizes neurons, and more weakly astrocytes and microglia, in normal brain tissue. It intensely labels Lewy bodies in Parkinson disease and related neurodegenerative disorders. It also labels a subset of neurofibrillary tangles in Alzheimer disease and the Parkinsonism dementia complex of Guam (PDCG). It labels thorn-shaped astrocytes and oligodendroglial coiled bodies in PDCG; oligodendroglial inclusions in multiple system atrophy; Pick bodies in Pick disease; nuclear and cytoplasmic inclusions in Huntington disease; and intraneuronal and glial inclusions in amyotrophic lateral sclerosis. In summary, LRRK2 is constitutively expressed in neurons and also in glial cells of human brain. It strongly associates with pathological inclusions in several neurodegenerative disorders.

Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.

We have developed a non-invasive method of diagnosing Alzheimer's disease (AD), which can also predict the risk of its future onset. It is based on measuring salivary levels of amyloid-β protein terminating at position 42 (Aβ42). Brain deposits of this peptide are characteristic of AD. Biomarker studies indicate that such brain deposits commence a decade or more prior to clinical onset of the disease. We report here that Aβ42 is produced in all peripheral organs tested, thus establishing the generality of its production. We used this information to develop simple and sensitive tests to determine salivary Aβ42 levels. The levels were first stabilized by adding thioflavin S as an anti-aggregation agent and sodium azide as an anti-bacterial agent. We then quantitated the Aβ42 in a series of samples with ELISA type tests. Control cases showed almost identical levels of salivary Aβ42 regardless of sex or age. All AD cases secreted levels of Aβ42 more than double those of controls. Individuals at elevated risk of developing AD secreted levels comparable to the AD cases. The results establish that salivary Aβ42 levels can be used to diagnose AD as well as to predict the risk of its future onset.