Soluble amyloid-beta (Abeta) peptide converts to structures with high beta-sheet content in Alzheimer's disease (AD). Soluble Abeta is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF Abeta levels may provide unique insights into Abeta metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our Abeta microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF Abeta in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of Abeta deposition, significant changes in the ISF Abeta40/Abeta42 ratio developed without changes in Abeta1-x. These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting Abeta production, we found that ISF Abeta half-life was short ( approximately 2 hr) in young mice but was twofold longer in mice with Abeta deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of Abeta synthesis reveals a portion of the insoluble Abeta pool that is in dynamic equilibrium with ISF Abeta. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.

Circadian rhythms are essential to health. Their disruption is associated with metabolic diseases in experimental animals and man. Local metabolic rhythms represent an output of tissue-based circadian clocks. Attempts to define how local metabolism is temporally coordinated have focused on gene expression by defining extensive and divergent "circadian transcriptomes" involving 5%-10% of genes assayed. These analyses are inevitably incomplete, not least because metabolic coordination depends ultimately upon temporal regulation of proteins. We therefore conducted a systematic analysis of a mammalian "circadian proteome." Our analysis revealed that up to 20% of soluble proteins assayed in mouse liver are subject to circadian control. Many of these circadian proteins are novel and cluster into discrete phase groups so that the liver's enzymatic profile contrasts dramatically between day and night. Unexpectedly, almost half of the cycling proteins lack a corresponding cycling transcript, as determined by quantitative PCR, microarray, or both and revealing for the first time the extent of posttranscriptional mechanisms as circadian control points. The circadian proteome includes rate-limiting factors in vital pathways, including urea formation and sugar metabolism. These findings provide a new perspective on the extensive contribution of circadian programming to hepatic physiology.

Studies have shown that clusterin (also called apolipoprotein J) can influence the structure and toxicity of amyloid-␤ (A␤) in vitro. To determine whether endogenous clusterin plays a role in influencing A␤ deposition, structure, and toxicity in vivo, we bred PDAPP mice, a transgenic mouse model of Alzheimer's disease, to clusterin ؊/؊ mice. By 12 months of age, PDAPP, clusterin ؊/؊ mice had similar levels of brain A␤ deposition as did PDAPP, clusterin ؉/؉ mice. Although A␤ deposition was similar, PDAPP, clusterin ؊/؊ mice had significantly fewer fibrillar A␤ (amyloid) deposits than PDAPP mice expressing clusterin. In the absence of clusterin, neuritic dystrophy associated with the deposited amyloid was markedly reduced, resulting in a dissociation between fibrillar amyloid formation and neuritic dystrophy. These findings demonstrate that clusterin markedly influences A␤ structure and neuritic toxicity in vivo and is likely to play an important role in Alzheimer's disease pathogenesis.A myloid-␤ (A␤) peptides are predominantly 39-43 aa in length and are derived from the amyloid precursor protein (APP) through endoproteolytic cleavage. Abundant evidence suggests that the conversion of A␤ from soluble to insoluble forms in the brain is a key event in the pathogenesis of Alzheimer's disease (AD). Genetic and biochemical evidence supporting this idea is that all known mutations that cause early-onset forms of familial AD or 〈␤-related cerebral amyloid angiopathy map to three genes [APP, presenilin-1 (PS-1), and PS-2] (1, 2). Most of these mutations result in relative overproduction of A␤ 42 , a particularly amyloidogenic form of A␤, which over time increases the probability of A␤ aggregation. Some mutations in APP within the A␤-coding region that result in familial cerebral amyloid angiopathy do not seem to increase A␤ production but increase its propensity for aggregation and toxicity (3, 4). Although these mutations have given insight into a central role for A␤ in both AD and cerebral amyloid angiopathy, cases of ''sporadic,'' late-onset AD (age Ͼ60 years), which accounts for most AD cases, are not associated with increased A␤ production or altered A␤ sequence. The probability that A␤ will aggregate into different insoluble forms in the brain can be influenced by A␤-binding proteins, a process which occurs after A␤ generation. One example of a protein that seems to influence A␤ in this way is apolipoprotein E (apoE). The apoE4 isoform of apoE is the only proven genetic risk factor for both late-onset AD and cerebral amyloid angiopathy, and studies suggest that apoE influences A␤ structure, clearance, and neuritic toxicity both in vitro and in vivo with no clear effect on A␤ production (5-7). Whether apolipoproteins other than apoE influence A␤ aggregation and toxicity in vivo is unknown, although a good candidate for such effects is apolipoprotein J, also known as clusterin.The two most abundantly expressed apolipoproteins in the central nervous system that are present at similar concentrations are apoE and cluste...

Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective loss of dopamine (DA) neurons and the presence of alpha-synuclein (AS) aggregates as Lewy bodies (LBs) in the remaining substantia nigra (SN) neurons. A continuing puzzle in studying PD pathogenesis is that although AS is expressed throughout the brain, LBs and selective dopaminergic cell loss lead to characteristic clinical signs of PD, suggesting that there is a link between AS aggregation and DA metabolism. One potential candidate for this link is the monoamine oxidase (MAO) metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), as neither DA nor DA metabolites other than DOPAL are toxic to SN neurons at physiological concentrations. We tested DOPAL-induced AS aggregation in a cell-free system, in vitro in DA neuron cultures and in vivo with stereotactic injections into the SN of Sprague-Dawley rats by Western blots, fluorescent confocal microscopy and immunohistochemistry. We demonstrate that DOPAL in physiologically relevant concentrations, triggers AS aggregation in the cell-free system, and in cell cultures resulting in the formation of potentially toxic AS oligomers and aggregates. Furthermore, DOPAL injection into the SN of Sprague-Dawley rats resulted in DA neuron loss and the accumulation of high molecular weight oligomers of AS detected by Western blot. Our findings support the hypothesis that DA metabolism via DOPAL can cause both DA neuron loss and AS aggregation observed in PD.