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Copy number mutations implicate excess production of α-synuclein as a possibly causative factor in Parkinson's disease (PD). Using an unbiased screen targeting endogenous gene expression, we discovered that the β2-adrenoreceptor (β2AR) is a regulator of the α-synuclein gene (SNCA). β2AR ligands modulate SNCA transcription through histone 3 lysine 27 acetylation of its promoter and enhancers. Over 11 years of follow-up in 4 million Norwegians, the β2AR agonist salbutamol, a brain-penetrant asthma medication, was associated with reduced risk of developing PD (rate ratio, 0.66; 95% confidence interval, 0.58 to 0.76). Conversely, a β2AR antagonist correlated with increased risk. β2AR activation protected model mice and patient-derived cells. Thus, β2AR is linked to transcription of α-synuclein and risk of PD in a ligand-specific fashion and constitutes a potential target for therapies.

Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1) as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11) translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.

INTRODUCTION: A wide array of post‐translational modifications of the tau protein occurs in Alzheimer's disease (AD) and they are critical to pathogenesis and biomarker development. Several promising tau markers, pT181, pT217, and pT231, rely on increased phosphorylation within a common molecular motif threonine‐proline‐proline (TPP). METHODS: We validated new and existing antibodies against pT217, pT231, pT175, and pT181, then combined immunohistochemistry (IHC) and immunoassays (ELISA) to broadly examine the phosphorylation of the tau TPP motif in AD brains. RESULTS: The tau burden, as examined by IHC and ELISA, correlates to Braak stages across all TPP sites. Moreover, we observed regional variability across four TPP motif phosphorylation sites in multiple brains of sporadic AD patients. DISCUSSION: We conclude that there is an elevation of TPP tau phosphorylation in AD brains as disease advances. The regional variability of pTPP tau suggests that examining different phosphorylation sites is essential for a comprehensive assessment of tau pathology. [ABSTRACT FROM AUTHOR]

α‐Synuclein phosphorylation at serine‐129 (pS129) is a widely used surrogate marker of pathology in Parkinson's disease and other synucleinopathies. However, we recently demonstrated that phosphorylation of S129 is also a physiological activator of synaptic transmission. In a feed‐forward fashion, neuronal activity triggers reversible pS129. Here, we show that Parkinson's disease‐linked missense mutations in SNCA impact activity‐dependent pS129. Under basal conditions, cytosol‐enriched A30P, H50Q, and G51D mutant forms of α‐synuclein exhibit reduced pS129 levels in rat primary cortical neurons. A53T pS129 levels are similar to wild‐type, and E46K pS129 levels are higher. A30P and E46K mutants show impaired reversibility of pS129 after stimulation. For the engineered profoundly membrane‐associated α‐synuclein mutant "3K" (E35K + E46K + E61K), de‐phosphorylation was virtually absent after blocking stimulation, implying that reversible pS129 is severely compromised. Importantly, pS129 excess resulting from proteasome inhibition is also associated with reduced reversibility by neuronal inhibition, kinase inhibition, or phosphatase activation. Our findings suggest that perturbed pS129 dynamics are probably a shared characteristic of pathology‐associated α‐synuclein, with possible implications for synucleinopathy treatment and diagnosis. Synopsis: Perturbed phospho‐S129 dynamics are a shared characteristic of various models of α‐synuclein dyshomeostasis, with possible implications for synucleinopathy treatment and diagnosis.α‐Synuclein solubility and pS129 accumulation are inversely proportional.α‐Synuclein dyshomeostasis impairs the reversibility of polo‐like‐kinase 2 (Plk2)‐ and protein phosphatase 2A (PP2A)‐modulated activity‐dependent pS129.Impaired pS129 reversibility is likely to be an early pathological change that precedes α‐synuclein aggregation. [ABSTRACT FROM AUTHOR]

Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta-(1-40)-Lys-biotin (FAbetaB). Liquid chromatography/mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.

Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta-(1-40)-Lys-biotin (FAbetaB). Liquid chromatography/mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.

In a Flemish kindred, an Ala(692)-->Gly amino acid substitution in the amyloid beta-protein precursor (AbetaPP) causes a form of early-onset Alzheimer's disease (AD) which displays prominent amyloid angiopathy and unusually large senile plaque cores. The mechanistic basis of this Flemish form of AD is unknown. Previous in vitro studies of amyloid beta-protein (Abeta) production in HEK-293 cells transfected with cDNA encoding Flemish AbetaPP have shown that full-length [Abeta(1-40)] and truncated [Abeta(5-40) and Abeta(11-40)] forms of Abeta are produced. In an effort to determine how these peptides might contribute to the pathogenesis of the Flemish disease, comparative biophysical and neurotoxicity studies were performed on wild-type and Flemish Abeta(1-40), Abeta(5-40) and Abeta(11-40). The results revealed that the Flemish amino acid substitution increased the solubility of each form of peptide, decreased the rate of formation of thioflavin-T-positive assemblies, and increased the SDS-stability of peptide oligomers. Although the kinetics of peptide assembly were altered by the Ala(21)-->Gly substitution, all three Flemish variants formed fibrils, as did the wild-type peptides. Importantly, toxicity studies using cultured primary rat cortical cells showed that the Flemish assemblies were as potent a neurotoxin as were the wild-type assemblies. Our results are consistent with a pathogenetic process in which conformational changes in Abeta induced by the Ala(21)-->Gly substitution would facilitate peptide adherence to the vascular endothelium, creating nidi for amyloid growth. Increased peptide solubility and assembly stability would favour formation of larger deposits and inhibit their elimination. In addition, increased concentrations of neurotoxic assemblies would accelerate neuronal injury and death.

We report here that a monomeric de novo designed alpha-helix-turn-alpha-helix peptide, alpha t alpha, when incubated at 37 degrees C in an aqueous buffer at neutral pH, forms nonbranching, protease resistant fibrils that are 6-10 nm in diameter. These fibrils are rich in beta-sheet and bind the amyloidophilic dye Congo red. alpha t alpha fibrils thus display the morphologic, structural, and tinctorial properties of authentic amyloid fibrils. Surprisingly, unlike fibrils formed by peptides such as the amyloid beta-protein or the islet amyloid polypeptide, alpha t alpha fibrils were not toxic to cultured rat primary cortical neurons or PC12 cells. These results suggest that the potential to form fibrils under physiologic conditions is not limited to those proteins associated with amyloidoses and that fibril formation alone is not predictive of cytotoxic activity.

Synthetic amyloid beta-protein (A beta) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of "low molecular weight A beta" (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid beta-protein (Abeta). The presence of neuritic plaques containing abundant Abeta-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier Abeta assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight Abeta (LMW Abeta) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW Abeta and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Abeta, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific Abeta assembly intermediates formed during the process of fibrillogenesis.

Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid beta-protein (Abeta). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Abeta production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Abeta fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364-22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Abeta (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed.

Aggregation and deposition of the 40-42-residue amyloid beta-protein (Abeta) are early and necessary neuropathological events in Alzheimer's disease. An understanding of the molecular interactions that trigger these events is important for therapeutic strategies aimed at blocking Abeta plaque formation at the earliest stages. Heparan sulfate proteoglycans may play a fundamental role since they are invariably associated with Abeta and other amyloid deposits at all stages. However, the nature of the Abeta-heparan sulfate proteoglycan binding has been difficult to elucidate because of the strong tendency of Abeta to self-aggregate. Affinity co-electrophoresis can measure the binding of proteoglycans or glycosaminoglycans to proteins without altering the physical state of the protein during the assay. We used affinity co-electrophoresis to study the interaction between Abeta and the glycosaminoglycan heparin and found that the aggregation state of Abeta governs its heparin-binding properties: heparin binds to fibrillar but not nonfibrillar Abeta. The amyloid binding dye, Congo red, inhibited the interaction in a specific and dose-dependent manner. The "Dutch" mutant AbetaE22Q peptide formed fibrils more readily than wild type Abeta and it also attained a heparin-binding state more readily, but, once formed, mutant and wild type fibrils bound heparin with similar affinities. The heparin-binding ability of aggregated AbetaE22Q was reversible with incubation in a solvent that promotes alpha-helical conformation, further suggesting that conformation of the peptide is important. Studies with another human amyloidogenic protein, amylin, suggested that its heparin-binding properties were also dependent on aggregation state. These results demonstrate the dependence of the Abeta-heparin interaction on the conformation and aggregation state of Abeta rather than primary sequence alone, and suggest methods of interfering with this association.