Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Geometrical principles of homomeric β‐barrels and β‐helices: Application to modeling amyloid protofilaments

Examples of homomeric β‐helices and β‐barrels have recently emerged. Here we generalize the theory for the shear number in β‐barrels to encompass β‐helices and homomeric structures. We introduce the concept of the “β‐strip,” the set of parallel or antiparallel neighboring strands, from which the whole helix can be generated giving it n‐fold rotational symmetry. In this context, the shear number is interpreted as the sum around the helix of the fixed register shift between neighboring identical β‐strips. Using this approach, we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α‐hemolysin, T4 phage spike, cylindrin, and the HET‐s(218‐289) prion. From reported dimensions measured by X‐ray fiber diffraction on amyloid fibrils, the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in‐register β‐strands folded into a “β‐strip helix.” Results suggest both stabilization of an individual β‐strip helix and growth by addition of further β‐strip helices can involve the same pair of sequence segments associating with β‐sheet hydrogen bonding at the same register shift. This process would be aided by a repeat sequence. Hence, understanding how the register shift (as the distance between repeat sequences) relates to helical dimensions will be useful for nanotube design.     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.

     

Pre‐synaptic localization of the γ‐secretase‐inhibiting protein p24α2 in the mammalian brain

Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α₂ reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α₂ in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α₂ in the mouse brain. Immunostaining showed that p24α₂ is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α₂ in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α₂ is condensed at active zone‐docked SV. During development, p24α₂ expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α₂ in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α₂ in the regulation of Aβ generation and secretion in the brain.

     

Structural characterization of α‐synuclein in an aggregation prone state

The relation of α‐synuclein (αS) aggregation to Parkinson's disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of αS in an aggregation‐prone state, we studied the structural properties of αS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that αS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of αS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu‐rich C‐terminal region, an increased probability for proximity between the NAC‐region and the C‐terminal region and a lower probability for interactions between the N‐ and C‐terminal regions.     

Identification of ciliary neurotrophic factor receptor α as a mediator of neurotoxicity induced by α‐synuclein

Accumulating evidence suggests that extracellular α‐synuclein (eSNCA) plays an important role in the pathogenesis of Parkinson's disease or related synucleinopathies by inducing neurotoxicity directly or indirectly via microglial or astroglial activation. However, the mechanisms by which this occurs remain to be characterized. To explore these mechanisms, we combined three biochemical techniques – stable isotope labeling of amino acid in cell cultures (SILAC), biotin labeling of plasma membrane proteins followed by affinity purification, and analysis of unique proteins binding to SNCA peptides on membrane arrays. The SILAC proteomic analysis identified 457 proteins, of which, 245 or 172 proteins belonged to membrane or membrane associated proteins, depending on the various bioinformatics tools used for interpretation. In dopamine neuronal cells treated with eSNCA, the levels of 86 membrane proteins were increased and 35 were decreased compared with untreated cells. In peptide array analysis, 127 proteins were identified as possibly interacting with eSNCA. Of those, seven proteins were overlapped with the membrane proteins that displayed alterations in relative abundance after eSNCA treatment. One was ciliary neurotrophic factor receptor, which appeared to modulate eSNCA‐mediated neurotoxicity via mechanisms related to JAK1/STAT3 signaling but independent of eSNCA endocytosis.     

Monoclonal antibodies selective for α‐synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α‐synuclein transgenic mice with the disease‐causing A30P mutation

Inclusions of intraneuronal alpha‐synuclein (α‐synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α‐synuclein is a central feature of the disease pathogenesis. Among the different α‐synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α‐synuclein oligomers were generated. These antibodies, which do not bind amyloid‐beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α‐synuclein transgenic mice than linear epitope antibodies. An oligomer‐selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α‐synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α‐synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer‐selective α‐synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.     

Long‐term administration of α‐tocopherol in captive Asian elephants (Elephas maximus)

After the loss of an African elephant (Loxodonta africana) in February 1989 at the Denver Zoological Gardens (DZG) with very low circulating serum α‐tocopherol, a long‐term study was initiated with three Asian elephants (Elephas maximus) to evaluate the effect of an oral micellized, water‐soluble, natural source d‐α‐tocopherol supplement. Baseline α‐tocopherol levels were evaluated and found to be approximately 3.75‐fold less than those reported for semi‐free‐ranging Asian Nepalese work camp and free‐ranging African elephants. The DZG elephants were then administered a liquid d‐α‐tocopherol (Emcelle^®) at 2.2 IU/kg body weight orally once daily. Serum samples were obtained and analyzed at 1, 2, 8, and 12 months and then annually for 96 months. The oral vitamin E supplement significantly elevated serum levels above baseline and were found to be comparable with levels reported for semi–free‐ranging and free‐ranging elephants. Zoo Biol 20:245–250, 2001. © 2001 Wiley‐Liss, Inc.     

Long‐lasting pathological consequences of overexpression‐induced α‐synuclein spreading in the rat brain

Increased expression of α‐synuclein can initiate its long‐distance brain transfer, representing a potential mechanism for pathology spreading in age‐related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpression‐induced α‐synuclein transfer were assessed over a 1‐year period after injection of viral vectors carrying human α‐synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long‐lasting pathological changes. Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression‐induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by α‐synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpression‐induced spreading, even if temporary, causes long‐lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder α‐synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation.     

Diet‐induced obesity accelerates the onset of terminal phenotypes in α‐synuclein transgenic mice

Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.

     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.