Marcia B. Podlisny

Isolation of Low-Molecular-Weight Proteins from Amyloid Plaque Fibers in Alzheimer's Disease

During aging of the human brain, and particularly in Alzheimers disease, progressive neuronal loss is accompanied by the formation of highly stable intra‐ and extraneuronal protein fibers. Using fluorescence‐activated particle sorting, a method has been developed for purifying essentially to homogeneity the extracellular amyloid fibers that form the cores of senile plaques. The purified plaque cores each contain 60–130 pg of protein. Their amino acid composition shows abundant glycine, trace proline, and ∼50% hydrophobic residues; it resembles that of enriched fractions of the paired helical filaments (PHF) that accumulate intraneuronally in Alzheimers disease. Senile plaque amyloid fibers share with PHF insolubility in numerous protein denaturants and resistance to proteinases. However, treatment of either fiber preparation with concentrated (88%) formic acid or saturated (6.8 M) guanidine thiocyanate followed by sodium dodecyl sulfate causes disappearance of the fibers and releases proteins migrating at 5–7.000 and 11–15.000 Mr which appear to be dimerically related. Following their separation by size‐exclusion HPLC, the proteins solubilized from plaque amyloid and PHF‐enriched fractions have highly similar compositions and, on dialysis, readily aggregate into higher Mr polymers. Antibodies raised to the major low‐Mr protein selectively label both plaque cores and vascular amyloid deposits in Alzheimer brain but do not stain neurofibrillary tangles, senile plaque neurites, or any other neuronal structure. Thus, extraneuronal amyloid plaque filaments in Alzheimers disease are composed of hydrophobic low‐Mr protein(s) which are also present in vascular amyloid deposits. Current evidence suggests that such protein(s) found in PHF‐enriched fractions may derive from copurifying amyloid filament srather than from PHF.

Presenilin Proteins Undergo Heterogeneous Endoproteolysis between Thr291and Ala299and Occur as Stable N- and C-Terminal Fragments in Normal and Alzheimer Brain Tissue

Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimers disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.

Enhanced Production and Oligomerization of the 42-residue Amyloid β-Protein by Chinese Hamster Ovary Cells Stably Expressing Mutant Presenilins

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of early onset familial Alzheimers disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or mutant (M239V) PS2 genes were stably transfected into Chinese hamster ovary cells that overexpress the β-amyloid precursor protein (APP). The identity of the 43-45-kDa PS1 holoproteins was confirmed by N-terminal radiosequencing. PS1 was rapidly processed (t1/2 = 40 min) in the endoplasmic reticulum into stable fragments. Wild-type and mutant PS2 holoproteins exhibited similar half lives (1.5 h); however, their endoproteolytic fragments showed both mutation-specific and cell type-specific differences. Mutant PS1 or PS2 consistently induced a 1.4-2.5-fold increase (p < 0.001) in the relative production of the highly amyloidogenic 42-residue form of amyloid β-protein (Aβ42) as determined by quantitative immunoprecipitation and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in Aβ42/Aβtotal ratios, spontaneous formation of low molecular weight oligomers of Aβ42 was observed in media, suggesting enhanced Aβ aggregation from the elevation of Aβ42. We conclude that mutant PS1 and PS2 proteins enhance the proteolysis of β-amyloid precursor protein by the γ-secretase cleaving at Aβ residue 42, thereby promoting amyloidogenesis.

Certain Inhibitors of Synthetic Amyloid β-Peptide (Aβ) Fibrillogenesis Block Oligomerization of Natural Aβ and Thereby Rescue Long-Term Potentiation

Recent studies support the hypothesis that soluble oligomers of amyloid β-peptide (Aβ) rather than mature amyloid fibrils are the earliest effectors of synaptic compromise in Alzheimers disease. We took advantage of an amyloid precursor protein-overexpressing cell line that secretes SDS-stable Aβ oligomers to search for inhibitors of the pathobiological effects of natural human Aβ oligomers. Here, we identify small molecules that inhibit formation of soluble Aβ oligomers and thus abrogate their block of long-term potentiation (LTP). Furthermore, we show that cell-derived Aβ oligomers can be separated from monomers by size exclusion chromatography under nondenaturing conditions and that the isolated, soluble oligomers, but not monomers, block LTP. The identification of small molecules that inhibit early Aβ oligomer formation and rescue LTP inhibition offers a rational approach for therapeutic intervention in Alzheimers disease and highlights the utility of our cell-culture paradigm as a useful secondary screen for compounds designed to inhibit early steps in Aβ oligomerization under biologically relevant conditions.

The role of cell-derived oligomers of Aβ in Alzheimer's disease and avenues for therapeutic intervention

Burgeoning evidence suggests that soluble oligomers of Abeta (amyloid beta-protein) are the earliest effectors of synaptic compromise in Alzheimers disease. Whereas most other investigators have employed synthetic Abeta peptides, we have taken advantage of a beta-amyloid precursor protein-overexpressing cell line (referred to as 7PA2) that secretes sub-nanomolar levels of low-n oligomers of Abeta. These are composed of heterogeneous Abeta peptides that migrate on SDS/PAGE as dimers, trimers and tetramers. When injected into the lateral ventricle of rats in vivo, these soluble oligomers inhibit hippocampal long-term potentiation and alter the memory of a complex learned behaviour. Biochemical manipulation of 7PA2 medium including immunodepletion with Abeta-specific antibodies and fractionation by size-exclusion chromatography allowed us to unambiguously attribute these effects to low-n oligomers. Using this paradigm we have tested compounds directed at three prominent amyloid-based therapeutic targets: inhibition of the secretases responsible for Abeta production, inhibition of Abeta aggregation and immunization against Abeta. In each case, compounds capable of reducing oligomer production or antibodies that avidly bind Abeta oligomers also ameliorate the synaptotoxic effects of these natural, cell-derived oligomers.

Diffuse plaques contain C-terminal Aβ42 and not Aβ40: Evidence from cats and dogs ☆

Abstract Recent reports have suggested that β-amyloid (Aβ) species of variable length C-termini are differentially deposited within early and late-stage plaques and the cerebrovasculature. Specifically, longer C-terminal length A β 42 3 fragments (i.e., Aβ forms extending to residues 42 and/or 43) are thought to be predominant within diffuse plaques while both A β 42 3 and Aβ40 (Aβ forms terminating at residue 40) are present within a subset of neuritic plaques and cerebrovascular deposits. We sought to clarify the issue of differential Aβ deposition using aged canines, a partial animal model of Alzheimers disease that exhibits extensive diffuse plaques and frequent vascular amyloid, but does not contain neuritic plaques or neurofibrillary tangles. We examined the brains of 20 aged canines, 3 aged felines, and 17 humans for the presence of Aβ immunoreactive plaques, using antibodies to Aβ1–17, Aβ17–24, Aβ1–28, Aβ40, and Aβ42. We report that plaques within the canine and feline brain are immunopositive for Aβ42 but not Aβ40. This is the first observation of nascent AD pathology in the aged feline brain. Canine plaques also contained epitopes within Aβ1–17, Aβ17–24, and Aβ1–28. In all species examined, vascular deposits were immunopositive for both Aβ40 and Aβ42. In the human brain, diffuse plaques were preferentially Aβ42 immunopositive, while neuritic plaques and vascular deposits were both Aβ40 and Aβ42 immunopositive. However, not all neuritic plaques contain Aβ40 epitopes.

Passage of human amyloid β-protein 1–40 across the murine blood-brain barrier

Abstract Previous studies have suggested that the amyloid β-protein present in the brains of patients with Alzheimers disease may be derived in part from peripheral blood. We determined that after IV injection of synthetic amyloid β-protein 1–40 (Aβ), labeled with radioactive 125 I(I-Aβ), radioactivity accumulated in the brains of mice by a nonsaturable mechanism. Radioactivity also accumulated in the brain after the IV injection of radioiodinated reverse amyloid β-protein 40-1 (I-rAβ). Capillary depletion techniques, however, showed I-Aβ to have a much greater degree of association with brain capillaries than I-rAβ. Acid precipitation of radioactivity in CSF samples and recovery from cortical homogenates suggested the presence of intact I-Aβ within the CNS after peripheral administration. HPLC analysis of cortical homogenates confirmed the presence of intact I-Aβ. Gel electrophoresis of the CSF acid precipitates and of the HPLC fractions further verified the presence of intact blood-derived I-Aβ peptide in CNS. These results suggest that endogenous bloodborne Aβ can enter the CNS after associating with the capillary endothelium to accumulate intact within the parenchymal and CSF spaces of the brain.

Soluble derivatives of the β amyloid protein precursor of Alzheimer's disease are labeled by antisera to the β amyloid protein

Abstract The amyloid deposited in Alzheimers disease (AD) is composed primarily of a 39–42 residue polypeptide (βAP) that is derived from a larger β amyloid protein precursor (βAPP). In previous studies, we and others identified full-length, membrane-associated forms of the βAPP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble ∼125 and ∼105 kDa forms of the βAPP found in human cerebrospinal fluid are specifically labeled by several different antisera to the βAP. This finding indicates that both soluble derivatives contain all or part of the βAP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.

Transforming growth factor-beta bound to soluble derivatives of the beta amyloid precursor protein of Alzheimer's disease.

Transforming growth factors beta (TGF beta) are multifunctional polypeptides that participate in regulation of growth, differentiation and function of many cell types. The mature TGF beta molecule is a 25 kDa protein composed of two 12.5 kDa monomers linked by disulfide bonds. Human glioblastoma cells secrete biologically active TGF beta 2. Here we report that in addition to the free form of TGF beta 2, a stable complex between a approximately 110 kDa binding protein and TGF beta 2 was isolated from glioblastoma cell supernatant. This binding protein was purified and was found to show sequence identity to part of the beta amyloid precursor protein (beta APP), to be specifically labeled by several different antisera to beta APP, and to be affinity labeled with TGF beta by crosslinking. The complex formation between TGF beta and beta APP may have important implications in regulation of biological activity of the two proteins and in delivery or clearance of TGF beta and beta APP in the brain and other compartments.

Detection of soluble forms of the β-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimers disease (AD), Downs syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.