Barry D. Greenberg

Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester.

Fibrillar amyloid deposits are defining pathological lesions in Alzheimer’s disease brain and are thought to mediate neuronal death. Amyloid is composed primarily of a 39–42 amino acid protein fragment of the amyloid precursor protein (APP), called amyloid β-protein (Aβ). Because deposition of fibrillar amyloid in vitrohas been shown to be highly dependent on Aβ concentration, reducing the proteolytic release of Aβ is an attractive, potentially therapeutic target. Here, the turnover rate of brain Aβ has been determined to define treatment intervals over which a change in steady-state concentration of Aβ could be measured. Mice producing elevated levels of human Aβ were used to determine approximate turnover rates for Aβ and two of its precursors, C99 and APP. Thet½ for brain Aβ was between 1.0 and 2.5 hr, whereas for C99, immature, and fully glycosylated forms of APP695 the approximate t½ values were 3, 3, and 7 hr, respectively. Given the rapid Aβ turnover rate, acute studies were designed using phorbol 12-myristate 13-acetate (PMA), which had been demonstrated previously to reduce Aβ secretion from cells in vitro via induction of protein kinase C (PKC) activity. Six hours after intracortical injection of PMA, Aβ levels were significantly reduced, as measured by both Aβ40- and Aβ42-selective ELISAs, returning to normal by 12 hr. An inactive structural analog of PMA, 4α-PMA, had no effect on brain Aβ levels. Among the secreted N-terminal APP fragments, APPβ levels were significantly reduced by PMA treatment, whereas APPα levels were unchanged, in contrast to most cell culture studies. These results indicate that Aβ is rapidly turned over under normal conditions and support the therapeutic potential of elevating PKC activity for reduction of brain Aβ.

Degeneration of vascular muscle cells in cerebral amyloid angiopathy of Alzheimer disease

In cerebral amyloid angiopathy, the amyloid-beta (A beta) deposits lie primarily in the tunica media suggesting that smooth muscle cells play an important role in A beta deposition. To define this role, we conducted an immunocytochemical study of brain tissue from cases of Alzheimer disease with extensive cerebral amyloid angiopathy and cerebral hemorrhage. Antibodies specific to recombinant beta protein precursor (beta PP) and synthetic peptides homologous to various beta PP sequences from residue 18 to 689 of beta PP695 were used. Antibodies to actin, tropomyosin, alpha-actinin or desmin were used to label muscle cells. Antibodies to A beta sequences intensely recognized the extracellular amyloid deposit. Antibodies raised against beta PP sequences other than the A beta domain recognized smooth muscle cells. beta PP-immunoreactivity was reduced in regions of A beta deposits, since no muscle cells were recognized by cytoskeletal markers or observed ultrastructurally. In order to assess why A beta is deposited in the tunica media, we used biotin-labelled beta PP to determine if beta PP can be locally retained. We found beta PP bound to the tunica media of vessels but not other brain elements. These findings suggest A beta in blood vessels derives from degenerating beta PP-containing smooth muscle cells.

Morphological and biochemical analyses of amyloid plaque core proteins purified from Alzheimer disease brain tissue

Abstract— Amyloid plaque cores were purified from Alzheimer disease brain tissue. Plaque core proteins were solubilized in formic acid which upon dialysis against guan‐idinium hydrochloride (GuHCI) partitioned into soluble (∼15%) and insoluble (∼85%) components. The GuHCI‐soluble fraction contained β‐amyloid1‐40, whereas the GuHCI‐insoluble fraction was fractionated into six components by size exclusion HPLC: S1 (>200 kDa), S2 (200 kDa), S3 (45 kDa), S4 (15 kDa), S5 (10 kDa), and S6 (5 kDa). Removal of the GuHCI reconstituted 10‐nm filaments composed of two intertwined 5‐nm strands. Fractions S5 and S6 also yielded filamentous structures when treated similarly, whereas fractions S1–S4 yielded amorphous aggregates. Chemical analysis identified S4–S6 as multimeric and monomeric β‐amyloid. Immunochemical analyses revealed α1‐antichymotrypsin and non‐β‐amyloid segments of the β‐amyloid precursor protein within fractions S1 and S2. Several saccharide components were identified within plaque core protein preparations by fluorescence and electron microscopy, as seen with fluores‐cein isothiocyanate‐and colloidal gold‐conjugated lectins. We have shown previously that this plaque core protein complex is more toxic to neuronal cultures than β‐amyloid. The non‐β‐amyloid components likely mediate this additional toxicity, imposing a significant influence on the pathophysiology of Alzheimer disease.

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.

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.

Binding of vascular heparan sulfate proteoglycan to Alzheimer's amyloid precursor protein is mediated in part by the N-terminal region of A4 peptide

The exact mechanisms of deposition and accumulation of amyloid in senile plaques and in blood vessels in Alzheimers disease remain unknown. Heparan sulfate proteoglycans may play an important role in amyloid deposition in Alzheimers disease. Previous investigations have demonstrated high affinity binding between heparan sulfate proteoglycans and the amyloid precursor, as well as with the A4 peptide. In the current studies, a specific vascular heparan sulfate proteoglycan found in senile plaques bound with high affinity to two amyloid protein precursors (APP695 and APP770). Vascular heparan sulfate proteoglycan also bound the Alzheimers amyloid A4 peptide, and not other amyloid protein precursor regions studied, with high affinity. Both heparan sulfate glycosaminoglycan chains and chemically deglycosylated vascular heparan sulfate proteoglycan protein core bound to A4. High affinity interactions between vascular heparan sulfate proteoglycan and the A4 peptide may play a role in the process of amyloidogenesis in Alzheimers disease, by localizing the site of deposition of A4, protecting A4 from further proteolysis, or by promoting aggregation and fibril formation.

The amyloid precursor protein of Alzheimer disease is expressed as a 130 kDa polypeptide in various cultured cell types

The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP‐130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP‐228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP‐130 showed a pI of 6.2, while APP‐228 failed to focus in the pH range of 4.7–7.0. Sequential extractions of cells with buffer and with Triton X‐100 indicate that APP‐130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP‐130 and APP‐228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.

A roadmap for the prevention of dementia: The inaugural Leon Thal Symposium

AD research has exploded during the last 20 years, resulting in the identification of numerous possible pathogenic pathways and targets for intervention. Accompanying this mechanistic research, neuropathologic and imaging studies have revealed that the pathogenic process begins long before the first symptoms appear and that modifying the disease process, in contrast to treating symptoms, might likewise require intervention long before the disease becomes evident. The drug development and regulatory processes, however, have for the most part remained focused on treating symptoms rather than preventing disease or intervening in its progression. The traditional phase I, II, and III trials follow a common pathway, evaluating first safety and tolerability, followed by dose finding and early effectiveness, and culminating in efficacy trials designed to demonstrate that a drug is safe and has a beneficial clinical effect. This model, however, is not appropriate for evaluating preventive or disease-modifying treatments, where the clinical benefits might be so subtle and far into the future as to be unmeasurable within a reasonable period of time. Multiple, interrelated barriers to drug development The barriers to developing preventive therapies span scientific, structural, business, and economic issues. Although it is clear that the disease process that eventually results in neurodegeneration and dementia in AD begins early, the exact nature and timing of these early steps are not known. AD might even represent the culmination of a neurodevelopmental process that begins at birth; or it might be a reflection of the normal aging process that is accelerated in some people for unknown reasons. The absence of a complete animal model of the disease (as opposed to partial models of selective pathologies) has certainly dampened the discovery process. A better understanding of disease progression in the earliest stages should also lead to the identification of novel therapeutic targets for preventing disease. The answers to these questions will emerge only through increased efforts to understand the basic biology of AD and neurodegeneration, including greater reliability in identifying early/prodromal AD, cognitive measures that are sensitive to change in mild disease, and the effect of changing lifestyles, medications, and lifestyle adaptations as management of mild dementia undergoes further development. This will require support for very long-term longitudinal studies, yet inadequate funding for basic science has stymied progress in this fundamental area. In fact, although the burden to society from AD has increased steadily since 1990, funding for AD research has remained flat. Clearly, new funding streams need to be established to ensure progress in both basic and applied research. As the drug development process moves from the basic science laboratories into clinical trials, infrastructural and regulatory barriers, along with different economic considerations, become paramount. Key players at this stage of the process include pharmaceutical companies, the Food and Drug Administration (FDA), and clinical trial sites. Although drug development in general is risky, expensive, and time-consuming, developing preventive treatments increases the challenges many fold. As a result, demonstrating effectiveness in preventing a disease might require such lengthy trials that the patent life on a drug will be exceeded. Moreover, at this time, methods for designing and implementing prevention trials, including how to identify at-risk subjects and how to assess outcome for those trials, have not been fully established. Responding to these multiple challenges will require flexibility and cooperation on the part of all stakeholders: clinicians, researchers, regulatory agencies, federal and private funders, Congress, pharmaceutical companies, and the public. A comprehensive strategic response to this challenge will require consideration of how to manage the risks that each of these stakeholders face. But the risk of not developing such a strategy is far greater in terms of the public health and economic impact.

Rank-Order of Potencies for Inhibition of the Secretion of Aβ40 and Aβ42 Suggests That Both Are Generated by a Single γ-Secretase

The Alzheimer’s disease amyloid peptide Aβ has a heterogeneous COOH terminus, as variants 40 and 42 residues long are found in neuritic plaques and are secreted constitutively by cultured cells. The proteolytic activity that liberates the Aβ COOH terminus from the β-amyloid precursor protein is called γ-secretase. It could be one protease with dual specificity or two distinct enzymes. By using enzyme-linked immunosorbent assays selective for Aβ40 or Aβ42, we have measured Aβ secretion by a HeLa cell line, and we have examined the dose responses for a panel of five structurally diverse γ-secretase inhibitors. The inhibitors lowered Aβ and p3 secretion and increased levels of the COOH-terminal 99-residue β-amyloid precursor protein derivative that is the precursor for Aβ but did not alter secretion of β-amyloid precursor protein derivatives generated by other secretases, indicating that the inhibitors blocked the γ-secretase processing step. The dose-dependent inhibition of Aβ42 was unusual, as the compounds elevated Aβ42 secretion at sub-inhibitory doses and then inhibited secretion at higher doses. A compound was identified that elevated Aβ42 secretion at a low concentration without inhibiting Aβ42 or Aβ40 at high concentrations, demonstrating that these phenomena are separable pharmacologically. Using either of two methods, IC50 values for inhibition of Aβ42 and Aβ40 were found to have the same rank-order and fall on a trend line with near-unit slope. These results favor the hypothesis that Aβ variants ending at residue 40 or 42 are generated by a single γ-secretase.

Mutant and native human β-amyloid precursor proteins in transgenic mouse brain

Human β-amyloid precursor protein (βAPP) has been targeted to transgenic neurons using synapsin I promoter-based chimeric transgenes. Native human βAPP was introduced as well as βAPP containing mutations genetically linked to familial Alzheimers disease (AD) and to hereditary cerebral hemorrhage with amyloidosis-Dutch type. In mouse brain, human βAPP RNA was up to 60% as abundant as total endogenous βAPP RNA. Human βAPP gene expression was most abundant in the CA subfields of the hippocampus and in the piriform cortex. Correct processing of human βAPP at the β-secretase cleavage site was demonstrated in transgenic mouse brains. Despite a 40% increase in total βAPP immunoreactivity in lines expressing mutant human βAPP, no evidence of amyloid deposition was found in brains of mice up to 14 months in age. Higher levels of mutant human βAPP, increased age, or other factors may be necessary to elicit β-amyloid-related neuropathologies in the rodent brain.