Richard T. Carroll

Alzheimer's β‐Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia

Abstract: β‐Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimers disease. Cytokine‐producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV‐2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low‐density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time‐ and dose‐dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ‐stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.

High Levels of Amyloid-b Protein from S182 (Glu246) Familial Alzheimer's Cells

Most early-onset familial Alzheimer disease is associated with missense mutations in S182, a membrane protein on chromosome 14. We investigated amyloid-beta protein (A beta) precursor (A beta PP) metabolism in skin fibroblasts from S182 (Glu246)-affected individuals and unaffected family members. Steady-state A beta PP levels were similar among all lines as was the degree of increase in soluble A beta PP released upon stimulation of cells with either phorbol ester or serum. Among all lines studied, A beta levels were consistently detectable only in the medium of a single line of S182 (Glu246) cells, consistent with the conclusion that some S182 mutant lines may accumulate A beta in their conditioned media. Studies of cells from additional individuals and under other conditions will be required to establish this association of elevated A beta levels with S182 mutations.

Secretion and accumulation of Alzheimer's β-protein by cultured vascular smooth muscle cells from old and young dogs

Cultured smooth muscle cells isolated from beta-amyloid-affected blood vessels from old dogs accumulate beta-protein at early passages [5,24]. Now, we show that smooth muscle cells derived from amyloid-free brain blood vessels and peripheral arteries from old and young animals are induced by culture conditions to deposit intracellularly fibrillar and non-fibrillar beta-protein. Accumulation of beta-protein is associated with a higher secretion of beta-protein, but not with a higher secretion of beta-amyloid precursor protein (beta APP) or higher cellular content of beta APP. Gradual cessation of proliferative activity was observed in cultures that accumulate beta-protein.

Interleukin-1β dissociates β-amyloid precursor protein and β-amyloid peptide secretion

A heightened production of interleukin 1β(IL‐1β) has been reported in microglial‐associated amyloid deposits in Alzheimers disease (AD) brains. These plaques are composed predominantly of β/A4 peptide derived from β‐amyloid precursor protein (βAPP). We demonstrate that short‐term (1 h) IL‐1β‐treatment increases βAPPs secretion and concomitantly decreases cell‐associated βAPP in human H4 neuroglioma cells. Long‐term (5 h) IL‐1β treatment did not alter secreted or cell‐associated βAPP content. In contrast, the secretion of β/A4‐containing epitope was not affected by short‐term IL‐1β stimulation; however, long‐term IL‐1β treatment decreased the amount of β/A4‐containing epitope secreted from the cells. These results show that IL‐1β modifies the processing and secretion of βAPP to exacerbate perhaps the neuropathology of AD.

Accumulation of Alzheimer amyloid-β peptide in cultured myocytes is enhanced by serum and reduced by cerebrospinal fluid

Smooth muscle cells cultured from leptomeningeal vessels from old dogs with amyloid-angiopathy accumulate intracellular deposits that are immunoreactive for amyloid-β peptide (Aβ). We used this cellular model in the present study to examine the influence of sera and cerebrospinal fluid on intracellular accumulation of Aβ-immunoreactive deposits and on secretion of soluble Aβ into culture media. We found that sera from old dogs significantly increased the percentage of Aβ-positive smooth muscle cells in culture. The enhanced accumulation of Aβ was associated with (a) lower secretion of Aβ into media, (b) altered maturation of amyloid-β-precursor protein (AβPP) into AβPP751–770 with faster electrophoretic mobility, (c) increased accumulation of C-terminal fragments of AβPP (12–15 kD, 10kD and less), and (d) increased secretion of AβPP into culture media. These findings suggest that age- or disease-related serum factors increase accumulation of Aβ by affecting production and processing of AβPP. In contrast, cerebrospinal fluids reduced accumulation of Aβ. Involvement of Aβ-carrier proteins—apolipoprotein E and transthyretin—in accumulation of Aβ is demonstrated. Accumulation of Aβ in cultured smooth muscle cells—a model of β-amyloidosis—may be regulated by factors that alter production and processing of AβPP as well as the fate of soluble Aβ in extracellular space.

The Role of Arachidonic Acid in the Secretion of the Amyloid Precursor Protein (APP)a

We have studied the activation of human ml‐muscarinic receptors in a genetically engineered Chinese hamster ovary cell line (CHO‐ml) to determine which second messenger systems affect the secretion of APP via the non‐amyloidogenie route. Carbachol activation of the signaling pathways in CHO‐ml cells promotes APP secretion by activation of both protein kinase C (PKC)‐dependent or Ca++ ‐dependent second messenger pathways. Both pathways converge to increase the enzyme activity of phospholipase A2 (PLA2), the enzyme that releases arachidonic acid from cellular stores. Directly activating PLA2 with melittin, a peptide from bee venom, or by adding arachidonic acid directly to cultured cells increases the secretion of APP. Thus, our results indicate that arachidonic acid is yet another cellular second messenger involved in regulating the metabolism of APP in addition to PKC and cytoplasmic Ca++. Moreover, activation of PLA2 appears to be an obligatory event in increasing the secretion of APP from CHO‐ml cells by the various methods of activation that we have tried thus far.

Identification of the trypsin-like activity in commercial preparations of eel acetylcholinesterase

Electricus electrophorus acetylcholinesterase (AChE, EC 3.1.1.7) is reported to possess a trypsin-like activity. We found that purification of AChE removes over 99% of this protease activity, which resides in a single 25 kDa protein with an N-terminal sequence identical to bovine pancreatic trypsin. Digests of neuropeptides using purified eel AChE or bovine pancreatic trypsin gave identical peptide maps. These results indicate that the commercial preparation of eel AChE is contaminated by a trypsin, which is difficult to remove completely during AChE purification.

β-Amyloid levels predict cholinesterase activity in human cerebrospinal fluid

There is increasing evidence suggesting that beta-amyloid (Abeta) has a direct influence on cholinergic activity. In particular, Abeta has been shown to induce the expression of acetylcholinesterase in the brains of CT-100-expressing transgenic mice and in cell culture experiments. These data indicate a link exists between Abeta production and acetylcholinesterase expression in the human CNS. To test this hypothesis, Abeta levels and cholinesterase activity were measured in 110 human CSF samples. Abeta levels were found to have a significant and positive correlation with cholinesterase activity. This correlation was particularly strong in individuals > 50 years of age. These data support the hypothesis that Abeta can effect cholinergic activity and that this effect may be enhanced in the elderly.

Factors secreted by activated microglia and monocytes reduce amyloidogenesis in vascular smooth muscle cells

SMOOTH muscle cells cultured from amyloid-β-affected arteries accumulate amyloid-β peptide Aβ. We now show that accumulation of “Aβ” deposits in this model can be significantly reduced by culture in conditioned media from microglia and monocytes. Reduced Aβ accumulation was associated with (i) lower secretion of Aβ, (ii) increased secretion, but not cellular levels of amyloid-β-precursor protein (AβPP), and (iii) increased cell proliferation and metabolic activity. We suggest that improper regulation of AβPP metabolism by monokines may facilitate vascular amyloidogenesis.

Cell Surface Receptor Mediated Control of Amyloid Precursor Protein Secretion: Involvement of Pleiotropic Signal Transduction Cascades

Amyloid plaques associated with brain vasculature and parenchyma are the pathological hallmark of Alzheimer’s Disease (AD) (Joachim and Selkoe, 1992). The deposits contain a variety of proteins but are mainly composed of a peptide with 39 to 43 amino acids, the s-amyloid peptide (s/A4). s/A4 is derived from the proteolytic processing of a larger protein, the amyloid precursor protein (APP). APP is a family of membrane associated glycoproteins that are constitutively synthesized by a variety of cells including neurons and glia (Estus et al., 1992; Shoji et al., 1992; Busciglio et al., 1993). Proteolytic cleavage at a single site in its s/A4 region releases the majority of APP from cells and at the same time eliminates the production of s/A4 (Weidemann et al., 1989; Anderson et al., 1992). Alternatively, APP is processed by a second pathway that leads to the formation of intact, amyloidogenic s/A4 (Haass et al., 1991; Golde et al., 1992; Hung et al., 1992). Cells normally produce small amounts of s/A4, but genetic mutations (Cai et al., 1993a; Haass et al., 1993) and trauma (Roberts et al., 1991) can enhance the expression of the peptide.