Barbara E. Slack

Regulation of Amyloid Precursor Protein Release By Protein Kinase C in Swiss 3T3 Fibroblastsa

Release of the amyloid precursor protein (APP) of Alzheimers disease from Swiss 3T3 fibroblasts was stimulated in a concentration‐dependent manner by phorbol 12‐myristate 13‐acetate. In fibroblasts overexpressing protein kinase Co (PKCα), the EC50 for this response was 7 nM, while in control cells the EC50 was 63 nM. The effect of PMA was inhibited by the PKC antagonist H‐7 in control cells, but not in cells that overexpressed PKCα. Basal release of APP was higher in cells that overexpressed PKCα, and was not affected by the phosphatase inhibitor okadaic acid, although this compound doubled APP release from control cells. The results suggest that PKCα regulates APP processing in mammalian cells. Alterations in the activity of PKC have been reported to occur in Alzheimers disease and might potentially contribute to abnormalities of APP metabolism characteristic of this disorder.

Choline's phosphorylation in rat striatal slices is regulated by the activity of cholinergic neurons.

The mechanism by which populations of brain cells regulate the flux of choline (Ch) into membrane or neurotransmitter biosynthesis was investigated using electrically stimulated superfused slices of rat corpus striatum. [Me-14C]Ch placed in the superfusion medium for 30 min during a 1-h stimulation period was incorporated into tissue [14C] phosphorylcholine (PCh) and [14C]phosphatidylcholine (PtdCh). Stimulation also caused a profound inhibition of PCh synthesis and a 10-fold increase in [14C]ACh release into the medium; it failed to affect tissue [14C]ACh levels. This effect was not explained by changes in ATP levels nor in the kinetic properties of Ch kinase (E.C. 2.7.1.32) or Ch acetyltransferase (ChAT) (E.C.2.3.1.7). To investigate the mechanism of these effects, Ch uptake studies were performed with and without hemicholinium-3 (HC3), a selective inhibitor of high affinity Ch uptake. A two-compartment model accurately fit the observed data and yielded a K(m) for Ch uptake of 5 microM into cholinergic structures and 72 microM into all other cells. Using this model it was estimated that cholinergic neurons account for 60% of observed uptake of Ch at physiologic Ch concentrations, even though they represent fewer than 1% of the total cells in the slice. The model also predicts that an increase in Ch uptake within cholinergic neurons, reported to be associated with depolarization [4,27,32], would significantly inhibit Ch uptake into all other cells, and would account for the observed decrease in PCh synthesis.

Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line

Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis).(ABSTRACT TRUNCATED AT 250 WORDS)

Choline Metabolism, Membrane Phospholipids, and Alzheimer’s Disease

Alzheimer’s Disease (AD) and even apparently-normal aging are associated with defects in brain choline metabolism. This chapter describes these abnormalities as well as some of the experimental systems used to study their mechanisms and effects. Cell culture, superfused brain slices, in vivo microdialysis, postmortem tissue analysis, and in vivo magnetic resonance spectroscopy (MRS) studies have been employed. Available data suggest that these defects provide a biochemical basis for the special vulnerability of cholinergic neurons in AD.

Modulation of APP Processing by Neurotransmission

Brain amyloid deposits are invariant neuropathological hallmarks of Alzheimer’s disease (AD) and Down’s syndrome, and are sometimes also found in lesser amounts in brains of neuropsychologically normal, aged human subjects. AD-type brain amyloid consists of aggregated As peptides which are 39–43 amino acid residues in length. As is derived, by proteolytic processing, from a larger amyloid s-protein precursor (APP), which is a transmembrane glycoprotein that contains a single membrane spanning domain, a large N-terminal ectodomain and a short cytoplasmic C-terminal tail. The As domain is located within the ectodomain and extends with its hydrophobic C-terminal region 11-15 residues into the membrane. APP exists in various forms generated by alternative splicing of mRNA derived from a single gene on chromosome 21 (for review, see Kosik, 1992). APP is highly conserved and expressed at high levels in brain and, at lower levels, in many peripheral tissues. The biological function of APP is unclear but accumulating evidence suggests roles in cell adhesion (Schubert et al., 1989), in neurite outgrowth (Milward et al., 1992), as well as excitoprotective functions via the regulation of intracellular calcium concentrations (Mattson et al., 1993). Mature APP is rapidly degraded by various alternative proteolytic processing pathways. Proteolytic derivatives are secreted into the extracellular space and are found at high concentrations in human cerebrospinal fluid. Secreted APP derivatives include the large N-terminal ectodomain, termed APPs (Esch et al., 1990; Sisodia et al., 1990) and ~4KDa As-peptides (Haass et al., 1992; Shoji et al., 1992) that potentially can aggregate into amyloid. In addition to the secretory processing pathways, full-length APP can be internalized from the cell surface and targeted to the endosomal-lysosomal system (Haass et al., 1992), where multiple cleavage products are generated. Some of these contain the intact sA4 domain and thus are also potentially amyloidogenic (Golde et al., 1992; Estus et al., 1992). As is neurotoxic in some experimental systems (Yankner et al., 1990) and may induce apoptosis (Loo et al., 1993). Thus, APP processing pathways yielding either As or APPs are likely to have distinct cellular consequences: processing events that generate As may be toxic and are potentially amyloidogenic, whereas APP processing to yield APPs generate trophic and precludes APP’s role as an amyloidogenic molecule. It thus becomes important to understand the cellular mechanisms involved in the regulation of APP processing pathways.

Accelerated Cell Membrane Degradation in Alzheimer’s Disease Brain: Relationship to Amyloid Formation?

Alzheimer’s disease is a degenerative brain disease first described by the German psychiatrist Alois Alzheimer (Alzheimer, 1907). His initial report was based upon the histological examination of brain tissue from a 51 year old female patient who experienced a rapid progressive deterioration of memory and died four and a half years after the onset of the first symptoms with total loss of mental functions. The brain of this woman showed generalized atrophy without macroscopic lesions. Using a novel silver staining method developed by Bielschowsky, Alzheimer found that more than a quarter of the cortical neurons contained fibrillary tangles, and that this abnormality was accompanied by the extracellular deposition of “multiple miliary lesions”, containing a “unique substance” which was later recognized to be amyloid. In his initial report, Alzheimer also described severe loss of nerve cells, which was most prominent in the upper layers of the brain cortex. More recently, Terry and colleagues used computerized image processing to confirm the loss of cortical neurons, and showed that large cortical neurons are particularly affected (Terry et al., 1981). Neuronal atrophy and the presence of degenerating neurites in the immediate vicinity of amyloid plaques has generated two controversial hypotheses: First, that amyloid is toxic to neurons, and, second, that amyloid is a byproduct of neuronal atrophy formed by neurons during their degeneration.