Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.
Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.
Title: Acceleration of phosphatidylcholine synthesis and breakdown by inhibitors of mitochondrial function in neuronal cells: a model of the membrane defect of Alzheimer's disease Farber SA, Slack BE, Blusztajn JK Ref: FASEB Journal, 14:2198, 2000 : PubMed
Brain cells in Alzheimer's disease (AD) exhibit a membrane defect characterized by accelerated phospholipid turnover. The mechanism responsible for this defect remains unknown. Recent studies indicate that impairment of mitochondrial function is frequently observed in AD and may be responsible for certain aspects of its pathophysiology. We show that when PC12 cells are exposed to inhibitors of mitochondrial bioenergetics, the turnover of their major membrane phospholipid, phosphatidylcholine, is accelerated, producing a pattern of metabolic changes that mimics that observed in brains of AD patients. Abnormalities of mitochondrial function may therefore underlie the membrane defect in AD.
Title: Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging Hendrickson HS, Hendrickson EK, Johnson ID, Farber SA Ref: Analytical Biochemistry, 276:27, 1999 : PubMed
Phospholipase substrate analogs containing both a fluorescent BODIPY group and a quenching 2,4-dinitrophenyl (DNP) group were synthesized. They showed little fluorescence, but upon hydrolysis became fluorescent as the quenching group was removed. Two substrates were phosphatidylethanolamine analogs with a BODIPY-pentanoyl group at the sn-2 position and DNP linked to the amino head group. The third was a phosphatidylcholine analog with a BODIPY-labeled alkyl ether at the sn-1 position and a N-(DNP)-8-amino-octanoyl group at the sn-2 position. These compounds were evaluated as substrates for cytosolic (85 kDa) phospholipase A(2) (cPLA(2)) and plasma platelet-activating factor acetylhydrolase (rPAF-AH). Two were good substrates for cPLA(2) (specific activities: 18 and 5 nmol min(-1) mg(-1)) and all were good for rPAF-AH (specific activities: 17, 11, and 6 micro mol min(-1) mg(-1)). The minimal amount of enzyme detectable was 50 ng for cPLA(2) and 0.1 ng for rPAF-AH. These substrates were active in assays of PLA(2) in zebrafish embryo extracts and one was well suited for imaging of PLA(2) activity in living zebrafish embryos. Embryos were injected with substrate at the one- to four-cell stage and allowed to develop until early somitogenesis when endogenous PLA(2) activity increases dramatically; substrate persisted (12 h) and specifically labeled cells of the developing notochord.
Title: Muscarinic M1 receptor agonists increase the secretion of the amyloid precursor protein ectodomain Muller DM, Mendla K, Farber SA, Nitsch RM Ref: Life Sciences, 60:985, 1997 : PubMed
Amyloid deposits in Alzheimer's disease are composed of amyloid beta-peptides (A beta) that are derived from the larger amyloid precursor protein (APP). Proteolytic APP processing is activity-dependent, and it can be regulated by muscarinic acetylcholine receptors. In particular, muscarinic m1 receptor subtypes increase cleavage within the A beta domain, followed by the release of the soluble APP ectodomain (APPs). In this study, we show that the m1-selective agonist talsaclidine concentration-dependently increased APPs release from both transfected human astrocytoma cell lines and rat brain slices. This increase was blocked by atropine. In contrast, the M2 antagonist BIBN 99 failed to increase APPs release, and decreased it at higher concentrations. These results show that talsaclidine can effectively modulate alpha-secretase processing of APP in human cell lines and in brain tissue. The data suggest that talsaclidine may be a useful candidate drug to modulate APP processing in Alzheimer's disease.
Title: Choline's phosphorylation in rat striatal slices is regulated by the activity of cholinergic neurons Farber SA, Savci V, Wei A, Slack BE, Wurtman RJ Ref: Brain Research, 723:90, 1996 : PubMed
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.
beta A4 is the principal component of Alzheimer's disease brain amyloid. It is derived from proteolytic processing of amyloid beta-protein precursors (APP), a family of transmembrane glycoproteins. Secretion of APPs, a secreted proteolytic derivative that is cleaved within the beta A4 domain of APP, is increased many-fold by the activation of cell-surface receptors, like the muscarinic m1 and m3 receptor subtypes, which are coupled to protein kinase C. Concomitantly, their activation decreases the formation of both secreted soluble beta A4 and of endosomal-lysosomal C-terminal APP derivatives. These data suggest that muscarinic m1 and m3 receptors accelerate non-amyloidogenic APP processing and depress the formation of potentially amyloidogenic derivatives. Other receptors that stimulate APPs secretion include those for bradykinin, vasopressin, and interleukin-1 receptors. A similar control mechanism is present in rat brain tissue slices, in which the release of both APPs and endogenous neurotransmitters is increased by electrical depolarization. This increase is tetrodotoxin-sensitive and frequency-dependent, suggesting that APPs release may normally depend on neuronal activity. Taken together, our findings suggest that specific receptor agonists might be effective in reducing the formation of potentially amyloidogenic APP derivatives in vivo.
The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.
Title: Release of amyloid beta-protein precursor derivatives by electrical depolarization of rat hippocampal slices Nitsch RM, Farber SA, Growdon JH, Wurtman RJ Ref: Proc Natl Acad Sci U S A, 90:5191, 1993 : PubMed
Proteolytic processing of the beta-amyloid protein precursor (APP) is regulated by cell-surface receptors. To determine whether neurotransmitter release in response to neuronal activation regulates APP processing in brain, we electrically depolarized superfused rat hippocampal slices and measured soluble APP derivatives released into the superfusate. Electrical depolarization caused a rapid increase in the release of both neurotransmitters and amino-terminal APP cleavage products. These derivatives lacked the APP carboxyl terminus and were similar to those found in both cell culture media and human cerebrospinal fluid. Superfusate proteins including lactate dehydrogenase were not changed by electrical depolarization. The release of amino-terminal APP derivatives increased with increasing stimulation frequencies from 0 to 30 Hz. The increased release was inhibited by the sodium-channel antagonist tetrodotoxin, suggesting that action-potential formation mediates the release of large amino-terminal APP derivatives. These results suggest that neuronal activity regulates APP processing in the mammalian brain.
