The Journal of Neuroscience, October 1, 2003, 23(26):8844-8853
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Cellular/Molecular
In Vivo Assessment of Brain Interstitial Fluid with Microdialysis Reveals Plaque-Associated Changes in Amyloid-
Metabolism and Half-Life
John R. Cirrito,1
Patrick C. May,4
Mark A. O'Dell,1
Jennie W. Taylor,1
Maia Parsadanian,1
Jeffrey W. Cramer,5
James E. Audia,6
Jeffrey S. Nissen,6
Kelly R. Bales,4
Steven M. Paul,4,7
Ronald B. DeMattos,4 and
David M. Holtzman1,2,3
Departments of 1Neurology and 2Molecular Biology and Pharmacology and 3Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, Missouri 63110, 4Neuroscience Discovery Research and 5Drug Disposition-Lead Optimization, 6Discovery Chemistry Research, Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, Indiana 46285, and 7Departments of Pharmacology, Toxicology, and Psychiatry, Indiana University School of Medicine, Indianapolis, Indiana 46285
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Abstract
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Soluble amyloid-
(A) peptide converts to structures with high -sheet content in Alzheimer's disease (AD). Soluble A is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF A levels may provide unique insights into A metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our A microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF A in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of A deposition, significant changes in the ISF A40/A42 ratio developed without changes in A1-x. These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting A production, we found that ISF A half-life was short (
2 hr) in young mice but was twofold longer in mice with A deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of A synthesis reveals a portion of the insoluble A pool that is in dynamic equilibrium with ISF A. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.
Key words: Alzheimer; amyloid; interstitial fluid; microdialysis; half-life; gamma-secretase
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Introduction
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Genetic, biochemical, and animal model studies strongly suggest a central role for amyloid- (A) in the pathogenesis of Alzheimer's disease (AD) (Sisodia, 1999
; Golde et al., 2000; St. George-Hyslop, 2000; Selkoe, 2001). A is a 38-43 amino acid peptide derived from the amyloid precursor protein (APP). Early-onset forms of familial, autosomal dominant AD appear to be attributable to an overproduction of a more amyloidogenic form of A (A42), resulting in conversion of A, in early adult life, from soluble, nontoxic forms to both soluble and insoluble toxic forms with a high -sheet content (Tanzi et al., 1996). In contrast, the most common form of AD (late-onset, age >60) does not appear to result from A overproduction. Instead, other genetic [e.g., APOE (apolipoprotein E) allelic differences] and nongenetic factors appear to be important in determining whether and when A conformational changes occur (Wisniewski et al., 1997; Teplow, 1998; Holtzman, 2001). Thus, understanding not only mechanisms of A production but also factors that control A metabolism are likely to be critical for understanding AD pathogenesis, as well as for developing better diagnostic and treatment methods.
A major pathologic hallmark of AD is the deposition of the normally soluble A peptide into aggregated, extracellular structures called plaques (Price et al., 1998; Selkoe, 2001). Although it is not proven whether the seed, or nidus, of plaques originates extracellularly or intracellularly, recent evidence suggests that initial formation and growth of plaques can occur extracellularly (Meyer-Luehmann et al., 2003). Regardless of where plaques originate, it is likely that the subsequent A building blocks for plaques are derived from the brain interstitial fluid (ISF). A appears to be released from neurons into the extracellular space normally via synaptic activity (Kamenetz et al., 2003). Assessment of the ISF pool of A may provide unique insights into the regulation of A metabolism, A aggregation, and plaque formation. To date, however, there have been no methods that have permitted analysis of ISF A.
To this end, we developed an in vivo microdialysis technique to measure ISF A in awake, APP transgenic mice that develop age-dependent A aggregation and deposition (PDAPP mice) (Games et al., 1995). We compared the steady-state concentrations of several A species in the ISF of 3-month-old and 12- to 15-month-old PDAPP mice to those species within the CSF and tissue. These studies show that the development of A deposition is associated with changes in ISF A levels and ratios that differ in relation to tissue lysates and CSF. By combining our microdialysis technique with a potent 
-secretase inhibitor, we also demonstrate that the half-life of ISF A is short and is markedly influenced by the presence of A deposition. This strongly suggests that rapid inhibition of A production in vivo reveals an equilibrium between soluble and part of the insoluble pool of A. This rapidly dissociable, now measurable pool of A may provide new ways to diagnose the presence of A deposits and is likely to be the target of various anti-A treatments.
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Materials and Methods
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In vitro microdialysis. Microdialysis probes used for in vitro experiments had a 4 mm, 35 kDa molecular weight cutoff (MWCO) membrane (BR-4-10 probes; Bioanalytical Systems, West Lafayette, IN) and were connected to a microdialysis peristaltic pump (MAB20; SciPro, Sanborn, NY) using teflon (FEP) tubing (inner diameter, 0.12 mm; 1.2 µl/10 cm; SciPro). Before use, probes were washed with 0.15% bovine serum albumin (BSA)-artificial CSF (aCSF) perfusion buffer (in mM: 1.3 CaCl2, 1.2 MgSO4, 3 KCl, 0.4 KH2PO4, 25 NaHCO3, and 122 NaCl, pH 7.35) that was filtered through a 100 kDa MWCO membrane (Millipore, Billerica, MA). In vitro studies using samples of human CSF were collected by lumbar puncture from cognitively normal research volunteers from the Washington University Memory and Aging Project as described previously (Fagan et al., 2000). The study was approved by the Human Studies Committee at Washington University. Samples were stored at 4°C in polypropylene tubes.
Interpolated zero flow method. A 0.15% BSA-aCSF perfusion buffer was passed through a microdialysis probe immersed in human CSF. Flow rates ranged from 0.5 to 2.6 µl/min. The concentration of A1-x obtained in each microdialysis sample was measured by ELISA. The actual concentration of exchangeable A (eA) in each sample was assessed by plotting the sample A concentration versus the flow rate and then extrapolating back to a zero flow rate to determine the concentration of eA within the target CSF. For each flow rate, the percentage recovery was determined by the following: (Cx/E) * 100, where Cx is the concentration of A in the dialysate at a given flow rate, and E is the calculated concentration of eA in that sample.
Animals. All experimental procedures involving animals were performed in accordance with guidelines established by the Animal Studies Committee at Washington University. PDAPP mice homozygous for the APPV717F transgene (Games et al., 1995; Bales et al., 1997) were bred to Swiss Webster mice (Charles River Laboratories, Wilmington, MA) to produce PDAPP hemizygous transgenic mice. Animals were used at 3-4 months of age (young) or 12-15 months of age (middle-aged). C57BL/6 mice were purchased from Harlan Sprague Dawley (Indianapolis, IN).
In vivo microdialysis technique. Microdialysis probes used for in vivo experiments had a 4 mm, 38 kDa MWCO membrane (MBR-4-10 probes; Bioanalytical Systems) and were connected to a Univentor 864 syringe pump (SciPro). Before guide implantation surgery, probes were prepared as stated above. For microdialysis guide cannula implantation, PDAPP mice were anesthetized using 1.5-2.5% isoflurane. The head was shaved, and the skin was transected along the midline to expose the skull from several millimeters anterior and posterior to bregma and lambda. The animal was then placed in a small animal stereotaxic device equipped with dual manipulator arms and an anesthetic mask (David Kopf Instruments, Tujunga, CA). To ensure the skull was level for each animal, measurements and adjustments were made so that bregma and lambda were at equal heights (with 0.1 mm tolerance), as well as 2 points equidistant from midline. Bore holes (0.75 mm) were made above the left hippocampus (coordinates, bregma -3.1 mm, 2.4 mm lateral, -0.6 mm relative to dura mater) and above the right striatum (coordinates, bregma +0.2 mm, 2.2 mm lateral, -0.5 mm relative to dura mater). An additional bore hole was made into the right, posterior skull quadrant for an anchoring bone screw. MBR-10 guide cannulas (Bioanalytical Systems) were stereotactically inserted into the hippocampus (12° angle) and striatum (0° angle) and cemented into place using a binary dental cement. Once the cement was firm, the animal was removed from the stereotaxic device, and the wound was closed anterior and posterior to the guide assembly using 1.75 mm Michel suture clips (Fine Science Tools, Foster City, CA).
The guide cannula stylets were removed, and the flushed microdialysis probes were manually inserted through the guide cannula into the target regions. After probe insertion, mice were fitted with a plastic mouse collar and placed into cages designed to allow unrestricted movement of the animal without applying pressure to probe assembly (Raturn Cage System; Bioanalytical Systems). Animals were then allowed to awaken and remained awake for remainder of the experiment. To measure A1-x, microdialysis probes had a constant flow rate of 2.6 µl/min. Microdialysis samples were collected hourly or bihourly using a refrigerated fraction collector.
At the conclusion of the microdialysis experiment, animals were anesthetized with 60 mg/kg Nembutal. CSF was collected from the cisterna magna (DeMattos et al., 2002b). Plasma was sampled via cardiac bleed, and the animal was perfused with cold PBS-heparin. The brain was fixed overnight in 4% paraformaldehyde at 4°C and then transferred to 30% sucrose. Brains sections (40 µm) were cut on a freezing microtome. All probe placements were verified by cresyl violet staining. Microdialysis samples were kept at 4°C and analyzed the day of collection by ELISA. Data from probe placements outside the appropriate region were discarded.
To assess ISF eA via microdialysis, as well as tissue A levels in the same animal, a subset of mice received only unilateral hippocampal probes. To measure eA40 and eA42, a single fraction was collected from hours 4 to 10 after probe implantation at a flow rate of 0.5 µl/min. This protocol enriched the fraction for A and facilitated individual A species to be detected by ELISA. At the conclusion of the experiment, the implanted hemisphere was fixed and processed for histology to verify probe placement and quantify percentage A load by immunostaining, whereas the contralateral hemisphere was dissected for several brain regions and processed for biochemical analysis.
A quantification. Microdialysis samples, CSF, and tissues were analyzed for A using a denaturing, sandwich ELISA specific for human A1-x,A1-40,orA1-42 (see Fig. 2 A). The A1-x ELISA measures A1-x by using a central-domain monoclonal antibody (m266) against amino acids 13-28 as a capture antibody. To capture the different species of full-length A, C-terminal domain monoclonal antibodies specific for either A40 (m2G3) or A42 (m21F12) were used. All assays used a biotinylated N-terminal domain monoclonal antibody specific for an intact N terminus (amino acids 1-5) of human A (m3D6) as a reporting antibody, followed by streptavidin-poly-horseradish peroxidase-20 (Research Diagnostics, Flanders, NJ). C-Terminal variants of human A (A-CTV) are a calculation of the A40 and A42 ELISA values subtracted from the mean concentration from the A1-x ELISA. This would include any peptide species that start at amino acids 1 through 28 or longer but does not end in amino acid 40 or 42. Murine A40 and murine A42 were detected using either m266 or m21F12 as a capture antibody and biotinylated m2G3 or m266 to detect. Slow ELISA TMB (Sigma, St. Louis, MO) was used for colorimetric detection and analyzed on a BioTek (Winooski, VT) 600FL microtiter plate reader. A40 and A42 standard curves were generated from A peptide obtained from American Peptide (Sunnyvale, CA).
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Figure 2. A ELISA and in vivo microdialysis technique. A, A species detected by ELISA for human A1-x, A40, and A42. Top, A peptide sequence with epitopes recognized by each monoclonal (m) antibody. Bottom, A species detected by each ELISA assay; solid lines represent segments of peptide that must be present for detection, and dashed lines represent segments of peptide that may vary but will still be recognized by the various antibody combinations. B, Awake mouse implanted with dual microdialysis probes. Black arrow denotes guide cannula-probe assembly; white arrow denotes collar used to attach mouse to balance arm that prevents force from being applied to implanted assembly. C, D, Representative probe placements in the hippocampus and striatum, respectively. Note partial probe tracts within each section. Hashed line depicts probe location. D, Cresyl violet stain of tissue surrounding the microdialysis probe tract after 18 hr implantation. There is no morphological evidence of substantial gliosis or inflammation along the tract (arrows), nor is there evidence of neuronal degeneration within the dentate granule cells. F, In vivo percentage recovery at various flow rates as determined by the interpolated zero flow method. At 0.5 and 2.6 µl/min, the percentage recovery of eA is 56.3 ± 4.21 and 4.9 ± 0.90% (n = 4), respectively.
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All ELISA samples were diluted in a final buffer of 0.25% BSA-500 mM guanidine-200 mM Tris-PBS, pH 7.4 with protease inhibitors. Microdialysis sample concentrations were corrected for in vivo percentage recovery based on the flow rate at which the sample was collected (example, 2.6 µl/min = 4.9% recovery and 0.5 µl/min = 56.3% recovery). Hippocampal tissues were processed by a sequential A extraction using a 0.1 M carbonate buffer, pH 11.5, followed by a 5 M guanidine extraction of the pellet as described previously (DeMattos et al., 2002c). Tissue sample concentrations were normalized to protein concentration as measured by Micro BCA Assay (Pierce, Rockford, IL).
Acid-urea PAGE and Western blot. A single, 24 hr hippocampal microdialysis sample was collected at 0.5 µl/min from a 3-month-old PDAPP mouse. This sample was then immunoprecipitated with biotinylated m266, followed by streptavidin Sepharose beads (Amersham Biosciences, Piscataway, NJ). A was eluted from the pellet with 100% formic acid, diluted in loading dye, and loaded onto a 22% acid-urea polyacrylamide gel (DeMattos et al., 2001, 2002c). The gel was run at 275 V overnight in a glacial acetic acid running buffer and then transferred to nitrocellulose. The membrane was incubated overnight at room temperature with m3D6 and then probed with sheep anti-mouse horseradish peroxidase (Amersham Biosciences) for 3 hr, followed by SuperSignal West Pico Chemiluminescence (Pierce). Image was captured using the Kodak ImageStation 440CF (Eastman Kodak, Rochester, NY). Densitometry and standard curves were generated using the Kodak 1D Image Analysis software.
Determination of A load. Tissue sections (40 µm) were cut using a freezing sliding microtome. A was identified using a rabbit polyclonal antibody against pan-A (BioSource International, Camarillo, CA). Percentage A load was determined using stereological techniques in the cingulate cortex and hippocampus of three sections separated by 300 µm (Holtzman et al., 2000; DeMattos et al., 2002c). Quantification was performed anterior to probe placement.
ISF A half-life determination--secretase inhibitor treatment. ISF A1-x half-life was determined in animals implanted with microdialysis probes as above. One hour baseline samples were taken from hours 5-8 after probe implantation. At the beginning of hour 9, animals were briefly anesthetized with isoflurane and injected subcutaneously with vehicle or 3 mg/kg LY411575 (Wu et al., 1998) diluted in corn oil. Ten additional 1 hr samples were taken for each animal. At the conclusion of the experiment, animals were perfused and probe placement was verified. Microdialysis samples were analyzed the same day by ELISA.
To determine A half-life, time points between drug delivery and when A concentrations plateaued were analyzed. For first-order processes, the elimination rate of a molecule from the body is directly related to the slope of the semi-log plot of concentration versus time: a = -Ke/2.3, where a represents the slope, and Ke represents the elimination rate constant. The half-life (t1/2) of a molecule is the time necessary for the concentration to decrease by 50%. By definition of a first-order process, the half-life remains constant regardless of molecule concentration. The half-life can be expressed in terms of the elimination rate constant: t1/2 = 0.693/Ke.
APP Western. Tissue from 3-month-old PDAPP mice was harvested 8 hr after a 3 mg/kg subcutaneous injection of LY41175 or vehicle (corn oil). Animals were perfused with chilled PBS-heparin, and brains were dissected to isolate the hippocampus. Hippocampus was homogenized in radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris, 0.5% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 2.5 mM EDTA, and protease inhibitors, pH 8.0), and 40 µg of protein was loaded per lane of a reducing 10-20% Tris-tricine-SDS mini-gel. Proteins were separated at 80 mV and transferred to nitrocellulose. To detect APP C-terminal fragments (CTFs), the blot was probed with CT15 (a kind gift from Dr. Ed Koo, University of California, San Diego, La Jolla, CA), a rabbit polyclonal antibody against the terminal 15 amino acids of APP (Sisodia et al., 1993), followed by goat anti-rabbit HRP (Bio-Rad, Hercules, CA). To detect full-length APP, 6E10 (Signet Labs, Dedham, MA) was used, followed by sheep anti-mouse peroxidase (Amersham Biosciences). Signal was detected with SuperSignal West Pico Chemiluminescence (Pierce).
LY411575 brain exposure. Three- and 12-month-old PDAPP mice were injected subcutaneously with 3 mg/kg LY411575 in corn oil. Fifteen minutes after treatment, animals received a cardiac bleed and were immediately perfused with chilled PBS-heparin. Brains were removed and frozen at -80°C. Tissue was homogenized in acetonitrile (5:1 vol/gm wet weight), followed by C8 Empore solid phase extraction (3M, St. Paul, MN), and then analyzed for LY411575 content by liquid chromatography-tandem mass spectrometry similar to that described previously (Dovey et al., 2001).
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Results
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Characterization of A in human CSF using in vitro microdialysis
Microdialysis has been useful in dynamically assessing the concentration of a variety of molecules in the brain ISF and has provided insight into their metabolism (Benveniste, 1989; Benveniste and Huttemeier, 1990). Because the concentration of brain ISF A is likely to be an important, dynamically regulated pool of A that determines whether A aggregates, we began studies to determine whether ISF A could be measured. To address this issue, we first began A microdialysis studies in vitro. There are likely different forms of A present in any solution, some percentage of which will be of appropriate size to cross the microdialysis membrane (Fig. 1A). We refer to A that is measurable by microdialysis as exchangeable A (eA). eA is a component of the total soluble A within a sample that can diffuse across a 35 kDa membrane on the probe. A bound to extracellular matrix, cell membrane, and large proteins, as well as some multimeric forms of A, would be excluded.
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Figure 1. In vitro microdialysis to measure A. A, Diagram of exchangeable A. Triangles represent potential A binding molecules, e.g., apoE, clusterin, and  2M. Only highlighted A molecules are of the appropriate size to pass through a 35 kDA MWCO membrane on the microdialysis probe. B, Interpolated zero flow method to quantify the pool of measurable A1-x and A40 within samples of human CSF (n = 4). At 2.2 µl/min, the percentage recovery of eA was 9.74 ± 1.53% (mean ± SEM). C, In vitro percentage recoveries for each A species using the interpolated zero flow method. Each recovery point contains error bars and are overlapping for each species (n = 4). In vitro recovery of each A species by microdialysis is the same. D, The concentration of eA and total soluble A are highly correlated within a sample of human CSF (Pearson's r = 0.9487; p < 0.0001; n = 9). The mean concentrations of soluble A1-x and eA1-x were 30.47 ± 4.23 ng/ml (mean ± SEM) and 196.2 ± 28.65pg/ml, respectively. E, F, Human CSF immunoprecipitated (IP'd) for A has undetectable levels of total soluble A and eA. Human CSF spiked with an amount of exogenous A40 peptide expected to double A concentration resulted in a 2.1-fold increase in total soluble A and a 1.9-fold increase in eA.
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During microdialysis, the perfusion buffer in the probe is flowing at a defined rate. Thus, molecules in the external solution do not have sufficient time to reach equilibrium with the internal dialysate buffer. The result is that the concentration of A, and other molecules, in the collected microdialysis sample are a fraction of the measurable A in solution. If experimental conditions remain the same, then the percentage of the target molecule recovered within the dialysate buffer will remain constant between samples. Once determined, the percentage recovery coefficient can then be applied to all samples collected under identical conditions.
To determine the in vitro percentage recovery of A within samples of human CSF, we used two techniques: the interpolated zero flow and interpolated zero flux methods (Jacobson et al., 1985; Menacherry et al., 1992). The zero flow method varies the flow rate of perfusion buffer. By varying the flow rates, a recovery curve can be plotted with extrapolation back to a theoretical zero flow rate that represents perfect recovery from the target solution. The mean in vitro percentage recovery of eA at 2.2 µl/min using the zero flow method was 10% at room temperature (Fig. 1 B). The in vitro recoveries for eA1-x, eA40, and eA42 were the same at all flow rates (Fig. 1C). The zero flux method varies the concentration of A within the microdialysis probe to assess at which concentrations A diffuses into or out of the membrane. Using this method, we obtained similar percentage recoveries as the zero flow method (data not shown).
To characterize the exchangeable pool of A in CSF, we determined the concentration of eA within individual samples of human CSF using the interpolated zero flow method and compared that with the concentration of total soluble A present in the samples as measured directly by ELISA (Fig. 1D). In a series of human CSF samples, the mean concentration of soluble A1-x was higher than the concentration of eA. Although eA was substantially lower than the total amount of soluble A in the CSF, values of eA and total soluble A were highly correlated (Fig. 1D). This suggests that, although the absolute concentration of soluble A differs from eA, the concentrations of eA and soluble A are directly proportional within human CSF under steady-state conditions. The reason that eA differs from measured soluble A is likely attributable to A-binding molecules that hinder the diffusion of 4 kDa A across the 35 kDa dialysis membrane. Potential candidate A-binding molecules include apolipoprotein E (Yang et al., 1997), -2-macroglobulin (Du et al., 1997), and clusterin (Ghiso et al., 1993; Matsubara et al., 1995), among others.
To further assess whether the concentration of eA directly reflects the concentration of total soluble A within a physiological sample, the concentration of eA was measured by in vitro microdialysis in a normal sample of human CSF, as well as in identical samples in which (1) A was removed by immunoprecipitation or (2) CSF A concentration was increased by adding exogenous A40. This simulated an acute decrease or increase of A within that sample. Values for soluble A were assessed directly by ELISA in CSF (Fig. 1E), and eA in the same sample was assessed by microdialysis followed by ELISA (Fig. 1F). CSF in which A was immunoprecipitated had virtually no detectable total soluble A or eA. CSF spiked with exogenous A40 (with an amount of A expected to be double the normal concentration) had twofold higher levels of both soluble A and eA. Thus, the concentration of eA directly reflects the concentration of soluble A under steady-state conditions, as well as after a sudden change.
Species of A analyzed
Because our various ELISA assays use monoclonal antibodies that recognize particular A epitopes, we were able to measure specific A species (Fig. 2A). The A1-x ELISA is a measurement of all A from amino acid 1 to amino acid 28 or greater. The A40 and A42 ELISA recognizes full-length A from amino acid 1 to either 40 or 42. As a result, a subtraction of the concentration of A40 and A42 from A1-x yields any A species in which the N terminus begins at position 1 and has a C terminus that does not end in position 40 or 42. These A C-terminal variants could include A1-38, A1-39, and A1-43.
Characterization of in vivo microdialysis technique
After validation of eA measurement using in vitro microdialysis (Fig. 1), we initiated an in vivo assessment of ISF eA in PDAPP mice. Microdialysis probes were implanted stereotactically under anesthesia. Figure 2, C and D, depicts representative probe placements in the hippocampus and striatum. The animals were permitted to awaken from the anesthetic and remained awake for the duration of the experiment (Fig. 2D).
Although insertion of a microdialysis probe does cause some acute and/or chronic injury to the surrounding tissue, we took measures to minimize this effect on our experiments. During probe insertion, there is some acute tissue injury, and the blood-brain barrier (BBB) integrity is compromised. However, the BBB has been shown to be rapidly repaired, and it progressively excludes smaller molecules as tight junctions reform. Small molecules, such as sucrose, are impermeable to the BBB within 3-4 after microdialysis probe implantation (Dykstra et al., 1992), whereas it only takes 30 min for larger molecules, such as Evan's blue (molecular weight of 960) (Morgan et al., 1996), to be impermeable. Chronic changes can include astrogliosis and inflammatory processes. These changes usually begin 24-36 hr after probe insertion and may effect long-term implantations by changing the local environment that the microdialysis probe membrane contacts. We limited our experimental window to hours 4-18 after probe insertion so there is time after the initial injury for the BBB to exclude large molecules; however, the experiment concludes before substantial chronic processes have occurred.
As assessed by cresyl violet staining (Fig. 2E), tissue surrounding a microdialysis probe implanted for 18 hr shows some tissue displacement, but, in the area immediately adjacent to the probe, neurons are intact and there is no marked glial activation. This was confirmed by the absence of an increase in GFAP staining (data not shown). We also found no evidence of activated caspase-3 immunostaining in any of the tissue analyzed, nor was there evidence of local A accumulation or removal as a result of probe insertion (data not shown). In PDAPP animals implanted with microdialysis probes for 18 hr (longest time point) that were given no other manipulations, the ISF eA concentration after 18 hr was not significantly different from the baseline concentration (mean concentration for hours 4-8), suggesting that these limited environmental changes did not adversely effect the ISF eA concentration in these experiments.
In vivo measurement of interstitial fluid A in PDAPP mice
The in vivo percentage recovery of eA for both 3- and 12- to 15-month-old PDAPP mice were the same (Fig. 2F), as was the recovery of eA1-x, eA40, and eA42 (data not shown). The recovery coefficients at each flow rate were applied to the A concentration obtained in dialysate samples to calculate the actual in vivo concentration of eA. The percentage recovery between 3- and 12- to 15-month-old mice did not differ significantly (p = 0.769; repeated-measures ANOVA with Huynh and Feldt adjustment).
To assess the baseline concentration of regional eA, microdialysis probes were implanted into the hippocampus and striatum of 3- and 12- to 15-month-old animals, 2 hr fractions of dialysis buffer were collected over a period of 8-12 hr, and A1-x was measured by ELISA (Fig. 3A). The value of ISF eA for a given animal was determined by taking the mean concentration of the sample fractions from hours 5-8 to control for slight variations between time points. Measurements of ISF eA for up to 18 hr after probe implantation were stable. Three-month-old PDAPP animals, which have not developed A deposits as plaques, had an eA1-x concentration in the hippocampus and striatum of 730 and 210 pg/ml, respectively (for actual A values, see Table 1). The hippocampus and striatum of 12- to 15-month-old mice with A deposition had eA1-x concentrations of 630 and 140 pg/ml, respectively. Regional eA1-x did not differ significantly between age groups (p = 0.6244); however, there was a trend for older mice (which have A deposits) to have a lower concentration of eA1-x in the striatum (p = 0.0839). Although the mean concentration of hippocampal eA1-x was lower in older mice, this difference was not significant (p = 0.4457). The ratio of hippocampal to striatal eA1-x in both 3-month-old and 12- to 15-month-old animals was 3.5:1, which is similar to the ratios of A1-x in brain lysates of these regions (ratio of 5.3:1; data not shown). The concentration of ISF eA1-x within the hippocampus and striatum of both age groups correlated significantly (Fig. 3C).
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Figure 3. In vivo concentration of eA in young and middle-aged PDAPP mice. A, ISF eA concentrations in hippocampus and striatum of 3- and 12- to 15-month-old PDAPP mice. Three-month-old mice have 727.7 ± 92.1 pg/ml (n = 21; mean ± SEM) and 207.9 ± 31.8 pg/ml (n = 6) of eA1-x in the hippocampus and striatum, respectively. Twelve- to 15-month-old mice have 626.4 ± 93.3 pg/ml (n = 21) and 142.1 ± 19.5 pg/ml (n = 13) of eA in those regions. B, The concentration of eA species in young and aged mice. The concentration of eA1-x does not change significantly with age. eA40 increases significantly in the hippocampus with age (p = 0.0429), whereas eA42 does not decrease (p = 0.6116). C, The concentration of eA1-x within the hippocampus and striatum are highly correlated (Pearson's r = 0.7202; p < 0.001; n = 19). D, A microdialysis sample was analyzed by acid- urea electrophoresis, followed by Western blot analysis to visualize the various A species present. On the basis of the peptide standard curves, A42,A40, and A38 represent 15.6, 31.6, and 52.8%, respectively, of the ISF A detected. No other A species were detected. This confirms that A-CTV is a major pool of A in the ISF and that A38 is the predominant species. E, The concentration of endogenous murine ISF eA40 and eA42 was measured in 3-month-old (n = 6) and 12-month-old (n = 6) C57BL/6 mice. There was no change with age in the concentration of murine eA40 (p = 0.718) or eA42 (p = 0.609) in wild-type mice.
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In a subset of 3- and 12- to 15-month-old mice, the hippocampal mean eA40 and eA42 concentrations were also determined using a microdialysis protocol with a slower flow rate designed to enrich the samples for A to facilitate measurement. The concentration of hippocampal eA40 in 3-month-old and 12- to 15-month-old PDAPP was 220 pg/ml (n = 6) and 350 pg/ml (n = 4) respectively, whereas eA42 was 95 pg/ml (n = 6) and 75 pg/ml (n = 4) respectively. Although hippocampal eA1-x values did not differ between age groups, the concentration of eA40 increased significantly in 12- to 15-month-old mice (p = 0.0429), whereas eA42 levels did not change with age (p = 0.6116). Together, this suggests that A C-terminal variants (A1-38,A1-39, and A1-43) were likely decreasing in older mice. To confirm that A C-terminal variants are present in ISF and to determine the major species within that pool, we performed acid-urea gel analysis, followed by Western blotting of eA from a microdialysis sample. This revealed that A38 is the major A C-terminal variant present in ISF in 3-month-old (Fig. 3D) and 12- to 15-month-old mice (data not shown). A38 has been shown previously to be a major A species in brain and CSF, as well (Weggen et al., 2001; Wiltfang et al., 2002; Pype et al., 2003).
ISF murine A40 and A42 was also measured by microdialysis in 3- and 12-month-old wild-type mice (C57BL/6). The concentrations of murine eA40 and eA42 were not altered between the groups (Fig. 3E). That murine eA does not change with age and previous findings that human A production does not change with age in PDAPP mice (Johnson-Wood et al., 1997) suggest that the changes in A species between young and middle-aged PDAPP mice are not age related but instead are related to A deposition.
Relationship between ISF eA and tissue A
A40 and A42 levels in CSF and tissue were also assessed in 3-month-old and 12- to 15-month-old animals (Table 1). Similar to previously published results (DeMattos et al., 2002b), CSF A42 levels decreased significantly with age (p = 0.0266), whereas in hippocampal brain lysates, carbonate and guanidine extractable A42 levels markedly increased (4.5-fold and 78-fold; p = 0.0043 and p = 0.0057, respectively). The carbonate buffer used is an alkaline, nondenaturing buffer, whereas guanidine is a chaotropic, denaturing buffer that solubilizes most, if not all, of the A aggregates. The increase in soluble A in tissue lysates (e.g., carbonate) subsequent to plaque formation has been interpreted by some as an increase in extracellular, soluble A in the brain that may contribute to its toxicity. However, in contrast to the increased concentration of soluble A42 in tissue lysates with age, the concentration of A42 in ISF does not significantly change with age or the presence of A deposits. This suggests that much of the soluble A in tissue lysates may not be truly "soluble" and diffusible in the extracellular space. Much of this tissue-soluble pool is likely intracellular, associated with membranes, or loosely associated to A aggregates. Thus, we conclude that soluble A in the brain lysate, in the presence of plaques, does not reflect soluble ISF eA.
To further compare A metabolism between different brain compartments, we compared the ratios of A40/A42 in ISF, CSF, and brain lysates. In both ISF and CSF, the A40/A42 ratio significantly increased in 12- to 15-month-old animals (Fig. 4). Interestingly, this increased ratio in ISF is attributable to an increase in A40, whereas in CSF, it is attributable to a decrease in A42. In contrast, in 12- to 15-month-old PDAPP mice, carbonate and guanidine extracted tissue lysates had a markedly decreased A40/A42 ratio, primarily attributable to a tremendous increase in aggregated A42. That A40 and A42 are changing in different patterns in ISF, CSF, and tissue underscores the difference in metabolism among these three compartments.
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Figure 4. Differences in A40/A42 ratios between ISF, CSF, and brain tissue with age. In both the ISF and CSF fluids, the A40/A42 ratio increases significantly with age (p = 0.0466, n = 10; and p = 0.0413, n = 9, respectively), primarily attributable to an increase in A40 within ISF and a decrease in A42 in CSF. In contrast, carbonate (Carb) and guanidine (Guan) of hippocampal tissue reveals a significant decrease in the A40/A42 ratio with age (p < 0.0001 and p < 0.001, respectively; n = 10). This change coincides with a tremendous deposition of A42 in older mice. Inset, Enlargement of hippocampal tissue lysate data.
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Relationships between ISF A, CSF A, and plaque load
To determine how various pools of CNS A are related, a variety of samples were collected from a cohort of mice. A values were determined in hippocampal ISF, CSF, and soluble and insoluble hippocampal tissue lysates. For each of these samples, A1-x, A40, and A42 were analyzed. The amount of A deposited in plaques in the hippocampus (percentage A load) was also quantified. These results enabled us to analyze correlations between the various compartments of A in individual mice (Fig. 5). In 3-month-old PDAPP mice, there was no correlation between ISF and CSF A levels (Fig. 5A), suggesting that, in the absence of insoluble A in plaques, A levels in these pools are independent. In contrast to these findings, there was a significant correlation between ISF and CSF A levels in 12- to 15-month-old mice (Fig. 5B). All of the mice at this age have biochemical or pathological (plaques) evidence of A aggregation, suggesting that the presence of insoluble A is either associated with, or directly causes, a change in ISF or CSF A metabolism, resulting in a new equilibrium between ISF eA, CSF A, and deposited insoluble A. In 12- to 15-month-old PDAPP mice, there was a significant correlation between the concentration of CSF A1-x and A plaque load (Fig. 5C) as noted previously (DeMattos et al., 2002b). There was also a trend for the hippocampal ISF A and A load to correlate (Fig. 5D). This suggests that the pool of aggregated, insoluble A directly influences two distinct pools of soluble, extracellular A.
Half-life of A in the brain interstitial fluid
It has been speculated for some time that the accumulation of both soluble and insoluble A in a -sheet conformation (e.g., oligomers and fibrils) in the brain may alter the metabolism of A in the other soluble pools within the CNS. One way to directly assess this issue is to determine the in vivo half-life of different pools of A in mice with or without A aggregates. Because in vivo microdialysis allows for a dynamic assessment of the interstitial fluid space, we estimated the in vivo t1/2 of ISF eA1-x after rapid inhibition of A production via a potent -secretase inhibitor.
Microdialysis probes were implanted into the hippocampus of 3-month-old or 12-month-old PDAPP mice. For each animal, the baseline concentration of ISF eA was determined by taking eight 1 hr microdialysis samples. At the end of hour 8, a potent, brain-penetrant -secretase inhibitor, LY411575 (Wu et al., 1998), was injected subcutaneously. Ten additional 1 hr samples were taken after treatment with either the drug or vehicle (corn oil). Within the first hour of treatment, both age groups had decreased concentrations of eA1-x. In 3- and 12-month-old animals, eA levels reached 20% of baseline by 6 hr after treatment (Fig. 6A). There was no significant difference in the new baseline A concentrations after treatment between the two age groups. Interestingly, the t1/2 of ISF A after LY411575 treatment was markedly different between young and middle-aged mice (p = 0.0045). Analysis of the t1/2 for eA in the hippocampal ISF was 1.97 ± 0.623 hr in young mice and 3.78 ± 0.866 hr in aged mice (Fig. 6B). A semi-log plot of baseline concentration versus time was linear at both ages (Fig. 6B), suggesting first-order kinetics for A metabolism within the brain interstitial fluid. ISF eA levels in vehicle-injected, 3- and 12-month-old mice remained relatively steady for the 18 hr of microdialysis and did not vary dramatically after injection at hour 9 (Fig. 6A).
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Figure 6. Half-life of ISF eA in young and middle-aged PDAPP mice. A, Effect of LY411575 on hippocampal eA1-x concentration in 3- and 12-month-old PDAPP mice. During microdialysis, animals received a 3 mg/kg subcutaneous injection of LY411575 or vehicle (corn oil). Concentrations of eA1-x dropped to 21.16 ± 11.46% (mean ± SEM; n = 4) and 22.34 ± 6.23% (n = 5) of baseline by 4 and 8 hr in young and middle-aged mice, respectively (p = 0.0045; repeated-measures ANOVA with Huynh and Feldt adjustment). Three-month-old (n = 5) and 12-month-old (n = 4) vehicle-injected mice do not show a significant change in A concentration after treatment. B, A semi-log plot of percentage baseline of eA versus time is linear (young, y = -0.229x + 2.11, r2 = 0.936; middle-aged, y = -0.099 + 2.10, r2 = 0.9173), suggesting first-order kinetics of A elimination in both age groups. The half-life of A within the ISF of young mice is 1.97 ± 0.623 hr (n = 4) and in aged mice is 3.78 ± 0.866 hr (n = 5). C, Concentration of LY411575 in whole brain of 3- and 12-month-old PDAPP mice 15 min after 3 mg/kg subcutaneous injection (n = 6). LY411575 enters the brain equally in both age groups with a concentration that was 220-fold higher than the IC50 needed to inhibit -secretase activity in cell culture. D, A Western blot probed for full-length and C-terminal fragments of APP. Tissue samples are from 3-month-old PDAPP mice injected with 3 mg/kg LY411575 or vehicle and killed at 8 hr after treatment (n = 8). Full-length APP levels do not change between these groups (top panel); however, both -CTF and -CTF increased in LY411575-treated mice (bottom panel), as expected with -secretase cleavage inhibited.
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LY411575 reaches peak concentrations in the brain 15 min after a 3 mg/kg subcutaneous injection. In 15 min, we found that brain levels of LY411575 were 200-fold in excess of the IC50 necessary in cell lines for -secretase inhibition (data not shown), and drug entry into the brain was equal in both 3- and 12-month-old PDAPP mice (Fig. 6C). As expected, a Western blot for APP C-terminal fragments in hippocampal tissue at the end of the experiment showed an increase in -CTF and -CTF, consistent with the -secretase cleavage being inhibited by LY411575 (Fig. 6D). The amount of full-length APP did not change in response to treatment. Together, these results not only demonstrate the remarkably short half-life of ISF eA, but that, concomitant with the formation of A aggregates, there is a marked change in ISF eA metabolism that becomes apparent once synthesis is blocked.
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Discussion
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Brain ISF eA is likely the pool of A that mediates its normal functions, if any, as well as being the major precursor to conformationally altered forms of A in the brain (e.g., plaques and oligomers). This study demonstrates that soluble, ISF A can be measured on a dynamic time scale, exclusive from the other CNS compartments. This provides a method to determine whether an acute manipulation to an individual animal alters an A pool likely to be critical in AD pathogenesis. We demonstrated that the overall concentration of ISF eA1-x does not change dramatically with age and A deposition in PDAPP mice (Fig. 3). There is, however, a substantial change in A metabolism associated with A deposition, as evidenced by altered relationships between A species (e.g., A40, A42, and A C-terminal variants) and between CNS compartments. We also demonstrate that the in vivo half-life of ISF eA is short in 3-month-old animals (2 hr). Importantly however, in 12- to 15-month-old PDAPP mice that have A deposits, the apparent t1/2 of ISF eA is doubled. This, coupled with previous in vitro studies (Maggio et al., 1992), strongly suggests that, in addition to a large amount of insoluble A that is primarily immobile, that there is a pool of extracellular A in dynamic equilibrium between soluble and insoluble forms of A. This pool can be visualized with use of a -secretase inhibitor and appears to be associated, but not locked onto, fibrils (Fig. 7, see model). It is this mobile pool that appears to be revealed by inhibition of A production. The ability to now assess the effects on ISF eA by endogenous molecules, as well as potential treatments such as A-degrading enzymes (Iwata et al., 2000; Farris et al., 2003), A-chelators (Cherny et al., 2001), and anti-A antibodies, may provide a useful approach to understanding pathogenesis, as well as evaluating A-related diagnostic and treatment strategies.
Altered A metabolism with age and A aggregation
The relative concentrations of hippocampal and striatal ISF eA mirror the levels of human APP expression and total tissue A levels in young PDAPP mice. With A conformational change, the concentration of ISF eA1-x does not change dramatically; however, eA40 increased significantly, whereas eA42 did not change. This was accompanied by an apparent decrease in ISF A C-terminal variants, which appear to consist predominantly of A38. Thus, age-related and, more likely, A deposition-related alterations in ISF A metabolism are A species specific. Interestingly, the concentration of ISF A42 did not change with A deposition. It has been postulated that the concentration of ISF A would decrease as a consequence of aggregation. A deposition, however, is a chronic process, and, at any given time, it is likely that the amount of newly synthesized A42 that becomes insoluble is a small percentage of the ongoing A42 produced and released into ISF. Thus, the small overall mass of depositing versus produced A42, at any given time, does not appear to be detectable in ISF under basal conditions.
ISF eA is a pool of A distinct from CSF and soluble A in tissue lysates. ISF eA reflects local A production, release, degradation, and clearance, whereas CSF A more likely reflects A metabolism from the entire CNS, not a specific region. CSF production and removal also occurs via different pathways than ISF production and removal (Cserr et al., 1992; Zhang et al., 1992). Tissue lysates include all forms of extracellular and intracellular A, including some A that may be "loosely" associated with insoluble A aggregates yet is soluble after tissue lysis, even in mild salt and alkaline buffers (Simmons et al., 1994). The clear demarcation between these compartments is evident by their differing ratios of A40/A42. Interestingly, with age and deposition, A40/A42 ratios change in distinct, yet different, patterns in each CNS compartment, in part because the individual A species in each compartment are changing in different ways. For example, in 12- to 15-month-old versus 3-month-old PDAPP mice, both CSF A40 and A42 decrease; however, ISF eA40 increases. These changes in individual A species suggest that there is a fundamental change in A metabolism in PDAPP mice likely attributable to the presence of insoluble A aggregates because changes in murine A do not occur in age-matched wild-type mice.
Although A levels within each CNS compartment are regulated in a particular manner, with A conformational change, a new relationship between the ISF and CSF is observed. A significant correlation of A levels emerges when comparing ISF and CSF in 12- to 15-month-old but not 3-month-old mice (Fig. 5). This suggests that, with time and deposition, a new equilibrium between ISF and CSF develops that does not exist in younger mice. ISF A1-x concentration did not differ between young and middle-aged mice, suggesting no net change in A elimination. However, it is conceivable that, in the presence of A deposits, A elimination from ISF involves not only local degradation, ISF clearance, and blood-brain barrier transport but also greater ISF to CSF transport than in younger mice.
Interstitial fluid A half-life
Dynamic sampling via microdialysis enabled us to follow ISF eA concentrations in 3- and 12- to 15-month-old PDAPP mice before and after treatment with a potent -secretase inhibitor that rapidly blocks CNS A production. The half-life for ISF eA in young mice is relatively short, 2 hr. This short t1/2 for ISF A in young mice suggests that A is rapidly and constantly produced, cleared, and replenished within the extracellular space. Intriguingly, in older mice with the appearance of A aggregates, ISF eA has a significantly longer, twofold increase in half-life or elimination rate. A production has been shown not to change in young versus older PDAPP mice (Johnson-Wood et al., 1997). Thus, inhibition of A synthesis revealed a major difference in A metabolism between young and middle-aged PDAPP mice that was not obvious under basal conditions. This extended half-life, however, does not correspond with an increased concentration of ISF A1-x. If the concentration of ISF A1-x were increased, this would suggest a fundamental change in A elimination via degradation, clearance, or both in young versus middle-aged mice. The absence of this increase in ISF eA1-x suggests another explanation for the prolonged t1/2 in old mice. The data show that, after inhibition of A production in mice with A deposits, the concentration of eA drops. As this occurs, it appears that the dynamic equilibrium between extracellular soluble and insoluble A can be measured and uncovered. In vitro studies have shown that A aggregates contain a readily dissociable pool of A, or "docked A," as well as a long-lasting or stable "locked" pool of A (Maggio et al., 1992; Esler et al., 2000). In vitro, as the concentration of A in solution decreases, this docked pool can quickly dissociate from fibrils. In vivo, when A production is inhibited and ISF A levels begin to decrease, it is likely that this associated, docked pool can return to solution over a finite period of time, as occurs in vitro, causing this pool of A to dissociate from fibrils and become soluble. This would result in a prolonged apparent half-life of ISF A in animals with A deposition.
Model of A-aggregation induced changes in A metabolism in vivo
We propose that, in the absence of A deposition in the brain, A is readily cleared from the interstitial fluid by several mechanisms (Fig. 7) (Cserr et al., 1992; Iwata et al., 2000; Shibata et al., 2000; Farris et al., 2003). We postulate that an alteration in A metabolism, probably related to the presence of A deposits, leads to changes in the equilibrium of different A species within brain compartments. This change in equilibrium results in a new correlation between some of these compartments. When A production is inhibited, we hypothesize that a new equilibrium is established between ISF A and a loosely associated pool of docked, deposited A that can reenter the soluble eA pool. This docked pool may represent conformationally altered forms of A, such as protofibrils (Hartley et al., 1999; Walsh et al., 1999) and oligomers (Kayed et al., 2003), that have been shown in vitro and after injection in vivo (Walsh et al., 2002) to be extremely toxic. If so, inhibition of A production with -secretase inhibitions may rapidly decrease this toxic pool. From a diagnostic standpoint, the presence of this docked pool may also be very important. It appears that A aggregation and plaques begin to build up in the human brain 10-20 years before the onset of clinical symptoms of AD (preclinical AD) (Morris and Price, 2001). Therefore, if visualization of this prolonged t1/2 in the presence of A aggregates can be assessed in humans (for example via assessment of plasma A) (DeMattos et al., 2002a), it may provide a novel, non-invasive method to detect the presence of A aggregation and deposition in vivo.
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Footnotes
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Received June 11, 2003;
revised August 4, 2003;
accepted August 4, 2003.
This work was supported by National Institutes of Health Grants AG13956, AG11355, and AG20222, MetLife Foundation, and Eli Lilly and Co. We thank the Washington University General Clinical Research Center (funded by United States Public Health System Grant MO1 RR00036).
Correspondence should be addressed to Dr. David M. Holtzman, Washington University School of Medicine, Department of Neurology, 660 South Euclid Avenue, Box 8111, St. Louis, MO 63110. E-mail: holtzman{at}neuro.wustl.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/238844-10$15.00/0
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Abstract
Introduction
