There is increasing evidence that soluble amyloid-β peptide (Aβ) uptake into neurons is an early event in the pathogenesis of Alzheimer's disease (AD). Identification of the early events leading to neuronal dysfunction is key to developing therapeutic strategies, but relative roles of receptors and factors modulating uptake are poorly understood. Studies have shown that transforming growth factor β (TGFβ), particularly TGFβ2, can influence the targeting of Aβ to cells in vitro. TGFβ2 can target Aβ to neurons in organotypic hippocampal slice cultures (OHSC). We examine a specific mechanism for TGFβ2-mediated targeting of Aβ to neurons. The receptor-associated protein (RAP), a low-density lipoprotein receptor-related protein (LRP) antagonist, can attenuate the cellular targeting of Aβ both in vitro and in vivo and prevent Aβ/TGFβ2-induced memory retention deficits. Using both in vitro and in vivo methods, we identify LRP as playing a role in TGFβ2-mediated Aβ uptake, neurodegeneration, and spatial memory impairment. © 2004 Wiley-Liss, Inc.

The low-density lipoprotein receptor-related protein (LRP) is a multifunctional receptor mediating internalization and degradation of lipoproteins and protease/protease inhibitor complexes (Strickland et al., 1995), α2-macroglobulin (A2M) (Borth, 1992), Apolipoprotein E (ApoE) (Herz et al., 1988), and the amyloid precursor protein (APP) (Kounnas et al., 1995). Interestingly, A2M, ApoE, and APP are all genetically associated with Alzheimer's disease (AD) (Strittmatter et al., 1993; Blacker et al., 1998) and found in senile plaques (Rebeck et al., 1995). Evidence for a role for LRP in AD comes from genetic studies (Kang et al., 1997) and confirmation by five other laboratories (Baum et al., 1998; Hollenbach et al., 1998; Kamboh et al., 1998; Lambert et al., 1998; Wavrant-DeVrieze et al., 1999). The impact of LRP polymorphisms is unclear, however, and levels of LRP protein do not always correlate with cognitive decline in AD (McIlroy et al., 2001; Sanchez-Guerra et al., 2001; Causevic et al., 2003). The mechanisms by which LRP and its ligands may contribute to AD pathogenesis are unknown but it is suggested to play a role in the balance between amyloid-β peptide (Aβ) synthesis and clearance (Arelin et al., 2002).

Generation and deposition of Aβ in the brain are believed to be crucial events in AD pathogenesis (Selkoe, 1994), whereas sequestration and clearance of Aβ limits amyloid deposition (Ladu et al., 1994; Rebeck et al., 1995; Kang et al., 1997). For example, A2M can complex with Aβ, leading to Aβ uptake and degradation through the LRP-mediated pathway (Du et al., 1997; Narita et al., 1997). Because LRP is highly expressed in the central nervous system (CNS) (Wolf et al., 1992), internalization of ApoE-enriched lipoprotein particles by way of the LRP may impact neuronal membrane remodeling (Holtzman et al., 1995). Interactions between ApoE and Aβ are also thought to be critical in AD pathogenesis and Aβ deposition (Rebeck et al., 1993; Ladu et al., 1994). This interaction seems to involve LRP, because ApoE-enhanced soluble Aβ uptake into synaptosomes is inhibited by the LRP antagonist receptor-associated protein (RAP) (Gylys et al., 2003).

Recent studies have revealed a potential link between LRP and TGFβs (Hussaini et al., 1996; Schneider and Nimpf, 2003), and LRP-1 has been shown recently to be identical to TGFβ receptor V (Huang et al., 2003). TGFβ expression is altered in AD (Chao et al., 1994; Flanders et al., 1995; Vawter et al., 1996; Lesne et al., 2003) and TGFβ affects Aβ accumulation (Frautschy et al., 1996; Wyss-Coray et al., 1997) and Aβ clearance by activated microglia (Wyss-Coray et al., 2001). TGFβ isoforms 1, 2, and 3 are expressed within neurons, astrocytes, and microglia (Constam et al., 1992; Krieglstein et al., 1995); TGFβ isoforms and receptors have a different regional distribution in the brain (Pelton et al., 1991) and they are considered to be independent regulatory molecules (Sanford et al., 1997). TGFβs are expressed in a number of CNS insults, including traumatic injury (Klempt et al., 1992; Lindholm et al., 1992; Logan et al., 1992), hypoxic injury (del Rio-Hortega, 1932,) and neurodegenerative diseases such as Parkinson's disease (Vawter et al., 1996) and AD (Chao et al., 1994; Flanders et al., 1995; Lippa et al., 1995; Peress and Perillo, 1995; Vawter et al., 1996). Recent studies show that the presence of ApoE and TGFβ1 can trigger vascular β-amyloidosis by increasing intracellular formation of stable deposits of Aβ/ApoE (Mazur-Kolecka et al., 2003).

Although much of the focus in TGFβ research has been on isoform 1 (Van der Wal et al., 1993; Frautschy et al., 1996), recent studies on isoform 2 have led to some intriguing findings that define specific roles for TGFβ2. The TGFβ2 isoform is likely most relevant to AD pathogenesis because both protein and mRNA are elevated. TGFβ2 is localized in neurofibrillary tangle (NFT)-bearing neurons, astrocytes (Flanders et al., 1995), and plaque neurites (Peress and Perillo, 1995). Interestingly, TGFβ2 is a binding protein for soluble APP derivatives (Bodmer et al., 1990) and both TGFβ1 and 2 can bind Aβ and accelerate oligomerization (Mousseau et al., 2003). This is consistent with the many forms of amyloid-producing diseases, including AD and prion neurodegenerative diseases, that exhibit early neuronal loading of TGFβ2 (Tashiro et al., 1998). Previous results from our lab demonstrate important functional differences among the TGFβ isoforms in their ability to alter cellular distribution and degradation of Aβ (Harris-White et al., 1998). TGFβ exacerbation of Aβ neurotoxicity has been previously reported to occur in the absence of TGFβ receptors (Mousseau et al., 2003). As the role of TGFβ2 and its receptors in Aβ-dependent toxicity in vitro and in vivo remain unclear, the current study examines a mechanism for altered uptake of Aβ by TGFβ2 into neurons through the LRP pathway, and effects on neurodegeneration and cognition in vivo.

MATERIALS AND METHODS

Animals

Surgical and animal procedures were carried out with strict adherence to the current guidelines set out in the NIH Guide for the Care and Use of Laboratory Animals. Accordingly, all experiments involving animals were approved by the appropriate UCLA and VA institutional committees. ICR mice (Jackson ImmunoResearch) were used for organotypic hippocampal slice culture (OHSC) studies and C57/bl6 mice (Jackson ImmunoResearch) were used for in vivo studies.

Organotypic Hippocampal Slice Culture

Hippocampal slice cultures were prepared according to the method of Stoppini et al. (1991) with some modification. Briefly, slice cultures were prepared from 6–7-day-old ICR mouse pups. Slices were cut at 400 μm on a Stoelting tissue chopper and transferred to Costar membrane inserts (0.4 μm). Initially, slice culture media consisted of minimal essential medium (MEM) + HEPES (50%; Gibco, Grand Island, NY), heat-inactivated horse serum (25%; Sigma, St. Louis, MO), and Hank's balanced salt solution (25%; Sigma) containing a total of 6.5 mg/ml glucose and penicillin-streptomycin (50 U/ml-0.05 mg/ml). After the first 4 days in culture, slice media was replaced gradually with a serum-free media prepared by replacing the horse serum with the supplement TCM (final concentration 2%; ICN Pharmaceuticals, Costa Mesa, CA). The exchange of serum-containing media with serum-free media was as follows: (1) 75% serum media/25% serum-free media on Day 4 in vitro; (2) 50% serum media/50% serum-free media on Day 6 in vitro; and (3) 100% serum-free media on Day 7. Experimental treatment of OHSC began on Day 7. Final concentrations of reagents were: 10 μg/ml Aβ 40 (2.2 nM); 1 μg/ml Aβ 42 (220 pM); 10 ng/ml TGFβ2 (400 pM); and 250 nM RAP. Treatment remained on OHSCs for 4 days and media was changed as normal (no additional treatment added) for 7 additional days. Slices were either fixed or frozen 11 days from the start of treatment.

Aβ Preparation

Aβ oligomers were prepared by monomerizing lyophilized Aβ with hexafluoroisopropanal (HFIP), followed by evaporation and reconstitution in 4 mM HEPES buffer. After 24-hr incubation at 4°C to enhance aggregation of monomer, Aβ42 oligomers were diluted to final pump concentration (for ICV infusion) with 4 mM HEPES buffer, pH 8.0 (5 μg/100 μL) or diluted in culture media (in vitro studies). Aβ was obtained from C. Glabe (University of California, Irvine) and TGFβ2 from R & D Systems (Minneapolis, MN).

Surgery

Aβ pump solution (100 μL) was infused into the right ventricle through a stainless steel cannula (coordinates to Bregma in mm were: lateral: +0.1 posterior: −0.05, and ventral: −0.25; ventral coordinate from dura mater: −0.2) of female C57/bl6 mice using mini-osmotic Alzet pumps (1002; Durect Corporation, Cupertino, CA) over a 2-week period. Stainless steel cannulas were cut to a custom length (2.7 mm; Plastics One, Roanoke, VA). Per hour delivery was 12.5 ng Aβ42 (5 μg total), 0.058 ng RAP (0.0195 μg total), and 0.3 ng TGFβ2 (10 ng total) in 4 mM HEPES, pH 8.0. The pump released 0.25 μL/hr into a ventricular volume of 35 μL (140-fold dilution), and 18 μl of new cerebrospinal fluid (CSF) was produced hourly, resulting in projected Aβ concentrations of 80 nM in CSF during infusion. After ventricular penetration into neuropil and normal catabolism, soluble Aβ levels of about 5 μg/g protein were maintained in circumventricular brain areas. We were attempting to mimic soluble brain parenchyma Aβ levels known to correlate well with cognitive decline and neurodegeneration in both AD brain and animal models. This produces low levels of soluble Aβ that enable assessment of factors that enhance or modulate toxicity without amyloid plaques.

Immunocytochemistry and Image Analysis

At the conclusion of the OHSC experiments, slices were submersion-fixed in 4% paraformaldehyde for 1 hr followed by three rinses in Tris-buffered saline (TBS). Slices were then cryopreserved in increasing concentrations of sucrose (10, 15, and 20%), sectioned at 10 μm on a crysostat (Microm Instruments Model HM505E) and mounted onto gelatin-coated slides. At the conclusion of the in vivo studies, animals were perfused with cold HEPES buffer containing protease inhibitors. Half of the brain was dissected and snap frozen in liquid nitrogen for biochemistry. The other half brain was immersion fixed in 4% paraformaldehyde and paraffin embedded; 10-μm sections were cut on a microtome (Microm Instruments Model HM335E), and then mounted onto poly-L-lysine/gelatin-coated glass slides.

For Aβ immunohistochemistry of OHSC (10G4 antibody against Aβ 5-13; Yang et al., 1994), slices were pretreated with 70% trichloroacetic acid (6 min) and rinsed with TBS. Endogenous peroxidase activity was suppressed using a peroxidase suppressor buffer (Vector Labs) for 30 min at room temperature and nonspecific binding sites were blocked with Superblock Blocking Buffer (Pierce, Rockford, IL) for 1 hr at room temperature. The 10G4 antibody was applied (overnight at 4°C) to slices at a 1:500 dilution in TBS containing 0.1% Tween 20, 3% bovine serum albumin (BSA), and 8 mM sodium azide. Slices were then rinsed and incubated with a biotinylated anti-mouse secondary antibody (1:500 in TBS plus Tween 20; 1 hr at room temperature). Slices were rinsed and incubated with ABC solution (Elite ABC kit; Vector Labs) for 45 min at room temperature. Diaminobenzidine-metal enhanced chromogen (Pierce) was used to reveal 10G4 antibody binding, and the specificity of Aβ staining was verified by preincubating 10G4 antibody in the presence of Aβ1–40 peptide before immunostaining. Preabsorption of 10G4 antibody was carried out by adding 1 μL of antibody to 50 μL of 3% BSA in TBS containing 30 μg Aβ40 peptide. The solution was incubated overnight at 4°C, brought up to 800 μL with 3% BSA in TBS, and centrifuged at 14,000 × g for 30 min at 4°C. The resulting supernatant was used for immunocytochemistry. To identify Aβ in mouse brain sections, sections were incubated overnight at 4°C (1:100) with DAE polyclonal antibody (anti-Aβ1–13) made against synthetic peptides Aβ1–13 and named after the first three amino acids of the Aβ peptide, Asp-Ala-Glu (Lim et al., 2000), or with anti-Aβ42 antibody (1:50; BioSource International., Camarillo, CA). For fluoro-Aβ studies, the fluorescein signal was amplified with tyramide signal amplification (TSA) Direct system (NEN, Boston, MA).

All histologic and immunohistochemical images were acquired with a Nikon E400 Eclipse microscope equipped with a SPOT digital camera. Images were analyzed on a PC computer using Image Pro software. Throughout the image analysis process, all sections for all treatments were carried out with identical microscope light and condenser settings. Each capture was exposed to the same computer subroutine to minimize biases of the microscopist. Most importantly, the density slice thresholding was maintained constant throughout analysis. Data was exported from Image Pro to Microsoft Excel for further analysis.

Fluoro-Jade

To quantitate degenerating cells, we stained brain sections with the degeneration stain Fluoro-Jade B (Histo-Chem, Jefferson, AR) (Schmued et al., 1997; Schmued and Hopkins, 2000).

ApoE Enzyme-Linked Immunosorbent Assay

OHSC were prepared from transgenic mice expressing human ApoE alleles E3 and E4 (Mahley et al., 1995) and treated with 10 ng/ml TGFβ2 (R & D) for 2 days. Human ApoE was measured from collected media by sandwich enzyme-linked immunosorbent assay (ELISA) using 2E1 monoclonal antibody to ApoE (Boehringer-Mannheim, , Indianapolis, IN) to capture and goat anti-human ApoE antibody to detect with alkaline phosphatase as reporter, as described previously (Gracia et al., 1994).

Western Blot

Frozen brain tissue was weighed and homogenized in 15× volume of TBS with protease inhibitors (0.05 mM fenvalerate, 0.05 mM cantharidin, 1 mM sodium vanadate, 1 mM sodium pyrophosphate, 0.02 mM phosphoramidone, 1 mM EGTA, 0.1 mM EDTA, 0.02 mM leupeptin, 0.0015 mM aprotinin, and 0.5 mM AEBSF) and phosphatase inhibitors (50 mM sodium fluoride and 0.25 mM phenylmethylsulfonylfluoride [PMSF]). Samples were sonicated on ice 3× 10 sec, transferred to centrifuge tubes, and centrifuged at 100,000 × g for 30 min at 4°C. Supernatant fractions were loaded onto a 10% Tris-glycine gel to evaluate NR2B levels (NMDAR2B; Chemicon, Temecula, CA) and the 25-kDa synaptosomal-associated protein (SNAP25; Sternberger Monoclonals Inc., Lutherville, MD). Protein content was determined to equalize protein concentrations across samples. Bands were visualized using ECL techniques (Amersham, Piscataway, NJ) with subsaturating exposures and quantified using a BioRad scanning densitometer.

Morris Water Maze

Mice were subjected to the Morris water maze in a paradigm similar to that described for Aβ-infused rats (Winkler et al., 1994; Frautschy et al., 2001) with the following modifications for mouse. A 1.525-m diameter white tank with nontoxic white paint was used for contrast with black mice, with a platform diameter of 12.5 cm and water temperature was maintained at 25°C. Distal stationary cues were placed around the walls of the room, and no proximal, mobile, or auditory cues were present during trials. Before the first trial of each day, mice were placed on the platform for 60 sec for spatial orientation. Mice were then removed from the platform, and their heads covered with a drape until they were placed in a random start position facing the tank wall. Initially, mice were trained for 1.5 days in a visible platform test (four trials/block, one block in the morning and another block in the afternoon; three blocks total) for elimination of animals with motor or visual deficits. Acquisition of spatial memory was evaluated subsequently in six blocks of a hidden platform test over three days (four trials/block, 60 sec maximum, two blocks/day). After a 3-day delay after the last acquisition testing, retention was evaluated in a probe test with platform removed from the tank and swim paths evaluated for 20 sec. Normal mice swim to and persistently cross the correct former location of the platform to an extent assumed to be proportional to the strength of their memory, whereas animals with hippocampal dysfunction show a weaker tendency to swim in the correct quadrant with no spatial bias to the quadrant of the pool to which they had been trained (i.e., NE or SW). We allowed a 3-day delay in probe trial, because preliminary studies showed Aβ had little impact on retention unless probe trials were delayed (unpublished observations). Percent time or path in quadrants, path lengths, and acquisition and swim speeds were determined by HVS Image software and video tracking system (HVS Image Ltd., Buckingham, UK).

Statistical Analysis

Stat View statistical analysis software was used to carry out analysis of variance (ANOVA), post hoc analyses, and t-testing.

RESULTS

In Vitro: OHSC

Using fluorescein isothiocyanate (FITC)-labeled Aβ42 to treat OHSC, we confirmed our previous observation that TGFβ2 targets exogenous Aβ to cells in the CA1 subfield of the OHSC. Compared to treatment with FITC-Aβ42 alone, which resulted in relatively more diffuse fluorescence throughout the OHSC, the presence of TGFβ2 drove a rapid (24-hr) uptake of exogenous FITC-Aβ42 into CA1 neurons (data not shown).

Extending these findings, we explored a mechanism for TGFβ2-mediated targeting of Aβ to neurons via induction of ApoE and uptake by the LRP receptor. After 4 days of exposure, TGFβ2 nearly doubled ApoE release from OHSC (100 ± 8.1% for control vs. 182.2 ± 24.1% for TGFβ2-treated OHSC; P = 0.03, t-test). Using OHSC, we show that the LRP receptor antagonist RAP (250 nM) (Bu, 1998) can effectively block TGFβ2-mediated targeting of Aβ to CA1 hippocampal neurons. Figure 1 shows a Western blot of the Aβ42 preparation 2 weeks and 4 weeks after it was prepared. The blot shows predominately monomer and dimer in the 4–8 kDa range at 2 weeks. Figure 2 shows representative micrographs for immunolocalization of Aβ in Aβ + TGFβ2-treated slices and attenuation of staining by RAP. To quantitate this effect, we counted Aβ-immunoreactive cells in the CA1 pyramidal layer (800 × 100 μm area) and show that RAP does significantly attenuate TGFβ2-mediated targeting of Aβ to neurons (Fig. 2B; P = 0.004, t-test). Furthermore, RAP can block Aβ + TGFβ2-induced neurodegeneration in the slice cultures (Fig. 3), as indexed by Fluoro-Jade-positive cell counts in the CA1 region of the OHSC (800 × 100 μm area; treatment ANOVA P = 0.0058, for homogeneity of variance, data was square root transformed). No Fluoro-Jade-positive cells were found in the group treated only with TGFβ2 (not shown on graph). Fluoro-Jade is a dead neuron-specific probe that correlates well with propidium iodide (Schmued et al., 1997) and can be used to either count dead neurons or estimate total death per area. We also quantitated SNAP25 by Western blot analysis. There was a trend for protection of SNAP25 levels by RAP but the results did not reach significance (data not shown).

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Western blot analysis of Aβ42 preparation. Aβ was monomerized then incubated for 24 hr at 4°C in 4 mM HEPES (pH 8). To evaluate stability of soluble Aβ in pump, we incubated samples at 37°C for 2 weeks (infusion period) and 4 weeks and ran samples on a tricine gel for Western blot analysis After 2-week incubation (the infusion period), all Aβ immunoreactivity (ir) was in the 4–8 kDa range with the low salt, high pH buffer preventing Aβ42 aggregates. After the infusion period, however, soluble Aβ preparation would not have remained stable, because longer incubations (4 weeks) at 37°C resulted in some aggregates (arrow) and higher molecular weight oligomer formation (arrowhead).

In Vivo: ICV Infusion Model

ICV infusion of Aβ with TGFβ2 resulted in widespread parenchymal plaque-like deposition and neuronal labeling of Aβ. Figure 4A–F shows Aβ immunostaining (left, anti-DAE) and cresyl violet (right) staining of the temporal cortex from representative brain sections from these mice. Figure 4A/B and 4C/D are representative micrographs from two mice infused with Aβ + TGFβ2. Figure 4E/F show the same region in a mouse infused with Aβ + TGFβ2 + RAP. In Figure 4A and 4C, neuronal labeling of neurons in layer III/IV of the temporal cortex is easily visible. Cresyl violet staining of adjacent tissue sections reveals darkly stained and abnormally shaped cells in the same region as the Aβ staining. We quantitated cell-associated DAE immunoreactivity (DAEir) in the same region shown in Figure 4A–F (Fig. 4G). ANOVA of DAEir revealed a significant treatment effect (P = 0.0023 for square root-transformed values). DAEir was increased in mice infused with Aβ + TGFβ2 (P = 0.022 vs. Aβ alone) and coinfusion of RAP with Aβ + TGFβ2 attenuated this increase (P = 0.014 vs. Aβ + TGFβ2). We did not observe CA1 hippocampal pyramidal cell Aβ loading using the DAE antibody, but we did observe neuron staining using an Aβ42-specific antibody (BioSource International). Aβ was infused into the lateral ventricle at a low dose. Where Aβ ends up in the brain can be influenced by proximity to the infusion cannula, direction and volume of CSF flow, and other clearance mechanisms at work in the ventricular space. The hippocampal Aβ42 staining was located in the CA2/3 area close to the ventricle. As in the cortex, there were many darkly stained and abnormally shaped cells stained with cresyl violet in the hippocampus.

Preliminary and published data (Frautschy et al., 2001; Nakamura et al., 2001) demonstrate delayed effects of Aβ on cognition. We therefore waited 12 weeks from the start of ICV infusion before testing mice for cognitive deficits. Figure 5 shows the schedule (Fig. 5A) and results of Morris water maze testing in the mouse ICV infusion study. Latencies to find a visible platform were similar across all treatment groups with no significant differences found between groups (Fig. 5B). Two mice were removed from the study (one mouse from the HEPES group and one from the TGFβ2 group) after failing to find the platform after three blocks, due to visual or motor deficits. After exclusion of visible platform failures (1% of animals), the group sizes were as follows: HEPES, n = 7; RAP, n = 11; TGFβ2, n = 8; Aβ, n = 8; Aβ + TGFβ2, n = 6, Aβ + RAP, n = 10; and Aβ + TFβ2 + RAP, n = 8). Regression analysis of acquisition data showed significant learning in all ICV-infused groups of mice (Fig. 5C). Figure 5D shows the interaction bars for combined blocks 5 and 6 of the acquisition training. By the end of the training, there were no significant differences in latencies between groups (P = 0.28). A 3-day delay followed by a 20-sec probe trial (removal of the platform) revealed striking treatment differences in retention memory. Probe trial data was analyzed for percent of time in the target quadrant (Fig. 5E) and percent of path in the target quadrant (Fig. 5F). ANOVA for probe trial data revealed a significant treatment effect for both % time in target quadrant (data log transformed for analysis; P = 0.05) and % path in target quadrant (P = 0.04). These data demonstrated that compared to Aβ42 treatment alone, Aβ plus TGFβ2 infused mice showed deficits in retention memory (P = 0.01 for % time and P = 0.04 for % path) and these deficits were prevented by coinfusion with RAP (P = 0.001 for time and P = 0.002 for path).

After completion of behavior testing (14 weeks from start of ICV infusion), mice were perfused with cold HEPES buffer with protease inhibitors. The left side of the brain was dissected into regions and flash frozen in liquid nitrogen for biochemical analysis of synaptic markers. Spatial memory in rodents involves postsynaptic factors associated with NMDA receptor function and deficits in spatial memory tasks have been correlated with decreases in NMDA receptor subunit expression, particularly subunit NR2B (Clayton et al., 2002). Figure 6 shows the Western blot quantitation of NR2B levels in the entorhinal cortex of infused mice. ANOVA of NR2B data revealed a significant treatment effect (P = 0.04, data square root transformed). Infusion with Aβ + TGFβ2 reduced NR2B levels and coinfusion with RAP attenuated this effect (P = 0.024, Aβ + TGFβ2 vs. RAP + Aβ + TGFβ2).

DISCUSSION

These data argue for a crucial role for TGFβ2 in driving neuronal Aβ uptake and targeting and increasing neurodegeneration of Aβ both in vitro and in vivo. Data support the hypothesis that TGFβ2 is driving extracellular uptake of Aβ and increasing intracellular Aβ. That targeting and neurotoxicity are blocked by RAP demonstrate mediation of this effect through an LRP-like receptor. These results and our observed TGFβ2 induction of ApoE levels in OHSC media suggest that LRP receptor function or numbers could be altered. These data are consistent with observations in AD brain that LRP and ApoE are associated with senile plaques in AD (Rebeck et al., 1995). These data are also consistent with in vitro studies showing that high-density lipoprotein (HDL)-like particles containing ApoE or ApoJ can transport Aβ to cells, particularly neurons (Beffert et al., 1998), where they bind and are internalized by lipoprotein receptors such as LRP.

Our results show that ICV infusion of Aβ or Aβ + TGFβ2 did not affect acquisition of spatial information because all groups of mice learned to locate a submerged platform in the Morris water maze. Retention of spatial learning, however, was impaired in the Aβ + TGFβ2-infused mice. Because retention of spatial memory learning is impaired after a 3-day interval without impairment of acquisition, TGFβ2 alters Aβ effects on the processing of spatial memory learning. Previous studies using Morris water maze testing have found similar results with acquisition and retention trials (Minetti et al., 1996). It is not uncommon that acquisition deficits are missed but probe deficits are detected, and this may result from acquisition tests being confounded by many variables such as mice not using spatial cues exclusively (e.g., egocentric and scanning strategies) to learn platform position (van Groen et al., 2002; Shukitt-Hale et al., 2004). Larger retention deficits in probe trials may be due also to the sensitivity of this test to detect pure spatial memory deficits. Alternatively, the probe test may be sensitive to detecting Aβ-specific deficits. For example, Westerman et al. (2002) showed that although acquisition impairments remained constant from middle age to old age in APP Swedish mutation mice accumulating Aβ, the probe retention deficits worsened.

In the present study, the first trial of each block in the acquisition phase was carried out immediately after mouse orientation on the platform, and the retention test was carried out after a 3-day interval. This may imply that TGFβ2 alters Aβ effects on the processing of spatial memory learning, having no impact on short-term spatial memory, but impairing retrieval or late stages of consolidation. Further characterization of the severity of spatial deficits will require tests with enhanced sensitivity such as multiple probe trial maze testing (Gallagher et al., 1993).

The factors and circumstances that induce neuron dysfunction and death in the AD brain are unclear. There is controversy as to whether Aβ in the form of plaques is neurotoxic or whether more soluble or intracellular Aβ correlates with severity of dementia. Aβ is present in intracellular compartments and evidence is rapidly growing in favor of intracellular accumulation of Aβ as an early event in AD pathology (Kim et al., 2003; Shie et al., 2003). D'Andrea et al. (2001) showed in AD brain that Aβ42 first accumulates in the perikaryon of pyramidal neurons as discrete granules that are cathepsin D (lysosome) positive. Lysis of these cells resulted in local, radial dispersion of their cytoplasmic contents in the extracellular space. Additional studies have confirmed Aβ accumulation in lysosomal compartments (Frautschy et al., 1998), and that loss of lysosomal membrane integrity is an early step in Aβ pathogenesis and may explain alteration in compartmentalizing extracellular and cytoplasmic components in the AD brain (Yang et al., 1998). Aβ binds avidly to LRP receptor ligands such as A2M and ApoE, and LRP-mediated clearance of Aβ can lead to Aβ in the lysosome. Furthermore, neuronal cell death in AD has been shown to correlate with ApoE uptake and intracellular Aβ stabilization (LaFerla et al., 1997).

ApoE likely serves multiple roles in the CNS, one of which may be to clear Aβ from the extracellular space through receptors like LRP. What is unclear is whether this function is neurotoxic or neuroprotective and in humans, the answer may depend on the isoform of ApoE. It has been suggested that ApoE4 promotes depolymerization of microtubules and thus ApoE may participate in maintaining integrity of the cytoskeleton (Mahley et al., 1995; Nathan et al., 1995). In studies looking at the different human isoforms of ApoE, there seems to be a differential accumulation of ApoE3 and E4 in neurons. Both isoforms are retained intact but ApoE3 shows greater cellular accumulation (Ji et al., 1998). The intracellular fate of ApoE is unknown but retention of ApoE within the cell is due most likely to association with the slow endosome to lysosome transport and may involve heparan sulfate proteoglycans (HSPGs) as chaperones (Ji et al., 1998). Previous studies have implicated the failure to degrade Aβ in the late endosome or secondary lysosome as a mechanism for the accumulation of Aβ in AD (Yang et al., 1998). Uptake through the LRP/ApoE pathway may indeed be a normal pathway for clearance of Aβ in normal human brain (Kang et al., 2000). Alterations in ApoE levels or LRP receptor function/number by factors such as TGFβ2, however, could cause this system to fail with neurotoxic results. In this study, we have shown that TGFβ2 increased ApoE release into OHSC media, yet the mechanism for this increase is unclear. Little is known about TGFβ2 in the CNS but previous studies have shown that TGFβ1 can increase ApoE mRNA in macrophages (Zuckerman et al., 1993) but does not increase ApoE mRNA or transcription in neurons and astrocytes (Laping et al., 1994).

Neuronal immunoreactivity for TGFβ2 seems specific for NFT neurons in Alzheimer's disease (Flanders et al., 1995); however, the mechanism of induction and the role of TGFβ2 in AD are unknown. TGFβs have been shown to play a role in synaptic plasticity and dendritic arborization and TGFβ2 is concentrated in the subsynaptic portions of mature muscle fibers (McLennan and Koishi, 1994; McLennan et al., 1998; Murakami et al., 1999) and this distribution is characteristic of proteins involved in synaptic function (Duclert and Changeux, 1995). Synaptic effects may be mediated by crosstalk between the TGFβ signaling pathway and another important signaling pathway, the Wnt pathway (Salinas, 2003). Wnt proteins are a large family of secreted glycoproteins and some of the first evidence that Wnts regulate the synapse came from studies utilizing Wnt-7A mutant mice. Wnt-7A has been shown to regulate microtubule organization and the clustering of synaptic proteins (Salinas, 2003). Wnt-7A signaling can increase microtubule stability through inhibition of glycogen synthase kinase-3β (GSK-3β) (Lucas et al., 1998). TGFβ-mediated MAP kinase activation controls Wnt-7A gene expression and Wnt-mediated signaling (Tuli et al., 2003). In different contexts, Wnts and TGFβ signaling can interact either antagonistically or synergistically (Salinas, 2003). Preliminary data from our lab shows that in addition to inducing morphologic degenerative changes in the OHSC model, Aβ + TGFβ2 can increase AT8 (hyperphosphorylated tau) staining of pyramidal neurons in the CA1–CA2 region. NFT formation has been associated with abnormal tau phosphorylation due to decreased protein phosphatase or increased protein kinase activity, such as GSK-3β, in AD (Mandelkow et al., 1993; Iqbal et al., 1994). TGFβ2, highly localized in NFT-bearing neurons is AD, could destabilize microtubules by inhibiting Wnt-7A signaling. Aβ injected into the dorsal hippocampus has also been shown to induce a loss of Wnt signaling and a deficit in spatial memory (De Ferrari et al., 2003). Precisely how Wnt signaling causes inhibition of GSK-3β is unclear but it seems to involve two members of the LRP receptor family, LRP5 and LRP6 (Herz and Bock, 2002). As suggested by our in vitro and in vivo data, signaling interactions between the LRP and TGFβ pathways could alter normal Aβ clearance pathways.

In addition to Wnt signaling, LRP is known to physically associate with NMDA receptors via the postsynaptic density-95 (PSD95) (Gotthardt et al., 2000). Influx of calcium due to LRP-mediated activation of NMDA receptor channels may affect a variety of downstream signaling cascades and provide a mechanism for changing local synaptic plasticity (Tashiro et al., 1998). Alterations in brain LRP expression or activity may therefore impact neuronal homeostasis, synaptic plasticity, and amyloid deposition and thereby modify the pathogenesis of AD. In the current study, NR2B, a subunit of the NMDA receptor, is reduced in mice infused with Aβ + TGFβ2 and these mice exhibit memory retention deficits. A reduction in NR2B protein expression has been shown to correlate with deficits in Morris water maze performance (Clayton et al., 2002). It was demonstrated recently that LRP levels are reduced in AD brain (Kang et al., 2000), but it remains to be determined how this relates to the progression of AD or if low LRP levels are simply age-related or are an end result of extensive neuron loss.

The results of the current study provide in vitro and in vivo evidence that TGFβ2 can alter uptake of Aβ through the LRP receptor. Our results lead us to postulate that alterations in LRP function or numbers contribute to Aβ pathogenesis by impeding appropriate Aβ degradation. Changes in signaling and structural changes in the synaptic membrane can lead to the demonstrated accumulation of toxic Aβ and deficits in memory retention. More studies are needed to determine the precise interactions of TGFβ2, Aβ, ApoE, and the LRP receptor.

Acknowledgements

This work was supported by National Institutes of Health (grant AG022080 to M.E.H.-W. and grant AG10685 to S.A.F.), Veterans Administration Merit Award (to M.E.H.-W.), and a career development award from the Claude D. Pepper Older Americans Independence Counsel (to M.E.H.-W.). We thank G. Cole for helpful discussions and F. Yang and P. Chen for help with tissue processing. We also thank HVS Image newsgroup and particularly Dr. A. Blokland, Maastricht University, Netherlands, Dr. M.M. Nicolle at Mayo Clinic, FL, and Dr. B. Shukitt-Hale, Tufts University for helpful interpretation of Morris water maze data.

REFERENCES

Arelin K, Kinoshita A, Whelan CM, Irizarry MC, Rebeck GW, Strickland DK, Hyman BT. 2002. LRP and senile plaques in Alzheimer's disease: colocalization with apolipoprotein E and with activated astrocytes. Brain Res Mol Brain Res 104: 38–46.
10.1016/S0169-328X(02)00203-6
CAS PubMed Web of Science® Google Scholar
Baum L, Chen L, Ng HK, Chan YS, Mak YT, Woo J, Chiu HFK, Pang CP. 1998. Low density lipoprotein receptor related protein gene exon 3 polymorphism association with Alzheimer's disease in Chinese. Neurosci Lett 247: 33–36.
10.1016/S0304-3940(98)00294-8
CAS PubMed Web of Science® Google Scholar
Beffert U, Aumont N, Dea D, Lussier-Cacan S, Davignon J, Poirier J. 1998. Beta-amyloid peptides increase the binding and internalization of apolipoprotein E to hippocampal neurons. J Neurochem 70: 1458–1466.
10.1046/j.1471-4159.1998.70041458.x
CAS PubMed Web of Science® Google Scholar
Blacker D, Wilcox MA, Laird NM, Rodes L, Horvath SM, Go RC, Perry R, Watson B Jr, Bassett SS, McInnis MG, Albert MS, Hyman BT, Tanzi RE. 1998. Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nat Genet 19: 357–360.
10.1038/1243
CAS PubMed Web of Science® Google Scholar
Bodmer S, Podlisny MB, Selkoe DJ, Heid I, Fontana A. 1990. Transforming growth factor-β bound to soluble derivatives of the β-amyloid precursor protein of Alzheimer's disease. Biochem Biophys Res Commun 171: 890–897.
10.1016/0006-291X(90)91229-L
CAS PubMed Web of Science® Google Scholar
Borth W. 1992. Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J 6: 3345–3353.
10.1096/fasebj.6.15.1281457
CAS PubMed Web of Science® Google Scholar
Bu G. 1998. Receptor-associated protein: a specialized chaperone and antagonist for members of the LDL receptor gene family. Curr Opin Lipidol 9: 149–155.
10.1097/00041433-199804000-00012
CAS PubMed Web of Science® Google Scholar
Causevic M, Ramoz N, Haroutunian V, Davis KL, Buxbaum JD. 2003. Lack of association between the levels of the low-density lipoprotein receptor-related protein (LRP) and either Alzheimer dementia or LRP exon 3 genotype. J Neuropathol Exp Neurol 62: 999–1005.
10.1093/jnen/62.10.999
CAS PubMed Web of Science® Google Scholar
Chao CC, Hu S, Frey WH, Ala TA, Tourtellotte WW, Peterson PK. 1994. Transforming growth factor β in Alzheimer's disease. Clin Diagn Lab Immunol 1: 109–110.
CAS PubMed Web of Science® Google Scholar
Clayton DA, Mesches MH, Alvarez E, Bickford PC, Browning MD. 2002. A hippocampal NR2B deficit can mimic age-related changes in long-term potentiation and spatial learning in the Fischer 344 rat. J Neurosci 22: 3628–3637.
10.1523/JNEUROSCI.22-09-03628.2002
CAS PubMed Web of Science® Google Scholar
Constam DB, Philipp J, Malipiero UV, ten Dijke P, Schachner M, Fontana A. 1992. Differential expression of transforming growth factor-beta1, -beta2, and -beta3 by glioblastoma cells, astrocytes, and microglia. J Immunol 148: 1404–1410.
CAS PubMed Web of Science® Google Scholar
D'Andrea MR, Nagele RG, Wang HY, Peterson PA, Lee DHS. 2001. Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease. Histopathology 38: 120–134.
10.1046/j.1365-2559.2001.01082.x
CAS PubMed Web of Science® Google Scholar
De Ferrari GV, Chacon MA, Barria MI, Garrido JL, Godoy JA, Olivares G, Reyes AE, Alvarez A, Bronfman M, Inestrosa NC. 2003. Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by beta-amyloid fibrils. Mol Psychiatry 8: 195–208.
10.1038/sj.mp.4001208
CAS PubMed Web of Science® Google Scholar
del Rio-Hortega P. 1932. Microglia. In: W Penfield, editor. Cytology and cellular pathology of the nervous system. New York: Hoeber. p 483–534.
Google Scholar
Du Y, Ni B, Glinn M, Dodel RC, Bales KR, Zhang Z, Hyslop PA, Paul SM. 1997. α₂-Macroglobulin as a beta-amyloid peptide-binding plasma protein. J Neurochem 69: 299–305.
10.1046/j.1471-4159.1997.69010299.x
CAS PubMed Web of Science® Google Scholar
Duclert A, Changeux JP. 1995. Acetylcholine receptor gene expression at the developing neuromuscular junction. Physiol Rev 75: 339–368.
10.1152/physrev.1995.75.2.339
CAS PubMed Web of Science® Google Scholar
Flanders KC, Lippa CF, Smith TW, Pollen DA, Sporn MB. 1995. Altered expression of transforming growth factor-beta in Alzheimer's disease. Neurology 45: 1561–1569.
10.1212/WNL.45.8.1561
CAS PubMed Web of Science® Google Scholar
Frautschy SA, Calderon L, Yang F, Cole GM. 1996. Rodent models of Alzheimer's disease: a comparison of transgenic APP mouse and rat A beta infusion approaches. Neurobiol Aging 17: 311–321.
10.1016/0197-4580(95)02073-X
PubMed Web of Science® Google Scholar
Frautschy SA, Horn DL, Sigel JJ, Harris-White ME, Mendoza JJ, Yang FS, Saido TC, Cole GM. 1998. Protease inhibitor coinfusion with amyloid beta-protein results in enhanced deposition and toxicity in rat brain. J Neurosci 18: 8311–8321.
10.1523/JNEUROSCI.18-20-08311.1998
CAS PubMed Web of Science® Google Scholar
Frautschy SA, Hu W, Kim P, Miller SA, Chu T, Harris-White ME, Cole GM. 2001. Phenolic anti-inflammatory antioxidant reversal of A beta-induced cognitive deficits and neuropathology. Neurobiol Aging 22: 993–1005.
10.1016/S0197-4580(01)00300-1
CAS PubMed Web of Science® Google Scholar
Gallagher M, Burwell R, Burchinal M. 1993. Severity of spatial learning impairment in aging: development of a learning index for performance in the Morris water maze. Behav Neurosci 107: 618–626.
10.1037/0735-7044.107.4.618
CAS PubMed Web of Science® Google Scholar
Gotthardt M, Trommsdorff M, Nevitt MF, Shelton J, Richardson JA, Stockinger W, Nimpf J, Herz J. 2000. Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction. J Biol Chem 275: 25616–25624.
10.1074/jbc.M000955200
CAS PubMed Web of Science® Google Scholar
Gracia V, Fiol C, Hurtado I, Pinto X, Argimon JM, Castineiras MJ. 1994. An enzyme-linked immunosorbent assay method to measure human apolipoprotein E levels using commercially available reagents: effect of apolipoprotein E polymorphism on serum apolipoprotein E concentration. Anal Biochem 223: 212–217.
10.1006/abio.1994.1576
CAS PubMed Web of Science® Google Scholar
Gylys KH, Fein JA, Tan AM, Cole GM. 2003. Apolipoprotein E enhances uptake of soluble but not aggregated amyloid-beta protein into synaptic terminals. J Neurochem 84: 1442–1451.
10.1046/j.1471-4159.2003.01643.x
CAS PubMed Web of Science® Google Scholar
Harris-White ME, Chu T, Balverde Z, Sigel JJ, Flanders KC, Frautschy SA. 1998. Effects of transforming growth factor-beta (isoforms 1–3) on amyloid-beta deposition, inflammation, and cell targeting in organotypic hippocampal slice cultures. J Neurosci 18: 10366–10374.
CAS PubMed Web of Science® Google Scholar
Herz J, Hamann U, Rogne S, Myklebost O, Gausepohl H, Stanley KK. 1988. Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J 7: 4119–4127.
10.1002/j.1460-2075.1988.tb03306.x
CAS PubMed Web of Science® Google Scholar
Hollenbach E, Ackermann S, Hyman BT, Rebeck GW. 1998. Confirmation of an association between a polymorphism in exon 3 of the low-density lipoprotein receptor-related protein gene and Alzheimer's disease. Neurology 50: 1905–1907.
10.1212/WNL.50.6.1905
CAS PubMed Web of Science® Google Scholar
Holtzman DM, Pitas RE, Kilbridge J, Nathan B, Mahley RW, Bu G, Schwartz AL. 1995. Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line. Proc Natl Acad Sci USA 92: 9480–9484.
10.1073/pnas.92.21.9480
CAS PubMed Web of Science® Google Scholar
Huang SS, Ling TY, Tseng WF, Huang YH, Tang FM, Leal SM, Huang JS. 2003. Cellular growth inhibition by IGFBP-3 and TGF-beta1 requires LRP-1. FASEB J 17: 2068–2081.
10.1096/fj.03-0256com
CAS PubMed Web of Science® Google Scholar
Hussaini IM, Lamarre J, Lysiak JJ, Karns LR, VandenBerg SR, Gonias SL. 1996. Transcriptional regulation of LDL receptor-related protein by IFN-gamma and the antagonistic activity of TGF-beta(1) in the RAW 264.7 macrophage-like cell line. J Leukoc Biol 59: 733–739.
10.1002/jlb.59.5.733
CAS PubMed Web of Science® Google Scholar
Iqbal K, Alonso AC, Gong CX, Khatoon S, Singh TJ, Grundke-Iqbal I. 1994. Mechanism of neurofibrillary degeneration in Alzheimer's disease. Mol Neurobiol 9: 119–123.
10.1007/BF02816111
CAS PubMed Web of Science® Google Scholar
Ji ZS, Pitas RE, Mahley RW. 1998. Differential cellular accumulation/retention of apolipoprotein E mediated by cell surface heparan sulfate proteoglycans. Apolipoproteins E3 and E2 greater than E4. J Biol Chem 273: 13452–13460.
10.1074/jbc.273.22.13452
CAS PubMed Web of Science® Google Scholar
Kamboh MI, Ferrell RE, DeKosky ST. 1998. Genetic association studies between Alzheimer's disease and two polymorphisms in the low density lipoprotein receptor-related protein gene. Neurosci Lett 244: 65–68.
10.1016/S0304-3940(98)00141-4
CAS PubMed Web of Science® Google Scholar
Kang DE, Pietrzik CU, Baum L, Chevallier N, Merriam DE, Kounnas MZ, Wagner SL, Troncoso JC, Kawas CH, Katzman R, Koo EH. 2000. Modulation of amyloid beta-protein clearance and Alzheimer's disease susceptibility by the LDL receptor-related protein pathway. J Clin Invest 106: 1159–1166.
10.1172/JCI11013
CAS PubMed Web of Science® Google Scholar
Kang DE, Saitoh T, Chen X, Xia Y, Masliah E, Hansen LA, Thomas RG, Thal LJ, Katzman R. 1997. Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer's disease. Neurology 49: 56–61.
10.1212/WNL.49.1.56
PubMed Web of Science® Google Scholar
Kim HJ, Chae SC, Lee DK, Chromy B, Lee SC, Park YC, Klein WL, Krafft GA, Hong ST. 2003. Selective neuronal degeneration induced by soluble oligomeric amyloid beta protein. FASEB J 17: 118–120.
10.1096/fj.01-0987fje
CAS PubMed Web of Science® Google Scholar
Klempt ND, Sirimanne E, Gunn AJ, Klempt M, Singh K, Williams C, Gluckman PD. 1992. Hypoxia-ischemia induces transforming growth factor beta 1 mRNA in the infant rat brain. Brain Res Mol Brain Res 13: 93–101.
10.1016/0169-328X(92)90048-G
CAS PubMed Web of Science® Google Scholar
Kounnas MZ, Moir RD, Rebeck GW, Bush AI, Argraves WS, Tanzi RE, Hyman BT, Strickland DK. 1995. LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation. Cell 82: 331–340.
10.1016/0092-8674(95)90320-8
CAS PubMed Web of Science® Google Scholar
Krieglstein K, Rufer M, Suter-Crazzolara C, Unsicker K. 1995. Neural functions of the transforming growth-factors-beta. Int J Dev Neurosci 13: 301–315.
10.1016/0736-5748(94)00062-8
CAS PubMed Web of Science® Google Scholar
Ladu MJ, Falduto MT, Manelli AM, Reardon CA, Getz GS, Frail DE. 1994. Isoform-specific binding of apolipoprotein E to beta-amyloid. J Biol Chem 269: 23403–23406.
10.1016/S0021-9258(17)31529-6
CAS PubMed Web of Science® Google Scholar
LaFerla FM, Troncoso JC, Strickland DK, Kawas CH, Jay G. 1997. Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Abeta stabilization. J Clin Invest 100: 310–320.
10.1172/JCI119536
CAS PubMed Web of Science® Google Scholar
Lambert JC, Wavrant-De Vrieze F, Amouyel P, Chartier-Harlin MC. 1998. Association at LRP gene locus with sporadic late-onset Alzheimer's disease. Lancet 351: 1787–1788.
10.1016/S0140-6736(05)78749-3
CAS PubMed Web of Science® Google Scholar
Laping NJ, Morgan TE, Nichols NR, Rozovsky I, Young-Chan CS, Zarow C, Finch CE. 1994. Transforming growth factor-beta 1 induces neuronal and astrocyte genes: tubulin alpha 1, glial fibrillary acidic protein and clusterin. Neuroscience 58: 563–572.
10.1016/0306-4522(94)90081-7
CAS PubMed Web of Science® Google Scholar
Lesne S, Docagne F, Gabriel C, Liot G, Lahiri DK, Buee L, Plawinski L, Delacourte A, MacKenzie ET, Buisson A, Vivien D. 2003. Transforming growth factor-beta 1 potentiates amyloid-beta generation in astrocytes and in transgenic mice. J Biol Chem 278: 18408–18418.
10.1074/jbc.M300819200
CAS PubMed Web of Science® Google Scholar
Lim GP, Yang F, Chu T, Chen P, Beech W, Teter B, Tran T, Ubeda O, Ashe KH, Frautschy SA, Cole GM. 2000. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease. J Neurosci 20: 5709–5714.
10.1523/JNEUROSCI.20-15-05709.2000
CAS PubMed Web of Science® Google Scholar
Lindholm D, Castren E, Kiefer R, Zafra F, Thoenen H. 1992. Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation. J Cell Biol 117: 395–400.
10.1083/jcb.117.2.395
CAS PubMed Web of Science® Google Scholar
Lippa CF, Smith TW, Flanders KC. 1995. Transforming growth factor-β: neuronal and glial expression in CNS degenerative diseases. Neurodegeneration 4: 425–432.
10.1006/neur.1995.0051
CAS PubMed Web of Science® Google Scholar
Logan A, Frautschy SA, Gonzalez A-M, Sporn MB, Baird A. 1992. Enhanced expression of transforming growth factor B1 in the rat brain after a localized cerebral injury. Brain Res 587: 216–225.
10.1016/0006-8993(92)91000-5
CAS PubMed Web of Science® Google Scholar
Lucas FR, Goold RG, Gordon-Weeks PR, Salinas PC. 1998. Inhibition of GSK-3beta leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium. J Cell Sci 111: 1351–1361.
10.1242/jcs.111.10.1351
CAS PubMed Web of Science® Google Scholar
Mahley RW, Nathan BP, Bellosta S, Pitas RE. 1995. Apolipoprotein E: impact of cytoskeletal stability in neurons and the relationship to Alzheimer's disease. Curr Opin Lipidol 6: 86–91.
10.1097/00041433-199504000-00005
CAS PubMed Google Scholar
Mandelkow EM, Biernat J, Drewes G, Steiner B, Lichtenberg-Kraag B, Wille H, Gustke N, Mandelkow E. 1993. Microtubule-associated protein tau, paired helical filaments, and phosphorylation. Ann N Y Acad Sci 695: 209–216.
10.1111/j.1749-6632.1993.tb23054.x
CAS PubMed Web of Science® Google Scholar
Mazur-Kolecka B, Frackowiak J, Le Vine H 3rd, Haske T, Evans L, Sukontasup T, Golabek A. 2003. TGFbeta1 enhances formation of cellular Abeta/apoE deposits in vascular myocytes. Neurobiol Aging 24: 355–364.
10.1016/S0197-4580(02)00095-7
CAS PubMed Web of Science® Google Scholar
McIlroy SP, Dynan KB, Vahidassr DJ, Lawson JT, Patterson CC, Passmore P. 2001. Common polymorphisms in LRP and A2M do not affect genetic risk for Alzheimer disease in Northern Ireland. Am J Med Genet 105: 502–506.
10.1002/ajmg.1474
CAS PubMed Web of Science® Google Scholar
McLennan IS, Koishi K. 1994. Transforming growth factor-beta-2 (TGF-beta-2) is associated with mature rat neuromuscular junctions. Neurosci Lett 177: 151–154.
10.1016/0304-3940(94)90889-3
CAS PubMed Web of Science® Google Scholar
McLennan IS, Koishi K, Zhang M, Murakami N. 1998. The non-synaptic expression of transforming growth factor-beta 2 is neurally regulated and varies between skeletal muscle fibre types. Neuroscience 87: 845–853.
10.1016/S0306-4522(98)00180-8
CAS PubMed Web of Science® Google Scholar
Minetti A, Arolfo MP, Virgolini MB, Brioni JD, Fulginiti S. 1996. Spatial learning in rats exposed to acute ethanol intoxication on gestational day 8. Pharmacol Biochem Behav 53: 361–367.
10.1016/0091-3057(95)02035-7
CAS PubMed Web of Science® Google Scholar
Mousseau DD, Chapelsky S, De Crescenzo G, Kirkitadze MD, Magoon J, Inoue S, Teplow DB, O'Connor-McCourt MD. 2003. A direct interaction between transforming growth factor (TGF)-betas and amyloid-beta protein affects fibrillogenesis in a TGF-beta receptor-independent manner. J Biol Chem 278: 38715–38722.
10.1074/jbc.M304080200
CAS PubMed Web of Science® Google Scholar
Murakami N, McLennan IS, Nonaka I, Koishi K, Baker C, Hammond-Tooke G. 1999. Transforming growth factor-beta 2 is elevated in skeletal muscle disorders. Muscle Nerve 22: 889–898.
10.1002/(SICI)1097-4598(199907)22:7<889::AID-MUS12>3.0.CO;2-B
CAS PubMed Web of Science® Google Scholar
Nakamura S, Murayama N, Noshita T, Annoura H, Ohno T. 2001. Progressive brain dysfunction following intracerebroventricular infusion of beta(1–42)-amyloid peptide. Brain Res 912: 128–136.
10.1016/S0006-8993(01)02704-4
CAS PubMed Web of Science® Google Scholar
Narita M, Holtzman DM, Schwartz AL, Bu G. 1997. α₂-Macroglobulin complexes with and mediates the endocytosis of beta-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. J Neurochem 69: 1904–1911.
10.1046/j.1471-4159.1997.69051904.x
CAS PubMed Web of Science® Google Scholar
Nathan BP, Chang K-C, Bellosta S, Brisch E, Ge N, Mahley RW, Pitas RE. 1995. The inhibitory effect of apolipoprotein E4 on neurite outgrowth is associated with microtubule depolymerization. J Biol Chem 270: 19791–19799.
10.1074/jbc.270.34.19791
CAS PubMed Web of Science® Google Scholar
Pelton RW, Saxena B, Jones M, Moses HL, Gold LI. 1991. Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 115: 1091–1105.
10.1083/jcb.115.4.1091
CAS PubMed Web of Science® Google Scholar
Peress NS, Perillo E. 1995. Differential expression of TGF-β1, 2 and 3 isotypes in Alzheimer's disease: a comparative immunohistochemical study with cerebral infarction, aged human and mouse control brains. J Neuropathol Exp Neurol 54: 802–811.
10.1097/00005072-199511000-00007
CAS PubMed Web of Science® Google Scholar
Rebeck GW, Harr SD, Strickland DK, Hyman BT. 1995. Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Ann Neurol 37: 211–217.
10.1002/ana.410370212
CAS PubMed Web of Science® Google Scholar
Rebeck GW, Reiter JS, Strickland DK, Hyman BT. 1993. Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions. Neuron 11: 575–580.
10.1016/0896-6273(93)90070-8
CAS PubMed Web of Science® Google Scholar
Salinas PC. 2003. Synaptogenesis: Wnt and TGF-beta take centre stage. Curr Biol 13: 60–62.
10.1016/S0960-9822(02)01429-X
CAS PubMed Web of Science® Google Scholar
Sanchez-Guerra M, Combarros O, Infante J, Llorca J, Berciano J, Fontalba A, Fernandez-Luna JL, Pena N, Fernandez-Viadero C. 2001. Case-control study and meta-analysis of low density lipoprotein receptor-related protein gene exon 3 polymorphism in Alzheimer's disease. Neurosci Lett 316: 17–20.
10.1016/S0304-3940(01)02342-4
CAS PubMed Web of Science® Google Scholar
Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, Cardell EL, Doetschman T. 1997. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development 124: 2659–2670.
10.1242/dev.124.13.2659
CAS PubMed Web of Science® Google Scholar
Schmued LC, Albertson C, Slikker W Jr. 1997. Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 751: 37–46.
10.1016/S0006-8993(96)01387-X
CAS PubMed Web of Science® Google Scholar
Schmued LC, Hopkins KJ. 2000. Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874: 123–130.
10.1016/S0006-8993(00)02513-0
CAS PubMed Web of Science® Google Scholar
Schneider WJ, Nimpf J. 2003. LDL receptor relatives at the crossroad of endocytosis and signaling. Cell Mol Life Sci 60: 892–903.
10.1007/s00018-003-2183-Z
CAS PubMed Web of Science® Google Scholar
Selkoe DJ. 1994. Cell biology of the amyloid beta-protein precursor and the mechanism of Alzheimer's disease. Annu Rev Cell Biol 10: 373–403.
10.1146/annurev.cb.10.110194.002105
CAS PubMed Web of Science® Google Scholar
Shie FS, LeBoeur RC, Jin LW. 2003. Early intraneuronal Abeta deposition in the hippocampus of APP transgenic mice. Neuroreport 14: 123–129.
10.1097/00001756-200301200-00023
CAS PubMed Web of Science® Google Scholar
Shukitt-Hale B, McEwen JJ, Szprengiel A, Joseph JA. 2004. Effect of age on the radial arm water maze-a test of spatial learning and memory. Neurobiol Aging 25: 223–229.
10.1016/S0197-4580(03)00041-1
PubMed Web of Science® Google Scholar
Stoppini L, Buchs PA, Muller D. 1991. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37: 173–182.
10.1016/0165-0270(91)90128-M
CAS PubMed Web of Science® Google Scholar
Strickland DK, Kounnas MZ, Argraves WS. 1995. LDL receptor-related protein: a multiligand receptor for lipoprotein and proteinase catabolism. FASEB J 9: 890–898.
10.1096/fasebj.9.10.7615159
CAS PubMed Web of Science® Google Scholar
Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. 1993. Apolipoprotein E: High-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci USA 90: 1977–1981.
10.1073/pnas.90.5.1977
CAS PubMed Web of Science® Google Scholar
Tashiro H, Doh-ura K, Iwaki T. 1998. Differential expression of transforming growth factor-beta isoforms in human prion diseases. Neuropathol Appl Neurobiol 24: 284–292.
10.1046/j.1365-2990.1998.00117.x
CAS PubMed Web of Science® Google Scholar
Tuli R, Tuli S, Nandi S, Huang X, Manner PA, Hozack WJ, Danielson KG, Hall DJ, Tuan RS. 2003. TGF-beta 1-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and MAP kinase and Wnt signaling crosstalk. J Biol Chem 278: 41227–41236.
10.1074/jbc.M305312200
CAS PubMed Web of Science® Google Scholar
Van der Wal EA, Gomez-Pinilla F, Cotman CW. 1993. Transforming growth factor-beta1 is in plaques in Alzheimer and Down pathologies. Neuroreport 4: 69–72.
10.1097/00001756-199301000-00018
CAS PubMed Web of Science® Google Scholar
van Groen T, Kadish I, Wyss JM. 2002. The role of the laterodorsal nucleus of the thalamus in spatial learning and memory in the rat. Behav Brain Res 136: 329–337.
10.1016/S0166-4328(02)00199-7
PubMed Web of Science® Google Scholar
Vawter MP, Dillon-Carter O, Tourtellotte WW, Carvey P, Freed WJ. 1996. TGFbeta1 and TGFbeta2 concentrations are elevated in Parkinson's disease in ventricular cerebrospinal fluid. Exp Neurol 142: 313–322.
10.1006/exnr.1996.0200
CAS PubMed Web of Science® Google Scholar
Wavrant-DeVrieze F, Lambert JC, Stas L, Crook R, Cottel D, Pasquier F, Frigard B, Lambrechts M, Thiry E, Amouyel P, Tur JP, Chartier-Harlin MC, Hardy J, Van Leuven F. 1999. Association between coding variability in the LRP gene and the risk of late-onset Alzheimer's disease. Hum Genet 104: 432–434.
10.1007/s004390050980
CAS PubMed Web of Science® Google Scholar
Westerman MA, Cooper-Blacketer D, Mariash A, Kotilinek L, Kawarabayashi T, Younkin LH, Carlson GA, Younkin SG, Ashe KH. 2002. The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer's disease. J Neurosci 22: 1858–1867.
10.1523/JNEUROSCI.22-05-01858.2002
CAS PubMed Web of Science® Google Scholar
Winkler J, Conner DJ, Frautschy SA, Behl C, Waite JJ, Cole GM, Thal LJ. 1994. Lack of long-term effects after β-amyloid protein injections in rat brain. Neurobiol Aging 15: 601–607.
10.1016/0197-4580(94)00054-9
CAS PubMed Web of Science® Google Scholar
Wolf BB, Lopes MB, VandenBerg SR, Gonias SL. 1992. Characterization and immunohistochemical localization of alpha 2- macroglobulin receptor (low-density lipoprotein receptor-related protein) in human brain. Am J Pathol 141: 37–42.
CAS PubMed Web of Science® Google Scholar
Wyss-Coray T, Lin C, Yan F, Yu GQ, Rohde M, McConlogue L, Masliah E, Mucke L. 2001. TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat Med 7: 612–618.
10.1038/87945
CAS PubMed Web of Science® Google Scholar
Wyss-Coray T, Masliah E, Mallory M, McConlogue L, Johnson-Wood K, Lin C, Mucke L. 1997. Amyloidogenic role of cytokine TGB-β1 in transgenic mice and in AD. Nature 389: 603–606.
10.1038/39321
CAS PubMed Web of Science® Google Scholar
Yang AJ, Chandswangbhuvana D, Margol L, Glabe CG. 1998. Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid A beta 1-42 pathogenesis. J Neurosci Res 52: 691–698.
10.1002/(SICI)1097-4547(19980615)52:6<691::AID-JNR8>3.0.CO;2-3
CAS PubMed Google Scholar
Yang F, Mak K, Vinters HV, Frautschy SA, Cole GM. 1994. Monoclonal antibody to the C-terminus of beta-amyloid. Neuroreport 5: 2117–2120.
10.1097/00001756-199410270-00032
CAS PubMed Web of Science® Google Scholar
Zuckerman SH, Evans GF, O'Neal L. 1993. Exogenous glucocorticoids increase macrophage secretion of apo E by cholesterol-independent pathways. Atherosclerosis 103: 43–54.
10.1016/0021-9150(93)90038-V
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume77, Issue2

15 July 2004

Pages 217-228

Role of LRP in TGFβ2-mediated neuronal uptake of Aβ and effects on memory

Abstract