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Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer’s disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1-40 transgenes in amyloid β protein precursor (APP) mouse models. Expression of BRI2-Aβ1-40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 amino acid peptide, Bri23, released from BRI2 by normal processing is present in human cerebrospinal fluid (CSF), inhibits Aβ aggregation in vitro, and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo.
Familial British and Danish dementias (FBD and FDD, respectively) are neurodegenerative dementias pathologically characterized by parenchymal preamyloid and amyloid deposits, cerebral amyloid angiopathy (CAA), neuronal loss and neurofibrillary tangles (Ghiso et al., 2006). Two mutations in the ITM2b gene encoding the BRI2 protein have been identified as the cause of FBD and FDD. BRI2 is a 266 amino acid (aa) long type 2 transmembrane protein of unknown function. It is expressed at high levels in the brain and cleaved by furin or furin-like proteases at its carboxyl terminus to produce a 23 aa peptide (Bri2-23) (Kim et al., 1999; Choi et al., 2004) (Fig. 1B). Disease causing mutations result in the production of COOH-terminally extended 277 aa mutant BRI2 proteins, which are cleaved at the normal furin processing site to generate distinct 34 aa peptides (ABri in FBD and ADan in FDD) that accumulate in the brains of affected patients (Vidal et al., 1999; Vidal et al., 2000). Notably, synthetic ABri and ADan undergo rapid aggregation and fibrillization into amyloid and, they are neurotoxic (Gibson et al., 2005; Ghiso et al., 2006). Thus, there are clear pathological and clinical similarities between FBD, FDD and AD. Indeed, genetic analyses of FBD, FDD and familial forms of AD support an unifying pathologic mechanism in which accumulation of amyloidogenic peptides triggers a complex pathological cascade leading to neurodegeneration (Golde, 2003).
Our interest in the BRI2 protein developed in the course of studies using BRI2-Aβ fusion proteins to express individual Aβ peptides (McGowan et al., 2005; Kim et al., 2007). By crossing BRI2-Aβ1-40 or BRI-Aβ1-42 transgenic mice with Tg2576 mice, we previously demonstrated that Aβ1-40 and Aβ42 have opposing effects on amyloid deposition (McGowan et al., 2005; Kim et al., 2007). To study the anti-amyloidogenic effect of Aβ1-40 in another APP mouse model, we utilized recombinant adeno-associated virus 1 (rAAV1)-mediated gene transfer to deliver the BRI2-Aβ1-40 and BRI2 transgenes to the brains of P0 TgCRND8 hAPPKM670/671NL+V717F APP mice (Levites et al., 2006a). This methodology of gene transfer, which we have termed somatic brain transgenesis, leads to consistent widespread and permanent expression of the transgene in forebrain and hippocampal neurons (Fig. 1A), and enables one to rapidly and cost-effectively evaluate the effects of transgene expression on the amyloid deposition phenotype (Levites et al., 2006a).
rAAV1 expressing BRI2, BRI2-Aβ1-40, BRI2del244-266, non-specific single-chain variable fragment (scFv ns), or enhanced green fluorescent protein (eGFP), under the control of the cytomegalovirus enhancer/chicken β actin (CBA) promoter were generated by calcium-phosphate transfection of pAM/CBA-pI-WPRE-BGH, rAAV1 cis plasmid pH21 (AAV1 helper plasmid) and pFΔ6 into a HEK293 cell line. rAAV1-scFv ns construct was reported previously (Levites et al., 2006a). At 48 hours after transfection, cells were lysed in the presence of 0.5% sodium deoxycholate and 50U/ml benzonase (Sigma) by repeated rounds of freeze/thaws at −80°C and −20°C. The virus was isolated using a discontinuous Iodixanol gradient, and then affinity purified on a HiTrap HQ column (Amersham). Samples were eluted from the column and buffer exchanged to PBS using an Amicon Ultra 100 Centrifugation device (Millipore). The genomic titer of each virus was determined by quantitative PCR using the ABI 7900 (Applied Biosystems). The viral DNA samples were prepared by treating the virus with DNase I (Invitrogen), heat inactivating the enzyme, then digesting the protein coat with Proteinase K (Invitrogen), followed by a second heat-inactivation. Samples were compared against a standard curve of supercoiled plasmid.
TgCRND8 mice expressing mutant human APP (KM670/671NL and V717F) gene under the control of hamster prion promoter were reported previously (Chishti et al., 2001). Hemizygous male TgCRND8 mice were crossed with female B6C3F1 wild-type mice. Tg2576 mice expressing mutant human APP (KM670/671NL) gene under the control of hamster prion promoter were reported previously (Hsiao et al., 1996). Hemizygous female Tg2576 mice were mated with male B6SJL wild-type mice. The injection procedures were performed as described previously (Passini et al., 2003; Broekman et al., 2006; Levites et al., 2006a). Briefly, P0 pups were cryoanesthetized on ice for 5 min. 2 μl of AAV1 construct (1×1012 genome particles/ml) was bilaterally injected into the cerebral ventricle of newborn mice using a 10 ml Hamilton syringe with a 30 gauge needle. The pups were placed on a heating pad until they recovered from cryoanesthesia then returned to their mother for further recovery. Negative control groups (total n=20) were non-injection (n=4), PBS injection (n=4), eGFP (n=5) and non-specific scFv (n=7) groups. Experimental groups were BRI2-Aβ1-40 (n=11), BRI2 (n=8), and BRI2del244-266 (n=13). Biochemical and histochemical Aβ loads in the control groups were equivalent. All animal procedures were approved by Mayo Clinic Institutional Animal Care and Use Committee in accordance with NIH guidelines.
Hemibrains were immersion fixed in 10% formalin then processed for paraffin embedding. Brain tissue sections (5μm) were immunostained with the anti-total Aβ antibody (33.1.1, 1:1000 (Levites et al., 2006b)) on a DAKO autostainer. The cortical Aβ plaque burden and the number of ThioS positive plaques were quantified as previously reported (Kim et al., 2007). Three to six sagittal sections per brain, 50 μm apart, were analyzed.
For brain Aβ ELISAs from TgCRND8 mice, hemi-forebrains were homogenized in 2% SDS with 1x protease inhibitor cocktail (Roche) dissolved in H2O then ultra-centrifuged at 100,000g for 1hr. The SDS-insoluble Aβ were extracted using 70% formic acid (FA). For brain Aβ ELISAs from 2 months old Tg2576 mice, hemi-forebrains were homogenized in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 0.5% Deoxycholate, 1% Triton X-100, 150mM NaCl, and 50mM Tris-HCl) then ultracentrifuged at 100,000g for 1hr. To measure the endogenous mouse Aβ levels, hemi-forebrains of non-transgenic littermates of the TgCRND8 mice expressing BRI2 were homogenized in 0.2% diethylamine (DEA) buffer containing 50 mM NaCl and 1x protease inhibitor cocktail (Roche). Endogenous mouse Aβ levels were measured using the previous validated rodent specific Aβ ELISA system as previously reported (Eckman et al., 2006). For plasma Aβ analysis, blood was collected in EDTA-coated tubes following cardiac puncture. Blood samples were centrifuged at 3000 rpm for 10min at 4°C and then the plasma was aliquoted and stored at −80°C until used. Aβ levels were determined by human Aβ end-specific sandwich ELISAs as previously described (Kim et al., 2007).
To test whether mice generate anti-Aβ antibody responses, anti-Aβ IgG antibody titers were determined by standard ELISA techniques, as described previously (Das et al., 2001). Briefly, microtitre plates (Maxi Sorp, Dynatech) were coated with aggregated Aβ42 at 2μg/well. After washings, serial dilutions of plasma (1:500 dilution) were added and incubated overnight at 4°C. Following washes with PBS/0.1% Tween-20, plasma IgG was detected using a anti-mouse IgG antibody conjugated with HRP (1:2000, Sigma) and TMB substrate (KPL).
Snap frozen forebrain samples were homogenized in 2% SDS buffer with 1x protease inhibitor cocktail (Roche). The homogenate was centrifuged at 100,000g for 1 hr at 4°C. Protein concentration in supernatants was determined using the BCA Protein Assay kit (Pierce). Protein samples (20μg) were run on Bis-Tris 12% XT gels (Biorad) with XT-MES buffer or Bis-Tris 4–12% XT gels (Biorad) with XT-MOPS buffer and transferred to 0.2μm nitrocellulose membranes. Blots were microwaved for 2 min in 0.1 M PBS twice and probed with the antibody 82E1 (anti-Aβ1-16, 1:1000, IBL), CT20 (anti-APP C-terminal 20 amino acids, 1:1000, T. Golde) and ITM2b (GenWay). Blots were stripped and reprobed with anti β-actin (1:1000, Sigma) as a loading control. Relative band intensity was quantified using ImageJ software (NIH).
Synthetic Aβ1-42 and Aβ1-40, treated with HFIP and dried (Bachem), and Bri2-23 peptides (Bachem) were dissolved in DMSO then diluted in TBS at molar ratios as indicated. Aβ1-42 and Bri2-23 peptide mixtures were either incubated for 3 hours at 0°C or 37°C without shaking. Mixtures were run on 4–20% Tris-HCl gels under nondenaturing conditions and transferred to 0.4 μm PVDF membrane as previously described (Klug et al., 2003; Kim et al., 2007). The blot was probed with Ab9 (anti-Aβ1-16, 1:1000, T. Golde). Relative band intensity was quantified using ImageJ software (NIH).
Bri2-23 peptides (Bachem) were reconstituted in 1 mg/mL Tris-HCl (pH 8.0). The lyophilized synthetic Aβ1-42 (Mayo Clinic Peptide Synthesis Facility) was dissolved at 0.5 – 2.0 mM in 20 mM NaOH 15 min prior to size exclusion chromatography on Superdex 75 HR 10/30 column (Amersham Pharmacia) to remove any pre-formed Aβ aggregates. The concentration of monomeric Aβ was determined by UV absorbance with a calculated extinction coefficient of 1450 cm−1M−1 at 276 nm (Rangachari et al., 2006). Aβ1-42 aggregation reactions were initiated in siliconized eppendorf tubes by incubating 25 – 50 μM of freshly purified Aβ1-42 monomer in 10 mM Tris-HCl and 150 mM NaCl (pH 8.0) buffer without agitation at 37°C. Monomeric Aβ1-42 aggregation process in the presence or absence of Bri2-23 peptide were monitored using a thioflavin T assay as previously reported (Rangachari et al., 2006). Atomic force microscopy images were obtained with a NanoScope III controller with a Multimode AFM (Veeco Instruments Inc, Chadds Ford PA) as described previously (Nichols et al., 2005). Images are shown in amplitude mode, where increasing brightness indicates greater damping of cantilever oscillation.
Conditioned media or CSF was filtered through a 0.45 μM syringe filter to remove large particulate matter. A fifty microliter aliquot of the sample was injected into an Agilent 1100 Series HPLC with a Zobax Eclipse XDB-C8 column and running buffer of acetonitrile/H2O (ACN:H2O) with 0.1% trifluoroacetic acid (TFA) at a flow rate of one milliliter a minute. Initial solvent composition was 20:80 ACN/H2O, this composition was held for 3 minutes then linearly ramped up to 37:63 ACN/H2O over the next 7 minutes. A fraction was collected between 9.4 minutes and 10.4 minutes (as the BRI-23 standard was seen to elute at 9.8 minutes) for a total of one milliliter.
The collected fraction was then blown down in nitrogen at 37°C to approximately 100 μL in volume. A one microliter aliquot of this concentrated sample was applied to a Bio-Rad gold array chip and allowed to air dry. After the sampled dried, one microliter of saturated α-Cyano-4-hydroxycinnamic acid (MALDI matrix) in 70:20:10 ACN:H2O:MeOH w/0.1% TFA was applied on top of dried sample and allowed to air dry. This was then analyzed on a Bio-Rad Ciphergen ProteinChip SELDI time-of-flight system. A laser intensity of 750 μJ was used to collect spectra from 3975 laser shots which were averaged into the final spectra. The finished spectra were baseline corrected.
One-way analysis of variance (ANOVA) with post hoc Holm-Sidak multiple comparison test or two-tailed Student’s t-test was used for statistical comparison (SigmaStat 3.0 version). If the data did not meet the parametric test assumptions, non-parametric statistics was performed, either Kruskal-Wallis Test (One Way Analysis of Variance on Ranks) followed by post hoc Dunn’s multiple comparison procedures or Mann-Whitney Rank Sum Test (SigmaStat 3.0 version). Variability of the estimates was reported as standard error of the mean (s.e.m).
The effects of the virally delivered BRI2-Aβ1-40 transgene were compared to effects of the rAAV1 delivered human BRI2 transgene and a non-injection control (Fig. 1B). Expression of BRI2 was intended to serve as a second control, as we had established that rAAV1-hGFP delivery and mock virus delivery did not alter Aβ deposition in the CRND8 APP mouse model (Levites et al., 2006a). Three months after rAAV1 mediated transgene delivery, mice were killed and brain Aβ deposition was analyzed using both biochemical and histochemical methods. These analyses revealed a dramatic suppressive effect of both the BRI2-Aβ1-40 and BRI2 transgenes on parenchymal Aβ1-40 and Aβ1-42 accumulation as measured by biochemical and histochemical assessments of Aβ levels (Fig. 1C–E).
The reduction in Aβ deposition observed in the mice expressing the rAAV1 BRI2-Aβ1-40 transgene was consistent with our previous transgenic mice studies (Kim et al., 2007), whereas the reduction of Aβ deposition observed in the mice expressing BRI2 was unexpected. Previous studies had demonstrated a potential interaction between BRI2 and APP and noted that BRI2 overexpression increased APP CTFβ and reduced Aβ secretion in cultured cells (Fotinopoulou et al., 2005; Matsuda et al., 2005). As in the studies of BRI2-Aβ transgenic mice crossed into Tg2576 mice (McGowan et al., 2005; Kim et al., 2007), we found no evidence for alterations in the steady state levels of APP or APP CTFβ in TgCRND8 mice expressing the virally delivered BRI2-Aβ1-40 or BRI2 transgenes (Fig. 2A,B). Moreover, levels of endogenous rodent Aβ levels in the brains of the non-transgenic littermates of the TgCRND8 mice expressing the BRI2 transgene were not altered (Fig. 2C). BRI2-Aβ1-40 expression slightly increased plasma Aβ40 levels, attributable to brain to plasma efflux of Aβ1-40; plasma Aβ1-42 levels were not significantly changed by BRI2 expression (Fig. 2D). Because of the rapid onset of Aβ depositon in TgCRND8 mice, it is not possible to measure steady state Aβ levels; therefore we conducted additional experiments in Tg2576 mice to determine whether BRI2 altered steady state Aβ prior to plaque deposition. rAAV1 mediated delivery of BRI2 to P0 Tg2576 did not lower steady state Aβ levels in brains of 2 month old mice. (Fig. 2E). As anti-Aβ antibodies reduce Aβ deposition in mice and expression of virally encoded Aβ peptides in the periphery has been shown to generate an anti-Aβ response, we examined whether central nervous system (CNS) delivery of the transgene induced a humoral immune response to Aβ. There was no evidence for an anti-Aβ titer in any of the rAAV1 injected mice (Fig. 2F). Collectively, these results in TgCRND8, wild type TgCRND8 littermates and Tg2576 mice demonstrate that the reduction of Aβ accumulation by BRI2 and BRI2-Aβ1-40 transgenes was not likely to be attributable to alterations in APP processing resulting altered Aβ production or induction of an anti-Aβ immune response.
To understand the mechanism by which BRI2 reduced Aβ accumulation, we tested whether the Bri2-23 peptide could directly inhibit Aβ1-42 fibrillogenesis in vitro. Several methods were used including a native gel assay previously employed to demonstrate that Aβ1-40 inhibits Aβ1-42 aggregation (Kim et al., 2007). When Aβ1-42 aggregation was assessed using the native gel assay we observed loss of the monomeric Aβ1-42 signal and the appearance of high-molecular weight (HMW) aggregates (Fig. 3A lane 10). Addition of Bri2-23 or Aβ1-40 to the reaction resulted in retention of the monomeric Aβ1-42 signal suggesting direct inhibition of aggregation (Fig. 3A compare lanes 10 to lanes 4, 6 and 8). Quantification by an ELISA capable of only recognizing monomeric Aβ confirmed that the Bri2-23 peptide retained Aβ1-42 in its monomeric state and this effect was more robust with increasing concentrations of Bri2-23 (Fig. 3B). To further analyze the effect of Bri2-23 on Aβ aggregates, we examined the effect of equimolar concentrations (25 μM or 50 μM) of the Bri2-23 peptide on monomeric Aβ1-42 aggregation into Aβ1-42 fibrils or protofibrils using Thioflavin T (ThT) fluorescence assay. Prolonged incubations of Bri2-23, by itself, did not result in aggregation or β-sheet formation as assessed by ThT fluorescence assay, change in CD spectra or insolubility (Data not shown) (Gibson et al., 2005). Co-aggregation of Aβ42 and Bri2-23 demonstrates that Bri2-23 appears to initially increase the rate of aggregate formation during the first 12 hours of incubation, but inhibits fibril formation at later time points (Fig. 3C). After 120–200 hours of incubation, Bri2-23 inhibited Aβ1-42 aggregation by 46% ±9% (n =6, p=0.0004). Atomic force microscopy (AFM) imaging confirmed the inhibitory effects of Bri2-23 on Aβ1-42 aggregation in these assays (Fig. 3D). These data show that Bri2-23 has a complex effect on aggregation of monomeric Aβ1-42; however, both assays are consistent with a net inhibitory effect of Bri2-23 peptide on amyloid formation presumably through inhibition of a later stage in fibril assembly.
These observations suggested that the anti-amyloidogenic effect of the BRI2 protein is mediated by an interaction between Bri2-23 and Aβ. To further test this idea in vivo, we generated a cDNA that expresses a truncated BRI2 protein lacking the Bri2-23 peptide (BRI2del244-266, Fig. 4A) and used rAAV gene transfer to deliver this construct to newborn TgCRND8 mice. Transgene positive mice were killed at 3 months of age and biochemical and histochemical Aβ loads were examined. Analyses of Aβ loads showed no significant difference between BRI2del244-266 and the control groups (Fig. 4B–D). Western blot analyses of brain lysates demonstrated that the somatic brain transgenic methodology produced roughly equivalent expression levels from the BRI2 and BRI2del244-266 constructs and somewhat higher levels from BRI2-Aβ1-40 (Fig. 4E). These later data and the lack of anti-amyloidogenic effect from BRI2del244-266 demonstrate that the Bri2-23 peptide sequence is critical for the inhibitory effect of BRI2 in vivo. Together with the data demonstrating that Bri2-23 directly inhibits Aβ aggregation in vitro, these data support an anti-amyloidogenic function for BRI2 mediated by the Bri2-23 peptide.
Our mouse data suggested that endogenous BRI2 could function, at least in part, by secretion of the Bri2-23 peptide as an anti-amyloidogenic binding partner of Aβ. To date, studies of normal BRI2 processing and secretion relied mainly on epitope tagged versions of the Bri2-23 peptide (Kim et al., 1999; Choi et al., 2004). First generation antibodies were not sensitive enough to detect the Bri2-23 peptide in biological samples. Thus, we developed a high performance liquid chromatography/Mass spectrometry (HPLC/MS)-based assay to detect secreted Bri2-23. We validated this methodology by detecting untagged Bri2-23 peptide secretion from H4 cells transfected with BRI2 but not BRIdel244-266 (Fig. 4F). We then tested normal human CSF and were able to detect the endogenously secreted Bri2-23 peptide in all samples tested (Fig. 4G). This finding strengthens the notion that the anti-aggregation effects of Bri2-23 peptide in our experiments may be physiologically relevant to human AD, FDD and FBD.
We have used somatic brain transgenic technology to deliver the BRI2 and BRI2-Aβ1-40 transgenes to the brains of APP mouse models. The studies with BRI2-Aβ1-40 confirmed previous studies obtained using conventional transgenic mice expressing BRI2-Aβ1-40 (McGowan et al., 2005; Kim et al., 2007). Thus, the somatic brain transgenic BRI2-Aβ1-40 studies provide additional validation for this rapid cost effective method of manipulating gene expression in the brain (Levites et al., 2006a).
The novel result from these studies was the finding that BRI2 suppresses Aβ deposition in APP CRND8 transgenic mice to an equivalent extent as Aβ1-40. Though it is not possible to completely rule out subtle effects on Aβ generation that could influence deposition, we found no evidence that the suppressive effect was mediated by alterations in APP processing or Aβ production. Instead, we find that the suppressive effect of BRI2 is likely to be mediated by inhibition of Aβ aggregation by the secreted peptide. We demonstrate that expression of the BRI2del244-266 construct that lacks a secreted peptide sequence has no effect on Aβ deposition following expression in vivo. BRI2del244-266 encodes a protein containing the region of BRI2 previously shown to interact with APP and interfere with APP processing in cell culture. Coupled with their inhibition of aggregation in vitro, we conclude that the Aβ1-40 and Bri2-23 peptides are directly responsible for reduced Aβ deposition in our experiments rather than any other part of the BRI2 protein scaffold on which they were delivered. Notably, in FDD brains, Aβ and the ADan peptide are co-deposited and bind to each other in vitro (Tomidokoro et al., 2005). These later findings suggest that the FDD-linked BRI2 mutation may corrupt a normally protective anti-amyloidogenic mechanism resulting in co-aggregation of the mutant peptide with a normal binding partner. In support of our observations, Bri2-23 contains the sequence FENKF that is homologous to peptide-based Aβ aggregation inhibitors incorporating a FxxxF motif (Sato et al., 2006). Moreover, solid state NMR analysis demonstrated direct binding of an 8 amino acid peptide containing the sequence FEGKF with the glycine zipper (G33xxxG37) segment of Aβ1-40, a sequence proposed to be critical for formation and stability of β-sheet structure (Liu et al., 2005; Sato et al., 2006).
Beyond the genetic link to FDD and FBD, little is known about the function of BRI2 and its homologues. BRI2 is encoded by the ITM2b gene located on chromosome 13q14.3, and is a member of a gene family consisting of BRI1 (ITM2A) and BRI3 (ITM2C) (Vidal et al., 2001; Akiyama et al., 2004; Choi et al., 2004). Orthologs are only found in higher eukaryotes. The BRI proteins share ~50% identify at the amino acid level, and are all expressed at modest (BRI1) to extremely high levels in the brain (BRI2, BRI3). They are relatively small (~260 aa) type 2 membrane proteins with single transmembrane domains, extracellular BRICHOS domains and furin cleavage sites near their carboxyl termini. At their carboxyl termini, they encode small peptides that, for BRI2 and BRI3, have been shown to be released and secreted following the furin cleavage (Kim et al., 1999; Wickham et al., 2005). Based on limited data, others have proposed that the BRICHOS domain targets the protein to the secretory pathway, performs an intramolecular chaperone-like function, and assists the specialized intracellular protease processing system (Sanchez-Pulido et al., 2002). Very recently BRI2 has been shown to be undergo sequential cleavage by ADAM10 to release its ectodomain and intramembrane proteolysis by SPPL2a and b (Martin et al., 2007). BRI2 has also been shown to undergo axonal transport (Choi et al., 2004). Nevertheless, other than the genetic link between BRI2 and FBD and FDD, almost nothing is known about the function of the BRI proteins (Ghiso et al., 2006).
Further study of BRI2 and the Bri2-23 peptide as well as analogous peptides released from the BRI2 homologues (which contain the conserved FxxxF motif) will be required to fully understand their anti-amyloidogenic action and other functions. The robust inhibitory effect of BRI2 on Aβ deposition in vivo and aggregation in vitro BRI2 indicates that BRI2 is a novel factor that modulates Aβ aggregation and deposition. These data support a novel approach to AD therapy or prevention based on increasing levels of BRI2 and more specifically the Bri2-23 peptide in the brain.
Support for this research was provided by: the NIH grants NIA RO1 AG18454 (T.G), the Robert H. and Clarice Smith and Abigail Van Buren Alzheimer’s Disease Research Program (T.G); Robert and Clarice Smith Postdoctoral Fellowship (BDM and VMM); A Development Award 0535185N from the American Heart Association (VR); and The Mayo Foundation. We would like to thank Dr. Eileen McGowan for providing the BRI2 cDNA. The authors acknowledge the technical assistance of Linda Rousseau, Virginia Phillips, Monica Casey-Castanedes, and John Gonzales, in the Neuropathology Laboratory at Mayo Clinic Jacksonville which is supported by the NIA grants AG25711, AG17216, AG03949 to D.D.