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Amyloid beta (Aβ) has been identified as a key component in Alzheimer’s disease (AD). Significant in vitro and human pathological data suggest that intraneuronal accumulation of Aβ peptides plays an early role in the neurodegenerative cascade. We hypothesized that targeting an antibody-based therapeutic to specifically abrogate intracellular Aβ accumulation could prevent or slow disease onset. Aβ42-specific intracellular antibodies (intrabodies) with and without an intracellular trafficking signal were engineered from a previously characterized single-chain variable fragment (scFv) antibody. The intrabodies, one with an endoplasmic reticulum (ER) targeting signal and one devoid of a targeting sequence, were assessed in cells harboring a doxycycline-regulated mutant human amyloid precursor protein (hAPPswe) transcription unit for their abilities to prevent Aβ peptide egress. Adeno-associated virus (AAV) vectors expressing the engineered intrabodies were administered to young adult 3xTg-AD mice, a model that develops amyloid and Tau pathologies, prior to the initial appearance of intraneuronal Aβ. Chronic expression of the ER-targeted intrabody led to partial clearance of Aβ42 deposits and interestingly, in reduced staining for a pathologic phospho-Tau epitope (Thr231). This approach may provide insights into the functional relevance of intraneuronal Aβ accumulation in early AD and potentially lead to the development of new therapeutics.
The accumulation of intraneuronal amyloid-beta (Aβ) occurs during initial stages of the AD pathophysiologic cascade, yet this disease process remains relatively understudied as compared to classic amyloid plaque and neurofibrillary tangle pathologies. Significant in vitro and human pathological data suggest that intraneuronal Aβ peptides play an early triggering role in AD-related neurodegeneration. Masters and colleagues first reported marked staining of intraneuronal Aβ in pyramidal neurons of the hippocampus and entorhinal cortices of AD patients . More recently, intracellular Aβ staining was detected prior to the appearance of paired helical filament-positive structures, further indicating that intraneuronal Aβ is one of the earliest documented AD-related changes. This alteration has also been suggested by Chiu et al. to strongly correlate with cell damage and apoptotic cell death in AD patients . Similar observations have been made in mouse AD models that neuronally overexpress Aβ peptides and in primary neuronal cultures transduced with viral vectors expressing human APP [3, 4]. Moreover, familial AD mutations in APP lead to different profiles of intracellular Aβ accumulation, where the Swedish APP mutation results in a 2–3 fold increase in intracellular Aβ levels as compared to cells expressing the wild-type human APP gene .
Increased oxidative stress, another early event in the AD pathologic cascade, exhibits a mechanistic connection with intracellular Aβ. Experimental application of an oxidative stressor, such as H2O2, to cells expressing human APP results in enhanced intracellular Aβ levels and a concomitant decrease in full-length APP and carboxy-terminal fragments. In this prior study, APP gene expression was unchanged, suggesting that oxidative stress fosters intracellular Aβ peptide generation via alteration of APP proteolytic processing . These data, in aggregate, point to intracellular Aβ accumulation as being not only a sentinel cellular process, but also a potentially viable therapeutic target.
To address the latter, we engineered a previously characterized Aβ-specific single-chain variable fragment (scFv) antibody  to specifically and efficiently abrogate the downstream pathologic effects of intracellular Aβ accumulation. ScFv’s are composed of the minimal antibody-binding site formed by non-covalent association of the VH and VL variable domains joined by a flexible polypeptide linker (reviewed by ). Further antibody engineering makes it possible to manipulate the genes encoding scFv’s for antibody binding site expression within mammalian cells, while in-frame fusion of the scFv gene to intracellular targeting signals facilitates specific subcellular localization [9, 10]. These intracellular antibodies, termed intrabodies, are capable of modulating target protein function by blocking or stabilizing macromolecular interactions; by modulating enzyme function through substrate sequestration, active site occlusion or active/inactive conformation stabilization; and/or by diverting proteins to alternative intracellular compartments (reviewed by  and ). In the present study, Aβ-specific intrabodies with differing intracellular trafficking characteristics were engineered into recombinant adeno-associated virus (rAAV) vectors. Focal stereotactic infusion of a rAAV vector expressing an endoplasmic reticulum-targeted anti-Aβ scFv into the hippocampi of young adult triple-transgenic Alzheimer’s disease (3xTg-AD) mice resulted in significant suppression of amyloid and Tau pathologies, indicating specific subcellular targeting of these promising therapeutics has the potential to disrupt downstream intraneuronal Aβ-associated pathological processes.
To facilitate the analysis of anti-Aβ42 intrabody expression and subcellular localization in vitro, a stably transfected human amyloid precursor protein Swedish mutant (hAPPswe) cell line was generated. Of note, eukaryotic cells exhibit evidence of toxicity when exposed to specific forms of proteolytically derived peptides of hAPP . Hence, we utilized a previously described doxycycline-regulated pBIG2i vector system to strictly control hAPPswe expression and avoid transgene-related toxicity during generation of stably transfected clonal cell lines . Following addition of the tetracycline homologue doxycycline (Dox), expression of the chimeric reverse tetracycline transactivator (rtTA) is feed-forward-activated via a tetracycline (Tet) operator-controlled synthetic thymidine kinase (TK*) promoter, while a synthetic CMV promoter (CMV*) is simultaneously up-regulated to drive expression of the human APPswe transgene and the downstream reporter gene, enhanced green fluorescent protein (eGFP), via an internal ribosomal entry site (IRES). This construct, designated pBIG2i(hAPPswe) and schematically illustrated in Fig. 1A, was transfected into baby hamster kidney (BHK) cells and placed under hygromycin selection (600 μg/ml). Positive BHK-hAPPswe clones were expanded and co-immunocytochemistry was performed for eGFP and hAPPswe/Aβ (using the 6E10 monoclonal antibody) on cells incubated in the absence or presence of 0.5 or 2 μg/ml Dox. Cells were visualized using phase contrast and multi-color fluorescence microscopy. Neither eGFP nor hAPPswe/Aβ proteins were detectable in the absence of Dox (Fig. 1B–E). Robust expression of both eGFP and hAPPswe/Aβ was apparent when BHK-hAPPswe cells were incubated in the presence of 0.5 and 2 μg/ml Dox (Fig. 1F–M). Co-expression of the two transgenes was significant, but there appeared to be a minor subset of cells that exhibited preferential expression of eGFP or hAPPswe.
Biosynthesis and post-translation modification of amyloid precursor protein (APP) involves subcellular trafficking through the secretory pathway of the cell, initiating within the endoplasmic reticulum (ER) . Here, APP undergoes a number of proteolytic processing events that are mediated by the α-secretase, which is a component of the non-amyloidogenic pathway, or the β- and γ-secretase complexes, which liberate pathogenic Aβ peptides as a result of the amyloidogenic processing pathway . Available evidence suggests that when α-secretase cleaves the APP molecule this precludes the pathological generation through β-secretase activity of Aβ fragments 1 to 40 and 1 to 42 [17–20]. Under circumstances where β-secretase cleavage is enhanced or α-secretase cleavage is diminished, pathological Aβ accumulation is augmented. Derivation of an anti-Aβ therapeutic that could encounter and undermine the pathogenic activity of Aβ at the point of its initial generation could significantly impact disease progression. To this end, we engineered a previously obtained single-chain antibody that specifically binds to the highly fibrillogenic Aβ42 peptide  into an “intrabody” to be differentially trafficked within an expressing neuron. The original antibody, termed Aβ42.2, effectively prevented Aβ deposition when it was administered passively to amyloidogenic mice prior to amyloid formation . The Aβ42.2 monoclonal antibody, was subsequently converted into a single-chain antibody fragment, and was extensively characterized . The resultant scFv, ScFv42.2, retained its selectivity for Aβ42, as was demonstrated by pull-down with Aβ fibrils and by ELISA. When expressed intracranially via AAV injection into the cerebral ventricles of newborn mice, scFv42.2 prevented amyloid formation in CRND8 mice.
In the present study, two recombinant adeno-associated virus (rAAV) vectors were constructed (Fig. 2A): one expressing an Aβ42-specific intrabody (IB) sequence with a c-myc epitope tag at the C-terminus to facilitate immunocytochemical detection (rAAV-scFvAβIB), and a second expressing the same c-myc tagged intrabody but with an endoplasmic reticulum targeting signal (KDEL) inserted in-frame between the intrabody coding sequence and c-myc epitope (rAAV-scFvAβKDELIB). The individual transgenes were placed under the transcriptional control of the human cytomegalovirus (CMV) promoter. A SV40-derived polyadenylation signal was included at the 3′ end of the transcription unit, which in total was flanked by AAV genome-derived inverted terminal repeat sequences (ITR). The resulting rAAV-scFvAβIB and rAAV-scFvAβKDELIB plasmids were transiently transfected into BHK-hAPPswe cells incubated in the presence of 2 μg/ml Dox, while non-transfected, Dox-treated BHK-hAPPswe cells were used as negative controls. Forty-eight hours post-transfection, co-immunocytochemistry was performed for eGFP, hAPP/Aβ, and the c-myc epitope tag, and images were obtained by confocal fluorescence microscopy (Fig. 2B–R). In parallel, the intrabody plasmids were co-transfected individually with a hAAPswe-expressing plasmid (pCMV-hAPPswe) into the neuroblastoma-derived cell line, Neuro2A (Fig. 2Y-Ar). Both anti-Aβ42 intrabodies were readily detectable within transfected BHK-hAPPswe and Neuro2A cells by immunocytochemistry. Moreover, the extent of co-localization of anti-Aβ42 intrabody and hAPP/Aβ staining was substantial, suggesting that each was residing within similar subcellular compartments.
To more definitively assess the subcellular localization of each intrabody, immuno-electron microscopy was performed. The rAAV-scFvAβIB and rAAV-scFvAβKDELIB plasmids were transiently transfected into BHK cells, while non-transfected BHK cells were used as negative controls. Forty-eight hours post-transfection, cell monolayers were fixed and processed for immuno-electron microscopy using a c-myc epitope-specific antibody for detection of the engineered intrabodies. BHK cells expressing rAAV-scFvAβKDELIB exhibited a qualitative enhancement in electron-dense signal localizing to ER-related ultrastructures (indicated by black arrows) as compared to rAAV-scFvAβIB-expressing cells and non-transfected control cells (Fig. 3A–C). The relative intensities of ER-associated signal could not be attributed to differential levels of intrabody expression as quantitative real-time RT-PCR analysis of transfected cultures indicated that transcripts encoding each intrabody were expressed at similar levels (data not shown).
To assess relative steady-state levels of the intrabodies at 48 h after transfection of BHK cells, Western blotting was performed. Incubation of blots with a myc epitope-specific antibody led to the detection of low, but similar, levels of each intrabody at the expected size of 36 kDa (Fig. 3D). To ensure that molecular engineering did not lead to ablation of target antigen recognition, the Aβ42-binding activity of each intrabody was tested by ELISA. BHK cells were transiently transfected with the rAAV-scFvAβIB and rAAV-scFvAβKDELIB plasmids, and 48 h later supernatants and cell lysates were collected and applied to 96-well plates coated with Aβ42 peptide. Non-transfected cell supernatants and lysates were used as negative controls. Intrabody binding to the Aβ42-coated microtiter plates was detectable in lysates generated from cells transfected with rAAV-scFvAβIB and rAAV-scFvAβKDELIB plasmids. Supernatants from these cultures harbored significantly less Aβ42 binding activity, suggesting that both anti-Aβ42 intrabodies were retained within the transfected cell (Fig. 3E). We separately transfected Dox-induced BHK-APPswe cells with rAAV plasmids encoding the intrabodies or rAAV-scFvPhe control plasmid and analyzed culture supernatants for Aβ40 and Aβ42 by ELISA. Only cultures transfected with the ER-targeted anti-Aβ42 intrabody construct exhibited a statistically significant diminution in secreted Aβ42 (Fig. 3F). While culture supernatants from cells transfected with the non-targeted intrabody showed a trending decrease in Aβ42 levels, this decrease did not reach statistical significance as compared to non-transfected or rAAV-scFvPhe transfected-cultures. Due to the Dox-regulated expression of the Swedish APP mutant in these cells, only Aβ42 is detectable, while Aβ40 concentrations lie at baseline levels.
To determine whether chronic Aβ42-specific intrabody expression in vivo could abrogate amyloid-related pathology, and whether subcellular targeting of the intrabody would influence its effectiveness in doing so, we subsequently packaged the rAAV-scFvAβIB and rAAV-scFvAβKDELIB plasmids into serotype 2 virions and delivered these constructs intrahippocampally to triple-transgenic Alzheimer’s disease mice (3xTg-AD). The 3xTg-AD mouse, created in the LaFerla laboratory, develops intracellular Aβ, amyloid plaques and neurofibrillary tangles (NFTs) in a progressive and age-related pattern [21–23]. Two month-old male 3xTg-AD mice were stereotactically infused with rAAV-scFvAβIB, rAAV-scFvAβKDELIB, or an eGFP-expressing rAAV vector control (rAAV-eGFP) unilaterally into the CA1 layer of the hippocampal formation (n=6 per group). An equivalent volume of saline was identically infused into the contralateral hippocampus to serve as a no-vector control. Mice at this age exhibit neither pathological nor behavioral signs of AD [23, 24]. Nine months post-transduction, animals were sacrificed and brains were sectioned for further analyses. Co-immunocytochemistry for intrabody expression and GRP94, an ER-localized protein, and subsequent staining of cellular nuclei with DAPI revealed confocal co-registration of fluorescent signals for GRP94 and the rAAV vector-expressed intrabodies, but not with the nuclear stain (Fig. 4A–R). We also performed triple immunocytochemistry for the myc epitope tag of the intrabodies, Aβ42 and phospho-Tau (as detected by the AT180 antibody) and used confocal microscopy to visualize the spatial relationship of intrabody expression and the accumulation of Aβ42 and phospho-Tau. The photomicrographs show, especially for the ER-targeted Aβ42 intrabody, the staining patterns/intensities for Aβ42 and phospho-Tau are altered in regions expressing the intrabodies (Fig. 4S-Ag). Unfortunately, the fluorescein isothiocyanate (FITC) pre-conjugated primary antibody used to detect the c-myc epitope led to higher background signals in neuronal processes for this triple immunocytochemical assessment, but even given this technical caveat, relative proximity information relating to localization of intrabody, Aβ42 and phospho-Tau could be gleaned.
To determine the extent to which AAV-vectored Aβ42-specific intrabody expression altered the severity of AD-related amyloid and Tau pathologies in 3xTg-AD mice and whether differential subcellular targeting of the intrabody altered therapeutic outcome, we subsequently performed immunohistochemistry for human APP, extracellular Aβ42, and phospho-Tau. Although the intrabodies were designed to be specific for Aβ42, it was possible that chronic expression in vivo could impact the accumulation of the human APPswe transgene product, which would suggest that the specificity of the intrabodies is not absolute. This potential caveat may prove disadvantageous therapeutically given APP plays a likely role in normal neuronal physiology and its untoward removal could lead to yet unknown deleterious consequences (reviewed by ).
Immunohistochemical assessment using the human APP-specific Y188 antibody indicated that chronic Aβ42-specific intrabody expression did not overtly alter the pattern or intensity of hAPPswe staining within the transduced CA1 layer of the 3xTg-AD hippocampus (Fig. 5A–P). Immunohistochemical staining of hippocampi from AAV vector-infused 3xTg-AD mice using the Aβ42-specific 12F4 monoclonal antibody and the phospho-Tau specific AT180 antibody demonstrated that Aβ42-specific intrabody expression (cytoplasmically or ER-localized) qualitatively impacted amyloid and Tau pathologies when saline control hemispheres were compared to contralateral vector-injected hemispheres (Fig. 5Q-Av). Stereologic examination of vector-infused hippocampal regions revealed inter-treatment group quantitative differences in relation to the severity of extracellular Aβ42 deposition and appearance of a pathologically relevant phospho-Tau epitope (Fig. 6). Although rAAV-scFvAβIB-treated mice collectively exhibited trending decreases in extracellular Aβ42 and phospho-Tau burden within their vector-injected hemispheres relative to saline-injected contralateral hippocampi, these differences did not reach statistical significance. Mice receiving rAAV-scFVKDELIB manifested statistically significant differences in both extracellular Aβ42 and intracellular phospho-Tau staining compared to controls. Negative control groups (rAAV-eGFP and saline-injected 3xTg-AD mice) did not exhibit notable differences in the severities of the AD-related pathologies analyzed. We do not believe that intrabody-mediated epitope masking accounts for the apparent diminution in extracellular Aβ42. If that were the case, it would not be expected a priori that the staining for the phospho-epitope of Tau at residue Thr231 using the AT180 antibody would have significantly decreased. Our findings further confirmed that therapeutic approaches designed to abrogate Aβ accumulation can influence Tau-related pathological outcomes in 3xTg-AD mice [26–28].
The marked prevention of extracellular Aβ42 deposition and delay in appearance of a disease-related phospho-epitope of Tau in 3xTg-AD mice observed in the present study supports the feasibility of chronically expressed anti-Aβ intracellular scFv antibodies to interdict AD pathogenesis in vivo. Other investigators have pursued viral vector-based approaches to deliver scFv’s as a means to target extracellular Aβ. Levites and colleagues compared three scFv’s specific to three distinct linear epitopes: Aβ1–16, monomeric Aβ40, and monomeric Aβ42 . CRND8 AD mice were given intraventricular injections at P0 and sacrificed 3 to 5 months later. At 3 months each scFv demonstrated a decrease in plaque load, a decrease in SDS-soluble Aβ40 and 42 levels, as well as promoted the efflux of Aβ-scFv complexes into the plasma. Fukuchi et al. used serotype-2 rAAV vectors to deliver scFv’s that recognized tetrameric Aβ to the brains of Tg2576 AD mice [29, 30]. A decrease in Aβ load was observed via immunohistochemistry in scFv-injected mice compared to PBS-infused control animals. Controversy does exist as to how scFv’s promote the removal and/or degradation of target antigens from the extracellular space in vivo given their lack of an Fc segment, which is classically recognized by cognate receptors on brain microglia for facilitation of phagocytosis. However, despite the unknowns, these prior in vivo studies provide confidence that the general methods of viral vectored Aβ-specific scFv’s are well-tolerated and effective in diminishing amyloid-related pathology in AD mouse models.
Perhaps most relevant to our approach to employ modified scFv’s for intracellular Aβ targeting relates to the work from Lecerf et al.  and more recently by Paganetti et al.  and Lynch et al. . The former identified human scFv intrabodies capable of interacting in situ with huntingtin thereby reducing its ability to aggregate . These scFv’s were bound to the N-terminal residues of huntingtin and maintained the normally aggregated protein in a soluble complex that subsequently underwent normal protein turnover. Specificity of binding was further determined by fusing the anti-huntingtin scFv with a nuclear localization signal and the subsequent retargeting of soluble huntingtin to cell nuclei. Prevention of specific aggregate formation in cellular models of Huntington’s disease suggested that intracellular scFv’s may represent a viable therapy for this disease as well as other neurodegenerative diseases with abnormal protein processing/accumulation/aggregation such as Aβ in AD. To that end, Paganetti and colleagues demonstrated that intrabodies designed to bind to an epitope proximal to the β-secretase cleavage site of human APP significantly blocked Aβ generation in a cell culture model . In fact, these authors also found that ER targeting of the intrabody resulted in more efficient blocking of APP processing. More recently, Lynch et al. isolated and tested scFv-based intrabodies specific for the nonamyloid component (NAC) of α-synuclein . The authors demonstrated that an anti-NAC intrabody could redirect synuclein trafficking intracellularly and showed that a neural progenitor cell line stably expressing one of these intrabodies exhibited significantly decreased mutant synuclein aggregation.
Although 3xTg-AD mice receiving the ER-targeted anti-Aβ42 intrabody exhibited evidence of improved AD-related pathological status as compared to control animals, this strategy portends a number of potential caveats. The possibility exists that intrabody-mediated retention of Aβ42 intracellularly could lead to increased APP processing that would yield more of the fibrillogenic Aβ42 species . This may result in enhanced intracellular aggregation and cellular dysfunction. Moreover, we are aware that high concentrations of intrabody/Aβ complexes intralumenally could inherently block normal cellular protein maturation, ultimately leading to cellular stress. Excessive aggregation could also lead to proteasomal activity inhibition . In the present study, we did not observe overt loss of neurons within the transduced CA1 layer of the hippocampus. However, if such an outcome were to occur, a weaker promoter could be employed in the rAAV vector backbone that would lead to lower levels of intrabody expression, proteasome-targeting sequences could be appended to the intrabodies to facilitate degradation , or even regulatable rAAV vectors could be engineered to finely control intrabody gene expression. A variety of regulated rAAV vector platforms have been developed and shown to be effective in vivo [37, 38].
Given the advances in brain-wide dissemination via convection-enhanced delivery (CED) of AAV vector particles, we believe that an intrabody-based approach has significant therapeutic merit in the future. CED was first described by Bobo and colleagues  and has steadily gained acceptance for widespread distribution of small-molecule therapeutics and viral vectors within the brain [40, 41]. This local infusion technique uses bulk flow to enable the delivery of small and large molecules to clinically significant volumes of targeted tissues, offering an improved volume of distribution (Vd) compared to simple diffusion. We envision at least pan-hippocampal diffusion of the intrabody-expressing AAV vector would be required as well as delivery to entorhinal cortex, which are regions affected earliest in AD. Moreover, AAV vectors have the ability to retrogradely migrate along axons, enabling dissemination to distal brain regions, if necessary and carefully monitored during infusion using advanced imaging techniques .
Gaining an enhanced understanding of mechanisms relating to target antigen clearance and the subversive role of intracellular Aβ in disease pathophysiology, as well as devising approaches that will not significantly overburden a compromised proteasomal machinery within AD-afflicted neurons provide significant challenges. However, the growing literature detailing the successful implementation of intrabody-based therapeutics in preclinical models of neurodegenerative diseases and tumorigenesis presents ample justification for optimization of the platform to enhance its safety profile and for future development and testing in a clinical setting.
The gene encoding the Swedish mutant form of human amyloid precursor protein (hAPPswe) was removed from its parental vector (kindly provided by Dr. William Van Nostrand) and cloned into the pBIG2i vector . The pBIG2i(hAPPswe) construct was stably transfected into BHK cells and placed under hygromycin selection (600 μg/ml). Expression of the hAPPswe gene was induced with doxycycline (2 μg/ml or 0.5 μg/ml) and inducible hAPPswe expression was subsequently confirmed by immunocytochemistry. Three separately generated cell clones were isolated and analyzed.
PCR was used to amplify the anti-Aβ42 213scFv antibody sequence from the parental pSecTag construct (Invitrogen, Carlsbad, CA) to remove its artificial Igκ secretion signal (while retaining an endogenous leader peptide) and facilitate its cloning into a cytomegalovirus (CMV) immediate-early promoter-containing shuttle plasmid, pBSFBRmcs . To complete the rAAV-scFvAβKDELIB vector, the intermediate plasmid was subsequently digested with AflIII and EcoRI and the following phosphorylated double-stranded linker encoding the KDEL ER-targeting sequence was ligated into the plasmid: sense strand – 5′-CATGTAAGGACGAGCTGTGAG-3′ and anti-sense strand – 5′-AATTCTCTCAGCTCGTCCTTA-3′. The CMV promoter-driven intrabody expression cassettes were excised out of the respective pBSFBRmcs intermediates with NotI and cloned into a NotI-cut pFBGR rAAV plasmid (kindly provided by Dr. R. Kotin). Each resultant rAAV plasmid was packaged into serotype 2 rAAV virions along with an enhanced green fluorescent protein encoding rAAV control vector, rAAV-egfp, using a baculovirus-based method . A previously described rAAV vector plasmid expressing a phenobarbital-specific scFv (scFvPhe) was used in a subset of in vitro studies as a negative control .
Dox-treated (2 μg/ml) or non-treated BHK-hAPPswe cells were plated on glass cover slips and transiently transfected with the rAAV plasmids encoding the anti-Aβ42 intrabodies or scFvPhe control plasmid. Neuro2A cells were plated identically and transiently co-transfected with one of the scFv plasmids described above with pCMV-hAPPswe, a plasmid that expresses the Swedish mutant of human APP. The cells were stained for expression of the scFv’s (anti-c-myc; Rockland Immunochemicals, Gilbertsville, PA) and expression of hAPPswe/Aβ (6E10; Signet, Dedham, MA). The expression of eGFP was also noted. Confocal fluorescent images were obtained at 488 nm, 568 nm, and 647 nm.
Companion BHK-hAPPswe cultures were plated in 6-well dishes, transiently transfected with 1.5 μg intrabody-expressing plasmids or rAAV-scFvPhe control plasmid per well, and lysates analyzed by Western blotting using the 9E10 anti-myc epitope antibody (1:500, Sigma). An anti-β-actin antibody (1:3000; Sigma, St. Louis, MO) was used to re-probe blots to assess loading consistency. Blots were developed using the Perkin-Elmer Western Lightning Kit (Perkin-Elmer, Waltham, MA).
Companion BHK-hAPPswe cultures were plated and transiently transfected with intrabody-expressing plasmids for ELISA-based assessment of their respective Aβ42 binding activities. Following transfection, cells were incubated for 48 h at 37°C. Supernatants were collected and lysates generated. Microtiter plates (Corning, NY) were coated using 500 ng of Aβ42 peptide per well (Tocris Cookson, Ellisville, MO). Plates were washed followed by addition of a dilution series of cell supernatants or lysates in PBS added in triplicate, or a positive control rabbit anti-Aβ antibody of (1:5000, Chemicon International, Temecula, CA) to appropriate wells. Appropriate secondary antibodies were added (1:3000; goat anti-c-myc, Novus Biologicals, Littleton, CO or 1:1000; goat anti-rabbit, Jackson Laboratories, West Grove, PA) and plates were developed using 3,3,5,5,-tetramethyl benzidine (Sigma-Aldrich) and phosphate citrate buffer (Sigma-Aldrich). Plates were analyzed at an absorbance of 450 nm using a Bio-Rad Model 550 microplate reader (Bio-Rad, Hercules, CA).
Dox-treated BHK-hAPPswe cultures (2 μg/ml) were plated in 24-well dishes, transiently transfected with 0.4 μg intrabody-expressing plasmids or rAAV-scFvPhe control plasmid per well, and 48 h later culture supernatants were analyzed using ELISA kits specific for amyloid-beta 40 and 42 according to manufacturer’s instructions (Covance, Berkeley, CA).
BHK cells were plated into a 12-well tissue culture plate onto glass coverslips at a density of 1×105 cells/well. Cells were transfected the following day, and after a 24-h incubation, cells were fixed, permeabilized, and incubated with blocking solution (1% BSA, 2% NHS, 0.1% fish gelatin, 0.01% Triton X-100). The primary mouse monoclonal 9E10 anti-c-myc (1:100, Sigma, St. Louis, MO) and biotinylated goat anti-mouse secondary antibody (1:2000, Vector Laboratories, Burlinghame, CA) were used. Coverslips were post-fixed with 2% glutaraldehyde, dehydrated and embedded in Epon in preparation for electron microscopy. Ultrathin sections were counterstained with uranyl acetate followed by lead citrate and examined using a Hitachi 7100 transmission electron microscope. Images for single-chain antibody localization were taken using a MegaView III digital camera and AnalySIS (Soft Imaging Systems, Lakewood, Colorado) software. Images were captured at 10,000X and 30,000X magnification.
Recombinant AAV vectors (3 × 109 transduction units) were stereotactically delivered into 2 month-old male and female 3xTg-AD mice (n=6 per condition) in accordance with approved University of Rochester animal use guidelines as described previously . At 11 months of age, vector-injected mice were sacrificed and perfused with 4% paraformaldehyde.
Paraformaldehyde-fixed brains removed, sectioned into 30-μm coronal sections using a sliding microtome, and processed for immunocytochemistry as previously described . Brain sections were incubated overnight at 4°C with primary antibodies specific for the ER marker GRP94 (rabbit anti-mouse, 1:500 dilution, Abcam) and the c-myc epitope tag incorporated into the intrabody sequences (mouse anti-c-myc, 1:1000 dilution, 9E10, Sigma). Fluorescently labeled secondary antibodies (goat anti-rabbit Alexa 488 for GRP94 and goat anti-mouse Alexa 568 for c-myc, 1:2500 dilution, Invitrogen) were subsequently used. Sections were washed and then histochemically stained with 4′,6-diamidino-2-phenylindole (DAPI). For AD-related protein co-localization analyses, the following primary antibodies were employed: rabbit anti-Aβ42 (1:1000, Invitrogen), anti-human phosphorylated Tau AT180 (Pierce, Rockford, IL; 1:200), and pre-FITC conjugated 9E10 (1:1000). The secondary antibodies Alexa 568 for anti-Aβ42 and Alexa 450 for phospho-Tau were then used. Imaging was performed using a Zeiss Scanning confocal microscope (Carl Zeiss Inc., Minneapolis, MN).
The following antibodies were used at the designated working dilutions: anti-amyloid precursor protein A4, corresponding to the NPXY motif of hAPP, (Clone Y188; AbCam, Cambridge, MA, 1:750); anti-amyloid beta 1–42 clone 12F4 reactive to the C-terminus of Aβ42 (Covance, Berkeley, CA, 1:1000) and anti-human phosphorylated Tau AT180, specific for hTau phosphorylated at the Thr231 residue (Pierce, Rockford, IL; 1:200). For Aβ peptide-specific detection, the sections were treated with 70% formic acid for 15 min. for epitope retrieval. The sections were further processed for immunohistochemistry as previously described . Sections were viewed using an Olympus AX-70 microscope and motorized stage (Olympus, Center Valley, PA) and the MCID 6.0 Imaging software (Interfocus Imaging, Cambridge, England).
Quantification of positively stained targets was performed as previously described . Three consecutive images per tissue section (10 sections per mouse) were obtained at 40X magnification in the CA1 region of the hippocampus using an Olympus AX-70 microscope equipped with a motorized stage (Olympus, Melville, NY). Sections corresponding to 2.5 mm to 2.9 mm posterior from Bregma were analyzed. Estimated target numbers were determined for each area using MCID 6.0 Elite Imaging Software (Imaging Research, Inc.).
Data were analyzed by means of Student T-test or analysis of variance (ANOVA), followed by post-hoc comparison using Bonferroni’s method in the GraphPad Prism v.4.0 (GraphPad Prism Software, San Diego, CA) data analysis software package. P<0.05 was considered statistically significant.
Supplemental Figure 1. Representative photomicrographs of AT180-stained 3xTg-AD mice receiving hippocampal infusions of rAAV-vectored intrabody constructs. Coronal brain sections from 11 month-old 3xTg-AD animals that were unilaterally injected with rAAV-eGFP (A-D), rAAV-scFvAβIB (I-L), rAAV-scFvAβKDELIB (Q-T) or saline (E-H, M-P, U-X) were processed for immunohistochemical analyses of human hyperphosphorylated Tau (AT180 antibody). Representative images of the infused hippocampus from 4 mice from each treatment group (animal number designations are shown) are displayed at 10X. Scale bar in Panel X indicate 500 μm
The authors wish to thank Dr. Linda Callahan (University of Rochester) for immunohistochemistry and microscopy advice, Sarah Woods (University of Rochester) for stereological assessments, and Karen L. de Mesy Bentley (University of Rochester) for electron microscopy services and advice. Supported by NIH R01-AG020204 to HJF, and NIH R01-AG023593 and NIH R21-AG031878 to WJB.