Decreased neuronal TGF-β signaling in vivo promotes age-related neurodegeneration.

Because TGF-βs can be neuroprotective (7, 8) and appear to be essential for maintaining neuronal integrity and survival in vivo (12), we tested whether reduced TGF-β signaling in neurons leads to neurodegeneration in the CNS. We generated transgenic mice expressing kinase-deficient TβRII (TβRIIΔk), which functions as a potent inhibitor of TGF-β signaling and has been used in other transgenic mice to specifically inhibit signaling in pancreatic or skin cells (28, 29). We expressed TβRIIΔk under control of regulatable tetracycline operator sequences (tetO) in neurons by crossing tetO-TβRIIΔk mice with previously described prion promoter–driven transactivator tTA (Prnp-tTA) mice (30). Prnp-tTA mainly restricts tetO-driven transgene expression to neurons in the cortex and hippocampus (30). Double-transgenic TβRIIΔk/Prnp-tTA mice expressed TβRIIΔk mRNA in a doxycycline-regulatable manner (Figure ​(Figure2A).2A). In subsequent experiments, transgene expression was left on (i.e., no doxycycline added) throughout the lifetime of the mice. Primary mixed hippocampal/cortical neurons from TβRIIΔk/Prnp-tTA mice showed increased cell death compared with control cultures (Figure ​(Figure2B),2B), demonstrating transgene bioactivity as well as a trophic function for TGF-β signaling in neurons. In contrast, the number of glial cells, which represented about 5% of cells in these cultures, was unchanged (data not shown).

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Figure 2

Reduced neuronal TGF-β signaling increases neurodegeneration in vivo.

(A) RNase protection assay shows TβRIIΔk mRNA expression and inhibition of transgene expression in transgenic mice caused by feeding doxycycline-containing food pellets. Arrows on the left indicate undigested (UD) riboprobes; arrows on the right indicate the corresponding protected fragments. TβRII represents the wild-type receptor. D, digested riboprobe. Each lane represents 1 animal. (B) TβRIIΔk-expressing primary neurons show reduced survival in culture. Primary neuron cultures were stained with BODIPY-FL-Fallacidin and Hoechst after 4 days in culture, and the number of viable and dead cells was counted based on the absence or presence of pyknotic nuclei. Data are mean ± SEM of triplicate wells of 3 mice per genotype. (C–K) Sagittal brain sections of 34-month-old (C–K) as well as 4-month-old (E, H, and K) TβRIIΔk/Prnp-tTA or Prnp-tTA mice were immunolabeled for MAP2 (C–E), synaptophysin (F–H), and NeuN (I–K), and percent immunoreactive area (IR) of the neuropil (C–H) or mean number of labeled cells (I–K) in the neocortex was determined by confocal microscopy and computer-aided image analysis. Scale bars: 50 μm. Images are examples from cases with clearly visible degeneration. Data are mean ± SEM (n = 5–7 mice per genotype). *P < 0.05, ***P < 0.005; Student’s t test.

Chronic expression of TβRIIΔk in neurons resulted in age-dependent neurodegeneration (Figure ​(Figure2,2, C–K, and Table ​Table1).1). Brains of 34-month-old double-transgenic TβRIIΔk/Prnp-tTA mice showed significant decreases in microtubule-associated protein 2–immunoreactive (MAP2-immunoreactive) dendrites (Figure ​(Figure2,2, C–E), synaptophysin-immunoreactive presynaptic terminals (Figure ​(Figure2,2, F–H), and NeuN-positive neurons (Figure ​(Figure2,2, I–K) compared with littermate control mice. Consistent with this neurodegeneration, GFAP immunoreactivity was significantly increased (Table ​(Table1).1). A trend toward neurodegeneration was already observed in the brains of mice at 4 and 10 months of age (Figure ​(Figure2,2, E and K, and Table ​Table1). 1).

Table 1

Effect of neuronal TGF-β signaling on neurodegeneration and astrocytosis

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Decreased neuronal TGF-β signaling in hAPP transgenic mice increases cerebral Aβ accumulation and dendritic degeneration.

To study the potential effect of decreased TGF-β signaling in the pathogenesis of AD, we first measured levels of endogenous mouse Aβ in old TβRIIΔk/Prnp-tTA and control mice in the absence of familial AD mutations. At 34 months of age, TβRIIΔk/Prnp-tTA mice showed a clear trend toward higher endogenous Aβ₄₀ and Aβ₄₂ levels (as measured by ELISA) compared with Prnp-tTA mice, but this did not reach statistical significance (Aβ₄₀, TβRIIΔk/Prnp-tTA, 377.4 ± 75.7; Prnp-tTA, 281.5 ± 13.5; P = 0.5; Aβ₄₂, TβRIIΔk/Prnp-tTA, 233.2 ± 19.3; Prnp-tTA, 209.0 ± 31.5; P = 0.5). In addition, no Aβ deposits were detected by immunohistochemistry in any of the mice. We therefore crossed a mouse model for AD (PDGF-hAPP, line J20; ref. 31) with double-transgenic TβRIIΔk/Prnp-tTA mice, and analyzed Aβ deposition and neurodegeneration. Twenty-month-old hAPP mice with decreased neuronal TGF-β signaling (TβRIIΔk/Prnp-tTA/hAPP mice) showed significantly more Aβ deposition and thioflavin-S–positive amyloid deposits than did Prnp-tTA/hAPP littermate controls (Figure ​(Figure3,3, A–D, and Table ​Table2).2). Quantification of Aβ immunostaining demonstrated a 2-fold increase in both total Aβ (anti-Aβ_1–5 antibody) and Aβ₄₂ immunostaining and a 60% increase in the number of thioflavin-S–positive plaques in TβRIIΔk/Prnp-tTA/hAPP mice compared with Prnp-tTA/hAPP mice (Figure ​(Figure3,3, B–D). These histologic findings were confirmed by ELISA measurements of Aβ in hippocampal and cortical brain homogenates. Levels of Aβ_1–42 and Aβ_1–x (which approximates total Aβ) were 2- to 3-fold higher in both brain regions in 20-month-old TβRIIΔk/Prnp-tTA/hAPP compared with Prnp-tTA/hAPP mice (Table ​(Table2).2). Aβ deposition did not significantly differ at 14 months of age (Figure ​(Figure3,3, B and D), and Aβ levels were similar at 2 and 8 months of age (Figure ​(Figure3,3, F–H, and Table ​Table2),2), indicating that age, or time, is an important contributing factor to increased Aβ deposition with decreased neuronal TGF-β signaling. No obvious differences in vascular amyloid were observed at any age between the different groups of mice (data not shown).

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Figure 3

Decreased neuronal TGF-β signaling in old hAPP mice increases Aβ levels, amyloid deposition, and dendritic degeneration.

(A) Representative images of hippocampi of 20-month-old TβRIIΔk/Prnp-tTA/hAPP and Prnp-tTA/hAPP mice stained with an antibody against Aβ₄₂. Scale bar: 500 μm. (B–D) Quantification of immunoreactive area occupied by staining with antibody against Aβ_1–5 (B), antibody against Aβ₄₂ (C), and thioflavin-S (D) of sagittal sections from 14- and 20-month-old TβRIIΔk/Prnp-tTA/hAPP and Prnp-tTA/hAPP mice (n = 5–7 mice per genotype). (E) Expression of TβRIIΔk in neurons of aged hAPP mice reduced MAP2 immunoreactivity in the hippocampus. Sagittal brain sections of 20-month-old mice were stained with MAP2, and percent immunoreactive area of the neuropil was determined by confocal microscopy and computer-aided image analysis. Each symbol represents 1 mouse. (F–H) Quantification of Aβ_1–x (F), Aβ₄₂ (G), and percent Aβ₄₂ (H) via ELISA in hippocampus (squares) and cortex (circles) of TβRIIΔk/Prnp-tTA/hAPP (black symbols) and Prnp-tTA/hAPP (gray symbols) mice aged 2, 8, and 20 months (n = 5–7 mice per genotype). Note the cutoff in the y axes. (I–L) Signal intensities of total APP (I), shAPPα and shAPPβ (K), and CTFs (L) were quantified and normalized against total APP levels (K and L) or protein amounts loaded (I). Representative Western blots (J) of cell pellets (hAPP, CTF, and actin) or supernatants (shAPPα and shAPPβ) probed with antibodies against total APP, CTFs, actin, shAPPα, and shAPPβ. *P < 0.05; Student’s t test (B–D and F–H), Tukey-Kramer test (E).

Table 2

Effect of neuronal TGF-β signaling on cerebral Aβ accumulation

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To explore the possible cause for this increase in pathology with age, we studied a number of previously described mechanisms that influence Aβ accumulation. There were no significant differences in hAPP protein expression between TβRIIΔk/Prnp-tTA/hAPP and Prnp-tTA/hAPP mice at 2 (Figure ​(Figure3I),3I), 14, or 20 months of age, indicating that the increase in Aβ accumulation in TβRIIΔk/Prnp-tTA/hAPP mice is not simply due to increased hAPP transgene expression. There were also no significant differences in the expression of insulin-degrading enzyme (IDE; TβRIIΔk/Prnp-tTA/hAPP, 0.73 ± 0.08; Prnp-tTA/hAPP, 0.88 ± 0.17; P = 0.5) and neprilysin (TβRIIΔk/Prnp-tTA/hAPP, 0.17 ± 0.02; Prnp-tTA/hAPP, 0.17 ± 0.01; P = 0.8), 2 Aβ-degrading enzymes involved in cerebral Aβ accumulation in mice (32, 33). There were also no significant differences in apoE levels (TβRIIΔk/Prnp-tTA/hAPP, 0.78 ± 0.09; Prnp-tTA/hAPP, 0.7 ± 0.05; P = 0.5). Lastly, microglial activation, which has been linked to Aβ clearance, was not altered by TβRIIΔk expression as measured by CD68 expression (TβRIIΔk/Prnp-tTA/hAPP, 1.24 ± 0.13; Prnp-tTA/hAPP, 1.28 ± 0.10; P = 0.8). Together, these data indicate that TβRIIΔk expression does not significantly affect hAPP transgene expression or Aβ clearance by neprilysin, IDE, apoE, or activated microglia in 14- to 20-month-old hAPP mice and that other mechanisms may be responsible for the development of disease.

One possibility is that reduced TGF-β signaling in neurons might directly alter amyloid precursor protein (APP) processing and lead to increased production of Aβ. While this is easiest studied in cell culture (as described below), we measured the relative levels of major APP cleavage fragments of hAPP in mouse brains using immunoblotting. No significant differences in the levels of α- or β-secretase–cleaved C-terminal hAPP fragments (CTFs) or soluble hAPP (shAPP) were detected in 2- to 14-month-old TβRIIΔk/Prnp-tTA/hAPP and Prnp-tTA/hAPP mice (Figure ​(Figure3,3, J–L). Taken together, these results suggest that reduced TGF-β signaling does not significantly affect APP processing in our mice.

Because TβRIIΔk/Prnp-tTA mice showed age-dependent neuro­degeneration (Figure ​(Figure2,2, C–K), we tested whether the presence of hAPP/Aβ accelerates neuronal damage. Indeed, inhibition of neuronal TGF-β signaling in 20-month-old Prnp-tTA/hAPP mice led to a decrease in MAP2-immunoreactive dendrites compared with hAPP mice (Figure ​(Figure3E).3E). In both cortex and hippocampus, MAP2 immunoreactivity correlated inversely with Aβ deposition (data not shown).

Human postmortem brain tissue.

Midfrontal cortex tissue samples form neurologically unimpaired subjects (n = 11) and from subjects with AD (n = 18) were obtained from the Department of Pathology, University of California, San Diego. Diagnosis of AD was confirmed by pathological and clinical criteria. Tissue was frozen at autopsy and stored at –80°C until use. The average age of subjects was 80.5 ± 2.4 years in the AD group and 79.0 ± 9.0 years in the control group (P = 0.8). Average postmortem delay was 9.6 hours and was not significantly different between groups. One AD case and 3 control cases were excluded from the analysis due to protein degradation of the sample as assessed by coomassie staining of SDS-PAGE gels.

Cells and tissue culture.

B103 rat neuroblastoma cells stably transfected with human wild-type APP or a neo plasmid (34) and NG108-15 neuroblastoma cells (ATCC; HB-12317) were cultured in DMEM supplemented with 10% FBS and 5% heat-inactivated horse serum. Cells were seeded at 50,000 cells/well in 12-well plates and transiently transfected with plasmids encoding for TβRIIΔk, GFP (Invitrogen), pBluescript (Invitrogen), PAI-1-luciferase (55) or SBE-luciferase (56), and constitutively active Smad3 (36) or dominant-negative Smad3 (36) using Superfect (Qiagen) according to the manufacturer’s instructions. GFP-expressing cells were counted after 12, 36, and 60 hours in culture. Luciferase activity was measured after 24 hours with a LMax plate photometer (Molecular Devices) using a Luciferase Assay System (Promega) according to the manufacturer’s instructions. Solid anhydrous SB-505124 (Tocris Bioscience) was reconstituted in DMSO to a concentration of 1 mM and further diluted to 1 μM immediately prior to use.

Postnatal primary neuronal and astroglial cell cultures.

Mixed hippocampal-neocortical primary neuronal cultures and primary astroglial cultures were prepared from P1 and P3 mice, respectively, generated through matings between heterozygous Prnp-tTA and tetO-TβRIIΔk mice as described previously (12). Primary neurons were used at 2 or 4 days in culture. After fixation in 4% paraformaldehyde, cells were labeled with BODIPY-FL-phallacidin (Invitrogen) to stain the actin cytoskeleton according to the manufacturer’s instructions. Cultures were costained with Hoechst 33342 (Invitrogen) to label DNA and examined with an Olympus IX70 inverted fluorescence microscope connected to a Coolsnap HQ camera (Photometrics). Images were processed and analyzed using Metamorph Imaging software (version 6.1r1; Universal Imaging Corp., Molecular Devices) and Adobe Photoshop version 6.0.1. The experimenter was blinded to the genotypes of the mice.

Protein extraction and immunoblotting.

Human tissue was dissected into gray and white matter, and gray matter was lysed in extraction buffer consisting of 20 mM Tris-HCl (pH 7.4), 10 mM EDTA, 10 mM EGTA, 1% sodiumdodecylsulphate, and 1 tablet complete proteinase inhibitor (Roche Diagnostics) per 50 ml extraction buffer. Equal amounts of protein were subjected to SDS-PAGE under reducing conditions in 3%–8% NuPage Novex gels (Invitrogen), transferred to nitrocellulose membranes (Bio-Rad), and probed with antibodies against TβRII (Upstate USA Inc.; 4 mg/ml). Mice were flush-perfused, and brains were dissected into hippocampus and cortex and homogenized in a buffer containing 1 mM HEPES, pH 8.8; 5 mM benzamidine; 2 mM β-mercaptoethanol; 3 mM EDTA; 0.5 mM magnesiumsulphate; and 0.05% sodiumazide. Pellets were further homogenized in equal volumes of RIPA buffer, and proteins were separated on 4%–12% Bis-Tris NuPage Novex gels (Invitrogen). Supernatants were probed with antibodies against the N-terminal (diluted 1:1,000, 8E5; Elan) and pellets with antibodies against the C-terminal (diluted 1:1,000; kindly provided by T. Golde, Mayo Clinic College of Medicine, Jacksonville, Florida, USA) fragments of APP. To adjust for loading differences, blots were stripped and reprobed with an antibody against actin (diluted 1:250; Chemicon International), and gels were stained with GelCode Blue Stain Reagent (Pierce) to check for protein degradation. To confirm the loading of equal amounts of neurons, blots were stripped and reprobed with an antibody against neuron-specific enolase (diluted 1:1,000; Lab Vision Corp.). Binding of secondary antibodies was visualized by ECL (Amersham Biosciences), and signal intensities were quantified with Metamorph Imaging software. Relative receptor levels were calculated by normalizing to actin levels.

Fluorescence-activated cell sorting.

Cells were transiently transfected with TβRIIΔk, GFP and pBluescript. Twelve hours after transfection, cells were dissociated, spun, resuspended in growth medium, and filtered through a 40-μm nylon mesh. Sorting and analysis were carried out on a FACSVantage SE flow cytometer (BD). Dead cells and GFP-negative cells were excluded by gating on forward and side scatter. Mock-transfected cells were used to set background fluorescence. Viable GFP-positive cells were sorted into growth medium and immediately replated at 70% confluency in 12-well plates. Twelve hours after replating, cells were incubated with serum-free neurobasal medium supplemented with N2 for 5–7 days. Supernatants and pellets were collected separately for Aβ and APP measurements, respectively.

RNA extraction and analysis.

Total RNA from snap-frozen hemibrains was isolated with TRI reagent (Molecular Research Center Inc.) and analyzed by solution hybridization RNase protection assay as previously described (17). The [³²P]-antisense riboprobes used to identify specific mRNAs were murine β-actin (nt 480–559 protected; GenBank accession no. M18194) and TβRII (nt 425–828 protected; GenBank accession no. S69114), which protects a shorter segment of mRNA in TβRIIΔk (nt 425–601 protected).

Aβ ELISA.

Snap-frozen hippocampi and cortices were homogenized in 5 M guanidine-HCl buffer, and Aβ peptides were quantified by ELISA as described previously (17). Cell culture conditioned media were centrifuged to pellet dead cells and stored at –80°C. Samples were thawed on ice immediately prior to use.

Immunohistochemistry.

Fixed hemibrains were equilibrated in 30% sucrose in phosphate-buffered saline and cut into 40-μm sections with a freezing microtome (Leica Microsystems). Thioflavin-S staining was carried out as described previously (17). Microtome sections were immunoperoxidase labeled with a VECTASTAIN Elite ABC Kit (Vector Laboratories) using anti-Aβ_1–5 (1 μg/ml, 3D6; Elan), anti-Aβ_1–42 (1 μg/ml, 21F12; Elan), biotinylated donkey anti-mouse (diluted 1:3,000; Vector Laboratories), and DAB (Sigma-Aldrich). The percentage area of the hippocampus covered by 3D6 or 21F12 immunoreactivity or by thioflavin-S staining was determined with Metamorph Imaging software using an Olympus microscope connected to a Coolsnap HQ camera. For each mouse, 3–4 sections were analyzed, and the average was used to calculate group means. Similarly, brain sections were labeled with CD68 to detect activated microglia (diluted 1:10; Serotec), GFAP (diluted 1:1,000; Dako) to detect reactive astrocytes, and NeuN (diluted 1:1,000; Chemicon International) to detect neurodegeneration and relative immunoreactivity, and mean cell numbers were quantified using the previously described selector method (57).

For MAP2-immunoreactive postsynaptic dendrites and synaptophysin-immunoreactive presynaptic terminals, brain sections were labeled with anti-MAP2 (1 μg/ml; Roche Diagnostics) and anti-Syn (diluted 1:800; Roche Diagnostics), respectively. Secondary antibody FITC-conjugated horse anti-mouse IgG (diluted 1:75; Vector Laboratories) was added and sections were imaged with confocal microscopy via the selector method (57). Briefly, counts per unit area were obtained by computer-aided image analysis morphometry in 2–4 sections per mouse at 3 images per section, and digitized images 0.5 μm thick were thresholded for immunolabeled area. The area occupied by MAP2- or synaptophysin-immunoreactive terminals of defined signal intensity were quantified and expressed as a percentage of the total image area (57, 58). This approach for the quantitative assessment of neurodegeneration has been validated in various experimental models of neurodegeneration (59–61) and in diseased human brain (62).

For TβRII, cryosections were fixed in 100% methanol and stained with antibodies against TβRII (4 μg/ml; Upstate USA Inc.), followed by postfixation in 4% paraformaldehyde and labeling with antibodies against NeuN (diluted 1:1,000; Chemicon International) or GFAP (diluted 1:1,000; Santa Cruz Biotechnology Inc.).

We would like to thank Stanley B. Prusiner (University of California, San Francisco) for providing us with the Prnp-tTA mice. We thank L. Head (University of California Irvine, Irvine, California, USA) for providing us with Parkinson’s disease, Pick’s disease, Progressive Supranuclear Palsy, Lewy Body dementia, and Frontotemperal dementia brain tissue and T. Golde (Mayo Clinic College of Medicine, Jacksonville, Florida, USA) for providing us with the CTF-specific APP antibody. This work was supported by NIH grants AG20603 and NS40994 (T. Wyss-Coray), the John Douglas French Alzheimer’s Foundation (I. Tesseur) and the Alzheimer’s Association (I. Tesseur and T. Wyss-Coray).