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Proc Natl Acad Sci U S A. Jul 6, 2010; 107(27): 12299–12304.
Published online Jun 21, 2010. doi:  10.1073/pnas.1007181107
PMCID: PMC2901482

Dissecting the involvement of tropomyosin-related kinase A and p75 neurotrophin receptor signaling in NGF deficit-induced neurodegeneration


NGF, the principal neurotrophic factor for basal forebrain cholinergic neurons (BFCNs), has been correlated to Alzheimer's disease (AD) because of the selective vulnerability of BFCNs in AD. These correlative links do not substantiate a comprehensive cause–effect mechanism connecting NGF deficit to overall AD neurodegeneration. A demonstration that neutralizing NGF activity could have consequences beyond a direct interference with the cholinergic system came from studies in the AD11 mouse model, in which the expression of a highly specific anti-NGF antibody determines a neurodegeneration that encompasses several features of human AD. Because the transgenic antibody binds to mature NGF much more strongly than to proNGF and prevents binding of mature NGF to the tropomyosin-related kinase A (TrkA) receptor and to p75 neurotrophin receptor (p75NTR), we postulated that neurodegeneration in AD11 mice is provoked by an imbalance of proNGF/NGF signaling and, consequently, of TrkA/p75NTR signaling. To test this hypothesis, in this study we characterize the phenotype of two lines of transgenic mice, one in which TrkA signaling is inhibited by neutralizing anti-TrkA antibodies and a second one in which anti-NGF mice were crossed to p75NTRexonIII(−/−) mice to abrogate p75NTR signaling. TrkA neutralization determines a strong cholinergic deficit and the appearance of β-amyloid peptide (Aβ) but no tau-related pathology. In contrast, abrogating p75NTR signaling determines a full rescue of the cholinergic and Aβ phenotype of anti-NGF mice, but tau hyperphosphorylation is exacerbated. Thus, we demonstrate that inhibiting TrkA signaling activates Aβ accumulation and that different streams of AD neurodegeneration are related in complex ways to TrkA versus p75NTR signaling.

Keywords: Alzheimer, β-amyloid, proNGF, signaling unbalance

Decreased neurotrophic support of NGF (1) to cholinergic neurons in the basal forebrain (BFCNs), caused by failure in its retrograde transport or by processing defects (25), has been associated with Alzheimer's disease (AD) (6) because of the selective vulnerability of BFCNs in AD (7).

However, these correlative links between the NGF signaling system and AD do not provide evidence for a comprehensive cause-and-effect mechanism linking NGF signaling or processing deficits to the overall AD neurodegeneration and to the production and accumulation of amyloid-β (Aβ) and tau.

Studies in the AD11 mouse model (8) demonstrated that neutralizing NGF activity in the brain could have consequences beyond direct interference with the cholinergic system, leading to pathological amyloid precursor protein (APP) and tau processing (9). AD11 mice express a highly specific anti-NGF antibody (10, 11) in the adult brain, which induces a progressive, NGF-dependent neurodegeneration encompassing several neuropathological features of human AD, including accumulation of Aβ and neuronal expression of hyperphosphorylated, truncated, and insoluble tau (1216).

The AD11 model uncovered a mechanism whereby neurotrophic deficits are an upstream driver of Aβ/tau accumulation as well as of BFCN atrophy (3). The NGF-binding properties of the anti-NGF mAb αD11 expressed in the AD11 brain provide a mechanistic clue to explain the neurodegenerative process: mAb αD11 binds mature NGF almost irreversibly, with an affinity 1,000-fold higher than for proNGF (11). Thus, we suggested (3) that the preferential binding of NGF by mAb αD11 would create an imbalance between NGF and proNGF, leaving the latter free to act in the functional “absence” of mature NGF. This imbalance in proNGF/NGF signaling would create a signaling imbalance through p75 neurotrophin receptor (p75NTR) versus tropomyosin-related kinase A (TrkA) receptors, with proNGF activating proneurodegenerative, proamyloidogenic pathways (Fig S1). This scheme leads to predictions that can be tested experimentally: Blocking TrkA signaling in the mouse brain should favor Aβ accumulation, whereas blocking p75NTR signaling should exert a protective effect.

To test this hypothesis, in this study we describe the phenotypic characterization of two lines of transgenic mice: one, transgenic MNAC13 (TgMNAC13), in which TrkA signaling is inhibited by the expression of a neutralizing anti TrkA antibody, and a second line in which AD11 anti-NGF mice were crossed to p75NTRexonIII(−/−) mice (AD12 mice) to abrogate p75NTR signaling.


Neutralization of TrkA Activity Determines Early Cholinergic Deficit and Late Aβ Accumulation.

Transgenic mice expressing the anti-TrkA MNAC13 antibody were derived by the neuroantibody approach (17) exploiting the neutralizing anti-TrkA mAb MNAC13 (18), which binds the extracellular domain of TrkA and thereby effectively inhibits TrkA activation by NGF in vitro and in vivo (18, 19). DNA sequences coding for the chimeric mouse/human anti-TrkA MNAC13 antibody chains (Fig S2A) were used to derive double-transgenic anti-TrkA mice (Fig S2B). The TgMNAC13 line was selected among different founders on the basis of expression levels of both chains. TgMNAC13 mice, unlike TrkA−/−-null mice (20), are born normally and thrive to adulthood. The expression of the transgenic antibody chains in the adult brain was demonstrated by real-time qualitative RT-PCR (qRT-PCR), showing limited interindividual variability (Fig. S2C). Immunohistochemistry confirmed the presence of both transgenic light and heavy chains in different tissues such as spleen (Fig. S2 D, E, H, and I) and brain (Fig. S2 F, G, J, and K) of adult mice.

Phenotypic analysis of TgMNAC13 mice was performed at 2, 6, and 14 mo of age. The overall aspect of TgMNAC13 mice did not differ substantially from WT mice.

In 2-mo-old TgMNAC13 mice, the number of medial septum and diagonal band BFCNs was 40% lower than in age-matched controls (Fig. 1 A, B, and D; P < 0.05). Thereafter, the number of BFCNs remained constantly low (Fig.1 C and D). The number of BFCNs was reduced more severely in 2-mo-old TgMNAC13 mice than in age-matched AD11 mice, which at this age show only a 20% decrease with respect to controls (Fig. 1D and ref. 13).

Fig. 1.
Cholinergic deficit in anti-TrkA TgMNAC13 transgenic mice. (A and B) ChAT immunostaining in the basal forebrain of (A) 2-mo-old WT mice, (B) 2-mo-old TgMNAC13 mice, and (C) 6-mo-old TgMNAC13 mice. (D) Quantification of ChAT-immunoreactive neurons in the ...

Brains of TgMNAC13 mice were analyzed for abnormal expression and accumulation of Aβ peptide, with AD11 mice as a reference. In AD11 mice, Aβ first appears in the 6-mo-old hippocampus (Fig. 2 A and C). By dual confocal microscopy analysis, Aβ-immunoreactive material appears to be present both inside and outside cells, in close contact with dystrophic neurites, which are identified in Fig. 2 as MAP-2–immunoreactive dendrites (Fig. 2 D–F and J and Fig. S3). In aged AD11 mice, Aβ accumulates in extracellular deposits (14). Interestingly, Aβ-immunoreactive clusters also were found in the hippocampal radial layer of 14-mo-old TgMNAC13 mice (Fig. 2B), although their number is significantly lower than in age-matched AD11 mice (compare Fig. 2 A, B, and L). Also, confocal analysis of double-stained sections shows that in TgMNAC13 mice Aβ accumulation occurs both intracellularly and extracellularly (Fig. 2 GI and K and Fig. S3) in close contact with dystrophic neurites. The appearance of Aβ in TgMNAC13 mice is delayed in comparison with AD11 mice, because no Aβ immunoreactivity is seen in younger anti-TrkA mice (Fig. 2L). Specific labeling of Aβ clusters in the TgMNAC13 hippocampus was obtained with three different anti-Aβ antibodies (Methods).

Fig. 2.
Aβ peptide staining and memory deficits in TgMNAC13 mice. (AC) Immunoenzymatic reaction showing Aβ-immunoreactive accumulation (stained with anti-Aβ mAb 4G8) in the CA1 region of the hippocampus of 14-mo-old (A) AD11 and ...

Finally, the presence of tau pathology, with particular regard to tau hyperphosphorylation and abnormal somatodendritic localization, was investigated by immunohistochemistry and Western blot. Unlike AD11 mice, no abnormal localization of tau was seen inTgMNAC13 mice at all ages tested, and tau was not found to be hyperphosphorylated (Fig. S4).

Memory Deficits in TgMNAC13 Mice.

Visual memory tasks were analyzed in TgMNAC13 mice using the object recognition test (ORT) at 2 and 6 mo of age. At both ages, during the sample phase, mice explored two identical objects for the same period (Kruskal–Wallis test: P > 0.05; Fig. S5A), as expected. During the test, unlike WT mice, TgMNAC13 mice showed no preference for the unfamiliar object, spending the same time in exploring the two objects (paired t test: P > 0.05; Fig. S5B), a clear sign of visual memory impairment quantified by a lower discrimination index (Kruskal–Wallis test: P < 0.05; Fig. 2M). TgMNAC13 mice show a memory deficit earlier than AD11 mice (Fig. 2M), in line with their accelerated cholinergic deficit.

Crossing anti-NGF to p75NTRexonIII(−/−) Mice to Abrogate p75NTR Signaling: Rescue of Cholinergic Deficit.

The contribution of p75NTR signaling to the progress of neurodegeneration observed in anti-NGF mice was studied in the AD12 line of mice obtained by crossing the anti-NGF–expressing AD10 transgenic line (21) to p75NTRexonIII(−/−) mice (22) (Fig. S6A). AD10 mice express anti-NGF antibodies systemically and have a phenotype that can be superimposed on that observed in AD11 mice (21), but they are more efficient breeders than AD11 mice and therefore are more suitable for cross-breeding experiments. The crossbreeding of anti-NGF AD10 and p75NTRexonIII(−/−) mice yielded AD12 mice, which are homozygous for the p75-null mutation and express anti-NGF antibodies systemically. AD12 mice are characterized by areas of alopecia (Fig. S6B), absent in WT, AD10, and p75NTRexonIII(−/−) mice (Fig. S6C).

In 2-mo-old AD12 mice, the number of choline acetyltransferase (ChAT)-immunoreactive neurons is decreased; this reduction is significantly different not only from WT and p75NTRexonIII(−/−) mice but also from AD10 mice (Fig. 3 AD and F; Kruskal–Wallis test: P < 0.05). In 2-mo-old AD12 mice, ChAT-immunoreactive neurons appeared shrunken, with fragmented ChAT labeling in dendrites. At 6 mo of age, on the contrary, the number of ChAT-immunoreactive neurons in AD12 mice returned to normal, equal to that in WT and p75NTRexonIII(−/−) mice (Fig. 3 E and F) and significantly higher than in age-matched AD10 mice (Fig. 3F; Kruskal–Wallis test: P < 0.05). The number of ChAT-immunoreactive neurons remained stably high thereafter (Fig. 3F). AD12 mice displayed a progressive deficit in visual memory performance tested by the ORT (Fig. S7; paired t test, P < 0.05, unfamiliar vs. familiar object). The persisting memory deficit in the presence of a rescue of cholinergic deficit might suggest a contribution from other neurodegenerating systems in AD12 mice, as discussed later.

Fig. 3.
Cholinergic and Aβ phenotype in anti-NGF AD10 × p75NTRexonIII(−/−) (AD12) mice. (AD) ChAT immunostaining in 2-mo-old (A) WT mice, (B) p75NTRexonIII(−/−) mice, (C) AD10 mice, and (D) AD12 mice. ( ...

Absence of Aβ Expression and Deposition in AD12 Mice.

The intracellular and extracellular expression of Aβ was investigated in 2- to 15-mo-old AD12 mice by using a panel of anti-Aβ antibodies. At 2 mo of age, none of the mice of the various genotypes showed the presence of Aβ in the hippocampus (Fig. 3K). At 6 mo of age, immunoreactive Aβ material was found, as expected, in close contact with dystrophic neurites of AD10 mice (Fig. 3 I and K), and this expression increased at 15 mo of age (Fig. 3K). Remarkably, Aβ immunoreactivity was completely absent in the hippocampus of 6-mo-old (Fig. 3 J and K) and 15-mo-old (Fig. 3K) AD12 mice, as it was in the hippocampus of WT (Fig. 3 G and K) and p75NTRexonIII(−/−) mice (Fig. 3 H and K). Thus, interfering with p75NTR signaling prevents the accumulation of Aβ, which occurs in anti-NGF–expressing AD10 mice.

Tau-Related Pathology Is Exacerbated in AD12 Mice.

In anti-NGF mice (both AD10 and AD11), tau pathology follows a clear regional and temporal progression, starting at 2 mo of age in the entorhinal cortex and progressing subsequently to other cortical regions and the hippocampus (Figs. 4 and and55 and Fig. S5) (13). Surprisingly, in 2-mo-old AD12 brains, anti-phospho-tau mAb AT8 intensely labels numerous neurons in the retrosplenial cortex (Fig. 4D), parietal cortex (Fig. 4E), hippocampus (Fig. 4F), red nuclei (Fig. 4G), substantia nigra [both in the pars compacta and in the pars reticulata; Fig. 4H)], and subthalamic nuclei (not shown). Phospho-tau staining is present in the neuronal soma and dendrites (Fig. 4I). Phospho-tau–immunoreactive neurons are totally absent in the retrosplenial cortex of 2-mo-old anti-NGF mice (Fig. 4C), WT mice (Fig. 4A), and p75NTRexonIII(−/−) mice (Fig. 4B) and in their hippocampus, thalamus, and brainstem (not shown).

Fig. 4.
Hyperphosphorylated tau expression in 2-mo-old AD12 mice. Immunostaining for hyperphosphorylated tau with mAb AT8 in the retrosplenial cortex of (A) WT mice, (B) p75NTRexonIII(−/−) mice, (C) AD10 mice, and (D) AD12 mice. In AD12 mice, ...
Fig. 5.
Tau phenotype in AD12 mice. (A) AT8 neuronal labeling in the cortex of 6-mo-old AD10 mice. (B) Microglial expression and faint neuronal labeling of phospho-tau (mAb AT8) in 6-mo-old AD12 mice. (C) mAb AT8-positive tufted astroglial cell in the hippocampal ...

From 4 to 6 mo of age onward, phospho-tau–specific mAb AT8 labels neurons throughout the cortex and hippocampus in AD10 mice (Fig. 5A) (8, 13, 21). In 4- to 6-mo-old AD12 mice, however, we found a lower number of AT8-immunoreactive neurons, faintly labeled, in all brain regions described (Fig. 5B). Instead, massive phospho-tau labeling was observed throughout wide brain regions in cells that appeared morphologically to be microglial or glial cells (Fig. 5 BD). mAb AT8-immunoreactive glial cells were absent in WT mice (Fig. S8A). The morphology of some of the AT8-positive cells in AD12 mice was characteristically reminiscent of tufted cells, particularly in the CA1 region of the hippocampus (a mean of 42 ± 1 cells by neurostereological counts from bregma −1.28 to bregma 2.12) (Fig. 5C), and was further characterized by spheroidal swellings and fragmentation reminiscent of microglial processes (Fig. 5D). These mAb AT8-immunoreactive cells were shown to include both microglia and astrocytes. Microgliosis in AD12 mice was revealed using the F4/80 antibody (Fig. S8B). Dual confocal analysis showed that some (70%) of the phospho-tau–immunoreactive cells also react with antibodies against the microglia marker CD45 (Fig. 5 EG). When antibodies to the astrocytic marker glial fibrillary acid protein (GFAP) were used, dual confocal analysis revealed that, in AD12 mice, a proportion of phospho-tau–immunoreactive cells are GFAP positive, with phospho-tau– and GFAP-immunoreactive material colocalized in granular structures in the cytoplasm (Fig. 5K). In WT (Fig. 5H), AD10 (Fig. 5I), and p75NTRexonIII(−/−) (Fig. 5J) mice, on the other hand, GFAP-labeled astrocytes are not phospho-tau immunoreactive, and their morphology is very different from that of phospho-tau/GFAP-immunoreactive astrocytes in AD12 mice (Fig. 5K).

Western blot analysis with the phospho-tau–specific AT270 antibody showed a statistically significant increase of soluble phosphorylated tau (Fig. S8C and Fig. 5L) and, to a lesser extent, of insoluble phospho-tau (Fig. S8C and Fig. 5L), in brains of 6-mo-old AD12 mice compared with age-matched WT and anti-NGF mice.

The intense and widespread neuronal phospho-tau immunoreactivity observed in the retrosplenial cortex and other cortical regions of 2-mo-old AD12 mice (Fig. 4), followed by a lower number of AT8-immunoreactive neurons and a concomitant increase of phospho-tau–positive microglia and astrocytes in the cortex of 6-mo-old AD12 mice, suggests that a neuronal death process might occur in the AD12 retrosplenial cortical region. Indeed, a neurostereological analysis of the retrosplenial cortex labeled by immunohistochemistry with the NeuN neuronal marker demonstrated a 30% reduction of NeuN neurons in the retrosplenial cortex of 6-mo-old AD12 mice as compared with WT mice (Fig. 5 MQ).


Despite intensive research, no generally accepted mechanism has been formulated causally linking the AD triad (cholinergic deficit, Aβ, and tau pathologies) into one unified conceptual scheme. Following the demonstration that interference with NGF signaling leads to progressive neurodegeneration that recapitulates many hallmarks of sporadic AD (reviewed in ref. 3), mounting evidence from different sources (2, 4, 2325) indicates that NGF deprivation could represent an upstream driver for sporadic AD cases or a subset of such cases (3, 26).

Within this framework, because mAb αD11 binds mature NGF with a 1,000-fold higher affinity than proNGF (11), we hypothesized that the comprehensive neurodegeneration observed in the AD11 model is the result of a proNGF/NGF signaling disequilibrium (Fig S1) that would entail a TrkA/p75NTR signaling unbalance.

To verify this hypothesis, we asked two questions in this study. (i) Would direct interference with TrkA signaling induce the same comprehensive neurodegenerative phenotype that is observed in AD11 mice? (ii) Would interference with p75NTR signaling lead to the rescue of (some aspects of) neurodegeneration in AD11 mice?

The selective inhibition of TrkA signaling in TgMNAC13 mice leads to a strong cholinergic deficit, which appears earlier than in AD11 mice and confirms the efficacy of TrkA neutralization, and to a significant Aβ phenotype, albeit a milder phenotype than observed in AD11 mice. Tau pathology, which is significantly present in AD11 mice, was not observed in TgMNAC13 neurons at any of the stages analyzed.

In AD12 mice, in which p75NTR has been knocked out in the anti-NGF background, the cholinergic deficit, induced by anti-NGF antibodies, after transient expression, is completely rescued. Even more significantly, the expression of Aβ is completely abolished at all time points analyzed, demonstrating that p75NTR signaling, possibly by proNGF, is involved in the activation of amyloidogenesis by anti-NGF antibodies.

Quite unexpectedly, AD12 mice display a striking tau phenotype characterized not only by an anticipation of the AD11 phenotype but also by qualitative differences, with involvement of brain regions (retrosplenial cortex, subthalamic nuclei, substantia nigra, and red nuclei of the brainstem) that are spared in AD11 mice at all ages. Interestingly, phospho-tau is present in AD12 substantia nigra neurons (both in the pars compacta and reticulata), which are reported to be dependent on BDNF (2729) and not on NGF. In normal conditions, BDNF dependency is thought to be limited to the pars compacta of the substantia nigra. However, the pars reticulata of the substantia nigra also constitutively expresses the BDNF receptor TrkB (30) and has been reported to degenerate in BDNF-deficient mice (31). Our data suggest that when p75NTR signaling is abolished in the anti-NGF context, BDNF signaling might be unbalanced also. In brain areas of AD12 mice where tau was highly phosphorylated, such as the retrosplenial cortex, neuronal loss coincided temporally with a generalized infiltration of phospho-tau–immunoreactive microglial cells and an increase of phospho-tau–immunoreactive astrocytes. The memory deficit in AD12 mice that persisted even in the presence of the rescue of cholinergic deficit probably is related to the neuronal loss deriving from the tau pathology.

On the whole, these results support our hypothesis that an imbalance in proNGF/NGF signaling and the consequent TrkA/p75NTR signaling imbalance (Fig S1) contribute to the progressive neurodegeneration induced by anti-NGF antibodies in the AD11 model. More generally, the results provide independent evidence, in addition to that already obtained in vivo (14) and in vitro (25, 32), linking the activation of pathological amyloid and tau processing to alterations in the signaling of NGF ligands and receptors. In particular, results show that TrkA and p75NTR signaling are linked in complementary and distinct ways to the amyloid and tau neurodegeneration streams (Table S1). These results could be relevant for the etiology of sporadic AD, suggesting neurotrophic deficit as an upstream driver of neurodegeneration and linking cholinergic, Aβ, and tau abnormalities into one comprehensive “multiple-pathway” mechanism of neurodegeneration (3) involving parallel, rather than serial, pathways (33). In this respect, the amyloid and tau streams of neurodegeneration would be linked by separate mechanisms to a common upstream neurotrophic signaling imbalance, differentially involving NGF receptors signaling (3).

Several aspects of the results described in this study deserve further comment. On one hand, our findings demonstrate that TrkA is implicated directly in triggering Aβ formation and accumulation in the intact brain, extending on the receptor side earlier observations in anti-NGF AD11 mice (14) and in NGF-deprived neuronal cultures (34). The involvement of p75NTR in mediating cellular responses to Aβ, although controversial, has been suggested previously (24, 35); the result in AD12 mice provides a direct demonstration for the involvement of p75NTR in APP processing and Aβ production.

An evolution from neuronal to glial expression of insoluble phosphorylated tau similar to that of the tau phenotype observed in AD12 mice has been found in the human tauopathies, supranuclear palsy and corticobasal degeneration (36). Microglia in AD12 mice show other morphological evidence of deterioration, such as cell aggregation, spheroidal swelling, and process fragmentation, typical of senescent microglia in age-related neuropathological conditions (37).

Although the AD12 phenotype clearly demonstrates that the cholinergic deficits and Aβ production observed in AD11 mice are rescued and that tau pathology is exacerbated, a mechanistic interpretation must consider that the p75NTR protein is not totally absent in these mice, and its transmembrane and intracellular domains are expressed as a truncated protein. Thus, it is possible that (i) the intracellular domain of p75NTR may constitutively signal or compete for signaling effectors of other pathways (38), (ii) the absence of p75NTR extracellular domain could allow binding of neurotrophin 3 to TrkA (39, 40), and (iii) absence of p75NTR expression might decrease the localization of NGF in the basal forebrain (41). The AD12 phenotype may depend in complex ways on some of these factors, but the conclusion that the neurodegenerative phenotype in AD11 mice is affected by p75NTR signaling, with a distinct effect on tau and APP processing (Table S1), has a strong experimental basis.

In conclusion, the data confirm that alterations in NGF receptor signaling, possibly secondary to an imbalance in NGF processing, can drive separate streams of neurodegeneration, showing that TrkA neutralization leads to Aβ production, whereas abrogating p75NTR signaling is protective of anti-NGF–induced amyloidogenesis (Fig. S1). With respect to the proNGF/NGF imbalance hypothesis, it will be interesting to verify whether experimentally increasing the proNGF/NGF ratio in transgenic mice will have a direct impact on Aβ formation, as the scheme in Fig S1 illustrates.


Derivation of TgMNAC13 Mice.

The variable regions of the light and heavy chains of anti-TrkA mouse mAb MNAC13 (17) were cloned from hybridoma cells and reassembled with human K and γ1 constant regions as described previously (8). Both light- and heavy-chain genes were placed under the transcriptional control of the early region of the human cytomegalovirus promoter, in two separate plasmids (Fig. S2A). The two transcriptional units were cut from the corresponding plasmids with the enzymes shown in Fig. S2A and were individually microinjected into fertilized mouse eggs (BD strain). We obtained three founder mice for the transgenic light chain and two founder mice for the heavy chain. Founders of each line were crossed to obtain double-transgenic mice. The TgMNAC13 line was selected for further study because double-transgenic TgMNAC13 mice thrive to adulthood and show a normal breeding activity when intercrossed (up to 12 pups/litter). Mice were genotyped by PCR on genomic DNA (Fig S2) as described previously (8) with oligonucleotides described in SI Methods.

The expression of the mRNA coding for the transgenic antibody chains was verified by real-time qRT-PCR, as described in SI Methods.

WT mice of the BD strain and AD11 mice (8) were used as negative and positive controls, respectively.

Derivation of AD12 Mice.

The AD12 line of mice was obtained by crossing the AD10 anti-NGF line of mice with p75NTRexon III (−/−) knockout mice (22). C57BL × SJL AD10 anti-NGF mice were generated as previously described (21). P75NTRexonIII(−/−) mice were purchased from Jackson Laboratory and were backcrossed five times into the C57BL × SJL background (the background of AD10 mice) before breeding with AD10 anti-NGF mice, yielding AD12 mice homozygous for the p75-null mutation. The genetic characterization of AD12 mice was performed by PCR analysis on genomic DNA. Primers are described in SI Methods.

Phenotypic Analysis of TgMNAC13 and AD12 Transgenic Mice.

All experiments with transgenic (AD10, AD11, AD12, and TgMNAC13) and nontransgenic lines of mice were conducted according to national and international laws for laboratory animal welfare and experimentation (EEC Council directive 86/609, OJ L 358, 12 December 1987).


Two-, 6- and 14-mo-old TgMNAC13 mice and 2-, 6-, and 15-mo-old AD12 mice and littermates [nontransgenic, p75NTRexonIII(−/−) and anti-NGF mice] were anesthetized and intracardially perfused, and brains were processed to detect ChAT in BFCNs, Aβ in the hippocampus, and phosphorylated tau using the primary antibodies described in SI Methods and the protocols previously described (8, 15). Confocal and neurostereological morphometric analysis was performed as described in SI Methods.

Western blot analysis.

The biochemical analysis of total tau and phosphorylated tau was performed on soluble and insoluble fractions extracted from brains of 6-mo-old WT, AD10, and AD12 mice, according to (42) (SI Methods).

Object recognition test.

The test for visual memory in 2- and 6-mo-old TgMNAC13 and AD12 mice was performed as described (12). A discrimination index was calculated as the difference between the time spent exploring the unfamiliar and the familiar object divided by the total time spent exploring the objects [(nf)/(f + n)], where n represents time spent exploring the unfamiliar object, and f represents the time spent exploring the familiar object.

Statistical Analysis.

Statistical analyses were performed using the Sigmastat v. 3.5 program (Systat Software). The α was set at 0.05, and normality and equal variance tests were performed first. A t test and Mann-Whitney rank sum test were used for the comparison between two groups. Kruskal–Wallis ANOVA was used for multiple comparisons.

Supplementary Material

Supporting Information:


We thank Sabina Giannotta, Giada Pastore, Rossella Brandi, and Paolo Capelli for their technical contributions in early phases of the characterization of TgMNAC13 and AD12 mice. This work was supported in part by Telethon Grant GGP05234 and by European Union Memories Project Grant 037831.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1007181107/-/DCSupplemental.


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