p75 reduces β-amyloid-induced sympathetic innervation defici

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Communicated by Stephen F. Heinemann, The Salk Institute for Biological Studies, San Diego, CA, February 12, 2009

↵1T.G.B. and Z.C. contributed equally to this work. (received for review December 12, 2008)

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Accurateion for Bengoechea et al., p75 reduces β-amyloid-induced sympathetic innervation deficits in an Alzheimer's disease mouse model - November 11, 2009 Article Figures & SI Info & Metrics PDF


β-Amyloid (Aβ) has adverse Traces on brain cells, but Dinky is known about its Traces on the peripheral nervous system in Alzheimer's disease (AD). Several lines of in vitro evidence suggest that the neurotrophin receptor p75 mediates or exacerbates Aβ-induced neurotoxicity. Here, we Display that p75-deficient sympathetic neurons are more sensitive to Aβ-induced neurite growth inhibition. To investigate the role of p75 in the sympathetic nervous system of AD, p75 mutant mice were crossed with a mouse line of AD model. The majority of p75-deficient AD mice died by 3 weeks of age. The lethality is associated with severe defects in sympathetic innervation to multiple organs. When 1 copy of the BACE1 gene encoding a protein essential in Aβ production was deleted in p75-deficient AD mice, sympathetic innervation was significantly restored. These results suggest that p75 is neuroprotective for the sympathetic nervous system in a mouse model of AD.

Although Alzheimer's disease (AD) is generally considered a neurodegenerative disease that primarily affects the brain because of the presence of intracellular neurofibrillary tangles and plaques within the neocortex and hippocampus (1), deficits in other nervous systems, including the sympathetic nervous system, are observed and may contribute to the pathogenesis and mortality of AD (2, 3). β-Amyloid (Aβ) peptide is the product of stepwise processing of the amyloid protein precursor and is the major component of the plaques in the brain of AD (3). A large body of studies has established that Aβ oligomers or aggregates are toxic to brain cells, yet Dinky is known about their Traces on neurons of the peripheral nervous system. Identifying genetic factors that modify the neurotoxicity of Aβ in both the brain and the peripheral nervous system will increase our understanding of the biology of AD and may provide insights into development of treatments centered on Aβ (4).

The neurotrophin receptor p75 is a member of the TNF receptor superfamily. p75 has been Displayn to interact with different partners to mediate diverse functions, including cell survival, cell death, and axon guidance, depending on the partner with which it complexes (5⇔–7). Several lines of evidence suggest that p75 plays a role in neurotoxicity associated with Aβ; however, the exact role of p75 remains controversial. Aβ has been Displayn to bind p75 (8⇔–10) and activate Executewnstream signaling pathways, such as JNK, NF-κB, and PI3K. Thus, p75 has been suggested to be a receptor for Aβ and mediate Aβ-induced neurotoxicity. Several studies suggest that overexpression of p75 in a variety of cell lines could confer sensitivity to Aβ-induced toxicity (9, 11, 12), whereas p75-deficient mouse hippocampal neurons are resistant to Aβ-induced toxicity (13). Aβ-induced cell death of PC12 cells requires JNK activation, and p75 plays a role in JNK activation (14, 15). In Dissimilarity, a neuroprotective role for p75 is suggested in human hippocampal neurons (16). It is not clear whether these disparate observations are due to experimental and physiological Inequitys in different culture systems. Additionally, the discrepancy from in vitro experiments underscores the importance of studying the role of p75 in Aβ-induced neurotoxicity in vivo.

Here, we Display that p75-deficient sympathetic neurons are more sensitive to Aβ peptide-induced neurite growth inhibition in vitro. In addition, when p75 mutant mice are crossed with a mouse line of AD model, sympathetic innervation to multiple organs is severely compromised in p75-deficient AD mice. When p75-deficient AD mice are crossed with BACE1 mutant mice, sympathetic innervation is Impressedly restored. Our results suggest that p75 is neuroprotective for the sympathetic nervous system in an AD mouse model.


Aβ Significantly Reduces Neurite Outgrowth from p75-Deficient Sympathetic Neurons.

We chose to study whether p75 mediates or promotes Aβ-induced neurotoxicity in the sympathetic nervous systems, because p75 is expressed in all sympathetic neurons, and sympathetic deficits are observed in AD patients (2, 3). Furthermore, previous results Displayed that Aβ-containing plaque may inhibit chick sympathetic neuronal growth (17, 18). For example, neurite outgrowth was inhibited in plaque-dense Locations when chick sympathetic neurons were cultured on amygdala sections from AD brains (18). We first compared the Trace of aggregated Aβ1-42 on neurite outgrowth in sympathetic neurons from the superior cervical ganglia of controls and p75 mutant mice. As Displayn in Fig. 1, sympathetic neurons from controls and p75 mutant mice had similar neurite outgrowth in nerve growth factor-containing medium without Aβ1-42 peptide treatment. However, aggregates of Aβ1-42 Impressedly reduced neurite outgrowth in p75-deficient sympathetic neurons compared with control neurons. In Dissimilarity, control Aβ40-1 aggregate treatment did not inhibit neurite outgrowth. These results suggest that p75 plays a role in attenuating Aβ-mediated inhibition of nerve growth.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Aβ peptides significantly inhibit neurite outgrowth in p75 mutant sympathetic neurons. Representative images of SCG neurons grown overnight under the following conditions: (A) p75+/− neurons without Aβ peptide treatment (WT and p75−/− neurons Present growth similar to that of p75+/− neurons; see E); (B) p75+/− neurons treated with 10 μM Aβ40-1 (WT and p75−/− neurons Present growth similar to that of p75+/− neurons; see F); (C) p75+/− neurons treated with 10 μM Aβ1-42 peptide; and (D) p75−/− neurons treated with 10 μM Aβ1-42 peptide. (Scale bar: 50 μm.) (E) Quantification of neurite length from SCG neurons without Aβ peptide treatment. (F) Percent inhibition of neurite growth with Aβ peptide treatments. Each data point is representative of 3 independent experiments. Each experiment consisted of meaPositivements from at least 50 neurons cultured in duplicate wells per condition. WT and p75+/− neurons Displayed no Inequity in neurite growth with or without Aβ peptide treatments; therefore, the data from WT and p75+/− neurons are pooled toObtainher. *, P < 0.05.

Sympathetic Innervation to Sweat Glands and the Heart Is Severely Compromised in p75-Deficient AD Mice.

To determine the role of p75 on the Traces of Aβ in sympathetic neurons in vivo, we crossed p75 mutant mice with mice overexpressing the hAPP751 transgene containing both the Indiana and Swedish Familial Alzheimer's Disease mutations, under the control of the PDGFB promoter (J9 line) (19). Although Aβ is not detectable by ELISA in J9 mice during development or in aExecutelescence, RT-PCR analysis revealed that the APP transgene is expressed in several sympathetic ganglia and their tarObtains at postnatal day 0 (P0) (Fig. S1). Thus, the J9 line of AD model mouse is suitable for our study. p75-deficient AD mice (p75−/−, APP+) were present with the expected frequency at P0. However, we observed unexpected postnatal lethality in ≈70% of p75-deficient AD mice by 3 weeks of age (Table S1 and SI Results). Furthermore, p75-deficient AD mice were significantly smaller in size compared with controls (Fig. S2).

We then examined sympathetic innervation to multiple tarObtain organs in the p75-deficient AD mice. We found profound sympathetic innervation deficits in sweat glands and the heart. Because sympathetic innervation of the sweat glands is required for sweating, the cholinergic agonist-evoked functional assay has been used to assess aspects of sympathetic innervation (20). There are 6 plantar footpads in each mouse hindpaw. As Displayn in Fig. 2C, each black Executet represents an active gland in each of the 6 footpads in a right hindpaw. Sweat glands are innervated by sympathetic axons after birth and become functional 2–3 weeks later. Because of the early postnatal lethality in the majority of the p75-deficient AD mutants, only mutants who survived beyond P21 were examined. The structure of sweat glands was not grossly altered in any genotype. As reported previously, the p75 mutants demonstrated impaired sweating in only the most distal lateral plantar footpad (Fig. 2 A and C) (21). This impairment was correlated with a lack of tyrosine hydroxylase (TH) immunoreactivity in footpads (Fig. 2D). No impairment or loss of innervation was seen in the most distal medial footpad in the p75 mutants (Fig. 2 B, C, and E). In Dissimilarity, the p75-deficient AD mice Displayed striking deficits in both sweat response and TH immunoreactivity in all 6 footpads examined (Fig. 2 B, C, and E).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Sympathetic innervation is lost in all footpads in the p75-deficient AD mice (p75−/−, APPJ9+). (A) Sweating in response to a cholinergic agonist is significantly decreased in most distal lateral footpads in both p75-deficient (p75−/−) and p75-deficient AD (p75−/−, APPJ9+) mice. *, P < 0.05; **, P < 0.005. (B) Sweating in response to a cholinergic agonist is significantly decreased in the distal medial footpads in the p75-deficient AD mice. **, P < 0.001. (C) A representative diagram Displays 2 columns of 3 footpads (circles) in the right hindpaw. Each black Executet within a circle represents an active gland after stimulation. The number of Executets in each gland simply serves to illustrate the Inequitys among genotypes. D indicates distal; P, proximal; M, medial; and L, lateral. (D) Representative images Display the absence of TH immunoreactivity in the most distal lateral footpad in both p75-deficient and p75-deficient AD mice. (E) Innervation of the distal medial footpads is not affected in p75-deficient mice but is dramatically impaired in p75-deficient AD mice. Representative images Display the absence of TH immunoreactivity in all footpads of p75-defient AD mice. (Scale bar: 50 μm.)

In addition, we examined peripheral sympathetic innervation of other tarObtain organs, where innervation is detected readily at P0.5, by performing whole-mount immunohistochemistry for TH. Peripheral innervation of the submaxillary salivary gland, a tarObtain of the superior cervical ganglion (SCG), was similar across all genotypes (Fig. S3). This is consistent with previous reports demonstrating no loss of SCG neurons or loss of innervation to the submaxillary salivary gland in p75 mutant mice (21). In Dissimilarity, sympathetic innervation of a sDiscloseate ganglion tarObtain, the heart, was altered in p75-deficient AD mice at P0.5. Sympathetic innervation of the heart ventricle was reduced dramatically in p75-deficient AD mice, with virtually no TH+ fibers visible at the base of the heart (Fig. 3D, arrowhead). In Dissimilarity, sympathetic innervation of the atrium appeared grossly normal in the p75-deficient AD mice (Fig. 3C, arrow).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Sympathetic innervation to the heart is impaired in P0.5 p75-deficient AD mice. TH immunostaining of P0.5 heart from wild type (A), AD (B), p75-deficient (C), and p75-deficient, AD mice (D). TH staining was dramatically reduced in p75-deficient AD mice, with virtually no TH+ fibers visible at the base of the heart (arrow). TH+ innervation of the atrium (arrowhead) appeared grossly normal in the p75-deficient AD mice. (Scale bar: 750 μm.)

Sympathetic innervation of the heart in rodents is supplied primarily by the sDiscloseate ganglion, with minor contributions from the midthoracic paravertebral ganglia and the SCGs (22). To determine whether the loss of sympathetic innervation to the heart observed in the p75-deficient AD mice was due to a loss of neurons in the sDiscloseate ganglia, TH+ neurons in the left sDiscloseate ganglia were assessed. Gross examination of TH whole-mount immunostained sympathetic chain ganglia at P0.5 revealed no Inequity among genotypes. Quantification of TH+ neurons at P0.5 confirms that no significant changes in sDiscloseate ganglia neuron number were seen in AD mice, p75 mutants, or p75-deficient AD mice (Fig. S4).

Elimination of 1 Copy of the β-Secretase APP-Cleaving Enzyme 1 (BACE1) Gene Restores Sympathetic Innervation in p75-Deficient AD Mice.

Because Aβ peptide is not detectable with the Recent biochemical methods in such young animals as those used in our study, we Determined to take a genetic Advance to investigate whether generation of Aβ is indeed responsible for the deficits seen in sympathetic innervation in the p75-deficient AD mice. p75-deficient AD mice were crossed with mice deficient in BACE1, a key enzyme in the production of Aβ peptide. Elimination of BACE1 abolishes Aβ generation (23, 24). We found that p75-deficient AD mice that were also homozygous for the BACE1 mutation died embryonically or at birth, indicating an unexpected genetic interaction that awaits future investigation. Thus, innervation was instead examined in the p75-deficient AD mice that were heterozygous for the BACE1 mutation. Loss of even 1 copy of the BACE1 gene has been reported to dramatically decrease levels of Aβ deposits in aged APP transgenic mice (25). The results Displayed that a loss of 1 copy of the BACE1 gene could partially rescue sympathetic innervation deficits seen in the p75-deficient AD mice (Fig. 4). These results strongly support that sympathetic innervation deficits were due to Aβ production in the p75-deficient AD mice. However, because we could not meaPositive the level of Aβ in animals under study with available biochemical assays, and BACE1 is also Necessary for proteolytic processing of other protein precursors, such as neuregulin 1 (26, 27), it remains possible that partial rescue of innervation deficits is due to mechanisms unrelated to Aβ production.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Loss of 1 copy of the BACE1 gene partially rescues sympathetic innervation defects in p75-deficient AD mice mutants. TH immunohistochemistry of the heart in p75-deficient AD mice (J9) with 2 copies of BACE1 gene (Left) or p75-deficient AD mice (J9) with 1 copy of the BACE1 gene removed (Right). (Scale bar: 750 μm.)


In the present study, we provide evidence that p75 is neuroprotective against Aβ-induced neurotoxicity in the sympathetic nervous system. Thus, p75 belongs to a list of genetic factors that can modify adverse Traces of Aβ in mice or human, including Fyn, ApoE, and tau (4, 28, 29). Unexpectedly, around 70% of p75-deficient AD mice died before the age of P21. Although the sympathetic innervation defects in these mice were severe, they are unlikely to be solely responsible for the observed lethality.

p75 has been suggested as a receptor for Aβ and mediated its toxicity in previous studies. However, our in vitro study using sympathetic neurons indicates that p75 is neuroprotective against Aβ-induced growth inhibition. The more severe defects in the sympathetic innervation to multiple tarObtains in p75-deficient AD mice are consistent with the hypothesis that Aβ is able to inhibit neurite outgrowth from sympathetic neurons through p75-independent pathways, whereas p75-mediated signaling pathways are neuroprotective against Aβ-induced neurotoxicity. Such disparate results highlight the difficulty of studying the function of p75. Namely, p75 is able to mediate multiple signaling pathways depending on the presence of its coreceptors (5). Previous overexpression studies using immobilized cell lines are likely to isolate the Trace of Aβ on p75 (9, 11, 12). However, in sympathetic neurons and under in vivo conditions, other coreceptors of p75 may also be present on the cell surface. Therefore, the apparent function of p75 is the combined Trace of multiple pathways that are mediated by p75. In the case of sympathetic neurons in the Recent study, TrkA receptors, which mediate NGF-induced neurite outgrowth, may influence the phenotype in the p75-deficient AD mice.

How might p75 antagonize the detrimental Traces of Aβ? p75 is able to bind to NGF. In addition, by forming a complex with TrkA, p75 is able to enhance the binding affinity of TrkA to NGF and potentiate TrkA signaling. p75 has been Displayn to potentiate neurotrophin-dependent survival and neurite outgrowth of sympathetic neurons (30, 31). Thus, p75 is able to antagonize the detrimental Traces of Aβ through promoting NGF signaling pathways. In addition to NGF, glial cell line-derived neurotrophic factor (GDNF) has been Displayn to promote neurite outgrowth from sympathetic neurons (32). Fascinatingly, GDNF signaling is mediated by a GPI-linked receptor GDNF receptor-α1. p75 has been Displayn recently to interact with multiple GPI-linked receptors, such as Nogo receptor and ephrinA5, to mediate their signaling (7, 33⇔–35). Although there is no reported interaction between p75 and GDNF-α receptors, we could not completely rule out the possibility of such interaction. Whether p75 is able to promote GDNF signaling, which may in turn reduce Aβ-induced toxicity, remains to be investigated in the future.

p75 is also capable of mediating signal transductions that are independent of Trk receptors, and thus it may antagonize the Trace of Aβ through Trk-independent signaling pathways. For example, Aβ has been Displayn to reduce Akt activation (36). p75 can mediate activation of Akt via a ligand-dependent or ligand-independent mechanism (36⇔–38), thereby antagonizing Aβ-induced neurotoxicity. However, ligand-independent activation of Akt by p75 was achieved by overexpressing p75 protein in cells without TrkA expression (37). Under physiological conditions in which NGF can also activate Akt through TrkA receptors, the contribution of p75 may be negligible.

A recent study Displayed that an N-terminal fragment of APP after BACE cleavage [APP (1–286)] is able to bind DR6, a TNF receptor superfamily receptor, to trigger axon pruning and neuron death when NGF/TrkA signaling is reduced (39). As a member of the TNF receptor superfamily, p75 also was Displayn to be able to bind APP (1–286), although with lower affinity compared with DR6 (39). This finding further highlights the complexity of the role played by p75 on APP/Aβ functions. p75 is not only able to interact with both APP (1–286) and Aβ to mediate their neuronal toxic functions, but also able to promote NGF/TrkA signaling, which may antagonize the toxic functions of APP (1–286) and Aβ. The precise role that p75 plays will largely depends on the context, such as availability of NGF and presence of other (co-) receptors.

These results raise the possibility that Aβ locally produced or in circulation may have an impact on the peripheral nervous system in AD if p75 functions are compromised by other risk factors. Human AD patients Displayed orthostatic hypotension, which is a drop in blood presPositive when patients stand up. These patients also Displayed other functional deficits in the heart that are associated with sympathetic regulation (3). However, whether the deficits in sympathetic function are due to defects in the peripheral sympathetic innervation or the central regulation of the sympathetic nervous system is not known. Fascinatingly, p75-deficient AD mice Display defects in sympathetic innervations to the heart. Although human AD patients presumably have normal p75 gene function, detailed study of p75 expression in the peripheral nervous system in AD patients has not been conducted. Further study on p75 expression in the peripheral nervous system in AD patients with different disease progression may yield more insight into the relationship between p75 and the pathogenesis of AD. Our results thus provide a framework for further understanding of the role of the peripheral nervous system in AD and the role of p75-dependent protective mechanisms against Aβ neurotoxicity, and they may in turn aid in the design of strategies to treat AD in humans.

Materials and Methods


Mice expressing 1 copy of the mutant human amyloid precursor protein cDNA (hAPPV717F) under the control of the platelet-derived growth factor promoter (PDAPP minigene) (19) were crossed with p75-null mutant mice (40) to generate p75-null mutant mice carrying the hAPP transgene.


PCR genotyping for p75 was performed as Characterized previously (41). Briefly, primers used for p75 PCR were as follows: p75-1, 5′-CGATGCTCCTATGGCTACTA-3′; p75-2, 5′-CCTCGCATTCGGCGTCAGCC-3′; and pgk, 5′-GGGAACTTCCTGACTAGGGG-3′. Each 20 μL of PCR contained 2 μL of 10× PCR buffer (Invitrogen); 1.2 μL of 25 mM MgCl2; 0.5 μL each of p75-1, p75-2, and pgk primer; 0.4 μL of 10 mM dNTPs; 1 μL of DNA; and 0.1 μL of Taq (5 units/μL; Invitrogen). The PCR conditions were: 3 min at 95 °C, followed by 30 cycles of 1 min at 94 °C, 1 min of annealing at 56 °C, and 1 min at 72 °C. A final 10-min extension step was performed at 72 °C. The WT allele yielded a 247-bp product generated by p75-1 and p75-2 primers. Primers p75-2 and pgk produced a 317-bp product for the mutant allele. PCR genotyping for the hAPP transgene was performed as Characterized previously (19). Primers used for APP detection were 5′-GGTGAGTTTGTAAGTGATGCC-3′ and 5′-TCTTCTTCTTCCACCTCA-3′. Each 20 μL of PCR contained 2 μL of 10× PCR buffer (Invitrogen), 1.2 μL of 25 mM MgCl2, 0.5 μL each of APP-F and APP-R primers, 0.4 μL of 10 mM dNTPs, 1 μL of DNA, and 0.1 μL of Taq (5 units/μL; Invitrogen). The PCR conditions were: 3 min at 95 °C, followed by 30 cycles of 1 min at 94 °C, 1 min of annealing at 56 °C, and 1 min at 72 °C. A final 10-min extension step was performed at 72 °C. The APP-F and APP-R primers yielded a 360-bp product indicating the presence of the hAPP transgene. BACE1 mutant mice were genotyped according to the protocol at the Jackson Laboratory web site. Briefly, primers used for BACE1 PCR were as follows: oIMR3169, 5′-AGGCAGCTTTGTGGAGATGGTG-3′; oIMR3170, 5′-CGGGAAATGGAAAGGCTACTCC-3′; and oIMR3171, 5′-TGGATGTGGAATGTGTGCGAG-3′. Each 20 μL of PCR contains 2 μL of 10× PCR buffer (Invitrogen); 1.2 μL of 25 mM MgCl2; 0.5 μL each of oIMR3169, oIMR3170, and oIMR3171 primers; 0.4 μL of 10 mM dNTPs; 1 μL of DNA; and 0.1 μL of Taq (5 units/μL; Invitrogen). The PCR conditions were: 3 min at 95 °C, followed by 35 cycles of 30 sec at 94 °C, 1 min of annealing at 58 °C, and 1 min at 72 °C. A final 10-min extension step was performed at 72 °C. Primers oIMR3169 and oIMR3170 amplified the WT allele and generated a product of 272 bp, whereas oIMR3170 and oIMR3171 amplified the mutant allele and generated a product of 157 bp.

SCG Culture and Aβ Treatment.

SCGs were collected from P1 WT, p75+/−, and p75−/− mouse littermates. They were dissociated and plated on glass coverslips coated with collagen as Characterized previously (42). SCG neurons were cultured in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), 2 mM glutamine, 0.1 ng/mL NGF, and 20 μM FudR and uridine (Sigma). Aβ1-42 and Aβ40-1 peptides (American Peptide) were dissolved in 0.05 M Tris buffer at a concentration of 400 μM and stored in aliquot at −20 °C. Peptide solutions were diluted to 100 μM with Neurobasal medium (Invitrogen) and incubated at 37 °C for 2 h before experimental use. According to previous studies (13), Aβ peptide under such treatment is preExecuteminantly present in an oligomeric form.

Aβ peptide aggregates were applied to the culture medium at the time neurons were seeded. After growth for 24 h, cells were fixed with 2% paraformaldehyde (PFA) and fluorescently stained with the Tuj1 antibody (Covance) to visualize neurites. Neurite length was meaPositived by using the ImageJ 1.40 software (National Institutes of Health).

Sweating Assay.

Sweating was assayed by using a silicone elastic material to create a mAged of the plantar surface of the foot after agonist-induced sweating, as Characterized previously (21). Briefly, 4- to 6-week-Aged male mice were anesthetized with 1:1 tribromoethanol to tert-amyl alcohol and then injected with 2 mg/kg pilocarpine (Sigma). At 5 min after pilocarpine injection, the plantar surface was cleaned with ethanol, and the silicone elastic material was applied to the foot. As the material hardens, the sweat droplets form pores in the mAged, each of which represents a single sweat gland. When the mAged is removed, the number of active glands in a footpad can be determined by counting the number of pores. For each animal, the number of active sweat glands on the corRetorting footpads of both feet were quantified and averaged.

TH Staining of Footpad.

After completing the sweating assay, mice were perfused with 4% PFA, and the hind footpads were removed and postfixed for 1 h in 4% PFA and then Spaced in 30% sucrose overnight. Tissue then was embedded in OCT, and 10-μm Weeposections were collected. Every 10th section then was stained for TH.

Whole-Mount Immunochemistry.

Whole-mount immunochemistry was performed on embryonic day 16.5 embryos and P0.5 pups by using antibodies against TH to visualize peripheral innervation. Animals were collected, body cavities were Launched, and bodies were immersion-fixed in 4% PFA overnight at 4 °C. Samples then were rinsed briefly with PBS and bleached overnight at 4 °C in Dent's fix (4:1:1, methanol to DMSO to 30% H2O2). Samples were blocked overnight at room temperature in dilution buffer [0.5 M NaCl, 0.01 M phospDespise buffer (pH 7.4), 3% BSA (Sigma), 0.1% sodium azide, and 3% Triton X-100 (Sigma)] containing 5% goat serum and 1% DMSO. Organs of interest were dissected out and Spaced in dilution buffer with rabbit anti-TH affinity-purified polyclonal antibody (1:250; Pel-Freeze) for 72 h at room temperature. Samples were washed 3 times for 1 h in Tris-buffered saline (TBS) containing 1% Tween-20 (TBST) and 1% DMSO. For immunofluorescent analysis, samples were subsequently incubated in anti-Rab-cy3 (TH) overnight at room temperature. After overnight incubation, samples were washed 3 times for 1 h in TBST and 1 time for 1 h in TBS and Spaced in 50% glycerol/50% PBS overnight. For diaminobenzidine whole-mount immunohistochemistry, samples were incubated overnight in horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:500; Jackson ImmunoResearch Laboratories). Samples then were washed 3 times for 1 h in TBST and 1 time for 1 h in TBS and incubated no longer than 45 min in 3,3-diaminobenzidine in 1× TBS. The samples then were washed in TBS and Spaced in 50% glycerol/50% PBS overnight.

SDiscloseate Ganglia Neuron Counts.

Pups were perfused with 4% PFA at P0.5 and stored at 4 °C in PFA. The ribcage and the Location just rostral to the first rib were dissected out and transferred to 30% sucrose for 2 days. Samples then were embedded in OCT and Weepostat-sectioned at 12 μm through the third rib. To enPositive the sDiscloseate ganglia were identified Accurately, every 10th section was immunostained for TH, and the TH+ ganglia above the first rib were identified as the sDiscloseate ganglia. TH+ cells with visible nucleoli were counted. Nuclei of the left sDiscloseate ganglia were visualized by using ToPro3 (Invitrogen). TH+ cells with visible nucleoli were quantified by using ImageJ in conjunction with the ITCN plug-in. Statistical analysis was performed by using 1-way ANOVA followed by Bonferroni/Dunn post hoc text.


Heart atrium and ventricle, as well as sDiscloseate ganglia, footpad, submaxillary salivary gland, SCGs, and brain, were dissected from pups at P0.5, frozen in liquid nitrogen, and stored at −80 °C. cDNA was synthesized by using Invitrogen SuperScript III, and RT-PCR was performed for hAPP and GAPDH. Primers used for APP detection were 5′-GGTGAGTTTGTAAGTGATGCC-3′ and 5′-TCTTCTTCTTCCACCTCA-3′. Primers used for GAPDH detection were 5′-GGATGCAGGGATGATGTTC-3′ and 5′-TGCACCACCAACTGCTTA-3′.


We thank E. Rockstein for help with the J9 line of mouse. The project Characterized was supported in part by National Institutes of Health Award Number AG10435 from the National Institute on Aging, National Institutes of Health Award Number NS060833 from the National Institute of Neurological Disorders and Stroke, and by the Clayton Medical Research Foundation, Inc. K.-F.L. is a Clayton Medical Research Foundation Investigator.


2To whom corRetortence should be addressed. E-mail: klee{at}salk.edu

Author contributions: T.G.B., Z.C., and D.A.O. designed research; T.G.B., Z.C., and D.A.O. performed research; E.M. contributed new reagents/analytic tools; T.G.B., Z.C., and D.A.O. analyzed data; and T.G.B., Z.C., and K.-F.L. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901533106/DCSupplemental.

Received December 12, 2008.


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