Developmental patterns of DR6 in normal human hippocampus and in Down syndrome
© Iyer et al.; licensee BioMed Central Ltd. 2013
Received: 9 December 2012
Accepted: 4 April 2013
Published: 24 April 2013
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© Iyer et al.; licensee BioMed Central Ltd. 2013
Received: 9 December 2012
Accepted: 4 April 2013
Published: 24 April 2013
Death receptor 6 (DR6) is highly expressed in the human brain: it has been shown to induce axon pruning and neuron death via distinct caspases and to mediate axonal degeneration through binding to N-terminal β amyloid precursor protein (N-APP).
We investigated the expression of DR6 during prenatal and postnatal development in human hippocampus and temporal cortex by immunocytochemistry and Western blot analysis (118 normal human brain specimens; 9 to 41 gestational weeks; 1 day to 7 months postnatally; 3 to 91 years). To investigate the role of N-APP/DR6/caspase 6 pathway in the development of hippocampal Alzheimer’s disease (AD)-associated pathology, we examined DR6 immunoreactivity (IR) in the developing hippocampus from patients with Down syndrome (DS; 48 brain specimens; 14 to 41 gestational weeks; 7 days to 8 months postnatally; 15 to 64 years) and in adults with DS and AD.
DR6 was highly expressed in human adult hippocampus and temporal cortex: we observed consistent similar temporal and spatial expression in both control and DS brain. Western blot analysis of total homogenates of temporal cortex and hippocampus showed developmental regulation of DR6. In the hippocampus, DR6 IR was first apparent in the stratum lacunosum-moleculare at 16 weeks of gestation, followed by stratum oriens, radiatum, pyramidale (CA1 to CA4) and molecular layer of the dentate gyrus between 21 and 23 gestational weeks, reaching a pattern similar to adult hippocampus around birth. Increased DR6 expression in dystrophic neurites was detected focally in a 15-year-old DS patient. Abnormal DR6 expression pattern, with increased expression within dystrophic neurites in and around amyloid plaques was observed in adult DS patients with widespread AD-associated neurodegeneration and was similar to the pattern observed in AD hippocampus. Double-labeling experiments demonstrated the colocalization, in dystrophic neurites, of DR6 with APP. We also observed colocalization with hyper-phosphorylated Tau and with caspase 6 (increased in hippocampus with AD pathology) in plaque-associated dystrophic neurites and within the white matter.
These findings demonstrate a developmental regulation of DR6 in human hippocampus and suggest an abnormal activation of the N-APP/DR6/caspase 6 pathway, which can contribute to initiation or progression of hippocampal AD-associated pathology.
Death receptors (DRs) belong to the tumor necrosis factor receptor superfamily and are known to induce apoptosis (programmed cell death) via the intracellular portion of the receptor referred to as the ‘death domain.’ [1–3]. A growing body of evidence indicates that DRs may mediate a variety of biological functions, including nonapoptotic functions [1, 4–7].
Tumor necrosis factor TNFRSF21 (death receptor 6, DR6) is a relatively new member of the DR family, which has been found to induce apoptosis when overexpressed [8, 9] (for review see [3, 7]). Recently, Nikolaev and colleagues  reported that the N-terminal fragment of amyloid precursor protein (N-APP) may act as a ligand of DR6 and trigger axon pruning and neurodegeneration via caspase 6, suggesting a role for this receptor in the neurodegeneration observed in Alzheimer’s disease (AD; for review see ). Interestingly, different signaling cascades have been shown to be involved in degeneration of the axon (caspase 6) and neuronal cell body (caspase 3) [10, 12]. Although there is still discussion about the formation of the N-APP fragment [13, 14] and the structural features of the potential DR6-APP signaling complex [15, 16], the identification of this APP-dependent pathway involving DR6 highlights the potential role of this receptor in specific types of disease-associated axonal degeneration. In addition, recent studies suggest additional physiological functions of DR6 during brain development [5, 6].
DR6 is expressed in most human tissues [7, 8]. Interestingly, DR6 mRNA expression is high in adult brain and particularly enriched in regions like the hippocampus that are vulnerable in AD . The downstream DR6 effector, caspase 6, has been shown to be activated early in AD and to be associated with mild cognitive impairment [17–19]. However, the expression pattern and cellular localization of DR6 protein during human brain development remains uncharacterized.
In this study, we examined the expression of DR6 in the developing human hippocampus and temporal cortex to get a better insight into the role of this receptor in prenatal human brain development. In addition, we investigated DR6 expression in the developing hippocampus of patients with Down syndrome (DS) prior to establishment of AD neurodegeneration and in DS patients with AD pathology compared with age-matched control, and in hippocampal specimens obtained from patients with sporadic AD with severe, endstage pathology. Elucidation of complex pathogenetic pathways characterizing the earliest stage of the detrimental processes that result in neurodegeneration represents an essential first step towards a therapeutic intervention, which could be able to block these pathological processes and, eventually, prevent the onset of the disease in DS patients.
Cases included in this study
Number of cases
9 to 20 GW
21 to 41 GW
1 to 6 days
2 weeks to 7 months
3 to 65 years
18; 3 stage II
30 to 67 years
14 to 20 GW
9/6 5 not determined
21 to 41 GW
7 to 12 days
2 to 8 months
15 to 64 years
7; 3 stage V; 2 VI
77 to 90 years
6; 3 stage V; 3 VI
Additionally, we obtained adult brain tissue at autopsy from eleven control subjects (without evidence of degenerative changes, and lacking a clinical history of cognitive impairment), three patients with posttraumatic brain injury, six patients with DS (Braak Neurofibrillary Staging: V and VI), and nine patients with AD (three with Braak stage II, without signs of cognitive impairment and six with Braak stage V and VI) (Table 1). All subjects were pathologically staged according to Braak and Braak criteria . Subjects without known cause of death were excluded. All autopsies were performed within 24 h after death.
One or two representative paraffin blocks per brain (hippocampus and temporal cortex) were sectioned, stained, and assessed. Formalin-fixed, paraffin-embedded tissue was sectioned at 6 μm and mounted on precoated glass slides (Star Frost, Waldemar Knittel GmbH, Braunschweig, Germany). Sections of all specimens were processed for hematoxylin eosin, Luxol fast blue, and Nissl stains, as well as for immunocytochemical stains for a number of markers, listed next. We performed a silver impregnation (Bielschowsky) staining in all adult brains.
Glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4,000; monoclonal mouse; DAKO; 1:50), vimentin (mouse clone V9, DAKO; 1:400), neuronal nuclear protein (NeuN; mouse clone MAB377, IgG1; Chemicon, Temecula, CA, USA; 1:1,000), neurofilament protein (mouse clone 2 F11, Neomarkers, Fremont, CA, USA; 1: 200), synaptophysin (mouse clone Sy38; DAKO; 1:200; polyclonal rabbit, DAKO; 1:200), human leukocyte antigen(HLA)-DP, DQ, DR (major histocompatibility complex class II, MHC-II; mouse clone CR3/43; DAKO, Glostrup, Denmark, 1:400), and CD68 (mouse clone PG-M1, DAKO, Glostrup, Denmark; 1:200) were used in the routine immunocytochemical analysis.
To detect death receptor 6 (DR6), we used a polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100); to detect APP, a monoclonal antibody (mouse clone 22C11, Chemicon, Temecula, CA, U.S.A;1:50,000); and to detect caspase 6, a polyclonal rabbit antibody (Abcam, Cambridge, MA, USA;1:500).
Single-label immunocytochemistry was developed using the Powervision kit (Immunologic, Duiven, The Netherlands). 3,3-Diaminobenzidine (Sigma, St. Louis, USA) was used as chromogen. Sections were counterstained with hematoxylin.
For double labeling of DR6 with amyloid-β (clone M0872, DAKO; 1:200), GFAP, or synaptophysin, sections were, after incubation with the primary antibodies overnight at 4°C, incubated for 2 h at room temperature with Alexa Fluor® 568-conjugated anti-rabbit and Alexa Fluor®-conjugated 488 anti-mouse IgG or anti-goat IgG (1:100, Molecular Probes, The Netherlands). Sections were then analyzed using a laser scanning confocal microscope (Leica TCS Sp2, Wetzlar, Germany).
For double labeling of DR6 with APP or Tau (Clone AT8, Innogenetics Alpharetta, GA, USA; 1:5,000), or caspase 6 or caspase 3 (polyclonal rabbit, Signaling Technology Danvers, MA, USA; 1:100), sections were incubated with Brightvision poly-alkaline phosphatase-anti-rabbit (Immunologic, Duiven, The Netherlands) for 30 min at room temperature, and washed with PBS. Sections were washed with Tris–HCl buffer (0.1 M, pH 8.2) to adjust the pH. Alkaline phosphatase activity was visualized with the alkaline phosphatase substrate kit I Vector Red (SK-5100, Vector laboratories Inc., CA, USA). To remove the first primary antibody (DR6), sections were incubated at 121°C in citrate buffer (10 mM NaCi, pH 6.0) for 10 min. Incubation with the second primary antibody was performed overnight at 4°C. Sections with primary antibody other than rabbit were incubated with post antibody blocking from the Brightvision+ system (containing rabbit-anti-mouse IgG; Immunologic, Duiven, The Netherlands). Alkaline phosphatase activity was visualized with the alkaline phosphatase substrate kit III Vector Blue (SK-5300, Vector laboratories Inc., CA, USA). Sections incubated without the primary antibodies or with the primary antibodies, followed by heating treatment were essentially blank. Images were analyzed with a Nuance VIS-FL Multi-spectral Imaging System (Cambridge Research Instrumentation; Woburn, MA), as described previously [21, 22].
All labeled tissue sections were evaluated by two independent observers, blind to clinical data, for the presence or absence of various histopathological parameters and specific immunoreactivity for the different markers. The intensity of DR6 staining was evaluated, as previously described [23, 24], using a using a semi-quantitative scale ranging from 0 to 4 (0: negative; 1: weak; 2: moderate; 3: strong; 4: very strong staining). Different sub-areas of the hippocampus (CA1 to CA4 and dentate gyrus) were examined: the score represents the predominant intensity found in each case.
We measured optical density in control and DS hippocampus (as previously described ) for DR6 in the CA1. Sections were digitized using an Olympus microscope equipped with a DP-10 digital camera (Olympus, Japan). Images from consecutive, nonoverlapping, fields (magnification, 20×) were collected using image acquisition and analysis software (Phase 3 Image System integrated with Image Pro Plus; Media Cybernetics, Silver Spring, MD). The absolute pixel staining density and the background from fields lacking labeling was determined. A mean optical density value for the CA1 was calculated, expressed as a ratio with the mean optical density of the background and comparison was made between patients. Statistical analyses were performed with SPSS for Windows (SPSS 11.5, SPSS Inc., Chicago, IL, USA). Data were analyzed using a two-tailed Student’s t test or a nonparametric Kruskal-Wallis test, followed by a Mann–Whitney test to assess the difference between groups. A value of P < 0.05 was defined statistically significant.
For immunoblot analysis we used frozen brain specimens (control cortex: 13 to 41 GW; 1 day postnatally; 2 and 7 months postnatally; 3, 4, and 7 years; and adult cortex (46, 50, 50 years); as well as control hippocampus: 19 GW; 2 months postnatally; 2 years; and adult hippocampus). The frozen specimens were homogenized in lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, 0.4 mg/ml Na orthovanadate, 5 mM EDTA (pH 8.0), 5 mM NaF, and protease inhibitors (cocktail tablets, Roche Diagnostics, Mannheim, Germany). Protein content was determined using the bicinchoninic acid method . For electrophoresis, equal amounts of protein (50 μg/lane) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide). Separated proteins were transferred to nitrocellulose paper by electroblotting for 1 h and 30 min (BioRad, Transblot SD, Hercules, CA). After blocking for 1 h in Tris-buffered saline with Tween (TBST; 20 mM Tris, 150 mM NaCl, 1% Tween, pH 7.5)/5% nonfat dry milk, blots were incubated overnight at 4°C with rabbit anti-DR6 (1:1,000), mouse anti-β-tubulin (1:30,000, monoclonal mouse, Sigma, St. Louis, MO, USA), or APP (1:50,000). After several washes in TBST, the membranes were incubated in TBST and 5% nonfat dry milk, containing the goat anti-rabbit or rabbit anti-mouse coupled to horse radish peroxidase (1:2,500; Dako, Denmark) for 1 h. After washing in TBST, immunoreactivity was visualized using ECL PLUS Western blotting detection reagent (GE Healthcare Europe, Diegen, Belgium). To quantify the blots, band intensities were measured densitometrically using Scion Image for Windows (beta 4.02) image-analysis software. A ratio of the integrated band density (IntDen) of the protein of interest to the IntDen of the reference protein was used to normalize band intensities.
Summary of DR6 immunoreactivity in human fetal hippocampus in control and Down syndrome
Down syndrome (GW)
Stratum pyramidale (CA1 to CA4)
Molecular layer–dentate gyrus
Occasionally, DR6 IR was detected in dystrophic neurites in elderly patients (Braak stage II), without cognitive decline (not shown). The pattern of DR6 IR in the hippocampus of AD patients (Braak stage V-VI) was similar to that observed in DS with AD pathology (Additional file 3: Figure S3), with strong DR6 IR in dystrophic neurites in and around amyloid plaques (Additional file 3: Figure S3 B-D) and colocalization with APP and hyperphosphorylated Tau (Additional file 3: Figure S3 E-H). Spectral analysis of double labeling of DR6 with hyperphosphorylated Tau or with APP confirmed the colocalization in both AD and DS adult hippocampus (Additional file 4: Figure S4).
Increasing evidence supports the concept that neurological disorders of the adult brain could represent a disorder of aberrant neural development. According to this ‘developmental hypothesis,’ even neurodegenerative disorders might have ‘fetal’ origins (for reviews see [11, 28, 29]).
Recent studies have provided evidence of a physiological function for N-APP in developmental pruning, which, together with other mechanisms, could also contribute to neurodegeneration in AD (for review see ). In particular, it has been suggested that activation of DR6 is required for initiation or progression of axonal degeneration . However, the expression pattern and cellular localization of DR6 protein in developing brain is still largely uncharacterized and it is not clear whether, or to what extent, the N-APP-DR6 pathway might be abnormally activated in AD.
This study provides the first description of the expression pattern and cellular localization of DR6 in human hippocampus and neocortex during the pre- and early postnatal development. The period of prenatal development studied (9 to 40 GW) includes all the critical stages of human hippocampal and cortical development [30–32].
Developmental regulation of DR6 expression was observed in both neocortex and hippocampus by immunocytochemistry and was confirmed by Western blot analysis, revealing relatively high expression levels in adult brain tissue. In agreement with previous observations at the mRNA level , DR6 was particularly enriched in the hippocampus. DR6 IR was already detectable in the SLM at 16 GW and prominent IR was observed throughout the different hippocampal subfields by 40 GW. The expression of DR6 during the prenatal stages of hippocampal development supports the suggested physiological role of this receptor in shaping neuronal connections, in particular contributing to axonal pruning (for review see ), which deserves further investigation in experimental models.
An attractive hypothesis, provided by in vitro and in vivo studies using DR6 knockout mice, is that activation of DR6 by N-APP may, via caspase 6, contribute to degenerative processes . To evaluate whether the N-APP/DR6/caspase 6 pathway is abnormally activated in AD and whether its activation might precede the development of AD, we studied the components of this pathway in DS hippocampus. AD-associated neuropathology is a consistent feature in DS patients and DS represents an opportunity to understand the link between early aging and neurodegenerative processes [33, 34]. Immunocytochemical evaluation of APP (using an antibody that recognizes N-APP) provided evidence of expression prior to establishment of AD pathology. In addition, an increased expression of DR6 in dystrophic neurites (similarly to cases of early AD pathology; Braak stage II) was detected in a 15-year-old DS patient. Detection of a premature neuritic pathology in DS is consistent with previous evidence supporting the occurrence of ongoing neurodegeneration prior to the establishment of widespread AD neurodegeneration [35–38]. In adult DS cases with frank dementia as well as significant AD pathology, caspase 6 expression was strongly increased within the hippocampus. Interestingly, we detected colocalization of DR6 and caspase 6 (as well as hyperphosphorylated Tau and APP) in plaque-associated dystrophic neurites. These observations support the possible involvement of the N-APP/DR6/caspase 6 pathway in the development and progression of AD-associated pathology in DS patients. Accordingly, a similar aberrant pattern of DR6 expression was detected in sporadic AD cases with severe, endstage pathology. The presence of abnormal DR6 expression within the white matter suggests that there is also a role of this pathway in the development of white matter abnormality reported in DS brain [38, 39]. In particular, a recent study  suggests that myelination is impaired in DS hippocampus. Interestingly, a role for DR6 signaling in oligodendrocyte maturation and myelination has recently been suggested [5, 40, 41]. Thus, although we did not observed detectable difference in the expression pattern of DR6 in DS patients without AD pathology, the role of DR6 in the delayed myelination in DS hippocampus requires further evaluation.
Immunocytochemical studies of postmortem fetal tissue represent one of the few available approaches for studying protein expression during human brain development, providing information about their temporal and spatial distribution that can be used to interpret functional experimental data. However, one limitation in these studies is the availability of brain tissue. An ideal experimental design (including postnatal ages ranging between 10 and 40 years of age, prior to the establishment of widespread AD neurodegeneration) is difficult to achieve, and frozen representative material is not available at all developmental ages. Another important aspect that should be taken into consideration when analyzing DS brain is that individuals with DS develop dementia with clinical and neuropathologic features similar, but not identical, to those of adults with AD without DS (that is including complex developmental abnormalities) [42–45]. Moreover, it has been suggested that oxidative stress and inflammation present early events in DS brain pathology .
An important issue that requires clarification is represented by the still discussed mechanism of formation of N-APP fragment and its physiological role in human brain. Guo and co-authors  argue in their study that APPβ and total APP are both highly stable. Other recent studies report that APP can be processed by the metalloprotease meprin β, resulting in specific N-APP fragments, which, however, did not exert significant cytotoxicity [13, 46]. Moreover, recently, a novel amyloid precursor protein-processing pathway that generates secreted N-terminal fragments has been described .
Our study cannot resolve these complex issues; however, it provides evidence of strong DR6 expression in human hippocampus with increased IR in dystrophic neurites in DS brains in parallel with the evolution of AD-associated lesions. The detection of increased DR6 expression before endstage AD pathology and dementia are established suggests that this aberrantly increased expression of DR6 could represent an early marker of AD neuronal degeneration. Thus, premature activation of DR6 may contribute to accelerate the formation of dystrophic neurites and more extensive AD pathology through apoptosis mechanisms. However, axonal degeneration might occur via multiple pathways, which can converge on a common downstream target. Recently, a nicotinamide mononucleotide adenylyltransferase 1-sensitive pathway has been described . Moreover, a novel apoptotic pathway that mediates the DR6 apoptotic signal to mitochondrial dysfunction has been reported. .
Our findings demonstrate a developmental regulation of DR6 in human hippocampus and highlight the potential role of DR6 in specific types of disease-associated neuronal degeneration. Thus, future investigation targeting DR6 in experimental models might be worthwhile to further develop our current understanding of the role of DR6 signaling pathways in the initiation or progression of hippocampal AD-associated pathology.
amyloid precursor protein
death receptor 6
N-terminal β-amyloid precursor protein
phosphate buffered saline
sodium dodecylsulfate-polyacrylamide gel electrophoresis
standard error of the mean
Tris-buffered saline with TWEEN.
This work was supported by the EU FP7 project DEVELAGE (Grant Agreement N 278486; EA, AI, IM, and GGK). None of the authors has any conflict of interest to disclose.
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