Metabotropic Glutamate Receptor 5 in Down's Syndrome ...

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Metabotropic Glutamate Receptor 5 in Down's Syndrome Hippocampus During Development: Increased Expression in Astrocytes A.M. Iyer1,#, J. van Scheppingen1,#, I. Milenkovic2, J.J. Anink1, D. Lim3, A.A. Genazzani3, H. Adle-Biassette4, G. G. Kovacs2 and E. Aronica1,5,6,* 1

Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, The Netherlands; 2Institute of Neurology, Medical University of Vienna, Austria; 3Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale ‘‘Amedeo Avogadro’’, Novara, Italy; 4Inserm UMR 676, Physiopathology and Neuroprotection of the developing brain, Robert Debre´ Hospital, Paris, France; 5SEIN – Stichting Epilepsie Instellingen Nederland, Heemstede, The Netherlands; 6Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, The Netherlands Abstract: Metabotropic glutamate receptor 5 (mGluR5) is highly expressed throughout the forebrain and hippocampus. Several lines of evidence support the role of this receptor in brain development and developmental disorders, as well as in neurodegenerative disorders like Alzheimer’s disease (AD). In the present study, the expression pattern of mGluR5 was investigated by immunocytochemistry in the developing hippocampus from patients with Down's syndrome (DS) and in adults with DS and AD. mGluR5 was expressed in developing human hippocampus from the earliest stages tested (9 gestational weeks), with strong expression in the ventricular/subventricular zones. We observed a consistent similar temporal and spatial neuronal pattern of expression in DS hippocampus. However, in DS we detected increased prenatal mGluR5 expression in white matter astrocytes, which persisted postnatally. In addition, in adult DS patients with widespread ADassociated neurodegeneration (DS-AD) increased mGluR5 expression was detected in astrocytes around amyloid plaque. In vitro data confirm the existence of a modulatory crosstalk between amyloid- and mGluR5 in human astrocytes. These findings demonstrate a developmental regulation of mGluR5 in human hippocampus and suggest a role for this receptor in astrocytes during early development in DS hippocampus, as well as a potential contribution to the pathogenesis of ADassociated pathology.

Keywords: Alzheimer’s disease, astrocytes, development, Down's syndrome, hippocampus, mGluR5. INTRODUCTION Metabotropic glutamate receptors (mGluRs) are a family of eight G-protein linked receptors that regulate a variety of intracellular signaling systems critically affecting hippocampus-dependent synaptic plasticity and memory [1, 2]. mGluRs are classified into three distinct groups on the basis of their agonist affinity and G-protein coupling. Group I includes mGluR1 and mGluR5 subtypes, which are coupled to phosphoinositide hydrolysis [3, 4]. mGluR5 is expressed in both neurons and astrocytes and plays an important role in the regulation of the glio-neuronal cross-talk under both physiological and pathological conditions [5-8]. Moreover, several lines of evidence indicate that mGluR5 is involved in early developmental processes, including proliferation, differentiation, and survival of neural progenitors ([9-11]; for review see [12]). In rat brain mGluR5 mRNA is highly expressed during early postnatal development [13], and remains high in zones of active neurogenesis, such as the adult olfactory bulb [14, 15]. We have recently investigated the *Address correspondence to this author at the Dept. (Neuro) Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands, Tel: 31-20-5662943; Fax: 31-20-5669522; E-mail: [email protected] #

The first two authors contributed equally to this study. 1567-2050/14 $58.00+.00

expression and cell-specific distribution of group I mGluRs during prenatal human cortical development, showing that mGluR5 is expressed during early stages of cortical development, with strong expression in the ventricular/subventricular zone [16]. A more recent study showed a transient expression of mGluR5 in neural progenitors within the prenatal human fetal hippocampus suggesting stagespecific roles for this receptor in activity-dependent forms of synaptic plasticity [17]. A growing body of evidence indicates that a dysfunction of mGluR5 activity may play a role in developmental disorders (such as such as Fragile X syndrome; FXS), as well as in neurodegenerative disorders like Alzheimer’s disease (AD) [4, 7, 12, 18-21]. FXS and Down’s syndrome (DS), both characterized by prominent cognitive impairments, share disturbances in common pathways regulating the local protein synthesis through phosphorylation of Fragile X mental retardation protein (FMR) [21]. Importantly, brains of individuals with DS develop AD-related neuropathological alterations [22-24]. mGluR5 also plays a key role in amyloid precursor protein (APP) processing [20, 25-27]. Moreover, amyloid- (A) has been shown to induce over-expression of mGluR5 in astrocytes resulting in alterations of Ca2+ homeostasis and the expression of this receptor has been found to be upregulated in hippocampal astrocytes of AD patients in © 2014 Bentham Science Publishers

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proximity of A plaques [28-30]. A previous study reported a prenatal upregulation of mGluR5 in the cerebral cortex of DS patients [31], however, the expression pattern of mGluR5 in the hippocampus of DS patients remains uncharacterized. In the present study, we have investigated the expression and cell-specific distribution of mGluR5 during prenatal and postnatal human hippocampal development in controls, as well as in the developing hippocampus of DS patients prior to establishment of AD neurodegeneration and in DS cases with AD end-stage pathology. METHODS Human Material The subjects included in this study were selected from the databases of the Department of Neuropathology of the Academic Medical Center, University of Amsterdam, The Netherlands; the Department of Neuropathology of the Institute of Neurology, Medical University of Vienna, Austria (in the frame of a project approved by the Ethical Committee of the Medical University of Vienna, entitled “Molecular neuropathologic examinations of neurodegeneration-related proteins in Down-syndrome, Ek Nr. 1316/2012); the National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders (NICHD; University of Maryland, Baltimore, MD); the Netherlands Brain Bank (NBB). Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. Tissue was obtained and used in a manner compliant with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee of the AMC. We included brains of fetuses at different gestational weeks (9-41 GW), neonates and children from control and DS patients (Table 1). Fetal brain was obtained from spontaneous or medically induced abortions with appropriate maternal written consent for brain autopsy. Gestational ages were based on obstetric data, fetal and brain weights and standard fetal anthropometric measurements. We performed a careful histological and immunohistochemical analysis and evaluation of clinical data (including genetic data, when available). We excluded cases with other chromosomopathies, major central nervous system (CNS) malformations, brains with postmortem autolysis, severe hypoxic/ischemic encephalopathy, intraventricular hemorrhages, severe hydrocephalus and meningitis or ventriculitis. We only included as control cases, specimens displaying a normal hippocampal and cortical structure for the corresponding age and without any significant brain pathology. Additionally (Table 1), we obtained adult brain tissue at autopsy from 11 controls (without evidence of degenerative changes, and lacking a clinical history of cognitive impairment), 6 patients with DS (neurofibrillary tangle, NFT stage: V and VI) and 3 patients (NFT stage II, without signs of cognitive impairment). All cases were pathologically staged according to published and recommended criteria (NFT stage; [32]. All autopsies were performed within 24 h after death. Tissue Preparation One or two representative paraffin blocks per case (hippocampus and temporal cortex) were sectioned, stained and

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assessed. Formalin fixed, paraffin-embedded tissue was sectioned at 6 m and mounted on pre-coated 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 stainings for a number of antigens described below. Table1.

Cases included in this study. Controls

Age Range (gw/d/wk/yrs)

N= Cases

Sex (m/f)

9-23 gw

52

29/20/3nd

24-32 gw

22

14/8

33-41 gw

8

4/4

1-15 d

9

3/6

2 -8 m

12

8/4

1-15 yrs

4

2/2

25-65 yrs

14; 3 stage II

5/9

Down‘s Syndrome 14-23 gw

27

14/11/2nd

24-32 gw

5

2/3

33-41 gw

4

2/2

1-15 d

4

3/1

2 -8 m

4

3/1

1-15 yrs

4

2/2

50-64 yrs

6; 3 stage V; 3 VI

4/2

gw: weeks of gestation; gw/d/wk/yrs: postnatal days/weeks years; nd: not determined; m = male; f = female; neurofibrillary tangle, NFT stage: II-VI.

Immunocytochemistry Glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4000; monoclonal mouse; DAKO; 1:50), vimentin (mouse clone V9, DAKO; 1:400), 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), A (Mouse clone 6F/3D; DAKO; 1:200), phosphorylated Tau (pTau; mouse clone AT8; Innogenetics, Alpharetta, GA, USA; 1:5000), were used in the routine immunocytochemical analysis. For the detection of mGluR5 we used two antibodies (polyclonal rabbit ab53090, from Abcam, 1:100; polyclonal rabbit from Upstate Biotechnology, Lake Placid, NY; 1:100). Characterization of these antibodies in human brain tissue has been documented previously [16, 17, 33-35]. Singlelabel immunocytochemistry was developed using the Powervision kit (Immunologic, Duiven, The Netherlands). 3,3-

mGluR5 in Down's Syndrome

Diaminobenzidine (Sigma, St. Louis, USA) was used as the chromogen. Sections were counterstained with hematoxylin. For double-labeling, sections were incubated with Brightvision poly-alkaline phosphatase (AP)-anti-Rabbit (Immunologic, Duiven, The Netherlands) for 30 minutes at room temperature, and washed with PBS. Sections were washed with Tris-HCl buffer (0.1 M, pH 8.2) to adjust the pH. AP 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, 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--mouse IgG; Immunologic, Duiven, The Netherlands). AP activity was visualized with the AP 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. For double-labeling immunofluorescent staining of mGluR5 with A [or GFAP, HLA-DR , vimentin or nestin (monoclonal mouse, R&D systems, Minneapolis, MN; 1:500)] 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® 488 anti-mouse IgG (1:100, Molecular Probes, The Netherlands). Sections were then analyzed by means of a laser scanning confocal microscope (Leica TCS Sp2, Wetzlar, Germany). Evaluation of Immunostaining 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 mGluR5 staining was evaluated, as previously described [36, 37], using a semi-quantitative scale ranging from 0 to 3 (0: negative; 1: weak; 2: moderate; 3: strong staining). The score represents the predominant cell staining intensity found for each case. The frequency of positive glial cells was also evaluated using a semi-quantitative scale ranging from 1 to 3, including score 1 (rare, immunolabeling observed in 50% of cells) to assess the relative number of positive glial cells within the alveus/white matter. As proposed before [3638], the product of these two values (intensity and frequency scores) was taken to give the overall score (total labeling score). We performed optical density (OD) measurements in control and DS hippocampus (as previously described [39]) for mGluR5 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, 20x) 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

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background from fields lacking labeling was determined. A mean optical density (OD) value for the CA1 was calculated, expressed as a ratio of the mean optical density (ODR) 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 analysed using two-tailed Student’s t-test or a nonparametric Kruskal-Wallis test, followed by a Mann-Whitney test to assess the difference between groups. The value of P < 0.05 was defined statistically significant. Western Blot Analysis For immunoblot analysis we used frozen brain specimens from control, AD and AD-DS 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 (Smith et al., 1985). For electrophoresis, equal amounts of proteins (50 μg/lane) were separated by sodium dodecylsulfatepolyacrylamide gel electrophoretic (SDS-PAGE) analysis (7.5% 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 TBST (20 mM Tris, 150 mM NaCl, 1 % Tween, pH 7.5) / 5% non-fat dry milk, blots were incubated overnight at 4°C with anti-mGluR5 antibody (polyclonal rabbit from Upstate Biotechnology, Lake Placid, NY; 1:500) or -tubulin (1:30000, monoclonal mouse, Sigma, St. Louis, MO, USA). After several washes in TBST, the membranes were incubated in TBST / 5% non-fat dry milk, containing the goat anti-rabbit or rabbit anti-mouse coupled to horseradish peroxidase (1:2500; Dako, Denmark) for 1 h. After washes in TBST, immunoreactivity was visualized using ECL PLUS western blotting detection reagent (GE Healthcare Europe, Diegen, Belgium). For the quantification of the blots, the band intensities were measured densitometrically using the 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 for normalization. Cell Cultures For cell culture experiments (astrocyte-enriched human cultures), fetal brain tissue (14-17 weeks of gestation) was obtained from spontaneous or medically induced abortions with appropriate maternal written consent for brain autopsy. Resected tissue samples were collected in Dulbecco’s modified Eagle’s medium, DMEM/HAM F10 (1:1) medium (Gibco, Grand Island, NJ). Cell isolation was performed as previously described [5, 40]. Secondary astrocyte cultures were established by trypsinizing confluent cultures and subplating onto PLL-precoated 6 wellplates or 75 cm2 flasks (0.5-1 X 106 cell/ml; for Western blot analysis or for the generation of serial passages respectively). In the present study astrocytes were used for Western blot analysis at passage 3-4. A 1-42 (A42) was purchased either from Bachem or from Innovagen (Lund, Sweden). A42 oligomers were prepared as described by Lim et al., [29]. The


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peptide was snap frozen and kept at -80°C. A42 was applied at the final concentration of 100 nM for 24 h. RESULTS mGluR5 Expression in the Developing Control Hippocampus The hippocampal expression of mGluR5 was investigated at different prenatal ages, ranging from 9 to 41 GW, as well as at postnatal ages (1day to 8 months and 1-15 yrs; Table 2; Fig. 1, 6). Strong mGluR5 immunoreactivity (with nuclear/perinuclear staining) was detected at early stages of development (9-19 GW) in the ventricular/subventicular zone (VZ/SVZ) and in the dentate gyrus (DG; Table 2; Fig. 1 A-E). Co-localization of mGluR5 with neuronal precursor markers (nestin and vimentin) was observed within the VZ (Fig. 1D). Around mid-gestation, mGluR5 was still detected in the VZ/SVZ, but increased expression was observed in the stratum pyramidale (SP; Table 2; Fig. 1F, H, K). mGluR5 was observed in the neuropil and in cell somas and this pattern of immunoreactivity (with cytoplasmic neuronal labeling) persisted at later prenatal and postnatal ages (Table 2; Fig. 2; Fig. 6 A). At older prenatal and early postnatal ages mGluR5 immunoreactivity was still observed in the VZ-SVZ zone in clusters of cells (Table 2; Fig. 1J; Fig. 2). Only weak mGluR5 was occasionally observed in astroglial cells within the hippocampal white matter (Table 2; Fig. 6B). mGluR5 Expression in the Developing Down's Syndrome (DS) Hippocampus In DS hippocampus the expression pattern of mGluR5 in VS/SVZ, DG and SP was similar to that observed in agematched controls (Fig. 3; Table 2). Strong mGluR5 immunoreactivity (with nuclear/perinuclear staining) was detected at early stages of development in VZ/SVZ, whereas increased expression was observed in the stratum pyramidale (with cytoplasmic neuronal labeling) at later prenatal and Table 2.

postnatal ages (Table 2; Fig. 3 and Fig. 4A, F). However, around mid-gestation we observed higher level of mGluR5 expression in astroglial cells within the hippocampal white matter which persisted postnatally (Table 2; Fig. 3G, L; Fig. 4D-E, G-H; Fig. 6B). We acknowledge limitations to the interpretation of these results. One major limitation in the studies using postmortem fetal tissue is the availability of brain tissue, because the number of cases with permission for brain autopsy at early developmental stages is limited, cases with other concomitant cerebral pathologies have to be excluded, and frozen representative material is often not available. mGluR5 Expression in DS Hippocampus with Alzheimer’s Disease (AD) Pathology In the hippocampus of DS with AD pathology, a prominent expression of mGluR5 in astrocytes was observed throughout the different hippocampal regions, particularly around A deposits (Fig. 4I-K). In contrast, in adult control hippocampus, no detectable immunoreactivity was observed in glial cells (not shown). Double labeling experiments confirmed the increased expression of mGluR5 in astrocytes of DS-AD patients in proximity of A plaques (Fig. 5A-C). Only occasionally co-localization with a microglial marker (HLA-DR) was observed (Fig. 5D). Western blot analysis could be only performed in adult DS-AD cases of which frozen material was available (n=3). Expression of mGluR5 was increased in patients with AD pathology, compared to controls (Fig. 7A-B). mGluR5 in Cultured Human Astrocytes A has been shown to induce an upregulation of mGluR5 in rodent astrocytes [28-30]. To investigate whether A may regulate the expression of this receptor also in human astrocytes, astrocyte-enriched human cell cultures were treated with A42 oligomers (two independent cultures).

Summary of mGluR5 immunoreactivity in human fetal hippocampus in control and Down's syndrome.

Hippocampal Complex

Stages Gestational Week (Staining Intensity: +/-;+;++; n/c/np) Control

Stages Gestational Week (Staining Intensity:+/-;+;++;n/c/np) Down's Syndrome

Ventricular/subventricular zone (VZ/SVZ)

9-23 (++); n/c 24-32 (++); n/c 33-41 (+/-;+);c

9-23 (++);n/c 24-32 (++);n/c 33-41 (+/-; +);c

Stratum pyramidale (CA1-CA4)

14-23 (+/-;+);n/c 24-32 (+);n/c/np 33-41 (++); c/np

14-23 (+/-;+)n/c 24-32 (+);n/c/np 33-41 (++);c/np

Dentate gyrus

14-23 (+;++);n/c 24-32 (+;++);n/c/np 33-41 (++); c/np

14-23 (+;++);n/c 24-32 (+;++);n/c/np 33-41 (++); c/np

Alveus/white matter (astrocytes)

14-23 (-) 24-32 (-;+/-) 33-41 (-;+/-)

14-23 (+;++) 24-32 (+;++) 33-41 (+;++)

Staining was evaluated using a semi-quantitative scale: -, no; +/-, weak; +, moderate; ++, strong staining); n= nuclear staining; c=cytoplasmic staining; np: neuropil staining



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Fig. (1). mGluR5 immunoreactivity (IR) at different gestational ages (GW) in the hippocampus. Panels A-B: mGluR5 IR in the hippocampus at 14 GW. The hippocampus shows strong mGluR5 expression in the ventricular zone (VZ; arrow in A; insert in B; nuclear/perinuclear staining). Panels C-E: mGluR5 IR in the hippocampus at 19 GW. mGluR5 IR is detected in the dentate gyrus (DG), VZ (D) and stratum pyramidale (SP; E; nuclear/perinuclear staining). Inserts in D: confocal images showing co-localization of mGluR5 (red) with nestin and vimentin (green) in VZ. Panels F-H: mGluR5 IR in the hippocampus at 23 GW. mGluR5 IR is detected in DG, VZ (G) and SP (F). Panel I: mGluR5 IR in VZ at 27 GW. Panels J-K: mGluR5 IR in VZ (J) and SP (K) at 32GW. Hematoxylin counterstain. Scale bar in K: A, C, F: 400 m; B, D: 80 m; E, H, K: 40 m G-J: 30 m.


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Fig. (2). mGluR5 immunoreactivity (IR) in the postnatal hippocampus. Panels A-C: postnatal hippocampus (2 weeks) showing mGluR5 IR throughout the different hippocampal subfields. mGluR5 IR is observed in the VZ (B), as well as in stratum pyramidale (CA1; C). Panels D and E: 7 months (D) and adult hippocampus (E) with prominent mGluR5 IR throughout the hippocampus. Insert a in D: mGluR5 IR in glial cells in the VZ. Insert b in D: mGluR5 IR in CA1. Hematoxylin counterstain. Scale bar in E: A, D, E: 400 m; B: 40 m; C: 30 m.

Western Blot analysis. demonstrated increased expression of mGluR5 in cultured human astrocytes exposed to 100 nM A42 oligomers (Fig. 8A-B). DISCUSSION Increasing evidence supports a role of group 1 mGluRs in age-associated cognitive deficits, and mGluR5 may represent a molecular link between FXS and DS [20, 26, 27]. DS represents one of the most common genetic causes of cognitive impairment and aged individuals with DS often develop progressive AD neuropathology [22-24]. Immunocytochemical studies of post-mortem tissue represent one valuable approach for studying neurotransmitter receptor expression during human brain development prior to the establishment of neurodegeneration, providing information that can be used to interpret functional experimental data. This study provides the first description of the expression pattern and cellular localization of mGluR5 in control and DS hippocampus during the pre- and early postnatal development. In agreement with previous observations in prenatal human cortex [16], mGluR5 was detected during the early stages of hippocampal development, with strong expression in the VZ/SVZ zone and co-localization with neural progenitor markers, such as vimentin and nestin. These observations are in line with the high expression of this receptor reported in developing rodent brain [13, 41, 42] as well as in zones of active neurogenesis in the postnatal rat brain [9, 14,15]. Accordingly, in vitro studies support the role of mGluR5 in the regulation of proliferation and survival of neural progenitor cells [10, 43-45]. This regulation may involve the generation

of oscillatory intercellular calcium waves [46], as well as the activation of key pathways, such as the mitogen-activated protein kinase (MAPK) signaling cascade [47]. Interestingly, during the early developmental stages we observed a nuclear expression of mGluR5. This nuclear expression has been previously reported also in zones of active neurogenesis of the embryonic and postnatal brain [9] and has been shown to mediate sustained Ca2+ oscillatory responses [48], playing a key role in the regulation of intranuclear signaling pathways associated with development, plasticity, and survival [49]. In agreement with previous reports in human tissues [16, 17], around mid-gestation mGluR5 expression increased within the SP with detection of cytoplasmic neuronal labeling, which persisted at later prenatal and postnatal ages. In DS hippocampus we observed a similar developmental pattern of mGluR5 expression with prenatal expression in VS/SVZ and increased neuronal expression at later prenatal and postnatal ages. However, in contrast to age matched controls, in DS hippocampus around mid-gestation we observed higher level of mGluR5 expression in astroglial cells within the hippocampal white matter and this astroglial expression persisted postnatally [50]. This receptor upregulation does not simply reflect the presence of reactive astrocytes. Accordingly, in a recent study using the same cohort we could not detect significant differences in the expression of GFAP between DS patients and controls [50]. A growing body of evidence indicates that mGluR5 critically regulates the function of glial cells and may play an important role in different pathological conditions, including also neurodegeneration (for reviews see [7, 51]. Glial mGluRs (including mGluR5) have been shown to regulate glial cell proliferation [52],



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Fig. (3). mGluR5 immunoreactivity (IR) at different gestational ages (GW) in Down's syndrome hippocampus. Panels A: mGluR5 IR in the hippocampus at 14 GW. The hippocampus shows strong mGluR5 IR in the ventricular zone (VZ; insert; nuclear/perinuclear staining). Panels B-D: mGluR5 in the hippocampus at 19 GW. mGluR5 IR is detected in the dentate gyrus (DG), VZ (C) and stratum pyramidale (SP; D; nuclear/perinuclear staining), as well as alveus/fimbria (Alv/Fim; insert in B). Panels E-H: mGluR5 IR in the hippocampus at 23 GW. mGluR5 IR is detected in VZ (E) and SP (F) and astroglial staining is observed in the alveus (Alv; arrows in G). Panel H: absence of mGluR5 IR in control alveus (23 GW). Panel I: confocal image showing co-localization of mGluR5 (red) with GFAP (green) in alveus (DS 23 GW). Panels J-L: mGluR5 IR in the hippocampus at 35 GW, showing IR throughout the different hippocampal subfields. mGluR5 IR is observed in VZ (K), as well as in the alveus (arrows in L) and in the stratum pyramidale (SP; M).Hematoxylin counterstain. Scale bar in M: A: 80 m; B, J: 400 m; C-E, H, K-L: 40 m; F-G: 25 m; D, I, M: 30 m.


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Fig. (4). mGluR5 immunoreactivity (IR) in control and Down's syndrome (DS) early postnatal and adult hippocampus. Panels A-D: postnatal hippocampus (1 day-2 weeks) showing mGluR5 IR throughout the different hippocampal subfields. mGluR5 IR is observed in VZ (B), as well as in stratum pyramidale (CA1; C) and in the alveus with expression in astrocytes (arrows in E). Panels F-H: mGluR5 IR in the hippocampus at 8 months. mGluR5 IR is detected throughout the different hippocampal subfields with strong expression in glial cells within alveus/fimbria (Alv/Fim; G-H). Panels I-K: adult hippocampus of Down's syndrome (50 yrs) showing mGluR5 IR throughout the different hippocampal subfields with increased IR in the CA1 (Arrows in I). Panels J and K: high magnification of CA1 (K) showing strong mGluR5 expression in astrocytes surrounding amyloid plaques (arrows). Hematoxylin counterstain. Scale bar in K: A, F, I: 400 m; B-E, G-H: 40 m; J: 160 m; K: 30 m.



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Fig. (5). mGluR5 immunoreactivity (IR) in Down's syndrome with Alzheimer’s disease (AD) pathology. Panel A: increased expression of mGluR5 (red; arrows) around an amyloid plaque (blue; asterisk); insert in A shows colocalization (purple) of mGluR5 (red) with GFAP (blue). Panel B: confocal image showing expression of mGluR5 (red) around A deposits (green). Panel C: confocal image showing colocalization (yellow) of mGluR5 (red) with GFAP (green). Panels D: confocal image of mGluR5 (red) with a microglial/macrophages marker (HLA-DR; green); the large majority of microglial cells are not expressing mGluR5, only occasionally co-localization is observed (arrow). Scale bar in D: A: 40 m; B: 30 m; C-D: 20 m.

Fig. (6). Evaluation of mGluR5 immunoreactivity in hippocampus of control and Down's syndrome (DS) during development. A: relative optical density ratio (ODR) of mGluR5 immunoreactivity in CA1. B: immunoreactive score (IR) of mGluR5 in the white matter (alveus). Values represent the mean ± SEM of samples at different prenatal and postnatal ages. *p < 0.05, compared to control.


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Fig. (7). Western blot analysis of mGluR5 in hippocampus of control and Down's syndrome with Alzheimer’s disease pathology (DSAD). (A): representative blots of mGluR5 (~ 140 kDa) in total homogenates of hippocampus of control (n= 3) and DS-AD (n= 3). (B): densitometric analysis values of DS-AD (optical density units, O.D., normalized with the optical density of -tubulin) are expressed relative to controls (mean ± SEM of two independent experiments; *p < 0.05, compared to control).

Fig. (8). Expression levels in cultured human astrocytes after exposure to A42. (A): Representative immunoblot of mGluR5 in human astrocytes 24h after exposure to A42 (100 nM; 3-5 treated); (B) densitometric analysis: values (optical density units relative to the optical density of -tubulin) are mean ± SEM of two independent cultures (n= 4 untreated; n= 6 treated) and are expressed relative to the levels in unstimulated cells (*p < 0.05, compared to control).

production and release of different growth factors and cytokines [53-55], as well as the expression glutamate transporter proteins [5, 56]. The upregulation of mGluR5 was still observed in the hippocampus of DS patients with AD and was more prominent around A deposits. This is in line with the recent observations of mGluR5 upregulation in astrocytes around amyloid plaques in patients with AD [28, 29]. In vitro studies [28-30]; present study) demonstrate that A is responsible for the induction of mGluR5 in astrocytes and this results in alterations of Ca2+ homeostasis [29, 30], which may critically contribute to the toxicity of A protein and disease progression [29, 30]. In particular, we have shown that A exposure leads to transcriptional remodeling of the Ca2+-signalling inducing an up-regulation of key components of the astroglial Ca2+ signalling machinery, including mGluR5, IP3R1 and IP3R2; this re-programming appears to be mediated by the Ca2+-dependent phosphatase calcineurin, a transcriptional switch and its downstream target NF-kB

[29, 30]. Interestingly, we also show that in hippocampus of AD patients, mGluR5 is over-expressed with calcineurin in GFAP-positive astrocytes in proximity to amyloid plaques and co-localized with the nuclear NF-kB subunit p65 [29]. Further research on the role of other mGluR (group II and III) subtypes in human brain tissue, as well as in animal models of DS is needed to further elucidate the role of these receptors and their signalling pathways in DS, as well as in other developmental disorders sharing a dysregulation of converging signalling pathways. Particularly interesting could be the evaluation of Homer isoforms, scaffolding proteins which closely interact with mGluR5 modulating its function, under both physiological and pathological conditions [57, 58]. In summary, our findings demonstrate a developmental regulation of mGluR5 in human hippocampus and highlight the potential contribution of mGluR5 upregulation in astro-


cytes to the early disease pathogenesis in DS patients. Future investigation targeting mGluR5 in DS experimental models might be worthwhile to further develop our current understanding of the role of this receptor as link between neurodevelopmental disorders and neurodegeneration.


[16] [17]

CONFLICT OF INTEREST

[18]

The authors confirm that this article content has no conflict of interest.

[19]

ACKNOWLEDGEMENTS This work was supported by the EU FP7 project DEVELAGE (Grant Agreement N 278486; EA, AI, IM, and GGK). We are grateful to Shauna J. Thompson for her technical help. REFERENCES [1] [2] [3] [4] [5]

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