Up-Regulation of Cation-Independent Mannose 6-Phosphate Receptor and Endosomal-Lysosomal Markers in Surviving Neurons after 192-IgG-Saporin Administrations into the Adult Rat Brain
2006; Elsevier BV; Volume: 169; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.051208
ISSN1525-2191
AutoresCheryl A. Hawkes, Doreen Kabogo, A. Amritraj, Satyabrata Kar,
Tópico(s)Adenosine and Purinergic Signaling
ResumoThe cation-independent mannose 6-phosphate receptor (CI-MPR) is a single transmembrane domain glycoprotein that plays a major role in the trafficking of lysosomal enzymes from the trans-Golgi network to the endosomal-lysosomal (EL) system. Because dysfunction of EL system is associated with a variety of neurodegenerative disorders, it is possible that the CI-MPR may have a role in regulating neuronal viability after toxicity/injury. In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons causes a transient up-regulation of CI-MPR protein levels in surviving neurons of the basal forebrain and frontal cortex but not in the brainstem region, which was relatively spared by the immunotoxin. This was accompanied by a parallel time-dependent increase in other EL markers, ie, cathepsin D, Rab5, and LAMP2 in the basal forebrain region, whereas in the frontal cortex the levels of cathepsin D, and to some extent Rab5, were increased. Given the critical role of the EL system in the clearance of abnormal proteins in response to changing conditions, it is likely that the observed increase in the CI-MPR and components of the EL system in surviving neurons after 192-IgG-saporin treatment represents an adaptive mechanism to restore the metabolic/structural abnormalities induced by the loss of cholin-ergic neurons. The cation-independent mannose 6-phosphate receptor (CI-MPR) is a single transmembrane domain glycoprotein that plays a major role in the trafficking of lysosomal enzymes from the trans-Golgi network to the endosomal-lysosomal (EL) system. Because dysfunction of EL system is associated with a variety of neurodegenerative disorders, it is possible that the CI-MPR may have a role in regulating neuronal viability after toxicity/injury. In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons causes a transient up-regulation of CI-MPR protein levels in surviving neurons of the basal forebrain and frontal cortex but not in the brainstem region, which was relatively spared by the immunotoxin. This was accompanied by a parallel time-dependent increase in other EL markers, ie, cathepsin D, Rab5, and LAMP2 in the basal forebrain region, whereas in the frontal cortex the levels of cathepsin D, and to some extent Rab5, were increased. Given the critical role of the EL system in the clearance of abnormal proteins in response to changing conditions, it is likely that the observed increase in the CI-MPR and components of the EL system in surviving neurons after 192-IgG-saporin treatment represents an adaptive mechanism to restore the metabolic/structural abnormalities induced by the loss of cholin-ergic neurons. The insulin-like growth factor-II (IGF-II) receptor, which is identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), is a 250-kd multifunctional glycoprotein containing a large extracellular domain, a single transmembrane region, and a small cytoplasmic tail.1Kornfeld S Structure and function of the mannose 6-phosphate/insulin-like growth factor II receptors.Annu Rev Biochem. 1992; 61: 307-330Crossref PubMed Scopus (934) Google Scholar, 2Jones JI Clemmons DR Insulin-like growth factors and their binding proteins: biological actions.Endocr Rev. 1995; 16: 3-34PubMed Google Scholar, 3Ghosh P Dahms NM Kornfeld S Mannose 6-phosphate receptors: new twists in the tale.Nat Rev Mol Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (801) Google Scholar, 4Dore S Kar S Quirion R Rediscovering an old friend, IGF-I: potential use in the treatment of neurodegenerative diseases.Trends Neurosci. 1997; 20: 326-331Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 5Dahms NM Hancock MK P-type lectins.Biochim Biophys Acta. 2002; 1572: 317-340Crossref PubMed Scopus (182) Google Scholar, 6Hawkes C Kar S The insulin-like growth factor-II/mannose-6-phosphate receptor: structure, distribution and function in the central nervous system.Brain Res Rev. 2004; 44: 117-140Crossref PubMed Scopus (124) Google Scholar The receptor is widely expressed in various tissues including the brain and recognizes, via distinct sites, two different classes of ligands: M6P-containing molecules such as lysosomal enzymes, and IGF-II, a mitogenic polypeptide with structural homology to IGF-I and insulin.2Jones JI Clemmons DR Insulin-like growth factors and their binding proteins: biological actions.Endocr Rev. 1995; 16: 3-34PubMed Google Scholar, 7Morgan DO Edman JC Standring DN Fried VA Smith MC Roth RA Rutter WJ Insulin-like growth factor II receptor as a multifunctional binding protein.Nature. 1987; 329: 301-307Crossref PubMed Scopus (636) Google Scholar, 8MacDonald RG Pfeffer SR Coussens L Tepper MA Brocklebank CM Mole JE Anderson JK Chen E Czech MP Ullrich A A single receptor binds both insulin-like growth factor II and mannose-6-phosphate.Science. 1988; 239: 1134-1137Crossref PubMed Scopus (258) Google Scholar A subpopulation of the CI-MPR is located at the plasma membrane, where it regulates internalization of IGF-II and various exogenous M6P-containing ligands for subsequent clearance or activation. However, the majority of the receptors are expressed in the trans-Golgi network/endosomal compartments and are involved in the intracellular trafficking of a battery of lysosomal enzymes including cathepsins B and D.1Kornfeld S Structure and function of the mannose 6-phosphate/insulin-like growth factor II receptors.Annu Rev Biochem. 1992; 61: 307-330Crossref PubMed Scopus (934) Google Scholar, 2Jones JI Clemmons DR Insulin-like growth factors and their binding proteins: biological actions.Endocr Rev. 1995; 16: 3-34PubMed Google Scholar, 5Dahms NM Hancock MK P-type lectins.Biochim Biophys Acta. 2002; 1572: 317-340Crossref PubMed Scopus (182) Google Scholar, 9Hille-Rehfeld A Mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes.Biochim Biophys Acta. 1995; 1241: 177-194Crossref PubMed Scopus (216) Google Scholar, 10Braulke T Type-2 IGF receptor: a multiple-ligand binding protein.Horm Metab Res. 1999; 31: 242-246Crossref PubMed Scopus (135) Google Scholar Given the evidence that defects in the synthesis/targeting of lysosomal enzymes or dysfunction of the endosomal-lysosomal (EL) system are associated with a variety of neurodegenerative disorders, often with progressive cognitive decline,11Nixon RA Mathews PM Cataldo AM The neuronal endosomal-lysosomal system in Alzheimer's disease.J Alzheimers Dis. 2001; 3: 97-107Crossref PubMed Scopus (106) Google Scholar, 12Bahr BA Bendiske J The neuropathogenic contributions of lysosomal dysfunction.J Neurochem. 2002; 83: 481-489Crossref PubMed Scopus (162) Google Scholar, 13Wraith JE Lysosomal disorders.Semin Neonatol. 2002; 7: 75-83Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 14Tardy C Andrieu-Abadie N Salvayre R Levade T Lysosomal storage diseases: is impaired apoptosis a pathogenic mechanism?.Neurochem Res. 2004; 29: 871-880Crossref PubMed Scopus (19) Google Scholar it is possible that the CI-MPR may have a role in regulating neuronal viability. In fact, a number of studies have shown that loss of CI-MPR function can induce cell proliferation in a variety of cancers.15Oates AJ Schumaker LM Jenkins SB Pearce AA DaCosta SA Arun B Ellis MJ The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), a putative breast tumor suppressor gene.Breast Cancer Res Treat. 1998; 47: 269-281Crossref PubMed Scopus (104) Google Scholar, 16Scott CD Firth SM The role of the M6P/IGF-II receptor in cancer: tumor suppression or garbage disposal?.Horm Metab Res. 2004; 36: 261-271Crossref PubMed Scopus (44) Google Scholar Conversely, a protective role for the receptor has been suggested by two lines of evidence: cultured PC12 cells that are resistant to β-amyloid-mediated toxicity show an up-regulation of the CI-MPR17Li Y Xu C Schubert D The up-regulation of endosomal-lysosomal components in amyloid beta-resistant cells.J Neurochem. 1999; 73: 1477-1482PubMed Google Scholar and overexpression or activation of the CI-MPR can block cell death induced by the mutant herpes simplex virus 1 or retinoic acid.18Zhou G Roizman B Cation-independent mannose 6-phosphate receptor blocks apoptosis induced by Herpes simplex virus 1 mutants lacking glycoprotein D and is likely the target of antiapoptotic activity of the glycoprotein.J Virol. 2002; 76: 6197-6204Crossref PubMed Scopus (27) Google Scholar, 19Louafi F Stewart CE Perks CM Thomas MG Holly JM Role of the IGF-II receptor in mediating acute, non-genomic effects of retinoids and IGF-II on keratinocyte cell death.Exp Dermatol. 2003; 12: 426-434Crossref PubMed Scopus (6) Google Scholar Nevertheless, very little is currently known about the role of the CI-MPR in regulating neuronal viability after toxicity/injury or in any of the neurodegenerative disorders associated with dysfunction of the EL system. Assimilated evidence suggests that CI-MPR protein and mRNA are widely distributed in the adult rat brain including cortex, striatum, and hippocampus.20Lesniak M Hill J Kiess W Rojeski M Pert C Roth J Receptors for insulin-like growth factors I and II: autoradiographic localization in rat brain and comparison to receptors for insulin.Endocrinology. 1988; 123: 2089-2099Crossref PubMed Scopus (231) Google Scholar, 21Couce M Weatherington A McGinty JF Expression of insulin-like growth factor-II (IGF-II) and IGF-II/mannose-6-phosphate receptor in the rat hippocampus: an in situ hybridization and immunocytochemical study.Endocrinology. 1992; 131: 1636-1642Crossref PubMed Scopus (39) Google Scholar, 22Kar S Chabot JG Quirion R Quantitative autoradiographic localization of [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in developing and adult rat brain.J Comp Neurol. 1993; 333: 375-397Crossref PubMed Scopus (180) Google Scholar, 23Nagano T Sato M Mori Y Du Y Takagi H Tohyama M Regional distribution of messenger RNA encoding in the insulin-like growth factor type 2 receptor in the rat lower brainstem.Mol Brain Res. 1995; 32: 14-24Crossref PubMed Scopus (9) Google Scholar, 24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar, 25Konishi Y Fushimi S Shirabe T Immunohistochemical distribution of cation-dependent mannose 6-phosphate receptors in the mouse central nervous system: comparison with that of cation-independent mannose 6-phophate receptors.Neurosci Lett. 2005; 378: 7-12Crossref PubMed Scopus (11) Google Scholar At a cellular level, the receptor is localized primarily in neurons and their processes, although its presence on glial cells under normal condition has not been excluded.24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar, 25Konishi Y Fushimi S Shirabe T Immunohistochemical distribution of cation-dependent mannose 6-phosphate receptors in the mouse central nervous system: comparison with that of cation-independent mannose 6-phophate receptors.Neurosci Lett. 2005; 378: 7-12Crossref PubMed Scopus (11) Google Scholar A variety of experimental approaches, such as electrolytic lesioning of the entorhinal cortex26Kar S Baccichet A Quirion R Poirier J Entorhinal cortex lesion induces differential responses in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in the rat hippocampal formation.Neuroscience. 1993; 55: 69-80Crossref PubMed Scopus (57) Google Scholar or intradentate injection of colchicine27Breese CR D'Costa A Rollins YD Adams C Booze RM Sonntag WE Leonard S Expression of insulin-like growth factor-1 (IGF-1) and IGF-binding protein 2 (IGF-BP2) in the hippocampus following cytotoxic lesion of the dentate gyrus.J Comp Neurol. 1996; 369: 388-404Crossref PubMed Scopus (72) Google Scholar have been shown to increase CI-MPR mRNA and/or its binding sites in selective layers of the hippocampal formation, whereas penetrating cortical injury28Walter HJ Berry M Hill DJ Cwyfan-Hughes S Holly JM Logan A Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity.Endocrinology. 1999; 140: 520-532Crossref PubMed Scopus (88) Google Scholar and cerebral ischemia29Lee WH Clemens JA Bondy CA Insulin-like growth factors in response to cerebral ischemia.Mol Cell Neurosci. 1992; 3: 36-43Crossref PubMed Scopus (108) Google Scholar, 30Stephenson D Rash K Clemens J Increase in insulin-like growth factor II receptor within ischemic neurons following cerebral infarction.J Cereb Blood Flow Met. 1995; 15: 1022-1031Crossref PubMed Scopus (27) Google Scholar elevate receptor expression in neurons and/or glial cells only in the affected areas. Although these results underscore a role for the CI-MPR in lesion-induced plasticity, its association to the EL system, the major site of receptor action, remains to be defined. In addition, it is not clear whether increased levels of the receptor are associated with degenerating neurons and/or surviving neurons that undergo structural reorganization as a compensatory adjustments after surgical/pharmacological lesion. We have recently reported that a subset of the CI-MP receptors are located in cholinergic as well as noncholinergic neurons in the basal forebrain region of the adult rat brain.24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar The majority of these forebrain cholinergic neurons express low-affinity neurotrophin receptors (p75NTR), which are known to be selectively vulnerable to 192-IgG-saporin, a ribosomal toxin coupled to a monoclonal antibody against the rat p75NTR. Noncholinergic cell groups of the basal forebrain and p75NTR-negative cholinergic neurons remain unaffected by 192-IgG-saporin treatment.31Heckers S Ohtake T Wiley RG Lappi DA Geula C Mesulam MM Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor.J Neurosci. 1994; 14: 1271-1289Crossref PubMed Google Scholar, 32Rossner S Cholinergic immunolesions by 192 IgG-saporin-useful tool to simulate pathogenic aspects of Alzheimer's disease.Int J Dev Neurosci. 1997; 15: 835-850Crossref PubMed Scopus (39) Google Scholar, 33Wiley RG Toxin-induced death of neurotrophin-sensitive neurons.Methods Mol Biol. 2001; 169: 217-222PubMed Google Scholar This immunotoxin has been used extensively to study the behavioral and neurochemical sequelae of cholinergic hypofunction, but its influence on surviving neurons remains unclear.32Rossner S Cholinergic immunolesions by 192 IgG-saporin-useful tool to simulate pathogenic aspects of Alzheimer's disease.Int J Dev Neurosci. 1997; 15: 835-850Crossref PubMed Scopus (39) Google Scholar, 33Wiley RG Toxin-induced death of neurotrophin-sensitive neurons.Methods Mol Biol. 2001; 169: 217-222PubMed Google Scholar, 34Torres EM Perry TA Blockland A Wilkinson LS Wiley RG Lappi DA Dunnet SB Behavioural, histochemical and biochemical consequences of selective immunolesions in discrete regions of the basal forebrain cholinergic system.Neuroscience. 1994; 63: 95-122Crossref PubMed Scopus (295) Google Scholar, 35Perry T Hodges H Gray JA Behavioural, histological and immunocytochemical consequences following 192 IgG-saporin immunolesions of the basal forebrain cholinergic system.Brain Res Bull. 2001; 54: 29-48Crossref PubMed Scopus (50) Google Scholar, 36Hawkes C Jhamandas JH Kar S Selective loss of basal forebrain cholinergic neurons by 192 IgG-saporin is associated with decreased phosphorylation of Ser9 glycogen synthase kinase-3b.J Neurochem. 2005; 95: 263-272Crossref PubMed Scopus (23) Google Scholar In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons is accompanied by a time-dependent increase in the levels of the CI-MPR as well as other markers of the EL system in neurons of the affected areas that survive the immunotoxin treatment. These results provide the very first evidence that up-regulation of the CI-MPR and EL system may act as an adaptive mechanism to restore metabolic and structural abnormalities in neurons that survive toxicity/injury. Adult male Sprague-Dawley rats (225 to 275 g; Charles River Canada, Montreal, QC, Canada) were used in all studies and handled in accordance with the University of Alberta policy on the handling and treatment of laboratory animals. 192-IgG-saporin was obtained from Advanced Targeting Systems (San Diego, CA). Polyacrylamide electrophoresis gels (4 to 20%) were purchased from Invitrogen (Burlington, ON, Canada), and the enhanced chemiluminescence kit was obtained from Amersham (Mississauga, ON, Canada). Polyclonal anti-choline acetyltransferase (ChAT) antiserum was from Chemicon Int. (Temecula, CA), and anti-cathepsin D, anti-lysosomal associated membrane protein 2 (LAMP2), and anti-Rab5 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Donkey anti-goat Texas Red, donkey anti-rabbit fluorescein isothiocyanate, and donkey anti-mouse fluorescein isothiocyanate-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Anti-actin, anti-vesicular acetylcholine transporter (VAChT), anti-microtubule-associated protein-2 (MAP2), and Hoechst 33258 were from Sigma (Mississauga, ON, Canada), and anti-ED1 was from Serotec (Raleigh, NC); anti-p75NTR was from Promega (Madison, WI), and anti-GFAP was from Zymed (San Francisco, CA). Antiserum against the CI-MPR was a generous gift from Dr. R.G. MacDonald (University of Nebraska Medical Centre, Omaha, NE). All other reagents were from Sigma Chemical or Fisher Scientific (Montreal, QC, Canada). Rats were anesthetized by sodium pentobarbital (65 mg/kg i.p.) and mounted on a stereotaxic frame. Each animal received a bilateral injection of either 192-IgG saporin (0.4 μg/ml; 5 μl/ventricle) or an equivalent volume of saline through a 26-gauge Hamilton syringe into the lateral ventricles at coordinates anteroposterior −1.4 mm, mediolateral +1.8 mm, and dorsoventral −3.5 mm, relative to bregma. The cannula was left in place for 3 minutes after injection to allow for diffusion of the substrate. Animals were sacrificed at 4, 7, 14, 28, 60, and 90 days (10 to 12 animals per group) after surgery, and brain tissues were collected for Western blotting or immunohistochemistry as described earlier.36Hawkes C Jhamandas JH Kar S Selective loss of basal forebrain cholinergic neurons by 192 IgG-saporin is associated with decreased phosphorylation of Ser9 glycogen synthase kinase-3b.J Neurochem. 2005; 95: 263-272Crossref PubMed Scopus (23) Google Scholar Saline-treated control and 192-IgG-saporin-injected adult rats (four to six animals per group) were deeply anesthetized with 4% chloral hydrate and then perfused intracardially with phosphate-buffered saline (PBS) (0.01 mol/L, pH 7.4), followed by 4% paraformaldehyde or Bouin's solution. Brains were sectioned (20 μm) on a cryostat and collected in a free-floating manner. Sections from the basal forebrain, frontal cortex, and brainstem areas were incubated overnight with anti-ChAT (1:250), anti-VAChT (1:250), or anti-CI-MPR (1:750), rinsed with PBS, exposed for 1 hour with anti-goat or anti-rabbit secondary antibody and developed using the enhanced glucose-oxidase method.24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar Sections were dehydrated, mounted with Permount, and then examined under bright field using a Zeiss Axioskop-2 microscope. For double-immunofluorescence labeling, tissue sections were incubated overnight with anti-CI-MPR (1:500) in combination with anti-ChAT (1:100), anti-cathepsin D (1:250), anti-GFAP (1:500), anti-ED1 (1:100), anti-Rab5 (1:250), anti-LAMP2 (1:250), or anti-MAP2 (1:500) antibodies. Other brain sections were incubated with anti-cathepsin D (1:250), anti-Rab5 (1:250), or anti-LAMP2 (1:250) in combination with anti-GFAP (1:500) or anti-ED1 (1:100). In addition, some brain sections were exposed to a combination of anti-ChAT (1:1000) or anti-VAChT (1:250) with anti-cathepsin D (1:250), anti-p75NTR (1:250), anti-Rab5 (1:250), or anti-LAMP2 (1:250) antibodies. After incubation in primary antibody, sections were rinsed three times with PBS and then exposed to the appropriate fluorescent secondary antibodies for 2 hours. To determine whether CI-MPR was expressed in surviving and/or dying neurons, some brain sections from control and 192-IgG-saporin-treated animals were incubated with anti-CI-MPR antiserum and then stained with 500 ng/ml Hoechst 33258 for 20 minutes. Sections were then coverslipped and examined under a Zeiss Axioskop-2 fluorescence microscope. Control and treated animals from different groups (six animals per group) were decapitated, their brains rapidly removed, and areas of interest [ie, septum/diagonal band of Broca (DBB), cortex, and brainstem] were dissected out and homogenized in RIPA lysis buffer [20 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate, 1 mmol/L ethylenediaminetetraacetic acid, 1% Igepal CA-630, 50 mmol/L NaF, 1 mmol/L NaVO3, 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Proteins from the brain homogenates were separated by 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked with 8% nonfat milk, and incubated overnight at 4°C with anti-ChAT (1:500), anti-CI-MPR (1:10 000), anti-cathepsin D (1:500), anti-Rab5 (1:5000), or anti-LAMP2 (1:500) antibodies. Membranes were then incubated for 1 hour at 22°C with the appropriate secondary antibody and visualized using an enhanced chemiluminescence detection kit. Blots were stripped and reprobed with anti-actin (1:1000) to ensure equal protein loading. All blots were quantified using an MCID image analysis system as described earlier,24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar and the data presented as mean ± SEM were analyzed using one-way analysis of variance followed by Newman-Keuls posthoc analysis with significance set at P < 0.05. The immunotoxin 192-IgG-saporin was well tolerated by adult male rats with no fatalities or significant weight loss throughout the 90-day experimental paradigm. As expected, the toxin induced an extensive bilateral loss of ChAT-immunoreactive cell bodies in the basal forebrain areas [ie, septum, vertical and horizontal limbs of DBB, and nucleus basalis magnocellularis] from day 4 after injection onwards (Figure 1, A, D, and G; Table 1). A few residual ChAT-positive cholinergic neurons were evident in the nucleus basalis magnocellularis but not in other areas of the basal forebrain. The degeneration of cholinergic neurons in the basal forebrain region was accompanied by a concomitant loss of ChAT-positive fibers in the frontal cortex throughout the 90-day experimental paradigm (Figure 1, B, E, and H; Table 1). However, the cholinergic motoneurons of the brainstem, which do not express the p75NTR, were unaffected by 192-IgG-saporin treatment, as reported in other studies (Figure 1, C, F, and I).31Heckers S Ohtake T Wiley RG Lappi DA Geula C Mesulam MM Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor.J Neurosci. 1994; 14: 1271-1289Crossref PubMed Google Scholar, 32Rossner S Cholinergic immunolesions by 192 IgG-saporin-useful tool to simulate pathogenic aspects of Alzheimer's disease.Int J Dev Neurosci. 1997; 15: 835-850Crossref PubMed Scopus (39) Google Scholar These immunohistochemical results were supplemented by Western blot data showing a significant reduction in ChAT enzyme levels in the septum/DBB (Figure 1J) and frontal cortex (Figure 1K) but not in the brainstem (Figure 1L) from 7 days onwards after administration of 192-IgG-saporin (Table 1).Table 1Summary of Changes in Various EL Markers at Different Time Points Following 192 IgG-Saporin Treatment Open table in a new tab To determine the possible alterations in CI-MPR levels after administration of 192-IgG-saporin, we first established the localization of the receptor in the basal forebrain, frontal cortex, and brainstem regions of saline-treated control rats. Our immunohistochemical experiments revealed that CI-MPR, as reported earlier,24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar, 25Konishi Y Fushimi S Shirabe T Immunohistochemical distribution of cation-dependent mannose 6-phosphate receptors in the mouse central nervous system: comparison with that of cation-independent mannose 6-phophate receptors.Neurosci Lett. 2005; 378: 7-12Crossref PubMed Scopus (11) Google Scholar exhibits a widespread distribution in the aforesaid brain regions, with relatively high immunoreactivity in the medial septum, DBB, nucleus basalis magnocellularis, deep cortical layers, and the brainstem nuclei (Figure 2, A–C). In keeping with our earlier study,24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar receptor labeling in the cortex was evident in most layers with varying degrees of intensity, ie, high in layers IV to VI, moderate in layers II to III, and almost absent in layer I. To evaluate the influence of 192-IgG-saporin treatment on CI-MPR receptor levels, we performed immunohistochemical staining and Western blot analysis using a specific CI-MPR antiserum.24Hawkes C Kar S Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype.J Comp Neurol. 2003; 458: 113-127Crossref PubMed Scopus (52) Google Scholar Our results clearly show that CI-MPR immunoreactivity was enhanced in both neuronal cell bodies, dendrites, and axons, in the medial septum/DBB (Figure 2D), in nucleus basalis magnocellularis, and throughout the frontal cortex (Figure 2E) from days 4 to 28 after injection and then returned to levels similar to saline-treated control rats by day 60 of 192-IgG-saporin administration (Figure 2, G and H; Table 1). The CI-MPR staining in the brainstem, however, remained unchanged throughout the 90-day experimental period (Figure 2, C, F, and I). These findings were supported by our Western blot analysis, which revealed a significant increase in receptor levels from 4 to 28 days in the septum/DBB (Figure 2J) and from 7 to 28 days in the frontal cortex (Figure 2K) of 192-IgG-saporin-treated rats compared with saline-treated control rats (Table 1). By contrast, receptor levels were not significantly altered in the brainstem region of the immunotoxin-treated rats at any time during the experimental paradigm (Figure 2L). Given the evidence that glial cells are activated after 192-IgG-saporin-induced death of the basal forebrain cholinergic neurons,37Hollerbach EH Haas CA Hildebrandt H Frotscher M Naumann T Region-specific activation of microglial cells in the rat septal com-plex following fimbria-fornix transaction.J Comp Neurol. 1998; 390: 481-496Crossref PubMed Scopus (21) Google Scholar, 38Lemke R Roßner S Schliebs R Leukemia inhibitory factor expression is not induced in activated microglia and reactive astrocytes in response to rat basal forebrain cholinergic lesion.Neurosci Lett. 1999; 267: 53-56Crossref PubMed Scopus (7) Google Scholar we sought to determine whether the increase in CI-MPR levels is associated with either reactive astrocytes or microglia in 14-day post-treated rats. Our results clearly showed that both GFAP-positive reactive astrocytes (Figure 3, A and B) and ED1-positive activated microglia (Figure 3, E and F) were evident in the basal forebrain, but not in the cortical region (data not shown), of the immunotoxin-treated rats. Additionally, double-labeling experiments revealed that neither reactive astrocytes (Figure 3, B–D) nor microglia (Figure 3, F–H) expressed CI-MPR immunoreactivity in the basal forebrain region of the treated rats. In subsequent experiments, using nuclear marker for apoptosis Hoechst 3325839Ekdahl CT Zhu C Bonde S Bahr BA Blomgren K Lindvall O Death mechanisms in status epilepticus-generated neurons and effects of additional seizures on the their survival.Neurobiol Dis. 2003; 14: 513-523Crossref PubMed Scopus (49) Google Scholar (Figure 3, I–K) and the neuronal marker MAP2 (Figure 3, L–N), we found that increased CI-MPR expression is associated with surviving neurons. To evaluate CI-MPR alteration in relation to the cholinergic system, we first d
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