ABSTRACT
The Golgi apparatus of motor neurons (GA) is fragmented in sporadic amyotrophic lateral sclerosis (ALS), in familial ALS with SOD1 mutations, and in mice that express SOD1G93A of familial ALS, in which it was detected months before paralysis. In paralyzed transgenic mice expressing SOD1G93A or SOD1G85R, mutant proteins aggregated not only in the cytoplasm of motor neurons, but also in astrocytes and oligodendrocytes. Furthermore, aggregation of the G85R protein damaged astrocytes and was associated with rapidly progressing disease. In order to gain insight into the functional state of the fragmented GA, we examined the effects of S0D1 mutants G93A and G85R in Chinese Hamster Ovary Cells (CHO). In contrast to cells expressing the wt and G93A, the G85R expressers had no SOD1 activity. However, cells expressing both mutants, and to a lesser degree the wt, showed decreased survival, fragmentation of the GA, and dysfunction of the secretory pathway, which was assessed by measuring the amount of cell surface co-expressed CD4, a glycoprotein processed through the GA. The G93A and wt proteins were partially recovered in detergent insoluble fractions; while the recovery of G85R was minimal. Both mutants showed equal reductions of cell survival and function of the secretory pathway, in comparison to the wt and cells expressing mutant alsin, a protein found in rare cases of fALS. These results are consistent with the conclusion that the two SOD1 mutants, by an unknown mechanism, promote the dispersion of the GA and the dysfunction of the secretory pathway. This and other in vitro models of mutant SOD1 toxicity may prove useful in the elucidation of pathogenetic mechanisms.
Subject(s)
Amyotrophic Lateral Sclerosis , Golgi Apparatus/pathology , Secretory Vesicles/metabolism , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Blotting, Western , CHO Cells , Cell Death , Cell Survival , Cricetinae , Golgi Apparatus/metabolism , Humans , Mutagenesis , Point Mutation , Superoxide Dismutase/metabolismABSTRACT
Mice expressing the G93A and other mutations of Cu,Zn superoxide dismutase (SOD1(G93A)) are valid models for the familial form of amyotrophic lateral sclerosis (FALS) with SOD1 mutations and, probably, for sporadic ALS. Adult mice become progressively paralyzed and show most of the histopathological lesions reported in sporadic ALS, i.e. neuronal loss, astrogliosis, ubiquitin and Lewy body-like inclusions, dystrophic axons and fragmentation of the Golgi apparatus (GA) of motor neurons. In transgenic mice, the mutant protein and ubiquitin aggregate within pathological 13 nm thick filaments [Stieber A, Gonatas JO, Gonatas NK. J Neurol Sci 2000;173:53-62]. This immunocytochemical and quantitative study of mice expressing SOD1(G93A) establishes the chronological order and cellular localization of aggregates of SOD1 and their correlation with fragmentation of the GA. Young asymptomatic mice expressing SOD1(G93A) showed aggregates of mutant SOD1 within neurites, prior to the detection of SOD1 in the perikarya of spinal cord motor neurons and astrocytes. Both dendrites and the periaxonal oligodendroglial cytoplasm, surrounding atrophic axons, contained SOD1 as revealed by immunoelectron microscopy The perikarya of a small percentage of spinal cord motor neurons contained both fragmented GA and aggregates of SOD1; however, about 50% of motor neurons with fragmented GA did not contain SOD1 in the perikaryon, suggesting that aggregates of mutant protein may not directly cause fragmentation of the GA. The mechanism of the putative toxic effect by the mutant protein remains to be clarified. The isolation and biochemical characterization of the filamentous aggregates of mutant protein and ubiquitin from spinal cords of transgenic mice expressing mutations of the SOD1 gene may offer clues on pathogenetic mechanisms.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Astrocytes/metabolism , Axons/metabolism , Dendrites/metabolism , Mutation/physiology , Nerve Tissue Proteins/genetics , Neurons/metabolism , Oligodendroglia/metabolism , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Astrocytes/ultrastructure , Axons/ultrastructure , Dendrites/ultrastructure , Immunoenzyme Techniques , Immunohistochemistry , Mice , Mice, Transgenic , Microscopy, Electron , Nerve Tissue Proteins/metabolism , Neurons/ultrastructure , Oligodendroglia/ultrastructure , Spinal Cord/metabolism , Spinal Cord/pathologyABSTRACT
Transgenic mice that express the G93A mutation of human Cu,Zn superoxide dismutase (SOD1(G93A)), found in familial amyotrophic lateral sclerosis (FALS), showed clinical symptoms and histopathological changes of sporadic ALS, including fragmentation of the neuronal Golgi apparatus (GA). The finding of fragmented neuronal GA in asymptomatic mice, months before the onset of paralysis, suggests that the GA is an early target of the pathological processes causing neuronal degeneration. Transgenic mice expressing human SOD1(G93A) have aggregates of mutant protein and ubiquitin in neuronal and glial cytoplasm; they appeared first in the neuropil and later in the perikarya of motor neurons, where they were adjacent to fragmented GA. The aggregates of SOD1(G93A) appeared in neuronal perikarya of asymptomatic mice containing fragmented GA. The numbers of neurons with deposits of SOD1(G93A) and fragmented GA progressively increased with age. Immuno-electron microscopy using colloidal gold showed labeling of ubiquitin and SOD1 over 13 nm thick cytoplasmic filaments. Spinal cord extracts showed a 20-fold increase of SOD1(G93A) in transgenic mice compared to the wild-type protein in controls. The results suggest a causal relationship between the aggregation of mutant SOD1 and ubiquitin, fragmentation of the Golgi apparatus of motor neurons and neurodegeneration.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Golgi Apparatus/metabolism , Superoxide Dismutase/metabolism , Ubiquitins/metabolism , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Disease Progression , Golgi Apparatus/ultrastructure , Mice , Mice, Transgenic , Microscopy, Electron , Neurons/metabolism , Neurons/ultrastructure , Spinal Cord/metabolism , Spinal Cord/ultrastructureABSTRACT
Fragmentation of the Golgi apparatus (GA) of motor neurons was first described in sporadic amyotrophic lateral sclerosis (ALS) and later confirmed in transgenic mice expressing the G93A mutation of the gene encoding the enzyme Cu,Zn superoxide dismutase (SOD1(G93A)) found in some cases of familial ALS. In these transgenic mice, however, the fragmentation of the neuronal GA was associated with cytoplasmic and mitochondrial vacuoles not seen in ALS. The present new series of transgenic mice expressing 14-17 trans gene copies of SOD1(G93A), compared to 25 copies in the mice we studied previously, showed consistent fragmentation of the GA of spinal cord motor neurons, axonal swellings, Lewy-like body inclusions in neurons and glia, but none of the cytoplasmic or mitochondrial vacuoles originally reported. Thus, this animal model recapitulates the clinical and most neuropathological findings of sporadic ALS. Neurofilaments (NF) accumulate in axons and, less often, in neuronal perikarya in most cases of sporadic ALS and they have been implicated in its pathogenesis. In order to investigate whether fragmentation of the neuronal GA also occurs in association with accumulation of perikaryal NFs, we studied the organelle in transgenic mice expressing the heavy subunit of human neurofilaments (NF-H) which developed a motor neuronopathy resembling ALS. The neuronal GA of mice expressing NF-H, however, was intact despite massive accumulation of NFs in both perikarya and axons of motor neurons. In contrast, in transgenic mice expressing SOD1(G93A), the GA was fragmented despite the absence of accumulation of perikaryal NFs. These findings suggest that, in transgenic mice with neuronopathies caused by the expression of mutant SOD1(G93A) or the human NF-H, the GA and the perikaryal NFs are independently involved in the pathogenesis. The evidence suggests that the GA plays a central role in the pathogenesis of the vast majority of sporadic ALS and in FALS with SOD1 mutations.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Golgi Apparatus/pathology , Motor Neurons/pathology , Neurofilament Proteins/metabolism , Superoxide Dismutase/metabolism , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Golgi Apparatus/ultrastructure , Mice , Mice, Transgenic , Microscopy, Electron , Motor Neurons/ultrastructure , Spinal Cord/pathology , Spinal Cord/ultrastructureABSTRACT
The Golgi apparatus (GA) of innervated rat and chicken skeletal muscle is present in a typical perinuclear location, and in subsynaptic areas where it disperses after denervation. It was suggested that the subsynaptic segments of the GA are linked with functions involved in the maturation and targeting of synaptic proteins. Similarly, the GA of rat myocardium is found in a perinuclear location and between myofibrils, adjacent to the T system of tubules. These findings raise the question whether the GA of polarized cells is present in a typical perinuclear location, for the performance of general "housekeeping" functions, and in distal areas, for the mediation of specialized functions. Astrocytes may contain GA within their long cytoplasmic processes which are difficult to identify in thin sections. To ensure the astrocytic origin of GA in otherwise unidentifiable small processes, we used transgenic mice expressing the rat MG160 medial Golgi sialoglycoprotein only in the GA of astrocytes, and visualized the GA with monoclonal antibody 10A8 (mAb10A8) which reacts only with rat MG160. Thus, we identified cisternae of the GA in distal perisynaptic and subependymal processes, in perivascular foot plates of cerebral astrocytes, and in processes of the Müller glia in the retina. A similar strategy may be adopted in future investigations aiming at the detection of elements of the GA in distal processes of neurons and oligodendrocytes. The functional implications of GA in perisynaptic astrocytic processes and other processes are unknown. However, the isolation and molecular characterization of the perisynaptic subset of astrocytic Golgi may be feasible, since others have purified the astrocytic glutamate transporter 1 (GLT1) from crude synaptosomal fractions in which astrocytic processes are probably unavoidable contaminants.
Subject(s)
Astrocytes/ultrastructure , Ependyma/cytology , Golgi Apparatus/ultrastructure , Receptors, Cell Surface , Retina/cytology , Animals , Antibodies, Monoclonal , Antibody Specificity , Astrocytes/chemistry , Golgi Apparatus/chemistry , Membrane Proteins/analysis , Membrane Proteins/immunology , Mice , Mice, Transgenic , Microglia/ultrastructure , Microscopy, Electron , Neuropil/ultrastructure , Oligodendroglia/ultrastructure , Rats , Receptors, Fibroblast Growth Factor , Sialoglycoproteins/analysis , Sialoglycoproteins/immunology , Synapses/chemistry , Synapses/ultrastructureABSTRACT
Several diseases involving a variety of cells and tissues are associated with defective enzymes of the Golgi apparatus (GA). An intact GA of neurons is crucial for the physiological function of axons and presynaptic terminals since proteins destined for fast axoplasmic transport are processed by the organelle. Despite the obvious importance of the GA of neurons, its function and involvement in pathological reactions have not been studied systematically. The purpose of this paper is to draw attention to the contribution of the neuronal GA in pathology using two paradigms: (1) the involvement of the neuronal GA in the pathogeneses of amyotrophic lateral sclerosis (ALS), in an animal model of ALS, and in Alzheimer's disease; and (2) the elucidation of a retrograde transport pathway involving the neuronal transgolgi network, in vitro and in vivo, and the participation of this pathway in the toxicity and/or endocytosis of ricin and other toxic or non-toxic ligands.
Subject(s)
Alzheimer Disease/pathology , Amyotrophic Lateral Sclerosis/pathology , Golgi Apparatus/pathology , Ricin/toxicity , Animals , Humans , ImmunohistochemistryABSTRACT
The Golgi apparatus (GA) of spinal cord motor neurons is fragmented in sporadic amyotrophic lateral sclerosis (ALS), and in asymptomatic and symptomatic transgenic mice expressing the G93A mutation of the gene of the human Cu,Zn superoxide dismutase, found in certain cases of familial ALS (FALS) [Gonatas NK (1994) Am J Pathol 145:751-761; Mourelatos Z, et al. (1996) Proc Natl Acad Sci USA 93:5472-5477]. A similar fragmentation of the GA has been described in cells treated with microtubule-depolymerizing drugs, where the organelle is functional and contains both Golgi stacks and trans-Golgi network (TGN), the compartment of exit and targeting of proteins processed by the GA. To gain a better definition of the structure of the fragmented neuronal GA in ALS, four cases of sporadic ALS with numerous Bunina bodies in spinal cord motor neurons were stained with antibodies against human TGN and against the lumenal and cytoplasmic domains of MG160, a protein of the medial cisternae of the GA. The fragmented GA was stained with the three antibodies, indicating the presence of both Golgi stacks and TGN. Furthermore, the staining of the fragmented GA by the antiserum against the cytoplasmic domain of MG160 indicates that the fragmentation of the GA is not the result of a terminal and global cytoplasmic lytic event. The Bunina bodies were not stained by the anti-MG160 antibodies, suggesting that they are not derived from the GA. The perikarya of neurons with fragmented GA showed normal immunoreactivity with antibodies against the heavy neurofilament subunit and alpha-tubulin. However, because of the lack of appropriate antibodies the localization of proteins such as spectrin, ankyrin, centractin and others which link the microtubules with the GA were not done. The findings support the hypothesis that, in ALS, the fragmented neuronal GA is functional. Additional work with animal models of ALS may establish whether the fragmentation of the GA is a sign of early degeneration or a compensatory reaction of the injured motor neuron.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Golgi Apparatus/pathology , Motor Neurons/pathology , Receptors, Cell Surface , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/metabolism , Animals , Antibody Specificity , Female , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Humans , Inclusion Bodies/chemistry , Inclusion Bodies/pathology , Male , Middle Aged , Motor Neurons/chemistry , Neurofilament Proteins/analysis , Rabbits , Receptors, Fibroblast Growth Factor , Sialoglycoproteins/analysis , Sialoglycoproteins/immunology , Spinal Cord/pathologyABSTRACT
MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown. In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia. In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus. This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.
Subject(s)
Golgi Apparatus/metabolism , Receptors, Cell Surface , Sialoglycoproteins/metabolism , Amino Acids , Animals , Antibodies/metabolism , Arginine , Binding Sites , Biological Transport , Biotin/analogs & derivatives , CHO Cells , Cell Membrane/metabolism , Cricetinae , Culture Media , Cytoplasm/metabolism , Female , Humans , Immunoenzyme Techniques , Microscopy, Immunoelectron , Precipitin Tests , Protein Processing, Post-Translational , Rabbits , Rats , Receptors, Fibroblast Growth Factor , Sequence Deletion , Sialoglycoproteins/biosynthesis , Sialoglycoproteins/genetics , Structure-Activity Relationship , Succinimides , TransfectionABSTRACT
The amino acid sequence of MG160, a membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, is more than 90% identical with CFR, a fibroblast growth factor (FGF) binding protein of chicken membranes, and with ESL-1, a ligand for E-selectin of plasma membranes of myeloid cells; furthermore, MG160, isolated by immunoaffinity chromatography from rat brain membranes, binds to basic FGF. The gene for MG160 has been assigned to human chromosome 16q22-23. To characterize this protein further in the human, its cDNA was cloned and sequenced. The protein has a large luminal domain composed of an initial proline-glutamine-rich segment, encoded by an uninterrupted exonic sequence of several CAG-CAA repeats. Expansion of CAG repeats has been implicated in the etiology of several neurodegenerative diseases. The proline-glutamine-rich segment is followed by 16 cysteine-rich repeats that contain five potential asparagine-linked glycosylation sites, which are conserved in the human, rat, mouse, and chicken. The large intralumenal domain of the protein is followed by a single transmembrane domain and a 13-amino-acid cytoplasmic carboxy-terminal tail, which is identical to that in the chicken, rat, and mouse. The overall amino acid identifies between MG160, CFR, and ESL-1 range from 88% to 95%. In several human fetal and adult tissues, three mRNA transcripts for MG160 of 10 kb, 5 kb, and 3.8 kb were identified by Northern blot analysis of poly(A)-selected mRNAs. These transcripts may represent alternatively spliced mRNAs of the protein or mRNAs encoding closely related proteins of the Golgi apparatus and/or plasma membranes.
Subject(s)
DNA, Complementary/genetics , Golgi Apparatus/chemistry , Membrane Glycoproteins/genetics , Receptors, Cell Surface , Sialoglycoproteins/genetics , Adult , Alternative Splicing , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Conserved Sequence , E-Selectin/metabolism , Exons/genetics , Fibroblast Growth Factors/metabolism , Humans , Molecular Sequence Data , Organ Specificity , Protein Binding , RNA, Messenger/analysis , Receptors, Fibroblast Growth Factor , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Trinucleotide Repeats/geneticsABSTRACT
A polyclonal antiserum raised against the carboxy-terminal 17 amino acids of the rat p185c-neu (anct) reacted with a 140 kDa polypeptide in membranes of synaptosome fractions from neocortex and hippocampus of 11-day-old and adult rats. The same antiserum reacted with a 185 kDa polypeptide in microsome membranes from rat pheochromocytoma cells (PC12). By light microscopic immunocytochemistry, the anct antibodies against the 140 kDa protein were localized in the neuropile of brain, cerebellum and spinal cord of 11-day-old and adult rats. Especially prominent staining was obtained in the CA2-CA3 zones of the hippocampus, and in the substantia gelatinosa in the spinal cord. The finely granular and diffuse pattern of the immunostain was consistent with synaptic localizations. Interestingly, antibodies against the entire endodomain of p185c-neu (a-Bacneu) were localized in granular structures, probably representing axo-somatic and axo-dendritic synapses, on a subset of pyramidal neurons of the CA3 zone. By immunoelectron terminals in the giant mossy fiber type in the CA3 and CA4 regions. The immunolocalization of the anct antibodies was restricted in segments of the presynaptic membrane facing the synaptic cleft which include the active zone. The identify and function of the 140 kDa membrane protein of rat brain presynaptic terminals, detected by the anct antibodies, is unknown. The 140 kDa protein may be related to p185c-neu, a tyrosine kinase, or to other known or unknown kinases.
Subject(s)
Membrane Proteins/analysis , Nerve Tissue Proteins/analysis , Presynaptic Terminals/chemistry , Receptor, ErbB-2/analysis , Amino Acid Sequence , Animals , Antibody Specificity , Blotting, Western , Cerebral Cortex/chemistry , Cerebral Cortex/ultrastructure , Cross Reactions , Hippocampus/chemistry , Hippocampus/ultrastructure , Membrane Proteins/immunology , Molecular Sequence Data , Molecular Weight , Nerve Tissue Proteins/immunology , PC12 Cells , Rats , Rats, Inbred Lew , Receptor, ErbB-2/immunology , Synaptosomes/chemistryABSTRACT
We report the primary structure of MG-160, a 160 kDa membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of rat neurons, pheochromocytoma (PC-12), and several other cells. The cDNA encodes a polypeptide of 1,171 amino acids with an M(r) of 133,403. An intralumenal cleavable signal peptide is followed by a Pro-Gln-rich segment and 16 contiguous, approx. 60-residue-long, regularly spaced cysteine-rich segments showing sequence identities ranging from 15 to 35%. The lumenal domain is followed by a single membrane spanning domain and a short carboxy-terminal cytoplasmic tail. The protein contains 5 potential NXT glycosylation sites. The sequence of MG-160 shows no homologies with enzymes and other membrane proteins of the Golgi apparatus. MG-160 displays a so far unique feature for a membrane protein of the Golgi apparatus: namely, an upstream, open reading frame (uORF), encoding 58 amino acids, located in front of the major open reading frame (ORF). Most vertebrate mRNAs containing uORF or AUG codons in front of the major ORF encode growth factors and cell surface receptors (Geballe and Morris 1994). In that regard a 90% identity between the primary structure of MG-160 and a receptor for acidic and basic fibroblast growth factors (CFR), isolated from chicken embryos (Burrus et. al., 1992), may be relevant. Immunoreactivity for MG-160 has been detected in the Golgi apparatus of neural and other cells of 2-day-old chicken embryos and adult chicken; furthermore, recombinant human basic fibroblast growth factor (bFGF) binds MG-160 purified from rat brain. MG-160 shows no sequence similarity with members of the family of fibroblast growth factor receptors (FGFR) involved in signal transduction. These findings are consistent with the hypothesis that MG-160 is involved in the traffic and processing of endogenous or autocrine FGFs. This is the first example of an intrinsic membrane protein of the Golgi apparatus which binds a growth factor and may be involved in its regulation.
Subject(s)
Fibroblast Growth Factor 2/metabolism , Golgi Apparatus/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface , Receptors, Fibroblast Growth Factor/genetics , Sialoglycoproteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chick Embryo , DNA, Complementary , Fibroblast Growth Factor 2/genetics , Intracellular Membranes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/ultrastructure , Molecular Sequence Data , PC12 Cells , Protein Binding , Rats , Sequence Homology, Amino Acid , Sialoglycoproteins/genetics , Sialoglycoproteins/ultrastructureABSTRACT
The Golgi apparatus (complex) is at the center stage of important functions of processing and transport of plasma membrane, lysosomal, and secreted proteins. The involvement of the Golgi apparatus in the pathogenesis of chronic degenerative diseases of neurons is virtually unknown. In the present study, fragmentation and atrophy of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis (ALS), has been detected with organelle specific antibodies. Approximately 30% of motor neurons in five ALS patients showed a fragmented Golgi apparatus whereas only about 1% of motor neurons from seven controls with neurologic or systemic disease showed a similar change. Morphometric studies are consistent with the hypothesis that the alteration of the Golgi apparatus is an early event in the pathogenesis of the neuronal degeneration in ALS. Immunocytochemical studies with antibodies against alpha tubulin, tau, and phosphorylated subunits of neurofilament polypeptides did not disclose differences in the staining of neurons with fragmented or normal Golgi apparatus, suggesting that the alteration of the organelle is not secondary to a gross lesion of the cytoskeleton. However, these observations do not rule out the hypothesis that the fragmentation of the Golgi apparatus is secondary to subtle changes of the polypeptides involved in the attachment of membranes of the organelle to the cytoskeleton.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Golgi Apparatus/ultrastructure , Motor Neurons/ultrastructure , Antibodies/immunology , Antibody Specificity , Cytoskeleton/ultrastructure , Humans , Immunohistochemistry , Organelles/immunology , Spinal Cord/pathologyABSTRACT
Monoclonal antibody (MAb) 2H1, raised in mice immunized with membrane fractions from cultured rat pheochromocytoma cells (clonal line PC-12), detects a polypeptide from rat brain and PC-12 cell membranes of 60-65 KD apparent molecular mass. The polypeptide has been localized by immunoelectron microscopy in the rough endoplasmic reticulum (RER) of neurons. By light microscopic immunocytochemistry, several rat tissues and two rat-derived cultured cell types show selective patterns of staining with 2H1. In the central nervous system, the antibody stains neuronal cytoplasm; in the spleen, staining is seen only in certain cells of the marginal zone of the white pulp, and in lymph nodes, in plasma cells, and in areas populated by monocytes and macrophages. Whereas astrocytes and adrenal medullary cells in situ are virtually unstained with 2H1, primary cultures of astrocytes and PC-12 cells, which are derived from adrenal medullary cells, stain intensely with 2H1. The strong staining of cultured astrocytes and PC-12 cells with 2H1 suggests that the levels of the 60-65 KD polypeptide are up-regulated during cell proliferation and growth. Only a few hepatocytes stain with 2H1; intestinal epithelial and pancreatic cells are not stained with 2H1. The organelle-specific antibody 2H1 may prove a useful probe in structural and functional studies of membranes of the rough endoplasmic reticulum in neurons, and in certain cells of the immune system.
Subject(s)
Adrenal Gland Neoplasms/pathology , Endoplasmic Reticulum/metabolism , Lymph Nodes/cytology , Membrane Proteins/metabolism , Neurons/metabolism , Pheochromocytoma/pathology , Adrenal Gland Neoplasms/metabolism , Adrenal Gland Neoplasms/ultrastructure , Animals , Antibodies, Monoclonal/immunology , Astrocytes/metabolism , Astrocytes/physiology , Astrocytes/ultrastructure , Brain/metabolism , Brain/physiology , Brain/ultrastructure , Cell Line , Cell Membrane/metabolism , Cell Membrane/physiology , Cell Membrane/ultrastructure , Endoplasmic Reticulum/physiology , Endoplasmic Reticulum/ultrastructure , Immunohistochemistry/methods , Lymph Nodes/metabolism , Lymph Nodes/ultrastructure , Membrane Proteins/immunology , Microscopy, Immunoelectron , Molecular Weight , Neurons/physiology , Neurons/ultrastructure , Pheochromocytoma/metabolism , Pheochromocytoma/ultrastructure , Plasma Cells/cytology , Plasma Cells/metabolism , Plasma Cells/ultrastructure , Rats , Rats, Inbred Lew , Spleen/cytology , Spleen/metabolism , Spleen/ultrastructureABSTRACT
We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.
Subject(s)
Golgi Apparatus/ultrastructure , Sialoglycoproteins/metabolism , Animals , Antibodies/immunology , Anura , Autopsy , Cattle , Chickens , Golgi Apparatus/metabolism , Humans , Immunohistochemistry/methods , Mice , Microscopy, Electron/methods , Rabbits , Rats , Rats, Inbred Strains , Sialoglycoproteins/immunologyABSTRACT
Many studies have established the central involvement of the Golgi apparatus in the transport and processing of plasma membrane, lysosomal, and secreted proteins. The Golgi apparatus of neurons is also involved in the axoplasmic flow of fast-moving macromolecules and in the orthograde, retrograde, and transsynaptic transport of exogenous ligands. Markers of the Golgi apparatus, based on traditional methods of enzyme cytochemistry, are not applicable to human tissues obtained at autopsy. For that reason, the Golgi apparatus of brain cells has not been examined adequately in diseases of the human nervous system. Here we report that an antiserum raised against MG-160, a 160-kDa sialoglycoprotein of medial cisternae of the Golgi apparatus of several rat cells, is a specific and easily reproducible immunocytochemical marker of the Golgi apparatus of human neurons and other cells obtained at autopsy. Application of this probe in amyotrophic lateral sclerosis has shown a fragmentation of the Golgi apparatus in motor neurons similar to that induced by depolymerization of microtubules. We suggest that the fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis has functional implications because significant reductions of secretion of insulin and immunoglobulins have been observed in islet cells and plasma cells, respectively, treated with microtubule-disrupting agents.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Golgi Apparatus/ultrastructure , Motor Neurons/ultrastructure , Aged , Aged, 80 and over , Aging , Blotting, Western , Golgi Apparatus/immunology , Humans , Intracellular Membranes/immunology , Intracellular Membranes/ultrastructure , Microtubules/physiologyABSTRACT
A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.
Subject(s)
Golgi Apparatus/analysis , Proteins/analysis , Animals , Antibodies, Monoclonal , Cattle , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Immunoenzyme Techniques , Intestine, Small/analysis , Intestine, Small/ultrastructure , Microscopy, Electron , Molecular Weight , Pancreas/analysis , Pancreas/ultrastructureABSTRACT
A monoclonal antibody (mAb 10A8), derived from mice immunized with fractions of the Golgi apparatus from rat brain neurons, was exploited to isolate and partially characterize a novel glycoprotein of 160 kDa apparent molecular mass which was localized by immunoelectron microscopy in medial cisternae of the Golgi apparatus of neurons, glia, pituitary cells, and rat pheochromocytoma (PC 12). The yield of immunoaffinity purified protein was 0.9 microgram/g of rat brain and represented 3% of the Golgi protein; the protein contained asparagine-linked carbohydrates and sialic acid and N-acetylglucosamine residues; unreduced protein had a greater electrophoretic mobility (130 kDa) consistent with the presence of intrachain disulfide bonds. The bulk of the glycoprotein resided within the membrane and/or luminal face of the Golgi cisternae. After extraction with Triton X-114, the glycoprotein was found in both aqueous and detergent phases. The monoclonal antibody did not inhibit the activities of Golgi enzymes or the uptake of nucleotide sugars by intact Golgi vesicles. The findings indicate that the 160-kDa glycoprotein is a specific constituent of medial Golgi cisternae. The results of this study lend support to the hypothesis that the distributions of glycosyltransferases in the Golgi apparatus are cell specific, since in neurons this sialic acid containing glycoprotein is found in medial rather than in trans and/or in the trans Golgi reticulum cisternae, where sialyltransferases have been localized in other cells. Alternatively, resident neuronal Golgi sialoglycoproteins may acquire sialic acid in trans elements of the apparatus and then shuttle back in medial cisternae.