Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis.

The ubiquitin-proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G9 3A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild-type or dominant-negative mutant ubiquitin (Ub(K48R)). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9-12% of total ubiquitin. Mice expressing the G9 3A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G9 3A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild-type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.

Disruption of the structure of the Golgi apparatus and the function of the secretory pathway by mutants G93A and G85R of Cu, Zn superoxide dismutase (SOD1) of familial amyotrophic lateral sclerosis.

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.

The involvement of the neuronal Golgi apparatus and trans-Golgi network in the human olivary hypertrophy.

We studied the Golgi apparatus (GA) and trans-Golgi network (TGN) in the human olivary hypertrophy by immunohistological methods with organelle specific antibodies against the medial cisternae of the organelle (MG160) and the trans-Golgi network (TGN46). The GA and TGN of enlarged neurons in the inferior olivary nuclei in the early stages after central tract lesions lost the normal network-like configuration, and they were reduced to numerous small disconnected granules (fragmentation). In chronic stages after lesions, the GA and TGN of vacuolated or enlarged neurons showed a variety of morphological profiles, such as normal-looking patterns, fragmentation, reduction in number, and aggregation around nuclei or at a distance in the cytoplasm. In patients with multiple system atrophy, the GA and TGN of the neurons in the inferior olivary nuclei showed almost similar findings to those seen in the chronic stages after brainstem lesions. These results suggest that the GA and TGN are affected in degenerating neurons by anterograde transneuronal mechanisms.

Aggregates of mutant protein appear progressively in dendrites, in periaxonal processes of oligodendrocytes, and in neuronal and astrocytic perikarya of mice expressing the SOD1(G93A) mutation of familial amyotrophic lateral sclerosis.

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.

Fragmentation of the Golgi apparatus of the ballooned neurons in patients with corticobasal degeneration and Creutzfeldt-Jakob disease.

We investigated immunohistologically the Golgi apparatus (GA) and the trans-Golgi network (TGN) of the cortical ballooned neurons (BNs) in two patients with corticobasal degeneration and in five with Creutzfeldt-Jakob disease. We observed that the BNs showed fragmentation of the GA and the TGN, accompanied by a reduction in the number of fragmented Golgi elements and by a unique perinuclear distribution. This is the first report of the abnormalities of GA in cortical BNs. These findings suggests that the GA and the TGN may play some roles in the BN formation.

The mitotic phosphorylation cycle of the cis-Golgi matrix protein GM130.

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Balpha regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.

Fragmentation of the Golgi apparatus of the anterior horn cells in patients with familial amyotrophic lateral sclerosis with SOD1 mutations and posterior column involvement.

The Golgi apparatus (GA) of the anterior horn cells in the spinal cord was examined by immunohistological methods with an antibody against the MG-160 protein, a conserved intrinsic membrane sialoglycoprotein of the medial cisternae of the GA, in three patients with familial amyotrophic lateral sclerosis (FALS) with posterior column involvement. Large motor neurons in the anterior horns were markedly reduced in number and 10 of total 14 remaining large motor neurons showed fragmentation and a reduction in the number of the elements of the GA. The fragmentation of the GA was identical to that previously reported in motor neurons of the spinal cord and motor cortex from patients with sporadic ALS and in transgenic mice expressing the G93A mutation of the gene encoding the Cu/Zn superoxide dismutase months before the onset of paralysis. This is the first report of fragmented GA of the anterior horn cells in patients with FALS with posterior column involvement. The findings suggest that the GA is a common target in the neuronal degeneration in sporadic and FALS.

Aggregation of ubiquitin and a mutant ALS-linked SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus.

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.

The neuronal Golgi apparatus is fragmented in transgenic mice expressing a mutant human SOD1, but not in mice expressing the human NF-H gene.

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.

The golgi apparatus is present in perisynaptic, subependymal and perivascular processes of astrocytes and in processes of retinal Müller glia.

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.

Fragmentation of the Golgi apparatus of Betz cells in patients with amyotrophic lateral sclerosis.

The Golgi apparatus (GA) of the large pyramidal motor neurons in the cerebral cortex (Betz cells), was examined in sixteen patients with sporadic amyotrophic lateral sclerosis (ALS), in one patient with familial ALS (FALS), and in ten non-ALS age matched controls including one patient with Huntington's disease and one patient with a brain infarct. The GA was immunostained with an antibody against the MG-160 protein, a conserved sialoglycoprotein of the medial cisternae of the organelle. In ALS, 13.2% of counted Betz cells had fragmented GA in contrast to 0.6% in the ten non-ALS controls. The fragmentation of the GA of Betz cells was identical to that previously reported in spinal cord motor neurons from patients with sporadic ALS and in transgenic mice expressing the G93A mutation of the gene encoding the Cu/Zn superoxide dismutase. The striking morphological similarity between the fragmentation of the GA observed in Betz cells and in spinal cord motor neurons suggests that a similar pathogenic mechanism is responsible for both, and that the fragmentation of the GA of the spinal cord motor neurons is not a consequence of deafferentation due to the degeneration of the Betz cells.

Environmental factors modulate the size and the secretory activity of the notochord. A study of the Golgi apparatus in avian embryos.

In this study we examined the Golgi apparatus of avian notochord transplants excised from 2-day-old (E2) chick embryos and grafted isochronically into a chick host either in a medial-ventral position, next to the host notochord, or in a superficial position under the ectoderm laterally or dorsally to the neural tube. The operated embryos were examined from E2 to E8. The diameters, the cytoplasmic vacuolization and the immunostained Golgi apparatus were identical between the endogenous and ventrally grafted notochords, as well as between host and superficially transplanted notochords when observed at E2. In contrast, from E4 to E8, the size of the notochords grafted dorsally or laterally to the neural tube significantly smaller than the host, while the cytoplasmic vacuolization and the degree of fragmentation of the Golgi apparatus were significantly less than in the host notochords. These results show that environmental and position-specific factors influence the developmental program and the secretory activity of the notochordal cells.

The involvement of the Golgi apparatus in the pathogenesis of amyotrophic lateral sclerosis, Alzheimer's disease, and ricin intoxication.

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.

The fragmented neuronal Golgi apparatus in amyotrophic lateral sclerosis includes the trans-Golgi-network: functional implications.

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.

Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia.

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.

Activity of Hippocampal CA1 Neurons in Alzheimer's Disease is not Affected by the Presence of Adjacent Neuritic Plaques.

Alzheimer's disease (AD) is neuropathologically characterized by neuritic plaques (NPs) and neurofibrillary tangles and functionally by a decreased metabolic rate of neurons. Our previous studies showed that in brain areas which are extensively affected by plaques and tangles, i.e. the CA1 area of the hippocampus and the hypothalamic tuberomamillary nucleus, neuronal protein synthetic ability is significantly lower in AD patients than in controls. However, the presence of tangles as shown by Bodian staining appeared not to be directly related to decreased protein synthetic ability of neurons. In order to study to what extent the metabolic function of neurons might be affected by the other neuropathological hallmark of AD, i.e. NPs, which are presumed to contain neurotoxic compounds, we studied eight severely demented AD patients matched for the ApoE genotype (ApoE 3/3). Using an image analysis system, the size of the neuronal Golgi apparatus (GA) and of the cell profile area was measured as a parameter for protein synthetic activity in the CA1 area of these patients. NPs were stained by Bodian, and subsequently the distance of each neuron with an immunostained GA to the nearest NP was measured. Our results showed that neither NP density nor the distance between NPs and neurons correlated with the protein synthetic ability of neurons as judged by the size of the GA. Based on these results we suggest that in AD the presence of NPs and decreased neuronal protein synthetic ability are basically two independent phenomena

The Golgi sialoglycoprotein MG160, expressed in Pichia pastoris, does not require complex carbohydrates and sialic acid for secretion and basic fibroblast growth factor binding.

MG160, a type I membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, shows high homology (over 90%) with CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand of the cell surface of murine myeloid cells. When Chinese Hamster Ovary (CHO) cells were stably transfected with a cDNA lacking the transmembrane and C-terminus cytoplasmic domain of MG160 (delta TMCT), a fully processed protein of 160 kDa apparent molecular mass was recovered in the culture medium. When these cells were treated with tunicymycin, a 130- to 140-kDa protein was immunoprecipitated from the culture medium. A construct lacking the signal sequence, the single transmembrane, and the cytoplasmic domains of MG160 (delta TMCT-) was integrated at the HIS Pichia pastoris genome site using the expression vector pPIC 9 which possesses a yeast compatible signal sequence (Invitrogen). Recombinant protein accumulated in the medium to approximately 10 mg/L. The yeast recombinant protein lacked complex carbohydrates and sialic acid but bound 125I bFGF. Similarly, rat MG160 subjected to deglycosylation by peptide:N-glycosidase F (PNGase) bound 125I bFGF.

Cloning and sequence analysis of the human MG160, a fibroblast growth factor and E-selectin binding membrane sialoglycoprotein of the Golgi apparatus.

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.

Syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the Golgi apparatus.

Coronavirus mouse hepatitis virus (MHV) possesses a membrane glycoprotein (M) which is targeted to the Golgi apparatus (GA). We used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the GA during infection of L2 murine fibroblasts with MHV-A59. Twenty-four hours after infection the GA was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. Two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (S), induced fragmentation of the GA. However, the GA migrated only partially to the centers of syncytia during infection with these mutants. Revertant viruses, in which the above mutation was corrected, had fusion properties and GA staining similar to wtMHV-A59. Experiments with brefeldin A (BFA), which induces redistribution of the GA into the rough endoplasmic reticulum (RER), revealed that an intact GA for a period of 4-16 hr postinfection, is required for coronavirus replication and syncytia formation. Thus, during MHV infection, syncytia formation is associated with fragmentation of the GA, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. The fragmentation of the GA, however, may occur without the formation of syncytia. Therefore, two distinct mechanisms may be responsible for the fragmentation of the GA and its subsequent migration to the center of syncytia.

The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

Dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase have been found in members of certain families with familial amyotrophic lateral sclerosis (ALS). To better understand the contribution of SOD1 mutations in the pathogenesis of familial ALS, we developed transgenic mice expressing one of the mutations found in familial ALS. These animals display clinical and pathological features closely resembling human ALS. Early changes observed in these animals were intra-axonal and dendritic vacuoles due to dilatation of the endoplasmic reticulum and vacuolar degeneration of mitochondria. We have reported that the Golgi apparatus of spinal cord motor neurons in patients with sporadic ALS is fragmented and atrophic. In this study we show that spinal cord motor neurons of transgenic mice for an SOD1 mutation display a lesion of the Golgi apparatus identical to that found in humans with sporadic ALS. In these mice, the stacks of the cisternae of the fragmented Golgi apparatus are shorter than in the normal organelle, and there is a reduction in Golgi-associated vesicles and adjacent cisternae of the rough endoplasmic reticulum. Furthermore, the fragmentation of the Golgi apparatus occurs in an early, presymptomatic stage and usually precedes the development of the vacuolar changes. Transgenic mice overexpressing the wild-type human superoxide dismutase are normal. In familial ALS, an early lesion of the Golgi apparatus of motor neurons may have adverse functional effects, because newly synthesized proteins destined for fast axoplasmic transport pass through the Golgi apparatus.