The prion-like nature of amyotrophic lateral sclerosis.

The misfolding, aggregation, and deposition of specific proteins is the key hallmark of most progressive neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). ALS is characterized by the rapid and progressive degenerations of motor neurons in the spinal cord and motor cortex, resulting in paralysis of those who suffer from it. Pathologically, there are three major aggregating proteins associated with ALS, including TAR DNA-binding protein of 43kDa (TDP-43), superoxide dismutase-1 (SOD1), and fused in sarcoma (FUS). While there are ALS-associated mutations found in each of these proteins, the most prevalent aggregation pathology is that of wild-type TDP-43 (97% of cases), with the remaining split between mutant forms of SOD1 (~2%) and FUS (~1%). Considering the progressive nature of ALS and its association with the aggregation of specific proteins, a growing notion is that the spread of pathology and symptoms can be explained by a prion-like mechanism. Prion diseases are a group of highly infectious neurodegenerative disorders caused by the misfolding, aggregation, and spread of a transmissible conformer of prion protein (PrP). Pathogenic PrP is capable of converting healthy PrP into a toxic form through template-directed misfolding. Application of this finding to other neurodegenerative disorders, and in particular ALS, has revolutionized our understanding of cause and progression of these disorders. In this chapter, we first provide a background on ALS pathology and genetic origin. We then detail and discuss the evidence supporting a prion-like propagation of protein misfolding and aggregation in ALS with a particular focus on SOD1 and TDP-43 as these are the most well-established models in the field.

Propagated protein misfolding: New opportunities for therapeutics, new public health risk.

There is now good consensus that propagated protein misfolding is the underlying mechanism for the infectious prion diseases (Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk). Over the past decade it has become increasingly clear that other diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis may progress via the same mechanism, involving a disease-specific polypeptide rather than the prion protein. Recent literature in these non-prion neurodegenerative diseases also points to the existence of multiple "strains" that express themselves differently in different contexts, resulting in different disease phenotypes. The probable cause of these neurodegenerative diseases is now referred to collectively as "propagated protein misfolding." Propagated protein misfolding raises many opportunities for new therapeutics and diagnostics. However, it also raises the theoretical risk of iatrogenic transmission, although experimental support for this notion is limited at present.

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions.

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 - 30 detectable by the 3F4 antibody against human PrP109 - 112. We recently identified a new PK-resistant PrP species, designated PrP*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 - 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP*20 but also a small amount of 3F4-detected PK-resistant PrP27 - 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP*20 to the occurrence of the 3F4-detected PrP27 - 30 was observed. Our study suggests that an increase in the level of PrP*20 characterizes the early stages of prion diseases.

The failure of Daudi cells to express the cellular prion protein is caused by a lack of glycosyl-phosphatidylinositol anchor formation.

The cellular prion protein (PrPc) is a glycosyl-phosphatidylinositol (GPI)-linked cell surface protein, which is expressed at high density on nervous tissues and at lower levels on most other solid-organ tissues. It is also expressed on peripheral blood mononuclear cells (PBMC) of all lineages. In lymphocytes, its level of expression is dependent upon the state of cell activation, and polyclonal anti-PrP antisera partially block lectin-induced T-cell activation, suggesting a functional role of the protein in this process. Using the monoclonal antibody (mAb) 3F4 we examined PrPc surface immunoreactivity on leukaemic cell lines of T- and B-cell origin, and unexpectedly observed a complete lack of PrPc cell-surface expression in Daudi cells, while all other cell lines displayed discernible reactivity. We demonstrated the intracellular presence of PrP-specific mRNA and PrP protein. The lack of surface PrPc is unrelated to the well-known defect of beta2-microglobulin (beta2m) expression in Daudi cells as other beta2m-deficient cells, such as the melanoma cell line F0-1 and spleen cells from beta2m gene-deleted mice, were not deficient in cell-surface PrPc. Daudi cells failed to bind antibodies directed against all GPI-linked cell surface proteins. In somatic hybridization experiments using murine spleen cells as partners, we observed de novo expression of human PrPc, CD55 and CD59, thus demonstrating in Daudi cells the availability of these gene products for GPI linkage and cell-surface expression.

Opposing actions of native and oxidized lipoprotein on motor neuron-like cells.

Lipoproteins are present in the central nervous system and surrounding vasculature and possibly mediate effects relevant to neuronal physiology and pathology. To determine the effects of lipoproteins on motor neurons, native low density lipoproteins (LDL) and oxidized LDL (oxLDL) were applied to a motor neuron cell line. Oxidized LDL, but not native LDL, resulted in a dose- and time-dependent increase in reactive oxygen species and neuron death. Oxidized LDL-induced toxicity was attenuated by a calcium chelator, antioxidants, caspase inhibitors, and inhibitors of macromolecular synthesis. In addition to being nontoxic, application of native LDL attenuated reactive oxygen species formation and neuron loss following glucose deprivation injury. Together, these data demonstrate a possible neuroprotective role for unmodified lipoproteins and suggest oxidized lipoproteins may amplify oxidative stress and neuron loss.

Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E.

Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.

Acute and persistent infection of human neural cell lines by human coronavirus OC43.

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.

GM2 promotes ciliary neurotrophic factor-dependent rescue of immortalized motor neuron-like cell (NSC-34).

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblastoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, beta 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.

The lipid peroxidation product 4-hydroxynonenal impairs glutamate and glucose transport and choline acetyltransferase activity in NSC-19 motor neuron cells.

Both oxidative stress and excitotoxicity are implicated in the pathogenesis of a number of neurodegenerative disorders, such as amyotrophic lateral sclerosis. We previously reported increased modification of proteins by 4-hydroxynonenal (HNE), a product of membrane lipid peroxidation, in the spinal cords of patients with amyotrophic lateral sclerosis relative to controls. In the current study, we examined the functional consequences of protein modification by HNE in a cell line with a motor neuron phenotype, NSC-19. Treatment of NSC-19 cells with FeSO4, which catalyzes lipid peroxidation, or HNE induced concentration-dependent decreases in glucose and glutamate transport. Vitamin E and propyl gallate blocked the impairment of glucose and glutamate transport caused by FeSO4 in these cells, but not that caused by HNE, whereas glutathione blocked the effects of FeSO4 as well as HNE. Both FeSO4 and HNE caused an increase in the number of apoptotic nuclei in NSC-19 cultures, but this occurred subsequent to the impairment of glucose and glutamate transport. Reductions in choline acetyltransferase activity were also observed in FeSO4- or HNE-treated NSC-19 cells before induction of apoptosis. Our results suggest that, prior to cell death, oxidative stress and HNE down-regulate cholinergic markers and impair glucose and glutamate transport in motor neurons, the latter of which may lead to excitotoxic degeneration of the cells.

Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.

X-linked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease-associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase-3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions.

Involvement of aminopeptidase N (CD13) in infection of human neural cells by human coronavirus 229E.

Attachment to a cell surface receptor can be a major determinant of virus tropism. Previous studies have shown that human respiratory coronavirus HCV-229E uses human aminopeptidase N (hAPN [CD13]) as its cellular receptor for infection of lung fibroblasts. Although human coronaviruses are recognized respiratory pathogens, occasional reports have suggested their possible neurotropism. We have previously shown that human neural cells, including glial cells in primary cultures, are susceptible to human coronavirus infection in vitro (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). However, the only reported expression of hAPN in the nervous system is at the level of nerve synapses. Therefore, we asked whether hAPN is utilized as a cellular receptor for infection of these human neural cell lines. Using flow cytometry, we were able to show the expression of hAPN on the surfaces of various human neuronal and glial cell lines that are susceptible to HCV-229E infection. An hAPN-specific monoclonal antibody (WM15), but not control antibody, inhibited the attachment of radiolabeled HCV-229E to astrocytic, neuronal, and oligodendrocytic cell lines. A correlation between the apparent amount of cell surface hAPN and the level of virus attachment was observed. Furthermore, the presence of WM15 inhibited virus infection of these cell lines, as detected by indirect immunofluorescence. These results indicate that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus.

Prion protein expression in human leukocyte differentiation.

The cellular isoform of the prion protein (PrPC) is a small glycoprotein attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol anchor. This molecule is involved in the pathogenesis of prion diseases in both humans and animals. We have characterized the expression patterns of PrPC during human leukocyte maturation by flow cytometry with monoclonal antibodies to PrPC, the glycan moiety CD15, and the stem cell marker CD34. We observe that prion protein is present on CD34+ bone marrow (BM) stem cells. Although lymphocytes and monocytes maintain PrPC expression throughout their differentiation, PrPC is downregulated upon differentiation along the granulocyte lineage. In vitro retinoic acid-induced differentiation of the premyeloid line HL-60 into granulocyte-like cells mimics the suppression of PrPC in granulocyte differentiation, as both PrPC mRNA and protein are downregulated. These data suggest that selected BM cells and peripheral mononuclear cells may support prion agent replication, because this process is dependent on availability of PrPC. Additionally, retinoic acid-induced extinction of PrPC expression in HL-60 cells provides a potential model to study PrP gene regulation and protein function. Finally, these data suggest the existence of cell-specific glycoforms of PrPC that may determine cellular susceptibility to infection by the prion agent.

Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases.

Apoptosis is a fundamental process required for normal development of the nervous system and is triggered during neurodegenerative disease. To dissect the molecular events leading to neuronal cell death, we have developed a cell-free model of neuronal apoptosis. The model faithfully reproduces key elements of apoptosis, including chromatin condensation, DNA fragmentation, caspase activation/processing, and selective substrate cleavage. We report that cell-free apoptosis is activated in premitochondrial, mitochondrial, and postmitochondrial phases by tamoxifen, mastoparan, and cytochrome c, respectively, allowing a functional ordering of these proapoptotic modulators. Furthermore, this is the first report of mitochondrial-mediated activation of cell-free apoptosis in a cell extract. Although Bcl-2 blocks activation at the premitochondrial and mitochondrial levels, it does not affect the postmitochondrial level. The cell-free system described here provides a valuable tool to elucidate the molecular events leading to neuronal cell death.

Multiple sclerosis: Fas signaling in oligodendrocyte cell death.

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas-ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase-mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 non-activating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.

Ganglioside characterization of a cell line displaying motor neuron-like phenotype: GM2 as a possible major ganglioside in motor neurons.

We have examined ganglioside compositions and the presence of sulfated glucuronyl glycolipids of immortalized motor neuron-like cell lines, neuroblastoma-spinal cord (NSC) hybrid cell lines established by fusing mouse neuroblastoma N18TG2 with motor neuron-enriched embryonic spinal cord cells. Among NSC cell lines, only NSC-34 aggregates acetylcholine receptors on co-cultured myotube and expresses a receptor for S-laminin, a neuromuscular junction specific basal lamina protein. GM2, which is only a minor ganglioside component of CNS, was the major component in NSC-34 occupying almost 75% of total gangliosides, whereas GD1a and GM3 were major species in the parental N18TG2, which had only 8.5% GM2. These results indicated that NSC lines have unique ganglioside pattern that is distinctive from other nervous tissues, and this pattern, especially that of NSC-34 cells, might reflect the characteristics of mouse spinal motor neuron gangliosides. Sulfated glucuronyl paragloboside was demonstrated to be present in N18TG2, however, it could not be detected in either of NSC cell lines. Even though the pathogenesis of amyotrophic lateral sclerosis remains unknown, autoimmunological participation has been suggested. Because high-titered antibody against GM2 has been observed in a patient with amyotrophic lateral sclerosis-like disease, GM2 which is possibly expressed on the surface of motor neurons might serve as a potential target antigen in this disorder.

Fibromyalgia is common in a postpoliomyelitis clinic.

OBJECTIVE: To determine prospectively the occurrence and clinical characteristics of fibromyalgia in patients serially presenting to a postpolio clinic. Fibromyalgia may mimic some of the symptoms of postpoliomyelitis syndrome, a disorder characterized by new weakness, fatigue, and pain decades after paralytic poliomyelitis. DESIGN: Case series. SETTING: A university-affiliated hospital clinic. PATIENTS: One hundred five patients were evaluated with a standardized history and physical examination during an 18-month period. Ten patients were excluded because of the absence of past paralytic poliomyelitis. INTERVENTIONS: Patients with fibromyalgia were treated with low-dose, nighttime amitriptyline hydrochloride or other conservative measures. MAIN OUTCOME MEASURES: Patients with fibromyalgia had diffuse pain and 11 or more of 18 specific tender points on examination (American College of Rheumatology criteria, 1990). Patients with borderline fibromyalgia had muscle pain and five to 10 tender points on physical examination. RESULTS: Ten (10.5%) of 95 postpolio patients met the criteria for fibromyalgia, and another 10 patients had borderline fibromyalgia. All patients with fibromyalgia complained of new weakness, fatigue, and pain. Patients with fibromyalgia were more likely than patients without fibromyalgia to be female (80% vs 40%, P < .04) and to complain of generalized fatigue (100% vs 71%, P = .057), but were not distinguishable in terms of age at presentation to clinic, age at polio, length of time since polio, physical activity, weakness at polio, motor strength scores on examination, and the presence of new weakness, muscle fatigue, or joint pain. Approximately 50% of patients in both the fibromyalgia and borderline fibromyalgia groups responded to low-dose, nighttime amitriptyline therapy. CONCLUSIONS: (1) Fibromyalgia occurs frequently in a postpolio clinic. (2) Fibromyalgia can mimic some symptoms of postpoliomyelitis syndrome. (3) Fibromyalgia in postpolio patients can respond to specific treatment.

A human glial hybrid cell line differentially expressing genes subserving oligodendrocyte and astrocyte phenotype.

We have developed a series of immortal human-human hybrid cell lines that express phenotypic characteristics of primary oligodendrocytes, by fusing a 6-thioguanine-resistant mutant of the human rhabdomyosarcoma RD with adult human oligodendrocytes by a lectin-enhanced polyethylene glycol procedure. Hybrids were selected in an aminopterin-containing media. In contrast to the tumor parent cells, a hybrid clone M03.13 expressed surface immunoreactivity for galactosyl cerebroside and intracellular immunoreactivity for myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). Serum deprivation or chronic treatment with a protein kinase C activator 4-beta-phorbol 12-myristate 13-acetate (PMA), but not dibutyl cyclic adenosine monophosphate induced coordinate up-regulation or de novo induction of oligodendrocyte phenotypic markers with concomitant down-regulation of GFAP expression. Consistent with immunohistochemical studies, northern blot analysis demonstrated that both MBP and PLP mRNA were up-regulated in MO3.13 cells by PMA treatment. M03.13 cells provide an immortalized clonal model system suitable for study of gene expression subserving oligodendrocyte and astrocyte phenotypes.

Intracellular calcium concentrations during metabolic inhibition in the motoneuron cell line NSC-19.

Changes in the concentrations of intracellular free calcium ([Ca2+]i) and adenine nucleotides were determined in response to metabolic inhibitors in the motoneuron cell line NSC-19. The NADH dehydrogenase inhibitor amobarbital (Amytal) and the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) were used to alter energy metabolism. Exposure of cells to 5 mM Amytal did not significantly change ATP concentrations but produced transient elevations of [Ca2+]i of approximately 80 nM, which were reduced by 32% when cells were studied in Ca(2+)-free solutions. CCCP (10 microM) caused a transient reduction in ATP concentration of 33%. CCCP also produced sustained elevations of [Ca2+]i of about 280 nM, which were reduced by 47% when in Ca(2+)-free solutions. In spite of the sustained elevation of [Ca2+]i induced by CCCP, NSC-19 showed no reduction in cell viability after 48 h compared with controls. Ruthenium red, a blocker of Ca2+ uptake by mitochondria, had little effect on the CCCP-induced [Ca2+]i increment. KCl or glutamate did not produce significant changes in [Ca2+]i, indicating that these cells do not possess significant numbers of voltage-dependent Ca2+ channels or excitatory amino acid receptor-gated channels. [Ca2+]i values in these cells were modified by changes in extracellular Ca2+ concentrations. In Ca(2+)-containing solutions, inhibition of Na+/Ca2+ exchange by amiloride and bepridil led to increased [Ca2+]i, as did blockade of Ca2+ ATPase by vanadate, suggesting that membrane transporters are important in Ca2+ efflux in NSC-19. The present studies indicate that exposure of NSC-19 cells to Amytal and CCCP produces Ca2+ increments by release from internal stores, as well as by transmembrane influx. These results demonstrate that small increments in [Ca2+]i can be produced by metabolic inhibitors or other compounds and that such changes are not associated with immediate cell death. Changes in [Ca2+]i could potentially result in abnormal cell function secondary to altered action of Ca(2+)-dependent enzymes.

Amyloid precursor protein gene expression in neural cell lines: influence of DNA cytosine methylation.

Transcription of the gene encoding amyloid precursor protein (APP) varies in a cell-specific and developmentally regulated manner. The 5' region of this gene possesses a high frequency of CpG dinucleotides as well as copies of a GC-rich sequence, a potential trans factor binding element. These findings raise the possibility that DNA cytosine methylation could participate in the regulation of APP gene expression. We examined APP mRNA/18S rRNA ratio in three neural cell lines (N18TG2, SN6, SN17) cultured in 5-azacytidine (5-AZA), an inhibitor of maintenance methylase which results in loss of cytosine methylation in proliferating cells. Culture in 5-AZA globally reduced methylation in genomic DNA as assessed by an increase in HpaII restriction sites, reduced cytosine methylation in the APP gene as assessed by Southern blotting of HpaII digests, and increased APP mRNA steady state abundance in all studied cell lines. Cell lines re-acquired APP gene methylation 48 h after removal of 5-AZA from media. These results indicate that in vitro alteration of DNA methylation can affect APP gene expression, and suggest that the APP gene in neuronal cell lines may be rapidly inactivated in vitro, perhaps to neutralize its potential toxicity.