Biomedical subjects

N R Cashman

Publications and source records attributed to N R Cashman.

At least 19 recordsLinked to original sources

PubMed1999

Processing of the beta-amyloid precursor protein in ex vivo human brain cells.

We studied distribution and processing of the Alzheimer's beta-amyloid precursor protein (betaAPP) in immediately ex vivo human brain cells obtained during neurosurgical procedures. Immunoblotting and flow cytometry studies revealed that brain cells supported betaAPP as a transmembrane holoprotein. Brain cells in short-term suspension culture were competent to process betaAPP into Abeta as shown by [35S]methionine pulse-chase studies. Brain cell Abeta was immunoprecipitated as SDS-stable dimers and higher-order multimers. Cleavage of cell surface betaAPP with trypsin prior to metabolic labeling reduced cellular Abeta by approximately 50%. We conclude that plasmalemmal betaAPP in human brain cells is a source of cellular Abeta, presumably via endosomal-lysosomal processing.

Adolescent↗

PubMed1999

A multicenter, randomized, double-blinded trial of pyridostigmine in postpolio syndrome.

BACKGROUND: Postpoliomyelitis syndrome (PPS) is likely due to degeneration and dysfunction of terminal axons of enlarged postpolio motor units. Age-related decline in growth hormone and insulin-like growth factor (IGF-I) may be a contributing factor. Neuromuscular junction abnormalities and decreased IGF-I levels may respond to the anticholinesterase pyridostigmine, with consequent improvement in strength, fatigue, and quality of life. OBJECTIVES: To determine the effect of pyridostigmine in PPS on health-related quality of life, isometric muscle strength, fatigue, and serum IGF-I levels; and to assess the safety of pyridostigmine in PPS. METHODS: The study was a multicenter, randomized, double-blinded, placebo-controlled trial of a 6-month course of pyridostigmine 60 mg three times per day in 126 PPS patients. The primary data analysis compared mean changes of outcomes between treatment and control groups at 6 months using an intention to treat approach. Secondary analyses included a comparison of outcomes at 6 and 10 weeks, and in compliant patients. RESULTS: The study showed no significant differences in pyridostigmine and placebo-treated patients with regard to changes in quality of life, isometric strength, fatigue, and IGF-I serum levels at 6 months in the primary analysis and in compliant patients. There were no differences in outcomes at 6 and 10 weeks between groups. However, very weak muscles (1 to 25% predicted normal at baseline) were somewhat stronger (p = 0.10, 95% CI of difference -9.5 to 73.3%), and in compliant patients IGF-I was somewhat increased (p = 0.15, 95% CI of difference -6.4 to 44.8 ng/mL) at 6 months with the medication. Pyridostigmine was generally well tolerated. CONCLUSIONS: This study showed no significant differences between pyridostigmine and placebo-treated PPS patients on measures of quality of life, isometric strength, fatigue, and serum IGF-I.

Aged↗

PubMed1999

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.

Aldehydes↗

PubMed1999

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.

Animals↗

PubMed1999

Biological markers in the diagnosis and treatment of ALS.

The care of patients with amyotrophic lateral sclerosis (ALS), which has classically focused on treatment of symptomatology, has now entered an encouraging new era of therapy targeted at the pathophysiology of the disease. However, an objective measure of disease progression and therapeutic response is sorely needed. Quantitative neuromuscular examinations, measurement of pulmonary function, disability scales, and even survival, are limited by variability due to a number of poorly controlled factors. Quantitative electromyography, positron emission tomography scanning, and magnetic cortical stimulation, provide potential objective indicators of disease progression, but require a large number of patients and a long observation period for adequate statistical power. We have examined the role of magnetic resonance spectroscopic imaging in detecting acute changes in motor cortical metabolism in response to riluzole therapy. N-acetylaspartate (NAA), the most prominent signal in proton spectra of normal brain, is a neuron-specific molecule. ALS patients were found to experience a significant increase in the NAA/creatine ratio within 3 weeks of initiation of riluzole therapy. As glutamate can trigger the generation of reactive oxygen species in neurons, we speculate that acute changes in NAA levels may reflect oxidative injury to mitochondria where NAA is synthesised. The advent of a useful test for upper motor neuron metabolic compromise may provide an objective, non-invasive, short duration measure with which to screen the efficacy of potential therapeutic agents for ALS.

Amyotrophic Lateral Sclerosis↗

PubMed1999

Adaptive resistance to nitric oxide in motor neurons.

Nitric oxide (NO) is a free radical produced actively by mammalian cells, including neurons. Low levels of NO can function in intercellular signaling, but high levels are cytotoxic. This cytotoxic potential suggests that cells at risk for NO damage, such as neurons, might have NO resistance mechanisms to prevent cell death, and adaptive resistance to NO-releasing compounds has been reported for some non-neuronal cell types. Here we show that immortalized mouse motor neurons (NSC34 cells) respond to sub-lethal fluxes of pure NO by activating adaptive resistance mechanisms that counteract cytotoxic NO exposure. This adaptive NO resistance is reversible and is paralleled by the induction of the oxidative stress enzyme heme oxygenase 1 (HO-1). An inhibitor of both HO-1 and heme-dependent guanylate cyclase (tin-protoporphyrin IX) greatly sensitized NO-pretreated NSC34 cells to the NO challenge. However, readdition of cyclic GMP (in the form of the 8-bromo derivative) restored rather little resistance, and a more selective guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxaline-1-one (at 10 microM), did not have the sensitizing effect. Therefore, the inducible HO-1 pathway contributes substantially to adaptive NO resistance, while cyclic GMP seems to play at most a small role. A similar adaptive resistance to NO was observed in primary rat spinal chord motor neurons. The activation of NO resistance in motor neurons may counteract age- or disease-related neurodegeneration.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Carcinogens↗

PubMed1999

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.

Astrocytoma↗

PubMed1999

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.

Cell Line↗

PubMed1999

Do the benefits of currently available treatments justify early diagnosis and announcement? Arguments for.

Both a biologic imperative and an ethical imperative exist for providing the diagnosis of amyotrophic lateral sclerosis (ALS) as soon as possible and involving patients and their families in therapeutic decisions. The participation of excitotoxic mechanisms in ALS and the availability of riluzole, which may slow the rate of progression of ALS, provide the biologic rationale for early therapy in ALS. A neuroprotective agent, such as riluzole, is more effective at an early stage of disease, when more undamaged neurons remain. The development of effective therapy for ALS also provides the ethical basis for early announcement of diagnosis. When no primary treatment was available, an accepted approach was one of "protecting" the patient from the diagnosis, which was one of exclusion, requiring almost 100% certainty before announcement. The availability of a primary therapy, albeit not a cure, means that a diagnosis of ALS can now be offered as the most likely diagnosis with 90-95% certainty. The logical corollary of this new model is that patients must be involved immediately, to help decide when therapy is appropriate and to balance the relative personal significance of therapeutic gains versus adverse effects.

Amyotrophic Lateral Sclerosis↗

PubMed1998

Recovery of N-acetylaspartate in corticomotor neurons of patients with ALS after riluzole therapy.

Riluzole, a glutamate antagonist, has been shown to be efficacious in the treatment of patients with amyotrophic lateral sclerosis (ALS), allowing prolonged survival and time to tracheostomy. The efficacy of riluzole in thought to result from reduced glutamate excitotoxicity on motor neurons of patients with ALS, but this has never been demonstrated directly in vivo. N-acetylaspartate (NAA), a compound that is readily measured in vivo using proton magnetic resonance spectroscopy, can be used as a surrogate marker for neuronal loss or sublethal injury. To determine whether riluzole reverses sublethal corticomotoneuron damage in patients with ALS we measured NAA/creatine (Cr) relative intensity ratios in the motor cortex before and after treatment with riluzole 50 mg bid. After 3 weeks of riluzole therapy in 11 patients NAA/Cr increased from 2.14 +/- 0.26 to 2.27 +/- 0.24 (p = 0.044), whereas, in 12 untreated patients NAA/Cr decreased from 2.17 +/- 0.20 to 2.08 +/- 0.20 (p = 0.099). Thus the change in NAA/Cr between the treated and untreated groups was 0.22 +/- 0.095 (p = 0.008). The magnitude of increase in NAA/Cr in those treated was not correlated with age, sex, duration of treatment or disease, the presence of probable or definite upper motor neuron (UMN) signs, bulbar features, or pre-treatment NAA/Cr. We conclude that magnetic resonance spectroscopy can provide a novel surrogate measure of neuronal integrity that demonstrates reversal of sublethal UMN injury in patients with ALS within weeks of initiating riluzole therapy.

Adult↗

PubMed1998

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.

Antibodies, Monoclonal↗

PubMed1998

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.

CD13 Antigens↗

PubMed1997

A prion primer.

By biological and medical criteria, prions are infectious agents; however, many of their properties differ profoundly from those of conventional microbes. Prions are "encoded" by alterations in protein conformation rather than in nucleic acid or amino acid sequence. New epidemic prion diseases (bovine spongiform encephalopathy and new variant Creutzfeldt-Jakob disease) have recently emerged under the active surveillance of the modern world. The risk of contracting prion disease from blood products or other biologicals is now a focus of worldwide concern. Much has been discovered about prions and prion diseases, but much remains to be done.

Global Health↗

PubMed1997

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.

Adenosine Triphosphate↗

PubMed1996

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.

Adult↗

PubMed1996

Alzheimer's beta-amyloid precursor protein is expressed on the surface of immediately ex vivo brain cells: a flow cytometric study.

Beta-amyloid precursor protein (beta APP) is ubiquitously expressed, but deposition of the beta APP proteolytic fragment A beta is virtually restricted to the brain, suggesting cell-specific processing of this molecule. Our laboratory has investigated expression of beta APP in mechanically dissociated, unfixed, immediately ex vivo cells from various mouse and rat organs by flow cytometry. Epitopes of predicted extracellular domains of beta APP recognized by the N-terminal 22C11 monoclonal antibody (mAb) and the juxtamembrane 4G8 mAb were not detectable on the surface of lymphoid cells, hepatocytes, or kidney cells. In contrast, surface 22C11 and 4G8 beta APP immunoreactivity was abundant on intact (propidium iodide-excluding) dissociated brain cells. The predicted C-terminal intracellular beta APP determinant recognized by the mAb Jonas was not detectable on the surface of intact brain cells, but was present in ethanol-permeabilized cells, consistent with a transmembrane configuration of beta APP in brain cells. Trypsinization of intact brain cells abolished cell surface immunoreactivity for 22C11, which was then reestablished by short-term culture. Augmentation of 22C11 and 4G8 surface immunoreactivity occurred when brain cells were cultured short-term in phenylarsine oxide, a general endocytosis inhibitor. By double staining protocols of brain cells with mAbs directed against beta APP ectodomain epitopes and the neuronal surface proteins Thy-1 or neural cell adhesion molecule (NCAM), we observed that all Thy-1+ and NCAM+ cells (approximately 50%) were immunoreactive for surface beta APP, but that some beta APP+ cells (approximately 20%) were negative for these neuronal markers. Our data suggest that neurons and a subpopulation of other brain cells, unlike peripheral cells, can support beta APP as a type 1 intrinsic membrane molecule with an intact ectodomain, and that beta APP surface abundance is regulated by an equilibrium between membranes vesicle insertion and endocytotic internalization. Transmembrane beta APP holoprotein may be a critical determinant of brain-predominant processing of beta APP to A beta, and may participate in a receptor/transducer function unique to brain cells.

Alzheimer Disease↗