[Axonal involvement in multiple sclerosis. Current concepts]. / Afectación axonal en la esclerosis múltiple. Conceptos actuales.

INTRODUCTION: Axon pathology in multiple sclerosis is an emerging concept, not because it is unknown but because it has been forgotten. However, clinical, functional and pathological aspects have clearly shown that it is damaged at a very early stage in development of the plaque of demyelination. There is sufficient clinical, radiological and pathological evidence to permit definition of axonal damage as the central element of the pathology and clinical features of multiple sclerosis. DEVELOPMENT AND CONCLUSIONS: Throughout this article we will see how the axon is affected in multiple sclerosis, how this affects the inflammatory response and which parameters allow us to measure axonal damage and its relation to disability. Finally we will see how a new physiopathogenic concept of multiple sclerosis appears, based on the axonal lesion, and how this fits current clinico-pathological concepts better.

Afectación axonal en la esclerosis múltiple. Conceptos actuales / Axonal involvement in multiple sclerosis. Current concepts

Introducción. La patología del axón en la esclerosis múltiple es un concepto emergente, no por desconocido sino por olvidado. Pero tanto a nivel clínico, como funcional y patológico, es un hecho plenamente demostrado que está dañado en fases muy precoces del proceso de desarrollo de la placa de desmielinización. Existen suficientes evidencias clínicas, radiológicas y patológicas que permiten definir el daño axonal como el elemento central en la patología y la clínica de la esclerosis múltiple. Desarrollo y conclusiones. A lo largo de la presente exposición veremos cómo se afecta el axón en la esclerosis múltiple, cómo determina la respuesta inflamatoria esta afectación axonal y cuáles son los parámetros que nos permiten medir el daño axonal y su relación con la discapacidad. Por último, veremos cómo se vislumbra una nueva concepción fisiopatogénica de la esclerosis múltiple basada en la lesión axonal y cómo ésta se adapta mejor a los conceptos clínico-patológicos actuales (AU)

[A critical review of the current pathogenesis of multiple sclerosis and possible future trends]. / Revisión crítica de la patogenia actual de la esclerosis múltiple y futuras direcciones posibles.

INTRODUCTION: Multiple sclerosis (MS) is a disorder in which the pathogenesis is not clearly understood, but where the disability caused and its progression determine the seriousness of the condition. At present, inflammation due to T lymphocytes is thought to be the cause of the clinical features and resulting disability. The main feature is demyelination, but it has not been possible to show by immunology studies, neuroimaging studies or therapeutic trials that the binomial inflammation-demyelination correlates with the disability. Therefore other possibilities of pathogenesis which provide a better explanation for the clinical findings in MS may be considered. DEVELOPMENT: We may start with the hypothesis of the existence of gliotoxic and neurotoxic factors in a patient with MS. These, by means of electrical inhibition mechanisms, autoimmune phenomena and loss of the normal neurone-oligodendrocyte-astrocyte relationship, or a non-specific immune response may give place to inflammation with secondary demyelination on the one hand and to axonopathy with alterations in the mechanisms of remyelination due to alterations in the signals between the axon and the oligodendrocyte on the other. CONCLUSIONS: When these three factors are considered, a better explanation is found for the factors which determine the progression of the disability, exhaustion of the inflammatory response, deficient remyelination and the poor correlation between demyelination and the progression of the disease. At the same time, new possibilities for approaches to the treatment and investigation of MS appear.

Distinct patterns of urokinase receptor (uPAR) expression by leukemic cells and peripheral blood cells.

The urinary type plasminogen activator, urokinase (uPA) is localized on the cell surface through the binding of a specific receptor, the uPA receptor (uPAR). The uPA localization enhances plasmin formation on the cell surface and facilitates cell migration. The cellular and tissue distribution of uPAR is not fully established. We have analyzed uPAR expression in nine leukemic cell lines of distinct lineages and maturational states and correlated this with expression of plasminogen receptors, tissue-type plasminogen activator (tPA) receptors and LDL receptor-related protein (LRP). The most immature and least differentiated cell line (an erythro-myeloid cell line) and cells of lymphoid lineage, did not express uPAR, whereas cells differentiated along the myelo-monocytic pathway displayed this receptor. Plasminogen and tPA receptors were expressed by all leukemic cell lines and by all nucleated peripheral blood cells but B and T lymphocytes were negative for cell surface expression of both uPAR and LRP while monocytes and neutrophils were positive for expression of both uPAR and LRP. PMA stimulation induced surface expression of uPAR in lymphocytes but did not induce expression of LRP by these cells. In contrast, lymphoid cell lines were negative for uPAR expression even after PMA stimulation, indicating differences in regulation of uPAR expression between lymphocytes and lymphoid cell lines. The pattern of uPAR expression on leukemic cell lines was also studied on bone marrow blast cells from leukemic patients. Only the most mature myeloid cells expressed uPAR on their surfaces. In contrast, M3 leukemic cells and other blast cells displaying lymphoid markers such as TdT (+) and/or CD2 (+) did not express intracellular or cell-surface associated uPAR, indicating an heterogeneity among these promyelocytic cells and suggesting that uPAR may be a useful marker for leukemia typing. Myeloid blast cells from some patients contained intracellular pools of uPAR but displayed no receptor on the cell surface, suggesting that translocation may be a mechanism regulating uPAR expression in these cells. The comparison of uPAR expression between these cell lines and peripheral blood cells and it correlation with plasminogen receptors, tPA receptors and LRP expression offers new insights regarding potential mechanisms for regulation of uPA-uPAR-mediated pericellular proteolysis.

A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica.

A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of beta-glucan by Zymolyase digestion. The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub-apical locations, suggesting that it participates directly in the mycelial wall architecture.

Ethanol increases cytochrome P4502E1 and induces oxidative stress in astrocytes.

We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its induction by ethanol, and the concomitant generation of free radical species. Double immunofluorescence using anti-CYP2E1 and anti-glial fibrillary acidic protein showed that CYP2E1 was distributed over the cytoplasm and processes, although labeling was more pronounced over the nuclear membrane. Immunogold labeling confirmed this pattern of distribution. Addition of 25 mM ethanol to the astrocyte culture medium for 14 days resulted in an increase in the CYP2E1 content, as determined by confocal microscopy and dot blot. In addition, ethanol induced a dose-dependent increase in the formation of reactive oxygen species that was partially prevented by incubating the astrocytes with anti-CYP2E1. Alcohol also induced a dose-dependent increase in malonaldehyde and hydroxynonenal formation and a depletion of the glutathione (GSH) content. These results suggest that ethanol induces oxidative damage in astrocytes, which could explain some of the toxic effects of ethanol on these cells, such as cytoskeletal alterations. This assumption is supported here by the fact that an increase in GSH content prevents the deleterious effects of alcohol on the cytoskeleton of astrocytes. These results suggest the importance of oxidative stress as a mechanism involved in alcohol-induced neural and brain damage.

Ammonia prevents activation of NMDA receptors by glutamate in rat cerebellar neuronal cultures.

Acute ammonia toxicity is mediated by activation of NMDA receptors and is prevented by chronic moderate hyperammonaemia. The aim of this work was to assess whether the protective effect of chronic hyperammonaemia is due to impaired activation of the NMDA receptor. It is shown that chronic hyperammonaemia in rats decreases the binding of [3H]MK-801 to synaptosomal membranes from the hippocampus but not the amount of NMDAR1 receptor protein as determined by immunoblotting. In primary cultures of cerebellar neurons, long-term treatment with 1 mM ammonia also decreased significantly the binding of [3H]MK-801. These results suggest that ammonia impairs NMDA receptor activation. To confirm this possibility we tested the effect of long-term treatment of the cultured neurons with 1 mM ammonia on three well known events evoked by activation of the NMDA receptor: neuronal death induced by glutamate, increase in aspartate aminotransferase activity and increase in free intracellular [Ca2+]. Long-term treatment with ammonia prevented noticeably the effects of glutamate or NMDA on all these parameters. These results indicate that long-term treatment of neurons with 1 mM ammonia leads to impaired function of the NMDA receptor, which cannot be activated by glutamate or NMDA. Activation of protein kinase C by a phorbol ester restored the ability of the NMDA receptor to be activated in neurons treated with ammonia. This suggests that ammonia impairs NMDA receptor function by decreasing protein kinase C-dependent phosphorylation.

Prenatal exposure to ethanol alters plasma membrane glycoproteins of astrocytes during development in primary culture as revealed by concanavalin A binding and 5'-nucleotidase activity.

We have investigated the effect of prenatal exposure to ethanol on the extent of binding and surface distribution of the lectin concanavalin A (con A) on rat cortical astrocytes during the periods of proliferation and differentiation in primary culture. The enzymatic activity of the plasma membrane glycoprotein 5'-nucleotidase was also assessed. The cells were obtained from control fetuses (no exposure to ethanol) and from fetuses prenatally exposed to ethanol. The main findings were: 1) both proliferating and differentiating control astrocytes showed two distinct types of surface con A receptors that could correspond to high- and low-affinity binding sites; 2) the extent of con A binding was greater in mature than in proliferating control cells; 3) the distribution of con A on cell surface components changed with differentiation; 4) the activity of 5'-nucleotidase showed a substantial increment during the period of differentiation; and 5) prenatal exposure to ethanol clearly decreased the ability of astrocytes to bind con A, altered the surface distribution of the receptors for this lectin, and decreased the activity of 5'-nucleotidase. These effects were more marked in proliferating cells. In conclusion, it is shown that the extent of con A labeling and the activity of 5'-nucleotidase in astrocytes are dependent on the stage of cell differentiation and that prenatal exposure to ethanol alters the plasma membrane structure of these cells during development.

Evolution of several cytoskeletal proteins of astrocytes in primary culture: effect of prenatal alcohol exposure.

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein alpha-tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of alpha-tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti alpha- and anti-beta tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin. In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed "in vivo".

Asymmetric incorporation of [14C]cyanate and of fluorescein isothiocyanate in mamillary body of conditioned rats.

A marked decrease in overall learning capacity has been observed in rats injected with cyanate. Therefore it was of interest to test whether learning influenced carbamylation of brain proteins. Incorporation of [14C]cyanate into proteins of the mamillary body was selectively modified following operant conditioning of the rat, so that trained rats showed an asymmetric image with higher levels of incorporation in the right side than in the left side, as compared to control rats. These results were confirmed using fluorescein isothiocyanate. The asymmetry persisted once the learning had been well established.

Inhibition by N-acetyl-aspartate of aspartate binding to a proteolipid fraction from rat cerebral cortex.

Many roles have been suggested for N-acetyl-aspartate in brain function because of it being located almost exclusively in that organ. However, its true role remains to be demonstrated. We show here that N-acetyl-aspartate: 1) binds to a hydrophobic protein fraction from the cerebral cortex of the rat, which specifically binds L-aspartate, L-glutamate, and gamma-amino-butyric acid; and 2) has a marked inhibitory effect on the aspartate binding sites of this proteolipid fraction. Structural analogs of N-acetyl-aspartate, i.e. N-carbamyl-aspartate and N-methyl-aspartate also inhibit the L-aspartate binding by the brain protein fraction used.

Effects of N-acetyl aspartate, aspartate, and glutamate on cAMP and cGMP levels in developing rat cerebral cortex.

N-Acetyl-aspartate (N-Ac-Asp) incubated with minced cerebral cortex caused a dose-dependent increase in the levels of cAMP and cGMP. This effect was followed during postnatal development. N-Ac-Asp elicits the greatest increase in cAMP in 5-day-old and in cGMP in 40-day-old rats. The levels of cyclic AMP were always higher than those of cGMP. We also studied the effects of L-aspartate (Asp) and L-glutamate (Glu) on the levels of cyclic nucleotides in the cerebral cortex minces of rats different ages, and observed that both amino acids produced the maximum increase in cAMP at 10 days, whereas in the case of cGMP the maximal effect of Asp occurs earlier than 20 days and of Glu after 40 days. In the adult rat, the N-Ac-Asp effect on cAMP was greater than that produced by either Asp or Glu, whereas the levels of cGMP were similarly affected by all three. The data show a peak response of cAMP and cGMP to N-Ac-Asp, Asp, and Glu during cortical maturation. Because this response varies with postnatal time, N-Ac-Asp, and Glu may act upon different receptor sites.