Stéphane Binet

Quantitative analysis of monoclonal immunoglobulins in serum of patients with amyotrophic lateral sclerosis

In a prospective study, we analysed the presence of monoclonal immunoglobulin (moIg) in the serum from 30 patients with amyotrophic lateral sclerosis (ALS) and 30 matched controls using a sensitive Western blot technique. The incidence of serum moIg was 60% in the ALS group and 13.3% in the control group. Most ALS sera contained 2 or 3 monoclonal components. They were IgG (72.7%) and IgM (27.3%). These results corroborate the concept of a probable association between ALS and serum moIg.

Characteristics of Microtubules at the Different Stages of Neuronal Differentiation and Maturation

The developing nervous system has proved to be a very powerful tool to analyze how MT are involved in basic biological processes such as cell proliferation, cell migration, cell shaping, and transport. A better knowledge of the basic events occurring during neurogenesis also affords us the possibility of establishing the basis of experiments and trying to solve unanswered and important questions. Despite the considerable value of cell culture, we need to use more discrete regions of the developing brain in situ in order to analyze the MT and their modifications into cells developing their natural environment. One major problem remains the question of the mode of assembly and disassembly, that is, the behavior of MT in living cells. Dynamic instability and/or treadmilling are accurate interpretations of the dynamics of MT at least in vitro or in cell culture, but we do need more information on what happens in situ and in vitro. One of the main tasks of cell biologists is to devise satisfactory tests to approach this fundamental question. In this view, pharmacological manipulation of embryos treated in whole-embryo culture systems might be a possible way. Microtubules are ubiquitous cell components. However, the extensive heterogeneity of MAP and tubulin in the CNS confers on the neurons a wide range of capabilities of assembly of these proteins and suggests that the neuron has a unique potential of a relation between MT composition and cell function. We have seen that each major event during neurogenesis is related to a specific series of modifications of the MT components. It remains to be determined if there is a causal or just a correlative relationship between the appearance of specific isotypes and the occurrence of specific events and/or functions. We have also to determine the exact spatial and temporal relations among the different isotypes of MT proteins, tubulin, and MAP. Is there a close correspondence between a tubulin and a MAP isotype? Can the appearance of one isotype of tubulin influence the appearance and the assembly of a specific MAP, or vice versa? Recent results obtained with the Tyr- and Glu-MT shed light on these questions and suggest a whole series of possibilities for cells to modulate the structure, behavior, and function of MT in specific domains of the neuron or in specific regions of the brain, by only a minute modification of the molecule of tubulin. Microtubule protein heterogeneity raises also a number of questions.(ABSTRACT TRUNCATED AT 400 WORDS)

Modifications of microtubule proteins in ALS nerve precede detectable histologic and ultrastructural changes.

Fast axonal transport is altered in nerves from amyotrophic lateral sclerosis (ALS) patients. Microtubules are involved in axonal transport. We analyzed the possibility of an involvement of microtubule modifications underlying the alterations in transport using biochemical and morphologic analysis of intercostal nerves from ALS and control patients. In intercostal nerves displaying no morphologic signs of acute neuronal degeneration, two-dimensional gels showed modifications of the group of β tubulins and abnormal spots of proteins, some appearing to be closely related to tau proteins. These results suggest that microtubule proteins are modified in ALS before ultrastructural axonal degeneration, but the significance of these abnormalities remains hypothetical.

Biochemical basis of microtubule cold stability in the peripheral and central nervous systems.

Cold-stable, cold-labile and unpolymerized tubulins extracted from thalamic nuclei (soma-enriched fraction) and various nerves (both central and peripheral: axon-enriched fractions) appear different when analyzed by high-resolution isoelectric focusing. Cold-labile tubulin appears identical to unpolymerized tubulin. The axonal fractions contain fewer tubulin isotypes than the soma-enriched fraction; the peripheral axonal fraction has fewer isotypes than the central fraction. Cold-stable tubulin exhibits a specific pattern characterized by the abundance of two isotypes of alpha-tubulin, 7 and 8, and one beta-tubulin, isotype 9, with slightly different patterns of the axon-enriched fractions from the central and peripheral nervous systems. Our results suggest that the cold stability of microtubules is based on biochemical properties of tubulin, and confirm the domain specificity of the heterogeneity of tubulin.

In situ appearance of the cold-stable microtubules in the growing axons of the tectal plate of mouse investigated immunocytochemically after polyethyleneglycol (PEG) embedding

Tubulin immunostaining of semi-thin sections after polyethylene glycol embedding was used in the tectal plate of the embryonic mouse at 10 days postmating to analyze the effects of cold treatment on the microtubules of the different cell types seen at this stage. Three sets of microtubules are observed. In the radially oriented bipolar columnar cells, dense bundles of microtubules are present in the ventricular processes between the cell nucleus and the ventricular surface. In the mitotic cells, located just at the surface of the ventricle, microtubules are among condensed chromosomal figures. In the apical region, the intermediate zone, tangentially oriented axonal profiles contain dense bundles of microtubules among tangentially oriented young neurons. Cold treatment does not modify the organization of the cells. However, it depolymerizes whole cytoplasmic and mitotic microtubules of the bipolar cells and a large number of microtubules in the growing axons. In the axonal profiles, the cold-stable fraction of microtubules displays the appearance of short fragments. Some of these are regularly organized, suggesting that they could be the remnants of the same individual microtubule. These fragments are approximately 1 micron long and seem to represent nearly 10% of the total microtubules in the axons. These cold-stable fragments might fulfill a function in the axon analogous to the microtubule organizing centers in the perikaryon and their presence can explain some properties of the growing axons suggested by previous studies on the guidance of neurites and growth cones as well as on the growth of isolated axons.

In situ response to vinka alkaloids by microtubules in cultured post-implanted mouse embryos

The response of microtubules to treatment with vinca alkaloids was investigated in vivo and in situ in the embryonic nervous system of mice. For this purpose we used rotatory cultures of post-implanted embryos in a serum medium containing the alkaloid combined with immunofluorescence using a tubulin-specific polyclonal antibody on high molecular weight polyethylene glycol embedded semithin sections. In mitotic cells, kinetochore microtubules were seen to be more resistant to the action of vinca alkaloids than interpolar microtubules. Increasing drug concentrations induced an increasing rate of mitosis together with an increasing rate of disassembly of the cytoplasmic microtubule complex, suggesting a probable relation between these events. In bipolar neuroepithelial cells at interphase, a small pool of microtubules was resistant to the vinca alkaloids. These microtubules were located near the centriolar apparatus associated with the primary cilium; they were short, curly and bent. Disruption of the cytoplasmic microtubule complex did not alter the shape of the bipolar neuroepithelial cells. In the axonal profiles, a drug-stable pool of microtubules were not disrupted by the alkaloids and were also short. They seem to act as microtubule organizing centres. These observations suggest vinca alkaloids seem to act in vivo much more by inducing, at a given concentration, the disruption of a particular group of microtubules without altering the others. The fact that these drugs affect the number, but not the length, of the microtubules raises the hypothesis that these drugs act on microtubules by a mechanism similar to that described as dynamic instability.

Heterogeneity of cold-stable and cold-labile tubulin in axon- and soma-enriched portions of the adult mouse brain

Microtubules from the optic nerve (axonal tubulin) and lateral geniculate nucleus (cell tubulin) were separated by cold treatment and the cold-soluble fraction was purified in the presence of Taxol. Isoforms of cold-stable and cold-soluble tubulin were resolved by the use of high-resolution isoelectric focusing. The cold-soluble fraction of axonal tubulin has only 14 of the 20 isotypes seen in the same fraction of cell tubulin. The two cold-stable fractions have 20 isotypes but axonal tubulin has a specific pattern of isotypes 1, 2 and 5. Cold-stable fractions of both axonal and cell tubulin display the existence of an intensely stained alpha-isotype, isotype 7, which seems associated with the property of cold stability. Our results highly favor the hypothesis of a physiological role of the heterogeneity of tubulin in neuronal microtubules.

Modifications of tubulin heterogeneity during axonal growth in the embryonic nervous system

Tectal plates of mouse embryos were used to analyze the heterogeneity of tubulin with high resolution isoelectric focusing, at time of appearance of the young neurons and axons. In the cold-labile pool of tubulin, isotypes 6 and 7 appear, and isotypes 1, 3, 11, 13 increase their relative quantities. The increase of the prominence of the alpha-group with regard to the beta-group in the cold-stable pool of tubulin raises the question as to the exact role of tubulin in the cold stability. A role of other specific microtubule components also appears probable.

Spatial organization of microtubules in various types of cells in the embryonic tectal plate of mouse using immunofluorescence after PEG embedding

The spatial organization of microtubules in mitotic as well as in interphase cells and in axons has been investigated in situ in the embryonic nervous system of mice using high molecular weight polyethylene glycol‐embedded semithin sections and immunofluorescence with a tubulin‐specific polyclonal antibody.

Modifications of tubulin heterogeneity during embryonic and postnatal stages in a specific region of mouse brain

An auditory nucleus (the inferior colliculus of the mouse) was used to study the modifications of the heterogeneity of tubulin which occur at various stages of development and maturation. Both the cold-stable (CS) and cold-labile (CL) fractions of tubulin were analyzed by high-resolution isoelectric focusing. Our results suggest that tubulin heterogeneity is modified at critical stages of development and maturation, with specific variations of the two fractions. Stage E10 corresponding to the appearance of the first young neurons and axonal profiles is marked by the modification of the CS fraction with the emergence of isotypes alpha 5 to alpha 8 and beta 12 and beta 17. Stage E12 is characterized by the modifications of the CL fraction, particularly the beta-group; at this stage the first dendrites become visible. At birth, all isotypes increase in both the CL and CS fractions. At stages P7-P10 transient modifications of a group of both CS and CL fractions and of the beta group of CS fraction occur. These are associated with the emergence of isotypes beta 17 to beta 20 in the CL fraction. This period precludes the initial period of functional maturation of the auditory system which occurs from P10 to P20. During this period, the CL fraction (alpha and beta group) remains unmodified, whereas all isotypes of the CS fraction, except 3-4, display complex variations with an initial decrease until P12, an increase until P17, and a final decrease until P20.(ABSTRACT TRUNCATED AT 250 WORDS)