Mutation Research, 256 (1991) 149-168 © 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50

149

MUTAGI 00158

Involvement of microtubules in modifications associated with cellular aging Martine Raes Laboratoire de Biochimie Cellulaire, Facult~s Unit,ersitaires Notre-Dame de la Paix, B-5000 Namur (Belgium) (Accepted 19 June 1991)

Keywords: Ageing; Microtubules; Microtubule associated proteins; Fibroblasts; Neuronal cells; Signalling systems

Summary Microtubules are ubiquitous cellular components involved in the control of cell structure and functions, such as cell division, regulation of shape and polarity, intracellular transport, etc. Consequently, any alteration affecting them in structure or function has a good chance of affecting the cell and generally leads to cell dysfunctions. This has been shown for instance, after treatment with microtubuleinteracting drugs. Cellular aging is also characterized by the appearance of various cell dysfunctions, but the possible involvement of the microtubules in the aging process, although a rather tempting hypothesis, has not yet been extensively investigated. In this paper, I will first rapidly review the different components that build, organize and control the microtubules in normal cells, independently of the aging process. I will then consider the possible involvement of the microtubules in the aging process, more particularly in models of cells aging in vitro and in aging neuronal cells, which have been the most extensively investigated. There is some evidence for alterations in the microtubule organization both in cells aging in vitro and in the aging brain. But the interpretation of these data awaits further experiments, taking into account the latest progress in tubulin genetics and in microtubule biochemistry. Microtubules could also represent one of the cellular targets affected after signal transduction and could thus be involved in the resulting cellular responses. This hypothesis will be discussed, as it offers new insights into the regulation of microtubule organization, dynamics and functions in normal cells, which will be worthwhile to investigate during the aging process.

Microtubules are universal components of the cell and together with the microfilaments and the intermediate filaments they constitute the cy-

Correspondence: Dr. M. Raes, Laboratoire de Biochimie Cellulaire, Facult6s Universitaires Notre-Dame de la Paix, B-5000 Namur (Belgium).

toskeleton. One of their functions as part of the cytoskeleton is to frame intracellular scaffoldings sustaining the cell, but this structural role, which has been known for more than 100 years, is only one aspect of their function. Microtubules are also highly dynamic and labile structures, able to reorganize very rapidly: by this property, they contribute to the cell polarity and are implicated

150

in the regulation of cell shape, cell movement and division (Kirschner, 1989). Finally, given their association with very peculiar proteins, the microtubule-based motor proteins such as dynein, kinesin, etc. they are part of the transport system for organelles and chromosomes in the cell. Microtubules are thus ubiquitous cell components, interacting with nearly all the other cellular organelles, from the nucleus to the cytoplasmic membrane, and are implicated in various important cellular functions. As a consequence, any event affecting - directly or indirectly - the microtubules themselves or their organization is accompanied by more or less important cell dysfunctions. This has been clearly shown, for instance, with drugs interacting with tubulin, the major constituent of microtubules, such as colchicine, nocodazole or taxol. It is thus tempting to speculate that if microtubules are to some extent altered with cellular aging, this alteration could also drastically impair normal cell function, contributing to cell aging and finally to cell degeneration and death. In this paper, we will try to answer the following questions: are there any data suggesting a possible alteration of the microtubules and their organization, in aging organisms or cells? And if there are, what is their relevance to the aging process? Before considering microtubules and aging, we will briefly summarize some of the fundamental data on the biochemistry and molecular biology of tubulins and microtubules in normal cells. Given the plethora of reports in this basic domain of cell biology, we will refer here only to some of the most significant reviews and papers published on the subject over the last few years. This introduction is essential at least for three reasons. First, tubulin and microtubule (MT) regulation is a complex phenomenon which is not completely understood in normal cells and as a consequence, looking for possible dysfunctions in cellular aging is not an easy task. Secondly, MT organization and function rely on many different associated proteins as well as organelles such as the centrosome; any alteration affecting either one of these proteins or one of these organelles with aging could thus also affect the MTs. Thirdly, despite undeniable progress over the last years in the biochemistry and even in the genetics of some

of the MT-associated proteins, we have to recognize that our knowledge of the role of these proteins remains quite fragmentary: some of these proteins, for instance dynamin, have been described and characterized very recently and their functions have to be ascertained; moreover, one cannot exclude the discovery of some still unkown MT-associated proteins, for instance in the mitotic spindle. Evaluating the importance of these proteins on microtubule organization and function with aging will thus require a great deal of further investigations. There is general agreement about the importance of MTs in normal cell functioning and division, in differentiation and morphogenesis, but the molecular mechanisms underlying their implication in these phenomena are far from being understood. Hence, their possible involvement in aging is a very tempting hypothesis (Rao and Cohen, 1990) but one that has been poorly investigated. As reviewed in this paper, there have been some preliminary often descriptive works on microtubules with aging, in vivo and in various models in vitro. But the recent progress in the understanding of MT dynamics raises new questions which are awaiting further investigations into the cell biology of aging. Microtubules

in normal

cells

Microtubule structure and assembly Microtubules are made of tubulins, c~-Tubulin and /3-tubulin associate into stable heterodimers. The tubulin dimer can bind 2 molecules of GTP: one is rapidly exchanged for a similar GTP or G D P molecule, but the second one is harder to exchange. The heterodimers linearly polymerize into protofilaments and the latter - generally 13 - associate through lateral interactions to form a tubule of 25 nm diameter. Because the tubulin subunits are arranged in a specific orientation in the polymer, microtubules are polar structures, with unequal ends, the plus or fast growing end and the other one, the minus end (Bershadsky and Vasiliev, 1988; Mandelkow and Mandelkow, 1989, 1990). Tubulin polymerization is an intricate phenomenon under complex regulation: it can be followed in vitro and is described by a sigmoidal curve starting with a preliminary lag period. The

151

reason for this behavior is that elongation is preceded by a nucleation step. The nature of the required nuclei remains unclear. However, a new tubulin isotype discovered in the yeast Aspergillus, quite different from c~-and /3-tubulins and called y-tubulin, has been proposed to play a role in MT nucleation, but y-tubulin genes have not yet been identified in vertebrate cells (for a review, see Murphy, 1991). Another protein, centrophilin, has recently been described by Tousson et al. (1991) and could be involved in MT nucleation in the mitotic spindle. During elongation, microtubules grow by the irreversible addition of tubulin subunits, concomitantly with a delayed nucleotide (GTP) hydrolysis and a conformational change (Carlier, 1991). The nucleotide hydrolysis lowers the critical concentration of the free subunits required for net polymerization, but this effect is more pronounced at the plus end than at the other end, so that MTs preferentially grow at their plus ends. Moreover in the cell, MT ends are not necessarily free: the minus ends are most of the time protected by the so-called microtubule organizing centers (MTOCs) and only the plus ends are free in their majority (Mandelkow and Mandelkow, 1989, 1990; Avila, 1990) (Fig. 1). Once assembled, microtubules are generally not in a simple equilibrium with their subunits although they maintain an overall steady state but they undergo phase transitions between extended periods of growth and shrinkage; in other words, at any instant, the total population of microtubules consists of slowly growing tubules and rapidly shrinking tubules, and this is observed over a wide range of free subunit concentrations. Individual microtubules have been shown in vivo and in vitro to switch spontaneously from the slowly growing to the rapidly shrinking state. This unique dynamic behaviour has been called dynamic instability by Mitchison and Kirschner (1984) But MT assembly, structure and functions are also governed by some particular properties of the tubulins that will be considered in the following section.

The tubulins Understanding the regulation of microtubule assembly and functions is complicated by two

A

CS

B

•

p-IVl~

÷

Fig. 1. Simplified diagram comparing the MT organization and polarity in interphase (A) and mitotic (B) cells. (A) In interphase cells, MTs build up the so-called MT cytoplasmic complex (MTCC): their minus ends are embedded in the pericentriolar material of the centrosome (or cell center) while the plus ends are oriented towards the plasma membrane. Some of the free plus ends could be captured and stabilized by cortical capping structures. The MT-based motor proteins may be involved in these interactions between MTs and plasma membrane. (B) Diagram of the mitotic spindle at metaphase: the spindle is made of 2 half-spindles, both of them composed of astral (A-MT), polar (P-MT) and kinetochore MTs (K-MT); all these MTs are oriented with the minus ends towards the centrosome and the plus ends away from it; polar MTs emanating from the opposite poles display a region of overlap, where they are antiparallel and probably crosslinked by MAPs. For the clarity of the diagram, only the plus ends are shown. N: nucleus; c: centrosome; dotted area: pericentriolar material; + : plus end; cs: capping structure.

features of the tubulins: first, their molecular heterogeneity into isotubulins and secondly, their susceptibility to various posttranslational modifications. The a- and /3-tubulin subunits of the MTs both represent families of structurally and biochemically different isoforms, which correspond to different genes and display specific posttrans-

152 lational modifications. The /3-tubulins have been more extensively studied and the number of their genes ranges from 1 to 6, depending on the animal species considered. Different combinations of tubulin isoforms are generally expressed in different tissues and cell types. Tubulin isoforms may even be non-uniformly distributed within the same cell, with partial exclusion of some of them in the MTs, for instance in differentiating rat PC12 cells in culture; P12 cells are a clonal cell line that extend neurites in response to nerve growth factor (Joshi and Cleveland, 1989). The functional significance of the various isotypes is not yet completely understood but the concept of one isotype-one function, first hypothesized, clearly does not fit any more with the recently accumulated data. For instance, tubulin isoforms by themselves generally display a limited capacity to segregate into distinct populations of MTs; however, limited amounts of one isotype can influence the properties of MT assembly and a threshold effect has been described, which means that a particular isotype could affect MT assembly and functions only if it accumulates to a critical percentage. Finally the assembly, properties and functions of MTs could be determined, not by a particular a- or /3-tubulin isoform, but rather by their combination in a particular heterodimer that would be the real functional basis of the MTs (for further reading, see MacRae and Langdon, 1989; Murphy, 1991). However it is not yet clear how each cell type regulates the expression of the different tubulin genes, the synthesis of its tubulin dimers as well as the stoichiometry of the a- and /3-subunits. Cleveland (1989) proposed a general model for an autoregulated /3-

tubulin synthesis, based on selective changes in cytoplasmic /3-tubulin m R N A stability. In this model, unpolymerized tubulin (a or /3) binds (directly or through an activated binding factor) to the nascent amino-terminal tetrapeptide of /3-tubulin. This binding, by still unknown mechanisms, could activate a RNAase which will degrade the /3-tubulin m R N A associated with the ribosome. The amino-terminal tetrapeptide (MetArg-Glu-Iie) is conserved among all the /3-tubufins sequenced to date. Similar mechanisms could also regulate a-tubulin synthesis, given the presence of another highly conserved amino-terminal tripeptide. However this model does not explain how the cell regulates the expression of the different isoforms. Finally, all tubulins undergo various posttranslational modifications, such as phosphorylation, which is a common regulatory mechanism, but also rather unusual modifications such as tyrosination, acetylation or polyglutamylation, affecting specific sites on a- and /3-tubulins. There is now good evidence for a destabilizing effect of tyrosination and acetylation. Tyrosination has been more particularly analyzed in details and clearly regulates the dynamic activity of the MTs. For instance, stable arrays of MTs present in erythrocytes, sperm or some neuronal cells in culture contain higher levels of detyrosinated tubulin than the other MTs within the same cells (MacRae and Langdon, 1989; Murphy, 199l). The microtubule-associated prote&s or MAPs

The MT-associated proteins or MAPs can be classified in different ways and according to various criteria. In this paper, we will consider them

TABLE 1 THE MAIN STRUCTURAL MAPs M r (kDa) 35O 325 300 55-65 200

Family species MAP1A MAP1B MAP2(A,B) tau family 200 K family(including MAP3 and MAP4)

Primary source brain brain and non-neuronal cell types brain brain neuronaland various non-neuronal tissues and cultured cells

Type fibrous fibrous fibrous fibrous fibrous

75

buttonin syncolin

urchin eggs erythrocytelineage

non-fibrous non-fibrous

_

153

in two main groups based on their function: on the one hand the structural or skeletal MAPs (Table 1) and on the other hand the mechanochemical MAPs capable of transducing energy, also called the MT motor proteins (Table 2). The structural MAPs Structural MAPs bind to the MTs and stabilize them against disassembly; they also mediate the interactions of the MTs with other cell components. The best studied MAPs associated with cellular microtubules are those isolated from the brain and they are generally classified into 2 classes: the high molecular weight MAPs (MAP1A, MAP-2, etc.) from 200,000 to 300,000 and the tau family (40,000 to 60,000). Thanks to recombinant DNA technology, these proteins have been recently sequenced, with characterization of different domains (tubulin binding, etc.) (for reviews, see Bershadsky and Vasiliev, 1988; Wiche, 1989; Matus, 1990; Cleveland, 1990; Olmstead, 1991; Wiche et al., 1991). MAPs have been known for a long time to stabilize MTs in vitro. They also control MT stability in vivo and act as probable targets of intracellular regulation signals, such as variations in the concentration of Ca or AMPc in ways probably similar to those involving the actin-binding proteins of the microfilaments (Forscher, 1989). Our understanding of the functions of the MAPs has improved over the last years thanks to recombinant DNA technology: MAPs have been directly expressed in living cells, from cloned cDNA. These experiments showed that tau and MAP-2 can induce the bundling of normally separated MTs in fibroblasts in culture and hence influence the cell morphology.

The investigation of the expression of the different mRNAs coding for the MAPs during brain development showed that the expression of the MAPs is developmentally regulated and also revealed partitioning of some MAPs in different cell types, which suggests an important role of MAPs in cell differentiation and in development (Meininger and Binet, 1989). MAPs are also involved in the control of cell morphology, a role which has been particularly well documented in neuronal morphology (Matus, 1988; Black and Bass, 1989; Sargent, 1989): in neurons, the dendrites as well as the cell body contain high levels of MAP2 while most axons do not; axons on the other hand are generally rich in tau protein whereas dendrites are not. Interestingly, MTs are more widely spaced in dendrites than in axons. This suggests that the bridges formed by the MAPs may serve as spacers between MTs and other cellular components (for a review, see Wiche et al., 1991). Finally, most of the MAPs undergo specific phosphorylations that could modulate their functions and for instance play an important role in the mitogenic signaling pathway. A MAP kinase has recently been described, which phosphorylates MAP2 and which is activated by a variety of growth factors as well as by simple disruption of the MTs (Shinohara-Gotoh et al., 1991). The high molecular weight MAPs and the tau family proteins described above are filamentous or fibrous MAPS, which are mainly encountered in neuronal cell types. Recently, some non-fibrous MAPs, possessing structures other than filamentous, have been described in non-neuronal cell types, but their function remains presently unknown. Buttonin was first described by Hirokawa

TABLE 2 THE MAIN MECHANO-CHEMICAL MAPs Relative molecular mass

Family species

Primary source

550,000-1,200,000 300,000- 600,000

M A P 1 C or cytoplasmic dynein axonemic dynein kinesin

brain, liver, testis, H e L a cells, etc. cilia, flagella, brain sea urchin eggs, bovine adrenal glands, H e L a cells, chick embryo fibroblasts, etc. brain

-

dynamin

154 and Hisanaga (1987); this spherical protein of 75 kDa decorates the spindle MTs in sea urchin eggs, forming button-like structures, and is thought to play a role in spindle formation and stabilization. Syncolin, another non-fibrous MAP, was shown in cells of the erythrocytic lineage in the chicken (for a review, see Wiche et al., 1991).

The mechano-chemical MAPs Dynein ATPase was the first MT-based motor protein, purified from Tetrahymena cilia. It took some time, however, to isolate and characterize biochemically some cytoplasmic motors, involved in organelle transport and possibly in mitosis, both phenomena associated with MT-based movements. Two cytoplasmic motors are presently well characterized, kinesin and dynein (for a review see Porter and Johnson, 1989; McIntosh and Porter, 1989; Vale, 1990; Scholey, 1990; Vale and Goldstein, 1990; Vallee and Shpetner, 1990; Schroer and Sheetz, 1991; Wiche et ai.,1991). Kinesin is a multimeric protein, resulting from the assembly of several heavy (110-135 kDa) and light (60-75 kDa) polypeptide chains. Dynein is mainly composed of high-Mr polypeptides, called heavy chains (300-450 kDa), although several polypeptides of smaller apparent Mr have been reported to copurify with the heavy chains. However, the subunit composition of both proteins varies according to the source and the animal species (for a review, see Wiche et al., 1991). The 2 proteins are thus quite different in their structural, biochemical and enzymatic properties. For instance, kinesin moves towards the MT plus ends, thus from the cell center to the periphery (anterograde movement); dynein moves towards the MT minus ends, towards the centrosome (retrograde movement). Kinesin and dynein, as well as myosin, seem to generate movement by similar molecular mechanisms involving ATP hydrolysis. On the one hand in vitro organelle transport assays suggest that both kinesin and dynein act as efficient organelle transport motors. This has been shown on isolated salt-washed organelles, from highly oriented squid giant axons, a model developed by Allen (Alien et al., 1982) which has contributed to our better understanding of cell movement, but also from chick embryo fibroblasts. On the other hand

the involvement of dynein and kinesin in the mitosis associated movements remains controversial. However, kinesin analogs have been recently described in yeast and Drosophila. For instance, Meluh and Rose (1990) have identified one of these analogs in yeast; it is a protein encoded by gene KR3 of S. cereL,isiae, which could act as a microtubule motor in the mitotic spindle and contribute to the movement of chromosomes. Finally, Vallee and his collaborators (see for instance, Vallee and Bloom, 1991) described another potential mechano-chemical MAP, called dynamin. This MAP consists mainly of a 100-kDa potypeptide associated with as yet unindentified components and seems involved in cellular MT sliding, and in particular in mitotic spindle elongation. According to recent data (Vallee and Bloom, 1991) dynamin may be involved in neuronal slow axonal transport.

Microtubule organization and MTOCs In an interphase cell in culture, MTs radiate out into the cell periphery, emerging from the cell center or centrosome, the major MTOC in almost all animal cells (Fig. 1A). MTOCs are structures capable of organizing MTs in oriented (parallel or radiate) arrays. In vitro experiments have shown that MTOCs are also able to nucleate MTs, control their structure as well as their array. The maximal number of MTs in the array, for instance, is determined by the MTOC itself. MTOCs are generally classified into 2 groups: on the one hand MTOCs themselves containing MTs, such as the basal bodies of cilia and flagella or the centrosomes with their 2 centrioles, on the other hand kinetochores associated with the chromosomes in mitotic cells or some special areas of the nuclear and plasma membranes having nucleating properties. The biochemistry of all these MTOCs is far from being elucidated (for a review, see Cande, 1990; Huang, 1990). For instance, in the centrosome it is not the well-defined centrioles, but rather the amorphous pericentriolar material that nucleates the MTs. The composition of this material has still not been completely unraveled. Immunological studies using antibodies have contributed to a definition of this composition and have revealed some cross-

155 reactivity in animal and plant cells, which suggests that some components could be highly conserved during evolution; antibodies specific for MAP1A, kinesin and dynein also recognize the centrosome. The biochemical analysis of the MTOCs is also progressing and some polypeptide components have recently been characterized, such as centrophilin (Tousson et al., 1991); this polypeptide seems to be associated with nucleating sites in the mitotic spindle and shuttles along the spindle MTs. Centrosome activity is well known to vary according to the cell cycle. The MT nucleating activity increases significantly during mitosis and the phosphorylation of some centrosomal polypeptides is also probably implicated in this regulation. Microtubules and MAPs in neuronal cells

MTs and MAPs have been particularly investigated in neuronal cells. In these highly specialized cells, their spatial organization as well as their functions display some peculiarities that we will briefly review. The nerve cell is a highly polarized cell, characterized by a cell body sprouting into various

~'.'_-

~c'

-

÷

~

neuritic processes: numerous postsynaptic short and branched dendrites and one presynaptic, long, unbranched axon (Black and Baas, 1989). The distinction between axon and dendrites can be made on different grounds, such as their morphology at the cellular and subcellular levels, but they also have specific functions (Sargent, 1989). Axon-dendrite differences extend in addition to the cytoskeleton and more particularly to the MTs and their associated proteins. In axons, all MTs are oriented with their plus ends away from the cell body (Fig. 2). They serve as scaffolds guiding the fast axonal transport (Okabe and Hirokawa, 1989; Sheetz et al., 1989). This transport is manifested at the subcellular level as anterograde or retrograde movements of membrane-bounded organelles along the MTs; the nature of the association between MTs and intracellular organelles has not been completely elucidated (Kelly, 1990). The anterograde movement supplies the axon terminus with the neurotransmitters and other secretory materials synthesized in the cell body. The corresponding organelles are conveyed by kinesin (Okabe and Hirokawa, 1989; Vallee and Bloom, 1991). In the retrograde movement, organelles are conveyed towards the

\

÷

÷

Fig. 2. Simplified diagram showing the organization and polarity of M T s in a typical neuronal cell. In the axon, the minus ends of the MTs are oriented towards the cell body and located proximal to the centrosome; the plus ends are oriented away from the cell body and found distally. Anterograde transport ( • ) is carried out by a plus end-directed motor (such as kinesin) and retrograde transport ( , - - ) by a minus end-directed motor (such as dynein). In dendrites, MTs display mixed polarities. N: nucleus; c: centrosome; dotted area: pericentriolar material; - : minus end; + : plus end.

156

cell body by dynein (Vallee et al., 1989; Vallee and Sphetner, 1990; Vailee and Bloom, 1991). The latter movement appears to be a specialization of endocytic and degradative pathways present in most of the cells. The third MT-based motor protein, dynamin, may play a role in slow axonal transport. Unlike fast axonal transport, there is no general agreement on the identity of the transported material and its nature is still controversial (for a review, see Vallee and Bloom, 1991). In dendrites, surprisingly, the orientation of the MTs is not uniform (Fig. 2): at the level of their midbodies, MTs have both plus and minus ends pointing outwards; their distal region, however, contains MTs of uniform polarity, with the plus end pointing away from the cell body. This has been shown in cultured rat nerve cells (Baas et al., 1988). The organization of the MTs in dendrites is not yet completely understood, but according to Baas et al. (1988), there is the possibility that 2 populations of MTs overlap. The MTs with the distal plus ends may be assembled as usual in the cell body while the other set of MTs may originate somewhere in the dendrite from some yet unknown nucleating component. Centrosome-associated material has been reported in the dendrite, but not in the axon. Given the non-uniform polarity of the dendritic MTs, MT-dependent transport in dendrites remains unclear (Black and Baas, 1989). Finally, axons and dendrites also display different sets of structural MAPs, as already pointed out in the section devoted to these proteins: tau is concentrated in the axon and MAP2 is confined to the dendrites. The structural MAPs, and more particularly tau and MAP2, have been well investigated in the nerve cells and obviously play an important role in the control of cell polarity, plasticity and morphology in the nervous system during development and differentiation as well as in the adult organism (see for instance, Matus, 1988; Meininger and Binet, 1989; Goedert et al., 1991). Thanks to molecular cloning, research into the biochemistry and genetics of both tau and MAP2 has made considerable progress. Tau is a heterogenous MAP represented by at least 6 isoforms, generated from a single gene by alternative splicing mRNA. The tau isoforms differ in the length of their amino acid sequence

and their phosphorylation degree. Tau phosphorylation leads to reduced affinity for the MTs and is likely to modulate its functions. Finally the various tau isoforms are expressed in a manner that is stage-specific and cell type-specific in the nervous system: the human fetal brain displays a pattern of tau isoforms which is different from the more complicated adult pattern. Different neuronal cell types also express the various isoforms in varying relative amounts. MAP2 refers to 3 different proteins of related amino acid sequence which are produced from a single gene: MAP2A, MAP2B and MAP2C. As for the tau isoforms, the expression of MAP2 also varies according to the type of neuritic process, the localization in the brain and the developmental stage of the nervous system. MAP2 function is also modulated by phosphorylation. The confinement of MAP2 to the dendritic compartment has been attributed to the sequestration in this compartment of the corresponding mRNA.

MTs and aging As shown in the first part of this paper, MT structure, regulation and functions are complex and rely on many different molecular and cellular components. MTs are also implicated in fundamental cellular functions, such as controlling the polarity and morphology of the cell, contributing to intracellular organelle and chromosome movement, mitosis, etc., and hence their involvement in differentiation and development is widely accepted. It is thus tempting to postulate that altered functions of the cytoskeleton in general and of the microtubules in particular could also play a key role in age-related changes (Wagner, 1989; Rao and Cohen, 1990). There have been some investigations trying to test the hypothesis of a possible alteration of MTs with aging. Most of the work concerns fibroblasts in culture, oocytes and eggs (in vivo and in vitro) and, of course, the nervous system. But unraveling the molecular mechanisms underlying the observed alterations is a much more difficult task, given the peculiarities in the genetics and biochemistry of the tubulins (isoforms, posttranslational modifications, etc.), due to the complexity of the organization and regulation of the MTs,

157 but also because our knowledge of all the components involved in MT structure and function is quite recent and still progressing. The main steps in the 'life' of a microtubule are represented in Fig. 3. The way these steps are regulated is unknown for most of them. But the figure clearly shows that an alteration affecting one of these steps has a good chance of affecting the MTs to some extent.

Microtubules in fibroblasts aging in r'itro The cytoskeleton, and hence the MTs, are built into 3-dimensional scaffolds and are therefore difficult to investigate by classical morphological techniques such as transmission electron microscopy. Moreover the requisite preparative steps are often damaging to the labile MTs. Most a-TUBULIN GENES ~ ~-TUBUL~

1

TRANSC~IPTION

mRNAs

B-TUBULIN GENES ~ B-TUBULIN mRNAs

~

TRANSLATION

o.-ISOTUBULINS

~

1

B-ISOTLrBULINS

l

l

FOLDING

FOLDING

~ ............................ lr . . . . . . . . . . . . . . . . . . . . . . ~

SPECIFICDIMER FORMATION / POS17-RANSLATIONAL MODIFICATIONS ~ ~ I ~ ~ GTP BINDING

!

~[. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PROTOFILAMENTFORMATION (through longitudinal interactions)

1 OR

SPIRALINTERMEDIATE

SHEEr FORMATION (through lateralinteractions) ~....................................................... T POSITRANSLATIONAL MODIFICATIONS ~

~

[ | .T

~INTF~ACTION WITH STRUCTURAL MAPS

l

MICROTUBULEFORMATION / POSITRANSLA TIONAL MODIFICATIONS ~ ~ ] ~

T

~ INTERACTION WITH STRUCTURAL MAPs AND llt~'OCs

SPATIALORGANIZATION (,,,i~ dy~n~c insta~itlty) MT ± TUBULIN Int~spha~ M'rcc < ................. • Mitoticspindle

Fig. 3. Schematic representation of the successive steps thal lead from tubulin gene expression to functional microtubules and to their cellular organization (according to M a c R a e and Langdon (1989) with some modifications).The degradation of tubulins and M A P s has not been considered in the figure, although degradatlve processes may also play a regulatory role.

of the comparative ultrastructural investigations of the MTs in young and old cells are thus rather disappointing (see for instance, Johnson, 1984). The development of immunocytochemical methods combined with the availability of antitubulin antibodies has brought some new insights. Interestingly, 3 teams working respectively on golden hamster fibroblasts (Raes et al., 1983), mouse fibroblasts (Van Gansen et al., 1984) and human skin fibroblasts (Wang and Gundersen, 1984) came to similar conclusions: in vitro aging is accompanied by the appearance of cells with increased size, low proliferative potential, modified but extended cytoskeleton and MT organization, and loss of cell polarity. Raes and Remacle (1983) called these cells type 2 cells, opposed to the type 1 cells, the predominant fibroblast-like cells in young cultures. In the type 2 cells, the MTs show a well developed stellate pattern, centered around the nucleus and radiating from the cell center (Fig. 4B). Some areas of the cytoplasm seem completely devoid of any MT, while others seem to concentrate the MTs into cytoplasmic channels. The MT-based movement of the cytoplasmic organelles could then be limited to those MT-rich channels, which is perhaps a way for these extremely large cells to face problems of communication between the nucleus and the cell periphery. It is interesting to point out that in all the investigated fibroblast strains, the MT cytoplasmic complex (MTCC) of the type 2 cells is represented by a rather well developed array of MTs, compared with the type 1 cells, which argues against a possible fragility of the MTCC with aging. Similar observations were made for the microfilaments (Raes et al., 1983; Wang and Gundersen, 1984) and for the intermediate filaments (Wang, 1985); the latter, according to Wang (1985), are stabilized into large bundles in aging fibroblasts via cross-bridges. Although the possible relationship between MT networks and proliferation control is still controversial and varies according to the cell type (Otto, 1987), an increased stability of the MTs is often associated with a hampering of cell division, while MT lability favors DNA synthesis. MT lability also seems crucial for organelle and chromosome movement (Coue et al., 1991). A stabilized and extended MT network in old cells could thus affect both

158

Fig. 4. Microtubules in young (A) and old (B) cultures of golden hamster embryonic fibroblasts. MTs were immunolabeled as described in Raes et al. (1983) and according to the method of De Brabander et al. (1977). The lifespan completed was 48%in the young and 95% in the old cultures. Young cultures are mainly characterized by typical fibroblast-like cells or type 1 cells. In these cells (A), the M T C C is formed by filaments radiating at the cell center from the perinuclear region towards the cell periphery. In old cultures (B), type 1 cells are scarce and large, apolar non-fibroblastic cells, type 2 cells, become the predominant phenotype. In the latter, the M T C C is well developed but delimits areas completely devoid of MTs; the cell center also seems less defined. Bar: 50 ~ m .

cell division and intracellular motility. However, cell shape and spreading, which indeed differ in type 1 and 2 cells, often determine the cytoskeleton rather than the reverse and this kind of investigation does not provide any explanation for the morphological modifications observed with cellular aging. Therefore a more physiological and dynamic

approach was undertaken, in order to put forward any possible qualitative difference affecting the tubulins or the MTs in aging cells. Young and old cultures were treated, for increasing periods of time, with various concentrations of nocodazole, a microtubule inhibitor inducing their depolymerization (Raes et al., 1983). At high concentrations and after long incubations, MTs were

Fig. 5. Microtubule depolymerization in type 1 (A) and type 2 (B) cells treated with 0.1 / x g / m l nocodazole for 1 h. In the type 1 cell, MTs depolymerize mainly centripetally, from the cell periphery to the cell center; a population of short MTs radiating from the cell center remains polymerized. In type 2 cells, a wave of centrifugal depolymerization clearly disorganizes the M T C C at the cell center even though some stable MTs remain at the cell center; surprisingly, n u m e r o u s stable MTs resist nocodazole treatment in the cortical area. Bar: 20/xm.

159 more or less completely depolymerized in both cell types. But milder conditions induced only a partial depolymerization and revealed unexpected differences. In type 1 ceils, the M T depolymerization was centripetal, starting from the cell edge and going to the nucleus. This is generally observed since the MTs are stabilized at their minus ends by embedding in the pericentriolar material of the centrosome (Fig. 5A). But in type 2 ceils, surprisingly, a centrifugal depolymerization wave was clearly observed, with some stable MT remnants remaining in the cortical area of the ceils (Fig. 5B). Hence, these experiments did not provide evidence for an increased susceptibility of the MTs to nocodazole in type 2 cells, but rather suggested anomalies in their organization. Van Gansen et al. (1984) performed similar depolymerization experiments on mouse fibroblasts aging in vitro, but observed no difference between young and old cells; however they used rather drastic conditions (colchicine instead of nocodazole, higher drug concentration, longer incubation time, etc.), which induced almost the complete depolymerization of the MTs in both cell types. Raes and Remacle (1987) also compared the behavior of aging hamster and human WI-38 fibroblasts towards mild nocodazole treatment. In old WI-38 cells, 20-25% of the type 2 cells displayed a clear centrifugal depolymerization of their MTs, but the majority (75-80%) had a mixed behavior with a depolymerizing wave starting from the periphery in some areas with a concomitant centrifugal depolymerization around the nucleus. In less than 1% of the type 2 ceils, polymerization was strictly centripetal, as for the type 1 cells. Hence, though alterations of the MT organization can indeed be shown with nocodazole treatment in both hamster and human cells, the phenomenon seems less clear-cut in the latter. As the effects of nocodazole are fully reversible, recovery experiments were performed in hamster cells completely devoid of any polymerized tubulin (Raes et al., 1984). This time, both cell types behaved similarly and after nucleation in the pericentriolar area, MTs clearly polymerized from the cell center to the cell periphery. Some unstable free MTs also appeared transiently in both cell types, all over the cytoplasm.

MTs thus have the same polarity in type 1 and 2 cells. Their dynamic properties also seem intact since they reacted similarly in the presence of taxol, a MT stabilizing drug (Raes et al., 1984). Then how to explain the centrifugal depolymerization after nocodazole treatment in type 2 ceils? Two explanations seem most likely: first, a decreased efficiency of the centrosome and second, a stabilization of the interactions between the MTs and the plasma membrane. The first hypothesis was investigated by an extended ultrastructural study of the centrosome in both cell types. The fundamental structure of the centrioles seemed unaltered in the type 2 cells, but the pericentriolar material generally appeared less dense (Raes et al., 1984). Although this is difficult to ascertain given the poorly defined structure of this materi,al, it would reinforce the idea of a slackening of its potential to stabilize the surrounding MTs. Moreover, when the cell reenters the interphase, the pericentriolar m a t e r i a l u n d e r g o e s similar m o r p h o l o g i c a l changes, with a decrease in the abundance of the electron-dense pericentriolar material. This reduction is accompanied by physiological changes, such as the decrease in the capacity to nucleate MTs as well as biochemical changes, for instance a lower activity of a centrosome-associated protein kinase (Cande, 1990; Huang, 1990). The ultrastructural study of the centrosome concomitantly with the high degree of polyploidy observed in type 2 cells led to the following question: could the hypothesized decrease in the organizing power of the centrosome explain the low division potential of the type 2 cells and their high polyploidy? In order to answer that question, time lapse sequence photographic studies were undertaken (Raes et al., 1984). The results can be summarized as follows: in both cell types mitosis was found to fail at some stage, although this event was seldom observed in type 1 cells: in type 1 cells, aborted mitosis led to binucleate ceils and in type 2 cells it resulted in giant multinucleate cells. These results are in agreement with the data of Wang and Gundersen (1984) on human skin fibroblasts. In conclusion, in hamster fibroblasts aging in vitro, MTs themselves seem unaltered at first glance but their stabilization by the centrosome could be lowered. During inter-

se, cells manage to maintain a well-developed 'CC. But in the presence of nocodazole or ing mitosis, where the balance between poly•ization and depolymerization of MTs has to :uned very sharply, troubles appear. Of course hypothesis, which integrates the behavior of a cell types in various conditions (Fig. 6), ds some further evidence at the molecular ',1. Symons (1988) investigated specifically the 'dynamics and chromosome movement during ,itro aging of fibroblasts: the average spindle ;th and the MT content were higher in aged s, but no difference was detected in spindle amics after colchicine treatment. These res do not contradict the hypothesis presented ve. The progress in the domain of the biomistry of the centrosome over the last years .,rs some new exciting prospects. Centrosomes n young and old cells could be purified and ir in vitro nucleating as well as organizing ential compared. Comparative immunological biochemical studies are now possible, at least ;ome extent. For instance, differences in the ~sphorylation of some of the pericentriolar tides would be worth looking for. But unforately our comprehension of the MT dynamics, motoring and the kinetochore function in the 9tic spindle remains incomplete, despite great ~rt and many publications in the domain. It is ,ossible and it is beyond the scope of this er to summarize the literature on M T dynamin the mitotic spindle. The reader is therefore ',rred to some of the recent reviews and papers the subject (see for instance, Mitchison, 1988; ~bsky and Borisy, 1989; Sluder, 1990; Sawin Mitchison, 1990, 1991; Cassimeris and non, 1991; Rieder, 1991; Vallee, 1990; etc.). s partial comprehension of M T dynamics in mitotic spindle does not facilitate the interration at the molecular level of the aborted 3ses observed in the aging hamster type 2 cells es et al., 1984). Belmont et al. (1990) recently wed some interesting cell cycle-dependent nges in M T dynamics: MTs are released from trosomes in both interphase and mitotic cells, more frequently in the latter, and according hese authors, this release may turn out to play important role in spindle dynamics. The cen~ome of aging hamster type 2 ceils could be

unable to tune exactly this release of MTs along the cell cycle. However the observations of Belmont et al. (1990) were made on a particular cell type, Xenopus eggs, and on cytoplasmic extracts of these cells. It remains to be proven that increased MT release from the centrosome is a general feature of all mitotic cells. A second hypothesis to explain the centrifugal depolymerization wave in type 2 cells, and in particular the cortical M T resistant remnants after nocodazole treatment, is increase a n d / o r stabilization of the interactions between MTs and the plasma membrane. This hypothesis has not been investigated at all. MTs could interact with the plasma m e m b r a n e through the MT-based motor proteins, kinesin and dynein. Evidence of these interactions came from subcellular localization, immunofluorescent studies and in vitro assays of vesicle motility (for a review, see Schroer, 1991). But to date, no quantitative study has been performed. Hence the possible implication of the motor proteins in a possible stabilization of the M T - p l a s m a m e m b r a n e interactions with aging awaits the further characterization of the molecular mechanisms involved. However, if we want to understand the MT organization and how it can be affected in aging cells, we must also approach the problem by biochemical and molecular analyses. As shown in Fig. 3, many open questions arise. Does the balance between different isoforms change with cell aging? What about the posttranslational modifications, such as tyrosination, known to stabilize the MTs or the phosphorylation of some of the MAPs? Posttranslational modifications, enzymatic or not, occur with aging (see for instance, Zappia et al., 1988; Harding et al., 1989) but have not yet been investigated in the peculiar domain of the MTs. Do the MT-based motor proteins keep their transducing properties unaltered? These questions, given the progress in tubulin genetics and microtubule biochemistry, should deserve some more attention from the cell biologists involved in cellular aging.

Microtubules and aging of the nert~ous system Normal aging of the brain has long been known to be associated with morphological and structural changes (for a review, see Schleibel and

161

TYPE 1Cr~.~ S

TYPE 2 CI:~.~-S A

B

C

D

~

E iq

Fig. 6. Comparative diagram summarizing the behavior of MTs in type 1 and 2 cells in hamster cells aging in vitro: in interphase cells (A), nocodazole-treated cells (B) and cells recovering after nocodazole treatment (C). Arrows show the main direction of depolymerization (B) or repolymerization (C). In the diploid type 1 cells, most of the mitoses are successfully achieved (D) and give rise to two diploid type 1 daughter cells (E); in a typical tetraploid type 2 cell, mitosis starts but aborts (D), giving rise to a giant octaploid cell (E); one of the hypotheses to explain this result in the type 2 cells is decreased control of the centrosome over the minus ends of the MTs located near it (see the arrows). The nucleus is not represented and the differences in cell size between the 2 cell types are disregarded. The centrosome (c) is surrounded by pericentriolar material (dotted area).

:ibel, 1981; Terry, 1981), but also functional fications (see for instance, Ordy, 1981; Katz1988; Ingram et al., 1988; Mattson, 1989). e are some indications that the cytoskeleton ,onents, and in particular the MTs and 's, are involved in some of the alterations iated with aging or with age-associated )logies of the brain.

plication of the MTs and MAPs in structural rtions of the brain fferent structural alterations have been de~d with aging, such as the reduction of denarborization, the atrophy of some neuronal ~odies, etc., but in this paper we will focus attention on neurofibrillary pathology, ein the MTs and some of their associated ituents have been more particularly impli• Affected neuronal cells display neurofibrilangles, composed of paired helical filaments :). Bundles of PHFs are also found in the 3phic neurites of the neuritic (senile) plaque, :ond age-related histological lesion. These ~tions are actually age-related and occur in ally all humans over time, but in some dissuch as the well-known age-linked senile eimer's dementia, the phenomenon seems to ~e all the existing control and regulatory anisms and turns into premature and acceld brain degeneration. The differences with ; are thus basically quantitative and topolic (Selkoe, 1986; Iqbal et al., 1986; Wis;ki et al., 1989). tFs represent only a part of the neurofibril)athology of the brain but many investigators tried to unravel their composition• Their temical analysis has been rather difficult be.. they are quite insoluble and available in •,d amounts. But thanks to numerous bioical and immunological investigations, tau, ff the main structural MAPs in the brain, has found to be a component of the Alzheimer's 3. However, as stressed by Wiche et al. (1991), aer the occurrence of tau in PHFs is a cause ther a consequence of the disease remains a ,~r of speculation. Le disease is also characterized by a neuritic ~phy that occurs in areas of the brain devoid urofibrillary tangles or senile plaques. There

is some evidence of a possible correlation between neuritic dystrophy and the occurrence of clinical dementia. Surprisingly, the dystrophic neurites can also be revealed with antibodies against tau. Therefore, as stated by Kosik (1990), tau reactivity is probably the single common denominator that most clearly defines the neurofibrillary pathology. Moreover, Alzheimer's tau behaves differently from its normal counterpart: it migrates less rapidly in acrylamide gels, is less soluble, etc. The molecular mechanisms leading to these changes are not yet completely unraveled, but 3 possible modifications are considered likely: alternative splicing (leading for instance to the expression of a juvenile isoform), ubiquitination and increased phosphorylation. In normal neurons, tau is enriched in the axonal compartment, but scarce in the somato-dendritic compartment (cell body and dendrites), although it is synthesized in the latter. According to Kosik (1990), this segregation in the axonal compartment, could involve a cycle of phosphorylation-dephosphorylation, the loss of phosphate favoring the binding of tau to the axonal MTs. In Alzheimer's neurons, excessive tau phosphorylation, among other modifications, has been described. There is at present no explanation for this observation, but abnormal phosphorylation could lead to aberrant tau functions, such as auto-assembly or co-assembly with as yet unidentified components into PHFs; abnormal sprouting from the somato-dendritic compartment into dystrophic dendrites is also possible. As a consequence, normal MTs are no longer stabilized by tau, they become scarce, which is typical of affected neurons, and MT-based motility, such as axonal transport, becomes impaired. According to Kosik (1990), Alzheimer's neurofibrillary lesions could be a disorder of neuronal polarity, with all the adverse consequences on the functions of the central nervous system. Moreover, recent data suggest that the MTs themselves are also involved (for a review, see Di Patre, 1991): anomalous interactions of /3-tubulin with guanosine triphosphate have been described; repolymerization experiments after colchicine treatment in fibroblasts from Alzheimer's patients show delayed MT reappearance; finally, decreased transcription of mRNAs coding for neu-

163 rofilament and tubulin proteins have been reported in the brains of these patients. MTs and some of their associated MAPs may thus account for some aspects of the disease. However, the cause of Alzheimer's disease remains unknown and the primary mechanisms leading to its appearance are still hypothetical (see for instance, Volicer and Crino, 1990; Bosman et al., 1991).

Implication of MTs and MAPs in the functional alteration of neuronal cells Aging of the nervous system is also associated with various functional alterations. Many hormonal and neurotransmitter responses decrease. This has been described in the aging human brain (Ordy, 1981; Roth, 1984; Scarpace and Abrass, 1988; Azmitia et al., 1988), but also in the brains of old animals, for instance in monkeys (Wenk et al., 1989). Do the MTs and the MAPs account for some of these age-related functional alterations? The hypothesis is of course attractive, but has so far been approached mainly from a theoretical point of view. Neuronal function and structure are directly correlated and their control is developmentally regulated: during development, the neuroarchitecture is achieved by processes of cell proliferation and migration, followed by neuronal differentiation and neurite outgrowth, and finally synaptogenesis with cessation of outgrowth. In the adult organism, the neuroarchitecture is not definitive and may change adaptively and undergo remodeling, with formation of new synapses and breaking or modification of old ones; in other words, the adult brain is characterized by a remarkable feature, its plasticity. The cytoskeleton and the MTs as well as the MAPs are involved in most of these phenomena as already pointed out above (Matus, 1988; Meininger and Binet, 1989; Black and Baas, 1989; Sargent, 1989; etc.). Finally, as a result of normal aging processes or specific age-related neuropathologies, neuritic arbors degenerate and cell loss occurs, at least in some parts of the brain. Mattson (1989) developed an interesting hypothesis: common cellular signaling mechanisms would control the neuroarchitecture and function from neuronal development to age-related neurodegeneration. Signaling systems mediate the neuronal responses

to the numerous neurotransmitters and neuronal growth factors, by modifying cellular targets, such as the components of the cytoskeleton, including the MTs and the MAPs. According to Mattson (1989), neurodegeneration in aging and disease results from an imbalance in growth factors and neurotransmitters a n d / o r in their corresponding intracellular signaling messengers; this imbalance will in turn affect cytoskeletal integrity, leading to neuritic degeneration and loss of axonal transport. Different second messengers could be involved: according to Gibson and Peterson (1987), Harris and DeLorenzo (1987) and Mattson (1989), calcium and some of the calcium/calmodulin-dependent kinases, such as calmodulin kinase II, are good candidates and may indeed play a significant role in specific cytoskeletal alterations of normal aging and selected neurodegenerative diseases. MTs and MAPs are prone to phosphorylation and are indeed modulated by calcium. A similar hypothesis has been put forward by Butcher and Woolf (1989) for Alzheimer's disease. According to these authors, the disease could be triggered by altered regulatory mechanisms governing the patterns of cytoskeletal protein expression in structurally plastic neurons of the mature nervous system. That signaling mechanisms play a role in neuroarchitecture and neuronal function has been substantiated by experimental data in vivo and in vitro (see for instance, Azmitia et al., 1988; Mattson, 1989). There is also increasing agreement on the modulation of cytoskeletal structure and function by signaling mechanisms: actin and microfilament dynamics are modulated by calcium and polyphosphoinositide turnover (Forsher, 1989; Rao and Cohen, 1990); tubulins, MTs and MAPs are the substrates for different kinases, although the actual regulation and role of these tubulin and MAP kinases in MT-dependent activities remain unclear (see for instance, MacRae and Langdon, 1989). There are some preliminary data suggesting that intracellular messengers and in particular calcium may control microfilaments and MTs in growth cone motility (Lankford et ai., 1990). The growth cone is the enlarged tip of a nerve fiber and is essential for the elongation of axons and dendrites. But the hypothesis of a direct correlation between unbalanced signaling

164 mechanisms and cytoskeletal dysfunction with aging has not yet been tested. Are there any experimental data suggesting an implication of MTs and MAPs in functional alterations of the brain? The available data are incredibly scarce. Matus and Green (1987) isolated MTs from the brain of very old rats; MAP1 and MAP2 were found only in a degraded form, this degradation happening mainly during the MT isolation procedure. Their study suggests an increased susceptibility with age of both MAPs to a brain cathepsin D-like protease. As MAP1 and MAP2 are known to promote MT assembly, their excessive degradation with age could be related to defective MT assembly, which is known to occur in age-linked degeneration conditions. More surprisingly, even neuronal functions where the role of MTs and MAPs is now widely accepted (Vallee and Bloom, 1991) have not been investigated thoroughly in aging neuronal cells. This is the case for fast axonal transport, a basic function of neuronal cells. Our knowledge of the molecular mechanisms involved in that kind of transport has improved impressively over the last years, with the latest discoveries on cytoplasmic dynein and kinesin. But all this new knowledge has unfortunately not yet been exploited in aging studies. In a preliminary study, Goemaere-Vanneste et al. (1988) followed the axonal transport of different molecular forms of acetylcholinesterase in the rat sciatic nerve during aging; only the rapid axonal transport of one molecular form seemed reduced with aging. Decrease of fast axonal transport has already been described in the past (as reviewed in Goemaere-Vanneste et al., 1988). But it is time now to unravel the molecular mechanisms involved in this decrease and to search for possible alterations of the MTs and MAPs, taking into account all the presently available data on the complex molecular machinery of fast axonal transport.

Prospects As clearly shown hereabove, the study of MTs and MAPs in the aging nervous system is still in its infancy. Several authors have developed hypothetical models where MTs and MAPs may play some role, but these models need to be substantiated by experimental data.

First, thanks to the development of genetic, immunologic and biochemical approaches, it has become possible to follow the expression of isotubulins and MAPs, as well as the levels of various posttranslational modifications in tubulins, MTs and MAPs. This has been done as a function of differentiation, development and maturation of the nervous system; variations have been put forward although their physiological significance is not always clear. It would be worthwhile continuing this kind of study in aging neuronal cells and brain. Secondly, new techniques have been developed to understand neuronal functions, such as fast axonal transport. This will be exemplified for in vitro and in vivo investigations. In vitro, some elegant variants of molecular cytochemistry have been designed: fluorescent tubulin is incorporated in the cells and can be followed dynamically as a function of time; the labeled cytoskeleton can also be photobleached in a narrow zone; the subsequent behavior of the fluorescent MTs and of the bleached areas yields information about the movement of polymerized tubulin (see for instance, Hollenbeck, 1990). Similar experiments can be performed by injecting unactivated nonfluorescent tubulin; this is locally activated by UV light and followed by low-light videomicroscopy (see for instance, Sawin and Mitchison, 1991). Surprisingly, only the latter appoach detected a poleward MT flux in mitotic spindles. In vivo, Nagahiro et al. (1990) have described a non-invasive autoradiographic method to measure axonal transport in rat brain. These approaches, amongst many others, could be applied to compare MT dynamics or axonal functions in young and old cells or animals. Finally, as stressed by Azmitia et al. (1988), neuronal tissue culture models have their drawbacks and limitations, but they provide a useful tool in studying the aging of the nervous central system and the temporal sequence of events leading to the degeneration and final death of the neuronal cells, associated with the aging process. Neuronal tissue culture models are particularly suitable for the investigation of MT and MAP organization and functions in aging cells. Experimental approaches could be designed to test the hypothesis of a correlation between unbalanced signaling systems and MT dysfunction

165 in aging neuronal cells, as developed by Mattson (1989).

Microtubules and aging in other systems When reviewing the literature on MT organization and aging, one particular cell type is often cited, the oocyte. Given the development of therapeutic techniques such as in vitro fertilization for the treatment of some kinds of infertility, oocytes and their MTs have been particularly investigated as a function of in vivo and in vitro aging. Oocytes are 'aged' in vitro by incubation for 24-48 h in suitable media. Several studies on human a n d / o r murine oocytes (Webb et al., 1986; Eichenlaub-Ritter et al., 1986, 1988; Picketing et al., 1988) have suggested an increased fragility of the cytoskeleton and in particular the MTs, as a function of both in vivo and in vitro aging, leading to high frequencies of aberrant oocytes. However, further work using larger numbers of oocytes is indispensable to confirm these preliminary studies. Conclusions and general prospects

Reviewing MT organization and function during cellular aging is a difficult task, and the data available at present, on possible age-related changes of MTs and cytoskeletal components in general, are rather limited. However, as stressed by several authors, the hypothesis of age-related alterations of these cellular components is attractive, because it offers a unified explanation for many functional age-related changes affecting different tissues and cells. Only the MTs were considered in this paper, but the MAPs and MTOCs were also taken into account, because all these cellular components function as a whole. There is some evidence of alterations in the organization and in the functions of MTs with cellular aging. The data generally do not favor any clear-cut increase in the fragility of the MTs; however, the organization and the control of MT dynamics seem to some extent impaired, specially in stressing conditions, such as drug treatments, or physiological processes that require an extreme fine-tuning of the MT polymerization and depolymerization events, such as mitosis. Unfortunately, given the corn-

plexity of M T dynamics, it is difficult to explain these alterations at the molecular level and to discover the identity of the cellular component responsible for them: is unpolymerized or polymerized tubulin involved? Or is it the MAPs or the MTOCs? On the one hand, any interpretation has so far been hampered by the fact that independently of the aging process - our knowledge and understanding of MT dynamics and functions are far from being complete, despite remarkable progress over the last years. On the other hand, this recent progress, mainly in the genetics and biochemistry of MTs, MAPs and MTOCs, as well as the development of techniques and analytical tools in the specific domain of MTs, has not been thoroughly applied in the cell biology of aging. Almost nothing is known about isotubulin isoforms or posttranslational modifications in MTs and MAPs with cellular aging. Tubulin mutants have recently been characterized, which offer new approaches for understanding MT organization and function (Noegel and Schleicher, 1991); such mutants may also give some clues for the interpretation of MT alteration in aging cells. Finally, the study of the interactions between signaling systems and the cytoskeleton offers exciting prospects in neuronal aging, but also in our understanding of the ageassociated decrease in the proliferation potential of dividing cells, such as the fibroblasts. The involvement of MTs in cell aging clearly needs to be substantiated by further experimental data. It is however more than likely that alterations in the MT organization and functions will indeed be described in the future, in different cell types and tissues and for different animals. This does not mean that MT alteration is the primary cause of aging, explaining all the events associated with aging. But MT alteration, together with the alterations of other cellular components (enzymes, DNA, organelles, etc.), could participate in a multistep aging process, as described by Toussaint et al. (1991): when a threshold of alteration is reached - whatever the affected cell component - the cell is doomed to degeneration and final death. In conclusion, MTs, but also microfilaments and intermediate filaments, are attractive targets for cell biologists involved in cellular aging, espe-

166

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