Neuronal polarity

Neuronal

polarity

Ann Marie Craig, Mark Jareb and Gary Banker University

of Virginia

School of Medicine,

Charlottesville,

Virginia,

USA

The axonal and somatodendritic domains of neurons differ in their cytoskeletal and membrane composition, complement of organelles, and capacity for macromolecular synthesis. Recently there has been progress in elucidating the cellular mechanisms that underlie the establishment and maintenance of neuronal polarity, including microtubule organization and the sorting, transport, and anchoring of membrane proteins.

Current Opinion

in Neurobiology

Introduction

1992,

tablishment neurons.

The polarity of neurons, which is integral to their function, is a result of the segregated distribution of molecules and organelles. The polarized distribution of cytoskeletal proteins-tau (axonal), MAP2 (somatodendritic); cell adhesion molecules-Ll, TAG1 (axonal); receptors and channels-Na+ channels (axonal), glycine, y-aminobutyric acid (GABA)*, and a-amino-3-hydroxy5methyl4isoxazole propionic acid (AMPA)-selective glutamate receptors (somatodendritic); and the growthassociated protein, GAP43 (axonal), serves to define the structural and physiological properties of axons and dendrites and their corresponding synaptic specializations. Other proteins, such as neurofilament subunits and MAPlb, are not segregated but are selectively phosphorylated in the axonal compartment. Ribosomes and mRNA are excluded from the axon, and within the somatodendritic domain different mRNA.s are differentially distributed. Microtubules are uniformly oriented with ( + ) ends distal in axons, but present in both polarity orientations in dendrites. What are the molecular mechanisms responsible for generating and maintaining this complex cellular orgamzation? This question has been addressed mainly using cell culture systems, where individual cells and their processes can be visualized, stained, and manipulated during development. Fetal rat hippocampal neurons in low density culture have been particularly well characterized with respect to both compartmentalization in mature cells and the development of neuronal polarity [ 11, During development, the cells initially extend four or five apparently equivalent ‘minor processes’. After a day or two in culture, one of the processes begins a phase of rapid growth, eventually becoming the cell’s axon. Later, at about 4 days in culture, the other processes mature into dendrites. A similar pattern of development appears to hold for some other types of neurons in culture, includ[ 21. This review will discuss ing cerebellar macroneurons the evidence for mechanisms that could underlie the es-

2:602+06

and maintenance

The development

of regional

of neuronal

differences

polarity

The primary determinants that govern neurite outgrowth and the differentiation of neurites into axons or dendrites are not known. The observations of Caceres and Kosik [2] in cerebellar neurons point to the selective stabilization of microtubules as of particular importance. Their work suggests that the microtubule-associated protein, tau, selectively binds to the microtubules in one of the minor processes, stabilizing them and allowing this process to grow rapidly and become the axon. In support of this hypothesis, Caceres et al. [3*] recently showed that the addition of tau antisense oligonucleotides to cerebellar neurons after 3 days in culture, when they had established an axon, resulted in retraction of the axon but continued growth of other processes. In previous papers [4,5] Caceres and colleagues reported that tau binds selectively to axonal microtubules, and that the developing axon, but not minor processes, stain with antibodies to acetylated and detyrosinated tubulin, post-translationally modified forms indicative of stable microtubules. Recent papers from two other laboratories using hippocampal or cortical cultures report that both minor processes and the developing axon stain for acetylated and detyrosinated tubulin [ 6*,7]. Perhaps quantitative fluorescence methods, such as that described by Keith [RI, might offer a more direct approach to resolve local differences in microtubule stability and their role in axonal differentiation. Other laboratories, including those of Higgins and Prochiantz, have emphasized that external factors, such as interactions with glial cells or with extracellular matrix molecules, can dramatically alfect the number, length, and identity of neuronal processes (for recent papers, see [9,10*,11-131). Even sensory neurons, which in situ lack dendrites, can develop dendrites and segregate marker

Abbreviations AMPA--a-amino-3-hydroxy-5-methyl+isoxazole

propionic

acid; CABA-y-aminobutyric

MAP-microtubule-associated

602

@

Current

Biology

in

protein.

Ltd ISSN 0959-4388

acid; GPI-glycosyl-phosphatidylinositol;

Neuronal polarity Craig, Jareb and Banker

proteins to axonal or dendritic domains conditions are manipulated appropriately Cytoskeletal

when [ 14.1.

culture

polarity

Two of the most obvious differences between axonal and dendritic cytoskeletons are their complement of microtubule-associated proteins (MAPS) and the orientation of their microtubules (reviewed in [ 15,161). A provocative report by Baas et al. [17**] raises the possibility that these two differences may be related, i.e. MAPS may affect microtubule polarity orientation. While it has long been known that MAPS promote microtubule stability and bundling, it is generally thought that microtubule-organizing centers determine polarity orientation. But when tau is overexpressed in insect Sf9 cells, which leads to the formation of long cellular processes [ 181, the dense arrays of microtubules in these processes are uniformly oriented ( + > end out, as in axons [ 179’1. Even in the cell body, microtubules are bundled with a uniform polarity orientation and do not appear to be as sociated with any observable nucleating structure. Could MAP2 induce bundles of ( - ) end out microtubules or microtubules with mixed polarity orientation, as occurs in dendrites? Clearly, tau is not the last MAP that Sf9 cells will see. Protein

sorting

One hypothesis that has driven a number of recent studies is that neurons and epithelial cells use common mechanisms to segregate proteins to polarized domains. The key observation that led to this hypothesis was made by Dotti et al. [19], who found that vesicular stomatitis virus G protein and fowl plague virus hemagluttinin, which in epithelial cells are sorted to the apical and basolateral domain, respectively, are sorted to the axonal and somatodendritic domains in cultured hippocampal neurons. If it is assumed that common mechanisms underlie the sorting, then the same amino acid sequences that in epithelia target proteins either to the apical or basolateral domains may target neuronal proteins to axons or dendrites. One approach to investigate this hypoth esis is to choose a protein of known polarity in one cell type, then transfect the gene encoding it into the other cell type and examine its distribution. Using this method, G Pietrini, YJ Suh, L Edelman G Rudnick and MJ Caplan (unpublished data) found that the GABA transporter, which in neurons is axonal, is expressed on the apical surface of transfected epithelial cells. Another approach is simply to compare the distribution of proteins common to both cell types. The transferrin receptor, a basolateral protein in epithelial cells, is polarized to the somatodendritic domain of cultured hippocampal neurons [20]. On the other hand, Pietrini et al. [21] showed that the Na+,K+ -ATPase, which is restricted to the basolateral surface of epithelial cells, is present in both axons and dendrites of cultured hippocampal neurons. This apparent exception to the sorting hypothesis may not be as serious as it seems. Recent studies have shown that the Na+ ,Kf -ATPase is delivered uniformly to both apical and basolateral domains in epithelial cells, but is

selectively stabilized in the basolateral domain [ 221. Parenthetically, this result shows that a polarized distribution need not imply polarized sorting. These data are consistent with the idea that neurons and epithelia sort proteins by similar mechanisms, but the paucity of examples is noteworthy. Yet another approach to test the hypothesis is to ask whether the specific signals that determine polarized protein sorting in epithelial cells are also recognized in neurons. The best known of these sorting signals is the glycosyl-phosphatidylinositol (GPI) anchor, a post-translational modification that links proteins to the plasma membrane and, in epithelial cells, directs them to the apical domain [ 231. Transfection experiments could be used to test directly whether the presence of a GPI anchor is alone sufficient to target proteins to the axon. While such experiments have not yet been performed, the distribution of several endogenous GPI-anchored neuronal proteins has been examined. Dotti et al. [24*] report that Thy-l is expressed axonally in cultured hippocampal neurons. Another GPI-anchored protein, the cell adhesion molecule TAG-1 (axonin-1), has previously been shown to be localized to axons [25-271. Other observations, however, appear to contradict this hypothesis. Earlier reports indicate that Thy-1 is present in both axons and dendrites of neurons in situ [28,29]. Morethat over, Faivre-Sarrailh et al. [30.*] have demonstrated F3/Fll, another GPI-linked protein, is axonal in cerebellar granule cells, but uniformly expressed in Golgi cells. The distribution of F3/Fll sheds doubt on the idea that the GPI anchor acts as a specific axonal targeting signal and hence on the ‘epithelial-neuronal sorting hypothesis’. In addition, it raises the puzzling question of how the segregation of a single molecule can be differentially controlled in different neuronal cell types. Polarized

transport

and anchoring

of proteins

and organelles One of the possible cues for directional transport in nerve processes is microtubule polarity orientation. Ax ons contain ( + > end distal microtubules, whereas dendrites contain microtubules of both polarity orientations, with the percentage of ( - ) end distal microtubules decreasing with distance from the cell body. It has been suggested that certain organelles move preferentially to ward one end of microtubules: secretory vesicles towards the ( + ) end, which would bias their transport to the axonal domain, and ribosomes and Golgi elements towards the ( - ) end, which would effectively exclude them from axons [ 151. This directional movement could presumably occur if organelles associate with different microtubulebased motors. Parton et al. [ 31.1 have shown that labeled endosomes move bi-directionally in proximal dendrites, but only toward the cell body in distal dendrites or axons, consistent with the idea that endosomes are transported to the ( - ) end of microtubules. Late endosomes, which are normally restricted to the cell body, enter axons and minor processes under conditions that lower intracellular pH, a treatment that is thought to enhance ( + > end over ( - ) end directed motor activity [32]. This result also drives home the point that the subcellular localiza-

603

604

Neuronal and glial cell biology

tion of organelles, even those conlined to the cell body, may well reflect dynamic rather than static processes. While there is no direct evidence that polarized protein transport depends on microtubule polarity orientation, it is reasonable to ask whether there is a correlation between the appearance of differentially oriented microtubules and the segregation of specific proteins. In cultured hippocampal neurons, all processes initially contain only ( + ) end distal microtubules, with ( - ) end distal microtubules appearing in dendrites at about the same time they begin to show a characteristic dendritic morphology [33]. Killisch et al. [34] have shown that GABA* receptor subtypes are initially present in both minor processes and the nascent axon of cultured hippocampal neurons, but become selectively localized to dendrites in mature neurons. A similar ontogeny has also been shown for the dendritic proteins MAP2 and vesicular stomatitis virus G protein, and for the axonal proteins Thy-l and fowl plague virus hemagglutinin. In contrast, the synaptic vesicle proteins synapsin I and synaptophysin, as well as GAP43, are preferentially sorted to axons as soon as an axon can be distinguished by its greater length [35], even though all processes at this stage contain only ( + > end distal microtubules. Ribosomes are also excluded from axons at this stage of development (J Deitch and G Banker, unpublished data). These observations are consistent with the idea that microtubule polarity orientation may be important for the polarized transport of some neuronal proteins, but different mechanisms may be required to explain the polarized localization of other proteins early in development. Another question that must be considered when studying intracellular transport is the role of diffusion. In the first quantitative study of macromolecular diffusion along nerve processes, Popov and Poo [36*] showed that, as expected, the diffusion of labeled dextrans is strongly size dependent. Unexpectedly, even the largest dextran (radius of gyration = 70 nm) exhibited diffusion. Prevous work in other cell types had suggested that particles the size of ribosomes or larger were prevented from diffusing. The ability of dextrans to diffuse freely between the cell body and the axon, as reported by Popov and Poo [36*], raises once again the problematic question of how constituents such as ribosomes and mRNA are excluded from the axon. Although there are few direct data in neurons, the answer may be that ribosomes and mRNAs are anchored to the cytoskeleton, as reported for other cell types [37,38]. It is generally presumed that the restricted distribution of membrane proteins is due to anchoring to the submembranous cytoskeleton. Novel optical methods are offering a more direct means of studying this phenomenon. For example, antibody-coated gold particles, which can be visualized using video-enhanced differential interference contrast microscopy, have been used to demonstrate the active transport and trapping of membrane proteins at the edge of growth cones [39]. It seems safe to predict that laser optical tweezers, which have been used to demonstrate barriers to lateral movement of membrane glycoproteins in non-neuronal cells [40], will also soon be applied to study this problem in neurons.

Spectrins and ankyrins are presumed to form the link between membrane proteins and cytoskeletal components (reviewed in [41]). Different spectrin and ankyrin isoforms are differentially distributed in neurons [42*,43]. By comparing specific antibody staining in normal and ankyrinR,-deficient mutant mice, Kordeli and Bennett [42*] have clearly demonstrated that ankyrinRo is selectively associated with neuronal cell bodies and dendrites, and that an isoform distinct from both ankyr&, and ankyrinB is present at nodes of Ranvier. Once the neuronal forms of these proteins have been more fully characterized, it should be possible to determine whether the localization of specific ankyrin and spectrin isoforms determines the localization of specific membrane proteins or vice versa.

Local synthesis

of proteins

and lipids

Another mechanism that could contribute to the establishment and maintenance of regional differences in neurons is local macromolecular synthesis. With regard to protein synthesis, axons appear to lack the necessary machinery. In contrast, dendrites contain abundant polyribosomes, although until recently there had been no direct evidence that mRNAs were actually translated in dendrites. Using a novel technique that permits the axons and dendrites of cultured hippocampal neurons to be harvested free of contamination by cell bodies, Torre and Steward [44*] have now shown that isolated dendrites incorporate labeled amino acids into proteins. A series of in situ hybridization studies over the past few years have helped to identify mRNAs that might be translated in dendrites (for a review, see [45] ). They indicate that most mRNAs are largely excluded from dendrites; only a select few-including those that encode MAP2 and the cl-subunit of Ca*+/calmodulin-dependent protein kinase-extend far into the dendritic tree. The local translation of these messages could in part determine the localization of the proteins they encode. In addition to the mRNAs in dendrites, BCl, a small untranslated RNA, which apparently associates with proteins to form a ribonucleoprotein particle, is also concentrated in dendrites [46,47]. It is presumed that this RNA plays some role in dendritic protein synthesis. Membrane lipids can also be synthesized within neuronal processes, as Vance et al. [48-l have recently demonstrated. By growing sympathetic neurons in Campenot chambers (which the authors modestly refer to as compartmented cultures), they were able to assess lipid synthesis independently in either cell bodies and proximal axons or in distal axons alone. When appropriate precursors were added to the axonal compartment, they were incorporated into phosphatidyl choline, phosphatidyl ethanolamine, and sphingomyelin. In contrast, incorporation of labeled amino acids into proteins was detected only in the cell body compartment. These results suggest that some of the membrane lipids required for neurite elongation may be synthesized within the axon, and offer a possible mechanism that could serve to generate differences in lipid composition of the cell surface in different regions of the cell.

Neuronal polarity Craig, lareb and Banker

Conclusion Progress continues in defining the cellular mechanisms responsible for the establishment of cytoskeletal polarity and for the sorting, transport, and anchoring of membrane proteins in neurons. New evidence raises the possibility that MAPS may influence microtubule polarity orientation in neuronal processes, and that this in turn may control directed transport of organelles. The hypothesis that neurons and epithelial cells use common mechanisms to sort proteins remains tantalizing, but has yet to be conclusively tested.

Acknowledgements We would like to thank our colleagues H Asmussen, D Benson, T Esch, R Kleiman, M Martinic, G Ruthel and 0 Steward, for many helpful discussions. We also thank those investigators who allowed us to discuss their results before publication. Our research on neuronal polarity is supported by NIH grants NS17112 and NS23092; AM Craig was supported by a fellowship from the MRC of Canada, and M Jareb by NIH training grant HD07323.

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AM Craig, M Jareb and G Banker, Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.