Gangliosides as Modulators of Dendritogenesis in Normal and Storage Disease-affected Pyramidal Neurons

Steven U. Walkley, Mark Zervas and Samson Wiseman

Department of Neuroscience, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY 10461, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Pyramidal cells initiate the formation of dendritic arbors in a prolific burst of neurite outgrowth during early cortical development. Although morphologically mature pyramidal neurons do not normally sprout additional primary dendrites, the discovery of ectopic dendritogenesis in neuronal storage diseases has revealed that these cells do retain this ability under appropriate stimulation. The capacity for renewal of dendritogenesis has been found to exhibit a species gradient with human > cat, dog, sheep > mouse. A consistent metabolic feature of ectopic dendrite-bearing pyramidal neurons is a heightened intracellular expression of GM2 ganglioside. Elevated expression of this same glycosphingolipid has also been found to correlate with normal dendritogenesis. Immature neurons in developing cat and ferret cortex exhibit high levels of GM2 ganglioside immunoreactivity coincident with normal dendritic sprouting and a similar relationship has now been shown for human cortical development. Ultrastructural studies of all three species revealed GM2 localized to vesicles in a manner consistent with Golgi synthesis and exocytic trafficking to the somatic–dendritic plasmalemma. We propose that GM2 ganglioside functions in glycosphingolipid-enriched microdomains (lipid rafts) in the plasmalemma to promote dendritic initiation through modulation of specific membrane proteins and/or their associated second messenger cascades.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
During development of the cerebral cortex postmitotic neurons migrate to their final location within the maturing cortical plate and undergo a dramatic burst of dendrite outgrowth. Primary dendrites sprout prolifically from neuronal perikarya and from proximal apical processes, and within days to weeks, depending on the species, individual neurons form characteristic dendritic arbors that persist for the lifetime of the cell. Although some distal dendritic branches may continue to slowly elongate (Buell and Coleman, 1979Go), there is no evidence for sprouting of new, primary dendrites in normal mature pyramidal neurons after the initial period of dendritic differentiation (Marin-Padilla, 1992Go). There is, however, one family of diseases — the neuronal storage disorders — in which cortical pyramidal cells and a few other select cell types do exhibit a renewal of primary dendrito- genesis. New dendritic membrane is produced principally at the axon hillock, emerging either as a single dendritic spine-covered enlargement (meganeurite) or as a prolific tuft of new dendritic neurites (Purpura and Suzuki, 1976Go) [reviewed by Walkley (Walkley, 1998Go)]. Over time ectopic dendrites become richly invested with spines and synapses, and thus resemble normal adjacent basilar dendrites. In spite of the ectopic location (at the axon hillock or meganeurite), these new dendrites appear to be an integral part of the overall dendritic arbor since experimental reversal of the storage process does not necessarily lead to their disappearance (Walkley et al., 1987Go; Walkley, 1998Go).

A central question in the study of ectopic dendritogenesis in storage diseases has been whether there are common mech- anisms underlying this disease-associated phenomenon and the sprouting of primary dendrites in normal developing brain. Studies examining cortical neurons undergoing normal and ectopic dendritic sprouting indicate that elevated expression of one specific glycosphingolipid — GM2 ganglioside — links these two events [reviewed by Walkley et al. (Walkley et al., 1995Go)]. This particular ganglioside has been found elevated in all neuronal storage diseases with ectopic dendritogenesis whereas normal mature neurons and neurons in storage diseases not characterized by ectopic dendritogenesis do not exhibit significant expression of GM2 ganglioside. Ectopic dendrito- genesis has been found to be most abundant in GM2 ganglio- sidosis, a disorder in which GM2 elevation is the direct result of a catabolic defect (Siegel and Walkley, 1994Go). There is also evidence that intraneuronal elevation of GM2 ganglioside pre- cedes the outgrowth of ectopic dendrites in storage diseases (Goodman et al., 1991Go). These findings are consistent with GM2 elevation being a cause, rather than a consequence, of new dendritogenesis.

In examining normal developing neurons in the same species (domesticated cat) used to document the correlation between GM2 ganglioside and ectopic dendritogenesis, it was found that a heightened expression of GM2 again correlated with dendrite outgrowth (Goodman and Walkley, 1996Go). That is, postmigrat- ory cortical neurons expressed GM2 ganglioside coincident with normal dendritic sprouting and, after dendritic maturation was complete in the early postnatal period, GM2 levels dropped to negligible amounts. Subsequent studies in the ferret documented not only the same correlation in this species but also showed that no other ganglioside had a similar pattern of expression (Zervas and Walkley, 1999aGo). In the present study we compare these findings with an examination of GM2 expression in developing human cerebral cortex during normal dendritogenesis, and with an evaluation of murine models of neuronal storage diseases for GM2 expression and ectopic dendrite growth.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Normal human cerebral cortex at 16, 17, 18, 22 and 23 weeks of gestation was obtained from the AECOM Human Fetal Tissue Repository through a tissue banking protocol approved by the Committee on Clinical Investigation of the Albert Einstein College of Medicine and the City of New York Health and Hospitals Corporation. Gestational age was determined by multiple parameters including the date of the last menstrual period, uterine size, ultrasonography and fetal foot length (Hern, 1984Go). Tissues were fixed in 4% paraformaldehyde for a minimum of 24 h, followed by storage in 0.1 M phosphate buffer (PB) at 4°C.

Brain tissue also was obtained from three murine models of GM2 gangliosidosis: Tay-Sachs disease or {alpha}-subunit knockout (Yamanaka et al., 1994Go), Sandhoff disease or ß-subunit knockout (Sango et al., 1995Go) and a combined {alpha}–ß double knockout (Sango et al., 1996Go). A murine model of Niemann-Pick disease type A (Horinouchi et al., 1995Go) also was evaluated by Golgi and immunocytochemical methods. This disorder in humans is known to exhibit secondary elevation of gangliosides (Kamoshita et al., 1969Go) and a feline model of this disease has been found to exhibit ectopic dendritogenesis (Walkley and Baker, 1984Go). Access to the mouse models of storage diseases was generously provided by Drs R. Proia, E. Schuchmann and K. Suzuki. Animals were deeply anesthetized with pentobarbital and tissues fixed by intracardiac perfusion with 4% paraformaldehyde, followed by 24 h of immersion fixation and sub- sequent storage at 4°C in PB.

Rapid Golgi staining was carried out on human and murine tissues according to published methods (Zervas and Walkley, 1999aGo). Immuno- cytochemical studies used an antibody to GM2 ganglioside (generously provided by P. Livingston) and peroxidase labeling carried out on 35 µm Vibratome sections according to previously published methods (Walkley, 1995Go; Zervas and Walkley, 1999aGo). Results of Golgi and immunocyto- chemical staining on normal human brain were compared with earlier studies on cortical development in the cat (Goodman and Walkley, 1996Go) and the ferret (Zervas and Walkley, 1999aGo). Data from murine models of storage diseases were compared with our earlier studies on storage diseases in animal models [reviewed by Walkley (Walkley, 1998Go)] and with archival material kindly provided by Drs D. Purpura and K. Suzuki.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Cerebral Cortical Development

Nissl staining of human cortical tissues at 16 weeks of gestation revealed the presence of specific cell layers consistent with previous studies at this stage of development in the human brain (Honig et al., 1996Go; Marin-Padilla, 1970Go, 1992Go). The undiffer- entiated cortical plate (uCP), a compact layer of undifferentiated neurons, was found immediately beneath the superficial and cell-sparse marginal zone (MZ). Immediately below the uCP was a 300-µm-wide band of differentiating cortical plate (CP) neurons representing presumptive layers V–VI (Figure 1AGo). Golgi staining of this area revealed the presence of bipolar neurons, many of which exhibited small neuritic sprouts from perikarya and proximal portions of the apical and basilar processes (Figure 1BGo). Below this layer of differentiating CP neurons was the subplate (SP), a broad band of cells ~800 µm wide. The SP appeared somewhat denser in its upper half and consisted of vertically elongated cells (presumptive migratory neurons) interspersed with other cells of more rounded and/or nonvertical orientation. Myelinated axons were seen traversing the area beneath the SP and above the dense palisades of cells of the ventricular zone (VZ). Immunocytochemical staining for GM2 ganglioside revealed fine punctate structures in cells immediately beneath the uCP and more conspicuous immunoreactive punctae in presumptive layers V/VI just above the SP (Figure 1CGo). The SP, as well as the MZ, revealed occasional GM2-immunoreactive cells which appeared principally rounded and with multiple processes.



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Figure 1.  Human cortical development at 16–23 weeks of gestation. (A) Illustration of the distribution of GM2 immunoreactivity (red stipples) in Nissl-stained developing cerebral cortex at 16, 17, 18, 22 and 23 weeks. (MZ, marginal zone; uCP, undifferentiated cortical plate; CP, differentiating cortical plate; SP, subplate; WM, white matter; VZ, ventricular zone.) (B) Photomicrographs of Golgi-impregnated neurons as seen in the differentiating CP at each age. (C) Photomicrographs of GM2 immunoreactivity within the CP at each age (arrows indicate labeled punctae within neurons). Calibration bar in C at 23 weeks = 20 µm and applies to all the photomicrographs in B and C.

 
Changes in the laminar pattern of the cerebral cortex observed between 17 and 23 weeks of gestation were again consistent with published studies. The uCP appeared as a 100- µm-wide band of compact cells just beneath the MZ while the differentiated portion of the CP expanded in size from ~500 µm at 17 weeks to 1000 µm by 23 weeks (Figure 1AGo). These changes, coupled with similar increases in the thickness of the SP, resulted in dramatic expansion of the cerebral cortex. Golgi staining of the differentiating CP over this time revealed a persistent increase in the dendritic complexity of neurons (Figure 1BGo). Neurons changed from a bipolar morphology similar to those observed at 16 weeks to cells of recognizable pyramidal morph- ology with prominent apical and basilar dendritic arbors by 23 weeks of gestation. GM2 immunoreactivity in CP neurons exhibited a punctate pattern similar to that at 16 weeks, with the number of labeled neurons increasing as the thickness of the differentiated portion of the CP expanded (Figure 1A,CGo). By 23 weeks, neurons of layers V and VI were intensely labeled while neurons lying between these cells and the unstained uCP were more finely stippled with GM2-positive punctae. Ultra- structural studies of neurons exhibiting GM2 labeling revealed that the immunoreactivity was confined to vesicular structures near the Golgi apparatus, in the cytoplasm of perikarya and prox- imal dendrites, and occasionally near the plasmalemma (Figure 2Go). A few cells within the MZ and SP also were GM2-labelled, as had been observed at 16 weeks. Some cells in these regions at later time-points exhibited glial morphology whereas others (scattered in the SP) appeared as neurons with widely ramifying dendrites.



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Figure 2.  An electron micrograph of a GM2-immunoreacted human CP neuron (N, nucleus) at 23 weeks gestation showing the presence of GM2-immunoreactive vesicles (dark granular stain, arrows) at the Golgi apparatus (G) and near the plasmalemma (P). Calibration bar = 0.5 µm.

 
Previously reported studies of ganglioside expression in the cat and ferret cerebral cortex are fully consistent with those reported above for the human cortex (Goodman and Walkley, 1996Go; Zervas and Walkley, 1999aGo) (Figure 3Go). GM2 immuno- reactivity in these two species was found in layer V–VI neurons during early cortical development, with this staining spreading into the supragranular layers as they formed. Dendritic differ- entiation was found to begin during late gestation and to reach completion in the early postnatal period (4–6 weeks of age). Thus, GM2 immunoreactivity appeared in neurons at the time dendritogenesis began and then diminished coincident with dendritic arbor maturation. The cerebral cortex of adult cats and ferrets had GM2 staining limited to rare punctae in occasional neurons and somewhat greater staining in glia. In addition to GM2 labeling of cortical neurons in an inside-out pattern characteristic of cortical development, occasional neurons in the MZ and SP of both the cat and ferret also demonstrated GM2 immunoreactivity in a manner similar to that described here in human tissue. The GM2-immunoreactive SP neurons exhibited long radiating spine-covered dendrites which disappeared over the first few postnatal weeks (Zervas and Walkley, 1999aGo) (L. Goodman et al., unpublished data).



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Figure 3.  Photomicrographs of ferret and cat cortical development. (A) GM2-immunoreactive neurons (arrows) and (B) Golgi-impregnated layer II/III CP neurons in a 21-day-old ferret. (C) GM2-immunoreactive neurons (arrows) and (D) Golgi-impregnated layer V CP neurons in a 1-day-old cat. Calibration bar in D = 40 µm and applies to all parts of the figure.

 
Neuronal Storage Diseases

Golgi staining in mouse models of GM2 ganglioside storage diseases (Tay-Sachs, Sandhoff and double knockouts) and in Niemann-Pick disease type A revealed occasional swollen axon hillocks and small aspiny meganeurites, but most neurons appeared essentially normal in somatic–dendritic morphology (Figure 4GoA–C). At most, an occasional short spine-like protrusion was observed at the axon hillock region of mouse pyramidal neurons. The absence of ectopic dendrite growth did not seem to be due to longevity of the disease state since cats living to ages equivalent to those of mice with the same disease (e.g. Sandhoff and Niemann-Pick type A diseases) exhibited significant numbers of axon hillock neurites. There was also no absence of GM2 immunostaining in cortical neurons as each of the mouse models examined in this study exhibited conspicuous elevations of this ganglioside in pyramidal neuron perikarya (Figure 4GoD–F). In contrast to these findings, cortical pyramidal neurons in human storage diseases (Purpura and Suzuki, 1976Go) and in non-rodent animal models [reviewed by Walkley (Walkley, 1998Go)] exhibited prolific dendrite growth (Figure 5Go). Dendritic neurites emerged from axon hillocks and/or from meganeurites and, particularly in human diseases, these processes often possessed spines and resembled the adjacent normal basilar dendrites. The presence of ectopic dendrites in storage diseases in humans and non-rodent animal models correlated closely with an accumulation of GM2 ganglioside. In contrast, normal mature neurons in all species revealed little or no GM2 immuno- reactivity (Siegel and Walkley, 1994Go; Walkley, 1995Go).



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Figure 4.  Photomicrographs of Golgi impregnations and GM2-immunostaining of a 10-week-old normal mouse (A,E) and mouse models of neuronal storage diseases: (B,F) Tay-Sachs disease (20 months); (C,G) Sandhoff disease (4 months); (D,H) Niemann-Pick disease type A (6 months). The normal adult mouse shows normal cortical pyramidal neuron morphology (A) and a lack of GM2 immunoreactivity in neurons (E). All of the murine disease models show normal pyramidal neuron morphology (B–D) and abundant GM2 immunoreactivity (F–H). Calibration bar in H = 15 µm and applies to all parts of the figure.

 


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Figure 5.  Ectopic dendrite growth in human GM2 gangliosidosis (AB variant) at 3.5 years (A–C), canine GM2 gangliosidosis at 2 years (D), feline Niemann-Pick disease type A at 7 months (E) and ovine GM1 gangliosidosis at 2 years (F). The inset in A shows a close-up of a meganeurite (B). The inset in B shows a close-up of a spine-covered ectopic dendrite (C). Arrows in D–F indicate axon hillock neuritic tufts. Calibration bar in F = 12 µm and applies to A and D–F.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Ectopic dendritogenesis was originally discovered in a case of GM2 gangliosidosis, AB variant, and subsequently demonstrated in other types of neuronal storage diseases affecting children (Purpura and Suzuki, 1976Go; Purpura, 1978Go). Later studies revealed dendritic sprouting on cortical pyramidal neurons in feline, canine and ovine models of a wide variety of genetic storage diseases ranging from the gangliosidoses to {alpha}-manno- sidosis [reviewed by Walkley (Walkley, 1998Go)]. These studies showed that only a few types of neurons (e.g. cortical pyramidal neurons) have the capacity to sprout ectopic dendrites and that the same types are consistently involved in different diseases. In addition to cell type differences, there also appear to be species differences in the capacity for renewal of dendrito- genesis. Ectopic dendrites on cortical pyramidal neurons in human storage diseases are generally more numerous and longer than those seen in those that affect cats or dogs (e.g. compare A–C with D–F in Figure 3Go). Additionally, as reported here, pyramidal neurons in murine models of GM2 gangliosidosis and Niemann-Pick disease type A revealed no evidence of ectopic dendrites even though these diseases in humans and cats exhib- ited the phenomenon. This finding is consistent with studies of a wide array of murine storage diseases, including GM1 ganglio- sidosis, various mucopolysaccharidoses and Niemann-Pick type C, all of which lacked evidence for significant disease-associated ectopic dendritic sprouting (Zervas and Walkley, 1999bGo). Thus there appears to be an overall species gradient in the potential for renewed dendritogenesis after cortical maturation, with human > cat, dog, sheep > mouse.

Studies of human cortical development reported here provide additional evidence in support of the link between GM2 ganglioside expression and dendritic sprouting. The presence of robust GM2 immunoreactivity in human cortical neurons during dendritic differentiation was remarkably similar to that reported in the cat and ferret. For these species, GM2 expression was first observed at the time of initial dendrite outgrowth on lower CP pyramidal neurons, spread into more superficial layers as they formed and then diminished as dendritic arbors became fully established in the early postnatal period (Goodman and Walkley, 1996Go; Zervas and Walkley, 1999aGo). Thus, for human and other non-rodent brain tissue, during both normal development and in storage diseases, the correlation between GM2 ganglioside expression and dendritogenesis is remarkably consistent. The relationship between gangliosides and dendritogenesis in the rodent brain, however, will require additional study. Rodent cortical neurons in culture have been shown to transiently express GM2 ganglioside during dendrite outgrowth, but appear to exhibit less GM2 immunoreactivity than ferret cultures under similar conditions (K. Dobrenis et al., unpublished data). Similarly, high-performance thin-layer chromatography analysis has revealed a lower level of expression of gangliosides in the mature rodent brain, based on both dry weight and cell number, in comparison with the human brain (Hess et al., 1976Go). Absence of demonstrable ectopic dendrite growth in murine storage disease models suggests possible significant differences in post- natal mechanisms controlling dendritic plasticity, or alternatively, in the role of gangliosides in normal dendritic differentiation in this species.

The consistent correlation between intraneuronal elevation of GM2 ganglioside and outgrowth of primary dendrites on cortical pyramidal neurons in humans and other non-rodent species raises the question of how a glycosphingolipid might function to influence dendritogenesis. Gangliosides are sialic-acid-contain- ing glycolipids found in many cell types and organs but enriched in brain. In spite of considerable investigation, the function of gangliosides is not well understood. Early studies focused on these molecules as possible receptors, but the current prevailing view is that they more likely act as modulators of receptors and/ or their second messenger cascades (Hakomori, 1990Go, 1995Go; Zeller and Marchase, 1992Go; Yates and Rampersaud, 1998Go). Fluctuation in expression of individual types of gangliosides and/or their synthetic enzymes during brain development has been cited as evidence of their importance during this period (Tetamanti, 1971Go; Vanier et al., 1971Go; Yu et al., 1988Go; Skaper et al., 1989Go; Yamamoto et al., 1996Go). Although GM2 ganglioside has rarely been part of these studies, it has been described as an oncofetal antigen since it was found to occur in certain tumors and in human fetal brain with peak expression at 22 weeks gestation (Irie et al., 1976Go; Tai et al., 1983Go).

Gangliosides are known to be synthesized sequentially in the Golgi/trans-Golgi network (TGN) by a series of membrane- bound glycosyltransferases, and individual gangliosides are believed to be transported to the plasmalemma by exocytic vesicles where they are inserted into its outer leaflet (Yusuf et al., 1984Go; Sandhoff and Schwarzmann, 1989Go) (Figure 6AGo). Little is currently known about the control of ganglioside synthesis, the mechanisms of differential transport of gangliosides away from the Golgi or the process of somatic–dendritic exocytosis. Consistent with the proposed scheme for ganglioside synthesis and trafficking, ultrastructural studies of immature human cor- tical neurons, as well as of those of the cat and ferret, show the presence of GM2-immunoreactive vesicles in close association with the Golgi/TGN, within the somatic–dendritic cytoplasm and near the plasmalemma. GM2-laden vesicles were not found to be present in axons of any of the species investigated. Whether GM2-labelled vesicles within neuronal perikarya con- tain other molecules destined for the cell surface is unknown. However, recent studies have suggested that calcium-evoked exocytic activity in dendrites of hippocampal neurons plays a role in dendritic plasticity through delivery of postsynaptic membrane and plasmalemmal proteins (Maletic-Savatic and Koothan, 1998; Maletic-Savatic and Malinow, 1998Go).



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Figure 6.  Illustration showing (A) the proposed scheme for synthesis and trafficking of GM2 ganglioside and (B) the possible ways in which GM2 ganglioside might act to influence dendritic initiation. (A) Immature cortical neuron showing synthesis, trafficking and recycling of GM2 ganglioside. (1) GM2 ganglioside is believed to be synthesized in the Golgi/TGN and subsequently trafficked via exocytic vesicles to the external leaflet of the somatic–dendritic plasmalemma. (2) Removal of GM2 ganglioside from the plasmalemma likely occurs through endocytosis and transport within the endosomal–lysosomal system. (3) GM2 may exit endosomes or lysosome and directly recycle to the Golgi apparatus or, alternatively, be degraded in the lysosome. (B) Patch of somatic–dendritic plasmalemma showing possible interactions of GM2 ganglioside with other membrane constituents. (1–2) GM2 may be associated with membrane rafts that act as platforms for attachment of GPI-anchored proteins, small GTPases (e.g. Rho-A) and related signal transduction molecules, all of which have been implicated in dendritic initiation. (3) GM2 may modulate dimerization, autophosphorylation and/or downstream signaling of specific receptor tyrosine kinases, some of which (e.g. TrkB) have been implicated in dendritic initiation. See text for details.

 
Following delivery to the plasmalemma, gangliosides are believed to be eventually recycled by way of an endocytic pathway to lysosomes (Sandhoff and Kolter, 1996Go) (Figure 6AGo). Here acid glycohydrolases catabolize gangliosides and related membrane components for subsequent recycling. Failure to degrade GM2 ganglioside due to the absence of functional ß-hexosaminidase leads to GM2 gangliosidosis (e.g. Tay-Sachs and Sandhoff diseases). GM2 ganglioside is also elevated in other types of storage diseases (mucopolysaccharidoses, Niemann- Pick types A and C, etc.), possibly due to secondarily induced degradative defects and/or to alterations in the ganglioside synthetic pathway (Walkley, 1998Go).

Although ganglioside function remains largely enigmatic, studies in recent years offer possible insights into how they might affect signaling across cell membranes. Implicated in these studies are possible associations between gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins, signal transduction molecules and growth factor receptors (Figure 6BGo). Gangliosides and other glycosphingolipids, in association with cholesterol, are believed to form lipid microdomains or rafts in the plasmalemma and to act as platforms for the attachment of proteins involved in signal transduction (Simons, 1997; Brown and London, 1998Go; Jacobson and Dietrich, 1999Go; Prinetti et al., 1999Go). Studies have shown that signaling molecules implicated in dendritogenesis, including GPI-anchored proteins, tyrosine kinases of the Src family and receptor tyrosine kinases (Trks), may be associated with these raft structures (Wu et al., 1997Go) [reviewed by Brown and London (Brown and London, 1998Go)]. As neurons mature and ganglioside expression patterns change during development, it is likely that the ganglioside constituents of lipid rafts would also change, leading to developmentally linked differences in raft function.

Transport and sorting of raft-associated GPI-anchored proteins from the Golgi/TGN to the plasmalemma may be dependent on an interaction with glycosphingolipids [reviewed by Futerman (Futerman, 1995Go)]. Evidence for such coupling is based on altered transport of GPI-anchored proteins secondary to inhibition of GSL synthesis. One type of GPI-anchored protein in neurons, CPG15, has recently been shown to be associated with dendritic sprouting (Nedivi et al., 1998Go; Corriveau et al., 1999Go). A product of candidate plasticity gene 15 (cpg15), this protein is located on the cell surface of Xenopus neurons, where it is believed to function in an activity-dependent manner to enhance dendritic complexity. A truncated form of CPG15 (neuritin) also has been associated with neurite outgrowth in rat brain, and neuritin mRNA expression is increased by treatment with brain-derived neurotrophic factor (BDNF) and other neurotrophins, as well as by neuronal activity (Naeve et al., 1997Go; Corriveau et al., 1999Go). In parallel to the Xenopus studies, overexpression of neuritin in rat hippocampal and cortical neurons caused enhanced neuritic outgrowth.

Gangliosides have been reported to be associated in rafts with signal transduction molecules like small GTPases and nonreceptor tyrosine kinases (Figure 6BGo). For example, GM3 ganglioside has been found in rafts containing Rho-A and c-Src in a melanoma cell line (Yamamura et al., 1997Go). This same ganglioside co-immunopurifies with the GPI-anchored surface glycoprotein CD59 and Src-family protein-tyrosine kinases in leukocytes (Kniep et al., 1994Go). The Src-family kinase Lyn has been found in rat brain to immunoprecipitate with GD3 ganglio- side (Kasahara et al., 1997Go) and in rat basophilic leukemia cells with GD1b (Minoguchi et al., 1994). As with the GPI-anchored protein CPG15, some of these second messengers have also been reported linked to dendritogenesis (Chen et al., 1996Go). Overexpression in immature rat cortical neurons of certain small GTPases (Rho-A, as well as Rac and Cdc42) leads to an increase in the number of primary dendritic sprouts (Threadgill et al., 1997Go). Additionally, some of these same GTPases have been implicated in the control of dendritogenesis in neurons of Drosophila (Cdc42) (Gao et al., 1999Go) and in retinal cells of Xenopus (Cdc42 and Rac1) (Ruchhoeft et al., 1999Go).

In addition to the above studies, other reports suggest that gangliosides in the outer membrane leaflet of the plasmalemma are in functional association with receptor tyrosine kinases (Figure 6BGo). It has been reported that gangliosides modify the function of these receptors by affecting receptor dimerization, tyrosine phosphorylation and/or their downstream signaling cascades, and that different gangliosides may have graded or even opposite effects [reviewed by Bremer and by Yates and Rampersaud (Bremer, 1994Go; Yates and Rampersaud, 1998Go)]. For example, several types of gangliosides have been shown to inhibit dimerization of the platelet-derived growth factor recep- tor as well as the kinase activity of this receptor in several cell types (Hynds et al., 1995Go; Yates et al., 1995Go; Sachinidis et al., 1996Go). GM1 ganglioside has been shown in immunoprecipi- tation studies to be closely associated with TrkA and to augment NGF-stimulated neurite outgrowth (Mutoh et al., 1995Go; Rabin and Moccheti, 1995; Farooqui et al., 1997Go). Similar studies have shown that GM1 enhancement of dimerization of TrkA receptors promotes survival of PC12 cells after withdrawal of trophic support (Ferrari et al., 1995Go). GM1 ganglioside has also recently been implicated in dimerization and tyrosine phosphorylation of TrkB in cultured cerebellar granule cells (Pitto et al., 1998Go). Studies linking individual types of Trks and gangliosides may be of particular relevance since the neurotrophins BDNF and NT4 (ligands for TrkB) have been reported to cause exuberant dendritogenesis when applied to immature cortical neurons (McAllister et al., 1995Go, 1996Go). Based on these and related reports, neurotrophins have emerged as likely pivotal elements in morphological and synaptic plasticity in the brain (McAllister et al., 1999Go).

From the above discussion it can be seen that three types of experimental manipulations involving mammalian cortical neurons result in an exuberant sprouting of primary dendrites. These involve the overexpression in rat cortical neurons of the GPI-anchored protein, neuritin (Naeve et al., 1997Go), and of Rho- family GTPases (Threadgill et al., 1997Go), and the application of TrkB ligands to ferret cortical slices (McAllister et al., 1995Go). There is also a fourth example whereby mammalian cortical neurons have been experimentally manipulated to undergo new dendritic sprouting. This study utilized the indolizadine alkaloid, swainsonine, to inhibit lysosomal {alpha}-mannosidase and induce a phenotypic replica of genetic {alpha}-mannosidosis in young adult cats (Walkley et al., 1988Go). As a result, pyramidal neurons in layers III and V accumulated GM2 ganglioside and underwent ectopic dendritic sprouting identical to inherited {alpha}-mannosidosis. The first three studies cited above involved immature neurons manipulated in vitro at a time when they were undergoing normal dendritic differentiation, whereas the swainsonine study involved fully mature postnatal neurons studied in vivo. Induc- tion of dendritic sprouting following overexpression of the GTPases and neuritin were suggested to be possible downstream events in a neurotrophin signalling pathway involving BDNF and TrkB (Naeve et al., 1997Go; Threadgill et al., 1997Go). But how might GM2 ganglioside fit into this scenario? The ferret cortical slices used in the TrkB studies by McAllister and colleagues were done at P14 — a time when GM2 is enriched in these neurons (Zervas and Walkley, 1999aGo). Likewise, the E17/18 dissociated rat cortical cultures showing increased dendrite outgrowth follow- ing transfection with Rho-related GTPases and with neuritin were also at a stage during treatment (3–7 days in vitro) when these neurons have been found to express GM2 ganglioside (Dobrenis and Walkley, 1992Go). These findings, coupled with in vivo studies linking GM2 expression with both normal and ectopic dendritogenesis, are consistent with GM2 ganglioside playing a pivotal role in priming neurons for dendritic out- growth. The swainsonine model further suggests that compon- ents of a regulatory mechanism with which GM2 presumably interacts in immature neurons (during normal development) are retained to some degree in young adult neurons.

An overall view that emerges from these studies is that the initiation of primary dendrites on neurons is likely controlled by multiple layers of interrelated regulatory mechanisms, with more highly evolved neurons having simpler mechanisms overlain by additional regulatory elements. Significant differences in such mechanisms according to species, developmental age and cell type are likely. Expression of regulatory molecules may be enhanced and/or temporally prolonged in some animals (e.g. primates) and more restricted in others (e.g. rodents). Changes in expression of one regulatory element in a control mechanism may alter the function of others. Conceivably, such switching could involve the regulation of dendritic sprouting at one stage of development and control over other aspects of dendritic plasticity at later stages. Understanding how such mechanisms initially craft and then maintain the unique dendritic arbors displayed by individual types of neurons likely will require attention not only to growth factors and their receptors, related membrane proteins and second messenger systems, but also to the specific glycolipid microenvironment of the membranes in which these molecules reside.


    Notes
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The authors thank M. Gondré and Drs K. Dobrenis and D. Siegel for helpful discussions, M. Huang for excellent technical assistance, and Drs Proia, Purpura, Schuchman and Suzuki for tissues. This work was supported by NS18804 and NS37027 from the NIH (S.U.W.).

Address correspondence to Steven U. Walkley, Department of Neuro- science, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA. Email: walkley{at}aecom.yu.edu.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
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