Pyramidal neurons with ectopic dendrites in storage diseases exhibit increased GM2 ganglioside immunoreactivity

Neuroscience Vol. 68, No. 4, pp. 1027 1035, 1995

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Pergamon

Elsevier Science Ltd 0306-4522(95)00208-1

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P Y R A M I D A L N E U R O N S WITH ECTOPIC D E N D R I T E S IN S T O R A G E DISEASES EXHIBIT I N C R E A S E D GM2 GANGLIOSIDE IMMUNOREACTIVITY S. U. W A L K L E Y Department of Neuroscience, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. Abstract--Cortical pyramidal neurons in several types of neuronal storage diseases have been shown by Golgi staining to sprout axon hillock-associated dendritic processes. Based on the relative incidence of this ectopic dendritogenesis, and on quantitative analyses of gangliosides in these same tissues, it has been proposed that abnormal accumulation of a specific metabolic product, GM2 ganglioside, is the pivotal event leading to re-initiation of dendritic sprouting [Siegel D. A. and Walkley S. U. (1994) J. Neurochem. 62, 185~1862]. In the present study, a monoclonal antibody was used to determine the cellular location of this ganglioside within the cerebral cortex of animal modelsof storage diseases with and without ectopic dendrite growth. Diseases exhibiting ectopic dendritogenesis included inherited and swainsonine-induced (juvenile-onset) a-mannosidosis, mucopolysaccharidosis type I, Niemann Pick disease type C, and GMI and GM2 gangliosidosis. Conditions lacking ectopic dendrite growth included adult-onset swainsonine-induced a-mannosidosis, fucosidosis, neuronal ceroid lipofuscinosis (Batten disease) and normal, mature brain. Immunocytochemical staining for GM2 ganglioside indicated that diseases exhibiting new dendritic sprouting, with the exception of GMI gangliosidosis, exhibited abundant GM2-1ike immunoreactivity within the cortical pyramidal cell population, whereas diseases without dendritic sprouting had GM2-1ike immunoreactivity limited to glia and/or to non-pyramidal neurons. Cortical tissues from normal animals at comparable ages and processed by similar procedures exhibited occasional glial cell staining but little or no neuronal labelling. Mechanisms by which normal cortical pyramidal neurons regulate dendritic initiation are poorly understood. However, it is known that this event is developmentally restricted, occurring only during early brain development. An exception, however, is in certain types of neuronal storage diseases in which mature pyramidal neurons have been shown to sprout new, ectopic dendrites. Results of the present study show that GM2 ganglioside is elevated within these neurons undergoing ectopic dendritogenesis and that pyramidal cells in storage diseases lacking this phenomenon also lack increases in this ganglioside. These findings, in conjunction with other recent reports, provide compelling evidence that GM2 ganglioside plays a pivotal role in regulating dendritic initiation on cortical pyramidal neurons.

N e u r o n a l storage disorders are diseases in which n e u r o n s chronically a c c u m u l a t e material within lysosomes, m o s t often due to defective activity of a specific lysosomal hydrolase. 27 M o s t storage diseases are inherited as a u t o s o m a l recessive c o n d i t i o n s a n d m a n y are well d o c u m e n t e d in terms o f enzyme deficiencies a n d in some cases molecular genetic defects. 18 In a d d i t i o n to n a m i n g by e p o n y m s , storage diseases are c o m m o n l y referred to according to the p r i m a r y metabolic p r o d u c t f o u n d a c c u m u l a t i n g in cells. T a y - S a c h s disease, for example, is a ganglioside storage disease with G M 2 ganglioside being the p r i m a r y storage product; H u r l e r ' s disease is a m u c o p o l y s a c c h a r i d o s i s with storage of specific gly-

Abbreviations: GM I, Gal,/~ I---~3GalNAc/~ l---~4(NeuAc~ 2---,

3)Galfl 1---,4Glc/~1---~l'Cer; GM2, GalNAc/~ 1---~ 4(NeuAc~ 2---*3)Gal/31--,4Glc/~ 1----~l'Cer; GM2-LIR, GM2-1ike immunoreactivity; ganglioside abbreviations and nomenclature follow that of Svennerholm. 29

cosaminoglycans. Both are due to defective activity of specific lysosomal hydrolases, fl-hexosaminidase a n d ~-L-iduronidase, respectively. O t h e r examples o f storage diseases include B a t t e n disease a n d N i e m a n n - P i c k disease type C, for which lysosomal hydrolase defects are n o t believed responsible for the characteristic i n t r a n e u r o n a l storage. 19,2° In a d d i t i o n to the presence of the p r i m a r y storage material occurring as a direct consequence o f the initial metabolic d e r a n g e m e n t , a variety o f secondary a n d tertiary storage p r o d u c t s m a y also be f o u n d in greater t h a n n o r m a l a m o u n t s within C N S tissues in these diseases. This e x p a n d i n g cascade of metabolic alterations a n d storage o f u n m e t a b o l i z e d c o m p o u n d s within n e u r o n s ultimately c o m p r o m i s e s cell function, t h o u g h the m e c h a n i s m s responsible for this dysfunction have n o t been fully elucidated. 28'33 Child r e n affected by these diseases m o s t often a p p e a r n o r m a l at b i r t h b u t later develop slowly progressive neurological deterioration consisting of m e n t a l

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r e t a r d a t i o n / d e m e n t i a , m o v e m e n t disorders, sensory i m p a i r m e n t s and seizures. 27 N e u r o n a l storage diseases are also k n o w n to occur in animals, including cats, dogs a n d a variety of livestock. As these are a u t o s o m a l recessive conditions, it has in m a n y instances been possible to identify obligate heterozygote animals for developm e n t o f breeding colonies. 33 Storage diseases have been f o u n d as acquired rather t h a n inherited conditions only rarely a n d most well-known is the ingestion by livestock of certain plants (Swainsona, Astragalus spp.) c o n t a i n i n g the alkaloid "swainsonine". ~° Swainsonine is a reversible inhibitor of lysosomal e - m a n n o s i d a s e a n d produces a phenotypic replica o f genetic ~-mannosidosis. 6"35 Inherited a n d induced a n i m a l models of n e u r o n a l storage diseases have been used extensively in recent years to explore the p a t h o b i o l o g y a n d t r e a t m e n t o f this family of diseases. Golgi studies of b r a i n tissue from storage diseases in m a n a n d animals have revealed t h a t some types of neurons, particularly cortical p y r a m i d a l cells, exhibit a unique response to the disease process: they sprout new dendrites. These neuritic processes emerge primarily from the axon hillock or from a greatly e x p a n d e d hillock region referred to as a m e g a n e u rite. 22 The new dendritic m e m b r a n e often possesses spines a n d receives synaptic inputs resembling those on n o r m a l dendrites, including p r o m i n e n t asymmetrical synapses. 4j'42 This p h e n o m e n o n of ectopic dendritogenesis has not been observed in o t h e r kinds o f neurological diseases a n d indeed is absent in some storage diseases like Batten disease. 43 Recently, we have s h o w n t h a t pyramidal n e u r o n s with ectopic dendrites in young cats with the storage disease, ~-mannosidosis, exhibit intracellular acc u m u l a t i o n of G M 2 ganglioside, whereas other pyramidal cells w i t h o u t new dendrites lack detectable a m o u n t s of this metabolic product. 8 F u r t h e r m o r e , q u a n t i t a t i v e analysis of gangliosides in cortical tissues from a variety of n e u r o n a l storage diseases revealed t h a t increased levels of G M 2 ganglioside in cerebral cortex o f affected animals correlate with the degree of new dendrite g r o w t h on p y r a m i d a l neurons. 25 These studies, in concert with the finding that ectopic dendritogenesis is m o r e c o m m o n in G M 2 gangliosidosis t h a n in o t h e r storage diseases, 25'37 support the hypothesis t h a t G M 2 ganglioside m a y be

i m p o r t a n t in the regulation o f dendritogenesis by pyramidal neurons. In the present report we have extended earlier studies by using a m o n o c l o n a l antibody to examine the cellular location of G M 2 ganglioside in cerebral cortex in a variety of storage diseases in animals exhibiting or lacking ectopic dendrite growth. O u r results d e m o n s t r a t e a striking correlation between a b n o r m a l l y elevated levels o f G M 2 ganglioside within cortical pyramidal n e u r o n s and the presence of new dendritic sprouting. A preliminary report of this work has appeared. 36

EXPERIMENTAL PROCEDURES

This study utilized tissues from the cerebral cortex of cats and dogs with well-characterized forms of neuronal storage diseases (Table l). With the exception of swainsonine-induced ~-mannosidosis described below, all of the diseases studied were autosomal recessive in inheritance and affected animals were derived from breeding colonies. Each colony was developed following discovery of spontaneous disease cases and donation of heterozygote breeding stock. Tissues and/or disease models were obtained as follows: GM1 gangliosidosis, Dr H. Baker, Auburn University; mucopolysaccharidosis I, Dr M. Haskins, University of Pennsylvania; Niemann-Pick C, Dr M. A. Thrall, Colorado State University; Batten disease, Dr A. Siakotos, University of Indiana; fucosidosis, Dr M. Ferraro, University of Sydney. Colonies of animals with GM2 gangliosidosis and ~-mannosidosis are available at the Albert Einstein College of Medicine. For storage diseases caused by lysosomal hydrolase deficiencies, diagnosis was carried out shortly after birth by measuring lysosomal enzyme activity in peripheral tissues (tail tissue biopsy or leukocytes) according to routine protocols. In the case of Batten disease, diagnosis was made by positive identification of storage material in an ultrastructural analysis of cortical biopsy tissue in young animals. For Niemann-Pick disease type C, affected kittens were identified on the basis of decreased ability of fibroblast cultures to esterify exogenously supplied cholesterol? Three affected animals in mid to late disease were used for each disease except feline mucopolysaccharidosis type I, for which a single animal was studied. For the study of induced e~-mannosidosis, the ~-mannosidase inhibitor, swainsonine, was partially purified from dried Astragalus plants and administered orally in daily measured doses to purpose-bred normal cats according to published procedures. 4° Disease was initiated in two cats at three weeks and 2.5 years of age and swainsonine administration (100 inhibitory units/g body wt/day) continued for four months, at which time the animals were killed. For tissue collection in this study, animals were given an overdose of pentobarbital followed by perfusion with cold fixative (4% paraformaldehyde-O.l% glutaraldehyde) in 0.I M phosphate buffer (pH 7.2). The brain was removed

Table 1. Genetic animal models of neuronal storage diseases used in this study Disease type GM 1 gangliosidosis GM2 gangliosidosis Mucopolysaccharidosis (type 1) Niemann Pick disease (type C) Batten disease -Mannosidosis Fucosidosis

Enzyme defect

Major storage product

Refs

fl-galactosidase fl-hexosaminidase

GM 1 ganglioside GM2 ganglioside

1 4

~-L-iduronidase

glycosaminoglycans

11

unknown unknown e-l>mannosidase e-L-fucosidase

cholesterol protein oligosaccharides oligosaccharides

3, 15 13 31 12

GM2 ganglioside and dendritogenesis and immersed in the same fixative for up to 6 h, followed by storage in cold phosphate buffer. All procedures using animals were performed in full accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. Every effort was made to minimize the number of animals used and any discomfort occurring as a consequence of the disease state. After fixation, tissues were examined by Golgi impregnation and by immunocytochemical staining using a mouse monoclonal immunoglobulin M antibody (MoAb 10-11) to GM2 ganglioside, ~7 which was a gift from Dr Philip Livingston. A mouse monoclonal immunoglobulin G antibody to GM2 ganglioside (Matreya, Inc., Pleasant Gap, PA)

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was also used on some tissue samples and gave similar neuronal labelling but less glial cell labelling. Blocks of cerebral cortex were impregnated by the Golgi rapid or Golgi-chloral hydrate methods using published procedures, 32 mounted in celloidin and cut on a sliding microtome for light microscopy. Immunocytochemical staining procedures have also been published. 8 Briefly, 40-/lm-thick Vibratome sections were reacted overnight with the monoclonal antibody against GM2 ganglioside, followed by biotinylated secondary antibody (1 h) and peroxidase-conjugated avidin (Vector Laboratories, Burlingame, CA; 1 h), with histochemical demonstration of peroxidase using 3,3'diaminobenzidine (Sigma, St Louis, MO). Saponin (0.02%) was used in the primary and secondary antibody steps to aid in tissue penetration. In some cases a preincubation step in

Fig. I. Photomicrographs of typical Golgi-impregnated lamina II/III pyramidal neurons from the storage diseases used in this study. (A) GM2 gangliosidosis. (B) GM1 gangliosidosis. (C) Niemann-Pick disease type C. (D) Mucopolysaccharidosis type I. (E) Batten disease. (F) Fucosidosis. Long arrows indicate ectopic neurites or spines (A-D), short arrows normal or enlarged axon hillocks lacking ectopic neurites or spines (E,F). Note that neurons in B and E exhibit meganeurites but that these differ. The meganeurite in E is covered with spines and short neurites, whereas the one in E is non-spiny. Scale bar in F = 25 #m, and applies to all panels.

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I% sodium borohydride (10 min) was added to chemically reduce unreacted aldehydes and thereby lower non-specific staining. RESULTS

Golgi studies demonstrated the growth of ectopic dendrites on a large percentage of pyramidal neurons in cerebral cortex of cats with GM1 and GM2 gangliosidosis, and on smaller numbers of these cells in Niemann-Pick type C and mucopolysaccharidosis type I, in confirmation of earlier studies (Fig. IA-D). 16'21'38'39'42 Young animals with c~mannosidosis induced by swainsonine, as well as kittens with genetic ~-mannosidosis, developed ectopic dendrite growth on a small percentage of pyramidal neurons, as expected (Fig. 3A). 4° Adultonset swainsonine-induced ~-mannosidosis, although exhibiting widespread intraneuronal storage, was characterized by an absence of new dendrite growth on cortical pyramidal neurons (Fig. 3C). Golgi studies of the canine model of Batten disease, again in confirmation of earlier studies, 2'43 revealed swollen pyramidal neurons which sometimes exhibited nonspiny meganeurites, but new dendrite growth was not seen (Fig. 1E). Likewise, Golgi-impregnated pyramidal neurons in ~anine fucosidosis exhibited no ectopic dendritogenesis in spite of widespread intraneuronal storage (Fig. IF). Golgi studies of cerebral cortex of normal cats and dogs at similar ages to the diseased animals revealed pyramidal neurons exhibiting the typical dendritic spine-free zones over the somata, axon hillock and proximal basilar and apical dendrites. No ectopic neurites or spines were detected as part of a normal staining pattern. Immunocytochemical staining for GM2 ganglioside in cerebral cortex from storage diseases exhibiting new dendrite growth routinely revealed abundant staining of pyramidal neurons. The only exception was GM1 gangliosidosis. The cerebral cortex of cats with GM2 gangliosidosis exhibited widespread distribution of GM2-1ike immunoreactivity (GM2-LIR) involving all types of cortical cells (Fig. 2A). In contrast, GM2 staining of cortical tissues from the cat with GM 1 gangliosidosis failed to label neuronal cell bodies, although glia exhibited some staining (Fig. 1B). Cortical tissues from cats with Niemann-

Pick disease type C and mucopolysaccharidosis type 1, like GM2 gangliosidosis, revealed widespread labelling of neurons (Fig. 2C, D). Cats with inherited or early-onset swainsonine-induced ~-mannosidosis exhibited GM2-LIR in intermixed populations of pyramidal and non-pyramidal cells scattered throughout cortical tissue (Fig. 2E). Non-pyramidal neurons exhibiting GM2-LIR were most numerous and were distributed across all cortical laminae; labelled pyramidal neurons, identified by the presence of prominent apical dendrites, were observed less often and appeared restricted to mid-level cortical areas. Cats with adult-onset swainsonine-induced ~-mannosidosis revealed GM2-LIR limited to non-pyramidal neurons and glial cells (Fig. 2F). In this case the overall number of GM2-positive non-pyramidal neurons did not appear significantly different from that observed in the animal with earlier disease onset; rather it appeared that pyramidal cells labelled in the younger animal were unlabelled in the adult staining pattern (Fig. 3B, D). GM2 ganglioside antibody staining in the canine models of Batten disease and fucosidosis revealed dense staining of numerous astrocytic-like cells and rare unidentified small cells, but pyramidal neurons were consistently unlabelled (Fig. 2G,H). Normal animals of similar ages to the diseased animals used in this study revealed little GM2-LIR. Occasional neurons exhibited small, immunoreactive punctate structures in their cytoplasm, whereas glial cells appeared more diffusely labelled or exhibited abundant punctate labelling (Fig. 21). DISCUSSION

When Purpura and Suzuki22 first discovered the phenomenon of ectopic dendritogenesis in Tay-Sachs disease and other ganglioside storage diseases, they proposed that alterations in ganglioside metabolism may in some way be associated with the aberrant production of new dendritic-like membrane. It was later discovered that ectopic dendrite growth was a prominent feature of a wide variety of neuronal s t o r a g e diseases t6,34,38,39,42 and that only a limited set of neurons (principally cortical pyramidal neurons and multipolar cells of the amygdala and claustrum) appeared capable of initiating growth of new,

Fig. 2. Photomicrographs of GM2-LIR in cerebral cortex from the storage diseases used in this study. All cortical cells in GM2 gangliosidosis (A) exhibited GM2-LIR, whereas in GM 1 gangliosidosis(B) glial cells (short arrow) but not neurons (long arrow) exhibited staining. Most cortical neurons in Niemann-Pick disease type C (C) and in mucopolysaccharidosis type 1 (D) exhibited GM2-LIR, although glial staining was less prominent than the gangliosidoses.In inherited (E) and juvenile-onset swainsonineinduced ~-mannosidosis, deep pyramidal neurons sometimes exhibited GM2-LIR (long arrow), as did many non-pyramidal neurons (short arrow) and glia (not shown). In swainsonine-inducedc~-mannosidosis of adult onset, only non-pyramidal neurons (F, arrow) and glia exhibited GM2-LIR. In canine Batten disease (G) and in fucosidosis (H), all pyramidal neurons and most non-pyramidal neurons failed to exhibit GM2-LIR (long arrows), but astrocytes and other glial cells were heavily stained (short arrows). In cerebral cortex of normal, adult cats glial cells often exhibited GM2-LIR (I, short arrow), whereas neurons appeared negative or exhibited only small numbers of labelled punctae within their cytoplasm (I, long arrow). Scale bar in I = 35 #m and applies to all panels.

GM2 ganglioside and dendritogenesis

Fig. 2.

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Fig. 3. Photomicrographs of cerebral cortex from cats with e-mannosidosis induced during the early postnatal period (A, B) or in adulthood (C, D). (A) Golgi-impregnated layer III pyramidal neuron showing typical axon hillock neurite growth (arrow) as was observed on a small percentage of pyramidal neurons. (B) Low power view of cerebral cortex of cat shown in A, immunostained for GM2 ganglioside. Note the presence of both pyramidal-like neurons with apical dendrites (long arrows) and rounded, non-pyramidal-like neurons (short arrows) distributed throughout the cortex. (C) Typical Golgi-stained pyramidal neuron in adult-onset e-mannosidosis showing absence of axon hillock neurites. (D) Immunostaining for GM2 ganglioside in the cerebral cortex of the animal shown in C revealed GM2-LIR limited to non-pyramidal-like neurons (short arrows), as pyramidal-like neurons exhibited no staining. Scale bar in C = 30#m (also applies to A); bar in D = 120pm (also applies to B). primary dendrites in each of these diseases. 32,33 The restricted distribution of neurons with ectopic dendrites vis-gt-vis the more widespread intraneuronal ganglioside storage, coupled with the subsequent discovery that ectopic dendritogenesis also occurred in non-lipidoses like e-mannosidosis, 38questioned the role of gangliosides as neuritogenic agents. Comparative studies on the incidence of new dendrite growth on pyramidal neurons in feline models of neuronal storage disorders clearly indicated major differences between the gangliosidoses and other storage diseases. G M 2 gangliosidosis was found to be most susceptible to new dendrite growth, with almost 90% of pyramidal neurons exhibiting this change by six months of age, whereas only 60% were affected in GM1 gangliosidosis at this age. 37 A much lower incidence of affected pyramidal neurons has been found in storage diseases without primary ganglioside storage (Niemann-Pick types A and C, approximately 15% and 40% respectively, and mucopolysaccharidosis type I, 20%). 25 All of these diseases in humans, however, had been reported to exhibit some degree of ganglioside storage secondary to the pri-

mary metabolic defect and neurons contained membranous storage inclusions consistent with the presence of gangliosides. The discovery that the small percentage of pyramidal neurons with ectopic dendrites in ct-mannosidosis also contained storage bodies exhibiting membranous materials rather than the typical clear or floccule-filled vacuoles of non-neurite bearing cortical cells4° gave additional support to a role for gangliosides in ectopic dendritogenesis. This membranous storage material was later found to contain G M 2 ganglioside. 8 It was subsequently shown that ~-mannosidosis and all other storage diseases in cats that exhibited ectopic dendritogenesis also had altered ganglioside patterns within cerebral cortex characterized by elevated levels of G M 2 ganglioside. 24'~5 The relative elevations in G M 2 ganglioside were roughly proportional to the measured incidence of pyramidal neurons with new dendrite growth. The present study has extended these earlier findings and demonstrated that G M 2 ganglioside is elevated within the pyramidal cell population in storage diseases exhibiting ectopic dendritogenesis

GM2 ganglioside and dendritogenesis (GM2 gangliosidosis, mucopolysaccharidosis type I, Niemann-Pick type C, inherited and early-onset swainsonine-induced ~-mannosidosis), but is not increased in pyramidal neurons in storage diseases lacking new dendrite growth (Batten disease, fucosidosis, and adult-onset swainsonine-induced a-mannosidosis). These findings support the hypothesis that elevated expression of GM2 ganglioside is a pivotal event in regulating dendritic initiation on pyramidal neurons. 25The single exception to the staining pattern was feline GM1 gangliosidosis, in which neuronal labelling for GM2 ganglioside was absent even though ectopic dendrite growth was present. Since quantitative ganglioside analysis has shown that GM2 ganglioside is elevated in these tissues,25the lack of GM2-LIR in neurons in this case may be a technical problem caused by poor antibody penetration or by masking of GM2 ganglioside secondary to the massive increase of GM 1 ganglioside in these tissues. Absence of GM2 ganglioside elevation in pyramidal neurons in GM 1 gangliosidosis, if true, would suggest that GM1 ganglioside also possesses neuritogenic potential and this notion has been argued elsewhere on the basis of other experiments.~4 Under this circumstance, one possibility is that GM 1 accomplishes this function through a mechanism more appropriately driven by GM2 ganglioside, but driven here by G M I due to its overwhelming abundance in the tissue. In support of GM2 ganglioside as the primary agent responsible for neuritogenesis is the finding that a higher incidence of neuritogenesis occurs in GM2 gangliosidosis than in GMI gangliosidosis. 37 An alternative view, that elevated levels of individual gangliosides act non-specifically to affect cell processes, is also possible but goes against current thinking on the function of gangliosides.7'23'26'44 The absence of GM2-LIR in pyramidal neurons in canine models of Batten disease and fucosidosis is consistent with GM2 ganglioside playing a role in dendritic initiation, since these diseases also lacked ectopic dendritogenesis. Pyramidal neurons in cerebral cortex of dogs have been shown to sprout new dendrites in GM2 gangliosidosis,5 and thus are not unlike pyramidal neurons in humans and cats in this regard. In neither Batten disease nor fucosidosis did it appear that gangliosides or other glycolipids were abundantly increased, so poor antibody penetration or other technical limitations appear an unlikely explanation for the lack of GM2-LIR in neurons. Pyramidal neurons in these diseases have not been reported to contain storage inclusions resembling the ganglioside-laden membranous cytoplasmic bodies 3° of the gangliosidoses and Niemann-Pick disease, ~5 zebra bodies of the mucopolysaccharidoses, H or membranous bodies of ~-mannosidosis.33 Batten disease is characterized in dogs, as in humans, as a neurodegenerative disease with many surviving pyramidal neurons exhibiting large non-spiny meganeurites containing inclusions filled largely with protein.~9 The axon hillock expansions appear to only manifest

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cell volume increases and do not exhibit spines, neurites or evidence of new synapses. 2 Swainsonine-induced ~t-mannosidosis exhibited changes in ganglioside metabolism in cerebral cortex and these correlated closely with the degree of ectopic dendrite growth. 25 When disease was induced in young animals, occasional pyramidal neurons and abundant non-pyramidal cells exhibited significant GM2-LIR. However, when the disease was induced in an adult animal only non-pyramidal neurons exhibited significant levels of GM2-LIR. Likewise, ectopic dendrite growth was found to be absent in adult-onset ct-mannosidosis in this and in a previous study, n° Why this apparent difference in ganglioside metabolism occurs in early- vs late-onset disease is unknown. Indeed, why GM2 ganglioside levels increase within neurons in any storage disease other than GM2 gangliosidosis is also not known. The studies reported here on neuronal storage diseases are consistent with GM2 ganglioside playing a specific role in the recapitulation of dendritogenesis on cortical neurons. Pyramidal neurons have been shown to undergo dendritogenesis, i.e. sprouting of new dendritic processes rather than lengthening of existing dendrites, only in two c i r c u m s t a n c e s ~ u r i n g normal, early brain development and in the diseases described here. A role for GM2 ganglioside in ectopic dendritogenesis in storage diseases would be considerably strengthened if a similar role for this ganglioside had been shown during early brain development. Recent studies have suggested that this is indeed the case, in that GM2-LIR appears in cortical neurons during early brain development in cats coincident with the normal phase of dendritogenesis, and subsequently diminishes after maturation of the normal dendritic tree. 9 CONCLUSIONS

Studies examining diseases in which pyramidal neurons sprout new dendrites have identified a common metabolic feature not present in normal, mature brain: elevated levels of GM2 ganglioside. The present study has used an antibody to GM2 ganglioside to show that increases in this ganglioside occur within the same neuronal population that sprouts new dendrites and that pyramidal neurons in diseases lacking ectopic dendrite growth also lack increases in GM2 immunoreactivity. These findings, in conjunction with other recent reports on this ganglioside, are consistent with GM2 ganglioside playing a pivotal role in regulation of dendritic initiation on cortical pyramidal neurons.

Acknowledgements--I would like to thank Drs K. Dobrenis and D. Siegel for their helpful comments on the manuscript and M. Huang and S. Wurzelmann for excellent technical assistance. I thank Drs H. J. Baker, M. Ferrara, M. Haskins, A. Siakotos and M. A. Thrall for access to their animal models and Dr P. Livingston for the antibody to GM2 ganglioside. The work was supported by the NIH (NS 18804, NS30163).

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