Activity of sialidases in fetal brain axonal growthcones and during postnatal development

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Pergamon

Int[ J[ Devl Neuroscience\ Vol[ 06\ No[ 0\ pp[ 04Ð19\ 0888 Þ 0888 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:88 ,08[99¦9[99

PII ] S9625Ð4637"87#99950Ð5

ACTIVITY OF SIALIDASES IN FETAL BRAIN AXONAL GROWTH CONES AND DURING POSTNATAL DEVELOPMENT J[ HERNANDEZ!R\$ A[ BOYZO$ and R[ MERCADOC% $ Department of Physiology\ Biophysics and Neurosciences\ Centro de Investigacion y de Estudios Avanzados del IPN\ Fac Medicina\ Universidad Autonoma de Queretaro\ Mexico % Laboratory of Biochemistry\ Centro Nacional de Rehabilitacion!Ortopedia\ Mexico "Received 12 June 0887 ^ revised 18 September 0887 ^ accepted 5 October 0887# Abstract*In the present work the cytosolic\ membrane!bound and the total activities of brain sialidases were measured in fetal axonal growth cone particles and in various brain regions during brain development[ The developmental pro_le showed an important activity in the prenatal and perinatal periods as well as in speci_c di}erentiating structures like the axonal growth cones from the fetal brain[ Interestingly membrane! bound activity was higher than the cytosolic activity\ starting from 49Ð59) at birth and increasing thereafter[ Cytosolic activity was almost at adult levels at birth and did not show a further signi_cant increase thereafter[ Our results strongly suggest the commitment of membrane!bound sialidase activity in early neurodi}erentiating phenomena like axogenesis\ probably regulating the turnover of glycoconjugates like gangliosides at the presynaptic period\ since high activity was observed in neuroblast|s derived mem! branes and in the perinatal period[ Þ 0888 ISDN[ Published by Elsevier Science Ltd[ All rights reserved Key words] sialidases\ axonal growth cones\ development[

INTRODUCTION Polysialilated compounds are abundant in the central nervous system "CNS#[ Sialic acid "Neu! raminic acid\ NANA\ or Acid 4!Neuraminic# occupies a peripheral position in sialoglycoconjugates such as sialoglycosphingolipids and sialoglycoproteins that are externally located in the cell membrane[04 They also form a part of polysaccharide chains in membranes[ These compounds appear to be related to di}erent cell!membrane functions through changes in macromolecular structure due to their negative electric charge[ Immunogenesis\ cell adhesion and signalization during neurodi}erentiation and the binding of cationic compounds\ are some examples[ Processes of sialilation!desialilation seem to be of relevance in the expression of adhesion molecules undergoing a transition from the anodic to the cathodic condition[2 Sialic acid is known to predominate in the N!CAM embryonic molecule as compared to adult N!CAM\09\03 and possibly modi_es cell adhesion conditions during neurite formation[ Neuraminidase "Sialidase\ N!acetyl!neuraminate glycohydrolase\ EC 2[1[0[07# catalyses the hydrolysis of neuraminic acid from sialoglycoproteins and gangliosides[ In the CNS this enzyme colocalizes in membranes with sialoglycomolecules\7\02\19 so the activity of sialidases through desi! alilation must be critical in the expression of sialoglycoconjugates functions[ If their activity is related to the metabolism of some molecules like gangliosides\ adhesion factors and receptors that participate in key phenomena during neurogenesis\ it must be expressed at signi_cant levels and in speci_c di}erentiating structures like the axonal growth cones "AGC# in early periods of CNS di}erentiation[ In the present work\ the activity of total\ cytosolic or membrane!bound sialidase was determined in axonal growth cones from the fetal rat brain and in various brain regions during subsequent development[ EXPERIMENTAL PROCEDURES Male Wistar adult rats were used weighing 079Ð199 g and nuliparous females about 049 g were adapted to stable environmental conditions for two weeks ] temperature 1020>C and humidity\ 49Ð  To whom all correspondence should be addressed[ Laboratory of Neurontogeny\ Department of Physiology\ Biophysics and Neurosciences\ Av[ Instituto Politecnico Nacional 1497\ Col[ Zacatenco\ 96299 Mexico\ D[ F[ Mexico[ Tel[] 9941 4 636 6999 Ext[ 4013 ^ Fax ] 9941 4 636 6094 ^ E!mail ] jorgehÝ_sio[cinvestav[mx 04

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59)\ light periods of 01 h "96]99Ð08]99 h#[ Afterwards females were mated and appearance of a vaginal plug was taken as the _rst day of gestation[ On gestation day 06\ a caesarean section was performed under pentobarbital anaesthesia "39 mg:Kg\ i[p[# and the fetal brain was removed[ A pool of fetal brain "without cerebellum# tissue was used to isolate axonal growth cone particles "AGC# following the method of Pfenninger et al[8 slightly modi_ed in our laboratory\ as follows ] brain tissue was homogenized with seven strokes in a Thomas pestle glass homogenizer at 699 rpm in 09 vol[ of 9[21 M sucrose\ containing in mM ] MgCl1\ 0 and TES "N!TrisðhydroxymethylŁ methyl! 1!1!aminoethane sulfonic acid#!NaOH\ 0\ pH 6[2[ To remove nuclei and cell debris the homogenate was centrifuged at 0599×g for 04 min[ The low speed supernatant solution was loaded onto a discontinuous sucrose density gradient "1[55\ 0[9 and 9[74 M#\ then centrifuged at 139\999×g for 0 h\ using a Beckman 59 Ti vertical rotor[ Three fractions were separated\ the fraction enriched with AGC was collected at the interface between the load and 9[74 M sucrose and diluted with tris! acetate bu}er 9[94 M\ pH 6[3 solution then it was centrifuged at 119\999×g for 39 min[ The _nal pellet was resuspended in tris!acetate bu}er 9[94 M\ pH 6[3[ Morphological validation of AGC has been done by electron microscopy with about 74) purity[5 To obtain cerebral cortex\ cerebellum and whole brain at di}erent developmental periods after birth\ rats were sacri_ced by cervical dislocation and decapitated\ the skull was opened and brain tissue dissected out on a cold plate and immediately homogenized in 09 vol[ of a cold Tris acetate bu}er solution 9[94 M\ pH 6[3[ To separate membrane!bound and cytosolic fractions the method of Venerando et al[10 was followed ] samples of 1Ð2 g of brain tissue were homogenized in 09 vol[ of 9[21 M sucrose\ 9[0 mM EDTA in potassium phosphate bu}er "PBS# solution 0 mM\ pH 6[1 in a polytron homogenizer "Kinematics\ GmbH\ Model PT09:B94# three strokes\ for 29 s at 799 rpm and 29 s at top speed[ The suspension was centrifuged at 049\999×g for 0 h at 3>C in a Beckman ultracentrifuge LS!64 with a vertical rotor 59 Ti[ The supernatant solution was collected and processed as described below[ The pellet was washed twice with an equal vol[ of PBS and was homogenized with a Thomas pestle glass homogenizer "clearance 9[0Ð9[4 mm#\ then centrifuged three times at 049\999×g for 0 h at 3>C in a Beckman ultracentrifuge LS!64 with a vertical rotor 59 Ti[ The supernatant solution was collected and processed as described below[ The pellet was washed twice with an equal vol[ of PBS and was homogenized with a Thomas pestle glass homo! genizer "clearance 9[0Ð9[04 mm#\ then centrifuged three times at 049\999×g for 0 h at 3>C in a Beckman ultracentrifuge LS!64 with a vertical rotor 59 Ti[ The pellets were homogenized in bidestilled water and then used to determine the enzyme activity of the membrane bound sialidases[ All supernatant solutions containing the brain cytosolic fraction were collected and used as the source for sialidase soluble activity[ Enzymatic activity of sialidases was determined in AGC\ whole tissue homogenates and soluble and membrane fractions of the enzyme[ The method used was that of Pitto et al[00 brie~y ] tissue aliquots were incubated in acetate bu}er pH 2[8 at 26>C for 04 min in a _nal volume of 199 ml in the presence of methyl umbelliferil N!acetilneuraminic acid as the substrate[ To stop the enzymatic reaction\ 2 ml of cold glycine bu}er 9[1 M at pH 09[4 was used[ The product of the reaction\ the methyl umbelliferil was quanti_ed in a spectrophoto~uorometer "Perkin Elmer LS!49B# at 254 nm excitation and 334 nm emission wavelengths[ Speci_c activity of sialidase is expressed as nmol NANA liberated per mg protein per h[ Proteins were determined according to Lowry et al[4

RESULTS Total sialidase activity assessed since the fetal period up to adulthood in whole brain "WB#\ cerebral cortex "CC#\ cerebellum "C# and in AGC fractions\ is shown in Fig[ 0"A#\ "B# and "C#[ It is interesting to note that the enzyme activity in the whole fetal brain accounted for more than 49) as compared to the adult brain activity[ Moreover\ the activity in discrete di}erentiating structures of the fetal brain\ the AGC was 14Ð24) as compared to the adult brain[ At birth the enzyme activity increased to about 59) in the WB and to 69) in C[ The developmental pattern di}ered between regions ] in WB and in C it remained almost the same up to day 4 ^ progressing in the former to reach the adult "74 nmol:mg protein:h#[ In CC there was a slight increase from birth up to day 4 and reached adult values thereafter[ In Fig[ 1 we show the total\ cytosolic and the membrane!

Developmental pattern of sialidases activity

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Fig[ 0[ Developmental pattern of total sialidase activity in ] "A# whole brain "without cerebellum# "# x2S[D[ from 4 Ð01 experiments^ "B# cerebellum "e# x2S[D[ from 3Ð00 experiments^ and "C# cerebral cortex "r# x2S[D[ from 5Ð06 experiments[ In parenthesis the total enzyme activity percentage compared to adult values is shown[ Experiments were done in triplicate samples[ E Total activity in AGC[  P ³ 9[90 ^  P ³ 9[94 ^  P ³ 9[990 "ANOVA#[

bound activities in fetal AGC[ The latter was signi_cantly higher "P ³ 9[990# than the cytosolic\ and it represented 50) of the total activity in this neuroblastic tissue[ Membrane!bound and cytosolic enzyme developmental patterns in the other regions studied are shown in Fig[ 2"A#\ "B# and "C#\ including the activity in AGC for comparison[ There were clearly two di}erent developmental patterns[ Cytosolic enzyme was always signi_cantly lower than the membranous one "P ³ 9[990# starting in the fetal whole brain as in AGC particles\ and it represented the 18Ð34) of the adult cytosolic activity at birth[ This fraction of the enzyme activity\ practically reached the adult value "74Ð87)# at birth and did not increase steadily\ showing only some up and downs\ between days 2 and 7[ Opposite to the cytosolic fraction\ the membrane!bound sialidase activity\ which was already higher in the fetal AGC particles remained signi_cantly elevated during the ontogenetic period\ being at birth a little more than half that in the adult[ It increased slightly up to day 7 in the WB

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J[ Hernandez!R et al[

Fig[ 1[ Sialidases activity in fetal axonal growth cones[ x2S[D[ from 4Ð09 experiments made in triplicate samples[ The cytosolic and membrane!bound enzyme activity percentage compared to adult values is also shown[  P ³ 9[990 "ANOVA#[

and C\ reaching twice as much an activity in the former as compared to birth and about 29) over that in the C[ In CC the membranous activity also increased but mostly from the prenatal period up to day 4 ^ thereafter only minor changes were noted up to adulthood[ DISCUSSION The overall developmental pattern of total sialidases activity clearly suggest a functional relation! ship of these enzymes in the metabolism of sialoglycoconjugates during early CNS development\ both in brain and cerebellum[ In speci_c neurodi}erentiating structures like AGC\ which are responsible for axonal growth and path _nding for the establishment of neuronal circuitry\3\07\11 the activity of sialidases is already at a signi_cant level\ suggesting a regulatory role on the functional expression of membrane functional molecules at this early period of neurogenesis[ This interpret! ation is strengthened by the fact that analysing the enzyme patterns of the membrane!bound fraction vs the cytosolic\ their prenatal level is also high in AGC\ by birth reaching about 49) or more of the adult activity[ Interestingly the cytosolic activity attains almost adult values "74Ð86)# at birth[ The membrane bound enzyme on the contrary is at mid!level and progressively increases[ These di}erent developmental patterns suggest that these type of sialidases are involved in di}erent metabolic and functional events during neural development[ Schiller et al[05 observed more than 49) of sialidase activity in the trout|s CNS\ just before hatching with an important increase in the period of neurodi}erentiation[ Other authors reported desialilation processes correlating with extensive synaptogenesis and in later periods like myelogenesis[6 In the chick\ a low level of activity was observed in the embryonic period with its maximum at hatching[1 Carubelli and Tulsiani\0 reported a sharp increase of sialidase activity 2Ð3 days prior to birth in the rat liver\ but low levels in the CNS during the _rst postnatal week[ Variations in the reported pattern of sialidases development may be due to di}erent methods and species used[ Our data\ however\ seem to support the idea that membrane!bound activity could be mostly correlated to early cell di}erentiating phenomena like sialic acid movements from gangliosides or to rearrangements of membrane sialocompounds for regulation of surface charges[01 Indeed\ there seems to be a pronounced lipid!bound sialic acid accumulation in the prenatal and early postnatal period[ This line of interpretation would also be in agreement with results reported by Yavin\12 concerning Gt and Gq ganglioside levels of 25) of the total brain content at birth as compared to 39) in the adult\ associated mainly with non!synaptic interneuronal connections and neuritic sprouting[ Suzuki06 and Tettamanti08 reached a similar conclusion reporting 14Ð29) of gangliosides adult levels at birth associated with early presynaptic events of neural di}erentiation[ Phillips et al[09

Developmental pattern of sialidases activity

08

Fig[ 2[ Developmental pattern of sialidase activity in ] "A# whole brain "without cerebellum#\ cytosolic "# and membrane! bound "Ž#^ "B# brain cerebellum\ cytosolic "e# and membrane!bound "E#^ and "C# cerebral cortex\ cytosolic "r# and membrane!bound "R#[ x2S[D[ from 3Ð7 experiments made in triplicate samples[ In parenthesis the sialidases activity percentage compared to adult values is shown[ T\ t\ membrane!bound and cytosolic sialidase activity in AGC respectively[  P ³ 9[90 ^  P ³ 9[94 ^  P ³ 9[990 "ANOVA#[

propose a regulatory mechanism of N!CAM sialilation during development and regeneration of CNS\ that could play a role in neurite extension and synaptic modulation[ In the present work we report a high activity of membrane!bound sialidase in fetal AGC which strongly suggests a regulatory role of these enzymes on sialoglycoconjugate|s metabolism possibly involved in the process of axogenesis and related to the expression of desialilated glycoconjugates[ It remains to be established on which membrane speci_c macromolecules sialidases are acting\ according to polysialogangliosides\ polisialoglycoproteins or other sialilated carbohydrates con! forming the structure of the axonic growth cone[ Other sialidases activities like lysosomal or those expressed at more neutral pH values are currently being studied in our laboratory during the same developmental period in order to obtain a more integrated picture of their ontogenetic role[ Acknowled`ement*The authors are grateful to Mr Ignacio Vargas for excellent technical assistance[

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