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Effect of experimental hyperphenylalaninemia on the postnatal rat brain
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Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 04\ No[ 0\ pp[ 18Ð25\ 0886 Copyright Þ 0886 ISDN[ Published by Elsevier Science Ltd Printed in Great Britain[ All rights reserved 9625Ð4637:86 ,06[99¦9[99
PII] S9625Ð4637"85#99970Ð9
EFFECT OF EXPERIMENTAL HYPERPHENYLALANINEMIA ON THE POSTNATAL RAT BRAIN GALINA A[ USHAKOVA\ % HELENA A[ GUBKINA\ VICTORIA A[ KACHUR and EUGENE A[ LEPEKHIN $ Department of Biophysics and Biochemistry\ Dniepropetrovsk State University\ Dniepropetrovsk\ Ukraine^ $International Center for Molecular Physiology of The National Academy of Sciences of Ukraine\ Dniepropetrovsk\ Ukraine "Received 07 March 0885^ revised 18 July 0885^ accepted 0 Au`ust 0885# Abstract*The molecular mechanism of the disturbance to brain development caused by phenylketonuria remains mostly unknown[ We have studied three molecular markers that re~ect the development of neurons\ glia and the extracellular matrix of the postnatal rat brain in an animal model of hyperphenylalaninemia\ in order to elucidate the possible mechanism by which increased phenylalanine in~uences brain development[ The contents of NCAM\ GFAP and hyaluronate!binding activity were compared in cerebellum and telencephalon of normal rats and those subjected to high phenylalanine[ No statistically signi_cant changes were found in telencephalon when experimental animals were compared to controls[ In the hyper! phenylalaninemic cerebellum\ the developmental dynamic of NCAM content "represented by two peaks at about postnatal days 4 and 11 during normal development# is dramatically altered[ The GFAP content in the cerebellum of treated rats exceeded those in controls signi_cantly during late developmental stages "postnatal days 17Ð24#[ Hyaluronate!binding activity in the extracellular protein fraction from treated rat cerebellum was increased compared to normal rat at the early stages of development only "postnatal day 6#[ These results suggest that high serum phenylalanine may lead to permanent brain dysfunction through a disturbance of a wide range of developmental events[ Þ 0886 ISDN Key words] hyperphenylalaninemia\ brain\ NCAM\ GFAP\ hyaluronate\ binding activity[
Phenylketonuria "PKU# is an inherited de_ciency of the enzyme phenylalanine hydroxylase "L! phenylalanine\ tetrahydropteridine] oxygen oxidoreductase!3!hydroxylating^ EC 0[03[2[0#\ which leads to increased serum "and brain# phenylalanine and\ as a consequence\ to abnormal brain development[ This genetic defect leads to imbecility\ if the diagnosis is not made directly after birth[ Molecular mechanisms for the way in which phenylalanine interferes with brain development are not clearly understood to date\ but there is evidence that phenylalanine a}ects every phase of brain development[00 The most crucial e}ect\ the permanent brain damage that leads to mental retardation\ occurs during the early period of brain development and may be connected to improper neuron di}erentiation and synaptogenesis[09\00\20 Such a genetic defect is not known to occur naturally in animals and is modeled in several ways for neurochemical analysis[ The most successful chemical model of hyperphenylalaninemia "HPA# is based on the simultaneous injection of postnatal rat pups with a!methylphenylalanine "an inhibitor of phenylalanine hydroxylase#\ together with phenyl! alanine[04\10 Such treatment leads to the permanent increase of phenylalanine in developing brain17 that induces enduring behavioral de_cit10\20 and thus is used widely as model for human phenyl! ketonuria[ The signi_cant abnormalities were found in cerebrum of patients with PKU\29 while only few changes have been observed in the cerebellum or other brain regions of those patients[7\25 In experimental rat HPA\ the dramatic changes were observed both in the neocortex\00\10\20 and in the cerebellum[8\04\05\39 Currently\ known events associated with HPA are the loss of myelin due to an increase of its turnover\01\26 disintegration of the myelin sheath00 and the reduction of synaptic terminals[20 The latter may be one of the main causes of mental retardation due to HPA\ because dynamic integrative capacities of the central nervous system are based on the proper formation of the synaptic network[03 At the same time\ many aspects of synaptogenesis are mediated by cellÐcell and cellÐextracellular %To whom correspondence should be addressed at] Department of Biophysics and Biochemistry\ Dniepropetrovsk State University\ 61 Gagarin Ave[\ Dniepropetrovsk 219514\ Ukraine[ Tel[] ¦279!451!358!179^ Fax] ¦279!451!354!412[ Abbreviations] ECM\ extracellular matrix^ EDTA\ ethylenediamine tetraacetate^ ELISA\ enzyme!linked immunosorbent assay^ GFAP\ glial _brillary acidic protein^ HA\ hyaluronate^ HABA\ hyaluronate!binding activity^ HPA\ hyp! erphenylalaninemia^ NCAM\ neural cell adhesion molecule^ PKU\ phenylketonuria^ PMSF\ phenylmethylsulfonyl ~uoride[ 18
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matrix interactions\18 but the detailed molecular mechanism of cell communication is still not understood[ The neural cell adhesion molecule "NCAM# belongs to the immunoglobulin superfamily of adhesion proteins and is believed to be involved in a variety of cellÐcell interactions in nervous tissue\ including cell migration\11 the association of axons with pathways and targets23 and synaptogenesis[08 Moreover\ the results of in vivo studies demonstrated brain structural and functional disturbances due to abnormal NCAM expression[3\27 Thus\ the quanti_cation of NCAM in developing brain under conditions of HPA may be useful for understanding the pathogenesis of HPA during brain development[ The extracellular matrix "ECM# of the developing central nervous system is an important factor that a}ects neuronal migration\ as well as the extension of axons and formation of synapses[24 Hyaluronate "HA# is a major component of the ECM that plays a key role in the regulation of morphogenesis and di}erentiation of neural tissues\ including both cell proliferation and migration during development[ The structural location and function of hyaluronate in the brain are probably mediated by its interaction with speci_c HA!binding proteins and proteoglycans[28 The HA!binding domains of reported hyaluronate!binding proteins "versican\ chondroitin sulfate proteoglycan\ GHAP and CD33# are strongly conservative[1 This fact suggests that these proteins have similar functions in immobilization of hyaluronic acid within the extracellular space[ The quantitative measurement of total HA!binding activity during brain development may re~ect the process of ECM formation[ There are many data indicating the participation of astrocytes in a variety of important physio! logical and pathological processes in the brain[5 Alterations in astroglial cells are accompanied by changes in the concentration of the glial _brillary acidic protein "GFAP# * the constituent com! ponent of intermediate glial _laments[2 The expression of GFAP is regulated during development as well as under di}erent physiological and pathological states\ including synapse formation and remodeling and chemically induced damage of the CNS[13\21 Thus\ if high phenylalanine is toxic to the developing brain and this e}ect is connected with myelin destruction and synapse reduction\09 the content of GFAP in the developing brain under HPA conditions may be changed[ In order to study the e}ect of HPA on the development of cells\ we have studied quantitatively] NCAM "neuronal marker# and GFAP "astroglial marker# content and HA!binding activity "ECM marker# in two regions of the rat brain "cerebellum and telencephalon# during postnatal development of rats subjected to HPA[
EXPERIMENTAL PROCEDURES Hyperphenylalaninemic model The animal model of PKU "experimental HPA# was prepared as described by Matsuo and Hommes[17 One hundred and _fty!two Wistar rat pups from 05 litters were used to reproduce experimental HPA in the following manner[ Pups from each litter were divided into two groups\ at least four animals per group[ One group was subjected to the HPA regimen from postnatal days 4 to 24\ and the second group was used as a control[ The rat pups were injected intraperitoneally twice a day at 01 hr intervals\ with a 9[8) NaCl solution containing 9[02 M L!phenylalanine and 9[95 M DL!a!methylphenylalanine "Research Organics\ Cleveland\ Ohio\ U[S[A[# at 9[1 ml per 09 g body weight for the _rst four injections and 9[3 ml per 09 g body weight for the remains[ The control pups were injected similarly with 9[8) NaCl solution[ The validity of experimental HPA model was evaluated by the control of pups body weight that is a proved hallmark of HPA[02\12 Brain extract preparation The pups were sacri_ced by decapitation on postnatal days 6\ 03\ 11\ 17 and 24[ The brains were removed and two areas "cerebellum and telencephalon# dissected and homogenized in 09 vol[ of cold bu}er A "14 mM TrisÐHCl pH 6[3\ 0 mM EDTA\ 1 mM 1!mercaptoethanol\ 9[1 mM PMSF and 9[90) merthyolate#[ The suspensions were centrifuged for 59 min at 099\999 g[ After being washed with bu}er A\ the pellets were extracted consecutively with the same bu}er\ containing
E}ect of hyperphenylalaninemia on brain
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either 1) Triton X!099 "membrane fraction# or 3 M urea "extracellular: cytoskeletal fraction#\ respectively[ All procedures were conducted at 3>C[ NCAM and GFAP quanti_cation These were performed by competitive ELISA[ The content of NCAM was measured in the membrane fraction using polyclonal rabbit antisera against rat brain NCAM obtained in our laboratory[ The antisera reacted with three main NCAM polypeptides] 019\ 039 and 079 kDa\ regardless of the sialylation degree19 "Fig[ 0#[ The _lament GFAP content was measured in the urea fraction using polyclonal rabbit antisera against human GFAP obtained in our laboratory[ Western immunoblotting of the urea!soluble fraction from rat brain with anti!GFAP antisera detected one band of 49 kDa polypeptide "Fig[ 1#[ Hyaluronate!binding activity determination The total HA!binding activity "HABA# was detected in extracellular:cytoskeletal fractions by an enzyme!linked carbohydrate!binding assay[4 Brie~y\ the HABA was measured in protein samples immobilized on the surface of microtiter plates using a hyaluronateÐhorseradish peroxidase con! jugate at pH 4[9 and in the presence of 19 mM NaCl and was expressed as ng of bound HA per mg of protein[ The protein content was determined according to Bradford0 with bovine serum albumin as a standard[ A triplicate assay was done for each sample[ The statistical signi_cance of the discovered alter! ations was calculated by MannÐWhitney|s U!test[ RESULTS Effect of HPA re`imen The intraperitoneal injections of the mixture of L!phenylalanine and DL!a!methylphenylalanine led to pronounced growth retardation of pups "Fig[ 2# that may serve as a sign of model validity[02\12 Moreover\ hyperphenylalaninemic pups appeared unwell and less agile[
Fig[ 0[ Western blot analysis of telencephalon 1) Triton X!099 extract for NCAM[ Twenty micrograms of protein were loaded per well and SDS!electrophoresed under reducing conditions in 6) polyacrylamide gel[ After electrophoresis\ separated proteins were electrophoretically transferred to nitrocellulose paper and immunostained with rabbit anti!NCAM "0]499#[ The antisera reacts with three NCAM polypeptides] 019\ 039 and 079 kDa[
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Fig[ 1[ Western blot analysis of mesencephalon 3 M urea extract for GFAP[ Thirty micrograms of protein were loaded per well and SDS!electrophoresed under reducing conditions in 01\4) polyacrylamide gel[ After electrophoresis\ separated proteins were electrophoretically transferred to nitrocellulose paper and immunostained with rabbit anti!GFAP "0]0999#[ The antisera reacts with GFAP polypeptide 49 kDa[
Fig[ 2[ Rat pup body weight during postnatal development "Ž\ control rats^ ,\ hyperphenylalaninemic rats#[ Hyperphenylalaninemic conditions were created by intraperitoneal injections with 9[8) NaCl solution containing 9[02 M L!phenylalanine and 9[95 M DL!a!methylphenylalanine[ Rats were injected twice a day at 01 hr intervals from postnatal days 4Ð24[ Control animals were similarly injected with 9[8) NaCl solution[
Content of NCAM The developmental dynamic of the NCAM concentrations in the cerebellum of the normal rats has been described previously07 as a curve having peaks at about postnatal days 4 and 11[ The results obtained here have the same pattern of NCAM content changes in the control rats[ Under hyperphenylalaninemic conditions\ NCAM concentrations increased up to postnatal day 03 and
E}ect of hyperphenylalaninemia on brain
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Fig[ 3[ The NCAM content in cerebellum during postnatal rat development "Ž\ control rats^ ,\ hyper! phenylalaninemic rats#[ The developmental dynamic of NCAM content is signi_cantly altered under hyperphenylalaninemic conditions[
remained increased through postnatal day 24 "Fig[ 3#[ The NCAM content in the telencephalon of hyperphenylalaninemic rats had no statistically signi_cant changes compared to control animals "data not shown#[ Content of _lament GFAP The pattern of GFAP changes in cerebellum and telencephalon was di}erent during postnatal development of the rat brain[ The _lament GFAP content increased up to postnatal day 11 with further stabilization in the telencephalon of control rat pups[ No statistically signi_cant di}erences in GFAP content in telencephalon were observed under HPA conditions "data not shown#[ A single peak of GFAP content was observed on postnatal day 11 with a pronounced decrease of this value on postnatal days 17 and 24 in the cerebellum of control animals[ In the hyper! phenylalaninemic rat pups\ GFAP content was signi_cantly increased in the cerebellum on postnatal days 17 and 24 "Fig[ 4#[ Hyaluronate!binding activity The highest amount of HABA was found in the extracellular fraction of both cerebellum and telencephalon of the control rat pups on postnatal day 6 with a following reduction "more than four!fold# to postnatal day 24[ However\ the dynamic of HABA in the telencephalon was not a}ected by hyperphenyalalinemia "data not shown#[ In the hyperphenylalaninemic cerebellum\ HABA was increased signi_cantly on postnatal day 6 in comparison to control animals\ and there were no di}erences between experimental and control animals\ at later stages of the brain development "Fig[ 5#[ DISCUSSION The data obtained allow us to suggest that increased phenylalanine causes dramatic changes in brain cellular events during development[ The lack of e}ects of HPA on the telencephalon and the well!pronounced e}ect on the cerebellum may be explained by di}erent times of maturation[ At the time of the hyperphenylalaninemic treatment\ the development of the rat telencephalon is near the end\ while the cerebellum is developing intensively[ The dramatic HABA decrease during postnatal development of both brain areas studied probably
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Fig[ 4[ Filament GFAP content in cerebellum during postnatal rat development "Ž\ control rats^ ,\ hyperphenylalaninemic rats#[ Statistically signi_cant "P³9[94# changes of GFAP content under hyper! phenylalaninemic conditions " # were observed on postnatal days 17 and 24[
Fig[ 5[ Hyaluronate!binding activity in extracellular fractions of the cerebellum during postnatal rat development "Ž\ control rats^ ,\ hyperphenylalaninemic rats#[ Statistically signi_cant "P³9[94# increase of HABA under hyperphenylalaninemic conditions " # was only found on postnatal day 6[
re~ects the formation of extracellular matrix via hyaluronic acid immobilization:insolubilization[ Free HA is believed to play an important role in cell migration by providing a highly hydrated and easily penetrated matrix[14 The ratio of bound to soluble HA increases in the maturing rat brain[15 Therefore\ the insolubilization of HA by HA!binding proteins follows neuronal cell migration and may switch developmental processes from migration to di}erentiation by the creation of tight "non! penetrable# extracellular matrix[ It has been shown that hyaluronate!binding region of chondroitin sulfate proteoglycan inhibits neural cell migration\22 while another hyaluronate!binding protein * versican * appeared in the marginal zone of the embryonal rat telencephalon which is the place of
E}ect of hyperphenylalaninemia on brain
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_nal destination of the migrating neurons[16 The increase of HABA in hyperphenylalaninemic cerebellum at about postnatal day 6 "when massive granule cell migration occurs# may prevent this migration\ interfering with layer formation[ No doubt\ the ECM alteration during early postnatal brain development may lead to the serious consequences for brain maturation[ Another possibility for a disturbance of cerebellar granule cell migration may be related to the alteration of the NCAM developmental pro_le in the cerebellum under HPA conditions[ It has been previously shown that the migration of granule cells coincides in time with the upregulation of NCAM and normally occurs at about postnatal day 6[07 The peak of NCAM content appeared to be shifted to postnatal day 03 under the HPA\ which probably re~ects a delay in the onset of migration[ It is worth noting that\ in reeler mice "characterized by pronounced perturbation of cell migration#\ a delay in NCAM appearance in ontogenesis has been demonstrated[6 At the same time\ it is clear that alterations in intercellular and cellÐextracellular matrix adhesion mediated by NCAM may interfere not only with cell migration\ but also with other NCAM!dependent developmental events in brain[ It is well known that the nervous system responds to diverse injuries by the activation of astrocytes[ One of the generally accepted signs of astrocyte activation is the upregulation of GFAP[ 5 The increase of GFAP at postnatal days 17 and 24 indicates that at least some astroglial cells become activated under HPA[ It may occur as a result of the break in myelin sheath formation\ that has been shown in hyperphenylalaninemic brain[09 Moreover\ our _ndings concerning GFAP content changes in HPA cerebellum resemble those in myelin!de_cient Jimpy mice[06 Therefore\ we can conclude that the e}ects of high phenylalanine on the postnatal rat brain are re~ected by a number of developmental events] "0# an immediate e}ect on hyaluronate!binding activity in extracellular matrix formation^ "1# a dramatic change in postnatal NCAM content^ and "2# an increase of GFAP content during later stages of postnatal cerebellum development[ We believe that these changes may lead to the retardation of brain development and cause permanent brain dysfunction[ Acknowled`ements*This study was supported by Volkswagen Sti}tung "grant I:57401#[ We thank Dr Hommes for the gift of DL!a!methylphenylalanine and Mr S[ Pulin for his help in the English text revision[
REFERENCES 0[ Bradford M[ "0865# Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein!dye binding[ Anal[ Biochem[ 61\ 137Ð143[ 1[ Bignami A[\ Hosley M[ and Dahl D[ "0882# Hyaluronic acid and hyaluronic acid!binding proteins in brain extracellular matrix[ Anat[ Embryol[ 077\ 308Ð322[ 2[ Dahl D[ and Bignami A[ "0874# Intermediate _laments in nervous tissue[ Cell Muscle Motil[ 5\ 64Ð85[ 3[ Davis J[ Q[ and Bennett V[ "0882# Ankyrin!binding activity of nervous system cell adhesion molecules expressed in adult brain[ J[ Cell Sci[ Suppl[ 06\ 098Ð006[ 4[ Dolzhenko M[ I[\ Lepekhin E[ A[ and Berezin V[ A[ "0883# A novel method for evaluation of carbohydrate!binding activity] enzyme!linked carbohydrate!binding assay "ELCBA#[ Biochem[ Mol[ Biol[ Intl 23\ 150Ð160[ 5[ Eddleston M[ and Mucke L[ "0882# Molecular pro_le of reactive astrocytes * implications for their role in neurologic disease[ Neuroscience 43\ 04Ð25[ 6[ Godfraind C[\ Schachner M[ and Go.net A[ M[ "0877# Immunohistological localization of cell adhesion molecules L0\ J0\ N!CAM and their common carbohydrate L1 in the embryonic cortex of normal and reeler mice[ Brain Res[ 369\ 88Ð 000[ 7[ Hajek M[\ Hejcmanova L[ and Pradny J[ "0882# Proton in vivo spectroscopy of patients with hyperphenylalaninaemia[ Neuropediatrics 13\ 000Ð001[ 8[ Hogan R[ N[ and Coleman P[ D[ "0870# Experimental hyperphenylalaninemia] dendritic alterations in cerebellum of rat[ Expl Neurol[ 63\ 123Ð133[ 09[ Hommes F[ A[ "0889# Demyelination in hyperphenylalaninemia[ In myelination and dysmyelination[ Ann[ NY Acad[ Sci[ 594\ 338Ð341[ 00[ Hommes F[ A[ "0880# On the mechanism of permanent brain dysfunction in hyperphenylalaninemia[ Biochem[ Med[ Metab[ Biol[ 35\ 166Ð176[ 01[ Hommes F[ A[\ Eller A[ G[ and Taylor E[ H[ "0871# The e}ect of phenylalanine on myelin metabolism in adolescent rats[ In Inborn Errors of Metabolism in Humans "eds Cockburn F[ and Gitzermann R[#\ pp[ 082Ð088[ MTP Press\ Lancaster[ 02[ Hommes F[ A[ and Moss L[ "0881# Myelin turnover in hyperphenylalaninaemia[ A re!evaluation with the HPH!4 mouse[ J[ Inher[ Metab[ Dis[ 04\ 132Ð140[ 03[ Hopkins W[ G[ and Brown M[ C[ "0874# Development of Nerve cells and their Connections[ Cambridge University Press\ Cambridge[ 04[ Huether G[ and Neuho} V[ "0870# Use of alpha!methylphenylalanine for studies of brain development in experimental phenylketonuria[ J[ Inherit[ Metab[ Dis[ 3\ 56Ð57[
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05[ Huether G[\ Neuho} V[ and Kaus R[ "0872# Brain development in experimental hyperphenylalaninaemia] disturbed proliferation and reduced cell numbers in the cerebellum[ Neuropediatrics 03\ 01Ð08[ 06[ Imamoto K[ "0874# Astrocytal changes in the white matter of Jimpy mice] immunohistochemistry using antisera to glial _brillary acidic protein[ Arch[ Histol[ Jpn[ 37\ 300Ð308[ 07[ Jorgensen O[ S[ "0870# Neuronal membrane D1!protein during rat brain ontogeny[ J[ Neurochem[ 26\ 828Ð835[ 08[ Jorgensen O[ S[ "0884# Neural cell adhesion molecule "NCAM# as a quantitative marker in synaptic remodeling[ Neurochem[ Res[ 19\ 422Ð436[ 19[ Krivko I[ M[\ Rusakov D[ A[\ Savina S[ V[\ Skibo G[ G[ and Berezin V[ A[ "0882# Lateral patterns of the neural cell adhesion molecule on the surface of hippocampal cells developing in vitro[ Neuroscience 44\ 380Ð387[ 10[ Lane J[ D[ and Neuho} V[ "0879# Phenylketonuria] clinical and experimental considerations revealed by the use of animal models[ Naturwissenschaften 56\ 116Ð122[ 11[ Linnemann D[ and Bock E[ "0878# Cell adhesion molecules in neural development[ Dev[ Neurosci[ 00\ 038Ð062[ 12[ Loo Y[ H[\ Scotto J[ and Wisniewski H[ M[ "0867# Myelin de_ciency in experimental phenylketonuria] contribution of the aromatic acid metabolites of phenylalanine[ Adv[ Expl Med[ Biol[ 099\ 342Ð358[ 13[ Malloch G[ D[ A[\ Clark J[ B[ and Burnet F[ R[ "0876# Glial _brillary acidic protein in the cytoskeletal and soluble fractions of developing rat brain[ J[ Neurochem[ 37\ 188Ð295[ 14[ Margolis R[ U[ and Margolis R[ K[ "0878# Nervous tissue proteoglycans[ Dev[ Neurosci[ 00\ 165Ð177[ 15[ Margolis R[ U[\ Margolis R[ K[\ Chang L[ and Preti C[ "0864# Glycosaminoglycans in brain during development[ Biochemistry 03\ 74Ð77[ 16[ Marin!Padilla M[ "0867# Dual origin of the mammalian neocortex and evolution of the cortical plate[ Anat[ Embryol[ 041\ 098Ð015[ 17[ Matsuo K[ and Hommes F[ A[ "0877# The development of the muscarinic acetylcholine receptor in normal and hyperphenylalaninemic rat cerebellum[ Neurochem[ Res[ 02\ 756Ð769[ 18[ Moonen G[\ Selak I[ and Grau!Wagemans M[!P[ "0876# In vitro analysis of glialÐneuronal communication during cerebellum ontogenesis[ In GlialÐNeuronal Communication in Development and Re`eneration "eds Althaus H[ H[ and Seifert W[#\ pp[ 214Ð227[ NATO ASI Series\ Springer!Verlag\ Berlin[[ 29[ Mostafawy A[\ Nagle J[ B[\ Newill A[ and Braatz E[ "0869# A study of cerebral atrophy in phenylketonuria[ Son! oencephalographic examination of 34 PKU patients[ Neuropadiatrie 1\ 104Ð114[ 20[ Nigam M[ P[ and Labar D[ R[ "0868# The e}ect of hyperphenylalaninemia on size and density of synapses in rat neocortex[ Brain Res[ 068\ 084Ð087[ 21[ O|Callaghan J[ P[ "0877# Neurotypic and gliotypic proteins as biochemical markers of neurotoxicity[ Neurotoxicol[ Teratol[ 09\ 334Ð341[ 22[ Perris R[ and Johansson S[ "0889# Inhibition of neural crest cell migration by aggregating chondroitin sulfate pro! teoglycans is mediated by their hyaluronan!binding region[ Dev[ Biol[ 026\ 0Ð01[ 23[ Rutishauser U[\ Acheson A[\ Hall A[ K[\ Mann D[ M[ and Sunshine J[ "0877# The neural cell adhesion molecule "NCAM# as a regulator of cellÐcell interactions[ Science 139\ 42Ð46[ 24[ Sanes J[ R[ "0878# Extracellular matrix molecules that in~uence neural development[ Ann[ Rev[ Neurosci[ 01\ 380Ð405[ 25[ Tada K[\ Narisawa K[\ Arai N[\ Ogasawara Y[ and Ishizawa S[ "0879# A sibling case of hyperphenylalaninemia due to a de_ciency of dihydropteridine reductase] biochemical and pathological _nding[ Tohoku J[ Expl Med[ 021\ 012Ð020[ 26[ Taylor E[ H[ and Hommes F[ A[ "0872# E}ect of experimental hyperphenylalaninemia on myelin metabolism at later stages of brain development[ Intl J[ Neurosci[ 19\ 106Ð117[ 27[ Tomasiewicz H[\ Ono K[\ Yee D[\ Thompson C[\ Goridis C[\ Rutishauser U[ and Magnuson T[ "0882# Genetic deletion of a neural cell adhesion molecule variant "N!CAM!079# produces distinct defects in the central nervous system[ Neuron 00\ 0052Ð0063[ 28[ Toole B[ P[ "0880# Proteoglycans and hyaluronate in morphogenesis and di}erentiation[ In Cell Biolo`y of Extracellular Matrix "ed[ Hay E[ D[#\ pp[ 294Ð230[ Plenum Press\ New York[[ 39[ Wen G[ Y[\ Wisniewski H[ M[\ Shek J[ W[\ Loo Y[ H[ and Fulton T[ R[ "0879# Neuropathology of phenylacetate poisoning in rats] an experimental model of phenylketonuria[ Ann[ Neurol[ 6\ 446Ð455[
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 04\ No[ 0\ pp[ 18Ð25\ 0886 Copyright Þ 0886 ISDN[ Published by Elsevier Science Ltd Printed in Great Britain[ All rights reserved 9625Ð4637:86 ,06[99¦9[99
PII] S9625Ð4637"85#99970Ð9
EFFECT OF EXPERIMENTAL HYPERPHENYLALANINEMIA ON THE POSTNATAL RAT BRAIN GALINA A[ USHAKOVA\ % HELENA A[ GUBKINA\ VICTORIA A[ KACHUR and EUGENE A[ LEPEKHIN $ Department of Biophysics and Biochemistry\ Dniepropetrovsk State University\ Dniepropetrovsk\ Ukraine^ $International Center for Molecular Physiology of The National Academy of Sciences of Ukraine\ Dniepropetrovsk\ Ukraine "Received 07 March 0885^ revised 18 July 0885^ accepted 0 Au`ust 0885# Abstract*The molecular mechanism of the disturbance to brain development caused by phenylketonuria remains mostly unknown[ We have studied three molecular markers that re~ect the development of neurons\ glia and the extracellular matrix of the postnatal rat brain in an animal model of hyperphenylalaninemia\ in order to elucidate the possible mechanism by which increased phenylalanine in~uences brain development[ The contents of NCAM\ GFAP and hyaluronate!binding activity were compared in cerebellum and telencephalon of normal rats and those subjected to high phenylalanine[ No statistically signi_cant changes were found in telencephalon when experimental animals were compared to controls[ In the hyper! phenylalaninemic cerebellum\ the developmental dynamic of NCAM content "represented by two peaks at about postnatal days 4 and 11 during normal development# is dramatically altered[ The GFAP content in the cerebellum of treated rats exceeded those in controls signi_cantly during late developmental stages "postnatal days 17Ð24#[ Hyaluronate!binding activity in the extracellular protein fraction from treated rat cerebellum was increased compared to normal rat at the early stages of development only "postnatal day 6#[ These results suggest that high serum phenylalanine may lead to permanent brain dysfunction through a disturbance of a wide range of developmental events[ Þ 0886 ISDN Key words] hyperphenylalaninemia\ brain\ NCAM\ GFAP\ hyaluronate\ binding activity[
Phenylketonuria "PKU# is an inherited de_ciency of the enzyme phenylalanine hydroxylase "L! phenylalanine\ tetrahydropteridine] oxygen oxidoreductase!3!hydroxylating^ EC 0[03[2[0#\ which leads to increased serum "and brain# phenylalanine and\ as a consequence\ to abnormal brain development[ This genetic defect leads to imbecility\ if the diagnosis is not made directly after birth[ Molecular mechanisms for the way in which phenylalanine interferes with brain development are not clearly understood to date\ but there is evidence that phenylalanine a}ects every phase of brain development[00 The most crucial e}ect\ the permanent brain damage that leads to mental retardation\ occurs during the early period of brain development and may be connected to improper neuron di}erentiation and synaptogenesis[09\00\20 Such a genetic defect is not known to occur naturally in animals and is modeled in several ways for neurochemical analysis[ The most successful chemical model of hyperphenylalaninemia "HPA# is based on the simultaneous injection of postnatal rat pups with a!methylphenylalanine "an inhibitor of phenylalanine hydroxylase#\ together with phenyl! alanine[04\10 Such treatment leads to the permanent increase of phenylalanine in developing brain17 that induces enduring behavioral de_cit10\20 and thus is used widely as model for human phenyl! ketonuria[ The signi_cant abnormalities were found in cerebrum of patients with PKU\29 while only few changes have been observed in the cerebellum or other brain regions of those patients[7\25 In experimental rat HPA\ the dramatic changes were observed both in the neocortex\00\10\20 and in the cerebellum[8\04\05\39 Currently\ known events associated with HPA are the loss of myelin due to an increase of its turnover\01\26 disintegration of the myelin sheath00 and the reduction of synaptic terminals[20 The latter may be one of the main causes of mental retardation due to HPA\ because dynamic integrative capacities of the central nervous system are based on the proper formation of the synaptic network[03 At the same time\ many aspects of synaptogenesis are mediated by cellÐcell and cellÐextracellular %To whom correspondence should be addressed at] Department of Biophysics and Biochemistry\ Dniepropetrovsk State University\ 61 Gagarin Ave[\ Dniepropetrovsk 219514\ Ukraine[ Tel[] ¦279!451!358!179^ Fax] ¦279!451!354!412[ Abbreviations] ECM\ extracellular matrix^ EDTA\ ethylenediamine tetraacetate^ ELISA\ enzyme!linked immunosorbent assay^ GFAP\ glial _brillary acidic protein^ HA\ hyaluronate^ HABA\ hyaluronate!binding activity^ HPA\ hyp! erphenylalaninemia^ NCAM\ neural cell adhesion molecule^ PKU\ phenylketonuria^ PMSF\ phenylmethylsulfonyl ~uoride[ 18
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matrix interactions\18 but the detailed molecular mechanism of cell communication is still not understood[ The neural cell adhesion molecule "NCAM# belongs to the immunoglobulin superfamily of adhesion proteins and is believed to be involved in a variety of cellÐcell interactions in nervous tissue\ including cell migration\11 the association of axons with pathways and targets23 and synaptogenesis[08 Moreover\ the results of in vivo studies demonstrated brain structural and functional disturbances due to abnormal NCAM expression[3\27 Thus\ the quanti_cation of NCAM in developing brain under conditions of HPA may be useful for understanding the pathogenesis of HPA during brain development[ The extracellular matrix "ECM# of the developing central nervous system is an important factor that a}ects neuronal migration\ as well as the extension of axons and formation of synapses[24 Hyaluronate "HA# is a major component of the ECM that plays a key role in the regulation of morphogenesis and di}erentiation of neural tissues\ including both cell proliferation and migration during development[ The structural location and function of hyaluronate in the brain are probably mediated by its interaction with speci_c HA!binding proteins and proteoglycans[28 The HA!binding domains of reported hyaluronate!binding proteins "versican\ chondroitin sulfate proteoglycan\ GHAP and CD33# are strongly conservative[1 This fact suggests that these proteins have similar functions in immobilization of hyaluronic acid within the extracellular space[ The quantitative measurement of total HA!binding activity during brain development may re~ect the process of ECM formation[ There are many data indicating the participation of astrocytes in a variety of important physio! logical and pathological processes in the brain[5 Alterations in astroglial cells are accompanied by changes in the concentration of the glial _brillary acidic protein "GFAP# * the constituent com! ponent of intermediate glial _laments[2 The expression of GFAP is regulated during development as well as under di}erent physiological and pathological states\ including synapse formation and remodeling and chemically induced damage of the CNS[13\21 Thus\ if high phenylalanine is toxic to the developing brain and this e}ect is connected with myelin destruction and synapse reduction\09 the content of GFAP in the developing brain under HPA conditions may be changed[ In order to study the e}ect of HPA on the development of cells\ we have studied quantitatively] NCAM "neuronal marker# and GFAP "astroglial marker# content and HA!binding activity "ECM marker# in two regions of the rat brain "cerebellum and telencephalon# during postnatal development of rats subjected to HPA[
EXPERIMENTAL PROCEDURES Hyperphenylalaninemic model The animal model of PKU "experimental HPA# was prepared as described by Matsuo and Hommes[17 One hundred and _fty!two Wistar rat pups from 05 litters were used to reproduce experimental HPA in the following manner[ Pups from each litter were divided into two groups\ at least four animals per group[ One group was subjected to the HPA regimen from postnatal days 4 to 24\ and the second group was used as a control[ The rat pups were injected intraperitoneally twice a day at 01 hr intervals\ with a 9[8) NaCl solution containing 9[02 M L!phenylalanine and 9[95 M DL!a!methylphenylalanine "Research Organics\ Cleveland\ Ohio\ U[S[A[# at 9[1 ml per 09 g body weight for the _rst four injections and 9[3 ml per 09 g body weight for the remains[ The control pups were injected similarly with 9[8) NaCl solution[ The validity of experimental HPA model was evaluated by the control of pups body weight that is a proved hallmark of HPA[02\12 Brain extract preparation The pups were sacri_ced by decapitation on postnatal days 6\ 03\ 11\ 17 and 24[ The brains were removed and two areas "cerebellum and telencephalon# dissected and homogenized in 09 vol[ of cold bu}er A "14 mM TrisÐHCl pH 6[3\ 0 mM EDTA\ 1 mM 1!mercaptoethanol\ 9[1 mM PMSF and 9[90) merthyolate#[ The suspensions were centrifuged for 59 min at 099\999 g[ After being washed with bu}er A\ the pellets were extracted consecutively with the same bu}er\ containing
E}ect of hyperphenylalaninemia on brain
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either 1) Triton X!099 "membrane fraction# or 3 M urea "extracellular: cytoskeletal fraction#\ respectively[ All procedures were conducted at 3>C[ NCAM and GFAP quanti_cation These were performed by competitive ELISA[ The content of NCAM was measured in the membrane fraction using polyclonal rabbit antisera against rat brain NCAM obtained in our laboratory[ The antisera reacted with three main NCAM polypeptides] 019\ 039 and 079 kDa\ regardless of the sialylation degree19 "Fig[ 0#[ The _lament GFAP content was measured in the urea fraction using polyclonal rabbit antisera against human GFAP obtained in our laboratory[ Western immunoblotting of the urea!soluble fraction from rat brain with anti!GFAP antisera detected one band of 49 kDa polypeptide "Fig[ 1#[ Hyaluronate!binding activity determination The total HA!binding activity "HABA# was detected in extracellular:cytoskeletal fractions by an enzyme!linked carbohydrate!binding assay[4 Brie~y\ the HABA was measured in protein samples immobilized on the surface of microtiter plates using a hyaluronateÐhorseradish peroxidase con! jugate at pH 4[9 and in the presence of 19 mM NaCl and was expressed as ng of bound HA per mg of protein[ The protein content was determined according to Bradford0 with bovine serum albumin as a standard[ A triplicate assay was done for each sample[ The statistical signi_cance of the discovered alter! ations was calculated by MannÐWhitney|s U!test[ RESULTS Effect of HPA re`imen The intraperitoneal injections of the mixture of L!phenylalanine and DL!a!methylphenylalanine led to pronounced growth retardation of pups "Fig[ 2# that may serve as a sign of model validity[02\12 Moreover\ hyperphenylalaninemic pups appeared unwell and less agile[
Fig[ 0[ Western blot analysis of telencephalon 1) Triton X!099 extract for NCAM[ Twenty micrograms of protein were loaded per well and SDS!electrophoresed under reducing conditions in 6) polyacrylamide gel[ After electrophoresis\ separated proteins were electrophoretically transferred to nitrocellulose paper and immunostained with rabbit anti!NCAM "0]499#[ The antisera reacts with three NCAM polypeptides] 019\ 039 and 079 kDa[
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Fig[ 1[ Western blot analysis of mesencephalon 3 M urea extract for GFAP[ Thirty micrograms of protein were loaded per well and SDS!electrophoresed under reducing conditions in 01\4) polyacrylamide gel[ After electrophoresis\ separated proteins were electrophoretically transferred to nitrocellulose paper and immunostained with rabbit anti!GFAP "0]0999#[ The antisera reacts with GFAP polypeptide 49 kDa[
Fig[ 2[ Rat pup body weight during postnatal development "Ž\ control rats^ ,\ hyperphenylalaninemic rats#[ Hyperphenylalaninemic conditions were created by intraperitoneal injections with 9[8) NaCl solution containing 9[02 M L!phenylalanine and 9[95 M DL!a!methylphenylalanine[ Rats were injected twice a day at 01 hr intervals from postnatal days 4Ð24[ Control animals were similarly injected with 9[8) NaCl solution[
Content of NCAM The developmental dynamic of the NCAM concentrations in the cerebellum of the normal rats has been described previously07 as a curve having peaks at about postnatal days 4 and 11[ The results obtained here have the same pattern of NCAM content changes in the control rats[ Under hyperphenylalaninemic conditions\ NCAM concentrations increased up to postnatal day 03 and
E}ect of hyperphenylalaninemia on brain
22
Fig[ 3[ The NCAM content in cerebellum during postnatal rat development "Ž\ control rats^ ,\ hyper! phenylalaninemic rats#[ The developmental dynamic of NCAM content is signi_cantly altered under hyperphenylalaninemic conditions[
remained increased through postnatal day 24 "Fig[ 3#[ The NCAM content in the telencephalon of hyperphenylalaninemic rats had no statistically signi_cant changes compared to control animals "data not shown#[ Content of _lament GFAP The pattern of GFAP changes in cerebellum and telencephalon was di}erent during postnatal development of the rat brain[ The _lament GFAP content increased up to postnatal day 11 with further stabilization in the telencephalon of control rat pups[ No statistically signi_cant di}erences in GFAP content in telencephalon were observed under HPA conditions "data not shown#[ A single peak of GFAP content was observed on postnatal day 11 with a pronounced decrease of this value on postnatal days 17 and 24 in the cerebellum of control animals[ In the hyper! phenylalaninemic rat pups\ GFAP content was signi_cantly increased in the cerebellum on postnatal days 17 and 24 "Fig[ 4#[ Hyaluronate!binding activity The highest amount of HABA was found in the extracellular fraction of both cerebellum and telencephalon of the control rat pups on postnatal day 6 with a following reduction "more than four!fold# to postnatal day 24[ However\ the dynamic of HABA in the telencephalon was not a}ected by hyperphenyalalinemia "data not shown#[ In the hyperphenylalaninemic cerebellum\ HABA was increased signi_cantly on postnatal day 6 in comparison to control animals\ and there were no di}erences between experimental and control animals\ at later stages of the brain development "Fig[ 5#[ DISCUSSION The data obtained allow us to suggest that increased phenylalanine causes dramatic changes in brain cellular events during development[ The lack of e}ects of HPA on the telencephalon and the well!pronounced e}ect on the cerebellum may be explained by di}erent times of maturation[ At the time of the hyperphenylalaninemic treatment\ the development of the rat telencephalon is near the end\ while the cerebellum is developing intensively[ The dramatic HABA decrease during postnatal development of both brain areas studied probably
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Fig[ 4[ Filament GFAP content in cerebellum during postnatal rat development "Ž\ control rats^ ,\ hyperphenylalaninemic rats#[ Statistically signi_cant "P³9[94# changes of GFAP content under hyper! phenylalaninemic conditions " # were observed on postnatal days 17 and 24[
Fig[ 5[ Hyaluronate!binding activity in extracellular fractions of the cerebellum during postnatal rat development "Ž\ control rats^ ,\ hyperphenylalaninemic rats#[ Statistically signi_cant "P³9[94# increase of HABA under hyperphenylalaninemic conditions " # was only found on postnatal day 6[
re~ects the formation of extracellular matrix via hyaluronic acid immobilization:insolubilization[ Free HA is believed to play an important role in cell migration by providing a highly hydrated and easily penetrated matrix[14 The ratio of bound to soluble HA increases in the maturing rat brain[15 Therefore\ the insolubilization of HA by HA!binding proteins follows neuronal cell migration and may switch developmental processes from migration to di}erentiation by the creation of tight "non! penetrable# extracellular matrix[ It has been shown that hyaluronate!binding region of chondroitin sulfate proteoglycan inhibits neural cell migration\22 while another hyaluronate!binding protein * versican * appeared in the marginal zone of the embryonal rat telencephalon which is the place of
E}ect of hyperphenylalaninemia on brain
24
_nal destination of the migrating neurons[16 The increase of HABA in hyperphenylalaninemic cerebellum at about postnatal day 6 "when massive granule cell migration occurs# may prevent this migration\ interfering with layer formation[ No doubt\ the ECM alteration during early postnatal brain development may lead to the serious consequences for brain maturation[ Another possibility for a disturbance of cerebellar granule cell migration may be related to the alteration of the NCAM developmental pro_le in the cerebellum under HPA conditions[ It has been previously shown that the migration of granule cells coincides in time with the upregulation of NCAM and normally occurs at about postnatal day 6[07 The peak of NCAM content appeared to be shifted to postnatal day 03 under the HPA\ which probably re~ects a delay in the onset of migration[ It is worth noting that\ in reeler mice "characterized by pronounced perturbation of cell migration#\ a delay in NCAM appearance in ontogenesis has been demonstrated[6 At the same time\ it is clear that alterations in intercellular and cellÐextracellular matrix adhesion mediated by NCAM may interfere not only with cell migration\ but also with other NCAM!dependent developmental events in brain[ It is well known that the nervous system responds to diverse injuries by the activation of astrocytes[ One of the generally accepted signs of astrocyte activation is the upregulation of GFAP[ 5 The increase of GFAP at postnatal days 17 and 24 indicates that at least some astroglial cells become activated under HPA[ It may occur as a result of the break in myelin sheath formation\ that has been shown in hyperphenylalaninemic brain[09 Moreover\ our _ndings concerning GFAP content changes in HPA cerebellum resemble those in myelin!de_cient Jimpy mice[06 Therefore\ we can conclude that the e}ects of high phenylalanine on the postnatal rat brain are re~ected by a number of developmental events] "0# an immediate e}ect on hyaluronate!binding activity in extracellular matrix formation^ "1# a dramatic change in postnatal NCAM content^ and "2# an increase of GFAP content during later stages of postnatal cerebellum development[ We believe that these changes may lead to the retardation of brain development and cause permanent brain dysfunction[ Acknowled`ements*This study was supported by Volkswagen Sti}tung "grant I:57401#[ We thank Dr Hommes for the gift of DL!a!methylphenylalanine and Mr S[ Pulin for his help in the English text revision[
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