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Research Report
Altered development of neuronal progenitor cells afterstimulation with autistic blood sera
Bozena Mazur-Koleckaa,⁎, Ira L. Cohenb, Edmund C. Jenkinsc,Wojciech Kaczmarskia, Michael Floryd, Janusz Frackowiaka
aDepartment of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd,Staten Island, NY 10314, USAbBehavioral Assessment and Research Laboratory, New York State Institute for Basic Research in Developmental Disabilities, Staten Island,NY 10314, USAcDepartment of Cytogenetics, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USAdResearch Design and Analysis Service, New York State Institute for Basic Research in Developmental Disabilities, Staten Island,NY 10314, USA
Article history:Accepted 18 June 2007Available online 27 July 2007
Changes of brain structure and functions in people with autism may result from alteredneuronal development, however, no adequate cellular or animal models are available tostudy neurogenesis in autism. Neuronal development can bemodeled in culture of neuronalprogenitor cells (NPCs) stimulated with serum to differentiate into neurons. Because serafrom people with autism and age-matched controls contain different levels of numerousbiologically active factors, we hypothesized that development of human NPCs induced todifferentiate into neurons with sera from children with autism reflects the altered earlyneuronal development that leads to autism. The control and autistic serawere collected fromsiblings aged below 6 years that lived in the same environment. The effect of sera ondifferentiation of NPC neurospheres into neuronal colonies was tested in 72-h-long culturesby morphometry, immunocytochemistry and immunoblotting. We found that sera fromchildrenwith autismsignificantly reducedNPCs' proliferation, but stimulated cellmigration,development of small neurons with processes, length of processes and synaptogenesis.These results suggest that development of network of processes and synaptogenesis – thespecific events in the brain during postnatal ontogenesis – are altered in autism. Furtherstudies in this cell culture model may explain some of the cellular alterations described inautistic patients.
Unknown developmental defects of brain growth, structure,and function cause autism spectrum disorder (ASD), definedclinically as an alteration of cognitive, linguistic, social, and
mr.state.ny.us (B. Mazur-
er B.V. All rights reserved
emotional functions (Acosta and Pearl, 2003). Among theneurodevelopmental alterations that could underlie the pa-thophysiology of autism, the most frequently observed areincreased volumes of whole brain, parieto-temporal lobe, andcerebellar hemisphere, as well as abnormal sizes of amygdala,
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hippocampus, and corpus callosum (Brambilla et al., 2003;Courchesne and Pierce, 2005b). Moreover, altered cyto-architectural organization of cerebral cortex minicolumns(Casanova et al., 2002), and changes in cell sizes and celldensity in the limbic system and cerebellum have beenreported (Acosta and Pearl, 2003). These neuropathologicalabnormalities can be caused by errors that occurred earlyin ontogeny, as suggested by the increased frequency ofmalformations associated with ASD that are caused bydevelopmental insults as early as the 4th to 6th week ofembryogenesis (Miller et al., 2005).
Pre-programmed neurogenesis, i.e., neuronal prolifera-tion, migration, differentiation, growth and circuit organiza-tion, may be strongly affected by factors present in thecellular microenvironment, such as neurotrophins, neuro-peptides, regulatory proteins and neurotransmitters, thelevels of which are altered in the brain, cerebrospinal fluid,and blood of individuals with autism (Acosta and Pearl,2003). Neonatal blood spots of individuals who later dev-eloped autism and/or mental retardation, contained elevatedlevels of brain-derived neutrophic factor, neurotrophin 4/5,vasoinhibitory peptide, and the calcitonin gene-relatedpeptide (Nelson et al., 2001; Miyzaki et al., 2004; Tsai, 2005).Blood of some autistic patients contains altered levels ofserotonin, a monoamine neurotransmitter that regulatesneurogenesis, neuronal differentiation, neuropil formation,axon myelination, and synaptogenesis (Anderson et al., 1990;Chugani et al., 1999; Chugani, 2002; Whitaker-Azmitia, 2005).Altered levels of other neurotransmitter that participate inneuronal integration and interneuron migration such asGABA have been detected in autistic youngsters (Dhosscheet al., 2002). Plasma from autistic patients containedincreased amounts of total nitrite (Zoroglu et al., 2003), ametabolite of nitric oxide, a diffusible intercellular messen-ger expressed by nitrergic neurons participating in sensorymotor function (Moreno-Lopez et al., 1996), and synapticformation and remodeling (Holscher, 1997). Nitric oxide,which has been suggested to be a physiological inhibitor ofneurogenesis (Moreno-Lopez et al., 2004), is increased in redblood cells in autistic patients (Sogut et al., 2003). Theseexamples of alterations in body fluid concentrations of somefactors confirm that the internal environment in people withautism is disrupted. Most authors conclude that alteredlevels of these factors in the periphery may reflect changesin brain development that underlie autistic pathology,although the mechanisms of such periphery–brain inter-actions during early development and during postnatal lifeare unclear. Not fully functional blood–brain barrier duringearly brain development may be responsible for disruptionof serotonin terminals in the brain through a negative feed-back function due to high levels of serotonin in the blood(Whitaker-Azmitia, 2005). In rats exposed prenatally tothalidomide or valproic acid, regarded as an animal modelof autism, increased concentrations of serotonin were de-tected simultaneously in both the brain and blood on post-natal day 35 (Narita et al., 2002), suggesting that autism-inducing factors have the same continuous effect on themonoamine system in the brain and the periphery duringdevelopment and in postnatal life. Hence, the autism-inducing factors may influence development of neurons
and neuronal network (neurogenesis) that takes place duringearly brain development, postnataly and also during adultlife (Johansson et al., 1999; Nunes et al., 2003; De Graaf-Petersand Hadders-Algra, 2006; Quinones-Hinojosa et al., 2006;Taupin, 2006).
The critical steps of early neurogenesis can be studied incultures of neuronal progenitor cells (NPCs) isolated fromhuman fetal tissues or from mature brain. These cells main-tain the ability to differentiate into distinct brain cell lineages,i.e., neurons and glia (Gage et al., 1995; Carpenter et al., 1999;Temple and Alvarez-Buylla, 1999; Gage, 2000). Self-renewaland differentiation of NPCs into specific neuronal phenotypescan be stimulated in culture with specific growth factors,cytokines, and neurotransmitters (Weiss et al., 1996; Cameronet al., 1998; Sogut et al., 2003; Temple and Alvarez-Buylla,1999; Gage, 2000; Ostenfeld and Svendsen, 2003). Develop-ment and differentiation of NPCs in culture can be triggeredby blood serum that contains a mixture of numerousregulatory factors (Johe et al., 1996). Because the localenvironment is the predominant determinant of NPC differ-entiation (Cao et al., 2002a), we hypothesize that serafrom people with autism – the sera that show altered levelsof regulatory proteins – will differently affect the develop-ment of human NPCs (HNPCs) in culture. The alterations ofneurogenesis in culture may reflect alterations of neuronaldevelopment during postnatal life in people with autism. Totest this hypothesis, the development of neurospheres ofHNPCs into colonies of neurons was studied after stimulationwith sera from individuals with autism and age-matchedcontrols.
Our results suggest that the altered body environment inautism could influence migration of NPCs, development ofnetwork of processes and synaptogenesis – the specific eventsin ontogenesis that characterize postnatal brain development.Our new cell culture model may be the tool to identify thespecific cellular mechanisms involved in the altered neuro-genesis thatmay lead to the structural and functional changesthat are observed in autism.
2. Results
2.1. Electrophoretic patterns of serum protein
Capillary zone electrophoresis was used to semiquantitate thetotal amount of serum proteins and five main protein frac-tions, i.e., gamma, beta, alpha 1 and alpha 2 globulins, andalbumins. Electropherograms from control and autism casesrevealed similar patterns without individual peaks withinglobulin fractions (representative electropherograms shownin Fig. 1A). Total serum protein levels and protein levels in fivemain fractions in control and autistic age-matched cases didnot differ significantly (Fig. 1B).
2.2. Characterization of cells grown from neurospheresof HNPC
Neurospheres of HNPCs cultured for 72 h in control conditions(NBM enriched with 1% human serum from healthy donors)formed colonies of cells that all were immunoreactive for
Fig. 1 – Amounts of blood serum proteins: gamma (γ), beta (β), alpha 1 (α1) and alpha 2 (α2) globulins, and albumin (alb) tested bycapillary zone electrophoresis. Representative electropherogram (A) and amounts of proteins in sera collected from control andautistic subjects (B). Results represent means±SD from double-run samples.
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neurofilament 200, a marker of mature neurons (Fig. 2). Cellsin colonies were a morphologically heterogenous population,with numerous small cells with processes, and some largerflat cells, usuallywithout processes. Small cells were classifiedas those with a nuclear index, calculated as the area ofnucleus/the area of cell body, above 0.25, while large cellswere those with a nuclear index below 0.25. The averagenuclear indexes of small and large cells were 0.37±0.09 and0.09±0.02, respectively. Small cells usually expressed strongimmunoreactivity for neurofilament 200, which was localizedin cell bodies and processes. Large cells usually showed weakimmunoreactivity for neurofilament 200 localized in cellbodies (Fig. 2, cells marked with asterisks).
Fig. 2 – The scheme of the study of neuronal development triggeage-matched controls. Free-floating neurospheres of HNPCs werproliferation (EXPANSION). Differentiation of HNPCs into neurongrowth factors (DIFFERENTIATION). Images show free-floating neneurospheres (phase-contrast microscopy) and a colony of differclarity of the scheme, proportions between sizes of the images wcomposite images (floating neurospheres and a neuronal colony)the image of a neuronal colony shows morphology of small and
2.3. Proliferation of HNPCs
BrdU applied simultaneously with 1% of serum was incor-porated into nuclei of dividing HNPCs in neurospheres thatwere in the S phase, and at the end of the 24-h culture boththose dividing cells and their progeny were labeled with BrdU.Hence, our results represent the effect of tested serum on allthe phases of the cell cycle. Cultures grown in control con-ditions, i.e., in NBM supplemented with 1% serum from thecontrol group, contained about 24% labeled cells. Neuro-spheres cultured in autistic conditions, i.e., in NBM supple-mented with 1% serum from the autistic group, showed asignificantly (p=0.03) reduced number of BrdU-labeled cells
red in culture with sera from children with autism or frome expanded in medium with growth factors that maintains was induced with 1% serum in fresh medium deprived ofurospheres, planted neurosphere and cell migrating fromentiated cells (immunostaining for neurofilament 200). Forere changed, and borders of individual images in thewere partly erased using Adobe Photoshop software. Inset inlarge ( * ) cells magnified 3.5×.
Fig. 3 – Human NPCs labeled with BrdU (brown nuclei) during the first 2 h of treatment with 1% sera from control cases (A) andage-matched individuals with autism (B). The graph shows means from 5 to 7 experiments+SD, *pbbbb0.05.
Table 1 – Morphometric evaluation of cell colonies thatdeveloped from neurospheres of human NPCs afterstimulation with sera from boys: control and with autism
Controls Autism p a
Number of cases 9 11Colony increase index (Ix) b 76±38 106±50 0.009Cell density (n/mm2)c 142±53 105±46 0.001Small cells with processes (%)c 63±12 70±10 0.002Number of processes per a cellwith processes (n)
1.41±0.3 1.55±0.29 0.056
Length of processes per a cellwith processes (mm)
0.08±0.02 0.1±0.02 0.0012
Number of processes per anaverage cell (n)
0.89±0.29 1.1±0.3 0.0015
Length of processes per anaverage cell (mm)
0.053±0.02 0.07±0.02 0.0001
Results represent means±SD.a Statistical significance of controls versus age-matched childrenwith autism.b The colony increase index (Ix)=the area of a colony after 72 h/areaof the original neurosphere 2 h after planting.c Cell density, cells with processes, and processes were evaluated infour randomly selected areas (one per each quarter of a colony) in 15%of the peripheral radial zone of colonies in the periphery of colonies.
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(Fig. 3). No morphological features of BrdU toxicity in colonieswere observed.
2.4. Morphometric studies of colonies of HNPCs grownfrom neurospheres
Morphometric evaluation was performed in 4–12 cultures ofHNPCs stimulated with each serum control or autistic. Theability of neurospheres to adhere to pre-coated plastic inmedia supplemented with sera was evaluated as the fractionof neurospheres planted at time 0 that developed into cellcolonies during 72 h of culture. The fractions of neurospheresthat developed into colonies were similar in both the controland autistic groups, and were equal to 0.75±0.13 and 0.76±0.15, respectively.
The ability of HNPCs to migrate from neurospheres and toform colonies of neuron-like cells was evaluated bymeasuringthe area of each neurosphere that anchored 2 h after plantingand, subsequently, the areas of cellular colonies that grewfrom each individual neurosphere during 72 h of culture. Toreduce the possible effect of the size of the neurosphere on itsability to form a colony, only neurospheres of the size between0.010 mm2 and 0.075 mm2 were counted. The average sizes ofneurospheres measured 2 h after planting were similar in thecontrol and autistic groups and were equal to 0.032±0.02 and0.03±0.02, respectively.
The dynamics of growth of each colony was expressed asthe colony increase index (Ix)=the area of a colony after 72 h /the area of the original neurosphere 2 h after planting. Theaverage Ix in the autistic group was significantly higher(p=0.009) than the average Ix in the control group (Table 1).
Cell densities in colonies, development of morphologicallydifferent subpopulations of cells, number and length of cellprocesseswere evaluated in four randomly selected areas (oneper each quarter of a colony) in 15% of the peripheral radialzone of colonies, i.e., in the regions that contained low densityof cells that migrated the farthest from each neurosphere andwere the most differentiated. The radial zone was measuredfrom the center of each colony. The total areas evaluated incolonies growing in control and autistic conditions were about30 mm2.
Cell densities in colonies (calculated as number of cells permm2) in colonies grown in “autistic” conditions were lowerthan in control conditions (p=0.001) (Table 1).
Development of morphologically different subpopulationsof cells in colonies was evaluated as percentages of small cells
developing processes and large cells. Colonies that developedin “autistic” conditions contained significantly more smallcells that develop processes (p=0.002) (Table 1).
The number of processes going out from all cells in selectedareas was counted. The total length of processes wasmeasured from cell body to the process tip, including allbranches, within selected areas. An average cell that differ-entiated in the control conditions developed 1.41 processes ofa total length of 0.08 mm. The number of processes per cell inthe subpopulation that developed processes in colonies grownin the autistic conditions was similar (p=0.06), while thelength of processes was significantly increased (p=0.0012)(Table 1). Thus, the neuron that differentiated in the autisticconditions developed the same number of processes, but theywere longer than in neurons that differentiated in controlconditions.
Formation of a network of processes in whole colonieswas assessed as the number of processes, and the length ofprocesses, calculated per total cells present in tested areas,i.e. cells which developed and did not develop processes.Both the number and the length of processes per total cells
Fig. 4 – SV2, a synaptic vesicle protein, detected in HNPCs grown fromneurospheres for 72 h in neural progenitor basalmediumsupplemented with 1% of human blood serum from control cases (control) and age-matched individuals with autism. Arepresentative image of cells immunostained for SV2 (left panel), immunoblots (middle panel) and densitometric evaluations oftotal SV2-immunoreactive material from four double-run samples (right panel) presented as fractions of actin levels (Ix).Positive controls (pc ) represent brain lysates from 22-day-old and 33-year-old controls. *pbbbb0.02.
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were significantly increased in colonies that were formedin the presence of sera from the children with autism, ascompared to colonies stimulated by sera from the controlchildren (Table 1).
2.5. Synaptogenesis in colonies of HNPCs grown fromneurospheres
Numerous cells in colonies showed immunoreactivity for syn-aptic vesicle protein SV2 that formed clusters associated withcell surfaces and in cell processes (Fig. 4).
Synaptogenesis was evaluated by measuring the levels ofsynaptic vesicle protein (SV2) in cell lysates detected byimmunoblotting with mAb against SV2. In brain lysate froma 33-year-old control case used as positive control for themAbagainst SV2, the antibody recognized abundant proteins ofsizes above 70 kDa, which represent glycosylated forms of SV2protein showing a distinctive heterogenous mobility in SDS–polyacrylamide gels (Feany et al., 1992). In brain lysates usedas positive controls, mAb against SV2 recognized proteins inthe range of 60–80 kDa and about 205 kDa. In cell lysates, theanti-SV2-immunoreactive protein was detected as 60–80, 105,and 205 kDa bands (Fig. 4). The total level of SV2 protein, i.e.,all SV2-immunoreactive material at all molecular sizes (allbands) was calculated as a fraction of actin levels and pre-sented as Ix. The levels of actin, recognized as a single 45-kDaband, were not significantly different between tested cultures(Fig. 4). The levels of total SV2-immunoreactive material afternormalization for actin-immunoreactive material were in-creased about 60% in cells grown in the presence of sera fromsubjects with autism (p=0.02). Apart from the quantitativedifferences recognized with SigmaGel software that wereweakly recognizable by human eye, the representative immu-noblots showed qualitative differences – the 105- and 205-kDabands immunoreactive for SV2, that were clearly detectableonly in cells grown in the presence of sera from subjects withautism (Fig. 4).
3. Discussion
The differences in several features of neurogenesis triggeredin cultures of HNPCs by sera from children with autism versusage-matched controls suggest that cell proliferation, migra-
tion, formation of processes and synaptogenesis – the pro-cesses that characterize postnatal brain development (DeGraaf-Peters and Hadders-Algra, 2006) – are altered in autismand that such alterations can be detected for the first time incell culture.
3.1. HNPCs as a model to study neuronal development
Neurospheres of mammalian NPCs, which differentiate intoneurons after deprivation of growth factors and exposureto appropriate differentiating factors, are a commonly usedmodel to study in cell culture genetic and epigenetic agentsin etiopathology of numerous diseases (Bhattacharyya andSvendsen, 2003; Kawakita et al., 2006; Pagani et al., 2006;Phillips et al., 2005; Riaz and Bradford, 2005). Previously, wedemonstrated that components of extracellular environmentassociated with Down syndrome and Alzheimer's diseaseinfluence development and differentiation of the HNPCs inculture (Mazur-Kolecka and Frackowiak, 2006; Mazur-Koleckaet al., 2006). Now, we demonstrated that extracellular factorspresent in serum affected development of HNPCs; however,we cannot exclude the possibility that the HNPCs used inthese experiments had the genetic background associatedwith autism (Bartlett et al., 2005), i.e., isolated from a fetus thatcould develop into a child with autism. This issue cannot beaddressed until genetic markers of autism are confirmed.
3.2. Proliferation of HNPCs and colony growth
The reduced proliferation of HNPCs in autistic conditionssuggests a reduced ability of HNPCs for self-renewal but alsoan accelerated differentiation of post-mitotic progenitors intoneural phenotypes. Moreover, the increased colony growthindexes and tendency to the reduction of cell density inautistic conditions suggest enhanced migration of HNPCsfrom neurospheres. Both proliferation and migration of NPCmay have a profound impact on brain development. In theontogeny of children with autism, the periods of acceleratedbrain growth followed by abnormally slow growth and apremature arrest of growth in certain regions have beendemonstrated (Aylward et al., 2002; Courchesne and Pierce,2005b). Hence, we have initiated further studies of cell cycle,cell renewal and cell death rates in cultures stimulated withsera from children with autism.
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Reduced proliferation of HNPCs may result in reduced self-renewal of NPCs in an autistic brain. Multipotential NPCspersist in the brain into adulthood (Ostenfeld and Svendsen,2003) and were demonstrated to generate new neuronsthroughout life, e.g., those located in the periventricularzone generate neurons destined for the olfactory bulb (Gageet al., 1995; McKay, 1997; Kukekov et al., 1999). Abnormalresponse to smell (that may result from altered neurogenesisin the olfactory bulb) has been reported by parents of toddlerswith autism (Rogers et al., 2003). The reduced proliferationof NPCs in the autistic brain may impair processes known tobe associated with increased proliferation of NPCs, e.g.,learning and memory (Gould et al., 1999; Aimone et al., 2006;Shimazu et al., 2006; Taupin, 2006; Nilsson et al., 1999; Shorset al., 2001). The poor memory for complex visual and verbalinformation and spatial working memory characterize child-ren with autism (Williams et al., 2006).
3.3. Development of small neurons with processes
Colonies that grew in autistic conditions showed a higherpercentage of small cells with processes. The pathologicalconsequences of development of such a population of neuronsare difficult to predict, although it may be speculated that anautistic environment promotes development of particularlineages of neurons or those destined for a specific site. Anincreased density of small immature-appearing neurons wasdescribed in the limbic system in autistic brain (Courchesne,1997; Bauman, 1999). A disrupted number (Cohen, 2006) and/orfunction of specific neuronal cell lineages, e.g., serotoninergicneurons, are suggested to be a causative factor in autism(Scott and Deneris, 2005) but lack of experimental data makesthis concept impossible to verify at present. HNPCs are knownto differentiate into several distinct neuronal subtypes (Car-penter et al., 1999), hence, long-term cultures may identifydifferences in induction of distinct neural phenotypes inautistic and control conditions.
3.4. Development of network of processes
Individual neurons differentiating in autistic conditions dev-eloped similar numbers of processes as in control conditions,but these processes were longer. Because the percentage ofneurons with processes was also increased, as we discussedabove, the networks of processes were more dense in coloniesgrew in autistic than in control conditions. Moreover, inautistic conditions, the cells produced more synaptic vesicleprotein SV2, suggesting increased synaptogenesis. Moreover,the appearance of the high molecular bands pattern suggestschanged glycosylation of SV2 (Feany et al., 1992) in autisticconditions, but the biological significance of this fact is notclear. All of the above may result in vivo in formation of moreconnections between cells and with more in-coming and out-going impulses, yielding consolidation of a functionally spcificneural network or, on the contrary, an informative chaos(Cohen, 2006). Developmental dysfunction of the neuralconnections between the limbic structures and other parts ofthe brain aswell as delayedmaturation and dysfunction of thefrontal circuitry was suggested in autism (Minshew et al., 1993,1999; Zilbovicius et al., 1995). The local over-connectivity
within the frontal lobe and poorly synchronized and reducedactivity between the frontal cortex and other systems had alsobeen suggested (Courchesne and Pierce, 2005a).
All the findings discussed above were not associated withoverexpression of specific proteins or monoclonal antibodiesin our cohort of donors with autism, as indicated by electro-pherograms of sera. Because a number of diseases associatedwith acute and chronic inflammation and autoimmune reac-tions, as well as cancer, and some genetic diseases (Paganaand Pagana, 1998) would be manifested by altered levels ofspecific protein fractions we assumed that sera donors werehealthy.
3.5. Conclusions
We attempted to develop a cell culture model suitable tostudy neurogenesis in autism that could be used in researchon etiology and pathomechanisms of autism. The specificchanges of differentiation of HNPCs after stimulationwith serafrom children with autism that we observed may model somealterations of postnatal neurogenesis. The results suggest thatcell proliferation, migration as well as formation of a networkof processes and synaptogenesis – the events that occur duringpostnatal brain development (De Graaf-Peters and Hadders-Algra, 2006) – were altered in the “autistic environment” thatwe generated. Aberrant neuronal and glial plasticity duringpostnatal brain development has been postulated in autism(Zoghbi, 2003; Dong and Greenough, 2004) but the responsiblemechanisms are not known. Hypothetically, the biologicalfactors, which cause autism, may act continuously in onto-genesis, i.e. throughout the pre-natal and post-natal life,changing the peripheral and brain environments and influ-encing neurogenesis. However, the unknown factors thatinfluence postnatal neurogenesis and brain plasticity may beeven more critical in the development of autism.
Regardless of all limitations associatedwith the cell culturemodel, small number of patients, uncertain selection criteria,preliminary nature of the study, etc., this model may be usedto identify the mechanisms responsible for the alteredneuronal differentiation in culture in autistic conditions, andmay be helpful in understanding pathogenesis in autism.Further studies with the use of sera from older children mayexplain if neurogenesis in the autistic conditions is altered orshifted in time in comparison with control conditions. Iden-tification of the alterations of neurogenesis that are specificfor autism needs additional testing of sera from children withdevelopmental disabilities other than autism because autismis frequently associated with other neurodevelopmentaldisorders (Acosta and Pearl, 2003). The study will also beexpanded to children with autism from distinct geographicalregions to evaluate the influence of external environmentalfactors on serum activity.
4. Experimental procedures
4.1. Sample collection
Our investigation involving human subjects has been ap-proved by the Institutional Review Board of the New York
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State Institute for Basic Research in Developmental Disabil-ities, in accordance with the NIH Guide and Federal WideAssurance FWA00006105.
Diagnosis of ASD was carried out using the Autism Dia-gnostic Observation Schedule–Generic (ADOS-G) (Lord et al.,2000). The ADOS-G is filled out based on observations madeduring the assessment. The ADOS-G protocol involves a seriesof tasks presented by a trained person. The sophistication ofthe task varies according to the language level of the personassessed. The reactions of the proband are coded, and adiagnostic algorithm is then employed to arrive at a diagnosisof autism, ASD, or neither of these. The examination takesabout 30 to 45 min to complete, depending on the moduleused. Sensitivity and specificity for ASD vs. other diagnosesare quite good for all modules.
Diagnosis was confirmed using the Autism DiagnosticInterview–Revised (ADI-R) (Lord et al., 1994), a briefer versionof the ADI, through parent interview. The ADI-R takes abouttwo h to administer and can be used to classify children asyoung as 2 years of age if their mental age is greater than18 months. Sensitivity and specificity are excellent (96% and92%, respectively). For children with a mental age less than18 months, DSM-IV criteria and ADOS-G ratings were used fordiagnosis.
Blood samples were collected from children in familiesfrom the same population located in the NYC area to limitinfluences of the external environmental factors, e.g., airpollution and water contaminations. Variety of dietary,differences in child care, maternal and children diets, ex-posures to customs and preferences associated with house-hold, and other non-specific environmental factors, etc., werenot controlled because relevant conditions associated withdevelopment of autism are not established yet. Samples ofsera selected for this study comprise two experimentalgroups: control (n=9 white males, the age range from 1 yearand 5months to 5 years and 4months, the average age 3 yearsand 8 months) and autistic (n=11 white males, the age rangefrom 2 year and 3months to 4 years and 9months, the averageage 3 years and 2 months). Control samples were obtainedfrom normal children that were evaluated by the samemethods as children with autism. All controls had siblingswith autism at different age and gender, but only two relatedpairs of brothers at the similar agewere selected for study. Theremaining blood donors were unrelated. Most donors of bothcontrol and with autism were re-evaluated during develop-ment. Blood samples in autistic group were collected fromchildren that met ADOS-G and DSM-IV criteria for autism. TheADI-R scores in autism group were: QA in Reciprocal SocialInteraction 18.1±6.3, range 8–26; QA in Communication 13.6±4.3, range 10–22; Restricted, Repeat & Stereo 5.9±2.8, range3–12; Abn of Dev before 36 months 3.8±0.8, range 3–5. Theywere additionally characterized with the use of the VinelandAdaptive Behavior Scale (Sparrow et al., 1984), and the scoreswere: Communication 70.8±14.3, range 56–134; daily LivingSkills 65.6±5.5, range 56–73; Socialization 64.8±9, range55–104; Motor 69.5±10.4, range 53–94. The level of IQ –known not to be a diagnostic feature for autism – was nottested and was not used as a criterion for selection of seradonors. Blood was collected from healthy children that didnot have seizures and gastrointestinal disturbances, none of
them had fragile X syndrome or Rett syndrome and allwere free of medication, particularly antidepressants, neuro-leptics, seizure medications and stimulants. The healthstatus was additionally confirmed by a capillary zone electro-phoresis of serum proteins. All sera were collected in thesame protocol and were stored as coded anonymous samplestogether at −20 °C in the Laboratory of Cytogenetics of IBRDD.Before experiments, sera were thawed once and were addedto the culture medium in the amount needed for all tests.Aliquots of serum-supplemented media were filtered (0.1-μmfilter) to remove possible contamination with bacteria, myco-plasma, etc.
4.2. Capillary zone electrophoresis
Capillary zone electrophoresis of serum proteins was used toscreen for the health status of donors, as well as to evaluateprotein fractions (mainly globulins) in sera of children withautism and age-matched controls. This is a highly sensitivemethod that requires a minimal quantity of serum sample forfast and automated protein separation and produces digitalabsorbance data appropriate for mathematical analysis.
The analyses were performed in uncoated fused-silicacapillaries (75-μm i.d.; effective length, 50 cm) on a BeckmanCoulter P/ACE MDQ Glycoprotein system equipped with asingle-wavelength ultraviolet absorbance detector and aninterference filter at 204 nm. Serum samples were diluted20-fold with Paragon 100 buffer (Beckman Coulter) that wasalso used in electrode vials. Diluted samples were electro-injected into a capillary tube, and the proteins were separatedat 23.5 kV at 37 °C for 20min, as was adapted from themethoddescribed by Gay-Bellile et al. (2003). Each sample of serumwas run twice, and protein fractions were measured onelectropherograms as independent absorption peaks in AU.Results in each experimental group were pooled and analyzedstatistically.
4.3. Cell culture
HNPCs (Cambrex Bio Science Walkersville, Inc., Walkersville,MD) originating from the fetal cerebral cortex (16–20 weeks ofgestational age) co-express markers specific for the neuronallineage – β-tubulin III, and for the astrocytic lineage – glialfibrillary acidic protein (GFAP), similarly as it has been des-cribed in other NPC lines during early neurogenesis (Sergent-Tanguy et al., 2006) and in neuronal stem cells in thesubventricular zone of human brain (Sanai et al., 2004). Neuro-spheres of these HNPCs can be stimulated to develop coloniesof neuron-like cells that express neuronal markers: neuro-filament 200 and NeuN (Mazur-Kolecka et al., 2006). Cells inneuronal colonies express immunoreactivities for synap-tophysin and synaptic vesicle protein SV2 and developprocesses that are immunoreactive for tau and microtubule-associated protein 2 (MAP-2) (Mazur-Kolecka et al., 2006).HNPCs were expanded in culture conditions recommendedby the supplier, i.e., in uncoated T75 flasks (105 cells per flask)as free-floating aggregates (neurospheres) in neural progeni-tor basal medium (NBM) supplemented with human recom-binant basic fibroblast growth factor, human recombinantepidermal growth factor, neural survival factor-1, and genta-
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micin/amphotericin-B (SingleQuots provided by Cambrex) at37 °C in 5% CO2 (Fig. 2: “Expansion”). The growth factors had tobe added to maintain viability and proliferative activity ofHNPCs. Half of the medium was changed every 4 days. Every10–14 days, neurospheres were collected into 15-ml tubes,centrifuged at 120×g for 5 min, and dispersed mechanically bypassing through a 75-μm mesh. Mechanical dispersionallowed retention of the cell–cell contact, resulting in fasterrecovery and higher expansion rates, as described (Svendsenet al., 1998). To obtain the number of cells sufficient forexperiments, neurospheres were expanded in culture for 4–6 weeks; that is, they were mechanically dissociated andpassaged 3–5 times.
In order to trigger differentiation, one or two neurospheresfrom the same passage 24 h after the last mechanical dis-persion were planted per well in 96-well plates pre-coatedwith polyethylenimine (Cambrex) and cultured in NBMwithout growth factors but supplemented with 1% of serum,as described (Mazur-Kolecka et al., 2006). Supplementation ofNBM with human serum collected from healthy donorstriggered differentiation in “control conditions” while serumfrom age-matched people with autism triggered differentia-tion in “autistic conditions”.
4.4. Cell proliferation
Proliferating HNPCs were detected as the cells incorporatingbromodeoxyuridine (BrdU) during 2 h of labeling, as recom-mended (Cao et al., 2002b). Neurospheres were plated onplastic coverslips pre-coated with polyethylenimine in 24-wellplates. Culture was in NBM without growth factors, supple-mented with 1% of human serum collected from control orautistic subjects, andwith 10 μg/ml bromodeoxyuridine (BrdU)(Sigma) for 2 h. Then neurospheres were washed 3 times andcultured in NBM without growth factors, supplemented with1% of serum for the next 24 h. During that time, neurosphereswere flatted into a monolayer of cells attached to surface.Incorporated BrdU was detected with sheep anti-BrdU anti-body (1:200) from Biodesign (Saco, ME) after pretreatment with10 mM citrate buffer, pH 6.0, and boiling in microwave oven3×5 min. The percentages of cells with BrdU-immunoreactivenuclei were estimated among 200–300 cells inmonolayer. Thismethod allows cells to remain attached to the surface duringprocedure and preserves a good morphology of cells growingin monolayer for evaluation and counting, as described(Mazur-Kolecka et al., 2006).
4.5. Morphometric studies
Neurogenesis in cell culture was evaluated by the ability ofHNPCs to form colonies of neuronal cells. The morphometricstudies were performed as described previously (Mazur-Kolecka et al., 2006). 1–2 neurospheres were planted per wellin a 96-well plate and cultured in 0.15 ml medium containing1% tested serum for 2 h for adherence. Then, an image of eachanchored neurosphere was taken using the COHU high-performance color CCD video camera mounted on the NikonDiaphot inverted phase-contrast microscope and Mochasoftware (Jandel Sci). After 72 h, cultures were fixed andimmunostained for neurofilament 200 with polyclonal anti-
body diluted 1:300 (Sigma, Saint Louis, MO). A series of imagesthat covered the whole area of each colony growing from eachindividual neurosphere was taken using a 10× objective lens.Composites of images were generated using Adobe Photoshopsoftware. The sizes of neurospheres (2 h after planting), thesizes of each resulting colony of differentiating cells (after72 h), the areas covered by cells in the whole colony, thenumber of cells, nuclear sizes, cell sizes, and the numbers andlength of cell processes were measured with the Image Jsoftware (NIH) after converting the grayscale images into blackand white images.
4.6. Synaptogenesis
Synaptic vesicles were detected with mAb against a synapticvesicle marker SV2 (a keratan sulfate proteoglycan) diluted1:100 (Developmental Studies Hybridoma Bank, University ofIowa, Iowa City, IA).
Cell lysates were collected after 72 h of culture in 1% serumfrom control or autistic subjects. Lysates were prepared in10 mM Tris buffer, pH 7.5, containing 0.15 M NaCl, 0.65%Nonidet P-40, and a mixture of protease inhibitor cocktail(Roche Molecular Biochemicals, Germany). The protein con-tent was measured with the BCA protein assay (Pierce).Induction of synaptogenesis in colonies was estimated basedon the expression of synaptic vesicles protein. 15-μg samplesof cell lysates were subjected to SDS–PAGE in 8% gels, blottedonto nitrocellulose, and developed with mAb against asynaptic vesicle marker SV2 (from DHB, IA) and mAb anti-actin (Affinity BioReagents) which was used to normalize theamount of SV2 protein. Actin is a ubiquitous cytoskeletalprotein that forms the three-dimensional network inside allhuman cell and is commonly used to ensure equal proteinloading.
The lysates of the control brains (frontal cortex), from 22-day-old and 33-year-old donors (IBR Brain Bank, and theHarvard Brain Tissue Resource Center, respectively) were usedas positive controls for the mAb against SV2. Densitometricmeasurements were performed with SigmaGel software( Jandel).
4.7. Statistics
Results for the control and autistic groups were statisticallyevaluated by Student's t-test (Stat View for Windows, SASinstitute, 5.0.1). Pb0.05was considered statistically significant.
Acknowledgments
The authors thank Michal Kolecki, M.S., for his voluntarywork on computer image analysis and transformation of allimages into files suitable for morphometric studies. Mono-clonal antibody against synaptic vesicles SV2 was obtainedfrom the Developmental Studies Hybridoma Bank (Universityof Iowa, Iowa City, IA). The antibody was developed by K.M.Buckley, under the auspices of the NICHD, and was main-tained by the University of Iowa, Department of BiologicalSciences, Iowa City, IA 52242. The brain samples wereprovided by the Harvard Brain Tissue Resource Center,
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which is supported in part by PHS grant number MH/NS31862. This work was supported by funds from the New YorkState Office of Mental Retardation and Developmental Dis-abilities (NYS OMRDD); the March of Dimes Birth DefectsFoundation (I.L. Cohen, P.I. – #12-FY03-42); and the NYSSpecial Legislative Grant for Autism Research (B. Mazur-Kolecka, P.I. – #M40438).
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