The Variation of Aneuploidy Frequency in the Developing and Adult Human Brain Revealed by an Interphase FISH Study
National Center of Mental Health, Russian Academy of Medical Sciences, Moscow, Russia (YBY,IYI,VVM,IVS,VMV) and Institute of Pediatrics and Children's Surgery, Russian Ministry of Health, Moscow, Russia (SGV)
Correspondence to: Y.B. Yurov, National Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe sh.2, 119152, Moscow, Russia. E-mail: y_yurov{at}yahoo.com; i_yurov{at}mail.ru
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Summary |
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(J Histochem Cytochem 53:385390, 2005)
Key Words: developing and adult human brain aneuploidy DNA probes FISH
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Introduction |
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There are only a limited number of molecular cytogenetic studies of the human brain using interphase FISH. The use of FISH was reported for examination of the interphase nuclei chromosomal complement in the adult human brain (Yang et al. 2001). A significant fraction of the hippocampal pyramidal and basal forebrain neurons in Alzheimer's disease was demonstrated to have fully or partially tetraploid chromosome complement. This imbalance in chromosome complement was proposed to be the direct cause of neuronal loss in Alzheimer's disease. Molecular cytogenetic study of postmortem brain of schizophrenic patients using multicolor FISH (mFISH) has been performed. A statistically significant level of aneuploidy (up to 4% of neurons) was detected in postmortem brain samples of some patients with schizophrenia (prefrontal cortex, Brodmann's area 10). These data have indicated the possibility of a low-level of chromosomal mosaicism being involved in the pathogenesis of schizophrenia (Yurov et al. 2001
). There is only one preliminary study using mFISH and organotypic cell cultures of the fetal human brain that indicates that the developing human brain contains a large proportion of chromosomally abnormal neuronal cells (Yurov et al. 2003
). Systematic studies of chromosome variations in non-dividing neuronal cells in the human fetal brain and comparative analysis of the aneuploidy level in adult and developing human brain have not been performed to date.
A resource of human DNA probes for the molecular cytogenetic detection of chromosomal aberrations has been developed. This collection was initially designed for identification of the most common chromosome aberrations in fetal and postnatal uncultured cells as well as metaphase spreads obtained by cell culturing. The DNA probe collection includes the set of centromeric and pericentromeric DNA probes for all human chromosomes, telomeric and subtelomeric probes, and band-specific DNA probes for a large number of human chromosome regions (Yurov et al. 2002). The original collection, containing a broad spectrum of DNA probes, was found to be applicable for different chromosome complement studies (Soloviev et al. 1995
, 1998
; Vorsanova et al. 1986
,1994
; Yurov et al. 1996a
,b
, 2002
). The collection includes more than 800 selected and accurately tested DNA probes as recombinant plasmid, cosmid, YAC, and PAC clones specifically marking different regions of all 24 human chromosomes (Yurov et al. 2002
). For this pilot FISH study of chromosome variations in the human brain, chromosome-enumerating DNA probes for chromosomes 1, 13, 18, 21, X, and Y from the original collection of probes were selected.
Taking into account that chromosomal complement instability in developing neuronal cells could have substantial effect on normal brain development and functions, direct studies of chromosomal complement in the human brain are of great significance. The aim of the present study was the comparative analysis of chromosomal complement variations in uncultured as well as cultured neuronal cells of developing and adult human brain samples using interphase mFISH.
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Materials and Methods |
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Six samples (five female and one male) of postmortem adult brain tissues were processed as described previously (Yurov et al. 2001). Fifty milligrams of these samples was homogenized in 2 ml of phosphate-buffered saline (PBS), pH 7.3, with 0.1% Nonidet P-40, using a glass-Teflon homogenizer, and centrifuged at 1000 x g for 5 min, followed by fixation of pellets in ice-cold methanol-glacial acetic acid mixture (3:1) three times for 20 min. The resulting suspensions of nuclei were then dropped onto the wet surfaces of microscope slides. The slides were allowed to dry overnight and were dehydrated by ethanol series before being used for FISH experiments. The quality of slides with the fixed nuclei was estimated by the criterion of the absence of surrounding cytoplasm. Another fixation step of cell suspensions was performed, if necessary.
Fetal brain tissue samples were obtained from 12 fetuses (8 female and 4 male) at the time of selective termination of intrauterine pregnancy from healthy females. Six samples (four female and two male) were used for direct preparations of tissue for FISH analysis without cultivation in vitro. Another six samples (four female and two male) were used for preparation of organotypic fetal brain cultures followed by FISH analysis. The criteria of inclusion were age from 25 to 35 years, gestation age 911 weeks, negative test for the most common infectious diseases, absence of systemic and genetic diseases in both parents, and absence of treatment by drugs with known teratogenic effects. Gestational age was determined according to a number of parameters (date of last menstrual period, uterine size, and ultrasonography; and after abortion, by measurement of fetal foot length).
The fetal tissue was transferred to a laminar-flow hood. Brain samples were rinsed with Earle's buffered saline solution (EBSS) (No. 24010-035, Gibco Invitrogene, SARL; Cergy Pontois Cedex, France). Medulla oblongata was chosen as a target fetal brain tissue because of its relatively large size to avoid possible contamination by other surrounding embryonic tissues. Medulla oblongata was isolated from the surrounding tissue by a transverse cut through the brain stem at the level of the pons and of the lower medulla using a dissecting microscope (Wild/Leitz M32, Leitz-Wetzlar; Wetzlar, Germany). Meninges and major blood vessels were removed. The isolated medulla was placed in a sterile plastic petri dish with 2 ml EBSS, and transversely cut 300400-µm slices were made by hand with razor blades. The slices were then divided into 20 pieces, rinsed in EBSS, and transferred into Pyrex glass bottles containing 10 ml of medium, with rubber silicon stoppers. Some tissue pieces were processed for FISH studies similarly to autopsy samples of the adult brain. Samples were cultivated in a high-speed mini-roller under rotation at 60 rpm. Tissue cultures were maintained for up to 30 days without changing medium according to the protocol previously described for organotypic cultures of postnatal rat hippocampus (Victorov et al. 2001
). One hundred ml of medium consists of 86.3 ml MEM (Gibco, No. 32260-018); 5 ml fetal bovine serum (Gibco, No.10106-169); 5 ml human umbilical blood serum (Paneco; Moscow, Russia); 2 ml of 40% glucose; 1 ml of 200 mM L-glutamine; 0.5 ml of insulin; and 0.2 ml of antibiotic/antimyotic (Gibco, No.15240-039). Cells in cultures were able to grow, differentiate, and establish histotypical organization and glialneuronal relationships by the end of 4 weeks in vitro. Nissl staining of tissue culture sections demonstrated good survival of neurons and glial cells. Neurons were immunochemically identified using antibodies against III ß-tubulin; glial fibrillary acidic protein (GFAP) immunostaining identified astrocytes (VM Vostrikov, unpublished data). The cell cultures (ages 34 weeks) were processed for FISH studies according to standard cytogenetic fixation procedures using hypotonic treatment and fixation with methanol-acetic acid (3:1).
Chromosome 1 (D1Z1)-, 18 (D18Z1)-, 13/21 (D13Z1/D21Z1)-, X (DXZ1)-, and Y (DYZ3)-specific DNA probes were cloned in the Laboratory of Cytogenetics of the National Center of Mental Health, Moscow, Russia and prepared as described previously (Vorsanova et al. 1986,1994
; Yurov et al. 1996b
,2002
; Alexandrov et al. 1988
; Soloviev et al. 1995
,1998
). This set of probe A mixtures of: (a) biotinylated chromosome 1specific probe, Cy3-labeled chromosome Yspecific probe, fluorescein-labeled chromosome Xspecific probe; (b) biotinylated chromosome 1specific probe, Cy3-labeled chromosome 13/21specific probe, fluorescein-labeled chromosome Xspecific probe; and (c) Cy3-labeled chromosome 18specific probe, biotinylated chromosome Yspecific probe, fluorescein-labeled chromosome Xspecific probe; each at a final concentration of 50 ng/10 µl of hybridizing solution (10% dextran sulfate and 2x SSC in 55% formamide, pH 7) was used. The in situ hybridization and detection protocols were performed as described previously (Soloviev et al. 1994
,1998
; Yurov et al. 1996b
). Aliquots of DNA probe (10 µl per slide) were placed onto the slides, covered by coverslips, and denatured for 5 min at 72C. After 4 hr or overnight hybridization at 37C, the slides were washed for 15 min in 50% formamide, 2x SSC (pH 7) at 42C, followed by two washes for 5 min each in 2x SSC, 0.1% Tween 20 at 37C. The slides were then incubated with 5 µg/ml 7-amino-4-methyl-3-coumarinylacetic acid (AMCA)-avidin (Vector Laboratories, Burlingame, CA) to detect biotin-labeled DNA probes. Finally, interphase nuclei were counterstained with diluted (1:10) 4',6-diamidino-2-phenylindole (DAPI) to allow simultaneous observation of nuclei and hybridized biotin-labeled probe after application of AMCA-avidin.
For epifluorescence microscopy, a Leitz Orthoplan microscope (Leica Mikroskopie und Systeme, Leitz-Wetzlar; Wetzlar, Germany) equipped with a 100 W lamp was used with the following filter sets: A (No. 513,596) for DAPI fluorescence; I3 (No. 513,719) or GR (No. 513,821) for propidium iodide fluorescence and fluorescein isothiocyanate (FITC) signals; GR (No. 513,821) for both fluorescein and cyanine signals; and N2 (No. 513,609) for cyanine signals. All images were observed with the Plan-Neofluar (Leica Mikroskopie und Systeme, Leitz-Wetzlar; Wetzlar, Germany) x40/1.30 or x63/1.300.60 oil immersion lenses.
No fewer than 500 nuclei (in a case of adult brain samples) and 1000 nuclei (in a case of embryonic brain samples) were scored for each sample for each probe. Only intact and undamaged nuclei free of cytoplasm were analyzed. Nuclei with low signal intensities, diffuse signals, or absence of signals on both homolog chromosomes were considered to be hybridization failures and were not scored. Two small focal (or paired) signals of the same color and the same intensity, separated by a distance of less than the area of one signal, were considered to be a split signal from one chromosome. Interphase nuclei with one large signal of the same color and increased intensity of fluorescence with the absence of a second hybridization signal in an interphase nucleus were considered to be the over-positioning of two signals and were not scored.
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Results |
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Discussion |
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The data obtained provide evidence that a relatively small but significant population of aneuploidy cells is present in fetal as well as in adult human brain in vivo. The number of chromosomally abnormal cells is dramatically increased during the cultivation of human embryonic brain cells in vitro. Therefore, we propose that fetal neuronal cells are characterized by an increased frequency of nonspecific aneuploidy involving different chromosomes. The analysis of one chromosome-specific DNA probe shows that 95% of neuronal cells could be considered as karyotypically normal. However, multicolor FISH analysis of several chromosomes in one FISH experiment and the application of a probe set for the chromosomes mentioned above strongly indicated that the total number of chromosomally abnormal neuronal cells is higher. We have estimated the total (cumulative) number of aberrant nuclei for chromosomes 1, 13/21, 18, X, and Y to be 22%. One can propose that the number of aneuploidy frequency in fetal human brain cells involving all 23 chromosome pairs is substantially higher. Therefore, our data provide evidence for large-scale genomic (chromosomal) variations in developing neuronal cells. The adult human brain, in contrast, contains significantly lower amounts of aneuploid neuronal cells. The data obtained indicate that fetal and adult brains differ in aneuploidy frequency. Therefore, one can suggest that the selective loss of chromosomally abnormal neuronal cells takes place during ontogenesis. However, a relatively small but significant amount of aneuploid neurons could survive and function in the adult brain. Thus, the high level of chromosomal aneuploidy in embryonic human neuronal cells could be considered to be a cause of genetic mosaicism in fetal brain with conservation of some anueploidy cells during ontogenesis, leading to cryptic chromosomal mosaicism in adult neurons of human brain.
Organotypic cultures of human fetal brain are generally used for the study of human neurodeveloping mechanisms of neurocytotoxicity and neuroprotection. Neuronal embryonic brain cells in cultures are able to grow, differentiate, and establish glialneuronal relationships. The advantage of this ability is the high survival rate of neurons and glial cells in vitro. Despite the growing number of molecular investigations of human fetal brain organotypic cultures, studies of chromosome variations in the developing human brain have not yet been performed. Our results regarding the dramatic increase of aneuploidy incidence in fetal brain cells cultured in vitro allow us to propose that nondisjunction of chromosomes could also take place in vivo during the differentiation of neuronal cells. The frequency of aneuploidy in uncultured fetal brain cells is lower than in cultured neuronal cells. Therefore, it is reasonable to suggest that conditions of cultivation in vitro probably artificially stimulate division of differentiated neuronal cells and provoke conflict between these two fundamental processes (cell division and cell differentiation). Differentiated neuronal cells could not normally replicate their DNA and pass cell cycles. The high incidence of aneuploidy could also be explained by the inability of partially or fully differentiated neuronal cells to pass correctly a mitotic division. Assuming that the increase of aneuploidy frequency takes place in developing human brain in vivo, one can speculate that the neuronal loss happens during prenatal and neonatal development. Therefore, early childhood is directly related to the phenomenon of increased levels of aneuploidy in differentiated neuronal cells. The dynamic of chromosome variations with a decrease in aneuploidy frequencies in non-cultured embryonic (non-differentiated) brain cells and highly differentiated adult brain cells in comparison to differentiated organotypic brain cell cultures is in agreement with this proposal.
The application of the one-color FISH technique to the study of chromosome complement of interphase nuclei in the adult human brain has demonstrated that a significant fraction of the hippocampal pyramidal and basal forebrain neurons in Alzheimer's disease have tetraploid chromosome complement. This imbalance in chromosome complement has been proposed as one of the causes of neuronal loss in Alzheimer's disease (Yang et al. 2001). Molecular cytogenetic study of postmortem brain of schizophrenic patients with mFISH application was performed in our earlier studies. A statistically significant level of aneuploidy (up to 4% of neurons) was detected in the postmortem brain of schizophrenia patients. Low-level chromosomal mosaicism was suggested to be involved in the pathogenesis of schizophrenia (Yurov et al. 2001
). Regarding previous as well as present studies of chromosome complement in human brain, we have concluded that a low percentage of mosaicism of aneuploidy does exist in the fetal and adult human brain. Recently, central nervous system of mouse, both during development and in adulthood, was proposed to have genetic mosaic features (euploid population intermixed with a smaller genetically diverse aneuploid population) (Rehen et al. 2001
). The occurrence of this mosaicism may have relevance to stem cell biology, mammalian cloning, genomics, neurogenetics, and neuropsychiatric diseases. These permanent genomic changes may also contribute to physiological and behavioral variations among individuals not accounted for by classical genetics. The presence of chromosomally abnormal neuronal cell populations in the form of extremely low-level mosaicism could significantly affect normal brain development and functions. The real amount of chromosomally abnormal neuronal cells in the normal adult human brain is unknown and should be determined in more-detailed studies with scoring of chromosomes in a larger population of cells. Taking into account that one neuron forms about one thousand connections with other neuronal cells, the presence of a small number of aneuploid neurons could substantially and negatively affect normal development and brain functions. "Cryptic" chromosomal mosaicism in the neurons of brain is not necessarily associated with any specific phenotypic appearance; however, it could have an unfavorable effect on normal brain development and functions as well as relevance to some neuropsychiatric diseases. Therefore, extended studies of chromosomal complement instability in the fetal and adult human brain should be continued.
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Acknowledgments |
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Footnotes |
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Received for publication May 31, 2004; accepted September 2, 2004
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