Jurjen W. Westra

Aneuploid mosaicism in the developing and adult cerebellar cortex

Neuroprogenitor cells (NPCs) in several telencephalic proliferative regions of the mammalian brain, including the embryonic cerebral cortex and postnatal subventricular zone (SVZ), display cell division “defects” in normal cells that result in aneuploid adult progeny. Here, we identify the developing cerebellum as a major, nontelencephalic proliferative region of the vertebrate central nervous system (CNS) that also produces aneuploid NPCs and nonmitotic cells. Mitotic NPCs assessed by metaphase chromosome analyses revealed that 15.3% and 20.8% of cerebellar NPCs are aneuploid at P0 and P7, respectively. By using immunofluorescent analysis of cerebellar NPCs, we show that chromosome segregation defects contribute to the generation of cells with an aneuploid genomic complement. Nonmitotic cells were assessed by fluorescence‐activated cell sorting (FACS) coupled with fluorescence in situ hybridization (FISH), which revealed neuronal and nonneuronal aneuploid populations in both the adult mouse and human cerebellum. Taken together, these results demonstrate that the prevalence of neural aneuploidy includes nontelencephalic portions of the neuraxis and suggest that the generation and maintenance of aneuploid cells is a widespread, if not universal, property of central nervous system development and organization. J. Comp. Neurol. 507:1944–1951, 2008.

Neuronal DNA content variation (DCV) with regional and individual differences in the human brain

It is widely assumed that the human brain contains genetically identical cells through which postgenomic mechanisms contribute to its enormous diversity and complexity. The relatively recent identification of neural cells throughout the neuraxis showing somatically generated mosaic aneuploidy indicates that the vertebrate brain can be genomically heterogeneous (Rehen et al. [2001] Proc. Natl. Acad. Sci. U. S. A. 98:13361–13366; Rehen et al. [2005] J. Neurosci. 25:2176–2180; Yurov et al. [2007] PLoS ONE:e558; Westra et al. [2008] J. Comp. Neurol. 507:1944–1951). The extent of human neural aneuploidy is currently unknown because of technically limited sample sizes, but is reported to be small (Iourov et al. [2006] Int. Rev. Cytol. 249:143–191). During efforts to interrogate larger cell populations by using DNA content analyses, a surprising result was obtained: human frontal cortex brain cells were found to display “DNA content variation (DCV)” characterized by an increased range of DNA content both in cell populations and within single cells. On average, DNA content increased by ∼250 megabases, often representing a substantial fraction of cells within a given sample. DCV within individual human brains showed regional variation, with increased prevalence in the frontal cortex and less variation in the cerebellum. Further, DCV varied between individual brains. These results identify DCV as a new feature of the human brain, encompassing and further extending genomic alterations produced by aneuploidy, which may contribute to neural diversity in normal and pathophysiological states, altered functions of normal and disease‐linked genes, and differences among individuals. J. Comp. Neurol. 518:3981–4000, 2010.

Normal Human Pluripotent Stem Cell Lines Exhibit Pervasive Mosaic Aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC – including induced pluripotent - lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.

Aneuploid Cells Are Differentially Susceptible to Caspase-Mediated Death during Embryonic Cerebral Cortical Development

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cells karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.

A reevaluation of tetraploidy in the Alzheimer's disease brain.

Alzheimer’s disease (AD) is characterized by extensive neuronal death in distinct brain regions, including the frontal cortex and hippocampus, although the specific mechanisms of neuronal degeneration in AD remain a topic of intense scientific pursuit. One model for cell death in AD postulates that abortive cell cycle events in neurons, including tetraploidy, precede neuronal death, and novel therapeutics based on suppressing cell cycle re-entry are being pursued. Using DNA content fluorescence-activated cell sorting combined with fluorescence in situ hybridization and immunostaining, we analyzed neuronal nuclei from postmortem human brain samples from the frontal cortex and hippocampus of nondiseased and AD patients for evidence of tetraploidy. Here, we show that tetraploid nuclei are similarly prevalent in AD and control brains and are exclusively non-neuronal, contrasting with an absence of tetraploid neurons. Our findings demonstrate that neuronal tetraploidy is nonexistent in the AD brain and intimate a reevaluation of neuronal cell cycle re-entry as a therapeutic target for AD.

A Reply to Iourov et al.

not constitutive of but rather create mosaic brains with myriad forms of chromosome gain and/or loss, intermixed amongst euploid cells, including normally occurring trisomy 21 [13] . However, the well-established pathological associations of constitutive aneuploidy in genetic diseases like Down syndrome (trisomy 21) and cancer suggest the possibility that alterations in normal mosaic aneuploidy might contribute to brain diseases and disorders [2, 3, 14, 15] . In particular, a disease that might be impacted by alterations in mosaic aneuploidy is Alzheimer’s disease (AD), in view of the precocious occurrence of neuropathological similarities in adult Down syndrome brains that are phenocopies of much older sporadic AD brains [16, 17] . In a completely independent line of study, Herrup and colleagues [18, 19] provided evidence for tetraploid neurons by using fluorescence in situ hybridization (FISH) on AD brains, an approach that was state of the art in many respects, to support the cell ‘cycle-related neuronal death’ (CRND) hypothesis. A question that has slowly evolved over the ensuing years and has remained unresolved is the relationship between FISH signals reported as evidence for CRND and the normally occurring neural aneuploidy/ aneusomy. This ambiguity reflects, in part, the fact that the existence of normal neural aneuploidy has neither immediately nor universally been accepted, and remains largely unappreciated. Key impediments to further clarification have been the technical challenges in studying neural aneuploidy/aneusomy as it relates to these issues: (1) techniques for interrogating postmitotic neurons, (2) distinctions between aneusomy and aneuploidy, and (3) rigorous quantitation. These three issues also speak to the We thank our colleagues for their Commentary on Westra et al. [1] , and here provide a brief response. Aneuploidy is chromosomal gain and/or loss from haploid multiples [2] , which is distinct from polyploid cells that are not aneuploid. Since the first report of somatic, mosaic aneuploidy in the normal mammalian CNS [3] , multiple independent groups [4–6] have replicated the essential findings: neural aneuploidy is present throughout the developing and mature vertebrate nervous system, including humans. Elegant confirmation using cutting-edge cytogenetic techniques by Yurov and colleagues (see their Commentary ‘Genomic Landscape of the Alzheimer’s Disease Brain: Chromosome Instability – Aneuploidy, but Not Tetraploidy – Mediates Neurodegeneration’ in the present issue for references) has left no doubt about the normal existence of neural aneuploidy or, more correctly, ‘aneusomy’ – whereby a limited number of chromosomes are interrogated rather than the entire complement of chromosomes – in human nonmitotic cells. Moreover, this general phenomenon has been shown to exist in other cell lineages as well [3, 7] . Mechanistic studies have revealed that neural aneuploidy can be produced by chromosomal segregation ‘defects’ in neural stem and progenitor cells [8] ; its occurrence in normal cells begs the appropriateness of the word ‘defect’. Neural aneuploidy/aneusomy can alter gene expression amongst cells of the same lineage [9] and involves functioning neurons that are integrated into active synaptic networks [10] . It is present throughout the neuraxis [11] , and occurs in all vertebrates thus far examined [3–5, 12, 13] . Critically, these forms of aneuploidy/aneusomy are Received: March 31, 2010 Accepted: May 21, 2010 Published online: September 3, 2010 D i s e a s e s