Altered cleavage plane orientation with increased genomic aneuploidy produced by receptor-mediated lysophosphatidic acid (LPA) signaling in mouse cerebral cortical neural progenitor cells.

The brain is composed of cells having distinct genomic DNA sequences that arise post-zygotically, known as somatic genomic mosaicism (SGM). One form of SGM is aneuploidy-the gain and/or loss of chromosomes-which is associated with mitotic spindle defects. The mitotic spindle orientation determines cleavage plane positioning and, therefore, neural progenitor cell (NPC) fate during cerebral cortical development. Here we report receptor-mediated signaling by lysophosphatidic acid (LPA) as a novel extracellular signal that influences cleavage plane orientation and produces alterations in SGM by inducing aneuploidy during murine cortical neurogenesis. LPA is a bioactive lipid whose actions are mediated by six G protein-coupled receptors, LPA1-LPA6. RNAscope and qPCR assessment of all six LPA receptor genes, and exogenous LPA exposure in LPA receptor (Lpar)-null mice, revealed involvement of Lpar1 and Lpar2 in the orientation of the mitotic spindle. Lpar1 signaling increased non-vertical cleavage in vivo by disrupting cell-cell adhesion, leading to breakdown of the ependymal cell layer. In addition, genomic alterations were significantly increased after LPA exposure, through production of chromosomal aneuploidy in NPCs. These results identify LPA as a receptor-mediated signal that alters both NPC fate and genomes during cortical neurogenesis, thus representing an extracellular signaling mechanism that can produce stable genomic changes in NPCs and their progeny. Normal LPA signaling in early life could therefore influence both the developing and adult brain, whereas its pathological disruption could contribute to a range of neurological and psychiatric diseases, via long-lasting somatic genomic alterations.

DNA damage signaling recruits the Rtt107-Slx4 scaffolds via Dpb11 to mediate replication stress response.

The DNA damage checkpoint kinase Mec1(ATR) is critical for maintaining the integrity of replication forks. Though it has been proposed to promote fork repair, the mechanisms by which Mec1 regulates DNA repair factors remain unclear. Here, we found that Mec1 mediates a key interaction between the fork protein Dpb11 and the DNA repair scaffolds Slx4-Rtt107 to regulate replication stress response. Dissection of the molecular basis of the interaction reveals that Slx4 and Rtt107 jointly bind Dpb11 and that Slx4 phosphorylation is required. Mutation of Mec1 phosphorylation sites in Slx4 disrupts its interaction with Dpb11 and compromises the cellular response to replisomes blocked by DNA alkylation. Multiple fork repair factors associate with Rtt107 or Slx4, supporting that Mec1-dependent assembly of the Rtt107-Slx4-Dpb11 complex functions to coordinate fork repair. Our results unveil how Mec1 regulates the Slx4 and Rtt107 scaffolds and establish a mechanistic link between DNA damage signaling and fork repair.

A new method of embryonic culture for assessing global changes in brain organization.

While dissociated, reaggregated cells and organotypic slice cultures are useful models for understanding brain development, they only partially mimic the processes and organization that exist in vivo. Towards bridging the gap between in vitro and in vivo paradigms, a method for culturing intact brain tissue was developed using whole cerebral cortical hemispheres in which the anatomical and cellular organization of nervous system tissue is preserved. Single, free-floating telencephalic hemispheres were dissected from embryonic mice and placed into defined culture medium on an orbital shaker. Orbital shaking was necessary for optimal growth, and cortices grown under these conditions closely approximated in vivo parameters of cell division, differentiation, migration and cell death for up to 24 h. In addition to wild-type cultures, the method was compatible with genetically altered tissues. One particular advantage of this method is its ability to reveal global anatomical alterations in the embryonic brain following exposure to soluble growth factors. This method should thus be helpful for assessing a wide range of soluble molecules for their systemic effects on the embryonic brain.

Constitutional aneuploidy in the normal human brain.

The mouse brain contains genetically distinct cells that differ with respect to chromosome number manifested as aneuploidy (Rehen et al., 2001); however, the relevance to humans is not known. Here, using double-label fluorescence in situ hybridization for the autosome chromosome 21 (chromosome 21 point probes combined with chromosome 21 "paint" probes), along with immunocytochemistry and cell sorting, we present evidence for chromosome gain and loss in the human brain. Chromosome 21 aneuploid cells constitute approximately 4% of the estimated one trillion cells in the human brain and include non-neuronal cells and postmitotic neurons identified by the neuronspecific nuclear protein marker. In comparison, human interphase lymphocytes present chromosome 21 aneuploidy rates of 0.6%. Together, these data demonstrate that human brain cells (both neurons and non-neuronal cells) can be aneuploid and that the resulting genetic mosaicism is a normal feature of the human CNS.

Enamel fluorosis prevalence after a 7-year interruption in water fluoridation in Jaú, São Paulo, Brazil.

OBJECTIVES: This paper analyzes the impact of a 7-year interruption in water fluoridation on the prevalence of enamel fluorosis in Jaú, state of São Paulo, Brazil. METHODS: Fluorosis prevalence (TF index) was evaluated in permanent maxillary central incisors of children (9-14 years old) that were 36 (n=81; cohort -36), 27 (n=81; cohort -27), and 18 months old (+/-1 month; n=89; cohort -18) in October 1991, when the breakstarted, and 18months old(+/-1 month;n=70; cohort 18) after that date. Children brushed their teeth prior to examination, which was conducted under natural light by three calibrated examiners (agreement 87.8-93.8%, kappa 0.72-0.85). RESULTS: The fluorosis prevalence (TF> or =1) was 7.41 percent, 3.70 percent, 7.87 percent, and 18.57 percent, respectively, for cohorts -36, -27, -18, and 18. The difference between cohort 18 and the other groups was statistically significant (Kruskall-Wallis test, P=.05). CONCLUSIONS: These results suggest that the fluoridated water is not an important risk factor for enamel fluorosis, since the prevalence of enamel fluorosis was low in the cohorts -36, -27, and -18 when fluoridated water was used.