Nanoscale structural mapping as a measure of maturation in the murine frontal cortex.

Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanomechanical and nanostructural features of the murine dorsolateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with histochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cortical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases.

Transgenic mice as a model of pre-clinical Alzheimer's disease.

At diagnosis, Alzheimer's disease (AD) brains are extensively burdened with plaques and tangles and display a degree of synaptic failure most likely beyond therapeutic treatment. It is therefore crucial to identify early pathological events in the progression of the disease. While it is not currently feasible to identify and study early, pre-clinical stages of AD, transgenic (Tg) models offer a valuable tool in this regard. Here we investigated cognitive, structural and biochemical CNS alterations occurring in our newly developed McGill-Thyl-APP Tg mice (over-expressing the human amyloid precursor protein with the Swedish and Indiana mutations) prior to extracellular plaque deposition. Pre-plaque, 3-month old Tg mice already displayed cognitive deficits concomitant with reorganization of cortical cholinergic pre-synaptic terminals. Conformational specific antibodies revealed the early appearance of intracellular amyloid ß (Aß)-oligomers and fibrillar oligomers in pyramidal neurons of cerebral cortex and hippocampus. At the same age, the cortical levels of insulin degrading enzyme -a well established Aß-peptidase, were found to be significantly down-regulated. Our results suggest that, in the McGill-Thy1-APP Tg model, functional, structural and biochemical alterations are already present in the CNS at early, pre-plaque stages of the pathology. Accumulation of intraneuronal neurotoxic Aß-oligomers (possibly caused by a failure in the clearance machinery) is likely to be the culprit of such early, pre-plaque pathology. Similar neuronal alterations might occur prior to clinical diagnosis in AD, during a yet undefined 'latent' stage. A better understanding of such pre-clinical AD might yield novel therapeutic targets and or diagnostic tools.

Metazoan origins of DNA replication: regulation through dynamic chromatin structure.

DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein-protein and protein-DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis-acting sequences that serve as replication origins and the trans-acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed.

High intracellular concentrations of amyloid-beta block nuclear translocation of phosphorylated CREB.

The beta-amyloid peptide (Abeta) is considered responsible for the pathogenesis of Alzheimer's disease. Despite the magnitude of reports describing a neurotoxic role of extracellular Abeta, the role for intracellular Abeta (iAbeta) has not been elucidated. We previously demonstrated that in rat pheochromocytoma cells expression of moderate levels of Abeta results in the up-regulation of phospho-extracellular signal-regulated kinases (ERK1)/2 along with an elevation of cyclic AMP-response element (CRE)-regulated gene expression; however, the effect of high intracellular levels of Abeta were not examined. Towards this goal we generated constructs that endogenously produce different expression levels of iAbeta in a human cell line. We show a bimodal response to Abeta in a neural human cell line. A moderate increase of endogenous Abeta up-regulates certain cyclic AMP-response element-binding protein (CREB) responsive genes such as presenilin 1, presenilin 2, brain-derived neurotrophic factor, and mRNA and protein levels by CREB activation and Synapsin 1 nuclear translocation. On the other hand, high-loads of iAbeta resulted in sustained hyper-phosphorylation of CREB that did not translocate to the nucleus and did not stimulate activation of CRE-regulated gene expression. Our study suggests that variations in levels of iAbeta could influence signaling mechanisms that lead to phosphorylation of CREB, its nuclear translocation and CRE-regulated genes involved in production of Abeta and synaptic plasticity in opposite directions.