Noelia López-Sánchez

A novel hypothesis for Alzheimer disease based on neuronal tetraploidy induced by p75NTR

Cumulative evidence indicates that neuronal cell cycle re-entry represents an early and critical event in AD, recapitulating known hallmarks of the disease including tau hyperphosphorylation and production of Aβ peptide-containing plaques. Neurons that duplicate their DNA are rarely observed to undergo mitosis, and they remain for long time as tetraploid cells, in accordance with the chronic course of the disease. We have recently shown that cell cycle re-entry and somatic tetraploidization occurs during normal development in a subpopulation of RGCs, giving rise to enlarged neurons with extensive dendritic trees. Tetraploization in these neurons occurs in response to the activation of the neurotrophin receptor p75NTR by an endogenous source of NGF. In contrast, BDNF inhibits G2/M transition in tetraploid RGCs, preventing their death by apoptosis. In AD both proNGF and p75NTR are overexpressed, and AD-associated oxidative conditions have been shown to enhance proNGF function. This suggests that p75NTR could be a trigger for neuronal tetraploidization in AD, being the p75NTR-mediated pathway a putative target for therapeutical intervention. Functional changes in affected neurons, derived from tetraploidy-associated hypertrophy, could compromise neuronal viability. The known decline of BDNF/TrkB expression in AD could facilitate G2/M transition and apoptosis in tetraploid neurons.

Mitochondrial c-Jun NH2-Terminal Kinase Prevents the Accumulation of Reactive Oxygen Species and Reduces Necrotic Damage in Neural Tumor Cells that Lack Trophic Support

In response to different stress signals, the c-Jun NH2-terminal kinase (JNK) can trigger cell death. However, JNK also facilitates the survival and cell cycle progression of tumor cells by mechanisms that are poorly defined. Here, we show that schwannoma RN22 cells can survive and proliferate under serum-free conditions although serum withdrawal rapidly induces mitochondrial fission and swelling. Although the morphologic changes observed in the mitochondria did not trigger cytochrome c release, they were accompanied by an increase in the mitochondrial membrane potential (ΔΨM) and of immunoreactivity for active JNK in these organelles. Pharmacologic inhibition of JNK provoked a further increase of the ΔΨM, an increase in reactive oxygen species (ROS) production, and a sustained decrease in cell viability due to necrosis. This increase in necrosis was prevented by the presence of ROS scavengers. Immunoreactivity for active JNK was also observed in the mitochondria of neuroblastoma 1E-115 and neuroblastoma 2a neuroblastoma cell lines on serum withdrawal, whereas active JNK was barely detected in serum-deprived fibroblasts. Accordingly, the reduction in neural tumor cell viability induced by JNK inhibition was largely attenuated in serum-deprived fibroblasts. These data indicate that local activation of JNK in the mitochondria can protect against necrotic cell death associated with ROS production, facilitating the growth of neural tumor cells subjected to serum deprivation. (Mol Cancer Res 2007;5(1):47–60)

Lengthening of G2/mitosis in cortical precursors from mice lacking β-amyloid precursor protein

The beta-amyloid precursor protein (APP) is expressed within the nervous system, even at the earliest stages of embryonic development when cell growth and proliferation is particularly important. In order to study the function of APP at these early developmental stages, we have studied the development of the cerebral cortex in both wild type and App-/- mutant mice. Here, we demonstrate that APP mRNA is expressed in cortical precursor cells and that APP protein is concentrated within their apical domains during interphase. However, during mitosis, APP re-localizes to the peripheral space surrounding the metaphase plate. In APP-deficient cortical precursors, the duration of mitosis is increased and a higher proportion of cortical precursor cells contained nuclei in late G2. We conclude that during cortical development APP plays a role in controlling cell cycle progression, particularly affecting G2 and mitosis. These observations may have important implications for our understanding of how APP influences the progression of Alzheimers disease, since degenerating cortical neurons have been shown to up-regulate cell cycle markers and re-enter the mitotic cycle before dying.

Genetic Evidence for p75NTR-Dependent Tetraploidy in Cortical Projection Neurons from Adult Mice

A subpopulation of chick retinal projection neurons becomes tetraploid during development, an event prevented by blocking antibodies against p75 neurotrophin receptor (p75NTR). We have used an optimized flow cytometric assay, based on the analysis of unfixed brain cell nuclei, to study whether p75NTR-dependent neuronal tetraploidization takes place in the cerebral cortex, giving rise to projection neurons as well. We show that 3% of neurons in both murine neocortex and chick telencephalic derivatives are tetraploid, and that in the mouse ∼85% of these neurons express the immediate early genes Erg-1 and c-Fos, indicating that they are functionally active. Tetraploid cortical neurons (65–80%) express CTIP2, a transcription factor specific for subcortical projection neurons in the mouse neocortex. During the period in which these neurons are born, p75NTR is detected in differentiating neurons undergoing DNA replication. Accordingly, p75NTR-deficient mice contain a reduced proportion of both NeuN and CTIP2-positive neocortical tetraploid neurons, thus providing genetic evidence for the participation of p75NTR in the induction of neuronal tetraploidy in the mouse neocortex. In the striatum tetraploidy is mainly associated with long-range projection neurons as well since ∼80% of tetraploid neurons in this structure express calbindin, a marker of neostriatal-matrix spiny neurons, known to establish long-range projections to the substantia nigra and globus pallidus. In contrast, only 20% of tetraploid cortical neurons express calbindin, which is mainly expressed in layers II–III, where CTIP2 is absent. We conclude that tetraploidy mainly affects long-range projection neurons, being facilitated by p75NTR in the neocortex.

Sortilin participates in light-dependent photoreceptor degeneration in vivo.

Both proNGF and the neurotrophin receptor p75 (p75NTR) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75NTR co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75NTR, and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75NTR and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the “pro” domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75NTR/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies.

Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.

Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti‐inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time‐lapse microscopy and flow‐cytometric cell sorting.

Flow cytometric analysis of DNA synthesis and apoptosis in central nervous system using fresh cell nuclei.

The use of flow cytometry in vertebrate nervous tissues is hampered by the morphological complexity and high level of interconnectivity intrinsic to their cellular constituents. Here we describe a simplified procedure for the identification and quantitative analysis of neural cells by flow cytometry based on the isolation and immunolabeling of fresh cell nuclei. We have applied this procedure for the quantitative analysis of apoptosis and DNA synthesis in the embryonic brain.

Neuronal tetraploidization in the cerebral cortex correlates with reduced cognition in mice and precedes and recapitulates Alzheimer's-associated neuropathology

A controversy exists as to whether de novo-generated neuronal tetraploidy (dnNT) occurs in Alzheimers disease. In addition, the presence of age-associated dnNT in the normal brain remains unexplored. Here we show that age-associated dnNT occurs in both superficial and deep layers of the cerebral cortex of adult mice, a process that is blocked in the absence of E2F1, a known regulator of cell cycle progression. This blockage correlates with improved cognition despite compromised neurogenesis in the adult hippocampus was confirmed in mice lacking the E2f1 gene. We also show that the human cerebral cortex contains tetraploid neurons. In normal humans, age-associated dnNT specifically occurs in the entorhinal cortex whereas, in Alzheimer, dnNT also affects association cortices prior to neurofibrillary tangle formation. Alzheimer-associated dnNT is likely potentiated by altered amyloid precursor protein (APP) processing as it is enhanced in the cerebral cortex of young APPswe/PS1deltaE9 mice, long before the first amyloid plaques are visible in their brains. In contrast to age-associated dnNT, enhanced dnNT in APPswe/PS1deltaE9 mice mostly affects the superficial cortical layers. The correlation of dnNT with reduced cognition in mice and its spatiotemporal course, preceding and recapitulating Alzheimer-associated neuropathology, makes this process a potential target for intervention in Alzheimers disease.

Strand-specific CpG hemimethylation, a novel epigenetic modification functional for genomic imprinting

Abstract Imprinted genes are regulated by allele-specific differentially DNA-methylated regions (DMRs). Epigenetic methylation of the CpGs constituting these DMRs is established in the germline, resulting in a 5-methylcytosine-specific pattern that is tightly maintained in somatic tissues. Here, we show a novel epigenetic mark, characterized by strand-specific hemimethylation of contiguous CpG sites affecting the germline DMR of the murine Peg3, but not Snrpn, imprinted domain. This modification is enriched in tetraploid cortical neurons, a cell type where evidence for a small proportion of formylmethylated CpG sites within the Peg3-controlling DMR is also provided. Single nucleotide polymorphism (SNP)-based transcriptional analysis indicated that these epigenetic modifications participate in the maintainance of the monoallelic expression pattern of the Peg3 imprinted gene. Our results unexpectedly demonstrate that the methylation pattern observed in DMRs controlling defined imprinting regions can be modified in somatic cells, resulting in a novel epigenetic modification that gives rise to strand-specific hemimethylated domains functional for genomic imprinting. We anticipate the existence of a novel molecular mechanism regulating the transition from fully methylated CpGs to strand-specific hemimethylated CpGs.