Isotope ratios, IRD and planktonic foraminifera content during marine isotope stage 5 of sediment cores MD99-2303, MD99-2304 and MD95-2010

Variable climatic and oceanographic conditions characterized the last interglacial at high northern latitudes, probably related to changes in the strength of the Atlantic Meridional Overturning Circulation (AMOC). The magnitudes of these changes are comparable to the Holocene variability, and were thus significantly subdued compared to glacial climate changes. A thermal optimum occurred during the early part of the interglacial, followed by a period of reduced Atlantic inflow to the northernmost Nordic Seas. Subsequently, a new period with increased strength of the AMOC occurred. Significant amounts of Ice-Rafted Debris (IRD) were deposited in the northernmost Nordic Seas before any major change of the global ice volume. This implies an early onset of local ice sheet growth, probably the result of enhanced inflow of Atlantic water to the northernmost Nordic Seas contemporary with a Northern Hemisphere summer insolation minimum. Contrasting sea-land conditions provided large moisture fluxes towards land, giving rise to rapid, early glacial growth. Throughout the glacial part of Marine Isotope Stage (MIS) 5, millennial-scale cold events occurred along the axis of the warm water transport, from the subtropics all the way to the northernmost Nordic Seas. Correlation of IRD events from sites in the Fram Strait, on the Voring Plateau, and in the North Atlantic provides evidence that the major Northern Hemisphere ice sheets at times responded coherently to the same forcing. The widespread distribution of these events highlights the importance of the oceanic influence on the regional climate system.

Advanced morphometric techniques applied to the study of human brain anatomy

El desarrollo de las técnicas de imágenes por resonancia magnética han permitido el estudio y cuantificación, in vivo, de los cambios que ocurren en la morfología cerebral ligados a procesos tales como el neurodesarrollo, el envejecimiento, el aprendizaje o la enfermedad. Un gran número de métodos de morfometría han sido desarrollados con el fin de extraer la información contenida en estas imágenes y traducirla en indicadores de forma o tamaño, tales como el volumen o el grosor cortical; marcadores que son posteriormente empleados para encontrar diferencias estadísticas entre poblaciones de sujetos o realizar correlaciones entre la morfología cerebral y, por ejemplo, la edad o la severidad de determinada enfermedad. A pesar de la amplia variedad de biomarcadores y metodologías de morfometría, muchos estudios sesgan sus hipótesis, y con ello los resultados experimentales, al empleo de un número reducido de biomarcadores o a al uso de una única metodología de procesamiento. Con el presente trabajo se pretende demostrar la importancia del empleo de diversos métodos de morfometría para lograr una mejor caracterización del proceso que se desea estudiar. En el mismo se emplea el análisis de forma para detectar diferencias, tanto globales como locales, en la morfología del tálamo entre pacientes adolescentes con episodios tempranos de psicosis y adolescentes sanos. Los resultados obtenidos demuestran que la diferencia de volumen talámico entre ambas poblaciones de sujetos, previamente descrita en la literatura, se debe a una reducción del volumen de la región anterior-mediodorsal y del núcleo pulvinar del tálamo de los pacientes respecto a los sujetos sanos. Además, se describe el desarrollo de un estudio longitudinal, en sujetos sanos, que emplea simultáneamente distintos biomarcadores para la caracterización y cuantificación de los cambios que ocurren en la morfología de la corteza cerebral durante la adolescencia. A través de este estudio se revela que el proceso de “alisado” que experimenta la corteza cerebral durante la adolescencia es consecuencia de una disminución de la profundidad, ligada a un incremento en el ancho, de los surcos corticales. Finalmente, esta metodología es aplicada, en un diseño transversal, para el estudio de las causas que provocan el decrecimiento tanto del grosor cortical como del índice de girificación en adolescentes con episodios tempranos de psicosis. ABSTRACT The ever evolving sophistication of magnetic resonance image techniques continue to provide new tools to characterize and quantify, in vivo, brain morphologic changes related to neurodevelopment, senescence, learning or disease. The majority of morphometric methods extract shape or size descriptors such as volume, surface area, and cortical thickness from the MRI image. These morphological measurements are commonly entered in statistical analytic approaches for testing between-group differences or for correlations between the morphological measurement and other variables such as age, sex, or disease severity. A wide variety of morphological biomarkers are reported in the literature. Despite this wide range of potentially useful biomarkers and available morphometric methods, the hypotheses and findings of the grand majority of morphological studies are biased because reports assess only one morphometric feature and usually use only one image processing method. Throughout this dissertation biomarkers and image processing strategies are combined to provide innovative and useful morphometric tools for examining brain changes during neurodevelopment. Specifically, a shape analysis technique allowing for a fine-grained assessment of regional thalamic volume in early-onset psychosis patients and healthy comparison subjects is implemented. Results show that disease-related reductions in global thalamic volume, as previously described by other authors, could be particularly driven by a deficit in the anterior-mediodorsal and pulvinar thalamic regions in patients relative to healthy subjects. Furthermore, in healthy adolescents different cortical features are extracted and combined and their interdependency is assessed over time. This study attempts to extend current knowledge of normal brain development, specifically the largely unexplored relationship between changes of distinct cortical morphological measurements during adolescence. This study demonstrates that cortical flattening, present during adolescence, is produced by a combination of age-related increase in sulcal width and decrease in sulcal depth. Finally, this methodology is applied to a cross-sectional study, investigating the mechanisms underlying the decrease in cortical thickness and gyrification observed in psychotic patients with a disease onset during adolescence.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation

We have isolated a dominant mutation, night blindness a (nba), that causes a slow retinal degeneration in zebrafish. Heterozygous nba fish have normal vision through 2–3 months of age but subsequently become night blind. By 9.5 months of age, visual sensitivity of affected fish may be decreased more than two log units, or 100-fold, as measured behaviorally. Electroretinographic (ERG) thresholds of mutant fish are also raised significantly, and the ERG b-wave shows a delayed implicit time. These defects are due primarily to a late-onset photoreceptor cell degeneration involving initially the rods but eventually the cones as well. Homozygous nba fish display an early-onset neuronal degeneration throughout the retina and elsewhere in the central nervous system. As a result, animals develop with small eyes and die by 4–5 days postfertilization (pf). These latter data indicate that the mutation affecting nba fish is not in a photoreceptor cell-specific gene.

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Germ-line mutations of the BRCA1 gene predispose women to early-onset breast and ovarian cancer by compromising the gene’s presumptive function as a tumor suppressor. Although the biochemical properties of BRCA1 polypeptides are not understood, their expression pattern and subcellular localization suggest a role in cell-cycle regulation. When resting cells are induced to proliferate, the steady-state levels of BRCA1 increase in late G1 and reach a maximum during S phase. Moreover, in S phase cells, BRCA1 polypeptides are hyperphosphorylated and accumulate into discrete subnuclear foci termed “BRCA1 nuclear dots.” BRCA1 associates in vivo with a structurally related protein termed BARD1. Here we show that the steady-state levels of BARD1, unlike those of BRCA1, remain relatively constant during cell cycle progression. However, immunostaining revealed that BARD1 resides within BRCA1 nuclear dots during S phase of the cell cycle, but not during the G1 phase. Nevertheless, BARD1 polypeptides are found exclusively in the nuclear fractions of both G1- and S-phase cells. Therefore, progression to S phase is accompanied by the aggregation of nuclear BARD1 polypeptides into BRCA1 nuclear dots. This cell cycle-dependent colocalization of BARD1 and BRCA1 indicates a role for BARD1 in BRCA1-mediated tumor suppression.

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Cerebral deposition of the amyloid β protein (Aβ) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of Aβ (Aβ40) accounts for ≈90% of all Aβ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (Aβ42), accounting for only ≈10% of secreted Aβ, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of Aβ42 production is a prime therapeutic goal. The same protease, γ-secretase, is assumed to generate the C termini of both Aβ40 and Aβ42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit γ-secretase, on β-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various Aβ40- and Aβ42-specific antibodies, we demonstrate a much stronger inhibition of the γ-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the Aβ40 and Aβ42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general β-amyloid precursor protein metabolism, including Aβ40 production, but specifically block the generation of the pathogenic Aβ42 peptide.

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid β (Aβ) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes Aβ deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of Aβ in the pathogenesis of the disease.

Evidence for phosphorylation and oligomeric assembly of presenilin 1

Pathogenic mutations in presenilin 1 (PS1) are associated with ≈50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12,13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20–23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage λ protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

Transgenic Drosophila expressing human amyloid precursor protein show γ-secretase activity and a blistered-wing phenotype

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer’s disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (βA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogaster is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of βA4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the βA4 region, transmembrane domain, and cytoplasmic tail, we observed βA4 release in flies and fly-tissue culture cells. Consequently, we showed a γ-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independently of βA4 generation.

Synchronization of oscillatory responses in visual cortex correlates with perception in interocular rivalry

In subjects suffering from early onset strabismus, signals conveyed by the two eyes are not perceived simultaneously but in alternation. We exploited this phenomenon of interocular suppression to investigate the neuronal correlate of binocular rivalry in primary visual cortex of awake strabismic cats. Monocularly presented stimuli that were readily perceived by the animal evoked synchronized discharges with an oscillatory patterning in the γ-frequency range. Upon dichoptic stimulation, neurons responding to the stimulus that continued to be perceived increased the synchronicity and the regularity of their oscillatory patterning while the reverse was true for neurons responding to the stimulus that was no longer perceived. These differential changes were not associated with modifications of discharge rate, suggesting that at early stages of visual processing the degree of synchronicity rather than the amplitude of responses determines which signals are perceived and control behavioral responses.

Expression of the Recessive Glomerulosclerosis Gene Mpv17 Regulates MMP-2 Expression in Fibroblasts, the Kidney, and the Inner Ear of Mice

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Reverse genetic analysis of Caenorhabditis elegans presenilins reveals redundant but unequal roles for sel-12 and hop-1 in Notch-pathway signaling

Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer’s disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, we used a reverse genetic strategy to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions displayed new phenotypes not observed in animals bearing either single deletion. These new phenotypes—germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects—resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1.

Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanism-based pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.