Carme Auladell

The early development of thalamocortical and corticothalamic projections in the mouse.

The initial ingrowth of corticothalamic and thalamocortical projections was examined in mice at embryonic and perinatal stages. Fibers, in fixed brains, were labeled with the carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocianine perchlorate (DiI). By E13, the corticofugal fibers had entered the lowest intermediate zone through which they ran, turned over the corpus striatum, and left the cortex. The fibers were arranged in scattered bundles throughout the corpus striatum. At E14 corticofugal axons reached the internal capsule and at E14.5–E15 they established contact within the thalamus. Meanwhile, the thalamocortical afferents reached the neocortex at E13. At this time fibers ran tangentially within the intermediate zone, immediately underneath the cortical plate. By E14, the fibers had started to invade the subplate and, by E15, thalamocortical fibers had begun their radial growth into the cortex. Such radial growth proceeded steadily, invading each cortical layer as it differentiated cytoarchitectonically from the dense cortical plate. The first retrogradely labeled cells were detected at the cortical plate at E15. By the day of birth (E20), thalamocortical fibers had formed a dense branching system within layers VI and V. Our observations indicate that, in mice, the thalamic axons reach the cortex before corticothalamic projections enter the thalamic nuclei. Moreover, the results suggest that the pathway followed by each fiber system is different. By DiI injections into the internal capsule we have also determined that subplate cells are the first to send axons to the thalamus.

Early alterations in energy metabolism in the hippocampus of APPswe/PS1dE9 mouse model of Alzheimer's disease

The present study had focused on the behavioral phenotype and gene expression profile of molecules related to insulin receptor signaling in the hippocampus of 3 and 6 month-old APPswe/PS1dE9 (APP/PS1) transgenic mouse model of Alzheimers disease (AD). Elevated levels of the insoluble Aβ (1-42) were detected in the brain extracts of the transgenic animals as early as 3 months of age, prior to the Aβ plaque formation (pre-plaque stage). By the early plaque stage (6 months) both the soluble and insoluble Aβ (1-40) and Aβ (1-42) peptides were detectable. We studied the expression of genes related to memory function (Arc, Fos), insulin signaling, including insulin receptor (Insr), Irs1 and Irs2, as well as genes involved in insulin growth factor pathways, such as Igf1, Igf2, Igfr and Igfbp2. We also examined the expression and protein levels of key molecules related to energy metabolism (PGC1-α, and AMPK) and mitochondrial functionality (OXPHOS, TFAM, NRF1 and NRF2). 6 month-old APP/PS1 mice demonstrated impaired cognitive ability, were glucose intolerant and showed a significant reduction in hippocampal Insr and Irs2 transcripts. Further observations also suggest alterations in key cellular energy sensors that regulate the activities of a number of metabolic enzymes through phosphorylation, such as a decrease in the Prkaa2 mRNA levels and in the pAMPK (Thr172)/Total APMK ratio. Moreover, mRNA and protein analysis reveals a significant downregulation of genes essential for mitochondrial replication and respiratory function, including PGC-1α in hippocampal extracts of APP/PS1 mice, compared to age-matched wild-type controls at 3 and 6 months of age. Overall, the findings of this study show early alterations in genes involved in insulin and energy metabolism pathways in an APP/PS1 model of AD. These changes affect the activity of key molecules like NRF1 and PGC-1α, which are involved in mitochondrial biogenesis. Our results reinforce the hypothesis that the impairments in both insulin signaling and energy metabolism precede the development of AD amyloidogenesis.

Role of cell cycle re-entry in neurons: a common apoptotic mechanism of neuronal cell death.

Currently, there is no effective treatment for neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Thus, a major focus of neuroscience research is to examine the mechanisms involved in neuronal loss in order to identify potential drug targets. Recent results indicate that DNA damage and re-entry into the cell cycle may constitute a common pathway in apoptosis in neurological diseases. The role of the cell cycle in such disorders is supported by data on the brain of patients who showed an increase in cell-cycle protein expression. Indeed, studies performed in neuronal cell preparations indicate that oxidative stress could be the main mechanism responsible for cell cycle re-entry. DNA damage and repair after oxidative stress may activate the enzyme ataxia telangiectasia mutated, which is a cell-cycle regulator. Once the cell cycle is activated, the increase in the expression of transcription factor E2F-1 could induce neuronal apoptosis. Furthermore, the potential routes involved in E2F-1 induced apoptosis could be p53-dependent or p53-independent. Under this E2F-1 hypothesis of cell death, multiple mitochondria-dependent pathways may be activated, including caspase and caspase-independent signaling cascades. Finally, given that cyclin-dependent kinase inhibitory drugs have neuroprotective and anti-apoptotic effects in experimental models, their potential application for the treatment of neurological disorders should be taken into account.

Zac1 is expressed in progenitor/stem cells of the neuroectoderm and mesoderm during embryogenesis: Differential phenotype of the Zac1-expressing cells during development

Zac1, a new zinc‐finger protein that regulates both apoptosis and cell cycle arrest, is abundantly expressed in many neuroepithelia during early brain development. In the present work, we study the expression of Zac1 during early embryogenesis and we determine the cellular phenotype of the Zac1‐expressing cells throughout development. Our results show that Zac1 is expressed in the progenitor/stem cells of several tissues (nervous system, skeleton, and skeletal muscle), because they colocalize with several progenitor/stem markers (Nestin, glial fibrillary acidic protein, FORSE‐1, proliferating cell nuclear antigen, and bromodeoxyuridine). In postnatal development, Zac1 is expressed in all phases of the life cycle of the chondrocytes (from proliferation to apoptosis), in some limbic γ‐aminobutyric acid‐ergic neuronal subpopulations, and during developmental myofibers. Therefore, the intense expression of Zac1 in the progenitor/stem cells of different cellular lineages during the proliferative cycle, before differentiation into postmitotic cells, suggests that Zac1 plays an important role in the control of cell fate during neurogenesis, chondrogenesis, and myogenesis. Developmental Dynamics 233:667–679, 2005.

Expression pattern of Zac1 mouse gene, a new zinc-finger protein that regulates apoptosis and cellular cycle arrest, in both adult brain and along development.

Using in situ hybridization, we analyzed the expression pattern of the Zac1 gene in mouse brain during the embryonic and postnatal development. Zac1 is a new gene that regulates extensive apoptosis and cell cycle arrest through unrelated pathways. At embryonic stages, strong expression was observed in brain areas with active proliferation (ventricular zone and numerous neuroepithelius) and in nervous system (neural retina and neural tube). In addition, some areas with differentiation activity were noticeably labeled such as arcuate nucleus and amygdaloid region of the brain together with other embryonic sites (hindlimb, forelimb and somites). From P0 onwards, the expression appeared in some proliferative areas, such as subventricular zone and cerebellum (external granular layer and Purkinje cells) and in some synaptic plasticity areas, such as the dorso and ventromedial hypothalamic nuclei, arcuate nucleus, ventral thalamic nucleus.

Growth, Degrowth and Regeneration as Developmental Phenomena in Adult Freshwater Planarians

Freshwater planarians are Platyhelminthes belonging to the class Turbellaria, order Seriata, suborder Tricladida, infra-order Paludicola. The term Tricladida (or triclads) refers to the three main branches into which their digestive system are divided; Paludicola means members are inhabitants of freshwater habitats. Freshwater planarians are the best known planarians due to there easy culture and ease of handling under laboratory conditions and, because they have been, and still are, the most widely used turbellarian in experimental research, particularly with regards to regeneration (see Bronsted, 1969, and Gremigni, 1988, for general references).

Current Research Therapeutic Strategies for Alzheimer’s Disease Treatment

Alzheimers disease (AD) currently presents one of the biggest healthcare issues in the developed countries. There is no effective treatment capable of slowing down disease progression. In recent years the main focus of research on novel pharmacotherapies was based on the amyloidogenic hypothesis of AD, which posits that the beta amyloid (Aβ) peptide is chiefly responsible for cognitive impairment and neuronal death. The goal of such treatments is (a) to reduce Aβ production through the inhibition of β and γ secretase enzymes and (b) to promote dissolution of existing cerebral Aβ plaques. However, this approach has proven to be only modestly effective. Recent studies suggest an alternative strategy centred on the inhibition of the downstream Aβ signalling, particularly at the synapse. Aβ oligomers may cause aberrant N-methyl-D-aspartate receptor (NMDAR) activation postsynaptically by forming complexes with the cell-surface prion protein (PrPC). PrPC is enriched at the neuronal postsynaptic density, where it interacts with Fyn tyrosine kinase. Fyn activation occurs when Aβ is bound to PrPC-Fyn complex. Fyn causes tyrosine phosphorylation of the NR2B subunit of metabotropic glutamate receptor 5 (mGluR5). Fyn kinase blockers masitinib and saracatinib have proven to be efficacious in treating AD symptoms in experimental mouse models of the disease.

Ultrastructural localization of RNA in the chromatoid bodies of undifferentiated cells (neoblasts) in planarians by the RNase–gold complex technique

Undifferentiated cells of planarians (Platyhelminthes, Turbellaria), also called neoblasts, are totipotent stem cells, which give rise to all differentiated cell types, while maintaining their own density by cell proliferation. Neoblasts are the only somatic cells of planarians bearing chromatoid bodies in their cytoplasm; these organelles disappear as differentiation takes place. Studies on germinal cells of several groups of organisms have shown that chromatoid bodies contain substantial amounts of RNA. To test its presence in neoblasts, we have used an RNase–gold technique. We found chromatoid bodies labeled with RNase–gold particles. Heterogeneity in the density of the label, may be correlated with the functionality and complexity of these organelles. The gold marker was also present over the nucleus and rough endoplasmic reticulum, but mitochondria, secretory granules, and the extracellular space were devoid of label. This specific localization of RNA in planarian chromatoid bodies supports earlier findings on germ cells and embryonic cells in a variety of organisms, indicating that chromatoid bodies are information‐storage structures, essential during the process of cell differentiation.

Postnatal development of zinc-rich terminal fields in the brain of the rat.

The appearance and distribution of zinc-rich terminal fields in the rat forebrain was analyzed at 12 stages of postnatal development using the selenium method. Zinc stain was detected in neonates in piriform, cingulate, and motor cortices, septal area, and hippocampal formation. In the neocortex, a laminar pattern appeared progressively following an inside-out gradient: layer VI at postnatal day 0 (P0), layer V at P1, layers Va and Vb at P5, layer II-III at P9, and layer IV at P12. In the hippocampal formation the layered pattern in the dentate molecular layer appeared at P1-P3, and in the hilus and mossy fibers the stain was observed at P5. Patches in the caudate-putamen were sharply delimited at P1-P3. At these ages, staining was observed in the amygdaloid complex. In the thalamic and hypothalamic nuclei, stain appeared at P5-P7. Thus, a general increase in vesicular zinc over different telencephalic areas was determined until P15-P21, which was followed by a slight decrease thereafter (at P41). The increased stain in zinc-rich terminal fields is consistent with the development of telencephalic circuits. The rise in zinc might be relevant for the establishment and maturation of these circuits. On the other hand, the decrease in staining for zinc at later stages might be due to methodological problems but it might also reflect pruning of supernumerary connections and programmed cell death affecting zinc-rich circuits.

Developmental Expression of ZnT3 in Mouse Brain: Correlation between the Vesicular Zinc Transporter Protein and Chelatable Vesicular Zinc (CVZ) Cells. Glial and Neuronal CVZ Cells Interact

We examine the expression pattern of ZnT3 in the cerebral and cerebellar areas of mouse brain throughout development. During embryogenesis and early postnatal stages, ZnT3 transcripts were detected in several areas. Label was clear in areas related to proliferation and differentiation. As development proceeded, the label gradually disappeared in these areas and increased in the chelatable vesicular zinc (CVZ) system. To assess whether ZnT3 was expressed in all CVZ cells, its distribution pattern was studied through postnatal stages using a retrograde zinc transport method. While the ZnT3 expression pattern and the distribution of CVZ cells coincided from P12 to adulthood, this coincidence was not detected in early postnatal days. Moreover, immunohistochemical procedures highlighted a differential phenotype within CVZ cells throughout postnatal development. These findings suggest the presence of different CVZ cell subpopulations throughout brain development and, consequently, the existence of distinct chelatable vesicular zinc pools.