86,599 research outputs found
Single-molecule localization to study cytoskeletal structures, membrane complexes, and mechanosensors
No full textIn the last decades, a promising breakthrough in fluorescence imaging was represented by the advent of super-resolution microscopy (SRM). Super-resolution techniques recently became a popular method to study sub-cellular structures, providing a successful approach to observe cytoskeletal and focal adhesion proteins. Among the SR techniques, single-molecule localization microscopy plays a significant role due to its ability to unveil structures and molecular organizations in biological systems. Furthermore, since they provide information at the molecular level, these techniques are increasingly being used to study the stoichiometry and interaction between several membrane channel proteins and their accessory subunits. The aim of this review is to describe the single-molecule localization-based techniques and their applications relevant to cytoskeletal structures and membrane complexes in order to provide as future prospective an overall picture of their correlation with the mechanosensor channel expression and activity
Lifespan of neurons is uncoupled from organismal lifespan.
No full textNeurons in mammals do not undergo replicative aging, and, in absence of pathologic conditions, their lifespan is limited only by the maximum lifespan of the organism. Whether neuronal lifespan is determined by the strain-specific lifetime or can be extended beyond this limit is unknown. Here, we transplanted embryonic mouse cerebellar precursors into the developing brain of the longer-living Wistar rats. The donor cells integrated into the rat cerebellum developing into mature neurons while retaining mouse-specific morphometric traits. In their new environment, the grafted mouse neurons did not die at or before the maximum lifespan of their strain of origin but survived as long as 36 mo, doubling the average lifespan of the donor mice. Thus, the lifespan of neurons is not limited by the maximum lifespan of the donor organism, but continues when transplanted in a longer-living host
Fate restriction and developmental potential of cerebellar progenitors. Transplantation studies in the developing CNS.
No full textThe generation of cell diversity from undifferentiated progenitors is regulated by interdependent mechanisms, including cell intrinsic programs and environmental cues. This interaction can be investigated by means of heterochronic/heterotopic transplantation, which allows to examine the behaviour of precursor cells in an unusual environment. The cerebellum provides an ideal model to study cell specification, because its neurons originate according to a well-defined timetable and they can be are readily recognised by morphological features and specific markers. Cerebellar progenitors transplanted to the embryonic cerebellum develop fully mature cerebellar neurons, which often integrate in the host circuitry in a highly specific manner. In extracerebellar locations, cerebellar progenitors preferentially settle in caudal CNS regions where they exclusively acquire cerebellar identities. By contrast, neocortical precursors preferentially settle in rostral regions and fail to develop hindbrain phenotypes. The phenotypic repertoire generated by transplanted cerebellar progenitors is strictly dependent on their age. Embryonic progenitors originate all mature cerebellar cells, whereas postnatal ones exclusively generate later-born types, such as molecular layer interneurons and granule cells. Together, these observations foster the hypothesis that neural progenitors are first specified towards region-specific phenotypes along the rostro-caudal axis of the neural tube. Thereafter, the developmental potential of progenitor cells is progressively restricted towards later generated types. Such a progressive specification of precursor cells in space and time is stably transmitted to their progeny and it cannot be modified by local cues, when these cells are confronted with heterotopic and/or heterochronic environments
ENGRAFTMENT AND DIFFERENTIATION OF NEOCORTICAL PROGENITOR CELLS TRANSPLANTED TO THE EMBRYONIC BRAIN IN UTERO.
No full textTransplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ss-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CN
Vitamin D receptor expression in human brain tumors.
No full textVitamin D receptor (VDR) has important effects not only on physiological processes related to Ca2+ metabolism but also on cell growth and differentiation. VDR is a member of the Steroid-Thyroid Receptors Superfamily (STRS). Work in our and other laboratories has shown that several other members of the STRS (androgen, estrogen, glucocorticoid, and progesterone receptors) are present in astrocytomas and glioblastomas. We now report the finding of VDR-like mRNA in human anaplastic astrocytomas and glioblastomas. VDR mRNA levels, as determined by a method, developed in our laboratory, based on the polymerase chain reaction, are significantly higher in glioblastomas compared to both low and high grade astrocytomas. We discuss the biological and clinical implications of our result
Cerebellar granule cells transplanted in vivo can follow physiological and unusual migratory routes to integrate into the recipient cortex.
No full textCNS repair by cell transplantation requires new neurons to integrate into complex recipient networks. We assessed how the migratory route of transplanted granule neurons and the developmental stage of the host rat cerebellum influence engraftment. In both embryonic and postnatal hosts, granule cells can enter the cerebellar cortex and achieve correct placement along their natural migratory pathway. Donor neurons can also reach the internal granular layer from the white matter and integrate following an unusual developmental pattern. Although the frequency of correct positioning declines in parallel with cortical development, in mature recipients correct homing is more frequent through the unusual path. Following depletion of granule cell precursors in the host, more granule neurons engraft, but their ability for achieving correct placement is unchanged. Therefore, while the cerebellar environment remains receptive for granule cells even after the end of development, their full integration is partially hindered by the mature cortical architecture
G-CSF administration to adult mice stimulates the proliferation of microglia but does not modify the outcome of ischemic injury.
No full textRecent evidence suggests that adult bone marrow stem cells reduce tissue damage and promote repair following CNS ischemic injury. Since granulocyte-colony stimulating factor (G-CSF) mobilizes hematopoietic stem cells to the circulating compartment, here we tested whether administration of this drug modifies the outcome of a permanent occlusion of the middle cerebral artery in adult mice. To elucidate the behavior and fate of blood-borne cells in the ischemic brain, we produced chimeric animals, in which hematopoietic derivatives are genetically tagged. G-CSF administration enhances the proliferation of microglia in the uninjured CNS but has no effect on the amount of hematopoietic cells that infiltrate the ischemic tissue and on the size of the lesion. The blood-borne elements acquire different mesodermal identities but fail to adopt neural phenotypes, even though they occasionally fuse with Purkinje neurons. These results indicate that G-CSF treatment does not exert a significant beneficial effect on the ischemic injury. (C) 2010 Elsevier Inc. All rights reserved
Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery
No full textHerpes simplex virus thymidine kinase (HSV-TK) is widely used in gene therapy. The enzymatic activity of HSV-TK may be traced in vivo by specific radiopharmaceuticals in order to image transgene expression. However, most of these radiopharmaceuticals are toxic per se or after activation by HSV-TK, and therefore do not represent ideal molecules for clinical applications and repeated imaging. Unlike human cytosolic TK, HSV-TK is not enantioselective and can efficiently phosphorylate both D and L enantiomers of beta-thymidine. Here we show that, after phosphorylation by HSV-TK, tritiated L-beta-thymidine (LT) is selectively retained inside the cells in vitro and in vivo. We used the in vivo accumulation of radioactive phosphorylated LT to image the HSV-TK-positive cells inside a transplantable murine brain tumour after inoculation of cells producing retroviruses carrying HSV-TK. Owing to their unnatural enantiomeric conformation, phosphorylated LT metabolites are very poorly processed by mammalian enzymes, thus leading to increased cellular retention and minimal toxicity. The ability to image cells expressing the HSV-TK gene by using radiolabelled LT, without damaging the cells accumulating the phosphorylated L-nucleoside, will be important to monitor the levels and spatial distribution of therapeutic vectors carrying HSV-TK
The contribution of surgical brain mapping to the understanding of the anatomo-functional basis of syntax : a critical review
No full textA wide range of studies on language assessment during awake brain surgery is nowadays available. Yet, a consensus on a standardized protocol for intraoperative language mapping is still lacking. More specifically, very limited information is offered about intraoperative assessment of a crucial component of language such as syntax. This review aims at critically analyzing the intraoperative studies investigating the cerebral basis of syntactic processing. A comprehensive query was performed on the literature, returning a total of 18 studies. These papers were analyzed according to two complementary criteria, based on the distinction between morphosyntax and syntax. The first criterion focused on the tasks and stimuli employed intraoperatively. Studies were divided into three different groups: group 1 included those studies that overtly aimed at investigating morphosyntactic processes; group 2 included studies that did not explicitly focus on syntax, yet employed stimuli requiring morphosyntactic processing; and group 3 included studies reporting some generic form of syntactic deficit, although not further investigated. The second criterion focused on the syntactic structures of the sentences assessed intraoperatively, analyzing the canonicity of sentence structure (i.e., canonical versus non-canonical word order). The global picture emerging from our analysis indicates that what was investigated in the intraoperative literature is morphosyntactic processing, rather than pure syntax. The study of the neurobiology of syntax during awake surgery seems thus to be still at an early stage, in need of systematic, linguistically grounded investigations
Activation and Inactivation of Homomeric KvLQT1 Potassium Channels
No full textAbstractThe voltage-gated potassium channel protein KvLQT1 (Wang et al., 1996. Nature Genet. 12:17–23) is believed to underlie the delayed rectifier potassium current of cardiac muscle together with the small membrane protein minK (also named IsK) as an essential auxiliary subunit (Barhanin et al., 1996. Nature. 384:78–80; Sanguinetti et al., 1996. Nature. 384:80–83). Using the Xenopus oocyte expression system, we analyzed in detail the gating characteristics of homomeric KvLQT1 channels and of heteromeric KvLQT1/minK channels using two-electrode voltage-clamp recordings. Activation of homomeric KvLQT1 at positive voltages is accompanied by an inactivation process that is revealed by a transient increase in conductance after membrane repolarization to negative values. We studied the recovery from inactivation and the deactivation of the channels during tail repolarizations at −120mV after conditioning pulses of variable amplitude and duration. Most measurements were made in high extracellular potassium to increase the size of inward tail currents. However, experiments in normal low-potassium solutions showed that, in contrast to classical C-type inactivation, the inactivation of KvLQT1 is independent of extracellular potassium. At +40mV inactivation develops with a delay of 100ms. At the same potential, the activation estimated from the amplitude of the late exponential decay of the tail currents follows a less sigmoidal time course, with a late time constant of 300ms. Inactivation of KvLQT1 is not complete, even at the most positive voltages. The delayed, voltage-dependent onset and the incompleteness of inactivation suggest a sequential gating scheme containing at least two open states and ending with an inactivating step that is voltage independent. In coexpression experiments of KvLQT1 with minK, inactivation seems to be largely absent, although biphasic tails are also observed that could be related to similar phenomena
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