Mitogen and stress-activated kinases 1/2 regulate ischemia-induced hippocampal progenitor cell proliferation and neurogenesis.

Pathophysiological conditions such as cerebral ischemia trigger the production of new neurons from the neurogenic niche within the subgranular zone (SGZ) of the dentate gyrus. The functional significance of ischemia-induced neurogenesis is believed to be the regeneration of lost cells, thus contributing to post-ischemia recovery. However, the cell signaling mechanisms by which this process is regulated are still under investigation. Here, we investigated the role of mitogen and stress-activated protein kinases (MSK1/2) in the regulation of progenitor cell proliferation and neurogenesis after cerebral ischemia. Using the endothelin-1 model of ischemia, wild-type (WT) and MSK1(-/-)/MSK2(-/-) (MSK dKO) mice were injected with BrdU and sacrificed 2 days, 4 weeks, or 6 weeks later for the analysis of progenitor cell proliferation, neurogenesis, and neuronal morphology, respectively. We report a decrease in SGZ progenitor cell proliferation in MSK dKO mice compared to WT mice. Moreover, MSK dKO mice exhibited reduced neurogenesis and a delayed maturation of ischemia-induced newborn neurons. Further, structural analysis of neuronal arborization revealed reduced branching complexity in MSK dKO compared to WT mice. Taken together, this dataset suggests that MSK1/2 plays a significant role in the regulation of ischemia-induced progenitor cell proliferation and neurogenesis. Ultimately, revealing the cell signaling mechanisms that promote neuronal recovery will lead to novel pharmacological approaches for the treatment of neurodegenerative diseases such as cerebral ischemia.

Human neural responses elicited to observing the actions of others.

Monkey electrophysiological and human neuroimaging studies indicate the existence of specialized neural systems for the perception and execution of actions. To date, the dynamics of these neural systems in humans have not been well studied. Here, we investigated the spatial and temporal behavior of human neural responses elicited to viewing motion of the face, hand, and body. Scalp event-related potentials (ERPs) were recorded in 20 participants viewing videotaped mouth (opening, closing), hand (closing, opening), and body stepping (forward, backward) movements. ERP peak differences within the movements of each body part were compared using topographical maps of voltage, voltage difference, and Student's t-test at ERP peak latencies. Predominantly temporoparietal negative ERPs occurred to motion of all body parts within 200 ms postmovement onset. Hand closure elicited a significantly greater negativity than opening, particularly in the left hemisphere. Vertex positive ERPs within 300 ms postmovement onset were elicited to hand and body motion. A significantly greater positivity occurred for the body stepping forward relative to stepping backward. The ERP topography was consistent with observed activation foci in human neuroimaging studies. Our data indicate that the neural activity of a system dedicated to the perception of high-level motion stimuli can rapidly differentiate between movements across and within body parts.

Loss of functional caveolae during senescence of human fibroblasts.

Primary human fibroblasts have a finite replicative lifespan in culture that culminates in a unique state of growth arrest, termed senescence that is accompanied by distinct morphological and biochemical alterations. Senescent cell responses to extracellular stimuli are believed to be altered at a point after receptors are bound by ligand, leading to improper integration of the signals which initiate DNA replication. In this study we demonstrate that one of the key organizing membrane microdomains for receptor signaling, caveolae, are absent in senescent cells. A comparison of young and senescent cells indicated that senescent cells contained a higher total amount of caveolins 1 and 2 but had significantly less of both proteins in the caveolar fraction. Additionally, caveolar fractions from senescent cells completely lacked the tyrosine-kinase activity associated with functional caveolae. Furthermore, old cells had little caveolar protein exposed to the outer plasma membrane as estimated by using an in vivo biotinylation assay and no detectable caveolin 1 on the cell surface when processed for immunofluoresence and confocal microscopy. Together, these data suggest that a fundamental loss of signal integration at the plasma membrane of senescent cells is due to the loss of signaling competent caveolae.

Gestures maintain spatial imagery.

Recent theories suggest alternatives to the commonly held belief that the sole role of gestures is to communicate meaning directly to listeners. Evidence suggests that gestures may serve a cognitive function for speakers, possibly acting as lexical primes. We observed that participants gestured more often when describing a picture from memory than when the picture was present and that gestures were not influenced by manipulating eye contact of a listener. We argue that spatial imagery serves a short-term memory function during lexical search and that gestures may help maintain spatial images. When spatial imagery is not necessary, as in conditions of direct visual stimulation, reliance on gestures is reduced or eliminated.

Decreased expression and activity of the immediate-early growth response (Egr-1) gene product during cellular senescence.

Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in culture before reaching the end of their proliferative life span, an event termed in vitro cellular senescence. Considerable evidence suggests that altered expression of key genes involved in the mitogenic response may be responsible for the inability of senescent cells to proliferate. Here we examined the expression and activity of the early growth response-1 (egr-1) gene, an "immediate-early" gene that is believed to link extracellular mitogenic signals to cell-cycle progression. We found that egr-1 was strongly downregulated in senescent HDFs at the level of mRNA, protein, and DNA binding activity. Decreased DNA binding activity of Egr-1 in vitro corresponded to decreased transcriptional activation in vivo. To further understand the mechanism of egr-1 downregulation, we examined the potential role of the serum response elements (SREs) present in the egr-1 promoter. Electrophoretic mobility shift studies using young and old cell nuclear extracts showed a marked decrease in serum response factor (SRF) binding activity to the SRE in old compared to young cells. Loss of SRF binding activity has been correlated with the loss of expression of another growth-related immediate-early gene (c-fos). These results suggest a common mechanism for the downregulation of c-fos, egr-1, and other SRE-dependent, mitogen-responsive genes during cellular senescence.

Regulation of transcription factor activity during cellular aging.

Several lines of evidence suggest that the limited replication potential of normal human cells is due to the presence of an intrinsic genetic programme. This "senescence programme" is believed to reduce the incidence of cancer by limiting the growth of most of the transformed cells arising in vivo, although some cells do escape senescence becoming both immortalized and transformed. Here we review the literature that describes the senescence process in terms of gene expression and the regulation of gene expression by a variety of mechanisms affecting transcription factor activity. We focus on regulation of the c-fos gene through posttranslational modification of the serum response factor (SRF) as an example of altered gene expression during cellular aging.

Identification and localization of a kainate binding protein in the frog inner ear by electron microscopy immunocytochemistry.

A kainate binding protein (KBP) was studied in Rana pipiens inner ear using monoclonal and polyclonal antibodies against affinity purified KBP from frog brain. The KBP identified and analyzed in inner ear tissue homogenates, with one- and two-dimensional immunoblots, was similar to the affinity purified KBP and to the antibody-identified frog brain KBP. As brain KBP, inner ear KBP had 5 main components in the molecular weight dimension, centered at Mr = 48,000; however, inner ear KBP had a greater abundance of the higher molecular weight components. Light and electron microscopy observations showed KBP immunostaining at two locations: (1) in the dendrites of the eight nerve afferent fibers contacting sensory hair cells, with the postsynaptic density being more intensely stained; and (2) on the cytoplasmic membrane of fibroblasts present in the inner ear connective tissue which displayed intense immunostaining. The presence of kainate (KA) binding sites in the inner ear was assessed using in vitro receptor autoradiography. [3H]KA binding sites were found in connective tissue areas confirming the immunocytochemistry results. The postsynaptic localization of the KBP in afferent endings, strongly supports it as being a component of the KA receptor complex. However, its presence on fibroblasts situated in the inner ear connective tissue makes its function hypothetical. The dual presence of the KBP on non-neuronal cells as well as at postsynaptic membrane sites suggests the existence of a family of proteins involved in KA binding and KA receptors with a complex organization.

Distribution of a putative kainic acid receptor in the frog central nervous system determined with monoclonal and polyclonal antibodies: evidence for synaptic and extrasynaptic localization.

A frog brain kainic acid receptor (KAR) was studied using monoclonal and polyclonal antibodies against the affinity-purified receptor. Immunocytochemistry was done on sections of the frog CNS, and the distribution of immunostaining was compared with the distribution of high- and low-affinity 3H-kainic acid (3H-KA) binding sites determined with in vitro receptor autoradiography. These studies showed (1) similar distributions of high- and low-affinity 3H-KA binding sites, (2) identical patterns of immunostaining with the polyclonal antibodies and 2 monoclonal antibodies, and (3) an antibody binding distribution which closely matched that of 3H-KA binding, suggesting that the antibodies recognize the primary KAR in frog brain. In the frog brain, an anteroposterior gradient of immunostaining was observed, with the telencephalon intensely and uniformly immunoreactive. Other areas intensely immunoreactive included the cerebellum, the infundibulum, the tectal and posterior commissures, and the laminar nucleus of the torus semicircularis. The optic tectum showed selective staining of the plexiform layers 3 and 5-7. The pattern of staining was punctate and appeared to be associated with nerve fibers, among them dendritic arborizations. Electron microscopic observations showed staining at the cytoplasmic side of postsynaptic membranes. Extra-synaptic staining was observed as patches on the surface of unmyelinated nerve processes.

Solubilization and partial purification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites from rat brain.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.

Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping.

Monoclonal antibodies (mAb) and a polyclonal antiserum were produced against a kainic acid receptor (KAR) purified from frog brain. Several of the mAb and the antiserum immunoprecipitated [3H]kainic acid binding activity from solubilized preparations of frog brain and labeled a group of proteins on immunoblots that migrated at Mr = 48,000. These results confirm that the ligand binding subunit of the frog brain KAR is contained in the Mr = 48,000 proteins. Immunoblots from different frog tissues demonstrated that the antibody reactivity was highly concentrated in the frog nervous system with no detectable immunoreactivity observed in non-neuronal tissues. The purified KAR was radioiodinated and subjected to two-dimensional gel electrophoresis and autoradiography. A series of proteins was detected at Mr = 48,000 with isoelectric points from 5.5 to 6.3. The anti-KAR mAb and the antiserum reacted with the same group of proteins from frog whole brain after separation by two-dimensional gel electrophoresis. Peptide maps of the 125I-labeled KAR separated by two-dimensional gel electrophoresis demonstrated that the group of proteins clustered at Mr = 48,000 is homologous. mAb KAR-B1 reacted on immunoblots with a protein in rat brain with a Mr = 99,000. This protein comigrated with an unreduced form of the KAR in frog brain. It was present in rat cerebral cortex, hippocampus, and cerebellum but was not detected in thalamus, globus pallidus, or brain stem, nor was it detected in rat non-neuronal tissues. The presence of the Mr = 99,000 immunoreactive polypeptide in discrete areas of rat brain suggests that this protein may be part of a mammalian KAR or a related receptor.