[Show abstract][Hide abstract] ABSTRACT: Rod outer segment membrane guanylate cyclase (ROS-GC1) is a bimodal Ca(2+) signal transduction switch. Lowering [Ca(2+)](i) from 200 to 20 nM progressively turns it "ON" as does raising [Ca(2+)](i) from 500 to 5000 nM. The mode operating at lower [Ca(2+)](i) plays a vital role in phototransduction in both rods and cones. The physiological function of the mode operating at elevated [Ca(2+)](i) is not known. Through comprehensive studies on mice involving gene deletions, biochemistry, immunohistochemistry, electroretinograms and single cell recordings, the present study demonstrates that the Ca(2+)-sensor S100B coexists with and is physiologically linked to ROS-GC1 in cones but not in rods. It up-regulates ROS-GC1 activity with a K(1/2) for Ca(2+) greater than 500 nM and modulates the transmission of neural signals to cone ON-bipolar cells. Furthermore, a possibility is raised that under pathological conditions where [Ca(2+)](i) levels rise to and perhaps even enter the micromolar range, the S100B signaling switch will be turned "ON" causing an explosive production of CNG channel opening and further rise in [Ca(2+)](i) in cone outer segments. The findings define a new cone-specific Ca(2+)-dependent feature of photoreceptors and expand our understanding of the operational principles of phototransduction machinery.
Cellular Physiology and Biochemistry 01/2012; 29(3-4):417-30. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have reported antibodies bound to cells in postmortem Alzheimer's disease (AD) brains, which are only rarely observed in the brains of healthy, age-matched controls. This implies that brain-reactive autoantibodies exist in the sera of AD individuals and can gain access to the brain interstitium. To investigate this possibility, we determined the prevalence of brain-reactive antibodies in sera from AD patients, patients with other neurodegenerative diseases, age-matched, non-demented controls and healthy younger individuals via immunohistochemistry and western blot analysis. Surprisingly, western analyses revealed that 92% of all human sera tested contain brain-reactive autoantibodies. When sera were used to probe western blots of human, pig, or rat brain membrane proteins, a number of comparably-sized protein targets were detected, suggesting cross-species reactivity. While the presence of brain-reactive autoantibodies was nearly ubiquitous in human sera, some autoantibodies appeared to be associated with age or disease. Furthermore, the intensity of antibody binding to brain tissue elements, especially the surfaces of neurons, correlated more closely to the serum's autoantibody profile than to age or the presence of neurodegenerative disease. However, while the blood-brain barrier (BBB) in control brains remained intact, BBB breakdown was common in AD brains. Results suggest a high prevalence of brain-reactive antibodies in human sera which, in the common context of BBB compromise, leads us to propose that these antibodies may contribute to the initiation and/or pathogenesis of AD and other neurodegenerative diseases.
Brain research 07/2010; 1345:221-32. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Early pathological features of Alzheimer's disease (AD) include synaptic loss and dendrite retraction, prior to neuronal loss. How neurons respond to this evolving AD pathology remains elusive. In the present study, we used single- and double-label immunohistochemistry to investigate the relationship between neuronal vimentin expression and local brain pathology. Vimentin was localized to neuronal perikarya and dendrites in AD brain, with vimentin-immunopositive neurons prevalent in regions exhibiting intra- and extracellular beta-amyloid(1-42) (Abeta42) deposition. Neuronal co-localization of vimentin and Abeta42 was common in the cerebral cortex, cerebellum and hippocampus. Additionally, neurons in affected brain regions of AD transgenic (Tg2576) mice and in brain tissue subjected to mechanical injury expressed vimentin, while those in comparable regions of control mouse brain did not. Finally, we show that neurons in human fetal brain express vimentin concurrently with periods of rapid neurite extension. Overall, our results suggest that neurons express vimentin as part of an evolutionarily conserved, damage-response mechanism which recapitulates a developmental program used by differentiating neurons to establish dendrites and synaptic connections.
Brain research 10/2009; 1298:194-207. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deposition of beta-amyloid (Abeta) peptides in the walls of brain blood vessels, cerebral amyloid angiopathy (CAA), is common in patients with Alzheimer's disease (AD). Previous studies have demonstrated Abeta peptide deposition among vascular smooth muscle cells (VSMCs), but the source of the Abeta and basis for its selective deposition in VSMCs are unknown. In the present study, we examined the deposition patterns of Abeta peptides, Abeta40 and Abeta42, within the cerebrovasculature of AD and control patients using single- and double-label immunohistochemistry. Abeta40 and Abeta42 were abundant in VSMCs, especially in leptomeningeal arteries and their initial cortical branches; in later-stage AD brains this pattern extended into the microvasculature. Abeta peptide deposition was linked to loss of VSMC viability. Perivascular leak clouds of Abeta-positive material were associated primarily with arterioles. By contrast, control brains possessed far fewer Abeta42- and Abeta40-immunopositive blood vessels, with perivascular leak clouds of Abeta-immunopositive material rarely observed. We also demonstrate that VSMCs in brain blood vessels express the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), which has high binding affinity for Abeta peptides, especially Abeta42. These results suggest that the blood and blood-brain barrier permeability provide a major source of the Abeta peptides that gradually deposit in brain VSMCs, and the presence and abundance of the alpha7nAChR on VSMCs may facilitate the selective accumulation of Abeta peptides in these cells.
Brain Research 09/2008; 1234:158-71. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ROS-GC1 membrane guanylate cyclase is a Ca(2+) bimodal signal transduction switch. It is turned "off" by a rise in free Ca(2+) from nanomolar to the semicromolar range in the photoreceptor outer segments and the olfactory bulb neurons; by a similar rise in the bipolar and ganglion retinal neurons it is turned "on". These opposite operational modes of the switch are specified by its Ca(2+) sensing devices, respectively termed GCAPs and CD-GCAPs. Neurocalcin delta is a CD-GCAP. In the present study, the neurocalcin delta-modulated site, V(837)-L(858), in ROS-GC1 has been mapped. The location and properties of this site are unique. It resides within the core domain of the catalytic module and does not require the alpha-helical dimerization domain structural element (amino acids 767-811) for activating the catalytic module. Contrary to the current beliefs, the catalytic module is intrinsically active; it is directly regulated by the neurocalcin delta-modulated Ca(2+) signal and is dimeric in nature. A fold recognition based model of the catalytic domain of ROS-GC1 was built, and neurocalcin delta docking simulations were carried out to define the three-dimensional features of the interacting domains of the two molecules. These findings define a new transduction model for the Ca(2+) signaling of ROS-GC1.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the possibility that soluble, blood-borne amyloid beta (Abeta) peptides can cross a defective blood-brain barrier (BBB) and interact with neurons in the brain. Immunohistochemical analyses revealed extravasated plasma components, including Abeta42 in 19 of 21 AD brains, but in only 3 of 13 age-matched control brains, suggesting that a defective BBB is common in AD. To more directly test whether blood-borne Abeta peptides can cross a defective BBB, we tracked the fate of fluorescein isothiocyanate (FITC)-labeled Abeta42 and Abeta40 introduced via tail vein injection into mice with a BBB rendered permeable by treatment with pertussis toxin. Both Abeta40 and Abeta42 readily crossed the permeabilized BBB and bound selectively to certain neuronal subtypes, but not glial cells. By 48 h post-injection, Abeta42-positive neurons were widespread in the brain. In the cerebral cortex, small fluorescent, Abeta42-positive granules were found in the perinuclear cytoplasm of pyramidal neurons, suggesting that these cells can internalize exogenous Abeta42. An intact BBB (saline-injected controls) blocked entry of blood-borne Abeta peptides into the brain. The neuronal subtype selectivity of Abeta42 and Abeta40 was most evident in mouse brains subjected to direct intracranial stereotaxic injection into the hippocampal region, thereby bypassing the BBB. Abeta40 was found to preferentially bind to a distinct subset of neurons positioned at the inner face of the dentate gyrus, whereas Abeta42 bound selectively to the population of large neurons in the hilus region of the dentate gyrus. Our results suggest that the blood may serve as a major, chronic source of soluble, exogenous Abeta peptides that can bind selectively to certain subtypes of neurons and accumulate within these cells.
Brain Research 05/2007; 1142:223-36. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ATP is an obligatory agent for the atrial natriuretic factor (ANF) and the type C natriuretic peptide (CNP) signaling of their respective receptor guanylate cyclases, ANF-RGC and CNP-RGC. Through a common mechanism, it binds to a defined ARM domain of the cyclase, activates the cyclase and transduces the signal into generation of the second messenger cyclic GMP. In this presentation, the authors review the ATP-regulated transduction mechanism and refine the previously simulated three-dimensional ARM model (Duda T, Yadav P, Jankowska A, Venkataraman V, Sharma RK. Three dimensional atomic model and experimental validation for the ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase. Mol Cell Biochem 2000;214:7-14; reviewed in: Sharma RK, Yadav P, Duda T. Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism. Can J Physiol Pharmacol 2001;79: 682-91; Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 2002;230:3-30). The model depicts the ATP-binding dependent configurational changes in the ARM and supports the concept that in the first step, ATP partially activates the cyclase and primes it for the subsequent transduction steps, resulting in full activation of the cyclase.
[Show abstract][Hide abstract] ABSTRACT: The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.
[Show abstract][Hide abstract] ABSTRACT: Odorant transduction is a biochemical process by which the odorant signal generates the electric signal. The cilia of the olfactory neuroepithelium are the sites of this process. This study documents the detailed biochemical, structural and functional description of an odorant-responsive Ca2+ -modulated membrane guanylate cyclase transduction machinery in the cilia. Myristoylated (myr)-neurocalcin delta is the Ca2+ -sensor component and the cyclase, ONE-GC, the transduction component of the machinery. Myr-neurocalcin delta senses increments in free Ca2+, binds to a defined domain of ONE-GC and stimulates the cyclase. The findings enable the formulation of an odorant transduction model in which three pivotal signaling components--Ca2+, myr-neurocalcin delta and ONE-GC--of the transduction machinery are locked. A glaring feature of the model is that its Ca2+ -dependent operational principle is opposite to the phototransduction model.
Molecular and Cellular Biochemistry 01/2005; 267(1-2):107-22. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.
[Show abstract][Hide abstract] ABSTRACT: Rod outer segment membrane guanylate cyclase (ROS-GC) transduction system is a central component of the Ca(2+)-sensitive phototransduction machinery. The system is composed of two parts: Ca(2+) sensor guanylate cyclase activating protein (GCAP) and ROS-GC. GCAP senses Ca(2+) impulses and inhibits the cyclase. This operational feature of the cyclase is considered to be unique and exclusive in the phototransduction machinery. A combination of reconstitution, peptide competition, cross-linking, and immunocytochemical studies has been used in this study to show that the GCAP1/ROS-GC1 transduction system also exists in the photoreceptor synaptic (presynaptic) termini. Thus, the presence of this system and its linkage is not unique to the phototransduction machinery. A recent study has demonstrated that the photoreceptor-bipolar synaptic region also contains a Ca(2+)-stimulated ROS-GC1 transduction system [Duda, T., et al. (2002) EMBO J. 21, 2547-2556]. In this case, S100beta senses Ca(2+) and stimulates the cyclase. The inhibitory and stimulatory Ca(2+)-modulated ROS-GC1 sites are distinct. These findings allow the formation of a new topographic model of ROS-GC1 transduction. In this model, the catalytic module of ROS-GC1 at its opposite ends is flanked by GCAP1 and S100beta modules. GCAP1 senses the Ca(2+) impulse and inhibits the catalytic module; S100beta senses the impulse and stimulates the catalytic module. Thus, ROS-GC1 acts as a bimodal Ca(2+) signal transduction switch in the photoreceptor bipolar synapse.
[Show abstract][Hide abstract] ABSTRACT: beta-Amyloid(1-42) (A beta 42), a major component of amyloid plaques, accumulates within pyramidal neurons in the brains of individuals with Alzheimer's disease (AD) and Down syndrome. In brain areas exhibiting AD pathology, A beta 42-immunopositive material is observed in astrocytes. In the present study, single- and double-label immunohistochemistry were used to reveal the origin and fate of this material in astrocytes. Our findings suggest that astrocytes throughout the entorhinal cortex of AD patients gradually accumulate A beta 42-positive material and that the amount of this material correlates positively with the extent of local AD pathology. A beta 42-positive material within astrocytes appears to be of neuronal origin, most likely accumulated via phagocytosis of local degenerated dendrites and synapses, especially in the cortical molecular layer. The co-localization of neuron-specific proteins, alpha 7 nicotinic acetylcholine receptor and choline acetyltransferase, in A beta 42-burdened, activated astrocytes supports this possibility. Our results also suggest that some astrocytes containing A beta 42-positive deposits undergo lysis, resulting in the formation of astrocyte-derived amyloid plaques in the cortical molecular layer in brain regions showing moderate to advanced AD pathology. These astrocytic plaques can be distinguished from those arising from neuronal lysis by virtue of their smaller size, their nearly exclusive localization in the subpial portion of the molecular layer of the cerebrocortex, and by their intense glial fibrillary acidic protein immunoreactivity. Overall, A beta 42 accumulation and the selective lysis of A beta 42-burdened neurons and astrocytes appear to make a major contribution to the observed amyloid plaques in AD brains.
Brain Research 06/2003; 971(2):197-209. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study documents the identity of a calcium- regulated membrane guanylate cyclase transduction system in the photoreceptor-bipolar synaptic region. The guanylate cyclase is the previously characterized ROS-GC1 from the rod outer segments and its modulator is S100beta. S100beta senses increments in free Ca(2+) and stimulates the cyclase. Specificity of photoreceptor guanylate cyclase activation by S100beta is validated by the identification of two S100beta-regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are specific for S100beta and not for another regulator of ROS-GC1, guanylate cyclase-activating protein 1. One site comprises amino acids (aa) Gly962-Asn981, the other, aa Ile1030-Gln1041. The former represents the binding site. The latter binds S100beta only marginally, yet it is critical for control of maximal cyclase activity. The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane-bound guanylate cyclase is regulated by small Ca(2+)-sensor proteins.
The EMBO Journal 07/2002; 21(11):2547-56. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Until now, ROS-GC1 signal transduction system was thought to be exclusive to photoreceptors in the retina. Two recent reports, however, now show that this is not the case. In one, the ROS-GC1 signal transduction system has been identified and characterized in pinealocyte neurons. This signaling is modulated by norepinephrine. However, the response of the individual pinealocyte neuron to the norepinephrine signal depends on whether the GCAP1-linked (results in hyperpolarization) or S100beta-linked (results in depolarization) pathway is operational in the pinealocyte. The GCAP1-linked pathway results in hyperpolarization, while the S100beta-linked pathway, in depolarization. The two pathways are mutually exclusive. In the other report, the calcium-modulated ROS-GC1:GCAP1 signaling system has been discovered in mitral cells of the olfactory bulb. These findings raise the possibility that a common theme of calcium-modulated ROS-GC signaling may be utilized in a wide variety of neurosensory cells. This idea is also supported from evolutionary and functional perspectives.
Molecular and Cellular Biochemistry 02/2002; 230(1-2):117-24. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background The α2-adrenergic receptor (α2-AR) expressed in the bovine retina has been demonstrated to be of the α2D subtype. The bovine α2D-adrenergic receptor (α2D/A-AR) gene has been cloned and characterized. This report describes the induction of this gene by phorbol- 12, 13-myristate acetate (PMA), an activator of protein kinase C (PKC). Results Treatment of the bovine retina for 60 min with PMA (1 μ m) resulted in significant and similar increases in α2D/A-AR mRNA level and gene transcription. This indicates that PMA causes α2D/A-AR gene induction and that this induction takes place directly at the transcriptional level. In C6 cells, treatment with PMA at a concentration which was as low as 0.1 μ m induced endogenous α2D/A-AR mRNA after 60 min. Luciferase reporter assays in C6 cells mapped the PMA-responsive element to a region between −247 bp and −163 bp on the α2D/A-AR promoter. Electrophoretic mobility shift assays showed an increased binding of nuclear factor(s) from PMA-treated bovine retina to this promoter region. Competition assays indicate that an AP-2 element may be involved in the PMA-dependent induction. Conclusion These findings demonstrate for the first time, the direct induction of the α2D/A-AR gene by PMA and support a role for an AP-2 element in the induction mechanism.
Genes to Cells 01/1999; 4(3):161-173. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this report, 5 amino acid residues (aa) in the third cytoplasmic loop of the a2D-adrenergic receptor are identified which (individually or together) alter its ligand-binding characteristics. An important structural discrepancy exists in the third cytoplasmic loop of the a2D-ARs encoded by the rat cDNA and the rat gene - five aa are different. The newly identified bovine receptor as well as the mouse receptor contained the 5 aa identical to that encoded by the rat cDNA. Site-directed mutation of these residues to those of the rat gene encoded receptor resulted in alteration of binding characteristics: significant changes in the ability of the mutant receptor to bind to a number of agonists and antagonists were observed - ranging from a decrease by half in the case of oxymetazoline, to near total loss of binding in the case of prasozin. Thus, the mutant receptor was no longer pure a2D-AR. This indicated a hitherto unrealized role of the third cytoplasmic loop in defining the ligand-binding characteristics of the receptor, and also suggested that the rat gene sequence was most probably in error.
Molecular and Cellular Biochemistry 01/1997; 177(1):125-129. · 2.33 Impact Factor