Imre Lengyel

Autonomous activity of CaMKII is only transiently increased following the induction of long-term potentiation in the rat hippocampus

A major role has been postulated for a maintained increase in the autonomous activity of CaMKII in the expression of long‐term potentiation (LTP). However, attempts to inhibit the expression of LTP with CaMKII inhibitors have yielded inconsistent results. Here we compare the changes in CaMKII autonomous activity and phosphorylation at Thr286 of αCaMKII in rat hippocampal slices using chemical or tetanic stimulation to produce either LTP or short‐term potentiation (STP). Tetanus‐induced LTP in area CA1 requires CaMKII activation and Thr286 phosphorylation of αCaMKII, but we did not observe an increase in autonomous activity. Next we induced LTP by 10 min exposure to 25 mm tetraethyl‐ammonium (TEA) or 5 min exposure to 41 mm potassium (K) after pretreatment with calyculin A. Exposure to K alone produced STP. These protocols allowed us to monitor temporal changes in autonomous activity during and after exposure to the potentiating chemical stimulus. In chemically induced LTP, autonomous activity was maximally increased within 30 s whereas this increase was significantly delayed in STP. However, in both LTP and STP the two‐fold increase in autonomous activity measured immediately after stimulation was short‐lived, returning to baseline within 2–5 min after re‐exposure to normal ACSF. In LTP, but not in STP, the phosphorylation of αCaMKII at Thr286 persisted for at least 60 min after stimulation. These results confirm that LTP is associated with a maintained increase in autophosphorylation at Thr286 but indicate that a persistent increase in the autonomous activity οf CaMKII is not required for the expression of LTP.

Modulation of the phosphorylation and activity of calcium/calmodulin- dependent protein kinase II by zinc

Calcium/calmodulin‐dependent protein kinase II (CaMPK‐II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn2+ has multiple functional effects on CaMPK‐II. Zn2+ generated a Ca2+/CaM‐independent activity that correlated with the autophosphorylation of Thr286, inhibited Ca2+/CaM binding that correlated with the autophosphorylation of Thr306, and inhibited CaMPK‐II activity at high concentrations that correlated with the autophosphorylation of Ser279. The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn2+ binding, generated an increased mobility form of CaMPK‐II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn2+ converts CaMPK‐II into a different form than the binding of Ca2+/CaM. In certain nerve terminals, where Zn2+ has been shown to play a neuromodulatory role and is present in high concentrations, Zn2+ may turn CaMPK‐II into a form that would be unable to respond to calcium signals.

Uncontrolled Zinc- and Copper-Induced Oligomerisation of the Human Complement Regulator Factor H and Its Possible Implications for Function and Disease

Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8-7.0 microM was unaffected until [Zn] increased to 20 microM, whereupon the radius of gyration, RG, values increased from 9 to 15 nm at [Zn]=200 microM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 microM. At [Zn] above 60 microM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed.

Pathological Phosphorylation Causes Neuronal Death: Effect of Okadaic Acid in Primary Culture of Cerebellar Granule Cells

Abstract: We have investigated the role of protracted phosphatase inhibition and the consecutive protracted protein phosphorylation on neuronal viability. We found that in primary cultures of cerebellar granule neurons, the protracted (24‐h) inhibition of the serine/threonine protein phosphatases 1 and 2A (EC 3.1.3.16) by treatment of the cultures with okadaic acid (OKA; 5–20 nM) caused neurotoxicity that could be inhibited by the protein kinase inhibitor l‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7) or by the previous down‐regulation of the neuronal protein kinase C (PKC; ATP:protein phosphotransferase; EC 2.7.1.37). PKC was down‐regulated by exposure of the cultures for 24 h to 100 nM phorbol 12‐myristate 13‐acetate (TPA). The effect of the drugs used in the viability studies on the pattern of protein phosphorylation was measured by quantitative autoradiography. In particular, the 50‐ and 80‐kDa protein bands showed dramatic changes in the degree of phosphorylation: increase by OKA and brief TPA treatment; decrease by H7 or 24 h of TPA treatment; and inhibition of the OKA‐induced increase by H7 or 24 h of TPA treatment. The results suggest that the protracted phosphorylation, in particular that mediated by PKC, may lead to neuronal death and are in line with our previous suggestion that prolonged PKC translocation is operative in glutamate neurotoxicity.

S100B-mediated inhibition of the phosphorylation of GFAP is prevented by TRTK-12.

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.

Calcium/calmodulin-stimulated protein kinase II is present in primary cultures of cerebral endothelial cells

Abstract: Calcium/calmodulin‐stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long‐term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α‐subunit of CaM‐PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α‐CaM‐PK II can act as one of the key enzymes of calcium‐mediated intracellular signaling in the cerebral endothelial cells and suggest that α‐CaM‐PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.

Subretinal pigment epithelial deposition of drusen components including hydroxyapatite in a primary cell culture model

Purpose Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruchs membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruchs membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Zinc Binding to the Tyr402 and His402 Allotypes of Complement Factor H: Possible Implications for Age-Related Macular Degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein–protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

Significance Proteins and lipids accumulating in deposits external to the retinal pigment epithelium (RPE) represent a barrier to metabolic exchange between the retina and the choroidal capillaries. With time, these deposits can lead to age-related macular degeneration (AMD), the most common cause of blindness in the elderly in the developed world. It remains unclear how sub-RPE deposits are initiated and grow to clinically relevant features. Using a combination of high-resolution analytical techniques, we found that tiny hydroxyapatite (bone mineral) spherules with cholesterol-containing cores are present in all examined sub-RPE deposits, providing a scaffold to which proteins adhere. If the spherules are important in initiating sub-RPE deposit formation, this finding may provide attractive new approaches for early identification and treatment of AMD. Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5–20 μm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch’s membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch’s membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits.

Kinetics of protein phosphorylation in microvessels isolated from rat brain: modulation by second messengers

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer‐aided x‐ray film densitometry. With the use of this method, Ca2+‐calmodulin (CAM)‐, Ca2+/phospholipid (PK C)‐, cyclic GMP (cGMP)‐, and cyclic AMP (cAMP)‐dependent protein kinases were detected. CAM‐dependent protein ki‐nase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP‐dependent protein kinase was much higher than that of the cAMP‐dependent one. Autophosphoryla‐tion of both the α‐ and β‐subunits of CAM‐dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothe‐lial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood‐brain barrier.