Dynamic Microtubules in Alzheimer's Disease: Association with Dendritic Spine Pathology.

Alzheimer's disease (AD) is the most common incurable neurodegenerative disorder that affects the processes of memory formation and storage. The loss of dendritic spines and alteration in their morphology in AD correlate with the extent of patient's cognitive decline. Tubulin had been believed to be restricted to dendritic shafts, until recent studies demonstrated that dynamically growing tubulin microtubules enter dendritic spines and promote their maturation. Abnormalities of tubulin cytoskeleton may contribute to the process of dendritic spine shape alteration and their subsequent loss in AD. In this review, association between tubulin cytoskeleton dynamics and dendritic spine morphology is discussed in the context of dendritic spine alterations in AD. Potential implications of these findings for the development of AD therapy are proposed.

Neuroprotective Effect of σ1-Receptors on the Cell Model of Huntington's Disease.

Huntington's disease is a hereditary neurodegenerative disease that primarily affects striatal neurons. Recent studies demonstrated abnormalities in calcium regulation in striatal neurons in Huntington's disease, which leads to elimination of synaptic connections between cortical and striatal neurons. In the present study, we focused on the neuroprotective properties of σ1-receptor, because one of its main functions is associated with modulation of calcium homeostasis in cells. The application of selective σ1-receptor agonists to the corticostriatal cell culture restores synaptic connections between the cortical and striatal neurons. Based on the obtained data, we assume that σ1-receptor is a promising target for the development of drugs for the therapy of Huntington's disease.

Neuroprotective properties of anti-apoptotic BCL-2 proteins in 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia. An early feature of the AD pathology is the dysregulation of intracellular Ca2+ signaling in neurons. In particular, increased Ca2+ release from endoplasmic reticulum-located Ca2+ channels, including inositol-1,4,5-trisphosphate type 1 receptors (IP3R1) and ryanodine receptors type 2 (RyR2), have been extensively reported. Known for its anti-apoptotic properties, Bcl-2 also has the ability to bind to and inhibit the Ca2+-flux properties of IP3Rs and RyRs. In this study, the hypothesis that the expression of Bcl-2 proteins can normalize dysregulated Ca2+ signaling in a mouse model of AD (5xFAD) and thereby prevent or slow the progression of AD was examined. Therefore, stereotactic injections of adeno-associated viral vectors expressing Bcl-2 proteins were performed in the CA1 region of the 5xFAD mouse hippocampus. In order to assess the importance of the association with IP3R1, the Bcl-2K17D mutant was also included in these experiments. This K17D mutation has been previously shown to decrease the association of Bcl-2 with IP3R1, thereby impairing its ability to inhibit IP3R1 while not affecting Bcl-2's ability to inhibit RyRs. Here, we demonstrate that Bcl-2 protein expression leads to synaptoprotective and amyloid-protective effects in the 5xFAD animal model. Several of these neuroprotective features are also observed by Bcl-2K17D protein expression, suggesting that these effects are not associated with Bcl-2-mediated inhibition of IP3R1. Potential mechanisms for this Bcl-2 synaptoprotective action may be related to its ability to inhibit RyR2 activity as Bcl-2 and Bcl-2K17D are equally potent in inhibiting RyR2-mediated Ca2+ fluxes. This work indicates that Bcl-2-based strategies hold neuroprotective potential in AD models, though the underlying mechanisms requires further investigation.

ATP modulates the function of inositol 1,4,5-trisphosphate-gated channels at two sites.

The inositol 1,4,5-trisphosphate (IP3) receptor, a Ca(2+)-permeable channel, plays a key role in intracellular Ca2+ signaling. The effects of ATP on the IP3 receptor at the single-channel level were characterized after channel incorporation into planar lipid bilayers. ATP alone was not sufficient to open the IP3-gated channel, but addition of ATP or nonhydrolyzable ATP analogs in the presence of IP3 increased the frequency of channel openings 4.8-fold and increased the average duration of channel openings 2.5-fold; channel conductance was unchanged. High concentrations of ATP (> 4 mM) decreased channel activity most probably by competing with IP3-binding site. Allosteric modulation of IP3-induced Ca2+ release by ATP may contribute to the maintenance of cell viability during periods of energy starvation.

Inositol 1,4,5-tripshosphate receptor, calcium signalling and Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder that has no cure. HD primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Htt(exp) and MSN neurodegeneration remains elusive. My laboratory discovered that mutant Htt(exp) protein specifically binds to the carboxy-terminal region of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular Ca2+ release channel. Moreover, we found that Htt(exp) association with InsP3R1 causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in primary MSN. Mutant Htt(exp) has also been shown to activate Ca2(+)-permeable NR2B-containing NMDA receptors. All these results suggested that deranged neuronal Ca2+ signaling may play an important role in pathogenesis of HD. In support of this idea, we demonstrated a connection between abnormal Ca2+ signaling and apoptosis of MSN cultured from YAC128 HD mouse model. These results indicate that InsP3R and other Ca2+ signaling proteins should be considered as potential therapeutic targets for treatment of HD.

Caffeine-induced inhibition of inositol(1,4,5)-trisphosphate-gated calcium channels from cerebellum.

Effects of the xanthine drug caffeine on inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels from canine cerebellum were studied using single channels incorporated into planar lipid bilayers. Caffeine, used widely as an agonist of ryanodine receptors, inhibited the activity of InsP3-gated Ca channels in a noncooperative fashion with half-inhibition at 1.64 mM caffeine. The frequency of channel openings was decreased more than threefold after addition of 5 mM caffeine; there was only a small effect on mean open time of the channels, and the single channel conductance was unchanged. Increased InsP3 concentration overcame the inhibitory action of caffeine, but caffeine did not reduce specific [3H]InsP3 binding to the receptor. The inhibitory action of caffeine on InsP3 receptors suggests that the action of caffeine on the intracellular Ca pool must be interpreted with caution when both ryanodine receptors and InsP3 receptors are present in the cell.

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.

Potassium channels in aortic microsomes: conductance, selectivity, barium-induced blockage and subconductance states.

The activity of potassium channels of canine aortic sarcoplasmic reticulum was measured using the planar lipid bilayer-fusion technique. The channels have a conductance of 208 pS (400/100 mM K+ in cis/trans solutions) and potassium-to-sodium permeability ratio of 7.7 Ba2+ ions produced two main effects: one is the interruption of channel currents for tens to hundreds of milliseconds in a voltage-dependent manner, and the other is the appearance of a second conductance level with amplitude about 60% of the main level.

The pharmacology of intracellular Ca(2+)-release channels.

Two classes of intracellular Ca(2+)-release channels, the ryanodine receptor and the inositol (1,4,5)-trisphosphate (IP3) receptor, are essential for spatio-temporal Ca2+ signalling in cells. Heparin and caffeine have been widely used to study these channels. It was originally thought that caffeine acts solely as an agonist for the ryanodine receptor and heparin acts solely as an inhibitor for the IP3 receptor. However, recent experiments indicate that these compounds have multiple effects, and are discussed in this review by Barbara Ehrlich and colleagues. In the same concentration range, caffeine activates the ryanodine receptor and inhibits the IP3 receptor, and heparin inhibits the IP3 receptor and activates the ryanodine receptor. More specific pharmacological tools that are suitable for studies of ryanodine and IP3 receptors are now beginning to emerge.

Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium.

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.

Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells.

The inositol (1,4,5)-trisphosphate receptor (InsP3R) mediates Ca2+ release from intracellular stores in response to generation of second messenger InsP3. InsP3R was biochemically purified and cloned, and functional properties of native InsP3-gated Ca2+ channels were extensively studied. However, further studies of InsP3R are obstructed by the lack of a convenient functional assay of expressed InsP3R activity. To establish a functional assay of recombinant InsP3R activity, transient heterologous expression of neuronal rat InsP3R cDNA (InsP3R-I, SI- SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP3R retained specific InsP3 binding properties (Kd = 60 nM InsP3) and were specifically recognized by anti-InsP3R-I rabbit polyclonal antibody. Density of expressed InsP3R-I was at least 20-fold above endogenous InsP3R background and only 2-3-fold lower than InsP3R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP3R formed a functional InsP3-gated Ca2+ channel with 80 pS conductance using 50 mM Ba2+ as a current carrier. Mean open time of recombinant InsP3-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP3R-I were very similar to properties of native rat cerebellar InsP3R recorded in identical experimental conditions. Recombinant InsP3R also retained bell-shaped dependence on cytosolic Ca2+ concentration and allosteric modulation by ATP, similar to native cerebellar InsP3R. The following conclusions are drawn from these results. (a) Rat neuronal InsP3R-I cDNA encodes a protein that is either sufficient to produce InsP3-gated channel with functional properties identical to the properties of native rat cerebellar InsP3R, or it is able to form a functional InsP3-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP3R in a heterologous expression system provides an opportunity for future detailed structure-function characterization of this vital protein.

Molecular basis of proton block of L-type Ca2+ channels.

Hydrogen ions are important regulators of ion flux through voltage-gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation for the modulatory effect of H+ ions on open channel flux and the competition between H+ ions and permeant divalent cations. The characteristics of H+ interactions advanced our picture of the functional interplay between P-region glutamates, with important implications for the mechanism of Ca2+ selectivity and permeation.

Theoretical analysis of calcium wave propagation based on inositol (1,4,5)-trisphosphate (InsP3) receptor functional properties.

In the presence of inositol (1,4,5)-trisphosphate (InsP3) repetitive waves of elevated cytosolic free Ca2+ (Ca waves) that travel through cellular cytoplasm are observed. Investigation of this phenomenon stimulated the view of cellular cytoplasm as 'an excitable medium composed of Ca release processes (InsP3R), coupled by a common stimulatory signal (Ca) through diffusion' [Lechleiter JD. Clapham DE. (1992) Molecular mechanisms of intracellular calcium excitability in Xenopus laevis oocytes. Cell, 69, 283-294]. Using a kinetic model of InsP3R gating, an analytical expression for the amplitude of Ca wave propagating through this excitable medium has been obtained. The amplitude of the Ca wave is determined by the combination of cell-specific parameters and the functional properties of a single InsP3R. An analytical expression for Ca wave propagation velocity has been also obtained using the Luther equation for diffusion-driven autocatalytic reaction. Both equations provided reasonable estimations for Ca wave amplitude (1.3 microM free Ca) and the velocity of the wave propagation (21 microns/s) for Ca waves in Xenopus oocytes when numerical values of parameters were used. The duration of refractory period has been shown to be determined mainly by the activity of CaATPase. Obtained results provide an insight into the mechanisms underlying the process of Ca wave propagation and define the interrelationship between different factors involved in this process. Some experimentally testable predictions can be done based on the analytical expressions obtained for Ca wave amplitude, the velocity of Ca wave propagation and the duration of refractory period.

The inositol trisphosphate receptor of Xenopus oocytes.

Xenopus laevis oocytes (stages V and VI) are a widely used model system for the study of Ca2+ signaling. The properties of the Xenopus oocyte InsP3 receptor (InsP3R) are of paramount importance for our thinking about this system and for our efforts to model Ca2+ dynamics in the oocyte cytosol. The recent data regarding the molecular structure, the regulation and the functional properties of the Xenopus oocyte InsP3R are summarized in this review. The main properties of the Xenopus oocyte InsP3R are compared with the properties of the cerebellar InsP3R and are shown to be remarkably similar. The density of the InsP3R in Xenopus oocyte cytoplasm is estimated to a value between 1.1-4.1 x 10(14) tetrameric InsP3R/l. The use of these numbers in a quantitative model of Ca2+ wave propagation leads to values of Ca2+ wave amplitude (0.8-1.5 microM Ca2+) and velocity of the wave propagation (12-24 microns/s) that are in excellent agreement with the values observed experimentally. The density of InsP3Rs in Purkinje cells of the cerebellum is estimated to be about 20,000-fold higher, but in other types of neurons and in peripheral tissues the InsP3R density is estimated to be of the same order of magnitude as, or up to 20-fold higher than, in Xenopus oocytes. The implications of differences in InsP3R density for Ca2+ signaling are discussed.

Classification of PDZ domains.

A diverse family of PDZ domains has been identified, but the rules that govern their ligand specificity are not clear. Here we propose a novel classification of PDZ domains based on the nature of amino acids in the two critical positions in the PDZ domain fold. Using these principles, we classified PDZ domains present in the SMART database. Using yeast two-hybrid, in vitro pull-down and plasmon surface resonance assays, we demonstrated that in agreement with their position in the proposed classification the Mint1-1, hINADL-5, and PAR6 PDZ domains display similar dual ligand specificity. The proposed classification helps to organize PDZ domain containing proteins.

Intracellular calcium release channels.

Two classes of intracellular calcium release channels have been identified, the ryanodine receptor and the inositol 1,4,5-trisphosphate (InsP3) receptor. Characterizations of each receptor at the single channel level has shown that the channel types are functionally distinct. These two channels differ in the relative density and localization within the cell, in the mechanism of activation, in the regulation by Ca and ATP, and the sensitivity to pharmacological agents. The differences in the properties and regulation of the two types of intracellular calcium release channels are important for determining cell-specific responses to stimuli and allows the cell to adjust Ca signalling to the needs of the physiological state of the cell.

Calcium signaling and neurodegeneration.

Neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and spinocerebellar ataxias (SCA) are very important both for fundamental science and for practical medicine. Despite extensive research into the causes of these diseases, clinical researchers have had very limited progress and, as of now, there is still no cure for any of these diseases. One of the main obstacles in the way of creating treatments for these disorders is the fact that their etiology and pathophysiology still remain unclear. This paper reviews results that support the so-called "calcium hypothesis of neurodegenerative diseases." The calcium hypothesis states that the atrophic and degenerative processes in the neurons of AD, PD, ALS, HD, and SCA patients are accompanied by alterations in calcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calcium signaling is one of the early-stage and key processes in the pathogenesis of these diseases. Based on the results we reviewed, we conclude that the calcium channels and other proteins involved in the neuronal calcium signaling system are potential drug targets for AD, PD, ALS, HD, and SCA therapy.

Regulation of store-operated channels by scaffold proteins in a431 cells.

Store-operated channels are major calcium influx pathways in nonexitable cells. Homer scaffold proteins are well known for their role in regulating calcium signaling. Here we report on a detailed single-channel level characterization of native store-operated channels regulated by Homer scaffold proteins in A431 carcinoma cells. By applying the single-channel patch-clamp technique, we found that different types of store-operated calcium channels have different sensitivities to Homer proteins.