Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

Actin bundle architecture and mechanics regulate myosin II force generation

The actin cytoskeleton is a soft, structural material that underlies biological processes such as cell division, motility, and cargo transport. The cross-linked actin filaments self-organize into a myriad of architectures, from disordered meshworks to ordered bundles, which are hypothesized to control the actomyosin force generation that regulates cell migration, shape, and adhesion. Here, we use fluorescence microscopy and simulations to investigate how actin bundle architectures with varying polarity, spacing, and rigidity impact myosin II dynamics and force generation. Microscopy reveals that mixed-polarity bundles formed by rigid cross-linkers support slow, bidirectional myosin II filament motion, punctuated by periods of stalled motion. Simulations reveal that these locations of stalled myosin motion correspond to sustained, high forces in regions of balanced actin filament polarity. By contrast, mixed-polarity bundles formed by compliant, large cross-linkers support fast, bidirectional motion with no traps. Simulations indicate that trap duration is directly related to force magnitude and that the observed increased velocity corresponds to lower forces resulting from both the increased bundle compliance and filament spacing. Our results indicate that the microstructures of actin assemblies regulate the dynamics and magnitude of myosin II forces, highlighting the importance of architecture and mechanics in regulating forces in biological materials.     

Presenilin-1 and intracellular calcium stores regulate neuronal glutamate uptake

Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin-1 (PS1) as a regulator of glutamate transport. PS1-deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild-type neurons. Gamma-secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss-of-function, accomplished by PS1 deficiency or gamma-secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF-96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss-of-function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage-gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.     

Dysregulation of intracellular calcium homeostasis is responsible for neuronal death in an experimental model of selective hippocampal degeneration induced by trimethyltin

Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca(2+)-binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca(2+) overload. The present study was aimed at determining whether intracellular Ca(2+) homeostasis dysregulation is involved in the TMT-induced neurodegeneration and if intracellular Ca(2+)-buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time- and concentration-dependent [Ca(2+)](i) increases that were primarily due to Ca(2+) release from intracellular stores although Ca(2+) entry through Ca(v)1 channels also contributed to [Ca(2+)](i) increases in the early phase of TMT action. Cell pre-treatment with the Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (2 muM) significantly reduced the TMT-induced neuronal death. Moreover, CR(+) neurons responded to TMT with smaller [Ca(2+)](i) increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca(2+) homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR(+) neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca(2+) overload.     

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca⁺⁺ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca⁺⁺ could bind the Nav1.1 C-terminal region with micromolar affinity.     

Essential role for autophagy protein VMP1 in maintaining neuronal homeostasis and preventing axonal degeneration

Vacuole membrane protein 1 (VMP1), the endoplasmic reticulum (ER)-localized autophagy protein, plays a key role during the autophagy process in mammalian cells. To study the impact of VMP1-deficiency on midbrain dopaminergic (mDAergic) neurons, we selectively deleted VMP1 in the mDAergic neurons of VMP1^fl/fl/DAT^CreERT2 bigenic mice using a tamoxifen-inducible CreERT2/loxp gene targeting system. The VMP1^fl/fl/DAT^CreERT2 mice developed progressive motor deficits, concomitant with a profound loss of mDAergic neurons in the substantia nigra pars compacta (SNc) and a high presynaptic accumulation of α-synuclein (α-syn) in the enlarged terminals. Mechanistic studies showed that VMP1 deficiency in the mDAergic neurons led to the increased number of microtubule-associated protein 1 light chain 3-labeled (LC3) puncta and the accumulation of sequestosome 1/p62 aggregates in the SNc neurons, suggesting the impairment of autophagic flux in these neurons. Furthermore, VMP1 deficiency resulted in multiple cellular abnormalities, including large vacuolar-like structures (LVSs), damaged mitochondria, swollen ER, and the accumulation of ubiquitin⁺ aggregates. Together, our studies reveal a previously unknown role of VMP1 in modulating neuronal survival and maintaining axonal homeostasis, which suggests that VMP1 deficiency might contribute to mDAergic neurodegeneration via the autophagy pathway.Subject terms: Neuroscience, Pathogenesis     

Long-lasting alterations in neuronal calcium homeostasis in an in vitro model of stroke-induced epilepsy

Altered calcium homeostatic mechanisms have been implicated in the development of acquired epilepsy in numerous models. Stroke is one of the leading brain injuries that cause acquired epilepsy, yet little is known concerning the molecular mechanisms underlying stroke-induced epileptogenesis. Recently an in vitro model of stroke-induced epilepsy was developed and characterized as a powerful tool to study the pathophysiology of injury and stroke-induced epileptogenesis. Using this glutamate injury-induced epileptogenesis model, we have investigated the role of altered calcium homeostatic mechanisms in the development and maintenance of stroke-induced epilepsy. Epileptic neurons manifested elevated intracellular calcium levels compared to control neurons independent of neuronal activity and seizure discharge for the remainder of the life of the neurons in culture. In addition, epileptic neurons were found to have alterations in the ability to reduce intracellular calcium levels following a calcium load. These long-term epileptic changes in calcium homeostasis were dependent on calcium during the initial glutamate injury. The data demonstrate that significant alterations in calcium homeostatic mechanisms occur in association with stroke-induced epilepsy and suggest that these changes may play a role in both the induction and maintenance of the epileptic phenotype in this model.     

Hebb and homeostasis in neuronal plasticity

The positive-feedback nature of Hebbian plasticity can destabilize the properties of neuronal networks. Recent work has demonstrated that this destabilizing influence is counteracted by a number of homeostatic plasticity mechanisms that stabilize neuronal activity. Such mechanisms include global changes in synaptic strengths, changes in neuronal excitability, and the regulation of synapse number. These recent studies suggest that Hebbian and homeostatic plasticity often target the same molecular substrates, and have opposing effects on synaptic or neuronal properties. These advances significantly broaden our framework for understanding the effects of activity on synaptic function and neuronal excitability.     

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.     

Review: Endocrine pathways to regulate calcium homeostasis around parturition and the prevention of hypocalcemia in periparturient dairy cows

Calcium homeostasis is crucial for the normal function of the organism. Parathyroid hormone, calcitriol and calcitonin play critical roles in the homeostatic regulation of calcium. Serotonin and prolactin have also been shown to be involved in the regulation of calcium homeostasis. In modern dairy cows, the endocrine pathways controlling calcium homeostasis during non-lactating and non-pregnant physiological states are unable to fully support the increased demand of calcium required for milk synthesis at the onset of lactation. This review describes different endocrine systems associated with the regulation of calcium homeostasis in mammalian species around parturition with special focus on dairy cows. Additionally, classic and novel strategies to reduce the incidence of hypocalcemia in parturient dairy cows are discussed.     

NPC1 and NPC2 regulate cellular cholesterol homeostasis through generation of low density lipoprotein cholesterol-derived oxysterols

Mutations in the Niemann-Pick disease genes cause lysosomal cholesterol accumulation and impaired low density lipoprotein (LDL) cholesterol esterification. These findings have been attributed to a block in cholesterol movement from lysosomes to the site of the sterol regulatory machinery. In this study we show that Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) mutants have increased cellular cholesterol, yet they are unable to suppress LDL receptor activity and cholesterol biosynthesis. Cholesterol overload in both NPC1 and NPC2 mutants results from the failure of LDL cholesterol tobothsuppresssterolregulatoryelement-bindingprotein-dependent gene expression and promote liver X receptor-mediated responses. However, the severity of the defect in regulation of sterol homeostasis does not correlate with endoplasmic reticulum cholesterol levels, but rather with the degree to which NPC mutant fibroblasts fail to appropriately generate 25-hydroxycholesterol and 27-hydroxycholesterol in response to LDL cholesterol. Moreover, we demonstrate that treatment with oxysterols reduces cholesterol in NPC mutants and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease genotype. Our findings support a role for NPC1 and NPC2 in the regulation of sterol homeostasis through generation of LDL cholesterol-derived oxysterols and have important implications for the treatment of NPC disease.     

Interaction of SiO2 nanoparticles with neuronal cells: Ionic mechanisms involved in the perturbation of calcium homeostasis

SiO₂ nanoparticles (NPs), in addition to their widespread utilization in consumer goods, are also being engineered for clinical use. They are considered to exert low toxicity both in vivo and in vitro, but the mechanisms involved in the cellular responses activated by these nanoobjects, even at non-toxic doses, have not been characterized in detail. This is of particular relevance for their interaction with the nervous system: silica NPs are good candidates for nanoneuromedicine applications. Here, by using two neuronal cell lines (GT1–7 and GN11 cells), derived from gonadotropin hormone releasing hormone (GnRH) neurons, we describe the mechanisms involved in the perturbation of calcium signaling, a key controller of neuronal function. At the non-toxic dose of 20 μg mL⁻¹, 50 nm SiO₂ NPs induce long lasting but reversible calcium signals, that in most cases show a complex oscillatory behavior. Using fluorescent NPs, we show that these signals do not depend on NPs internalization, are totally ascribable to calcium influx and are dependent in a complex way from size and surface charge. We provide evidence of the involvement of voltage-dependent and transient receptor potential-vanilloid 4 (TRPV4) channels.     

Mitochondria-rough-ER contacts in the liver regulate systemic lipid homeostasis

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Flavin homeostasis in the mouse retina during aging and degeneration

Involvement of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in cellular homeostasis has been well established for tissues other than the retina. Here, we present an optimized method to effectively extract and quantify FAD and FMN from a single neural retina and its corresponding retinal pigment epithelium (RPE). Optimizations led to detection efficiency of 0.1 pmol for FAD and FMN while 0.01 pmol for riboflavin. Interestingly, levels of FAD and FMN in the RPE were found to be 1.7- and 12.5-fold higher than their levels in the retina, respectively. Both FAD and FMN levels in the RPE and retina gradually decline with age and preceded the age-dependent drop in the functional competence of the retina as measured by electroretinography. Further, quantifications of retinal levels of FAD and FMN in different mouse models of retinal degeneration revealed differential metabolic requirements of these two factors in relation to the rate and degree of photoreceptor degeneration. We also found twofold reductions in retinal levels of FAD and FMN in two mouse models of diabetic retinopathy. Altogether, our results suggest that retinal levels of FAD and FMN can be used as potential markers to determine state of health of the retina in general and more specifically the photoreceptors.     

Carboxyl-terminal fragment of Alzheimer's APP destabilizes calcium homeostasis and renders neuronal cells vulnerable to excitotoxicity.

Numerous lines of evidence indicate that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (Abeta). However, the possible role of other cleaved products of APP is less clear. We have previously shown that a recombinant carboxy-terminal 105 amino acid fragment (CT 105) of APP induced strong nonselective inward currents in XENOPUS: oocyte; it also revealed neurotoxicity in PC12 cells and primary cortical neurons, blocked later phase of long-term potentiation in rat hippocampus in vivo, and induced memory deficits and neuropathological changes in mice. We report here that the pretreatment with CT 105 for 24 h at a 10 microM concentration increases intracellular calcium concentration by about twofold in SK-N-SH and PC 12 cells, but not in U251 cells, originated from human glioblastoma. In addition, the calcium increase and toxicity induced by CT 105 were reduced by cholesterol and MK 801 in SK-N-SH and PC 12 cells, whereas the toxicity of Abeta(1-42) was attenuated by nifedipine and verapamil. CT 105 rendered SK-N-SH cells and rat primary cortical neurons more vulnerable to glutamate-induced excitotoxicity. Also, conformational studies using circular dichroism experiments showed that CT 105 has approximately 15% of beta-sheet content in phosphate buffer and aqueous 2,2, 2-trifluoroethanol solutions. However, the content of beta-sheet conformation in dodecyl phosphocholine micelle or in the negatively charged vesicles, is increased to 22%-23%. The results of this study showed that CT 105 disrupts calcium homeostasis and renders neuronal cells more vulnerable to glutamate-induced excitotoxicity, and that some portion of CT 105 has partial beta-sheet conformation in various environments, which may be related to the self-aggregation and toxicity. This may be significantly possibly involved in inducing the neurotoxicity characteristic of AD.     

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.