An endoplasmic-reticulum-specific apoptotic pathway is involved in prion and amyloid-beta peptides neurotoxicity

Prion (PrP) and amyloid-beta (Abeta) peptides are involved in the neuronal loss that occurs in Prion disorders (PrD) and Alzheimer's disease (AD), respectively, partially due to Ca(2+) dysregulation. Besides, the endoplasmic reticulum (ER) stress has an active role in the neurotoxic mechanisms that lead to these pathologies. Here, we analyzed whether the ER-mediated apoptotic pathway is involved in the toxic effect of synthetic PrP and Abeta peptides. In PrP106-126- and Abeta1-40-treated cortical neurons, the release of Ca(2+) through ER ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP(3)R) receptors induces ER stress and leads to increased cytosolic Ca(2+) and reactive oxygen species (ROS) levels and subsequently to apoptotic death involving mitochondrial cytochrome c release and caspases activation. These results demonstrate that the early PrP- and Abeta-induced perturbation of ER Ca(2+) homeostasis is a death message that leads to neuronal loss, suggesting that the regulation of ER Ca(2+) levels may be a potential therapeutical target for PrD and AD.     

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid β (Aβ) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt–Jakob disease, while Aβ is involved in the pathogenesis of Alzheimer's disease. Synthetic fragments of both proteins, PrP106–126 and β25–35 (β25–35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106–126 and β25–35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca²⁺]_i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca²⁺]_i concentration as observed after treatment with PrP106–126 and β25–35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106–126 and β25–35.     

Nordihydroguaiaretic acid protects hippocampal neurons against amyloid β-peptide toxicity, and attenuates free radical and calcium accumulation

Recent findings indicate that amyloid β-peptide (Aβ) can be neurotoxic by a mechanism involving an increase in the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i) and the generation of free radicals. In the present study, the lipoxygenase inhibitor/antioxidant nordihydroguaiaretic acid (NDGA) protected cultured rat hippocampal neurons against the toxicity of Aβ in a concentration-dependent manner. Measurements of cellular oxidation (using the oxidation-sensitive dye 2,7-dichlorofluorescin) and intracellular free Ca²⁺ levels (using the Ca²⁺ indicator dye fura-2), showed that NDGA suppressed Aβ-induced accumulation of reactive oxygen species (ROS) and Ca²⁺; Ca²⁺ responses to glutamate were also suppressed by NDGA. NDGA prevented neuronal injury and accumulation of ROS induced by iron, indicating a role for NDGA as an antioxidant in NDGA-mediated neuroprotection. Another lipoxygenase inhibitor (AA861) also protected against Aβ and iron toxicity whereas the the 5-lipoxygenase-activating protein inhibitor L655,238 and the cyclooxygenase inhibitor indomethacin were ineffective. These findings suggest that NDGA can interupt a neurodegenerative pathway relevant to the pathophysiology of Alzheimer's disease.     

Amyloid β peptide induces necrosis rather than apoptosis

Amyloid β peptide (AβP), a major component of Alzheimer's disease plaques, is toxic to rat pheochromocytoma PC12 cells and to rat cortical neurons. A reduction in cell survival could be detected after 24 h incubation with 0.01 to 20 μM of the 25–35 peptide fragment (β25–35) of AβP. To study the mechanism of cell death induced by AβP, the morphological as well as the biochemical features of neuronal cell death were analyzed. To distinguish between necrosis and apoptosis, PC12 cell death caused by β25–35 was compared to that induced by serum deprivation, a process known to be apoptotic in these cells. The DNA-degradation pattern of AβP treated cells appeared random rather than at distinct internucleosomal sites as with apoptosis. Electron microscopic studies of NGF-treated PC12 cells and cortical primary cultures exposed to 20 μM β25–35 revealed immediate cellular damage such as vacuolization of the cytoplasm, breakdown of Golgi-apparatus and other membrane systems, and neurite disintegration. This was followed by total collapse of the cytoplasm and cell lysis. These data show that AβP toxicity occurs via a necrotic rather than an apoptotic pathway.     

Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease

Mitochondria are the major source of energy for the normal functioning of brain cells. Increasing evidence suggests that the amyloid precursor protein (APP) and amyloid beta (Aβ) accumulate in mitochondrial membranes, cause mitochondrial structural and functional damage, and prevent neurons from functioning normally. Oligomeric Aβ is reported to induce intracellular Ca²⁺ levels and to promote the excess accumulation of intracellular Ca²⁺ into mitochondria, to induce the mitochondrial permeability transition pore to open, and to damage mitochondrial structure. Based on recent gene expression studies of APP transgenic mice and AD postmortem brains, and APP/Aβ and mitochondrial structural studies, we propose that the overexpression of APP and the increased production of Aβ may cause structural changes of mitochondria, including an increase in the production of defective mitochondria, a decrease in mitochondrial trafficking, and the alteration of mitochondrial dynamics in neurons affected by AD. This article discusses some critical issues of APP/Aβ associated with mitochondria, mitochondrial structural and functional damage, and abnormal intracellular calcium regulation in neurons from AD patients. This article also discusses the link between Aβ and impaired mitochondrial dynamics in AD.     

Otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat embryo cortical neurons in vitro

In the frame of a repositioning programme with cholinergic medicines in clinical use searching for neuroprotective properties, we surprisingly found that spasmolytic antimuscarinics otilonium and pinaverium exhibited neurotoxic effects in neuronal cultures. We decided to characterize such unexpected action in primary cultures of rat embryo cortical neurons. Neurotoxicity was time- and concentration-dependent, exhibiting approximate EC₅₀ values of 5 μM for both drugs. Seven antimuscarinic drugs endowed with a quaternary ammonium, and another 10 drugs with different cholinergic activities, carrying in their molecule a ternary ammonium did not exhibit neurotoxicity. Both drugs caused a concentration-dependent blockade of whole-cell inward currents through voltage-activated calcium channels (VACCs). Consistent with this, they also blocked the K⁺-elicited [Ca²⁺]_c transients. Neither antioxidant catalase, glutathione, n-acetylcysteine, nor melatonin protected against neurotoxicity of otilonium or pinaverium. However cyclosporine A, a blocker of the mitochondrial permeability transition pore, prevented the neurotoxic effects of otilonium and pinaverium monitored as the fraction of cells undergoing apoptosis. Furthermore, the caspase-9 and caspase-3 inhibitor Ac-LEHD-CHO mitigated the apoptotic neuronal death of both drugs by around 50%. Data are compatible with the hypothesis that otilonium and pinaverium elicit neuronal death by activating the intrinsic mitochondrial-mediated signaling pathway of apoptosis. This may have its origin in the mitigation of Ca²⁺ entry and the uncoupling of the Ca²⁺-dependent generation of mitochondrial bioenergetics, thus causing the opening of the mitochondrial mPTP to elicit apoptotic neuronal death.     

Apoptosis induced by Aβ25–35 peptide is Ca2+‐IP3 signaling‐dependent in murine astrocytes

Although the accumulation of the neurotoxic peptide β‐amyloid (Aβ) in the central nervous system is a hallmark of Alzheimer's disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca²⁺ dysregulation, induced by a neurotoxic fragment (Aβ25–35), caused apoptosis in a concentration‐dependent manner, leading to cytoplasmic Ca²⁺ mobilization from extra‐ and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP₃ receptor activation. This mechanism was related to Aβ‐mediated apoptosis by the intrinsic pathway because the expression of pro‐apoptotic Bax was accompanied by its translocation in cells transfected with GFP‐Bax. Aβ‐mediated apoptosis was reduced by BAPTA‐AM, a fast Ca²⁺ chelator, indicating that an increase in intracellular Ca²⁺ was involved in cell death. Interestingly, the Bax translocation was dependent on Ca²⁺ mobilization from IP₃ receptors because pre‐incubation with xestospongin C, a selective IP₃ receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca²⁺ homeostasis induces ER depletion of Ca²⁺ stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments.     

Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

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Cytoplasmic gelsolin increases mitochondrial activity and reduces Aβ burden in a mouse model of Alzheimer's disease

Accumulation of amyloid-β (Aβ) peptides is thought to be a critical event in the pathology of Alzheimer's disease (AD), because they induce multiple neurotoxic effects, including mitochondrial dysfunction and apoptotic cell death. Therefore the reduction of Aβ is considered a primary therapeutic target. Gelsolin, an Aβ binding protein, has been shown to inhibit apoptosis, although the underlying mechanism is unclear. To clarify these effects, we manipulated cytoplasmic gelsolin levels through viral-directed overexpression in the brain of APP/Ps1 transgenic mice. We observed that gelsolin reduces brain Aβ burden in the APP/Ps1 mice, possibly by enhancing Aβ clearance via megalin. The reduction in brain Aβ levels was accompanied by an inhibition of nitric oxide production and cell death, not only in the choroid plexus but also in the cerebral cortex. Notably, overexpressed gelsolin restored the impaired mitochondrial activity in the APP/Ps1 mice, resulting in the increase of cytochrome c oxidase activity. By contrast, RNA interference to block gelsolin expression, confirmed that cytoplasmic gelsolin acts as a modulator of brain Aβ levels and its neurotoxic effects. We conclude that gelsolin might prevent brain amyloidosis and Aβ-induced apoptotic mitochondrial changes. These findings make cytoplasmic gelsolin a potential therapeutic strategy in AD.     

Erratum to: Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum: [Brain Research 903 (2001) 247–252]

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (α_1A), N-type (α_1B), and L-type (α_1C and α_1D) Ca²⁺ channels were observed in the hippocampus at days 1–5 after ischemic injury. However, increased expression of N-type Ca²⁺ channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca²⁺ channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca²⁺ channels may prove useful for future investigations.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

Acidic pH promotes the formation of toxic fibrils from β-amyloid peptide

Beta-amyloid (Aβ) peptides, the major component of senile plaques in brains of patients with Alzheimer’s disease (AD), were found in the low pH organelles (i.e. endosome/lysosome) of cultured neuronal cells. Since acidic pH values have been shown to promote the self-assembly of Aβs, which seems to be a prerequisite for their neuropathogenicity, elucidating the aggregation behavior of Aβs in acidic environments and their subsequent effects on neuronal cells may be crucial for understanding the neurodegenerative process of AD. In this study, the extent and rate of aggregation of Aβ_1–42 peptides at pH values of 5.8 and 7.4, as well as the structure and neurotoxic effects of these aggregates, were examined. We showed that Aβ_1–42 peptides formed large and complex fibrils much more efficiently at acidic rather than neutral pH. Furthermore, only the pH 5.8 Aβ aggregates induced significant apoptotic death of PC12 cells, as indicated by a decrease in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) reduction and an increase in phosphatidylserine externalization. Taken together, our results suggest that the Aβs present in the acidic organelles may form neurotoxic fibrils more easily than those in the neutral cellular compartments.     

Application of quantitative LC–MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Aβ) biosynthetic intermediate protein APP–βCTF

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Aβ) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and γ-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP–βCTF, or also called C99, which then undergoes further cleavage by γ-secretase to form Aβ. Inhibitors of γ-secretase have been observed to yield a so-called ‘Aβ rise’ phenomenon whereby low inhibitor concentrations result in an increase in Aβ levels while high inhibitor concentrations result in lower Aβ levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP–βCTF substrate relative to γ-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP–βCTF, γ-secretase enzyme and various inhibitors resulting in the ‘Aβ rise’. An on-line liquid chromatography mass spectrometry (LC–MS) method employing the ‘surrogate peptide’ methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case ^C100Aβ17–28 (LVFFAEDVGSNK) that can be readily detected by LC–MS. The absolute quantitative assessment of C100 was accomplished using synthetic Aβ17–28 to generate calibration curves over a 0.001–1 μM range and 15N isotopically labeled Aβ1–40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Aβ17–28 for the analysis.     

Insulin protects against amyloid β-peptide toxicity in brain mitochondria of diabetic rats

This study compared the status of brain mitochondria isolated from 12-week streptozotocin (STZ)-diabetic rats versus STZ-diabetic animals treated with insulin during a period of 4 weeks. Brain mitochondria isolated from 12-week citrate (vehicle)-treated rats were used as control. For that purpose, several mitochondrial parameters were evaluated: respiratory indexes (respiratory control ratio (RCR) and ADP/O ratio), transmembrane potential (ΔΨm), repolarization lag phase, repolarization level, ATP, glutathione and coenzyme Q (CoQ) contents, production of H₂O₂, ATPase activity, and the capacity of mitochondria to accumulate Ca²⁺. Furthermore, the effect of Aβ_1–40 was also analyzed.We observed that STZ-induced diabetes promoted a significant decrease in mitochondrial CoQ9, ATPase activity, and a lower capacity of mitochondria to accumulate Ca²⁺ when compared with control and insulin-treated diabetic rats. The presence of 4 μM Aβ_1–40 induced a significant decrease in RCR in the three groups of rats. However, this peptide induced a significant increase in the repolarization lag phase and a significant decrease in the repolarization level in control and diabetic animals without insulin treatment. Furthermore, this peptide exacerbated significantly the production of H₂O₂ in STZ-diabetic rats, this effect being avoided by insulin treatment.Our data show that although diabetes induces some alterations in brain mitochondrial activity, those alterations do not interfere significantly with mitochondria functional efficiency. Similarly, insulin does not affect basal mitochondria function. However, in the presence of amyloid β-peptide, insulin seems to prevent the decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in oxidative stress, improving or preserving the function of neurons under adverse conditions, such as Alzheimer's disease.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

Fibrillar Alzheimer’s amyloid peptide A β1–42stimulates low density lipoprotein binding and cell association, free radical production and cell cytotoxicity in PC12 cells

Amyloid forming peptides are known to disturb vital cellular functions and induce cell death. However, the mechanisms by which fibrillogenic peptides induce cytotoxic effects in various cells has not been established. In this study the effects on low density lipoprotein binding and uptake of fibrils of the Alzheimer’s amyloid β-peptide (Aβ_1–42), which is known to play a central role in the pathogenesis of Alzheimer’s disease, were investigated in pheochromocytoma PC12 cells. Fibrillar Aβ_1–42at μmol concentrations increased low-density lipoprotein (LDL) binding and cell asociation by 460% and 200% respectively, and LDL degradation by about 62%. Approximately 49% and 34% of Aβ fibril stimulated LDL cell association and degradation was inhibited by anti-LDL receptor antibodies. The soluble form of Aβ had no effect on any of these measures of LDL metabolism. The observed increased glutathione reductase activity, DNA fragmentation (TUNEL assay) and decreased DNA synthesis ([³H] thymidine incorporation assay) in cells treated with Aβ_1–42fibrils alone or together with LDL relative to controls, suggests that the interaction of fibrils with LDL receptors might be one possible pathway which contributes to fibril cytotoxicity.     

Metabolic inhibition potentiates AMPA-induced Ca fluxes and neurotoxicity in rat cerebellar granule cells

The effects of partial metabolic inhibition (induced by 2 h exposure to low concentrations of cyanide (NaCN)) on the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced excitotoxicity and elevation of free cytoplasmic Ca²⁺ levels ([Ca²⁺]_i) were studied in glucose-deprived primary cultures of cerebellar granule cells. Co-application of AMPA plus NaCN caused a marked increase of cell death, with morphological features of both necrotic and apoptotic cell death as estimated by the capacity of cultured cerebellar granule cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan (MTT method), and by measuring the amount of DNA fragmentation in neurons using an ELISA test for histone-bound DNA fragments, respectively. Cell morphology was assessed by confocal microscopy of propidium iodide-stained cultures. No toxic effects were observed when AMPA or a low concentration of NaCN (0.1–0.3 mM; in the presence of NMDA receptor antagonist MK-801; 10 μM) were applied alone. The neurotoxic actions induced by AMPA plus NaCN were preceded and accompanied by a significant elevation of [Ca²⁺]_i, as well as by depletion of neuronal ATP stores. The marked enhancement in the functional responsiveness of AMPA receptors in energetically compromised neurons suggests that at least under certain conditions AMPA receptors may play an important role in excitotoxic processes which might be of relevance for the slowly developing neuronal death seen in several neurodegenerative diseases.     

Thapsigargin enhances carotid body chemosensory discharge in response to hypoxia in zero [Ca]e: evidence for intracellular Ca release

To test the hypothesis that Ca²⁺ is released from intracellular store in the carotid body glomus cells during hypoxia, we stimultaneously measured chemosensory discharge and tissue PO₂ of perfused-superfused cat carotid body before and during flow interruption in the presence and absence of extracellular [Ca²⁺ with and without thapsigargin (1–10 μM). Ca²⁺-free solution increased the latency of sensory response, and decreased the rate of rise and peak activity but thapsigargin significantly influenced these responses, without affecting oxygen consumption. Since thapsigargin depletes the intracellular Ca²⁺ store, and since Ca²⁺ is needed for the sensory discharge, these results suggest that intracellular release and influx of Ca²⁺ occur during hypoxia.     

Stimulation induced changes in extracellular free calcium in normal cortex and chronic alumina cream foci of cats

Changes in extracellular [Ca²⁺]₀ (δ Ca) were measured with ion selective microelectrodes in the sensorimotor cortex of cats, surrounding alumina cream lesions and in the contralateral homotopic cortex. The lesions were produced by topical application of alumina cream 6 months-6 years prior to experiments. In normal cortex, stimulus induced reductions of [Ca²⁺]₀ were found to be maximal (up to 0.45 mM) in depths of 200–300 μm below the cortical surface. At depths of 600 μm and more below the cortical surface, [Ca²⁺]₀ usually rose by up to 0.2 mM above baseline. In the vicinity of the chronic lesion as well as in contralateral cortex [Ca²⁺]₀ fell initially during stimulation in all depths. close to the lesion δ Ca was as high as 0.8 mM and sites of maximal δ Ca were found to be located deeper in the cortex. About 5 mm from the scar as well as in the contralateral homotopic cortex, maximum δ Ca levels were found in a depth of 200–300 μm. It is suggested that Ca²⁺ dependent mechanisms are involved in epileptogenesis in chronic epileptic foci.     

Ca-independent activity of Ca/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells

We investigated the involvement of Ca²⁺-independent activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the α isoform of CaM kinase II (Nb2a/α cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca²⁺-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca²⁺-independent activity. The autophosphorylation and appearance of Ca²⁺-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (αT286A kinase) or Asp (αT286D kinase) was examined. αT286A kinase was not converted to a Ca²⁺-independent form, and αT286D kinase had Ca²⁺-independent activity significantly as an autophosphorylated kinase. Cells expressing αT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing αT286D kinase had much longer neurites than Nb2a/α cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca²⁺-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.