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Lysosomes serve as cellular degradation and signaling centers that coordinate the turnover of macromolecules with cell metabolism. The adaptation of cellular lysosome content and activity via the induction of lysosome biogenesis is therefore key to cell physiology and to counteract disease. Previous work has established a pathway for the induction of lysosome biogenesis in signaling-inactive starved cells that is based on the repression of mTORC1-mediated nutrient signaling. How lysosomal biogenesis is facilitated in signaling-active fed cells is poorly understood. A recent study by Malek et al. (2022) partially fills this gap by unraveling a nutrient signaling-independent pathway for lysosome biogenesis that operates in signaling-active cells. This pathway involves the receptor-mediated activation of phospholipase C, inositol (1,4,5)-triphosphate (IP3)-triggered release of calcium ions from endoplasmic reticulum stores, and the calcineurin-induced activation of transcription factor EB (TFEB) and its relative TFE3 to induce lysosomal gene expression independent of calcium in the lysosome lumen. These findings contribute to our understanding of how lysosome biogenesis and function are controlled in response to environmental changes and cell signaling and may conceivably be of relevance for our understanding and the treatment of lysosome-related diseases as well as for aging and neurodegeneration. [ABSTRACT FROM AUTHOR]

Summary: Commercialization of Brassica napus. L (oilseed rape) meal as protein diet is gaining more attention due to its well‐balanced amino acid and protein contents. Phytic acid (PA) is a major source of phosphorus in plants but is considered as anti‐nutritive for monogastric animals including humans due to its adverse effects on essential mineral absorption. The undigested PA causes eutrophication, which potentially threatens aquatic life. PA accounts to 2‐5% in mature seeds of oilseed rape and is synthesized by complex pathways involving multiple enzymes. Breeding polyploids for recessive traits is challenging as gene functions are encoded by several paralogs. Gene redundancy often requires to knock out several gene copies to study their underlying effects. Therefore, we adopted CRISPR‐Cas9 mutagenesis to knock out three functional paralogs of BnITPK. We obtained low PA mutants with an increase of free phosphorus in the canola grade spring cultivar Haydn. These mutants could mark an important milestone in rapeseed breeding with an increase in protein value and no adverse effects on oil contents. [ABSTRACT FROM AUTHOR]

In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] generation on the mechanical-stimulation-induced Ca2+ response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P3 ([IP3]i), as indicated by the Ins(1,4,5)P3 biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P3 wave as [IP3]i increased in the stimulated region. A small transient [IP3]i increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P3 responses and resultant Ca2+ releases. The purinergic receptor blocker suramin completely blocked increases in [IP3]1 and the Ca2+ release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P3 in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP3]i increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P3-induced Ca2+ release was larger than that of Ca2+ entry in the Ca2+ response to mechanical stimulation in HSY-EA1 cells. [ABSTRACT FROM AUTHOR]

Abstract: The development of chronic pain involves increased sensitivity of peripheral nociceptors and elevated neuronal activity in many regions of the central nervous system. Much of these changes are caused by the amplification of nociceptive signals resulting from the modulation and altered expression of specific ion channels and receptors in the central and peripheral nervous system. Understanding the processes by which these ion channels and receptors are regulated and how these mechanisms malfunction may lead to new treatments for chronic pain. Here we review the contribution of the Ca2+-permeable acid-sensing ion channel (ASICCa) in the development and persistence of chronic pain, and the potential underlying mechanisms. Accumulating evidence suggests that ASICCa represents an attractive new target for developing effective therapies for chronic pain. [Copyright &y& Elsevier]

Abstract: As part of our continuing effort aimed at the development of selective, efficacious and centrally active M1 muscarinic agonists for the treatment of Alzheimer’s presenile dementia, a series of N-arylthioureas substituted 3-morpholino arecoline derivatives 9(a–j) were synthesized by using N-benzyl amino ethanol coupling with α-bromo acetyl pyridine followed by reduction and cyclization to develop a new class of M1 receptor agonists. Subsequently the synthesized compounds were subjected to in vitro radioligand M1 receptor affinity studies, IP3 formation studies and also to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Derivatives with chloro (9f) and methoxy (9c) groups on the para position of the benzene ring attached to the nitrogen of thiourea showed several fold high affinity for the M1 receptor (in vitro) among all the synthesized molecules 9(a–j), and also significantly elevated IP3 levels and as well elicited beneficial effects in vivo in memory and learning models in rats (rodent memory evaluation, plus and Y maze studies). [Copyright &y& Elsevier]

Abstract: Calcium (Ca2+) is involved in a myriad of cellular functions in the brain including synaptic plasticity. However, the role of intracellular Ca2+ stores in memory processing remains poorly defined. The current study explored a role for glutamate-dependent intracellular Ca2+ release in memory processing via blockade of metabotropic glutamate receptor subtype 1 (mGluR1) and inositol (1,4,5)-trisphosphate receptors (IP3Rs). Using a single-trial discrimination avoidance task developed for the young chick, administration of the specific and potent mGluR1 antagonist JNJ16259685 (500nM, immediately post-training, ic), or the IP3R antagonist Xestospongin C (5μM, immediately post-training, ic), impaired retention from 90min post-training. These findings are consistent with mGluR1 activating IP3Rs to release intracellular Ca2+ required for long-term memory formation and have been interpreted within an LTP2 model. The consequences of different patterns of retention loss following ryanodine receptor (RyR) and IP3R inhibition are discussed. [Copyright &y& Elsevier]

Abstract: Activation of ionotropic glutamate (Glu) receptors, such as N-methyl-d-aspartate receptors, is shown to modulate the gene transcription mediated by the transcription factor activator protein-1 (AP1) composed of Fos and Jun family proteins in the brain, while little attention has been paid to the modulation of AP1 expression by metabotropic Glu receptors (mGluRs). In cultured rat cortical neurons, where constitutive expression was seen with all groups I, II and III mGluR subtypes, a significant and selective increase was seen in the DNA binding activity of AP1 120min after the brief exposure to the group II mGluR agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) for 5min. In cultured rat cortical astrocytes, by contrast, a significant increase was induced by a group I mGluR agonist, but not by either a group II or III mGluR agonist. The increase by DCG-IV was significantly prevented by a group II mGluR antagonist as well as by either an intracellular Ca2+ chelator or a voltage-sensitive Ca2+ channel blocker, but not by an intracellular Ca2+ store inhibitor. Moreover, DCG-IV significantly prevented the increase of cAMP formation by forskolin in cultured neurons. Western blot analysis revealed differential expression profiles of Fos family members in neurons briefly exposed to DCG-IV and NMDA. Prior or simultaneous exposure to DCG-IV led to significant protection against neuronal cell death by NMDA. These results suggest that activation of the group II mGluR subtype would modulate the gene expression mediated by AP1 through increased intracellular Ca2+ levels in cultured rat cortical neurons. [Copyright &y& Elsevier]

Abstract: Prolonged intrathecal (i.t.) administration of morphine results in tolerance to morphine-induced antinociception. We found that co-administration of selective metabotropic glutamate receptor subtype 5 antagonist MPEP with morphine could suppress the loss of morphine-induced antinociception and inhibit the development of tolerance to morphine-induced antinociceptive effect. Whereas, the specific metabotropic glutamate receptor subtype 5 agonist CHPG does the opposite. As the activation of NMDA receptor after chronic morphine administration has been verified, we suppose there is an enhanced activation of mGluR5 during the development of tolerance to morphine-induced antinociception. Activation of mGluR5 may mobilize the release of intracellular Ca2+ and activate PKC, leading to morphine-induced antinociception suppression. We conclude that mGluR5 contributes to the development of tolerance to morphine-induced antinociception after chronic morphine exposure. [Copyright &y& Elsevier]

Abstract: Many receptors for neurotransmitters and hormones rely upon members of the Gqα family of heterotrimeric G proteins to exert their actions on target cells. Gα subunits of the Gq class of G proteins (Gqα, G11α, G14α and G15/16α) directly link receptors to activation of PLC-β isoforms which, in turn, stimulate inositol lipid (i.e. calcium/PKC) signalling. Although Gqα family members share a capacity to activate PLC-β, they also differ markedly in their biochemical properties and tissue distribution which predicts functional diversity. Nevertheless, established models suggest that Gqα family members are functionally redundant and that their cellular responses are a result of PLC-β activation and downstream calcium/PKC signalling. Growing evidence, however, indicates that Gqα, G11α, G14α and G15/16α are functionally diverse and that many of their cellular actions are independent of inositol lipid signalling. Recent findings show that Gqα family members differ with regard to their linked receptors and downstream binding partners. Reported binding partners distinct from PLC-β include novel candidate effector proteins, various regulatory proteins, and a growing list of scaffolding/adaptor proteins. Downstream of these signalling proteins, Gqα family members exhibit unexpected differences in the signalling pathways and the gene expression profiles they regulate. Finally, genetic studies using whole animal models demonstrate the importance of certain Gqα family members in cardiac, lung, brain and platelet functions among other physiological processes. Taken together, these findings demonstrate that Gqα, G11α, G14α and G15/16α regulate both overlapping and distinct signalling pathways, indicating that they are more functionally diverse than previously thought. [Copyright &y& Elsevier]

Abstract: Defining the underlying causes of psychiatric disorders has provided an ongoing and intractable problem. The analysis of the genetic basis of manic depression, in particular, has been impeded by the absence of a suitable model system and by the lack of candidate causative genes. One recent approach to overcome these problems has involved identifying those genes which control the sensitivity to anti-manic drugs in a model organism. Characterisation of the role of these genes and their encoded proteins in this model has allowed the analysis of their mammalian homologues to elucidate the therapeutic role of these drugs and the possible aetiology of manic depression. This approach has been used successfully with the cellular slime mould, Dictyostelium discoideum. This article introduces the use of model systems for pharmacogenetics research. It describes the identification of prolyl oligopeptidase in D. discoideum as a modulator of inositol phosphate signalling, and the subsequent identification of a common mechanism of action of three anti-manic drugs in mammalian neurons. The use of pharmacogenetics in model systems will provide a powerful tool for the ongoing analysis of both the treatment and cause of psychiatric disorders. [Copyright &y& Elsevier]

Abstract: In the human lens, stimulation of tyrosine-kinase coupled growth factor receptors such as epidermal growth factor receptor (EGFR) can induce calcium release from endoplasmic reticulum (ER) stores. The present study investigated the impact of calcium store inactivation on EGFR signalling, cell growth and death in a well-characterised human lens cell line (FHL124). FHL124 cells were routinely cultured in Eagle''s minimum essential medium (EMEM) supplemented with 10% foetal calf serum (FCS) and seeded on 24-well plates (DNA and protein synthesis), tissue culture dishes (growth assay, western immunoblot), and glass coverslips (immunocytochemistry). DNA and protein synthesis rates were quantified by measuring the incorporation of 3H-thymidine and 35S-methionine into FHL124 cells in serum-free EMEM or EMEM supplemented with thapsigargin (Tg) (100nm and 1μm). Longer-term growth was assessed by quantifying the increase in area over time of a circular patch of seeded cells. EGFR was identified using anti-EGFR mouse monoclonal antibody and visualised by fluorescence microscopy with ALEXA 488 conjugated secondary antibody. Programmed cell death was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay method. Activation of the mitogen-activated protein kinase (MAPK) signalling protein extracellular signal-regulated kinase (ERK) and the cell cycle proteins CDK2 and P27kip1 were detected by western immunoblot techniques. Inactivation by ≥100nm Tg inhibited both protein and DNA synthesis although the effect on the latter was greatest. The cell cycle activator CDK2 was reduced by Tg, while the inhibitor P27kip1 was increased along with the percentage of apoptotic cells. A single, maximal epidermal growth factor (EGF) (10ngml−1) exposure induced receptor internalization and increased ERK phosphorylation. Both internalisation and ERK activation were unaffected by the presence of Tg. However, reduced internalisation and ERK activation followed repeated EGF applications in the presence of Tg. Additionally, ERK activation by submaximal EGF concentrations was reduced by store depletion. An intact endoplasmic reticulum calcium store therefore plays a significant role in human lens cell survival and growth. [Copyright &y& Elsevier]

Cytosolic Ca2+ controls a wide range of cellular events. The versatility of this second messenger depends on its ability to form diverse spatial and temporal patterns, including waves and oscillations. Ca2+-signaling patterns are thought to be determined in part by the subcellular distribution of inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P3Rs] but little is currently known about how the localization of the Ins(1,4,5)P3R itself is regulated. Here, we report that the recruitment of GFP-tagged Ins(1,4,5)P3Rs in the vicinity of tight junctions in Madin-Darby canine kidney (MDCK) cells requires the N-terminal domain. Stable expression of this domain in polarized MDCK cells induced a flattened morphology, affected cytokinesis, accelerated cell migration in response to monolayer wounding and interfered with the cortical targeting of myosin IIA. In addition, downregulation of myosin IIA in polarized MDCK cells was found to mimic the effects of stable expression of the N-terminal part of Ins(1,4,5)P3R on cell shape and to alter localization of endogenous Ins(1,4,5)P3Rs. Taken together, these results support a model in which the recruitment of Ins(1,4,5)P3Rs at the apex of the lateral membrane in polarized MDCK cells, involves myosin IIA and might be important for the regulation of cortical actin dynamics. [ABSTRACT FROM AUTHOR]

Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] originating in the vascular smooth-muscle cells (VSMCs) has been shown to modulate the Ca2+ stores in endothelial cells (ECs). However, the reverse is not found, suggesting that Ins(1,4,5)P3 movement might be unidirectional across gap junctions at the myoendothelial junction (MEJ), or that distribution of the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3-R] is different between the two cell types. To study trans-junctional communication at the MEJ, we used a vascular-cell co-culture model system and selectively modified the connexin composition in gap junctions in the two cell types. We found no correlation between modification of connexin expression and Ins(1,4,5)P3 signaling between ECs and VSMCs. We next explored the distribution of Ins(1,4,5)P3-R isoforms in the two cell types and found that Ins(1,4,5)P3-R1 was selectively localized to the EC side of the MEJ. Using siRNA, selective knockdown of Ins(1,4,5)P3-R1 in ECs eliminated the secondary Ins(1,4,5)P3-induced response in these cells. By contrast, siRNA knockdown of Ins(1,4,5)P3-R2 or Ins(1,4,5)P3-R3 in ECs did not alter the EC response to VSMC stimulation. The addition of 5-phosphatase inhibitor (5-PI) to ECs that were transfected with Ins(1,4,5)P3-R1 siRNA rescued the Ins(1,4,5)P3 response, indicating that metabolic degradation of Ins(1,4,5)P3 is an important part of EC-VSMC coupling. To test this concept, VSMCs were loaded with 5-PI and BAPTA-loaded ECs were stimulated, inducing an Ins(1,4,5)P3-mediated response in VSMCs; this indicated that Ins(1,4,5)P3 is bidirectional across the gap junction at the MEJ. Therefore, localization of Ins(1,4,5)P3-R1 on the EC side of the MEJ allows the ECs to respond to Ins(1,4,5)P3 from VSMCs, whereas Ins(1,4,5)P3 moving from ECs to VSMCs is probably metabolized before binding to a receptor. This data implicates the MEJ as being a unique cell-signaling domain in the vasculature. [ABSTRACT FROM AUTHOR]

ITPKB phosphorylates inositol 1,4,5-trisphosphate into inositol 1,3,4,5-tetrakisphosphate and controls signal transduction in various hematopoietic cells. Surprisingly, it has been reported that the ITPKB messenger RNA level is significantly increased in the cerebral cortex of patients with Alzheimer’s disease, compared with control subjects. As extracellular signal-regulated kinases 1/2 activation is increased in the Alzheimer brain and as ITPKB is a regulator of extracellular signal-regulated kinases 1/2 activation in some hematopoietic cells, we tested whether this increased activation in Alzheimer’s disease might be related to an increased activity of ITPKB. We show here that ITPKB protein level was increased 3-fold in the cerebral cortex of most patients with Alzheimer’s disease compared with control subjects, and accumulated in dystrophic neurites associated to amyloid plaques. In mouse Neuro-2a neuroblastoma cells, Itpkb overexpression was associated with increased cell apoptosis and increased β-secretase 1 activity leading to overproduction of amyloid-β peptides. In this cellular model, an inhibitor of mitogen-activated kinase kinases 1/2 completely prevented overproduction of amyloid-β peptides. Transgenic overexpression of ITPKB in mouse forebrain neurons was not sufficient to induce amyloid plaque formation or tau hyperphosphorylation. However, in the 5X familial Alzheimer’s disease mouse model, neuronal ITPKB overexpression significantly increased extracellular signal-regulated kinases 1/2 activation and β-secretase 1 activity, resulting in exacerbated Alzheimer’s disease pathology as shown by increased astrogliosis, amyloid-\(β_{40}\) peptide production and tau hyperphosphorylation. No impact on pathology was observed in the 5X familial Alzheimer’s disease mouse model when a catalytically inactive ITPKB protein was overexpressed. Together, our results point to the ITPKB/inositol 1,3,4,5-tetrakisphosphate/extracellular signal-regulated kinases 1/2 signalling pathway as an important regulator of neuronal cell apoptosis, APP processing and tau phosphorylation in Alzheimer’s disease, and suggest that ITPKB could represent a new target for reducing pathology in human patients with Alzheimer’s disease with ITPKB expression.