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Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca²⁺ sensor that responds to ER Ca²⁺ store depletion and activates Ca²⁺ channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca²⁺ store depletion. These oligomers rapidly dissociated when ER Ca²⁺ stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER-PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca²⁺ store depletion but failed to form puncta at ER-PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 μm to reach ER-PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca²⁺ gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (i) dissociation of Ca²⁺, (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER-PM junctions, and (iv) activation of PM Ca²⁺ channels.

Acquired drug resistance is a major challenge in the management of cancer, which underscores the need for discovery and development of novel therapeutic strategies. We report here the mechanism of the anti-cancer activity of a small coordinate complex composed of the rare earth metal praseodymium (Pr) and mercaptopyridine oxide (MPO; pyrithione). Exposure of cancer cells to relatively low concentrations of the conjugate Pr-MPO (5 µM) significantly impairs cell survival in a p53-independent manner and irrespective of the drug resistant phenotype. Mechanistically, Pr-MPO-induced cell death is caspase-independent, not inhibitable by necrostatin, but associated with the appearance of autophagy markers. However, further analysis revealed incomplete autophagic flux, thus suggesting altered integrity of lysosomal machinery. Supporting the lysosomal targeting activity are data demonstrating increased lysosomal Ca 2+ accumulation and alkalinization, which coincides with cytosolic acidification (drop in pH c from 7.75 to 7.00). In parallel, an increase in lysosomal activity of glycosidase alpha acid (GAA), involved in passive glycogen breakdown, correlates with rapid depletion of glucose stores upon Pr-MPO treatment. This is associated with swift cataplerosis of TCA cycle intermediates, loss of NAD + /NADH and increase in pyruvate dehydrogenase (PDH) activity to compensate for pyruvate loss. Addition of exogenous pyruvate rescued cell survival. Notably, lysosomal impairment and metabolic catastrophe triggered by Pr-MPO are suggestive of Zn 2+ -mediated cytotoxicity, which is confirmed by the ability of Zn 2+ chelator TPEN to block Pr-MPO-mediated anti-tumor activity. Together, these results highlight the ability of the small molecule lanthanide conjugate to target the cells’ waste clearing machinery as well as mitochondrial metabolism for Zn 2+ -mediated execution of cancer cells, which could have therapeutic potential against cancers with high metabolic activity.

To address hepatotropic body distribution and toxicity, transfection systems based on protein architecture have been proposed. Attenuated anthrax toxin (aATx) has provided the backbone for a first in class transfection system that, in the wild, uses intraluminal vesicles (ILVs) as an intermediary compartment during the translocation of large molecules into the cytosol. Small interfering (si)RNA molecules non-covalently attached to a carrier (LFn-PKR) would not be predicted to be an aATx translocase (protective antigen (PA)) substrate. Previously, siRNA has been shown to be delivered to the cytosol using this system. Here, the localisation of P-labelled siRNA delivered using aATx was quantified directly and related to siRNA activity. In addition, inhibition of ILV formation by hypertonic sucrose or wheatgerm agglutinin (WGA) was shown to inhibit the aATx-mediated cytosolic translocation of siRNA. MCF-7 cells were used to establish siRNA intracellular distribution in relation to pharmacological activity by targeting STAT3 gene expression. After Lipofectamine-mediated transfection using 100 nM P-labelled siRNA, 45 ± 3.2% (±SD; = 3) of the cell associated siRNA was found in the cytosol. After the transfection of 100 nM P-labelled siRNA using aATx, 77 ± 2.5% (±SD; = 3) of the cell associated siRNA was found in the cytosol and resulted in a reduction in STAT3 expression of 64.04 ± 14.17% (±SD; = 3) relative to an untreated control by Western analysis. Further, 25 μg/mL of WGA inhibited 75.23 ± 0.06% (±SD; = 3) of the knockdown attributed to a non-WGA-treated control. Relative to the control, treatment with 200 mM sucrose resulted in a reduction of 74.58 ± 7.76% (±SD; = 3) of target gene knockdown. These data indicated that the insertion of the PA pore into endosomal membrane did not weaken the endosomal limiting membrane, leading to vesicular bursting during transfection and ILVs played critical role in translocase activity.

Valosin-containing protein (VCP) is a ubiquitously expressed type II AAA + ATPase protein, implicated in both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This study aimed to explore the impact of the disease-causing VCP R191Q/wt mutation on mitochondrial function using a CRISPR/Cas9-engineered neuroblastoma cell line. Mitochondria in these cells are enlarged, with a depolarized mitochondrial membrane potential associated with increased respiration and electron transport chain activity. Our results indicate that mitochondrial hypermetabolism could be caused, at least partially, by increased calcium-induced opening of the permeability transition pore (mPTP), leading to mild mitochondrial uncoupling. In conclusion, our findings reveal a central role of the ALS/FTD gene VCP in maintaining mitochondrial homeostasis and suggest a model of pathogenesis based on progressive alterations in mPTP physiology and mitochondrial energetics.

De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/− NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells – two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/− developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.

Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.

Studying the complex mechanisms underlying breast cancer metastasis and therapy response necessitates relevant in vivo models, particularly syngeneic models with an intact immune system. Two syngeneic spontaneously metastatic sublines, D2A1-m1 and D2A1-m2, were generated from the poorly metastasising BALB/c-derived D2A1 cell line by serial in vivo passaging. In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes, such as enhanced proliferation, migration and invasion, are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 subline is associated with an increased ability to recruit an activated tumour stroma. The metastatic D2A1-m1 and D2A1-m2 cell lines provide additional syngeneic models for investigating the different steps of the metastatic cascade and thereby represent valuable tools for breast cancer researchers. Finally, this study highlights that morphology and cell behaviour in 2D cell-based assays cannot be used as a reliable predictor of metastatic behaviour in vivo.

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium—some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.

Background MicroRNAs (miRNAs) are short non-coding RNAs that are emerging as important post-transcriptional regulators of neuronal and synaptic development. The precise impact of miRNAs on presynaptic function and neurotransmission remains, however, poorly understood. Results Here, we identify miR-27b—an abundant neuronal miRNA implicated in neurological disorders—as a global regulator of the presynaptic transcriptome. miR-27b influences the expression of three quarters of genes associated with presynaptic function in cortical neurons. Contrary to expectation, a large majority of these genes are up-regulated by miR-27b. This stimulatory effect is mediated by miR-27b-directed silencing of several transcriptional repressors that cooperate to suppress the presynaptic transcriptome. The strongest repressive activity appears to be mediated by Bmi1, a component of the polycomb repressive complex implicated in self-renewal of neural stem cells. miR-27b knockdown leads to reduced synaptogenesis and to a marked decrease in neural network activity, which is fully restored by RNAi-mediated silencing of Bmi1. Conclusions We conclude that silencing of Bmi1 by miR-27b relieves repression of the presynaptic transcriptome and supports neurotransmission in cortical networks. These results expand the repressive activity of Bmi1 to genes involved in synaptic function and identify a unique post-transcriptional circuitry that stimulates expression of synaptic genes and promotes synapse differentiation.

INTRODUCTION Pharmacological restoration of septin filament integrity has the potential to provide symptomatic benefit and disease modification in Alzheimer's disease (AD). METHODS REM127, a septin modulator, was assessed in mild‐to‐moderate AD (EudraCT: 2022‐000080‐43) in a phase 2a trial (n = 14). Primary endpoints: safety and tolerability; exploratory endpoints: pharmacokinetics, cerebrospinal fluid (CSF) biomarkers, electroencephalography (EEG), and functional outcomes. RESULTS In participants on active therapy, dose‐dependent increases in serum aminotransferase were observed, leading to study discontinuation. CSF hyperphosphorylated tau (P‐tau181), endpoints reflecting synaptic function and cognitive outcomes, were changed significantly (p < 0.05) to normal compared to placebo. DISCUSSION REM127 triggers off‐target liver adverse effects. Anticipated on‐target outcomes suggest septin modulation has symptomatic benefit and modifies processes underlying AD. Results are considered exploratory as statistical power is constrained due to the small sample size caused by early termination. Further investigation of the therapeutic concept using an optimized septin molecular glue with an improved safety profile is warranted. Highlights Septin 6/7 molecular glue REM127 was assessed in symptomatic participants with Alzheimer's disease (AD). REM127 triggers off‐target effects suggesting liver adverse effects. REM127 brain exposure was consistent with saturated target engagement. Biomarker and cognitive outcomes were changed consistent with therapeutic benefit. Septin modulation may restore synaptic function and mitigate pathology in AD.