Tetraspanin-8 sequesters syntaxin-2 to control biphasic release propensity of mucin granules.

Agonist-mediated stimulated pathway of mucin and insulin release are biphasic in which rapid fusion of pre-docked granules is followed by slow docking and fusion of granules from the reserve pool. Here, based on a cell-culture system, we show that plasma membrane-located tetraspanin-8 sequesters syntaxin-2 to control mucin release. Tetraspanin-8 affects fusion of granules during the second phase of stimulated mucin release. The tetraspanin-8/syntaxin-2 complex does not contain VAMP-8, which functions with syntaxin-2 to mediate granule fusion. We suggest that by sequestering syntaxin-2, tetraspanin-8 prevents docking of granules from the reserve pool. In the absence of tetraspanin-8, more syntaxin-2 is available for docking and fusion of granules and thus doubles the quantities of mucins secreted. This principle also applies to insulin release and we suggest a cell type specific Tetraspanin/Syntaxin combination is a general mechanism regulating the fusion of dense core granules.

Defects in lipid homeostasis reflect the function of TANGO2 in phospholipid and neutral lipid metabolism.

We show that TANGO2 in mammalian cells localizes predominantly to mitochondria and partially at mitochondria sites juxtaposed to lipid droplets (LDs) and the endoplasmic reticulum. HepG2 cells and fibroblasts of patients lacking TANGO2 exhibit enlarged LDs. Quantitative lipidomics revealed a marked increase in lysophosphatidic acid (LPA) and a concomitant decrease in its biosynthetic precursor phosphatidic acid (PA). These changes were exacerbated in nutrient-starved cells. Based on our data, we suggest that TANGO2 function is linked to acyl-CoA metabolism, which is necessary for the acylation of LPA to generate PA. The defect in acyl-CoA availability impacts the metabolism of many other fatty acids, generates high levels of reactive oxygen species, and promotes lipid peroxidation. We suggest that the increased size of LDs is a combination of enrichment in peroxidized lipids and a defect in their catabolism. Our findings help explain the physiological consequence of mutations in TANGO2 that induce acute metabolic crises, including rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, often leading to fatality upon starvation and stress.

The ulcerative colitis-associated gene FUT8 regulates the quantity and quality of secreted mucins.

Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.

Reactive oxygen species triggers unconventional secretion of antioxidants and Acb1.

Nutrient deprivation triggers the release of signal-sequence-lacking Acb1 and the antioxidant superoxide dismutase 1 (SOD1). We now report that secreted SOD1 is functionally active and accompanied by export of other antioxidant enzymes such as thioredoxins (Trx1 and Trx2) and peroxiredoxin Ahp1 in a Grh1-dependent manner. Our data reveal that starvation leads to production of nontoxic levels of reactive oxygen species (ROS). Treatment of cells with N-acetylcysteine (NAC), which sequesters ROS, prevents antioxidants and Acb1 secretion. Starved cells lacking Grh1 are metabolically active, but defective in their ability to regrow upon return to growth conditions. Treatment with NAC restored the Grh1-dependent effect of starvation on cell growth. In sum, starvation triggers ROS production and cells respond by secreting antioxidants and the lipogenic signaling protein Acb1. We suggest that starvation-specific unconventional secretion of antioxidants and Acb1-like activities maintain cells in a form necessary for growth upon their eventual return to normal conditions.

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

GRASP55 and UPR Control Interleukin-1ß Aggregation and Secretion.

Signal-sequence-lacking interleukin (IL)-1ß, is cleaved by caspase-1 to mature mIL-1ß, which is secreted, without entering the endoplasmic reticulum. We report that macrophages of GRASP55-/- mice are defective in mIL-1ß secretion and retain it as intracellular aggregates. Intriguingly, GRASP55-/- macrophages are defective in the IRE1α branch of the unfolded protein response. This finding fits well with our data that inhibition of IRE1α also impairs mIL-1ß secretion and causes its accumulation in intracellular aggregates. PERK inhibition, on the other hand, controls caspase-1-mediated conversion of proIL-1ß to mIL-1ß. These findings reveal translation-independent functions of PERK and IRE1α: PERK controls the production of mIL-1ß, which is then followed by GRASP55 and IRE1α activity to keep mIL-1ß in a secretion-competent form.

Sodium channel TRPM4 and sodium/calcium exchangers (NCX) cooperate in the control of Ca2+-induced mucin secretion from goblet cells.

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.

KChIP3 coupled to Ca2+ oscillations exerts a tonic brake on baseline mucin release in the colon.

Regulated mucin secretion from specialized goblet cells by exogenous agonist-dependent (stimulated) and -independent (baseline) manner is essential for the function of the epithelial lining. Over extended periods, baseline release of mucin can exceed quantities released by stimulated secretion, yet its regulation remains poorly characterized. We have discovered that ryanodine receptor-dependent intracellular Ca2+ oscillations effect the dissociation of the Ca2+-binding protein, KChIP3, encoded by KCNIP3 gene, from mature mucin-filled secretory granules, allowing for their exocytosis. Increased Ca2+ oscillations, or depleting KChIP3, lead to mucin hypersecretion in a human differentiated colonic cell line, an effect reproduced in the colon of Kcnip3-/- mice. Conversely, overexpressing KChIP3 or abrogating its Ca2+-sensing ability, increases KChIP3 association with granules, and inhibits baseline secretion. KChIP3 therefore emerges as the high-affinity Ca2+ sensor that negatively regulates baseline mucin secretion. We suggest KChIP3 marks mature, primed mucin granules, and functions as a Ca2+ oscillation-dependent brake to control baseline secretion. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

A diacidic motif determines unconventional secretion of wild-type and ALS-linked mutant SOD1.

The nutrient starvation-specific unconventional secretion of Acb1 in Saccharomyces cerevisiae requires ESCRT-I, -II, and -III and Grh1. In this study, we report that another signal sequence lacking cytoplasmic protein, superoxide dismutase 1 (SOD1), and its mutant form linked to amyotrophic lateral sclerosis (ALS), is also secreted by yeast upon nutrient starvation in a Grh1- and ESCRT-I-, -II-, and -III-dependent process. Our analyses reveal that a conserved diacidic motif (Asp-Glu) in these proteins is necessary for their export. Importantly, secretion of wild-type human SOD1 and the ALS-linked mutant in human cells also require the diacidic residues. Altogether, these findings reveal information encoded within the cytoplasmic proteins required for their unconventional secretion and provide a means to unravel the significance of the cytoplasmic versus the secreted form of mutant SOD1 in the pathology of ALS. We also propose how cells, based on a signal-induced change in cytoplasmic physiology, select a small pool of a subset of cytoplasmic proteins for unconventional secretion.

Role of Kif15 and its novel mitotic partner KBP in K-fiber dynamics and chromosome alignment.

Faithful segregation of the genetic material during the cell cycle is key for the continuation of life. Central to this process is the assembly of a bipolar spindle that aligns the chromosomes and segregates them to the two daughter cells. Spindle bipolarity is strongly dependent on the activity of the homotetrameric kinesin Eg5. However, another kinesin, Kif15, also provides forces needed to separate the spindle poles during prometaphase and to maintain spindle bipolarity at metaphase. Here we identify KBP as a specific interaction partner of Kif15 in mitosis. We show that KBP promotes the localization of Kif15 to the spindle equator close to the chromosomes. Both Kif15 and KBP are required for the alignment of all the chromosomes to the metaphase plate and the assembly of stable kinetochore fibers of the correct length. Taken together our data uncover a novel role for Kif15 in complex with KBP during mitosis.

ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion.

The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior.

Reduced secretion and altered proteolytic processing caused by missense mutations in progranulin.

Progranulin (GRN) is a secreted growth factor involved in various cellular functions, and loss-of-function mutations are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43 positive pathology. Most FTLD-related GRN mutations are nonsense mutations resulting in reduced GRN expression. Nonsynonymous GRN missense mutations have been described as risk factor for neurodegenerative brain diseases, but their pathogenic nature remains largely elusive. We identified a double missense mutation in GRN leading to amino acid changes p.D33E and p.G35R in an FTLD patient from Turkish origin. Biochemical and cell biological analysis of the double-mutation together with 2 so-far uncharacterized GRN missense mutations (p.C105R and p.V514M) revealed a reduced secretion efficiency of the GRN p.D33E/p.G35R and p.C105R proteins. Furthermore, loss of the conserved cysteine residue affects protein folding and altered proteolytic processing by neutrophil elastase and proteinase 3. Our data indicate that the described variants may cause a loss-of-function, albeit to a lesser extent than GRN null mutations, and hence could be considered as low-penetrant risk factors for neurodegenerative diseases.

TANGO1 recruits ERGIC membranes to the endoplasmic reticulum for procollagen export.

Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process.

Brain-specific tryptophan hydroxylase, TPH2, and 5-HTTLPR are associated with frontal lobe symptoms in Alzheimer's disease.

A prospective, longitudinal study was set up to investigate possible genetic associations of behavioral and psychological signs and symptoms of dementia (BPSD) in Alzheimer's disease (AD) with two candidate genes in the serotonergic neurotransmitter pathway: serotonin transporter (SLC6A4) and brain-specific tryptophan hydroxylase (TPH2). Patients with probable AD (n = 249) were diagnosed according to NINCDS/ADRDA criteria. During follow-up, autopsy-confirmation of clinical diagnosis was obtained in 32 AD patients. Taking into account follow-up behavioral assessments by means of validated behavioral assessment scales (Middelheim Frontality Score and Behave-AD), behavioral ratings reflecting the highest scores on any behavioral item ever observed since dementia onset were calculated and applied for statistical analyses. A functional insertion/deletion polymorphism in the promoter region of SLC6A4 (5-HTTLPR) and 10 selected SNPs within TPH2 were genotyped. Single-marker allelic association analyses (TPH2, 5-HTTLPR) were performed. TPH2 allelic analyses revealed significant associations with frontal lobe symptoms, as well as with diurnal rhythm disturbances. 5-HTTLPR S allele carriers had an increased risk to display loss of insight and judgment, another frontal lobe symptom. The present prospective, longitudinal study showed that mainly frontal lobe symptoms were significantly associated with TPH2 and 5-HTTLPR polymorphisms, pointing toward a role of the serotonergic neurotransmitter system in the pathophysiology of frontal lobe symptoms in AD.

Genetic association of CR1 with Alzheimer's disease: a tentative disease mechanism.

CR1 is a novel Alzheimer's disease (AD) gene identified by genome-wide association studies (GWAS). Recently, we showed that AD risk could be explained by an 18-kilobase insertion responsible for the complement component (3b/4b) receptor 1 (CR1)-S isoform. We investigated the relevance of the CR1 isoforms to AD in a Canadian dataset. Also, we genotyped rs4844610 tagging the GWAS-significant CR1 single nucleotide polymorphisms. Individuals with F/S genotype had a 1.8 times increased risk for AD compared with F/F genotype (p-adjusted = 0.003), while rs4844610 was only marginally significant (p-adjusted = 0.024). The analyses of brain samples demonstrated that the CR1-S isoform is expressed at lower protein levels than CR1-F (p < 0.0001) hence likely associated with increased complement activation. Intriguingly, our neuropathological results show that the pattern of CR1 expression in neurons is different between the F/F and F/S genotypes (filiform vs. vesicular-like profiles). Furthermore, double labeling studies supported a differential distribution of CR1 in neurons (endoplasmic reticulum intermediate compartment vs. lysosomes). These observations indicate that the CR1-S and CR1-F isoforms could be processed in different ways in neurons. In conclusion, our results support that the CR1-S isoform explains the GWAS signals and open a novel prospect for the investigation of CR1-related disease mechanisms.

Both common variations and rare non-synonymous substitutions and small insertion/deletions in CLU are associated with increased Alzheimer risk.

BACKGROUND: We have followed-up on the recent genome-wide association (GWA) of the clusterin gene (CLU) with increased risk for Alzheimer disease (AD), by performing an unbiased resequencing of all CLU coding exons and regulatory regions in an extended Flanders-Belgian cohort of Caucasian AD patients and control individuals (n = 1930). Moreover, we have replicated genetic findings by targeted resequencing in independent Caucasian cohorts of French (n = 2182) and Canadian (n = 573) origin and by performing meta-analysis combining our data with previous genetic CLU screenings. RESULTS: In the Flanders-Belgian cohort, we identified significant clustering in exons 5-8 of rare genetic variations leading to non-synonymous substitutions and a 9-bp insertion/deletion affecting the CLU ß-chain (p = 0.02). Replicating this observation by targeted resequencing of CLU exons 5-8 in 2 independent Caucasian cohorts of French and Canadian origin identified identical as well as novel non-synonymous substitutions and small insertion/deletions. A meta-analysis, combining the datasets of the 3 cohorts with published CLU sequencing data, confirmed that rare coding variations in the CLU ß-chain were significantly enriched in AD patients (OR(MH) = 1.96 [95% CI = 1.18-3.25]; p = 0.009). Single nucleotide polymorphisms (SNPs) association analysis indicated the common AD risk association (GWA SNP rs11136000, p = 0.013) in the 3 combined datasets could not be explained by the presence of the rare coding variations we identified. Further, high-density SNP mapping in the CLU locus mapped the common association signal to a more 5' CLU region. CONCLUSIONS: We identified a new genetic risk association of AD with rare coding CLU variations that is independent of the 5' common association signal identified in the GWA studies. At this stage the role of these coding variations and their likely effect on the ß-chain domain and CLU protein functioning remains unclear and requires further studies.

Potent amyloidogenicity and pathogenicity of Aß43.

The amyloid-ß peptide Aß42 is known to be a primary amyloidogenic and pathogenic agent in Alzheimer's disease. However, the role of Aß43, which is found just as frequently in the brains of affected individuals, remains unresolved. We generated knock-in mice containing a pathogenic presenilin-1 R278I mutation that causes overproduction of Aß43. Homozygosity was embryonic lethal, indicating that the mutation involves a loss of function. Crossing amyloid precursor protein transgenic mice with heterozygous mutant mice resulted in elevated Aß43, impairment of short-term memory and acceleration of amyloid-ß pathology, which accompanied pronounced accumulation of Aß43 in plaque cores similar in biochemical composition to those observed in the brains of affected individuals. Consistently, Aß43 showed a higher propensity to aggregate and was more neurotoxic than Aß42. Other pathogenic presenilin mutations also caused overproduction of Aß43 in a manner correlating with Aß42 and with the age of disease onset. These findings indicate that Aß43, an overlooked species, is potently amyloidogenic, neurotoxic and abundant in vivo.

Amyloid precursor protein mutation E682K at the alternative ß-secretase cleavage ß'-site increases Aß generation.

BACE1 cleaves the amyloid precursor protein (APP) at the ß-cleavage site (Met(671) -Asp(672) ) to initiate the generation of amyloid peptide Aß. BACE1 is also known to cleave APP at a much less well-characterized ß'-cleavage site (Tyr(681) -Glu(682) ). We describe here the identification of a novel APP mutation E682K located at this ß'-site in an early onset Alzheimer's disease (AD) case. Functional analysis revealed that this E682K mutation blocked the ß'-site and shifted cleavage of APP to the ß-site, causing increased Aß production. This work demonstrates the functional importance of APP processing at the ß'-site and shows how disruption of the balance between ß- and ß'-site cleavage may enhance the amyloidogenic processing and consequentially risk for AD. Increasing exon- and exome-based sequencing efforts will identify many more putative pathogenic mutations without conclusive segregation-based evidence in a single family. Our study shows how functional analysis of such mutations allows to determine the potential pathogenic nature of these mutations. We propose to classify the E682K mutation as probable pathogenic awaiting further independent confirmation of its association with AD in other patients.

Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

The CALHM1 P86L polymorphism is a genetic modifier of age at onset in Alzheimer's disease: a meta-analysis study.

The only established genetic determinant of non-Mendelian forms of Alzheimer's disease (AD) is the ε4 allele of the apolipoprotein E gene (APOE). Recently, it has been reported that the P86L polymorphism of the calcium homeostasis modulator 1 gene (CALHM1) is associated with the risk of developing AD. In order to independently assess this association, we performed a meta-analysis of 7,873 AD cases and 13,274 controls of Caucasian origin (from a total of 24 centers in Belgium, Finland, France, Italy, Spain, Sweden, the UK, and the USA). Our results indicate that the CALHM1 P86L polymorphism is likely not a genetic determinant of AD but may modulate age of onset by interacting with the effect of the ε4 allele of the APOE gene.