BBA - Molecular Basis of Disease (v.1822, #10)

Higher susceptibility to amyloid fibril formation of the recombinant ovine prion protein modified by transglutaminase by Angela Sorrentino; Concetta Valeria L. Giosafatto; Ivana Sirangelo; Carmela De Simone; Prospero Di Pierro; Raffaele Porta; Loredana Mariniello (1509-1515).
Prion proteins are known as the main agents of transmissible spongiform encephalopathies affecting humans as well as animals. A recombinant ovine prion protein was found to be in vitro able to act as an effective substrate for a microbial isoform of transglutaminase, an enzyme catalyzing the formation of isopeptide bonds inside the proteins. We proved that transglutaminase modifies the structure of the prion protein by leading to the formation of three intra-molecular crosslinks and that the crosslinked protein form is more competent in amyloid formation compared to the unmodified one. In addition, the crosslinked prion protein was shown also to be more resistant to proteinase K digestion. Our findings suggest a possible use of transglutaminase in stabilizing the prion protein three-dimensional structure in order to investigate the molecular basis of the conversion of the protein into its pathological form.► Microbial transglutaminase as enzymatic tool to modify the ovine prion protein (PrP). ► TG-modified PrP is stabilized by 3 intra-molecular crosslinks. ► TG-modified PrP form is more prone in amyloid formation. ► TG-modified PrP is more resistant to proteinase K digestion.
Keywords: Transglutaminase; Intra‐molecular crosslink; Prion protein; Amyloid fibril; Proteinase K; Polyamine;

Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia by Thomas J. McCorvie; Ying Liu; Andrew Frazer; Tyler J. Gleason; Judith L. Fridovich-Keil; David J. Timson (1516-1526).
Deficiency of UDP-galactose 4′-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD+. p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD+. These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.Display Omitted► The K161N variant of human GALE is more resistant to denaturation ► K161N has very low detectable NAD+ and greatly reduced enzymatic activity ► The D175N variant is less stable and slightly less active than the wild type ► GALE's activities with UDP-Gal and UDP-GalNAc are not necessarily correlated
Keywords: Type III galactosemia; Yeast model; GALE; Disease-associated mutation; UDP-galactose 4′-epimerase; Differential scanning fluorimetry;

Catalase, a target of glycation damage in rat liver mitochondria with aging by Hilaire Bakala; Maud Hamelin; Jean Mary; Caroline Borot-Laloi; Bertrand Friguet (1527-1534).
Aging is characterized by progressive decline of major cell functions, associated with accumulation of altered macromolecules, particularly proteins. This deterioration parallels age-related dysfunction of mitochondria, thought to be a major determinant of this decline in cell function, since these organelles are both the main sources of reactive oxygen species and targets for their damaging effects. To investigate the link between glycation damages that accumulate with aging and the status of mitochondrial antioxidant enzymes, we identified, by mass spectrometry after two dimensional-gel electrophoresis and western blotting, advanced glycation endproduct-modified matrix proteins in rat liver mitochondria. Catalase appeared to be the only antioxidant enzyme markedly glycated in old rats. Immunogold labeling performed on isolated mitochondria confirmed the mitochondrial matrix location of this enzyme. The content of catalase protein in mitochondrial extract increased with aging whereas the catalase activity was not significantly modified, in spite of a significant increase rate of glycation. Treatment of catalase with the glycating agent fructose led to significant time-dependent inactivation of the enzyme, while methylglyoxal had no noticeable effect. Catalase was co-identified with unglycated glutathione peroxidase-1 in the mitochondrial extracts. Taken together, these results indicate that both anti-oxidant enzymes catalase and glutathione peroxidase-1 housed in liver mitochondria, exhibited a differential sensitivity to glycation; moreover, they lend support to the hypothesis that glycation damages targeting catalase with aging may severely affect its activity, suggesting a link between glycation stress and the age-related decline in antioxidant defense in the mitochondria.► AGE-modified catalase was identified during aging in rat liver mitochondria. ► A mild decrease in catalase activity was measured. ► Glycation of catalase with fructose led to its inactivation. ► Catalase and GPX-1 colocalization suggests a synergy of both facing age-related oxidative stress.
Keywords: Aging; Glycation; Catalase; Mitochondrion; Liver; Antioxidant enzyme;

Presence of mutation m.14484T>C in a Chinese family with maternally inherited essential hypertension but no expression of LHON by Hao Guo; Xin-Ying Zhuang; A-Mei Zhang; Wen Zhang; Yong Yuan; Li Guo; Dandan Yu; Juan Liu; Da-Kuan Yang; Yong-Gang Yao (1535-1543).
► Presence of m.14484T>C in a Chinese family with essential hypertension but no LHON. ► The arterial stiffness of members carrying m.14484T>C was significantly increased. ► m.14484T>C caused mitochondrial dysfunction in cells from affected members. ► Mitochondrial DNA mutation is involved in essential hypertension. ► Patients with primary LHON mutation should undergo evaluation of blood pressure.
Keywords: Essential hypertension; mtDNA; m.14484T>C; Chinese; Arterial stiffness; LHON;

4-Hydroxy-2-oxoglutarate aldolase inactivity in primary hyperoxaluria type 3 and glyoxylate reductase inhibition by Travis J. Riedel; John Knight; Michael S. Murray; Dawn S. Milliner; Ross P. Holmes; W. Todd Lowther (1544-1552).
Mutations in the gene encoding for 4-hydroxy-2-oxoglutarate aldolase (HOGA) are associated with an excessive production of oxalate in Primary Hyperoxaluria type 3 (PH3). This enzyme is the final step of the hydroxyproline degradation pathway within the mitochondria and catalyzes the cleavage of 4-hydroxy-2-oxoglutarate (HOG) to pyruvate and glyoxylate. No analyses have been performed to assess the consequences of the mutations identified, particularly for those variants that produce either full-length or nearly full-length proteins. In this study, the expression, stability, and activity of nine PH3 human HOGA variants were examined. Using recombinant protein produced in Escherichia coli as well as transfected Chinese hamster ovary (CHO) cells, it was found that all nine PH3 variants are quite unstable, have a tendency to aggregate, and retain no measurable activity. A buildup of HOG was confirmed in the urine, sera and liver samples from PH3 patients. To determine how HOG is cleaved in the absence of HOGA activity, the ability of N-acetylneuraminate aldolase (NAL) to cleave HOG was evaluated. NAL showed minimal activity towards HOG. Whether the expected buildup of HOG in mitochondria could inhibit glyoxylate reductase (GR), the enzyme mutated in PH2, was also evaluated. GR was inhibited by HOG but not by 2-hydroxyglutarate or 2-oxoglutarate. Thus, one hypothetical component of the molecular basis for the excessive oxalate production in PH3 appears to be the inhibition of GR by HOG, resulting in a phenotype similar to PH2.► The PH3 HOGA mutations lead to unstable and inactive proteins. ► N-acetylneuraminate aldolase does not contribute to HOG cleavage. ► Glyoxylate reductase is inhibited by HOG. ► Glyoxylate reductase inhibition is one component of the molecular basis for PH3.
Keywords: Hydroxyproline; Oxalate; Calcium oxalate stone disease;

Background: In our earlier work, a reduction of cholesterol content increased the extracellular glutamate level in rat brain nerve terminals (synaptosomes) that was a result of the lack of transporter-mediated glutamate uptake. The aim of this study was to assess transporter-mediated release of glutamate from cholesterol-deficient synaptosomes. In stroke, cerebral hypoxia/ischemia, and traumatic brain injury, the development of neurotoxicity is provoked by enhanced extracellular glutamate, which is released from nerve cells mainly by glutamate transporter reversal — a distinctive feature of these pathological states. Methods: Laser scanning confocal microscopy, spectrofluorimetry, radiolabeled assay, and glutamate dehydrogenase assay. Results: Cholesterol acceptor methyl-β-cyclodextrin (15 mM) reduced the cholesterol content in the synaptosomes by one quarter. Transporter-mediated glutamate release from synaptosomes: 1) stimulated by depolarization of the plasma membrane; 2) by means of heteroexchange with competitive transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate; 3) in low [Na+] medium; and 4) during dissipation of the proton gradient of synaptic vesicles by the protonophore cyanide‐p‐trifluoromethoxyphenyl‐hydrazon (FCCP); was decreased under conditions of cholesterol deficiency by ~ 24, 28, 40, and 17%, respectively. Conclusions: A decrease in the level of membrane cholesterol attenuated transporter-mediated glutamate release from nerve terminals. Therefore, lowering cholesterol may be used in neuroprotection in stroke, ischemia, and traumatic brain injury which are associated with an increase in glutamate uptake reversal. This data may explain the neuroprotective effects of statins in these pathological states and provide one of the mechanisms of their neuroprotective action. However, beside these disorders, lowering cholesterol may cause harmful consequences by decreasing glutamate uptake in nerve terminals.►Lowering cholesterol reduces transporter-mediated glutamate release from nerve terminals. ►Lowering cholesterol attenuates glutamate release by heteroexchange and in low N+ medium. ►Lowering cholesterol decreases glutamate release during dissipation of the proton gradient of synaptic vesicles. ►Lowering cholesterol may be used for neuroprotection after stroke, cerebral hypoxia/ischemia. ►A decrease in transporter-mediated glutamate release may be mechanism of neuroprotective effect of statins.
Keywords: Neuroprotection; Cholesterol; Methyl-β-cyclodextrin; Ambient glutamate; Transporter-mediated glutamate release; Synaptosomes;

Functional effects of congenital myopathy-related mutations in gamma-tropomyosin gene by Katarzyna Robaszkiewicz; Elżbieta Dudek; Andrzej A. Kasprzak; Joanna Moraczewska (1562-1569).
Missense mutations in human TPM3 gene encoding γ-tropomyosin expressed in slow muscle type 1 fibers, were associated with three types of congenital myopathies—nemaline myopathy, cap disease and congenital fiber type disproportion. Functional effects of the following substitutions: Leu100Met, Ala156Thr, Arg168His, Arg168Cys, Arg168Gly, Lys169Glu, and Arg245Gly, were examined in biochemical assays using recombinant tropomyosin mutants and native proteins isolated from skeletal muscle. Most, but not all, mutations decreased the affinity of tropomyosin for actin alone and in complex with troponin (± Ca2 +). All studied tropomyosin mutants reduced Ca-induced activation but had no effect on the inhibition of actomyosin cross-bridges. Ca2 +-sensitivity of the actomyosin interactions, as well as cooperativity of myosin-induced activation of the thin filament was affected by individual tropomyosin mutants with various degrees. Decreased motility of the reconstructed thin filaments was a result of combined functional defects caused by myopathy-related tropomyosin mutants. We conclude that muscle weakness and structural abnormalities observed in TPM3-related congenital myopathies result from reduced capability of the thin filament to fully activate actin–myosin cross-bridges.► Molecular mechanism of tropomyosin-related congenital myopathies is proposed. ► Tropomyosin mutants affect assembly and regulation of actin thin filaments. ► A decrease of Ca-dependent activation of actomyosin is common in all myopathies. ► Ca- and myosin-induced activation is required for normal actin motility. ► Mutations located in the central tropomyosin region have the most severe effects.
Keywords: Congenital myopathy; Nemaline myopathy; Cap disease; Fiber type disproportion; Thin filament; γ-tropomyosin;

Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations by Virginie Agier; Patricia Oliviero; Jeanne Lainé; Caroline L'Hermitte-Stead; Samantha Girard; Sandrine Fillaut; Claude Jardel; Frédéric Bouillaud; Anne Laure Bulteau; Anne Lombès (1570-1580).
Deleterious consequences of heterozygous OPA1 mutations responsible for autosomal dominant optic atrophy remain a matter of debate. Primary skin fibroblasts derived from patients have shown diverse mitochondrial alterations that were however difficult to resolve in a unifying scheme. To address the potential use of these cells as disease model, we undertook parallel and quantitative analyses of the diverse reported alterations in four fibroblast lines harboring different OPA1 mutations, nonsense or missense, in the guanosine triphosphatase or the C-terminal coiled-coil domains. We tackled several factors potentially underlying discordant reports and showed that fibroblasts with heterozygous OPA1 mutations present with several mitochondrial alterations. These included defective mitochondrial fusion during pharmacological challenge with the protonophore carbonyl cyanide m‐chlorophenyl hydrazone, significant mitochondrial elongation with decreased OPA1 and DRP1 proteins, and abnormal mitochondrial fragmentation during glycolysis shortage or exogenous oxidative stress. Respiratory complex IV activity and subunits steady-state were decreased without alteration of the mitochondrial deoxyribonucleic acid size, amount or transcription. Physical link between OPA1 protein and oxidative phosphorylation was shown by reciprocal immunoprecipitation. Altered cristae structure coexisted with normal response to pro-apoptotic stimuli and expression of Bax or Bcl2 proteins. Skin fibroblasts with heterozygous OPA1 mutations thus share significant mitochondrial remodeling, and may therefore be useful for analyzing disease pathophysiology. Identifying whether the observed alterations are also present in ganglion retinal cells, and which of them underlies their degeneration process remains however an essential goal for therapeutic strategy.► Heterozygous OPA1 mutations are associated with defective mitochondrial fusion. ► Mitochondrial morphology may be elongated despite defective mitochondrial fusion. ► Mutant OPA1 cells have altered mitochondrial dynamic response to nutritional stress. ► Mitochondrial activities are significantly altered in mutant OPA1 cells. ► Physical link between OPA1 and OXPHOS proteins is widespread.
Keywords: Mitochondrial compartment; Mitochondrial fusion; Oxidative phosphorylation; Energy metabolism; Mitochondrial disease;

Clostridium perfringens alpha-toxin induces the release of IL-8 through a dual pathway via TrkA in A549 cells by Masataka Oda; Ryota Shiihara; Yuka Ohmae; Michiko Kabura; Teruhisa Takagishi; Keiko Kobayashi; Masahiro Nagahama; Masahisa Inoue; Tomomi Abe; Koujun Setsu; Jun Sakurai (1581-1589).
A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.Display Omitted ► Clostridium perfringens alpha-toxin induces the release of IL-8 from A549 cells. ► The toxin activates ERK1/2-NF−κB and p38 MAPK cascades via TrkA. ► The ERK1/2-NF−kB cascade participates in expression of IL-8 mRNA. ► The p38 cascade plays an important role in stabilization of IL-8 mRNA. ► TrkA is a therapeutic drug target of inflammation with alpha-toxin.
Keywords: Alpha-toxin; A549 cell; TrkA; IL-8; ERK1/2; p38 MAPK;

Overexpression of HGF transgene attenuates renal inflammatory mediators, Na+-ATPase activity and hypertension in spontaneously hypertensive rats by Freddy Romero-Vásquez; Maribel Chávez; Mariela Pérez; José L. Arcaya; Alberto J. García; Jaimar Rincón; Bernardo Rodríguez-Iturbe (1590-1599).
Renal inflammation and oxidative stress are constantly present in experimental hypertension. Since the spontaneously hypertensive rat (SHR) has reduced levels of hepatocyte growth factor (HGF), which suppresses the activation of the proinflammatory nuclear transcription factor kappa B (NF-κB), we speculated that HGF deficiency could play a key role in the pathogenesis of hypertension in the SHR. To test this hypothesis we increased HGF in the SHR by HGF gene delivery. We found that kidneys of 15-week-old SHR had an important reduction in HGF mRNA and protein expression. Adult SHRs were randomly assigned to receive weekly hydrodynamic injection (1 mg/kg) of a naked plasmid containing human HGF (hHGF) gene associated with a cytomegalovirus promoter (pCMV-HGF) or empty vector (pcDNA3.1) during 6 weeks. WKY rats treated with pcDNA3.1 and pCMV-HGF served as controls. The kidneys in the hypertensive SHR untreated and treated with the empty vector had increased NF-κB activation, elevated mRNA and protein expression of RANTES, MCP-1 and IL-6 and increased oxidative stress. Activity of Na+-ATPase was increased while activity of Na+, K+-ATPase was normal. hHGF gene therapy normalized renal NF-κB activity, proinflammatory cytokines, antioxidant status (GSH, SOD and CAT), Na+-ATPase activity, reduced renal injury and ameliorated hypertension. Our results suggest that reduction in HGF production plays a major role in the pathogenesis of hypertension in the SHR and increasing HGF is a potential therapeutic target in the treatment of hypertension.►HGF gene therapy reduces hypertension. ►hHGF gene therapy normalized renal NF-κB activity and proinflammatory cytokines. ►hHGF gene therapy normalized renal antioxidant status and Na+-ATPase activity. ►Kidney of SHR had an important reduction in HGF mRNA and protein expression.
Keywords: Hypertension; Gene therapy; Renal inflammation; Oxidative stress; NF-κB; Na+-ATPase;

Alteration of colonic stem cell gene signatures during the regenerative response to injury by Laurie A. Davidson; Jennifer S. Goldsby; Evelyn S. Callaway; Manasvi S. Shah; Nick Barker; Robert S. Chapkin (1600-1607).
Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n − 3 polyunsaturated fatty acids (n − 3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creERT2 mice were fed diets enriched with n − 3 PUFA vs n − 6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5 days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5+ stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5+ stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n − 3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5+ stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.► Colon stem cell kinetics is altered in the colitis/wounding model. ► Lgr5 + stem cells in the colon are highly susceptible to DSS-induced damage. ► Diet modulates stem cell associated genes, Ascl2, Axin2 and Eph3.
Keywords: Lgr5; Dextran sodium sulfate; Fish oil; Wnt signaling; Colon;

The anti-atherogenic cytokine, TGF-β, plays a key role during macrophage foam cell formation by modulating the expression of key genes involved in the control of cholesterol homeostasis. Unfortunately, the molecular mechanisms underlying these actions of TGF-β remain poorly understood. In this study we examine the effect of TGF-β on macrophage cholesterol homeostasis and delineate the role of Smads-2 and ‐3 during this process. Western blot analysis showed that TGF-β induces a rapid phosphorylation-dependent activation of Smad-2 and ‐3 in THP-1 and primary human monocyte-derived macrophages. Small interfering RNA-mediated knockdown of Smad-2/3 expression showed that the TGF-β-mediated regulation of key genes implicated in the uptake of modified low density lipoproteins and the efflux of cholesterol from foam cells was Smad-dependent. Additionally, through the use of virally delivered Smad-2 and/or Smad-3 short hairpin RNA, we demonstrate that TGF-β inhibits the uptake of modified LDL by macrophages through a Smad-dependent mechanism and that the TGF-β-mediated regulation of CD36, lipoprotein lipase and scavenger receptor-A gene expression was dependent on Smad-2. These studies reveal a crucial role for Smad signaling, particularly Smad-2, in the inhibition of foam cell formation by TGF-β through the regulation of expression of key genes involved in the control of macrophage cholesterol homeostasis.► Anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation. ► The role of Smads in the control of macrophage cholesterol homeostasis was studied. ► Smads were found to play a key role in the TGF-β-mediated uptake of modified LDL. ► A dominant role of Smad2 was identified in the regulation of gene expression. ► The TGF-β-Smad axis may represent a powerful anti-foam cell therapeutic target.
Keywords: Foam cell; Atherosclerosis; Cholesterol; TGF-β; Macrophage;

Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7 by Pieter Steenhuis; Joshua Froemming; Thomas Reinheckel; Stephan Storch (1617-1628).
CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.► The lysosomal membrane glycoprotein CLN7 is proteolytically cleaved in lysosomes. ► Proteolytic cleavage can be mediated by cathepsin L and occurs in luminal loop L9. ► CLN7 cleavage occurs gradually and cleavage products are relatively stable. ► Disease-associated mutations in CLN7 result in increased proteolytic cleavage.
Keywords: CLN7/MFSD8; Cathepsin L; Proteolytic cleavage; Lysosomal storage disorder; Neurodegeneration;

scyllo-Inositol (SI) is an endogenous inositol stereoisomer known to inhibit aggregation and fibril formation of the amyloid-beta peptide (Aβ). Human clinical trials using SI to treat Alzheimer disease (AD) patients have shown potential benefits. In light of the growing therapeutic potential of SI, the objective of our study was to gain a more thorough understanding of the mechanism of action. In addition to Aβ plaques, a prominent pathological feature of AD is the extensive accumulation of autophagic vacuoles (AVs) suggesting dysfunction in this degradation pathway. Using the TgCRND8 mouse model for AD, we examined SI treatment effects on various components of the autophagic pathway. Autophagy impairment in TgCRND8 mice occurs in the latter stages of the pathway where AV-lysosome fusion and lysosomal degradation take place. SI treatment attenuated this impairment with a decrease in the size and the number of accumulated AVs. We propose that the beneficial effects of SI–Aβ interactions may resolve autophagic deficiencies in the AD brains.► scyllo-Inositol (SI) rescues autophagic vacuole accumulation in TgCRND8 mice. ► SI rescues abnormalities in the latter stages of autophagy in TgCRND8 mice. ► Autophagy rescue may be a mechanism of action for SI–amyloid-β interaction.
Keywords: scyllo-Inositol; Alzheimer disease; Amyloid-beta peptide; TgCRND8; Autophagy; Lysosome;

DHEA-S inhibits human neutrophil and human airway smooth muscle migration by Cynthia J. Koziol-White; Elena A. Goncharova; Gaoyuan Cao; Martin Johnson; Vera P. Krymskaya; Reynold A. Panettieri (1638-1642).
Airway diseases such as asthma, emphysema, and chronic bronchitis are, in part, characterized by reversible airflow obstruction and inflammation. In severe disease, marked decreases in lung function are associated with airway smooth muscle proliferation and airway neutrophilia. Inhaled glucocorticoids attenuate increased airflow obstruction and airway inflammation that occur, in part, due to increased smooth muscle migration and proliferation, as well as the airway neutrophilia. Glucocorticoids, however, have adverse side effects and, in some patients, are ineffective despite high doses. Recent research has explored the effects of non-traditional steroids on attenuation of inflammation associated with airway diseases. These non-traditional steroids have improved side effect profiles in comparison to glucocorticoid therapy. Our studies assessed effects of dehydroepiandrosterone-3-sulfate (DHEA-S) on migration of both human peripheral blood neutrophils (PMN) and human airway smooth muscle cells (HASM). DHEA-S dose-dependently inhibited chemotaxis of PMN and HASM while having no effect on the phosphorylation levels of Akt, ERK1/2, p38 MAPK or PKC, canonical positive regulators of cell migration. These studies demonstrate direct effects of DHEA-S on cell migration, thereby suggesting that DHEA-S may attenuate airway inflammation and cell migration.► DHEA-S attenuates migration of airway smooth muscle and neutrophils. ► DHEA-S has little effect on PDGF-induced Akt phosphorylation. ► Non-traditional steroids may offer novel therapy for airway disease.
Keywords: Airway inflammation; DHEA-S; Airway remodeling;