Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Prominent tauopathy and intracellular ß-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. METHODS: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. RESULTS: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. CONCLUSIONS: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aß accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Prominent tauopathy and intracellular ß-amyloid accumulation triggered by genetic deletion of cathepsin D: Implications for Alzheimer disease pathogenesis.

Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. Methods: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model characterized by pronounced lysosomal dysfunction (Krabbe A). Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. Results: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatDKO mice were found to develop prominent tauopathy by just ~ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology in aged JNPL3 mice. CatDKO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (~ 1250%) are present in CatD-KO mice, but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. Conclusions: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, CatD-KO mice are the only model to develop detectable Aß acumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

New insights into the function and pathophysiology of the ectodomain sheddase A Disintegrin And Metalloproteinase 10 (ADAM10).

The 'A Disintegrin And Metalloproteinase 10' (ADAM10) has gained considerable attention due to its discovery as an 'α-secretase' involved in the nonamyloidogenic processing of the amyloid precursor protein, thereby possibly preventing the excessive generation of the amyloid beta peptide, which is associated with the pathogenesis of Alzheimer's disease. ADAM10 was found to exert many additional functions, cleaving about 100 different membrane proteins. ADAM10 is involved in many pathophysiological conditions, ranging from cancer and autoimmune disorders to neurodegeneration and inflammation. ADAM10 cleaves its substrates close to the plasma membrane, a process referred to as ectodomain shedding. This is a central step in the modulation of the functions of cell adhesion proteins and cell surface receptors. ADAM10 activity is controlled by transcriptional and post-translational events. The interaction of ADAM10 with tetraspanins and the way they functionally and structurally depend on each other is another topic of interest. In this review, we will summarize findings on how ADAM10 is regulated and what is known about the biology of the protease. We will focus on novel aspects of the molecular biology and pathophysiology of ADAM10 that were previously poorly covered, such as the role of ADAM10 on extracellular vesicles, its contribution to virus entry, and its involvement in cardiac disease, cancer, inflammation, and immune regulation. ADAM10 has emerged as a regulator controlling cell surface proteins during development and in adult life. Its involvement in disease states suggests that ADAM10 may be exploited as a therapeutic target to treat conditions associated with a dysfunctional proteolytic activity.

Rab GTPase regulation of phagosome-lysosome fusion is bypassed in the presence of micromolar Ca2.

Several ATP- and cytosol-dependent fusion processes between membranes of the endocytic and exocytic pathways have been biochemically reconstituted. Here, we present a phagosome-lysosome fusion reaction that is driven by micromolar concentrations of Ca2+ in the absence of ATP and cytosol. Investigating classical fusion and Ca2+-driven fusion (CaFu) side-by-side in vitro, using the same membrane preparations, we show that CaFu is faster than standard fusion (StaFu), leads to larger fusion products and is not blocked by established inhibitors of StaFu. A Ca2+ concentration of ∼120 µM supports maximal membrane attachment, and 15 µM Ca2+ supports maximal membrane fusion, indicating that Ca2+ has both a membrane-binding activity and a fusion-promoting activity. StaFu and CaFu are inhibited by a mutant form of α-SNAP (NAPA) that does not support soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) activation, and both are inhibited by a mixture of the cytosolic domains of three cognate Q-SNARE proteins, demonstrating a role of SNAREs in Ca2+-driven membrane merger. CaFu is independent of the Ca2+-regulated proteins synaptotagmin-7, calmodulin, and annexins A2 and A7. We propose that CaFu corresponds to the last step of phagosome-lysosome fusion, when a raised Ca2+ concentration from the compartment lumen activates SNAREs for fusion.

Lysosome-targeted multifunctional lipid probes reveal the sterol transporter NPC1 as a sphingosine interactor.

Lysosomes are catabolic organelles involved in macromolecular digestion, and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases, many of which have lipid accumulation phenotypes. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions, and their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photocrosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and to a lesser extent LIMP-2/SCARB2, bind to sphingosine and showed that their absence leads to lysosomal sphingosine accumulation which hints at a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

Mechanisms of amyloid-ß34 generation indicate a pivotal role for BACE1 in amyloid homeostasis.

The beta­site amyloid precursor protein (APP) cleaving enzyme (BACE1) was discovered due to its "amyloidogenic" activity which contributes to the production of amyloid-beta (Aß) peptides. However, BACE1 also possesses an "amyloidolytic" activity, whereby it degrades longer Aß peptides into a non­toxic Aß34 intermediate. Here, we examine conditions that shift the equilibrium between BACE1 amyloidogenic and amyloidolytic activities by altering BACE1/APP ratios. In Alzheimer disease brain tissue, we found an association between elevated levels of BACE1 and Aß34. In mice, the deletion of one BACE1 gene copy reduced BACE1 amyloidolytic activity by ~ 50%. In cells, a stepwise increase of BACE1 but not APP expression promoted amyloidolytic cleavage resulting in dose-dependently increased Aß34 levels. At the cellular level, a mislocalization of surplus BACE1 caused a reduction in Aß34 levels. To align the role of γ-secretase in this pathway, we silenced Presenilin (PS) expression and identified PS2-γ-secretase as the main γ-secretase that generates Aß40 and Aß42 peptides serving as substrates for BACE1's amyloidolytic cleavage to generate Aß34.

Consecutive functions of small GTPases guide HOPS-mediated tethering of late endosomes and lysosomes.

The transfer of endocytosed cargoes to lysosomes (LYSs) requires HOPS, a multiprotein complex that tethers late endosomes (LEs) to LYSs before fusion. Many proteins interact with HOPS on LEs/LYSs. However, it is not clear whether these HOPS interactors localize to LEs or LYSs or how they participate in tethering. Here, we biochemically characterized endosomes purified from untreated or experimentally manipulated cells to put HOPS and interacting proteins in order and to establish their functional interdependence. Our results assign Rab2a and Rab7 to LEs and Arl8 and BORC to LYSs and show that HOPS drives LE-LYS fusion by bridging late endosomal Rab2a with lysosomal BORC-anchored Arl8. We further show that Rab7 is absent from sites of HOPS-dependent tethering but promotes fusion by moving LEs toward LYSs via dynein. Thus, our study identifies the topology of the machinery for LE-LYS tethering and elucidates the role of different small GTPases in the process.

LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

Targeting cardiomyocyte ADAM10 ectodomain shedding promotes survival early after myocardial infarction.

After myocardial infarction the innate immune response is pivotal in clearing of tissue debris as well as scar formation, but exaggerated cytokine and chemokine secretion with subsequent leukocyte infiltration also leads to further tissue damage. Here, we address the value of targeting a previously unknown a disintegrin and metalloprotease 10 (ADAM10)/CX3CL1 axis in the regulation of neutrophil recruitment early after MI. We show that myocardial ADAM10 is distinctly upregulated in myocardial biopsies from patients with ischemia-driven cardiomyopathy. Intriguingly, upon MI in mice, pharmacological ADAM10 inhibition as well as genetic cardiomycyte-specific ADAM10 deletion improves survival with markedly enhanced heart function and reduced scar size. Mechanistically, abolished ADAM10-mediated CX3CL1 ectodomain shedding leads to diminished IL-1ß-dependent inflammation, reduced neutrophil bone marrow egress as well as myocardial tissue infiltration. Thus, our data shows a conceptual insight into how acute MI induces chemotactic signaling via ectodomain shedding in cardiomyocytes.

Cathepsin D: Analysis of its potential role as an amyloid beta degrading protease.

Proteolysis catalyzed by the major lysosomal aspartyl protease cathepsin-D (CTSD) appears to be of pivotal importance for proteostasis within the central nervous system and in neurodegeneration. Neuronal Ceroid Lipofuscinosis (NCL) type 10 is caused by a lack of CTSD leading to a defective autophagic flow and pathological accumulation of proteins. We previously demonstrated a therapeutic-relevant clearance of protein aggregates after dosing a NCL10 mouse model with recombinant human pro-cathepsin-D (proCTSD). Similar results could be achieved in cells and mice accumulating α-synuclein. Prompted by these positive effects and our in vitro findings showing that cathepsin-D can cleave the Alzheimer's Disease (AD)-causing amyloid beta peptides (Aß), we envisaged that such a treatment with proCTSD could similarly be effective in clearance of potentially toxic Aß species. We demonstrated that CTSD is able to cleave human Aß1-42 by using liquid chromatography-mass spectrometry. Intracerebral dosing of proCTSD in a NCL10 (CTSD knockout) mouse model revealed uptake and processing of CTSD to its mature and active form. However, the re-addition of CTSD did not obviously affect intracellular APP processing or the generation of soluble APP and Aß-species. ProCTSD treated HEK cells in comparison with untreated cells were found to contain comparable levels of soluble and membrane bound APP and Aß-species. Also, the early intracranial application (P1 and P20) of proCTSD in the 5xFAD mouse model did not change Aß pathology, plaque number and plaque composition and neuroinflammation, however we observed an increased level of Aß1-42 in the CSF. Our data confirm proteolytic cleavage of human Aß1-42 by CTSD but exclude a prominent role of CTSD in APP processing and Aß degradation in our in vitro and in vivo models.

Adam10-dependent Notch signaling establishes dental epithelial cell boundaries required for enamel formation.

The disintegrin and metalloproteinase Adam10 is a membrane-bound sheddase that regulates Notch signaling and ensures epidermal integrity. To address the function of Adam10 in the continuously growing incisors, we used Keratin14 Cre/+;Adam10 fl/fl transgenic mice, in which Adam10 is conditionally deleted in the dental epithelium. Keratin14 Cre/+;Adam10 fl/fl mice exhibited severe abnormalities, including defective enamel formation reminiscent of human enamel pathologies. Histological analyses of mutant incisors revealed absence of stratum intermedium, and severe disorganization of enamel-secreting ameloblasts. In situ hybridization and immunostaining analyses in the Keratin14 Cre/+;Adam10 fl/fl incisors showed strong Notch1 downregulation in dental epithelium and ectopic distribution of enamel-specific molecules, including ameloblastin and amelogenin. Lineage tracing studies using Notch1 CreERT2 ;R26 mT/mG mice demonstrated that loss of the stratum intermedium cells was due to their fate switch toward the ameloblast lineage. Overall, our data reveal that in the continuously growing incisors the Adam10/Notch axis controls dental epithelial cell boundaries, cell fate switch and proper enamel formation.

Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals.

Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O-glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

Phagosomal signalling of the C-type lectin receptor Dectin-1 is terminated by intramembrane proteolysis.

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal ß-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses.

Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models.

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/ß-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus.

Intravitreal gene therapy restores the autophagy-lysosomal pathway and attenuates retinal degeneration in cathepsin D-deficient mice.

Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

S-palmitoylation determines TMEM55B-dependent positioning of lysosomes.

The spatiotemporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo. TMEM55B (also known as PIP4P1), as an integral lysosomal membrane protein, is a component of such a complex that mediates the retrograde transport of lysosomes by establishing interactions with the cytosolic scaffold protein JIP4 (also known as SPAG9) and dynein-dynactin. Here, we show that TMEM55B and its paralog TMEM55A (PIP4P2) are S-palmitoylated proteins that are lipidated at multiple cysteine residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes retention of the mutated protein in the Golgi. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor protein-dependent sorting of TMEM55B. Our data suggest a critical role for S-palmitoylation in the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

Inhibition of ADAM17 impairs endothelial cell necroptosis and blocks metastasis.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.