Short-term regulation of TSFM level does not alter amyloidogenesis and mitochondrial function in type-specific cells.

BACKGROUND: Mitochondrial Ts translation elongation factor (TSFM) is an enzyme that catalyzes exchange of guanine nucleotides. By forming a complex with mitochondrial Tu translation elongation factor (TUFM), TSFM participates in mitochondrial protein translation. We have previously reported that TUFM regulates translation of beta-site APP cleaving enzyme 1 (BACE1) via ROS (reactive oxygen species)-dependent mechanism, suggesting a potential role in amyloid precursor protein (APP) processing associated with Alzheimer's disease (AD), which led to the speculation that TSFM may regulate APP processing in a similar way to TUFM. METHODS AND RESULTS: Here, we report that in cultured cells, knockdown or overexpression TSFM did not change protein levels in BACE1 and APP. Besides, the levels of cytoplasmic ROS and mitochondrial superoxide, in addition to ATP level, cell viability and mitochondrial membrane potential were not significantly altered by TSFM knockdown in the short term. Further transcriptome analysis revealed that expression of majority of mitochondrial genes were not remarkably changed by TSFM silencing. The possibility of TSFM involved in cardiomyopathy and cancer development was uncovered using bioinformatics analysis. CONCLUSIONS: Collectively, short-term regulation of TSFM level in cultured cells does not cause a significant change in proteins involved in APP processing, levels in ROS and ATP associated with mitochondrial function. Whereas our study could contribute to comprehend certain clinical features of TSFM mutations, the roles of TSFM in cardiomyopathy and cancer development might deserve further investigation.

Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.

Identification of PS1/gamma-secretase and glutamate transporter GLT-1 interaction sites.

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.

Chicoric Acid Ameliorated Beta-Amyloid Pathology and Enhanced Expression of Synaptic-Function-Related Markers via L1CAM in Alzheimer's Disease Models.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease. The accumulation of amyloid-beta (Aß) plaques is a distinctive pathological feature of AD patients. The aims of this study were to evaluate the therapeutic effect of chicoric acid (CA) on AD models and to explore its underlying mechanisms. APPswe/Ind SH-SY5Y cells and 5xFAD mice were treated with CA. Soluble Aß1-42 and Aß plaque levels were analyzed by ELISA and immunohistochemistry, respectively. Transcriptome sequencing was used to compare the changes in hippocampal gene expression profiles among the 5xFAD mouse groups. The specific gene expression levels were quantified by qRT-PCR and Western blot analysis. It was found that CA treatment reduced the Aß1-42 levels in the APPswe/Ind cells and 5xFAD mice. It also reduced the Aß plaque levels as well as the APP and BACE1 levels. Transcriptome analysis showed that CA affected the synaptic-plasticity-related genes in the 5xFAD mice. The levels of L1CAM, PSD-95 and synaptophysin were increased in the APPswe/Ind SH-SY5Y cells and 5xFAD mice treated with CA, which could be inhibited by administering siRNA-L1CAM to the CA-treated APPswe/Ind SH-SY5Y cells. In summary, CA reduced Aß levels and increased the expression levels of synaptic-function-related markers via L1CAM in AD models.

New precision medicine avenues to the prevention of Alzheimer's disease from insights into the structure and function of γ-secretases.

Two phase-III clinical trials with anti-amyloid peptide antibodies have met their primary goal, i.e. slowing of Alzheimer's disease (AD) progression. However, antibody therapy may not be the optimal therapeutic modality for AD prevention, as we will discuss in the context of the earlier small molecules described as "γ-secretase modulators" (GSM). We review here the structure, function, and pathobiology of γ-secretases, with a focus on how mutations in presenilin genes result in early-onset AD. Significant progress has been made in generating compounds that act in a manner opposite to pathogenic presenilin mutations: they stabilize the proteinase-substrate complex, thereby increasing the processivity of substrate cleavage and altering the size spectrum of Aß peptides produced. We propose the term "γ-secretase allosteric stabilizers" (GSAS) to distinguish these compounds from the rather heterogenous class of GSM. The GSAS represent, in theory, a precision medicine approach to the prevention of amyloid deposition, as they specifically target a discrete aspect in a complex cell biological signalling mechanism that initiates the pathological processes leading to Alzheimer's disease.

Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.

The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.

In silico and in vitro analyses of a novel FoxO1 agonist reducing Aß levels via downregulation of BACE1.

AIMS: FoxO1 is an important target in the treatment of Alzheimer's disease (AD). However, FoxO1-specific agonists and their effects on AD have not yet been reported. This study aimed to identify small molecules that upregulate the activity of FoxO1 to attenuate the symptoms of AD. METHODS: FoxO1 agonists were identified by in silico screening and molecular dynamics simulation. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were used to assess protein and gene expression levels of P21, BIM, and PPARγ downstream of FoxO1 in SH-SY5Y cells, respectively. Western blotting and enzyme-linked immunoassays were performed to explore the effect of FoxO1 agonists on APP metabolism. RESULTS: N-(3-methylisothiazol-5-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl) acetamide (compound D) had the highest affinity for FoxO1. Compound D activated FoxO1 and regulated the expression of its downstream target genes, P21, BIM, and PPARγ. In SH-SY5Y cells treated with compound D, BACE1 expression levels were downregulated, and the levels of Aß1-40 and Aß1-42 were also reduced. CONCLUSIONS: We present a novel small-molecule FoxO1 agonist with good anti-AD effects. This study highlights a promising strategy for new drug discovery for AD.

Effects of DHA (omega-3 fatty acid) and estradiol on amyloid ß-peptide regulation in the brain.

In the early stages of sporadic Alzheimer's disease (SAD), there is a strong correlation between memory impairment and cortical levels of soluble amyloid-ß peptide oligomers (Aß). It has become clear that Aß disrupt glutamatergic synaptic function, which can in turn lead to the characteristic cognitive deficits of SAD, but the actual pathways are still not well understood. This opinion article describes the pathogenic mechanisms underlying cerebral amyloidosis. These mechanisms are dependent on the amyloid precursor protein and concern the synthesis of Aß peptides with competition between the non-amyloidogenic pathway and the amyloidogenic pathway (i.e. a competition between the ADAM10 and BACE1 enzymes), on the one hand, and the various processes of Aß residue clearance, on the other hand. This clearance mobilizes both endopeptidases (NEP, and IDE) and removal transporters across the blood-brain barrier (LRP1, ABCB1, and RAGE). Lipidated ApoE also plays a major role in all processes. The disturbance of these pathways induces an accumulation of Aß. The description of the mechanisms reveals two key molecules in particular: (i) free estradiol, which has genomic and non-genomic action, and (ii) free DHA as a preferential ligand of PPARα-RXRα and PPARÉ£-RXRα heterodimers. DHA and free estradiol are also self-regulating, and act in synergy. When a certain level of chronic DHA and free estradiol deficiency is reached, a permanent imbalance is established in the central nervous system. The consequences of these deficits are revealed in particular by the presence of Aß peptide deposits, as well as other markers of the etiology of SAD.

Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex.

The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.

Proteolytic cleavage of amyloid precursor protein by ADAM10 promotes proliferation and migration via activating MAPKs pathway in tongue squamous cell carcinoma in vitro.

APP, well-studied in the development of Alzheimer's disease, has been recently identified as the key gene correlated with TSCC. Here, we investigate the function of APP and its proteolytic cleavage by ADAM10 in the pathogenesis of TSCC. A total of 63 TSCC patients and 30 healthy controls were included and the results of IHC assay showed high expressions of ADAM10 and APP in TSCC tissues compared to paired para-carcinoma tissues. Interestingly, APP expression in TSCC patients was correlated with ADAM10 expression and their combined expression was related to the poor patients' survival. We found that APP was ɑ-cleaved in TSCC cells to form sAPPα, and the serum level of sAPPα but not sAPPß in TSCC patients was higher than healthy controls. Both overexpression with full-length APP and sAPPα promoted TSCC cell proliferation, migration and invasion. Downregulation of APP or ADAM10 by siRNA decreased the generation of sAPPα and inhibited the activity of ERK1/2 and p38 pathways, thereby reducing TSCC cell proliferation, migration and invasion. Treatment with ERK1/2 or p38 agonist or sAPPα overexpression reversed the effects of APP or ADAM10 knockdown. In conclusion, our data demonstrated the pathogenic roles of APP cleaved by ADAM10 to activate ERK1/2 and p38 pathways in TSCC cells. Both high expressions of ADAM10 and APP were related to poor prognosis. Targeting APP cleaved by ADAM10 might be a potential strategy in TSCC treatment.

The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity.

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

m6A modified BACE1-AS contributes to liver metastasis and stemness-like properties in colorectal cancer through TUFT1 dependent activation of Wnt signaling.

BACKGROUND: Liver metastasis is one of the most important reasons for high mortality of colorectal cancer (CRC). Growing evidence illustrates that lncRNAs play a critical role in CRC liver metastasis. Here we described a novel function and mechanisms of BACE1-AS promoting CRC liver metastasis. METHODS: qRT-PCR and in situ hybridization were performed to examine the BACE1-AS level in CRC. IGF2BP2 binding to m6A motifs in BACE1-AS was determined by RIP assay and S1m-tagged immunoprecipitation. Transwell assay and liver metastasis mice model experiments were performed to examine the metastasis capabilities of BACE1-AS knockout cells. Stemness-like properties was examined by tumor sphere assay and the expression of stemness biomarkers. Microarray data were acquired to analyze the signaling pathways involved in BACE1-AS promoting CRC metastasis. RESULTS: BACE1-AS is the most up-regulated in metastatic CRC associated with unfavorable prognosis. Sequence blast revealed two m6A motifs in BACE1-AS. IGF2BP2 binding to these two m6A motifs is required for BACE1-AS boost in metastatic CRC. m6A modified BACE1-AS drives CRC cells migration and invasion and liver metastasis both in vitro and in vivo. Moreover, BACE1-AS maintains the stemness-like properties of CRC cells. Mechanically, BACE1-AS promoted TUFT1 expression by ceRNA network through miR-214-3p. CRC patients with such ceRNA network suffer poorer prognosis than ceRNA-negative patients. Depletion of TUFT1 mimics BACE1-AS loss. BACE1-AS activated Wnt signaling pathway in a TUFT1 dependent manner. BACE1-AS/miR-214-3p/TUFT1/Wnt signaling regulatory axis is essential for CRC liver metastasis. Pharmacologic inhibition of Wnt signaling pathway repressed liver metastasis and stemness-like features in BACE1-AS over-expressed CRC cells. CONCLUSION: Our study demonstrated BACE1-AS as a novel target of IGF2BP2 through m6A modification. m6A modified BACE1-AS promotes CRC liver metastasis through TUFT1 dependent activation of Wnt signaling pathway. Thus, targeting BACE1-AS and its downstream Wnt signaling pathways may provide a new opportunity for metastatic CRC intervention and treatment.

Human Presenilin-1 delivered by AAV9 rescues impaired γ-secretase activity, memory deficits, and neurodegeneration in Psen mutant mice.

Mutations in the Presenilin (PSEN1 and PSEN2) genes are the major cause of early-onset familial Alzheimer's disease (FAD). Presenilin (PS) is the catalytic subunit of the γ-secretase complex, which cleaves type I transmembrane proteins, such as Notch and the amyloid precursor protein (APP), and plays an evolutionarily conserved role in the protection of neuronal survival during aging. FAD PSEN1 mutations exhibit impaired γ-secretase activity in cell culture, in vitro, and knockin (KI) mouse brains, and the L435F mutation is the most severe in reducing γ-secretase activity and is located closest to the active site of γ-secretase. Here, we report that introduction of the codon-optimized wild-type human PSEN1 cDNA by adeno-associated virus 9 (AAV9) results in broadly distributed, sustained, low to moderate levels of human PS1 (hPS1) expression and rescues impaired γ-secretase activity in the cerebral cortex of Psen mutant mice either lacking PS or expressing the Psen1 L435F KI allele, as evaluated by endogenous γ-secretase substrates of APP and recombinant γ-secretase products of Notch intracellular domain and Aß peptides. Furthermore, introduction of hPS1 by AAV9 alleviates impairments of synaptic plasticity and learning and memory in Psen mutant mice. Importantly, AAV9 delivery of hPS1 ameliorates neurodegeneration in the cerebral cortex of aged Psen mutant mice, as shown by the reversal of age-dependent loss of cortical neurons and elevated microgliosis and astrogliosis. These results together show that moderate hPS1 expression by AAV9 is sufficient to rescue impaired γ-secretase activity, synaptic and memory deficits, and neurodegeneration caused by Psen mutations in mouse models.

Presenilin-1, mutated in familial Alzheimer's disease, maintains genome stability via a γ-secretase dependent way.

Mutations in Presenilin-1 (PS1) account for over 80 % mutations linked to familial Alzheimer's disease (AD). However, the mechanisms of action of PS1 mutations in causing familial AD are not fully understood, limiting opportunities to develop targeted disease-modifying therapies for individuals carrying PS1 mutation. To gain more comprehensive insights into the impact of PS1 mutations on genome stability, we knocked down PS1 in SH-SY5Y, HMC3 and A549 cells. This revealed that PS1 knockdown (KD) dramatically induces genome instability (GIN) in all cell types, as indicated by the increased incidence of micronuclei, nucleoplasmic bridges and/or nuclear buds. Although amyloid ß (Aß) was able to induce GIN, PS1-KD was associated with decreased expression of Aß in SH-SY5Y cells, suggesting Aß is not the primary cause of GIN in PS1-KD cells. In contrast, inhibiting the PS1 γ-secretase activity by DAPT recapitulated GIN phenotype as seen in PS1-KD cells, indicating that the induction of GIN following PS1 KD can be attributed to the loss of γ-secretase activity. PS1 KD or γ-secretase inhibition markedly sensitizes SH-SY5Y to the genotoxicity of mitomycin C. Interestingly, overexpression of the wildtype PS1 dramatically increased GIN in SH-SY5Y. Collectively, our study demonstrates the potential of PS1 and its γ-secretase activity in maintaining genome stability, highlighting a novel potential link between PS1 loss-of-function or gain-of-function mutations and familial AD through GIN. Several mechanisms by which GIN induced by PS1 dys-expression may contribute to AD are discussed.

Nuclear factor kappa B (NF-κB) participates in the aluminum-induced down-regulation of miR29a/b1.

BACKGROUND: Studies have shown that aluminum (Al) is one of the environmental risk factors leading to Alzheimer's disease (AD), and Al exposure can cause elevated levels of BACE1mRNA, ß-secretase (BACE1), and amyloid beta (Aß) in vivo and in vitro. Previous studies by our research group have shown that this is partly caused by the negative regulation of BACE1 by miRNA29a/b1 (miR29a/b1). Despite the observed the role of nuclear factor kappa B (NF-κB) on many miRNAs, the upstream regulation of NF-κB protein on miR29 remains poorly understood. The purpose of this study was to better define the relationship between NF-κB and miR29a/b1 and the potentially relevant signaling pathways. METHODS: On the one hand, we constructed the animal model of Al exposure by the intraperitoneal injection of aluminum-maltolate (Al(mal)3) in rats. Conversely, NF- κB inhibitors were added to adrenal phaeochromocytoma (PC12) cells exposed to Al(mal)3. RESULTS: We verified that NF-κB shows an increasing trend with Al accumulation in the brain of rats, which is accompanied by a downward trend of miR29a/b1. Notably, the suppression of NF-κB significantly increased miR29a/b1 and affected the expression of BACE1mRNA and downstream proteins. CONCLUSION: Al-induced NF-κB can negatively regulate the expression of miR29a/b1, which then significantly enhances the expression of BACE1 and Aß plaques.

Stem cells-derived exosomes alleviate neurodegeneration and Alzheimer's pathogenesis by ameliorating neuroinflamation, and regulating the associated molecular pathways.

Amyloid beta (Aß) aggregation and tau hyper phosphorylation (p-tau) are key molecular factors in Alzheimer's disease (AD). The abnormal formation and accumulation of Aß and p-tau lead to the formation of amyloid plaques and neurofibrillary tangles (NFTs) which ultimately leads to neuroinflammation and neurodegeneration. ß- and γ-secretases produce Aß peptides via the amyloidogenic pathway, and several kinases are involved in tau phosphorylation. Exosomes, a recently developed method of intercellular communication, derived from neuronal stem cells (NSC-exos), are intriguing therapeutic options for AD. Exosomes have ability to cross the BBB hence highly recommended for brain related diseases and disorders. In the current study, we examined how NSC-exos could protect human neuroblastoma cells SH-SY5Y (ATCC CRL-2266). NSC-exos were derived from Human neural stem cells (ATCC-BYS012) by ultracentrifugation and the therapeutic effects of the NSC-exos were then investigated in vitro. NSC-exos controlled the associated molecular processes to drastically lower Aß and p-tau. A dose dependent reduction in ß- and γ-secretase, acetylcholinesterase, GSK3ß, CDK5, and activated α-secretase activities was also seen. We further showed that BACE1, PSEN1, CDK5, and GSK-3ß mRNA expression was suppressed and downregulated, while ADAM10 mRNA was increased. NSC- Exos downregulate NF-B/ERK/JNK-related signaling pathways in activated glial cells HMC3 (ATCC-CRL-3304) and reduce inflammatory mediators such iNOS, IL-1ß, TNF-α, and IL-6, which are associated with neuronal inflammation. The NSC-exos therapy ameliorated the neurodegeneration of human neuroblastoma cells SH-SY5Y by enhancing viability. Overall, these findings support that exosomes produced from stem cells can be a neuro-protective therapy to alleviate AD pathology.

A loss-of-function NCSTN mutation associated with familial Dowling Degos disease and hidradenitis suppurativa.

Dowling Degos disease (DDD) is a rare autosomal dominant genodermatosis characterized by acquired, slowly progressive reticulated pigmented lesions primarily involving flexural skin areas. Mutations in KRT5, POGLUT-1 and POFUT-1 genes have been associated with DDD, and loss-of-function mutations in PSENEN, a subunit of the gamma-secretase complex, were found in patients presenting with DDD or DDD comorbid with hidradenitis suppurativa (HS). A nonsense mutation in NCSTN, another subunit of the gamma-secretase, was already described in a patient suffering from HS and DDD but whether NCSTN could be considered a novel gene for DDD is still debated. Here, we enrolled a four-generation family with HS and DDD. Through Whole Exome Sequencing (WES) we identified a novel nonsense mutation in the NCSTN gene in all the affected family members. To study the impact of this variant, we isolated outer root sheath cells from patients' hair follicles. We showed that this variant leads to a premature stop codon, activates a nonsense-mediated mRNA decay, and causes NCSTN haploinsufficiency in affected individuals. In fact, cells treated with gentamicin, a readthrough agent, had the NCSTN levels corrected. Moreover, we observed that this haploinsufficiency also affects other subunits of the gamma-secretase complex, possibly causing DDD. Our findings clearly support NCSTN as a novel DDD gene and suggest carefully investigating this co-occurrence in HS patients carrying a mutation in the NCSTN gene.

Tspan15-ADAM10 signalling enhances cancer stem cell-like properties and induces chemoresistance via Notch1 activation in ICC.

BACKGROUND & AIMS: Notch1 activation promotes ICC progression and is associated with chemoresistance; however, therapies directly targeting Notch1 showed severe adverse effects. Notch1 activation is mediated by ADAM10, a molecular scissor that separates the target protein from its substrates in the cell membrane. Tspan15 regulates ADAM10 function, but the role of Tspan15 in ICC progression is unclear. METHODS: Tspan15, ADAM10, and Notch1 expression and activation in fresh surgical specimens from 80 ICC patients and ICC cells were evaluated by immunohistochemistry, RT-PCR, western blotting, and flow cytometry. RESULTS: Tspan15 expression was increased in ICC compared with adjacent liver tissue, and high Tspan15 expression was an independent factor for poor prognosis. In ICC with high Tspan15 expression, vascular invasion, lymph node metastasis, and haematogenous recurrence were increased. Tspan15 was co-expressed with ADAM10 in ICC, and associated with the expression of stemness and EMT markers. In ICC cells, Tspan15 induced ADAM10 activation by mediating the translocation of activated m-ADAM10 from the cytoplasm to the surface of the cell membrane, which further activated Notch1 by separating the intracellular domain of Notch1 from its extracellular domain, leading to enhancement of CSC-like properties and EMT. This signalling was associated with enhanced chemoresistance against gemcitabine and cisplatin. Inhibition of Tspan15 or ADAM10 is a promising therapeutic strategy in ICC, as Tspan15 or ADAM10 knockdown or treatment with ADAM10 inhibitor reduced chemoresistance and invasiveness by suppressing Notch1-mediated CSC-like properties and EMT. CONCLUSIONS: Tspan15-ADAM10-Notch1 signalling is associated with aggressive tumour progression and poor prognosis in ICC.

Glucocorticoid enhances presenilin1-dependent Aß production at ER's mitochondrial-associated membrane by downregulating Rer1 in neuronal cells.

Stress-induced release of glucocorticoid is an important amyloidogenic factor that upregulates amyloid precursor protein (APP) and ß secretase 1 (BACE1) levels. Glucocorticoid also contributes to the pathogenesis of Alzheimer's disease (AD) by increasing ER-mitochondria connectivity, in which amyloid ß (Aß) processing occurs rigorously because of its lipid raft-rich characteristics. However, the mechanism by which glucocorticoid enhances γ-secretase activity in the mitochondrial-associated membrane of ER (MAM) and subsequent accumulation of mitochondrial Aß is unclear. In this study, we determined how glucocorticoid enhances Aß production in MAM using SH-SY5Y cells and ICR mice. First, we observed that cortisol-induced Aß accumulation in mitochondria preceded its extracellular apposition by enhancing γ-secretase activity, which was the result of increased presenilin 1 (PSEN1) localization in MAM. Screening data revealed that cortisol selectively downregulated the ER retrieval protein Rer1, which triggered its maturation and subsequent entry into the endocytic secretory pathway of PSEN1. Accordingly, overexpression of RER1 reversed the deleterious effects of mitochondrial Aß on mitochondrial respiratory function and neuronal cell viability. Notably, we found that cortisol guided the glucocorticoid receptor (GR) to bind directly to the RER1 promoter, thus trans-repressing its expression. Inhibiting GR function reduced Aß accumulation at mitochondria and improved the outcome of a spatial memory task in mice exposed to corticosterone. Taken together, glucocorticoid enhances PSEN1-mediated Aß generation at MAM by downregulating Rer1, which is a potential target at early stages of AD pathogenesis.

Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo.

BACKGROUND: Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells. METHODS: To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo. RESULTS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo. CONCLUSIONS: These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.