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Lanthanide ions have attracted great interest owing to their optical and magnetic properties. Single-molecule magnet (SMM) behavior has been a fascinating science for thirty years. Moreover, chiral lanthanide complexes allow the observation of remarkable circularly polarized luminescence (CPL). However, the combination of both SMM and CPL behaviors in a single molecular system is very rare and deserves attention in the design of multifunctional materials. Four chiral one-dimensional coordination compounds involving 1,1′-Bi-2-naphtol (BINOL)-derived bisphosphate ligands and the Yb(III) centre were synthesized and characterized by powder and single-crystal X-ray diffraction. All the Yb(III)-based polymers displayed field-induced SMM behavior with magnetic relaxation occurring by applying Raman processes and near infrared CPL in the solid state.

International audience; The commercially available chlorodicarbonylrhodium(I) dimer was shown to be a highly active catalyst in the hydrosilylation of aldimines and ketimines under mild conditions. From these initial results, new procedures for the direct reductive amination of aldehydes and ketones employing this rhodium complex were developed with various primary and secondary amines in the presence of hydrosilanes.

Patient-derived cells hold great promise for precision medicine approaches in human health. Human dermal fibroblasts have been a major source of cells for reprogramming and differentiating into specific cell types for disease modeling. Postmortem human dura mater has been suggested as a primary source of fibroblasts for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols has not been fully established. In this study, cells derived from postmortem dura mater are directly compared to those from dermal biopsies of living subjects. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences were observed between cells of dermal and dural origin. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, slower growth rates, and a higher rate of karyotype abnormality. Dura mater-derived cells also failed to express fibroblast protein markers. When dermal fibroblasts and dura mater-derived cells from the same subject were compared, they exhibited highly divergent gene expression profiles that suggest dura mater cells originated from a mixed mural lineage. Given their postmortem origin, somatic mutation signatures of dura mater-derived cells were assessed and suggest defective DNA damage repair. This study argues for rigorous karyotyping of postmortem derived cell lines and highlights limitations of postmortem human dura mater-derived cells for modeling normal biology or disease-associated pathobiology.

Description, TSC1/hamartin haploinsufficiency increases risk for tauopathy by promoting tau acetylation and accumulation., Age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions are known as tauopathies. We previously linked a loss-of-function mutation in the TSC1 gene to tau accumulation and frontotemporal lobar degeneration. Now, we have identified genetic variants in TSC1 that decrease TSC1/hamartin levels and predispose to tauopathies such as Alzheimer’s disease and progressive supranuclear palsy. Cellular and murine models of TSC1 haploinsufficiency, as well as human brains carrying a TSC1 risk variant, accumulated tau protein that exhibited aberrant acetylation. This acetylation hindered tau degradation via chaperone-mediated autophagy, thereby leading to its accumulation. Aberrant tau acetylation in TSC1 haploinsufficiency resulted from the dysregulation of both p300 acetyltransferase and SIRT1 deacetylase. Pharmacological modulation of either enzyme restored tau levels. This study substantiates TSC1 as a novel tauopathy risk gene and includes TSC1 haploinsufficiency as a genetic model for tauopathies. In addition, these findings promote tau acetylation as a rational target for tauopathy therapeutics and diagnostic.

Patient-derived cells hold great promise for precision medicine approaches in human health. Fibroblast cells have been a major source of human cells for reprogramming and differentiating into specific cell types for disease modeling. Such cells can be isolated at various stages during life (presymptomatic, symptomatic, and postmortem) and thus can potentially be used to model different phases of disease progression. In certain circumstances, however, tissues are not collected during life and only postmortem tissues are the only available source of fibroblasts. Fibroblasts cultured from postmortem human dura mater of individuals with neurodegenerative diseases have been suggested as a primary source of cells for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols requires further characterization. In this study, cells derived from dermal biopsies performed in living subjects were compared to cells derived from postmortem dura mater. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences between the dermis and dura mater-derived cell lines were found. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, exhibited slower growth rates, failed to express fibroblast protein markers, and exhibited significant differences in gene expression profiles. In addition, dura mater-derived cells were found to exhibit a high rate of chromosomal abnormalities, particularly in the loss of the Y chromosome. Our study highlights potential limitations of postmortem human dura mater-derived cells for disease modeling, argues for rigorous karyotyping prior to reprograming, and brings into question the identity of dura mater-derived cells as belonging to a fibroblast lineage.

International audience; The enantiopure coordination polymer [Dy(hfac)3((S/R)-L)]n ([(S/R)-1]n) involving a BINOL-derived bisphosphate ligand (S/R)-L is reported. Paramagnetic NMR and computational investigations demonstrated the formation of mononuclear species [(S/R)-1] in CH2Cl2 solution and its optimized structure was determined. The experimental electronic circular dichroism signals of (S/R)-L were strongly enhanced after metal coordination [(S/R)-1]. Both polymeric and mononuclear structures displayed field-induced Single-Molecule Magnet (SMM) behaviour with similar multi-relaxation processes due to the retention of crystal-field splitting determined by CASSCF calculations, despite the strong structural transformation between the two media. The HOMO ? LUMO transition of the (S)-L ligand induced the classical Dy(iii) emission at 77 K that was correlated with the magnetic properties. [(S)-1]n is described as one-dimensional assembly of chiral luminescent Single-Ion Magnets (SIMs).

The coordination reaction of the [Dy(hfac)3(H2O)2] units (hfac− = 1,1,1,5,5,5-hexafluoroacetylacetonate) with the [8′-(Diphenoxylphosphinyl)[1,1′-binaphthalen]-8-yl]diphenoxylphosphine oxide ligand (L) followed by a crystallisation in a 1:3 CH2Cl2:n-hexane solvent mixture led to the isolation of a new polymorph of formula [(Dy(hfac)3((S)-L))3]n (1). The X-ray structure on single crystal of 1 revealed the formation of a mono-dimensional coordination polymer with three crystallographically independent DyIII centres, which crystallised in the polar chiral P21 space group. Ac magnetic measurements highlighted single-molecule magnet behaviour under both zero and 1000 Oe applied magnetic field with magnetic relaxation through quantum tunneling of the magnetisation (QTM, zero field only) and Raman processes. Despite the three crystallographically independent DyIII centres adopting a distorted D4d coordination environment, a single slow magnetic relaxation contribution was observed at a slower rate than its previously studied [(Dy(hfac)3((S)-L))]n (2) polymorph.

Additional file 1 Fig. S1. Enzymes that do not cleave PGRN are biologically active. a, 1 μM of mature CTSD was incubated with 400 ng recombinant human PGRN or 400 ng BSA at the indicated pH. The incubation was stopped after 60 min or 16 h, and reaction contents were subjected to Coomassie staining (60 min) or silver staining (16 h) respectively. Bands correlate to proteins indicated on the right. BSA was used as a control for CTSD activity. At lower pH of 3.4, CTSD self-digest leading to a decreased level of mature CTSD compared to pH 4.5 [52]. b, FRET-based enzyme activity assay at pH 4.5 was performed on all enzymes that do not cleave PGRN. The values obtained were normalized to timepoint t = 0 and plotted relative to activity. All enzymes show activity against the optimized FRET peptides compared to substrate only control. Fig. S2. Antibody validation for individual granulin peptides. a, Dot blots confirming the specificity of each custom antibody towards the granulin peptide. 2.5 μg of each peptide was used with each antibody (11,000 dilution). Paragranulin, p-Gran; granulin G, Gran-G; granulin F, Gran-F; granulin B, Gran-B; granulin A, Gran-A; granulin C, Gran-C; granulin D, Gran-D; granulin E, Gran-E. b, Peptide blocking experiment to demonstrate antibody specificity. 400 ng of rhPGRN was incubated with 1uM CTSL for 20 min. The antibody was incubated with or without the respective blocking peptide (10 or 100-fold molar ratio of peptide to antibody) and then incubated with the membrane overnight. Increase in peptide concentrations show a decrease in the bands of each antibody tested. Fig. S3. Lysosomal proteases unable to digest PGRN in vitro. Full western blot images of the proteases that do not cleave PGRN. 1 μM of each enzyme was incubated with 400 ng of PGRN for 20 min. C-terminal Gran-E antibody or anti-PGRN (Invitrogen) antibody was used to assess the results of the in vitro assay. a, Cysteine proteases Cathepsins C (CTSC), H (CTSH), X (CTSX), O (CTSO) and F (CTSF). b, Aspartyl proteases cathepsin D (CTSD). c, Serine protease Pro-X carboxypeptidase (PRCP) and Cathepsin A (CTSA). The lower molecular weight bands (indicated by *) correspond to the enzyme. Fig. S4. PGRN processing by lysosomal proteases in vitro at pH 6.5. 1 μM of each enzyme was incubated with 400 ng of PGRN for 20 min at pH 6.5. Anti-p-Gran, anti-Gran-F, and anti- Gran-E antibodies were used to assess the results of the in vitro assay. Cathepsin B (CTSV), cathepsin L (CTSL), cathepsin K (CTSK), cathepsin S (CTSS), cathepsin V (CTSV), asparagine endopeptidase (AEP), cathepsin G (CTSG), paragranulin (p-Gran), granulin F (Gran-F), granulin E (Gran-E). Fig. S5. Summary of PGRN processing into granulins by multiple proteases. Summary of the results of the in vitro assays. a, Processing of PGRN to granulins by different proteases is dependent on pH. The range of cleavage is represented in greyscale with no cleavage in white and complete cleavage in black. The range takes into account the amount of full-length PGRN processed into multi-granulin fragments and individual granulins within 20 min. b, Represented is the ability of each enzyme to liberate individual paragranulin (p) in red, granulin F (F) in green, and granulin E (E) in blue. The protease classes are also indicated. CTSE, cathepsin E; CTSV, cathepsin V; CTSL, cathepsin L; CTSB, cathepsin B; CTSK, cathepsin K; AEP, asparagine endopeptidase; CTSG, cathepsin G; CTSS, cathepsin S. Fig. S6. CTSL is highly efficient at liberating paragranulin and granulin E from PGRN. A time course analysis to determine efficiency of cleavage. 400 ng of recombinant human PGRN was incubated with 50 nM of enzyme for the time points indicated. P-Gran, Gran-F and Gran-E antibodies were used to assess the results of the assay. PGRN, progranulin; CTSL, cathepsin L; CTSB, cathepsin B. Fig. S7. Antibody specificity to detect both PGRN and granulin sized bands in wild-type and PGRN knock out iPSC cell lysates. a, Lysates from isogenic WTC11 and Pgrn KO iPSCs were probed with custom anti-granulin antibodies. b, Commercial antibodies from Invitrogen and Sigma were tested on the same iPSC cell lines. Fig. S8. Expression profile of the PGRN proteases in differentiated SH-SY5Y cells. a, qPCR analysis to assess the expression of candidate PGRN proteases in differentiated SH-SY5Y cells. The graph represents the mean expression value of each enzyme normalized to GAPDH. The absolute mean values are noted for each sample. All enzymes were run in triplicates with n = 4 biological replicates. b, Differentiated SH-SY5Y cells were subjected to treatment with AEP siRNA or scramble control. A separate quantification and statistical analysis of PGRN, Gran-F and mature AEP was performed, similar to that seen in Fig. 3 of the manuscript. c, PGRN protein is assessed with an Anti-Gran E antibody. PGRN KO SH-SY5Y cells are used to determine specificity of PGRN band. d, Quantification of c. Fig. S9. PGRN is processed by AEP to liberate individual granulins F and B. Recombinant human PGRN was incubated with and without AEP for 1 h at pH 4.5. The reaction was stopped, and the cleavage bands were separated by SDS-PAGE. a, Multiple cleavage bands and individual granulin sized bands can be visualized upon silver stain. b-e, Bands were cut and digested for mass spectrometry to identify the cleavage sites (also see Fig. 4c). Identified peptides are annotated with observed fragment ions. Previous and next amino acids are in () and carbamidomethylated Cys are colored red. The precursor mass of each peptide is as follows: b, 428.5229 m/z; c, 847.8607 m/z; d, 639.3205 m/z; e, 478.9133 m/z. Fig. S10. Progranulin and granulin F levels in the human brain. a, Western blot analysis of the levels of PGRN and Gran-F in the non-degenerating IOC regions of the control brain compared to the same region in FTLD-TDP-Pgrn subjects. b, quantification of PGRN and Gran-F level, normalized to actin. c, Western blot analysis of the levels of PGRN and Gran-F in the degenerating MFG regions of the control brain compared to the same region in FTLD-TDP-Pgrn subjects. d, quantification of PGRN and Gran-F level, normalized to actin. (**, p = 0.008). Unpaired, two-tailed student t-test was performed between the pairs. Error bars represent mean with standard deviation. e-g, Levels of PGRN and Gran-F in a and c are plotted against age. There is no correlation between age and level of PGRN and Gran-F. Fig. S11. AEP levels are significantly increased in a degenerating brain region of FTLD-TDP-Pgrn subjects. a, Western blot analysis of endogenous immature (pro AEP) and mature AEP levels in control brain. b, Quantification of pro and mature AEP levels between IOC and MFG regions in control subjects. Paired two-tailed student t-test was performed between the different groups. No significant (ns) difference was observed. c, Western blots of endogenous Pro and mature AEP in IOC and MFG brain regions of FTLD-TDP-Pgrn subjects. d, Levels of mature AEP in the degenerating MFG region are increased compared to the non-degenerating IOC region from the same FTLD-TDP-Pgrn samples (*, p = 0.01, n = 6). Paired two-tailed student t-test was performed between the groups. IOC, inferior occipital cortex; MFG, middle frontal gyrus; AEP, asparagine endopeptidase. Sample numbers listed below each Western blot correspond to subject number in Table 2.

Background Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases to process PGRN at a range of pH setpoints. Results In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in distinctive, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn showed increased PGRN processing to granulin F and increased AEP activity in degenerating brain regions but not in regions unaffected by disease. Conclusions This study demonstrates that multiple lysosomal proteases may work in concert to liberate multi-granulin fragments and granulins. It also implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage and granulin production may represent therapeutic strategies for FTLD-Pgrn and other progranulin-related diseases.

Age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions, including Alzheimer’s disease (AD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), are collectively known as tauopathies. The vast majority of human tauopathies accumulate non-mutant tau rather than mutant forms of the protein, yet cell and animal models for non-mutant tauopathies are lacking. We previously linked a monoallelic mutation in the TSC1 gene to tau accumulation and FTLD. Now, we have identified new variants in TSC1 that predisposed to other tauopathies such as AD and PSP. These new TSC1 risk variants significantly decreased the half-life of TSC1/hamartin in vitro. Cellular and murine models of TSC1 haploinsufficiency (TSC1+/-) accumulated tau protein that exhibited aberrant acetylation on six lysine residues. Tau acetylation hindered its lysosomal degradation via chaperone-mediated autophagy leading to neuronal tau accumulation. Enhanced tau acetylation in TSC1+/- models was achieved through both an increase in p300 acetyltransferase activity and a decrease in SIRT1 deacetylase levels. Pharmacological modulation of either enzyme restored tau levels. Together, these studies substantiate TSC1 as a novel tauopathy risk gene and advance TSC1 haploinsufficiency as a new genetic model for tauopathy. In addition, these results promote acetylated tau as a rational target for diagnostic and therapeutic modalities in multiple tauopathies.

Background - Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases at a range of pH setpoints. Results - In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in variable, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn show increased PGRN processing to granulin F, correlating with increased activity of AEP, in a region-specific manner. Conclusions - This study demonstrates that multiple lysosomal proteases may work in concert to liberate granulins and implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage may represent a new strategy to modulate PGRN and granulin levels in disease.