recombinant grn protein  (R&D Systems)

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: a , Representative immunoblots of mouse embryonic fibroblasts (MEF), isolated from wild-type (wt) and granulin knockout ( Grn ko) mice, probed for progranulin (PGRN), legumain (LGMN), and calnexin as loading control. MEF are either non-(-), control-( ctrl ) or Lgmn siRNA- transfected. Quantification of LGMN expression normalized to non-transfected wt levels (n=4). b , In vitro LGMN activity of non-(-), mock, ctrl and Lgmn siRNA-transfected Grn ko MEF normalized to non-transfected wt MEF (n=4). c , Representative immunoblot for LGMN in total brain homogenates of 6-month-old wt and Grn ko mice. Proform (LGMN p ), mature form (LGMN m ), and non-specific band (*) are indicated, β-actin verified equal loading. Quantification of the immunoblot signals normalized to wt (n=3 mice per genotype). d , In vitro LGMN activity of brain homogenates from 3-, 6-, 12-13-, and 20-24-month-old wt and Grn ko mice (n=4-6 per genotype and age group). e , Total brain mRNA of 3-, 6-, 12-13-, and 20-24-month-old mice normalized to wt (n=4-5). f , Representative immunoblot for LGMN in MACS-sorted microglia-, astrocyte- and neuron-enriched brain cell fractions of 4-5-month-old wt and Grn ko mice. LGMN p and LGMN m are indicated, calnexin verified equal loading for each fraction. g , Microglia-, astrocyte- and neuron-enriched fractions from 4-5-month-old Grn ko and wt mice analyzed for LGMN activity either normalized to wt microglia or wt of the respective cell type (insert) (n=3 mice per cell type and genotype). h , Lgmn mRNA levels of microglia isolated from 6-month-old wt and Grn ko mice normalized to wt (n=5). i, LGMN in vitro activity in lysates of cultured primary mouse microglia (n=3 mice per genotype). j , k , LGMN expression ( j ) and activity ( k ) analyzed in brain lysates of frontal cortex from FTLD/ GRN patients and pathology-negative control cases (see Extended Data Table 1). LGMN p and LGMN m are quantified in the immunoblot and normalized to the mean signal of control cases ( j ). Data are mean ± s.d. of biologically independent experiments; unpaired two-tailed t-test: ns not significant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. P values and statistical source data are provided.

Article Snippet: To investigate the inhibitory effect of PGRN on LGMN activity, 200 ng or the indicated amount of human recombinant PGRN (rPGRN, # 10826-H08H, Sino Biological Inc.) was either added to the activation assay or to the activity assay.

Techniques: Western Blot, Isolation, Knock-Out, Transfection, Expressing, In Vitro, Activity Assay, Cell Culture, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: a , Representative immunoblots of MEF wt, Grn ko and Grn ko stably transfected with a mouse Grn cDNA probed for PGRN, LGMN, and β-actin to verify equal loading. b , LGMN substrate turnover and quantification of LGMN activity normalized to MEF wt of biologically independent duplicates is shown as mean ± s.d.. c, Schematic of PTV:PGRN single dosing study in Grn ko/h TfR ki. d , LGMN activity of a Grn wt, Grn ko, and Grn ko treated with a single 10 mg/kg i.v. dose of PTV:PGRN for 7 d and 14 d. Data indicate the mean normalized to wt. (n=6 mice per genotype and treatment, 3-4-month-old). e , Representative immunoblot for LGMN in total brain homogenate of wt and Grn ko non-treated and treated mice. Proform (LGMNp), mature form (LGMNm), unspecific band (asterisk) are indicated, β-actin verified equal loading. Quantification of LGMN immunoblot signals normalized to wt (n=6 mice per genotype and treatment). Statistical significance was determined using one-way ANOVA and Tukey’s post hoc test: ns, not significant; * P <0.05, ** P <0.01, **** P <0.0001. P values and statistical source data are provided.

Article Snippet: To investigate the inhibitory effect of PGRN on LGMN activity, 200 ng or the indicated amount of human recombinant PGRN (rPGRN, # 10826-H08H, Sino Biological Inc.) was either added to the activation assay or to the activity assay.

Techniques: Western Blot, Stable Transfection, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: Representative immunoblots for PGRN and LGMN in microglia-, astrocyte-and neuron-enriched brain cell fractions isolated from 10-month-old mice. Enrichment of each cell fraction is verified by immunoblotting for IBA1, GFAP and TUJ. Equal amounts of protein were loaded for all cell types, calnexin expression varies between cell types. n=2 mice per cell type and genotype.

Article Snippet: To investigate the inhibitory effect of PGRN on LGMN activity, 200 ng or the indicated amount of human recombinant PGRN (rPGRN, # 10826-H08H, Sino Biological Inc.) was either added to the activation assay or to the activity assay.

Techniques: Western Blot, Isolation, Expressing

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: a , Schematic representation of pH-dependent auto- and not auto-catalytic LGMN maturation. N- and C-terminal pro-peptides (p1-p3) and the mature chain (c) with the active site residues (blue stars) are specified. Proform (LGMN p ), intermediate-form (LGMN i ) and mature-form (LGMN m ) are indicated. b , c , Activity analysis of in vitro auto-catalytically matured recombinant (r) LGMN incubated for 4 h at acidic pH. Either rPGRN or elastase-digested PGRN were added after ( b ) or before the 4 h activation step ( c ) (n=3). d , PGRN digest with elastase controlled by immunoblotting. e , f, Activation of rLGMN with/without rPGRN at acidic pH for indicated time points, monitored by immunoblotting. Quantification of the relative LGMN i level (% of total LGMN) ( e ) or activity ( f ) for each timepoint (n=3). g , LGMN activity after 2 h incubation with different concentrations of rPGRN at pH 4. The IC 50 was calculated using a non-linear curve fit, 95% confidence intervals (CI) are indicated (n=3). h , Immunoblot analysis of rPGRN turnover and granulin peptide generation by rLGMN. Stars indicate rLGMN independent bands. Data are mean ± s.d. of independent technical replicates, unpaired two-tailed t-test: ns, Strikingly, rLGMN activity was reduced by more than 50 % when rPGRN was added to pro-rLGMN during the activation step at acidic pH 4 , while elastase-digested rPGRN did not reduce rLGMN activation , suggesting that rPGRN, but not granulins, regulate the maturation of rLGMN. Autocatalytic maturation of pro-rLGMN was then monitored longitudinally at pH 4.0 with and without rPGRN. Proteolytic in vitro maturation and enzymatic activation of rLGMN under acidic conditions was significantly slowed in the presence of rPGRN . Moreover, rPGRN reduced autocatalytic activation of rLGMN in a dose-dependent manner with an IC50 of 39.28 nM . However, after full autocatalytic maturation , the catalytic activity of rLGMN was identical in the presence and absence of rPGRN . Thus, PGRN does not affect the catalytic activity of LGMN per se , but rather slows its autocatalytic maturation. Intriguingly, as suggested before , rLGMN efficiently generates granulin peptides in vitro . After 8 h of co-incubation of rPGRN with rLGMN, full length rPGRN was completely converted into single or multiple granulin peptides. At this time point also full conversion of rLGMN to the intermediate form was observed , further indicating that full length PGRN is needed to slow LGMN activation and that LGMN regulates its own maturation by processing of PGRN in a negative feedback loop. In vivo , the interaction of LGMN with PGRN is supported by reduced generation of granulins in Lgmn ko mice. Granulin peptides are readily detected within purified liver lysosomes of wt mice, but are substantially reduced in Lgmn ko mice . Thus, LGMN regulates its own maturation by processing of PGRN in a negative feedback loop.

Article Snippet: To investigate the inhibitory effect of PGRN on LGMN activity, 200 ng or the indicated amount of human recombinant PGRN (rPGRN, # 10826-H08H, Sino Biological Inc.) was either added to the activation assay or to the activity assay.

Techniques: Activity Assay, In Vitro, Recombinant, Incubation, Activation Assay, Western Blot, Two Tailed Test, In Vivo, Purification

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: a , Schematic representation of the media transfer experiment. HeLa cells were transfected with wt mouse LGMN, the proteolytically inactive LGMN C191A variant (mt) or mock (-) and 25% conditioned media supplemented to primary hippocampal mouse neurons for 24 h. ( b,c) , LGMN expression (LGMN p , LGMN i , and LGMN m ) ( b ) and secretion of LGMN p ( c ) by HeLa cells was verified by immunoblotting (n=3 independent experiments). ( c,d ) To monitor autocatalytic processing of LGMN, conditioned media was incubated at pH 7 or pH 4 for 4 h. d , LGMN activity of conditioned media incubated at pH 7 and pH 4 (n=3 independent experiments). ( e- g) , Primary neurons were analyzed after 24 h incubation with conditioned media. Presence of the LGMN proform was confirmed in culture media ( e ). LGMN uptake by neurons ( e ) and TDP-To provide evidence for a pathological link between PGRN reduction, LGMN activation, and enhanced TDP-43 processing in a human disease-relevant system, we generated human iPSC lacking PGRN ( GRN ko iPSC) and differentiated them into human induced pluripotent stem cell-derived microglia (hiMGL) and neurons (hiNE). GRN ko hiMGL showed a 3-4-fold increased expression and activity of LGMN compared to wt hiMGL but only a 1.5-fold increase of LGMN mRNA levels . Thus, the difference in LGMN mRNA levels between wt and GRN ko hiMGL was less pronounced than on LGMN protein or activity levels, further supporting a post-transcriptional regulation of LGMN activity upon PGRN deficiency. In line with our findings in mice , hiNE had almost no LGMN protein expression or activity and showed lower transcript levels than hiMGL . Furthermore, GRN ko hiNE did not show an increase of LGMN activity in contrast to Grn ko mouse neurons . To investigate a potential cross-talk between microglia and neurons upon PGRN deficiency, we co-cultured GRN wt hiNE with either GRN wt or ko hiMGL . In co-cultures of wt hiNE with GRN ko hiMGL, we observed increased mature and active LGMN in lysates . As a consequence, co-culture of GRN ko hiMGL with GRN wt hiNE showed enhanced TDP-43 processing, indicated by reduced TDP- 43 full length and enhanced TDP-43 fragments . To investigate if TDP-43 processing in the co-culture occurs predominantly within neurons, we selectively blocked neuronal LGMN activity by AAV9 (AAV-hSyn-Cst7) mediated neuronal expression of cystatin F (CSTF) prior to co-culturing with hiMGL . CSTF and CSTC are brain expressed , effective inhibitors of LGMN and other cysteine-proteases . Since CTSF showed stronger inhibitory effects in cell culture, we decided to use AAV-hSyn-Cst7. Immunofluorescence confirmed neuronal CTSF expression , which resulted in reduced LGMN maturation and strongly reduced activity . Furthermore, as a consequence of the inhibition of LGMN activity, TDP-43 processing was also reduced to levels seen in wt co-culture .

Article Snippet: To investigate the inhibitory effect of PGRN on LGMN activity, 200 ng or the indicated amount of human recombinant PGRN (rPGRN, # 10826-H08H, Sino Biological Inc.) was either added to the activation assay or to the activity assay.

Techniques: Transfection, Variant Assay, Expressing, Western Blot, Incubation, Activity Assay, Activation Assay, Generated, Derivative Assay, Cell Culture, Co-Culture Assay, Immunofluorescence, Inhibition

Journal: bioRxiv

Article Title: Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

doi: 10.1101/2024.01.16.575687

Figure Lengend Snippet: Suggested pathomechanism: In homeostasis, PGRN slows the maturation of LGMN, which is regulated in a negative feed-back loop via processing of PGRN into single granulins by LGMN. Upon PGRN loss-of-function, lysosomal function is impaired and microglia become hyperactivated. In this context, LGMN is not regulated by PGRN anymore and its protein levels and activity increase. The pro-form of LGMN is secreted by activated microglia and taken up by neurons (mechanism unknown), where it hyperprocesses TDP-43 and thereby drives TDP-43 pathology.

Article Snippet: To investigate the inhibitory effect of PGRN on LGMN activity, 200 ng or the indicated amount of human recombinant PGRN (rPGRN, # 10826-H08H, Sino Biological Inc.) was either added to the activation assay or to the activity assay.

Techniques: Activity Assay