TNFR-1 and GDF-15 Are Associated With Plasma Neurofilament Light Chain and Progranulin Among Community-Dwelling Older Adults: A Secondary Analysis of the MAPT Study.

There is growing evidence that cognitive decline can be affected by both nutritional aspects and inflammation. Plasma neurodegenerative biomarkers stand out as minimally invasive useful measures to monitor the potential risk of cognitive decline. This study aimed to investigate the associations between biomarkers of neurodegeneration, nutrition, and inflammation among community-dwelling older adults, and to verify if associations differed according to apolipoprotein E (APOE) ε4 status. This cross-sectional analysis included 475 participants ≥70 years old from the Multidomain Alzheimer Preventive Trial (MAPT), mean age 76.8 years (SD = 4.5), 59.4% women. Biomarkers of neurodegeneration (plasma amyloid-ß 42/40-Aß 42/40, neurofilament light chain-NfL, progranulin), nutrition (erythrocyte docosahexaenoic acid, eicosapentaenoic acid, omega-3 index; plasma homocysteine-Hcy, 25 hydroxyvitamin D), inflammation (plasma tumor necrosis factor receptor 1-TNFR-1, monocyte chemoattractant protein 1-MCP-1, interleukin 6-IL-6), and cellular stress (plasma growth differentiation factor 15-GDF-15) were assessed. Linear regression analyses were performed to investigate the associations between nutritional and inflammatory biomarkers (independent variables) and neurodegenerative biomarkers (dependent variables), with adjustments for age, sex, education, body mass index, physical activity, allocation to MAPT groups, and APOE ε4 status. After adjusting for confounders, Aß 42/40 was not associated with nutritional or inflammatory markers. NfL was positively associated with GDF-15, TNFR-1, IL-6, and Hcy. Progranulin was positively associated with GDF-15, TNFR-1, and MCP-1. Analyses restricted to APOE ε4 carriers (n = 116; 26.9%) or noncarriers were mostly similar. Our cross-sectional study with community-dwelling older adults corroborates previous evidence that inflammatory pathways are associated to plasma markers of neurodegeneration. Clinical Trials Registration Number: NCT00672685.

Are granulins copper sequestering proteins?

Granulins (GRN 1-7) are short (~6 kDa), cysteine-rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These peptides, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Previously we showed that GRN-3 and GRN-5 are fully disordered in the reduced form implicating redox sensitive attributes to the proteins. Redox-based modulations are often carried out by metalloproteins in mitigating oxidative stress and maintaining metal-homeostasis within cells. To probe whether GRNs play a role in metal sequestration, we tested the metal binding propensity of the reduced forms of GRNs -3 and - 5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu and no other divalent metal cations, with a greater specificity for Cu(I). Binding of Cu did not result in a disorder-to-order structural transition but partly triggered the multimerization of GRNs via uncoordinated cystines at both pH conditions. Overall, the results indicate that GRNs -3 and - 5 have surprisingly strong affinity for Cu in the pM range, comparable to other known copper sequestering proteins. The results also hint at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu-induced ROS cytotoxicity in cells. Together, this report uncovers metal-coordinating property of GRNs for the first time, which may have profound significance in their structure and pathophysiological functions.

In Vitro Physical and Functional Interaction Assays to Examine the Binding of Progranulin Derivative Atsttrin to TNFR2 and Its Anti-TNFα Activity.

TNFα/TNFR signaling plays a critical role in the pathogenesis of various inflammatory and autoimmune diseases, and anti-TNFα therapies have been accepted as the effective approaches for treating several autoimmune diseases. Progranulin (PGRN), a multi-faced growth factor-like molecule, directly binds to TNFR1 and TNFR2, particularly to the latter with higher affinity than TNFα. PGRN derivative Atsttrin is composed of three TNFR-binding domain of PGRN and exhibits even better therapeutic effects than PGRN in several inflammatory disease models, including collagen-induced arthritis. Herein we describe the detailed methodology of using (1) ELISA-based solid phase protein-protein interaction assay to demonstrate the direct binding of Atsttrin to TNFR2 and its inhibition of TNFα/TNFR2 interaction; and (2) tartrate-resistant acid phosphatase (TRAP) staining of in vitro osteoclastogenesis to reveal the cell-based anti-TNFα activity of Atsttrin. Using the protocol described here, the investigators should be able to reproducibly detect the physical inhibition of TNFα binding to TNFR and the functional inhibition of TNFα activity by Atsttrin and various kinds of TNF inhibitors.

Investigating the Conformational Response of the Sortilin Receptor upon Binding Endogenous Peptide- and Protein Ligands by HDX-MS.

Sortilin is a multifunctional neuronal receptor involved in sorting of neurotrophic factors and apoptosis signaling. So far, structural characterization of sortilin and its endogenous ligands has been limited to crystallographic studies of sortilin in complex with the neuropeptide neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of sortilin to binding biological ligands including the peptides neurotensin and the sortilin propeptide and the proteins progranulin and pro-nerve growth factor-ß. The results show that the ligands use two binding sites inside the cavity of the ß-propeller of sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of sortilin. Our findings improve our structural understanding of sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.

A Brief Overview of Progranulin in Health and Disease.

The purpose of this brief overview of the progranulin protein is to provide a sense of the range and extent of the roles of progranulin in normal physiology and pathology. Progranulin has received attention due to its role in neurodegeneration, where mutation of a single copy of GRN, the gene encoding progranulin, results in frontotemporal dementia, whereas viral delivery of progranulin to the brains of mice exhibiting Parkinson's or Alzheimer's disease phenotypes inhibits the progression of the neurodegenerative phenotypes. Of equal importance, progranulin protects tissues against the harmful effects of poorly controlled inflammation and promotes tissue regeneration after injury at a multitude of sites throughout the body. Progranulin is overexpressed by many types of cancer and contributes to their progression. Given suitable analytical methods and model systems, progranulin offers a wealth of research possibilities.

Data Mining: Applying the AD&FTD Mutation Database to Progranulin.

The online AD&FTD Mutation Database ( http://www.molgen.vib-ua.be/FTDmutations ) was conceived to meet the needs of a comprehensive knowledge base of genetic variations in genes associated with monogenic forms of Alzheimer's disease (AD) and frontotemporal dementia (FTD). Today, the AD&FTD Mutation Database provides curated, referenced information of 764 genetic variants in APP, PSEN1, and PSEN2 associated with AD and GRN, C9orf72, TBK1, MAPT, VCP, CHMP2B, TARDBP, and FUS associated with FTD and related diseases. In addition, the database stores demographic and clinicogenetic data of 1646 dementia families associated with these mutations. In FTD, the granulin (GRN) gene has the highest number of different mutations (79/231 = 34%) and the second highest number of associated FTD families after C9orf72. In addition to the detailed mutation and patient information, summary reports in tabular and graphical formats can be consulted. Further, all variants can be uploaded to the human genome browser for custom-designed analyses.

The Use of Caenorhabditis elegans to Study Progranulin in the Regulation of Programmed Cell Death and Stress Response.

The nematode Caenorhabditis elegans (C. elegans) has proven to be a powerful model organism for the study of many biological processes, with major implications for human health and disease. As progranulin is a pleiotropic, secreted protein with both cell autonomous and non-autonomous roles, a multicellular organism such as C. elegans is ideal for the investigation of its normal function and pathological effects. The C. elegans genome contains a progranulin-like gene known as pgrn-1. The nematode pgrn-1 encodes a protein with three cysteine-rich granulin domains, compared to the seven and a half granulins in the human protein. We have shown that C. elegans mutants lacking pgrn-1 appear grossly normal, but exhibit accelerated apoptotic cell engulfment as well as a stress resistance phenotype (Kao et al., Proc Natl Acad Sci U S A 108:4441-4446, 2011; Judy et al., PLoS Genet 9:e1003714, 2013). In addition, the roles of individual granulins can also be dissected in C. elegans (Salazar et al., J Neurosci 35:9315-9328, 2015). Here, we describe methods for studying apoptosis and stress response in C. elegans.

Structure dissection of zebrafish progranulins identifies a well-folded granulin/epithelin module protein with pro-cell survival activities.

The ancient and pluripotent progranulins contain multiple repeats of a cysteine-rich sequence motif of ∼60 amino acids, called the granulin/epithelin module (GEM) with a prototypic structure of four ß-hairpins zipped together by six inter-hairpin disulfide bonds. Prevalence of this disulfide-enforced structure is assessed here by an expression screening of 19 unique GEM sequences of the four progranulins in the zebrafish genome, progranulins 1, 2, A and B. While a majority of the expressed GEM peptides did not exhibit uniquely folded conformations, module AaE from progranulin A and AbB from progranulin B were found to fold into the protopypic 4-hairpin structure along with disulfide formation. Module AaE has the most-rigid three-dimensional structure with all four ß-hairpins defined using high-resolution (H-15 N) NMR spectroscopy, including 492 inter-proton nuclear Overhauser effects, 23 3 J(HN,Hα ) coupling constants, 22 hydrogen bonds as well as 45 residual dipolar coupling constants. Three-dimensional structure of AaE and the partially folded AbB re-iterate the conformational stability of the N-terminal stack of two beta-hairpins and varying degrees of structural flexibility for the C-terminal half of the 4-hairpin global fold of the GEM repeat. A cell-based assay demonstrated a functional activity for the zebrafish granulin AaE in promoting the survival of neuronal cells, similarly to what has been found for the corresponding granulin E module in human progranulin. Finally, this work highlights the remaining challenges in structure-activity studies of proteins containing the GEM repeats, due to the apparent prevalence of structural disorder in GEM motifs despite potentially a high density of intramolecular disulfide bonds.