Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN

TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer’s disease-associated H157Y variant

We have characterised the proteolytic cleavage events responsible for the shedding of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild type and H157Y human TREM2 and for the wild type murine orthologue. Crucially, we also show that the Alzheimer diseaseassociated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer’s and other neurological diseases.Funding from the Wellcome Trust and the Canadian Institutes of Health Research contributed to the support of this study

Bace1-dependent amyloid processing regulates hypothalamic leptin sensitivity in obese mice

Obesity places an enormous medical and economic burden on society. The principal driver appears to be central leptin resistance with hyperleptinemia. Accordingly, a compound that reverses or prevents leptin resistance should promote weight normalisation and improve glucose homeostasis. The protease Bace1 drives beta amyloid (Aβ) production with obesity elevating hypothalamic Bace1 activity and Aβ₁–₄₂ production. Pharmacological inhibition of Bace1 reduces body weight, improves glucose homeostasis and lowers plasma leptin in diet-induced obese (DIO) mice. These actions are not apparent in ob/ob or db/db mice, indicating the requirement for functional leptin signalling. Decreasing Bace1 activity normalises hypothalamic inflammation, lowers PTP1B and SOCS3 and restores hypothalamic leptin sensitivity and pSTAT3 response in obese mice, but does not affect leptin sensitivity in lean mice. Raising central Aβ₁–₄₂ levels in the early stage of DIO increases hypothalamic basal pSTAT3 and reduces the amplitude of the leptin pSTAT3 signal without increased inflammation. Thus, elevated Aβ₁–₄₂ promotes hypothalamic leptin resistance, which is associated with diminished whole-body sensitivity to exogenous leptin and exacerbated body weight gain in high fat fed mice. These results indicate that Bace1 inhibitors, currently in clinical trials for Alzheimer’s disease, may be useful agents for the treatment of obesity and associated diabetes

Structure-function studies of GDNF and other members of the TGF-beta superfamily

The glial cell line-derived neurotrophic factor (GDNF) family is a distant subclass of the TGF-beta superfamily. The GDNF family of ligands, consisting of GDNF, neurturin (NTN), artemin (ART) and persephin (PSP), are potent survival and differentiation factors for neurons in the central and peripheral nervous systems. These ligands signal through a tyrosine kinase receptor, Ret and an accessory receptor subunit, the GDNF family receptor alphas (GFRas). There are four GFRas known to date (GFRalpha1 to 4). GDNF preferentially binds to GFRalpha1, while NTN, ART, and PSP bind to GFRalpha2, 3 and 4, respectively, but with some degree of cross talk. It has been postulated that the ligand first associates with the GFRalpha and that only then is Ret recruited to the complex, becoming autophosphorylated on several cytoplasmic tyrosine residues. Phosphotyrosine residues on active Ret form a platform for the recruitment of multiple adaptor and effector proteins. There are two main isoforms of Ret, Ret9 and Ret51, differing at their C-terminal sequence. We have assessed the ability of different GDNF mutants to bind to GFRalpha1 and induce tyrosine phosphorylation in Ret. Hydrophobic and negatively charged residues in the tips of GDNF fingers 1 and 2, were found to be important for receptor interaction. Unexpectedly some of the mutants that lost their affinity for GFRalpha1 were still able to induce Ret tyrosine phosphorylation. These mutants however, were not able to activate Ret in cells not expressing GFRalpha1, indicating that GFRalpha1 was still required even if GDNF was unable to bind. These results led us to propose a model including two distinct binding sites for GDNF: one formed by GFRalpha1 alone, requires hydrophobic and acidic residues in finger 1 and 2, and another by a pre-associated GFRalpha1/Ret complex, which requires acidic residues in finger 1. Several mutations have been found in the GDNF gene of patients with Hirschsprung disease (HSCR). We have characterized the effects of these mutants on the ability of GDNF to bind to and activate its receptors. Although none of the four mutations analyzed appeared to affect the ability of GDNF to activate Ret, two of them resulted in a significant reduction in the binding affinity of GDNF for GFRalpha1. Indicating that, although none of the GDNF mutations identified so far in HSCR patients are per se likely to result in HSCR, two of these mutations may, in conjunction with other genetic lesions, contribute to the pathogenesis of this disease. We have demonstrated that Ret51 associates more strongly, than Ret9, with the ubiquitin ligase Cbl, leading to increased ubiquitylation and faster turnover of active Ret51. The association of Cbl with Ret is indirect and mediated through Grb2. A constitutive complex of Grb2 and Cbl can be recruited to both receptor isoforms via docking and tyrosine phosphorylation of Shc. However, Ret 51, but not Ret9, can in addition recruit Cbl via direct interaction of the Grb2/Cbl complex with phosphorylated Tyr-1096, unique to the Ret51. Interestingly, this same phosphotyrosine also allows Ret51 to recruit the adaptor protein CrkL, leading to prolonged activation of MAP kinases ERK1 and ERK2 upon activation of Ret51 in neuronal cells. Our results have therfore established distinct signaling mechanisms by Ret51 and Ret9. Taking advantage of the conserved pattern of cysteine residues in the TGF-beta superfamily, we sought to identify new members of this family, using a novel search engine called Motifer. We identified two genes, provisionally named Motifer Derived Factors (MDF451 and MDF628), in the public human genome database. The MDFs can only be found in genomes of primates and not in other species such as rodents. Both genes are expressed in human fetal brain and cerebellum, but so far we have been unable to isolate full-length cDNA for either of the two MDFs. Further analysis of upstream sequence in MDF628 revealed STOP codons in frame with the TGF-beta like reading frame, thereby ruling out the capacity of this gene to encode a TGF-beta family member. We are suggesting that the genes identified may have recently appeared in evolution, in a common ancestor of the primate lineage, perhaps by duplication of a GDNF/TGF-beta-like gene

Åldersindelad och åldersblandad undervisning : - En jämförande studie ur ett lärar- och ledningsperspektiv för de tidigare åren i skolan

Syftet med vårt examensarbete var att inhämta kunskap kring samt jämföra de olika organisa­tions­formerna åldersblandad och åldersindelad undervisning i år 1-3, med fo­kus på lärar- och led­ningsperspektiv. Våra frågeställningar löd: Vilka för- respek­ti­ve nack­delar finns med åldersblandad undervisning? Vilka för- respektive nackdelar finns med åldersindelad undervisning? Vad, vilka motiv, styr skolans val av organisa­tions­form? Samt vilka konsekvenser leder åldersblandad res­­pek­­tive ål­ders­indelad un­dervisning till för lärarrollen? Vår undersökning grundar sig i en fall­studie av två sko­lor, en F-6 skola i ett mindre samhälle samt en F-3 skola i en mellanstor stad, belägna i två intilliggande län i Mellansverige. Kvali­ta­tiva intervjuer använts som metod. Två rektorer samt nio lärare har inter­vjuats. Resultatet vi­sa­de att det finns en rad olika för- och nackdelar med de båda orga­nisa­tionsformerna, åldersblandad och ålders­in­delad undervisning. Resultatet visade även att det är eko­no­­mi, sviktande elevantal, pe­dagogiska skäl samt konkurrens från andra skolor som styr skolans val av organi­sationsform. Det framkom även att det är skillnader i lära­rens arbetssituation när man jämför de båda organisationsformerna med varandra

Distinct structural elements in GDNF mediate binding to GFRalpha1 and activation of the GFRalpha1-c-Ret receptor complex.

Ligand-induced receptor oligomerization is a widely accepted mechanism for activation of cell-surface receptors. We investigated ligand-receptor interactions in the glial cell-line derived neurotrophic factor (GDNF) receptor complex, formed by the c-Ret receptor tyrosine kinase and the glycosylphosphatidylinositol (GPI)-anchored subunit GDNF family receptor alpha-1 (GFRalpha1). As only GFRalpha1 can bind GDNF directly, receptor complex formation is thought to be initiated by GDNF binding to this receptor. Here we identify an interface in GDNF formed by exposed acidic and hydrophobic residues that is critical for binding to GFRalpha1. Unexpectedly, several GDNF mutants deficient in GFRalpha1 binding retained the ability to bind and activate c-Ret at normal levels. Although impaired in binding GFRalpha1 efficiently, these mutants still required GFRalpha1 for c-Ret activation. These findings support a role for c-Ret in ligand binding and indicate that GDNF does not initiate receptor complex formation, but rather interacts with a pre-assembled GFRalpha1- c-Ret complex

Four Systemic Lupus Erythematosus Subgroups, Defined by Autoantibodies Status, Differ Regarding HLA-DRB1 Genotype Associations and Immunological and Clinical Manifestations.

OBJECTIVE: The heterogeneity of systemic lupus erythematosus (SLE) constitutes clinical and therapeutical challenges. We therefore studied whether unrecognized disease subgroups can be identified by using autoantibody profiling together with HLA-DRB1 alleles and immunological and clinical data. METHODS: An unsupervised cluster analysis was performed based on detection of 13 SLE-associated autoantibodies (double-stranded DNA, nucleosomes, ribosomal P, ribonucleoprotein [RNP] 68, RNPA, Smith [Sm], Sm/RNP, Sjögren's syndrome antigen A [SSA]/Ro52, SSA/Ro60, Sjögren's syndrome antigen B [SSB]/La, cardiolipin [CL]-Immunoglobulin G [IgG], CL-Immunoglobulin M [IgM], and β2 glycoprotein I [β2 GPI]-IgG) in 911 patients with SLE from two cohorts. We evaluated whether each SLE subgroup is associated with HLA-DRB1 alleles, clinical manifestations (n = 743), and cytokine levels in circulation (n = 446). RESULTS: Our analysis identified four subgroups among the patients with SLE. Subgroup 1 (29.3%) was dominated by anti-SSA/Ro60/Ro52/SSB autoantibodies and was strongly associated with HLA-DRB1*03 (odds ratio [OR] = 4.73; 95% confidence interval [CI] = 4.52-4.94). Discoid lesions were more common for this disease subgroup (OR = 1.71, 95% CI = 1.18-2.47). Subgroup 2 (28.7%) was dominated by anti-nucleosome/SmRNP/DNA/RNPA autoantibodies and associated with HLA-DRB1*15 (OR = 1.62, 95% CI = 1.41-1.84). Nephritis was most common in this subgroup (OR = 1.61, 95% CI = 1.14-2.26). Subgroup 3 (23.8%) was characterized by anti-ß2 GPI-IgG/anti-CL-IgG/IgM autoantibodies and a higher frequency of HLA-DRB1*04 compared with the other patients with SLE. Vascular events were more common in Subgroup 3 (OR = 1.74, 95% CI = 1.2-2.5). Subgroup 4 (18.2%) was negative for the investigated autoantibodies, and this subgroup was not associated with HLA-DRB1. Additionally, the levels of eight cytokines significantly differed among the disease subgroups. CONCLUSION: Our findings suggest that four fairly distinct subgroups can be identified on the basis of the autoantibody profile in SLE. These four SLE subgroups differ regarding associations with HLA-DRB1 alleles and immunological and clinical features, suggesting dissimilar disease pathways