Differential Inhibition of LRRK2 in Parkinson's Disease Patient Blood by a G2019S Selective LRRK2 Inhibitor.

BACKGROUND: A common genetic mutation that causes Parkinson's disease (PD) is the G2019S LRRK2 mutation. A precision medicine approach that selectively blocks only excess kinase activity of the mutant allele could yield a safe and effective treatment for G2019S LRRK2 PD. OBJECTIVE: To determine the activity of a G2019S mutant selective leucine-rich repeat kinase 2 (LRRK2) kinase inhibitor as compared to a nonselective inhibitor in blood of subjects with genetic and idiopathic PD on two LRRK2 biomarkers, pSer935 LRRK2 and pThr73 Rab10. METHODS: Blood was collected from 13 subjects with or without a G2019S LRRK2 mutation with PD and one healthy control. Peripheral blood mononuclear cells were treated ex vivo with a novel G2019S LRRK2 inhibitor (EB-42168) or the nonselective inhibitor MLi-2. Quantitative western immunoblot analyses were performed. RESULTS: EB-42168 was 100 times more selective for G2019S LRRK2 when compared to wild-type (WT) LRRK2. Concentrations that inhibited phosphorylation of pSer935 LRRK2 by 90% in homozygous G2019S LRRK2 patients, inhibited pSer935 LRRK2 by 36% in heterozygous patients, and by only 5% in patients carrying only the WT allele. Similar selectivity was seen for pThr73 Rab10. MLi-2 showed an equivalent level of inhibition across all genotypes. CONCLUSIONS: These findings demonstrate that EB-42168, a G2019S LRRK2 selective inhibitor, lowers mutant G2019S LRRK2 phosphorylated biomarkers while simultaneously sparing WT LRRK2. Selective targeting of G2019S LRRK2 with a small molecule lays the foundation for a precision medicine treatment of G2019S LRRK2 PD. © 2021 ESCAPE Bio, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

R-spondin 1 protects against inflammatory bone damage during murine arthritis by modulating the Wnt pathway.

OBJECTIVE: During the course of different musculoskeletal diseases, joints are progressively damaged by inflammatory, infectious, or mechanical stressors, leading to joint destruction and disability. While effective strategies to inhibit joint inflammation, such as targeted cytokine-blocking therapy, have been developed during the last decade, the molecular mechanisms of joint damage are still poorly understood. This study was undertaken to investigate the role of the Wnt pathway modulator R-Spondin 1 (RSpo1) in protecting bone and cartilage in a mouse model of arthritis. METHODS: Tumor necrosis factor alpha (TNFalpha)-transgenic mice were treated with vehicle or Rspo1. Mice were evaluated for signs of arthritis, and histologic analysis of the hind paws was performed. Moreover, we determined the effect of Rspo1 on Wnt signaling activity and osteoprotegerin (OPG) expression in murine primary osteoblasts. RESULTS: The secreted Wnt pathway modulator RSpo1 was highly effective in preserving the structural integrity of joints in a TNFalpha-transgenic mouse model of arthritis by protecting bone and cartilage from inflammation-related damage. RSpo1 antagonized the Wnt inhibitor Dkk-1 and modulated Wnt signaling in mouse mesenchymal cells. In osteoblasts, RSpo1 induced differentiation and expression of OPG, thereby inhibiting osteoclastogenesis in vitro. In vivo, RSpo1 promoted osteoblast differentiation and bone formation while blocking osteoclast development, thereby contributing to the integrity of joints during inflammatory arthritis. CONCLUSION: Our results demonstrate the therapeutic potential of RSpo1 as an anabolic agent for the preservation of joint architecture.

The first propeller domain of LRP6 regulates sensitivity to DKK1.

The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.

R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6.

The R-Spondin (RSpo) family of secreted proteins act as potent activators of the Wnt/beta-catenin signaling pathway. We have previously shown that RSpo proteins can induce proliferative effects on the gastrointestinal epithelium in mice. Here we provide a mechanism whereby RSpo1 regulates cellular responsiveness to Wnt ligands by modulating the cell-surface levels of the coreceptor LRP6. We show that RSpo1 activity critically depends on the presence of canonical Wnt ligands and LRP6. Although RSpo1 does not directly activate LRP6, it interferes with DKK1/Kremen-mediated internalization of LRP6 through an interaction with Kremen, resulting in increased LRP6 levels on the cell surface. Our results support a model in which RSpo1 relieves the inhibition DKK1 imposes on the Wnt pathway.

PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif.

An emerging series of papers has identified new receptor proteins that predict seven-transmembrane pass topologies. We have consolidated this family to 11 human genes and have named the family PAQR, after two of the initially described ligands (progestin and adipoQ receptors). This protein family has ancient evolutionary roots, with identified homologs found in eubacteria. To date, published data indicate that the prokaryotic members of this family appear to encode hemolysin-type proteins, while in eukaryotes, PAQR proteins encode functional receptors with a broad range of apparent ligand specificities. We provide the complete human and mouse complement of this family, suggest a conserved structure/topology with invariant intracellular amino acid residues, and have measured mRNA expression levels for these genes across a range of human tissues.

The complete complement of C1q-domain-containing proteins in Homo sapiens.

The C-terminal domains of the A, B, C chains of C1q subcomponent of C1 complex represent a common structural motif, the C1q domain, that is found in a diverse range of proteins. We analyzed the human genome for the complete complement of this family and have identified a total of 31 independent gene sequences. The predominant organization of C1q-domain-containing (C1qDC) proteins includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 15 highly conserved residues within the C1q domain, among which 8 are invariant within the human gene set and these are predicted to cluster within the hydrophobic core of the protein. We suggest a 3-subfamily classification based on sequence homology. For some C1qDC-encoding genes, strict orthology has been retained throughout vertebrate evolution and these examples suggest a highly specific functional role for C1qDC proteins that has been under significant selective pressure. Alternatively, individual species have co-opted C1qDC proteins for roles that are highly specific to their biology, suggesting an evolutionary strategy of gene duplication and functional diversification. A more extensive analysis of the evolutionary relationship of C1qDC proteins reveals an ancient rooting, with clear members found in eubacterial species. Curiously, we have been unable to identify C1qDC-encoding genes in many eukaryotic genomcs, such as Sacchromyces cerivisae and C. elegans, suggesting that the retention or loss of this gene family throughout evolution has been sporadic.

Three new human members of the lipid transfer/lipopolysaccharide binding protein family (LT/LBP).

We have identified three novel, rarely expressed human genes that encode new members of the lipid transfer/lipopolysaccharide binding protein (LT/LBP) gene family based on sequence homology. BPI and other members of the LT/LBP family are structurally related proteins capable of binding phospholipids and lipopolysaccharides. Real-time PCR studies indicate that BPIL1 and BPIL3 are highly expressed in hypertrophic tonsils. In situ hybridization analysis of BPIL2 shows prominent expression in skin specimens from psoriasis patients. BPIL1 and BPIL3 map to Chromosome 20q11; thus, these novel genes form a cluster with BPI and two other members of the LT/LBP gene family on the long arm of human Chr 20. BPIL2maps to Chr 22q13. The exon/intron organization of all three genes is highly conserved with that of BPI, suggesting evolution from a common ancestor.