Non-cell autonomous and non-catalytic activities of ATX in the developing brain

The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non cell-autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX) as a non cell-autonomous regulator of neural stem cell proliferation. ATX (also known as ENPP2) is best known to catalyze lysophosphatidic acid (LPA) production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA

CCL24 regulates biliary inflammation and fibrosis in primary sclerosing cholangitis

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis

Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1).

Mutations in the depalmitoylating enzyme gene, PPT1, cause the infantile form of Neuronal Ceroid Lipofuscinosis (NCL), an early onset neurodegenerative disease. During recent years there have been different therapeutic attempts including enzyme replacement. Here we show that PPT1 is palmitoylated in vivo and is a substrate for two palmitoylating enzymes, DHHC3 and DHHC7. The palmitoylated protein is detected in both cell lysates and medium. The presence of PPT1 with palmitoylated signal peptide in the cell medium suggests that a subset of the protein is secreted by a nonconventional mechanism. Using a mutant form of PPT1, C6S, which was not palmitoylated, we further demonstrate that palmitoylation does not affect intracellular localization but rather that the unpalmitoylated form enhanced the depalmitoylation activity of the protein. The calculated Vmax of the enzyme was significantly affected by the palmitoylation, suggesting that the addition of a palmitate group is reminiscent of adding a noncompetitive inhibitor. Thus, we reveal the existence of a positive feedback loop, where palmitoylation of PPT1 results in decreased activity and subsequent elevation in the amount of palmitoylated proteins. This positive feedback loop is likely to initiate a vicious cycle, which will enhance disease progression. The understanding of this process may facilitate enzyme replacement strategies

Michaelis-Menten graph of PPT1 and PPT1 C6S.

<p>The Km and Vmax of PPT1 and PPT1 C6S were calculated using the Michaelis-Menten equation following plotting the results of enzymatic activity obtained with different concentrations of the substrate (N = 9) A) Michaelis-Menten graph derived from the cell lysates. B) Michaelis-Menten graph derived from the cell media.</p

PPT1 is palmitoylated by DHHC3 and DHHC7.

<p>A) PPT1 was co-transfected with HA tagged DHHC enzymes to HEK293 cells. The cells were metabolically labeled with 17-ODYA and PPT1 was immunoprecipitated with anti-PPT1 antibodies. A fluorescent tag was introduced by click chemistry and samples were separated by SDS-PAGE. Fluorescent scanning of the gels detected that PPT1 is palmitoylated by DHHD3 and DHHC7 (top panel, boxed). The relative amount of immunoprecipitated PPT1 is shown (middle panel). The expression of the DHHC enzymes in the cell lysates was verified by immunoblotting with anti-HA antibodies (low panel). B) PPT1 was co-transfected with HA-DHHC3 followed by metabolic labeling, immunoprecipitation of PPT1 and click chemistry. One reaction was treated with hydroxylamine and one reaction with PBS. The addition of hydroxylamine eliminated the fluorescent signal of PPT1 (upper panel) demonstrating that the signal is specific for palmitoylation. Similar amounts of PPT1 were immunoprecipitated and expressed (middle panels). The level of HA-DHHC3 expression in the cell lysates was detected by anti-HA antibodies (lower panel). C) PPT1 is palmitoylated on cysteine residue 6 and can be detected in the cell media. Empty vector, PPT1 or PPT1 C6S were co-expressed with HA-DHHC3 in HEK293, followed by metabolic labeling with 17-ODYA, immunoprecipitation and click chemistry. The control treatment results in a low background fluorescent signal probably due to low levels of expression of endogenous PPT1. The expression and amount of immunoprecipitated PPT1 and PPT1 C6S are similar as detected by immunoblotting with anti-PPT1 antibodies (middle panels). The expression of HA-DHHC3 in was detected by immunoblotting with anti-HA antibodies (lower panel). D) The fluorescence signal intensity for PPT1 and PPT1 C6S were quantified and normalized relative to the amount of immunoprecipitated PPT1 using four independent repeats. The fluorescent signal of PPT1 signal is almost five fold higher than that observed with PPT1 C6S. E) Palmitoylated PPT1 can be detected in cells and media and the signal is reduced following hydroxylamine treatment. PPT1 was co-expressed with HA-DHHC3 in HEK293 cells followed by metabolic labeling both cells and media were collected and PPT1 was immunoprecipitated and labeled using click chemistry. The fluorescent signal of PPT1 palmitoylation is shown in the upper panel. This signal is eliminated by the addition of hydroxylamine. The amount of immunoprecipitated PPT1 and the level of expression in the lysates are shown in the middle panels and in both cell and media by immunoblotting with anti-PPT1 antibodies. The expression of HA-DHHC3 was detected by immunoblotting with anti-HA antibodies in the lower panel. E) Palmitoylation of endogenous PPT1 in human embryonic stem cells. WIBR3 wildtype and 6C, PPT1 knockout clone generated using CRISPR/Cas9 were metabolically labeled with 17-ODYA, followed by immunoprecipitation of PPT1 and click chemistry. We could distinguish fluorescent band appeared in the–HA treatment, that band could be eliminated by hydroxylamine (+HA). The band did not appear in negative control of immunoprecipitation with beads only (-mAB) or in the immunoprecipitation from the PPT1 knockout 6C clone. The lower panel confirms that PPT1 was immunoprecipitated using Western blot.</p

Proteomics insights into infantile neuronal ceroid lipofuscinosis (CLN1) point to the involvement of cilia pathology in the disease

Mutations in the depalmitoylation enzyme, palmitoyl protein thioesterase (PPT1), result in the early onset neurodegenerative disease known as Infantile Neuronal Ceroid Lipofuscinosis. Here, we provide proteomic evidence suggesting that PPT1 deficiency could be considered as a ciliopathy. Analysis of membrane proteins from brain enriched for acylated proteins from neonate Ppt1 knock out and control mice revealed a list of 88 proteins with differential expression levels. Amongst them, we identified Rab3IP, which regulates ciliogenesis in concert with Rab8 and Rab11. Immunostaining analysis revealed that PPT1 is localized in the cilia. Indeed, an unbiased proteomics analysis on isolated cilia revealed 660 proteins, which differed in their abundance levels between wild type and Ppt1 knock out. We demonstrate here that Rab3IP, Rab8 and Rab11 are palmitoylated, and that palmitoylation of Rab11 is required for correct intracellular localization. Cells and brain preparations from Ppt1-/- mice exhibited fewer cells with cilia and abnormally longer cilia, with both acetylated tubulin and Rab3IP wrongly distributed along the length of cilia. Most importantly, the analysis revealed a difference in the distribution and levels of the modified proteins in cilia in the retina of mutant mice versus the wildtype, which may be important in the early neurodegenerative phenotype. Overall, our results suggest a novel link between palmitoylated proteins, cilial organization and the pathophysiology of Neuronal Ceroid Lipofuscinosis