Kif13b Regulates PNS and CNS Myelination Through the Dlg1 Scaffold

Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS

Niacin‐mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination

Abstract Charcot–Marie–Tooth (CMT) neuropathies are highly heterogeneous disorders caused by mutations in more than 70 genes, with no available treatment. Thus, it is difficult to envisage a single suitable treatment for all pathogenetic mechanisms. Axonal Neuregulin 1 (Nrg1) type III drives Schwann cell myelination and determines myelin thickness by ErbB2/B3‐PI3K–Akt signaling pathway activation. Nrg1 type III is inhibited by the α‐secretase Tace, which negatively regulates PNS myelination. We hypothesized that modulation of Nrg1 levels and/or secretase activity may constitute a unifying treatment strategy for CMT neuropathies with focal hypermyelination as it could restore normal levels of myelination. Here we show that in vivo delivery of Niaspan, a FDA‐approved drug known to enhance TACE activity, efficiently rescues myelination in the Mtmr2−/− mouse, a model of CMT4B1 with myelin outfoldings, and in the Pmp22+/− mouse, which reproduces HNPP (hereditary neuropathy with liability to pressure palsies) with tomacula. Importantly, we also found that Niaspan reduces hypermyelination of Vim (vimentin)−/− mice, characterized by increased Nrg1 type III and Akt activation, thus corroborating the hypothesis that Niaspan treatment downregulates Nrg1 type III signaling

Combined gene/cell therapies provide long-term and pervasive rescue of multiple pathological symptoms in a murine model of globoid cell leukodystrophy

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by deficient activity of β-galactocerebrosidase (GALC). The infantile forms manifest with rapid and progressive central and peripheral demyelination, which represent a major hurdle for any treatment approach. We demonstrate here that neonatal lentiviral vector-mediated intracerebral gene therapy (IC GT) or transplantation of GALC-overexpressing neural stem cells (NSC) synergize with bone marrow transplant (BMT) providing dramatic extension of lifespan and global clinical-pathological rescue in a relevant GLD murine model. We show that timely and long-lasting delivery of functional GALC in affected tissues ensured by the exclusive complementary mode of action of the treatments underlies the outstanding benefit. In particular, the contribution of neural stem cell transplantation and IC GT during the early asymptomatic stage of the disease is instrumental to enhance long-term advantage upon BMT. We clarify the input of central nervous system, peripheral nervous system and periphery to the disease, and the relative contribution of treatments to the final therapeutic outcome, with important implications for treatment strategies to be tried in human patients. This study gives proof-of-concept of efficacy, tolerability and clinical relevance of the combined gene/cell therapies proposed here, which may constitute a feasible and effective therapeutic opportunity for children affected by GL

Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathy

Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P-2, with a preference for PtdIns(3,5)P-2. A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P-2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P-2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth

Marker-independent Method for Isolating Slow-Dividing Cancer Stem Cells in Human Glioblastoma

Glioblastoma (GBM) is a devastating brain tumor with a poor survival outcome. It is generated and propagated by a small subpopulation of rare and hierarchically organized cells that share stem-like features with normal stem cells but, however, appear dysregulated in terms of self-renewal and proliferation and aberrantly differentiate into cells forming the bulk of the disorganized cancer tissues. The complexity and heterogeneity of human GBMs underlie the lack of standardized and effective treatments. This study is based on the assumption that available markers defining cancer stem cells (CSCs) in all GBMs are not conclusive and further work is required to identify the CSC. We implemented a method to isolate CSCs independently from cell surface markers: four patient-derived GBM neurospheres containing stem, progenitors, and differentiated cells were labeled with PKH-26 fluorescent dye that reliably selects for cells that divide at low rate. Through in vitro and in vivo assays, we investigated the growth and self-renewal properties of the two different compartments of high- and slow-dividing cells. Our data demonstrate that only slow-dividing cells retain the ability of a long-lasting self-renewal capacity after serial in vitro passaging, while high-dividing cells eventually exhaust. Moreover, orthotopic transplantation assay revealed that the incidence of tumors generated by the slow-dividing compartment is significantly higher in the four patient-derived GBM neurospheres analyzed. Importantly, slow-dividing cells feature a population made up of homogeneous stem cells that sustain tumor growth and therefore represent a viable target for GBM therapy development

p38γ MAPK acts in complex with Kif13b and Dlg1 in the CNS.

<p>(A) Dlg1 and p38γ co-immunoprecipitate from rat optic nerve lysates at P11. Two independent experiments. (B) Kif13b/MBS-GST pulls down Dlg1 and p38γ from rat optic nerve lysates at P11. Two independent experiments. (C,D) p38γ expression levels are reduced in <i>Kif13b</i>^<i>Fl/-</i> <i>CNP-Cre</i> optic nerve and spinal cord lysates at P30, with quantification, <i>p</i> = 0.0005, <i>n</i> = 4 animals per genotype, representative of four independent experiments. (E) p38γ expression is not reduced in <i>Dlg1</i>^<i>Fl/Fl</i> <i>CNP-Cre</i> spinal cords at P30.</p

Kif13b regulates myelin thickness in oligodendrocytes.

<p>(A) RT-PCR analysis on optic nerves shows reduction of <i>Kif13b</i> mRNA in the mutant (<i>n</i> = 5, <i>p</i> = 0.0004). (B) Western blot analysis on lysates from corpus callosum at P20 shows reduction of Kif13b protein expression in the mutant. (C) Ultrathin analysis and quantification of the g-ratio as a function of axonal diameter in <i>Kif13b</i>^<i>Fl/-</i> <i>CNP-Cre</i> and control optic nerves at P30. Red asterisks indicate fibers with similar diameter to be compared in the two genotypes (<i>Kif13b</i>^<i>Fl/-</i> <i>CNP-Cre</i>, 0.792 ± 0.005, 296 fibers; <i>Kif13b</i>^<i>Fl/+</i>, 0.814 ± 0.006, 345 fibers, <i>n</i> = 4 animals per genotype, <i>p</i> = 0.030; <i>Kif13b</i>^<i>+/-</i> 0.813 ± 0.004, 435 fibers, <i>n</i> = 5 animals per genotype). (D) Ultrathin analysis and quantification of the g-ratio as a function of axonal diameter in <i>Kif13b</i>^<i>Fl/-</i> <i>CNP-Cre</i> and control spinal cords at P30 (<i>Kif13b</i>^<i>Fl/-</i> <i>CNP-Cre</i>, 0.785 ± 0.003, 364 fibers; <i>Kif13b</i>^<i>Fl/+</i>, 0.803 ± 0.007, 403 fibers, <i>n</i> = 5 animals per genotype, <i>p</i> = 0.016). (E) Western blot analysis on lysates from optic nerves and spinal cords at P30 shows increased AKT phosphorylation at S473 in the mutant. Three independent experiments. Quantification of AKT phosphorylation levels from spinal cord lysates, <i>p</i> = 0.005, <i>n</i> = 3. (F) Pull down assay from P11 rat optic nerves using GST-Kif13b/MBS as a bait indicates Dlg1 and Kif13b interaction. Three independent experiments. (G) Western blot analysis on optic nerve and spinal cord lysates shows increased Dlg1 expression in <i>Kif13b</i> mutant oligodendrocytes. Three independent experiments. Bar in (C) and (D) is 1 μm.</p

Kif13b negatively regulates Dlg1 expression and activity.

<p>(A) Immunoprecipitation of Dlg1 from <i>Kif13b</i>^<i>Fl/Fl</i> <i>P0-Cre</i> nerves and controls at P20 followed by western blot analysis using an anti-phospho-serine antibody shows that Dlg1 is less phosphorylated in the mutant. Two independent experiments. (B) Immunoprecipitation of Dlg1 from <i>Dlg1</i>^<i>Fl/Fl</i> <i>P0-Cre</i> nerves indicates that the band at 140–150 KDa is Dlg1. The residual Dlg1 protein in mutant nerves could result from non-recombined fibroblasts within the nerve as described in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref009" target="_blank">9</a>]. (C) Immunoprecipitation of Dlg1 at P4 followed by western blot analysis shows that Dlg1 is less ubiquitinated in mutant nerves. Two independent experiments. (D) G-ratio value distribution shows that haploinsufficiency of <i>Dlg1</i> in the <i>Kif13b</i>^<i>Fl/Fl</i> <i>P0-Cre</i> background rescues the hypomyelination, <i>p</i> = 0.0093 and <i>p</i> = 0.0077, <i>n</i> = 4 animals per genotype, semithin section analysis. (E) Western blot analysis shows restored Dlg1 expression and AKT phosphorylation levels in <i>Kif13b</i>^<i>Fl/Fl</i>//<i>Dlg1</i>^<i>Fl/+</i>; <i>P0-Cre</i> nerve lysates, with quantification in (F), <i>p</i> = 0.0004 (Dlg1) and <i>p</i> = 0.020 (p-Akt), <i>n</i> = 3. The genetic reduction of 50% of Dlg1 is sufficient to reduce Dlg1 expression level in lysates from <i>Dlg1</i>^<i>Fl/+</i> <i>P0-Cre</i>.</p

Kif13b regulates myelination in PNS and CNS through the Dlg1 scaffold.

<p>The PI3K-AKT-mTOR signaling axis is one of the signaling pathways regulating myelination in both PNS and CNS, as recently reviewed [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref049" target="_blank">49</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref056" target="_blank">56</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref064" target="_blank">64</a>]. Our results suggest that in Schwann cells, Kif13b is a positive regulator of myelination. Kif13b promotes p38γ MAPK-mediated Dlg1 phosphorylation and ubiquitination. Dlg1, in complex with PTEN, is known to reduce AKT activation and thus negatively regulates myelination. Dlg1 loss is associated with increased myelin thickness and myelin outfoldings, as a result of increased PIP₃ and AKT phosphorylation levels [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref008" target="_blank">8</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref009" target="_blank">9</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002440#pbio.1002440.ref047" target="_blank">47</a>]. On the contrary, in oligodendrocytes, loss of Kif13b-mediated negative regulation of Dlg1 and the consequent increase in Dlg1 levels are associated with transient hypermyelination. Of note, we found that in the CNS Dlg1 is a promoter and not an inhibitor of myelination, and it likely modulates PI3K class I activity.</p