Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development.

During the later stages of lung development, two types of pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) alveolar epithelial cells, form distal lung saccules. Here, we highlight how fibroblasts expressing MAP-microtubule affinity regulating kinase 1 (Mark1) are required for the terminal stages of pulmonary development, called lung sacculation. In Mark1-knockout (KO) mice, distal sacculation and AECI flattening are significantly impaired. Fetal epithelial cells generate alveolar organoids and differentiate into pneumocytes when co-cultured with fibroblasts. However, the size of organoids decreased and AECI flattening was impaired in the presence of Mark1 KO fibroblasts. In Mark1 KO fibroblasts themselves, cilia formation and the Hedgehog pathway were suppressed, resulting in the loss of type I collagen expression. The addition of type I collagen restored AECI flattening in organoids co-cultured with Mark1 KO fibroblasts and rescued the decreased size of organoids. Mathematical modeling of distal lung sacculation supports the view that AECI flattening is necessary for the proper formation of saccule-like structures. These results suggest that Mark1-mediated fibroblast activation induces AECI flattening and thereby regulates distal lung sacculation.

Modulation of apical constriction by Wnt signaling is required for lung epithelial shape transition.

In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicular-saccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages.

[Canonical Wnt signaling pathway and current issues.]

The Wnt signaling pathways are classified into the ß-catenin dependent pathway, which regulates gene expression through ß-catenin, and the ß-catenin independent pathway, which does cytoskeletal rearrangement in a ß-catenin independent manner. The former is also called as the canonical Wnt signaling pathway and extensively studied in development, tumorigenesis, and regenerative research. Recently, novel mechanistic insights into the canonical Wnt signaling pathway have been clarified through the analysis of structure of Wnt-receptor complex, regulation of Wnt signaling at the cell surface membrane, and intracellular protein complex of Wnt complexes.

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations.

ALS-associated mutations in FUS disrupt the axonal distribution and function of SMN.

Mutations in the RNA binding protein fused in sarcoma/translated in liposarcoma (FUS/TLS) cause amyotrophic lateral sclerosis (ALS). Although ALS-linked mutations in FUS often lead to a cytosolic mislocalization of the protein, the pathogenic mechanisms underlying these mutations remain poorly understood. To gain insight into these mechanisms, we examined the biochemical, cell biological and functional properties of mutant FUS in neurons. Expression of different FUS mutants (R521C, R521H, P525L) in neurons caused axonal defects. A protein interaction screen performed to explain these phenotypes identified numerous FUS interactors including the spinal muscular atrophy (SMA) causing protein survival motor neuron (SMN). Biochemical experiments showed that FUS and SMN interact directly and endogenously, and that this interaction can be regulated by FUS mutations. Immunostaining revealed co-localization of mutant FUS aggregates and SMN in primary neurons. This redistribution of SMN to cytosolic FUS accumulations led to a decrease in axonal SMN. Finally, cell biological experiments showed that overexpression of SMN rescued the axonal defects induced by mutant FUS, suggesting that FUS mutations cause axonal defects through SMN. This study shows that neuronal aggregates formed by mutant FUS protein may aberrantly sequester SMN and concomitantly cause a reduction of SMN levels in the axon, leading to axonal defects. These data provide a functional link between ALS-linked FUS mutations, SMN and neuronal connectivity and support the idea that different motor neuron disorders such as SMA and ALS may be caused, in part, by defects in shared molecular pathways.

Binding of APC and dishevelled mediates Wnt5a-regulated focal adhesion dynamics in migrating cells.

Wnt5a is a representative ligand that activates the Wnt/beta-catenin-independent pathway, resulting in the regulation of cell adhesion, migration, and polarity, but its molecular mechanism is poorly understood. This report shows that Dishevelled (Dvl) binds to adenomatous polyposis coli (APC) gene product, and this binding is enhanced by Wnt5a. Dvl was involved in the stabilization of the plus end dynamics of microtubules as well as APC. Frizzled2 (Fz2) was present with Wnt5a at the leading edge of migrating cells and formed a complex with APC through Dvl. Fz2 also interacted with integrins at the leading edge, and Dvl and APC associated with and activated focal adhesion kinase and paxillin. The binding of APC to Dvl enhanced the localization of paxillin to the leading edge and was involved in Wnt5a-dependent focal adhesion turnover. Furthermore, this new Wnt5a signalling pathway was important for the epithelial morphogenesis in the three-dimensional culture. These results suggest that the functional and physical interaction of Dvl and APC is involved in Wnt5a/Fz2-dependent focal adhesion dynamics during cell migration and epithelial morphogenesis.

Wnt5a signaling controls cytokinesis by correctly positioning ESCRT-III at the midbody.

Wnts activate at least two signaling pathways, the ß-catenin-dependent and -independent pathways. Although the ß-catenin-dependent pathway is known to contribute to G1-S transition, involvement of the ß-catenin-independent pathway in cell cycle regulation remains unclear. Here, we show that Wnt5a signaling, which activates the ß-catenin-independent pathway, is required for cytokinesis. Dishevelled 2 (Dvl2), a mediator of Wnt signaling pathways, was localized to the midbody during cytokinesis. Beside the localization of Dvl2, Fz2, a Wnt receptor, was detected in the midbody with the endosomal sorting complex required for transport III (ESCRT-III) subunit, CHMP4B. Depletion of Wnt5a, its receptors, and Dvl increased multinucleation. The phenotype observed in Wnt5a-depleted cells was rescued by the addition of purified Wnt5a but not Wnt3a, which is a ligand for the ß-catenin-dependent pathway. Moreover, depletion of Wnt5a signaling caused loss of stabilized microtubules and mislocalization of CHMP4B at the midbody, which affected abscission. Inhibition of the stabilization of microtubules at the midbody led to the mislocalization of CHMP4B, while depletion of CHMP4B did not affect the stabilization of microtubules, suggesting that the correct localization of CHMP4B depends on microtubules. Fz2 was localized to the midbody in a Rab11-dependent manner, probably along stabilized microtubules. Fz2 formed a complex with CHMP4B upon Wnt5a stimulation and was required for proper localization of CHMP4B at the midbody, while CHMP4B was not necessary for the localization of Fz2. These results suggest that Wnt5a signaling positions ESCRT-III in the midbody properly for abscission by stabilizing midbody microtubules.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis.

OBJECTIVE: Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD. METHODS: We reviewed all previous studies on ANG in ALS and performed sequence experiments on additional samples, which allowed us to analyze data from 6,471 ALS patients and 7,668 controls from 15 centers (13 from Europe and 2 from the USA). We sequenced DNA samples from 3,146 PD patients from 6 centers (5 from Europe and 1 from the USA). Statistical analysis was performed using the variable threshold test, and the Mantel-Haenszel procedure was used to estimate odds ratios. RESULTS: Analysis of sequence data from 17,258 individuals demonstrated a significantly higher frequency of ANG variants in both ALS and PD patients compared to control subjects (p = 9.3 × 10(-6) for ALS and p = 4.3 × 10(-5) for PD). The odds ratio for any ANG variant in patients versus controls was 9.2 for ALS and 6.7 for PD. INTERPRETATION: The data from this multicenter study demonstrate that there is a strong association between PD, ALS, and ANG variants. ANG is a genetic link between ALS and PD.

Axin localizes to the centrosome and is involved in microtubule nucleation.

Axin is known to have an important role in the degradation of beta-catenin in the Wnt pathway. Here, we reveal a new function of Axin at the centrosome. Axin was localized to the centrosome in various cell lines and formed a complex with gamma-tubulin. Knockdown of Axin reduced the localization of gamma-tubulin and gamma-tubulin complex protein 2-components of the gamma-tubulin ring complex-to the centrosome and the centrosomal microtubule nucleation activity after treatment with nocodazole. These phenotypes could not be rescued by the reduction in the levels of beta-catenin. Although the expression of Axin rescued these phenotypes in Axin-knockdown cells, overexpression of Axin2, which is highly homologous to Axin, could not. Axin2 was also localized to the centrosome, but it did not form a complex with gamma-tubulin. These results suggest that Axin, but not Axin2, is involved in microtubule nucleation by forming a complex with gamma-tubulin at the centrosome.

CKAP4 Regulates Cell Migration via the Interaction with and Recycling of Integrin.

Cytoskeleton-associated protein 4 (CKAP4) is an endoplasmic reticulum protein that is also present in the cell surface membrane, where it acts as a receptor for Dickkopf1 (DKK1). In this study, we found that CKAP4 interacts with ß1 integrin and controls the recycling of α5ß1 integrin independently of DKK1. In S2-CP8 cells, knockdown of CKAP4 but not DKK1 enlarged the size of cell adhesion sites and enhanced cell adhesion to fibronectin, resulting in decreased cell migration. When CKAP4 was depleted, the levels of α5 but not ß1 integrin were increased in the cell surface membrane. A similar phenotype was observed in other cells expressing low levels of DKK1. In S2-CP8 cells, α5 integrin was trafficked with ß1 integrin and CKAP4 to the lysosome or recycled with ß1 integrin. In CKAP4-depleted cells, the internalization of α5ß1 integrin was unchanged, but its recycling was upregulated. Knockdown of sorting nexin 17 (SNX17), a mediator of integrin recycling, abrogated the increased α5 integrin levels caused by CKAP4 knockdown. CKAP4 bound to SNX17, and its knockdown enhanced the recruitment of α5ß1 integrin to SNX17. These results suggest that CKAP4 suppresses the recycling of α5ß1 integrin and coordinates cell adhesion sites and migration independently of DKK1.

CKAP4, a DKK1 Receptor, Is a Biomarker in Exosomes Derived from Pancreatic Cancer and a Molecular Target for Therapy.

PURPOSE: The survival rate of pancreatic ductal adenocarcinoma (PDAC) is poor; thus, novel molecularly targeted therapy and companion diagnostics are required. We asked whether cytoskeleton-associated protein 4 (CKAP4), a novel Dickkopf1 (DKK1) receptor, is a candidate for PDAC diagnosis and therapy.Experimental Design: Whether CKAP4 can be secreted with small extracellular vesicles (SEV) from PDAC cells was examined. It was also investigated whether CKAP4 can be detected in sera from patients with PDAC by ELISA using newly generated anti-CKAP4 mAbs and whether anti-CKAP4 mAbs can show antitumor activity in vivo. RESULTS: CKAP4 was secreted with SEVs from PDAC cells, and the SEVs exhibited the characteristics of exosomes. The secretion of CKAP4-containing exosomes was mediated by DKK1-dependent endocytosis routes and required exosome biogenesis molecules. Two ELISAs capable of detecting tumor-secreted CKAP4 were developed. The serum CKAP4 levels were higher in patients with PDAC than healthy control individuals. CKAP4 was highly detected in the sera of pancreatic tumor-bearing xenografted mice and patients with PDAC, whereas CKAP4 was barely detectable in sera from normal mice and postoperative patients. Anti-CKAP4 mAbs with different epitopes demonstrated the inhibitory activities for the binding of DKK1 and CKAP4, AKT activity, and proliferation and migration of PDAC cells. Anti-CKAP4 mAbs also suppressed xenograft tumor formation in immunodeficient mice and extended the survival of mice receiving intraperitoneal or orthotopic injection of PDAC cells. CONCLUSIONS: CKAP4 secreted in exosomes may represent a biomarker for PDAC. Anti-CKAP4 mAbs can contribute to the development of novel diagnostic methods and therapeutics.

Activation of the Dickkopf1-CKAP4 pathway is associated with poor prognosis of esophageal cancer and anti-CKAP4 antibody may be a new therapeutic drug.

Aberrant expression of the secretory protein Dickkopf1 (DKK1) is associated with poor prognosis of esophageal squamous cell carcinoma (ESCC), but the underlying mechanism of how DKK1 is involved in aggressiveness of ESCC is not clear. In this study, we show that cytoskeleton-associated protein 4 (CKAP4) functions as a DKK1 receptor in ESCC cells. Immunohistochemical analyses of ESCC revealed that both DKK1 and CKAP4 are minimally expressed in associated normal esophageal squamous mucosa of non-tumor regions, but strongly expressed in tumor lesions. Forty-six of 119 cases (38.7%) were positive for both DKK1 and CKAP4. Those expressing both proteins showed poor prognosis and relapse-free survival. Multivariate analysis demonstrated that expression of both proteins was the poor prognostic factor. The Cancer Genome Atlas data set indicated that the mRNA levels of DKK1 and CKAP4 are significantly elevated in the tumor lesions compared to non-tumor regions. DKK1 bound to CKAP4 at endogenous levels. DKK1 induced the internalization of CKAP4 from and its recycling to the plasma membrane. AKT was activated in ESCC cells in which DKK1 was highly expressed and CKAP4 was localized to the plasma membrane. Knockdown of either DKK1 or CKAP4 inhibited AKT activity and cell proliferation in vitro and xenograft tumor formation. Wild-type CKAP4 or DKK1, but not a DKK1 mutant that was unable to bind to CKAP4, rescued phenotypes induced by CKAP4 or DKK1 knockdown, respectively. The anti-CKAP4 antibody also inhibited AKT activity and suppressed xenograft tumor formation. In contrast, in ESCC cells in which DKK1 was marginally expressed, knockdown of CKAP4 or anti-CKAP4 antibody affected neither AKT activity nor cell proliferation. These findings suggest that the DKK1-CKAP4 pathway promotes ESCC cell proliferation and that CKAP4 might represent a novel therapeutic target for ESCCs expressing both DKK1 and CKAP4.

p63-Dependent Dickkopf3 Expression Promotes Esophageal Cancer Cell Proliferation via CKAP4.

Dickkopf3 (DKK3) is a secretory protein that belongs to the DKK family, but exhibits structural divergence from other family members, and its corresponding receptors remain to be identified. Although DKK3 has been shown to have oncogenic functions in certain cancer types, the underlying mechanism by which DKK3 promotes tumorigenesis remains to be clarified. We show here that DKK3 stimulates esophageal cancer cell proliferation via cytoskeleton-associated protein 4 (CKAP4), which acts as a receptor for DKK3. DKK3 was expressed in approximately 50% of tumor lesions of esophageal squamous cell carcinoma (ESCC) cases; simultaneous expression of DKK3 and CKAP4 was associated with poor prognosis. Anti-CKAP4 antibody inhibited both binding of DKK3 to CKAP4 and xenograft tumor formation induced by ESCC cells. p63, a p53-related transcriptional factor frequently amplified in ESCC, bound to the upstream region of the DKK3 gene. Knockdown of p63 decreased DKK3 expression in ESCC cells, and reexpression of DKK3 partially rescued cell proliferation in p63-depleted ESCC cells. Expression of ΔNp63α and DKK3 increased the size of tumor-like esophageal organoids, and anti-CKAP4 antibody inhibited growth of esophageal organoids. Taken together, these results suggest that the DKK3-CKAP4 axis might serve as a novel molecular target for ESCC.Significance: In esophageal cancer, findings identify DKK3 as a poor prognostic indicator and demonstrate CKAP4 inhibition as an effective therapeutic strategy. Cancer Res; 78(21); 6107-20. ©2018 AACR.

The Dickkopf1-cytoskeleton-associated protein 4 axis creates a novel signalling pathway and may represent a molecular target for cancer therapy.

Dickkopf 1 (DKK1) is a secreted protein and antagonizes oncogenic Wnt signalling by binding to the Wnt co-receptor, low-density lipoprotein receptor-related protein 6. DKK1 has also been suggested to regulate its own signalling, associated with tumour aggressiveness. However, the underlying mechanism by which DKK1 promotes cancer cell proliferation has remained to be clarified for a long time. The cytoskeleton-associated protein 4 (CKAP4), originally identified as an endoplasmic reticulum membrane protein, was recently found to act as a novel DKK1 receptor. DKK1 stimulates cancer cell proliferation when CKAP4 is expressed on the cell surface membrane. Although there are no tyrosine residues in the intracellular region of CKAP4, CKAP4 forms a complex with PI3K upon the binding of DKK1, leading to the activation of Akt. Both DKK1 and CKAP4 are frequently expressed in pancreatic and lung tumours, and their simultaneous expression is negatively correlated with prognosis. Knockdown of CKAP4 in cancer cells and treatment of mice with the anti-CKAP4 antibody inhibit Akt activity in cancer cells and suppress xenograft tumour formation, suggesting that CKAP4 may represent a therapeutic target for cancers expressing both DKK1 and CKAP4. This review will provide details of the novel DKK1-CKAP4 signalling axis that promotes cancer proliferation and discuss the possibility of targeting this pathway in future cancer drug development. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

CKAP4 is a Dickkopf1 receptor and is involved in tumor progression.

Dickkopf1 (DKK1) is a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). DKK1 may also regulate its own signaling to promote cancer cell proliferation, but the mechanism is not understood. Here, we identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. We determined that DKK1 binds CKAP4 and LRP6 with similar affinity but interacts with these 2 receptors with different cysteine-rich domains. DKK1 induced internalization of CKAP4 in a clathrin-dependent manner, further supporting CKAP4 as a receptor for DKK1. DKK1/CKAP4 signaling activated AKT by forming a complex between the proline-rich domain of CKAP4 and the Src homology 3 domain of PI3K, resulting in proliferation of normal cells and cancer cells. Expression of DKK1 and CKAP4 was frequent in tumor lesions of human pancreatic and lung cancers, and simultaneous expression of both proteins in patient tumors was negatively correlated with prognosis and relapse-free survival. An anti-CKAP4 antibody blocked the binding of DKK1 to CKAP4, suppressed AKT activity in a human cancer cell line, and attenuated xenograft tumor formation in immunodeficient mice. Together, our results suggest that CKAP4 is a potential therapeutic target for cancers that express both DKK1 and CKAP4.

Wnt5a signaling promotes apical and basolateral polarization of single epithelial cells.

Single epithelial-derived tumor cells have been shown to induce apical and basolateral (AB) polarity by expression of polarization-related proteins. However, physiological cues and molecular mechanisms for AB polarization of single normal epithelial cells are unclear. When intestinal epithelial cells 6 (IEC6 cells) were seeded on basement membrane proteins (Matrigel), single cells formed an F-actin cap on the upper cell surface, where apical markers accumulated, and a basolateral marker was localized to the rest of the cell surface region, in a Wnt5a signaling-dependent manner. However, these phenotypes were not induced by type I collagen. Rac1 activity in the noncap region was higher than that in the cap region, whereas Rho activity increased toward the cap region. Wnt5a signaling activated and inhibited Rac1 and RhoA, respectively, independently through Tiam1 and p190RhoGAP-A, which formed a tertiary complex with Dishevelled. Furthermore, Wnt5a signaling through Rac1 and RhoA was required for cystogenesis of IEC6 cells. These results suggest that Wnt5a promotes the AB polarization of IEC6 cells through regulation of Rac and Rho activities in a manner dependent on adhesion to specific extracellular matrix proteins.

FUS mutations in familial amyotrophic lateral sclerosis in the Netherlands.

OBJECTIVES: To assess the frequency of FUS mutations in 52 probands with familial amyotrophic lateral sclerosis (FALS) and to provide careful documentation of clinical characteristics. DESIGN: FUS mutation analysis was performed using capillary sequencing on all coding regions of the gene in a cohort of patients with FALS. The clinical characteristics of patients carrying FUS mutations were described in detail. SETTING: Three university hospitals in the Netherlands (referral centers for neuromuscular diseases). PATIENTS: Fifty-two probands from unrelated pedigrees with FALS. MAIN OUTCOME MEASURE: FUS mutations. RESULTS: We identified 3 mutations in 4 of 52 probands. We observed 2 previously identified mutations (p.Arg521Cys and p.Arg521His) and 1 novel mutation (p.Ser462Phe). In addition, a p.Gln210His polymorphism was identified in 1 proband and 3 healthy control subjects. Phenotypic analysis demonstrated that patients may lack upper motor neuron signs, which was confirmed at autopsy, and disease survival was short (<36 months for 8 of 10 patients). CONCLUSIONS: We discovered FUS mutations in Dutch patients with FALS and the occurrence of benign variations in the gene. Therefore, caution is warranted when interpreting results in a clinical setting. Although the phenotype associated with FUS mutations is variable, most patients predominantly demonstrate loss of lower motor neurons and have short disease survival.

Genome-wide association study identifies 19p13.3 (UNC13A) and 9p21.2 as susceptibility loci for sporadic amyotrophic lateral sclerosis.

We conducted a genome-wide association study among 2,323 individuals with sporadic amyotrophic lateral sclerosis (ALS) and 9,013 control subjects and evaluated all SNPs with P < 1.0 x 10(-4) in a second, independent cohort of 2,532 affected individuals and 5,940 controls. Analysis of the genome-wide data revealed genome-wide significance for one SNP, rs12608932, with P = 1.30 x 10(-9). This SNP showed robust replication in the second cohort (P = 1.86 x 10(-6)), and a combined analysis over the two stages yielded P = 2.53 x 10(-14). The rs12608932 SNP is located at 19p13.3 and maps to a haplotype block within the boundaries of UNC13A, which regulates the release of neurotransmitters such as glutamate at neuromuscular synapses. Follow-up of additional SNPs showed genome-wide significance for two further SNPs (rs2814707, with P = 7.45 x 10(-9), and rs3849942, with P = 1.01 x 10(-8)) in the combined analysis of both stages. These SNPs are located at chromosome 9p21.2, in a linkage region for familial ALS with frontotemporal dementia found previously in several large pedigrees.

GSK-3beta regulates proper mitotic spindle formation in cooperation with a component of the gamma-tubulin ring complex, GCP5.

Glycogen synthase kinase-3beta (GSK-3beta) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3beta in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the gamma-tubulin ring complex (gammaTuRC). GCP5 bound directly to GSK-3beta in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and gamma-tubulin in the gammaTuRC fraction of sucrose density gradients and disrupted gamma-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of gammaTuRC and the recruitment of gamma-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of gamma-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of gammaTuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3beta regulates the localization of gammaTuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.

GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome.

Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.