Endostatin, an angiogensis inhibitor, was discovered by Dr. Judah Folkman¡¦s group in 1997. From their series studies, they demonstrated that the angiogenesis inhibition approach, which abolished the formation of new blood vessels and led to starvation of cancer cells, is a safe, effective anticancer method without side effect and drug resistance. Phase clinical trial on endostatin was carried out in 1999 and completed in 2001, heralding the approaching of a new arsenal of cancer therapy drugs. Endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on other cells. Above all, cycled therapy of experimental cancer in rodents with endostatin led to tumor dormancy without drug resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E. coli expression vector to express and generate large quantities of recombinant GST-fused endostatin. Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting endothelial cell lines EA.hy926 with a half-maximal inhibition concentration (IC50) of 20 nM. In present study, we investigated whether GST-endostatin caused alterations in cytoskeleton in endothelial cells. By using a fluorescence dye to visualize the actin filament under confocal microscope, it was found that endostatin induced the corruption of actin network in endothelial cells. Western blot analysis revealed that GST-endostatin treatment caused downregulation of cytoskeleton proteins such as tubulin, vimentin and ECM-related signaling molecules such as focal adhesion kinase (FAK), mitogen activated protein kinse (MAPK), Erk in a dose-dependent manner. Moreover, GST-endostatin decreased the levels of cell survival factor such as AKT and NF-£eB. Since GST-endostatin induced sustained calcium rise, the effect of endostatin on protein kinase Cs (PKCs) were studied and revealed that endostatin reduced the levels of PKCK1¡BPKC eta¡BPKC iota and PKC lamda. Other than endothelial cell, the cytotoxicity of GST-endostatin in hepatoma cells were investigated since liver the primary expression site of collagen XVIII, precursor of endostatin. Unexpectedly, endostatin also inhibited the proliferation of hepatoma cells. Flow cytometry and nucleus staining indicated that GST-endostatin also induced apoptosis in hepatoma cells. Moreover, GST-endostatin exhibited differential cytotoxic effect against well-differentiated (such as HepG2, Hep3B) and poor differentiated (such as Mahlavu, Sk-hep-1) hepatoma cells that the IC50 for well differentiated hepatoma cells were 8-10 folds lower than for poor-differentiated cells. Above all, GST-endostatin inhibited the migration of SK-hep-1 and modulated the secretion of matrix-metalloproteinases (MMPs) by Mahlavu and SK-hep-1 cells. In summary, present study explored the role of alterations in cytoskeleon network in the cytotoxic mechanism of GST-endostatin. Moreover, the inhibitory effects of GST-endostatin on proliferation of hepatoma cells were reported for the first time.

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBPα e C/EBPβ, interagem com as mesmas. Demonstramos que C/EBPβaumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBPαe C/EBPβem tecido hepático cirrótico e tumoral. As expressões de C/EBPαem tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBPα and C/EBPβ interact with them. We have demonstrated that C/EBPβ increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBPα and C/EBPβ in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBPα expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

Les protéases Tolloïdes constituent un groupe de métalloprotéases extracellulaires comptant quatre membres chez les mammifères (BMP-1, mTLD, mTLL-1 et mTLL-2). Ces protéases jouent un rôle majeur dans le développement et la réparation tissulaire, ainsi que dans certaines pathologies comme les fibroses. En 2006, le premier inhibiteur endogène des protéases Tolloïdes a été identifié chez le xénope et le poisson zèbre. Il s'agit de la protéine Sizzled, qui appartient à la famille des secreted Frizzled-Related proteins (sFRPs). Le travail présenté dans ce manuscrit suggère que ce mécanisme d'inhibition des protéases Tolloïdes par les sFRPs n'est pas conservé chez les mammifères. En effet, trois des cinq sFRPs de mammifères ont été testées (sFRP1, sFRP2 et sFRP4), et aucune d'entre elles ne s'est avérée capable d'inhiber l'activité de la protéase BMP-1 humaine in vitro. Ce travail montre toutefois que les protéases BMP-1, mTLD et mTLL-1 humaines peuvent être inhibées de façon puissante et spécifique par la protéine Sizzled de xénope. Cette inhibition repose sur l'interaction du domaine Frizzled de Sizzled avec le domaine catalytique des protéases Tolloïdes. Plus particulièrement, les résidus Asp-92, Phe-94, Ser-43 et Glu-44 de Sizzled (dont certains ne sont pas présents chez les sFRPs de mammifères) jouent un rôle crucial dans cette inhibition. Enfin, nous nous sommes intéressés au variant long du collagène XVIII, qui comporte également un domaine Frizzled. Nous avons pu montrer que BMP-1 clive le collagène XVIII in vitro, libérant un fragment contenant le domaine Frizzled. Des expériences sont en cours pour déterminer si ce fragment est capable d'inhiber les protéases Tolloïdes / Tolloid proteinases constitute a group of extracellular metalloproteinases which includes four members in mammals (BMP-1, mTLD, mTLL-1, mTLL-2). These proteinases play major roles in development, tissue repair and related pathological conditions such as fibrosis. In 2006, the first endogenous inhibitor of Tolloid proteinases was identified in Xenopus and zebrafish. This inhibitor, called Sizzled, is a member of the secreted Frizzled- related proteins (sFRPs). The present study strongly suggests that inhibition of Tolloid proteinases activity by sFRPs is not conserved in mammals. Indeed, three of the five mammalian sFRPs were tested (sFRP1, sFRP2 and sFRP4) and none of them was found to inhibit human BMP-1 activity in vitro. In contrast, this study demonstrates that Xenopus Sizzled is a potent and specific inhibitor of human BMP-1, mTLD and mTLL-1. This inhibition involves an interaction between the Frizzled domain of Sizzled and the catalytic domain of Tolloid proteinases. More precisely, residues Asp-92, Phe-94, Ser-43 and Glu-44 of Sizzled (among which only Asp-92 is conserved in mammalian sFRPs) play a crucial role in Tolloid proteinase inhibition. Finally, we studied the longest isoform of collagen XVIII, which also contains a Frizzled domain. We found that BMP-1 can cleave collagen XVIII in vitro, resulting in a Frizzled domain-Containing fragment. Experiments are in progress to determine if this fragment can also inhibit Tolloid proteinase activity

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBPα e C/EBPβ, interagem com as mesmas. Demonstramos que C/EBPβaumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBPαe C/EBPβem tecido hepático cirrótico e tumoral. As expressões de C/EBPαem tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBPα and C/EBPβ interact with them. We have demonstrated that C/EBPβ increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBPα and C/EBPβ in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBPα expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

Abstract The tumor microenvironment (TME) is critically important for tumor development. The microenvironment consists of fibroblasts, endothelial and immune cells as well as extracellular matrix (ECM), proteases and various other soluble factors produced by the cells. It is challenging to develop methods that appropriately mimic the human microenvironment, but this effort is essential in order to reliably elucidate the properties of potential anti-tumor drugs. The aim of this study was to create new 3D organotypic invasion models based on human tissue that would be used to study the effects of the anti-angiogenic molecules arresten and endostatin on tongue squamous carcinoma cells. The classic way to study cancer invasion has been to use a collagen invasion model that is created by mixing rat type I collagen, matrix produced by mouse EHS tumor cells and human fibroblasts. Our research group has developed a novel human myoma tissue based invasion model, which is composed of several different cell types and molecules that are normally present in the human TME. We show how this model is suitable for invasion studies, not only for oral cancer, but for other invasive cell lines as well. There are several matrix-derived fragments that have been shown to possess anti-angiogenic activity. Arresten is a 26 kDa fragment that is cleaved from type IV collagen and is known to inhibit angiogenesis, the formation of new capillaries and tumor growth in vivo. However, its effect on the tumor microenvironment in addition to endothelial cells has not been studied. We show that arresten also directly affects oral cancer cells by decreasing their migration and invasion as well as tumor size, invasion and angiogenesis in in vivo mouse xenografts. Another inhibitor of angiogenesis, endostatin, is cleaved from type XVIII collagen. It has been shown to suppress angiogenesis and tumor growth without toxicity or side effects in mouse models. Our studies show that endostatin directly affects tongue squamous carcinoma cells by reducing their invasion and spreading in organotypic 3D assays and mouse tumor models. In summary, arresten and endostatin are anti-angiogenic as well as anti-invasive molecules and therefore potential cancer drugs. They seem to have a direct effect on carcinoma cells making the cells less invasive. The myoma model allows us to study the effects of anti-cancer molecules with a new prospective. / Tiivistelmä Syövän mikroympäristö on erittäin tärkeä syövän kehittymisen kannalta. Se koostuu fibroblasteista, endoteeli- ja immuunisoluista, soluväliaineesta, proteaaseista ja monista muista solujen tuottamista liukoisista molekyyleistä. On haastavaa kehittää uusia menetelmiä, jotka jäljittelisivät oikeaa ihmisen syövän mikroympäristöä, mutta se on välttämätöntä uusien syöpälääkkeiden tutkimiseksi. Väitöstutkimuksen tavoitteena oli kehittää kolmiulotteinen ihmisen myoomakudokseen perustuvan invaasiomalli, jonka avulla voisimme tutkia verisuonten kasvua estävien arresten ja endostatin molekyylien vaikutusta kielisyöpäsoluihin. Aiemin syövän invaasiota on tutkittu käyttämällä klassista kollageeni-invaasiomallia, joka tehdään sekoittamalla rotan tyypin I kollageeniä, hiiren sarkoomasolujen tuottamaa matriksia ja ihmisen fibroblasteja. Tutkimuksissamme kehitimme uuden invaasiomallin, joka perustuu ihmisen myoomakudokseen. Tutkimuksessa sen todettiin sisältävän monia erilaisia soluja ja molekyylejä, joita on normaalistikkin syövän mikroympäristössä. Lisäksi osoitimme, että se sopii invaasiotutkimuksiin monille syöpätyypeille. Soluvälitilamatriksista pilkotaan useita erilaisia molekyylejä joilla on osoitettu olevan angiogeneesia hillitseviä ominaisuuksia. Arresten on 26 kDa kokoinen polypeptidi, jota pilkotaan tyypin IV kollageenista. Sen tiedetään vähentävän angiogeneesia – uusien verisuonten muodostumista ja syövän kasvua in vivo. Sen vaikutuksia muihin kuin endoteelisoluihin ei ole kuitenkaan tutkittu. Tutkimuksissamme se vaikutti suoraan kielisyöpäsoluihin vähentäen niiden liikkumista ja invaasiota kolmiulotteisissa organotyyppisisssä malleissa ja hiirimallissa. Toinen tutkimamme angiogeneesin inhibiittori on endostatin, jota pilkotaan tyypin XVIII kollageenista. Sen tiedetään vähentävän angiogeneesia hiirimalleissa ilman toksisia sivuvaikutuksia. Me osoitimme tutkimuksissamme, että se vaikuttaa suoraan kielisyöpäsoluihin vähentäen niiden invaasiota ja leviämistä 3D organotyyppisissä malleissa sekä hiirikokeissa. Koska arresten ja endostatin ovat anti-angiogeenisiä ja anti-invasiivisia molekyylejä, ne ovat täten potentiaalisia syöpälääkkeitä. Ne näyttäisivät vaikuttavan suoraan syöpäsoluihin vähentämällä niiden invaasiota. Myoomainvaasiomalli mahdollistaa syöpää ehkäisevien molekyylien tutkimisen uudella ja todenmukaisemmalla tavalla.

Abstract Transgenic mice were used to evaluate the roles of collagens XV and XVIII in retinal vessel development and to examine the roles of collagen XV and laminin α4 in peripheral nerve development and function. Also, in vitro methods were used to study the functions of recombinant, full-length human collagen XV produced in insect cells. The lack of collagen XVIII alone was found to result in overproliferation of astrocytes in the mouse retina and deficient vascularization of the retina, which was ultimately rescued by the persistent hyaloid vessels. VEGF mRNA expression was appropriately regulated in the retina of the collagen XVIII deficient mice, which also showed reduced susceptibility to oxygen-induced neovascularization. Lack of collagen XV alone had no obvious effect on the mouse eye, but an abnormal migration of astrocytes onto the persistent hyaloid vessels could be seen in mice lacking both collagens XVIII and XV. Recombinant full-length human collagen XV was seen in rotary shadowing EM as an extended protein with numerous kinks and a globular domain at its N-terminus. The mean length of the molecules was 241 nm and they had a tendency to form aggregates. Collagen XV attaches to the collagen binding region of fibronectin and to a lesser extent to vitronectin and laminin. It was rapidly bound to cultured cells with a staining pattern co-localizing with fibronectin. Collagen XV was also found to inhibit the adhesion and migration of cells in vitro. Lack of collagen XV in mice was found to result in ultrastructural abnormalities in the sciatic nerves, such as polyaxonal myelination and more loosely packed C-fibres, mild impairment of BM assembly and mild motor dysfunction with a lower sensory nerve conduction velocity. Polyaxonal myelination and a failure in the segregation of axons were evident in laminin α4 null mice, which also had diminished amounts of C-fibres, BM abnormalities, diminished myelin and a greater myelin period compared with wild-type mice. They performed poorly in the round beam test and showed diminished compound muscle action potentials. A simultaneous lack of collagen XV and laminin α4 resulted in a permanent defect in the segregation of axons and C-fibre development. The performance of the double null mice on the round beam was poor relative to the other genotypes. / Tiivistelmä Tässä väitöskirjatyössä on tutkittu kollageenien XV ja XVIII merkitystä silmän verkkokalvon verisuonituksen kehittymiselle sekä kollageeni XV:n ja laminiini α4:n merkitystä ääreishermon kehittymiselle ja toiminnalle käyttäen hyväksi muuntogeenisiä hiiriä. Lisäksi tässä työssä on käytetty in vitro –menetelmiä hyönteissoluissa yhdistelmä-DNA-tekniikalla tuotetun kollageeni XV:n toiminnan tutkimiseksi. Kollageeni XVIII:n puutteen todettiin johtavan verkkokalvon astrosyyttien määrän lisääntymiseen sekä verkkokalvon suonituksen kehittymiseen epänormaalilla tavalla lasiaissuonista, jotka eivät olleetkaan hävinneet kehityksen aikana. VEGF:n lähetti-RNA:n ilmentyminen oli asianmukaisesti säädelty eri verkkokalvon alueilla kollageeni XVIII:n suhteen poistogeenisillä hiirillä, joilla kehittyi myös vähemmän uudissuonia matalalle hapen osapaineelle altistamisen jälkeen. Kollageeni XV:n puute ei johtanut havaittaviin muutoksiin silmässä, mutta molempien kollageenien XV ja XVIII puuttuessa havaittiin silmässä epänormaalia astrosyyttien soluvaellusta lasiaissuonten päälle. Tässä väitöskirjatyössä osoitetaan, että yhdistelmä-DNA-tekniikalla tuotettu kokopitkä ihmisen kollageeni XV näytti elektronimikroskopiassa pitkältä molekyyliltä, jonka pituus oli keskimäärin 241 nm ja joka sitoutui helposti toisiin kollageeni XV -molekyyleihin. Kollageeni XV sitoutuu fibronektiinin kollageenia sitovaan osaan sekä vitronektiiniin ja laminiiniin. Viljeltyihin soluihin kollageeni XV sitoutui siten, että vasta-ainevärjäyksellä todettiin sen paikallistuvan samoille alueille fibronektiinin kanssa. Kollageeni XV:n todettiin myös vähentävän solujen kiinnittymistä ja liikkumista. Hiirissä kollageeni XV:n puutoksen todettiin johtavan iskiahermoissa polyaksonaaliseen myelinisaatioon, C-säikeiden lievään kehityshäiriöön, tyvikalvon koostumuksen häiriöön, lieviin motorisiin vaikeuksiin ja matalampaan tuntohermojen johtonopeuteen. Laminiini α4 –puutteisilla hiirillä oli todettavissa myös polyaksonaalista myelinisaatiota ja lisäksi häiriö aksonien erottelussa. Niiden hermoista löytyi myös tyvikalvon epämuodostumia, vähemmän myeliiniä ja C-säikeitä sekä suuremmat myeliinijaksot verrattuna villityypin hiiriin. Ne selviytyivät huonosti kävelytestissä ja niiden lihasten aktiopotentiaalit olivat pienemmät kuin kontrollihiirillä. Samanaikainen kollageeni XV:n ja laminiini α4:n puutos johti pysyvään aksonien erottelun ja C-säikeiden kehittymisen häiriöön. Kävelytestissä tuplapoistogeeniset hiiret selviytyivät huonoiten muihin genotyyppeihin verrattuna.