Margaret Atiba-Davies

Konrad Noben-Trauth

TRPML3 and hearing loss in the varitint-waddler mouse

published or not. The documents may come

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A C C E P T E D M A N U S C R I P T

Introduction

The physiology of hearing has been studied for many decades and has revealed several attributes that are unique to the inner ear such as the tonotopic organization of the cochlea, the mechanosensitivity of the transducer channels, and cochlear amplification [START_REF] Hudspeth | Sensory transduction in the ear, Principles of Neural Science[END_REF]. The molecular characterization of these mechanisms through biochemical means has been hampered by the limiting number of sensory cells in the cochlea but has been facilitated more recently by the systematic application of genetic means in mouse, humans, and zebra fish [2 -4].

Sound is heard in the cochlea. Within this coiled compartmentalized cellular structure the stria vascularis, the organ of Corti and the spiral ganglion are the major domains that enable the perception, analysis, and transduction of acoustic stimuli.

When sound waves travel through the scala tympany they peak at frequency-specific sites along the basilar membrane causing an upward motion of the organ of Corti.

Actin-filled protrusions of the sensory outer hair cells, or stereocilia, are moved by the tectorial membrane overlying the organ of Corti (Fig. 1). Deflections of the stereocilia hair bundle stretch molecular springs in the stereocilia, and thereby mechanically gate ion channels located in the plasma membrane of the stereocilia. This results in an influx of K + and Ca 2+ cations, depolarization of the hair cell, and synaptic transmission to the cochlear nerve with neurons located in the spiral ganglion. A major source of the high concentration of K + in the endolymph and the establishment of the endocochlear potential are the pigmented intermediate cells located in the stria vascularis, which is a highly stratified epithelium outlining the cochlear lateral wall [START_REF] Wangemann | K+ cycling and the endocochlear potential[END_REF].

The TRPML gene family (also known as mucolipins) consists of the three mammalian genes TRPML1 (MCOLN1), TRPML2 (MCOLN2) and TRPML3 (MCOLN3). TRPML1 was first recognized as the gene mutated in mucolipidosis type IV (ML-IV) an autosomal recessive lysosomal storage disorder with clinical features that include psychomotor retardation and opthalmological defects (reviewed in [START_REF] Bach | Mucolipidosis type IV[END_REF]) [START_REF] Bargal | Identification of the gene causing mucolipidosis type IV[END_REF].

Genes homologous to Trpml3 also have been identified in Drosophila melanogaster (CG8743), Caenorhabditis elegans (CUP-5) and Leishmania major [8 -11].

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Phenotype of varitint-waddler mice

The varitint-waddler phenotype is the result of two mutations, Va and Va J , that occurred spontaneously on the distal region of mouse chromosome 3. Both mutations exhibit a semi-dominant mode of inheritance with a well-defined set of phenotypes.

The Va allele arose in an outcross of (C57black/C57brown)F1 and C57black mice [START_REF] Cloudman | The varitint-waddler mouse[END_REF] and mice homozygous or heterozygous for Va are deaf, and exhibit circling behaviour indicative of a vestibular defect. They also display a variegated and dilute coat color and homozygotes are embryonic lethal. The second allele, Va J , arose in a colony segregating Va and causes a less severe phenotype. Although homozygotes and heterozygotes are deaf they exhibit normal vestibular behaviour and display much reduced coat color variegation. In addition, Va J homozygotes are generally viable, although the extent of the viability depends on the genetic background [START_REF] Kim | Genetic Analyses of the Mouse Deafness Mutations Varitint-Waddler (Va) and Jerker (Espnje)[END_REF]. The homozygote phenotype appears to be more severe for both alleles.

Hearing Loss in varitint-waddler

Hearing tests conducted with varitint-waddler mice have demonstrated abnormalities with respect to cochlea functioning. Compound action potentials, which are generated by the auditory nerve exiting the cochlea were absent in both +/Va J and Va J /Va J . In addition the endocochlear potential, which is generated by strial intermediate cells was reduced in some, but not in all mutants. This finding was supported by data obtained from auditory-brain stem response analyses, which measures the activity response of the auditory nerve in response to discrete acoustic stimuli [START_REF] Kim | Genetic Analyses of the Mouse Deafness Mutations Varitint-Waddler (Va) and Jerker (Espnje)[END_REF]. At postnatal day (P)14 +/Va J heterozygotes showed some responses to sound stimuli, albeit at very high sound intensities (> 80 dB SPL), indicating that some residual hearing existed at this stage in these animals. At P21 response thresholds in Va J heterozygotes had increased to > 100 dB SPL. No response could be elicited from +/Va and Va J /Va J mutants at any age. The congenital hearing loss in Va J mutants also appeared independent of the genetic background. In a series of F2 outcrosses involving C3HeB/FeJ, CZECHII/Ei, BALB/cByJ, A/J, C57BL/6J and DBA/2J strains, F2

+/Va J and Va J /Va J showed no ABR responses [START_REF] Kim | Genetic Analyses of the Mouse Deafness Mutations Varitint-Waddler (Va) and Jerker (Espnje)[END_REF].

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Histological studies on inner ears of +/Va heterozygotes identified several affected structures. The tectorial membrane appeared thicker than in normal ears and failed to contact the hair cells in the organ of Corti and the cell density in the spiral ganglion was decreased [START_REF] Deol | The anomalies of the labyrinth of the mutants varitint-waddler, shaker-2 and jerker in the mouse[END_REF]. In the organ of Corti sensory hair cells and supporting cells appeared as an undifferentiated cell mass as early as P11. The stria vascularis had an abnormal appearance, showing nuclear and cytoplasmic irregularities within the marginal cell layer. Ultra -structural analyses based on transmission electron microscopy on the Va J allele refined the cellular defects [START_REF] Cable | Combined cochleo-saccular and neuroepithelial abnormalities in the Varitint-waddler-J (Va J ) mouse[END_REF]. In +/Va J heterozygotes the stria vascularis was normal, but in Va J /Va J homozygotes it appeared abnormal with reduced or absent pigmentation, suggesting the absence of the pigmented intermediate cells.

The organ of Corti of 14-day old +/Va J heterozygotes exhibited a mild phenotype, showing fused stereocilia on mainly inner hair cells. The apical surface of hair cells was also affected showing extrusions and fragmentation of the cuticular plate.

Defects were more pronounced in the homozygotes, which showed profound atrophy of the organ of Corti and fusion of stereocilia in all regions of the cochlea duct. At the base of inner and outer hair cells, affe rent and efferent synapses were reduced in number or absent. In both +/Va J and Va J /Va J the surrounding supporting cells, Reissner's and tectorial membranes were normal.

The earliest signs of abnormality of the stereocilia hair bundle were observed in

Va J homozygotes at embryonic day 17.5 (E17.5) [START_REF] Palma | Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice[END_REF]. At this stage the growing microvilli, at the apical surface of hair cells, from which the mature stereocilia develop, were arranged in an irregular fashion. At P5, hair cell stereocilia had grown to normal height but were disorganized and appeared as clumps atop some hair cells (Fig. 3). At P11 disorganization progressed further resulting in fusion, clumping or absence of stereocilia along the length of the organ of Corti.

Mutations in Trpml3 result in varitint-waddler

A positional cloning approach identified Trpml3 (Mcoln3) as the gene mutated in varitint-waddler [START_REF] Palma | Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice[END_REF].

Trpml3 has an open reading frame of 1,662 base pairs consisting of 12 exons, which encode a 553 amino acid protein, predicted to be 64 kDa in size.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

The TRPML3 amino acid sequence is 74% and 77% similar to TRPML1 and TRPML2, respectively, and is 63% similar to Drosophila CG8743 and 55% similar to C. elegans CUP-5. In addition, the putative channel pore region of TRPML3 is weakly similar to the TRPP2 cation channel (also known as PKD2), which is implicated in autosomal dominant polycystic kidney disease (Table 1), suggesting a conserved molecular function. Like all TRP channels TRPML3 is predicted to have six trans membrane domains (6TM), cytoplasmic amino and carboxy termini, an ion transport motif (PF00520), and a transient receptor potential like motif (TRPL, PS50272). The Va allele results from a 1225G>C transversion leading to an Ala419Pro substitution in the fifth trans membrane domain, which is a critical component of the pore region of the channel. Although no physiological data exist, it is likely that the Ala419Pro substitution is either a gain-of-function mutation, which severely alters the physiological properties of the channel or a dominant-negative allele, which renders a channel complex dysfunctional. The Va J mutation results from a 1085T>C transition that leads to an Ile362Thr substitution in the second extracellular loop. The Va J allele arose in a colony segregating the Va allele and given that the 1225G>C mutation was also detected in Va J , it implies that Va J arose in cis to Va. Owing to the milder phenotypes exhibited by Va J , the Ile362Thr substitution may act as an intragenic suppressor of the Ala419Pro allele.

Molecular function of TRPML3

To date there is only limited information about the molecular function of TRPML3. A recent study by Ventakachalam and colleagues showed that TRPML3 forms homo-and heterodimers with both TRPML1 and TRPML2 proteins when transiently co-expressed in HEK293 cells [START_REF] Venkatachalam | Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1[END_REF]. Under such conditions, TRPML3 homodimers localized predominantly to the endoplasmic reticulum, but formed heterodimers when co-expressed with either TRPML1 or TRPML2 and localized to In cochlear hair cells, the onset and site of the defects (stereocilia disorganization and fusion) as well as the localization to the plasma membrane of sterocilia [START_REF] Palma | Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice[END_REF] are consistent with the view that TRPML3 may function during e arly embryogenesis.

During the elongation of the stereocilia hair bundle, molecular links are established between individual stereocilia, which are modified and become restricted to the top region as the bundle grows and differentiates [START_REF] Goodyear | Development and properties of stereociliary link types in hair cells of the mouse cochlea[END_REF]. TRPML3 may play a role in regulating ion homeostasis along the stereocilia and mutant TRPML3 may thus lead to the selective loss of stereocilia links; hence the disorganized and splayed appearance of stereocilia in varitint-waddler mutants [START_REF] Steel | Varitint-waddler: a double whammy for hearing[END_REF].

Mutations in another member of the TRPML family, TRPML1, cause the lysosomal storage disorder mucolipidosis type IV, characterized by the accumulation of large vacuoles in the cytosol. TRPML1 localizes to late endosomes and lysosomes and loss of TRPML1 activity is thought to disrupt biogenesis of lysosomes during the final stages of endocytosis [START_REF] Bach | Mucolipin 1: endocytosis and cation channel--a review[END_REF]. On the molecular level, TRPML1 has been shown to be permeable to H + , leaking H + into the cytosol and thereby regulate the pH in lysosomes

Fig. 2 The varitint-waddler mouse

A representative Va J /Va J homozygous varitint-waddler mouse is shown. Note the pigmentation defects seen as white and dilute patches of fur on an agouti background.

Fig. 3 Stereocilia defects in varitint-waddler mouse

Organ of Corti surface preparations from 5-day old neonates stained with phalloidin to visualize the stereocilia hair bundle are shown. Note the disorganized hair bundle in the homozygous mutants (Va J /Va J ) compared to the normal bundle shape of +/+ littermates. Reproduced from Di Palma et al. 2002 [START_REF] Palma | Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice[END_REF].

Fig. 4 Schematic diagram of TRPML3

A schematic diagram of the predicted secondary structure with six trans membrane domains, amino and carboxy termini, and the location of the I362T and A419P amino acid substitutions is shown.

  TRPML3 in the cochlea has not yet been elucidated. The mutant phenotypes and the immuno-localization studies, however, provide interesting clues about its function. The motif structure of TRPML3 suggests a cell-autonomous A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT function, implying that TRPML3 acts in strial intermediate cells and inner ear hair cells.

[ 20 ]Fig. 1

 201 Fig. 1 Mouse cochlea ductA photomicrograph of a cross-section through the mouse cochlea duct at the middle turn is shown. The tectorial (tm) and basilar membranes (bm), stria vascularis (sv), organ of Corti (oc), and scala tympani (st) are indicated.

Acknowledgements

We thank Mark Schneider, Nelson Lopez and Tony Ricci for their insightful comments on the manuscript. MAD and KNT are supported by the intramural program at NIDCD.


Tatiana El-Bacha
email: tatiana_elbacha@yahoo.com.br

Victor Midlej

Ana Paula Pereira

Da Silva

Leandro Silva Da Costa

Marlene Benchimol

Antonio Galina

Andrea T Da Poian

Ana Paula

Ana Paula Pereira Da Silva

Leandro Silva Da Costa

Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus

Keywords: dengue virus infection, mitochondrial dysfunction, metabolism, energy charge, apoptosis, HepG2 cells

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T Introduction

Dengue virus (DV) infection affects 50-100 million people and it is considered the major human arbovirosis [START_REF] Gubler | The global emergence/resurgence of arboviral diseases as public health problems[END_REF][START_REF] Gubler | Dengue and dengue hemorrhagic fever[END_REF][START_REF] Mackenzie | Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses[END_REF]. Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS), the more severe manifestations of DV infection, hospitalize and kill several hundred thousand people annually and represent a serious public health, economic and social problem, especially in tropical areas of the globe [START_REF] Gubler | The global emergence/resurgence of arboviral diseases as public health problems[END_REF][START_REF] Gubler | Dengue and dengue hemorrhagic fever[END_REF][START_REF] Gúzman | Dengue and dengue hemorrhagic fever in the Americas: lessons and challenges[END_REF]. Although the clinical manifestations of DHF/DSS have already been described, the pathophysiology of this disease is still not completely understood, and the molecular mechanisms involved in organ and cellular dysfunction are not yet clear [START_REF] Gubler | Dengue and dengue hemorrhagic fever[END_REF][START_REF] Mackenzie | Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses[END_REF][START_REF] Gúzman | Dengue and dengue hemorrhagic fever in the Americas: lessons and challenges[END_REF].

Several lines of evidence support the idea that liver dysfunction is a characteristic of severe dengue infection. Analyses of liver autopsies from individuals with DHF revealed extensive areas of tissue damage, with foci of necrosis, steatosis and apoptosis, characterized by the presence of apoptotic bodies [START_REF] Couvelard | Report of a fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes and liver apoptosis[END_REF][START_REF] Huerre | Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children[END_REF]. Viral antigens were detected near the lesioned areas, suggesting an association between virus replication and hepatic damage [START_REF] Huerre | Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children[END_REF]. Additionally, it has been reported that cytokines production is implicated in liver dysfunction [START_REF] Gubler | Dengue and dengue hemorrhagic fever[END_REF][START_REF] Chaturvedi | Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis[END_REF].

Tissue injury is responsible for many of the clinical manifestations observed in DHF/DSS [START_REF] Couvelard | Report of a fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes and liver apoptosis[END_REF][START_REF] Huerre | Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children[END_REF], although metabolic disturbances may also be involved [START_REF] Marianneau | Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited[END_REF][START_REF] Catteau | Expression of dengue ApoptoM sequence results in disruption of mitochondrial potential and caspase activation[END_REF]. In accordance with these hypotheses, it has been shown that DV infection induces apoptosis in different cell types in culture [START_REF] Courageot | Mechanisms of dengue virus-induced cell death[END_REF]. Some studies have demonstrated the involvement of anti-and pro-apoptotic proteins of the Bcl-2 family [START_REF] Catteau | Expression of dengue ApoptoM sequence results in disruption of mitochondrial potential and caspase activation[END_REF][START_REF] Courageot | Mechanisms of dengue virus-induced cell death[END_REF][START_REF] Catteau | Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM[END_REF] and the participation of reactive oxygen species [START_REF] Lin | Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2virus-infected human liver cells[END_REF], suggesting that altered permeability of mitochondrial membranes is involved in the process of cell death following DV infection.

A C C E P T E D M A N U S C R I P T

For a series of viruses, including flaviviruses, the association between virus-induced cell death and the integrity of mitochondrial membranes has been extensively studied [START_REF] Everett | Viruses and Apoptosis: Meddling with Mitochondria[END_REF][START_REF] Verrier | Study of PTPC composition during apoptosis for identification of viral protein target[END_REF][START_REF] Piccoli | Mitochondrial dysfunction in hepatitis C virus infection[END_REF][START_REF] Kroemer | Mitochondrial control of cell death[END_REF]. Viral proteins that interact with mitochondria are able to modulate the permeability of mitochondrial membranes and the liberation of components from the inter-membrane space to cytoplasm and regulate the process of apoptosis [START_REF] Everett | Viruses and Apoptosis: Meddling with Mitochondria[END_REF][START_REF] Verrier | Study of PTPC composition during apoptosis for identification of viral protein target[END_REF][START_REF] Piccoli | Mitochondrial dysfunction in hepatitis C virus infection[END_REF]. In the case of pro-apoptotic viruses, the hallmark of cell damage is an abrupt decrease in mitochondrial membrane electrochemical potential (ΔΨ m ) [START_REF] Kroemer | Mitochondrial control of cell death[END_REF].

Although it is well known that altered permeability states can compromise mitochondrial physiology, ATP synthesis and consequently cell function, the study of mitochondrial bioenergetic dysfunction during virus infection has not been characterized in detail. In the case of DV, the possibility that mitochondrial bioenergetics and cellular energy state are associated with the deterioration of cell function during the course of infection has never been addressed.

In the present work it is demonstrated for the first time that DV infection of a human hepatoma cell line (HepG2) leads to alterations in the bioenergetic function of mitochondria, indicated by a decrease in ΔΨ m , altered respiratory properties and mitochondrial morphology. These events compromise cellular energy homeostasis and may be related to the pathophysiology of DV infection.

A C C E P T E D M A N U S C R I P T

Materials and Methods

Cell culture and virus propagation

HepG2 cells were acquired from American Type Cell Collection (USA). HepG2 is a differentiated human hepatocellular carcinoma cell line, which preserves many of the morphological and functional characteristics of hepatocytes [START_REF] Chen | Effect of eadysterone on glucose metabolism in vitro[END_REF][START_REF] Knowles | Human Hepatocellular carcinoma cell line secrete major proteins and hepatitis B surface antigens[END_REF][START_REF] Kosaki | Effect of dexamethasone on the alternative splicing of the insulin receptor mRNA and insulin action in HepG2 hepatoma cells[END_REF], thus representing an interesting model for the study of liver bioenergetics during DV infection.

A C C E P T E D M A N U S C R I P T

Oxygen Consumption

Oxygen consumption was measured in an oximeter (Yellow Springs Instruments Co., USA or Hansatech, UK) using a Clark type electrode, in a 1.0mL water jacket maintained under constant stirring, at 37ºC. Intact and permeabilized HepG2 cells were used for analyses of respiratory properties of mitochondria.

For measurements of oxygen consumption of intact cells, approximately 5 × 10 6 cells were suspended in culture medium (MEM) without fetal bovine serum, as described elsewhere [START_REF] Hutter | Senescenceassociated changes in respiration and oxidative phosphorylation in primary human fibroblasts[END_REF]. After recording basal respiration, 3µg/mL oligomycin was added to record oligomycin-independent oxygen consumption. Oxygen consumption in the presence of oligomycin represents the sum of proton leak through the inner mitochondrial membrane plus any non-mitochondrial oxygen consumption. The block of respiration observed after the addition of antimycin A reveals that the non-mitochondrial oxygen consumption in HepG2 cells is negligible (Fig. 1a). Maximum respiration was achieved by adding FCCP 200 nM. Optimum FCCP concentration was determined after FCCP titration (40-500 nM, data not shown).

For respiratory complexes activities, respiratory control ratio (RCR) and ADP/O ratio calculations, permeabilized HepG2 cells were used. In this case, cells were suspended in respiration media containing 0.25 M mannitol, 0.1% bovine serum albumin, 10 mM MgCl 2 , 10 mM Hepes, 10 mM KH 2 PO 4 , pH 7.2, as described elsewhere [START_REF] Sakurai | Oxidative stress and cytotoxicity induced by ferricnitrilotriacetate in HepG2 cells that express cytochrome P450 2E1[END_REF]. Immediately after the addition of cells (approximately 5 × 10 6 cells), digitonin (0.005%, w/v) was added, followed by respiratory modulators and substrates. Complex I maximum activity was determined after the addition of 5 mM pyruvate, 5 mM malate and 5 mM glutamate and 1 mM ADP. For RCR analyses, 1 µM rotenone and 12 mM succinate were added. After the presence of endogenous substrates on cell preparation, RCR, in each case, was calculated after the second addition of ADP. Following the first addition of ADP, endogenous adenylates are converted to ATP, an activator of succinate dehydrogenase [START_REF] Gutman | Control of succinate dehydrogenase in mitochondria[END_REF][START_REF] Bhuvaneswaran | Inactivation of coupled respiration of mitochondria by inorganic arsenate and partial restoration by ATP[END_REF] and respiratory control was clearly measured following the second addition of ADP. ADP/O ratios were calculated after each addition of ADP.

Mitochondrial membrane potential (ΔΨ m )

JC-1 dye ( 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; Molecular Probes) was used to measure changes in ΔΨ m following DV infection.

JC-1 has the property to selectively enter into mitochondria and according to the magnitude of ΔΨ m , its oligomeric state changes reversibly and so does its fluorescence properties. The ratio red:green fluorescence intensity of JC-1 gives an index of the ΔΨ m , where the higher the ratio, the higher the ΔΨ m [START_REF] Reers | J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential[END_REF]. Cells were incubated with 5 μg/ml JC-1 at 37 °C for 1 hour. Microscopic analyses of fluorescent images were performed using an epifluorescence microscope (Olympus BX40, Tokio, Japan) equipped with appropriate filters and using 40

× oil-immersion objectives (Ex 485 nm; Em JC-1 Red 546, Em JC-1 Green 532 nm).

HPLC of cellular nucleotides

HepG2 nucleotides were analyzed by reverse-phase ion-pair high-performance liquid chromatography (HPLC) in a LC-10AS chromatograph with a high-pressure phase mixer and a UV-visible detector (SPD-10A; Shimadzu, Japan) operated at 254 nm as described in [START_REF] Huang | Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography[END_REF]. Briefly, cells were disrupted with liquid N 2 and cellular proteins were Energy charge (EC) was calculated according to the following equation, as described in reference [START_REF] Atkinson | Cellular energy metabolism and its regulation[END_REF]:

EC = [(ATP)+0.5×(ADP)]/[(AMP)+(ADP)+(ATP)] (1)

Transmission electron microscopy

HepG2 cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2.

Cells were post-fixed on 1% OsO 4 in cacodylate buffer plus 5 mM CaCl 2 and 0.8% potassium ferricyanide, dehydrated in acetone series and infiltrated in Epon. Polymerization was carried out for 72 h. Thin sections were collected on copper grids, stained with uranyl acetate and lead citrate, and examined in a JEOL 1210 transmission electron microscope.

Statistical analysis

Statistical analyses were performed using Statgraphics version 4.0 (Statistical Graphics Corporation/Plus Ware). Results are presented as means (± SE). Paired and unpaired Student's t tests were used for comparison of mock-infected (control) and dengue virus-infected cells. Two-tailed P values < 0.05 were considered statistically significant.

A C C E P T E D M A N U S C R I P T

Results

DV infection induces alterations in mitochondrial respiratory properties in HepG2 cells

The effects of DV infection on the respiratory properties of mitochondria was investigated in intact and permeabilized HepG2 cells. In order to study the effects of DV infection on cellular respiration under physiological conditions, intact HepG2 cells were used. Figure 1a shows a typical record of oxygen consumption in intact HepG2 cells where it can be seen that both basal and oligomycin-independent respiration are increased in DVinfected cells. Basal respiration significantly increased from 3.62 nmol O 2 /10 6 cell × min -1 in mock-infected cells to 4.02 nmol O 2 /10 6 cell × min -1 in DV-infected cells (Fig. 1b; P<0.05). Additionally, oligomycin-independent respiration increased from 0.83 nmol O 2 /10 6 cell × min -1 to 1.09 nmol O 2 /10 6 cell × min -1 in infected HepG2 cells (Fig. 1c; P<0.05). FCCP addition to either mock-infected and infected cells stimulated maximum respiration in a similar way (Fig. 1a), suggesting that DV infection did not cause any alteration in the electron chain capacity of HepG2 cells.

Figures 2a and3a show typical records of oxygen consumption in mock-infected and DV-infected permeabilized cells using, respectively, NADH-linked or succinate as respiratory substrates. In agreement with the results obtained from intact cell, both basal and oligomycin-independent respiration was also increased in permeabilized cells (Fig. 2a).

Basal respiration increased from 0.60 nmol O 2 /10 6 cell × min -1 in mock-infected cells to 0.72 nmol O 2 /10 6 cell × min -1 in DV-infected cells (Fig. 2b; P<0.05), representing a 20% increase. There was no difference between Complex I respiration from mock-infected and infected HepG2 cells where oxygen consumption was, respectively, 1.54 and 1.61 nmol O 2 /10 6 cell × min -1 (Fig. 2a). Uncoupled or oligomycin-independent respiration raised from In agreement with the results from intact cells, following FCCP addition maximum respiration did not differ between mock-infected and infected cells (Fig. 2a).

Since complex I-driven respiration was better accessed under maximum conditions (using 1 mM ADP), ADP/O ratio was determined feeding Complex II. Figure 3a shows that after the addition of succinate, a substrate of Complex II, the rate of oxygen consumption in both cases rose significantly. It is well established that when cells are maintained in an appropriated medium containing reducing substrates that can donate hydrogen atoms (electrons and protons) to the respiratory electron chain, mitochondria start to respire at a slow rate due to the build up of a maximum proton gradient (state 2 or state 4 respiration) [START_REF] Chance | The respiratory chain and oxidative phosphorylation[END_REF]. Once ADP and Pi are added, respiration is increasingly stimulated due to ADP conversion to ATP by F o F 1 -ATP-synthase using the proton gradient established across the inner membrane by the electron transport chain (state 3 respiration) and ΔΨ m is partially reduced. When limiting amounts of ADP are added to mitochondria, state 3 respiration persists until all ADP is converted to ATP and, at this point, the proton gradient is increased again and oxygen consumption is reduced [START_REF] Chance | The respiratory chain and oxidative phosphorylation[END_REF]. Accordingly, figure 3a also shows that the rate of oxygen consumption in state 4 respiration was significantly higher in DV-infected cells. The calculated state 4 respiration rates were, respectively, 0.27 and 0.64 nmol O 2 /10 6 cell × min -1 for mock-infected and DV-infected HepG2 cells (P<0.05). The rates of state 3 respiration did not differ significantly between the two samples being 2.01 for mock-infected and 2.31 nmol O 2 /10 6 cell × min -1 for DV-infected cells. These results

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

11 are in agreement with the calculated rates of oxygen consumption using NADH-linked substrates (Fig. 2).

The RCR, which is indicative of the state of mitochondrial permeability, is shown in Figure 3b. The average RCR was 12.20 in mock-infected cells and 3.98 in virus-infected cells, suggesting that, even in permeabilized HepG2 cells, mitochondria had lost some of their natural intactness after DV infection. Figure 3c shows that the stoichiometry of the oxidative phosphorylation, ADP consumed per mol of oxygen atoms reduced to water (ADP/O ratio), was significantly decreased from 1.81 to 1.49 in DV-infected cells, indicating a decreased efficiency in ATP synthesis following infection.

The results with permeabilized HepG2 cells are in agreement with those obtained with intact cells. The increase in oligomycin-independent respiration (Figs. 1c and2c) and in state 4 respiration (Fig. 3a) along with the decrease in ADP/O ratio (Fig. 3c) reinforce the idea that DV-infection affects mitochondrial membrane permeability and thus the efficiency of ATP synthesis in HepG2 cells.

DV infection induces a decrease in ΔΨ m in HepG2 cells

In order to better investigate the altered respiratory properties of HepG2 cells following DV infection, ΔΨm was evaluated. Figure 4 shows fluorescence analyses of cells loaded with JC-1 dye. The red:green fluorescence intensity was 3-fold lower in virusinfected cells, suggesting that DV infection promoted a decrease in ΔΨ m (Fig. 4e). In addition, both red and green fluorescence intensities were decreased in virus-infected cells (Fig. 4c and4d), indicating a decreased incorporation of JC-1 into mitochondria and possibly an altered permeability state of mitochondrial membranes.

A C C E P T E D M A N U S C R I P T

Nucleotide content was analyzed to investigate whether the uncoupling of mitochondria and the decrease in ΔΨ m in DV-infected cells were interfering with ATP synthesis. The average amounts of AMP, ADP and ATP in mock-infected and virusinfected samples were, respectively, 0.3 and 1.7 nmols/10 6 cells; 2.6 and 5.0 nmols/10 6 cells; 18.7 and 15.5 nmols/10 6 cells (Fig. 5a). These differences corresponded to a 5-fold and a 2-fold increase in AMP and ADP levels, respectively, and a 20% decrease in ATP levels in virus-infected cells. All differences shown are significant (P<0.05).

The cellular adenylate pool composed of ATP, ADP and AMP includes the majority of the available chemical energy in cellular metabolism [START_REF] Atkinson | Cellular energy metabolism and its regulation[END_REF]. From the results

shown in Figure 4a, the total nucleotide pool was 22.5 nmols nucleotide/10 6 cells for mockinfected cells and 22.1 nmols nucleotide/10 6 cells for virus-infected cells (Fig. 5b). This result shows that infection did not alter the total amount of nucleotides, but instead their distribution. This altered distribution resulted in a decrease in the energy charge (EC) reflects the cellular state of free energy availability. An EC value of 1 indicates that all the available energy is present as ATP and an EC value of zero means that only AMP is present [START_REF] Atkinson | Cellular energy metabolism and its regulation[END_REF]. The calculated EC values were 0.93 and 0.81, respectively, for mock-infected and virus-infected cells (Fig. 5c), indicating that infection promoted a significant decrease (P<0.005) in this index.

DV infection induces ultrastructural alterations in HepG2 cells

Transmission electron microscopy analyses were performed to associate the alterations in mitochondrial bioenergetics with possible morphological changes. As shown in Figure 6, mock-infected cells exhibit mitochondria with characteristic electron-dense matrix (Fig. 6a; arrows), contrasting to swollen mitochondria with a loss of electron density in the matrix of DV-infected cells (Fig 6b ; arrows). In a total of 164 mitochondria counted in mock-infected cells, 95% presented normal ultrastructure. In DV-infected cells, 144 mitochondria were counted and 75% showed some degree of morphological alterations. In addition to the morphological changes in mitochondria, other ultrastructural alterations following DV infection can be pointed out in Figure 7, including the formation of membrane blebs (Fig. 7c and7d; arrow heads) and the localization of mitochondria around the nuclear membrane (Fig. 7c; arrows). Mock-infected cell, on the other hand, exhibit an intact cellular membrane (Fig. 7a). Electron dense particles seen in Figure 7b are compatible with DV particles [START_REF] Barth | Replication of dengue viruses in mosquito cell cultures-a model from ultrastructural observations[END_REF].

A C C E P T E D M A N U S C R I P T

Discussion

Virus infections elicit a number of cellular responses in the host that may create a hostile environment to the virus and therefore to control replication and minimize the deleterious effects of infection. Viruses, on the other hand, have developed strategies to curtail the cellular responses and ensure the success of infection. Among different hosts and viruses many of these responses involve alterations in energy metabolism, in particular an increased utilization of glucose molecules, alterations in the production of ATP and the modulation of mitochondrial functions [START_REF] Everett | Viruses and Apoptosis: Meddling with Mitochondria[END_REF][START_REF] Romero | Possible mechanism by which stress accelerates growth of virally derived tumors[END_REF][START_REF] Everett | Apoptosis: an innate immune response to virus infection[END_REF][START_REF] El-Bacha | Mayaro virus infection alters glucose metabolism in cultured cells through activation of the enzyme 6-phosphofructo 1-kinase[END_REF]. This last response is evident concerning the process of apoptosis, which most frequently includes alterations in mitochondrial integrity [START_REF] Everett | Viruses and Apoptosis: Meddling with Mitochondria[END_REF][START_REF] Verrier | Study of PTPC composition during apoptosis for identification of viral protein target[END_REF][START_REF] Everett | Apoptosis: an innate immune response to virus infection[END_REF]. The present work shows that cells infected with DV present mitochondrial dysfunction characterized by functional and ultrastructural alterations.

The respiratory properties of HepG2 cells were clearly altered by DV infection, which promoted an increase in cellular oxygen consumption along with a decrease in RCR and ADP/O (Figs. 1a, 1b; Figs. 2a, 2b and Fig. 3). A decrease in the RCR suggests that significant morphologic alterations of mitochondria may be undertaken in HepG2 cells after DV infection, which can be confirmed from the TEM analyses (Fig. 6). In addition, the decrease in ADP/O ratio (Fig. 3c) and the increase in both oligomycin-independent respiration (Figs. 1c and2c) and state 4 respiration (Fig. 3a) indicate that DV infection causes an uncoupling of the electron transfer chain from the phosphorylation of ADP, which results in a decreased efficiency for synthesis of ATP [START_REF] Brown | Control of respiration and oxidative phosphorylation in isolated rat liver cells[END_REF]. Both the decrease in RCR and in the ADP/O in virus-infected cells are due to the marked increase in the rate of state 4 respiration, since state 3 respiration was not significantly changed neither in Complex I nor

Complex II-linked substrates (Figs. 2a and3a). Additionally, the increase in oligomycin-

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

15 independent respiration also indicates an increase in proton leak, a feature of uncoupled oxidative phosphorylation in DV-infected cells (Figs. 1a, 1c and Figs. 2a and2c).

Mitochondrial uncoupling reflects a flux of protons from the inter-membrane space to the mitochondrial matrix through channels other than the F o F 1 ATP synthase and, therefore, not coupled to ATP synthesis. When mitochondrial uncoupling is physiological, there is an increase in electron transport and in oxygen consumption due to a small decrease in the ΔΨ m in order to maintain the electrochemical proton gradient and the synthesis of cellular ATP [START_REF] Brown | Control of respiration and oxidative phosphorylation in isolated rat liver cells[END_REF]. However, when mitochondrial uncoupling is important, one may expect a substantial decrease in ΔΨ m , as observed in Figure 4, with several consequences for cellular energy homeostasis, as occurred with HepG2 cells infected with DV. The decrease in ΔΨ m in intact cells infected with DV may be explained, in part, by an increase in proton leak, in accordance with the increase in oligomycin-independent respiration (Fig. 1c).

The effects of DV infection on the respiratory properties and mitochondrial membrane depolarization, which decreased the efficiency of ATP synthesis, also promoted a significant decrease in the steady-state ATP concentration of HepG2 cells (Fig. 5a). This decrease in the content of cellular ATP may indicate that energy demand is increased due to the synthesis of viral proteins. By this way, it can be speculated that DV infection also affects cellular regulatory mechanisms that compensate for mitochondrial inefficiency, such as an increased production of glycolytic ATP, as observed in various situations, like alphavirus infection [START_REF] El-Bacha | Mayaro virus infection alters glucose metabolism in cultured cells through activation of the enzyme 6-phosphofructo 1-kinase[END_REF].

As shown in Figure 5a, although the total nucleotide pool was not altered (Fig. 5b), infection induced a significant change in the relative amounts of ATP, ADP and AMP. This led to a decrease in EC in virus-infected cells (Fig. 5c). EC in hepatocytes is usually around

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 16
0.9 [START_REF] Atkinson | Adenosine triphosphate conservation in metabolic regulation[END_REF], a value similar to that observed for mock-infected cells in the present work. In liver cells, when ATP synthesis is impaired, the EC may reach extremely low levels such as 0.7 [START_REF] Raivio | Depletion of liver adenine nucleotides induced by D-fructose, Dose-dependence and specificity of the fructose effect[END_REF]. Thus, the EC of 0.8 observed in virus-infected cells indicates a metabolic stress. It has been shown that myocarditis induced by CoxsackieB3 virus promoted a decrease in cellular EC, and it was suggested that this alteration in energy balance is involved in the pathogenesis of the Coxsackie virus infection [START_REF] Waldenstrom | Coxsackie B3 myocarditis induces a decrease in energy charge and accumulation of hyaluronan in the mouse heart[END_REF]. Although the role of energy homeostasis in DV infection has never been addressed, it can be suggested that the deregulation of processes involved in ATP balance may be associated with the pathogenesis of DV infection.

The altered bioenergetic status caused by DV infection may be regarded as an initial sign of a more serious condition. Although cell viability was not altered in DV-infected cells in all m.o.i. used (data not shown), the small but significant decrease in cellular ATP and EC might be signaling an apoptotic stimulus, since it has been demonstrated that a transient decrease in the content of cellular ATP preceded the initiation of the apoptotic cascade in another cell type [START_REF] Izyumov | Wages of Fear": transient threefold decrease in intracellular ATP level imposes apoptosis[END_REF]. In agreement with these observations are the ultrastructural alterations in mitochondria in DV-infected cells (Fig. 6b), showing a loss of electron density in the matrix and mitochondrial swelling. This feature is characteristic of the permeability transition state, and, therefore, may indicate an increase in outer mitochondrial membrane permeability [START_REF] Kroemer | Mitochondrial control of cell death[END_REF][START_REF] Crompton | The mitochondrial permeability transition pore and its role in cell death[END_REF]. This association between mitochondrial structure and metabolic function has already been studied [START_REF] Gottlieb | Mitochondrial respiratory control is lost during growth factor deprivation[END_REF], however not in cells infected with DV. Although nuclear alterations typical of the apoptotic process have already been described in HepG2 cells infected with DV [START_REF] Marianneau | Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited[END_REF][START_REF] Marianneau | Differing infection patterns of dengue and yellow fever viruses in a human hepatoma cell line[END_REF], this is the first report of mitochondrial morphologic alterations. Additionally, other apoptotic features can be The mechanisms through which DV infection most contribute to the observed functional and morphological alterations remain open. Cellular proteins that interact directly with mitochondria, such as pro-or anti-apoptotic Bcl-2 family proteins, might be involved [START_REF] Marianneau | Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited[END_REF][START_REF] Courageot | Mechanisms of dengue virus-induced cell death[END_REF][START_REF] Everett | Viruses and Apoptosis: Meddling with Mitochondria[END_REF][START_REF] Lee | Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection[END_REF]. Additionally, reactive oxygen and nitrogen species may contribute to mitochondrial dysfunction, since DV infection increases oxidative stress both in vivo and in vitro [START_REF] Lin | Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2virus-infected human liver cells[END_REF][START_REF] Valero | Short report: increased level of serum nitric oxide in patients with dengue[END_REF][START_REF] Gil | Oxidative stress in adult dengue patients[END_REF].

Specific viral proteins, which interact with mitochondria, have the potential to affect mitochondrial membrane permeability and thus the process of apoptosis, as shown for other viruses [START_REF] Everett | Viruses and Apoptosis: Meddling with Mitochondria[END_REF][START_REF] Verrier | Study of PTPC composition during apoptosis for identification of viral protein target[END_REF][START_REF] Piccoli | Mitochondrial dysfunction in hepatitis C virus infection[END_REF]. Our observations that ADP/O ratio is decreased and oligomycinindependent respiration in increased after infection favor the hypothesis that viral proteins or viral sub-products could be interacting with mitochondrial membranes of HepG2 cells, altering their permeability, increasing proton leak and thus causing the described alterations in mitochondrial physiology. Indeed, further studies are needed to clarify the causal link between mitochondrial physiology and the apoptotic events in DV infection. It is important to mention that the fact that either basal and oligomycin-independent respiration are stimulated by DV infection rules out, at least at this stage of DV infection, an important impairment of respiratory electron chain complexes activities, as reported in some mitochondrial dysfunction as observed in sepsis in both human [START_REF] Svistunenko | Mitochondrial dysfunction in patients with severe sepsis: an EPR interrogation of individual respiratory chain components[END_REF] and animal [START_REF] Brealey | Mitochondrial dysfunction in a long-term rodent model of sepsis and organ failure[END_REF] studies.

In the present work we described for the first time functional and structural

  respiration, 0.1 mM ADP was added to induce state 3 respiration. Due to

A

  C C E P T E D M A N U S C R I P Tneutralized with 2.5 M K 2 CO 3 . Samples and standards were eluted from the column (Hypersil ODS, Supelco Co.) with a linear solvent gradient between phase A (10 mM tetrabutylammonium, 10 mM KH 2 PO 4 , 0.25% methanol) and phase B (5.6 mM tetrabutylammonium, 50 mM KH 2 PO 4 , 30% methanol). Identities of AMP, ADP, and ATP peaks were confirmed by spiking of samples with nucleotides standards. Nucleotides were quantified by external calibration of peak areas of standards.

  A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10 0.22 nmol O 2 /10 6 cell × min -1 to 0.37 nmol O 2 /10 6 cell × min -1 in infected HepG2 (Fig. 2c; P<0.05), indicating that even in permeabilized HepG2 cells, DV infection induces changes in the respiratory properties of mitochondria by altering mitochondrial membrane permeability.

  -infected cells, such as the localization of mitochondria around the nucleus and membrane blebs (Figs.7c and 7d), as previously shown for other cell types infected with DV[START_REF] Marianneau | Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited[END_REF][START_REF] Courageot | Mechanisms of dengue virus-induced cell death[END_REF][START_REF] Mosquera | Ultrastructural studies on dengue virus type 2 infection of cultured human monocytes[END_REF]. Accordingly, Hoechst fluorescence indicated an increase in the percentage of apoptotic cells from 5-10% in mock-infected cells to 15-20% in DV infected cells (data not shown).

Figure legends Figure 1 :

 legends1 Figure legends

Figure 2 :

 2 Figure 2: Dengue virus infection increases oxygen consumption and alters respiratory

Figure 3 :

 3 Figure 3: Dengue virus infection increases oxygen consumption and decreases RCR

Figure 4 :

 4 Figure 4: Dengue virus infection promotes a decrease in mitochondrial membrane potential of HepG2 cells. Cells were incubated with 5 μg/ml of the dye JC-1 for 1 h at

Figure 5 :

 5 Figure 5: Dengue virus infection affects nucleotide content and energy charge of

Figure 6 :

 6 Figure 6: Dengue virus infection promotes morphological alterations of mitochondria

Figure 7 :

 7 Figure 7: Dengue virus infection induces ultrastructural alterations in HepG2 cells.

Acknowledgments

We thank Dr. Alexandre Torres, Dr. Maria Augusta Arruda and Dr. Martha Sorenson for the valuable discussions to this manuscript. We also would like to thank the referees for the valuable contribution to this work. This work was support by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).


Thorsten Rieckmann

Lei Zhuang

Christa E Flück

Beat Trueb
email: beat.trueb@dkf.unibe.ch

Characterization of the first FGFRL1 mutation identified in a craniosynostosis patient

Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley-Bixler syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wildtype protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine motif and a histidine-rich sequence.

Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.

Introduction

Organogenesis and pattern formation are controlled by many cytokines and growth factors, including the fibroblast growth factors (FGFs). In human beings and mice, the FGFs form a large family of 22 structurally related proteins that bind with varying affinity to four different FGF receptors (FGFR1-FGFR4) [START_REF] Itoh | The Fgf families in humans, mice, and zebrafish: their evolutional processes and roles in development, metabolism, and disease[END_REF][START_REF] Itoh | Evolution of the Fgf and Fgfr gene families[END_REF][START_REF] Eswarakumar | Cellular signaling by fibroblast growth factor receptors[END_REF]. Binding to the receptors is stabilized by heparin and heparan sulfate chains. The FGFRs belong to the tyrosine kinase receptors and contain three extracellular immunoglobulin (Ig)-like domains, a single transmembrane domain and an intracellular tyrosine kinase domain. Upon binding of ligands and heparin, the receptors dimerize and transduce the signal to the interior of the cells by autophosphorylation of selected tyrosine residues at the cytoplasmic domain [START_REF] Mohammadi | Structural basis for fibroblast growth factor receptor activation[END_REF].

A while ago, we discovered an additional FGFR in a cartilage-specific cDNA library and termed it FGFRL1 [START_REF] Wiedemann | Characterization of a novel protein (FGFRL1) from human cartilage related to FGF receptors[END_REF]. Independently, two other research groups described the same receptor and termed it FGFR5 [START_REF] Kim | A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas[END_REF][START_REF] Sleeman | Identification of a new fibroblast growth factor receptor, FGFR5[END_REF]. Similar to the classical FGFRs, FGFRL1 contains three extracellular Ig-like domains and a single transmembrane domain. However, in contrast to the classical receptors, it lacks the intracellular tyrosine kinase domain, but instead contains a short cytoplasmic tail of ~100 amino acid residues with a peculiar histidine-rich motif. Conserved sequences for FGFRL1 are found in vertebrates from fish to man [START_REF] Wiedemann | The mouse Fgfrl1 gene coding for a novel FGF receptor-like protein[END_REF][START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF][START_REF] Trueb | Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor[END_REF] and in the cephalochordate amphioxus [START_REF] Beyeler | Fgfrl1, a fibroblast growth factor receptor-like gene, is found in the cephalochordate Branchiostoma floridae but not in the urochordate Ciona intestinalis[END_REF], but not in the urochordate Ciona or in other invertebrates. In vitro, recombinant FGFRL1 interacts with FGF2 and with heparin [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF]. However, in contrast to the classical FGFRs, it forms constitutive dimers at the surface of the cells even in the absence of FGF or heparin. These dimers promote cell adhesion in a way similar to other adhesion molecules located at cell-cell junctions [START_REF] Rieckmann | The cell surface receptor FGFRL1 forms constitutive dimers that promote cell adhesion[END_REF].

FGFRL1 is expressed primarily in cartilage and some muscles [START_REF] Trueb | Expression of FGFRL1, a novel fibroblast growth factor receptor, during embryonic development[END_REF]. Our current data suggest that FGFRL1 has a negative effect on cell growth and a positive effect on cell differentiation. When overexpressed in osteosarcoma cells, it inhibits cell proliferation [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF]. In ovary tumors, its synthesis is largely perturbed when compared to matched control tissues [START_REF] Schild | Aberrant expression of FGFRL1, a novel FGF receptor, in ovarian tumors[END_REF]. In a zebrafish model, inhibition of FGFRL1 synthesis with the help of morpholino constructs inhibited the proper development of the pharyngeal arches, suggesting a role for FGFRL1 in cartilage formation [START_REF] Hall | An essential role for zebrafish Fgfrl1 during gill cartilage development[END_REF].

Recently, we have prepared a mouse model with a targeted disruption of the Fgfrl1 gene [START_REF] Baertschi | Mice with a targeted disruption of the Fgfrl1 gene die at birth due to alterations in the diaphragm[END_REF]. These knock-out mice develop normally until birth, but die immediately after birth due to respiratory failure. The respiratory problems are explained by a reduction of the diaphragm muscle, which is not strong enough to inflate the lungs after birth. Thus, FGFRL1 plays a critical role in the development of cartilage and the diaphragm.

A C C E P T E D M A N U S C R I P T

Germline mutations in FGFRs cause a variety of skeletal disorders [START_REF] Wilkie | Bad bones, absent smell, selfish testes: the pleiotropic consequences of human FGF receptor mutations[END_REF][START_REF] Coumoul | Roles of FGF receptors in mammalian development and congenital diseases[END_REF].

Craniosynostosis, a relatively common malformation of the human skull, occurs in approximately one of 2500 individuals [START_REF] Coumoul | Roles of FGF receptors in mammalian development and congenital diseases[END_REF]. Some of these patients exhibit alterations in the genes for FGFRs, primarily in FGFR2 and FGFR1. Missense mutations, deletions, insertions and splice mutations have been identified causing a variety of syndromes such as Pfeiffer, Crouzon, Jackson-Weiss, Apert, Beare-Stevenson and Antley-Bixler. Mutations in FGFR3 mostly lead to malformations of the limbs as observed in chondrodysplasias, but can also cause Muenke syndrome and Crouzon syndrome with acanthosis nigricans.

In this report, we analyzed the FGFRL1 gene from 55 patients with different congenital skeletal disorders. We found one patient with a craniosynostosis syndrome who had a frameshift mutation in the C-terminal domain of the FGFRL1 sequence. Our in vitro studies indicate that the mutated FGFRL1 protein differs significantly from the wild-type protein in its subcellular localization.

Materials and methods

Genomic sequencing

Various genomic DNA samples were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, England): human random control DNA panel HRC-1, human craniosynostosis panel HC-1, Antley-Bixler BV0954, BV0956, NP0003.

Genomic DNA was also isolated from blood leukocytes of several patients followed at the University Children's Hospital of Bern. For these samples ethical approval and informed consent was obtained. DNA was extracted with the QIAamp DNA Blood Kit according to the supplier's recommendations (Qiagen, Hilden, Germany

Plasmids

The reading frame for human FGFRL1 [START_REF] Wiedemann | Characterization of a novel protein (FGFRL1) from human cartilage related to FGF receptors[END_REF] was inserted into the Bam HI / Xba I site of the expression vector pcDNA3. For stable transfections, cells were grown in 75 cm 2 cell culture flasks. At 70% optical confluence, the cells were transfected with selected plasmids utilizing Metafectene as described above. After 24 hours, the transfected cultures were split into two culture flasks and supplemented with 1 µg/ml puromycin (Invivogen, San Diego CA). On the following day, puromycin was increased to 2 µg/ml and then kept at this concentration until resistant colonies became visible (4 weeks). All individual colonies from one transfection were pooled to yield an oligoclonal puromycin resistant cell population.

Reporter gene assay

The FGF inducible responsive element FiRE [START_REF] Jaakkola | Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor family members in mesenchymal cells[END_REF] was amplified by PCR from genomic mouse DNA (NT039548.6 position 5787210-5787500) and cloned into the Sac I / Nhe I sites of the luciferase reporter vector pGL3 (Promega Catalys, Switzerland). The authenticity of the FiRE element was verified by DNA sequencing.

Subconfluent MC3T3-E1 cells were co-transfected with this FiRE-luciferase reporter vector (0.3 µg per 30 mm dish) and the wild-type or mutant FGFRL1 expression plasmids (1 µg per 30 mm dish) as described above. After 5 h, the transfection medium was replaced by medium supplemented with only 0.3% v/v fetal bovine serum. One day later, the cells were stimulated with FGF2 (15 ng/ml) and heparin (150 ng/ml). On the following day, the cells were washed with phosphate buffered

A C C E P T E D M A N U S C R I P T
saline (PBS), lysed and processed for measurement of luciferase activity using the Luciferase Assay System (Promega E45530).

The expression plasmids used for transfection harbored the cDNA sequences for human FGFRL1 (AJ277437) or for human FGFR1 lacking the tyrosine kinase domain (FGFR1∆C, see above) cloned into pcDNA3.1 (Invitrogen, Carlsbad CA). For RNAimediated down-regulation of the endogenous Fgfrl1 mRNA, synthetic oligonucleotides (Microsynth, Balgach, Switzerland) were ligated into the Bgl II / Hind III sites of the H1-RNA promoter vector pSuper (OligoEngine, Seattle, WA). These oligonucleotides were designed according to the pSuper protocol of the supplier and included the nucleotides GAAGAAGTGGACACTGAGC (sense, position 685-703)

and GCTCAGTGTCCACTTCTTC (antisense, position 703-685) of the mouse Fgfrl1 cDNA (AJ293947).

Northern blots

Total RNA was isolated from stably transfected cell lines by the guanidinium isothiocyanate method utilizing the RNeasy kit from Qiagen GmbH (Hilden, Germany). The RNA was separated on a 1% agarose gel and processed for Northern blotting as previously described [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF].

Antibodies

Monoclonal antibodies against human FGFRL1 were selected from a recombinant phage display library ( HuCAL Gold). To this end, the extracellular domain of human FGFRL1 was expressed in HEK293 cells [START_REF] Rieckmann | The cell surface receptor FGFRL1 forms constitutive dimers that promote cell adhesion[END_REF] and screened against 1.5 x 10 10 Fab fragments utilizing an automated panning process (Morphosys, Antibodies by Design, Martinsried, Germany). Selected antibodies were expressed in bacteria and purified by affinity chromatography. Polyclonal antibodies against human TGN46 and monoclonal antibodies against human LAMP1 (G1/137) were a kind gift from Dr. M. Spiess, University of Basel, Switzerland. Secondary antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA), namely goat anti-mouse IgG (Cy3 conjugated) and goat anti human IgG F(ab) 2 (Cy2 and Cy3 conjugated). All the antibodies were diluted to the recommended working concentrations in PBS containing 1 mg/ml bovine serum albumin, except for anti-LAMP1 that was diluted in PBS containing 1 mg/ml bovine serum albumin plus 0.5% w/v saponin.

Immunofluorescence

Cells on coverslips were fixed for 10 minutes in 4% w/v paraformaldehyde in PBS. After two washing steps with PBS, the cells were permeabilized for 10 min with 0.2% v/v Triton X-100 in PBS and rinsed twice with PBS. After blocking for 30 min

A C C E P T E D M A N U S C R I P T

with 1% w/v bovine serum albumin in PBS, the cells were incubated for 1 h with the primary antibody (1 µg/ml in the case of monoclonal antibodies against FGFRL1).

Following three washing steps with 0.1% v/v Tween 20 in PBS, the cells were incubated for 1 h with the secondary antibody. After washing as above, the cells were counterstained with 4',6'-diamino-2-phenylindole dihydrochloride (DAPI).

Alternatively, they were incubated for 15 min at 37°C with 100 µg/ml RNase A, rinsed twice with PBS and counterstained with acridine orange. Finally, the coverslips were mounted with Mowiol and inspected with a Nikon Eclipse E1000M microscope (Nikon Corp. Tokyo, Japan) or with a confocal LSM510 instrument equipped with an Axiovert 100M microscope (Carl Zeiss, Germany).

2.9. Ligand binding assay FGF2 (Invitrogen) was labeled with DyLight 547-NHS ester (Pierce, Rockford IL) according to the manufacturer's instructions and stabilized with 2 mg/ml bovine serum albumin. Living cells that had been transfected transiently or stably with different FGFRL1 constructs were chilled to 4°C and incubated for 1 h with the labeled FGF2 preparation (1 µg/ml) and heparin (7.5 µg/ml). The cells were then thoroughly rinsed with cold PBS and fixed with 4% w/v paraformaldehyde (see above). After a permeabilization step with 0.2% v/v Triton X-100, the cells were counterstained with monoclonal antibodies against FGFRL1 (see above) and inspected by epifluorescence microscopy.

Results

Identification of the first mutation in the FGFRL1 gene

Genomic DNA from 55 patients suffering from a broad spectrum of congenital skeletal malformations (including 22 patients with nonsyndromic craniosynostosis and 19 with short stature) as well as DNA from 109 Caucasian controls was analyzed for polymorphisms and mutations in the FGFRL1 gene. All protein coding exons were amplified with primers that annealed outside of the coding regions and the PCR fragments were subjected to direct DNA sequencing. Several polymorphisms were identified in the DNA of the patients as well as the controls. In addition, one patient was found with a frameshift mutation in exon 6 shortly before the end of the open reading frame. Downstream of the dinucleotide repeat (AC) n encoding the histidinerich tail, this patient had an insertion of the four nucleotides ACAC. Since the mutation occurred only on one allele, the sequence of the genomic DNA was superimposed starting at the insertion (Fig. 1A). After subcloning of the PCR product into the vector pUC19, the two alleles were isolated in a 1:1 ratio, each displaying a

A C C E P T E D M A N U S C R I P T

clean sequence, one without and the other with the 4 nucleotide insertion. The mutation caused an elongation of the encoded polypeptide by 47 residues before a stop codon was reached at amino acid position 551 (Fig. 1B). The insertion was not simply caused by a PCR artefact since it could be verified with a different primer pair.

Furthermore, none of the 109 controls showed a similar insertion at this locus.

The genomic DNA originated from a female patient who had been diagnosed with the Antley-Bixler craniosynostosis syndrome (OMIM 207410) [START_REF] Reardon | Evidence for digenic inheritance in some cases of Antley-Bixler syndrome?[END_REF]. The patient suffered from craniosynostosis and radio-ulnar synostosis and showed genital malformations including clitoromegaly and labial fusion. Since Antley-Bixler syndrome with genital anomalies may also be caused by P450 oxidoreductase mutations [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF][START_REF] Flück | Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome[END_REF][START_REF] Arlt | Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study[END_REF] and since a heterozygous P450 oxidoreductase mutation has already been described in this patient, we re-screened the genomic DNA for mutations in this gene. We found two mutations, the common A287P mutation in exon 8 that changed alanine 207 into proline, and a novel mutation at the 3' splice junction of exon 8 changing the donor sequence AGgtacca into AGatacca (Fig. 1C). In silico analysis revealed that translation of the corresponding polypeptide would proceed to the next stop codon 45 nucleotides (15 amino acids) downstream of the mutated site if this splice site were neglected by the splicing machinery. Subcloning of the PCR product from exon 8 further showed that each mutation occurred on a separate allele (Fig. 1C). Thus, the patient harbored both a heterozygous FGFRL1 mutation and two heterozygous P450 oxidoreductase mutations. Unfortunately, no DNA was available from the parents of this patient for a detailed genetic analysis, especially with regard to the origin of the FGFRL1 mutation.

Effect of FGFRL1 on FGF signaling

To gain mechanistic insight into the action of FGFRL1, a cell based reporter gene assay was performed. Transactivation activity of wild-type and mutant FGFRL1 was assessed when co-transfected with the FiRE luciferase reporter into osteoblastic cells. FiRE is a 170 bp FGF inducible responsive element derived from the syndecan-1 gene that is specifically activated by FGFs in mesenchymal cells [START_REF] Jaakkola | Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor family members in mesenchymal cells[END_REF]. Stimulation of FiRE transfected cells with 15 ng/ml FGF2 showed a ten-fold induction of luciferase activity (Fig. 2A). This induction was reduced to one third when the wildtype, full-length construct of FGFRL1 was co-transfected into the cells together with the reporter gene, suggesting that FGFRL1 exerts a negative effect on FGF signaling. A slightly stronger reduction was observed when the deletion construct FGFR1∆C lacking the intracellular tyrosine kinase domain was co-transfected together with the reporter gene. This FGFR1 variant has previously been demonstrated to exert a dominant negative effect on FGF signaling [START_REF] Ueno | A truncated form of fibroblast growth factor receptor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor[END_REF]. When the mutated FGFRL1 construct harboring the 4 nucleotide insertion from the reported

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10
craniosynostosis patient was utilized in the reporter assay, a significant reduction of FGF signaling was observed as well.

It is conceivable that downregulation of endogenous FGFRL1 mRNA will have a positive effect on FGF signaling when overexpression of FGFRL1 has a negative effect. RNA interference was utilized to investigate this possibility (Fig. 2B). Small interfering RNAs for the FGFRL1 mRNA were introduced into the osteoblastic cells by transfection of a pSuper construct that contained 19 nucleotides of the FGFRL1 cDNA. As expected, the presence of these interfering oligonucleotides led to a nearly two-fold increase in FiRE luciferase activity in comparison to control cells transfected with the empty pSuper vector and stimulated with FGF2 (Fig. 2B).

Subcellular localization of wild-type and mutant FGFRL1

Since there was barely any difference in the FGF2 mediated reporter assay between wild-type and mutant FGFRL1, we investigated the subcellular localization of wild-type and mutant proteins in cultivated SW1353 chondrosarcoma cells. To prepare a reagent that would be able to detect the untagged protein, we isolated monoclonal antibodies from a recombinant phage display library. The extracellular portion of FGFRL1 produced in HEK293 cells served as the specific antigen. Eight different antibodies were obtained that reacted with the non-reduced antigen on dot blots. However, none of the antibodies reacted with the antigen on a Western blot after reduction of disulfide-bonds with β-mercaptoethanol (not shown). This result suggests that all antibodies recognize a structural epitope of FGFRL1, which is stabilized by disulfide bonds.

The antibodies were used for visualization of the FGFRL1 protein in several cultivated cell lines including SW1353, MC3T3-E1, 3T3, CHO-K1, COS7 and VA13.

Surprisingly, none of the antibodies produced a clear signal with these cells. It is possible that the amount of endogenous FGFRL1 protein was too low to be detected by our antibodies. However, a prominent signal was obtained when the wild-type cDNA sequence for FGFRL1 was transfected into the cells. In this case, the signal was mainly confined to vesicular structures and the Golgi complex (Fig. 3). The signal in the Golgi co-localized with the trans-Golgi network protein TGN46 as demonstrated by double fluorescence experiments. Unexpectedly, the plasma membrane was barely stained with our antibodies. This result is in sharp contrast to our previous findings obtained with an FGFRL1-GFP fusion construct [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF]. In those experiments, the majority of the fluorescence signal emitted from the GFP-fusion protein had clearly localized to the plasma membrane. It is conceivable that the subcellular distribution of the GFP-fusion protein differed from that of the wild-type protein due to some structural differences. Relative to wild-type FGFRL1, the fusion

A C C E P T E D M A N U S C R I P T

protein lacked the last amino acid residues (469-504), but instead contained the GFP moiety at its C-terminus.

To address this hypothesis in detail, we deleted the sequence corresponding to the intracellular, C-terminal domain from our untagged wild-type protein. In fact, the resulting construct FGFRL1∆C encompassing residues 1-416 revealed a subcellular distribution very similar to that of the fusion protein FGFRL1-GFP (Fig. 3). An untagged construct corresponding to the FGFRL1 mutation found in the craniosynostosis patient revealed basically the same distribution. Moreover, when a 1:1 mixture of wild-type and mutated constructs was transfected into the HEK293 cells, immuno-reactive FGFRL1 protein was found primarily at the cell membrane, too (not shown). Such a mixture should mimic the situation as it occurs in the craniosynostosis patient suffering from the heterozygous frame-shift mutation.

Taken together, these results suggest that the wild-type FGFRL1 sequence contains one or several sequence motif(s) that control its subcellular distribution.

These motifs appear to be missing or shielded in the proteins encoded by FGFRL1∆C and by the cDNA from the craniosynostosis patient.

Motifs in the intracellular C-terminal sequence of FGFRL1

In contrast to the extracellular portion of the FGFRL1 sequence, the intracellular portion is not well conserved among different species (Fig. 4A). Nevertheless, a multiple sequence alignment identified three conserved motifs that might play a role in protein trafficking, namely (1) a dileucine sequence occurring 7-9 residues downstream of two acidic amino acids, (2) two tyrosine motifs that were arranged in tandem (YXXΦYXXΦ, where Φ is a bulky, hydrophobic amino acid), and (3) the histidine-rich region that mainly consists of His-Ser and His-Thr repeats. Dileucine and tyrosine based motifs are well known mediators of endocytosis and transmembrane protein trafficking [START_REF] Bonifacino | Signals for sorting of transmembrane proteins to endosomes and lysosomes[END_REF], but no similar function has yet been described for histidine-rich motifs. To study the influence of the three motifs on the subcellular distribution of FGFRL1, we prepared deletion constructs lacking the histidine-rich region (FGFRL1∆His) or the histidine-rich region plus the tandem tyrosine motif (FGFRL1∆His∆Tyr) (Fig. 4B). Furthermore, we disrupted the putative dileucine (L460S) and tyrosine (Y471A, Y475A) motifs by site directed mutagenesis. The truncated and the mutated constructs were transiently overexpressed in chondrosarcoma cells and detected with our antibodies (Fig. 5, left side). Disruption of the putative dileucine motif (L460S) did not have any effect on the subcellular distribution of FGFRL1 (not shown). However, deletion of the histidine-rich region (∆His) or the histidine-rich region plus the tyrosine motif ( ∆His∆Tyr) induced translocation of FGFRL1 to the plasma membrane. Site directed mutagenesis of either one of the two tyrosine residues alone (Y471A, Y475A) did not have any effect

A C C E P T E D M A N U S C R I P T

(not shown). However, when both tyrosines were mutated in concert, the resulting protein was relocalized to the plasma membrane.

The results obtained with transient transfections were confirmed by stably expressing the same constructs in HEK293 cells (Fig. 5, right side). Wild-type FGFRL1 and FGFRL1 carrying either one of the single mutations (L460S, Y471A, Y475A) were found to be localized almost exclusively in the trans-Golgi network and in vesicular structures. Deletion of the histidine-rich region or the histidine-rich region plus the tyrosine motif led to the translocation of the protein to the plasma membrane. This was particularly well observed at cell-cell contact sites. As noted with transient transfections, mutation of the two tyrosines in concert resulted in a phenotype similar to that of FGFRL1∆C. These results demonstrate for the first time that the C-terminus of FGFRL1 carries motifs, which efficiently regulate the subcellular distribution of the FGFRL1 protein.

Finally, the subcellular distribution of the mutant FGFRL1 protein from the craniosynostosis patient was analyzed (Fig. 5, bottom). In transiently transfected SW1353 cells as well as in stably transfected HEK293 cells, the FGFRL1 protein with the frameshift mutation was found predominantly at the cell membrane. The distribution differed significantly from that of the wild-type protein, but corresponded to that of the FGFRL1∆C construct. Thus, the frameshift mutation in the FGFRL1 gene of the craniosynostosis patient caused a significant redistribution of the FGFRL1 protein, probably due to shielding or functional loss of an important trafficking motif.

Endocytosis of cell surface FGFRL1

The virtual absence of wild-type FGFRL1 from the plasma membrane may be explained in two ways. Either, FGFRL1 reaches the plasma membrane but is efficiently and rapidly internalized thereafter. Alternatively, the intracellular trafficking machinery targets the protein directly to another compartment. To address this question, living HEK293 cells that stably expressed wild-type FGFRL1 were precooled and incubated at 4°C with our monoclonal antibodies (Fig. 6). The cells were fixed and a fluorescently labeled, secondary antibody was added. To ensure the structural integrity of the cell membrane, no detergent was added before the final washing steps. By confocal microscopy a spotty signal was observed at the periphery of the cells. This indicates that a fraction of the vesicles observed before must represent FGFRL1 complexes located at the cell surface. As expected, the trans-Golgi network was not stained in this experiment since the cells had not been permeabilized before addition of the antibodies.

In the next experiment, the antibodies were applied to the living cells at 37°C (Fig. 7). After three hours, the cells were fixed, permeabilized and co-stained with an

A C C E P T E D M A N U S C R I P T

antibody against the lysosomal marker protein LAMP1. Again, we found a spotty signal of vesicle-like structures. Some of these vesicles co-localized with LAMP1.

Thus, a fraction of the FGFRL1 protein must reach the plasma membrane from where it is rapidly internalized and transported to the lysosomes.

Retention of mutant FGFRL1 at the cell surface

The experiments with our monoclonal antibodies had suggested that mutant FGFRL1 from the Antley-Bixler patient stayed for a prolonged time at the cell surface where it might participate in FGF signaling. In an attempt to directly validate this observation, we performed an experiment with fluorescently labeled FGF ligand (Fig. 8). HEK293 cells were transiently transfected with wild-type and mutant FGFRL1 and chilled to 4°C. A fluorescently labeled preparation of FGF2 was added and, after an incubation period of 1 hour, the distribution of the label was examined under the microscope. As expected, cells expressing wild-type FGFRL1 revealed only background staining since they possessed barely any FGFRL1 at their cell surface.

Nevertheless, these cells did produce FGFRL1, which could be detected with our monoclonal antibodies in the Golgi complex, the endoplasmic reticulum and the nuclear membrane. In contrast, cells that had been transfected with the mutant FGFRL1 corresponding to the Antley-Bixler sequence bound relatively large amounts of FGF2 as revealed by the red label at the cell surface. In this case, the distribution was found to be very similar to that of a control experiment performed with the truncated version FGFRL1∆C. Analogous results were also obtained with stably transfected HEK293 cells (Fig. 8, lower part). Taken together, these experiments clearly demonstrate that mutant FGFRL1 is retained at the cell surface for a longer period of time than the wild-type protein and that the fraction of FGFRL1 at the cell surface is able to interact with extracellular FGF ligands.

Discussion

FGFRL1 (also known as FGFR5) is a novel member of the FGFR family that lacks the intracellular tyrosine kinase domain, but instead contains a short C-terminal domain of unknown function [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF]. We report here the first human mutation in this receptor. The mutation was identified in the C-terminal domain of FGFRL1 from a British patient that has previously been described by

A C C E P T E D M A N U S C R I P T

Recent findings suggest that the Antley-Bixler phenotype comprises two genetically different variants of the syndrome, an autosomal dominant form that segregates with mutations in one of the FGFR genes and an autosomal recessive form that segregates with mutations in the gene for P450 oxidoreductase [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF][START_REF] Flück | Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome[END_REF][START_REF] Arlt | Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study[END_REF].

Antley-Bixler patients with mutations in P450 oxidoreductase show steroid hormone abnormalities due to decreased activities of three enzymes that depend on electron transfer from P450 oxidoreductase, namely 21-hydroxylase, 17α-hydroxylase and aromatase. As a consequence, such patients present with hormonal and genital anomalies including hypoplastic labia majora and clitoromegaly in females. In contrast, Antley-Bixler patients with mutations in one of the FGFRs have normal steroid profiles and normal genitalia [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF][START_REF] Flück | Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome[END_REF][START_REF] Arlt | Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study[END_REF]. Mutations in FGFRs are also found in other craniosynostosis syndromes such as Pfeiffer, Apert, Jackson-Weiss and

Crouzon syndromes [START_REF] Wilkie | Bad bones, absent smell, selfish testes: the pleiotropic consequences of human FGF receptor mutations[END_REF][START_REF] Coumoul | Roles of FGF receptors in mammalian development and congenital diseases[END_REF]. However, patients with these syndromes do not suffer from abnormalities in steroid hormone biosynthesis.

Patient 14683 was found to have an abnormal steroid profile and ambiguous genitalia but no FGFR2 mutation [START_REF] Reardon | Evidence for digenic inheritance in some cases of Antley-Bixler syndrome?[END_REF]. The relatively common P450 oxidoreductase mutation A287P was identified on one allele, but no mutation was observed on the second allele (patient 16 in [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF]). We have now identified a novel splice site mutation on the second allele at exon/intron junction 8. Similar splice site mutations have been identified in other Antley-Bixler patients at exon/intron junctions 6 and 7 [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF][START_REF] Flück | Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome[END_REF].

Hence, patient 14683 is compound heterozygous for two P450 oxidoreductase mutations that may explain the disordered steroid profile and the genital anomalies.

In addition to the abnormal genitalia, patient 14683 suffers from craniosynostosis, brachycephaly, midface hypoplasia, proptosis, choanal stenosis, radio-ulnar synostosis and femoral bowing [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF][START_REF] Reardon | Evidence for digenic inheritance in some cases of Antley-Bixler syndrome?[END_REF]. These anomalies are typical hallmarks of Antley-Bixler syndrome, but so far no convincing explanation has been offered for the pathogenesis of the bone malformations through P450 oxidoreductase mutations.

Moreover, about one third of all the patients with P450 oxidoreductase mutations do not show any skeletal malformations at all.

We have now demonstrated that patient 14683 harbors a mutation in the FGFRL1 gene in addition to the P450 oxidoreductase mutations, offering an explanation for the bone phenotype. FGFRL1 binds FGF2 and other FGF ligands similar to all other FGFRs. Although the exact function of the novel receptor is not known, there is good evidence that it modulates FGF signaling. We have previously shown that overexpression of FGFRL1 in osteosarcoma cells inhibits cell growth and proliferation [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF]. In a reporter gene assay, we demonstrated here (Fig. 2) that it diminishes the stimulatory effect of FGF2 on transcription of an FGF inducible gene.

Taken together, our results suggest that FGFRL1 exerts a negative effect on FGF signaling.

A C C E P T E D M A N U S C R I P T

FGFRL1 is expressed in cartilaginous and bony tissues and in some muscles [START_REF] Trueb | Expression of FGFRL1, a novel fibroblast growth factor receptor, during embryonic development[END_REF]. Unexpectedly, mice with a targeted disruption of the Fgfrl1 gene exhibit only subtle alterations in their skeleton, yet they die perinatally due to respiratory failure [START_REF] Baertschi | Mice with a targeted disruption of the Fgfrl1 gene die at birth due to alterations in the diaphragm[END_REF]. The respiratory problems are explained by a severe reduction of the diaphragm muscle, which is not strong enough to inflate the lungs after birth. A more pronounced cartilage or bone phenotype would probably become overt in this mouse model after birth when the skeleton grows at maximal speed. However, our knock-out mice cannot be investigated at later stages as all of them die immediately after birth.

Neveretheless, newborn Fgfrl1 -/-mice exhibit subtle alterations in the shape of their head, including a dome-shaped skull with high front (S. Baertschi and B. Trueb, unpublished observation). Similar alterations have previously been noticed in mice with targeted mutations in Fgfr1 (P250R) or Fgfr2 (S250W) which had been created as animal models for human craniosynostosis [START_REF] Coumoul | Roles of FGF receptors in mammalian development and congenital diseases[END_REF]. Furthermore, Hall et al. [START_REF] Hall | An essential role for zebrafish Fgfrl1 during gill cartilage development[END_REF] studied the expression of Fgfrl1 in a zebrafish model. These authors used morpholino constructs to inhibit the expression of the novel receptor and found that the branchial arches of Fgfrl1 deficient animals failed to complete chondrogenesis.

Thus, Fgfrl1 is crucial for proper cartilage and bone formation, at least at some specific sites. in the mutant FGFRL1 protein such that they become non-functional. Without functional motifs, endocytosis of FGFRL1 will be delayed and the proportion at the plasma membrane will increase. A similar effect has recently been described for two point mutations of the FGFR2 receptor that occur in Apert syndrome [START_REF] Ahmed | Extracellular point mutations in FGFR2 elicit unexpected changes in intracellular signalling[END_REF]. After ligand stimulation, the wild-type FGFR2 protein underwent rapid endocytosis into lysosomes, whereas the modified protein with the S252W or P253R mutation remained at the cell membrane for an extended period of time, thereby leading to an elevation of intracellular signaling.

The mutant sequence of the ABS patient has several features in common with the Fgfrl1 sequences from rat and mouse. During evolution, the murine sequences appear to have suffered a similar frameshift mutation in the C-terminal domain when compared to the sequences from man, chicken, frog and fish [START_REF] Trueb | Characterization of FGFRL1, a novel FGF receptor preferentially expressed in cartilage[END_REF]. They contain only half of the histidine-rich domain but possess an extension of the polypeptide to amino acid residue 529. Nevertheless, the full-length protein from mice reveals a subcellular distribution virtually identical to that of human full-length FGFRL1 with the majority of the protein found in the endoplasmic reticulum, the Golgi and the secretory vesicles (Rieckmann and Trueb, unpublished). It remains to be determined why the murine protein does not display the altered subcellular distribution observed with the mutant FGFRL1 protein from the ABS patient.

Since genital abnormalities are usually not observed in craniosynostosis syndromes caused by FGFR mutations (Apert, Crouzon, Pfeiffer, Jackson-Weiss),

Reardon and coworkers proposed that Antley-Bixler syndrome with genital and steroid abnormalities might be explained by a "digenic disorder" involving one factor related to FGF signaling and another factor related to genital development [START_REF] Reardon | Evidence for digenic inheritance in some cases of Antley-Bixler syndrome?[END_REF].

However, so far all Antley-Bixler patients with verified FGFR2 mutations segregated completely from those with genital abnormalities and verified P450 oxidoreductase mutations [START_REF] Huang | Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis[END_REF]. The identification of an FGFRL1 mutation in combination with P450 oxidoreductase mutations in a patient with abnormal steroid profiles now fuels the hypothesis that Antley-Bixler syndrome might represent a digenic disorder in some patients. Another patient that may support this conclusion was described in 2004 by Monoclonal antibodies against FGFRL1 were added to living HEK293 cells that had been stably transfected with the wild-type FGFRL1 construct. After 3 hours, the cells were fixed, permeabilized and stained with a Cy3 conjugated secondary antibody (red). LAMP1 was co-stained with polyclonal antibodies followed by Cy2 conjugated secondary antibodies (green). A subfraction of the FGFRL1-positive vesicles was found to be positive for Lamp1 as shown by arrowheads. Images were taken by confocal microscopy. DIC, differential interference contrast. Bar = 10 µm.

2. 2 .

 2 Cell cultures All cell lines were obtained from the American Type Culture Collection: human osteosarcoma cells MG63 (CRL-1427), human chondrosarcoma cells SW1353 (HTB 94), human rhabdomyosarcoma cells A204 (HTB-82), human embryonic kidney cells HEK293 (CRL-1573), human lung fibroblasts VA13 (CCL 75.1), Chinese hamster ovary cells CHO-K1 (CCL-61), mouse embryonic fibroblasts 3T3 (CCL-163), and mouse osteoblast-like cells MC3T3-E1 (CRL-2593). Most of these cells were maintained at 37°C under an atmosphere of 5% v/v CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. The medium for SW1353 cells, CHO-K1 cells and HEK293 cells under puromycin selection was further enriched with nonessential amino acids. MC3T3-E1 cells were propagated in α-minimum essential medium (αMEM).

  The frameshift mutation observed in FGFRL1 from patient 14683 leads to a clearly different phenotype in cell culture experiments. As shown with monoclonal antibodies against FGFRL1 and with Cy3-labeled FGF2, wild-type FGFRL1 is barely located at the cell membrane, where it could interact with ligands, but is rather found in vesicular structures and the Golgi complex. In sharp contrast, mutant FGFRL1 is primarily located at the cell surface where it actively participates in FGF binding. The subcellular distribution of FGFRL1 appears to be controlled by two (or more) motifs situated in the intracellular, C-terminal domain of the protein, a histidine-rich region with 10 histidines distributed over a stretch of 23 residues and a tandem tyrosine motif YXXΦ, where X denotes any amino acid and Φ an amino acid with a bulky, hydrophobic side chain. Deletion of either one of these motifs results in the preferential localization of the receptor at the plasma membrane. Our experiments suggest that the two motifs function as signals for trafficking of FGFRL1 from the plasma membrane to endosomes and lysosomes. While such a function has not yet been described for the histidine-rich sequence, it is well documented for YXXΦ motifs[START_REF] Bonifacino | Signals for sorting of transmembrane proteins to endosomes and lysosomes[END_REF]. These motifs occur for instance in the transferrin receptor, in the trans-Golgi network protein TGN46 and in the lysosomal associated membrane proteins LAMP1 and LAMP2. The frameshift mutation observed in patient 14683 deletes part of the histidinerich motif and increases the length of the C-terminal domain of FGFRL1 by 45 amino acids. In cell culture experiments, the mutated protein exhibits an altered subcellular A C C E P T E D M A N U S C R I P T distribution similar to that of the truncated FGFRL1 version lacking the tyrosine and histidine motifs. It is therefore likely that the tyrosine and histidine motifs are shielded

AFig. 1 .

 1 Fig. 1. Mutations in FGFRL1 and P450 oxidoreductase in a craniosynostosis patient. (A) Partial sequence of exon 6 from FGFRL1 demonstrating heterozygosity for the 4 nucleotide insertion ACAC. After subcloning, the insertion is found only on one allele. (B) Alignment of the C-terminal amino acid sequences derived from the mutated allele of the patient and from a normal control. (C) Compound heterozygosity of the same patient for the P450 oxidoreductase mutation A287P (left side) and the splice mutation (right side). After subcloning, the A287P mutation is found on one allele, the splice mutation on the other. The nucleotide and derived amino acid sequences are given below the chromatograms.

Fig. 2 .

 2 Fig. 2.Effects of FGFRL1 on FGF signaling. A plasmid containing the luciferase reporter gene under the control of the FiRE enhancer was cotransfected with the expression vectors pcDNA3.1 (A) or pSuper (B) into MC3T3-E1 cells. One half of the culture dishes was stimulated with FGF2 (15 ng/ml), while the other half was left untreated. After 24 h, the cells were lysed and luciferase activity was determined.Stimulation of the cells with FGF2 led to a 10-fold induction of luciferase activity (A).This induction was partially reduced by FGFRL1 transcribed from wild-type DNA, from the DNA of the craniosynostosis patient or from the DNA of a dominant negative FGFR1 variant that lacked the intracellular kinase domain (FGFR1∆C). On the other hand, the induction was further enhanced by down-regulation of the endogenous mRNA for FGFRL1 via a hairpin RNA transcribed from the pSuper vector (B). All bars

Fig. 3 .

 3 Fig.3. Subcellular localization of FGFRL1. SW1353 chondrosarcoma cells were transfected with expression constructs corresponding to the wild-type sequence of FGFRL1, the truncated form FGFRL1∆C (residues 1-416) or the frameshift mutation of the craniosynostosis patient. After fixation and permeabilization, FGFRL1 was detected with monoclonal antibodies, followed by Cy3 conjugated secondary antibodies (red). The trans-Golgi network protein TGN46 was visualized with polyclonal antibodies, followed by Cy2 conjugated secondary antibodies (green).Scale bar = 10 µm. Wild-type FGFRL1 is found in the trans-Golgi network and in vesicle-like structures. The truncated and the mutated proteins are preferentially located at the plasma membrane.

Fig. 4 .

 4 Fig. 4. Motifs and signals found in the intracellular domain of FGFRL1. The FGFRL1 sequences from six different vertebrates are aligned in (A). Identical residues are boxed. The double leucine motif, the tyrosine based signal YXXΦ and the histidinerich sequence are marked. All the different constructs utilized in this study are presented in (B) together with the amino acid positions they encompass and the abbreviations used. TM, transmembrane domain.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Distribution of FGFRL1 in transfected cells. Various FGFRL1 constructs as depicted in Figure 4B were transiently transfected into SW1353 cells or stably transfected into HEK293 cells. After fixation and permeabilization of the cells, FGFRL1 was visualized with monoclonal antibodies, followed by Cy3 conjugated secondary antibodies (red). Nuclei were visualized by DAPI (left) or by acridine orange (right) and are presented in blue. SW1353 cells were inspected by conventional fluorescence microscopy, HEK293 cells were inspected by confocal microscopy. Wild-type FGFRL1 is primarily found in the trans-Golgi network and in vesicle-like structures. The truncated and mutated variants are located preferentially at the plasma membrane. Scale bar = 10 µm.

Fig. 7 .

 7 Fig. 7. Partial co-distribution of FGFRL1 and the lysosomal marker protein LAMP1.

Fig. 8 .

 8 Fig.8. Retention of mutant FGFRL1 at the cell surface. HEK293 cells that had been transiently or stably transfected with different FGFRL1 constructs as indicated were cooled to 4°C and incubated with a fluorescently labeled preparation of FGF2. The cells were fixed, permeabilized and incubated with antibodies against FGFRL1, followed by Cy2 conjugated secondary antibodies. The distribution of FGF2 (red) and FGFRL1 (green) was inspected by epifluorescence microscopy. Prominent staining of FGF2 at the cell surface was observed in cells transfected with the mutant construct corresponding to the ABS sequence and the FGFRL1∆C construct, but not in cells transfected with the wild-type construct.

Acknowledgements

This study was supported by grants from the Swiss National Science Foundation (3100A0-113806) and the Swiss Foundation for Research on Muscular Diseases.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T