Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease

1 Larger aggregates of mutant seipin in Celia’s Encephalopathy, a new protein misfolding neurodegenerative disease Alejandro Ruiz-Riquelmea, Sofía Sánchez-Iglesiasa, Alberto Rábanob, Encarna Guillén-Navarroc, Rosario Domingo-Jiménezd, Adriana Ramosa,†, Isaac Rosaa,f, Ana Senraa, Peter Nilssone, Ángel Garcíaa,f, David Araújo-Vilara,g* and Jesús R. Requenaa,g* aCIMUS Biomedical Research Institute, University of Santiago de Compostela- IDIS, 15782 Santiago de Compostela, Spain bNeuropathology Department and Tissue Bank, Fundación CIEN, 28031 Madrid, Spain cSection of Medical Genetics and Dysmorphology, Division of Pediatrics, Hospital Clínico Universitario Virgen de la Arrixaca, IMIB-Arrixaca. 30120 Murcia; UCAM-Catholic University of Murcia; CIBERER-ISCIII, Madrid, Spain dSection of Neuropediatrics, Division of Pediatrics, Hospital Clínico Universitario Virgen de la Arrixaca, IMIB-Arrixaca. 30120 Murcia; CIBERER-ISCIII, Madrid, Spain eAffinity Proteomics, SciLifeLab, School of Biotechnology, KTH – Royal Institute of Technology, SE 171-21Stockholm, Sweden fDepartment of Pharmacology, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain gDepartment of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain †Present address: Department of Psychiatry and Behavioural Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. *To whom correspondence should be addressed at: Room SS1D, CIMUS Biomedical Research Institute, University of Santiago de Compostela-IDIS, 15782 Santiago de Compostela, Spain Tel: +34 881815464; Email: [email protected] (J.R.R.); U.E.T.e.M, Lab 3, 2nd floor, CIMUS Biomedical Research Institute, University of Santiago de Compostela-IDIS, 15782 Santiago de Compostela, Spain. Tel: +34 881815426; Email: [email protected] (D.A.V.) 2 Abstract Celia’s Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at age 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. Keywords Seipin; BSCL2; Neurodegeneration; Lipodystrophy; Intranuclear inclusions; Progressive encephalopathy; Celia’s encephalopathy; Oligomerization; Phenotype rescue. Introduction Seipin is an endoplasmic reticulum (ER) resident protein highly expressed in brain and testicles (Windpassinger et al., 2004). It has 3 isoforms of 462, 398 and 287 amino acids (Cartwright and Goodman, 2012). Its predicted structure would encompass two transmembrane domains, a conserved core region and both amino- and carboxitermini facing the cytoplasm (Lundin et al., 2006). The function of seipin remains incompletely understood. There are evidences for a role of seipin in lipid droplet (LD) synthesis (Fei et al., 2011 Szymanski et al., 2007 and Yang et al., 2013a) and adipocyte differentiation (Payne, 2008, Yang, 2013a and Yang, 2013b). Previous studies have shown the existence of seipin oligomers of about 9 subunits in yeast related to the synthesis of lipid 3 droplets (Binns et al., 2010). In addition, oligomers of 12 subunits have also been described for human seipin (Sim et al., 2013a). Recently, new roles for seipin in nervous tissue have been proposed. Seipin might regulate excitatory synaptic transmission by modulating AMPA receptor levels (Wei et al., 2013) and synaptic vesicle exocytosis by directly regulating synaptic vesicle docking (Wei et al., 2014). Furthermore, a role for seipin in the mobilization of lipids to the developing brain has also been proposed (Hötta- Vuori et al., 2013). Mutations in the Seipin/BSCL2 gene cause either type 2 congenital generalized lipodystrophy (CGL2) (Magré et al., 2001) or dominant motor neuron diseases (Windpassinger et al., 2004). However, we recently reported on a novel mutation in the BSCL2 gene (c.985C>T) that results in a fatal neurodegenerative syndrome (Guillén-Navarro et al., 2013) henceforward called “Celia’s Encephalopathy” (MIM #615924). This mutation induces an alternative splicing event that results in skipping of exon 7 and a reading frame shift giving place to a new aberrant protein, hereafter called Celia seipin. To date, six patients have been identified. Two of them are homozygous for the c.985C>T mutation whereas the other four are compound heterozygous, carrying the c.985C>T mutation plus a lipodystrophic mutation (c.538G>T or c.507_511del). Both homozygous and compound heterozygous patients showed a similar neurological course, suffering from progressive encephalopathy starting at age 2-3, with a fatal outcome at age 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to the mutation (Guillén-Navarro et al., 2013). The pathogenic mechanism by which Celia seipin exerts its pathogenic effect is not completely understood. Our previous work has shown high expression level of BiP, an ER stress marker, in Celia seipin-overexpressing compared to control cells (Guillén-Navarro et al., 2013). ER stress has been extensively associated to several neurodegenerative diseases (Matus et al., 2011). Besides, we found ubiquitin positive intranuclear inclusions in the hypothalamus of the index case (Guillén-Navarro et al., 2013). Ubiquitin positive intranuclear inclusions have been associated to other neurodegenerative syndromes like Huntington’s disease (Sieradzan et al., 1999). Likewise, ER stress has been demonstrated in the N88S and S90L mutations of the seipin gene, both of which lead to motor neuropathies (Ito and Suzuki, 2007 and Ito et al. 2008). Moreover, cytoplasmic inclusion bodies with a cytoprotective function have been detected in association to these mutations (Ito et al., 2008 and Ito et al., 2012).On the other hand, mild mental retardation has been reported linked to CGL2 (Van Maldergem et al., 2002) suggesting an 4 impairment of brain functions of seipin. However, considering the short term lethal nature of Celia’s Encephalopathy, a different or more aggressive pathogenic mechanism has to be involved in this new disease. The results reported here provide new insights about the molecular mechanism involved in Celia’s Encephalopathy. Materials and Methods This study was approved by the local Ethics Review Panel of Xunta de Galicia (Spain) and was in accordance with the Declaration of Helsinki. Patient’s relatives gave informed consent for participation in the study and publication of clinical and biochemical information. Tissue samples Hypothalamus, and parietal and occipital cortex samples were obtained from the homozygous index case during autopsy, from two control cases (both of them males, 43 and 68 years at death, respectively) deceased by suicide, and from one control case (female, 6 years at death) deceased from an abdominal tumor, with autopsy performed in accordance with the Spanish legislation. Adipose tissue samples were obtained by biopsy of the abdominal area from a homozygous patient (index case), at 6 years of age, and from an age-matched healthy control. Small pieces of visceral adipose tissue were placed on a 60 mm dish (BD FalconTM, Mississauga, ON, Canada) containing Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) plus 30% fetal bovine serum (FBS) (Gibco, Life Technologies, Gaithersburg, MD, USA) and 50 μg/ml gentamicin (Sigma-Aldrich), and incubated at 37 ºC with 5% CO2 in a Water-Jacket CO2 incubator (NuAire, Plymouth, MN, USA). Preadipocytes were recognised by the presence of small lipid droplets in the fibroblast-like cells. Preadipocytes were tripsinized with TrypLE™ Express Stable Trypsin-like Enzyme with Phenol Red (Gibco) and cultured on 100 mm dishes in DMEM containing 10% FBS and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Cell culture and transfection HeLa, 3T3, HEK293 and COS-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Invitrogen) and 1% L-glutamine (Gibco). SH-SY5Y cells were grown in 1:1 Ham’s F12:Earle’s Balanced Salt Solution (Sigma- Aldrich) supplemented with 15% FBS (Gibco), 1% penicillin/streptomycin (Invitrogen), 1% L-glutamine (Gibco) and 1% non-essential amino-acids (NEAA) 5 (Gibco). Transfections were performed with TurboFect (Thermo-Fisher Scientific, Waltham, MA, USA) or Fugene 6 (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. cDNA A plasmid containing wild type human seipin fused to a myc tag (6 myc-wt seipin pCS2+MT) was a kind gift from D. Ito, Keio University, Japan. Myc fused Celia seipin expression plasmid were previously described (Guillén-Navarro et al., 2013). Plasmids containing wild type or Celia seipin fused to a FLAG tag were created as follows: seipin cDNA was amplified by PCR using primers designed for human wt seipin (forward: 5’-GCCGAATTCATGTCTACAGAAAAGGTAGACCA- 3’; reverse: 5’-GCCTCTAGAGGAACTAGAGCAGGTGGGG-3’) and Celia seipin (forward: 5’-GCCGAATTCATGTCTACAGAAAAGGTAGACCA-3’; reverse: 5’- GCCTCTAGAGGGGCTGCTGATCTGGTTT-3’), then digested with the restriction enzymes EcoRI (Thermo-Fisher Scientific) and XbaI (Thermo-Fisher Scientific), purified and inserted into p3XFLAG-CMV-14 (Sigma-Aldrich) plasmid. Correct cloning was verified by sequencing. Control plasmids were empty p3XFLAG-CMV-14 (Sigma-Aldrich) and pCS2+MT. Antibodies The following primary antibodies were used in this study: anti seipin (HPA042394) rabbit polyclonal antibody (1:40-1:500, The Human Protein Atlas [Uhlen, 2010]), anti c-myc (9E10) mouse monoclonal antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-FLAG rabbit polyclonal antibody (1:1000, Sigma-Aldrich), anti-GAPDH (1:10000-1:20000, Sigma-Aldrich), anti- Tubulin (1:5000, Sigma-Aldrich), anti BiP (1:1000, Cell Signaling), anti 14-3-3 pan (Thermo-Fisher Scientific). The following secondary antibodies were used: Alexa Fluor 555- conjugated anti-mouse IgG secondary antibody (Invitrogen), horseradish peroxidase-linked anti mouse IgG secondary antibody (GE Healthcare UK, Buckinghamsire, UK), horseradish peroxidase-linked anti rabbit IgG secondary antibody (Dako, Glostrup, Denmark). Density gradient fractionation Lysates (vide infra) from HeLa cells transfected with Myc-wt, Myc-Celia or both seipin isoforms (10:1 proportion) were loaded on the top of 10-60 % sucrose gradients (sucrose dissolved in PBS) (Supplementary Material, Fig. S3A) and centrifuged at 272109 g in a SW60Ti rotor (Beckmann Coulter, Brea, CA, USA), 16 h, 4ºC. 6 8 fractions of 325 μl were collected from the top, subjected to SDS-PAGE and analyzed by immunoblotting with 1:1000 anti c-myc (9E10) antibody (Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase-linked secondary antibody (GE Healthcare). The MW of the oligomers was estimated using the theoretical MW of Myc-wt seipin (58 kDa) and Myc-Celia seipin (46 kDa). According to the calibration curve (Supplementary Data, Fig. S3B) Myc-wt seipin oligomers have a MW of approximately 690 kDa. Dividing the estimated MW of Myc-wt seipin oligomers and the MW of each subunit we can conclude that the oligomers are composed of 12 subunits (690/58 = 11.89). Myc-Celia seipin aggregates have a MW of 1250-2000 kDa, being composed of between 27 and 44 subunits. This estimation is the result of 3 independent assays. Western Blot analysis HeLa cells were transfected with Myc-wt seipin, Myc-Celia seipin or empty vector. After 48 hours, cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM PMSF, 1mM DTT, 10 μg/ml leupeptin and 10 μg/ml aprotinin). Protein content was determined by using the Bio-Rad protein assay (Bio-Rad laboratories, Hercules, CA, USA). Protein samples were separated by SDS-PAGE and transferred to a PVDF transfer membrane (Millipore, Billerica, MA, USA). The membrane was probed with primary antibodies and subsequently with horseradish peroxidaselinked secondary antibodies. Immunoblots showed in Figure 2A and 2B were scanned and analyzed by densitometry using the Image J 1.48v software (National Institutes of Health, Bethesda, MD, USA). All blots shown are representative of a minimum of 3 experiments. BSCL2 expression study Total RNA was extracted from lymphocytes and reverse-transcribed as previously reported (Victoria et al., 2010). BSCL2 cDNA PCR details are available upon request. Expression of BSCL2 mRNA was quantified in a Light Cycler 2.0 (Roche Diagnostics, Sant Cugat del Vallés, Spain) using specific probes and oligonucleotide primers designed by Universal Probe Library (Roche Diagnostics) (Supplementary Data, Fig. S4B). Results were normalized to the RNA polymerase II and 18s genes, using the 2-ΔΔ CT method (Livak and Schmittgen, 2001). 7 Co-immunoprecipitation Lysates (vide supra) from transfected HeLa cells were subjected to coimmunoprecipitation using the Pierce c-Myc Tag IP/Co-IP Kit (Thermo-Fisher Scientific) according to the manufacturer’s instructions. 500 μg of lysates were incubated in spin columns with anti c-Myc agarose overnight at 4ºC. Myc immunoprecipitates were eluted with non-reducing sample buffer, boiled for 5 min, separated by SDS-PAGE and analyzed by immunoblotting with 1:1000 anti c-Myc antibody. Immunohistochemistry Paraffin sections of the posterior hypothalamus, at a coronal level including the mammillary body, and parietal and occipital cortices of the homozygous index case and of three control cases (two of them males, 43 and 68 years at death, respectively, and one female child, 6 years at death) were immunostained with anti-seipin antibody HPA042394 (The Human Protein Atlas). Primary antibody was incubated at 1:40-1:500 dilutions and heat-induced antigen retrieval was performed by pressure cooker heating using PT-Link (Dako). Staining was amplified and revealed by means of the Dako Envision system (Dako). Immunostaining and confocal imaging HeLa, SH-SY5Y, HEK293, COS7 and 3T3 cells were grown in EZ Millicell slides (Millipore) and transfected with Myc-wt seipin, Myc-Celia seipin or empty vector. After 48 h, cells were fixed with 1:1 Methanol-Acetone for 20 min at -20 ºC and permeabilized in 0.5% Triton X-100. Non-specific binding was blocked with 5% BSA. Slides were incubated with 1:1000 anti c-myc (9E10) antibody (Santa Cruz Biotechnology) at 4ºC overnight. The next day, after four washes, the slides were incubated with Alexa Fluor 555 (Thermo-Fisher Scientific) conjugated anti mouse secondary antibody and DAPI stain (Sigma-Aldrich) for 1 hour in darkness and mounted. Immunofluorescence staining was examined a Leica TCS SP5 confocal microscope and LAS AF Software (Leica, Mannheim, Germany). Proteomic analysis Proteomic analysis of preadipocytes was performed using high-resolution twodimensional gel electrophoresis (2-DE) for protein separation and matrixassisted laser desorption/ionization mass spectrometry (MALDI/MS) or nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-MS/MS) for the analysis of selected differentially regulated protein spots. Further details are available in Supplementary Data, S6. 8 Results Celia seipin forms intranuclear aggregates in the brain Our previous work showed the presence of intranuclear ubiquitin-positive inclusions in the brain of the index case, likely to contain misfolded, aggregated mutant seipin. To confirm the presence of seipin in these intranuclear ubiquitin positive inclusions we performed an immunohistochemistry study. Tissue samples from different brain regions from the index case (homozygous for the c.985C>T mutation) were probed with anti-seipin (Supplementary Data, Fig. S1) and antiubiquitin antibodies. We observed several scattered positively stained intranuclear inclusions in neurons of the posterior hypothalamic nuclei (Fig. 1A- C) and the parietal and occipital cortices (Supplementary Data, Fig. S2A-B) of the patient. As previously reported (Guillén-Navarro et al., 2013) ubiquitinreactive intranuclear inclusions were also frequently observed in neurons (Fig. 1B). These inclusions were mostly single and round-shaped, although occasional multiple inclusions were also observed. Some of the ubiquitinreactive inclusions were recognized by the seipin-specific antibody; ubiquitinreactive inclusions were found in a higher number probably because of a higher sensibility of the anti-ubiquitin antibody. No neuronal nuclear inclusions were identified in the control cases (Fig. 1D & Supplementary Data, Fig. S2C-D). Celia seipin exhibits aberrant oligomerization status It has been shown that human wt seipin forms oligomers of 12 subunits (Sim et al., 2013a). At least for the yeast seipin homolog FLD1, these oligomers are involved in the generation of lipid droplets at the ER-LD junctions (Szymanski et al., 2007). We reasoned that mutated seipin might fail to assemble correctly. Instead, aberrant aggregation would result in the observed inclusions. To test this hypothesis, protein extracts from HeLa cells overexpressing Myc-wt or Myc-Celia seipin were subjected to fractionation in 10 to 60% sucrose density gradients (Supplementary Data, Fig. S3A). Fractions of 325 μl were collected and analyzed by western blot (WB) (Fig. 2A). Wt seipin mainly appears in fraction 6. According to the calibration curve (Supplementary Data, Fig. S3B), this corresponds to a molecular weight (MW) of 690 kDa which means that wt seipin is made up of oligomers of 12 subunits. In contrast, Celia seipin mainly appears in fraction 8, which suggests that it forms much larger aggregates under similar conditions. These results suggest impaired oligomerization/aggregation of Celia seipin. 9 Both Celia and wt seipin are localized at the ER/nucleus continuum The fact that aberrant aggregates pf Celia seipin appear in the nucleus begs the question of how a protein that is theoretically located in the ER can reach that localization. However, previous studies have shown that mutant seipin can appear in a ring surrounding the cell nucleus (Payne et al., 2008, Sim et al., 2013a). This has been confirmed for A212P and L61P mutations, both of which lead to classic lipodystrophy, although this perinuclear localization has only been observed in C3H10T1/2 cells. On the other hand, we have previously detected, by microscopy and subcellular fractionation, the presence in the nuclear fraction of both wt and Celia seipin (Guillén-Navarro et al., 2013). Because of the continuity between the ER membrane and the outer nuclear envelope (Goyal et al., 2013 and Mauger, 2012), a perinuclear localization for an ER protein like seipin is not unreasonable. Perinuclear localization of Celia seipin would be consistent with the observed intranuclear deposition of aberrant aggregates. By immunofluorescence, we compared the subcellular localization of wt and Celia seipin in 5 different cell lines: HeLa, SH-SY5Y, 3T3, HEK293 and COS7. Both seipin isoforms appeared surrounding the nucleus in addition to their normal cytoplasmatic localization (Fig. 3). BSCL2 expression levels in heterozygous carriers Our previous findings point to a gain of toxic function for the new aberrant protein Celia seipin. Therefore, the absence of symptoms observed in heterozygous carriers for the c.985C>T mutation is strikingly paradoxical. We hypothesized that interaction of wt with Celia seipin might inhibit aberrant aggregation of the latter in heterozygous individuals, thus rescuing the disease phenotype. We therefore quantitated expression levels of RNA corresponding to both seipin isoforms in two heterozygous carriers (parents of the index case) by real time RT-PCR (Supplementary Data, S4A), to assess their relative abundance, as a first step to validate our hypothesis. We found a 10:1 ratio of wt seipin to Celia seipin (Fig. 4A). The difference with the expected Mendelian ratio of 1:1 might be explained by the degradation of the majority of the conceivably less stable Celia seipin mRNA, by nonsense-mediated mRNA decay (NMD), as suggested for other mutations (Frischmeyer & Dietz, 1999, Caputi, 2002). While our results were obtained in lymphocytes, it is reasonable to extrapolate them to brain, where a vast excess of wt seipin over Celia seipin would facilitate a protective neutralization of Celia seipin if wt seipin were able to interact with it. To complete a comprehensive picture of the expression of different BSCL2 transcripts, we compared expression of wt seipin in lymphocytes of 16 References Binns, D., Lee, S., Hilton, C.L., Jiang, Q.X. and Goodman, J.M., 2010. Seipin is a discrete homooligomer. Biochemistry, 49, 10747–10755 Caputi, M., Kendzior, R.J., and Beemon, K.L., 2002. A nonsense mutation in the fibrillin-1 gene of a Marfan syndrome patient induces NMD and disrupts an exonic splicing enhancer. Genes Dev., 16, 1754–1759 Cartwright, B.R. and Goodman, J.M., 2012. Seipin: from human disease to molecular mechanism. J. Lipid. Res., 53, 1042–1055 Cole, N.B., Murphy, D.D., Grider, T., Rueter, S., Brasaemle, D. and Nussbaum, R.L., 2002. Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alpha-synuclein. J. Biol. Chem., 277, 6344– 6352 Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P., 1997. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537–48 Dennis, G., Jr., Sherman, B. T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Lempicki, R.A., 2003. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol., 4, P3. Dudek, J., Benedix, J., Cappel, S., Greiner, M., Jalal, C., Müller, L. and Zimmermann, R., 2009. Functions and pathologies of BiP and its interaction partners. Cell. Mol. Life Sci., 66, 1556–1569 Fei, W., Li, H., Shui, G., Kapterian, T.S., Bielby, C., Du, X., Brown, A.J., Li, P., Wenk, M.R., Liu, P. et al., 2011. Molecular characterization of seipin and its mutants: implications for seipin in triacylglycerol synthesis. J. Lipid. Res., 52, 2136–2147 Frischmeyer, P.A. and Dietz, H.C., 1999. Nonsense-mediated mRNA decay in health and disease. Hum. Mol. Genet., 8, 1893–1900 Goyal, U. and Blackstone, C., 2013. Untangling the web: mechanisms underlying ER network formation. Biochim. Biophys. Acta, 1833, 2492– 2498. Guillén-Navarro, E., Sánchez-Iglesias, S., Domingo-Jiménez, R., Victoria, B., Ruiz-Riquelme, A., Rábano, A., Loidi, L., Beiras, A., González-Méndez, B., 17 Ramos, A. et al. 2013. A new seipin-associated neurodegenerative syndrome. J. Med.Genet., 30, 401–409 Han, S., Bahmanyar, S., Zhang, P., Grishin, N., Oegema, K., Crooke, R., Graham, M., Reue, K., Dixon, J.E., and Goodman, J.M., 2012. Nuclear envelope phosphatase 1-regulatory subunit 1 (formerly TMEM188) is the metazoanSpo7p ortholog and functions in the lipin activation pathway. J. Biol. Chem., 287, 3123–3137. Hötta-Vuori, M., Salo, V.T., Ohsaki, Y., Suster, M.L. and Ikonen, E., 2013. Alleviation of seipinopathy-related ER stress by triglyceride storage. Hum. Mol. Genet., 22, 1157–1166 Huang, D.W., Sherman, B.T. andLempicki, R.A., 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protocols, 4, 44–57. Ito, D. and Suzuki, N., 2007. Molecular pathogenesis of Seipin/BSCL2 related motor neuron diseases. Ann. Neurol., 61, 237–250 Ito, D. et al., 2008. Characterization of seipin/BSCL2, a protein associated with spastic paraplegia 17. Neurobiol. Dis. 31, 266–277 Ito, D., Yagi, T., Ikawa, M. and Suzuki, N., 2012. Characterization of inclusion bodies with cytoprotective properties formed by seipinopathy-linked mutant seipin. Hum. Mol. Genet., 21, 635–646. Jeninga, E.H., De Vroede, M.,Hamers, N., Breur, J.M., Verhoeven-Duif, N.M., Berger, R., Kalkhoven, E., 2012. A patient with congenital generalized lipodystrophy due to a novel mutation in BSCL2: indications for secondary mitochondrial dysfunction. JIMD reports, 4, 47–54 Layerenza, J.P., González, P., García de Bravo, M.M., Polo, M.P., Sisti, M.S. and Ves-Losada, A., 2013. Nuclear lipid droplets: A novel nuclear domain. Biochim. Biophys. Acta, 1831, 327–340 Licker, V., Turck, N., Kövari, E., Burkhardt, K., Côte, M., Surini-Demiri, M., Lobrinus, J.A., Sánchez, J.C. and Burkhard, P.R., 2014. Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson’s disease pathogenesis. Proteomics, 14, 784–794 Lin, M.T. and Beal, M.F., 2006. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature, 443, 787–795 18 Livak, K.J. andSchmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25, 402–408 Lundin, C., Nordström, R., Wagner, K., Windpassinger, C., Andersson, H., Von Heijne, G. and Nilsson, I., 2006. Membrane topology of the human seipin protein. FEBS Lett., 580, 2281–2284 Magré, J., Delépine, M., Khallouf, E., Gedde-Dahl, T., Van Maldergem, L., Sobel, E., Papp, J., Meier, M., Mégarbané, A., Bachy, A. et al., 2001. Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13. Nat. Genet. 28, 365–370 Margulis, J. and Finkbeiner, S., 2014. Proteostasis in striatal cells and selective neurodegeneration in Huntington’s disease. Front. Cell. Neurosci. 8, 218 Matus, S., Glimcher, L.H., and Hetz, C., 2011. Protein folding stress in neurodegenerative diseases: a glimpse into the ER. Curr. Opin. Cell. Biol., 23, 239–252 Mauger, J.P., 2012. Role of the nuclear envelope in calcium signaling. Biol. Cell., 104, 70–83 Mi, H., Dong, Q., Muruganujan, A., Gaudet, P., Lewis, S. and Thomas, P.D., 2010. PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium. Nucleic Acids Res., 38, D204–D210. Payne, V.A., Grimsey, N., Tuthill, A., Virtue, S., Gray, S.L., Dalla Nora, E., Semple, R.K., O’Rahilly, S. and Rochford, J.J., 2008. The human lipodystrophy gene BSCL2/Seipin may be essential for normal adipocyte differentiation. Diabetes. 57, 2055–2060 Shevchenko, A., Wilm, M., Vorm, O. and Mann, M., 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem., 68, 850–858. Sieradzan, K.A., Mechan, A.O., Jones, L., Wanker, E.E., Nukina, N. and Mann, D.M., 1999. Huntington’s disease intranuclear inclusions contain truncated, ubiquitinated huntingtin protein. Exp. Neurol. 156, 92–99 Sim, M.F.M., Talukder, M.M.U., Dennis, R.J., O’Rahilly, S., Edwarson, J.M. and Rochford, J.J., 2013a. Analysis of naturally occurring mutations in the 19 human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms. Diabetologia, 56, 2498–2506 Sim, M.F.M., Dennis, R.J., Aubry, E.M., Ramanathan, N., Sembongi, H., Saudek, V., Ito, D., O’Rahilly, S., Siniossoglou, S. and Rochford, J.J., 2013b. The human lipodystrophy protein seipin is an ER membrane adaptor for the adipogenic PA phosphatase lipin 1. Mol. Metab. 2, 38–46 Supek, F., Bošnjak, M., Škunca, N. and Šmuc, T., 2011. REVIGO summarizes and visualizes long lists of Gene Ontology terms. PLoS ONE, 6, e21800 Szymanski, K.M., Binns, D., Bartz, R., Grishin, N.V., Li, W.P., Agarwal, A.K., Garg, A., Anderson, R.G., and Goodman, J.M., 2007. The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology. Proc. Natl. Acad. Sci. U.S.A., 104, 20890–20895 Uhlen, M., Oksvold, P., Fagerberg, L., Lundberg, E., Jonasson, K., Forsberg, M., Zwahlen, M., Kampf, C., Wester, K., Hober, S. et al., 2010. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28 (12): 1248–50 Van Dijk, K.D., Berendse, H.W., Drukarch, B., Fratantoni, S.A., Pham, T.V., Piersma, S.R., Huisman, E., Brevé, J.J.P., Groenewegem, H.J., Jiménez, C.R. et al., 2012. The proteome of the locus ceruleus in Parkinson’s disease: relevance to pathogenesis. Brain Pathology, 22, 485–498 Van Maldergem, L., Magré, J., Khallouf, T.E., Gedde-Dahl, T., Delépine, M., Trygstad, O., Seemanova, E., Stephenson, T., Albott, C.S., Bonnici, F. et al., 2002. Genotype-phenotype relationships in Berardinelli-Seip congenital lipodystrophy. J. Med. Genet., 39, 722–733 Victoria, B., Cabezas-Agricola, J.M., González-Méndez, B., Lattanzi, G., Del Coco, R., Loidi, L., Barreiro, F., Calvo, C., Lado-Abeal, J. and Araújo-Vilar, D., 2010. Reduced adipogenic gene expression in fibroblasts from a patient with type 2 congenital generalized lipodystrophy. Diabetic Medicine, 27, 1178–1187 Waelter, S., Boeddrich, A., Lurz, R., Scherzinger, E., Lueder, G., Lehrach, H. and Wanker, E.E., 2001. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol. Biol. Cell, 12, 1393–1407 20 Wei, S., Soh, S.L., Qui, W., Yang, W., Seah, C.J., Guo, J., Ong, W.Y., Pang, Z.P. and Han, W., 2013. Seipin regulates excitatory synaptic transmission in cortical neurons. J. Neurochem., 124, 478–489 Wei, S., Soh, S.L., Xia, J., Ong, W.Y., Pang, Z.P. and Han, W., 2014. Motor neuropathy-associated mutation impairs Seipin functions in neurotransmission. J. Neurochem., 129, 328–338 Wen, Z., Nguyen, H.N., Guo, Z., Lalli, M.A., Wang, X., Su, Y., Kim, N.S., Yoon, K.J., Shin, J., Zhang, C. et al., 2014. Synaptic dysregulation in a human iPS cell model of mental disorders. Nature, 515, 414–418 Windpassinger, C., Auer-Grumbach, M., Irobi, J., Patel, H., Petek, E., Hörl, G., Malli, R., Reed, J.A., Dierick, I., Verpoorten, N. et al., 2004. Heterozygous missense mutations in the BSCL2 gene cause distal hereditary motor neuropathy and Silver syndrome. Nat. Genet., 36, 271–276 Yang, W., Thein, S., Guo, X., Xu, F., Venkatesh, B., Sugii, S., Radda, G.K. and Han, W., 2013a. Seipin differentially regulates lipogenesis and adipogenesis through a conserved core sequence and an evolutionary acquired C-terminus. Biochem. J., 452, 37–44 Yang, W., Thein, S., Wang, X., Bi, X., Ericksen, R.E., Xu, F. and Han, W., 2013b. BSCL2/seipin regulates adipogenesis through actin cytoskeleton remodeling. Hum. Mol. Genet., 23, 502–513 Xu, Z., Graham, K., Foote, M., Liang, F., Rizkallah, R., Hurt, M., Wang, Y., Wu, Y. and Zhou, Y., 2013. 14-3-3 protein targets misfolded chaperoneassociated proteins to aggresome. J. Cell Sci., 126, 4173–4186 Web References Duke University Medical Center, Light Microscopy Core Facility. http://microscopy.duke.edu/sampleprep/if.html (Accessed January 8, 2015) Legends to figures Figure 1. Immunohistochemical staining for seipin and ubiquitin in pathological and control cases. A-B: High magnification images of neuronal intranuclear inclusions in hypothalamus from a homozygous patient, reactive to seipin (HPA042394) (A) and to ubiquitin (B) antibodies. C-D: Low magnification images of the posterior hypothalamus of a homozygous patient (C) and a control case (D). The higher seipin-reactivity observed in neuronal bodies in the 21 control case compared with the homozygous patient could be caused by the lower expression level of Celia seipin. Scale bar: 100 μm. Figure 2. Density gradient fractionation of wt and Celia seipin. Homogenates of HeLa cells overexpressing (A) wt or Celia seipin and (B) wt and Celia seipin co-expressed at a 10:1 (wt:Celia) ratio were subjected to sucrose gradient fractionation. (C) Percentage of seipin in each gradient fraction, as determined by scanning of WBs. (D) Higher exposure time image of the band with MW = 50 in panel B (expression 10:1, wt:Celia). Figure 3. Subcellular localization of seipin in several cell lines. HeLa, 3T3, SH-SY5Y, HEK293 and COS7 cells grown on coverslips were transfected with wt seipin, Celia seipin or empty vector, immunostained with anti c-Myc (9E10) and imaged by confocal microscopy. Nuclei were stained with DAPI. Figure 4. BSCL2 expression levels in healthy heterozygous carriers. (A) Percentage of wt and Celia seipin in lymphocytes from heterozygous carriers. Total RNA extracted from lymphocytes was analyzed by real time RT-PCR. Wt seipin was measured as BSCL2 bearing exon 7 transcript whereas Celia seipin was measured as exon 7 skipped BSCL2 transcript (Supplementary Data, S3A– B). (B) Percentage of wt seipin in lymphocytes from heterozygous carriers compared to non-carriers (wt/wt). The difference to 100 % corresponds to a naturally expressed seipin isoform (287 aas) indistinguishable of Celia seipin through RT-PCR. Figure 5. Wt and Celia seipin interact. Equal amounts of lysates from transfected HeLa cells were immunoprecipitated with anti c-myc and examined for coimmunoprecipitation of FLAG-wt seipin by immunoblotting. Lysates were previously probed with antibodies to confirm the validity of the transfection. Figure 6. Proposed model for the pathogenic mechanism of Celia seipin in homozygotes (A) and phenotype rescue in heterozygous carriers (B). Please read the text for details. Table 1. List of differentially regulated proteins when comparing preadipocytes from the index case vs. control. Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Supplementary Data S8 Click here to download Supplementary Material: Supplementary Data S8.xlsx