Spastic paraplegia 11 (SPG11): Mutation spectrum, phenotype-genotype correlations and functional in vitro and in vivo studies Giovanni Stevanin, Nadia Soussi-Yanicostas, Elodie Martin, Reena P. Murmu, Sylvain Hanein, Christel Depienne, Emeline Mundwiller, Amir Boukhris, Paola S. Denora, Cyril Goizet, Frédéric Darios, Hamid Azzedine, Agnès Rastetter, Typhaine Esteves, Khalid H. El-Hachimi, Constantin Yanicostas, Alexandra Durr, Alexis Brice: Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière. GHU Pitié-Salpêtrière, 47 Bd de l'Hôpital, 75013 Paris. SPG11 / Neurosciences, Neurologie et Psychiatrie – 2007 programme State of the Art and Aims Expression profiles and subcellular Hereditary spastic paraplegias (HSP) localisation of spatacsin and Spastizin . Characterized by progressive spasticity in lower limbs, . Due to the dysfunction and/or degeneration of the upper motor neuron, Spatacsin and spastizin are proteins: . Pure forms present with pyramidal signs (brisk reflexes, Babinski sign, spasticity, motor deficit, +/- sphincter - expressed with similar profiles in the CNS (Hanein et al, 2008), disturbances) often associated with deep sensory loss, - strongly expressed during development in CNS and non CNS tissues, . Complex forms present with spasticity and numerous combinations of neurological and extra-neurological - with no major colocalisation with a specific cellular compartment, but given their distribution relative to microtubules signs such as ataxia, dysarthria, neuropathy, optic atrophy, retinitis pigmentosa, hearing loss or mental and vesicles, it is tempting to speculate that they play a role in axonal transport. retardation, . Genetic heterogeneity : > 45 loci have been mapped to date. Since partially colocalized with mitochondria, spastizin may have additional mitochondria-based functions. In 2007, identification of 10 different nonsense and frameshift mutations in a novel gene of unknown function, Figure: Intracellular distribution of KIAA1840 (SPG11), which segregated with the disease in 11 autosomal recessive (AR) HSP families. endogenous spatacsin or spastizin in human neuronal-like differentiated SH-SY5Y cells. Aims of the project: Spatacsin and spastizin show partial 1) Establish the spectrum of the mutations or rearrangements in the SPG11 gene by direct sequencing or MLPA in a large Spastizin co-localisations with cytoskeleton (α- collection of ARHSP families and isolated patients, within the European and Mediterranean SPATAX network, α-tubulin tubulin, and with vesicle-like 2) Establish genotype/phenotype correlations, to determine the frequency and nature of SPG11 mutations according to structures (γ adaptin, g,h,i). In blue, the associated phenotype, DAPI. Scale bar: 20 µm. In electron microscopy, spastizin co- 3) Approach the function of the SPG11 gene product (spatacsin) by the identification of partners, the study of its tissue localises partially with vesicle-like and subcellular expression profiles, structures (arrow head) and more 4) Model the disease by RNA interference in Zebrafish. rarely with mitochondria (arrow, colocalisation significant compared to dot distribution in cytosol). Identification of partners of SPG11; ZFYVE26 and KIAA0415, involved in SPG15 and SPG48 Modeling of SPG11 and SPG15 in vivo Identification of truncating mutations in a new gene (ZFYVE26/SPG15) responsible for an SPG11-like phenotype by positional Morpholino oligonucleotide targeting the first coding ATG or splice sites in each of the genes caused abnormal cloning in 3 families linked to chromosome 14 (Hanein et al, 2008). head and tail development associated with locomotor impairment and abnormal branching of spinal cord neurons at Mass spectrometry showed that Spastizin (SPG15) and Spatacsin (SPG11) are members of a common protein complex with the neuromuscular junction. KIAA0415, which is also involved in a new AR-HSP subtype, SPG48 (Slabicki et al, 2010). A Figure (right): In the Tg(Olig2:EGFP) transgenic line staining spinal motor neurons, tetramethylrhodamine conjugated α-bungarotoxin labeling of AchR clusters was used to verify proper differentiation of neuromuscular synapses Figure. KIAA0415 interacts with SPG11, SPG15, KFZp761E198 and C20orf29. (A) SDS-PAGE gels visualized by colocalization of motor axons and AchRs. SPG11 and obtained from the immunoprecipitation of KIAA0415-LAP and SPG11-LAP. Baits (marked in green) and SPG15 morphant embryos show truncated axons (arrows in C, D, and E) prey (marked in black) were identified by in-gel digestion and nanoLC-MS/MS analysis. Bands that are not disturbed axon trajectories; ectopic and aberrant motor axon branches marked represent unspecific background proteins or bait specific proteins. (B) The composition of (arrowheads in C, D, and E) while most AchR clusters are not localized on KIAA0415 protein complex analyzed as established by shotgun-LC-MS/MS. The number of matched axon branches (C’’, D’’, and E’’). Some clusters still colocalize with motor detected peptides and protein sequence coverage are shown. Results for bait proteins are marked in bold. neuron neurites (stars in C’’, D’’, and E’’). Scale bar, 50µm. B (C) Schematic representation of the exon-intron structure of KIAA0415. (D) Pedigree of the SPG48 family and electrophoregram showing the mutation. (Slabicki et al, 2010). C D Figure (Left): (A) Phenotypes of zspg11 and zspg15 morphant embryos, 48 hours post- Relative frequencies, clinical and mutation fecondation (hpf). Whereas embryos injected with 1 pmol of the control mismatch morpholino (MO) were phenotypically undistinguishable from spectra of SPG11 and SPG15 non-injected controls, injection of MO impairs embryonic development of the caudal region. Scale x44. (B) Proportion of deformed embryos following MO injection. The numbers of SPG11 accounts for ~21% of AR-HSP and is a major cause of complicated forms (~50-60% of patients with thin corpus non-injected controls and zspg11 and zspg15 morphant embryos are callosum [TCC] and mental retardation). indicated in parentheses. Each group is compared to its own control group of non-injected siblings. * p< 0.0001. HSP associated with cognitive Pure HSP or SUM TCC and TCC without cognitive impairment with + cases/ cognitive cognitive impairment (MRI not cerebellar tested cases impairment impairment without TCC done) signs (frequency) Conclusions and perspectives 62/97 10/38 6/22 1/30 1/21 80/208 SPG11 is the most frequent form of AR-HSP, (64%) (26%) (27%) (3%) (4%) (38%) SPG11 and SPG15 mutations are associated with mental retardation, peripheral neuropathy and thin corpus callosum at brain MRI, Figure: Brain MRI of a ARHSP-TCC patient Table: Frequency of SPG11 mutations according to the cinical signs in patients Spatacsin (SPG11) and Spastizin (SPG15) are members of the same protein complex and their subcellular expression is showing atrophy of the corpus callosum (left) and (Denora et al, 2009; Stevanin et al, 2008; UF de neurogénétique) white matter hyperintensities (right). compatible with their involvement in intracellular trafficking, Their knock down in zebrafish shows that they are important for proper development of motoneurons, These results call for further studies to understand the contribution of impaired trafficking to the pathology using the zebrafish models and a KO SPG11 model just produced at the Institut Cinique de la souris. Figure: Location of the mutations Publications in SPG11. Note that they are distributed in all exons of the Original articles: gene, which complicates the .Stevanin G, et al. Mutations in SPG11 are frequent in autosomal recessive spastic paraplegia with thin corpus callosum, cognitive decline and lower motor neuron degeneration. Brain, 131(Pt 3):772-84, 2008. routine diagnosis. .Boukhris A, et al. Hereditary spastic paraplegia with mental impairment and thin corpus callosum in Tunisia: SPG11, SPG15 and further genetic heterogeneity. Arch. Neurol., 65:393-402, 2008. .Hanein S, et al. Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal recessive spastic paraplegia including Kjellin syndrome. Am J Hum Genet 2008, 82:992-1002. .Erichsen AK, et al. SPG11, the most common type of recessive spastic paraplegia in Norway? Acta Neurol Scanda 2008, 117 (suppl.188):46-50. .Denora PS, et al. Screening of ARHSP-TCC patients expands the spectrum of SPG11 mutations and includes a large scale gene deletion. Human Mut 2009, 30:E500-E519 .Denora PS, et al. Spastic paraplegia with thinning of the corpus callosum and white matter abnormalities: further mutations and relative frequency of ZFYVE26/SPG15 in the Italian population. J Neurol Scie 2009, 277:22-25. .Boukhris A, et al. Tunisian hereditary spastic paraplegias: clinical variability supported by genetic heterogeneity. Clin Genet 2009,75:527-536. .Goizet C, et al. SPG15 is the second most common cause of hereditary spastic paraplegia with thin corpus callosum. Neurology 2009, 73:1111-1119. .Denora PS, et al. Identification of a de novo mutation in SPG11. Mov Disord 2010, 25: 501-503. SPG15 accounts for ~4% of AR-HSP and its frequency in complicated AR-HSP .S łabicki M, et al. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PloS-Biol 2010,8: e1000408. .Puech B, et al. Kjellin syndrome: long-term neuro-ophthalmologic follow-up and novel mutations in the SPG11 gene. Ophthalmol 2011, 118: 564-573. varies from 5% (Italy) to 11% (Continental Europe) and 25% (Tunisia). No .Murmu RP, et