Hereditary Spastic Paraplegias from phenotype to models back to phenotype

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1 Hereditary Spastic Paraplegias from phenotype to models back to phenotype Andrea Martinuzzi IRCCS E. Medea Polo Regionale Veneto Andrea Martinuzzi Primate Cortico- Spinal System 1

2 Highly polarized cell Motoneuron Target dependent trophism Axon length >1 m cell body volume / axon volume ratio >1000 Transcription/translation: soma Maximal energy consumption (mitochondrion dependent) at the synapse Axonal Trasport Along tracks microtubules (long range transports) actin (short range transports) Fast axonal Flux anterograde Kinesine dependent vescicoles, membranes e membranous organelles (mitochondria) retrograde Dyneine dependent endosomes, neurotrophic factors, organelles Slow axonal Flus Anterograde, involvement of cytoskeletal proteins, axonal elongation/renewal 2

Motoneuron Diseases I motoneuron (HSP) Reduced motor selectivity Reduced strength Spasticity pyramidal DTRs Delayed CCT II motoneuron (SMA) Reduced Strength

3 Motoneuron Diseases I motoneuron (HSP) Reduced motor selectivity Reduced strength Spasticity pyramidal DTRs Delayed CCT II motoneuron (SMA) Reduced Strength Reduced trophism Hypotonia denervation I & II motoneuron (ALS) Reduction of strength and motor selectivity pyramidal DTRs Hypotrohy & denervation Andrea Martinuzzi 3

4 Hereditary Spastic Paraplegias Neurodegenerative syndromes characterized by: progressive spasticity and weakness ot the lower limbs hyperactive DTR and Babinski response Mild sensory deficits (vibratory), sphyncteric disturbances (urinary urgency) Pure Signs are limited to progressive weakness and spasticity of lower limbs, with possible sphyncteric disturbances Complicated Besides the signs of the pure forms, presence of: other neurological signs neuropathy ataxia Mental impairment epilepsy optic atrophy Extraneurological signs cataract Epidemiology Prevalence / Individually rare (the most common form accounts for 40% of AD : 1-4/100,000) Variable age of onset (1-60 y) Generally good prognosis q.v. Functional prognosis variable (possible early loss of deambulation) 72 loci, 55 identified genes Dominant (20 loci, 12 genes), Recessive (47 loci, 37 genes), X linked (5 loci, 3 genes) Diaginic (mtdna: 3 genes) 4

5 Neuropathology Lenght dependent disto-proximal degeneration of long cortico-spinal traits Loss of axons in the ventral and lateral corticospinal tracts (+++ lumbar) Myelin loss in dorsal column No or minimal apoptotic loss of UMN No loss of LMN Autosomal Dominant LOCUS POSIZION GENE PHENOTYPE SPG3A 14q12-q21 Atlastin pure SPG4 2p21-p24 Spastin pure (some complicated) SPG6 15q11.2-q12 NIPA1 pure SPG8 8q24 KIAA0196 pure & complicated SPG9 10q23.3-q24.2 Unknown SPG10 12q13 KIF5A pure & complicated SPG12 19q13 RTN2 pure SPG13 2q24-q34 HSP60 pure SPG17 11q12-q14 BSCL2 complicated (Silver Syndr) SPG19 9q33-q34 Unknown pure SPG29 1p31.1-p21.1 Unknown complicated SPG31 2p12 REEP1 pure SPG33 10q24 ZFYVE27 pure SPG36 12q23-q24 Unknown complicated SPG37 8p21.1-q13.3 Unknown pure SPG38 4p16-p15 Unknown complicated SPG40 Unknown Unknown pure SPG41 11p14.1-p11.2 Unknown pure SPG42 3q25.31 SLC33A1 pure SPG72 5q31.2 REEP2 pure 5

6 Autosomal Recessive LOCUS POSIZION GENE PHENOTYPE SPG5 8q11-q13 CYP7B1 pure & complicated SPG7 16q24.3 Paraplegin pure & complicated SPG11 15q13-q15 Spatacsin complicated, TCC SPG14 3q27-q28 Unknown complicated SPG15 14q22-q24 Spastizin complicated (Kjellin syndrome) SPG18 8p12-p11.21 Erlin2 complicated SPG 20 13q12.3 Spartin complicated (Troyer syndrome) SPG21 15q21-q22 Maspardin complicated (MAST syndrome) SPG23 1q24-q32 Unknown complicated SPG24 13q14 Unknown pure SPG25 6q23-q24.1 Unknown SPG26 12p11.1-q14 B4GALNT1 complicated SPG27 10q q24.1 Unknown pure SPG28 14q21.3-q22.3 DDHD1 pure SPG30 2q37.3 KIF1A complicated SPG32 14q12-q21 Unknown complicated SPG35 16q23 FA2H complicated SPG39 19p13.3 NTE complicated SPG43 19p13.11-q12 C19orf12 complicated SPG44 1q42.13 GJC2 complicated SPG45 10q24.3-q25.1 Unknown complicated SPG46 9p21.2-q21.12 GBA2 complicated SPG47 1p13.2 AP4B1 complicated Autosomal Recessive LOCUS POSIZION GENE PHENOTYPE SPG48 7p22.1 KIAA0415 complicated SPG49 14q32.31 TECPR2 complicated SPG50 7q22.1 AP4M1 SPG51 15q21.2 AP4E1 complicated SPG52 14q12 AP4S1 SPG53 8p22 VPS37A complicated SPG 54 8p11.23 DDHD2 complicated SPG55 12q24.31 C12orf65 complicated SPG56 4q25 CYP2U1 complicated SPG57 3q12.2 TFG complicated SPG58 17p13.2 KIF1C SPG59 15q21.2 USP8 SPG60 3p21.33 WDR48 SPG61 16p12-p11.2 ARL6IP1 complicated SPG62 10q24.31 ERLIN1 SPG63 1p13.3 AMPD2 SPG64 10q24 ENTPD1 SPG66 5q32 ARSI SPG65 10q24.32 NT5C2 SPG67 2q33.1 PGAP1 SPG68 11q13.1 FLRT1 SPG69 1q41 RAB3GAP2 SPG70 12q13.3 MARS SPG71 5p13.3 ZFR 6

7 X-linked Forms LOCUS POSITION GENE PHENOTYPE SPG1 Xq28 L1CAM complicated SPG2 Xq22 PLP, DM20 complicated, Rarely pure SPG16 X q11.2 Complicated SPG22 Xq13.2 SLC16A2 SPG34 Xq25 Rarely pure SPGs WITH MITOCHONDRIAL INHERITANCE GENE PHENOTYPE MT-ATP6 Complicated (Mitochondrial infantile bilateral striatal necrosis and Mitochondrial complex v deficiency, Mitochondrial 1) MTTI Complicated (CPEO) MT-ND4 Complicated (LHON + Hereditary neuropathy) MT-CO3 Complicated (Leigh-like syndrome, Leber hereditary optic neuropathy) 7

8 OTHER SPGs POSIZION GENE PHENOTYPE 19q13.12 MAG Complicated (Central pontine myelinolysis, Wallerian degeneration) 9q22.31 BICD2 Complicated (Spinal muscular atrophy, lower extremity-predominant, 2 and 1, ad) 1q42.3 LYST Complicated (Chediak-higashi syndrome) Xq13.2 IFIH1 Complicated (Amyopathic dermatomyositis) Xq25 CCT5 Complicated (Neuropathy, hereditary sensory, with spastic paraplegia; Autosomal recessive sensory neuropathy with spastic paraplegia) 5p15.1 FAM134B Complicated (Hereditary sensory and autonomic neuropathy type iib) 19q13.2-q13.3 OPA3 Complicated (Optic atrophy 3 with cataract) EARLY ONSET POSITION GENE PHENOTYPE 11q13 SPOAN Complicated (SPG, optic atrophy, neuropathy) 9p13.2 EXOSC3 Complicated (Pontocerebellar hypoplasia) 2q33 Alsin Complicated (Infantile-onset ascending SPG) 2q31 GAD1 Complicated (West Nile fever) 8

9 Pathogenetic mechanisms Axonal Growth & maintenanance Axonal elongation Axonal Transport Lipid metabolism Proteins Signal/Receptors Supporting Glia Membranes ER Cargo: Endosomes Tracks: Cytoskeleton Energy: Mitochondria CYP7B1 NIPA1 Atlastin KIAA0196 Spastin Paraplegin ERLIN2 PLP1 REEP1 Maspardin Strumpellin HSP60 B4GALNT1 L1CAM Seipin Spatacsin? KIAA0415 KIF5A FA2H FA2H CYP7B1 Spastizin Spartin CYP2U1 NTE SLC33A1 Pathogenetic Mechanism: Age related onset Selective vulnerability Modelling: Animals Rodents Drosophila Zebra fish cells Systematic clinical study Problems Genotype/Phenotype relationship Treatment and therapy Relationship among various MND (IAPLS, JPLS, ALS) Research 9

10 In vivo pathogenetic paradox Age related onset: All proteins expressed since birth, but pathology has a specific clock surfacing later in life Selective vulnerability All proteins expressed broadly, but only subtypes of neurons are affected Experimental models Cells Localization studies Cellular function Functional interactions K-Down Drosophila Physiological function Overexpression Selected or general KO/KD Expression of mutated forms Interacting genes Response to modulating conditions (genetic or epigenetic) Andrea Martinuzzi 10

Drosophila is a complex organism There is a relevant structural and functional conservation among Human & Fly genes systematical genetic analysis can be employed to extract functional information a

11 Drosophila is a complex organism There is a relevant structural and functional conservation among Human & Fly genes systematical genetic analysis can be employed to extract functional information a great wealth of informations and experimenatl technique is available - it s CHEAP!!! The general strategy for using Drosophila as model organism is based on: Identification and isolation of genes homologue to Human genes of interest (e.g. involved in pathology) study their function taking advantage of the relative simplicity of the fly 11

12 Various types of genetic analysis are applicable in Drosophila: Loss of Function - dsrna-mediated genetic interference - somatic mosaicism Gain of Function - ectopic expression system UAS/Gal4 GAL4/UAS binary expression system GAL4 Line X UAS Target gene GAL4 Tissue-specific transcriptional activation of the gene gal4 Tissue-specific promoter UAS Expression construct 12

13 dsrna interference Gal4 line enhancer gal4 Hairpin ds RNA GAL4 UAS inverted repeat line UAS Gene X X eneg Drosophila as model for human pathology if Human gene known protein function unknown molecular mechanism unknown interacting genes undetermined 13

Analysis of Hereditary Spastic Paraplegia genes in Drosophila General Experimental strategy Identify and Isolate the Drosophila homologous gene map the gene on the chromosome

14 Analysis of Hereditary Spastic Paraplegia genes in Drosophila General Experimental strategy Identify and Isolate the Drosophila homologous gene map the gene on the chromosome search for existing mutants or creation of them Phenotype characterization of: loss of function mutants gain of function mutants mutated alleles mutants in putative interacting genes 14

15 Spastin SIN Triveneta Este 5 Dicembre 2009 Andrea Martinuzzi Drosophila and Human Spastin Alignement TM+ 40% MIT 55% AAA 70% 15

16 D-spastin binds to microtubules 76 KDa D-Spastin 55 KDa Tubulin Lysate Supernatant Pellet Microtubule co-sedimentation assay using Drosophila S2 cells transfected with D-spastin Dspastin is expressed at the Drosophila larva neuromuscular junction Larva CNS NMJ 6/7 Wild type overexpression RNAi 16

neurotrasmission D-spastin RNAi enhances

stability of NMJ microtubules Control NMJ

17 D-spastin dosage affects NMJ morphology and function Total synaptic area is reduced by neuronal expression of RNAi D-spastin overexpression decreases neurotrasmission D-spastin RNAi enhances neurotrasmission D-Spastin regulates the stability of NMJ microtubules Control NMJ on ventral longitudinal muscles 6 and 7 Overexpression RNAi 17

18 Can we generate an HSP disease model in flies? Such a model should mimic at least in part some features of the human disease: locomotor dysfunction adult onset of symptoms evidence of neurodegeneration progressive nature of the symptoms To create an HSP model we expressed pathogenic mutation K467R in a neuron specific fashion Dspastin K467R Dro PGNGKTLLARAVATECSATFLNISAASLTSKYVG Hum PGNGKTMLAKAVAAESNATFFNISAASLTSKYVG 18

Neuron specific D-spastin RNAi and overexpression of pathogenic mutation K467R induce similar

control RNAi K467R Climbing activity (%) 120 100 80 60 40 20 CRL RNAi K467R 0 1 4 7 10 13 16

37 39 41 43 45 47 49 51 Age (days) Survival Locomotor ability Knockdown of Dspastin and K467

19 Neuron specific D-spastin RNAi and overexpression of pathogenic mutation K467R induce similar shortening of lifespan and loss of motor ability Flies surviving (%) control RNAi K467R Climbing activity (%) CRL RNAi K467R Age (days) Age (days) Survival Locomotor ability Knockdown of Dspastin and K467 expression in the nervous system causes neurodegeneration RNAi Drosophila adult brain section K467R 19

20 Neuronal Expression of D-spastin K467R decreases synaptic area and increases acetylated tubulin levels K467RContr ol NMJ synaptic area µm * * 1 α AcTub Fluorescence Intensity normalized to HRP * * 1 α HRP Mutation K467R suppresses D-spastin rough eye phenotype Control D-spastin expression K467R expression D-spastin and K467R coexpression 20

21 Can drug treatment rescue pathological phenotypes in vivo? nocodazole Microtubule destabilizing drugs with different mechanism of action + Drug Pharmacological rescue of neurotrasmission in D- spastin mutant synapse 21

22 Vinblastine treatment increases the eclosion rate and suppresses shortening of adult lifespan and loss of motor ability in flies expressing Dspastin RNAi and Dspastin K467R Vinblastine treatment restores synaptic area and normal acetylated tubulin levels + vinblastine - vinblastine tcontrol K467R RNAi Control K467R RNAi NMJ synaptic area µm vinblastine Vinblastine Fluorescence Intensity normalized to HRP vinblastine +vinblastine

23 Conclusions D-spastin regulates microtubule stability Neuronal loss of D-spastin and neuronal expression of D-spastin K467R cause similar adult onset phenotypes reminiscent of the human disease Pathological mutation K467R acts as a dominant negative The microtubule targeting drug vinblastine suppresses the pathological effects induced by expression of D-spastin RNAi and D-spastin K467R Atlastin Andrea Martinuzzi 23

24 Homology between Human and Drosophila Atlastin GTP binding domain Cyt 56% identity 77% similarity D-atlastin is localized on the ER 24

25 D-atlastin is localized on the ER Endoplasmic Reticulum (ER) 25

26 Efficacy of RNA interference Loss of Datlastin causes fragmentation of the ER 26

27 FLIP analysis in muscles depleted of D-atlastin demonstrates discontinuity of ER membranes D-atlastin homo-oligomerizes and mediates tethering of ER membranes 27

28 Overexpression of D-atlastin induces fusion of ER membranes Datlastin overexpression induces secretory pathway blockade 28

29 Inhibition of D-atlastin GTP binding ability results in inactivation of the protein GTPase-deficient Datlastin K51A is unable to mediate membrane tethering 29

30 Datlastin reconstitution Detergent assisted insertion Mix protein in detergent with preformed liposomes Remove detergent with dialysis and/or Biobeads or Gel filtration Octylglucoside monomer Phospholipid monomer Octylglucoside micelle Extruded liposome Fusion Assay Model Rhodamine NBD ATL ATL 30

31 GTP-dependent liposome fusion by Datlastin Datlastin liposomes Datlastin liposomes + GTP + MgCl 2 GTP-dependent liposome fusion by Datlastin 31

32 Membrane fusion: Atlastin Orso et al., Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin. Nature Pendin et al., 2011 GTP-dependent packing of a three-helix bundle is required for atlastinmediated fusion. Proc NatlAcadSciU S A 32

Conclusions - Datlastin localizes to the ER - Datlastin induces ER fragmentation - Datlastin is not

distinct ER membranes - GTPase-deficient Datlastin is inactive because unable to selfassemble and

mediates ER membrane tethering through the formation of a trans-oligomeric complex between adjacent

diverse pathways ER biogenesis of Lipid Droplets HSP proteins spartin LDs and mitochondria

33 Conclusions - Datlastin localizes to the ER - Datlastin induces ER fragmentation - Datlastin is not involved in secretory traffic - Overexpression causes over-fusion of ER membranes - Datlastin tethers distinct ER membranes - GTPase-deficient Datlastin is inactive because unable to selfassemble and thus unable to link ER membranes Datlastin undergoes GTP-dependent homooligomerization and thereby mediates ER membrane tethering through the formation of a trans-oligomeric complex between adjacent ER membranes, which as a consequence are primed to progress to fusion Lipids as unifying elements of diverse pathways ER biogenesis of Lipid Droplets HSP proteins spartin LDs and mitochondria interaction DDHD1 DDHD2 spastin REEP2 TGF REEP1 reticulon2 atlastin Seipin erlin2 FA2H PNPLA6 CYP7B1 SLC33A1 CYP2U1 33

34 Experimental design IN VIVO ANALYSIS OF FLY MODELS IN VITRO ANALYSIS OF PATIENTS CELLS LIPIDOMIC PROFILES Blood sample Spastin binds to lipid droplets and affects lipid metabolism ChrisovalantisPapadopoulos, Genny Orso, Giuseppe Mancuso, MarijaHerholz, SentiljanaGumeni, NimeshaTadepalle, Christian Jüngst, Anne Tzschichholz, AstridSchauss, Stefan Höning, Aleksandra Trifunovic, Andrea Daga, Elena I. Rugarli. (PLoS Genetics in press). Dspastin dosage affects LD number and size in fat bodies and TAG levels in the larvae Dspastin dosage affects LD number and size in skeletal muscle and nerves 34

Lipid Droplets defects and mitochondria disruption in SPG5 patients (mutated in CYP7B1) Mitochondria LDs

SPG5 Patients MALDI-Spectra zoom area in the range of 700-900 m/z, the region of phosphatidylcholines cluster

Unsupervised PCA score plot with m/z cluster of hosphatidylcholines Supervised PLS-DA score plot with m/z

35 Lipid Droplets defects and mitochondria disruption in SPG5 patients (mutated in CYP7B1) Mitochondria LDs Mitochondria LDs CTRL Mitochondria LDs Mitochondria LDs SPG5 Plasma lipidic and Cholestenoic acid profile In SPG5 Patients MALDI-Spectra zoom area in the range of m/z, the region of phosphatidylcholines cluster with overlapping samples. Unsupervised PCA score plot with m/z cluster of hosphatidylcholines Supervised PLS-DA score plot with m/z cluster of Phosphatidylcholines Levels of (F) 26-HC, (G) 3β-HCA, (H) 3β,7α-diHCA and (I) 3β,7βdiHCA (ng/ml, mean ± SEM) in CSF from controls (black, n = 18), SPG5 patients (purple, n = 3), SPG5 carriers (green, n = 2). 35

36 Loss of D-REEP1 (SPG31) affects ER morphology and lipid pathway D-REEP1P19R pathological mutation affects the neuronal LDs size and number 36

/swisscheese results in neurodegeneration Electron microscopy reveals membrane defects in

37 Spartin/SPG20 Regulates Synaptic Growth and Neuronal Survival by Inhibiting BMP-Mediated Microtubule Stabilization Hooper 2010 Brain-specific deletion of neuronal NTE (SPG39) /swisscheese results in neurodegeneration Electron microscopy reveals membrane defects in sws mutants DSWS and MSWS can restore the wildtype function in glia and neurons. Akassoglu

Tissue-Autonomous Function of Drosophila Seipin (SPG17) in Preventing

Tian 2011 Drosophila spichthyin (NIPA1/SPG6) inhibits BMP signaling

38 Tissue-Autonomous Function of Drosophila Seipin (SPG17) in Preventing Ectopic Lipid Droplet Formation dseipin functions tissue-autonomously in both salivary gland and fat body. dseipin genetically interacts with lipogenic genes. Tian 2011 Drosophila spichthyin (NIPA1/SPG6) inhibits BMP signaling and regulates synaptic growth and axonal microtubules spict null mutants cause BMP-dependent NMJ overgrowth. Wang

39 ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H RETICULUM SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 GOLGI LOCALIZATION GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 PLASMA MEMBRANE NIPA1 MASPARDIN Alsin VPS37A KIAA0196 ENDOSOME ZFR EXOSC3 TECPR2 WDR48 NUCLEUS GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 CYTOSOL MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 MITOCHONDRION KIAA1840 ZFYVE26 LYSOSOME KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN CYTOSKELETON ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome ER STRUCTURE GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton 39

40 ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi GOLGI STRUCTURE GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi LIPID METABOLISM GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton 40

41 ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome MITOCHONDRION GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome MICROTUBULES GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton 41

42 ATLASTIN ZFYVE27 PNPLA6 SLC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 AP4B1 FAM134B AP4E1 B4GALNT1 MASPARDIN P4M1 AP4S1 golgi ER-GOLGI TRAFFICKING GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 AP4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi GOLGI-ENDOSOME-LYSOSOME TRAFFICKING GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton 42

ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi MEMBRANE

43 ATLASTIN ZFYVE27 PNPLA6 LC33A1 ARSI ARL6IP1 REEP2 PGAP1 ERLIN1 RTN2 CYP7B1 CYP2U1 BSCL2 ERLIN2 REEP1 FA2H reticulum SLC33A1 MASPARDIN AP4B1 P4M1 FAM134B AP4E1 AP4S1 B4GALNT1 golgi MEMBRANE TRAFFICKING GJC2 ENTPD1 L1CAM FLRT1 RAB3GAP2 MAG PLP1 SLC16A2 plasma membrane NIPA1 MASPARDIN Alsin VPS37A KIAA0196 endosome ZFR EXOSC3 TECPR2 WDR48 nucleus GAD1 MARS NT5C2 DDHD2 DDHD1 IFIH1 AP5Z1 AMPD2 TFG USP8 cytosol KIAA1840 ZFYVE26 lysosome MT-CO3 MT-ATP6 MTTI MT-ND4 PARAPLEGIN MT-ND4 SPARTIN OPA3 HSPD1 mitochondrion KIF5A LYST KIF1A KIF1C BICD2 CCT5 SPASTIN cytoskeleton Pathophysiology of HSP mutations Multiple metabolicpathwaysaffectedin relation to the different involved gene Neurodegeneration observed in most Stillnotdefinedwhereand howthe various pathways converge How muchthisisreflectedin human disease? 43

44 74 subjects HSP phenotype A standardized clinical evaluation protocol inclusive of Measures of body function (strength, tone, DTR, trophism, cognition) Measures of activity (walking, independence) Neurophysiological and DTI & NMR assessment EMG/ENG, EP, MRI Andrea Martinuzzi Demographics & genetics TABLE 1. DEMOGRAPHIC DATA Genotype n Gender (M) Age at onset (yrs) mean ± SD (range) Disease duration (yrs) mean ± SD (range) Age at the visit (yrs) mean ± SD (range) SPG3a ± 0.85 (0-2) 12.5 ± (2-38) ± (2-40) SPG ± (1-64) ± 9.02 (3-33) ± (7-79) SPG ± (8-54) ± (1-60) ± (34-71) SPG ± 9.2 (34-54) ± (6-32) ± (40-73) SPG ± (4-35) ± 8.5 (10-26) ± 9.02 (30-48) SPG ± (3-46) ± 7.55 (10-31) ± 8.6 (26-57) SPG ± 7.02 (8-22) ± 10.6 (15-36) ± 8.96 (29-45) SPG SPG ± 3.53 (36-41) 7.5 ± 2.12 (6-9) 46 ± 1.41 (45-47) TOTAL ± 18.98(0-64) ± 12.5 (1-60) ± (2-79) Andrea Martinuzzi 44

Age of onset by genotype Age at onset >50y 41-50y Decade of disease onset 31-40y 21-30y 11-20y SPG3a SPG4 SPG5 SPG7 SPG10 SPG11 SPG15 SPG31 SPG35 0-10y 0 5 10 15 20 25 n of patients Andrea Martinuzzi

45 Age of onset by genotype Age at onset >50y 41-50y Decade of disease onset 31-40y 21-30y 11-20y SPG3a SPG4 SPG5 SPG7 SPG10 SPG11 SPG15 SPG31 SPG y n of patients Andrea Martinuzzi Functional measures TABLE 2. CLINICAL DATA PARAMETER n mean ± SD Range SPRS ± (4-48) 6MWT (mt) ± ( ) FIM ± (55-126) MUSCLE TONE ± 0.98 (0-4) MUSCLE STRENGTH ± 12.3 (0-40) DTR ± 0.88 (0-4) R MEP LL mcct (ms) ± 4.37 ( ) L MEP LL mcct (ms) ± 4.93 ( ) LL ATROPHY Andrea Martinuzzi 45

SPRS score by genotype 25 Number of patients 20 15 10 SPG35 SPG31 SPG15 SPG11 SPG10 SPG7 SPG5 SPG4 SPG3a 5 0 0-13 14-26 27-39 40-52 SPRS score Andrea Martinuzzi Additional signs in the symptomatic

46 SPRS score by genotype 25 Number of patients SPG35 SPG31 SPG15 SPG11 SPG10 SPG7 SPG5 SPG4 SPG3a SPRS score Andrea Martinuzzi Additional signs in the symptomatic HSP patients GENOTYPE n Ataxia Dysarthri a Swallowing abnormaliti IDD (*) es Cognitive impariment (**) Epileps Deafness y Optic atrophy Early cataract Axonal Muscle atrophy neuropathy n. of Patients and (%) SPG3a (14.3) (28.6) 1 (14.3) SPG4 (5) 32 2 (6.3) 1 (3.1) - 1 (3.1) 5 (15.6) (9.4) - SPG5 7 2 (28.6) - 3 (42.9) (28.6) 1 (14.3) 1 (14.3) 4 (57.1) - SPG7 (6) 4 3 (75.0) (75) 3 (75.0) SPG SPG (81.8) 6 (54.5) 6 (54.5) SPG (100.0) 2 (66.7) 1 (33.3) 1 (33.3) 8 (72.7) 2 (66.7) 1 (33.3) (66.7) 2 (18.2) (18.2) 9 (81.8) 9 (81.8) 1 (33.3) (100.0) 3 (100.0) SPG SPG (100.0) (100.0) (100.0) (50.0) 1 (50.0) - TOTAL n (%) (30.0) 11 (15.7) 10 (14.3) 12 (17.1) 11 (15.7) 1 (1.4) 2 (2.9) 3 (4.3) 2 (2.9) 25 (35.7) 18 (25.7) (*) IDD: Intellectual Developmental Disorder. (**) Mild cognitive impariment Andrea Martinuzzi with ENB-2 <66 46

47 Multiple cluster Analysis of additionalsignsby genotype 3 Cluster 1(No additional signs): SPG3a, SPG4, SPG5, SPG10 and SPG31 Cluster 2: (ataxia, axonal neuropathy, dysarthria, dysphagia, IDD, muscle atrophy): SPG 11, SPG 15 Cluster 3: (ataxia, axonal neuropathy, cognitive impairment, muscle atrophy): SPG7 Single outlier: (IDD): SPG35 A1 A2 B1 B2 C1 C2 D1 D2 E1 E2 F1 F2 47

48 48

49 Summary of the clinical study Bimodal distribution of age at onset Weakness more prevalent than increased tone or DTRs SPRS resumes is a good proxy for clinical severity and disease duration Presence of additional signs identifies discrete clusters of SPGs Most patients cope well with the condition MER are costantly abnormal but do not correlate with SPRS or disease duration Advanced MRI may add to diagnostic and prognostic definition NeuroimagingCluesto the HSP pathophysiology A common modificationof structuralorderingof long and connectingtraits isfoundconsitentlyin all SPGs Albeitthe CST isthe structureallowingthe most efficientdiscriminationbetweenhsp and controls, FA/ADC alterations are found also elsewhere in the CNS Spectroscopyin ourhandsdidn tallowthe consistent identification of specific abnormalities 49

50 Conclusions The final pathway of motoneuronal degeneration seen in the various forms of MND recognises different pathogenetic mechanisms, and even among forms clinically similar the underlying molecular mechanism may be strikingly different There is no «gold-standard» model to study pathologies primarily affecting a very specialized and evolutionary recent system Modelling disease process in different organisms and systems may be the only option to gain complementary information useful to understand human pathology and devise effective treatments Andrea Martinuzzi With Special thanksto: Medea Veneto (clinical) Marinela Vavla Elisa Petacchi Gabriella Paparella Elena Carraro Medea Bosisio Mariateresa Bassi Grazia D Angelo Alessia Arnoldi Claudia Crimella Medea Veneto Molecular genetics lab Andrea Daga Genny Orso Marianna Fantin Diana Pendin Andrea Martinuzzi Neuroradiology(Fondazione Monasterio& ULSS7 Veneto) Domenico Montanaro Hana Hlavata Giuseppe Rossi Niccola Martino Alessandra Baratto 50

ASL-HSP France & RESEARCH a rear overview ( )

ASL-HSP France & RESEARCH a rear overview (1992-2012) Jean BENARD, Scientific Advisor, ASL-HSP France & EURO-HSP May 26, 2012 Louis Lumière International Center, 75020 Paris, France How to fight HSP? 1992: