Lysosomal Storage Disease Module 755 The Brain in

Lysosomal Storage Disease Module 755 The Brain in Health and Disease Sean Sweeney

Lysosomal Storage Disease (Amaurotic Idiocy) c. a. 45 autosomal recessive diseases Individually rare Collectively occur c. a. 1/8000 live births Cause death in early to late childhood (after normal infancy) Varying involvement of the nervous system All ‘store’ material in the lysosome due to defects in substrate degradation or biogenesis of the lysosome

The Lysosome subcellular electron dense organelle filled with c. a. 70 hydrolytic enzymes: will break down all biological macromolecules low p. H (~4. 0), membrane bound Considered the ‘gut’ or garbage disposal unit of cell Material for degradation trafficked to lysosome via endocytosis or autophagy Lysosomal enzymes trafficked to lysosome via M 6 P receptor pathway

Delivering material for degradation to the lysosome: endocytosis and autophagy Endosome to lysosome: decreasing p. H, membrane limited. Autophagy: controls cell size, used during caloric restriction, Phagocytosis: - degrades ‘dead’ cells, pathogens Autophagy and phagocytosis meet in the Phagolysosome Professional Phagocytes: macrophages, neutrophils

Endocytosis in the nervous system The polarised and extended structure of the neuron creates a trafficking problem for neurons: ‘lysosomes’ (as we know them!) not present at synapse. Late endosomal markers present: fuse with lysosomes in the soma

Delivering degradative enzymes and cofactors to the lysosome, the M 6 P/M 6 PR pathway. Mannose-6 -phosphate group added to lysosomal hydrolases via N-linked oligosaccharides as hydrolases transit through cis-golgi M 6 P recognised by M 6 P-receptors in trans-golgi: delivers them to late endosome Lower p. H causes dissociation M 6 PR then retrieved in late endosome and trafficked for re-use in trans-golgi (recognised via C-terminal tail).

General outline of LSD dysfunction: Mutations arising in hydrolytic enzyme, co-factor or factor essential of enzyme delivery to lysosome Also, factors essential for lysosome function and biogenesis (membrane proteins, channels and proteins of unknown function) plus factors for protein traffic to lysosome Material (substrate) continues to be delivered to lysosome resulting in ‘stored’ material, usually ‘primary’ and ‘secondary’ leads to swollen lysosomes Developmental dysfunction and early death: symptoms v. variable, varying involvement of different tissues

General Cellular Phenotype: Swollen, multilammellar ‘osmiophilic endosomes/lysosomes (function? p. H? ) Accumulation of lipofuscin/ceroid ‘ageing pigment’ Defects in autophagy (? ) Appearance of meganeurites (variable)

Cellular phenotype contd. Excessive synaptogenesis/dendritogenesis (MPS and sphingolipidoses) Shrinkage of the CNS (variable) Mistrafficking of cholesterol (cholesterol recycling? )

Why are symptoms and effects in different organs variable? tissue turnover rates? presence (or relative abundance) of substrate? sensitivity of cell type (neurons and polarity)? What is the ‘pathogenic cascade’? (volume of substrate not key!!!)

Classification : Mucopolysaccharidoses (variable nervous system involvement) Mucolipidoses (originally considered an MPS) Glycoproteinoses Glycogen storage Sphingolipidoses Lipid storage disorders Multiple enzyme defects Transport defects Batten Disease (Red = nervous system involvement)

Mucopolysaccharides • Defective metabolism and accumulation of GAGs • Most abundant polysaccharides • Long unbranchedstructure containing disaccharide units: • High viscosity + rigidity • Excellent lubricators and shock absorbers • Important component of cell membranes

Mucopolysaccharidoses: Enzyme Defective MPS-I: (Hurler, Sheie, Hurler/Sheie) iduronidase MPS-II: (Hunter) iduronate-2 -sulfatase MPS-III: (Sanfilippo) IIIA heparan-N-sulfatase IIIB N-acetyl-glucosaminidase IIIC Acetyl Co-A glucosamine N-acetyl transferase IIID N-acetyl-glucosamine-6 -sulfatase MPS-IV (Morquio) IVA N-acetyl-galactosamine 6 -sulfatase IVB ß-galactosidase MPS-VI (Maroteaux Lamy) N-acetyl-galatosamine 4 -sulfatase MPS-VII (Sly) ß-glucuronidase MPS-IX hyaluronidase

Sanfilippo Syndrome (MPS III) Four types: A, B, C, D, cannot break down Heparan sulfate Most common MPS, 1/70, 000 births hepatosplenomegaly (may resume normal size with age) Hyperactivity Speech delay Mental retardation Joint stiffness, bone defects (dystosis multiplex) Coarse features (dysmorphism Death in middle teens Screening: GAGs in urine Diagnostic: WBC enzyme assay or plasma enzyme assay Prognosis: No effective treatment to date.

Mucolipidosis (I-Cell disease) and MPS-IV Mucolipidosis-II I-Cell (Pseudo-Hurler): first described 1967 I = Inclusion, stored material mucolipid MPS and sphingolipid Occurrence: 1/640, 000 live births Symptoms: Developmental delay, psychomotor deterioration, dysmorphia, death in early childhood Genetic defect: N-acetylglucosaminyl-1 -phosphotransferase Prognosis: v. poor, limited treatment (nutritional), death by 10 years of age. Mucolipidosis-IV Storage material: mucolipids, MPS and sphingolipids Occurrence: carriers in Ashkenazim Jewish population, 1/90 to 1/100 Symptoms: Psychomotor retardation, corneal opacity, retinal degeneration, iron deficiency, improper stomach p. H (achloridia) Genetic defect: Mucolipin-1 (MCOLN 1), a TRP channel (TRPML-1) Involved in Fe 2+ efflux from lysosomes? (Dong et al. , (2008) Nature, 455, 992 -6) Prognosis: v. poor. Nutritional supplements, physcial and speech therapy

Sphingolipids: a major component of neural tissue Ceramide STRUCTURE OH CH 2 O H microdomains (? ) trafficking NH O SIGNALLING Sphingomyelin O OH CH 2 O NH P CH 3 O (CH 2)2 N+ CH 3 O- O Glycosphingolipids OH CH 2 O NH O Glc n Apoptosis proliferation stress - Sphingomyelin - Ceramide - Sphingosine-1 -phosphate - Cerebrosides - Gangliosides

Sphingolipids are tightly associated with cholesterol

The sphingolipidoses: Tay-Sachs (GM 2 -gangliosidosis) First described in 1880’s from ‘cherry-red’ spot in fundus (retina) (lipid deposition in bipolar ganglion cells) Infantile (death ~ 5 yrs), Juvenile (death between 5 and 15 yrs) and ‘Late-onset’ forms (v. rare) All present with increasing neurological and deterioration (ataxia, atrophy, spasticity) Occurrence: 1/27 to 1/30 Ashkenazim Jews are carriers, also: Acadians, Cajuns Genetic defect: Hexosaminidase A (HEXA) storage material: GM 2 ganglioside, globoside, glycolipids cf: Sandhoff Disease: HEXB mutations and GM 2 gangliosidosis (mutations in GM 2 activator protein) Glial Involvement! Prognosis: early death, ameliorated by treatment Enzyme Replacement Therapy Substrate Reduction Therapy Population Screening (model of genetic screening for recessive condition)

Other cellular defects: Niemann-Pick disease: occurrence: A, B collectively- 1/1000 Ashkenazim Jews are carriers, type C no ethnic distribution type A accounts for 85% of cases Symptoms: enlarged spleen and liver, enlarged lymph nodes, darkening of skin, neurologic impairment (not in B), cherry red spot genetic defect: A and B, mutant for sphingomyelinase Type C mutants: two loci, two proteins, multi-transmembrane protein (related to hedgehog receptor ‘patched’ and small co-protein(cholesterol binding protein/carrier? ). Homolog NPCL 1 involved in cholesterol absorption in gut. storage material: sphingomyelin, cholesterol and sphingolipids Diagnosis: ‘filipin’ staining cell biology (and diagnosis): mislocalised unesterified cholesterol, neurofibrillary tangles Endosomal trafficking jam? cholesterol and sphingolipids required to organise endosomal trafficking steps. Cholesterol recycled from lysosome. Drosophila models reveal cholesterol is ‘limited’

Batten disease A family of closely related disorders 9 forms: congenital, infantile, late infantile, juvenile adult AKA: Neuronal Ceroid Lipofuscinosis (NCL) Batten (1903) Incidence: global with hotspots for some loci Loci: ‘CLN’ genes CLN 1, CLN 2, CLN 3, CLN 5, CLN 6, CLN 8 CTSD cloned so far, others remain to be mapped. occurrence: most common childhood neurodegeneration 1/8000 livebirths Symptoms: visual defects, seizures, stumbling, echolalia, eventual loss of sight speech and motor skills, early death after blindness, dementia. storage material: Lipofuscin/ceroid, subunit C of mitochondrial ATP synthase Phenotype: multilamellar inclusions, selective brain cell death (glia mediated) infiltration of neuronal tissue with antibodies (defective BBB? ) Prognosis and treatment: anti-convulsives, therapy. Death in childhood

Locus Disease Protein deficiency Function CLN 1 infantile NCL palmitoyl protein thioesterase de-palmitoylation Lysosome CLN 2 late infantile NCL tripeptidyl peptidase protease lysosome CLN 3 juvenile NCL transmembrane protein ? lysosome CLN 4 adult (Kuf’s) Not identified CLN 5 late infantile NCL (Finnish variant) transmembrane protein CLN 6 late infantile variant transmembrane protein CLN 7 late infantile variant Not Identified CLN 8 EPMR transmembrane protein ER, ER/Golgi CTSD Ovine NCL cathepsin D protease lysosome ? LE/lysosome ER protein

Endocytosis in the nervous system Lysosomes (hydrolases!) not present at synapse Many of NCL proteins found at synapse NPC protein, others? Identification of proteins involved in neurodegeneration help to describe functions in the neuron

Treatment: BMT (membrane proteins) enzyme replacement (BBB? ) gene therapy substrate reduction- Miglustat (monosaccharide mimetic-imino sugar) Neuronal stem cells (membrane proteins? ) Chemical chaperone therapy Neuroinflammation

Economic cost ERT is current most effective treatment (non neurodegenerative LSDs): Disease Treatment Annual Cost (per patient in $) Gaucher ERT 145, 000 - 290, 000 Gaucher SRT 91, 000 Fabry ERT 156, 000 Hurler-Scheie (MPS-I) ERT 340, 000 Maroteaux-Lamy (MPS-VI) ERT 377, 000 Reasons: High regulatory costs Cost of research Lack of competition (Orphan Drug Act 1983, US)

Studying the Lysosomal Storage Diseases: Model Organisms Sheep (Batten) sheepdogs (Batten) mouse (Batten, Tay-Sachs, Sandhoff, NPC) zebrafish (Batten) Drosophila (MPS, NPC, Batten, others) C. elegans (MPS, NPC) Yeast (cerviseae, pombe) Batten, NPC Reverse Genetics (qv Tay-Sachs) Forward Genetics

The Drosophila neuromuscular junction: A model glutamatergic synapse http: //132. 236. 112. 18/fruitfly/shaker/physiology/

spinster synapses are overgrown spinster suppresses synaptic growth spinster mutants have a shortened lifespan

spinster encodes a twelve transmembrane transporter 4 transcripts = 12 TM domains 1 transcript = 8 TM domains

Spin localises to a low p. H late-endosomal compartment

A low p. H compartment is expanded in spin mutants

Loss of spinster induces a redistribution of cholesterol WT filipin spin 4/spin 5

spinster identifies a novel component of the late endosome/lysosome that when mutated gives rise to all of the hallmarks of lysosomal storage disease spinster potentially identifies a signalling pathway driving synaptic overgrowth

Summary Lysosomal storage disease are caused by defects in lysosomal hydrolases and proteins essential to lysosomal biogenesis/function LSD lysosomal defects give rise to swollen lysosomes, developmental and degenerative defects with varying involvement of the nervous system due to ‘storage’ of material in the lysosome. Lysosomal storage diseases identify proteins essential to lysosomal function LSDs cause death in childhood (generally) after normal infancy LSDs are essentially incurable, but some are treatable to varying degrees. Model organisms are helping to define the biology of the LSDs, in particular the ‘pathogenic cascade’