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 insubstrate 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 pH (~4.0), membrane bound

Considered the ‘gut’ or garbage disposal unit of cell

Material for degradation trafficked to lysosome via endocytosisor autophagy

Lysosomal enzymes trafficked to lysosome via M6P receptorpathway

Endosome to lysosome:decreasing pH, membranelimited.

Autophagy: controls cell size,used during caloric restriction,Phagocytosis:- degrades ‘dead’ cells, pathogensAutophagy and phagocytosismeet in the PhagolysosomeProfessional Phagocytes:macrophages, neutrophils

Delivering material for degradation to the lysosome:endocytosis and autophagy

Endocytosis in the nervous system

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The polarised and extendedstructure 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 co-factors to the lysosome, the M6P/M6PR pathway.

Mannose-6-phosphate group added to lysosomalhydrolases via N-linked oligosaccharides ashydrolases transit through cis-golgi

M6P recognised by M6P-receptors in trans-golgi:delivers them to late endosome

Lower pH causes dissociation

M6PR 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? pH?)

Accumulation of lipofuscin/ceroid ‘ageing pigment’

Defects in autophagy (?)

Appearance of meganeurites (variable)

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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)GlycoproteinosesGlycogen storageSphingolipidosesLipid storage disordersMultiple enzyme defectsTransport defectsBatten 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

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Mucopolysaccharidoses: Enzyme Defective

MPS-I: (Hurler, Sheie, Hurler/Sheie) iduronidaseMPS-II: (Hunter) iduronate-2-sulfataseMPS-III: (Sanfilippo)

IIIA heparan-N-sulfataseIIIB N-acetyl-glucosaminidaseIIIC Acetyl Co-A glucosamine

N-acetyl transferaseIIID N-acetyl-glucosamine-6-sulfatase

MPS-IV (Morquio)IVA N-acetyl-galactosamine 6-sulfataseIVB ß-galactosidase

MPS-VI (Maroteaux Lamy) N-acetyl-galatosamine 4-sulfataseMPS-VII (Sly) ß-glucuronidaseMPS-IX hyaluronidase

Sanfilippo Syndrome (MPS III)

Four types: A,B,C,D, cannot break down Heparan sulfateMost common MPS, 1/70,000 births

hepatosplenomegaly (may resume normal size with age)HyperactivitySpeech delayMental retardationJoint stiffness, bone defects (dystosis multiplex)Coarse features (dysmorphismDeath in middle teens

Screening: GAGs in urineDiagnostic: WBC enzyme assay or plasma enzyme assay

Prognosis: No effective treatment to date.

Mucolipidosis (I-Cell disease) and MPS-IV

Mucolipidosis-III-Cell (Pseudo-Hurler): first described 1967I = Inclusion, stored material mucolipid MPS and sphingolipidOccurrence: 1/640,000 live birthsSymptoms: 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 sphingolipidsOccurrence: carriers in Ashkenazim Jewish population, 1/90 to 1/100Symptoms: Psychomotor retardation, corneal opacity, retinal degeneration, iron deficiency, improper stomach pH (achloridia)

Genetic defect: Mucolipin-1 (MCOLN1), a TRP channel (TRPML-1)Involved in Fe2+ 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

O

OH

NH

CH2O H

O

OH

NH

CH2O P O (CH2)2 N+

O

O-

CH3

CH3

CH3

O

OH

NH

CH2O Glcn

Ceramide

Sphingomyelin

Glycosphingolipids

STRUCTURE

microdomains (?)trafficking

SIGNALLING

Apoptosis proliferation stress

- Sphingomyelin - Ceramide - Sphingosine - Sphingosine-1-phosphate - Cerebrosides - Gangliosides

Sphingolipids aretightly associated withcholesterol

The sphingolipidoses: Tay-Sachs (GM2-gangliosidosis)

First described in 1880’s from ‘cherry-red’ spot in fundus (retina) (lipid deposition in bipolarganglion cells)

Infantile (death ~ 5yrs), Juvenile (death between 5 and 15yrs) 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: GM2 ganglioside, globoside, glycolipids

cf: Sandhoff Disease: HEXB mutations and GM2 gangliosidosis(mutations in GM2 activator protein)

Glial Involvement!

Prognosis: early death, ameliorated by treatment

Enzyme Replacement TherapySubstrate Reduction Therapy

Population Screening (model of genetic screening for recessive condition)

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Other cellular defects:

Niemann-Pick disease:

occurrence: A, B collectively- 1/1000 Ashkenazim Jews are carriers, type C no ethnic distributiontype 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 sphingomyelinaseType C mutants: two loci, two proteins, multi-transmembrane protein (related tohedgehog receptor ‘patched’ and small co-protein(cholesterol binding protein/carrier?). Homolog NPCL1 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 endosomaltrafficking steps. Cholesterol recycled from lysosome.

Drosophila models reveal cholesterol is ‘limited’

Batten disease

A family of closely related disorders9 forms: congenital, infantile, late infantile, juvenileadultAKA: Neuronal Ceroid Lipofuscinosis (NCL)

Incidence: global with hotspots for some loci

Loci: ‘CLN’ genes CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 CTSDcloned 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

Batten (1903)

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Locus Disease Protein deficiency Function

CLN1 infantile NCL palmitoyl protein thioesterase de-palmitoylationLysosome

CLN2 late infantile NCL tripeptidyl peptidase proteaselysosome

CLN3 juvenile NCL transmembrane protein ?lysosome

CLN4 adult (Kuf’s) Not identified

CLN5 late infantile NCL transmembrane protein ?(Finnish variant) LE/lysosome

CLN6 late infantile variant transmembrane protein ER protein

CLN7 late infantile variant Not Identified

CLN8 EPMR transmembrane protein ER, ER/Golgi

CTSD Ovine NCL cathepsin D proteaselysosome

Endocytosis in the nervous system

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Lysosomes (hydrolases!)not present at synapse

Many of NCL proteins foundat synapse

NPC protein, others?

Identification of proteins involvedin neurodegeneration helpto describe functions in the neuron

Treatment:BMT (membrane proteins)

enzyme replacement (BBB?)

gene therapy

substrate reduction- Miglustat (monosaccharidemimetic-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 costsCost of researchLack 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

http://132.236.112.18/fruitfly/shaker/physiology/

The Drosophila neuromuscular junction: A model glutamatergic synapse

spinster synapses are overgrown

spinstersuppressessynaptic growth

spinster mutantshave a shortenedlifespan

spinster encodes a twelve transmembrane transporter

4 transcripts = 12 TM domains1 transcript = 8 TM domains

Spin localises to a low pH late-endosomal compartment

A low pH compartment is expanded in spin mutants

WT spin4/spin5

Loss of spinster induces a redistribution of cholesterol

filipin

spinster identifies a novel component of the late endosome/lysosome that when mutated givesrise 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 proteinsessential to lysosomal biogenesis/function

LSD lysosomal defects give rise to swollen lysosomes, developmental and degenerativedefects with varying involvement of the nervous system due to ‘storage’ of materialin 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’