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 pH (~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 M6P receptor pathway

Delivering material for degradation to the lysosome: endocytosis and autophagy
Endosome to lysosome: decreasing pH, 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
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Lower pH causes dissociation
M6PR then retrieved in late endosome and trafficked for re-use in trans-golgi
(recognised via C-terminal tail).
Delivering degradative enzymes and cofactors to the lysosome, the M6P/M6PR pathway.
Mannose-6-phosphate group added to lysosomal hydrolases via N-linked oligosaccharides as hydrolases transit through cis-golgi
M6P recognised by M6P-receptors in trans-golgi: delivers them to late endosome

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)
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

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Mucopolysaccharidoses: Enzyme Defective
MPS-I: (Hurler, Sheie, Hurler/Sheie) iduronidase
MPS-II: (Hunter) iduronate-2-sulfatase
MPS-III: (Sanfilippo)
IIIA
IIIB
IIIC heparan-N-sulfatase
N-acetyl-glucosaminidase
Acetyl Co-A glucosamine
IIID
MPS-IV (Morquio)
IVA
IVB
N-acetyl transferase
N-acetyl-glucosamine-6-sulfatase
N-acetyl-galactosamine 6-sulfatase
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 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
Ceramide
OH
CH
2
O H
NH
O
Sphingomyelin
OH
O CH
3
NH
CH
2
O P O (CH
2
O -
)
2
N +
CH
3
CH
3
O
Glycosphingolipids
OH
CH
2
O
NH
O
Glc n
STRUCTURE microdomains (?) trafficking
SIGNALLING
Apoptosis proliferation stress
- Sphingomyelin
- Ceramide
- Sphingosine
- Sphingosine-1-phosphate
- Cerebrosides
- Gangliosides

Sphingolipids are tightly associated with cholesterol

The sphingolipidoses: Tay-Sachs (GM2-gangliosidosis)
First described in 1880’s from ‘cherry-red’ spot in fundus (retina) (lipid deposition in bipolar ganglion 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 Therapy
Substrate 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 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 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 endosomal trafficking steps. Cholesterol recycled from lysosome.
Drosophila models reveal cholesterol is ‘limited’

Batten disease
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A family of closely related disorders
9 forms: congenital, infantile, late infantile, juvenile adult
AKA: Neuronal Ceroid Lipofuscinosis (NCL)
Incidence: global with hotspots for some loci
Loci: ‘CLN’ genes CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 CTSD cloned so far, others remain to be mapped.
Batten (1903) 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
CLN1
CLN2
CLN3
CLN4
CLN5
CLN6
CLN7
CLN8
CTSD
Disease infantile NCL
Protein deficiency palmitoyl protein thioesterase late infantile NCL tripeptidyl peptidase juvenile NCL adult (Kuf’s) transmembrane protein
Not identified late infantile NCL transmembrane protein
(Finnish variant) late infantile variant transmembrane protein late infantile variant Not Identified
EPMR
Ovine NCL transmembrane protein cathepsin D
Function de-palmitoylation
Lysosome protease lysosome
?
lysosome
?
LE/lysosome
ER protein
ER, ER/Golgi protease lysosome

Endocytosis in the nervous system
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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
Fabry
SRT
ERT
Hurler-Scheie (MPS-I) ERT
Maroteaux-Lamy (MPS-VI) ERT
Reasons:
High regulatory costs
Cost of research
Lack of competition (Orphan Drug Act 1983, US)
91,000
156,000
340,000
377,000

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

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

Spin localises to a low pH late-endosomal compartment

A low pH 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’