LYSOSOMAL STORAGE DISEASES

Presented by PRADEEP. M

Submitted to Dr. R. Singh

LSD

• To date 51 naturally occurring LSD • Most of them are autosomal recessive

gene traits• 9 inherited and 2 acquired mechanisms of

LSD known• No sex predilection • Brain lesions are prevalent (2/3rd of LSD)

HISTORY OF LSD

• Symptoms of some LSDs were described as early as the 1880s,

• Many had been described and classified before the lysosome was discovered in 1955 and before their biochemical and genetic basis was fully understood

• This is why they received common names (i.e.: Gaucher disease, name of discovering physician).

• Later, an additional, more clinically descriptive name often came into use (glucocerebrosidase deficiency)

HISTORY OF LSD

Ernest GAUCHER (1854-1919)

Gaucher cell 1882

LSD IN HUMAN IN INDIA (Sheth et.al., 2014)

• 387 children (34.8 %) • highest prevalence of • glycolipid storage disorders in 48 %,• mucopolysaccharide disorders in 22 %• defective sulfatide degradation in 14 % of the children.• glycogen degradation defect and protein degradation defect in 5 %

each• Lysosomal trafficking protein defect in 4 %, • Transport defect in 3 % of the patients.

• Higher incidence of Gaucher disease (16 %) followed by GM2 gangliosidosis that includes Tay-Sachs disease (10 %) and Sandhoff disease (7.8 %) and mucopolysaccharide disorders among all LSDs

Pathways for degradation of cellular materials

BIOCHEMICAL AND CELLULAR BASIS OF LSDS

Futerman AH & van Meer G (2004) 5:554-565

1 catalytic activity2 activator3 misfolding4 multienzyme complex5 glycosylation

6 M-6-P targetting7 other transport steps8 membrane transporters9 membrane regulators

General outline of LSD dysfunction:

• Mutations arising in hydrolytic enzyme, co-factor or

factor essential of enzyme delivery to lysosome

• 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

Effect of lysosomal storage diseases

• Direct consequence of abnormal accumulation of substrates or catabolites in the lysosomesSevere impairment of cellular structures and functions and abnormal extracellular matrix. They include alterations of– Signalling pathways, – Intracellular Ca2+ homeostasis, – Intracellular trafficking,– Dysmyelinogenesis– Formations of ectopic dendrites,– Retarded bone formation, and– Cloudy cornea

• Secondary changes are due to disrupted recycling– manifested in abnormal or shortage of cellular and extracellular

products– In the brain, there is a compromised recycling of neurotransmitter

receptors, altered signalling and dysmylinogenesis

LYSOSOMAL STORAGE DISEASE- DAMAGING EFFECTS IN NERVOUS SYSTEM

LSD- SUB-CATEGORIES

1. Sphingolipidoses

2. Glycoproteinoses

3. Mucopolysaccharidoses (variable nervous system involvement)

4. Mucolipidoses (originally considered an MPS)

5. Glycogen storage diseases

6. Lipid storage disorders

7. Multiple enzyme defects

8. Transport defects

9. Batten Disease (Ceroid lipofuscinoses)- proteinosis

(Blue = nervous system involvement)

1. SPHINGOLIPIDOSES

• Sphingolipids- 1. Sphingomyelins ( a phospholipid)

2. Glycosphingolipids

(cerebroside, globoside, sulfatides and gangliosides)

Ceramide= sphingosine + FA

Glycosphingolipid = ceramide + oligosaccharide

Cerebroside = glycosphingolipid with one sugar as side chain (gluc or

gal)

Globoside = glycosphingolipid with more than one sugar.. (glucose,

galactose or GalNAc)

Ganglioside = glycosphingolipid with one or more sialic acid (NANA)

linked to sugar side chain

STRUCTURE AND CATABOLISM OF SPHINGOLIPIDS

GM1 Gangliosidosis

GM2Gangliosidosis

(Tay-Sachs disease, Sandhoff, variant

AB)

Glucocerebrosidosis (Gaucher disease)

Sphingomyelinosis (Nieman-Pick typeA, B)

Galactosialidosis

Galactocerbrosidosis(Krabbe disease)

sialidase

αGalactosidase A

Sandhoff

Fabry disease

2. GLYCOPROTEINOSES glycoprotein contain hybrid type oligosaccharides

Alpha-Mannosidosis Beta- mannosidosis

Alpha-L-fucosidosis

MPS I (Hurler, Hurler-Scheie, Scheie)

MPS II (Hunter)

MPS III (San filipo Types A,B,C and D)

MPS IV (Morquio type A and B)

MPS VI (Maroteaux-Lamy)

MPS VII (Sly)

MPS IX (Hyaluronidase deficiency)

Multiple Sulfatase deficiency

3. MUCOPOLYSACCHARIDOSES Defective metabolism of glycosaminoglycans

MUCOPOLYSACCHARODOSIS- CLASSIFICATION

4. MUCOLIPIDOSES

• Features of both sphingolipidosis and mucopolysaccharidoses

Disease Enzyme deficient signs Species affected

Mucolipidoses I αNuraminidase Neurological signs, organomegaly, and skeletal abnormalities.

cats

ML II (I cell disease) phospho-N-acetylglucosamine transferase

-do- cat

Pseudo-Hurler polydystrophy(ML-III)

phospho-N-acetylglucosamine transferase

-do-

MUCOLIPIDOSES II – (I cell disease)

• Face; cat, 8 months of age. Left. Note the broad face with hypertelorism, thickened eye lids, and small ears. Right. Note frontal bossing

5. GLYCOGENOSES

• Glycogen storage disease type II (Pompe disease)– α1,4-glucosidase deficiency(Only lysosomal enzyme in glycogen catabolism others are metabolic pathways

(type III- Amylo1,6 glucosidase defiency- Cori disease;

type IV- Polyglucosan body disease) )

OTHER LYSOSOMAL STORAGE DISEASES

• Ceroid-lipofuscinoses– Neuronal ceroid-lipofuscinoses (Batten disease)– Autofluorescent neurons

• Laffora disease– Storage of polyglucosan

GENETICS OF LSD

• X linked diseases in human– Fabry disease (alpha- galactosidase dificiency)- affect both sex – MPS II – Adult lipofuscinoses

• All other are autosomal recessive

INDUCED STORAGE DISEASES

• Swainsonine toxicosis– An indolizidine alkaloid– Seen in plants- locoweeds, broom weed etc.– Inhibit αMannosidase- αMannosidoses– “locoism” , “pea-struck” – identical to genetic form

• Trachyandra poisoning– Ingestion of Trachyandra sps plants– Lipofuscinosis- central & peripheral neurones– sheep, horse, goat, pig– Reason not known

• Phalaris poisnoing– By ingestion of Phalaris sp - grass – Accumulation of granular pigment material – Sudden death form- by cardio toxic compounds– Stagger form

Phalaris poisoning

• A. Pigmentation in lateral geniculate body (arrow) • B. Neuronal accumulation of granular pigmented material (arrow head)

B

Drug induced LSD

• Amiodarone and chloroquine,• Induce phospholipidosis and MPS.

TAY-SACHS DISEASE

• Mutation- chromo 15- Hex A gene- hexaminidase deficiency- accu of GM2 ganglioside

Typical signs and symptoms of Fabry disease

Typical time at onset Signs and symptoms

Childhood and adolescence (≤16 years)

Neuropathic pain(burning sensation)Ophthalmological abnormalities (cornea verticillata and tortuous retinal blood vessels)Hearing impairmentDyshidrosis (hypohidrosis and hyperhidrosis)Hypersensitivity to heat and coldGastrointestinal disturbances and abdominal painLethargy and tirednessAngiokeratomasOnset of renal and cardiac signs, e.g. microalbuminuria, proteinuria, abnormal heart rate variability

Early adulthood (17–30 years)

Extension of any of the aboveProteinuria and progressive renal failureCardiomyopathyTransient ischaemic attacks, strokesFacial dysmorphism

Later adulthood (age >30 years)

Worsening of any of the aboveHeart disease (e.g. left ventricular hypertrophy, angina, arrhythmia and dyspnoea)Stroke and transient ischaemic attacksOsteopenia and osteoporosis

LSD IN DOGSAbnormality Breeds affected (RARE) Special tests Clinical features

Ceroid lipofuscinosis (Batten disease)

many breeds - Australian cattle dog, border collie, chihuahua, cocker spaniel, dalmatian, wire-haired dachshund, English setter, Tibetan terrier

none, diagnosis confirmed on post-mortem

signs usually start around 1 to 2 years of age, vary between breeds (see resources below), may include diminished eyesight, abnormal behaviour, incoordination, seizures

Fucosidosis English springer spaniel measure enzyme levels

signs by 6 to 12 months, include slow learning, anxiety and behaviour changes, which gradually progress over the next 18 months or so, to severe incoordination, dementia, visual problems

Glucocerebrosidosis (Gaucher's disease) Australian silky terrier measure enzyme

levelssigns by 4 to 8 months, include incoordination, tremors, hyperactivity, stiff gait

Glycogen storage disease type III (Cori's disease) Akita, German shepherd measure enzyme

levels

signs by 6 to 12 weeks, muscle tremors, incoordination, hypoglycemia (low blood sugar), seizures, death by 8 months

GM1 gangliosidosis Portuguese water dog, English springer spaniel

measure enzyme levels

signs by 2 to 4 months, include vision problems, lethargy, difficulty in walking, death occurs by 8 months.

GM2 gangliosidosis (type B - Tay-Sachs disease, type O - Sandhoff's disease)

German short-haired pointer (rare)

measure enzyme levels

signs by 6 to 9 months, include visual problems, abnormal behavior, incoordination, stiff gait

Mucopolysaccharidosis I Plott hound (rare) measure enzyme levels

signs often develop later, in adulthood; this is a connective tissue disorder and signs include musculoskeletal abnormalities, and heart disease due to thickened valves.

Sphingomyelinosis (Niemann-Pick disease) very rare measure enzyme

levelssigns by 2 to 5 months, include incoordination, exaggerated gait, dullness

Lafora diseaseMineature wire haired dachshund, bassett hound and beagle

Jerking movements, ataxia , blindness

CLINICAL SIGNS 0F LSD

• Cerebellar or cerebellovestibular signs such as tremor, ataxia, dysmetria, and nystagmus with progression to paresis and paralysis

• Visual impairment, ataxia, hypermetria, tremors, seizures, and behavioral abnormalities. Differ from other LSD by diffuse forebrain disease and central blindness can be among the earliest clinical signs

• Gangliosidoses in dogs and cats• Niemann-Pick disease types A and C• Globoid cell leukodystrophy• Canine Gaucher disease• Feline -mannosidosis

• Ceroid lipofuscinosis

Lysosomal storage diseases in dogs – MRI STUDY (Hasegawa D, Tamura S, Nakamoto Y, Matsuki N, Takahashi K, et al. (2013) Magnetic Resonance Findings of the Corpus Callosum in Canine and Feline Lysosomal Storage

Diseases. PLoS ONE 8(12)

• The corpus callosum was ‘not visualized’ or ‘partially visualized’ in all cases of the juvenile-onset group, including the Shiba Inu dogs. It was barely recognized on both the transverse and sagittal images (Figures 1C, D), and in the domestic shorthair cats with GM1 gangliosidosis (data not shown) and GM2 gangliosidosis (Figures 1E, F). In the late juvenile-onset group, visualization of the corpus callosum in the toy poodles (Figures 1G, H) and golden retriever (data not shown) with canine GM2 gangliosidoses was inconsistent (‘partially visualized’ or ‘visualized but atrophic’), although the sagittal plane was not obtained from 2 of the 3 cases. In those 2 groups, if the corpus callosum was evaluated as ‘partially visualized’, the visualized portion was significantly atrophic (Figure 1C) and shortened longitudinally (Figure 1H). On the other hand, the corpus callosum of the early adult-onset group, comprised of those with canine neuronal ceroid lipofuscinosis, was recognized clearly and consistently, although it was thinner than those of normal animals (Figures 1I, J).

Gross pathology

• Radial nerve enlargement- fucosidosis in dogs

GM1-gangliosidosis - vertebrae

• lumbar vertebrae from a 3-month-old normal Portuguese water dog (A) and his brother with GM1-gangliosidosis (B). In (B) the vertebrae are shorter and the intervertebral disks are irregular and wide.

Ceroidlipofuscinosis- sheep

• Gross appearance of aMiniature Poodle lung with Niemann-Pick disease, showing diffuse nodular appearance of lung on pleural and cut surface

HISTOPATHOLOGY

SPHINGOLIPIDOSIS

• GM1 gangliosidosis, type I, II and III

• GM2 gangliosidosis (Tay-Sachs type I, II, III and Sandhoff)

• Acid sphingomyelinase deficiency (Niemann-Pick A & B)

• Fabry disease- (alpha-galactosidase A deficiency)

• Farber disease (Ceramidase deficiency)

• Gaucher disease, type I, II and III

• Krabbe disease (galactosylceramidase deficiency)

GM1 GANGLIOSIDOSIS- CEREBRUM

Diffusely, neurons are moderately swollen with finely granular to microvacuolated cytoplasm. Multifocally, glial cells often contain large, discrete, clear vacuoles

GM1 GANGLIOSIDOSIS- CEREBRUM

Rarely within the white matter there are spheroids characterized by swollen, hypereosinophilic axons (arrow) X400

GM1 GANGLIOSIDOSIS- CEREBRUM

Within the white matter there are rare digestions chambers characterized by swollen axon sheaths containing gitter cells and cellular debris (arrow

GM1-gangliosides- Luxol Fast Blue stain

• Paraffin section of the brain of a 7-month-old English Springer Spaniel dog stained with Luxol Fast Blue demonstrating the storage of GM1-gangliosides.

GANGLIOSIDOSIS

High power electronmicrograph of membranous cytoplasmic bodies (open arrow) and zebra bodies (closed arrow) within a brain stem neuronal cell body

GM1 GANGLIOSIDOSIS- CEREBRUM

Intraneuronal concentric lamellar membranous whorls

Globoid cell leukodystrophy (krabb’s disease)

PAS - Mulinucleated macrophages ("globoid cells") and loss of myelinated fibers in a case of Krabbe's leukodystrophy (globoid cells are enlarged vacuolated macrophages typically localized around blood vessels)

Krabbe disease( globoid cell leukodystrophy or galactosylceramide lipidosis)

• Globoid cells with electron microscopical view(*) (enlarged lysosomes packed with twisted tubules)

α-Mannosidosis -cow

Cow cerebrum with a-mannosidosis illustrating• (A) enlarged neurons with vacuolated cytoplasm, hematoxylin and eosin (H&E).• B, Staining with Con A demonstrating the storage of oligosaccharides with terminal a-mannosyl

residues

MANNOSIDOSIS- CALF

neuronal cell bodies with vacuolated cytoplasm (arrows)

CEROID LIPOFUSCINOSIS- SPINAL CORD

• Frozen section of spinal cord of a 11=2-year-old Australian Blue Heeler dog with neuronal ceroid lipofuscinosis in spinal cord stained with Sudan black revealing the storage of ceroid lipofuscin within the neurons.

DIAGNOSIS

• Hematological and routine biochemical assessment is usually unremarkable

• Blood smear- storage vacuoles in leukocytes• Lymph node aspirates or biopsies can be diagnostically

helpful• Radiograph- bone deformities• CSF analyisis- vacuoles in macrophages / lymphocytes (eg.

globoid cell leukodystrophy and fucosidosis)

• Peripheral nerve biopsy- Globoid cell leukodystrophyl, g Niemann-Pick type A,47 -mannosidosis,2 and fucosidosis, fucosidosis

• Muscle biopsy- glycogen storage disease• MRI

Blood smear examination

• Blood smear of a patient with multiple sulfatase deficiency showing a lymphocyte and neutrophil stained with Wright-Giemsa. The cytoplasm of both cells contains metachromatic granules.

• Urine analysis (in MPS)– Fucosidosis - excrete fucoglycoconjugates– Cats with mannosidosis - mannose-rich

oligosaccharides– MPS spot test- staining urine on filter paper

with toluidine blue- give a crude indication of increased concentrations of glycosaminoglycans in urine

Confirming diagnosis- live animal

• Enzyme activity analysis- – homozygotes- 0-5% of normal activity– Heterzygotes- 50% of normal activity

(dried blood sample analysis using fluorescent substrate; Sewell et.al., 2012)

• 2o increase of activities of other enzyme when one is deficient

ANTI-NIEMANN-PICK DISEASE, TYPE C1 (NPC1) (C-TERM) ANTIBODY

• Rabbit antimouse antibody

Flow diagram- LSD diagnosis

Treatment:

• BMT (membrane proteins)

• Enzyme replacement (BBB?)

• Gene therapy

• Substrate reduction- Miglustat (monosaccharide• mimetic-imino sugar)

• Neuronal stem cells (membrane proteins?)

• Chemical chaperone therapy

conclusion

• Lysosomal storage diseases are one of the less identified animal diseases in India mainly due to lack of awareness. As these are mainly a genetic disease, early identification will help in taking proper preventive measures like excluding the related animals from breeding. More study needed to be conducted to evaluate the extend of the disease in the animal population

THANK YOU

• Sheth, Mehul Mistri, Frenny Sheth, Raju Shah, Ashish Bavdekar, Koumudi Godbole, Nidhish Nanavaty, Chaitanya Datar, Mahesh Kamate, Nrupesh Oza, Chitra Ankleshwaria, Sanjeev Mehta, andMarie Jackson (2014), Burden of Lysosomal Storage Disorders in India: Experience of 387 Affected Children from a Single Diagnostic Facility

JIMD Rep. 2014; 12: 51–63.