Lysosomes and

lysosomal

diseases

The lysosome is the terminal

degradative station of multiple

trafficking routes, including

endocytic and scavenging

pathways.

Prof. Dr. Nurten ÖZSOY

Learning goals

• Describe the functions of lysosomes and

how vesicles are targeted to them.

• Understand the biological functions of

lysosomes

• Learn about the lysosomal storage

diseases

• "Lysosome" was the name given because of enzymes'ability to "lyse" the cell

• Initially referred to as "suicide bags" since one of thefunctions of lysosomes is to rupture when the cell dies

• Lysosomes were discovered by the Belgian cytologistChristian de Duve in 1949

• Rupturing releases hydrolytic enzymes that digest allcomponents of the cell, including proteins, DNA, RNA,carbohydrates, lipids and cellulose.

Lysosomes

Nobel-prize

1974

Lysosomes are limited by a single 7–10 nm phospholipid-

bilayer. A unique feature of the lysosomal membrane is its

high carbohydrate content, due to heavily glycosylated

lysosomal membrane proteins. Glycosylations at their

luminal domains form a glycocalyx, which is suggested to

protect the membrane from the lytic enzymes within the

lysosome.

Lysosome Types• Primary

• Newly formed without digestive substrate. Lysosomes are

formed by budding off of the Golgi apparatus, and the

hydrolytic enzymes within them are formed in the

endoplasmic reticulum. The enzymes are tagged with the

molecule mannose-6-phosphate, transported to the Golgi

apparatus in vesicles, and then packaged into the lysosomes.

They can be distinguished from endosomes by the lack of

mannose-6-phosphate receptors (MPRs).

• Can be secreted by exocytosis

• Secondary

• active form with enzyme + substrate

• formed by vesicle fusion event

Functions of lysosomes

Lysosomes function as the digestive system of the cell, serving both to degrade

material taken up from outside the cell and to digest obsolete components of the cell

itself.

1. Release enzymes outside of the cell (exocytosis) which may serve the purpose of

destroying materials around the cell.

2. Break-down 'digestion' of materials from inside the cell (autophagy) i.e. by fusing

with vacuoles from inside the cell.

This could include digesting worn-out organelles so that useful chemicals locked-

up in their structures can be re-used by the cell.

3. Break-down 'digestion' of materials from outside the cell (heterophagy)

i.e. by fusing with vacuoles from outside the cell.

This could include breaking-down material taken-in by phagocytes, which include

many types of white blood cells- also known as leucocytes. Specific mechanisms

of heterophagy can be:

phagocytic - by which cells engulf extracellular debris, bacteria or other

particles - only occurs in certain specialized cells

pinocytic - by which cells engulf extracellular fluid

endocytic - by which cells take-up particles such as molecules that have

become attached to the outer-surface of the cell membrane.

Deposit Degradation and Recycling

Normal Degradation

Missing Enzyme - Accumulation

Autophagy is a process of self-degradation of cellular

components.

-The products of these degradative reactions are made

available to the cell

-It is estimated that 1 to 1.5 percent of the proteins within a

healthy liver cell are degraded via autophagy per hour as part

of a normal process of cellular renovation.

Autophagy is upregulated in response to signals such as:

starvation

growth factor deprivation

ER stress

pathogen infection.

Types of autophagy

• Macro-autophagy

• Micro-autophagy

• Chaperone-mediated autophagy

While each is morphologically distinct, all three culminate in

the delivery of cargo to the lysosome for degradation and

recycling

• During macroautophagy,

sequestration of a small

portion of the cytoplasm,

including soluble materials and

organelles, within a newly

generated double membrane

called the isolation membrane

(or phagophore) results in the

formation of an

autophagosome. The outer

membrane of the

autophagosome fuses with a

lysosome, generating a

structure called an

autolysosome, in which both

the inner membrane of the

autophagosome and the

enclosed contents are

degraded.

• Microautophagy involves the

direct uptake of cargo through

invagination of the lysosomal

membrane.

• Chaperone-mediated autophagy transports

individual unfolded proteins directly across the

lysosomal membrane. Targeted proteins are

translocated across the lysosomal membrane in

a complex with chaperone proteins (such as

Hsc-70) that are recognized by the lysosomal

membrane receptor lysosomal-associated

membrane protein 2A (LAMP-2A), resulting in

their unfolding and degradation.

In the cell there are two major degradation routes: the lysosomal

network and the ubiquitin-proteasome system. While the proteasome is

largely handling degradation of short-lived intracellular proteins,

lysosomes are degrading all kinds of macromolecules of intra- or

extracellular origin.

Lysosome Digestive Enzymes

•Acid hydrolases

• Enzymes are named on basis of substrate

• Protease - digests proteins

• Nuclease - digests nucleotides (DNA)

• Glycosidase - digests carbohydrates (sugars)

• Lipases - digests lipids (fats)

• Phospholipases - digests phospholipids

(membranes)

• Phosphatases - removes a phosphate group

• These enzymes work only at

low pH (highly acidic) levels.

The lysosome maintains this

pH differential by pumping

protons (H+ ions) from the

cytosol across the membrane

via proton pumps and

chloride ion channels.

• However because they can

only work at low pH levels

and the rest of the cell has a

neutral pH level, they can be

neutralized if they

accidentally escape from the

lysosome

•Acid Hydrolases

• Active at acid pH (5)

•Hydrogen ion pump in lysosomal membrane

• drives ions from cytoplasm into

lumenal space

• generates internal acidic environment

The lysosomal limiting membrane has multiple

functions including

• Acidification of the lysosomal matrix

• Sequestration of lysosomal enzymes

• Mediation of fusion events beween lysosomes and

other organelles

• Transport of degradation products to the

cytoplasm.

Lysosome-Associated Membrane Proteins

Lysosomal membrane proteins are usually highly

glycosylated proteins decorating the luminal surface of

lysosomal membranes. LAMP-1, LAMP-2 and Lysosomal

integral membrane protein 2 (LIMP-2) are the most

abundant components of this membrane.

Lysosomal membrane as the interface to regulate communication

between lysosomal lumen and the cytosol

LIMP-2/SCARB2 mediates transport of

the β-glucocerebrosidase to the lysosome

where receptor and ligand dissociate.

The multi-subunit v-type H+ ATPase

mediates transport of protons into the

lysosomal lumen and the chloride

channel ClC-7 drives chloride anion

transport into the lysosome using the

proton gradient.

DIRC2 is also subject to cathepsin-L-mediated proteolysis.

CD63 was shown to be involved in fusion

events with the plasma membrane.

Lysosomal storage diseases

• Undegraded material accumulates within the

lysosomes of affected individuals

• Most of these diseases result from deficiencies

in single lysosomal enzymes.

LSD Sub-Categories

• When a lysosomal enzyme (or another proteinthat directs it) is deficient or malfunctioning,the substrate it targets accumulates, interferingwith normal cellular activity.

Healthy cell vs. LSD cell with accumulated substrate

Morphology of storage compartments commonly observed in

lysosomal storage disorder cells

A: floccular-granular storage. B: lipid whorls. C: zebra bodies. D:

autophagic vacuoles.

For example, in the mucopolysacchridoses, A is typical of the

primary storage vacuoles observed early in the disease course,

whereas other storage compartments appear later in the disease

process due to autophagy (D) and the secondary disruption of lipid

metabolism (B and C).

Glycolipid structure — cerebrosides

• The carbohydrate component is linked by an O-

glycosidic bond to ceramide

• Cerebrosides contain a single sugar (Glu or Gal) or

few sugars; they are abundant in brain and myelin

Glycolipid structure — gangliosides

• Gangliosides are acidic glycosphingolipids

• They contain oligosaccharides with terminal, charged N-

acetyl neuraminic acids (NANA)

o Depending on the number of NANA sugars,

gangliosides are designated M, D, T, Q (e.g., GM).

o 'GM': a single (mono) sialic acid,

o GD, GT and GQ: two, three and four sialic acid residues

in the molecule, respectively

Ganglioside GM2

Ganglioside SulfatideGalactocerebroside

Biochemical and Cellular basis of

lysosomal storage disorders (LSD)

Biochemical and Cellular basis of LSDs

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

1. Reduced catalytic activity

2. Defective or absent activator

3. Misfolding (defective transport of a

lysosomal hydrolase out of the ER

because multienzyme complex cannot

form)

5. Defective glycosylation result in

reduced enzyme catalytic activity

6. Defective M-6-P targetting

7. Defects in other transport steps

8. Defects in membrane transporters

9. Defects in membrane regulators

Biochemical and Cellular basis of lysosomal

storage disorders1. Most mutations result in the delivery of a defective enzyme with a

reduced catalytic activity to lysosomes

2. Another (activator) protein required for optimal hydrolase activity isdefective or absent

3. A mutation that causes misfolding results in defective transport of alysosomal hydrolase out of the endoplasmic reticulum

4. Alternatively, defective transport of a lysosomal hydrolase out of the ERoccurs because a multi-enzyme complex that is required for transportcannot form (Cathepsin A / sialidase / -galactosidase )

5. In the Golgi, defective glycosylation could result in an enzyme withreduced catalytic activity

6. Alternatively, defective glycosylation with mannose-6-phosphate in theGolgi could produce an enzyme that cannot reach lysosomes

7. Defects in other transport steps from the Golgi could also lead to an LSD

8. Defects in integral lysosomal membrane proteins with transporter roles

9. Defects in proteins that are involved in other vital regulatory events oflysosomal function (LAMP2, lysosomal associated membrane protein 2)

LSD: Common phenotypical features and affected organs

Central nervous system: neurodegeneration,

Spleen, liver : hepato and splenomegaly,

Skeleton: Facial dysmorphy,

Peripheral nervous system: peripheral neuropathy

Heart – cardiomyopathy, valve disease

Kidney : renal failure, nephrolithiasis (kidney stones)

Skin : agiokeratomas (vascular lesion characterized by epidermal

hyperplasia (hyperkeratosis)

Eye: cataracts, corneal clouding, cherryred spot, retinal degeneration

Ear: Sensorineural deafness

Bone marrow: anemia

To have an autosomal recessive disorder, you inherit two mutated genes, one from

each parent. These disorders are usually passed on by two carriers. Their health is

rarely affected, but they have one mutated gene (recessive gene) and one normal gene

(dominant gene) for the condition.

With each pregnancy, two carriers have a 25 percent chance of having an unaffected

child with two normal genes (left), a 50 percent chance of having an unaffected child

who is also a carrier (middle), and a 25 percent chance of having an affected child

with two recessive genes (right).

• MPS I (Hurler) (iduronidase deficiency)

• MPS II (Hunter) (iduronate-2-sulfatase deficiency)

• MPS III (San filipo Types A, B, C and D) (heparan-N-sulfatase; N-acetyl-

glucosaminidase; acetyl Co-A glucosamine N-acetyl transferase; N-acetyl-

glucosamine-6-sulfatase deficiency)

• MPS IV (Morquio type A and B) (N-acetyl-galactosamine 6-sulfatase; -

galactosidase deficiency)

• MPS VI (Maroteaux-Lamy) (N-acetyl-galatosamine 4-sulfatase deficiency)

• MPS VII (Sly) (-glucuronidase deficiency)

• MPS IX (hyaluronidase deficiency)

• Multiple sulfatase deficiency

I - Defective metabolism of glycosaminoglycans

"the mucopolysaccharidoses "

Glycosaminoglycans: long unbranched polysaccharides containing a repeating disaccharide unit.

Typesof GAGs...

Due to the missing enzyme-missing degradation of different GAGs-

differnet type of MPS

MPS I (Hurler Syndrome)

A deficiency of L-

iduronidase leads to mental

retardation and structural

changes due to

accumulation of dermatan

sulfate and heparan sulfate

MPS II (Hunter Syndrome)

X-linked disease due to a

deficiency of iduronate sulfatase

MPS III (Sanfilippo Syndrome)

Deficiency in one of four

degradative enzymes leads to

severe mental retardation but

little structural change

MPS IV (Morquio Syndrome)

Deficiency of a galactose-6-

sulfatase or a beta-

galactosidase leads to

accumulation of keratan

sulfate with normal

intelligence but severe

deformity

• Aspartylglucosaminuria

- deficient activity of the N-aspartyl-beta-glucosaminidase

• Fucosidosis, type I and II

- deficiency of the enzyme alpha-L-fucosidase

• Mannosidosis

- deficient activity of the enzyme alpha (beta)-D-mannosidase.

• Sialidosis, type I and II

- deficient activity of the enzyme alpha-N-acetyl neuraminidase

II - Defective degradation of glycan portion of

glycoproteins

III - Defective degradation of glycogen

• Pompe disease- deficiency of acid alpha-glucosidase

JC Pompe discovered the disease in 1932. The affected gene was identified in 1965 by

Henri G. Hers and he characterized the disease as resulting from the buildup of

glycogen in cells. Researchers realized that the deficiency of the enzyme alpha

glucosidase was the cause. Pompe

diseaseBundle of muscle fibers

Normal muscle fiber (cell)

Affected muscle

fiber (cell)

Sceletal

muscle

Lysosomes are compartments inside each

cell where glycogen is broken down

In Pompe disease, the buildup of

glycogen causes the lysosomes to

expand until they take up so much

space that the muscle cell is damaged

Glycogen begins to leak out of the

lysosomes and cause more damage to the

surrounding muscle cells. This leads to

muscle weakness that gets worse over time

Symptoms

• Early onset

• Cardiac and

respiratory

infections/failure

• Other difficulties:

Floppiness, inability

to gain weight

• Death (within 1 year)

• Late onset

• Difficulty breathing

• Niemann-Pick A & B

- acid sphingomyelinase deficiency

• Fabry disease

- deficient activity of lysosomal enzyme α -galactosidase A

• Farber disease

- ceramidase deficiency

• Gaucher disease, type I, II and III

- characterized by the deposition of glucocerebroside in cells of the

macrophage-monocyte system.

• GM1 gangliosidosis, type I, II and III

- deficiency of acid β–galactosidase, accumulation of GM1 ganglioside,

oligosaccharides, and the mucopolysaccharide keratan sulfate

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

- deficiency of hexosaminidase A results in accumulation of GM2 in the brain

• Krabbe disease

- deficiency of a galactosylceramide beta-galactosidase (GALC), which

degrades galactosylceramide, a major component of myelin

• Metachromatic leukodystrophy, type I, II and III

- the accumulation of sulfatides

IV- Defective degradation of sphingolipid components

Some

sphingolipidoses

Disorder Lipid Accumulation Enzyme Deficiency Primary Organ Involvement

Generalized Gangliosidosis Ganglioside GM1 GM1 ganglioside:

b-galactosidase

brain, liver, skeleton

Tay-Sachs Disease Ganglioside GM2 Hexosaminidase A brain

Gaucher Disease Glucocerebroside Glucocerebrosidase brain, liver, spleen

Metachromatic

Leukodystrophy

-Sulfogalactocerebroside Arylsulfatase A brain

Krabbe Disease Galactocerebroside Galactosylceramide

b-galactosidase

brain

Sandoff-Jatzkewitz Disease Globoside and

Ganglioside GM2

b-Hexosaminidase

A and B

brain

Fabry Disease Ceramide trihexoside a-Galactosidase A kidney

Niemann-Pick Disease Sphingomyelin Sphingomyelinase brain, liver, spleen

Farber Disease Ceramide Ceramidase joints, liver, spleen

Fucosidosis Pentahexosylfucoglycolipid a-Fucosidase brain, nerves, skin

Sphingolipidoses

• Synthesis (Normal); Degradation (Defective).

• Substrate accumulates in organs.

• Progressive, early death.

• Phenotypic and genotypic variability.

• Autosomal recessive (mostly).

• Rare, Except in Ashkenazi Jewish.

• Usually only a single sphingolipid accumulates in the involved organs in

each disease

SPHINGOLIPIDOSES

• Diagnosis:

• Measure enzyme activity:

• Cultured fibroblasts or peripheral leukocytes.

• Cultured amniocytes or chorionic villi (prenatal).

• Histologic examination.

• DNA analysis.

• Treatment: e.g. for Gaucher disease:

• Replacement Therapy (e.g. recombinant human enzyme).

• Bone marrow transplantation.

Gaucher disease•Etiology: autosomal recessive inherited disease

•Epidemiology: most common lysosomal lipid storage disease

•Pathophysiology: deficiency of β-glucocerebrosidase → accumulation

of glucocerebroside in the brain, liver, spleen, bone marrow.

•Clinical features

• Vary according to the exact subtype of Gaucher disease

• Type I: non-neuronopathic Gaucher disease

• Type II: acute neuronopathic Gaucher disease

• Type III: chronic neuronopathic Gaucher disease

• Hepatosplenomegaly

• Bone: bone crises, osteoporosis, avascular necrosis of the femur

• Blood abnormalities: anemia, thrombocytopenia

• Pulmonary manifestations

• Growth delays

• Neurodegeneration

•Diagnosis:

• Reduced glucocerebrosidase activity in leukocytes or fibroblasts

• Accumulation of glucocerebrosidase in leukocytes or fibroblasts

• Gaucher cell: Lipid-rich macrophages with an enlarged cytoplasm with

inclusions that resemble crumpled tissue paper on microscopy.

•Treatment: recombinant glucocerebrosidase

Gaucher

Disease

Type 1

(Non-

Neuronopathic)

Enlarged

liver

Huge spleen

Easy bruising

due to low

blood platelets

Anemia

Bone damage

Krabbe disease•Etiology: autosomal recessive inherited disease

•Pathophysiology: lack of or reduced activity of

the lysosomal enzyme galactocerebrosidase (GALC) → accumulation of

psychosine, galactocerebroside (toxic myelin degradation products) +

formation of globoid cells → demyelination + destruction

of oligodendrocytes up to decerebration

•Clinical features

• Major mental and motor deficits

• Developmental delay

• Peripheral neuropathy

• Limb stiffness

• Opisthotonic posture - causes the back to become extremely

rigid and curved or arched. The heels and head usually bend or

flex backward as far as possible in response.• Loss of vision (atrophy of the optical nerve)

• Diagnosis

• Globoid cell: A large multinucleated cell of mesodermal origin that is

found clustered in the brain tissue

•Treatment

• Before symptom onset, stem cell transplantation may improve outcome.

• Only supportive treatment is possible after symptom onset.

Tay-Sachs disease•Etiology: autosomal recessive inherited disease

•Epidemiology: more common in the Ashkenazi Jewish population

•Pathophysiology: hexosaminidase A deficiency → intracellular

accumulation of GM2 ganglioside → progressive neurodegeneration

•Clinical features

• Developmental delay

• Rapid reduction of physical and mental ability beginning at 6

months of age; patients typically die around the age of 4

• “Cherry-red” spot on macula

• Hypotonia

• Seizures

• Hearing impairment

• Note: no hepatomegaly (compared to Niemann-Pick disease)

•Diagnostics: lysosomes with “onion skin” appearance

•Treatment: supportive

Fabry disease•Etiology: x-linked recessive inherited disease

•Epidemiology

• Typical onset is during childhood but may also appear in 60–80-year-old adults

• Mainly affects boys

•Pathophysiology: α-galactosidase A deficiency → accumulation of ceramidetrihexoside in

the endothelium of vessels, in the epithelium of many organs, and in smooth muscle cells →

disorder affecting many organ systems

•Clinical features

• Early symptoms

• Periodically occurring dysesthesia (painful, itchy sensation) in the hands and

feet, which presents as burning pain (Fabry crises)

• Anhidrosis or hypohidrosis (condition in which you aren't able to sweat

normally.

• Nonspecific gastrointestinal disturbances

• Angiokeratomas

• Corneal clouding

• Cataract

• Late symptoms

• Cardiomyopathy

• Cerebrovascular lesions

• Fabry nephropathy

•Treatment: enzyme replacement therapy with α-galactosidase A

Metachromatic leukodystrophy•Etiology: autosomal recessive inherited disease

•Pathophysiology: arylsulfatase A deficiency → cerebroside sulfate

accumulation in neural and non-neural tissue → central and peripheral

nervous system demyelination

•Clinical features:

• Infantile onset

• Motor regression and developmental delay

• Impaired memory

• Ataxia

• Hypotonia and hyporeflexia

• Optic nerve atrophy → loss of vision

• Flaccid paralysis (reduced muscle tone) followed by spastic

paralysis

• Childhood and juvenile onset

• Ataxia

• Dementia

• Peripheral neuropathy

•Treatment: supportive

Niemann-Pick disease

•Etiology: autosomal recessive inherited disease

•Epidemiology: more common in the Ashkenazi Jewish population

•Pathophysiology

• deficiency of sphingomyelinase → accumulation

of sphingomyelin

•Clinical features

• Progressive neurodegeneration

• “Cherry-red” spot in the macula

• Hepatosplenomegaly

•Diagnostics

• Light microscopy: lipid-laden macrophages (foam cells) in

the bone marrow, spleen, and liver

• Electron microscopy: Zebra bodies, which are abnormal

configuration of myelinoid membranes into parallel palisading

lamellae in the lysosomal cytoplasm.

•Treatment: mainly conservative

• lipid-laden macrophages (foam cells) in the bone marrow, spleen,

and liver

Juvenile

Infantile

Chronic Adult Adult

GM1Gangliosidosis

Phenotypes

Hypotonia

Hypotonia, commonly known as floppy baby syndrome, is a state of

low muscle tone often involving reduced muscle strength.

Coarse Facial Features

Cytoplasmic Vacuolation

Vacuolated

lymphocytes

Lymphocytes with multiple medium-sized vacuoles (top left).

Skeletal Survey – dysostosis

multiplex

Thickened calvarium; enlarged sella

A dysostosis is a disorder of the development of bone, in particular affecting ossification.

V - Defective degradation of polypeptides

• Pycnodysostosis

- mutation in the gene that codes the enzyme cathepsin K

VI - Defective degradation or transport of

cholesterol, cholesterol esters, or other complex

lipids

• Neuronal ceroid lipofuscinosis, type I, II, III and IV

(Batten disease)

VII - Multiple deficiencies of lysosomal enzymes

• Galactosialidosis

• Mucolipidosis, type II and III

VIII - Transport and trafficking defects

• Cystinosis

- Defective transport of the amino acid cystine out of lysosomes

• Danon disease

- LAMP-2 deficiency in humans

- fatal cardiomyopathy and myopathy

• Mucolipidosis type II - At least 8 hydrolytic enzymes are exported out of the

cell, instead of going to the lysosomes. Lack of hydrolytic enzymes in them

results in cells acumulating inclusion bodies, i.e., becoming I-cells (I for

inclusin). An I-cell is, thus, defective because it exports lysosomal enzymes.

This LSD is inherited as an autosomal recessive trait.

• Niemann-Pick type C

- a defect in cholesterol and glycolipid transport mechanisms.

• Infantile sialic acid storage disease

• Salla disease (sialic acid accumulation disorder)

I-cell disease is a rare lysosomal storage disease in which the acid

hydrolases normally found in lysosomes are absent, resulting in an

accumulation of substrates normally degraded by these enzymes.

[Note: I-cell disease is so-named because of the large inclusion

bodies seen in cells of patients with this disease]. In addition, high

amounts of lysosomal enzymes are found in the patient’s plasma and

urine, indicating that the targeting process to lysosomes (rather than

the synthetic pathway of these enzymes) is deficient. Individuals

with I-cell disease are lacking the phosphotransferase needed to

phosphorylate the mannose residues of potential lysosomal

enzymes, causing the enzymes to be secreted (by default), rather

than being targeted to lysosomal vesicles. I-cell disease is

characterized by skeletal abnormalities, restricted joint movement,

coarse (dysmorphic) facial features, and severe psychomotor

impairment. Currently, there is no cure, and death from

cardiopulmonary complications usually occurs by age 10 years.

CURRENT AND FUTURE STRATEGIES FOR THE

TREATMENT OF METABOLIC STORAGE DISORDERS

1. BONE MARROW TRANSPLANTATION

2. ENZYME REPLACEMENT THERAPY

3. SUBSTRATE REDUCTION THERAPY

4. MOLECULAR CHAPERONE THERAPY

5. GENE THERAPY ?

How common are lysosomal diseases?

a. 1 in 5000 live births

b. 1 in 50,000 live births

c. 1 in 100,000 live births

Answer: a.

With what frequency are children born world-wide

with a lysosomal disease?

a. Every 30 minutes

b. Every 2 hours

c. Once a day

Answer: a.