Cellular basis of Therapeutics

Question 1

What are glycolipid storage diseases​?

Answer 1

Glycolipid storage diseases​ are caused mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes.

In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and disease.

Question 2

What are the symptoms glycolipid storage diseases​?

Answer 2

• Delay in intellectual and physical development.
• Seizures.
• Facial and other bone deformities.
• Joint stiffness and pain.
• Difficulty breathing.
• Problems with vision and hearing.
• Anemia, nosebleeds, and easy bleeding or bruising.
• Swollen abdomen due to enlarged spleen or liver.

Question 3

What are glycolipids?

Answer 3

Glycolipids are lipids with a carbohydrate attached by a glycosidic bond. Their role is to maintain the stability of the cell membrane and to facilitate cellular recognition, which is crucial to the immune response and in the connections that allow cells to connect to one another to form tissues.

Question 4

What is glucosylceramide synthase (GCS)?

Answer 4

Glucosylceramide synthase (GCS) is an enzyme inherent to ceramide metabolism. The enzyme catalyzes the transfer of glucose to ceramide, the first committed step in glycolipid biosynthesis.

Question 5

Where are glycolipids synthesized?

Answer 5

Glycolipids are synthesized in the endoplasmic reticulum and golgi-apparatus where the majority are transported to membranes to maintain the bilayer.

Question 6

What are glycosphingolipids (GSLs)?

Answer 6

Glycosphingolipids (GSLs), a subclass of glycolipids found in the cell membranes of organisms from bacteria to humans, are the major glycolipids of animals.

GSLs are internalised, recycled or degraded to sphingosine​s.

Lysosomes have low pH and are the centres for degradation and recycling​.

Question 7

What are lysosomal storage diseases (LSDs)?

Answer 7

Lysosomal storage diseases (LSDs) are inborn errors of metabolism characterized by the accumulation of substrates in excess in various organs’ cells due to the defective functioning of lysosomes. They cause dysfunction of those organs where they accumulate and contribute to great morbidity and mortality.

Question 8

What do lysosomal hydolases​ have?

Answer 8

They have a ‘signal patch’​ which identifies them as​ lysosomal enzymes and ​M6P is attached to them in the Golgi.

Identified first by looking at I-cell patients.

Even though the hydrolases accumulated in the cell medium they were unable to correct other lysosomal storage diseases.​

Question 9

What is the role of Mannose-6-phosphate (M6P)?

Answer 9

The M6P is attached to the hydrolase so it binds to the M6P receptor in the Golgi.

The M6P Receptor complex is then targeted to the lysosome.​

Once the hydrolase is delivered the empty receptor recycles to the Golgi to pick up more hydrolases for transport to the lysosome.

Some M6P receptors also traffic from the cell surface and some M6P containing hydrolases escape due to inefficiencies in the sorting process.​

Question 10

Explain the biochemical and cellular basis of glycolipid storage disease​:

Answer 10

Most mutations result in the delivery of a defective enzyme with a reduced catalytic activity to lysosomes​.

​Another (activator) protein required for optimal hydrolase activity is defective or absent.​

​A mutation that causes misfolding results in defective transport of a lysosomal hydrolase out of the endoplasmic reticulum. ​

Alternatively, defective transport of a lysosomal hydrolase out of the ER occurs because a multi-enzyme complex that is required for transport cannot form (Cathepsin A / sialidase / β-Galactosidase).

In the Golgi, defective glycosylation could result in an enzyme with reduced catalytic activity​.

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

Defects in other transport steps from the Golgi could also lead to an Glycolpid Storage Disease. ​

​Defects in integral lysosomal membrane proteins with transporter roles eg Niemann-Pick C​.

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

Question 11

How is the severity of the phenotype related to the residual enzyme activity?

Answer 11

The severity of the phenotype is closely related to the residual enzyme activity.​

There is a ‘critical threshold’ of enzyme activity. ​

Above this level, enzyme activity can deal with substrate influx. ​

Below this, it cannot and there is accumulation of substrate. ​

It has been demonstrated that small changes in residual enzyme activity can have a profound effect on rate of accumulation of substrate. ​

In general, the lower the residual activity, the earlier the age at onset and the more severe the disease, although there is considerable overlap, for example, in Gaucher disease. ​

Therefore in diseases for which enzyme-based therapies are available, residual enzyme activity is of critical importance in determining response to treatment. ​

The higher the residual activity, the more satisfactory the response. ​

Question 12

What are the roles of the activator proteins?

Answer 12

Two genes are known to encode saposins:

One encodes the GM2 activator protein; its defective function results in the AB variant of GM2 gangliosidosis. ​

The second gene encodes prosaposin which is processed to four homologous saposins (A, B, C and D). ​

Deficiency of a saposin results in a clinical phenotype that may resemble a lysosomal storage disease. ​

Mutations in the coding region of Sap B cause a variant form of metachromatic leukodystrophy with sulphatide storage. ​

Sap C deficiency causes a variant form of Gaucher disease with glucosylceramide storage. ​

Deficiency of prosaposin results in a combined saposin deficiency with a very severe phenotype, as might be expected. ​

However, saposin deficiency often results in features of more than one disorder because each saposin activates more than one enzyme.​

Question 13

Give examples of primary GSL storage diseases:

Answer 13

• Gaucher (types 1,2 &3)​
• Fabry
• Tay-Sachs​
• Sandhoff ​
• GM1 gangliosidosis​

Question 14

Give examples of lysosomal diseases with secondary storage of GSLs in neurones:

Answer 14

• Niemann-Pick A ​
• Niemann-Pick C​
• MPSI, III A, III B, VI, VII​
• alpha-mannosidosis​

Question 15

What is Niemann-Pick C disease?

Answer 15

• Niemann-Pick type C is a rare inherited disease.
• Neurodegeneration and death due to a defective late endosomal protein, NPC1.​
• Defective NPC1 leads to intracellular storage of glycolipids and cholesterol. ​
• The genetic mutations of this type cause cholesterol and other fats to accumulate in the liver, spleen or lungs.
• The brain is eventually affected too.

Question 16

What are the three broad clinical phenotypes of Gaucher disease?

Answer 16

There are three broad clinical phenotypes:

• Type I Gaucher disease has no neurological involvement (non-neuronopathic).
• Types II and III have neurological involvement (neuronopathic).
• Patients with the neuronopathic forms of Gaucher disease have increased levels of the substrate glucosylceramide (GCS) in the brain.​

Question 17

What two sources is glucosylceramide derived from?

Answer 17

• Inside the CNS it is derived predominantly from gangliosides.
• Elsewhere it is derived predominantly from the breakdown of effete blood cells.

In type I patients, the degradation of blood cell derived glucosylceramide is blocked, but there is sufficient enzyme activity in the CNS to break down ganglioside-derived glucosylceramide, thus preventing its accumulation in the brain. ​

In types II and III, there is less residual enzyme activity, insufficient to degrade even ganglioside-derived GCS in the CNS.​

Question 18

How does accumulation of substrate result in disease? ​

Answer 18

Enlargement of the affected cell, resulting in enlargement of the respective organ. ​

Secondary biochemical and structural events have been reported which appear to be triggered by the primary storage event.

Question 19

What are the two types of morphological changes in neurons?

Answer 19

Meganeurites:

• Meganeurites are enlargements of the axon hillock and are of two types, ‘spiny’ and ‘non-spiny’ or smooth, depending upon their appearance ​
• The spiny appearance is conferred by the presence of new dendritic membrane. ​
• It is always associated with accumulation of ganglioside, predominantly GM2, irrespective of the primary disorder.​

Axonal spheroids:​

• Axonal spheroids are focal axonal enlargements and are usually seen in disorders in which ganglioside accumulates, either primarily or secondarily. ​
• The relationship is not that close, however, and spheroids do not contain storage bodies. ​
• In addition, the morphology of meganeurites is disease specific; that of spheroids is not. ​
• These morphological changes are reminiscent of that seen with inhibitors of axonal transport.​

Question 20

When does macrophage activation and/or cytokine release happen?

Answer 20

• Macrophage activation following storage is seen in many LSD. ​
• Raised concentrations of cytokines or chemokines have been found in patients with Gaucher disease.
• Macrophage activation has also been reported in the brain in animal models of some storage disorders and appears to be a major cause of neuronal death.​
• Macrophage activation may also affect the BBB.

Question 21

What are the functions of glycolipids in normal cells?

Answer 21

• Maintain stability of the membrane and to facilitate cell–cell interactions.
• Glycolipids can also act as receptors for viruses and other pathogens to enter cells.
• Cholesterol and glycolipids form lipid rafts which are lipid/protein platforms inside the cell.​

Question 22

How does cholesterol accumulate with glycolipids?

Answer 22

• Cholesterol can form H bonds withglycolipids.
• Depletion of cholesterol from lipid-storage disease cells relieves lysosomal-storage.
• Excessive cholesterol induces lysosomal-storage.

Question 23

What are the functions of glycolipid rafts?

Answer 23

• These are tiny platforms in the plane of the bilayer that form signalling and transport complexes.
• Involved in intracellular transport through the endocytic pathway and the Golgi.
• Compartmentalize in intracellular-signalling events.
• Glycolipid probes can be used to visualise rafts in live cells.
• Glycolipid storage diseases differ in the degree to which they alter lipid rafts in fibroblast lysosomes.
• Glycolipids can enter cells via specialised form of lipid raft endocytosis (caveoli).

Question 24

What is the retrograde pathway to the Golgi the glycolipid rafts follow?

Answer 24

Glycolipid rafts accumulate in the lysosome in storage diseases.

Question 25

How can altered trafficking be due to abnormal raft accumulation in GSL-storage diseases?

Answer 25

• Accumulating GSL in the lysosomes.​
• Accumulating cholesterol in the lysosomes.​
• Raft associated proteins may end-up in the lysosome rather than at the cell surface and be degraded.​
• This may impair intracellular trafficking leading to pathology.​

Question 26

What is a SNARE?

Answer 26

• Lipid raft proteins in vesicle transport.
• In order transport to happen the vesicle derived from an endosome/lysosome has a specific SNARE that must find the correct target SNARE on the surface of the Golgi. Each SNARE is like a ‘fishing rod’​
• SNAREs mediate specificity in the transport process by ensuring that only correctly targeted vesicles fuse is targetted to the presynaptic membrane.
• In order to fuse membranes must be ~1 nm apart.
• Long tails of SNAREs wrap around each other releasing energy which is used to expel water molecules.​
• Although fusion can occur slowly in vitro, in cells other proteins are recruited via lipid rafts to the fusion complex to speed it up.​
• After fusion the lysosome-SNARE has to separate from the Golgi-SNARE in order to recycle.
• A protein called NSF is an ATPase that separates the intertwined strands of the SNAREs so that the t-SNARE can be recycled after the fusion has taken place.

Question 27

What effect does cholesterol have on SNAREs?

Answer 27

• Cholesterol enriched regions accumulate in the lysosomal membranes of storage disease sequestering SNARES in lipid rafts.
• This locks SNAREs in assembled complexes and reduces their availability to interact with clathrin coats which is a process needed for SNARE re-distribution between membrans for future rounds of fusion.
• The net effect is a decrease in the rate of new fusion events.
• Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.