Charcot Marie Tooth (CMT) disease is one of the most commonly inherited neurodeg
ID: 210451 • Letter: C
Question
Charcot Marie Tooth (CMT) disease is one of the most commonly inherited neurodegenerative diseases. CMT is characterized by axon degeneration, which in turn leads to the loss of motor and sensory nerve functions.
A recent breakthrough has been the discovery that some mutations in dynein can lead to the development of CMT.
You are studying a sample of neuronal tissue from a young patient with CMT. One side of this patient’s family has suffered from CMT for many generations.
Genetic testing in this patient’s family has identified a mutation in dynein that is not expected to affect the ATPase enzymatic activity.
You prepare a cellular extract (CE1) from the patient’s neuronal tissue using a gentle method that preserves protein-protein interactions occurring in the cell.
As a control, you prepare a cellular extract (CE2) from tissue donated by a healthy volunteer using the same, gentle method.
Next, you fractionate CE1 and CE2 using differential centrifugation to separate microtubule-associated proteins from soluble and membrane-bound proteins. The resulting microtubule fraction will ONLY contain proteins that associate with microtubules.
You analyze various fractions from these extracts and the centrifugation experiments using gel electrophoresis and Western blotting with several different antibodies specific for tubulin, dynein, dynactin and Rab6. The results are shown below.
IMPORTANT: As you look at the gels, kind reminder that the samples were prepared in such a way that protein-protein interactions were preserved.
Refer specifically to the presence or absence of bands in the different lanes of each Western-blot shown on the previous page to support your answers to the following questions.
(A) Does the mutant dynein from the patient bind to microtubules? CIRCLE your answer and briefly explain your reasoning. (2 marks)
YES NO
How do you know? For full marks, refer specifically to the lane(s) in the gel(s) shown above. Please limit your answer to the lines below.
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(B) Does the mutant dynein from the patient bind to dynactin? CIRCLE your answer and briefly explain your reasoning. (2 marks)
YES NO
How do you know? For full marks, refer specifically to the lane(s) in the gel(s) shown above. Please limit your answer to the lines below.
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(C) Does the mutant dynein from the patient bind to Rab6 (or vesicles containing Rab6)? CIRCLE your answer and briefly explain your reasoning. (2 marks)
YES NO
How do you know? For full marks, refer specifically to the lane(s) in the gel(s) shown above. Please limit your answer to the lines below.
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Question 5 (3 marks): Specifically refer to your answers to 4(A)-(C) to support a reasonable hypothesis explaining why the mutant form of dynein in the patient’s family leads to neurodegeneration and the development of CMT.
Hint: Many neurodegenerative diseases, including CMT, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS or “Lou Gehrig’s Disease,” as highlighted by the recent “ice bucket challenge”), have been linked to specific defects in autophagy, the process whereby damaged organelles and cellular waste components accumulate in autophagosome vesicles that are then transported to the lysosome in the cell body for degradation and recycling. How could defects in dynein function impair autophagy and in turn lead to the degeneration of axons? Please limit your answer to the lines below.
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WB-A WB-B WB-C WB-D 1: CE2 (normal) 2: microtubule fraction from CE2 (normal) 3: CE1 (patient) 4: microtubule fraction from CE1 (patient) Anti-Tubulin Anti-Dynein Anti-Dynactin Anti-Rabe6Explanation / Answer
A) The answer is YES
Explanation: The microtubule fraction from patient sample (CE1) in lane 4 of WB-B shows the dyenin band similar to that of normal sample (CE2) in lane 2. This suggest that isolated microtubule fractions from CE1 has dyenin protein indicating interaction between microtubule and dyenin.
B) The answer is No
Explanation: The microtubule fraction from patient sample (CE1) in lane 4 of WB-C doesnot have dynactin band, whereas normal sample (CE2) in lane 2 of WB-C has dynactin band. This suggest that isolated microtubule fractions from CE1 has no dynactin protein indicating loss of interaction between dyenin and dynactin (in WB-B, we can see interaction between dyenin and microtubule, but in WB-C, dynactin is not present in microtubule fractions indicating dynactin is not bound to dyenin in patient samples).
C) The answer is No
Explanation: The microtubule fraction from patient sample (CE1) in lane 4 of WB-D doesnot have Rab6 band, whereas normal sample (CE2) in lane 2 of WB-D has dynactin band. This suggest that isolated microtubule fractions from CE1 has no rab-6 protein indicating loss of interaction between dyenin and rab6 (in WB-B, we can see interaction between dyenin and microtubule, but in WB-D, rab6 is not present in microtubule fractions indicating rab6 is not bound to dyenin in patient samples).
Question-5
From the above answer we observed that in CMT pateints there is a loss of interaction between dyenin to dynactin and rab6 proteins.
The autophagosome vesicles are transported in to the lysosome by dyenin complex attached to dynactin and rab6 (rab6 is associated with autophagosome vesicles) by moving along the microtubules in the cell. In patients the loss of interaction between dyenin and rab6 leads to impaired transport of vesicles to lysosome. These vesicles get accumulated in cells. These events lead to activation of apoptotic pathways which lead to death of neurons in CMT disease