Cytoskeletal Diseases 1

Question 1

Components of Cytoskeleton

Answer 1

1) Microtubules (25nm diameter)
2) Actin filaments (7nm diameter)
3) Intermediate filaments (10nm diameter)

Question 2

Actin

Answer 2

Monomer (G actin) polymerises into helical form (F actin) polymer, polar:

• plus end = fast growing and barbed
• minus end = pointed and slow going

3 main types:

• alpha muscle actin
• beta and gamma non-muscle actin

Question 3

Microtubules

Answer 3

Made of apha-beta tubulin dimers

polar:

• Beta tubulin = plus end (fast growing towards membrane)
• alpha tubulin = minus end (slow growing at centrosome)

Question 4

Intermediate filaments

Answer 4

elongated fibrous molecules assemble into filaments with a central rod domain

• non-dynamic
• filament protein varies depending on cell type (epithelial cells = keratin; muscle = desmin)

Tough, durable fibres found in cytoplasm and forming the nuclear lamina around nuclear envelope

Question 5

Tubulin organisation in skeletal muscle

Answer 5

Desmin in the skeletal muscle cells found at Costamers
- link the sarcolemma to the Z-discs along myofibril

Longitudinal transmission of force via crossbridges and titin along fibre

Lateral transmission of force

• Z-discs and costameres in membrane, to extracellular space
• lateral transmission to extracellular matrix reaches neighbouring muscle cells (minimising stress on sarcolemma during contraction/stretch)

Question 6

Costameres

Answer 6

Circumferential elements that physically couple peripheral myofibrils to the sarcolemma in periodic register with the Z-discs

Made of many proteins including Dystrophin

Question 7

Duchenne Muscular Dystrophe (DMD)

Answer 7

X-linked recessive

• affects 1/3500 male newborns
• 30% of cases from spontaneous mutation in dystrophin (DMD) gene

Caused by a lack of dystrophin due to DMD gene disruption:

• deleting a single exon to alter reading frame and code a premature STOP codon
• truncated/non-functional dystrophin produced

Question 8

Symptoms of DMD

Answer 8

Progressive weakness of muscles:

• delayed walking/running with frequent falls
• muscle necrosis, fibrosis
• loss of muscle function

Behavioural issues:

• Neurocognitive disease
• Speech delay

Question 9

Diagnosing DMD

Answer 9

First signs observed:

• gross motor delay, inability to keep up with peers
• muscle weakness
• trouble walking/running/climbing

Lengthy delay between first symptoms and definitive diagnosis (up to 8yrs)

Question 10

Dystrophin

Answer 10

A huge (427kDa) protein

• Actin binding domain
• 24 spectrin-like repeats: rod domain with 4 hinges
• cystein rich domain: binds B-dystroglycan (WW domain)
• C-terminal domain: binding sites for syntrophins & dystrobrevin

Question 11

Dystrophin forming part of costameres

Answer 11

• N-terminal domain binds gamma-actin
• WW domain binds B-dystroglycan (this binds to alpha-dystroglycan on sarcolemma and the extracellular membrane)
• C-terminus binds dystrobrevin and syntrophins
• Activates nNOS which produces Nitric Oxide (NO), promoting vasodilation
• links microtubules (otherwise they get disordered)
• Cysteine-rich domain binds ankyrin to locate sarcolemma
• Sarcoglycans in sarcolemma form strong interactions with Dystroglycan

Question 12

Importance of dystrophin in costameres

Answer 12

Important in linking cytoskeleton to extracellular matrix at costameres
- if lacking, muscle is more easily damaged and leaky

Helps to anchor proteins important in signalling

Question 13

Muscle histology in DMD mutant

Answer 13

• Leaky plasma membrane, elevated Ca2+ levels, muscle necrosis, build-up of connective and fatty tissue, loss of muscle
• variable muscle fibre sizes, central nuclei, fibres engulfed by macrophage (common treatment = anti-inflammatories)
• Z-line damage, spreading sarcomere damage

Question 14

Eccentric Muscle damage to sarcolemma

Answer 14

Damage visualised by Evans Blue Dye (EBD):
- Damage reduces force (maximal tetanic tension, fewer intact muscle fibres)

Damage leads to leakage of muscle contents:

• creatine kinase, myoglobulin, lactate dehydrogenase, even myosin fragments found in blood
• Ca2+ floods into muscle from extracellular space activating proteases to degrade other proteins
• White blood cells enter damaged cells

Question 15

Immune response following eccentric damage

Answer 15

after 4 days:
- full of macrophage & neutrophils engulfing dead tissue

after 10 days:
- muscle cells regenerated, central nuclei (typical of newly generated fibres) and smaller fibre diameter

Question 16

Stimulating muscle’s Satellite cells

Answer 16

Satellite cells = stem cells

Actviated by NO release:

• causes release of Hepatocyte Growth Factor (HGF) which stimulates satellite cells
• also induces release of Insulin Growth Factor 1 (IGF-1)

Question 17

Satellite Cells

Answer 17

Muscle stem cells that repair and grow muscle

• quiescent muscle stem cells lie under a layer of connective tissue that surrounds muscle fibre (basal lamina)
• Upon HGF stimulation, they divide and move within muscle to repair it (undergoes chemotaxis to site of myotrauma)
• they renew their own satellite cell population

Question 18

The plane of satellite cell division drives cell fate

Answer 18

Planar division = stem cells renewing
Asymmetric division = one of daughter cells starts to differentiate (Dystrophin regulates PAR proteins required for asymmetric division)

Balance of self-renewal and repair:

• DMD = symmetric division (lots of self-renewal, no repair)
• Normal = equal symmetric & asymmetric
• Aging = reduced stem cell count, increased lineage commitment

Question 19

Issues with using stem cell therapy for DMD

Answer 19

1) dystrophin = large and therefore difficult to deliver as a transgene
2) Targeting the most abundant tissue in body is complex (lots of different muscle groups)
3) Difficult to identify effective stem cell that targets muscle after systemic delivery (most injected cells die)

Question 20

Becker’s muscular dystrophe

Answer 20

Unlike DMD, mutation isn’t a deletion but instead the dystrophin is truncated/partially functioning

• milder disease than DMD
• 1/20000 males affected
• weakness in 30s, still walking with aid in 60s
• characteristic ‘mini-dystrophin’ with most of central domain missing

Question 21

Treating Becker’s muscular dystrophe

Answer 21

AAV & lentiviruses can be used to deliver transgenes such as mini-dystrophin

AAV (single stranded DNA virus) needs a serotype that targets muscles
- not targeting satellite cells means DNA doesn’t integrate into genome and is diluted out after satellite cell proliferation

Lentivirus (RNA) can target satellite cells persistently integrating into genome
- risk of oncogenesis or replication-competent virus

Question 22

Further treatment of muscular dystrophe

Answer 22

1) Using anti-sense oligonucleotides to induce exon skipping
- skip out mutated part of exon
- problem with delivery

2) CRISPR/Cas9 genome editing
- target specific genomic site and remove mutation
- low efficiency and problematic delivery

3) STOP codon readthrough: Ataluren
- binds premature STOP codon and replaces it with another amino acid

Question 23

Dilated Cardiomyopathy

Answer 23

Due to a lack of dystrophin, causes increased Ca2+ and NO

• triggers protein degradation, fibrosis, necrosis and macrophage activation (leads to ventricular dysfunction)
• increased Ca2+ through leaky PM and from SR causes myopathies with an arythmias

Question 24

Treating dystrophinopathies

Answer 24

1) Presymptomatic treatment
- ACE inhibitors, beta-blockers, mineralocorticoid receptor antagonists

2) Direct effect, symptomatic
- same as #1 plus diuretics and ivabradine
- pacemakers, ICD, heart transplant

3) Indirect effect, symptomatic
- scoliosis therapy
- pain relief, anasthesia management