Muscle – DMD, BMD Flashcards
What is botox?
toxin produced by bacterium Clostridium botulinum
How does botox lead to botulism?
botox inhibits fusion of synaptic vesicles with presynaptic membrane
inhibits ACh release by axon terminals, which leads to botulism
no endplate potential, therefore no muscle contraction – state of relaxation
What does botulism cause?
muscular paralysis
respiratory failure
death
What happens when people get botox?
freeze muscles in face – remove wrinkles and emotional responses
What are Duchenne (DMD) and Becker (BMD) Muscular Dystrophies characterized by?
- weakening of skeletal muscles
- progressive wasting of muscles
- mental retardation
What type of disease is DMD and BMD?
genetic diseases caused by mutations in dystrophin gene located on X chromosome
What is dystrophin protein?
cytoplasmic protein that plays role in linking actin cytoskeleton with basal lamina
How is DMD and BMD inherited?
from carrier mother
- ¼ chance of having son with DMD
- ¼ chance of having carrier daughter
What are symptoms of DMD and BMD?
- mostly affects boys
- first signs appear around ages 3-5, especially people with DMD
- waddling walk
- walking on tiptoes
- curvature of spine
- enlarged calf muscles – pseudohypertrophy
- progression of muscle weakness
- use of wheelchair between age of 8-12 (people with DMD)
- limited fine movements
- development of scoliosis (curvature of spine)
- breathing difficulties
- death occurs before age of 20 from heart failure or pneumonia
Where is the dystrophin gene?
on short arm of X chromosome
What does the dystrophin gene produce?
large dystrophin protein
Where is the actin binding domain of the dystrophin protien?
NH2 terminal
What are the regions of the dystrophin protein?
- 4 hinge regions
- 24 rod-like domains
- cysteine-rich domain
- actin binding domain
What happens if there is mutation in dystrophin gene?
results in stop codon – truncated protein
What types of mutation will result in stop codon in dystrophin gene?
any mutation (exon deletion, base substitution, etc.)
What happens to truncated dystrophin proteins?
don’t live long in the cell because it will be targeted by degradation enzymes
What happens if a mutation in the dystrophin gene does not result in stop codon?
synthesis of a shorter, but still functional, dystrophin protein
What does the dystroglycan-containing complex (dystrophin-associated protein complex) link?
links actin cytoskeleton to basal lamina
How does the dystroglycan-containing complex link actin cytoskeleton to basal lamina?
- dystrophin binds to actin cytoskeleton via its NH2 terminal
- dystrophin binds to 𝛽-dystroglycan via cysteine-rich domain
- 𝛽-dystroglycan binds to 𝛼-dystroglycan (extracellular)
- laminin-2 (component of basal lamina) binds to 𝛼-dystroglycan
When are muscle fibers more prone to injury and tears?
when performing tasks requiring muscle contraction
Describe the link between tears, contraction-induced injuries, and dystrophin.
- dystrophin is structural protein that makes sure there’s a cross-talk link between inside and outside of cell
- in DMD, no dystrophin – severed link between inside and outside of cell
- when muscle fibre twitches and contracts, muscle fibre eventually breaks down and dies
Describe the dystrophin protein and its fate in normal individuals.
functional dystrophin protein
- binds to actin via NH2 terminal
Describe the dystrophin protein and its fate in individuals with DMD.
truncated dystrophin protein
targeted by degradation enzymes
Describe the dystrophin protein and its fate in individuals with BMD.
partially functional dystrophin protein
- can still bind actin cytoskeleton (maintain link between basal lamina and actin cytoskeleton)
What does lack of dystrophin (DMD) result in?
infiltration of connective and adipose tissues
Where are connective tissues?
intercalated between muscle cells and muscle fascicles
What happens as a result of infiltration of connective and adipose tissues (due to DMD)?
clusters of nuclei of macrophages that have invaded tissue (to digest debris of muscle fibres) have degenerated
What is pseudohypertrophy? (ie. of large calf)
from outside, we are led to believe there are more muscle fibres, or they’re much larger
but, actually due to lots of connective tissue and adipose tissue that invades muscles – not ‘real’ hypertrophy
What is a mdx mouse?
mouse that carries spontaneous mutation in exon 23 of dystrophin gene – used as model for DMD
What is kyphosis in mdx mouse?
more prominent curvature of spine
How does kyphosis occur?
- muscles have weakened
- less muscle fibres – degenerate
- cannot maintain normal spinal shape
What are the 3 main therapeutic strategies for DMD?
- cell therapy
- pharmacological therapy
- gene therapy
What does pharmacological therapy due?
aim to reduce inflammation
What is cell therapy?
myoblast or stem cell transplantation into skeletal muscles
Is cell therapy successful?
not very successful in the past
How is cell therapy done?
skeletal muscles are large, therefore transplantation must be done in many sites
What is the downside of cell therapy?
generates inflammation due to transplantation done in many sites
What is gene therapy?
delivery of DNA encoding dystrophin
What is the challenge of delivering DNA encoding dystrophin in gene therapy?
- gene is too long to deliver
- need to find ways to chop gene and express its most significant sequences to end up with some kind of functional protein
What are the 2 methods used in gene therapy?
- exon skipping
- CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)
What does pharmacological therapy rely on?
relies on use of arginine butyrate
What does arginine butyrate do?
inhibits histone acetylases (which modulate rate of gene transcription) → increases gene transcription
What is utrophin?
homolog of dystrophin also expressed in skeletal muscle fibres
Describe pharmacological therapy in mdx mouse. State the results.
treat mdx with arginine butyrate and look at histology of muscle
much less connective tissue in mdx mouse
What is microdystrophin?
possible therapeutic strategy for DMD
In dystrophin-associated protein complex in mdx muscle expressing microdystrophin:
- deleted rod domain from R4 to R23 → left with NT, hinge 1, R1, R2, hinge 3, R24, hinge 4
- when DNA sequence encoding microdystrophin was made, some things were able to be restored
What is the effect of microdystrophin in mdx mouse?
microdystrophin gene delivery using AAVs restores dystrophin expression in mdx mouse
What do adeno-associated virus vectors (AAVs) do?
packages microdystrophin in order for it to reach the appropriate sites
What does microdystrophin delivery do in mdx mouse?
corrects dystrophic pathology in mdx mouse
no full-sized fields of macrophage nuclei
at motor level, improvements were marginal
What occurs during exon skipping?
- skip sequence where stop codon appears
- use antisense oligonucleotides to mask/block DNA sequence where stop codon appears
What are antisense oligonucleotides?
sequences of nucleotides that are somewhere around 20 nucleotides
Exon Skipping
- skip sequence where stop codon appears
- use antisense oligonucleotides to mask/block DNA sequence where stop codon appears
Multi-Exon Skipping Approach
- use multiple antisense oligonucleotides to skip exons
What occurs during multi-exon skipping?
use multiple antisense oligonucleotides to skip exons
What is a limitation of exon skipping?
skipping in critical regions
ie. mutation in exon, which encodes portion of cysteine-rich domain (green)
- without cysteine-rich domain, dystrophin cannot bind to beta dystroglycan
- non-functional protein
What is an out of frame mutation?
mutation that disrupts 3-letter reading pattern, creating words that don’t make sense and leading to unreadable sentence
What is the general idea of exon skipping?
converts unreadable sentence to sentence that makes sense
