Motor Neuron Disease/Amyotrophic Lateral Sclerosis (ALS) PART II

Question 1

Briefly summarise the common molecular pathway hypothesised for ALS

Answer 1

Nuclear proteins (TDP-43 + FUS) that mislocalise to cytolsol -(TARDBP/FUS/ageing)-> TDP-43 (+ve), FUS (+ve), ubiquitinated protein aggregates -> neurodegeneration

Question 2

Genetic relationship between ALS and fronto-temporal dementia (FTD)?

Answer 2

[mutation]

Expanded repeats in intron 1 of C9ORF72

[chromosome 9 locus]

NB: non-coding repeat expansions are present for myotonic dystropies, Fragile X, spino-cerebellar ataxias

Question 3

The most prevalent mutation in FALS?

Answer 3

C9ORF72 mutation

Question 4

Outcomes of C9ORF72 mutation?

Answer 4

[unknown function]- likely member of DENN proteins involved in membrane fusion

• reduced mRNA levels
• RNA foci accumulate in ALS
• RAN peptides: bi-directional transcrip/translat of expanded repeat sequences

Question 5

C9ORF72-specific pathology?

Answer 5

p62 +ve cytoplasmic and nuclear inclusions in hippocampus and cerebellum that are TDP-43 positive

Question 6

Putative mechanism of C9ORF72 pathogenesis?

Answer 6

Bi-directional transcription of expanded repeat containing sequences followed by translation of repeat associated non-AUG initiated (RAN) tranlation of aggregate prone dipeptide repeat peptides (DPRs)

NB: Poly GA is the most aggregatable, interfering w/ proteostasis

Question 7

Relationship between distribution of dipeptide repeat peptides (DPR) and neurodegeneration

Answer 7

Degeneration and loss of anterior horn cells occurs in the absence of DPR

[5 DPR species seen in unaffected regions: granule cells of hippocampus and cerebellum]

Question 8

Compare clinical characteristics of C9orf72 FALS cases with other FALS cases?

Answer 8

For C9prf72 FALS:

• slightly lower age of onset
• higher co-morbidity w/ FTD
• slightly reduced survival

Question 9

FALS genes: overall mechanisms?

Answer 9

Protein-quality control (waste disposal)

1. Unfolded protein response: increases levels of protein chaperones to facilitate folding
2. Protein degradation via the proteasome and autophagy (via lysosome aggrephagy)

Question 10

Relevance of VAPB and ALS?

Answer 10

VAPB is localised in motor neurons and is decreased in SALS spinal cord

VAPB mutation -> ER fragmentation -> ubiquitinated protein aggregates -> apoptosis

Question 11

Relevance of P4HB and ALS?

Answer 11

P4HB is an ER foldase, induced in ER stress and SALS]- it is a disease modifier/risk factor

Disease will vary between ppl with same mutation: Modifies disease onset/progression/penetrance

Question 12

Function of ER-associated protein degradation (ERAD) proteins?

Answer 12

ER protein export to the proteasome

Question 13

Superoxide dismutase 1 (SOD1): association w/ ALS and mechanism?

Answer 13

Associated with ALS 1

Binds to Derlin 1

Question 14

VCP association w/ ALS and mechanism?

Answer 14

Associated w/ ALS14 and IBMPFD (inclusion body myopathy, Paget’s disease, FTD)

Involved in ER protein export to proteasome

Question 15

Ubiquilin 2 association w/ ALS and mechanism?

Answer 15

Associated w X-linked FALS and SALS

Binds to poly-ubi chains and components of proteasome

Question 16

Define autophagy

Answer 16

Aggrephagy of misfolded or aggregated proteins. It’s activated by the failure of proteasomal degradation and molecular chaperones to resolve aggregate buildup

Question 17

Role of P62/SQSTM1?

Answer 17

Allelic disorder

is Pagets disease of bone

Question 18

Role of OPTN

Answer 18

ALS12 slow
progressing AR/AD
Allelic disorder is primary open
angle glaucoma.

Question 19

What did whole exome sequencing in ALS yield?

Answer 19

ID’d a novel ALS gene: TANK-binding Kinase 1 (TBK1)

Question 20

Function of TANK-binding kinase 1 (TBK-1)?

Answer 20

Phosphorylates OPTN (optineurin) and P62 and proteins involved in innate immunity

Enhances OPTN binding to autophagosome protein LC3 -> autophagic turnover of infectious bacteria (coated w/ ubiquitylated proteins)

Question 21

Implications from the discovery of TBK-1?

Answer 21

Strengthens importance of autophagy in ALS pathogenesis

Question 22

Riluzole MOA?

Answer 22

Anti-epileptic

Targets VDSC’s and reduces glutamate release

Question 23

What triggers ALS that is specific for motor neurons?

Answer 23

Constant excitatory activity (NMDArs) activated by glutamate and D-serine

D-serine is sig. elevated in SALS

Question 24

Function of D-amino acid oxidase (DAO)?

Answer 24

High levels of DAO required to metabolise and regulate D-serine levels

Question 25

What is the pathological DAO mutation and what condition does it confer?

Answer 25

R199W-DAO

Involved in FALS

Question 26

Effect of R199W-DAO?

Answer 26

• increased ubiquitinated protein aggregates
• autophagy (increased autophagosomes and LC3-II in motor neuron cell line)
• significant loss of spinal cord motor neurons (in trans-mice expressing R199WDAO)

Question 27

Overview of clinically effective ALS drugs?

Answer 27

• Riluzole
• Edaravone]- free radical scavenger used in stroke

[drugs tested aren’t clinically effective in large trials…except riluzole]

Question 28

Edaravone: Pros, Cons and its future?

Answer 28

PROS

• initial study-hint of effect
• second smaller study- normal resp function after 2 years from diagnosis
• treated pts declined 5.1 compared to 7.5 control

CONS

• prev studies show normal decline of 5.6. so 5.1 ain’t that great
• no effect on severely affected cases

FUTURE: longer studies (over 2 yrs) and test effect on survival

Question 29

ALS treatment, when the causal gene is known?

Answer 29

• targeted gene delivery/deletion using direct delivery of antisense oligonucleotides (ASOs) by adeno-associated viral (AAV) vectors to neutralise mutation]- SOD1 mutation had intrathecal injection w/ adenovirus gene delivery
• CRISPR-Cas9 technology to target mutant genes and re-introduce wild-type copies

Question 30

Potential future treatment targets (where the affected pathways are known)?

Answer 30

•Motor neuron susceptibility and trigger factors?
•Prevention of nuclear RNA foci (C9orf72 specific)?
•Prevention of cytosolic inclusion formation?
•Up-regulate UPR, UPS, autophagy?
•Upregulate molecular chaperones (HSPs)?
•Reduce D-serine levels?
•Designer Drug treatment: 15,000 patients and
7,500 controls Project MinE (US) to develop basis
for treatment from Whole Genome Sequencing,
metabolomics and proteomic analysis.