FTD and ALS

Question 1

what is ALS (name and five points)

Answer 1

• Loss of motor neurons in the CNS
• Progressive weakness and paralysis
• Death from respiratory failure
• 2-6 years from onset to death
• Approx. 15% can develop FTD.

Question 2

What is FTD

Answer 2

• Pathological heterogenous group of neurological dsiorders that are characterised by:
  o Neurodegeneration of frontal and/or temporal lobes
  o Personality and/or behavioural change
  o Language dysfunction
  o Young onset <65 years
  o Up to 15% can develop ALS.

Question 3

Mutations involved in FTD-ALS

Answer 3

most common mutation is C9orf72, but there are others incl. TARDBP and UBQLN2.

Question 4

SOD1 mutations in ALS

Answer 4

Superoxide dismutase 1
- antioxidant enzyme involved in protecting cells against ROS toxicity.
- toxic gain of function mutation
- targetd with ASOs

Question 5

GRN and FTD

Answer 5

Progranulin loss of function toxicity in FTD.
- targeted with AAV

Question 6

Ataxin-2

Answer 6

important regulator of TDP43
- can be targeted with ASO or AAV

Question 7

AAV as an in vivo gene therapy

Answer 7

can be internalised by cells but usually do not integrate into the host genome. BUT have a packaging limit of 4.7kB.

Question 8

main common pathology of FTD and ALS

Answer 8

TDP-43 - mislocalisatiion (hyperphosphorylated)
approx 50% of FTD
approx 98% of ALS

Question 9

TDP-43 gain of function toxicity

Answer 9

o Nuclear depletion and cytoplasmic deposition due to cleavage, hyperphosphorylation and ubiquitination of TDP-43
o TDP-43 burden associated with disease progression in ALS
o Insoluble! Non-functional

Question 10

TDP-43 loss of function toxicity effects (4)

Answer 10

 mRNA levels
 mRNA splicing
 mRNA transport
 stress granules (RNA-protein granules that form during cellular stress to prevent translation, thus preserving energy)

Question 11

indirect modulation of TDP-43

Answer 11

• using ataxin-2

which is a poly1-containing RNA binding protein.
* Involved in mRNA translation and SG assembly.
* Intermediate length polyQ expansions are a risk factor for ALS.
* Ataxin-2 is a modifier of both TDP-43 and FUS pathology in ALS.

Question 12

C9orf72 mutation

Answer 12

an intronic GGGGCC repeat expansion

Question 12

4 ways to target Atxn2

Answer 12

1. ASO (Success)
2. CRISPR-Cas13 ribonclueases - targeting RNA, no PAM site requirement
3. CRISPR-Cas13(rfx)
4. AAV9 delivery of Ataxin-2 miRNA

Question 12

C9orf72 haploinsufficiency

Answer 12

• Doesn’t cause phenotype or pathology, BUT does contribute to the disease
• Example: preclinical studies show that loss of C9orf72 leads to autophagy dysfunction and TDP-43 accumulation in neurons.

Question 12

DPR-mediated toxicity

Answer 12

• Expanded repeats undergo repeat-associated non-ATG (RAN) translation.
• This can lead to DPR proteins that form toxic aggregates in the brain.

Question 12

mutations in C9orf72 can lead to:

Answer 12

• LoF:
  1. haploinsufficiency of C9ORF72 gene.
• GoF:
  2. Repeat RNA mediated toxicity,
  3. Dipeptide repeat protein toxicity

Question 12

RNA mediated toxicity

Answer 12

• Repeat extension that is transcribed bidirectionally on the sense and anti-sense reading frames.
• leading to very unstable forms of RNA that aggregate in the form of G-quadruplexes cytoplasm, and sequester RNA-binding proteins such as TDP-43

Question 12

C9orf72 RAN translation: SENSE frame

Answer 12

alanine rich = poly(GA)
Pro rich = poly(GP)
Arg rich = poly(GR)

Question 12

C9orf72 RAN translation: ANTISENSE frame

Answer 12

Gly rich = poly(GP)
Ala rich = poly(AP)
Arg rich = poly(PR)

Question 12

Arg richness

Answer 12

these are toxic !
in flies - Overexpressing in the eye, clear degermation of ARGININE rich DPR, both in the eye and also in the adult CNS. Massive reduction in life span.

Question 12

what are DPRs?

Answer 12

dipeptide rich proteins

Question 13

most toxic DPRs

Answer 13

poly(GR)
poly(PR)

Question 14

C9orf72 targeting with ASOs:

Answer 14

Only variant 1 and 3 contain expansion repeat area. To avoid LoF toxicity, want to target upstream of the promoter for variant 2 to make sure it is still being transcribed and function BUT variant 1 and 3 are being silenced.

This process observed that the pathology was reduced in iPSC cells.

Question 15

limitations of ASOs for FTD/ALS

Answer 15

• Prohibitively expensive
• Administration requires multiple injections per year.
  Clinical safety does not always translate to a clinical setting (C9orf72)

Question 16

limitations of CRISPR/CAS13 for FTD/ALS

Answer 16

• Limited by packaging size limitations (AAV)
• Long-term expression of a bacterial protein may lead to an immune response.
• Off-target effects due to bystander RNA degradation when Cas13 active
  o Novel approach utilises human effector proteins to emulate Cas13 RNA targeting and may help reduce off targets and immune response.

Question 17

Limitations of miRNA for FTD/ALS

Answer 17

• Less specific CRISPR-Cas13 (multiple targets can be beneficial but can also mean more off-targeting silencing effects)
• Poor stability due to nuclease cleavage (requires chemical modification to increase stability)
• Low membrane permeability by itself (use of AAV or nonviral vectors)
• Off-target silencing effects (can be reduced with combination with ABs or peptides)