Week 5

Question 1

What is neuronal ceroid lipofuscinosis?

Answer 1

Aka Batten Disease

Lysosomal storage disorder

Hallmark: Accumulation of material in lysosome and neurodegeneration

Most common childhood neurodegenerative disorder (approx 1 in 20,000 live births)

Clinical signs: Blindness, refractory epilepsy, loss of motor and cognitive skills (or lack of development of those skills), premature death

No effective treatments for most forms of NCL, only palliative care

Enzyme replacement therapy approved treatment for CLN2 disease

Question 2

What do we know about the genetics of Neuronal ceroid lipofuscinosis?

Answer 2

Various clinical subtypes with:
- Different age of onset and rates of progression
- infantile, late infantile, juvenile (batten), adult (Kufs)

Caused by 13 different gene mutations
(CLN1-CLN14) but no CLN9

Autosomal recessive

Monogenetic

Question 3

What is the definition of a congenital disease?

Answer 3

Congenital anomalies can be defined as structural or functional anomalies that occur during intrauterine life

Also called birth defects, congenital disorders, or congenital malformations, these conditions develop prenatally and may be identified before or at birth, or later in life.

Question 4

What are NCLs?

Answer 4

Lysosomal Storage Disorders

Gangliosidoses
(Tay-Sachs, Sandhoff, Fabry, Niemann-Pick)

Mucopolysaccharidoses
(Hurler, Scheie, Hunter, Sanfillipo, Morquio, Gaucher, Mucolipodoses, Glycoproteinoses)

Neuronal Ceroid Lipofuscinoses
(Batten, Kufs, Parry)

Progessive neurodegenerative disorders:
- Many affect children and are fatal after prolonged disability

Question 5

What are some underlying causes of NCL?

Answer 5

Two types of CLN proteins

1) Lysosomal Enzymes
- Eg. CLN2 Novel pepstatin-insensitive lysosomal protease (TPP1)
- Eg. CLN10 Cathepsin D aspartate protease (CTSD)

2) Novel proteins of unknown function
- E.g. CLN3 which is a predicted transmembrane protein
- Most CLN proteins are expressed in the endolysosomal system, in ALL cells of the body

Question 6

How is NCL diagnosed?

Answer 6

HUMANS
- 1st Symptoms depend on the type of NCL - Loss of vision, siezure, motor decline, developmental delay, cognitive decline

ANIMALS
- 1st Symptoms depend on NCL type, animal, age of onset
- Ataxia - Trouble walking upstairs, corners, stumbling, holding up head
- Blindness - lag behind the herd (sheep)
- Deaf - walk in circles
- Cognition - loss of learned behaviours
- Also, loss of housebreaking, aggression, hallucinations (displayed as fly biting, hyperactivity, and epileptic fits

BOTH
- Electroencephalogram (EEG) - seizures
- Visual evoked potential/electroretinogram (VEP/ERG) - vision
- Computer tomography/magnetic resonance imaging (CT/MRI) - brain morphology
- Histology - storage material
- Ultrastructural studies (Electron microscope EM) - storage material
- Enzymatic assays - some forms of NCL are caused by mutations in lysosomal enzymes
- Genotyping - Most NCL genes are known

Question 7

What is the histology of NCL?

Answer 7

Autofluorescent granules (Ceroid)

Luxol fast blue granules (inclusion bodies)

Periodic acid-Schiff (PAS) granules (glycogen and oligosaccharides)

Sudan black-positive granules (triglycerides and lipids)

Question 8

What is the immunohistochemistry of NCL?

Answer 8

Either of the following can be seen accumulating in tissues:
- Subunit C of mitochondrial ATP synthase
- Saposins

Question 9

What enzymatic assays are associated with NCL?

Answer 9

Cathepsin D (CTSD)
- Lysosomal aspartic protease involved in limited proteolysis
- Gene known as CLN10

Tri-peptidyl peptidase 1 (TPP1)
- Remove three amino acids (as one unit) from the amino terminus of proteins
- Gene known as CLN2

Palmitoyl protein thioesterase 1 (PPT1)
- Removes fatty acids in covalent linkage to cysteine residues in proteins
- Gene known as CLN1

Question 10

What do we know about CLN10 disease?

Answer 10

Cathepsin D mutation

Congenital NCL

Question 11

What do we know about Human CLN10 disease?

Answer 11

Post-natal respiratory insufficiency and status epilepticus and death occurs within hours to weeks

Microcephalic, with extremely small atrophic brains

Loss of neurons in the cerebral cortex and cerebellum

Generalised activation of astrocytes and microglia

White matter appears to lack myeline

Most cells of the central nervous system are loaded with autofluorescent storage bodies, showing a granular ultrastructure, granular osmiophilic deposits

Accumulation of saponins

Various loss-of-function mutations found in the Cathepsin D gene in several families

No cure

Question 12

What do we know about Ovine CLN10 disease?

Answer 12

Extreme neuronal loss

Glial activation

Periodic acid schiff (PAS) staining showed infiltration of macrophages (dark pink) and inclusions (light pink)

Accumulation of saposin is throughout the cytoplasm

EM shows GRODS

All points to CLN1/PPT1/Infantile NCL but no mutation in CLN1
- So it is likely to be caused by a mutation in a gene not yet associated with NCL

Question 13

What do we know about Ovine CLN10 gene identifictation?

Answer 13

Tested lysosomal enzyme activity and found Cathepsin D levels at zero

Cathepsin D is present, as demonstrated by Western Blot

Sequencing showed a conserved aspartate (Asp295) has changed to aparagine and is homozygous in all affected lambs but not unaffected sheep/lambs

So Cathepsin D is made but it is likely to be enzymatically inactive

Question 14

What new discovery was made about Ovine CLN10 disease?

Answer 14

Mutation of Asp295 is Cathepsin D in sheep causes congenital NCL when homozygous

Cathepsin D is a lysosomal aspartic protease

In vivo substrates are unknown

This leads to the identification of a mutation in human cathepsin D in NCL patients. This is the first study on an NCL animal model that led to identification of a mutation in human NCL patients

Question 15

What do we know about Canine CLN10 disease?

Answer 15

Onset at 1-3 years with death at about 7 years

Behavioural changes and motor deficits

No visual impairment or seizures

Autofluorescence throughout CNS

GRODs (granular osmiophilic deposits)

Mutation results in only a relatively small loss of function (36% of normal activity)

Question 16

What do we know about the Murine CLN10 mutation?

Answer 16

Knockout created by insertion of neomycin cassette into Exon 4

No enzyme activity

Onset at 3 weeks and death at 4 weeks

Neuronal loss

Glial activation

Seizures

Retinal neurodegeneration

Autofluorescence throughout CNS

GRODs (granular osmiophilic deposits)

Question 17

What new discoveries have been made about Murine CLN10 mutation?

Answer 17

Thalamus affected before the cortex

The removal of the pro-apoptotic protein Bax did not significantly decreased neuronal cell loss supporting further the involvement of non-apoptotic death mechanisms in the neuronal degeneration of Ctsd-/- mice

Functional alterations in the pre-synapse before onset of clinical signs

Increased autophagic vesicles (involved in catabolism of cytoplasmic material and organelles)

Question 18

What do we know about the Zebrafish CLN10 morphant?

Answer 18

Complete loss of function

Micropthalmia (RPE microvilli affected)

Shorter body

Swim bladder not inflated

Hyper-pigmentation

Impaired yolk absorption

(all at 4dpf)

Onset at 4dpf and premature death at 10dpf

But also described as congenital myopathy

Question 19

What new discovery has been made about the zebrafish CLN10 morphant?

Answer 19

Microvilli of retinal pigmented epithelium reduced

Phenotype is caused by independent Morpholinos and can be rescued with cln10/cathepsin D mRNA injection

Question 20

What is the drosophila CLN10 mutation?

Answer 20

Loss-of-function created by P element excision

Autofluorescent material

GRODs

Periodic acid schiff and luxol fast blue inclusions

Modest age-dependent neurodegeneration

Phenotype present in ctsd-/- and ctsd/Df

No early death

Question 21

What is the yeast CLN10 mutation S cerevisiae?

Answer 21

Complete loss-of-function

Altered vacuolar morphology

Decreased vacuolar protease maturation

Decreased protein turnover

Decreased post-mitotic life span

Increased apoptotic and necrotic cell death

Question 22

What is the Yeast CLN10 mutation?

Answer 22

Catalytically active Cathepsin D/PEP4 protects against apoptosis

Catalytically inactive Cathepsin D/PEP4 protects agains necrosis

Complete loss of function

Increased sensitivity to mating pheromone

Question 23

What is the wider relevance of CLN10 disease?

Answer 23

Some activites of cathepsin D don’t require catalytic activity and these functions may be critical and other types of mutations may give different pathology

Structure function studies confirm that Asp295 is required for catalytic activity

Cathepsin D mutant mice have no phenotype at birth despite complete loss-of-function, but lambs and humans do. Perhaps long gestation in sheep and humans allows a threshold level of toxic insult to be reached before birth or difference is due to type of mutations or is species specific

Aspartly proteases (secretases) were potential therapeutic targets in Alzheimer’s Disease - dont want to target Cathespin D!

Question 24

What is CLN2 disease?

Answer 24

Tri-peptidyl peptidase 1 (TPP1) Mutation

Late infantile NCL

Question 25

What do we know about Human CLN2 disease?

Answer 25

Loss-of-function mutation in Tri-peptidyl peptidase Lysosomal enzyme/protease Cleaves peptides into groups of 3 amino acids Presents with epilepty or visual failure at 2-4 years old Rapid cognitive and motor deterioration Neuronal loss Periodic Acid Schiff (PAS)-positive, curvilinear profiles, accumulation of subunit C of mitochondrial ATP synthase Death between 8 and 12 years old

Question 26

What do we know about Canine CLN2 disease?

Answer 26

Longhaired Dachshund Frameshift in exon4 Onset of 7-9 months Death at 12 months Visual, behaviour and motor deficits, seizures Lamellar profiles and autofluorescence Experimental colony used for therapeutic testing Biomarkers and quantitative clinical assays being developed Being used for therapeutic testing

Question 27

What do we know about mouse CLN2 disease models?

Answer 27

2x Knockout Loss-of-function mutation in Tri-peptidyl peptidase - no enzyme function Presents at 7 weeks Autofluorenscent material (no EM or histology performed yet) Behaviour changes, motor deficits and seizures but NO visual deficit neuronal loss and glial activation in thalamus, cortex, cerebellum and spinal cord Used for therapeutic testing and mechanistic studies

Question 28

What new discovery has been made in the mouse CLN2 disease model?

Answer 28

The genetic modulation of the central apoptotic pathways involving p53 or Bcl-2 failed to alter the disease course in a mouse model of CLN2 disease, indicating that either neuronal death does not occur via apoptosis in this disease, or if it does, it occurs via pathways not involving p53 or Bcl-2

Question 29

What is the Zebrafish CLN2 disease model?

Answer 29

Early nonsense mutation Lack of enzyme activity Phenocopied by antisense morpholino injection Small eyes and head, curved body, large yolk and no jaw Hyperactive at 3dpf, loss of motor activity at 4dpf Accumulation of subunit C of mitochondrial ATP synthase, NO autofluorescence (EM not described) Widespread apoptosis in the CNS Being used for drug discovery and mechanistic studies Lack of proliferation

Question 30

What doe we know about the treatment of CLN2?

Answer 30

CLN2 is the only NCL with a treatment Brineura Enzyme Replacement Therapy (Biomarin) First tested on mouse and canine models Deliver enzyme through device into Cerebrospinal Fluid every other week Results suggest disease is slowed Retinal phenotype NOT improved as retina not targeted Patients are now receiving Brineura as eye injections, through compassionate use (expanded access outside of clinical trials)

Question 31

What is CLN3 disease?

Answer 31

CLN3 Mutation Juvenile NCL

Question 32

What is human CLN3 disease?

Answer 32

Juvenile onset at 4-8 years Death during their 20s or 30s Rapid vision loss, less rapid motor and cognitive deficits, seizures, behavioural, attention and sleeping difficulties, heart problems Mutation in CLN3 gene, also called Battening Many patients have the 1kb deleltion which deletes exon 7-8 Likely loss-of-function but 1kb deletion may cause some change of function Encoded protein is trans-membrane within the endosomes and Golgi storage of subunit C of mitochondrial ATP synthase, autofluorescence, fingerprint profile Glycerophosphodiester levels increase (plasma and CSF). Potential biomarker?

Question 33

What is the porcine CLN3 disease model?

Answer 33

Engineered exon 7-8 deletion Progressive brain pathology Vision impairment (ERG), loss of photoreceptors Motor impairments Being used for therapeutic testing

Question 34

What do we know about the mouse CLN3 disease model

Answer 34

2x knockout 2x knockin Onset varies from 8 weeks to 16 months depending on genotype Visual and motor deficits, seizures, behavioural changes Storage of subunit C of mitochondrial ATP synthase, autofluorescence, fingerprint, curvilinear and rectilinear profile (depending on genotype) Neuronal loss in thalamus, cortex, cerebellum, retina (depending on genotype) Used for therapeutic testing and mechanistic experiments but slow onset hampers research

Question 35

What new discoveries have been made in the mouse CLN3 disease model?

Answer 35

Oxidative stress, dysregulated autophagy, membrane or vesicle trafficking and mitochondrial function Glial activation precedes neuronal loss Selective loss of GABAergic interneurons Atypical immune response - GAD65 autoantibodies mean reduced glutamate Leady blood-brain barrier

Question 36

What is the zebrafish CLN3 disease model?

Answer 36

cln3 MO showed abnormal brain and heart morphology EEG showed seizures Loss of locomotion Early death Subunit C accumulation, glial activation, apoptosis New discoveries: - Lack of cell proliferation - Mitochondrial defects

Question 37

What is the Zebrafish mutant CLN3 disease model?

Answer 37

cln3 mutants are adult viable Sensitive to pro-convulsive drugs Changes in the proteome and metabolome (lipids) at 5 dpf Glycerophosphodiester levels increase

Question 38

What is the drosophila CLN3 disease model?

Answer 38

Generated by imprecised excision of a Minos transposable element Reduced lifespan No autofluorescence New discoveries - Hypersensitive to oxidative stress - Reactive oxygen species accumulate

Question 39

What is the Yeast CLN3 mutant?

Answer 39

S Cerevisiae Reduced nitric oxide production, alteration in vacuolar and cellular pH, basic amino acid homeostasis, trafficking and phospholipid distribution S Pombe Alterations in vacuolar pH and morphology, cell polarity, cell wall deposition, heat tolerance, osmoregulation, trafficking, Golgi size and number, glycolytic flux, amino acid and nucleotide homeostasis Modifier screen demonstrated that the 1kb deletion does not simply cause loss of function. Also causes change of function

Question 40

What are some animals with naturally occurring NCL mutation identified?

Answer 40

CLN10/CTSD - American Bulldog, Swedish Landrace Sheep CLN2/TPP1 - Longhaired Daschund CLN5 - Border collie, borderdale sheep, devon cattle CLN6 - South Hampshire sheep, merino sheep, australian shepherd dog CLN8 - English Setter ARSG - American Staffordshire Terrier ATP132A2 - Tibetan Terrier

Question 41

What animals have a naturally-occurring NCL mutation not yet identified?

Answer 41

Polish Owczarek, Nizinny/Polish lowland sheepdog Miniature Schnauzer Dachshund Chihuahuas Cocker Spaniel Dalmatian Japanese Retriever Welsh Corgi American pit bull terrier Labrador retriever Golden retriever Australian cattle dog Saluki

Question 42

What lessons have we learned from mice about NCL?

Answer 42

Pathogenic mechanisms shared between NCLs Astrocytic and microglial activation Apoptosis Synaptic Changes Selective vulnerability - localised changes Loss of cortical GABAergic interneurons lead to seizures

Question 43

What lessons have we learned from fish about NCL?

Answer 43

Disease models indicate new phenotypes - Cathepsin D MO - loss of microvilli - Cln2/Tpp1 mutant/MO - loss of proliferation - Cln3 MO - loss of proliferation, mitochondrial defects

Question 44

What lessons have we learned from flies about NCL?

Answer 44

Disease models generated but good viability i.e. not good disease models: ctsd, cln1, cln3 Modifier screen to find modifiers of the phenotype caused by over-expression of CLN3 indicates new pathways for CLN3 - Notch, JNK, stress signalling pathways

Question 45

How do organisms help us understand and treat human disease?

Answer 45

Gene Identification Drug discovery Therapeutic testing Molecular pathways Processes Protein function Expression patterns Sequence of pathology Device testing Pharmacodynamics

Question 46

What is the problem with experimental therapeutics?

Answer 46

Delivering the therapeutic to the whole brain in sufficient quantities

Question 47

What is Gene Therapy (GT) and enzyme replacement therapy (ERT)?

Answer 47

Being tested for use for enzyme deficiencies ERT approved for CLN2 disease - tested in mouse and dog for efficacy and primates for tolerability before going in to patients Enzymes pass from cell to cell via the mannose 6-Phosphate receptor pathway so they spread from the site of delivery Transmembrane proteins like CLN3 cannot move from cell to cell so therapeutic agent cannot spread from the site of delivery

Question 48

What will gene therapy and enzyme replacement therapy problems still include?

Answer 48

How to deliver it to brain and retina - vectors, time and rout for gene therapy; intracerebral or intrathecal for enzyme replacement therapy The disease lode already in the patient - can we reverse the disease, can 'rescued' cells function in a diseased environment Disease may become apparent in other tissues as patients live longer Reactivity against the viral vectors in some patients Likely reactivity if a second dose is given (either in the same tissue or a new tissue)

Question 49

What is the Canine CLN2 disease model use?

Answer 49

Longhaired dachshund - CLN2 mutation Recombinant protein (ERT) via AAV-mediated transduction of brain ependyma Assay toxicity, spread, levels, efficacy, survival, pharmacodynamics, device tolerability

Question 50

What are some other experimental therapeutics?

Answer 50

Stem Cell Therapy being tested for CLN3 disease Highly invasive surgery, risky cells to transplant, and need to take immunosuppressants Gene therapy is being tested for CLN3 disease (controversial as it is probably not a simple loss-of-function of CLN3 that causes disease) and CLN2 disease Enzyme replacement therapy being tested for CLN1 disease Antisense oligonucleotide (ASO) being developed for CLN3 disease but it mutation-specific Drugs being tested in various forms of NCL e.g. immunosuppressants (CLN3 disease), AMPA-antagonists, stop-codon readthrough, storage material depletion (Miglustat), cell death inhibition Anti-epileptic drugs often ineffective