Pathomechanism and potential treatments of diseases with trinucleotide repeat

expansion

陳瓊美 MD.PhD.

林口長庚醫學中心神經內科

Non-coding trinucleotide repeat diseases:Fragile X syndrome, Friedreich’s ataxia,Myotonic dystrophy, SCA8, SCA12

Polyglutamine diseases:SBMA, DRPLA, SCA1,2,3,6,7,17, HD

Polyalanine: ocular pharyngeal muscular dystrophy

Triplet repeat diseases

Common features of triplet repeat expansion diseases

• 1) the mutant repeats show both somatic and germline instability, frequently expanding rather than contracting in successive transmissions through the generations of a family;

• 2) an earlier age of onset and increasing severity of phenotypes in subsequent generations (a phenomenon known as anticipation) that correlates with larger repeat lengths;

• 3) the parental origin of the disease allele can often influence anticipation, with paternal transmission carrying a greater risk of expansion for many of the disorders except from congenital myotonic dystrophy.

Spinocerebellar ataxia (SCA)

A cluster of hereditary, late onset neurodegenerative disorders in which progressive neuronal loss and gliosis predominantly affect the cerebellum and related structures in the CNS.

Common clinical features: cerebellar ataxia. Other lesions: brainstem, spinal cord, and peripheral nerve system

28 autosomal dominant subtypes (SCA 1-8, 10-28, and, DRPLA) have been reported.

SCA

• The non-coding-repeat diseases are typically multi-system disorders involving dysfunction or degeneration of many different tissues.

• The size and variation of the repeat expansions are much greater in the non-coding trinucleotide-repeat diseases than in the polyglutamine diseases.

Non-coding trinucleotide repeat diseases

aa

>2306-53

FMR15’ 3’

CGG

Disease Gene/locus Protein Schematic representation Possible pathogenicmechanism

Fragile X syndrome

Fragile XE mentalretardation

>2006-35

FMR25’ 3’

GCC

X255’ 3’

>1007-34

GAA

Intron 1

DMPK5’ 3’

>505-37

CTG

DMAHPDMWDpoly A

SCA85’ 3’

110-25016-37

CTG

poly A

66-767-28

SCA125’ 3’

CAG

Friedreich’s ataxia

Myotonic dystrophy

Spinocerebellar ataxia type 8

Spinocerebellar ataxia type 12

FMR1Xq27.3

FMR2Xq26

X259q13-21.1

DMPK19q13

SCA813q21

SCA125q31-33

FMR1protein

FMR2protein

DMPK

None

PPP2R2B

Frataxin

Loss of function

Loss of function

Loss of function

Loss and/or gain of functionReduced DMPK expressionSilencing in the DM regionDominant effect on RNAprocessing

Loss of function?Abnormal RNA regulation?

Loss of function?Disruption in phosphataseactivity?

Non-coding trinucleotide repeat diseases

Clinical Features of the Myotonic Dystrophies

Clinical Features of the Myotonic Dystrophies

RNA-Mediated DM pathogenesis

RNA-Mediated DM pathogenesis

A, In situ hybridization of CAGG probe to DM2 muscle. B, In situ hybridization of CAGG probe to normal muscle. C, In situ hybridization of CAG probe to DM1 muscle.

RNA in situ hybridization of the expansion: RNA foci

CUG and CCUG repeat–containing foci accumulate in affected muscle nuclei suggests that a gain-of-function RNA mechanism underlies the clinical features common to both diseases. (Curr Opin Genet Dev 12:266–271.)

Modification of (iCUG)480-induced phenotypes by muscleblind

CUGBP1 enhances the eye and muscle phenotypes caused by (iCUG)480, and does not accumulate in nuclear foci.

Pathogenesis of myotonic dystophy

• Heterozygous loss of SIX5 in mice causes cataracts and cardiac conduction disease, and homozygous loss also leads to sterility and decreased testicular mass, reminiscent of DM1 in humans.

• Late bursts of skeletal muscle Na channel openings is recapitulated in Dmpk -/- and Dmpk +/- murine skeletal muscle.

• CTG repeat expansion leads to a decrease in DMPK mRNA levels by affecting splicing at the 3' end of the DMPK pre-mRNA transcript.

• Muscleblind-like protein 1 nuclear sequestration is a molecular pathology marker of DM1 and DM2. Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy.

RNA-Mediated non-coding triplet repeat diseases pathogenesis

DNA expansion

RNA with repeat motif Accumulates with sequestration of

RNA binding protein

Misregulation of Global splicing

Chromatic structure changeAnd decreased expression of Six5, DMPK and DMWD

Polyglutamine diseases

• A group of several progressive neurodegenerative disorders caused by expanding CAG repeats coding for polyglutamine.

• Mutant proteins do not share any homology outside the polyglutamine tract.

• Ubiquitous expression of all individual genes, only a certain subset of neurons is vulnerable in each disease.

• Probably caused by a gain of function mechanism.

• Intracellular aggregates: intranuclear inclusions (NIIs) or cytoplasmic.

• Selective neuronal vulnerability is probably influenced by aspects of the respective gene involved.

• A progressive increase in the expressivity of an mutation over subsequent generations is termed “anticipation”.

• Germline and somatic mutation length instability.• A larger CAG repeat length is associated with a

severe and early-onset clinical picture. • Anticipation results from length dependent

intergenerational mutation instability.• Somatic instability of the CAG mutation may play a

role in the cell-specificity of the pathological HD phenotype.

• Slippage during DNA replication or DNA repair

Polyglutamine diseases

Disease Gene Locus Protein CAG repeat size (Normal)

Disease

Spinobulbar muscular atrophy AR Xq13-21

Androgen receptor

9-36 38-62

Huntington’s disease HD 4p16.3 Huntingtin 6-35 36-121

Dentatorubral-pallidoluysian atrophy

DRPLA

12p13.31

Atrophin-1 6-35 49-88

Spinocerebellar ataxia type 1 SCA1 6p23 Ataxin-1 6-44 39-82

Spinocerebellar ataxia type 2 SCA2 12q24.1 Ataxin-2 15-31 36-63

Spinocerebellar ataxia type 3 SCA3 14q32.1 Ataxin-3 12-40 55-84

Spinocerebellar ataxia type 6 SCA6 19q13 1A-voltage-

dependent calcium channel subunit

4-18 21-33

Spinocerebellar ataxia type 7 SCA7 12q12-13

Ataxin-7 4-35 37-306

Spinocerebellar ataxia type 17 TBP 6q27 TATA-binding protein

29-42 46-63

Diseases caused by expanded polyglutamine tracts.

Clinical features of Huntington’s disease

• AD inheritance, the earlier the symptoms appear, the faster the disease progresses

• mood swings or becomes irritable, apathetic, passive, or hostile outbursts or deep bouts of depression. Range from antisocial personality, psychosomatic disorder, delusional disorder, and affective disorder to schizophrenia

• judgment, memory, and other cognitive functions. • may begin with uncontrolled movements in the

fingers, feet, face, or trunk--signs of chorea• In the later stages of HD, body weigh loss is a

frequent but not invariable symptom.

Huntington’s disease

Striatum Ventricle

(CAG)<36

HD gene (Huntingtin)

Exon 1Normal allele

(CAG)>36

Mutated allele

For a CAG count of 41, one person had onset at 25 years of age, another had onset at 81 years of age.

What is the mechanism underlie the triplet instability?

• The size of the repeat expansion is directly related to the occurrence and severity of the disease.

• Large repeats are subject to greater additional instability both in somatic and germinal cells and both cis- and trans-acting factors are involved.

• Age-dependent increase in mutation frequency.• Slippage during cell-division-dependent DNA

replication or • Repair of DNA lesions induced by TNR secondary

structures, or oxidative stress• Recombination of DNA

Somatic mosaicism of CAG mutation in HD mice

Tissue-specific differences in mutation load and magnitude of CAG repeat expansion

The greatest variability and largest mutation length changes occur in the striatum

The CAG repeat instability is expansion-biased and age-dependent

a

M M

150

72

7

3 mos 9 mos 15 mos 19 mos

Striatum

CAG repeat copy number of the progenitor mutation influences tissue-specific mutation length profiles.

Dramatic mutation length increases in human HD striata prior to pathological cell loss.

Expansion-biased size changes in HD mutation length occur earlier in striatal cells than other regions of the brain.

The striatum is absent of replication or meiotic recombination. Mismatch repair function is necessary for marked somatic mosaicism.

Smaller mutation lengths may have pathological consequences that are much more tissue-specific in nature, whereas longer mutation lengths could have more global cytotoxic consequences.

Summary

How does the mutant gene causeneuronal death?

Loss of gene function in polyglutamine diseases

• Inactivation or knock out of causative gene (HD or SCA3) does not generate a characteristic phenotype.

• Over-express normal Huntingtin can rescue some of the neuronal degeneration in HD models, suggesting loss of function may play a part of pathogenesis.

• Over-express wild-type ATXN3 in Drosophila protects neuron from toxicity initiated by other polyglutamine-expanded proteins.

Deubiquitinating function of ataxin-3

Inactivation of the mouse Atxn3 (ataxin-3) gene increases protein

ubiquitination, but not causes significant neurodegeration

•Intracellular aggregates (intranuclear and cytoplasmic inclusions) in the brains of HD patients and HD mice and cellular models.

•Mutant protein tend to form intracellular aggregates, especially those containing longer polyglutamine tract following proteolytic cleavage.

•Form a stable beta-pleated sheet via a so- called “polar zipper” held together by hydrogen bonds

•Intranuclear inclusions (NIIs) are mostly composed of N-terminal huntingtin fragments, ubiquitin, proteasome components heat shock proteins (chaperones) and transcription factors.

•NIIs in patient’s brain may interfere with nuclear activity and lead to neuronal dysfunction (transcription factors).

Intracellular aggregates

Beta Pleated Sheet Protein formed by hydrogen bond

Immunolabelled nuclear inclusions

HD 21m

Ctx

Str

HippCereb

HD

HD

4m 7m 24m

Molecular chaperones and transcription factor are recruited to polyglutamine aggregates

Are intracellular aggregates toxic or protective to neurons?

Evidence supporting toxic intracellular aggregates:• In vitro studies• In a Drosophila HD model, a bivalent artificial huntingtin binding polypeptide could delay and limit the appearance of aggregates and inhibit neuron degeneration.

Evidence supporting non-toxic intracellular aggregates:• Suppression of NII formation can increase cell death in vitro. • SCA1 transgenic mice with a deletion in the self-association region developed profound degenerative changes without NII formation.• The distribution of huntingtin aggregates does not correspond

well to the location of neuropathology in HD post-mortem brains.

Toxic fragment hypothesis of HD pathogenesis

• Huntingtin is cleaved within cells by a caspase or other protease into a short protein fragment containing the polyglutamine tract, which is toxic to cells. A number of NH2-terminal htt fragments of varying sizes have been found in the brains of HD patients.

• In vitro cell models: truncated huntingtin constructs vs full-length huntingtin constructs.

• In vivo mouse models: HD mouse models expressing truncated N-terminal fragments of mutant huntingtin have more rapidly progressing phenotypes than those expressing full-length mutant protein.

Double immunofluorescence labeling of a striatal section of an HD patient brain with mouse EM48 (mEM48) and EM121 or rabbit EM48 and 2166. and EM121 labeling. This suggests that htt aggregates in the human brain primarily consist of small NH2 -terminal htt fragments.

Proteolysis of full-length mutant huntingtin (htt) generates multiple N-terminal htt fragments that carry an expanded polyglutamine (polyQ) tract.

(a) Extracts of various brain regions [striatum (Str), cerebellum (Cereb) and cortex (Ctx)] from heterozygous HdhCAG150 (Huntington’s disease gene with 150 CAG repeats) knock-in mice at various ages.

(b) Schematic diagram showing thestructure of full-length htt. The N-terminal region of htt contains (polyQ) and polyproline (polyP) tracts. Proteolysis of htt generates multiple N-terminal htt fragments, which can accumulate in the nucleus, form aggregates and causecytotoxicity. The smaller N-terminal htt fragments are more prone to nuclearaccumulation and aggregation.

Proteolytic cleavage of pathogenic AT3 as aprerequisite for aggregation

Proteolytic cleavage of pathogenic AT3 as aprerequisite for aggregation

Interaction of huntingtin with cytoplasmic proteins

A number of huntingtin-interacting proteins: glyceraldehyde-3-phosphate dehydrogenase, calmodulin, huntingtin-interacting protein 1 (HIP1), huntingtin-interacting protein 2 (HIP2), huntingtin-associated protein 1 (HAP1), huntingtin-associated protein 40 (HAP40), cystathionine b-synthase.

Polyglutamine expansion causes a conformational change leading to altered protein-protein interactions and the gain of deleterious functions.

Colocalization of transfected HAP1-A with huntingtin and P150 in HEK 293 cells.

Interference with axonal transport

• Neuritic aggregates represent a physical barrier.

• In model of SBMA, mutant AR aggregates are able to cause axonal swellings that show accumulation of kinesin, a motor protein involved in transporting large intracellular organelles such as mitochondria.

Chaperones, the ubiquitin-proteasome pathway and polyglutamine pathogenesis

Misfolding of the mutant protein.

Nuclear aggregates of polyglutamine proteins are ubiquitinated. Ataxin-3 is a ubiquinating hydrolase

HSP40 and HSP70 heat shock proteins and components of the proteasome system are co-localized with aggregates.

Overexpressed chaperones in Drosophila models.

SCA1 mice crossbred with mice over-expressing a molecular chaperone afforded protection against neurodegeneration.

Fly expressing the pathologically active Q78 protein, but now also expressing the molecular chaperone Hsp70. Eye degeneration is suppressed

Transcriptional dysregulation caused by expanded polyglutamine

Nuclear localization of mutant protein is important in toxicity for SCA1, 3, 7, 12.

CBP [cAMP-responsive element binding protein (CREB)-binding protein], a cofactor for CREB-dependent transcriptional activation, co-localize with the mutant huntingtin.

Expanded polyglutamine interferes with CBP-activated gene transcription, and over expression of CBP rescues polyglutamine-induced neuronal toxicity in cell models and human HD.

Nuclear localization of Ataxin-3 Is Required for the Manifestation of Symptoms in SCA3

Transcription factor are recruited to polyglutamine aggregates

Fig. CBP localizes to nuclear inclusions in SCA3 cell model.

CREB/CBP-mediated gene transcription and mutant polyQHtt. Schematic representation of CRE-regulated genes, theiractivation and interference with this process by mutant polyQ proteins—in this case Htt.

Loss of CBP (a histone acetyltransferase) function as a key cellular defect in polyglutamine disease.

Model for HD Cellular Pathogenesis

Possible pathogenesis of neuronal dysfunction in polyglutamine diseases

Increased oxidative stress

Metabolic deficits

An interplay between excitotoxicity, metabolic deficits, and oxidative stress via mitochondrial dysfunction. Mitochondrial abnormalities:N-terminal mutant huntingtin on neuronal mitochondrial membranes.

Oxidative damage may be involved in HDpathogenesis

Increased oxidized glutathione, and 8-hydroxydeoxyguanosine (OH8dG) in nuclear DNA and mtDNAin striatum of HD patients and transgenic HD mice.

Aconitase, an indirect marker of increased oxidativestress is decreased in striatum of HD brains and transgenic HD mice.

Levels of malonaldehyde (a product of lipid peroxidation), 3-nitrotyrosine and heme oxygenase-1 are increased in striatum of HD brains.

Metabolic deficits

Weight loss in HD patients and transgenic mouse models.

Ingestion of 3-nitropropionic acid (3-NP) in man produces selective basal ganglia lesions and dystonia

Decreased cerebral metabolic rates have beenshown in the caudate and putamen as well as in frontal and parietal cortex of HD patients and carrier by PET and MRS.

Decreased complex II/III activity have been identified in caudate and putamen of HD patients, but not in cortex, cerebellum or fibroblasts. Reduction of aconitase activity and decreased complex IV activity in the striatum have been shown in R6/2 mice at ~12 weeks of age (Tabrizi et al., 2000).

Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines (Nat Neurosci. 2002 ): N-terminal mutant huntingtin on neuronal mitochondrial membranes.

Mitochondrial abnormalities

Mitochondrial defects in Huntington's disease

Potential therapeutics in HD

Inhibition of mutant protein aggregation

Over-expressing specific chaperons.

Transglutaminase inhibitor (cystamine).

IC2 antibody chemical compounds Congo red, thioflavine S, chrysamine G and Direct fast yellow.

Single-chain Fv intracellular antibody (intrabody). Bivalent artificial huntingtin binding polypeptide.

Trehalose

Over-expressing specific chaperons reduced protein aggregation and toxicity in transfected cells suppresses neurodegeration in invertebrate models of polyglutamine diseases (Warrick et al., 1999).

Brain-enriched chaperone, MRJ, that Inhibits huntingtin aggregation and toxicity Independently(Chuang, JBC, 2002).

SCA1 mice were crossbred with mice over-expressing a molecular chaperone (inducible HSP70), in which high levels of HSP70 indeed afford protection against neurodegeneration (Cummings et al., 2001).

Over-expressing specific chaperons

Brain-enriched chaperone, MRJ, that Inhibits huntingtin aggregation and toxicity (Chuang, JBC, 2002)

Approximately 61.8% of cells transfected with huntingtin alone contain aggregates, whereas only about 11.2% of cells transfected with both huntingtin and MRJ contain aggregates.

Inhibition of huntingtin fibrillogenesis by specificantibodies and small molecules: Implications for

Huntington’s disease therapy(Heiser et al., 2000)

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington’s disease (Lecerf et al., PNAS 2001;4764–4769)

Intrabody association with huntingtin via its N-terminal residues can significantly reduce in situ aggregation of expanded-repeat exon 1 analogues. BHK-21 cells transfected with HD-polyQ-GFP (normal control, Q25; pathogenic, Q72 or Q103) alone or cotransfected with C4 intrabody or control (ML3-9 sFv-HA) at an sFv to antigen plasmid ratio of 5:1

A bivalent Huntingtin binding peptidesuppresses polyglutamine aggregationand pathogenesis in Drosophila (Kanzantsev et al., 2002)

Transglutaminase involved in aggregate formation and its increased enzyme activity in HD brains have beenshown (Karpuj et al., 1999).

A transglutaminase inhibitor (cystamine) administrated to R6/2 mice after the appearance of abnormal movements extends survival, reduces motor abnormalities and meliorates weight loss (Karpuj etal.,2002).

Transglutaminase inhibitor (cystamine)

Enhencement of histone acetylase function and inhibition of histone deacetylase

Histone acetylation was reduced in cells expressing mutant polyglutamine. Reversal of hypoacetylation, which can be achieved by overexpression of CBP or by treatment with deacetylase inhibitors, reduced cell loss (McCampell et al., 2001).

Drosophila HD model, progressive neurodegeneration caused by expanded polyQ were arrested by feeding flies with histone deacetylase inhibitors (Steffan et al., 2001).

Similar results were shown in a mouse model expressing nuclear expanded polyQ (Hockly et al., 2003).

Suberoylanilide hydroxamic acid, a histonedeacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease (PNAS 18, 2003 vol. 100, 2041–2046)

Inhibition of caspase expression

Increased activity of caspase-1 in brains of HD patients and mice (Ona et al., 1999).

Expression of a capase-1 dominant negative mutant, delay disease onset and mortality in R6/2 mouse model (Ona et al., 1999).

In R6/2 mice, minocycline inhibit caspase-1, caspase-3 and inducible nitrite oxide synthase upregulation and delay mortality (Chen et al., 2000).

Minocycline inhibits caspase-independent and –dependent cell death pathways in models of Huntington's disease.

Reduction of glutamate-mediated excitotoxicity and restoration of mitochondrial energy production

Neuroprotective effects of creatine in a trangenice mouse model, R6/2 mice (Ferrante et al., 2000).

Cr improved neither the functional nor the neuromuscular status of the patients (Verbessem, 2003)

In transgenic mice, remacemide and CoQ10 transiently improve the motor performance, but not prolong the survival and change the appearance of inclusions (Schilling et al., 2001).

Remacemide hydrochloride, a non-competitive NMDA receptor antagonist and co-enzyme Q10 (CoQ10), in a 5-year clinical trial study.

Neuroprotective effects of pyruvate in the quinolinic acid rat model of Huntington's disease.(Ryu, 2003)

Amantadine: improved (Lucetti et al.), Not improved (O'Suilleabhain et al.)

Nicotinamide and acetyl-L-carnitine?

Increased survival and neuroprotective effects of BN82451 in a transgenic mouse model of Huntington's disease.

Protective effects of the antioxidant selenium on quinolinic acid-induced neurotoxicity in rats:

Effect of antioxidants (N-acetylcysteine)on 3-nitropionic acid-induced in vivo oxidative stress and striatal lesions.

Melatonin detoxifies the highly toxic hydroxyl radical as well as the peroxyl radical, peroxynitrite anion, nitric oxide, and singlet oxygen.

Antioxidant

• Pathogenic RNA repeats: an expanding role in genetic disease. Trends Genet. 2004;20(10):506-12.

• Mouse models of triplet repeat diseases.Methods Mol Biol. 2004;277:3-15.

• Myotonic dystrophy: RNA pathogenesis comes into focus. Am J Hum Genet. 2004 May;74(5):793-804.

• Huntingtin-protein interactions and the pathogenesis of Huntington's disease. Trends Genet. 2004 Mar;20(3):146-54.

• Recent advances in understanding the pathogenesis of polyglutamine diseases: involvement of molecular chaperones and ubiquitin-proteasome pathway. J Chem Neuroanat. 2003 Oct;26(2):95-101.

• Mechanisms of neuronal cell death in Huntington's disease.Cytogenet Genome Res. 2003;100(1-4):287-95.

• Pathogenesis of polyglutamine disorders: aggregation revisited.Hum Mol Genet. 2003 Oct 15;12 Spec No 2:R173-86. Review.

• Huntingtin aggregation and toxicity in Huntington's disease.Lancet. 2003 May 10;361(9369):1642-4. Review.

• Transcriptional abnormalities in Huntington disease.Trends Genet. 2003 May;19(5):233-8.

• Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington's disease and related disorders.Neuron. 2002 Aug 29;35(5):819-22. Review.

• Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity. Proc Natl Acad Sci U S A. 2002 Dec 10;99 Suppl 4:16412-8.

• Mechanisms of cell death in polyglutamine expansion diseases.Curr Opin Pharmacol. 2004 Feb;4(1):85-90.

References

Pathogenic mechanisms in FRDA:

Impaired transcription elongation of frataxin

The longer the repeats, the lower the level of frataxin and the more severe the phenotype.