A number of diseases are caused by polyglutamine/CAG expansions.

1. A number of diseases are caused by polyglutamine/CAG expansions. What is the evidence that these mutations confer gain of function? Discuss the role of aggregates in disease pathogenesis.

2. A number of diseases are caused by polyglutamine/CAG expansions.

3. SCA, spino-cerebellar ataxia

4. Common features of the diseases caused by unstable expansion of the CAG repeat within a gene • All late onset neurodegenerative diseases, all dominantly inherited (exception of Kennedy disease). • No other mutation in the gene has been found that causes the disease. • The expanded allele is transcribed and translated. • The trinucleotide repeat encodes a polyglutamine tract in the protein. • There is a critical repeat threshold size, below which the repeat is nonpathogenic and above which it causes disease. • The larger the repeat, above the threshold, the earlier is the average age of onset.

5. What is the evidence that these mutations confer gain of function?

6. Gain of function mutations • Gain of function is likely when only a specific mutation in a gene produces a given pathology. • Gain of function is likely to require a much more specific change than loss of function. • The mutational spectrum in gain-of-function conditions should be more restricted, and the same condition should not be produced by deletion or disruption of the gene. • Example: • Huntington disease. Phenotype is dominant suggests disorder results from a G of F mutation in which protein product of the disease allele has a new property or is expressed inappropriately.

7. Huntington Disease: G of F Mutation-Allelic Variants • Normal allelic variants: The HD gene has 67 exons covering 200kb. It is ubiquitously expressed as 2 transcripts, 10.3 kb and 13.6 kb, differ in size of 3’UTR. Gene contains a CAG repeat expanded within the HD gene on at least 1 chromosome of individuals with Huntington disease. HD gene lacks homology to any other characterised gene. CAG repeat length is highly polymorphic and normal CAG repeat size ranges 10 to 35 (median 18). Most common alleles in all populations contain repeats 15-20 CAG in length. • Pathogenic allelic variants: The mutation underlying HD is an expansion of a CAG/polyglutamine tract in the first exon. CAG repeat length in cases with HD is 36 or more. Individuals with adult-onset HD usually have CAG expansion from 40 to 55, whereas juvenile onset have CAG expansions greater than 60 often inherited from father. Inverse correlation between CAG repeat length and age of onset exists. (Refer to www.cmmt.ubc.ca/clinical/hayden).

8. Huntington Disease: G of F Mutation-Proteins • Normal Gene product: Huntingtin (HTT) protein of 3144 AA, predicted molec mass 348kd. Widely expressed, no obvious differences in regional distribution of mutant or WT protein. The polyglutamine tract starts residue 18 is followed by a polyproline region. Region downstream of CAG tract contains a HEAT repeat, a motif of ~ 40 loosely conserved AA repeated multiple times in tandem, proposed involved protein-protein interactions. • Abnormal gene product: The CAG repeat is translated into an uninterrupted stretch of glutamine residues that when expanded has altered structural and biochemical properties.

9. Discuss the role of aggregates in disease pathogenesis.

10. Neuropathology of HD and Expression of Mutant HTT • Neuropathologic features of HD include selective degeneration of neurons in the caudate and putamen. • Preferential degeneration of enkephalin containing neurons of the indirect pathway of movement control in the basal ganglia provides the basis for chorea. • Other regions of the brain can be affected including substantia nigra, hippocampus and various regions of the cortex. • Intraneural inclusions containing HTT protein also appear however, the expression of HTT protein and the pattern and timing of HTT-inclusions in brain do not correlate with the degeneration of the disease, not thought to be the primary determinants of the pathology.

11. Pathology of Aggregates (i) • Exact molecular mechanisms by which mutant HTT induces neuronal cell death in HD are not completely understood but may involve a gain of new toxic function and loss of normal function of the protein. • Lots studies, (since 1993) some conflicting findings: • Detailed postmortem analyses of brains of HD patients • HD transgenics • Knock-in mouse models • Techniques of the time, y2Hybrid systems, time lapse, laser microdissection, mass spectrometry. • Subcellular fractionation and immunolocalization studies suggest a role HTT in organelle transport, protein trafficking and regulation of energy metabolism. • Evidence from vertebrate and invertebrate models of HD indicate expression of expanded form of HTT results in impairment of axonal transport and mitochondrial function.

12. Pathology of Aggregates (ii) • Evidence from transgenic mice expressing full-length HD cDNA phenotypically representative of human HD, similar neurodegeneration. However polyglutamine aggregates in the form of neuronal intranuclear inclusions were found in regions of the brain typically spared in human HD suggesting polyglutamine aggregates may not be sufficient to cause neuronal loss in HD (Reddy et al, 1999). • Mutant HTT found to alter the activity of the N-methyl-d-aspartate (NMDA) type glutamate receptor, which implicated as a main mediator of excitotoxic neuronal death (Cowan, 2006). • Cleavage of HTT characterised in vitro, studies using protease inhibitorsshow proteolysis of HTT at the caspase-6 cleavage site important event in mediating neuronal dysfunction and neurodegeneration. Accumulation of caspase cleavage fragments are found as early pathological changes in brains of HD patients (Graham et al, 2006).

13. Pathology of Aggregates (iii) • More recently link between transcriptional dysregulation and mitochondrial impairment has been postulated in patients with HD, gene transcription regulated by PGC1A (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) is defective, resulting in reduced expression of mitochondrial and antioxidant genes regulated by PGC1A. Raised the possibility increasing PGC1A expression or function might be therapeutic option in HD (Greenamyre, 2007). • Using mass spectrometry-based method to quantify polyubiquitin chains found that Lys48-linked polyubiquitin chains accumulate early in pathogenesis in brains from transgenic mouse model of HD, from a knock-in model of HD and from human HD patients, establishing the ubiquitin-proteasome system dysfunction is a feature of HD pathology (Bennett et al, 2007).

14. In Conclusion • Expression of polyglutamine aggregate does not cause HD. • Polyglutamine aggregates have a role in neuronal cell death but are not the whole story in human HD. • Likely interaction of mutant HTT (gain of function mutation) with other proteins underlying key cellular functions that may have a role in the pathogenesis of HD.

15. References • Cowan CM, Raymond LA. Curr top Dev Biol. 2006;75:25-71. • Reddy et al, Philos Trans R Soc Lond B Biol Sci. 1999; 354(1386):1035-45. • Farrington et al. Cell transplant. 2006;15(4):279-94. • Graham RK, et al. Cell. 2006;125(6):1179-91. • Greenamyre JT. New Eng J Med. 2007;356:518-520. • Bennett EJ, et al. Nature. 2007;448:704-708. • GeneReviews • OMIM