Martina Owens 17.09.10

1. Review the neurodegenerative diseases which are associated with aggregate formation and discuss how these aggregates may contribute to pathology. 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. Key words: Neurodegenerative diseases, Aggregates, Parkinson disease, Alzheimer disease, spinocerebellar ataxias, Huntington disease, Martina Owens 17.09.10

2. Introduction • Neurodegenerative diseases are usually characterised by: • onset in late adulthood • slowly progressive clinical course • neuronal loss with regional specificity in the central nervous system (CNS) The formation of protein aggregates that are toxic to cells (especially neurons) is now recognised as a common feature of several adult-onset neurological diseases – toxic gain-of-function. • Neurodegenerative diseases associated with aggregate formation: • Parkinson’s disease • Alzheimer’s disease • Spinocerebellar ataxias • Huntington disease • Fragile X-associated tremor/ataxia syndrome (FXTAS) • Dentatorubral-pallidoluysian atrophy (DRPLA) • Creutzfeldt-Jakob disease (CJD)

3. Parkinson’s Disease (PD) Characterised by tremor at rest, muscle rigidity, and slowed movement (bradykinesia). Onset before age 20 years → juvenile-onset Parkinson disease Onset before age 50 years → early-onset Parkinson disease Onset after age 50 years are → late-onset Parkinson disease Gross depigmentation of the substantia nigra (harbours the neurons that produce the neurotransmitter, dopamine), due to neuronal loss, is the most evident feature of end-stage PD. Symptoms result from dopamine depletion in the striatum, and are associated with increased, disordered neuronal firing and loss of synchronisation in the basal ganglia. Neuronal degeneration in PD is not limited to the substantia nigra or dopaminergic neurons but extends as the disease progresses. A good response to levodopa and asymmetric onset of limb involvement are generally regarded as supporting diagnostic features.

4. Seven known genes have been implicated in PD: Disease susceptibility genes: NR4A2, SNCAIP and mitochondrial (NADH complex I).

5. Seven known genes have been implicated in PD: p.Gly2019Ser found in approximately 5%-7% of familial, autosomal dominant PD ~50% patients have a mutation in PARK2 Accounts for ~1-2% PD patients

6. Pathways to PD:

7. Alzheimer’s Disease (AD) Typically begins with subtle and poorly recognized failure of memory and slowly becomes more severe and, eventually, incapacitating. Other common findings include confusion, poor judgment, language disturbance, agitation, withdrawal, and hallucinations. The dementia in AD is associated with neurodegeneration that is characterised initially by synaptic injury followed by neuronal loss in the cerebral cortex. This loss results in gross atrophy of the affected regions, including degeneration in the temporal lobe and parietal lobe, and parts of the frontal cortex and cingulate gyrus.

8. Both amyloid plaques and neurofibrillary tangles are clearly visible by microscopy in brains of those afflicted by AD. Plaques are dense, mostly insoluble deposits of amyloid-beta peptide and cellular material outside and around neurons. Tangles (neurofibrillary tangles) are aggregates of the microtubule-associated protein tau which has become hyperphosphorylated and accumulate inside the cells themselves.

10. APP: integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation and neural plasticity. APP is proteolytically cleaved by ß-secretase and subsequently by gamma-secretase generating full length Beta-amyloid (Aß40 or Aß42) Gene located on chromosome 21 PSEN1 and PSEN2: PSENs are functionally involved in the gamma-secretase-mediated proteolytic cleavage of APP. Most mutations in the APP and presenilin genes increase the production of Aβ42, which is the main component of senile plaques. Early-onset AD:

11. PSEN protein structure. Boxes represent the transmembrane regions that are separated by hydrophilic loops.

13. Huntington Disease (HD) Imaging shows degeneration of the striatum as well as the cortex The mean age of onset for HD is 35 to 44 years. Juvenile HD is defined by the onset of symptoms before age 20 years and accounts for 5%-10% of HD cases Defining phenotype of HD is generally considered to be chorea and involuntary movements. Individuals with the disease frequently also display psychiatric symptoms, most commonly depression and irritability, as well as declines in cognitive abilities.

14. Caused, in most cases, by an expansion of a CAG (glutamine) repeat located in the DNA encoding the first exon of HTT (IT15): Longer repeats confer, on the average, earlier ages of symptom onset. Anticipation observed (paternal transmission)

15. Repeat Instability: Mechanisms Of Dynamic Mutations Replication: Replication across hairpin caused by repeat might result in expansions or deletions for nascent or template hairpins, respectively. Recombination: Homologous recombination between allelic repeats. Repair: Mismatch repair, double strand break repair, nucleotide excision repair Alternative DNA structures formed in repeated DNA

16. Function of Htt protein not known Essential for embryonic neurogenesis – knockout mice nullizygous = lethal Proposed role in cellular transport and neurotransmission. Also implicated in iron homeostasis, RNA biogenesis and membrane trafficking

17. HD disease pathways

18. Spinocerebellar Ataxias (SCAs) Clinical and molecular heterogeneous group of disorders Characterized by slowly progressive incoordination of gait and often associated with poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs, and different ataxias are known to affect different regions within the cerebellum

19. SCAs 1,2,3,6 and 7 comprise ~60% of cases Sporadic cases are most likely to have SCA2 or SCA6 SCA7 is distinguished from other SCAs by presence of retinal degeneration

20. Each, with the exception of SCA6 (which forms cytoplasmic aggregates that stain negative for ubiquitin), features the accumulation of the mutant protein in large intranuclear inclusions Expanded polyQ proteins are intrinsically prone to misfold and aggregate. The repeat length threshold for aggregation in vitro closely mirrors the repeat length known to cause disease - toxic to cells? Supporting a ‘toxic protein’ model is the fact that expanded CAG repeats engineered to be expressed at the mRNA level, but not at the protein level, tend to display little or no toxicity when introduced into cells or animals. Thus, polyQ protein aggregation, or at least a biochemical process associated with aggregation, seems integral to the disease process. However, inclusions are not always found in affected brain cells, and in some studies correlate with neuronal survival rather than neuronal cell death - possible that small oligomers of mutant protein may prove to be the toxic species, engaging in deleterious interactions with additional polyQ proteins and other cellular proteins.

21. Spinocerebellar ataxia (SCA) 1–3, SCA6–7, SCA17 and dentatorubral-pallidoluysian atrophy (DRPLA) harbour expansions of CAG repeats in the coding region (1) that are transcribed (2) and translated (3) into long polyglutamine (polyQ) tracts. The figure refers to putative pathogenic mechanisms in SCA1, SCA2, SCA3, SCA7, SCA17 and DRPLA, although the relevance of crucial cellular events in each disease remains to be established. The presence of an expanded polyglutamine tract induces an aberrant conformation of the protein, which can be partly rescued by molecular chaperones (4). The misfolded protein might undergo proteolytic cleavage by caspases (5), and the resulting polyQ-containing fragments might be ubiquitinated (Ub) (6) and degraded via the proteasome (7). Non-ubiquitinated polyQ fragments might aggregate in the cytoplasm (8) and trigger a mitochondrion-mediated apoptotic pathway (9, 10). Part of the polyQ-containing fragments (8) might be internalized into the nucleus (11) where they might aggregate. Aggregates might be partly rescued by molecular chaperones (heat-shock proteins, HSP) (12) or they might trigger cell death either directly (13) or through the formation of neuronal intranuclear inclusions (NIIs) (14). In addition to polyQ polypeptides, NIIs contain transcription (Tx) factors, ubiquitin (Ub), and other proteasome components. NIIs are hallmarks of neurodegeneration in the brains of patients with SCA1, SCA3, SCA7, SCA17 and DRPLA. By contrast, only cytoplasmic aggregates (8) can be observed in SCA2 neurons.

22. Fragile X Tremor Ataxia Syndrome (FXTAS) Principal clinical features of FXTAS are progressive intention tremor and gait ataxia, although motor involvement may be more widespread, with aspects of parkinsonism Associated with global loss of brain volume with attendant enlargement of ventricular volume, mild-to-severe white matter disease and, in particular, high-signal (T2-weighted)lesions in the middle cerebellar peduncles Immunocytochemical staining of post-mortem brain tissue from FXTAS cases as revealed the presence of nuclear inclusions in both neurons and astrocytes in most brain regions, with the numbers of inclusions strongly correlating with the size of the CGG repeat Inclusions are also found in tissues outside of the CNS in subjects with FXTAS (e.g. testicles and peripheral nerve ganglia)

23. Affects many older adult carriers of expanded CGG-repeat alleles in the permutation range (55–200 CGG repeats; 1) in the 5′ non-coding region of the fragile X mental retardation 1 (FMR1) gene The severity of both clinical and neuropathological phenotypes is correlated with the extent of the CGG-repeat expansion within the premutation range. FMRP plays an important role in synaptogenesis and synaptic plasticity Larger CGG-repeat expansions (200 CGG repeats; full mutation) are predominantly transcriptionally silent, resulting in the absence, or highly diminished levels of the FMR1 mRNA and protein (FMRP) The restriction of the FXTAS clinical phenotype to the premutation range, where the gene is active, coupled with multiple studies of the adverse consequences of expressing the CGG repeat in diverse human, animal and cell models, has given rise to the concept of a ‘toxic’ gain-of-function of the CGG-repeat containing RNA

24. The precise mechanisms involved in rCGG toxicity are unknown, Possible mechanisms:

25. Dentatorubral-pallidoluysian atrophy (DRPLA) DRPLA is characterized by marked, generalized brain atrophy and the presence of neuronal intranuclear inclusions Juvenile-onset (< 20 years) Early adult-onset (20–40 years) Late adult-onset (> 40 years) Late adult-onset DRPLA is characterized by ataxia, choreoathetosis and dementia. Early adult-onset DRPLA also includes seizures and myoclonus. Juvenile-onset DRPLA presents with ataxia and symptoms consistent with progressive myoclonus epilepsy Caused by the expansion of the polyglutamine region of the atrophin-1 (ATN1) gene Normal: 7-34 CAG repeats, affected individuals display 49-93 repeats Function of ATN1 is not clear, however it is believed to be a transcriptional co-repressor.

26. Conclusion • Nuerodegenerative diseases associated with aggregate formation: • Parkinson’s disease - intracellular inclusions known as Lewy bodies • Alzheimer’s disease - extracellular protein deposits • Spinocerebellar ataxias - nuclear inclusions • Huntington disease - nuclear inclusions • Fragile X-associated tremor/ataxia syndrome (FXTAS) – nuclear inclusions • Dentatorubral-pallidoluysian atrophy (DRPLA) • Creutzfeldt-Jakob disease (CJD) - extracellular protein deposits The precise role of aggregates in the pathogenesis of disease is still under investigation Cells have mechanisms to protect against the accumulation of misfolded and aggregated proteins including the ubiquitin system and the phagosome-lysosome system. Individuals with a mutation have accelerated formation of misfolded proteins which overwhelms the protection systems, ultimately leading to neuronal cell death. Most of the neurodegenerative disease of which aggregates are a feature are the result of gain-of-function mutations. However the autosomal recessive forms of PD result in UPS dysfunction, signalling defects and mitochondrial dysfunction.

27. References • GeneTests • Armstrong et al, 2008, Neuropathology, 28: 351 (aggregate composition) • Cookson and Bandmann, 2010, HMG, 19: R21 (PD) • Farrer M, 2006, Nat. Rev. Genet, 7:307 (PD) • Paulson H, 2009, J Neurophthalmol, 29(3):227 (SCAs) • Stevanin et al, 2000, EJHG, 8: 4 (SCAs) • Shao and Diamond, 2010, HMG, 19: R115 (polyQ diseases) • Johnson et al, 2010, HMG, 19: R98 (HD) • Crews and Masliah, 2010, HMG, 19:R12 (AD) • Garcia-Arocena and Hagerman, 2010, HMG R83 (FXTAS)