ATXN3 Human

What is the normal function of the ATXN3 protein?

ATXN3 encodes ataxin-3, a deubiquitinating enzyme (DUB) that plays a critical role in the ubiquitin-proteasome system. Ataxin-3 removes ubiquitin from proteins targeted for degradation, allowing ubiquitin to be recycled . Beyond this primary function, ataxin-3 is also involved in:

• Protein quality control mechanisms

• Autophagy regulation

• Transcription regulation

• Cytoskeletal regulation

• Stress responses

• Antiviral response

• DNA repair processes

The protein's deubiquitinating function appears central to most of these roles, with evidence suggesting that polyQ expansion may impair this activity in disease states .

What are the different isoforms of ATXN3 and how do they differ functionally?

ATXN3 undergoes alternative splicing, resulting in multiple isoforms with distinct properties:

Research has shown that these isoforms differ significantly in their:

• Protein stability and half-life

• Subcellular localization patterns

• Protein-protein interaction networks

• Involvement in cellular pathways (e.g., ataxin-3c shows stronger association with ERAD pathway proteins)

• Aggregation properties when containing polyQ expansions

Ataxin-3c appears to be the predominant isoform in human and mouse brain tissue, but all isoforms are expressed to some degree .

What cellular and animal models are available for studying ATXN3 function and SCA3 pathogenesis?

Multiple complementary models have been developed for ATXN3 research:

Cellular Models:

• Patient-derived iPSCs and differentiated neurons

• ATXN3-knockout cell lines created using TALENs or CRISPR/Cas9

• CRISPR/Cas9 modified ATXN3-Exon4-Luciferase reporter cell lines

• SCA3 patient-derived fibroblasts

• ATXN3 disease-specific human embryonic stem cells (hESCs)

Animal Models:

• Transgenic mice expressing full-length human mutant ATXN3

• AAV-based mouse models expressing human ATXN3 with expanded polyQ repeats

• ATXN3 knockout mice (which interestingly do not show overt abnormalities)

The AAV-based mouse model recapitulates several key disease features, including:

• Locomotor defects

• Cerebellar-specific neuronal loss

• PolyQ-expanded ATXN3 inclusions

• TDP-43 pathology

• Elevated neurofilament light in CSF

• Detectable expanded polyQ-ATXN3 in plasma

How can patient-derived iPSCs be used effectively to model SCA3?

Patient-derived iPSCs offer several advantages for SCA3 research:

Methodological approach:

• Obtain skin fibroblasts from SCA3 patients and reprogram them to iPSCs

• Confirm pluripotency through immunocytochemistry and PCR for pluripotency markers

• Verify the presence of expanded CAG repeats in the ATXN3 gene

• Differentiate iPSCs into neural lineages, particularly cerebellar neurons

• Characterize ATXN3 expression, aggregation, and cellular phenotypes

These cells maintain abnormal ATXN3 protein expression without changes in CAG repeat length during:

• At least 35 passages as iPSCs

• Up to 3 passages as neural stem cells

• After 4 weeks of neural differentiation

Research applications include:

• Studying mechanisms of neurodegeneration in a human genetic background

• Testing potential therapeutic compounds

• Examining autophagy and protein degradation pathways

• Drug discovery screening platforms

How do polyQ expansions in ATXN3 affect protein behavior and lead to neurodegeneration?

The polyQ expansion in ATXN3 alters protein behavior through multiple mechanisms:

• Protein Misfolding and Aggregation:

  • PolyQ expansion causes the protein to fold incorrectly

  • This leads to the formation of oligomers, protofibrils, and eventually fibrillar aggregates

  • These aggregates form inclusions in the nucleus and cytoplasm of neurons

• Altered Protein Stability:

  • Expanded polyQ ATXN3 shows increased stability compared to wild-type protein

  • This results in higher steady-state levels of the mutant protein

• Aberrant Protein Interactions:

  • PolyQ expansion alters interaction networks of ATXN3

  • Studies show different binding partners for wild-type versus expanded ATXN3

• Nuclear Localization:

  • Mutant ATXN3 shows increased nuclear localization

  • Nuclear toxicity is considered a key pathogenic mechanism

• Impaired DUB Function:

  • PolyQ expansion appears to decrease efficiency of deubiquitinating activity

  • This leads to altered levels of K48- and K63-ubiquitinated proteins in affected brain regions

The selective neurodegeneration pattern may be influenced by the interaction between these mechanisms and the native functions of ATXN3 in specific neuronal populations .

What role does autophagy play in SCA3 pathogenesis and how can it be modulated experimentally?

Autophagy plays a critical role in SCA3 pathogenesis and represents a potential therapeutic target:

Role in pathogenesis:

• Autophagy is a primary degradation pathway for wild-type ATXN3c and ATXN3aL isoforms

• Degradation of the ATXN3aS isoform occurs through both autophagy and proteasomal pathways

• Impaired autophagy may contribute to accumulation of mutant ATXN3 protein

Experimental modulation:

• Pharmacological activation:

  • Rapamycin treatment promotes autophagy and significantly reduces levels of mutant ATXN3 in neurally differentiated SCA3 iPSCs (p < 0.05)

  • Other autophagy inducers could potentially be assessed for therapeutic potential

• Monitoring autophagy:

  • Autophagy can be inhibited experimentally using bafilomycin A1

  • Proteasome inhibition can be achieved using lactacystin

  • These tools allow researchers to distinguish between degradation pathways

• Measuring autophagic flux:

  • LC3-II levels with and without lysosomal inhibitors

  • p62/SQSTM1 accumulation

  • Colocalization of ATXN3 aggregates with autophagy markers

Research suggests that enhancing autophagy may be a promising therapeutic strategy for SCA3, particularly as neural differentiation in iPSCs is accompanied by increased autophagy .

What techniques are most effective for detecting and quantifying different ATXN3 species (monomers, oligomers, aggregates)?

Detecting and quantifying ATXN3 species requires multiple complementary techniques:

For monomeric ATXN3:

• Western blotting with SDS-PAGE

• Immunoprecipitation followed by mass spectrometry

• ELISA-based assays for quantification

For oligomeric and aggregated species:

• Filter trap assays:

  • Useful for detecting large SDS-insoluble aggregates

  • Limited ability to distinguish different aggregation stages

• SDS-PAGE and SDS-AGE (agarose gel electrophoresis):

  • SDS-PAGE detects monomeric and some oligomeric species

  • SDS-AGE better resolves larger oligomeric species

• Native PAGE:

  • Preserves protein complexes and can detect oligomeric species

  • Useful for studying early aggregation events

• Fluorescence microscopy with aggregate-specific antibodies:

  • Allows visualization of inclusions in cells and tissues

  • Can be combined with high-content imaging for quantification

• Mass spectrometry techniques:

  • Reveal time-dependent aggregation of polyQ-expanded ATXN3

  • Can detect various aggregation stages leading to fibril formation

For comprehensive analysis, researchers should employ multiple techniques, as each has limitations in unequivocally showing all stages of ATXN3 aggregation .

How can ATXN3 CAG repeat size be accurately measured, and what challenges exist in repeat sizing?

Accurate measurement of ATXN3 CAG repeats presents several methodological challenges:

Standard techniques:

• Fragment length analysis:

  • Traditional method but shows high levels of inaccuracy

  • Can lead to misinterpretation when identifying modifiers of clinical phenotypes

• Repeat-primed PCR combined with fluorescent fragment-length assay:

  • Provides more accurate sizing of expanded repeats

  • Used in clinical testing for SCA3/MJD diagnosis

• Small-pool PCR:

  • Allows detection of somatic mosaicism

  • Can measure repeat instability in different tissues

• Next-generation sequencing:

  • Provides accurate sizing and can detect interruptions within repeats

  • Higher cost but increasingly used for research purposes

Key challenges include:

• Somatic mosaicism resulting in different repeat sizes across tissues and even within the same tissue

• Age-dependent somatic expansion that complicates interpretation

• Effects of single nucleotide polymorphisms (SNPs) like rs12895357 on the rate of somatic expansion

• Higher levels of somatic expansion in certain tissues (buccal cells show higher expansion than blood)

For research purposes, multiple sampling from different tissues and time points may be necessary to fully characterize CAG repeat dynamics in SCA3 .

What are the most promising therapeutic strategies for SCA3 based on ATXN3 biology?

Current research has identified several promising therapeutic approaches:

• Gene Silencing Approaches:

  • Antisense oligonucleotides (ASOs) targeting ATXN3 mRNA

  • RNAi-based therapies using siRNA or shRNA

  • These approaches have shown efficacy in reducing mutant ATXN3 levels in animal models

• Autophagy Modulation:

  • Rapamycin and other mTOR inhibitors promote degradation of mutant ATXN3

  • Other autophagy enhancers may have therapeutic potential

• Protein Aggregation Inhibitors:

  • Compounds that prevent misfolding or aggregation of expanded polyQ ATXN3

  • High-throughput screens have been developed to identify such compounds

• CRISPR/Cas9-based Approaches:

  • Targeted removal or correction of expanded CAG repeats

  • Still in early experimental stages

• Statin Derivatives:

  • Simvastatin has been shown to increase ATXN3 expression through SREBP1 binding to the ATXN3 promoter

  • This may help restore functional ATXN3 levels, though effects on expanded ATXN3 require further study

As these therapies advance toward clinical trials, there is an increased need for biomarkers to track disease progression and treatment efficacy .

What biomarkers can be used to monitor SCA3 disease progression and therapeutic efficacy?

Several biomarkers show promise for monitoring SCA3:

Fluid Biomarkers:

• PolyQ-ATXN3 levels:

  • Detectable in CSF, plasma, and urine of SCA3 patients

  • Correlates with cerebellar degeneration and locomotor deficits in SCA3 mice

  • Potential pharmacodynamic biomarker for therapies targeting ATXN3

• Neurofilament light (NFL):

  • Elevated in CSF of SCA3 patients and animal models

  • Indicator of axonal damage and neurodegeneration

  • Can distinguish SCA3 patients from controls

Imaging Biomarkers:

• MRI measures of cerebellar volume and atrophy

• Diffusion tensor imaging for white matter tract integrity

• PET imaging with tracers for neuroinflammation or protein aggregation

Functional Biomarkers:

• Quantitative measures of ataxia (SARA scale, gait analysis)

• Electrophysiological measurements (evoked potentials)

In mouse models, plasma polyQ-ATXN3 levels correlate with cerebellar degeneration measures, suggesting this could be a valuable biomarker for tracking disease progression and therapeutic response .

How do different ataxin-3 isoforms contribute to SCA3 pathogenesis, and what are the contradictions in current research?

The role of different ataxin-3 isoforms in SCA3 pathogenesis remains controversial:

Current understanding:

• Alternative splicing creates multiple ATXN3 isoforms (ataxin-3c, ataxin-3aL, ataxin-3aS)

• These isoforms differ in their C-termini, number of UIMs, and subcellular localization

• The expanded CAG repeat in ATXN3 appears associated with increased generation of the ataxin-3a transcript

Contradictory findings:

• Nuclear localization:

  • While ataxin-3aS shows increased nuclear localization, which is considered pathogenic

  • Some studies suggest nuclear localization is independent of polyQ repeat length

• Degradation pathways:

  • Some research indicates that all mutant ATXN3 is primarily degraded by autophagy

  • Other studies show isoform-specific degradation pathways (ataxin-3aS uses both autophagy and proteasome)

• Protective role of wild-type allele:

  • Some evidence suggests the normal ATXN3 allele has neuroprotective functions

  • Other studies find no significant modification of disease by wild-type ATXN3

• Aggregation properties:

  • Different studies report varying aggregation propensities for different isoforms

  • Contradictory findings regarding whether expanded polyQ affects all isoforms equally

Research gaps include the need for:

• Comprehensive profiling of isoform expression in different brain regions

• Studies of isoform-specific interactomes in disease contexts

• Better understanding of how isoforms affect each other's behavior in cells expressing both wild-type and mutant ATXN3

What methodological approaches can resolve contradictions in ATXN3 experimental data across different model systems?

Resolving contradictions in ATXN3 research requires methodological standardization and integrated approaches:

Standardization approaches:

• Consistent isoform expression:

  • Studies should clearly specify which ATXN3 isoform(s) are being investigated

  • Standardized nomenclature for isoforms (ataxin-3c, ataxin-3aL, ataxin-3aS)

  • Control for endogenous ATXN3 expression using knockout backgrounds

• Uniform CAG repeat sizing:

  • Use multiple methods for repeat sizing to ensure accuracy

  • Report both the number of pure CAG repeats and any interruptions

  • Consider somatic mosaicism and age-dependent expansion

• Cell type considerations:

  • Different cellular contexts may yield different results

  • Compare findings across multiple cell types relevant to SCA3

  • Prioritize neuronal models for disease-relevant mechanisms

Integration of multiple models:

Advanced analytical approaches:

• Single-cell analysis to account for cellular heterogeneity

• Systems biology approaches to model complex interaction networks

• Meta-analysis of published data to identify consistent findings

By implementing these methodological improvements, researchers can better reconcile contradictory findings and develop a more unified understanding of ATXN3 biology and SCA3 pathogenesis .

Normal vs. Pathological ATXN3 CAG Repeat Ranges

Source: Compiled from multiple references .

ATXN3 Protein Interaction Partners by Cellular Pathway

Source: Compiled from reference .

Comparison of Research Models for ATXN3/SCA3

Source: Compiled from references .