ATXN1 Antibody

What is ATXN1 and why are antibodies against it important for research?

ATXN1 is a ubiquitous polyglutamine protein encoded by the ATXN1 gene. In humans, the canonical protein comprises 815 amino acid residues with a molecular mass of 86.9 kDa. It functions primarily as a chromatin-binding transcriptional repressor that inhibits Notch signaling in the absence of Notch intracellular domain by acting as a CBF1 corepressor . ATXN1 is localized in both nucleus and cytoplasm and undergoes various post-translational modifications including ubiquitination, sumoylation, and phosphorylation .

Antibodies against ATXN1 are critical research tools because:

• They enable detection of normal and pathological forms of ATXN1

• They facilitate investigation of ATXN1's role in SCA1 pathogenesis

• They help elucidate ATXN1's functions in immune regulation and B cell activity

• They allow exploration of potential therapeutic approaches for ATXN1-related disorders

What are the main applications of ATXN1 antibodies in neurodegenerative disease research?

ATXN1 antibodies serve multiple crucial functions in neurodegenerative research, particularly in studying SCA1. Key applications include:

• Detecting ATXN1 oligomers that can propagate locally in vivo in mouse models of SCA1

• Monitoring the spread of ATXN1 pathology in brain tissues

• Evaluating the efficacy of passive immunotherapy targeting ATXN1 oligomers

• Distinguishing between monomeric and oligomeric forms of ATXN1 in experimental models

• Examining protein-protein interactions between ATXN1 and its binding partners like capicua (CIC)

What is the recommended ELISA protocol for detecting ATXN1 oligomers?

Based on established research methodologies, the following ELISA protocol is recommended for detecting ATXN1 oligomers:

• Coat plates with 10 μl of brain soluble fraction using 0.1 M sodium bicarbonate (pH 9.6) as coating buffer

• Incubate for 1 hour at 37°C

• Wash three times with TBS containing 0.01% Tween 20 (TBS-T)

• Block for 1 hour at 37°C with 10% BSA

• Wash three times with TBS-T

• Add primary antibodies (F11G3 at 1:500, A-11 at 1:1000, or 11750 at 1:2000) diluted in 5% nonfat milk in TBS-T

• Incubate for 1 hour at 37°C

• Wash three times with TBS-T

• Add HRP-conjugated secondary antibodies (anti-mouse IgM, anti-mouse IgG, or anti-rabbit IgG) diluted 1:10,000

• Incubate for 1 hour at 37°C

• Wash three times with TBS-T

• Develop with TMB-1 component substrate

• Stop reaction with 100 μl of 1M HCl

• Read absorbance at 450 nm

This protocol has been validated for detecting both oligomeric and monomeric forms of ATXN1 in experimental models.

How can researchers optimize Western blot conditions for ATXN1 detection?

Optimizing Western blot conditions for ATXN1 detection requires consideration of several factors:

• Sample preparation:

  • Use appropriate lysis buffers that preserve protein integrity

  • Include protease and phosphatase inhibitors to prevent degradation and maintain post-translational modifications

  • Employ gentle homogenization techniques to preserve oligomeric structures when studying aggregation

• Gel selection:

  • Use 8-10% SDS-PAGE gels for monomeric ATXN1 (86.9 kDa)

  • Consider gradient gels (4-12%) when examining both monomeric and oligomeric forms

• Transfer conditions:

  • Optimize transfer time and voltage based on protein size

  • Use PVDF membranes for better protein retention and sensitivity

• Antibody selection:

  • For monomeric ATXN1: Anti-ATXN1 antibodies (like 11750)

  • For oligomeric forms: Oligomer-specific antibodies (F11G3 or A-11)

  • Validate antibody specificity using appropriate controls (ATXN1 knockout tissues)

• Detection system:

  • Enhanced chemiluminescence provides suitable sensitivity for most applications

  • Consider fluorescent detection systems for more precise quantification

How do ATXN1 oligomers propagate in cellular and animal models of SCA1?

Research has demonstrated that ATXN1 oligomers exhibit a distinctive propagation pattern in SCA1 models:

• Local propagation mechanism: Unlike some other neurodegenerative disease proteins, ATXN1 oligomers propagate primarily to neighboring cells rather than through transsynaptic transmission. Evidence suggests a secretion and reuptake mechanism between adjacent cells .

• Seeding capability: ATXN1 oligomeric complexes can penetrate cells in culture and seed the formation of new ATXN1 oligomers, characteristic of amyloid formation .

• Propagation limitation: When injected into mouse models, ATXN1 oligomers induce formation of new oligomers only in areas proximal to the injection site, suggesting limited long-distance propagation .

• Therapeutic implications: The local propagation mechanism suggests that immunotherapy targeting extracellular ATXN1 oligomers might help arrest propagation to neighboring areas, though such treatment would have limited impact on intracellular oligomer formation .

This localized propagation pattern distinguishes ATXN1 from proteins involved in other neurodegenerative diseases that show more extensive transsynaptic spread.

What is the role of ATXN1 in immune function and how can antibodies help investigate this relationship?

Recent research has uncovered an unexpected immunomodulatory role for ATXN1:

• B cell regulation: ATXN1 ablation leads to dysregulation of B cell activity, specifically aberrant expression of key costimulatory molecules involved in proinflammatory T cell differentiation, including CD44 and CD80 .

• T cell polarization: ATXN1 deficiency promotes increased T helper type 1 (Th1) cell polarization, leading to more severe experimental autoimmune encephalomyelitis (EAE) in knockout mouse models .

• Signaling pathways: Comprehensive phosphoflow cytometry and transcriptional profiling link exaggerated proliferation of ATXN1-deficient B cells to activation of ERK and STAT pathways .

• Multiple sclerosis connection: ATXN1 has been nominated as a susceptibility locus for multiple sclerosis, with ATXN1-null mice developing more severe EAE compared to wildtype mice .

Antibodies against ATXN1 and its binding partners can help investigate these relationships by:

• Identifying ATXN1 expression patterns in immune cell populations

• Monitoring changes in ATXN1 levels during immune responses

• Examining ATXN1 interactions with transcriptional regulators in immune cells

• Analyzing how ATXN1 deficiency affects signaling cascades in B cells

How effective is passive immunotherapy targeting ATXN1 oligomers in SCA1 models?

Passive immunotherapy using antibodies targeting ATXN1 oligomers has shown limited but measurable efficacy in SCA1 models:

• Observed effects:

  • Mice treated with anti-oligomer antibodies showed fewer Purkinje cells containing ATXN1 oligomers

  • ELISA analysis confirmed decreased amounts of oligomers in cerebella of treated mice

  • Western blot analysis revealed a reduction in ATXN1 oligomers without affecting monomeric ATXN1 levels

• Mechanism of action:

  • The anti-oligomer antibody appears to arrest propagation of ATXN1 oligomer complexes by targeting extracellular oligomeric entities rather than directly targeting intracellular oligomers

  • This extracellular targeting mechanism is supported by observations that the antibody does not affect nuclear inclusion formation in the cortex

• Limitations:

  • Treatment provided modest benefits because neurons expressing polyQ ATXN1 continue to form their own toxic oligomeric entities

  • Non-oligomeric forms of PolyQ ATXN1 might also contribute to toxicity, limiting effectiveness

  • Complete halting or reversal of symptoms would likely require targeting the root cause - abnormal accumulation of polyQ ATXN1

• Future directions:

  • Combination therapies that both block oligomer propagation and reduce ATXN1 expression may prove more effective

  • This limitation likely extends to other disease-driving proteins in neurodegenerative disorders

How should researchers design experiments to differentiate between normal and pathological forms of ATXN1?

Designing experiments to differentiate between normal and pathological forms of ATXN1 requires careful consideration of several factors:

• Antibody selection:

  • Use antibodies that recognize epitopes outside the polyQ region to detect total ATXN1

  • Employ conformation-specific antibodies like F11G3 or A-11 to detect oligomeric forms

  • Include antibodies against post-translational modifications that influence toxicity

• Model systems:

  • Compare wildtype (Atxn1+/+), heterozygous (Atxn1+/-), and knockout (Atxn1-/-) models to assess gene dosage effects

  • Include knock-in models with expanded CAG repeats (Atxn1154Q/2Q) to study polyQ-dependent effects

  • Use cell-specific conditional knockout models to isolate tissue-specific functions

• Experimental readouts:

  • Assess both biochemical measures (protein levels, oligomer formation) and functional outcomes

  • In neurodegeneration models, correlate molecular changes with behavioral deficits

  • In immune function studies, measure B cell activation markers and T cell polarization

• Temporal considerations:

  • Monitor ATXN1 expression and modification patterns during disease progression

  • Assess acute versus chronic effects of ATXN1 manipulation

  • Track transcriptional changes as early indicators of pathology

Why might researchers observe different ATXN1 band patterns in Western blots?

Variability in ATXN1 band patterns during Western blot analysis can result from several factors:

• Post-translational modifications:

  • ATXN1 undergoes ubiquitination, sumoylation, and phosphorylation, each altering migration patterns

  • Phosphorylation at specific residues can change ATXN1 stability and interactions

  • Sample preparation methods may preserve or disrupt these modifications

• Protein aggregation states:

  • Monomeric ATXN1 appears at approximately 87 kDa

  • Oligomeric forms produce higher molecular weight bands

  • Sample heating and SDS concentration can affect oligomer stability

• Polyglutamine expansion:

  • CAG repeat length variations alter protein size

  • Normal ATXN1 vs. expanded polyQ ATXN1 show different migration patterns

  • Somatic instability may produce heterogeneous band patterns in affected tissues

• Proteolytic processing:

  • Partial proteolysis during sample preparation can generate fragments

  • Disease-specific cleavage events may produce distinctive degradation products

  • Inclusion of protease inhibitors is critical for consistent results

• Antibody epitope specificity:

  • Antibodies recognizing different regions of ATXN1 may detect distinct conformational species

  • N-terminal vs. C-terminal antibodies often produce different banding patterns

  • Conformational antibodies may selectively detect specific oligomeric forms

What controls should be included when using ATXN1 antibodies in experimental procedures?

To ensure reliable and interpretable results when using ATXN1 antibodies, researchers should include the following controls:

• Genetic controls:

  • ATXN1 knockout tissue/cells (Atxn1-/-) as negative controls

  • Heterozygous samples (Atxn1+/-) to assess gene dosage effects

  • Overexpression systems as positive controls with defined expression levels

• Antibody controls:

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls matched to the primary antibody class and species

  • Pre-absorption with recombinant ATXN1 to confirm specificity

• Experimental condition controls:

  • For ELISA: Include standard curves using recombinant ATXN1 protein

  • For Western blots: Include molecular weight markers and loading controls

  • For immunohistochemistry: Include parallel sections stained for cell-type specific markers

• Pathological state controls:

  • Compare wildtype ATXN1 with expanded polyQ forms

  • Include tissues from different disease stages to track progression

  • For immunotherapy studies, include both treated and untreated samples

• Cross-validation approaches:

  • Use multiple antibodies targeting different ATXN1 epitopes

  • Confirm antibody results with complementary techniques (mRNA analysis, mass spectrometry)

  • Apply both biochemical and functional readouts to assess ATXN1 status

How can ATXN1 antibodies contribute to understanding the protein's role in conditions beyond SCA1?

ATXN1 antibodies are valuable tools for exploring the protein's newly discovered roles in multiple conditions:

• Cancer research:

  • ATXN1 loss-of-function has been implicated in cancer development

  • Antibodies can help track ATXN1 expression changes in various cancer types

  • Immunohistochemical profiling of tumors may reveal prognostic correlations

• Alzheimer's disease connections:

  • ATXN1 loss-of-function is implicated in Alzheimer's disease pathogenesis

  • Antibodies can help investigate ATXN1's interactions with AD-related proteins

  • Dual labeling with ATXN1 and AD markers can reveal co-localization patterns

• Autoimmune disorders:

  • ATXN1 has been identified as a susceptibility locus for multiple sclerosis

  • Antibodies against ATXN1 can help evaluate expression in immune cells

  • Immunophenotyping B cells for ATXN1 levels may correlate with disease progression

• Developmental biology:

  • ATXN1's role as a transcriptional regulator suggests developmental functions

  • Antibodies can track ATXN1 expression patterns during embryonic development

  • Temporal and spatial expression profiling may reveal previously unknown functions

What methodological approaches can overcome challenges in detecting oligomeric versus monomeric ATXN1?

Differentiating between oligomeric and monomeric ATXN1 presents technical challenges that can be addressed through specialized approaches:

• Antibody-based strategies:

  • Use conformation-specific antibodies like F11G3 that preferentially recognize oligomeric ATXN1

  • Apply oligomer-specific antibodies like A-11 in parallel with total ATXN1 antibodies

  • Develop new antibodies targeting oligomer-specific epitopes that become exposed during aggregation

• Biochemical separation techniques:

  • Employ size exclusion chromatography to separate protein species by molecular weight

  • Use density gradient centrifugation to isolate different ATXN1 assemblies

  • Apply native gel electrophoresis to preserve oligomeric structures

• Advanced microscopy methods:

  • Implement super-resolution microscopy to visualize oligomeric structures

  • Use proximity ligation assays to detect oligomers through protein-protein interactions

  • Apply FRET (Förster Resonance Energy Transfer) to identify closely associated ATXN1 molecules

• Functional assays:

  • Develop cell-based assays that respond differently to monomeric versus oligomeric ATXN1

  • Measure seeding capability as an indicator of oligomeric activity

  • Assess differential binding to partner proteins like capicua (CIC)