BIN1 Antibody

What is BIN1 and why is it significant in neurological research?

BIN1 (Bridging Integrator 1) represents a key regulator of proinflammatory and neurodegenerative processes in the brain. The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer's disease . Research has established BIN1 as a homeostatic microglial regulator with a non-redundant role in activating proinflammatory responses upstream of important genes including Apoe, Trem2, and Tyrobp . Additionally, BIN1 functions upstream of PU.1 and IRF1, which are master regulators of microglial gene expression and transition to disease-associated microglia phenotype . The protein's critical role in neuroinflammation and its genetic association with AD risk make BIN1 antibodies essential tools for investigating neurodegeneration mechanisms.

How many BIN1 isoforms exist in the CNS and which antibodies detect them?

The CNS expresses at least 10 distinct BIN1 isoforms with different cellular distributions. Three isoforms (1, 2, and 3) are predominantly expressed in neurons and astrocytes, while four isoforms (6, 9, 10, and 12) are expressed in microglia . When selecting antibodies, researchers should consider which epitopes and isoforms need to be detected:

Different antibodies may yield varying results depending on the epitope accessibility and tissue processing methods .

How do BIN1 expression patterns change in Alzheimer's disease brain tissue?

BIN1 expression undergoes significant alterations in AD, with distinct isoform-specific changes:

These findings suggest that BIN1 may play a role in amyloid pathology, and that isoform-specific changes may contribute to Alzheimer's disease pathogenesis.

What are optimal protocols for BIN1 immunoblotting in brain tissue?

For effective BIN1 immunoblotting in brain tissue samples, follow these methodological guidelines:

• Protein extraction: Measure total protein by BCA assay (Pierce) to ensure equal loading .

• Gel selection and separation: Use a range of SDS-PAGE gels (Criterion Bio-Rad or Nu-PAGE Invitrogen) appropriate for BIN1 isoforms (ranging from approximately 45-75 kDa) .

• Transfer conditions: Electrically transfer to 0.45 μM nitrocellulose membranes .

• Blocking strategy: Block overnight with a solution of 1% bovine serum albumin (Calbiochem/EMD Millipore) and 2% Block Ace (AbD Serotec) in PBS .

• Primary antibody selection and dilution:

  • Rabbit monoclonal anti-BIN1 (Epitomics)

  • Mouse monoclonal anti-BIN1 (99D; Sigma-Aldrich)

  • Use appropriate concentrations based on manufacturer recommendations and validation studies

• Detection system: Use HRP-conjugated secondary antibodies with enhanced chemiluminescent detection reagents (Pierce) .

• Controls: Include housekeeping proteins such as GAPDH (HRP-conjugated) or β-Actin for normalization .

• Analysis: Obtain densitometry data using appropriate imaging software (e.g., Scion Image) .

For spot blot analysis, samples can be directly spotted onto membranes using a MINIFOLD I spot blot system (Whatman) .

What immunohistochemistry protocols are most effective for studying BIN1 in relation to AD pathology?

For optimal BIN1 immunohistochemistry in relation to AD pathology:

• Tissue preparation: Use human frontal cortex tissue sectioned at 50 μm and stored in 1X PBS with 0.02% sodium azide at 4°C for long-term storage .

• Double-staining approach:

  • First primary antibody (e.g., PHF-1, 1:1000 dilution): Incubate overnight at room temperature

  • Secondary antibody: Use biotinylated secondary antibody (Vector Laboratories)

  • Detection: Apply avidin-biotin complex (ABC; Vector Laboratories) and detect using 3′-diaminobenzidine and hydrogen peroxide (DAB; Vector Labs)

  • Intermediate fixation: Incubate in 37% formaldehyde at 37°C between stains when using multiple antibodies from the same host

  • Second primary antibody (e.g., BIN1 99D, 1:3000 dilution): Incubate overnight

  • Detection of second antibody: Use SG substrate (Vector Labs) for differential visualization

• For amyloid co-labeling: Use antibodies like mAb 3D6 or pAb M78 for detecting amyloid deposits .

• For high-resolution analysis: Employ confocal imaging to evaluate the precise relationship between BIN1 immunoreactivity and pathological features like amyloid deposits .

• For ultrastructural analysis: Consider pre-embedding immunogold-EM to determine BIN1 distribution at the ultrastructural level, particularly around amyloid deposits .

This approach allows for detailed analysis of BIN1 in relation to tau and amyloid pathology in AD tissue.

How can researchers investigate cell-type specific BIN1 expression and function?

To investigate cell-type specific BIN1 expression and function:

• Conditional knockout approaches:

  • Develop microglia-specific BIN1 conditional knockout mice using selective ablation of BIN1 alleles in microglia to study microglial-specific functions .

  • Analyze phenotypes under both basal conditions and neuroinflammatory challenge (e.g., LPS administration) .

• Primary culture systems:

  • Establish primary microglial cultures from control and BIN1-manipulated animals .

  • Assess cellular responses to inflammatory stimuli (e.g., LPS challenge) .

• Transcriptomic profiling:

  • Perform RNA sequencing on isolated cell populations to identify cell-type specific transcriptional changes associated with BIN1 manipulation .

  • Use pathway analysis to identify key regulatory networks affected by BIN1 .

• Functional assays:

  • Measure cytokine production in response to inflammatory challenge .

  • Assess type 1 interferon responses, particularly focusing on key genes like IFITM3 .

• Co-expression analysis:

  • Use antibodies targeting different BIN1 epitopes along with cell-type specific markers to identify which isoforms are expressed in which cell types .

  • Correlate findings with mRNA expression data from purified cell populations .

These approaches enable detailed investigation of BIN1's role in specific CNS cell types, particularly microglia, which are critical for neuroinflammatory responses in AD.

How does BIN1 regulation affect microglial neuroinflammatory responses?

BIN1 serves as a key regulator of microglial neuroinflammatory responses through multiple mechanisms:

• Proinflammatory gene regulation: BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production .

• Pathway modulation: Transcriptomic profiling reveals that BIN1 regulates key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways .

• Transcription factor control: BIN1 regulates transcription factors PU.1 and IRF1, which are master regulators of microglial gene expression .

• Type 1 interferon response: Loss of BIN1 impairs the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly affecting the upregulation of IFITM3, a critical type 1 immune response gene .

• Disease-associated microglia (DAM) genes: Microglia-specific BIN1 conditional knockout in vivo reveals novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling .

• Inflammatory response blunting: Loss of BIN1 in vitro profoundly impairs microglial ability to respond to LPS, resulting in a blunted proinflammatory response measured by cytokine production and gene expression .

These findings position BIN1 as a central regulator of neuroinflammatory processes relevant to Alzheimer's disease pathogenesis.

What is the significance of BIN1 accumulation near amyloid deposits?

The accumulation of BIN1 near amyloid deposits has significant implications for understanding AD pathology:

• Consistent association: BIN1 immunoreactivity is associated with over 90% of amyloid deposits in multiple transgenic mouse models of AD (5XFAD, PDAPP, APP/PS1, and Tg21 mice) .

• Distinct morphology: BIN1 forms irregular-shaped edematous immunoreactive patches within or juxtaposed to amyloid deposits .

• Multiple model confirmation: This phenomenon has been observed across various transgenic models including mouse models and a transgenic rat model (APP Swe/PS1 ΔE9; line TgF344-AD) .

• Ultrastructural localization: High-resolution analysis using pre-embedding immunogold-EM reveals that BIN1 immunogold particles decorate the tips of, but are not within, Aβ fibrils .

• Multiple isoform involvement: Proteomics analysis indicates that multiple BIN1 isoforms, including the brain-specific isoform 1, become insoluble in mouse models of AD amyloidosis .

• Full-length protein accumulation: Evidence suggests that full-length BIN1 molecules, rather than cleavage products, accumulate with Aβ in deposits .

This consistent association between BIN1 and amyloid deposits across multiple models suggests a potential role for BIN1 in amyloid pathology, though the exact functional significance remains under investigation.

What therapeutic potential do anti-BIN1 antibodies show for Alzheimer's disease?

Anti-BIN1 antibodies demonstrate promising therapeutic potential for Alzheimer's disease:

• Tau turnover promotion: Cell-penetrating BIN1 antibodies developed by LIMR scientists appear to promote tau turnover, inhibiting its expression and cellular deposition .

• Tauopathy targeting: Anti-BIN1 antibodies have been developed as a strategy to target tauopathy-based pathology in AD .

• Survival benefits: A survival benefit has been observed in early tests of anti-BIN1 administration in a tauopathy-based mouse model of AD .

• Mechanistic rationale: With elevated BIN1 expression identified as a risk factor in late-onset AD development, targeting BIN1 represents a mechanistically sound approach .

• Gut-brain axis consideration: Given emerging evidence of gut-brain interactions in neurodegenerative diseases, anti-BIN1 antibodies originally developed for inflammatory bowel disease may have relevant therapeutic effects in AD .

• Development status: Current work is at a preclinical stage, including ongoing mechanism studies and antibody humanization . The technology has intellectual property protection through U.S. Patent 10,494,424 (issued December 3, 2019) .

These findings suggest that anti-BIN1 antibodies may offer a novel approach to limit the development or progression of AD pathophysiology, particularly by targeting tau-related mechanisms.

What are common pitfalls in BIN1 antibody-based experiments?

Researchers should be aware of several potential pitfalls when working with BIN1 antibodies:

• Epitope accessibility issues:

  • The mAb 99D antibody (targeting the Myc-binding domain) may fail to visualize peri-deposit BIN1 accumulation in certain tissue processing methods, despite working well for human BIN1 detection and in paraffin-embedded sections .

  • The previously reported lack of BIN1 staining near amyloid deposits may reflect epitope loss due to sample processing requirements .

• Isoform detection challenges:

  • Some antibodies cannot detect specific isoforms (e.g., Epitomics anti-BIN1 antibody does not detect the larger iso1) .

  • Using multiple antibodies targeting different domains is necessary for comprehensive isoform analysis .

• Tissue processing considerations:

  • BIN1 immunostaining appears more robust in brain sections cleared by the CLARITY method .

  • Different fixation and processing methods may yield contradictory results regarding BIN1 distribution .

• Contradictory findings interpretation:

  • Studies may report opposing results regarding BIN1 association with amyloid plaques due to methodology differences .

  • A closer examination of BIN1 immunoreactivity within and tangential to amyloid plaques is required for accurate characterization .

• Sample preparation effects:

  • SDS-insoluble brain fractions may contain different BIN1 isoform profiles compared to soluble fractions .

  • Full characterization requires analysis of both soluble and insoluble protein pools .

Addressing these challenges requires careful experimental design with appropriate controls and validation using multiple antibodies and techniques.

How can researchers validate BIN1 antibody specificity?

To ensure robust research findings, comprehensive validation of BIN1 antibody specificity is essential:

• Multiple antibody approach:

  • Use antibodies raised against non-overlapping epitopes (e.g., N-terminal BAR domain antibodies like N-19 and 2F11, and C-terminal SH3 domain antibodies like BSH3) .

  • Verify consistent patterns across antibodies from different host species .

• Isoform-specific validation:

  • Test antibodies against lysates containing known BIN1 isoforms .

  • Confirm that antibody reactivity matches expected molecular weights for specific isoforms .

• Knockout/knockdown controls:

  • Validate specificity using BIN1 knockout or knockdown tissues when available .

  • Include appropriate negative controls (primary antibody omission, isotype controls) .

• Cross-model validation:

  • Confirm findings across multiple transgenic models (e.g., 5XFAD, PDAPP, APP/PS1) .

  • Verify results in both mouse and human tissues when possible .

• Peptide competition:

  • Perform peptide competition assays to confirm epitope specificity .

  • Pre-adsorb antibodies with purified antigen to demonstrate specific binding .

• Orthogonal methods:

  • Correlate immunostaining results with proteomics or transcriptomic data .

  • Confirm antibody findings using mRNA expression analysis when possible .

These validation approaches ensure that findings related to BIN1 expression and localization are reliable and reproducible.

What advanced analytical methods can resolve contradictory findings about BIN1 in AD?

To address contradictory findings about BIN1 in Alzheimer's disease, researchers should employ these advanced analytical approaches:

• Single-cell analysis:

  • Use single-cell RNA sequencing to identify cell-type specific BIN1 isoform expression patterns .

  • Employ multiplexed immunofluorescence to correlate BIN1 expression with specific cell types in tissue sections .

• High-resolution imaging techniques:

  • Utilize STED super-resolution microscopy to precisely localize BIN1 in relation to amyloid fibrils .

  • Apply pre-embedding immunogold-EM for ultrastructural analysis of BIN1 distribution .

  • Consider tissue clearing methods like CLARITY that may preserve epitopes lost in traditional processing .

• Quantitative proteomics:

  • Employ mass spectrometry to identify specific BIN1 peptides corresponding to different isoforms .

  • Analyze both soluble and insoluble fractions to capture the complete BIN1 profile .

• Systems biology integration:

  • Correlate BIN1 alterations with other AD risk genes and pathways .

  • Analyze BIN1 in the context of broader transcriptomic networks in AD .

• Longitudinal analysis:

  • Study BIN1 changes across disease progression in animal models .

  • Consider temporal relationships between BIN1 alterations and other AD pathological features .

• Human genetic correlation:

  • Integrate findings with human genetic data, including the rs59335482 insertion/deletion variant associated with increased BIN1 mRNA expression .

  • Correlate protein findings with known BIN1 risk variants for AD .

These advanced analytical approaches can help resolve apparently contradictory findings by providing higher resolution data and more comprehensive context for understanding BIN1's complex role in AD.

What are the most promising avenues for targeting BIN1 in therapeutic development?

Several promising approaches for targeting BIN1 in AD therapeutic development warrant further investigation:

• Cell-penetrating antibody development:

  • Continue refinement and humanization of murine anti-BIN1 mAb exhibiting anti-tau properties .

  • Optimize delivery methods to ensure sufficient CNS penetration .

• Isoform-specific targeting:

  • Develop approaches that selectively target disease-associated BIN1 isoforms while preserving beneficial functions .

  • Focus on the increased iso9 and decreased iso1 pattern observed in AD .

• Microglial BIN1 modulation:

  • Target BIN1's role in regulating microglial proinflammatory responses .

  • Develop compounds that enhance type 1 interferon responses impaired by BIN1 loss .

• BIN1-tau interaction targeting:

  • Design small molecules or peptides that disrupt pathological BIN1-tau interactions .

  • Focus on the survival benefit observed in tauopathy models treated with anti-BIN1 .

• Combination approaches:

  • Explore synergistic effects of targeting BIN1 alongside other AD risk genes .

  • Investigate BIN1 modulation in combination with anti-amyloid or anti-tau therapies .

• Biomarker development:

  • Develop assays to measure specific BIN1 isoform ratios as potential biomarkers .

  • Correlate BIN1 alterations with disease progression and treatment response .

These approaches leverage the growing understanding of BIN1's multifaceted roles in AD pathogenesis to develop novel therapeutic strategies.

What research questions remain unanswered about BIN1's role in neurodegeneration?

Despite significant advances, several critical questions about BIN1's role in neurodegeneration remain unanswered:

• Causal relationships:

  • Does altered BIN1 expression cause neurodegeneration or result from it?

  • What mechanisms drive the isoform-specific changes observed in AD?

• Cell-type specific functions:

  • How do neuronal, astrocytic, and microglial BIN1 isoforms differentially contribute to disease?

  • Which cell type's BIN1 alterations are most critical for AD pathogenesis?

• Temporal dynamics:

  • When do BIN1 alterations first appear in relation to other AD pathological features?

  • Could BIN1 changes serve as early biomarkers for disease onset or progression?

• Mechanistic understanding:

  • How precisely does BIN1 regulate tau pathology at the molecular level?

  • What explains BIN1's accumulation near but not within amyloid fibrils?

• Genetic risk translation:

  • How do BIN1 genetic risk variants alter protein function or expression?

  • Why does the rs59335482 insertion/deletion variant increase BIN1 mRNA expression?

• Therapeutic targeting specificity:

  • How can therapeutic interventions selectively target pathological BIN1 functions while preserving normal functions?

  • What are the long-term consequences of BIN1 modulation?

Addressing these questions will require integrated approaches combining genetic, molecular, cellular, and systems-level analyses to fully elucidate BIN1's role in neurodegeneration.

How might emerging technologies advance BIN1 antibody research?

Emerging technologies offer exciting opportunities to advance BIN1 antibody research:

• Spatial transcriptomics and proteomics:

  • Map BIN1 isoform expression patterns with spatial resolution across brain regions .

  • Correlate BIN1 distribution with other AD-related proteins and pathological features .

• CRISPR-based approaches:

  • Generate isoform-specific knockouts to dissect functions of individual BIN1 variants .

  • Develop cellular and animal models with human AD-associated BIN1 variants .

• Advanced imaging technologies:

  • Apply expansion microscopy for improved visualization of BIN1 distribution .

  • Use multiplexed ion beam imaging (MIBI) or CyTOF imaging to simultaneously visualize multiple proteins in relation to BIN1 .

• Human iPSC-derived brain organoids:

  • Study BIN1 function in 3D human cellular models carrying AD risk variants .

  • Test anti-BIN1 therapeutic approaches in patient-derived organoids .

• Bispecific antibody development:

  • Create antibodies targeting both BIN1 and other AD-related proteins (tau, Aβ) .

  • Develop antibodies that can distinguish between specific BIN1 isoforms .

• In vivo molecular imaging:

  • Develop PET ligands to track BIN1 alterations in living subjects .

  • Create molecular imaging probes for monitoring treatment effects on BIN1 .

These emerging technologies will enable more precise characterization of BIN1's roles in health and disease and facilitate the development of targeted therapeutic approaches.