LRP10 Antibody

What is LRP10 and why is it significant in neurological research?

LRP10 is a 76.2 kDa single-pass transmembrane protein belonging to the LDLR family with 713 amino acid residues in its canonical form. Its significance in neurological research has grown substantially with the identification of potentially pathogenic variants in the encoding gene in autosomal dominant forms of Parkinson's disease (PD), Parkinson's disease-dementia (PDD), and dementia with Lewy bodies (DLB). LRP10 accumulates in α-synuclein-positive neuronal Lewy bodies in diseased brains, despite typically being expressed in non-neuronal cells like astrocytes and neurovasculature in healthy individuals. This unusual localization pattern suggests LRP10 may play a role in disease pathogenesis, possibly through interaction with α-synuclein and participation in protein aggregation mechanisms . Recent studies have linked LRP10 function to intracellular vesicle transport pathways via its interactions with clathrin adaptors and the retromer complex, suggesting it might influence protein trafficking and degradation in neurodegenerative disorders .

What are the known isoforms of LRP10 and how can antibodies distinguish between them?

Human LRP10 has up to two reported isoforms with potential functional differences . Additionally, pathogenic variants such as the c.1424 + 5G > A LRP10 variant can produce truncated forms that may have dominant negative effects on wild-type LRP10 . When selecting antibodies, researchers should determine the epitope location and whether the antibody can detect all relevant isoforms or distinguish between them. Antibodies targeting different domains of LRP10 (N-terminal vs. C-terminal) may produce distinct staining patterns depending on the isoforms present in the sample. For studies involving patient-derived tissues with LRP10 variants, it is essential to verify whether the antibody can detect both wild-type and variant forms of the protein. Western blotting with appropriate controls should be performed to confirm antibody specificity for the isoforms of interest before proceeding with more complex applications.

What are the optimal methods for detecting LRP10 in neuronal tissue samples?

For detecting LRP10 in neuronal tissues, a multi-modal approach is recommended. Immunohistochemistry (IHC) with validated antibodies is essential for localizing LRP10 within specific cell types and subcellular compartments. When examining potential co-localization with Lewy bodies, double immunofluorescence staining with anti-α-synuclein and anti-LRP10 antibodies provides valuable spatial information . For quantitative analysis, Western blotting allows detection of different LRP10 species, including potential truncated forms resulting from splice variants. When performing Western blots, it's critical to optimize protein extraction conditions, as LRP10 is a membrane protein with post-translational modifications including glycosylation . For challenging samples like brain tissues, consider using specialized extraction buffers containing appropriate detergents (e.g., RIPA buffer supplemented with deoxycholate) to efficiently solubilize membrane-bound LRP10. Additionally, deglycosylation treatments may help resolve complex banding patterns when analyzing LRP10 by Western blot.

How should researchers optimize Western blot protocols specifically for LRP10?

Optimizing Western blot protocols for LRP10 requires addressing several protein-specific challenges. Begin with efficient extraction using buffers containing 1% Triton X-100 or RIPA buffer supplemented with protease inhibitors to prevent degradation. Given LRP10's transmembrane nature and glycosylation status, sample preparation should include either membrane enrichment protocols or total protein extraction with special attention to solubilization efficiency . During electrophoresis, use gradient gels (4-12% or 4-15%) to effectively resolve the full-length 76.2 kDa protein along with any truncated forms or splice variants, such as those resulting from the c.1424 + 5G > A mutation described in LBD patients . For transfer, semi-dry systems may be insufficient; use wet transfer systems with methanol-containing buffers for efficient transfer of this hydrophobic protein. During antibody incubation, extend primary antibody incubation times (overnight at 4°C) and optimize blocking conditions (5% BSA often performs better than milk for membrane proteins). When interpreting results, be aware that glycosylation can cause LRP10 to appear at higher molecular weights than predicted, and patient-derived samples may show aberrant high-molecular-weight species or truncated forms that require careful analysis and comparison with appropriate controls .

What approaches are effective for studying LRP10 trafficking and secretion mechanisms?

Studying LRP10 trafficking and secretion requires specialized techniques that preserve the protein's native localization and movement patterns. Live-cell imaging using fluorescently tagged LRP10 constructs (such as mCherry-LRP10) allows real-time visualization of trafficking events . When designing such constructs, consider whether N-terminal or C-terminal tags might interfere with trafficking signals, as mentioned in the literature where specific plasmids like pcDNATM3.1-mCherry-LRP10 have been successfully employed . For extracellular vesicle (EV) isolation to study LRP10 secretion, differential ultracentrifugation followed by density gradient separation provides the cleanest preparations. The literature indicates that LRP10 is secreted via EVs and this process is highly sensitive to autophagy inhibition by compounds such as Bafilomycin A1 .

When studying endocytosis of LRP10, clathrin-dependent mechanisms should be specifically examined, as wild-type LRP10 has been demonstrated to be internalized via this pathway . Pharmacological inhibitors of endocytic pathways (e.g., dynasore for dynamin-dependent processes) can help dissect the specific routes of LRP10 internalization. For studying autophagy effects on LRP10, researchers should note that Bafilomycin A1 treatment induced the formation of atypical LRP10 vesicular structures in neurons in brain organoids, suggesting a critical role for autophagy in LRP10 processing .

How can LRP10 antibodies be utilized to investigate its role in α-synuclein pathology?

Investigating LRP10's role in α-synuclein pathology requires multifaceted approaches centered around high-quality antibodies. Co-immunoprecipitation experiments using LRP10 antibodies can capture protein complexes containing both LRP10 and α-synuclein, helping to characterize their physical interactions in different cellular contexts. When designing such experiments, use gentle lysis conditions that preserve protein-protein interactions while ensuring sufficient solubilization of membrane-bound LRP10 . Proximity ligation assays (PLA) offer another powerful approach to visualize and quantify LRP10-α-synuclein interactions in situ with nanometer resolution.

For investigating how LRP10 influences α-synuclein levels and distribution, researchers can employ LRP10 overexpression or knockdown approaches followed by comprehensive α-synuclein analysis. Recent work has shown that LRP10 overexpression leads to time-dependent changes in intracellular α-synuclein levels and strongly promotes α-synuclein secretion . When performing these experiments, consider monitoring both intracellular and extracellular α-synuclein using antibodies against different α-synuclein forms (monomeric, oligomeric, phosphorylated). This approach revealed that LRP10 overexpression specifically induces monomeric α-synuclein secretion, potentially through ER and proteasomal pathways . Additionally, patient-derived cell models carrying LRP10 variants can provide valuable insights into pathological mechanisms, as demonstrated by studies showing that astrocytes carrying the c.1424 + 5G > A LRP10 variant secrete aberrant high-molecular-weight LRP10 species in EV-free media fractions .

What strategies should be employed when investigating LRP10 variants associated with neurological disorders?

Investigating LRP10 variants requires a comprehensive genetic and protein-level analysis strategy. Begin with genetic screening to identify variants of interest, such as the c.1424 + 5G > A variant associated with Lewy body diseases . For protein-level analysis, use antibodies that can detect both wild-type and variant forms, preferably selecting multiple antibodies targeting different epitopes to ensure complete coverage. When studying the pathogenic mechanisms of LRP10 variants, consider their potential dominant-negative effects. Research has shown that truncated patient-derived LRP10 protein species (LRP10 splice) can bind to wild-type LRP10, reduce wild-type LRP10 levels, and antagonize its effect on α-synuclein distribution .

To model variant effects in vitro, several approaches can be employed: (1) Overexpression of variant forms using constructs like pLVX-EF1α-LRP10 splice-IRES-NeoR or doxycycline-inducible systems like pCW57-LRP10 splice-2A-PuroR ; (2) CRISPR/Cas9 genome editing to introduce specific variants into cellular models; (3) Patient-derived iPSCs differentiated into relevant cell types. For functional studies, assess how variants affect LRP10's established functions: α-synuclein modulation, vesicular trafficking, and potential roles in autophagy. Advanced imaging techniques, such as super-resolution microscopy, can reveal subtle differences in subcellular localization between wild-type and variant LRP10. Additionally, proteomics approaches can identify altered interaction partners or post-translational modifications resulting from pathogenic variants.

How do experimental conditions affect LRP10 detection in different cellular compartments?

Experimental conditions significantly impact the detection of LRP10 across cellular compartments due to its complex trafficking patterns and sensitivity to cellular stress. For fixation methods, paraformaldehyde (typically 4%) is generally suitable, but membrane proteins like LRP10 may require optimization of fixation time to prevent epitope masking while maintaining membrane structure. Permeabilization agents should be selected based on the cellular compartment of interest: Triton X-100 provides thorough permeabilization for detecting intracellular LRP10, while milder detergents like saponin better preserve membrane structures when examining transmembrane regions .

LRP10 detection is particularly sensitive to cellular stress conditions. Research has shown that autophagy inhibition with Bafilomycin A1 induces the formation of atypical LRP10 vesicular structures in neurons . When designing experiments involving stress conditions, consider the following:

For subcellular fractionation studies, traditional protocols may need optimization as LRP10 distributes across multiple compartments, including the plasma membrane, endosomes, and potential association with the retromer complex. When isolating extracellular vesicles to study secreted LRP10, differential ultracentrifugation followed by density gradient separation provides the cleanest preparations, minimizing contamination with non-vesicular proteins .

What are the critical considerations when designing LRP10 knockout or knockdown experiments?

Designing effective LRP10 knockout or knockdown experiments requires careful consideration of several factors. For CRISPR/Cas9-mediated knockout approaches, guide RNA selection is critical. Prior research has utilized constructs such as pSpCas9-PuroR-LRP10gRNA to generate LRP10 knockout neuronal precursor cells (NPCs) . When designing guide RNAs, target conserved exons present in all splice variants to ensure complete knockout, while avoiding regions with high homology to other LDLR family members to prevent off-target effects.

For knockdown approaches using siRNA or shRNA, validate efficiency at both mRNA and protein levels, as LRP10 may have tissue-specific expression patterns and post-transcriptional regulation. When phenotyping LRP10 knockout/knockdown models, focus on pathways implicated in LRP10 function:

• α-synuclein processing and secretion: Recent work shows that LRP10 overexpression induces changes in α-synuclein intracellular levels over time and strongly promotes α-synuclein secretion .

• Vesicular trafficking: Assess endocytosis efficiency and clathrin-dependent pathways, as wild-type LRP10 is internalized via clathrin-dependent endocytosis .

• Autophagy: Given LRP10's sensitivity to autophagy inhibition, investigate autophagic flux in knockout models .

• T-cell homeostasis: For immunological studies, examine CD8 T-cell accumulation in peripheral lymphoid organs, as Lrp10 deletion enhances T-cell homeostatic expansion through IL7R signaling .

Importantly, consider potential compensatory mechanisms from other LDLR family members when interpreting results from long-term knockout models. For some applications, inducible knockout systems may provide advantages by allowing temporal control of LRP10 depletion, minimizing compensatory adaptations.

How can researchers utilize LRP10 antibodies to investigate its emerging role in T-cell regulation?

Recent discoveries have revealed LRP10's unexpected role in T-cell biology, particularly in regulating CD8 T-cell homeostasis and anti-tumor responses. When investigating this function, researchers should begin with comprehensive immune phenotyping of peripheral lymphoid organs from Lrp10-deficient models, focusing on CD8 T-cell subsets. Flow cytometry with LRP10 antibodies can verify knockout efficiency while simultaneously characterizing naive and central memory CD8 T-cell populations, which accumulate in peripheral lymphoid organs of Lrp10-deficient mice . For mechanistic studies exploring how Lrp10 regulates IL7 receptor expression, co-immunoprecipitation experiments using LRP10 antibodies can identify potential direct interactions with IL7R or intermediate signaling molecules.

T-cell activation experiments should incorporate temporal analysis of LRP10 expression, as T-cell activation induces Lrp10 expression, which then post-translationally suppresses IL7 receptor levels . When designing tumor studies, focus on highly immunogenic models (like MC38), where Lrp10-deficient mice demonstrate intrinsic resistance, rather than "immunologically cold" tumors (like B16F10), where no significant differences were observed . Tumor infiltration analysis should comprehensively characterize TILs (tumor-infiltrating lymphocytes), as the enhanced tumor resistance in Lrp10-deficient mice depends on CD8 T-cell infiltration. These cells display increased memory characteristics and reduced terminal exhaustion, suggesting potential implications for immunotherapy . Additionally, adoptive transfer experiments can distinguish between cell-intrinsic and microenvironment effects on T-cell function in Lrp10-deficient settings.

What protocols are recommended for investigating LRP10 in immune vs. neurological contexts?

Investigating LRP10 across immune and neurological contexts requires tailored approaches for each tissue environment. For neurological tissues, where LRP10 primarily localizes to astrocytes and neurovasculature in healthy controls but appears in neuronal Lewy bodies in disease states, multiplexed immunofluorescence with cell-type specific markers (GFAP for astrocytes, NeuN for neurons) alongside LRP10 antibodies enables precise cellular localization . In contrast, immunological tissues require flow cytometric analysis of freshly isolated cells, particularly when examining LRP10's role in T-cell biology.

For protein extraction, neurological tissues typically require stronger solubilization methods due to the presence of protein aggregates and lipid-rich environments. Immunoprecipitation protocols must be optimized differently: in neurological contexts, focus on potential interactions with α-synuclein and vesicular trafficking machinery , while in immunological contexts, investigate interactions with IL7R signaling components .

For functional studies, neurological investigations should center on protein secretion, vesicular trafficking, and autophagy processes, as LRP10 secretion is highly sensitive to autophagy inhibition . In contrast, immunological studies should examine homeostatic proliferation and IL7R-dependent signaling, as Lrp10 deletion enhances T-cell homeostatic expansion through IL7R signaling . A novel approach combining both fields would be to investigate whether LRP10's role in vesicular trafficking directly impacts immune receptor expression and turnover, potentially explaining its regulatory effect on IL7R levels.

What are the common false positives/negatives in LRP10 antibody applications and how can they be addressed?

False negatives commonly occur due to epitope masking in fixed tissues, particularly problematic for membrane proteins like LRP10. Test multiple antigen retrieval methods, including heat-induced epitope retrieval (citrate or EDTA-based) and enzymatic methods. Additionally, LRP10's sensitivity to autophagy inhibition and stress conditions can alter its detection profile . When analyzing patient samples, be aware that LRP10 variants like c.1424 + 5G > A produce truncated forms that may not be recognized by antibodies targeting C-terminal epitopes . For such scenarios, use antibodies targeting preserved regions or employ multiple antibodies to different epitopes. Western blotting validation should include positive controls from LRP10-overexpressing cells using plasmids like pLVX-EF1α-LRP10-IRES-NeoR to confirm the correct molecular weight pattern .

How can researchers validate the specificity of LRP10 antibodies across different experimental systems?

Validating LRP10 antibody specificity requires a multi-tiered approach across experimental systems. Begin with Western blot validation using positive controls (LRP10-overexpressing cells) and negative controls (LRP10 knockout cells or siRNA knockdown) . Use protein expression systems with plasmids like pLVX-EF1α-LRP10-IRES-NeoR or pCMVsport6-SNCA for overexpression controls . For knockout controls, CRISPR/Cas9 approaches with constructs like pSpCas9-PuroR-LRP10gRNA have been successfully employed .

For immunocytochemistry and immunohistochemistry validation, perform parallel staining in LRP10-overexpressing and knockout systems, examining subcellular localization patterns for consistency with LRP10's known distribution in the membrane and vesicular compartments . When working with patient samples, particularly those with potential LRP10 variants, validate antibody recognition of both wild-type and variant forms. This is especially important for variants like c.1424 + 5G > A that produce truncated proteins .

Cross-species validation is essential when working with animal models, as LRP10 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, and chimpanzee . Ensure that your selected antibody recognizes the species-specific form by testing in appropriate controls. For functional validation, assess whether the antibody can detect changes in LRP10 levels or localization under conditions known to affect LRP10, such as autophagy inhibition with Bafilomycin A1, which induces the formation of atypical LRP10 vesicular structures .

What strategies help resolve discrepancies between protein and mRNA expression data for LRP10?

Resolving discrepancies between LRP10 protein and mRNA expression requires understanding the potential post-transcriptional and post-translational regulatory mechanisms affecting this protein. Several methodological approaches can help address these discrepancies:

• Temporal analysis: LRP10 mRNA and protein levels may not correlate due to differences in half-life. Monitor both over time, particularly after stimuli like T-cell activation, which induces Lrp10 expression .

• Post-transcriptional regulation: Investigate potential microRNA regulation of LRP10 mRNA stability or translation efficiency by examining binding sites in the 3'UTR region.

• Post-translational regulation: LRP10 undergoes glycosylation and may experience regulated protein degradation. Examine these modifications using enzymatic deglycosylation followed by Western blotting, and assess protein stability using cycloheximide chase experiments.

• Splicing analysis: The c.1424 + 5G > A variant affects splicing, producing truncated LRP10 forms . Use RT-PCR with primers flanking potential splice sites to detect alternative transcripts not captured by standard qPCR.

• Compartmentalization effects: LRP10 secretion via EVs may create discrepancies between cellular protein levels and mRNA expression . Analyze both cellular and secreted fractions when comparing to mRNA data.

• Cell type heterogeneity: In tissues like brain, where LRP10 expression differs between cell types (astrocytes vs. neurons) , bulk RNA measurements may not match protein localization by immunohistochemistry. Use single-cell approaches or cell sorting to resolve these discrepancies.

• Detection antibody specificity: Ensure antibodies can detect all relevant isoforms or variants to avoid false discrepancies with mRNA data .

By systematically addressing these factors, researchers can better understand the complex relationship between LRP10 mRNA and protein expression in both physiological and pathological contexts.