SEZ6L2 Antibody

What is SEZ6L2 and why is it important in neurological research?

SEZ6L2 (Seizure Related 6 Homolog Like 2) is a type 1 transmembrane protein predominantly expressed in the brain, particularly in the hippocampus and cerebellum. It belongs to the seizure-related gene 6 (SEZ6) family, which contains SEZ6, SEZ6L, and SEZ6L2. These proteins have significant implications in neuronal development and function, particularly influencing synapse numbers and dendritic morphology .

SEZ6L2 has gained importance in neurological research due to its role as part of the AMPA receptor complex, where it acts as a scaffolding protein linking GluR1 to adducin . This function suggests a crucial role in synaptic transmission and plasticity. Additionally, SEZ6L2 and other family members have been linked to various neurological and psychiatric disorders, making them important targets for neuroscience research .

What are the key characteristics of SEZ6L2 protein that researchers should be aware of?

Researchers working with SEZ6L2 should be familiar with several key characteristics:

When designing experiments involving SEZ6L2, researchers should account for these characteristics, particularly the difference between calculated and observed molecular weights due to glycosylation .

What types of SEZ6L2 antibodies are available for research applications?

Multiple types of SEZ6L2 antibodies are available for research, each with specific characteristics suitable for different applications:

Additionally, conjugated versions of these antibodies (APC, Biotin, FITC, PE, HRP) are available for specialized applications such as flow cytometry and immunohistochemistry . When selecting an antibody, researchers should consider both the application requirements and the species of their experimental model.

What are the optimal conditions for using SEZ6L2 antibodies in Western blot applications?

For optimal Western blot results with SEZ6L2 antibodies, researchers should follow these methodological guidelines:

For polyclonal antibodies like 19428-1-AP:

• Recommended dilution: 1:1000-1:4000

• Sample preparation: Use brain tissue lysates (mouse, rat, or human, depending on antibody specificity)

• Expected band size: ~150 kDa (due to glycosylation, despite the calculated 98 kDa)

• Conditions: Reducing conditions are recommended

• Buffer system: Immunoblot Buffer Group 1 has shown good results

• Membrane: PVDF membrane provides better results than nitrocellulose for this protein

• Secondary antibody: Use species-appropriate HRP-conjugated secondary antibodies (anti-rabbit for polyclonal rabbit antibodies)

For monoclonal antibodies like MAB4916:

• Recommended concentration: 2 μg/mL

• Detection method: For best sensitivity, use HRP-conjugated secondary antibody with appropriate chemiluminescent substrate

Note that titration is necessary for each specific experimental system to obtain optimal results, as antibody performance can vary based on sample type and preparation method .

How can SEZ6L2 antibodies be effectively used in immunohistochemistry (IHC)?

For effective immunohistochemistry with SEZ6L2 antibodies, researchers should consider these methodological approaches:

For fixed frozen sections (particularly effective for neuronal tissues):

• Tissue preparation: Perfusion-fixed frozen sections of brain tissue provide optimal results

• Antibody concentration: 25 μg/mL has shown good results with monoclonal antibodies

• Incubation conditions: Overnight at 4°C for primary antibody

• Detection system: Anti-Rat HRP-DAB staining kit is recommended for rat monoclonal antibodies

• Counterstaining: Hematoxylin provides good nuclear contrast

• Regions of interest: Hippocampus shows strong SEZ6L2 expression and is ideal for positive controls

Additional considerations for paraffin-embedded tissues:

• Antigen retrieval: May be necessary to unmask epitopes after formalin fixation

• Background reduction: Use appropriate blocking solutions to minimize non-specific binding

• Controls: Include both positive controls (brain tissue) and negative controls (tissue without SEZ6L2 expression or primary antibody omission)

The specific protocol should be optimized based on the particular antibody and tissue being used, as fixation methods can significantly impact antibody performance in IHC applications.

What methods are used to detect anti-SEZ6L2 autoantibodies in clinical samples?

Detection of anti-SEZ6L2 autoantibodies in clinical samples involves several complementary techniques:

• Cell-Based Assay (CBA):

  • HEK293 cells transfected with SEZ6L2 are used as the primary detection method

  • Patient serum or CSF is incubated with the transfected cells

  • Bound antibodies are detected using fluorescently-labeled secondary antibodies

  • This is considered the gold standard method for detecting conformational epitope recognition

• Immunohistochemistry on rat brain sections:

  • Patient samples are applied to rat brain tissue sections (particularly cerebellum)

  • This helps confirm binding patterns consistent with SEZ6L2 distribution

  • Positive samples show characteristic patterns in cerebellar regions

• Immunoprecipitation from rat cerebellar neurons:

  • Can be used to confirm specificity of antibody binding

  • Helps identify the molecular weight of the target protein

• Immunoblot analysis:

  • Using purified FLAG-tagged SEZ6L2 protein

  • Confirms specificity and helps distinguish from other potential autoantibodies

For clinical diagnostic purposes, a titer of >1:10 in serum is typically considered positive for anti-SEZ6L2 antibodies, with some paraneoplastic cases showing titers as high as 1:1000 .

What is the relationship between SEZ6L2 antibodies and autoimmune cerebellar syndromes?

Anti-SEZ6L2 antibodies have been identified as causative agents in a recently discovered form of autoimmune cerebellar syndrome. The relationship is characterized by several key features:

Clinical presentation:

• Patients typically present with subacute gait ataxia, dysarthria, and mild extrapyramidal symptoms

• Median age of affected patients is around 62 years (range: 54-69), with no strong gender predominance

• Initial brain MRI may be normal, though some cases show cerebellar atrophy

• CSF analysis may show pleocytosis in some patients, while others have normal cell counts

Pathophysiological mechanism:

• SEZ6L2 antibodies recognize conformational epitopes on the protein

• IgG4 is the predominant antibody subclass in many patients

• The antibodies may disrupt the interaction between SEZ6L2 and AMPA glutamate receptor 1 (GluA1), as these proteins are binding partners

• This disruption potentially affects synaptic function in the cerebellum, leading to ataxia

Response to treatment:

• Limited response to conventional immunotherapy has been observed in some cases

• In paraneoplastic cases, treatment of the underlying malignancy has shown improvement in both neurological symptoms and antibody titers

This syndrome represents an important addition to the growing spectrum of antibody-mediated neurological disorders, with implications for diagnosis and management of patients with unexplained cerebellar ataxia.

How are SEZ6L2 antibodies implicated in paraneoplastic neurological syndromes?

SEZ6L2 antibodies have emerging significance in paraneoplastic neurological syndromes, with evidence from clinical cases and research:

Oncological associations:

• Anti-SEZ6L2 antibodies have been detected in patients with breast cancer presenting with cerebellar syndrome

• SEZ6L2 protein is overexpressed in various malignancies, including lung cancer, hepatocellular carcinoma, thyroid carcinoma, and cholangiocarcinoma

• This overexpression may trigger an autoimmune response targeting the protein

Clinical features of paraneoplastic anti-SEZ6L2 syndrome:

• Cerebellar ataxia with subacute onset

• MRI shows cerebellar atrophy particularly affecting the vermis and hemispheres

• High antibody titers (e.g., 1:1000) in serum

• Associated conditions may include recurrent pancreatitis (potentially linked to SEZ6L2 expression in pancreatic islet cells)

Treatment approach:

• Tumor-directed therapy has shown significant improvement in both neurological symptoms and SEZ6L2 antibody titers

• Immunotherapy alone may have limited efficacy

• Combined approaches with both tumor treatment and immunomodulation (IVIG, rituximab) have been used

These findings highlight the importance of thorough cancer screening in patients with unexplained cerebellar ataxia and positive anti-SEZ6L2 antibodies, particularly in older adults.

How do experimental conditions affect SEZ6L2 antibody performance in neuronal culture studies?

When using SEZ6L2 antibodies in neuronal culture experiments, several experimental conditions critically influence antibody performance and experimental outcomes:

Culture system considerations:

• Primary hippocampal neurons provide an optimal model given the high expression of SEZ6L2 in this region

• Timing of culture maturation is important, as SEZ6L2 expression patterns may change during neuronal development

• Fixation methods significantly impact epitope accessibility and antibody binding

Antibody application parameters:

• Antibody concentration: Requires careful titration for each experiment, with effective ranges typically between 1-25 μg/mL depending on the application

• Incubation time: Longer incubation periods (overnight at 4°C) often yield better results than short incubations at room temperature

• Blocking agents: Must be optimized to reduce background without interfering with specific binding

Readout considerations:

• Studies examining SEZ6L2 effects on synaptic cluster formation require high-resolution imaging methods

• When assessing the impact on AMPA receptor clustering, control experiments examining total and synaptic SEZ6L2 or GluA1 clusters are essential

• SEZ6L2 antibodies do not appear to alter the number of total or synaptic SEZ6L2 or GluA1 clusters on hippocampal neuron surfaces in some experimental paradigms

These methodological considerations are crucial for experiments investigating the role of SEZ6L2 in synapse formation, neurotransmitter receptor trafficking, or potential pathogenic mechanisms of anti-SEZ6L2 autoantibodies.

What are the key differences between research-grade SEZ6L2 antibodies and autoantibodies found in patients?

Research-grade SEZ6L2 antibodies and autoantibodies found in patients differ in several important aspects that researchers must consider:

Understanding these differences is crucial when designing experiments to study the pathogenic mechanisms of SEZ6L2 autoantibodies or when developing diagnostic assays for clinical applications.

How can researchers distinguish between the effects of different SEZ6 family members in experimental systems?

Distinguishing between the effects of SEZ6, SEZ6L, and SEZ6L2 in experimental systems requires careful methodological approaches:

Antibody selection strategies:

• Use antibodies validated for specificity against individual family members

• Test for cross-reactivity with all three family members using recombinant proteins

• Consider epitope mapping to ensure targeting of non-conserved regions

Genetic manipulation approaches:

• Gene knockout or knockdown should be validated by confirming specific reduction of target protein without affecting other family members

• Rescue experiments with individual family members can confirm specificity of observed phenotypes

• CRISPR/Cas9 genome editing allows for precise targeting of specific family members

Expression pattern analysis:

• Quantitative comparisons of expression levels across brain regions can help distinguish roles

• SEZ6L2 shows particularly strong expression in hippocampus and cerebellum

• Temporal expression patterns during development may differ between family members

Functional assays:

• SEZ6L2-specific function in AMPA receptor modulation can be assessed through GluA1 binding assays

• Comparative analysis of synaptic clustering between family members can reveal specific roles

• Electrophysiological recordings combined with specific antibody or genetic manipulation can identify distinct functional impacts

By implementing these approaches, researchers can minimize confounding effects from related family members and isolate the specific contributions of SEZ6L2 to neuronal function and pathology.

What is the potential role of SEZ6L2 antibodies in cancer research and diagnostics?

SEZ6L2 antibodies are emerging as important tools in cancer research and potential diagnostic applications, based on several lines of evidence:

Oncological relevance:

• SEZ6L2 is overexpressed in multiple cancer types, including lung cancer, hepatocellular carcinoma, thyroid carcinoma, and cholangiocarcinoma

• This overexpression may serve as a negative prognostic marker in several tumor entities

• In breast cancer, SEZ6L2 overexpression has been linked to paraneoplastic neurological syndromes

Research applications:

• SEZ6L2 antibodies enable detection and quantification of this protein in tumor specimens

• Immunohistochemical analysis with these antibodies can help assess expression patterns across different cancer types and stages

• Western blot applications can quantify expression levels and correlate with clinical outcomes

Potential diagnostic applications:

• Detection of anti-SEZ6L2 autoantibodies in patient serum may serve as a biomarker for occult malignancies

• In patients with unexplained cerebellar ataxia, testing for these antibodies could trigger appropriate cancer screening

• The presence of SEZ6L2 antibodies in tumor tissue might help classify tumors for therapeutic decisions

Therapeutic implications:

• In mouse models of lung adenocarcinoma, anti-SEZ6L2 antibodies showed positive effects on drug resistance and metastasis

• This suggests potential therapeutic applications beyond diagnostic uses

• Further research is needed to determine if targeting SEZ6L2 could represent a novel treatment approach

These findings highlight the expanding role of SEZ6L2 antibodies beyond neuroscience research into the field of oncology.

How do different methodologies for detecting SEZ6L2 antibodies compare in sensitivity and specificity?

Various methodologies for detecting SEZ6L2 antibodies exhibit different performance characteristics that researchers should consider:

For research applications, Western blot and immunohistochemistry using validated antibodies provide complementary information about SEZ6L2 expression . For clinical detection of autoantibodies, a combination of CBA and tissue immunohistochemistry offers the best balance of sensitivity and specificity .

Japanese researchers have validated that combining CBA with immunoblot analysis using FLAG-SEZ6L2 provides superior specificity in distinguishing SEZ6L2 autoantibodies from other autoantibodies in cerebellar ataxia patients .

What are the current hypotheses regarding the pathogenic mechanisms of anti-SEZ6L2 antibodies in neurological disorders?

Current research suggests several hypotheses regarding the pathogenic mechanisms of anti-SEZ6L2 antibodies in neurological disorders:

• Disruption of SEZ6L2-AMPA receptor interaction:

  • SEZ6L2 directly binds to AMPA glutamate receptor 1 (GluA1)

  • Anti-SEZ6L2 antibodies may inhibit this binding

  • This disruption could impair glutamatergic neurotransmission, particularly in the cerebellum

• Interference with synaptic organization:

  • SEZ6 family members influence synapse numbers and dendritic morphology

  • Anti-SEZ6L2 antibodies might disrupt these functions, leading to impaired synaptic transmission

  • This could explain the cerebellar ataxia observed in affected patients

• Altered cathepsin D transport:

  • SEZ6L2 plays a role in cathepsin D transport

  • Disruption of this function could lead to abnormal protein processing and degradation

  • This might contribute to neurodegeneration rather than a purely autoimmune mechanism

• Complement activation:

  • SEZ6 proteins are involved in complement regulation

  • Anti-SEZ6L2 antibodies might interfere with this function

  • Inappropriate complement activation could contribute to neuronal damage

• Direct cytotoxicity:

  • In some cases, anti-neuronal antibodies can cause direct cellular damage

  • The predominance of IgG4 subclass in many patients argues against this mechanism, as IgG4 typically does not activate complement or trigger antibody-dependent cellular cytotoxicity

As noted by researchers, "There is evidence for direct pathogenicity of the anti-SEZ6L2 antibodies in the development of cerebellar syndrome. Nonetheless, considering the various roles SEZ6L2 plays as part of the AMPA receptor and in cathepsin D transport, a degenerative mechanism could also be involved. Further research is needed to address this question."