NEUROD2 Antibody

What is NEUROD2 and why is it significant in neuroscience research?

NEUROD2 (Neurogenic Differentiation Factor 2) is a highly conserved basic helix-loop-helix (bHLH) transcription factor that plays crucial roles in neuronal development and function. It operates in tandem with other proteins like NEUROD1 to guide neural precursor cells toward differentiation into mature neurons . NEUROD2 is particularly significant because it's highly expressed during development and remains present in postmitotic neurons in adulthood, notably in the cerebellum, hippocampus, and cerebral cortex . Recent research has revealed that disruptions in NEUROD2 are associated with neurodevelopmental disorders including intellectual disability and autism spectrum disorders, making it an important focus for researchers investigating brain development and pathology .

What types of NEUROD2 antibodies are available for research applications?

Multiple NEUROD2 antibodies are available for research purposes, with different species origins and applications. Common examples include:

• Rabbit polyclonal antibodies such as ab104430 that have been validated for Western blot and immunohistochemistry on paraffin sections (IHC-P)

• Affinity-purified antibodies like Synaptic Systems' 498 003, which reacts with mouse and rat NEUROD2

When selecting an antibody, researchers should consider:

• The species reactivity (most are validated for mouse and rat tissues)

• The specific applications (Western blot, IHC-P, immunofluorescence)

• The immunogen used (typically recombinant proteins corresponding to specific regions of NEUROD2)

• Validation status in the specific experimental context you intend to use it

How does NEUROD2 function as a transcription factor and what are its target genes?

NEUROD2 functions as a bHLH transcription factor that typically acts as a heterodimer with other bHLH transcription factors. It can induce transcription from neuron-specific promoters containing conserved E-box DNA sequences . As a transcriptional regulator, NEUROD2 influences the expression of genes involved in neuronal excitability, synaptic transmission, and neurodevelopmental processes .

RNA sequencing studies in NEUROD2 knockout mice have revealed dysregulated expression of many genes associated with neuronal excitability and synaptic function. Interestingly, the human orthologs of these genes show strong associations with autism spectrum disorders (ASD) . Some of the target genes identified through ChIP-Seq analysis include those related to:

• Neuronal migration and positioning

• Synaptic structure and function

• Ion channels and neuronal excitability

• Cell survival pathways

What are the optimal protocols for using NEUROD2 antibodies in Western blot applications?

When using NEUROD2 antibodies for Western blot applications, researchers should consider the following protocol elements based on validated approaches:

Protein Preparation and Loading:

• Use reducing conditions for sample preparation

• Be aware that NEUROD2 has a predicted band size of 41 kDa, but observed bands may appear at 21 kDa, 41 kDa, and 50 kDa

Antibody Dilutions and Detection:

• For primary antibody: Dilution factors vary by antibody source; for example, some rabbit polyclonals have been validated at 1:5000 dilution

• For secondary antibody: Anti-rabbit IgG H&L (HRP) preadsorbed has been used successfully at 1:5000 dilution

• Detection method: ECL technique is commonly used

• Exposure time: May require extended exposure (4+ minutes for some applications)

Expected Results:

• Multiple bands may be observed (21 kDa, 41 kDa, 50 kDa)

• Verify specificity using NEUROD2 knockout tissues as negative controls

How should NEUROD2 antibodies be used for immunohistochemistry on brain tissue sections?

For optimal immunohistochemistry on brain tissue sections, the following protocol elements are recommended:

Tissue Preparation:

• Deparaffinize sections if working with paraffin-embedded tissues

• Perform heat-mediated antigen retrieval using citric acid

Blocking and Antibody Incubation:

• Block sections using 1% BSA at room temperature (21°C) for approximately 10 minutes

• Dilute primary antibody appropriately (e.g., 1:1000-1:2000 for certain rabbit polyclonals)

• Incubate with primary antibody at room temperature for approximately 2 hours

Region-Specific Considerations:

• CA1 hippocampus sections have been successfully stained with NEUROD2 antibodies in both mouse and rat tissues

• When studying cortical regions, be aware that NEUROD2 expression patterns may vary across developmental stages and cortical layers

Controls:

• Always include negative controls (primary antibody omission)

• If possible, include tissue from NEUROD2 knockout animals as definitive negative controls

• Consider dual-labeling with neuronal markers to confirm specificity in expected cell populations

What considerations are important when studying NEUROD2 expression during neurodevelopment?

When investigating NEUROD2 expression during neurodevelopment, researchers should consider:

Developmental Timeline:

• NEUROD2 is highly expressed during development but also remains present in postmitotic neurons in adulthood

• Expression patterns change throughout neurodevelopment, so precise developmental staging is critical

Regional Specificity:

• NEUROD2 has region-specific roles in the cerebellum, hippocampus, cerebral cortex, and amygdala

• Different brain regions may show distinct temporal patterns of NEUROD2 expression

Experimental Models:

• In vitro neuronal cultures: Time your experiments to capture relevant developmental windows

• In vivo studies: Consider both embryonic and postnatal timepoints

• Mouse models: Be aware that complete NEUROD2 knockout mice have severe neurodevelopmental defects and poor survival

Functional Roles:

• NEUROD2 contributes to terminal neuron localization within the cortical plate

• It influences neuronal migration, laminar positioning, and structural synaptic maturation of cortical projection neurons

• It plays roles in amygdala development and emotional learning

What are common issues when detecting NEUROD2 by immunostaining and how can they be resolved?

Problem: Weak or Absent Signal

• Solution approaches:

  • Optimize antigen retrieval: Heat-mediated retrieval using citric acid has been successful for NEUROD2 detection

  • Increase antibody concentration: Try a range of dilutions (e.g., 1:500-1:2000)

  • Extend primary antibody incubation time: Consider overnight incubation at 4°C instead of 2 hours at room temperature

  • Ensure tissue quality: Poor fixation or overfixation can mask epitopes

  • Try signal amplification methods (e.g., tyramide signal amplification)

Problem: High Background

• Solution approaches:

  • Increase blocking time or concentration (e.g., use 5% normal serum)

  • Reduce primary antibody concentration

  • Add detergents like Triton X-100 (0.1%) to reduce non-specific binding

  • Lengthen washing steps

  • Use more specific secondary antibodies (e.g., preadsorbed versions)

Problem: Unexpected Subcellular Localization

• Solution approaches:

  • Verify antibody specificity using knockout controls

  • Consider co-staining with nuclear markers (NEUROD2 is primarily nuclear)

  • Check fixation protocols that might affect nuclear permeability

  • Ensure permeabilization steps are adequate for nuclear antigen detection

How can researchers quantify NEUROD2-positive cells or expression levels accurately?

For Cell Counting Applications:

• Use standardized image acquisition parameters:

  • Fixed exposure settings

  • Consistent focal planes

  • Systematic sampling of brain regions

• Employ appropriate controls:

  • Positive controls (tissues with known NEUROD2 expression)

  • Negative controls (primary antibody omission or NEUROD2 knockout tissue)

  • Threshold controls to distinguish specific from non-specific staining

• Quantification methods:

  • Manual counting with blinded observers

  • Automated counting using software with validated parameters

  • Consider co-labeling with cell-type specific markers for subpopulation analysis

For Expression Level Quantification:

• Western blot quantification:

  • Use appropriate loading controls

  • Perform densitometry across multiple biological replicates

  • Consider the multiple band pattern (21 kDa, 41 kDa, 50 kDa)

• Immunofluorescence intensity:

  • Measure nuclear intensity in identified cells

  • Use standardized exposure settings

  • Normalize to background or control regions

• Statistical considerations:

  • Account for regional variability

  • Use appropriate statistical tests for the distribution of your data

  • Report both mean and variability metrics

What controls are essential when interpreting NEUROD2 antibody results?

Essential Controls for NEUROD2 Antibody Experiments:

• Specificity Controls:

  • Genetic validation: Use tissue from NEUROD2 knockout models as negative controls

  • Antibody validation: Test multiple antibodies targeting different epitopes

  • Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Technical Controls:

  • Primary antibody omission: To assess secondary antibody non-specific binding

  • Isotype controls: Use matched isotype antibodies at the same concentration

  • Positive controls: Include tissues with known NEUROD2 expression patterns

• Biological Controls:

  • Developmental controls: Compare tissues at different developmental stages when NEUROD2 expression changes

  • Regional controls: Include brain regions with known high and low expression

  • Cross-species validation: Confirm findings across multiple species when possible

• Functional Validation:

  • Correlate antibody staining with mRNA expression data

  • Perform complementary techniques (e.g., in situ hybridization)

  • Validate findings using genetic manipulation approaches

How can NEUROD2 antibodies be used to study neurodevelopmental disorders?

NEUROD2 antibodies can be powerful tools for studying neurodevelopmental disorders through several methodological approaches:

Disease Model Characterization:

• Compare NEUROD2 expression patterns in brain tissues from disease models (e.g., autism, intellectual disability) versus controls

• Analyze layer-specific alterations in cortical NEUROD2 expression in models of neurodevelopmental disorders

• Examine co-localization with other disease-related markers

Human Patient-Derived Samples:

• Study NEUROD2 expression in postmortem brain tissue from patients with neurodevelopmental disorders

• Analyze induced pluripotent stem cell (iPSC)-derived neurons from patients with NEUROD2 mutations

• Correlate NEUROD2 expression with clinical phenotypes

Functional Studies:

• Use NEUROD2 antibodies to monitor changes in expression following genetic or pharmacological interventions

• Examine how NEUROD2 disruption affects downstream molecular pathways implicated in neurodevelopmental disorders

• Investigate how NEUROD2 alterations affect synaptic markers and neuronal morphology

Research has demonstrated that disruption of NEUROD2 can cause a neurodevelopmental syndrome with features of intellectual disability and autism spectrum disorder . Mouse models with NEUROD2 deletion show abnormalities in cortical neuron migration, altered spine density, increased neuronal excitability, and behavioral deficits including social interaction problems, stereotypies, and hyperactivity .

What methodological approaches can be used to study the relationship between NEUROD2 and synaptic function?

To investigate the relationship between NEUROD2 and synaptic function, researchers can employ these methodological approaches:

Morphological Analysis:

• Use NEUROD2 antibodies in combination with synaptic markers to analyze:

  • Dendritic spine density and morphology

  • Excitatory versus inhibitory synapse distribution

  • Pre- and post-synaptic protein localization

• Quantification methods:

  • Confocal microscopy with high-resolution imaging

  • Structured illumination microscopy for subsynaptic structures

  • Electron microscopy for ultrastructural analysis

Functional Studies:

• Electrophysiological approaches:

  • Patch-clamp recordings to assess intrinsic excitability in NEUROD2-expressing or NEUROD2-deficient neurons

  • Analysis of excitatory and inhibitory postsynaptic currents

  • Field recordings to assess network properties

• Molecular analysis:

  • Co-immunoprecipitation to identify NEUROD2 interacting partners at synapses

  • Chromatin immunoprecipitation to identify synaptic genes regulated by NEUROD2

  • Transcriptomic analysis to identify synaptic genes affected by NEUROD2 manipulation

Research has shown that NEUROD2 knockout mice exhibit dysregulated expression of genes associated with neuronal excitability and synaptic function . At the cellular level, loss of NEUROD2 leads to increased intrinsic excitability in layer 5 neurons and dysregulation of spine density and turnover in apical dendrites .

How can researchers effectively study NEUROD2 target genes and regulatory networks?

To investigate NEUROD2 target genes and regulatory networks, researchers should consider these methodological approaches:

Genomic Approaches:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Use validated NEUROD2 antibodies to identify genomic binding sites

  • Analyze enriched DNA motifs to identify consensus binding sequences

  • Compare binding profiles across developmental stages or brain regions

• RNA sequencing after NEUROD2 manipulation:

  • Compare gene expression profiles in wild-type versus NEUROD2 knockout tissues

  • Perform conditional or temporal knockdown to identify direct versus indirect targets

  • Analyze cell type-specific responses to NEUROD2 deletion

Integrated Analysis:

• Compare ChIP-seq and RNA-seq data to identify:

  • Direct transcriptional targets with NEUROD2 binding sites

  • Genes showing expression changes upon NEUROD2 deletion

  • Biological pathways enriched among NEUROD2 targets

• Bioinformatic approaches:

  • Gene ontology analysis of NEUROD2-regulated genes

  • Network analysis to identify hub genes and regulatory circuits

  • Comparison with known neurodevelopmental disorder risk genes

Functional Validation:

• Reporter assays:

  • Test NEUROD2 binding sites in luciferase assays

  • Mutate E-box sequences to confirm direct regulation

  • Assess activity across developmental timepoints

• CRISPR-based approaches:

  • Delete specific NEUROD2 binding sites to test functional relevance

  • Perform CRISPR activation/interference at NEUROD2 target loci

  • Engineer mutations that mimic human patient variants

Research has demonstrated that genes dysregulated in NEUROD2 knockout mice have human orthologs strongly associated with autism spectrum disorders . Some promising candidate target genes regulated by NEUROD2 include gastrin-releasing peptide (GRP) and the small conductance, calcium-activated potassium channel, SK2, which may mechanistically link NEUROD2 levels to inhibitory synapse number and cellular excitability .

How can NEUROD2 antibodies help identify potential therapeutic targets for neurodevelopmental disorders?

NEUROD2 antibodies can facilitate the identification of therapeutic targets for neurodevelopmental disorders through several methodological approaches:

Target Pathway Identification:

• Use NEUROD2 antibodies to characterize expression patterns in:

  • Patient-derived tissues or cells

  • Animal models of neurodevelopmental disorders

  • Different neuronal populations affected in disorders

• Employ NEUROD2 antibodies to identify:

  • Downstream molecular pathways disrupted in NEUROD2-associated disorders

  • Potential compensatory mechanisms in partial NEUROD2 deficiency

  • Cell-type specific vulnerabilities to NEUROD2 dysfunction

Therapeutic Screening Applications:

• Develop high-content screening assays using NEUROD2 antibodies to:

  • Identify compounds that restore NEUROD2 expression in deficient models

  • Screen for drugs that normalize expression of NEUROD2 target genes

  • Monitor normalization of neuronal morphology or synaptic density

• Validation approaches:

  • Test candidate compounds in NEUROD2 haploinsufficient models

  • Assess rescue of electrophysiological abnormalities

  • Evaluate behavioral improvements in animal models

Research has shown that NEUROD2 regulates pathways critical for cortical excitatory neuron development and function, with heterozygous mutations sufficient to cause neurodevelopmental phenotypes (indicating haploinsufficiency) . This creates opportunities for therapeutic approaches that could enhance remaining NEUROD2 activity or target downstream pathways.

What insights do NEUROD2 knockout and mutation studies provide for understanding human neurodevelopmental disorders?

NEUROD2 knockout and mutation studies have provided critical insights into human neurodevelopmental disorders:

Cellular and Molecular Phenotypes:

Behavioral Phenotypes:
Mouse models with NEUROD2 disruption exhibit:

• Social interaction deficits

• Stereotypic behaviors

• Hyperactivity

• Occasional spontaneous seizures

These phenotypes closely mirror core and associated features of human neurodevelopmental disorders including ASD and intellectual disability.

Human Genetics:
Based on mouse model studies, researchers identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations . This demonstrates the translational value of model organism studies guided by NEUROD2 antibody-based research.

How do heterozygous versus homozygous NEUROD2 mutations differ in their phenotypic effects?

Understanding the differential effects of heterozygous versus homozygous NEUROD2 mutations provides important insights for researchers:

Heterozygous NEUROD2 Mutations:

• Are sufficient to cause neurodevelopmental phenotypes, indicating that NEUROD2 is haploinsufficient

• May lead to intellectual disability and ASD in humans

• In mice, heterozygous mutations recapitulate many of the defects seen in homozygous knockouts, though potentially with reduced severity

Homozygous NEUROD2 Mutations:

• Complete loss of NEUROD2 in mice results in severe neurodevelopmental defects

• Homozygous knockout mice show poor survival

• Cellular phenotypes include:

  • Severe alterations in cortical neuron migration and positioning

  • Disrupted synaptic development

  • Marked electrophysiological abnormalities

Methodological Implications:

• When designing experiments:

  • Consider that heterozygous models may better reflect human pathology

  • Use conditional knockout approaches to bypass early lethality of homozygous deletion

  • Include gene dosage analysis when interpreting phenotypes

• When analyzing patient mutations:

  • Characterize the functional impact of specific mutations (e.g., missense vs. truncating)

  • Consider domain-specific effects on NEUROD2 function

  • Correlate mutation type with clinical severity

• Experimental approaches:

  • Use NEUROD2 antibodies to quantify protein levels in heterozygous models

  • Compare transcriptional targets affected in heterozygous versus homozygous conditions

  • Evaluate rescue approaches in both conditions to assess therapeutic potential

Research has demonstrated that region-specific deletion of NEUROD2 in forebrain excitatory neurons recapitulates cellular and behavioral phenotypes found in constitutive knockout mice, revealing the region-specific contribution of dysfunctional NEUROD2 to neurodevelopmental symptoms .

How can single-cell approaches enhance our understanding of NEUROD2 function?

Single-cell approaches offer powerful methodological advantages for studying NEUROD2 function:

Single-Cell RNA Sequencing Applications:

• Cell type-specific analysis:

  • Identify specific neuronal populations expressing NEUROD2

  • Compare transcriptional profiles of NEUROD2-positive versus NEUROD2-negative cells

  • Track developmental trajectories of NEUROD2-expressing neurons

• Disease model applications:

  • Analyze cell-specific responses to NEUROD2 disruption

  • Identify compensatory mechanisms in specific populations

  • Discover cell-autonomous versus non-cell-autonomous effects

Single-Cell Protein Analysis:

• Mass cytometry (CyTOF) with NEUROD2 antibodies:

  • Simultaneously measure NEUROD2 with dozens of other proteins

  • Correlate NEUROD2 levels with cell state markers

  • Analyze signaling network responses to stimulation

• Single-cell western blot:

  • Quantify NEUROD2 protein levels in individual cells

  • Correlate with other transcription factors

  • Assess heterogeneity in expression levels

Spatial Transcriptomics:

• Combine NEUROD2 antibody staining with spatial transcriptomics to:

  • Map regional distribution of NEUROD2-expressing cells

  • Correlate NEUROD2 expression with local transcriptional environments

  • Identify spatially restricted target gene regulation

Recent studies have utilized single-cell approaches to understand the role of transcription factors in neuronal development . For NEUROD2 specifically, these approaches could help resolve outstanding questions about its cell type-specific functions and contributions to neurodevelopmental disorders.

What are the differential roles of NEUROD2 in excitatory versus inhibitory neuron development?

Understanding the differential roles of NEUROD2 in excitatory versus inhibitory neuron development requires specific methodological approaches:

Comparative Expression Analysis:

• Use NEUROD2 antibodies with cell type-specific markers to:

  • Quantify expression in glutamatergic versus GABAergic neurons

  • Analyze developmental trajectories in different neuronal lineages

  • Examine region-specific expression patterns

• Single-cell transcriptomics:

  • Profile NEUROD2 expression across neuronal subtypes

  • Identify cell type-specific co-expression networks

  • Compare regulatome across excitatory and inhibitory populations

Functional Impact Assessment:

• Cell type-specific manipulations:

  • Use Cre-Lox systems to delete NEUROD2 in specific neuronal populations

  • Compare phenotypes when deleted in excitatory versus inhibitory neurons

  • Assess non-cell-autonomous effects on circuit development

• Electrophysiological assessment:

  • Compare intrinsic properties of different neuronal types in NEUROD2 mutants

  • Analyze excitatory/inhibitory balance in neural circuits

  • Measure synaptic connectivity between affected populations

Research has shown that NEUROD2 promotes inhibitory synaptic drive while decreasing cell-intrinsic neuronal excitability of pyramidal neurons . Additionally, studies have demonstrated that specific deletion of NEUROD2 in forebrain excitatory neurons recapitulates cellular and behavioral phenotypes found in constitutive knockout mice , suggesting a particularly important role in excitatory neuron development and function.

How does NEUROD2 interact with other transcription factors in coordinating neurodevelopment?

To study how NEUROD2 interacts with other transcription factors in neurodevelopment, researchers should consider these methodological approaches:

Interaction Analysis:

• Co-immunoprecipitation with NEUROD2 antibodies to:

  • Identify protein-protein interactions with other transcription factors

  • Determine developmental stage-specific interactions

  • Analyze how mutations affect interaction partners

• Proximity labeling approaches:

  • BioID or APEX2 fused to NEUROD2 to identify proximal proteins

  • Compare interactomes across developmental stages

  • Identify context-specific cofactors

Combinatorial Binding Analysis:

• ChIP-seq co-localization studies:

  • Compare NEUROD2 binding sites with those of other neuronal transcription factors

  • Identify combinatorial binding motifs

  • Analyze cooperative and competitive binding relationships

• Sequential ChIP (Re-ChIP):

  • Identify genomic loci co-bound by NEUROD2 and partner factors

  • Assess how co-binding affects target gene expression

  • Compare co-binding patterns across development

Functional Cooperation:

• Transcriptional analysis in single and double knockouts:

  • Compare transcriptional changes in NEUROD2 versus partner factor knockouts

  • Identify synergistic versus additive effects on gene expression

  • Analyze epistatic relationships between transcription factors

• Rescue experiments:

  • Test if overexpression of partner factors can rescue NEUROD2 deficiency

  • Identify domains required for functional cooperation

  • Assess whether human mutations affect specific factor interactions