NLGN4X antibodies are employed in diverse experimental contexts, including neurobiology, oncology, and stem cell research. Key applications include:
Neuronal Tissue: Polyclonal antibodies (e.g., AF5158) detect NLGN4X in human brain cortex sections, highlighting neuronal cell bodies and processes .
Cancer Research: NLGN4X antibodies are used to study its downregulation in metastatic melanoma, where low expression correlates with HIF1A signaling and migratory phenotypes .
Pluripotent Stem Cells: Monoclonal antibodies (e.g., CSTEM30) enable flow cytometric analysis of NLGN4X expression in human iPSCs, aiding studies on synaptic differentiation .
Glioma Models: NLGN4X-specific TCR-engineered T cells (detected via NLGN4X antibodies) demonstrate efficacy in preclinical glioblastoma models, achieving a 44.4% objective response rate .
Melanoma: IHC studies reveal that NLGN4X expression inversely correlates with metastatic progression. High NLGN4X levels predict better survival and suppress HIF1A-driven migration .
Glioblastoma: NLGN4X is overexpressed in gliomas, making it a therapeutic target. Antibodies facilitate tracking of TCR-engineered T cells in intracerebroventricular delivery models .
Synaptic Function: NLGN4X interacts with neurexins to regulate excitatory synaptic transmission. Antibodies help map its localization in synapses and its role in ASD-linked mutations .
VBP1 Regulation: Loss of NLGN4X downregulates Von Hippel-Lindau Binding Protein 1 (VBP1), leading to HIF1A stabilization and oncogenic signaling in melanoma .
Monoclonal Antibodies: Clone CSTEM30 shows no cross-reactivity with other neuroligins (NLGN1-3, NLGN4Y), ensuring specificity in flow cytometry .
Polyclonal Antibodies: While robust in WB and IHC, they may require optimization to avoid non-specific binding .
NLGN4X (Neuroligin-4 X-linked) is a 110 kDa type I transmembrane glycoprotein belonging to the type B carboxyesterase/lipase family of proteins . It is postsynaptically expressed on neurons and plays a critical role in initiating excitatory presynapse maturation through its binding with specific isoforms of beta-neurexin . The human NLGN4X gene is located on the X-chromosome and encodes a protein with an 816 amino acid sequence comprising a 41 aa signal sequence, a 635 aa extracellular domain (ECD), a 21 aa transmembrane domain, and a 119 aa cytoplasmic tail .
NLGN4X has gained significant research attention because mutations in this gene have been associated with autism spectrum disorders (ASD), Asperger syndrome, and Tourette syndrome . Understanding the structure and function of NLGN4X is therefore critical for elucidating the neurobiological basis of these conditions.
Despite sharing 97% sequence homology, NLGN4X and NLGN4Y exhibit profound functional differences . NLGN4Y displays severe deficits in maturation, surface expression, and synaptogenesis compared to NLGN4X . Biochemical analyses reveal that NLGN4Y primarily exists in its immature form, whereas NLGN4X is detected in both mature and immature forms .
This functional difference is regulated by a single critical amino acid substitution in the extracellular domain - specifically, a proline at position 93 in NLGN4X corresponds to a serine in NLGN4Y (P93S) . When this amino acid is mutated in NLGN4X to match NLGN4Y (P93S), it results in decreased expression of the mature form, mimicking the NLGN4Y phenotype . The inability of NLGN4Y to compensate for NLGN4X functional deficits has implications for the male bias observed in NLGN4X-associated neurodevelopmental disorders .
NLGN4X antibodies serve multiple critical applications in neuroscience research:
Western Blotting: Used to detect NLGN4X in cell lysates, including those from neuronal cultures, demonstrating specificity for the ~110 kDa protein .
Immunohistochemistry (IHC): Applied to detect NLGN4X in fixed tissue sections, such as human brain cortex, where specific staining is localized to neuronal cell bodies and their processes .
Immunocytochemistry (ICC): Used to visualize NLGN4X expression and localization in cultured neurons .
Flow Cytometry: Employed to detect NLGN4X expression in cell populations, including human-induced pluripotent stem cells (hiPSCs) .
ELISA: Used for quantitative detection of NLGN4X in various sample types .
These applications enable researchers to investigate NLGN4X expression, localization, and function in various experimental contexts relevant to neurodevelopmental disorders.
When designing experiments to distinguish between NLGN4X and NLGN4Y, consider these methodological approaches:
Antibody Selection: Use antibodies specifically developed against unique epitopes of NLGN4X or NLGN4Y. Validate these antibodies by testing them against both proteins expressed in a heterologous system like HEK293T cells .
Western Blot Analysis: Exploit the different migration patterns - NLGN4X typically shows both mature and immature bands, while NLGN4Y predominantly shows the lower molecular weight immature band .
RT-PCR with Specific Primers: Design primers that target non-homologous regions between NLGN4X and NLGN4Y transcripts. Sequence verification of amplicons may be necessary for confirmation .
Sex-specific Controls: Include both male and female samples in your experimental design. NLGN4Y should only be detected in male-derived samples, providing an internal control for antibody specificity .
Surface Biotinylation Assays: To differentiate their trafficking properties, use surface biotinylation to compare the surface expression of NLGN4X versus NLGN4Y .
When reporting results, clearly document the antibody clone, epitope, and validation experiments performed to substantiate your findings.
Several cell models offer distinct advantages for investigating NLGN4X function and trafficking:
Human-derived Neuronal Cultures: Differentiated neurons from human induced pluripotent stem cells (hiPSCs) represent a physiologically relevant model that expresses endogenous NLGN4X . Male-derived lines additionally express NLGN4Y, enabling comparative studies .
Primary Rat/Mouse Hippocampal Neurons: These provide a well-established system for studying synaptogenesis. When using these models, consider microRNA-mediated knockdown of endogenous neuroligins (NLmiRs) to avoid potential heterodimerization with endogenous neuroligins when expressing human NLGN4X .
HEK293T Cells: Though non-neuronal, these cells are valuable for biochemical characterization, protein interaction studies, and trafficking experiments due to their high transfection efficiency and lack of endogenous neuroligin expression .
NTera-2 Cells: This human testicular embryonic carcinoma cell line has been validated for NLGN4X expression and can be useful for antibody validation and basic expression studies .
When selecting a model system, consider whether you need to study endogenous protein or if heterologous expression is sufficient, and whether you aim to investigate neuronal-specific functions like synaptogenesis.
For rigorous NLGN4X studies in autism research, implement these essential controls:
Genetic Controls:
Experimental Validation Controls:
Sample Controls:
Technical Controls:
These controls enhance experimental rigor and facilitate proper interpretation of results in the context of autism research.
For optimal NLGN4X detection via Western blotting, consider these methodological details:
Sample Preparation:
Gel Conditions:
Antibody Parameters:
Detection Considerations:
These optimized conditions should enable reliable detection of NLGN4X in Western blot applications while minimizing non-specific background.
For successful immunostaining of NLGN4X in brain tissue sections, follow this validated protocol:
This protocol has been validated for human cortical tissue and should yield specific staining of NLGN4X with minimal background.
Several complementary methods can reliably distinguish mature from immature forms of NLGN4X:
SDS-PAGE Mobility Analysis:
Glycosidase Treatment:
Treat protein samples with EndoH or PNGaseF enzymes
Immature ER-resident forms are typically EndoH-sensitive, while mature forms that have passed through the Golgi are EndoH-resistant but PNGaseF-sensitive
Subcellular Fractionation:
Separate ER, Golgi, and plasma membrane fractions
Immature NLGN4X predominantly localizes to ER fractions, while mature forms are found in Golgi and plasma membrane fractions
Surface Biotinylation Assays:
Immunofluorescence Microscopy:
Use antibodies against NLGN4X along with organelle markers
Co-localization with ER markers indicates immature forms, while surface or synaptic staining indicates mature forms
The ratio of mature to immature forms provides valuable information about NLGN4X processing efficiency, which is particularly relevant when studying autism-associated variants.
Autism-associated NLGN4X mutations profoundly impact protein trafficking and function through several mechanisms:
Impaired Protein Maturation:
Deficient Surface Trafficking:
Compromised Synaptogenic Activity:
Loss of Compensation Mechanism:
Altered Protein-Protein Interactions:
These molecular consequences provide critical insights into the pathogenic mechanisms underlying NLGN4X-associated neurodevelopmental disorders.
Several post-translational modifications (PTMs) critically regulate NLGN4X function:
N-Glycosylation:
Phosphorylation:
Proteolytic Processing:
Dimerization:
S-Palmitoylation:
While not specifically mentioned in the search results, S-palmitoylation has been documented for other neuroligins and may influence NLGN4X membrane localization and lateral mobility
Understanding these PTMs provides critical insights for researchers investigating NLGN4X function in normal development and disease states, and may inform therapeutic strategies targeting specific modifications.
To quantify NLGN4X-mediated synaptic effects, researchers can employ these sophisticated approaches:
Electrophysiological Recordings:
Whole-cell patch-clamp recordings to measure changes in excitatory postsynaptic currents (EPSCs) or inhibitory postsynaptic currents (IPSCs)
Paired recordings can assess the strength of specific synaptic connections
These techniques can directly measure functional consequences of NLGN4X expression or mutation
High-Content Imaging of Synaptogenesis:
Co-culture assays where NLGN4X-expressing non-neuronal cells are cultured with neurons to quantify induced presynaptic differentiation
Automated microscopy with synaptic marker quantification in primary neurons expressing wild-type or mutant NLGN4X
Use of synapse-specific markers like vGLUT1 (excitatory) or vGAT (inhibitory) to determine synapse type specificity
Live Imaging of Surface Trafficking:
pH-sensitive GFP tags (pHluorin) fused to NLGN4X to visualize surface insertion events in real-time
FRAP (Fluorescence Recovery After Photobleaching) to measure lateral mobility and clustering dynamics
Super-Resolution Microscopy:
STORM/PALM imaging to visualize nanoscale organization of NLGN4X at synapses
Dual-color super-resolution to measure co-localization with other synaptic proteins at nanometer resolution
Biochemical Assays:
These methodologies provide complementary data on how NLGN4X influences synaptic development, function, and plasticity in both physiological and pathological contexts.
To investigate differential interactions between NLGN4X/Y and neurexins, implement these specialized approaches:
In vitro Binding Assays:
Structural Studies:
Cell-Based Interaction Assays:
Proteomic Analysis:
Mutagenesis Studies:
These approaches provide complementary data on the molecular determinants of differential neurexin binding and signaling between NLGN4X and NLGN4Y.
Several factors can significantly influence NLGN4X detection:
Sample Preparation Issues:
Antibody-Related Factors:
Expression Level Variations:
Post-translational Modifications:
Technical Variables:
Understanding these variables is crucial for experimental design and troubleshooting when inconsistent results occur.
The interpretation of mature versus immature NLGN4X band patterns provides valuable insights into protein processing:
This interpretation framework allows researchers to extract mechanistic insights from what might otherwise appear to be simple differences in band patterns.
Researchers studying NLGN4X should be aware of these common pitfalls and their solutions:
Antibody Cross-Reactivity Issues:
Pitfall: Antibodies may cross-react with other neuroligin family members, particularly NLGN3 which shares significant homology .
Solution: Validate antibody specificity using overexpression systems with all neuroligin family members. The search results indicate that R&D Systems antibody showed <5% cross-reactivity with recombinant human NLGN3 in direct ELISAs .
Confusing NLGN4X and NLGN4Y Signals:
Improper Controls in Mutation Studies:
Overlooking Surface Expression:
Species Differences:
Expression System Artifacts:
Pitfall: Overexpression systems may saturate cellular machinery, creating artificial trafficking bottlenecks.
Solution: Include dose-response experiments and validate key findings in systems with endogenous expression levels when possible.
Awareness of these pitfalls and implementing appropriate controls will significantly enhance the rigor and reproducibility of NLGN4X research.
Several cutting-edge technologies offer promising avenues for advancing NLGN4X research:
CRISPR-Based Approaches:
CRISPR/Cas9 genome editing to generate isogenic human iPSC lines with NLGN4X mutations
CRISPRa/CRISPRi for endogenous gene modulation without overexpression artifacts
CRISPR base editors for precise introduction of autism-associated point mutations
Advanced Imaging Technologies:
Live super-resolution microscopy to track NLGN4X dynamics at synapses in real-time
Expansion microscopy for enhanced visualization of synaptic proteins
Lattice light-sheet microscopy for long-term imaging of NLGN4X trafficking with minimal phototoxicity
Organoid and 3D Culture Systems:
Brain organoids to study NLGN4X function in complex human neural networks
Microfluidic devices to examine NLGN4X's role in circuit formation
3D bioprinting of neural tissues with controlled NLGN4X expression patterns
Single-Cell Approaches:
Single-cell transcriptomics to profile cell type-specific NLGN4X expression
Single-cell proteomics to examine NLGN4X interactome heterogeneity
Patch-seq to correlate NLGN4X expression with electrophysiological properties
In Situ Structural Biology:
Cryo-electron tomography to visualize NLGN4X-neurexin complexes in their native environment
In-cell NMR to probe NLGN4X structural dynamics and interactions
Mass photometry for single-molecule characterization of NLGN4X complexes
These emerging technologies promise to overcome current limitations in understanding NLGN4X biology and pathology, potentially leading to therapeutic strategies for associated neurodevelopmental disorders.
NLGN4X research has significant potential to inform novel therapeutic strategies for autism spectrum disorders (ASD):
Protein Trafficking Enhancement:
Post-translational Modification Modulation:
Synaptic Function Modulation:
Compounds that enhance or mimic NLGN4X-neurexin interactions
Modulation of downstream signaling pathways to compensate for NLGN4X dysfunction
These approaches address the fundamental synaptic abnormalities in ASD
Gene Therapy Approaches:
CRISPR-based repair of specific NLGN4X mutations
Viral delivery of wild-type NLGN4X to overcome haploinsufficiency
Antisense oligonucleotides to modulate NLGN4X splicing or expression
Compensatory Mechanisms:
Targeting other neuroligin family members to compensate for NLGN4X dysfunction
Enhancing NLGN4Y function in males could provide sex-specific therapeutic strategies
This approach acknowledges the redundancy and compensation within the neuroligin family