PCDH19 Antibody

What is PCDH19 and why is it important in neuroscience research?

PCDH19 is a calcium-dependent synaptic cell-adhesion molecule primarily expressed in the brain and encoded by the X-linked PCDH19 gene. It has gained significant attention due to its association with epilepsy, particularly female-restricted epilepsy with mental retardation (EFMR) . The protein plays crucial roles in neuronal development, synaptic function, and activity-dependent signaling pathways. PCDH19 is distributed in the perinuclear region and along dendrites of hippocampal neurons under basal conditions, making it an important target for studying neuronal connectivity and function . Understanding PCDH19 function is essential for elucidating the mechanisms underlying hyperexcitability in epilepsy models and potentially developing targeted therapeutic approaches.

What are the optimal sample preparation techniques for PCDH19 antibody applications?

For effective PCDH19 detection, researchers should consider different sample preparation techniques based on the experimental goal. For immunocytochemistry (ICC) in primary neurons, standard 4% paraformaldehyde fixation followed by permeabilization with 0.1-0.3% Triton X-100 is effective . For flow cytometry applications, as demonstrated with SH-SY5Y neuroblastoma cells and Jurkat cells, non-permeabilizing conditions allow detection of surface-expressed PCDH19 . For Western blotting, tissue homogenization in RIPA buffer supplemented with protease inhibitors is recommended. In brain slice preparations, HCR3.0 protocol adaptations have proven effective for high-sensitivity RNA detection, where permeabilization with 1% DMSO and 1% Triton-X in PBS for 2 hours at 37°C maximizes antibody penetration . Importantly, when working with embryonic tissues, specialized extraction protocols may be necessary to preserve protein integrity, as PCDH19 expression varies significantly across developmental stages.

How can I validate PCDH19 antibody specificity for my experiments?

Validating PCDH19 antibody specificity requires multiple complementary approaches. First, compare staining patterns in wild-type and PCDH19 knockout models if available; PCDH19 KO mice serve as ideal negative controls . Second, perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific staining. Third, verify antibody reactivity across multiple applications (Western blot, ICC, flow cytometry) to confirm consistent detection patterns. For flow cytometry applications, always include appropriate isotype controls (e.g., MAB0041) followed by fluorophore-conjugated secondary antibodies to distinguish specific from non-specific binding . Additionally, comparing staining patterns with multiple antibodies targeting different PCDH19 epitopes can further confirm specificity, particularly when studying processed forms like PCDH19 CTF fragments which require C-terminal domain antibodies .

What expression patterns does PCDH19 show during embryonic brain development?

PCDH19 exhibits dynamic expression patterns during brain development. Quantification of PCDH19 protein in telencephalon lysates reveals a significant increase in production at late embryonic and early postnatal stages . At embryonic day 13.5 (E13.5), high-resolution RNA analysis using hybridization chain reaction (HCR) demonstrates that PCDH19 is highly expressed in the neocortical cortical plate, with lower levels in the (sub)ventricular zone (SVZ), and is nearly absent from the intermediate zone (IZ) . In the ganglionic eminence, PCDH19 shows low expression in the SVZ and is almost undetectable in the mantle zone. Interestingly, migrating interneurons display heterogeneous expression, with some cells showing higher expression (marked by white arrowheads in the original study) while others are negative . Western blot analysis of E13.5 mouse ventral telencephalon (VT), telencephalon (TEL), and distinct ganglionic eminence regions confirms PCDH19 protein production at the expected molecular weight of approximately 126 kDa .

How does neuronal activity regulate PCDH19 processing and distribution?

Neuronal activity dramatically alters PCDH19 processing and subcellular distribution through an NMDA receptor (NMDAR)-dependent mechanism. Under basal conditions, PCDH19 is primarily distributed in the perinuclear region and along dendrites of hippocampal neurons . When neurons are stimulated with bicuculline (BIC) to increase network activity or with NMDA, PCDH19 expression decreases along dendrites and increases in the nucleus, as visualized by immunocytochemistry using antibodies against the PCDH19 intracellular C-terminal domain . This redistribution is blocked by the NMDAR antagonist APV, confirming NMDAR dependence. Biochemically, NMDA treatment (50 μM, 30 min) causes a significant decrease of full-length PCDH19 (PCDH19 FL) in total lysate and plasma membrane fractions, accompanied by the appearance of three lower-molecular-weight fragments (CTF1, CTF2, and CTF3) at approximately 60, 55, and 45 kDa . CTF3, the most abundant fragment, increases significantly after 6 minutes of NMDA application and peaks after 20 minutes. This activity-dependent proteolytic processing represents a crucial step in PCDH19's role in synapse-to-nucleus signaling.

What is the mechanism of PCDH19 proteolytic cleavage and nuclear translocation?

PCDH19 undergoes a complex two-step proteolytic cleavage mediated by specific proteases. The metalloprotease ADAM10 generates membrane-bound protein stumps CTF1 and CTF2, while gamma secretase likely processes these fragments to produce the cytosolic fragment CTF3 . This proteolytic cascade is activated by NMDA receptor stimulation. The resulting CTF3 fragment can translocate to the nucleus due to a bipartite nuclear localization signal (NLS) composed of two basic regions (BR1BR2) located approximately 60 amino acids downstream of the transmembrane region . Specifically, this NLS sequence (aa 760–782 "RGKRIAEYSYGHQKKSSKKKKIS") contains multiple basic amino acids (shown in bold) that are crucial for nuclear entry. Mutation experiments confirm the importance of the first stretch of basic amino acids (RGKR to AGAA), as this modification prevents nuclear localization of CTF-V5 in both HEK cells and neurons . Subcellular fractionation experiments provide further evidence that endogenous CTF3 accumulates in the nuclear-aggregate-enriched fraction (P2) following NMDA treatment, confirming the physiological relevance of this nuclear translocation mechanism.

How does PCDH19 interact with GABA-A receptors in neurons?

PCDH19 directly interacts with GABA-A receptors (GABAARs) in both heterologous cells and rat neurons, representing a potential molecular mechanism linking PCDH19 dysfunction to epilepsy . Co-immunoprecipitation experiments demonstrate that PCDH19 binds to the GABAAR α1 subunit both when expressed alone or with β2 and γ2 subunits in HEK293T cells . This interaction is specific to PCDH19, as another delta protocadherin, PCDH9, which shares conserved motifs CM1 and CM2 with PCDH19, fails to co-immunoprecipitate with α1 . Furthermore, a truncated PCDH19 mutant lacking the distal portion of the intracellular tail containing CM1 and CM2 (PCDH19Δ879) maintains its ability to interact with α1, indicating that these conserved regions are not essential for GABAAR binding . Quantitative colocalization analysis in cultured hippocampal neurons estimates that approximately 30% of PCDH19 colocalizes with α1 subunits . This interaction has been confirmed in native tissue, as anti-PCDH19 antibodies successfully co-immunoprecipitate α1 subunits from both hippocampal neuron cultures and brain homogenates .

What chromatin interactions does PCDH19 CTF mediate and how can they be studied?

PCDH19 CTF enters the nucleus and associates with chromatin to regulate gene expression, particularly of immediate-early genes (IEGs). Chromatin immunoprecipitation (ChIP) experiments in mouse hippocampal slice homogenates using antibodies against PCDH19 CTF have demonstrated its association with promoters of LSD1 target IEGs, including Nr4a1, c-Fos, and Npas4 . To study these interactions, researchers should perform ChIP followed by qRT-PCR targeting specific promoter regions. The protocol involves crosslinking protein-DNA complexes with formaldehyde, sonicating to fragment chromatin, immunoprecipitating with anti-PCDH19 CTF antibodies, reversing crosslinks, and analyzing by qPCR with primers specific to target gene promoters. Importantly, PCDH19 CTF's chromatin association can be detected under basal conditions, suggesting a constitutive role in transcriptional regulation . For mechanistic studies, co-immunoprecipitation experiments have revealed that PCDH19 interacts with both ubiquitous LSD1 and neuronal-specific neuroLSD1 isoforms but not with unrelated nuclear proteins like NOVA1, indicating specific association with chromatin-modifying complexes .

What techniques are most effective for studying PCDH19 in interneuron migration?

Studying PCDH19's role in interneuron migration requires specialized techniques combining genetic manipulation with ex vivo and in vivo imaging approaches. Effective strategies include: (1) Creation of mouse models with altered PCDH19 expression, such as PCDH19-V5 overexpression lines or PCDH19 knockout lines using CRISPR/Cas9 technology with carefully designed sgRNAs ; (2) Embryonic brain slice cultures for ex vivo migration assays, where interneurons can be labeled and tracked over time; (3) Hybridization Chain Reaction (HCR3.0) for high-sensitivity RNA detection in tissue sections, which has been successfully adapted for PCDH19 visualization with customized probe design avoiding cross-reactivity with deleted regions in knockout models ; (4) Quantitative protein analysis across developmental stages and brain regions using Western blotting, which has revealed significant increases in PCDH19 production during late embryonic and early postnatal development ; and (5) Genotyping protocols using PCR with GoTAQ Polymerase and specific primers to identify genetically modified animals . These techniques collectively provide powerful tools for dissecting how PCDH19 contributes to the complex process of interneuron migration during cortical development.

How can researchers distinguish between different PCDH19 proteolytic fragments in experimental samples?

Distinguishing between PCDH19 proteolytic fragments requires careful consideration of antibody specificity and experimental conditions. The full-length PCDH19 (PCDH19 FL) has a molecular weight of approximately 126 kDa, while the proteolytic fragments CTF1, CTF2, and CTF3 run at approximately 60, 55, and 45 kDa, respectively, on SDS-PAGE . For effective detection of all forms, researchers should use antibodies targeting the PCDH19 intracellular C-terminal domain . Western blotting with gradient gels (4-12%) can improve separation of these fragments. To enhance detection of transient fragments, proteasome inhibitors like MG132 can be employed, as they tend to increase CTF abundance . For mechanistic studies of proteolytic processing, specific protease inhibitors should be used: ADAM10 inhibitors (e.g., GI254023X) will prevent the generation of CTF1 and CTF2, while gamma-secretase inhibitors (e.g., DAPT) will block the conversion to CTF3 . For subcellular localization studies, fractionation protocols separating cytosolic (S1), soluble nuclear (S2), and nuclear-aggregate-enriched (P2) fractions are effective, as CTF3 accumulates primarily in the P2 fraction following NMDA treatment .

What methodological approaches can be used to study the nuclear function of PCDH19 CTF in gene regulation?

Studying PCDH19 CTF's nuclear function in gene regulation requires multifaceted approaches combining molecular, cellular, and genomic techniques. First, subcellular fractionation followed by Western blotting can confirm the nuclear localization of endogenous PCDH19 CTF3 in the nuclear-aggregate-enriched fraction (P2) after NMDA treatment . For functional studies, chromatin immunoprecipitation (ChIP) with anti-PCDH19 antibodies followed by qRT-PCR for specific target promoters (e.g., Nr4a1, c-Fos, and Npas4) can reveal direct associations with chromatin . To investigate protein interactions, co-immunoprecipitation experiments with tagged versions (e.g., PCDH19-V5) can identify binding partners such as LSD1 and its neuronal isoform neuroLSD1 . For mechanistic insights, mutagenesis of the bipartite nuclear localization signal (particularly the basic regions at aa 760-782) can prevent nuclear entry, providing a tool to dissect nuclear versus non-nuclear functions . Gene expression analysis using qRT-PCR or RNA-seq following NMDAR activation, with or without PCDH19 knockdown/knockout, can identify downstream transcriptional targets. Finally, CRISPR-mediated genome editing to create specific PCDH19 mutations that affect cleavage or nuclear localization without disrupting other functions would provide powerful tools for dissecting the specific role of nuclear PCDH19 CTF in neuronal homeostasis.

How do mutations in PCDH19 contribute to epilepsy pathogenesis?

PCDH19 mutations are associated with female-restricted epilepsy with mental retardation (EFMR), now referred to as PCDH19-related epilepsy . The pathogenic mechanism likely involves disruption of multiple PCDH19 functions. First, PCDH19 participates in a synapse-to-nucleus signaling pathway that bridges neuronal activity with gene expression regulation . Mutations could disrupt this signaling cascade, preventing proper feedback regulation of neuronal excitability. Second, PCDH19 directly interacts with GABA-A receptors, which are central mediators of inhibitory neurotransmission and frequent targets in epilepsy research . Approximately 30% of PCDH19 colocalizes with the α1 subunit of GABA-A receptors in hippocampal neurons, suggesting that mutations might alter inhibitory neurotransmission, contributing to hyperexcitability . Third, PCDH19 CTF associates with chromatin and the chromatin remodeler lysine-specific demethylase 1 (LSD1) to regulate immediate-early genes (IEGs) expression . This provides a mechanism for PCDH19 to maintain neuronal homeostasis through negative feedback regulation of IEG expression. Disruption of this regulatory function could lead to aberrant gene expression and neuronal hyperexcitability characteristic of epilepsy . Finally, PCDH19 levels affect cortical interneuron migration during development, suggesting that mutations could also cause structural or circuit abnormalities that predispose to seizures .

What models are available for studying PCDH19-related epilepsy in the laboratory?

Several experimental models have been developed to study PCDH19-related epilepsy. Transgenic mouse lines include PCDH19-V5 overexpression models and PCDH19 knockout mice generated using CRISPR/Cas9 technology . These models allow for in vivo assessment of how PCDH19 alterations affect brain development and function. For cellular models, primary hippocampal neuron cultures treated with NMDA receptor agonists can be used to study activity-dependent PCDH19 processing and nuclear translocation . This system has revealed that PCDH19 undergoes NMDA-dependent proteolytic cleavage, generating C-terminal fragments that translocate to the nucleus and regulate gene expression . For molecular studies, heterologous expression systems (e.g., HEK293T cells) have been employed to examine interactions between PCDH19 and other proteins, such as GABA-A receptor subunits . Brain slice cultures from embryonic mice can be used to investigate how altering PCDH19 dosage affects cortical interneuron migration, which might contribute to circuit abnormalities in PCDH19-related epilepsy . Additionally, patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons offer a human cellular model for studying how specific PCDH19 mutations affect neuronal development and function, though this approach isn't explicitly mentioned in the provided search results.

What factors affect the detection sensitivity of PCDH19 in different experimental systems?

Multiple factors influence PCDH19 detection sensitivity across experimental systems. First, antibody selection is critical—antibodies targeting the PCDH19 intracellular C-terminal domain are essential for detecting proteolytic fragments (CTF1-3) , while those against extracellular domains may only recognize full-length protein. Second, expression level variations during development significantly impact detection sensitivity, with PCDH19 protein production increasing markedly during late embryonic and early postnatal stages . Third, subcellular localization affects detection—PCDH19 redistributes from dendrites to the nucleus upon neuronal activation , necessitating appropriate fractionation protocols for comprehensive analysis. Fourth, proteolytic processing generates multiple fragments (CTF1-3) with different stabilities; proteasome inhibitors like MG132 can enhance detection of these transient species . Fifth, sample preparation techniques greatly impact results—for RNA detection, specialized HCR3.0 protocols with extensive permeabilization (1% DMSO, 1% Triton-X) improve sensitivity , while for protein detection, different extraction buffers may preferentially solubilize certain PCDH19 forms. Finally, detection method sensitivity varies significantly—flow cytometry effectively detects surface PCDH19 in cell lines like SH-SY5Y and Jurkat , while Western blotting with gradient gels provides better resolution of differently sized PCDH19 fragments.

How can researchers optimize immunoprecipitation protocols for PCDH19 interaction studies?

Optimizing immunoprecipitation (IP) protocols for PCDH19 interaction studies requires several technical considerations. First, antibody selection is critical—for co-immunoprecipitation of GABA-A receptor α1 subunits, antibodies against PCDH19 have proven effective in both heterologous systems and brain tissue . For studying interactions with nuclear proteins like LSD1, antibodies recognizing the PCDH19 C-terminal fragment provide better results . Second, lysis conditions must preserve protein-protein interactions while effectively solubilizing membrane proteins; for membrane-associated PCDH19, non-ionic detergents like 1% Triton X-100 in physiological buffers work well, while nuclear interactions may require gentler conditions. Third, crosslinking with membrane-permeable reagents (e.g., DSP or formaldehyde) prior to lysis can stabilize transient interactions, particularly for the processed CTF fragments. Fourth, including appropriate controls is essential—using another delta protocadherin like PCDH9 as a negative control helps confirm specificity , while unrelated nuclear proteins like NOVA1 serve as controls for nuclear interactions . Fifth, validation across multiple systems enhances confidence in results—interactions observed in heterologous cells (HEK) should be confirmed in neurons and brain homogenates when possible . Finally, for chromatin interactions, chromatin immunoprecipitation (ChIP) protocols require optimization of crosslinking, sonication, and elution conditions to effectively capture PCDH19 CTF associations with target gene promoters like Nr4a1, c-Fos, and Npas4 .

What are the common pitfalls in designing PCDH19 knockout or overexpression systems?

Designing effective PCDH19 knockout or overexpression systems presents several challenges that researchers should anticipate. For CRISPR/Cas9-mediated knockout generation, careful sgRNA design is critical to ensure specificity and efficiency—researchers should follow established protocols for primer design, PCR template selection (e.g., pSpCas9-based vectors), and verification through gel electrophoresis . When creating overexpression constructs, researchers should consider adding epitope tags (e.g., V5) for easier detection while ensuring these tags don't interfere with protein function or localization . Genotyping strategies must be optimized for reliable identification of modified animals, typically using PCR with GoTAQ Polymerase and carefully designed primers . For functional studies, it's important to remember that PCDH19 expression varies significantly across developmental stages, with marked increases during late embryonic and early postnatal periods —this temporal variation must be considered when interpreting phenotypes. Additionally, researchers should be aware that PCDH19 undergoes complex post-translational modifications, including proteolytic processing that generates multiple fragments with distinct functions ; therefore, knockout strategies should target early exons to prevent expression of functional fragments. Finally, since PCDH19 plays roles in both neuronal development (affecting interneuron migration) and mature neuron function (through activity-dependent signaling) , conditional knockout systems may be necessary to dissect stage-specific functions.