WDR47 Antibody

What is WDR47 and what is its role in neuronal development?

WDR47 is a microtubule-associated protein particularly enriched in neurons in humans and mice . It functions as a crucial regulator of brain development through multiple mechanisms. WDR47 regulates the proliferation of late progenitors and neuronal migration in developing brains, while also influencing axonal and dendritic development in neuronal cultures . At the molecular level, WDR47 regulates cellular processes critical for proper brain development, including microtubule stability, intracellular transport, and autophagy . The gene is essential for mouse survival and brain morphogenesis in a dose-dependent manner, with complete loss leading to postnatal lethality and partial expression resulting in severe neuroanatomical abnormalities, including microcephaly and loss of commissural fibers .

What are the known structural domains of WDR47 protein?

WDR47 is a multidomain protein with a specific structural organization that determines its functionality:

• N-terminal region: Contains LISH and CTLH domains

• C-terminal region: Contains seven WD40 domains

The protein structure includes specific regions involved in homodimerization, particularly in the WDR47-NTD region. Structural modeling with Alphafold2-derived atomic models has revealed that amino acid R193 forms a salt bridge with E205 in the opposite monomer, which is critical for homodimerization . The various domains serve distinct functions, with N-terminal domains being critical for both neuronal migration and survival, while the WD40 repeats are at least partially dispensable for migration but essential for long-term neuronal survival .

What applications can WDR47 antibodies be used for?

Based on commercially available antibodies, WDR47 antibodies have been validated for multiple research applications:

These antibodies have been tested on various human samples including HeLa cells, K-562 cells, U-2 OS cell line, and human kidney tissue . When designing experiments, researchers should optimize antibody dilutions for their specific sample types and experimental conditions, as reactivity can be sample-dependent .

How do mutations in WDR47 affect protein expression and function?

Research has identified several bi-allelic variants in WDR47 that differentially impact protein expression and function. These variants have been associated with severe neurodevelopmental disorders including corpus callosum dysgenesis (CCD), microcephaly, and other neuroanatomical phenotypes .

Specific missense substitutions in WDR47 demonstrate various effects on protein expression:

The p.(Arg193His) variant shows the most dramatic effect, with nearly complete loss of protein expression in patient-derived fibroblasts due to protein instability and proteasomal degradation . This instability can be rescued with the proteasome inhibitor MG132, suggesting a specific mechanism of degradation for this mutant protein .

What are the methodological approaches to study WDR47 function through domain analysis?

Researchers have employed domain analysis to understand the specific functions of WDR47 regions:

• Creation of truncated constructs: Studies have utilized human WDR47 constructs lacking different domains (hWDR47-ΔLISH, -ΔCTLH, -ΔLISH/CTLH, and -ΔWD40) to determine domain-specific functions .

• In utero electroporation (IUE): This technique allows for the introduction of truncated WDR47 constructs into developing mouse brains to assess their ability to rescue phenotypes induced by Wdr47 deletion .

• Complementation assays: By introducing wild-type or truncated WDR47 constructs into Wdr47-deficient neurons, researchers can assess which domains are essential for specific functions, such as neuronal migration and survival .

Results from these approaches have revealed that:

• Both N- and C-terminal domains are indispensable for WDR47's pro-survival function

• N-terminal LISH and CTLH domains mediate functions in both neuronal migration and survival

• WD40 repeats are partially dispensable for migration but critical for long-term neuronal survival

How does WDR47 deficiency affect neuronal survival, and what cell death mechanisms are involved?

Loss of WDR47 leads to severe, early-onset neuronal death that manifests as corpus callosum dysgenesis in vivo . Research has identified the specific cell death mechanism through in vitro time-lapse imaging studies of primary neuronal cultures from Wdr47 knockout embryos.

Key findings on WDR47-related neuronal death:

• Temporal pattern: Wdr47-deficient neurons show a burst of neuronal death starting at DIV8 (days in vitro), leading to approximately 50% reduction in surviving neurons by DIV10 compared to wild-type controls .

• Cell death mechanism: The neuronal death is Caspase-dependent, as treatment with Qvd-Oph (a pan-Caspase inhibitor) fully rescues the survival of Wdr47 knockout neurons .

• Other cell death pathways: Treatment with Necrostatin (necroptosis inhibitor) and Ferrostatin (ferroptosis inhibitor) did not rescue the neuronal death phenotype, suggesting these pathways are not primarily involved .

This research demonstrates that WDR47 is required cell-autonomously to protect callosal projection neurons (CPNs) from apoptotic cell death, providing insight into the mechanism behind corpus callosum dysgenesis observed in patients with WDR47 mutations .

What are the optimal conditions for using WDR47 antibodies in Western blotting?

When using WDR47 antibodies for Western blotting, researchers should consider the following methodological details:

For optimal results, researchers should titrate the antibody in their specific testing system, as reactivity can be sample-dependent . When studying WDR47 variants, it's important to note that some mutations (particularly p.(Arg193His)) can dramatically reduce protein expression, potentially leading to very weak or absent bands in patient-derived samples .

How can researchers effectively evaluate WDR47 antibody specificity?

Ensuring antibody specificity is critical for accurate interpretation of results. For WDR47 antibodies, researchers should:

• Include appropriate controls:

  • Positive controls: Use cell lines with known WDR47 expression (HeLa cells, K-562 cells)

  • Negative controls: Consider using siRNA/shRNA-mediated knockdown or CRISPR-Cas9 knockout samples

  • For genetic studies: Compare patient samples with control samples, as done with the p.(Arg193His) variant in fibroblasts and the compound heterozygous variant (p.(Asp466His), p.(Lys592Arg)) in lymphoblastoid cell lines

• Verify protein size: Confirm that the detected band matches the expected molecular weight (100-102 kDa)

• Correlate protein with transcript levels: As demonstrated in patient studies, compare protein expression with transcript levels using qRT-PCR to distinguish between effects on protein stability versus transcription

• Use proteasome inhibitors: For suspected protein degradation (as with the p.(Arg193His) variant), treatment with proteasome inhibitors like MG132 can confirm if reduced levels are due to increased degradation

What considerations are important when using WDR47 antibodies for immunocytochemistry and immunohistochemistry?

For immunostaining applications with WDR47 antibodies, researchers should consider:

• Fixation and permeabilization:

  • For ICC: Standard PFA fixation followed by appropriate permeabilization for intracellular proteins

  • For IHC-P: Paraffin-embedded tissues have been successfully used with WDR47 antibodies at 1/40 dilution (Abcam ab121935)

• Antibody concentration:

  • For ICC: 2 μg/mL has been validated for U-2 OS cells with Abcam ab121935

  • Optimization may be required for different cell types

• Subcellular localization expectations:

  • WDR47 is a microtubule-associated protein, so staining patterns should be consistent with microtubule networks

  • In neurons, expect enrichment in cellular processes including axons and dendrites, given WDR47's role in axonal and dendritic development

• Co-localization studies:

  • Consider co-staining with microtubule markers to confirm appropriate localization

  • For studies of neuronal migration or axon development, combine with appropriate neuronal markers

How can researchers address challenges in detecting WDR47 protein in mutant samples?

When studying WDR47 variants, researchers may encounter difficulties detecting the protein, especially with mutations that affect protein stability. Based on the research with patient samples:

• For unstable variants (e.g., p.(Arg193His)):

  • Prevent protein degradation using proteasome inhibitors like MG132 prior to cell lysis

  • Use freshly prepared samples and include protease inhibitors in lysis buffers

  • Consider increasing sample concentration or using more sensitive detection methods

  • Optimize antibody concentration and incubation conditions

• For variants with moderate expression reduction:

  • Load higher amounts of protein

  • Use more sensitive detection substrates

  • Consider longer exposure times for Western blots

• For variants with no apparent effect on expression level:

  • Focus on functional assays to detect subtle changes in protein activity

  • Consider assessing protein-protein interactions or subcellular localization

What approaches can be used to study WDR47 function in neuronal models?

Based on the published research, several methodological approaches have been effectively used to study WDR47 function:

• In vivo approaches:

  • In utero electroporation (IUE) of Cre-expressing constructs in Wdr47 fl/fl embryos to achieve conditional knockout

  • Complementation assays with full-length or truncated WDR47 constructs to assess domain-specific functions

  • Analysis of neuronal migration by quantifying distribution of GFP-positive neurons in the cortical plate

  • Assessment of axonal projections by examining labeled neurons in the corpus callosum

• In vitro approaches:

  • Primary neuronal cultures from wild-type or Wdr47 knockout embryos

  • Time-lapse imaging to monitor neuronal survival over time (DIV3 to DIV10)

  • Cell death pathway analysis using specific inhibitors (Qvd-Oph for caspases, Necrostatin for necroptosis, Ferrostatin for ferroptosis)

  • Neuroblastoma cell lines (N2A) for overexpression studies of wild-type and mutant WDR47 constructs

These approaches provide complementary data on WDR47 function, from molecular mechanisms to physiological roles in developing neurons.

What are the key considerations for quantifying WDR47 protein levels in experimental and patient samples?

When quantifying WDR47 protein levels, especially when comparing wild-type and mutant proteins or patient samples, researchers should:

• Ensure equal loading:

  • Use appropriate loading controls (housekeeping proteins)

  • Confirm equal protein concentration using protein assays before loading

  • Consider normalized loading based on cell count for patient-derived samples

• Account for detection limitations:

  • For variants with severely reduced expression (like p.(Arg193His) with −72% reduction), ensure detection methods have sufficient sensitivity

  • Use quantitative Western blot analysis with standard curves when precise quantification is needed

• Compare protein with transcript levels:

  • Perform qRT-PCR to determine if reduced protein levels are due to transcriptional or post-transcriptional effects

  • Research with patient samples showed stable WDR47 transcript levels despite reduced protein levels for some variants, indicating post-transcriptional regulation

• Consider treatment conditions:

  • For suspected proteasomal degradation, compare protein levels with and without proteasome inhibitors

  • For the p.(Arg193His) variant, MG132 treatment rescued protein levels, confirming instability due to proteasomal degradation

How might WDR47 research contribute to understanding neurological disorders?

WDR47 research has significant implications for understanding neurological disorders:

• Neurodevelopmental disorders: Bi-allelic variants in WDR47 cause severe neurodevelopmental delay associated with corpus callosum dysgenesis, microcephaly, and other neuroanatomical phenotypes . Further research could identify additional patient populations with WDR47 mutations.

• Alzheimer's disease: A genome-wide association study identified WDR47 as a hub gene in Alzheimer's disease . Additional research could clarify how WDR47 dysfunction contributes to neurodegenerative processes.

• Broader microtubule-related disorders: WDR47 functions in a similar fashion to LIS1 (a gene associated with lissencephaly) but with distinctive properties . Comparative studies could reveal common pathways in microcephaly and migration disorders.

• Autophagy-related disorders: Given WDR47's role in autophagy , research could explore connections between WDR47 dysfunction and other autophagy-related neurological conditions.

Research examining WDR47 antibodies in patient samples could provide biomarkers for diagnosis and potentially identify targets for therapeutic intervention in these conditions.

What are the optimal experimental designs for studying WDR47 interactions with the microtubule cytoskeleton?

To study WDR47's interactions with microtubules, researchers should consider:

• Co-localization studies:

  • Use super-resolution microscopy to visualize WDR47 association with microtubules

  • Perform co-immunoprecipitation with tubulin and microtubule-associated proteins

  • Examine effects of microtubule-stabilizing or -destabilizing drugs on WDR47 localization

• Functional assays:

  • Assess microtubule stability in wild-type versus WDR47-deficient neurons

  • Examine effects of WDR47 variants on microtubule dynamics

  • Investigate WDR47's role in specific microtubule-dependent processes like intracellular transport

• Domain-specific analyses:

  • Determine which domains of WDR47 are required for microtubule association

  • Create domain-specific mutations to disrupt microtubule binding without affecting other functions

• In vivo modeling:

  • Use in utero electroporation to introduce WDR47 variants and assess effects on neuronal migration and axon development

  • Examine microtubule architecture in patient-derived cells carrying WDR47 mutations

Research has already established that WDR47's cell-autonomous function on neuronal survival partly relies on its effects on the microtubule cytoskeleton , making this an important direction for future investigation.