PSENEN Antibody

What is PSENEN and why are antibodies against it important in neurodegenerative disease research?

PSENEN (Presenilin Enhancer 2) is a 10 kDa protein that functions as an essential subunit of the γ-secretase complex, which catalyzes the intramembrane cleavage of integral membrane proteins including Notch receptors and amyloid precursor protein (APP) . Human PSENEN is 101 amino acids in length and is a multi-pass transmembrane protein with specific topology in the endoplasmic reticulum and Golgi apparatus .

PSENEN antibodies are vital tools because:

• They allow detection and visualization of this key component in the γ-secretase complex, directly involved in generating amyloid-beta peptides in Alzheimer's disease

• They enable researchers to study the assembly, maturation, and activity of the γ-secretase complex

• Recent research demonstrates PSENEN is not merely structural but plays crucial roles in presenilin (PSEN) endoproteolysis, complex stabilization, and regulation of γ-secretase activity

• PSENEN contributes to the catalytic mechanism of the enzyme beyond its structural role

What applications can PSENEN antibodies be used for in neuroscience research?

PSENEN antibodies can be utilized in multiple applications:

PSENEN antibodies have been successfully used to detect the protein in human Alzheimer's brain, with specific staining localized to the cytoplasm and neurites of neurons .

How do researchers validate the specificity of PSENEN antibodies?

Validating PSENEN antibody specificity is crucial for reliable results. Recommended approaches include:

• Testing in knockout models: Using PSENEN-/- fibroblasts to confirm absence of signal (gold standard)

• Peptide competition assays: Pre-incubating antibody with immunizing peptide to block specific binding

• Western blot analysis: Confirming detection at expected molecular weight (~10 kDa)

• Cross-species reactivity testing: Verifying reactivity across species (human PSENEN shares 96% amino acid sequence identity with mouse and rat)

• Immunofluorescence co-localization: Demonstrating expected subcellular localization in ER and Golgi

• Positive controls: Using known PSENEN-expressing cell lines (e.g., 293T, A549, SH-SY5Y, mouse kidney, rat lung)

Research has confirmed that a cysteine-free form of PSENEN (replacing C15 with alanine) retains functionality and can serve as a suitable control for antibody validation studies .

What are the recommended protocols for immunohistochemistry with PSENEN antibodies?

For effective immunohistochemistry using PSENEN antibodies:

Tissue preparation:

• Fix tissues in 4% paraformaldehyde or formalin

• Embed in paraffin and cut sections at 4-5 μm thickness

• For antigen retrieval, use heat-induced epitope retrieval with TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0

Staining protocol:

• Deparaffinize sections in xylene

• Rehydrate through graded alcohols (100%, 95%, 90%, 80%, 70%, 50%)

• Perform heat-induced antigen retrieval at 120°C for 10 minutes

• Cool to room temperature

• Block endogenous peroxidase activity

• Incubate with primary PSENEN antibody overnight at 4°C (dilution: 1:50-1:200)

• Apply HRP-labeled secondary antibody for 1 hour

• Develop with DAB substrate for approximately 5 minutes

• Counterstain with hematoxylin

Scoring system for PSENEN staining intensity (clinical samples):

• Staining intensity: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

• Area stained: 0 (0-5%), 1 (6-25%), 2 (26-50%), 3 (51-75%), 4 (>75%)

• Final score = intensity × area, where 1-4 = weakly positive (+), 5-8 = moderately positive (++), 9-12 = strongly positive (+++)

How can PSENEN antibodies be used to study the assembly and function of the γ-secretase complex?

PSENEN antibodies are valuable tools for investigating γ-secretase complex assembly and function:

Complex assembly analysis:

• Blue native PAGE combined with Western blotting using PSENEN antibodies can identify different complex states

• Research has shown that in PSENEN-/- fibroblasts, a trimeric complex forms consisting of full-length PSEN1, NCSTN, and APH1A, but lacks enzymatic activity

• Co-immunoprecipitation with PSENEN antibodies can pull down the entire complex for analysis

Activity assessment:

• PSENEN is essential for γ-secretase activity beyond PSEN endoproteolysis

• Studies demonstrate that even when expressing PSEN1 ΔE9 (which is active without endoproteolysis) in PSENEN-/- fibroblasts, no γ-secretase activity is detected

• Activity can be measured through Aβ production assays, with research showing Aβ40 production in PSENEN-/- fibroblasts is less than 1% of wild-type production

Topology and structure studies:

• Scanning cysteine accessibility method (SCAM) combined with PSENEN antibodies has revealed:

  • Hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity

  • Transmembrane domain 3 is not water-exposed

  • Glycine 22 and proline 27 border membrane domains and are crucial for complex formation

What is the role of PSENEN in presenilin endoproteolysis and how can antibodies help investigate this process?

PSENEN plays a critical role in presenilin endoproteolysis, which can be investigated using PSENEN antibodies:

Mechanism of action:

• Downregulation of PSENEN leads to decreased PSEN endoproteolysis

• This results in increased full-length PSEN and decreased PSEN amino- and carboxy-terminal fragments (NTF and CTF)

• The N-terminal part of hydrophobic domain 1 of PSENEN interacts with TMD4 of PSEN1 and is important for endoproteolysis

Research approaches:

• Compare PSEN1 processing in wild-type vs. PSENEN-knockout cells using Western blotting

• Perform rescue experiments with wild-type or mutant PSENEN in knockout cells

• Use cross-linking experiments with PSENEN antibodies to identify interaction sites

• A documented cross-linking experiment using SPDP (a cross-linker with 6.8Å spacer arm) successfully detected interaction between E49C PSENEN mutant and PSEN1 CTF

Functional implications:

• PSENEN is not only involved in PSEN endoproteolysis but also plays a role in the active complex

• Research shows that a conserved DYSLF motif in the C-terminus of PSENEN is crucial for complex assembly, stabilization of PSEN fragments, and γ-secretase activity

How do PSENEN antibodies contribute to understanding familial Alzheimer's disease (FAD) mechanisms?

PSENEN antibodies provide insights into FAD mechanisms through several approaches:

Conformational studies:

• N-terminal modifications to PSENEN can change PSEN conformation, resulting in increased Aβ42/Aβ40 ratio similar to FAD mutations

• This suggests PSENEN's conformation affects the pathological mechanism of FAD

Interaction analysis:

• γ-secretase modulators that decrease Aβ42 production bind mainly to PSENEN

• This highlights PSENEN's role in regulating complex activity and potential as a therapeutic target

Cross-linking experiments:

• PSENEN antibodies can detect conformational changes in the γ-secretase complex caused by FAD mutations

• Research has revealed that the loop of PSENEN and PSEN1 CTF are within 6.8Å of each other in the complex

Functional assays:

• PSENEN is essential for both γ-cleavage and ε-cleavage of APP

• In PSENEN-/- cells, AICD (APP intracellular domain) production is completely abolished, indicating total loss of γ-secretase function

What epitope targets are most effective for PSENEN antibodies in various applications?

Different PSENEN epitopes offer advantages for specific applications:

Several commercial antibodies target specific regions:

• Human PSENEN Antibody (MAB6859) targets Gly89-Gly98

• Some antibodies are raised against synthetic peptides from the C-terminus

• Recombinant fusion proteins containing amino acids 1-101 of human PSENEN are also used as immunogens

The full sequence of human PSENEN is: MNLERVSNEEKLNLCRKYYIGGFAFLPFLWLVNIFWFFREAFIVPAYTEQSQIKGYVWRSAVGFLFWVIVLTSWI TIFQIYRPRWGALGDYLSFTIPLGTP

How can researchers troubleshoot non-specific binding when using PSENEN antibodies?

When encountering non-specific binding with PSENEN antibodies:

Common issues and solutions:

• Antibody concentration issues:

  • Perform titration experiments to determine optimal concentration

  • Follow manufacturer's recommendations: typically 1:50-1:200 for IHC/IF, 1:500-1:1000 for WB

• Sample preparation optimization:

  • For brain tissue, use heat-induced epitope retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

  • Include adequate permeabilization for this transmembrane protein

  • Consider cross-linking experiments for preserving protein-protein interactions

• Validation approaches:

  • Use PSENEN-/- fibroblasts as negative controls

  • Perform peptide competition assays with immunizing peptide

  • Include multiple antibody controls (isotype control, secondary-only)

• Application-specific recommendations:

  • For WB: Use 10-15% gels due to PSENEN's small size (10 kDa)

  • For IF: Consider methanol fixation which may better preserve membrane proteins

  • For co-IP: Use mild detergents like CHAPSO (1%) to maintain complex integrity

Remember that PSENEN's small size and membrane localization present unique challenges for antibody-based detection.

How are PSENEN antibodies being used in cancer research?

Recent research has expanded the role of PSENEN beyond Alzheimer's disease to cancer research:

Renal cell carcinoma studies:

• PSENEN expression is being investigated as a prognostic marker in renal clear cell carcinoma (KIRC)

• Immunohistochemistry with PSENEN antibodies is used to analyze clinical samples

• Expression is semi-quantitatively assessed based on staining intensity and area

Methodological approach:

• Tumor samples can be divided based on median PSENEN expression

• GO functional analysis and KEGG analysis help explore biological functions of PSENEN

• Gene Set Variation Analysis (GSVA) is used to investigate cellular mechanisms

Current research hypothesizes that PSENEN may be a survival-related gene in certain cancers, opening new avenues for therapeutic targeting of the γ-secretase complex beyond neurodegenerative diseases .

How can PSENEN antibodies be utilized in screening potential γ-secretase inhibitors or modulators?

PSENEN antibodies play crucial roles in screening for γ-secretase inhibitors or modulators:

Compound screening approaches:

• Several γ-secretase inhibitors have been developed, including DAPT, DBZ, and BMS 299897

• PSENEN antibodies can confirm whether compounds affect complex assembly

• Blue native PAGE with PSENEN antibody detection can track complex formation changes

Target validation:

• Research shows γ-secretase modulators that decrease Aβ42 production bind mainly to PSENEN

• This highlights PSENEN as a direct target for therapeutic development

• Selective inhibitors targeting specific γ-secretase complexes (PSEN1 vs PSEN2) provide a feasible therapeutic window in preclinical models

Complex specificity:

• The γ-secretase complex consists of four essential proteins: PSEN, NCSTN, APH1, and PSENEN in 1:1:1:1 stoichiometry

• PSENEN antibodies help distinguish between complexes containing different PSEN (PSEN1/PSEN2) and APH1 (APH1A/APH1B) variants

• This is critical for developing selective inhibitors that avoid Notch-related side effects