shisa9a Antibody

What is Shisa9 and what specific functions does it perform in neural systems?

Shisa9 (also known as CKAMP44 or CKAMP44a) is a brain-specific type I transmembrane protein that serves as an auxiliary subunit of AMPA-type glutamate receptors. It plays a critical role in modulating synaptic transmission through several key mechanisms:

• Associates with AMPA receptors in synaptic spines

• Promotes AMPA receptor desensitization at excitatory synapses

• Modulates short-term plasticity at specific excitatory synapses

• Contains a C-terminal PDZ domain that interacts with postsynaptic scaffolding proteins

Functionally, Shisa9 belongs to a family of proteins that modulate both FGF and Wnt signaling pathways by blocking maturation and transport of their respective receptors to the cell surface. In neurons specifically, Shisa9 tunes the functional properties of AMPARs, affecting deactivation rates and recovery from desensitization, which ultimately influences synaptic strength and plasticity .

What are the recommended applications and experimental conditions for using Shisa9a antibodies?

Shisa9a antibodies can be utilized in multiple experimental approaches with specific recommended conditions:

For optimal results, antibodies should be stored at -20°C, with care taken to minimize freeze-thaw cycles. Most commercially available Shisa9 antibodies are supplied in PBS pH 7.4 with 0.02% sodium azide and remain stable for one year when properly stored .

How can researchers distinguish between Shisa9a and other Shisa family members in experimental systems?

Distinguishing between Shisa9a and other family members requires careful consideration of multiple factors:

Methodological approaches:

• Antibody selection: Choose antibodies raised against unique epitopes specific to Shisa9a. Antibodies targeting regions within amino acids 250-350 have shown good specificity for Shisa9 .

• Controls: Include positive controls (tissues known to express Shisa9a) and negative controls (tissues lacking Shisa9a expression).

• Cross-reactivity testing: Test antibodies against recombinant proteins of different Shisa family members to confirm specificity.

Considerations for zebrafish studies:

When working with zebrafish models, it's important to note that Shisa9 has two paralogs: Shisa9a (zgc:113574, zgc:194367) and Shisa9b (im:7137228). These require different antibodies for accurate detection .

Molecular verification:

For definitive identification, complement immunological approaches with molecular techniques:

• RT-PCR with isoform-specific primers

• Gene silencing experiments combined with antibody detection to confirm specificity

• Mass spectrometry analysis of immunoprecipitated proteins

What are the optimal protocols for co-immunoprecipitation of Shisa9 with its interaction partners?

The following optimized protocol for co-immunoprecipitation of Shisa9 complexes is derived from successful experiments in brain tissue:

Sample preparation:

• Homogenize mouse cortex or hippocampus with a potter and piston at 900 rpm in ice-cold homogenization buffer (25 mM HEPES/NaOH pH 7.4, 0.32 M sucrose, 1× protease inhibitor cocktail).

• Centrifuge homogenate at 1,000 g for 10 minutes at 4°C.

• Remove supernatant and centrifuge at 100,000 g for 2 hours to obtain P2-fraction.

• Resuspend pellet in HEPES buffer to 10 μg/μL protein concentration.

Membrane solubilization:

• Mix resuspended pellet with equal volume of lysis buffer containing 2% dodecylmaltoside (DDM).

• Incubate with rotation for 45 minutes at 4°C.

• Centrifuge at 20,000 g for 15 minutes at 4°C.

• Resuspend pellet in lysis buffer with 1% DDM and repeat incubation.

• Pool supernatants (approximately 6 mg protein in 1425 μL).

Immunoprecipitation:

• Add anti-Shisa9 antibody (12 μg) to pooled supernatants.

• Incubate overnight with rotation at 4°C.

• Add agarose-protein A/G beads and incubate for 1 hour at 4°C.

• Wash beads 4 times with lysis buffer containing 0.1% DDM.

• Elute proteins from beads with SDS sample buffer.

• Analyze by SDS-PAGE and immunoblotting for interaction partners .

This protocol has successfully demonstrated the interaction between Shisa9 and PSD95 in both hippocampus and cortex samples .

How do the C-terminal interactions of Shisa9 affect AMPA receptor function and what methodologies can be used to study this?

The C-terminal interactions of Shisa9, particularly through its PDZ-binding motif (EVTV), significantly impact AMPA receptor function in several ways:

Functional effects:

• Modulation of AMPA receptor deactivation kinetics

• Alteration of recovery from desensitization

• Regulation of short-term plasticity at excitatory synapses

• Influence on paired-pulse facilitation

Recommended methodologies:

1. Peptide competition assays:

• Use TAT-tagged mimetic peptides (e.g., TAT-Shisa9WT containing the PDZ-binding motif EVTV)

• Compare with control peptides (TAT-Shisa9ΔEVTV lacking the PDZ motif)

• Measure effects on AMPA receptor function through electrophysiological recordings

2. Electrophysiological approaches:

• Whole-cell patch-clamp recordings to measure AMPAR-mediated synaptic currents

• Analysis of current decay times and paired-pulse ratios

• Comparison of wild-type versus disrupted PDZ interactions

3. Molecular interaction studies:

• Direct two-hybrid assays with wild-type and truncated constructs

• Co-immunoprecipitation from heterologous expression systems

• Pull-down assays using biotinylated peptides and recombinant proteins

Research has shown that disrupting the PDZ interactions between Shisa9 and its binding partners affects hippocampal network activity, demonstrating the importance of these C-terminal interactions in regulating synaptic function .

What are the challenges in detecting Shisa9 in different brain regions and how can these be addressed?

Detecting Shisa9 in different brain regions presents several technical challenges:

Common challenges:

• Protein solubilization: The postsynaptic density is a protein-packed structure that is notoriously difficult to solubilize while maintaining protein complex integrity.

• Antibody specificity: Cross-reactivity with other Shisa family members can confound results.

• Expression levels: Shisa9 may be expressed at different levels across brain regions.

• Heterogeneity of neural circuits: Different neural circuits may express different interacting partners.

Recommended solutions:

For immunohistochemistry and immunofluorescence:

• Use antigen retrieval techniques for fixed tissues

• Optimize fixation protocols (4% paraformaldehyde for 24 hours)

• For immunofluorescence of paraffin-embedded sections, antibody concentrations of 2.5 μg/mL have shown good results

For biochemical detection:

• Use specialized detergents like dodecylmaltoside (DDM) at 1-2% for effective solubilization

• Employ more sensitive detection methods like immunoblotting rather than mass spectrometry

• Consider region-specific extraction protocols to account for different protein compositions

For validation:

• Include positive controls (e.g., rat brain tissue lysate for Western blot)

• Use multiple antibodies targeting different epitopes of Shisa9

• Complement with mRNA detection methods

Successful detection has been demonstrated in hippocampus and cortex, with Western blot analysis using 1 μg/mL antibody concentration showing clear bands in rat brain tissue lysate .

How can researchers effectively study Shisa9's role in neuropsychiatric conditions like depression?

Recent research has implicated Shisa9 in depression pathophysiology, particularly within the reward circuitry. Here are effective approaches to study this connection:

Cell-type specific analysis:

• Use RiboTag approach to dissect transcriptional profiles of specific neuronal populations (e.g., D1- vs D2-MSNs)

• Employ RNA sequencing following chronic social defeat stress (CSDS) or other depression models

• Target specific circuits like the ventral tegmental area (VTA) to nucleus accumbens (NAc) pathway

Circuit-specific manipulations:

• Use optogenetic activation of specific neural circuits (e.g., VTA to NAc)

• Monitor changes in Shisa9 expression in response to circuit activation

• Correlate with behavioral outcomes in depression models

Molecular and cellular assessments:

• Examine Shisa9 localization at excitatory synapses of specific neuronal populations

• Measure neuronal excitability in relation to Shisa9 expression levels

• Assess anxiety- and depression-like behaviors in relation to Shisa9 manipulation

Research has shown that Shisa9 is increased specifically in D1-MSNs in the nucleus accumbens of susceptible mice following chronic social defeat stress, and selective optogenetic activation of the VTA-NAc circuit increases Shisa9 expression in D1-MSNs. This suggests Shisa9 may function as a potential pro-depressive gene in D1-MSNs, making it a promising target for rapid-antidepressant development .

What validation methods should be employed to confirm Shisa9a antibody specificity?

Comprehensive validation of Shisa9a antibody specificity is critical for reliable research outcomes:

Recommended validation hierarchy:

1. Genetic controls:

• Test antibodies in tissues from Shisa9 knockout models

• Compare with wild-type tissues as positive controls

• Use tissues known not to express Shisa9 as negative controls

2. Preabsorption tests:

• Preincubate antibody with excess immunizing peptide

• Compare staining patterns with and without preabsorption

• Specific signal should be eliminated after preabsorption

3. Multiple antibody approach:

• Use antibodies raised against different epitopes of Shisa9

• Compare staining patterns for consistency

• Antibodies targeting regions AA 251-350 and those near the center of Shisa9 have shown good specificity

4. Heterologous expression systems:

• Transfect cells with Shisa9 expression constructs

• Include wild-type and mutant constructs (e.g., Shisa9ΔEVTV)

• Verify antibody detection in immunoblotting and immunocytochemistry

5. Molecular verification:

• Correlate protein detection with mRNA expression using in situ hybridization

• Confirm protein identity by mass spectrometry after immunoprecipitation

• Use RNA interference to confirm signal specificity

A complete validation should include at least three of these approaches to ensure robust antibody specificity.

What are the optimal experimental designs for studying Shisa9's interactions with PDZ domain-containing proteins?

To effectively study Shisa9's interactions with PDZ domain-containing proteins, consider the following experimental design recommendations:

In vitro interaction studies:

Yeast two-hybrid screening:

• Use Shisa9 cytoplasmic domain as bait

• Compare wild-type constructs with ΔEVTV mutants lacking the PDZ-binding motif

• Screen against mouse brain cDNA libraries

• Validate interactions with direct two-hybrid assays using empty bait vector as control

Biochemical validation:

• Peptide pull-down assays using biotinylated Shisa9 C-terminal peptides

• Compare wild-type peptides (biotin-HFPPTQPYFITNSKTEVTV) with truncated variants (biotin-HFPPTQPYFITNSKT)

• Use recombinant PDZ domain proteins as prey

• Include competition with excess non-biotinylated peptides

Cellular interaction studies:

Co-immunoprecipitation:

• Co-express HA-tagged Shisa9WT or HA-Shisa9ΔEVTV with V5-tagged interactors in HEK293T cells

• Immunoprecipitate with anti-HA antibody and immunoblot for V5-tagged proteins

• Include appropriate controls (empty vectors, unrelated proteins)

Peptide competition assays:

• Use TAT-tagged peptides to disrupt endogenous interactions

• Compare effects of TAT-Shisa9WT vs TAT-Shisa9ΔEVTV

• Measure functional outcomes using electrophysiology

This multi-faceted approach has successfully identified several PDZ domain-containing interactors of Shisa9, including PSD95, PSD93, PICK1, GRIP1, and Lin7b .

How can researchers investigate the functional significance of Shisa9 in synaptic plasticity?

Investigating Shisa9's role in synaptic plasticity requires integrating molecular manipulations with electrophysiological and behavioral assessments:

Electrophysiological approaches:

Ex vivo recordings:

• Prepare acute brain slices from regions of interest (hippocampus, cortex)

• Record AMPAR-mediated excitatory postsynaptic currents (EPSCs)

• Measure key parameters affected by Shisa9:

  • Current decay times

  • Paired-pulse ratios

  • Recovery from desensitization

  • Short-term plasticity

Experimental manipulations:

• Apply TAT-Shisa9WT or TAT-Shisa9ΔEVTV peptides to disrupt PDZ interactions

• Compare wild-type and Shisa9 knockout animals

• Use cell-type specific Shisa9 manipulations (e.g., in D1-MSNs vs D2-MSNs)

Molecular correlates:

Protein complex analysis:

• Immunoprecipitate AMPA receptor complexes before and after plasticity induction

• Quantify Shisa9 association with receptor complexes

• Examine PDZ scaffold recruitment in relation to synaptic activity

Subcellular localization:

• Track Shisa9 trafficking in response to neuronal activity

• Use live imaging with fluorescently tagged Shisa9

• Correlate with AMPA receptor surface expression