PPID Mouse
Description
Neurological and Behavioral Studies
-
Fear Extinction: Ppid knockdown in the basolateral amygdala (BLA) impaired fear extinction in mice, while overexpression enhanced it. This effect is mediated through glucocorticoid receptor interactions .
-
Stuttering Pathogenesis: A Ppid p.Pro270Ser knock-in mouse model revealed microstructural abnormalities in the corticospinal tract and cortico-striatal-thalamo-cortical loops, mirroring findings in humans with stuttering .
Mitochondrial Apoptosis and Cancer
PPID regulates mitochondrial permeability transition pores (mPTPs). Overexpression suppresses apoptosis in cancer cells by stabilizing hexokinase-2 on mitochondrial membranes, promoting chemoresistance . Inhibitors like cyclosporin A (CsA) disrupt this interaction, sensitizing tumors to therapy .
Ischemia-Reperfusion Injury
Cardioprotective roles of PPID were observed during hypoxia-reoxygenation stress, where it complexes with heat shock proteins to mitigate cellular damage .
PPID in Neurodevelopmental Disorders
Biochemical Assays
-
Rotarod Performance: Ppid mutant mice showed no motor deficits, confirming specificity to cognitive/behavioral phenotypes .
-
Y-Maze Spatial Memory: Intact spatial working memory in Ppid mutants, suggesting selective roles in fear circuitry .
Clinical and Therapeutic Implications
-
Cancer: PPID overexpression correlates with tumor survival, making it a target for cyclosporin analogs .
-
Neuropsychiatric Disorders: Modulating PPID activity could treat extinction deficits in PTSD or anxiety .
-
Stuttering: First genetic link between chaperone dysfunction and speech disorders, highlighting PPID’s role in neural circuit maturation .
Recombinant PPID Mouse Protein
Produced in E. coli, recombinant PPID (ENZ-1069) is utilized for:
Product Specs
Cyclophilin-D, a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family, plays a crucial role in protein folding. PPIases accelerate the cis-trans isomerization of proline imidic peptide bonds within oligopeptides, thereby facilitating protein folding. Cyclophilin-D exhibits PPIase activity and binds to the immunosuppressive drug cyclosporin-A. Notably, its overexpression is known to suppress apoptosis in cancer cells. This anti-apoptotic effect is mediated through a mitochondrial hexokinase-2 dependent mechanism.
Recombinant Mouse PPID, produced in E. coli, is a single, non-glycosylated polypeptide chain comprising 395 amino acids (1-370a.a.). With a molecular weight of 43.4 kDa, this protein is fused to a 25 amino acid His-tag at the N-terminus and purified using proprietary chromatographic techniques.
The PPID protein solution is provided at a concentration of 1 mg/ml in a buffer consisting of 20 mM Tris-HCl (pH 8.0), 10% glycerol, and 1 mM DTT.
For short-term storage (2-4 weeks), the product can be stored at 4°C. For extended storage, freezing at -20°C is recommended. To ensure optimal stability during long-term storage, adding a carrier protein (0.1% HSA or BSA) is advisable. Avoid repeated freeze-thaw cycles.
The purity of the protein is determined to be greater than 90.0% using SDS-PAGE analysis.
The specific activity, defined as the amount of enzyme required to cleave 1 µmol of suc-AAFP-PNA per minute at 37°C in Tris-HCl pH 8.0 using chymotrypsin, is greater than 700 nmol/min/mg.
Peptidyl-prolyl cis-trans isomerase D, PPIase D, 40 kDa peptidyl-prolyl cis-trans isomerase, Cyclophilin-40, CYP-40, Cyclophilin-related protein, CYP40, CYPD, PPID, Peptidylprolyl Isomerase D.
MGSSHHHHHH SSGLVPRGSH MGSEFMSHAS PAAKPSNSKN PRVFFDVDIG GERVGRIVLE LFADIVPKTA ENFRALCTGE KGTGSTTGKP LHFKGCPFHR IIKKFMIQGG DFSNQNGTGG ESIYGEKFED ENFHYKHDRE GLLSMANAGP NTNGSQFFIT TVPTPHLDGK HVVFGQVIKG LGVARTLENV EVNGEKPAKL CVIAECGELK EGDDWGIFPK DGSGDSHPDF PEDADIDLKD VDKILLISED LKNIGNTFFK SQNWEMAIKK YAKVLRYVDS SKAVIEKADR SRLQPIALSC VLNIGACKLK MSNWQGAIDS CLEALEMDPS NTKALYRKAQ GWQGLKEYDQ ALADLKKAQE IAPGDKAIQA ELLKVKQMIK AQKDKEKAVY AKMFA.
Q&A
What is PPID and why is it significant in mouse model research?
PPID (peptidylprolyl isomerase D, also known as cyclophilin 40) is a member of the tetratricopeptide repeat (TPR) protein family that has emerged as an important regulator of neural circuits associated with trauma-related behaviors. The gene is enriched in both excitatory and inhibitory neuronal populations in the basolateral amygdala (BLA) . Recent research has also implicated PPID in developmental stuttering, representing the first evidence linking a chaperone protein to this condition .
Mouse models enable controlled investigation of PPID's neurobiological functions through:
-
Precise genetic manipulation of expression levels
-
Assessment of behavioral consequences in standardized paradigms
-
Evaluation of molecular interactions with other signaling pathways
-
Translational modeling of human pathogenic variants
Which mouse strains are most commonly used in PPID research?
Research on PPID has primarily utilized two inbred mouse strains that exhibit robust phenotypic differences:
| Mouse Strain | PPID Expression Level in BLA | Fear Extinction Phenotype | Response to PPID Manipulation |
|---|---|---|---|
| C57BL/6J (B6) | Higher | Efficient | Modest effects when upregulated |
| 129S1/SvImJ (S1) | Lower | Impaired | Significant improvement when upregulated |
These strain differences have proven particularly valuable for quantitative trait loci (QTL) mapping to identify genetic factors influencing extinction learning. The S1 strain's extinction impairment is resistant to developmental cross-fostering, suggesting early developmental programming of this phenotype .
What behavioral paradigms are used to assess PPID function in mice?
The selection of appropriate behavioral assays depends on the specific aspect of PPID function being investigated:
-
Fear conditioning and extinction paradigms: The primary method for assessing PPID's role in emotional learning, measuring freezing behavior during acquisition, retrieval, and extinction of conditioned fear responses .
-
Ultrasonic vocalization (USV) recording: Particularly valuable for studying PPID mutations associated with stuttering, these recordings capture communication-related phenotypes using the pup separation assay methodology .
-
Y-maze testing: Used to evaluate spatial working memory, especially in models investigating broader cognitive effects of PPID manipulation .
When designing these experiments, researchers should standardize testing conditions and consider that physiological stress responses directly impact performance measures .
What methods are used to alter PPID expression in mouse models?
Multiple approaches have been developed to manipulate PPID expression with varying degrees of temporal and spatial precision:
-
Viral-mediated gene transfer:
-
CRISPR/Cas9 knock-in models:
-
Cross-strain approaches:
What controls are essential when manipulating PPID in mouse models?
| Control Type | Purpose | Implementation |
|---|---|---|
| Vehicle controls | Account for effects of injection/surgery | Identical procedure with delivery vehicle only |
| Non-targeting vectors | Control for non-specific effects of viral infection | Same viral backbone without target sequence |
| Wild-type littermates | Genetic background control for knock-in models | Use littermates from heterozygous breeding |
| Behavior-only groups | Distinguish behavioral from molecular effects | Include non-manipulated groups exposed to behavioral testing |
| Anatomical controls | Verify region-specificity | Target adjacent brain regions as anatomical controls |
Proper validation of manipulation efficiency through qPCR, western blotting, or immunohistochemistry is essential for interpreting behavioral results .
How is PPID distributed in the mouse brain?
PPID shows specific patterns of expression across brain regions and cell types:
For accurate quantification, researchers should employ multiple complementary techniques:
-
Immunohistochemistry for anatomical localization
-
Western blotting for protein level quantification
-
In situ hybridization for cell-type specific expression analysis
What neural circuits are affected by PPID manipulation?
PPID functions within key neural circuits implicated in emotional learning and speech production:
-
Amygdala circuits:
-
Corticospinal tract:
-
Cortico-striatal-thalamo-cortical loop:
How does PPID interact with stress hormone signaling?
A critical finding in PPID research is its functional interaction with glucocorticoid receptor (GR) signaling:
-
The extinction-facilitating effects of PPID upregulation are blocked by GR antagonists such as RU-486
-
This suggests PPID may function as a modulator of GR-mediated transcriptional activity
-
The relationship likely involves PPID's role as a co-chaperone in hormone receptor complexes
Researchers studying this interaction should:
-
Monitor stress levels during experiments
-
Consider circadian variation in corticosterone levels
-
Include appropriate pharmacological controls when manipulating either pathway
What molecular mechanisms underlie PPID's effects on neural plasticity?
Several mechanisms have been proposed for how PPID influences neural function:
-
Regulation of hormone receptor trafficking: Through its co-chaperone function in protein complexes involving Hsp90 .
-
Modulation of perineuronal nets (PNNs):
-
PNNs are extracellular matrix structures surrounding BLA parvalbumin-positive interneurons
-
They protect fear memories from extinction-induced plasticity
-
S1 mice (with lower PPID) show accelerated developmental emergence of PNNs
-
Degrading PNNs through chondroitinase ABC (ChABC) treatment facilitates extinction
-
-
Influence on neuronal excitability: PPID manipulation affects in vivo neuronal activity during extinction learning, suggesting direct effects on cellular physiology .
How should researchers address developmental factors in PPID studies?
PPID function appears to be developmentally regulated, requiring careful experimental design:
-
S1 mice already show impaired extinction at juvenile age (P17), when most strains typically show enhanced extinction ("fear erasure")
-
The development of BLA PNNs coincides with the transition from juvenile to adult extinction properties
-
Cross-fostering experiments (prenatal, postnatal, post-weaning) suggest early programming of extinction phenotypes
Recommended approaches include:
-
Testing at multiple developmental timepoints
-
Age-specific molecular profiling
-
Inducible genetic systems to dissociate developmental from acute effects
-
Consideration of early-life experiences that might affect stress reactivity
What imaging approaches best characterize PPID-related brain changes?
Advanced imaging techniques provide valuable insights into PPID's effects on brain structure and function:
-
Diffusion-weighted MRI:
-
Quantitative susceptibility mapping:
-
In vivo single-unit recordings:
-
Ex vivo imaging:
What are common technical challenges in PPID mouse research and how can they be addressed?
| Challenge | Potential Causes | Recommended Solutions |
|---|---|---|
| Variable viral expression | Injection placement, viral titer variation | Stereotaxic precision, titer standardization, post-hoc verification |
| Inconsistent behavioral results | Testing conditions, handling stress | Standardize testing protocols, habituate animals, blind experimenters |
| Strain-specific effects | Genetic background differences | Use multiple strains, consider QTL approaches, backcross to common background |
| Developmental compensation | Genetic redundancy, adaptive changes | Use inducible/acute manipulations, combine genetic and pharmacological approaches |
| Translating between species | Evolutionary differences in circuit organization | Focus on conserved molecular mechanisms, validate in human samples when possible |
How should researchers interpret strain differences in PPID expression and function?
Strain differences provide valuable insights but require careful interpretation:
-
B6 mice show higher baseline PPID expression and better extinction compared to S1 mice
-
Extinction training upregulates PPID in S1 mice but has different effects in B6 mice
-
Multiple genetic variants exist in the PPID sequence between strains, with functional significance that remains unclear
When interpreting these differences, researchers should:
What are the translational implications of PPID mouse research for human conditions?
PPID research in mice has direct relevance to human health conditions:
-
Anxiety and trauma-related disorders:
-
Developmental stuttering:
Future translational work should focus on:
-
Developing pharmacological modulators of PPID function
-
Identifying biomarkers based on PPID pathway activity
-
Exploring genetic variation in PPID and related genes in clinical populations
-
Testing whether PPID-related circuits represent common pathways across neuropsychiatric conditions
Product Science Overview
Peptidylprolyl Isomerase D (PPID), also known as Cyclophilin D, is an enzyme that belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) family. This family of enzymes catalyzes the cis-trans isomerization of proline imidic peptide bonds in polypeptides, which is crucial for protein folding and function .
PPID, like other cyclophilins, forms a β-barrel structure with a hydrophobic core. This β-barrel is composed of eight anti-parallel β-strands and is capped by two α-helices at the top and bottom. The β-turns and loops in the strands contribute to the flexibility of the barrel . The primary function of PPID is to facilitate the folding or repair of proteins by catalyzing the cis-trans isomerization of proline imidic peptide bonds .
PPID is involved in various biological processes, including mitochondrial metabolism, apoptosis, redox regulation, and inflammation. It plays a significant role in diseases and conditions such as ischemic reperfusion injury, AIDS, and cancer . In mice, PPID is expressed in several tissues, including the liver, muscle, and colon .
Recombinant expression of mouse PPID involves cloning the gene into an expression vector, which is then introduced into a host cell, such as E. coli or HEK293T cells. The host cells are cultured, and the recombinant protein is purified using various chromatographic techniques . The recombinant protein is often tagged with a marker, such as MYC/DDK, to facilitate purification and detection .
Recombinant PPID is used in various research applications, including studies on protein folding, signal transduction, and the development of therapeutic agents targeting PPIases. The enzyme’s role in mitochondrial metabolism and apoptosis makes it a valuable tool for studying cellular stress responses and related diseases .
