Recombinant Mouse Calcium-dependent secretion activator 2 (Cadps2), partial

What is Cadps2 and what is its primary function in neuronal systems?

Calcium-dependent activator protein for secretion 2 (Cadps2/CAPS2) is a member of the CAPS/CADPS family of proteins that facilitates secretory vesicle exocytosis. Research has demonstrated that CADPS2 plays a crucial role in the regulated release of neurotrophins, particularly brain-derived neurotrophic factor (BDNF), from neurons. CADPS2 is predominantly expressed in brain regions including the cerebellum, neocortex, and hippocampus, where it localizes to axon terminals . In the hippocampus, CADPS2 immunoreactivity is found primarily in the stratum lucidum of hippocampal subfield CA3, where mossy fibers from dentate granule cells project, and colocalizes with BDNF . Recent studies have also revealed CADPS2 expression in tyrosine hydroxylase and vesicular monoamine transporter 2 (VMAT2)-positive dopaminergic neurons in the midbrain, indicating a role in dopamine release from central neurons .

How are Cadps2-knockout mouse models created for research purposes?

Cadps2-knockout mice are generated through targeted disruption of the mouse Cadps2 gene. In the study by Sadakata et al., the Cadps2 gene was disrupted by deleting 0.9 kb of the first exon from its genomic sequence. This genetic modification was confirmed through multiple validation techniques:

• Southern blot hybridization to verify genomic structure changes

• RT-PCR to confirm absence of mRNA expression

• Western blot analysis to demonstrate absence of CADPS2 protein in the cerebellum, neocortex, and hippocampus of homozygous knockout mice

The genetic disruption follows Mendelian inheritance patterns, with wild-type (Cadps2+/+), heterozygous (Cadps2+/−), and homozygous (Cadps2−/−) pups born at the expected 1:2:1 ratio (66 +/+, 128 +/–, and 63 –/– of 257 animals analyzed) .

What behavioral phenotypes characterize Cadps2-knockout mice?

Cadps2-knockout mice exhibit several autistic-like behavioral phenotypes, making them valuable models for investigating neurodevelopmental disorders. Key behavioral abnormalities include:

• Impaired social interaction: These mice show deficits in social behaviors that resemble the social impairments seen in autism .

• Hyperactivity: Cadps2−/− mice demonstrate significantly higher locomotor activity, particularly during the dark cycle when mice are normally more active .

• Decreased exploratory behavior/increased anxiety in novel environments: Knockout mice show reduced exploration of novel objects and environments, suggesting heightened anxiety or fear responses .

• Reduced spatial memory: In the Morris water maze test, Cadps2−/− mice demonstrate impairment in retention of spatial memory, showing lower spatial accuracy in probe tests .

• Maternal behavior defects: Female knockout mice exhibit abnormal maternal care, with cross-fostering experiments showing that pups born to wild-type dams and fostered to Cadps2−/− dams tended to have poorer survival rates .

• Anxiety-like behavior: Standardized tests reveal increased anxiety-like behavior in CAPS2-KO mice, though with minimal impairment in learning and memory tests .

How does Cadps2 deficiency affect specific neuronal populations and brain development?

Cadps2 deficiency leads to significant alterations in neuronal development and brain architecture, with specific effects on different neuronal populations:

What are the molecular mechanisms of Cadps2-mediated neurotrophin release?

CADPS2 plays a critical role in the regulated release of neurotrophins, particularly BDNF, through several molecular mechanisms:

• Secretory vesicle exocytosis: CADPS2 facilitates the exocytosis of dense-core vesicles containing neurotrophins .

• Altered release kinetics: CAPS2 promotes regulated BDNF secretion by directly affecting BDNF release kinetics. This enhanced secretion is indispensable for proper development and function of hippocampal GABAergic neurons and synapses .

• Activity-dependent release: In cell culture experiments, spontaneous BDNF release (constitutive release plus spontaneous activity-dependent release) from neocortical dissociated cultures was significantly reduced in Cadps2−/− neurons compared to wild-type .

• KCl-stimulated release: Exogenous expression of wild-type CADPS2 in PC12 cells resulted in approximately 200% increase in KCl-stimulated BDNF release compared to cells without exogenous CADPS2 expression .

• Exon 3 function: Studies of CADPS2 with exon 3 skipping (observed in some autism patients) showed functional implications, suggesting this region is important for proper CADPS2 activity in neurotrophin release .

How does Cadps2 influence dopamine release in the central nervous system?

Recent research has expanded our understanding of CADPS2 function beyond neurotrophin release to include a role in dopamine secretion:

• Expression in dopaminergic neurons: CADPS2 is expressed in tyrosine hydroxylase and VMAT2-positive dopaminergic neurons of the midbrain and primary mesencephalic cell cultures .

• Association with dopamine-containing vesicles: Subcellular fractions rich in dopamine can be collected using immunoaffinity for CADPS2 from midbrain protein extracts, indicating a physical association between CADPS2 and dopamine-containing vesicles .

• Functional impact on dopamine release: Cell imaging using fluorescent false neurotransmitter FFN511 (a substrate for VMAT2) revealed decreased activity-dependent dopamine release in Cadps2-deficient cultures compared to wild-type cultures .

• Quantitative measurements: Dopamine enzyme immunoassay (EIA) showed significant differences in dopamine concentrations between control (11.41 ± 0.22) and anti-CADPS2 antibody (16.60 ± 0.23) immunoaffinity-purified fractions (p = 0.0022) .

What are the implications of Cadps2 dysfunction for neuropsychiatric disorders?

Research on Cadps2 has revealed important connections to neuropsychiatric disorders, particularly autism spectrum disorder:

• Autism association: Analysis of Cadps2-knockout mice revealed autistic-like phenotypes, including impaired social interaction, hyperactivity, and anxiety in novel environments .

• Human CADPS2 abnormalities: Examination of CADPS2 mRNA from blood samples of autism patients identified aberrant splicing, specifically exon 3 skipping, in 4 of 16 patients, suggesting a potential genetic component to some cases of autism .

• Network activity disruption: CAPS2-KO mice exhibit decreased frequency of hippocampal theta oscillations, which are critical for temporal coding of neuronal ensembles and modification of synaptic efficacy. This suggests that CADPS2-mediated secretion contributes to GABA or BDNF-related network activities that play a role in psychiatric behavior .

• GABAergic system abnormalities: The reduced BDNF secretion in CAPS2-KO mice leads to deficits in the development and function of hippocampal GABAergic interneurons, potentially contributing to anxiety-like behaviors observed in these animals .

What techniques are used to measure BDNF release in Cadps2 research?

Researchers employ several methodologies to quantify BDNF release in relation to CADPS2 function:

• Primary cell cultures: Dissociated neocortical cultures from wild-type and Cadps2-knockout mice are used to measure spontaneous BDNF release. The culture medium is collected after defined periods and BDNF content is measured using enzyme-linked immunosorbent assay (ELISA) .

• PC12 cell expression system: To investigate the effect of specific CADPS2 variants (such as exon 3 skipping) on BDNF release, researchers co-express BDNF with various CADPS2 constructs in PC12 cells. BDNF release into the culture medium is measured in response to high-KCl stimulation (to induce depolarization) and compared between constructs .

• Neurotrophin measurements: Neurotrophin (BDNF and NGF) content in culture medium is measured using ELISA and normalized to the culture area (pg·mm−2/μl) .

How can researchers evaluate dopamine release in relation to Cadps2 function?

Several techniques have been developed to investigate the relationship between CADPS2 and dopamine release:

• Immunoaffinity purification: Subcellular fractions containing dopamine are isolated using immunoaffinity for CADPS2. Midbrains are homogenized in buffer (50 mM HEPES pH 7.4, 5 mM EDTA, 0.32 M sucrose with protease inhibitors), and the supernatant after centrifugation is mixed with hypotonic solution. Immuno-magnet beads (Dynabeads) bound to anti-CADPS2 antibody or control IgG are used for selection, and immunoaffinity-purified fractions are eluted with 0.01 N HCl .

• Dopamine enzyme immunoassay (EIA): The eluted fractions are subjected to dopamine EIA using commercial kits (e.g., Dopamine Research EIA). Dopamine concentrations are determined based on absorbance at 450 nm compared to known standards .

• Fluorescent false neurotransmitter imaging: Cell imaging using FFN511 (a substrate for VMAT2) allows visualization and measurement of activity-dependent dopamine release in cultured neurons from wild-type and Cadps2-deficient mice .

What behavioral tests are most appropriate for assessing Cadps2 knockout phenotypes?

Based on research findings, several behavioral tests are valuable for characterizing Cadps2 knockout phenotypes:

• Open field test: Measures general locomotor activity and can detect the hyperactivity phenotype observed in Cadps2−/− mice, particularly during dark cycle periods .

• Novel object test: Evaluates exploratory behavior and anxiety in a novel environment. Cadps2−/− mice make significantly fewer contacts with novel objects compared to wild-type mice .

• Morris water maze: Assesses spatial learning and memory. While Cadps2−/− mice show similar escape latency to wild-type mice in visible platform trials (indicating normal motor function), they demonstrate impaired retention of spatial memory in probe tests .

• Eight-arm radial maze test: Evaluates spatial learning and exploratory behavior. Cadps2−/− mice show decreased locomotor activity and lower arm entries compared to wild-type mice .

• Cross-fostering experiments: Valuable for assessing maternal behavior defects in female Cadps2−/− mice .

• EEG recordings: Measurements of hippocampal EEGs during the awake phase reveal decreased theta oscillation frequency in the CA1 region of CAPS2-KO mice compared to wild-type littermates .

What are the optimal methods for examining neuronal development in Cadps2-deficient models?

Several approaches have proven effective for investigating neuronal development in the context of Cadps2 deficiency:

• Immunohistochemistry: Using antibodies against specific neuronal markers (e.g., parvalbumin, calbindin, tyrosine hydroxylase) to quantify different neuronal populations in brain sections from wild-type and knockout mice .

• Primary neuronal cultures: Establishing cultures from specific brain regions (cerebellum, hippocampus, midbrain) of wild-type and Cadps2-knockout mice to study neuronal development, morphology, and function in vitro .

• Rescue experiments: Administering BDNF injections at specific developmental timepoints (e.g., P5) to determine if neuronal deficits in Cadps2−/− mice can be reversed, confirming the causal relationship between reduced neurotrophin signaling and observed phenotypes .

• Triple immunostaining: Simultaneously detecting multiple markers (e.g., CADPS2, tyrosine hydroxylase, and MAP2) in primary mesencephalic cell cultures to characterize the expression pattern of CADPS2 in specific neuronal subtypes .

• Electrophysiological recordings: Measuring long-term potentiation (LTP) and other synaptic parameters to assess functional development of neural circuits in the absence of CADPS2 .

How should researchers interpret partial phenotypes observed in Cadps2 heterozygous mice?

Cadps2 heterozygous mice (Cadps2+/−) display intermediate phenotypes between wild-type and homozygous knockout mice, providing important insights into gene dosage effects:

• Protein level correlation: The partial phenotypes observed in heterozygotes correlate with the decrease in CADPS2 protein levels in these mice. Western blot analysis shows reduced CADPS2 protein in the cerebellum, neocortex, and hippocampus of heterozygous mice compared to wild-type, but higher levels than in homozygous knockouts .

• Behavioral assessments: In tests such as the 8-arm radial maze, heterozygotes show intermediate performance between wild-type and homozygous knockout mice .

• Dose-dependent effects: These observations suggest that CADPS2 function is dose-dependent, with partial reduction leading to milder but detectable phenotypes.

• Research implications: When designing studies using Cadps2 mouse models, researchers should consider including heterozygous groups to examine potential gene dosage effects, which may be relevant to human conditions where CADPS2 levels might be reduced but not absent.

What controls are essential when studying Cadps2 function in dopamine release?

When investigating CADPS2's role in dopamine release, several critical controls should be implemented:

• Antibody specificity controls: Use control rabbit IgG alongside anti-CADPS2 antibody in immunoaffinity purification experiments to account for non-specific binding .

• Multiple quantification methods: Employ complementary techniques such as dopamine EIA and fluorescent imaging to validate findings through different methodological approaches .

• Wild-type comparisons: Always include wild-type littermates as controls when studying Cadps2-deficient models to account for genetic background effects .

• Technical replicates: Perform experiments in triplicate to ensure statistical reliability of measurements, particularly for biochemical assays like dopamine quantification .

• Validation of neuronal identity: Use multiple markers (e.g., tyrosine hydroxylase and VMAT2) to confirm the dopaminergic identity of neurons expressing CADPS2 .

Table 1: Comparative Behavioral Phenotypes in Cadps2 Mouse Models

Table 2: Neuronal Population Changes in Cadps2-Knockout Mice