Recombinant Mouse Phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3), partial

What is the primary function of Pik3c3 in cellular processes?

Pik3c3 (also known as Vps34) plays crucial roles in both autophagy and endosomal trafficking pathways. As a class III phosphatidylinositol 3-kinase, it catalyzes the phosphorylation of phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PI3P), which serves as a signaling lipid essential for membrane trafficking processes. In the context of autophagy, Pik3c3 is critical for autophagosome formation, while in endosomal pathways, it regulates endosomal maturation and trafficking . The distinct dual functionality of Pik3c3 makes it a pivotal regulator of cellular homeostasis, particularly in neurons and immune cells where its deletion leads to significant functional abnormalities.

How does Pik3c3 regulate autophagy in mammalian cells?

Pik3c3 regulates autophagy by generating PI3P at sites of autophagosome formation. This process facilitates the recruitment of autophagy-related proteins containing PI3P-binding domains, which are essential for initiating the formation of isolation membranes that eventually develop into autophagosomes. In Pik3c3-deficient macrophages, impaired autophagy is evidenced by increased mitochondrial mass and compromised autophagosome-lysosome fusion . These defects lead to the accumulation of autophagic vacuoles and mitochondria, indicating the critical role of Pik3c3 in maintaining mitochondrial homeostasis through autophagy. Additionally, Pik3c3-deficient cells show increased mitochondrial potential as demonstrated by tetramethylrhodamine ethyl ester perchlorate (TMRE) staining, further confirming the expansion of functional mitochondrial content due to defective mitophagy.

What are the observable phenotypes of Pik3c3 deficiency in different cell types?

Pik3c3 deficiency manifests distinctly across different cell types:

• Macrophages: Pik3c3-deficient macrophages exhibit increased surface levels of MHC class I and II molecules, enhanced M1 (pro-inflammatory) polarization, and reduced M2 (anti-inflammatory) polarization . They display defective autophagy with increased mitochondrial mass.

• Sensory Neurons: Two distinct phenotypes emerge in Pik3c3-deficient sensory neurons. One class (14%) accumulates numerous large vesicles or vacuoles (200 nm to 2 μm in diameter) with a slight increase in lysosome numbers. Another class (79%) contains fewer vesicles but exhibits a dramatic increase in lysosomes (more than 15-fold) . These neurons show progressive degeneration, with large-diameter neurons degenerating more rapidly than small-diameter neurons.

• Cell Type-Specific Vulnerability: Large-diameter (TrkB/TrkC-positive) sensory neurons accumulate abnormal endosomes, vacuoles, and ubiquitinated aggregates, leading to rapid degeneration. Small-diameter neurons develop numerous lysosome-like organelles and can activate a non-canonical autophagy pathway, resulting in slower degeneration .

How are Pik3c3 conditional knockout (cKO) mice generated and validated?

Generation of Pik3c3 conditional knockout mice involves a Cre-loxP system that allows cell-specific deletion of the Pik3c3 gene. The process typically includes:

• Breeding Strategy: Female Pik3c3flox/flox mice are bred with male Advillin-Cre mice to generate Cre/+; Pik3c3flox/+ mice, which are further bred to obtain Cre/+; Pik3c3flox/flox (Pik3c3-cKO) mice .

• Genotyping: PCR primers are used to distinguish between wild-type, floxed, and deleted Pik3c3 alleles. The specific primers for detecting the Pik3c3flox allele are A1 (5′-GGCCACCTAAGTGAGTTGTG-3′), A2 (5′-GAAGCCTGGAACGAGAAGAG-3′), and A3 (5′-ATTCTGCTCTTCCAGCCACTG-3′). For detecting the deleted Pik3c3 allele, primers L1 (5′-AACTGGATCTGGGCCTATG-3′), L2 (5′-GAAGCCTGGAACGAGAAGAG-3′), and L3 (5′-CACTCACCTGCTGTGAAATG-3′) are used .

• Validation of Deletion Efficiency: Western blotting and immunofluorescence staining confirm the absence of Pik3c3 protein in targeted cells. For myeloid-specific Pik3c3 deletion, the efficiency reportedly ranges from 40-100% depending on the macrophage population, with approximately 90-100% efficiency in alveolar and peritoneal macrophages .

What methods are used to assess autophagy flux in Pik3c3-deficient cells?

Assessing autophagy flux in Pik3c3-deficient cells employs multiple complementary approaches:

• GFP-LC3 Transgenic System: Crossing Pik3c3-cKO mice with GFP-LC3 transgenic mice allows visualization of autophagosome formation. In Pik3c3-deficient neurons, the presence of numerous bright GFP-LC3 punctae (versus diffuse cytosolic fluorescence in controls) indicates autophagosome accumulation .

• LC3-II/LC3-I Ratio Analysis: Western blot analysis of LC3 conversion from LC3-I to LC3-II provides quantitative assessment of autophagosome formation. An increased LC3-II/LC3-I ratio may indicate either enhanced autophagy initiation or blockade of autophagosome degradation .

• Autophagy Flux Assay: This involves culturing cells with and without lysosomal inhibitors (e.g., Chloroquine) and measuring changes in LC3-II levels. In both control and Pik3c3-deficient DRG cultures, Chloroquine treatment increased the LC3-II/LC3-I ratio (from 35.3% to 100.3% in controls and from 52% to 99.4% in mutants), suggesting ongoing autophagosome formation despite Pik3c3 deficiency .

• Electron Microscopy: EM analysis confirms the presence of double- or multi-membrane-encircled autophagosomes and autolysosomes, providing structural evidence of autophagy activity .

How can researchers differentiate between PIK3C3-dependent and PIK3C3-independent autophagy pathways?

Differentiating between PIK3C3-dependent and PIK3C3-independent autophagy pathways requires a multi-faceted experimental approach:

• Genetic Approach: Generate double-knockout models by crossing Pik3c3-cKO mice with mice deficient in key autophagy genes (e.g., Atg7-cKO). If autophagosome formation persists in Pik3c3-deficient cells but is abolished in Pik3c3/Atg7 double-deficient cells, this indicates a PIK3C3-independent but ATG7-dependent alternative autophagy pathway .

• Cell Type-Specific Analysis: Perform colocalization studies with cell type-specific markers and autophagy markers (e.g., LC3). In sensory neurons, GFP-LC3 punctae were selectively present in small-diameter (CGRP-positive) neurons but absent from large-diameter (NF200 or parvalbumin-positive) neurons in Pik3c3-deficient conditions, indicating cell type-specific activation of alternative autophagy .

• Pharmacological Inhibitors: Use specific inhibitors of canonical autophagy components alongside Pik3c3 inhibitors. Differential responses to these inhibitors can help distinguish between pathways.

• Ultrastructural Analysis: Electron microscopy reveals distinct morphological features of autophagosomes formed through different pathways. The presence of double-membrane structures in Pik3c3-deficient cells suggests functional autophagosome formation despite Pik3c3 absence .

How does Pik3c3 deficiency in myeloid cells affect autoimmune disease models?

Pik3c3 deficiency in myeloid cells significantly impacts autoimmune disease models, particularly experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis:

• Reduced Disease Severity: Myeloid cell-specific Pik3c3-deficient animals show significantly reduced severity of EAE, which is primarily a CD4+ T-cell-mediated condition .

• Altered T Cell Dynamics: This protection is associated with reduced accumulation of myelin-specific CD4+ T cells in the central nervous system, indicating that Pik3c3 in myeloid cells indirectly regulates the pathogenic T cell response .

• Decreased Inflammatory Cytokine Production: Pik3c3-deficient myeloid cells produce less IL-1β, a critical pro-inflammatory cytokine involved in EAE pathogenesis .

• Altered Antigen Presentation: Despite increased MHC class I and II molecule expression on Pik3c3-deficient macrophages (suggesting enhanced antigen presentation capacity), these cells are less effective at promoting pathogenic T cell responses in vivo .

• Translational Potential: Administration of SAR405, a selective PIK3C3 inhibitor, delays EAE disease progression, suggesting therapeutic potential for PIK3C3 inhibitors in multiple sclerosis and other autoimmune diseases .

What are the neurological consequences of Pik3c3 deletion in sensory neurons?

Pik3c3 deletion in sensory neurons leads to severe neurological consequences:

How do Pik3c3 inhibitors affect cellular function in pathological conditions?

PIK3C3 inhibitors exert multiple effects on cellular function in pathological conditions:

How can molecular docking be used to design specific PIK3C3 inhibitors?

Molecular docking is a powerful computational approach for designing specific PIK3C3 inhibitors:

• Structural Analysis: The process begins with obtaining the crystal structure of PIK3C3, such as the PIK3C3 structure (PDB code 4OYS) complexed with SAR405 available from the Protein Data Bank .

• Binding Site Definition: The binding site of PIK3C3 is defined by identifying residues located within a specific distance (≤10 Å) from known inhibitors like SAR405 .

• 3D Structure Generation: Three-dimensional structures of PIK3C3, candidate inhibitors, and reference molecules (like ATP) are generated using molecular modeling software such as ACD/ChemSketch .

• Docking Simulation: Candidate compounds are docked onto the defined binding site using docking programs like iGEMDOCK. These programs assign formal charges and atom types (donor, acceptor, both, or nonpolar) to individual atoms of both compounds and proteins to simulate interactions .

• Energy-Based Scoring: An energy-based scoring function, such as piecewise linear potential, calculates intermolecular interaction energy between the protein and docked compounds to predict binding affinity and specificity .

• Structure-Activity Relationship Analysis: Results from docking simulations identify critical residues involved in molecular recognition, which can guide rational design of PIK3C3-specific inhibitors with minimized off-target effects.

What are the methodological challenges in studying PIK3C3-independent autophagy pathways?

Studying PIK3C3-independent autophagy pathways presents several methodological challenges:

• Genetic Model Complexity: Generating appropriate double-knockout models (e.g., Pik3c3/Atg7 double-conditional knockout mice) is technically challenging and yields rare viable offspring (approximately 1 in 32), making it difficult to obtain sufficient animals for statistical analysis .

• Cell Type Heterogeneity: PIK3C3-independent autophagy appears to be cell type-specific, occurring in small-diameter but not large-diameter sensory neurons. This heterogeneity necessitates precise cell identification and isolation techniques to avoid misleading results from mixed cell populations .

• Temporal Dynamics: The rapid neurodegeneration in Pik3c3-deficient models creates a narrow window for studying alternative autophagy before cell death occurs. Neurons from later stages (P5-P9) cannot survive dissociation and in vitro culture, limiting experimental options .

• Distinguishing Autophagosome Formation vs. Clearance: Increased LC3-II levels may indicate either enhanced autophagosome formation or blocked degradation. Autophagy flux assays with lysosomal inhibitors are essential but technically challenging in rapidly degenerating neurons .

• Overlapping Functions of PIK3C3: PIK3C3's involvement in both autophagy and endosomal pathways complicates interpretation of phenotypes. Effects observed after PIK3C3 inhibition may result from disruption of either or both pathways, requiring careful experimental design to differentiate the contributions .

How can researchers leverage the differential vulnerability of cell types to Pik3c3 deficiency?

The differential vulnerability of cell types to Pik3c3 deficiency offers unique research opportunities:

• Identification of Compensatory Mechanisms: Small-diameter neurons activate PIK3C3-independent autophagy pathways that delay degeneration. Investigating these compensatory mechanisms may reveal novel targets for enhancing cellular resilience in neurodegenerative diseases .

• Cell Type-Specific Therapeutic Strategies: Understanding why certain cell types (like large-diameter neurons) are more vulnerable to PIK3C3 deficiency can inform targeted therapeutic approaches that protect susceptible cells while leveraging innate resistance mechanisms of others .

• Biomarker Development: The distinct ultrastructural phenotypes in different cell types (vacuole accumulation versus lysosome proliferation) may serve as biomarkers for detecting early-stage pathology before clinical symptoms emerge.

• Experimental Design Considerations: When studying Pik3c3 function, researchers should carefully consider cell type-specific effects. Experiments should include appropriate controls and cell-type identification to avoid confounding results from cellular heterogeneity .

• Translational Applications: The stronger susceptibility of large-diameter sensory neurons to Pik3c3 deficiency parallels the selective vulnerability observed in certain neurodegenerative diseases. This parallel may be leveraged to develop more relevant disease models and targeted therapies .

What is the rationale for dual inhibition of PIK3C3 and FGFR in therapeutic applications?

The dual inhibition of PIK3C3 and FGFR represents a promising therapeutic strategy based on several principles:

• Complementary Pathway Targeting: PIK3C3 and FGFR regulate distinct but potentially synergistic cellular processes. While PIK3C3 controls autophagy and endosomal trafficking, FGFR mediates cell proliferation, survival, and differentiation signaling .

• Enhanced Efficacy: Simultaneous inhibition of both pathways may produce more robust therapeutic effects than single-pathway inhibition, potentially circumventing compensatory mechanisms that limit efficacy of single-target approaches.

• Rational Drug Design: Molecular docking studies help identify critical residues for inhibitor design and predict binding affinities. These computational approaches guide the development of dual-action inhibitors or optimized drug combinations .

• Therapeutic Applications: While the specific condition targeted by dual PIK3C3/FGFR inhibition is not fully elaborated in the search results, this approach likely has applications in cancer treatment, given that FGFR signaling is frequently dysregulated in various malignancies .

• Reduced Resistance Development: Targeting multiple pathways simultaneously may reduce the likelihood of resistance development, which commonly limits the long-term efficacy of single-target therapies.

How do Pik3c3 and autophagy contribute to immune cell differentiation and polarization?

Pik3c3 and autophagy significantly influence immune cell differentiation and polarization:

• Macrophage Polarization: Pik3c3-deficient macrophages display increased expression of M1 markers (CD86) and reduced expression of M2 markers (CD206) compared to controls, indicating that Pik3c3 normally constrains M1 polarization while promoting M2 polarization .

• Antigen Presentation Capacity: Pik3c3-deficient macrophages and dendritic cells exhibit increased surface levels of both MHC class I and class II molecules, suggesting enhanced antigen presentation capacity. This altered phenotype contributes to the homeostatic activation state of these cells .

• Inflammatory Cytokine Production: Myeloid cells lacking Pik3c3 show decreased IL-1β production, indicating that Pik3c3 normally promotes certain pro-inflammatory responses. This reduced IL-1β production contributes to the decreased severity of EAE in myeloid-specific Pik3c3-deficient mice .

• T Cell Regulation: Through effects on myeloid cell function, Pik3c3 indirectly regulates T cell responses. Myeloid cell-specific Pik3c3 deficiency results in reduced accumulation of myelin-specific CD4+ T cells in the CNS during EAE, demonstrating Pik3c3's role in facilitating pathogenic T cell responses .

• Autophagy Dependency: The effects of Pik3c3 deficiency on myeloid cell phenotype are dependent on the early machinery (initiation/nucleation) of the classical autophagy pathway, indicating that autophagy plays a crucial role in immune cell differentiation and function .

What future research directions could advance our understanding of Pik3c3 biology in neurodegeneration?

Several promising research directions could enhance our understanding of Pik3c3 biology in neurodegeneration:

• Characterization of PIK3C3-Independent Autophagy: Further investigation of the PIK3C3-independent autophagy pathway observed in small-diameter sensory neurons could reveal novel autophagy initiation mechanisms. Determining whether this pathway exists in other neuronal subtypes across the nervous system would be valuable .

• Temporal Dynamics of Neurodegeneration: Developing methods to study the temporal sequence of subcellular pathology following Pik3c3 deletion could identify the earliest events and potential intervention points. Time-course studies with conditional knockout models allowing temporal control of gene deletion would be informative .

• Cell Type-Specific Vulnerability Mechanisms: Investigating the molecular basis for the differential vulnerability of neuronal subtypes to Pik3c3 deficiency could identify protective factors in resistant neurons that might be therapeutically exploitable .

• Therapeutic Development: Expanding studies with PIK3C3 inhibitors like SAR405 to various neurodegeneration models could assess their therapeutic potential. Developing cell type-specific delivery methods could maximize therapeutic effects while minimizing off-target complications .

• Integrative Multi-omics Approaches: Applying transcriptomic, proteomic, and metabolomic analyses to Pik3c3-deficient neurons at various stages of degeneration could provide comprehensive insights into the molecular pathways affected by Pik3c3 deficiency and identify novel therapeutic targets.