ENO2 Mouse

What is ENO2 and what is its primary function in mouse neural tissue?

ENO2, also known as gamma-enolase or neuron-specific enolase (NSE), is an 80 kD glycolytic enzyme that catalyzes the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate in the glycolytic pathway. In mouse neural tissue, ENO2 serves multiple functions beyond its metabolic role, including promoting neuronal survival, differentiation, and axonal regeneration . It is primarily expressed in mature neurons and cells of neuronal origin, with a developmental switch from alpha-enolase to gamma-enolase occurring during neural tissue development . ENO2 is considered a neuronal marker and has been extensively studied for its roles in both normal neural function and in pathological conditions affecting the nervous system .

How does mouse ENO2 expression differ across brain regions?

ENO2 expression varies significantly across different mouse brain regions, with particularly high expression observed in the striatum compared to other regions such as the hippocampus . Comprehensive proteomic analyses have revealed that ENO2 has a closer association with proteasome complexes in the striatum than in the hippocampus, suggesting region-specific functions . The Allen Brain Atlas data indicates differential expression patterns of ENO2 across various brain tissues, reflecting its region-specific roles in neuronal function and metabolism . This regional specificity is important to consider when designing experiments targeting ENO2 in specific brain areas, as its functional interactions and expression levels may vary substantially between regions.

What are the established mouse models for studying ENO2 function?

Several established mouse models exist for studying ENO2 function, including ENO2 knockout mice available through the International Mouse Phenotyping Consortium (IMPC) . Cell line models derived from mouse neurons, such as the ST HDH Q7/7 cell line from E14 wildtype Huntingtin mouse embryos, are widely used for in vitro studies of ENO2 function . For in vivo studies of ENO2's role in motor neuron development, researchers have utilized zebrafish transgenic lines like Tg(mnx1:GFP), in which motor neurons are GFP-tagged, combined with morpholino-based ENO2 knockdown approaches . These models allow researchers to investigate the physiological and pathological roles of ENO2 in various contexts, from basic neuronal function to disease pathogenesis.

How can I effectively knock down or overexpress ENO2 in mouse neuronal cells?

For effective manipulation of ENO2 expression in mouse neuronal cells, lentiviral vector systems have proven to be highly efficient tools. When designing ENO2 knockdown experiments, siRNA approaches targeting specific regions of the ENO2 transcript have shown good efficacy . For overexpression studies, plasmid vectors containing the full-length mouse ENO2 coding sequence can be transfected into neuronal cell lines such as NSC34 . For in vivo studies, antisense morpholino oligonucleotides (MO) specifically targeting ENO2 can be used, with subsequent rescue experiments performed using wobble-ENO2-mRNA to confirm specificity . When validating knockdown or overexpression, it is essential to perform both RT-qPCR and Western blot analyses to confirm changes at both mRNA and protein levels, as post-transcriptional regulation may affect protein expression independent of transcript levels .

What are the optimal conditions for studying ENO2's role in mouse motor neuron development?

The optimal conditions for studying ENO2's role in mouse motor neuron development involve a combination of in vitro and in vivo approaches. For in vitro studies, NSC34 cells (a mouse motor neuron-like cell line) can be used with ENO2 knockdown or overexpression followed by neurite outgrowth assays . These cells should be cultured in standard conditions (DMEM with 10% FCS) and can be differentiated by incubation in serum-free DMEM containing specific growth factors and signaling molecules . For examining ENO2's interaction with extracellular phosphoglycerate kinase 1 (ePgk1), which is critical for neurite outgrowth, recombinant ePgk1 protein can be added to the culture medium . For in vivo studies, the zebrafish model system offers advantages due to transparent embryos that facilitate visualization of motor neuron development. Injection of ENO2-specific morpholinos followed by rescue with wild-type or mutant ENO2 mRNA allows for assessment of specific amino acid residues critical for ENO2's function in motor neuron development .

What are the key considerations for analyzing ENO2 protein interactions in mouse brain tissue?

When analyzing ENO2 protein interactions in mouse brain tissue, several methodological considerations are critical. First, tissue-specific extraction buffers should be optimized to preserve protein complexes - for brain tissue, buffers containing mild detergents like 0.5% NP-40 or 1% Triton X-100 with protease inhibitors are recommended . Region-specific analyses are essential, as ENO2 shows differential interactions across brain regions, particularly between the striatum and hippocampus . Co-immunoprecipitation with proteasome antibodies has successfully detected ENO2 as a proteasome-interacting protein, suggesting this approach is valuable for identifying novel interaction partners . For confirmation of interactions, reciprocal co-immunoprecipitation (using ENO2 antibodies to pull down suspected partners) should be performed. Advanced techniques like proximity ligation assays or FRET can provide spatial information about these interactions within cells. When studying the functional significance of interactions, point mutations of specific residues (such as D419S) can be employed to disrupt binding and assess functional consequences .

How does ENO2 function as a receptor for extracellular proteins in motor neurons?

ENO2 functions as a cell membrane receptor in motor neurons, particularly for extracellular phosphoglycerate kinase 1 (ePgk1) which is secreted by muscle cells. This interaction promotes neurite outgrowth of motor neurons (NOMN) . The binding mechanism involves specific amino acid residues, with the 419th aspartic acid (D419) being particularly critical for this interaction . When ePgk1 binds to membrane-localized ENO2, it triggers downstream signaling pathways that regulate actin dynamics through modulation of cofilin phosphorylation. Specifically, the ENO2-ePgk1 interaction normally decreases p-Cofilin levels, which inhibits growth cone collapse and promotes axonal growth . Mutation of the D419 residue to serine (D419S) significantly disrupts this interaction, leading to increased p-Cofilin levels and impaired neurite outgrowth both in vitro and in vivo . This receptor function appears to be specific to ENO2, as other enolase isozymes like ENO1 do not share this capability despite structural similarities .

What are the critical amino acid residues in mouse ENO2 for motor neuron axonal growth?

Research has identified the 419th aspartic acid (D419) as the most critical residue in mouse ENO2 for motor neuron axonal growth . Through systematic mutagenesis studies comparing ENO2 to ENO1 (which does not promote axonal growth), seven point mutations were constructed and tested . Among these, the D419S mutation (changing aspartic acid to serine at position 419) showed the most profound reduction in neurite length and disrupted the ENO2-ePgk1 interaction necessary for promoting neurite outgrowth . The E420K mutation also reduced neurite outgrowth but to a lesser extent than D419S . The functional significance of D419 was confirmed both in vitro using NSC34 cells and in vivo using zebrafish models, where embryos expressing ENO2-D419S failed to rescue the axonal growth defects caused by ENO2 knockdown, even in the presence of ePgk1 . These findings indicate that D419 is essential for the receptor function of ENO2 in binding ePgk1 and promoting downstream signaling required for axonal growth.

How can I effectively study the relationship between ENO2 and proteasome complexes in mouse striatum?

To effectively study the relationship between ENO2 and proteasome complexes in the mouse striatum, a multi-faceted approach is recommended. Begin with region-specific tissue isolation, carefully dissecting the striatum and comparable regions like the hippocampus for control comparisons . For proteasome complex purification, use gradient centrifugation or chromatography techniques optimized to preserve native protein complexes . Co-immunoprecipitation with antibodies against proteasome subunits can effectively pull down ENO2 from striatal extracts, and this should be validated with reciprocal immunoprecipitation using ENO2 antibodies . For cellular studies, the ST HDH Q7/7 cell line derived from mouse striatal neurons provides an excellent model system . These cells can be cultured at 33°C in DMEM with 10% FCS and differentiated to neuronal cells using a specific differentiation protocol involving serum-free DMEM supplemented with α-FGF, TPA, forskolin, and dopamine . For functional studies, proteasome activity assays in the presence or absence of ENO2 can reveal its modulatory effects on proteasome function. Additionally, proximity ligation assays can visualize the ENO2-proteasome interaction in situ within striatal neurons.

How does ENO2 contribute to cancer progression in mouse models?

ENO2 has been identified as a significant contributor to cancer progression in multiple mouse models through several mechanisms. In colorectal cancer models with microsatellite instability-high (MSI-H), ENO2 overexpression promotes cell migration, invasion, and epithelial-mesenchymal transition (EMT) . Functional experiments with ENO2 knockdown and overexpression have demonstrated its critical role in glycolysis, providing energy for rapidly dividing cancer cells . ENO2 also enhances the invasive capacity of cancer cells by promoting EMT through regulation of key markers – decreasing epithelial markers like E-cadherin while increasing mesenchymal markers such as N-cadherin and SLUG . Mechanistically, ENO2 appears to modulate these processes via the PI3K-AKT signaling pathway, a key regulator of cell survival and migration . Additionally, ENO2 has been implicated as a biomarker for clear cell renal cell carcinoma (ccRCC), where it regulates energy metabolism to promote cancer progression . These findings across multiple cancer types suggest that ENO2 plays a conserved role in promoting malignant transformation and progression in mouse models.

What methodologies are recommended for studying ENO2's role in glycolysis and EMT in mouse cancer models?

For studying ENO2's role in glycolysis and EMT in mouse cancer models, a comprehensive methodological approach is recommended. For glycolytic function assessment, begin with Seahorse XF analysis to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in ENO2-overexpressing or ENO2-knockdown mouse cancer cell lines . Complement this with lactate production assays and glucose consumption measurements to directly quantify glycolytic flux. For investigating EMT, Western blotting and immunofluorescence should be performed to assess key markers including E-cadherin, N-cadherin, vimentin, and SLUG in cells with modified ENO2 expression . Wound healing and transwell assays are effective for evaluating the effects of ENO2 on cell migration and invasion capabilities . For mechanistic studies, investigate the PI3K-AKT pathway activation status through phospho-specific antibodies against key signaling nodes, and validate using specific pathway inhibitors . For in vivo relevance, orthotopic implantation of ENO2-modified mouse cancer cells can be performed, followed by assessment of tumor growth, invasiveness, and metastatic potential. Single-cell RNA sequencing of resulting tumors can provide insights into heterogeneity and identify malignant subpopulations associated with ENO2 expression .

How can I design experiments to investigate ENO2 as a prognostic biomarker in mouse tumor models?

To design experiments investigating ENO2 as a prognostic biomarker in mouse tumor models, a systematic approach is necessary. First, establish baseline ENO2 expression profiles across different mouse tumor types using immunohistochemistry, Western blotting, and RT-qPCR to identify cancer types with variable ENO2 expression . Next, develop mouse tumor models with controlled manipulation of ENO2 expression through CRISPR/Cas9 gene editing or shRNA-mediated knockdown . For colorectal cancer models, consider using MSI-H mouse cancer cell lines like RKO or GP2D with modified ENO2 expression . Implement longitudinal studies tracking tumor growth, invasion depth, perineural invasion, and survival outcomes in relation to ENO2 expression levels . Collect tissues at different stages of tumor progression to correlate ENO2 expression with pathological features. For mechanistic insights, perform transcriptomic and proteomic analyses on tumors with varying ENO2 expression to identify associated pathways and potential therapeutic targets . Based on the data from ENO2-associated gene clusters in human cancers, develop and validate a prognostic nomogram incorporating ENO2 expression and other clinical parameters . Finally, evaluate treatment responses in ENO2-high versus ENO2-low tumors to determine if ENO2 status can predict therapeutic efficacy.

What are common challenges in detecting ENO2 protein in mouse neural tissue samples?

Several challenges commonly arise when detecting ENO2 protein in mouse neural tissue samples. First, tissue preservation is critical – rapid post-mortem changes can affect ENO2 stability, requiring immediate flash-freezing or fixation of samples . The cellular heterogeneity of brain tissue presents another challenge, as ENO2 expression varies significantly between neuronal and non-neuronal cells, potentially diluting signals in whole tissue lysates . When using immunohistochemistry, antibody specificity is crucial as cross-reactivity with other enolase isoforms (ENO1 and ENO3) can occur due to structural similarities . Optimization of extraction buffers is essential – standard RIPA buffers may disrupt ENO2's interactions with membrane components or protein complexes like the proteasome . For Western blotting detection, the presence of multiple ENO2 isoforms or post-translational modifications can result in multiple bands that require careful interpretation . When studying membrane-associated ENO2, standard extraction protocols may fail to efficiently isolate the membrane-bound fraction, necessitating specific membrane protein extraction methods . For detecting region-specific differences, microdissection techniques may be necessary to isolate specific brain areas where ENO2 shows differential expression or interactions .

How can I optimize antibody selection for mouse ENO2 detection in different experimental contexts?

Optimizing antibody selection for mouse ENO2 detection requires consideration of several experimental factors. For immunohistochemistry applications, monoclonal antibodies like NSE-P2 demonstrate high specificity for ENO2 without cross-reactivity to other enolase isoforms . When selecting antibodies, verify that they recognize the specific epitope of interest – for instance, studies focusing on the D419 residue may require antibodies whose binding is not affected by mutations at this position . For co-immunoprecipitation studies of ENO2 with proteasome complexes or other interaction partners, choose antibodies that do not interfere with protein-protein interaction domains . When detecting membrane-localized ENO2, select antibodies recognizing extracellular epitopes for non-permeabilized immunofluorescence studies . For Western blotting, antibodies recognizing denatured ENO2 epitopes are preferred, while native structure-recognizing antibodies work better for immunoprecipitation . Consider validating antibody specificity using ENO2 knockout or knockdown samples as negative controls . For multiplexed detection, select antibodies raised in different host species to avoid cross-reactivity in co-staining experiments. Always perform titration experiments to determine optimal antibody concentrations, as high concentrations may lead to non-specific binding while too low concentrations may yield insufficient signal-to-noise ratios.

What controls should be included when conducting ENO2 knockdown or mutation studies in mouse neuronal cells?

When conducting ENO2 knockdown or mutation studies in mouse neuronal cells, several essential controls should be included to ensure experimental validity. For siRNA knockdown experiments, include a non-targeting siRNA control with similar GC content to the ENO2-targeting siRNA to account for non-specific effects of the transfection process . When using lentiviral shRNA approaches, empty vector controls are essential to distinguish between ENO2-specific effects and those caused by viral transduction . For point mutation studies (such as D419S), include both wild-type ENO2 overexpression and empty vector controls to differentiate between effects of the mutation and those of overexpression itself . When performing rescue experiments following knockdown, use a wobble-mutated ENO2 construct that is resistant to the siRNA but encodes the same amino acid sequence as wild-type ENO2 . For functional assays following ENO2 manipulation, include positive controls known to affect the pathway of interest (e.g., cytoskeletal disruptors for neurite outgrowth assays) . When studying ENO2's role in specific signaling pathways like PI3K-AKT, include pathway inhibitor controls to confirm the specificity of observed effects . For long-term studies, monitor ENO2 expression levels throughout the experiment as compensatory mechanisms may restore expression over time despite initial knockdown. Finally, validate knockdown efficiency at both mRNA (RT-qPCR) and protein (Western blot) levels, as post-transcriptional regulation may result in discrepancies between transcript and protein reduction .