ALDH1A1 Human, Active

What is the primary biological function of ALDH1A1 in human cells?

ALDH1A1 performs multiple functions in human cellular metabolism. Its primary enzymatic activity involves catalyzing the oxidation of retinol (vitamin A) metabolite, retinal (retinaldehyde), to retinoic acid (RA) . This process is critical for RA signaling pathways that regulate numerous developmental and cellular processes. Beyond retinoid metabolism, ALDH1A1 plays a significant role in cellular detoxification by metabolizing highly reactive aldehydes, including acetaldehyde from ethanol metabolism and products of lipid peroxidation such as 4-hydroxynonenal and malondialdehyde generated during oxidative stress . In dopaminergic neurons, ALDH1A1 provides neuroprotection by oxidizing 3,4-dihydroxyphenylacetaldehyde, a toxic metabolite of dopamine .

Methodologically, researchers can investigate these functions using enzyme activity assays with specific substrates, gene knockout studies, and metabolic profiling of retinoid derivatives and aldehyde substrates.

How is ALDH1A1 expression regulated across different tissue types?

ALDH1A1 demonstrates tissue-specific expression patterns with regulatory mechanisms that vary by cell type. It is highly expressed in:

• Liver hepatocytes, where it plays a key role in retinal oxidation and alcohol metabolism

• Hematopoietic stem cells (HSCs), where it regulates self-renewal and differentiation

• Corneal stromal keratocytes, where expression increases during development of postnatal corneal transparency

• Retina of mice, rabbits, and chickens, though its retinal function remains unclear as Aldh1a1(-/-) knockout mice exhibit normal retinal development

Interestingly, selective ALDH1A1 expression has been observed in queen honey bees but not worker bees, suggesting a role in caste-selective metabolism and antioxidant response . Expression patterns can be analyzed through tissue-specific RT-PCR, immunohistochemistry (as demonstrated with liver tissue microarrays ), and single-cell RNA sequencing approaches.

What methodologies are most effective for detecting and measuring ALDH1A1 activity in research samples?

Several methodologies provide reliable detection and quantification of ALDH1A1:

• Aldefluor® Assay - This metabolism-based fluorescent assay allows for flow cytometric identification and isolation of cells with high ALDH activity (ALDH^bright cells). This method is particularly valuable for isolating potential cancer stem cells .

• Real-time PCR - For expression analysis at the mRNA level, as demonstrated in studies examining ALDH1A1 expression in peripheral blood .

• Immunohistochemistry (IHC) - Using specific antibodies to detect ALDH1A1 protein in tissue sections, as shown in liver tissue microarray analyses using rabbit anti-ALDH1A1 mAb .

• Spectrophotometric Enzyme Activity Assays - Direct measurement of enzyme kinetics using specific substrates like retinal or acetaldehyde.

• High-content cell-based assays - For screening ALDH1A1 inhibitors and measuring cellular activity, as implemented during drug discovery efforts .

When interpreting results, researchers should consider that some methods (particularly Aldefluor®) may detect activity from multiple ALDH isoforms rather than ALDH1A1 specifically.

How does ALDH1A1 contribute to cancer stem cell biology and what are the implications for targeting cancer-initiating cells?

ALDH1A1 has emerged as a critical marker and functional component of cancer stem cells (CSCs) or cancer-initiating cells (CICs):

ALDH^bright cells isolated from various carcinomas demonstrate profound tumorigenic potential—as few as 500 ALDH^bright cells from HLA-A2+ breast, head and neck, and pancreatic carcinoma cell lines were sufficient to establish tumors in immunodeficient mice . This indicates that ALDH activity, significantly attributed to ALDH1A1, identifies a subpopulation with stem-like properties.

The functional role of ALDH1A1 in CSCs appears multi-faceted:

• It potentially mediates retinoic acid signaling that regulates self-renewal and differentiation

• It provides cytoprotection through detoxification of reactive aldehydes

• It contributes to drug resistance, particularly against conventional chemotherapeutics

Targeting strategies have yielded promising results. ALDH1A1-specific CD8+ T cells can recognize and eliminate ALDH^bright cells in vitro and in vivo. Adoptive therapy with these immune cells has been shown to inhibit tumor growth and metastases in xenograft models . This approach represents a potential immunotherapeutic strategy to selectively target the cancer-initiating cell population responsible for treatment resistance and recurrence.

ALDH1A1 expression in epithelial cancers correlates with poor prognosis and tumor grade , further supporting its clinical significance in cancer progression.

What genetic variations in ALDH1A1 have been identified and how do they influence disease susceptibility?

Genetic analysis has revealed several significant ALDH1A1 variations with disease implications:

*GGTA haplotype includes SNPs rs4646547, rs1888202, rs7043217, and rs647880 in that order.

The mechanism linking ALDH1A1 variations to Parkinson's disease likely involves its role in detoxifying dopamine metabolites. Animal models suggest ALDH1A1 protects against PD by reducing toxic metabolites of dopamine . Interestingly, interaction analyses revealed that simultaneous presence of the CC genotype of rs7043217 (ALDH1A1) and the TT genotype of rs4767944 (ALDH2) confers elevated protection against PD (P = 4.68×10^-4, OR = 0.378) .

Methodologically, researchers can investigate these associations through tag-SNP analysis, haplotype construction, and gene-gene interaction analysis, as demonstrated in the Han Chinese population study that identified these associations .

What are the challenges and opportunities in developing selective ALDH1A1 inhibitors for therapeutic applications?

The development of ALDH1A1 inhibitors presents several unique challenges and opportunities:

Challenges:

• Selectivity - Achieving specificity against other ALDH isoforms, particularly ALDH2 and ALDH3A1, which share structural similarities with ALDH1A1

• Cellular potency - Many compounds with good in vitro potency exhibit moderate to low cellular activities, as seen in high ALDH1A1-expressing pancreatic cancer cell lines

• Off-target effects - ALDH1A1 expression in normal tissues, particularly liver hepatocytes, raises potential toxicity concerns

• Bioavailability - Developing inhibitors with appropriate pharmacokinetic properties for in vivo applications

Key Inhibitor Developments:
Several classes of ALDH1A1 inhibitors have been developed:

• Compound 1: A substrate-competitive inhibitor with 0.02 μM potency and selectivity against ALDH2 and ALDH3A1

• Tricyclic pyrimidinone (Compound 2): 4.6 μM potency, selectively disrupts ovarian cancer spheroid formation and sensitizes cells to cisplatin

• Compound 3: A tricyclic ALDH1A1-selective inhibitor that sensitizes multidrug-resistant ovarian cancer cells to paclitaxel or doxorubicin

• NCT-501 (Compound 4): A theophylline-derived inhibitor with in vivo efficacy in cisplatin-resistant head and neck squamous cell carcinoma xenografts

• Quinoline-based inhibitors (Compound 6): A hybrid design approach combining features of compound 4 and previously identified hits

Research opportunities include developing high-throughput screening assays, implementing cellular target engagement studies, and validating inhibitors in diverse disease models beyond cancer, including obesity, diabetes, and inflammatory conditions .

What is the role of ALDH1A1 in neurodegenerative diseases, particularly Parkinson's disease?

ALDH1A1 appears to play a neuroprotective role in Parkinson's disease (PD) through several mechanisms:

• Dopamine metabolite detoxification - ALDH1A1 oxidizes 3,4-dihydroxyphenylacetaldehyde, a toxic metabolite of dopamine, potentially protecting dopaminergic neurons from toxicity

• Genetic evidence - The tag-SNP rs7043217 of ALDH1A1 shows significant association with PD susceptibility, with the T allele serving as a risk factor (genotype frequency, P = 0.030; allele frequency, P = 0.013, OR = 1.258)

• Haplotype associations - Multiple ALDH1A1 haplotypes link to abnormalities in PD risk, most notably a 4-SNP GGTA module (rs4646547, rs1888202, rs7043217, rs647880) with P = 9.610×10^-8, OR = 6.420

• Expression changes - ALDH1A1 mRNA expression shows a trend of reduction (P = 0.084) in PD patients compared to controls

• Gene-gene interactions - Interactions between ALDH1A1 and ALDH2 variants suggest cooperative neuroprotective effects, as simultaneous presence of the CC genotype of rs7043217 (ALDH1A1) and the TT genotype of rs4767944 (ALDH2) confers elevated protection against PD (P = 4.68×10^-4, OR = 0.378)

These findings suggest that therapeutic strategies that enhance ALDH1A1 activity or expression might be neuroprotective in PD. Research approaches should include genetic association studies in diverse populations, functional validation in cellular and animal models, and exploration of small molecules that can modulate ALDH1A1 activity in the brain.

How can ALDH1A1 be effectively targeted in chemotherapy-resistant cancers?

ALDH1A1 appears to contribute to chemotherapy resistance through several mechanisms, and multiple targeting strategies have shown promise:

Mechanisms of ALDH1A1-mediated chemoresistance:

• Cytoprotection through detoxification of reactive aldehydes generated by chemotherapy

• Identification of a therapy-resistant cancer stem cell population (ALDH^bright cells)

• Potential involvement in drug metabolism pathways

Effective targeting strategies:

• Immunotherapeutic approaches - ALDH1A1-specific CD8+ T cells can recognize and eliminate ALDH^bright cells present in various carcinoma cell lines, xenografts, and surgically removed lesions. Adoptive therapy with these T cells has shown efficacy in inhibiting tumor growth and metastases in xenograft models .

• Small molecule inhibitors - Several ALDH1A1 inhibitors have demonstrated the ability to reverse chemoresistance:

  • Compound 2 (tricyclic pyrimidinone) sensitized ovarian cancer cells to cisplatin

  • Compound 3 sensitized multidrug-resistant ovarian cancer cells to paclitaxel and doxorubicin

  • NCT-501 (Compound 4) demonstrated efficacy in cisplatin-resistant head and neck squamous cell carcinoma xenografts

  • ALDH1A1 depletion was shown to reverse cisplatin resistance in human lung cancer cell line A549/DDP

• Combination approaches - Combining ALDH1A1 inhibitors with conventional chemotherapeutics has shown synergistic effects.

Research should focus on the optimal timing of ALDH1A1 targeting relative to chemotherapy administration, patient selection based on ALDH1A1 expression profiles, and development of combination regimens that effectively eliminate both ALDH^bright and ALDH^low cancer cell populations.

What considerations are important when designing experiments to study ALDH1A1 activity in human samples?

When designing experiments to study ALDH1A1 activity in human samples, researchers should consider:

• Isozyme specificity - ALDH1A1 is one of many ALDH isozymes. Ensure methods can distinguish between ALDH1A1 and other family members, particularly ALDH2 (mitochondrial) and ALDH3A1 .

• Substrate selection - For enzymatic assays, consider that ALDH1A1 has high affinity for both all-trans- and 9-cis-retinal, along with other aldehydes including acetaldehyde and products of lipid peroxidation .

• Sample preparation - ALDH1A1 is primarily cytosolic, unlike ALDH2 which is mitochondrial. Subcellular fractionation may be necessary to accurately measure isozyme-specific activity .

• Expression heterogeneity - ALDH1A1 expression can vary significantly between cell types within a tissue. Single-cell approaches or microdissection techniques may be necessary to account for this heterogeneity .

• Genetic variation - Consider screening for relevant ALDH1A1 polymorphisms (e.g., rs7043217) that may affect enzyme activity and disease associations .

• Functional validation - Complement activity measurements with functional assays relevant to the specific role being studied (e.g., stem cell properties, chemoresistance, retinoic acid production) .

• Controls and standards - Include appropriate positive and negative controls, such as known ALDH1A1 inhibitors (e.g., NCT-501) or samples from ALDH1A1 knockout models .

A robust experimental design would typically combine multiple approaches, such as enzyme activity assays, expression analysis (mRNA and protein), and functional assessments appropriate to the research question.

How can researchers effectively isolate and characterize ALDH1A1-expressing stem cell populations?

Isolation and characterization of ALDH1A1-expressing stem cell populations require specific methodological approaches:

Isolation techniques:

• Aldefluor® assay with flow cytometry - This metabolism-based assay remains the gold standard for isolating ALDH^bright cells. The assay utilizes a fluorescent substrate that is retained in cells with high ALDH activity. When combined with fluorescence-activated cell sorting (FACS), researchers can isolate viable ALDH^bright cells for further analysis .

• Combined marker strategy - For increased specificity, combine Aldefluor® with additional stem cell markers. For example, in hematopoietic stem cells, combining CD34 with ALDH activity can increase purity .

• Immunomagnetic separation - Using antibodies against ALDH1A1 (though less common than functional Aldefluor® assay).

Characterization approaches:

• Tumorigenic potential - Xenograft assays with limiting dilutions of sorted ALDH^bright cells. As demonstrated, as few as 500 ALDH^bright cells from various carcinoma cell lines were sufficient to establish tumors in immunodeficient mice .

• Self-renewal capacity - Serial sphere formation assays (e.g., mammospheres, tumorspheres) to assess stem-like properties .

• Differentiation potential - Assessing the ability of isolated cells to generate diverse cell types of the original tissue.

• Resistance profiling - Testing isolated ALDH^bright cells for resistance to conventional therapies like cisplatin, paclitaxel, or doxorubicin .

• Molecular profiling - RNA-seq, proteomics, or metabolomics analysis of isolated populations to identify associated pathways.

• Immunophenotyping - Comprehensive analysis of other stem cell markers and pathway components that may cooperate with ALDH1A1.

For validation, researchers should perform functional inhibition studies using ALDH1A1 inhibitors or genetic knockdown/knockout approaches to confirm the specific contribution of ALDH1A1 to the observed stem cell properties.

What is the clinical significance of ALDH1A1 expression in various human cancers?

ALDH1A1 expression has emerged as a clinically relevant biomarker across multiple cancer types:

• Prognostic significance - Immunohistochemical studies have revealed increased expression of ALDH1A1 protein in human epithelial cancers, which appears to be an indicator of poor prognosis and correlates with the histological grade of the tumor .

• Cancer stem cell marker - ALDH1A1 expression and activity identifies a subpopulation of cancer cells with stem-like properties (ALDH^bright cells) that demonstrate profound tumorigenic potential, even at low cell numbers (500 cells) .

• Treatment resistance indicator - High ALDH1A1 expression is associated with resistance to conventional treatments. For example:

  • ALDH1A1 depletion can reverse cisplatin resistance in human lung cancer cell line A549/DDP

  • ALDH1A1 inhibition sensitizes ovarian cancer cells to cisplatin, paclitaxel, and doxorubicin

• Therapeutic target - ALDH1A1-specific CD8+ T cells can recognize and eliminate ALDH^bright cells in vitro and in vivo, with adoptive therapy inhibiting tumor growth and metastases in xenograft models .

• Metastasis association - ALDH^bright cells are considered responsible for tumor recurrence and metastasis .

For clinical application, standardized assessment methods are needed, including threshold values for "high" versus "low" expression, consensus on antibody clones for IHC, and integrated analysis with other biomarkers. Furthermore, prospective clinical trials evaluating ALDH1A1-targeted therapies or ALDH1A1 as a companion diagnostic would advance the translational potential of these findings.

How does ALDH1A1 contribute to metabolic regulation and what are the implications for metabolic disorders?

ALDH1A1 plays several significant roles in metabolic regulation with implications for metabolic disorders:

• Protection against diet-induced obesity - Interestingly, Aldh1a1(-/-) knockout mice are protected against diet-induced obesity and insulin resistance, suggesting that ALDH1A1 may regulate the metabolic response to high-fat diet through mechanisms involving retinal metabolism .

• Retinoid metabolism - As a key enzyme in retinoid metabolism, ALDH1A1 regulates retinoic acid (RA) production, which impacts adipogenesis, glucose homeostasis, and energy expenditure. Knockout mice display reduced RA synthesis and increased serum retinal levels following retinol treatment .

• Liver metabolism - ALDH1A1 is highly expressed in liver hepatocytes where it plays a key role in oxidizing retinal and potentially influencing broader metabolic pathways .

• Alcohol metabolism - ALDH1A1 is involved in ethanol metabolism by eliminating acetaldehyde, a toxic metabolite. Polymorphic variants resulting in low enzyme activity have been associated with alcohol sensitivity in Caucasians .

• Antioxidant function - Through its ability to detoxify reactive aldehydes, ALDH1A1 may protect against oxidative stress-induced metabolic dysfunction .

These findings suggest that ALDH1A1 inhibition could potentially represent a therapeutic strategy for metabolic disorders, particularly obesity and diabetes . Research approaches should include:

• Metabolic phenotyping of tissue-specific ALDH1A1 knockout models

• Metabolomic analysis of ALDH1A1 deficiency states

• Investigation of ALDH1A1 inhibitors in metabolic disease models

• Exploration of genetic variations in human populations with metabolic disorders

What are the safety considerations when targeting ALDH1A1 in therapeutic applications?

When targeting ALDH1A1 for therapeutic purposes, several important safety considerations must be addressed:

• Expression in normal tissues - ALDH1A1 is expressed in multiple normal tissues, particularly:

  • Liver hepatocytes, where it plays important metabolic roles

  • Hematopoietic stem cells, where it regulates self-renewal and differentiation

  • Corneal stromal keratocytes, where it contributes to transparency

  • Dopaminergic neurons, where it provides neuroprotection

• Potential on-target toxicities:

  • Hepatotoxicity due to high expression in normal liver

  • Hematopoietic suppression affecting normal stem cell function

  • Visual disturbances due to corneal or retinal effects

  • Neurological effects through altered dopamine metabolism

• Metabolic effects - Given ALDH1A1's role in retinoid metabolism and protection against diet-induced obesity, inhibition might alter metabolic homeostasis .

• Alcohol metabolism interference - Potential interactions with alcohol consumption due to ALDH1A1's role in metabolizing acetaldehyde .

• Selectivity challenges - Ensuring specificity against other ALDH family members, particularly ALDH2 and ALDH3A1, to prevent off-target effects .

Mitigation strategies include:

• Developing highly selective inhibitors with limited distribution to non-target tissues

• Exploring tissue-specific delivery systems

• Implementing careful monitoring protocols in clinical studies

• Considering intermittent dosing schedules to allow recovery of normal stem cell populations

• Evaluating immunotherapy approaches that might offer greater specificity than small molecule inhibitors

Preliminary safety assessment in IHC analyses of liver tissue microarrays has been conducted to evaluate ALDH1A1 and HLA class I antigen expression in normal liver hepatocytes , highlighting the importance of understanding target expression in normal tissues.

What emerging technologies could advance our understanding of ALDH1A1 function and targeting?

Several cutting-edge technologies show promise for advancing ALDH1A1 research:

• CRISPR-Cas9 genome editing - Precise modification of ALDH1A1 regulatory elements or coding regions can help elucidate structure-function relationships and create improved disease models.

• Single-cell multi-omics - Integrating single-cell transcriptomics, proteomics, and metabolomics to understand ALDH1A1's role in cellular heterogeneity, particularly in cancer stem cell populations .

• Spatial transcriptomics/proteomics - Mapping ALDH1A1 expression in spatial context within tissues to understand microenvironmental influences on its expression and function.

• Organoid models - Patient-derived organoids can serve as platforms for testing ALDH1A1 inhibitors and studying ALDH1A1's role in stem cell dynamics in a physiologically relevant context.

• Chemoproteomics and activity-based protein profiling - These approaches can identify ALDH1A1 interaction networks and develop more selective inhibitors with fewer off-target effects .

• In vivo imaging of ALDH activity - Development of non-invasive imaging probes to visualize ALDH activity in living organisms could advance translational research.

• Artificial intelligence for drug design - Machine learning approaches to design novel ALDH1A1 inhibitors with improved selectivity and pharmacokinetic properties, building on existing compound series .

• Antibody-drug conjugates - Targeting ALDH1A1-expressing cells with antibody-drug conjugates could provide more selective delivery of cytotoxic agents to cancer stem cells.

These technologies could help address key questions, including the precise mechanism by which ALDH1A1 contributes to chemoresistance, the structural basis for developing isoform-selective inhibitors, and the biological significance of ALDH1A1 genetic variations.

What are the most promising therapeutic applications for ALDH1A1 modulation beyond cancer?

Based on current research, several non-cancer therapeutic applications for ALDH1A1 modulation show promise:

• Metabolic disorders - Given that Aldh1a1(-/-) knockout mice are protected against diet-induced obesity and insulin resistance, ALDH1A1 inhibition represents a potential approach for treating obesity and metabolic syndrome . This could function through mechanisms involving retinoid metabolism, which impacts adipogenesis and energy homeostasis.

• Neurodegenerative diseases - ALDH1A1's role in dopamine metabolism suggests potential applications in Parkinson's disease. The significant genetic associations between ALDH1A1 variants and PD risk support this direction . Rather than inhibition, enhancing ALDH1A1 activity might be neuroprotective in this context.

• Inflammatory conditions - ALDH1A1 expression in macrophages has been linked to inflammatory phenotypes in Crohn's disease, suggesting potential applications in inflammatory bowel disease and other inflammatory conditions .

• Alcohol use disorder - Given ALDH1A1's role in alcohol metabolism and the association of polymorphic variants with alcohol sensitivity, modulating its activity might influence alcohol consumption behavior or alcohol-related toxicity .

• Stem cell mobilization - Modulating ALDH1A1 activity could potentially enhance hematopoietic stem cell mobilization for transplantation purposes, given its high expression in HSCs .

• Ocular conditions - ALDH1A1's contribution to corneal transparency suggests potential applications in corneal injury and opacity conditions. Research indicates injury-induced corneal haze is associated with loss of ALDH1A1 expression .