Recombinant Mouse Acyl-CoA-binding domain-containing protein 5 (Acbd5)

What is the primary cellular function of Acbd5 in mice?

Acbd5 is a tail-anchored membrane protein localized at peroxisomes, with its C-terminus inserted in the peroxisomal membrane while its acyl-CoA binding domain faces the cytosol. Its primary function involves tethering peroxisomes to the endoplasmic reticulum (ER) by binding to the ER-resident proteins VAPA and VAPB. This interaction induces membrane contact site formation between peroxisomes and the ER, which is critical for several cellular processes including lipid metabolism. Beyond its tethering role, Acbd5 contributes to the regulation of anabolic and catabolic cellular lipid pathways through its acyl-CoA binding function .

How does Acbd5 differ from other acyl-CoA binding proteins?

Acbd5 belongs to a family of acyl-CoA binding proteins, but it is specifically distinguished by its peroxisomal localization and tethering function. While ACBD4 is another acyl-CoA binding protein that can promote peroxisome-ER associations through VAPB interaction, research has shown that ACBD4 expression does not significantly change to compensate for the lack of ACBD5 in knockout models. This suggests non-redundant functions despite similar domain structures . The peroxisomal ACBD4 isoform 2 is found in the same tissues as ACBD5 but appears to have distinct physiological roles, highlighting the specialized function of Acbd5 in maintaining peroxisome-ER contacts specifically for lipid metabolism regulation.

What are the available mouse models for studying Acbd5 deficiency?

There are currently two well-characterized mouse models for studying Acbd5 deficiency:

• The C57BL/6N-A tm1Brd Acbd5 tm1a(EUCOMM)Wtsi/WtsiCnbc mouse line (referred to as Acbd5−/−): This model exhibits efficient block in ACBD5 expression, verified through qRT-PCR of mRNA from different tissues and immunodetection in mouse embryonic fibroblasts .

• The B6.B6/129-Acbd5 em1#9PB/PB strain (Gly357* mice): This model was developed using CRISPR/Cas9 to introduce a premature stop codon (p.Gly357*) in exon 9 of the Acbd5 gene. Western blot analysis confirmed undetectable protein levels, indicating that the Gly357* allele functions as a null allele . This model was backcrossed to C57BL/6J to eliminate possible off-target mutations, creating a congenic strain with an estimated 99% of the donor genome eliminated by generation N7.

Both models demonstrate similar pathological features, including elevated very long-chain fatty acid (VLCFA) levels and progressive neurological defects, mirroring aspects of the human disorder.

How can researchers genotype Acbd5-deficient mouse models effectively?

For the Gly357* mouse model, a PCR-based genotyping approach using primers flanking the knock-in sequence can effectively differentiate between wild-type, heterozygous, and homozygous mutants. The specific primers reported are:

• Forward: 5′-TCAAACAATGGACACTTTCAGT-3′

• Reverse: 5′-GCTACTTCTGCCATCTTCTCC-3′

This PCR yields amplicons of 180 bp for the wild-type allele and 201 bp for the mutant Acbd5 Gly357* allele . For genotyping offspring from heterozygous matings, researchers should expect normal Mendelian ratios as Gly357* mice are viable.

For confirmation of Acbd5 expression levels, researchers should complement genotyping with qRT-PCR using primers targeting Acbd5 (Forward: 5′-GCCGTGAAGGTGATCCAGAG-3′, Reverse: 5′-CAGAATCCAGGCCGTGAAAG-3′) and Western blot analysis of tissues such as liver and spinal cord to confirm the absence of protein expression in homozygous mutants .

What are the tissue-specific lipid alterations in Acbd5-deficient mice?

Lipidomic analyses of Acbd5-deficient mice have revealed profound and tissue-specific alterations in lipid composition. In cerebellum, there is a notable accumulation of unusual ultra-long chain fatty acids (C > 32) in phosphocholines, which are not elevated in liver, indicating an organ-specific imbalance in fatty acid degradation and elongation pathways .

In the liver, the primary alteration is in ether lipid formation, with an accumulation of alkyldiacylglycerols. Unbiased LC-MS lipidomic analysis of spinal cords from Gly357* mice revealed dysregulation of 139 lipids, with the main affected classes being phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingolipids (SL) .

The lipid dysregulation in Acbd5-deficient mice is complex and extends beyond simple VLCFA accumulation:

These tissue-specific alterations suggest that Acbd5 plays differential roles in different organs, potentially related to the specific lipid metabolic requirements of each tissue .

How does Acbd5 deficiency affect neuronal function and lead to neurodegeneration?

Acbd5 deficiency leads to progressive neurodegeneration through multiple mechanisms:

• In retina, Acbd5-deficient mice exhibit decreases in photoreceptor outer segment and outer nuclear area, with targeted degeneration of the synaptic terminals of rods and cones, leading to concomitant loss of neurons .

• In cerebellum and spinal cord, there is severe neuropathology with neurodegeneration, gliosis, and myelin loss.

• At the cellular level, proteomic analysis of Gly357* mice revealed disruption in actin dynamics. This was confirmed in cultured cortical neurons from Gly357* mice treated with methyl-ester C24:0 (meC24:0), which displayed increased actin-enriched filipodia, an abundance of actin patches, and a 20% reduction in actin retrograde flow dynamics .

• The pathology affects multiple cell types, with protein dysregulations observed in neurons, oligodendrocytes, astrocytes, and microglia, all identified as targets of Acbd5 deficiency.

These findings suggest that VLCFA accumulation due to impaired β-oxidation in Acbd5-deficient mice may directly disrupt cellular processes or mediate further alterations in lipid composition, resulting in widespread cellular dysfunction and eventual neurodegeneration .

What are the optimal methods for measuring peroxisome-ER contacts in Acbd5 research?

For accurate visualization and quantification of peroxisome-ER contacts in Acbd5 research, serial section scanning electron microscopy (S³EM) has proven highly effective. This technique was used to verify the extensive reduction in peroxisome-ER membrane contacts in hepatocytes of Acbd5-deficient mice .

Alternative methods include:

• Fluorescence microscopy with co-localization analysis of peroxisomal and ER markers

• Proximity ligation assays to detect protein-protein interactions between peroxisomal and ER proteins

• Transmission electron microscopy to directly visualize membrane contact sites

When analyzing peroxisome-ER contacts, it is crucial to examine multiple cell types as the degree of contact reduction may vary by tissue. Additionally, researchers should consider assessing peroxisome membrane extension capacities under peroxisome proliferating conditions, as these capacities were reduced in Acbd5-deficient models .

What lipidomic approaches are most informative for characterizing Acbd5 deficiency?

The most informative lipidomic approaches for characterizing Acbd5 deficiency include:

• Unbiased LC-MS lipidomic analysis: This has been successfully used to identify dysregulation across multiple lipid species in tissues like spinal cord from Acbd5-deficient mice. The approach revealed alterations in 139 lipids, particularly affecting phosphatidylcholines, phosphatidylethanolamines, and sphingolipids .

• Targeted fatty acid analysis: Particularly focusing on very long-chain fatty acids (VLCFAs) such as C26:0, which typically show a 2-fold increase in Acbd5-deficient tissues .

• Tissue-specific comparative analyses: Due to the significant tissue-specific differences in lipid alterations, it is essential to analyze multiple tissues (particularly cerebellum, liver, and spinal cord) to capture the full spectrum of lipid changes.

• Analysis of lipid subspecies: Beyond broad lipid classes, detailed characterization of lipid subspecies with varying fatty acid compositions is crucial, as Acbd5 deficiency affects lipids containing VLCFA as well as those without VLCFA .

For comprehensive characterization, researchers should examine both accumulating lipids (such as those containing saturated and unsaturated VLCFA) and deficient lipid species (including various lysophospholipids), as both aspects contribute to the complex metabolic outcome of impaired Acbd5 function .

How effective is AAV-mediated gene therapy for Acbd5 deficiency in mouse models?

AAV-mediated gene therapy has shown remarkable efficacy in treating Acbd5 deficiency in mouse models. In the Gly357* mouse model, administration of an AAV vector expressing murine Acbd5 (AAV-GFP-P2A-Acbd5) effectively halted the severe progression of pathology and improved characteristic disease manifestations .

Key findings on the efficacy of this approach include:

• A single tail intravenous injection of the viral solution containing 1.06 × 10¹² gene copies for AAV-Acbd5 was sufficient to achieve therapeutic effects.

• The treatment was administered at 3 months of age, and by 6 months, significant improvements were observed compared to controls treated with AAV-GFP.

• The therapy successfully restored Acbd5 expression in Gly357* cells, as verified by Western blot analysis.

• Most importantly, AAV-mediated gene therapy was capable of preventing the escalating degeneration observed within the CNS, demonstrating that despite the multitude of cellular defects, the ataxia and neurodegeneration could be prevented through this approach .

This promising result suggests that AAV-mediated gene therapy could be a viable therapeutic strategy for human patients with ACBD5 deficiency, particularly if intervention occurs before extensive neurodegeneration has developed.

What experimental controls are critical when evaluating therapeutic interventions for Acbd5 deficiency?

When evaluating therapeutic interventions for Acbd5 deficiency, several critical experimental controls should be implemented:

• Vehicle or null vector controls: As demonstrated in the Gly357* mouse study, control groups should receive either vehicle alone or a null vector (such as AAV-GFP) to control for non-specific effects of the vector itself .

• Wild-type untransduced controls: These serve as baseline comparisons for normal phenotype and physiology.

• Pre-treatment and post-treatment assessments: Behavioral and biochemical parameters should be measured before treatment (e.g., at 3 months in the Gly357* study) and at defined intervals post-treatment to track progression or improvement .

• Blinded assessment: As implemented in the mouse behavior studies, evaluations should be conducted by investigators blinded to the treatment groups to prevent bias .

• Multi-parameter evaluation: Given the complexity of Acbd5 deficiency, therapeutic efficacy should be assessed across multiple parameters including:

  • Behavioral outcomes (motor function tests for ataxia)

  • Biochemical markers (VLCFA levels, lipid profiles)

  • Histological analysis (neurodegeneration, gliosis, myelin integrity)

  • Molecular markers (expression of Acbd5 and potential compensatory proteins)

• Dose-response studies: Though not explicitly mentioned in the search results, establishing optimal dosing would be critical for translational applications.

These controls ensure that observed therapeutic effects can be confidently attributed to the intervention rather than to experimental artifacts or natural disease fluctuations .

How might peroxisome-ER contact sites mechanistically influence lipid metabolism beyond simple VLCFA degradation?

The role of peroxisome-ER contact sites in lipid metabolism extends far beyond facilitating VLCFA degradation. These membrane contact sites likely serve as platforms for coordinated lipid synthesis, modification, and trafficking between organelles. In Acbd5-deficient mice, the drastically reduced peroxisome-ER contacts observed in hepatocytes correlate with complex lipid abnormalities that cannot be explained by impaired β-oxidation alone .

Several mechanistic pathways may be involved:

• Lipid exchange and trafficking: Peroxisome-ER contacts may facilitate the transfer of lipid intermediates between organelles for sequential processing steps in biosynthetic pathways, particularly for complex lipids that require enzymatic contributions from both organelles.

• Regulation of ether lipid synthesis: The observed accumulation of alkyldiacylglycerols in liver suggests dysregulation in ether lipid processing, which involves both peroxisomal and ER enzymes .

• Coordination of elongation and degradation: The imbalance between fatty acid elongation (ER) and degradation (peroxisome) pathways observed in Acbd5-deficient mice suggests that the contact sites may serve as regulatory hubs that coordinate these opposing processes.

• Calcium signaling: Peroxisome-ER contacts may influence calcium homeostasis, which in turn regulates multiple enzymes involved in lipid metabolism.

• Peroxisome biogenesis and membrane dynamics: Reduced capacity for peroxisome membrane extension under proliferating conditions in Acbd5-deficient mice suggests that these contacts may contribute to peroxisome growth and division .

Research approaches to address these questions could include proximity-dependent labeling techniques to identify proteins and lipids at the contact sites, and super-resolution microscopy combined with lipid probes to visualize lipid transfer events in real-time.

What is the relationship between VLCFA accumulation and disruption of actin dynamics in neurons from Acbd5-deficient mice?

The relationship between VLCFA accumulation and disruption of actin dynamics in neurons from Acbd5-deficient mice represents a critical mechanistic link in understanding the neurodegeneration associated with Acbd5 deficiency. Proteomic analysis of degenerating spinal cord in Gly357* mice revealed a signature of protein dysregulations highlighting disruption in actin dynamics .

Experimental evidence demonstrates that:

This suggests that VLCFA accumulation directly impacts actin cytoskeletal organization and dynamics . The mechanism could involve:

• Direct interaction of VLCFA with actin-binding proteins, altering their function

• Changes in membrane fluidity and organization due to VLCFA incorporation, affecting membrane-associated actin regulatory proteins

• Altered lipid raft composition, which serves as organizing centers for actin cytoskeletal assembly

• Disruption of signaling pathways that regulate actin dynamics

This relationship provides insight into how abnormal VLCFA metabolism within the CNS leads to neurodegeneration. Importantly, the fact that AAV-mediated Acbd5 expression can prevent these defects suggests that the actin cytoskeletal disruption is a reversible consequence of VLCFA accumulation rather than a permanent developmental abnormality .