Keywords
X-linked adrenoleukodystrophy, X-ALD, ABCD1, Very long chain fatty acids, VLCFA, Neurodegeneration, Demyelination, Myelopathy
Introduction
X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease that mostly affects males. There are three main forms with varying degrees of severity: cerebral adrenoleukodystrophy (cALD), Adrenomyeloneuropathy (AMN), and Addison’s disease.(Berger, Forss-Petter, & Eichler, 2014; Köhler, Curiel, & Vanderver, 2018; Kemp, Huffnagel, Linthorst, Wanders, & Engelen, 2016) The gene involved in X-ALD pathology, ABCD1 , encodes adenosine triphosphate (ATP)- binding cassette subfamily D member 1 (ABCD1) protein, a peroxisomal transmembrane protein. Defective ABCD1 protein fails to transport fatty acyl-CoAs, including very long chain fatty acid (VLCFA)-CoAs into peroxisomes from the cytoplasm for β-oxidation with consequent accumulation of VLCFAs in the cells,(Wiesinger, Kunze, Regelsberger, Forss-Petter, & Berger, 2013) leading to neuron damages. To date, the underlying pathology of X-ALD is poorly understood. A lack of reliable cALD animal models is a hurdle of X-ALD studies. In this review, we will summarize current knowledge of X-ALD, as well as pre-clinical and clinical studies. We will also introduce a novelABCD1- rabbit model created by our lab and discuss potential development of future X-ALD investigation.
Clinical and Biochemical Features of X-ALD
The diagnostic markers of X-ALD include an increase in VLCFAs in blood plasma, especially lignoceric acid (C24:0) and hexacosanoic acid (C26:0) in tissues and body fluid. Since the amount of shorter fatty acid, such as docosanoic acid (C22:0) or even shorter (carbon chain length <22), is little affected by dysfunctional ABCD1 proteins, C26:0/C22:0 is particularly an important index of X-ALD diagnosis. However, this index does not necessarily reflect disease severity. Recently, levels of 1-hexacosanoyl-2-hydroxy-sn -glycero-3-phosphocholine (C26:0 LysoPC) and C26:0-carnitine in dry blood spot are proposed to be more sensitive biomarkers that correlate with VLCFA levels in brain and spinal cord in X-ALD mice.(van de Beek et al., 2016) More work is needed to investigate the relationship between the levels of these two markers and disease progression.
Cerebral demyelination is a hallmark of cALD and can be diagnosed on magnetic resonance imaging (MRI).(Liberato et al., 2019; Paik, Kim, Yoon, & Kim, 2001) Demyelination may be present in the brain of cALD patients, commonly in the corpus callosum.(Nowak, Löbel, Wölfl, Schlegel, & Warmuth-Metz, 2015; Santosh Rai, Suresh, Bhat, Sekhar, & Chakraborti, 2013) Such lesions can lead to motor impairment, memory loss and seizures. Patients often have rapid spread of demyelination in the brain with death occurring within a few years.
In AMN, no such damage is evident in the brain although axonopathy is common in the spinal cord and peripheral nerves.(Castellano et al., 2016) Compared with cALD, symptoms of AMN are less severe. Patients usually experience weak muscle control and poor control of urination. Patients will experience difficulty with activities of daily living but will not necessarily progress to a vegetative state or succumb to the disease on disease onset. However, the chance of developing cerebral demyelination after AMN onset is not neglectable.(de Beer, Engelen, & van Geel, 2014)
Myelopathy and adrenal insufficiency are usually defined as common early symptoms of X-ALD.(Huffnagel, Laheji, et al., 2019) Most cALD cases, especially those occur in adulthood, already have myelopathy with gait disorder before cerebral demyelination.(Engelen, Kemp, & Poll-The, 2014) This comes to a thought that monitoring myelopathy level during early phase of the disease can somehow estimate the time or the probability of cALD onset. Recently, there are studies developing method to quantify progression of myelopathy in X-ALD.(Huffnagel, van Ballegoij, et al., 2019; van de Stadt et al., 2020) These studies aim to provide new markers in detecting disease progression in future clinical studies.
For adrenal insufficiency, elevation of plasma adrenocorticotropic hormone and hyperpigmentation are the characteristics. Rarely, it was reported that generalized skin hyperpigmentation could be the only sign of X-ALD. In that case, a boy that had hyperpigmentation at gums, elbows and knees was diagnosed with X-ALD subsequently, although he appeared to be healthy when this manifestation was observed.(Lee, Ko, & Lee, 2020) These reports have raised the awareness that patients who have signs of adrenal insufficiency, especially in young boys, should also be screened for X-ALD, in order to prevent delayed therapeutic treatments. Early diagnosis is also important for their siblings to have early DNA screening for X-ALD.
The main damaged site in cALD is the central nervous system (CNS). Microglia and macrophages play an important role in the progress of demyelination in cALD. Aberrant activation of microglia is considered to be involved in myelin degradation process,(Bergner et al., 2019; F. S. Eichler et al., 2008) and macrophages were found to have lost the plasticity in X-ALD patients.(Weinhofer et al., 2018) cALD macrophages lost the ability to switch to anti-inflammatory activation state to phagocytose destructive myelin, which may lead to progressive myelin damage. The blood-brain barrier permeability was also found to be altered at the demyelinating edge of the brain.(Musolino et al., 2015) These findings provide new insights for the development of novel therapeutic targets.
On cellular level, oxidative stress is proven to be another hallmark of X-ALD.(Deon, Marchetti, Donida, Wajner, & Vargas, 2016; Galea et al., 2012) Impaired mitochondrial oxidative phosphorylation and mitochondrial depletion have been demonstrated in X-ALD mice and cells.(Baarine, Beeson, Singh, & Singh, 2015; Fourcade, Ferrer, & Pujol, 2015) One example marker is the significant increase of oxidized glutathione, a strong antioxidant, in lymphocytes.(Petrillo et al., 2013) Heat shock proteins, which would response with cellular stress, was also found to be upregulated in X-ALD astrocyte before demyelination process.(Görtz et al., 2018)
X-ALD is a complex disease affecting different cell types and involving different pathological pathways. The level of ABCD1 gene mutation is not necessarily correlated with the severity of the disease,(Wiesinger, Eichler, & Berger, 2015) even a single mutation can lead to cALD, AMN, or Addison’s disease only.(Ozdemir Kutbay, Ozbek, Sarer Yurekli, & Demirbilek, 2019) It has also been reported that even in genetically identical twins with similar environmental impacts, different forms of X-ALD may occur.(Di Rocco, Doria-Lamba, & Caruso, 2001; Korenke et al., 1996) All these prove that genetic background is not the sole determinant of the phenotypic heterogeneity of X-ALD. There may be some underlying pathology that has not been clearly identified. In 2010, Singh and Pujol suggested a “Three-Hit Hypothesis” that described an ABCD1 mutation that was not the sole cause of X-ALD.(Singh & Pujol, 2010) The complex nature of the disease makes fully understanding its pathophysiology more difficult.
Current Clinical Treatments and Limitations
The first clinical case of X-ALD was reported in the early 1990s.(Aubourg et al., 1990) A 7.5-year-old boy diagnosed with cALD received a bone marrow transplant (BMT). The therapy was successful and favorable results persisted for at least eight years. BMT and allogeneic hematopoietic stem cell transplantation (HSCT) were shown to have successfully reduced levels of VLCFAs as well as oxidative stress and, more importantly, to have improved the patients’ performance in later trials performed during the early stage of cALD.(Kühl et al., 2017; Rockenbach et al., 2012; van Geel et al., 2015) Nonetheless although transplantation is an effective therapy for cALD, there remains a risk of mortality if performed during late-stage disease.(Bladowska et al., 2015; Jia et al., 2019; Kühl et al., 2018) Serious fatal complications such as graft-verse-host disease (GVHD) and graft failure can occur. Transplantation involves stressful surgery that may not be advisable during the middle and advanced stages of the disease. The availability of an HLA-matched donor is another barrier to transplantation. Patients may need to wait a long time for a suitable donor by which time the disease has advanced beyond early stage.
Another clinical trial with lentiviral hematopoietic stem cell (HSC) gene therapy.(Cartier et al., 2012; F. Eichler et al., 2017) The first reported case in 2009 involved two boys (7 and 7.5 years old) for whom no suitable bone marrow donor was available.(Cartier et al., 2009) Both patients enjoyed favorable therapeutic effects for up to 30 months. The advantage of HSC gene therapy over BMT is that the patient’s own stem cells are used, obviating the need to wait for a suitable donor and avoiding the risk of GVHD. This approach is currently in phase II/ III of clinical research. Nonetheless safety is a main concern of lentiviral HSC gene therapy. Due to the mechanism of lentiviral infection, there is a risk of developing insertional mutagenesis following gene therapy, which can trigger tumor formation.(Luis, 2020; Marcucci et al., 2018) Some modified viral vectors, such as Lenti-D lentivirus, were shown to reduce the risk of mutagenesis.(F. Eichler et al., 2017) More evaluations are required to further assess the safeness in clinical trials.
Dietary therapy is another approach to lower VLCFA level in patients. Lorenzo’s oil (LO), which is a mixture of glyceryl trioleate oil and glyceryl trierucate oil in a ratio of 4:1, has been found to reduce accumulation of VLCFAs by inhibiting endogenous synthesis.(Sassa, Wakashima, Ohno, & Kihara, 2014) Administration of LO can help significantly decrease plasma VLCFA levels.(Ahmed et al., 2016; Moser, Moser, Hollandsworth, Brereton, & Raymond, 2007; Stradomska, Drabko, Moszczyńska, & Tylki-Szymańska, 2014) It is a comparably safe treatment, but its limitation is that while it may be able to reduce VLCFA levels during the early stage of X-ALD and slow disease progression, it has not been proven effective for cALD. The effect of arresting disease progression, especially in patients who already have neurological involvement, is still questionable.(Deon et al., 2008; Majori et al., 2014)
Current Animal Models of X-ALD
The first X-ALD mouse model was created in 1997 in different laboratories by disrupting the ABCD1 gene.(Forss-Petter et al., 1997; Kobayashi, Shinnoh, Kondo, & Yamada, 1997; Lu et al., 1997) All the three ABCD1- mouse models showed some biochemical changes and late onset resembling AMN. VLCFA level was increased in various tissues compared with wild type (WT) mice. Accumulation of VLCFAs was evident in the brain with an approximate 5-fold increase in C26:0/C22:0 ratio in the model compared with WT mice. In a human sample, the ratio in a X-ALD brain demonstrated a 3- to 15- fold increase compared with normal samples, depending on the level of demyelination in different brain regions.(Kobayashi et al., 1997) These results were exciting because it showed that the animal model was moderately successful. Nonetheless surprisingly, theABCD1- mouse model showed no significant changes in blood plasma C26:0/C22:0 level. In contrast, in human patients, accumulated VLCFA levels can be easily measured in blood plasma, with an average 5-fold increase.(Moser, Moser, Singh, & O’Neill, 1984) This may imply that the accumulation of VLCFAs in mice is not as severe as that in humans.
No ABCD1- mice showed any behavioral changes at a young age. On the contrary, in humans, the most severe form of X-ALD, cALD, often has an onset in childhood, certainly below the age of 10 years. AMN often has an onset at ages 20 to 30 years. InABCD1 - mice, some neurological phenotypes in the spinal cord started to appear at 16-months of age, and clinical presentations such as decreased muscle strength were evident in 20-month-old mice,(Pujol et al., 2002) equating to over 50 years of age in humans. No demyelination was found in the brain.ABCD1- mice can be said to mimic AMN according to its onset age and the site of neuron degeneration.
Scientists have also endeavored to knockout both ABCD1 andABCD2 genes in mice.(Pujol et al., 2004) These double knockout mice have dysfunctional ABCD1 proteins, as well as its close homolog, ABCD2 proteins.(Kawaguchi & Morita, 2016; Morita & Imanaka, 2012) It was expected that this genetic modification would lead to more severe symptoms, similar to the characteristics of cALD. Excitingly, compared with single knockout ABCD1- mice, the double knockout mouse model exhibited earlier disease onset at 12-months of age. In addition, the phenotypes, such as enhanced VLCFA level in spinal cord and level of oxidative damage, were more obvious. Nonetheless disappointingly, and similar to ABCD1- mice, no demyelination was found in the brain. Both models developed AMN-like pathology (Table 1).
In 2017, a new zebrafish model of X-ALD was developed. Absence of ABCD1 proteins in zebrafish results in an elevated level of C26:0 by 1.5 to 2-fold in whole animal extracts.(Strachan et al., 2017) Altered myelin development was found in this new model. Excitingly, this model displays impaired motor function very early, within a week of life. In 2020, the deletion of the pmp-4 gene in worm, which is an orthologue ofABCD1, also demonstrated some AMN phenotypes, such as a 1.25-fold increase in C26:0 accumulated in lysophosphatidylcholine from whole body extract, axonal dysregulation, and impaired locomotion.(Coppa et al., 2020) Nonetheless further investigations with these two novel models of X-ALD are required. They may be a potential model for drug screening and molecular study of X-ALD but are not appropriate for assessing effect of therapy at this stage.
Apart from ABCD1 and ABCD2, studies suggested that double mutation of bubblegum (bgm) and double bubble (dbb) that cause acyl-CoA synthetases (ACS) dysfunction in Drosophila showed some X-ALD phenotypes, such as accumulation of VLCFAs and loss of neurons and neuron supporting cells in the brain.(Sivachenko et al., 2016) ACS is an enzyme that activates free fatty acids to become the substrates for ABCD1 transporter. It is particularly exciting that this model showed neuron impairment in the brain, which is potentially important for studying the relationship between evaluated VLCFA levels and neuron damages in the brain. More interestingly, due to the fact that X-ALD is not genotype-phenotype correlated, scientists used this fly model to study the gene-environment interactions by exposing the flies in different light patterns.(Gordon, Valdez, & Letsou, 2018) The results demonstrated that environmental stress can modulate the phenotype expressions in the same genetic background.
Pre-clinical studies of X-ALD for New Therapeutic Development
To understand the pathogenesis of X-ALD, reliable cell and animal models are essential to find out the causes of the disease. Apart from patient fibroblasts and patient-derived cell models such as a patient’s induced pluripotent stem cells (iPSCs),(Baarine, Khan, Singh, & Singh, 2015; Son et al., 2017; Yeon et al., 2019) researchers started to rely onABCD1- andABCD1-/ABCD2 -/- mouse models and their cells to study pathologies and develop promising therapeutic strategies for X-ALD.(Morita, Shinbo, Asahi, & Imanaka, 2012; Muneer et al., 2014) To date, different underlying pathological pathways have been proposed, and corresponding drug treatments and therapies investigated using X-ALD models. The ABCD1- mouse model is often used to assess biochemical signs, but theABCD1-/ABCD2- /-mouse model is commonly used to determine a therapeutic effect by studying the clinical signs. We summarize some examples of in vivo therapeutic studies that have targeted different pathways over the past few years [See Additional file 1].
Gene therapy is a direct approach for treating X-ALD. Studies showed that overexpression of the ABCD1 gene using adeno-associated virus (AAV) serotype 9 in an ABCD1- mouse model is promising. In these studies, level of VLCFAs inABCD1- mice in multiple tissues was lowered after injecting AAV intravenously.(Gong et al., 2015) Nonetheless blood plasma level of VLCFAs were unchanged. This may be a limitation of theABCD1- mouse model where there is no elevation in plasma VLCFAs compared with WT mice and hampers its reliability to evaluate the effect of the gene therapy. Studies also showed that injecting AAV intrathecally into ABCD1 - mice can lower 20% of VLCFAs in spinal cord, which is the main lesion site of AMN.(Gong et al., 2019) It is believed that this injection method will be more effective in the treatment for AMN.
Reducing VLCFA level is a simple concept for halting symptom development. Overexpression of ABCD2 has been an approach to compensate the function loss of ABCD1. In 2017 and 2020, studies showed that upregulation of ABCD2 by thyromimetics, such as sobetirome and its prodrug in ABCD1- mice significantly reduced VLCFA level in various tissues,(Hartley, Kirkemo, Banerji, & Scanlan, 2017; Hartley et al., 2020) up to 50% in peripheral tissues and 15-20% in the brain. In their studies, the ABCD1-mouse model did not show serious impairment of motor functions, so it was difficult to access its effectiveness in curing X-ALD. Nonetheless it is a promising approach that should be further studied in reliable X-ALD disease models.
Oxidative stress was suggested as a key factor of neurodegenerative and metabolic diseases inducing X-ALD. Drugs that target antioxidant pathways are thought to be effective in arresting X-ALD disease progression.(López-Erauskin et al., 2012; Marchetti et al., 2015) In the last few years, dimethyl fumarate (DMF) has been reported to successfully prevent oxidative damage to proteins and lipids,(Ranea-Robles et al., 2018) and more importantly, arrest axonal degeneration and restore normal motor activities inABCD1-/ABCD2 -/- mice.
Novel therapeutic strategies have also tried to target mitochondria related pathways to cure X-ALD. For example, a common drug for type 2 diabetes, Pioglitazone, was found to be a potential drug for X-ALD.(Morató et al., 2013) Pioglitazone works by regulating PPARγ/PGC-1α-dependent-pathways and thereby stimulating mitochondrial biogenesis in ABCD1- deficient cells. The mouse models showed therapeutic effects upon Pioglitazone treatment.
Researchers continue to search for methods to normalize redox balance in X-ALD mouse models. Most recently, high-dose biotin has been reported to help stimulate mitochondrial biogenesis and restore redox balance.(Fourcade et al., 2020; Sghaier et al., 2019)ABCD1-/ABCD2 -/- mice clinical presentations were improved upon such treatment. These successful trials support the concept that restoring mitochondrial functions are one of the key approaches in X-ALD treatment.
Apart from mitochondria, the endoplasmic reticulum (ER) has been proven to be another important organelle that is associated with VLCFA-induced oxidative stress.(van de Beek et al., 2017) Recent findings suggest that administration of a bile acid tauroursodeoxycholate (TUDCA), which is an ER stress inhibitor, can halt axonal degeneration and locomotor impairment inABCD1-/ABCD2-/ -mice.(Launay et al., 2017)
Autophagy dysfunction has been shown to be related to some neurodegenerative diseases such as Alzheimer’s and Parkinson’s.(Nixon, 2013; Orr & Oddo, 2013; Sarkar, 2013) Autophagy was shown to provide protection to VLCFA enriched cells.(Doria et al., 2019) Researchers have tried to target this mechanism to cure X-ALD. One example of attempts to restore autophagy in X-ALD mice is the use of Temsirolimus, an mTOR inhibitor. Results showed an ability to stop disease progression in double knockout mice models.(Launay et al., 2015)
Novel Clinical Trials and Development
The ABCD1- andABCD1-/ABCD2-/- murine models pave the way to new therapeutic development. From the pre-clinical studies, we learn that oxidative stress plays an important role in X-ALD pathogenesis. Recently, a phase II study of AMN suggested that administration of a combination of antioxidants, α-tocopherol, N-acetylcysteine and α-lipoic acid to patients improved their motor performance,(Casasnovas et al., 2019) which agreed with the pre-clinical trial in murine model.(López-Erauskin et al., 2011)
To date, BMT, HSCT and HSC gene therapies remain the only effective treatments for cALD. A recent successful case of HSCT reported in 2020 was of a 31-year-old man diagnosed with X-ALD at the early stage of the disease.(Ciftciler et al., 2020) For patients who do not have suitable donors, haploidentical allogenetic HSCT, together with post-transplant cyclophosphamide treatment or infusion of umbilical cord blood, are currently proposed and under investigation.(Chen et al., 2019; Fernandes et al., 2018; Jiang et al., 2015) Transplantation is undoubtedly beneficial, but a post-transplantation follow-up study showed that worsening in neurocognitive function can still occur in cALD survivors who have undergone HSCT at early stage. This observation is more likely to happen in younger boys, and patients with more advanced disease prior the time of transplantation.(Pierpont et al., 2017; Raymond et al., 2019)
There were reports that patients with advanced stage of cALD were treated by cord blood transplantation with reduced-intensity conditioning regimen.(Awaya et al., 2011; Niizuma et al., 2012) These trials not only demonstrated promising treatments for advanced stage of the disease with favorable effects in the follow-up period, but also provide an alternative therapy for patients who lack suitable donor for HSCT. More large-scale and robust studies should be carried out to further assess its efficacy.
On the other hand, there was a new therapeutic approach for patients who were in advanced stage of cALD in 2020. Disappointingly it was not successful. This study included three cALD patients who received Vorinostat orally, a drug that could stimulate ABCD2 expression. In this study, the drug failed to stop disease progression and all patients developed thrombocytopenia.(Zierfuss et al., 2020) Until now, treatments for late stage of cALD is still lacking.
In view of this, screening of X-ALD in babies is thought to be beneficial for timely treatment. In 2013, New York implemented newborn screening (NBS) to screen for newborn X-ALD patients, using dry blood spots as the testing samples.(Vogel et al., 2015) In 2016, X-ALD was added to the federal Recommended Uniform Screening Panel (RUSP), more states began NBS for X-ALD. An NBS pilot study was carried out in North Carolina and reported in 2020.(S. Lee et al., 2020) With the experience of NBS in the US, other countries such as Japan and Netherland also started to develop reliable protocols for screening X-ALD newborns.(Barendsen et al., 2020; Wu et al., 2017) With careful considerations, such as ethical and feasibility, this trend may spread to more countries in the future.
Challenges in the development of new therapies for X-ALD
Given that most current treatments are effective only at the very early stage of X-ALD, they are clearly not ideal for such a rapidly progressing disease. For cALD, the brain is the major affected site. This makes treatment more difficult because of the inability of drugs to penetrate the blood-brain barrier.(Banks, 2016) Drug distribution within the CNS is also another attribute that needed to be considered.(Warren, 2018)
Study of X-ALD is currently hampered by the lack of a suitable animal disease model. As mentioned before, the ABCD1-mouse model shows only symptoms that mimic AMN.ABCD1-/ABCD2-/ -double knockout mice show earlier onset and more severe biochemical alterations and are thought to be a better model for AMN studies. Nonetheless it is not perfect. There is no direct evidence that ABCD2 genotyping is associated with X-ALD phenotypes.(Maier et al., 2008) The absence of ABCD2 may cause alterations to other pathways that may cause phenotypes that are irrelevant to X-ALD.
Apart from animal models, scientists recently created a novelABCD1-/ABCD2-/- microglial model that successfully showed biochemical changes similar to those in X-ALD cells, such as accumulation of VLCFAs and mitochondrial modifications.(Raas et al., 2019) This would be a useful cell line to further investigate the pathology of X-ALD. Nevertheless, the accuracy of mimicking tissue cells in vivo is limited by 2D cell culture models.
Current studies tend to focus on the upstream pathology pathways, such as ABCD1 gene defect, VLCFA accumulation, mitochondria depletion and oxidative stress. All these biochemical characteristics can be studied in both X-ALD cells and animal models. The downstream pathologies that lead to cerebral demyelination in cALD, the life-threatening form of X-ALD, remain unknown. Although favorable therapeutic effects can be seen in AMN mouse models, we still cannot estimate the effectiveness in curing cALD. A reliable animal model of cALD is vital to study the underlying pathology and to develop novel therapies.
Perspectives
The difficulties in creating an ideal animal model for a complex disease such as X-ALD are understandable. Many factors apart from genetic involvement need to be considered when efforts to create a cALD animal model. It is possible that living environment, lifestyle and eating patterns should also be taken into account when developing new animal models of X-ALD. Much work remains to be done. The failure ofABCD1- andABCD1-/ABCD2-/- mouse models to develop cALD might imply some compensation mechanisms in response to the absence of ABCD1 proteins in rodents that differ to human beings.
In this regard, our lab tried to use rabbits (Oryctolagus cuniculus ) to create X-ALD model by knocking out ABCD1 using CRISPR/Cas9 technologies. Three male rabbits with mutations inABCD1 gene exon 1, namely Rabbit 1, 2 and 3, were successfully created. All of them have 4 to 4.5-fold increase of C26:0/C22:0 in blood plasma, which is more similar to human X-ALD compared with bothABCD1 - andABCD1 -/ABCD2 -/- mouse models. Nonetheless, no rabbits showed cALD phenotypes in the 2-year observational period. No behavioral and locomotion disability were observed, and body weights were steady. No abnormalities were found in brain MRI (Fig. 1 ). More examination is needed to evaluate the pathophysiological changes in this novel model.
Recently there was a case reporting a chimpanzee with an ABCD1mutation developed cALD.(Curiel et al., 2017) This fact gives hope that chimpanzee could be a suitable model for cALD. Despite the increasing ethical concerns, few numbers of offspring in long term and high cost, creating an X-ALD model using ABCD1-absent non-human primates may still be a direction for better disease studies.
Before the establishment of reliable X-ALD, particularly cALD animal models, 3D cell culture models may play a contributive role in the disease studies. Human PSC-derived brain organoid may serve as a disease model that mimics in vivo environment, offering an opportunity to better understand cell-to-cell interactions or metabolic profile in a diseased brain, and to develop new therapeutic interventions in near future.
Conclusions
There is currently no effective treatment for severe cALD that remains a rapidly progressing fatal disease. The full pathophysiology of X-ALD is poorly understood. The mechanism that drives ABCD1 mutation into different types of X-ALD remains a mystery. The absence of a reliable cALD animal model is the biggest hurdle to cALD pathological study and therapeutic development. Researchers have relied onABCD1- andABCD1-/ABCD2-/- mouse models in X-ALD studies but these fail to simulate cALD phenotypes in human. Although there are a number of promising therapeutic strategies being tested in mouse models, these novel interventions can only be applied to AMN phenotypes. There remains a lack of investigations for cALD (Fig. 2 ). Hopefully, the emerging new cell and animal models will provide potential means to investigate the disease pathophysiology and develop new therapies in the future.