Progress in Elucidating Pathophysiology of Mucolipidosis IV.

Abstract

Mucolipidosis IV (MLIV) is an autosomal-recessive disease caused by loss-of-function mutations in the MCOLN1 gene encoding the non-selective cationic lysosomal channel transient receptor potential mucolipin-1 (TRPML1). Patients with MLIV suffer from severe motor and cognitive deficits that manifest in early infancy and progressive loss of vision leading to blindness in the second decade of life. There are no therapies available for MLIV and the unmet medical need is extremely high. Here we review the spectrum of clinical presentations and the latest research in the MLIV pre-clinical model, with the aim of highlighting the progress in understanding the pathophysiology of the disease. These highlights include elucidation of the neurodevelopmental versus neurodegenerative features over the course of disease, hypomyelination as one of the major brain pathological disease hallmarks, and dysregulation of cytokines, with emerging evidence of IFN-gamma pathway upregulation in response to TRPML1 loss and pro-inflammatory activation of astrocytes and microglia. These scientific advances in the MLIV field provide a basis for future translational research, including biomarker and therapy development, that are desperately needed for this patient population.

Mucolipidosis IV Is a Hypomyelinating Leukodystrophy

1.1. Clinical Presentation of Mucolipidosis IV.

Mucolipidosis type 4 (MLIV) is an autosomal-recessive lysosomal disease that primarily affects the central nervous system (CNS). In the absence of functional TRPML1 protein (typical form), the disease course is characterized by a hypomyelinating leukodystrophy with brain iron accumulation that manifests with severely impaired psychomotor development, and later onset, gradual neurological decline paralleled by cerebellar degeneration and neuroaxonal injury [1–3] (Figure 1). Retinal dystrophy also develops in the first years of life and rapidly progresses in adolescence, leaving patients legally blind by the second decade [4] (Figure 1). Characteristic brain MRI abnormalities in childhood include marked hypoplasia of the corpus callosum, decreased subcortical white matter with variable T2 hyperintense lesions, ferritin deposition in the basal ganglia, and relative preservation of the cortical gray matter (Figure 2A). Over the first two decades, the cerebellum degenerates and eventually diffuse cerebral atrophy may become apparent [2, 3]. In the mildest form of disease (atypical form), in which residual TRPML1 activity can be demonstrated, an isolated retinal dystrophy develops across the first decade of life.

Figure 1.

Qualitative representation of disease progression and stages in MLIV. Patients typically present with psychomotor delay in the first year of life and continue to make slow developmental gains through ~6 years of age (Stage I). A plateau in development is reached between ~6–16 years of age during which time muscular hypertonicity and visual impairment continue to worsen (Stage II). In early adolescence around the time of puberty, patients begin to exhibit a precipitous decline in motor function that eventually leads to loss of all meaningful movement and the need for gastric and tracheostomy tube placement to sustain survival.

Figure 2.

Hypomyelination in the brain of MLIV patients and Mcoln1^−/− mice. Representative MRI imaging of a MLIV patient at 1 (A–C) and 16 years (D–F) of age demonstrating characteristic abnormalities including a hypoplastic corpus callosum (A–B and D–E; arrows), diminished subcortical white matter volume, and deposition of ferritin in the globi pallidi (F; arrows) while cortical volume is relatively preserved. Cerebellar volume is initially normal, but atrophy emerges over time (A and D; asterisks). G. Representative electron microscopy images of WT and Mcoln1^−/− mouse brain showing reduced thickness of the myelin sheath in Mcoln1^−/− mice [12]. Scale bar =500 nm.

Outside of the central nervous system (CNS), corneal opacities and achlorhydria with elevated gastrin levels are the only consistently reported signs of MLIV [4, 5]. Acute or chronic renal failure, which may limit survival in the third and fourth decade of life, has been observed, but only in a subset of patients. The only known kidney biopsy with signs of chronic kidney disease was taken from a 38-year-old patient and demonstrated focal segmental glomerulosclerosis (R. Schiffman, personal communication, April 15, 2020). Further studies are needed to understand the nature and natural history of this important aspect of disease.

The developmental stage at which MLIV first affects brain development is unknown, but a brain MRI and fetal autopsy in a single patient at 35 weeks of gestation revealed subcortical hypomyelination and iron accumulation in the basal ganglia (just accepted for publication in Metabolic Brain Disease, reference TBD). The cerebellum and brainstem, in contrast, were relatively preserved at this early time point. Despite the early manifestation of brain pathology, patients typically meet their developmental motor milestones up to six months of life and later come to medical attention around one year of age with axial hypotonia, delayed gross motor development, and corneal opacities [4, 6]. The brainstem largely controls motor movements in the first 6 months of life, which may explain the initial lack of neurological signs. Alternatively, subtle neurological abnormalities resulting from abnormal prenatal brain development may be present in the first months of life but have not been investigated. Future studies assisted by new wearable technologies (i.e., accelerometers) could analyze the variation, complexity, and fluency of general movements in infants with MLIV, aiming to identify early signs of motor dysfunction and provide a non-invasive method of screening patients [7].

MLIV disrupts development in motor and language domains with relative preservation of social function. While significant heterogeneity exists in developmental trajectories among patients, representative generalizations can be made. In regard to gross motor function, the majority of patients do not achieve independent ambulation, however, some are able to crawl in an uncoordinated fashion and take steps with a walker that provides full truncal support. Impaired finger articulation limits fine motor function, and most patients will only develop an inferior or modified pincer grasp. Reaching for objects, bringing food to the mouth, and limited bimanual manipulation of objects are generally achieved [4, 8], (author’s observations). Therapeutic interventions such as physical and occupational therapy do appear to boost development gains and help patients reach their maximum potential, though the functional limitations represent a ceiling that the majority of patients do not surpass.

Pyramidal and extrapyramidal tract dysfunction are prominent features of MLIV that negatively impact motor development and reveal progressive compromise of the central motor networks. Spasticity, diffuse hyperreflexia, and decreased force with volitional muscle activation emerge across the first 5 years of life and gradually increase in severity across the lifetime. The probable pathoanatomical basis of these pyramidal tract signs was recently revealed by brain MRI and MR spectroscopy data, which demonstrated an increase in mean diffusivity within the corticospinal tracts of MLIV patients and a decrease in the N-acetyl aspartate (NAA) to creatine or choline ratios in the frontal gray matter [1, 3]. The increased mean diffusivity, indicative of degeneration, was also shown to correlate with worsening motor function. Decreased levels of NAA, a neuron-specific metabolite, may suggest reversible or irreversible neuronal injury in the frontal lobe, where the motor cortex resides. Interestingly, cortical gray matter volumes remained constant or increased in longitudinal assessments of 5 MLIV patients under the age of 20 years, while subcortical white matter volumes decreased with time [3]. Taken together, these clinicoradiographic data suggest that early in the course of disease the cortical neurons are dysfunctional as evidenced by decreased NAA, but perhaps not degenerating since cortical gray matter volume is preserved. The eventual compromise and degeneration of descending corticospinal tracts, as evidenced by increased diffusivity, likely drives declining motor function and worsening pyramidal tract signs with age.

Extrapyramidal motor dysfunction in MLIV manifests as rigidity, dystonic posturing, postural instability, bradykinesia, tremor, truncal ataxia, and jerk nystagmus (authors observations). Signs are typically present in the first year of life, but worsening spasticity across the lifetime may mask rigidity, making the presence of extrapyramidal signs difficult to discern. Progression across the lifetime remains unstudied. The pattern of iron deposition in the basal ganglia and the progressive cerebellar denegation fit well with the listed signs. Extrapyramidal features are commonly found in other lysosomal disorders, but a clear description in MLIV is still needed in the literature.

Impaired language development in MLIV results from a combination of oral motor dysfunction and poorly defined cognitive component. Hearing remains intact and does not affect language acquisition. At the height of development, patients follow simple and multi-step commands, laugh at jokes, and exhibit appropriate social responses to verbal context. Verbal communication is typically limited to less than 10 understandable words, but many patients use hand signs and adaptive communication devices, demonstrating a more complex synthesis of expressive language. Thus, impairment of articulation stemming from restricted tongue mobility and oral motor coordination likely conceals the true level of cognitive language production.

A granular assessment of cognitive development in patients with MLIV is precluded by the degree of motor, expressive language, and visual impairment, which limit the applicability of available clinical instruments. Processing time is markedly slow, and a lag time between a verbal command and execution of the requested task is evident on routine exam. Interestingly, caregivers consistently report a mature sense of humor in patients and appropriate verbal and non-verbal responses to complex social situations, suggesting development of some higher cortical functions. Behavioral issues are not typical, and patients are consistently described as warm natured and engaging with a strong penchant for music [9].

Some MLIV patients exhibit absence seizures, but epilepsy is not a prominent feature of MLIV. Absence seizures are usually accompanied by the expected 3htz spike and wave signature on EEG, are easily controlled with AEDs, and abate by the second decade of life. The conspicuous absence of epilepsy in MLIV is likely explained by the relative preservation of cortical neuron populations similar to other white matter disorders.

MLIV patients begin to precipitously decline in neurological function during early adolescence around the time of puberty (Figure 1, author’s observations). Decreased neurological function may be attributable to multiple factors, including near complete loss of vision, increased body mass to muscle strength, or the effects of changing sex hormone levels. In the second and third decade, patients begin to aspirate, and food modifications or parenteral feeding become necessary. Currently, the oldest surviving patients with MLIV are in the 5th decade of life and exhibit severe hypertonicity with little spontaneous movement and are tracheostomy and g-tube dependent.

The slower course of neurological decline in MLIV contrasts with the more rapid neurodegeneration typical of many other lysosomal diseases, where prominent gray matter degeneration and seizures are common. The pathological mechanism(s) responsible for the disparity remains uncertain but may relate to differences in accumulation of toxic metabolite intermediates, redundant and compensatory cellular processes, and/or the emergent properties of unique genetic variants and developmental context. Interestingly, toxic metabolite accumulation does not appear to play a major role in the pathogenesis of MLIV, and manifestations of disease likely reflect the consequences of generalized lysosomal and endosomal dysfunction.

A spectrum of phenotypic severity has been reported in MLIV, ranging from the typical features described above, to patients exhibiting isolated retinal degeneration in the absence of other CNS symptoms. Residual function of the TRPML1 channel has been demonstrated in several MCOLN1 variants associated with milder phenotypes [10], while the most common variants associated with a typical disease course ablate TRPML1 function or preclude its translation. However, the authors have evaluated one patient homozygous for MCOLN1 null mutations who has achieved the ability to ambulate independently and displays milder neurological symptoms. These observations suggest that residual TRPML1 function underlies some of the reported genotype-phenotype correlations, but other unknown factors, which may include environment and/or unidentified genetic modifiers, may also play a role in determining disease severity.

In summary, several key correlations between the clinical and neuropathological features of MLIV guide our current understanding of the natural history. First, a hypomyelinating leukodystrophy manifests early in life, and may begin during prenatal development. Impaired myelination of CNS fiber tracts likely interferes with the maturation of neuronal networks by altering activity-dependent processes, such as synaptic pruning, ultimately resulting in altered network connectivity, impaired cognitive processing, and delayed neurodevelopment. Visual impairment progresses quickly and illustrates a specific susceptibility of the retina to loss of TRPML1 function. Extrapyramidal motor dysfunction appears early in life when iron deposition in the basal ganglia and progressive cerebellar degeneration emerge. In contrast, pyramidal motor dysfunction develops gradually over the lifetime and correlates with signs of corticospinal tract degeneration that precede cortical gray matter atrophy. These findings suggest that the sequela of neurodegeneration begins in the descending axons eventually triggering an irreversible compromise in neuronal integrity and subsequent degeneration of cell bodies in the cortex. The extent to which these pathological features are produced by cell autonomous mechanisms or result from secondary injury due to inflammation or other poorly defined disease processes is a critical knowledge gap in our understanding of disease pathogenesis and warrant further investigation.

1.2. Findings in the Mouse Model of Mucolipidosis IV.

With very limited human brain tissue samples available for research, the MLIV mouse model became a valuable source for studying the role of TRPML1 in brain development and myelination. Loss of Mcoln1 in mice recapitulates all major manifestations of human MLIV disease, including motor and cognitive dysfunction, brain and eye pathology, and impaired secretion of chloric acid by stomach parietal cells [11–14]. Mcoln1^−/− mice are indistinguishable from their wild-type and Mcon1^−/+ littermates at birth and develop cognitive and motor deficits by 2 months [12]. Motor dysfunction progresses with age as demonstrated by the rotarod and balance beam tests [15], resulting in rear limb paralysis by 7 months of age and premature death a few weeks later [14]. Histopathological studies of Mcoln1^−/− brain tissue at the terminal stage revealed hallmarks also seen in other mouse models of human lysosomal disorders. These include the build-up of autofluorescent material, neuronal accumulation of gangliosides and cholesterol, an increase in the autophagy substrate P62/SQSTRM, indicative of inhibited autophagic proteolysis, activation of glia, and decreased myelination [16]. Formation of axonal spheroids and partial loss of cerebellar Purkinje neurons is a prominent hallmark of MLIV brain pathology in the end-stage cerebellum [15, 16].

Analysis of brain tissue in younger Mcoln1^−/− mice demonstrated that many of the pathological hallmarks found in the brain at the terminal stage are present by the early symptomatic stage (2 months of age). These include activation of glia, reduced myelination and build-up of lysosomal material [12]. In fact, some of the brain pathological hallmarks, such as lysosomal material accumulation, are present during embryogenesis [17] and some, including glial activation and hypomyelination, develop soon after birth [18, 19]. This indicates that TRPML1 plays a role in brain development and that loss of TRPML1 in mice leads to neurodevelopmental aberrations as seen in MLIV patients. Remarkably, Mcoln1^−/− mice display no significant brain atrophy or neuronal loss through the course of disease with the exception of the above-mentioned partial loss of Purkinje cells in the cerebellum [12, 15]. Taken together with the data in patients, these findings suggest that TRPML1 plays an essential role in orchestrating early brain development, but that loss of TRPML1 function does not severely compromise neuronal integrity.

We used the Mcoln1^−/− mouse model to better understand the nature of the myelination deficits resulting from the loss of TRPML1 in an age-dependent and region-dependent manner and found decreased expression of the mature oligodendrocyte markers myelin-associated glycoprotein precursor (Mag), myelin basic protein (Mbp), myelin-associated oligodendrocyte basic protein (Mobp) and Myelin proteolipid protein (Plp) in the Mcoln1^−/− cortex during post-natal brain development [18]. The increase in the oligodendrocyte precursor marker Ng2 in Mcoln1^−/− brain at post-natal days 10 and 21 may indicate a compensatory mechanism to overcome deficient oligodendrocyte maturation and rules out loss of oligodendrocyte precursors. Similar to MLIV patients, Mcoln1^−/− mice have a dysgenic corpus callosum [11, 12]. Histopathological analysis using myelination and neurofilament markers shows that thinning of the corpus callosum in Mcoln1^−/− mice is caused by reduced myelination rather than by loss of neuronal fibers, as the thickness of the neurofilament-labeled fibers in the corpus callosum remains intact [11, 18]. Additionally, electron microscopy analysis of myelin sheath showed reduced thickness of the myelin sheaths (Figure 2B) [12]. Overall, these data showed that loss of myelin in MLIV is caused by hypomyelination, as has been described for hypomyelinating leukodystrophies [20]. It is important to note that levels of myelination markers were stably decreased in Mcoln1^−/− mice later in the course of disease (2 and 7 months of age), indicating that myelination delays are not overcome later in life in Mcoln1^−/− mice [11], further supporting a hypomyelination leukodystrophy rather than a delay in myelination.

In contrast with the cerebral cortex, we found no significant differences in mature myelination marker expression in the cerebellum [18]. TRPML1 expression rapidly increases in the mouse brain across post-natal development, reaching higher levels in the rostral brain regions (cortex), than in the caudal regions (cerebellum). Interestingly, this expression pattern correlates with the pattern of reduced myelination in Mcoln1^−/− mice. This indicates that the role of TRPML1 in normal myelination is region- and developmental period-specific.

Reduced expression of the myelination genes Mag, Mal, Mobp, Mog and Cnp has recently been shown in the cerebral cortex of pre-symptomatic and symptomatic Mcoln1−/− mice using RNAseq [21]. Importantly, expression of the human myelination genes MOG, MAG, MOBP, LGI4 and CNTNAP1 has also been reported in human MLIV brain tissue that has recently become available through the Maryland Tissue Bank. Remarkably, reduced levels of myelin proteins were only evident in the cerebral cortex but not in the cerebellum, the same pattern seen in the MLIV mouse [21]. This further highlights the similarity between mouse and human brain pathology in MLIV and highlights the value of the Mcoln1−/− mouse model for pre-clinical research.

The precise role of TRPML1 in oligodendrocyte maturation and myelination is currently unclear. The function of TRPML1 as a lysosomal iron-release channel [22] and deviations in the iron uptake pathways unique to oligodendrocytes suggest a scenario in which loss of TRPML1 may have additional implications specific to myelinating oligodendrocytes [11]. Iron is a key factor in energy production and brain development, and its role in myelin biosynthesis has been well documented. Further, iron deficiency leads to hypomyelination in both, human and animal models [23]. While iron is absorbed by endocytosis of Fe³⁺-transferrin in most tissues, oligodendrocytes rely on extracellular ferritin as a source of iron [23–26]. In the endolysosomal compartment, Fe³⁺ is liberated from the protein complex, converted to Fe²⁺ and absorbed into the cytoplasm via endolysosomal membranes. In the cytoplasm, Fe²⁺ is oxidized to Fe³⁺ and binds to cytoplasmic ferritin. From the cytoplasm, iron enters mitochondria for energy production, binds to transferrin for trafficking or exporting, serves as a cofactor for enzymatic reactions such as myelin production, or binds to cytoplasmic ferritin and other scavengers for storage. Hence, based on the role of TRPML1 in endocytic membrane trafficking [22, 27–30], it might regulate the delivery of Fe³⁺-ferritin to the lysosomes. Additionally, due to the ability of the TRPML1 channel to transfer divalent heavy metal ions, including Fe²⁺ [22], it could facilitate the direct transport of Fe²⁺ from lysosome to cytoplasm in addition to or in place of DMT1. Our data showing reduced levels of ferritin-bound ferric iron in Mcoln1^−/− brain by Perls’ stain indicates a loss of biologically available H-ferritin-bound iron and supports this hypothesis [11]. Given that TRPML1 is permeable to Fe²⁺ but not to Fe³⁺, it is also possible that loss of TRPML1 leads to lysosomal build-up of Fe²⁺, which is known to catalyze Fenton-like reactions that result in the formation of reactive oxygen species. In agreement with this, oxidative stress has been shown both in in vitro cellular models [31, 32], in a Drosophila model [33], and in the Mcoln1^−/− mouse brain [11].

In summary, while the detailed mechanisms of TRPML1 involvement in brain myelination require further investigation, clinical observations in MLIV patients and data obtained using the Mcoln1^−/− mouse model suggest that MLIV should be classified as a hypomyelinating leukodystrophy.

2. Neuroinflammation in Mucolipidosis IV

In health, microglia and astrocytes play central roles in maintaining brain homeostasis, including pathogen and debris clearance, synaptic pruning, and metabolic support, among others. When these cell types are diseased, or exposed to a diseased tissue microenvironment, they have the potential to either attenuate or potentiate disease progression. Since loss of TRPML1 function in MLIV affects all cells in the body, microglia and astrocytes in the MLIV brain may be both autonomously affected by the disease. On top of cell-autonomous changes caused by loss of TRPML1, they also face the multifactorial changes in the brain and systemic environments, including increased cytokines [34]. Studies using the Mcoln1^−/− mouse model have revealed that microglial activation and astrocyte reactivity are early and prominent brain pathological features occurring prior to onset of neurologic dysfunction in these mice [12]. Since these features appeared early in the disease course and in the absence of neuronal loss, these data suggest that dysregulation of microglia and astrocytes are potential drivers of functional loss in MLIV, prompting several studies to interrogate the changes of these cells in MLIV [34, 35].

A recent study by Cougnoux et al evaluated changes in acutely isolated microglia from Mcoln1^−/− mice and compared them to microglia isolated from wild-type mice and a mouse model of Fabry disease, a lysosomal storage disorder without CNS involvement [35]. Using a panel of pro-inflammatory markers, including iNOS and phosphorylated ERK, the authors demonstrated increased free radicals and a switch to glycolytic metabolism Mcoln1^−/− microglia, similar to their previous findings in microglia from NPC1 mice [36]. Using transcriptional profiling they reported that while microglia from Fabry disease mice showed similarity to wild-type controls, microglia isolated from Mcoln1^−/− mice displayed pro-inflammatory and transcriptional signatures that were similar to other lysosomal and neurodegenerative diseases, such as Niemann Pick Type C1 (NPC1), ceroid neuronal lipofuscinosis 3 (CLN3), amyotrophic lateral sclerosis (ALS) and Alzheimer’s Disease (AD).

Motivated by the early appearance of reactive astrocytes in the Mcoln1^−/− mouse model [12], Weinstock et al established a cell culture model to study the astrocyte autonomous response to loss of TRPML1 [19]. The authors found that Mcoln1^−/− astrocytes secrete a panel of cytokines that strongly overlap with those measured from Mcoln1^−/− cortical tissues. Interestingly, cytokine protein data from mouse tissue, as well as cytokine secretion and transcriptional profiling in astrocyte cultures, implicated interferon pathway signaling as a central molecular feature of MLIV. Up-regulation of the interferon pathway was also shown by RNAseq analysis in pre-symptomatic Mcoln1^−/− mouse brain [21] and isolated Mcoln1^−/− microglia [35]. Consistent with these findings in the MLIV mouse model, a proteomic analysis of a single human MLIV postmortem brain sample has also identified increased interferon pathway signaling at the terminal stage of the disease [21]. Therefore, based on studies of whole brain tissue and isolated astrocytes and microglia from MLIV mice and limited human brain data, dysregulation of interferon gamma signaling appears to be a key feature of the neuroinflammatory response in MLIV. While the details of how loss of TRPML1 is linked to interferon signaling remain unknown, it is intriguing that a recent study in natural killer (NK) cells found that activation of TRPML1 using the small molecule agonist MK6–83 decreased interferon-gamma signaling and granzyme B in response to stimulation by leukemia cells [37]. Moreover, siRNA silencing of TRMPL1 led to increased levels of granzyme B and interferon gamma, further supporting the role of TRPML1 in this response. Additionally, inhibition of phosphoinositide kinase PIKfyve, which regulates TRPML1 activity [28], also resulted in enhanced NK cell toxicity against leukemia cells [37].

It is interesting to note that while upregulation of interferon signaling has also been reported in other lysosomal diseases, including Gaucher and Krabbe, genetic ablation of Ifnar1, a key effector receptor of type I interferon signaling, failed to prolong survival in Gaucher model mice [38]. It is currently unclear whether inhibiting interferon signaling can produce any therapeutic benefit in MLIV. However, reported functional connections between TRPML1 and the interferon pathway suggest that it has potential as a therapeutic target or biomarker for MLIV and warrant future study (35).

Using a combination of phospho-protein multiplexed assays and transcriptional profiling, a study of Mcoln1^−/− astrocyte cultures [34] also revealed autonomously up-regulated signaling in the mitogen activated protein kinase (MAPK) and PI3K/Akt/mTOR pathways as well as upstream activation of the sphingosine-1-phosphate (S1P) pathway. The authors found that treating astrocytes with the S1P functional antagonist FTY720 (fingolimod) attenuated downstream phospho-protein signaling and cytokine secretion. Moreover, it had the capability to normalize lysosomal morphology in Mcoln1^−/− astrocyte cultures. Together, these findings indicated that astrocytes are autonomously affected by loss of TRPML1 and may represent a promising therapeutic target.

Given the robust neuroinflammatory phenotype observed in the Mcoln1^−/− mouse, including robust elevation of pro-inflammatory cytokines in the mouse brain, more recent work hypothesized that it may be possible to identify a blood cytokine signature associated with disease severity in MLIV patients. Interestingly, the authors found that cytokines were robustly increased in plasma isolated from MLIV patients compared to familial controls [39]. Interferon cytokines (IFN-α1, IP-10) were found to correlate with loss of motor function and tonicity in human patients. Additionally, the interferon pathway cytokine IP-10 was found to be upregulated with advancing severity in MLIV patients and increased in the blood and brains of Mcoln1−/− mice, suggesting it should further be investigated as a biomarker of MLIV severity.

Conclusions

Significant progress has been made in understanding mucolipidosis IV in recent years. Characterization of glial activation and hypomyelination, the most robust and early brain pathological features of this disease, has been a priority. These studies revealed an important role for TRPML1 in regulating brain myelination, changes in cytokine release in the response to TRPML1 loss of function and upregulation of interferon-gamma signaling. These important discoveries suggest several novel therapeutic targets for translational research in MLIV, a disease with no existing therapies and very high unmet medical need. Despite this progress, the underlying mechanisms behind oligodendrocyte dysfunction and pro-inflammatory activation of microglia and astrocytes are still not well understood, and future studies are needed to better understand the functional consequences of these glial phenotypes for CNS dysfunction and pathophysiology of MLIV. Future studies must be devoted to identifying reliable molecular biomarkers for tracking both disease progression and efficacy of investigational therapies. The new studies using the MLIV mouse model, human MLIV plasma samples and a single human MLIV postmortem brain and CSF sample, reviewed here, provided some valuable insights for future biomarker discovery research. Importantly, based on the human clinical manifestations and supporting data from the MLIV mouse model, we suggest classifying MLIV as a hypomyelinating leukodystrophy. Recognizing MLIV as a leukodystrophy will help to bring awareness among pediatric neurologists and other medical professionals which will ultimately facilitate diagnosis and supportive care for children with MLIV.

Highlights.

• Hypomyelinating leukodystrophy in MLIV manifests in infanthood.

• Extrapyramidal motor dysfunction appears early in life when iron deposition in the basal ganglia and progressive cerebellar degeneration emerge.

• Pyramidal motor dysfunction develops gradually over the lifetime and correlates with signs of corticospinal tract degeneration.

• Dysregulation of cytokines, with emerging evidence of interferon gamma pathway upregulation, present in both, the brain tissue and in periphery, provides new insights for biomarker discovery.