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. 2019 Mar;148(5):669-689.
doi: 10.1111/jnc.14462. Epub 2018 Aug 30.

Current Concepts in the Neuropathogenesis of Mucolipidosis Type IV

Free PMC article

Current Concepts in the Neuropathogenesis of Mucolipidosis Type IV

Lauren C Boudewyn et al. J Neurochem. .
Free PMC article


Mucolipidosis type IV (MLIV) is an autosomal recessive, lysosomal storage disorder causing progressively severe intellectual disability, motor and speech deficits, retinal degeneration often culminating in blindness, and systemic disease causing a shortened lifespan. MLIV results from mutations in the gene MCOLN1 encoding the transient receptor potential channel mucolipin-1. It is an ultra-rare disease and is currently known to affect just over 100 diagnosed individuals. The last decade has provided a wealth of research focused on understanding the role of the enigmatic mucolipin-1 protein in cell and brain function and how its absence causes disease. This review explores our current understanding of the mucolipin-1 protein in relation to neuropathogenesis in MLIV and describes recent findings implicating mucolipin-1's important role in mechanistic target of rapamycin and TFEB (transcription factor EB) signaling feedback loops as well as in the function of the greater endosomal/lysosomal system. In addition to addressing the vital role of mucolipin-1 in the brain, we also report new data on the question of whether haploinsufficiency as would be anticipated in MCOLN1 heterozygotes is associated with any evidence of neuron dysfunction or disease. Greater insights into the role of mucolipin-1 in the nervous system can be expected to shed light not only on MLIV disease but also on numerous processes governing normal brain function. This article is part of the Special Issue "Lysosomal Storage Disorders".

Keywords: TFEB; mTOR; autophagy; heterozygote; lysosomal disease; lysosome.

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Figure 1
Figure 1. Structure and functional motifs of mucolipin-1
Schematic depicting mucolipin-1's structure, including 6 transmembrane domains (TMD), a proteolytic cleavage site between TMDs 1 and 2, and a pore between TMDs 5 and 6. Protein interacting motifs are noted in purple. N terminal domains contain binding sites for AP1/AP3 proteins, PI(3,5)P2, ALG-2 binding motifs, and di-leucine lysosomal targeting domains. The pore is permeable to calcium, iron, zinc, manganese, magnesium, potassium, sodium, and hydrogen ions. The C terminus contains binding sites for PKA phosphorylation, palmitoylation for membrane targeting, AP2 binding sites, and another lysosomal targeting di-leucine sequence. (Kiselyov et al. 2005; Clapham et al. 2001; Zeevi et al. 2007; Li et al. 2017; Zhang et al. 2017b; Vergarajauregui et al. 2008b; Abe and Puertollano 2011; Dong et al. 2010; Vergarajauregui et al. 2009; Vergarajauregui and Puertollano 2006; LaPlante et al. 2002)
Figure 2
Figure 2. Aggregations of p62 in the Mcoln1-/- mouse brain
A) p62 aggregation in Mcoln1-/- mouse neocortex. p62 aggregates were not observed in WT (left panel), but aggregations positive for p62 can be detected in scattered neurons (NeuN+, p62 in neurons denoted by arrows) from all Mcoln1-/- mouse neocortical layers, shown here in cortical layer VI Mcoln1-/- (right panel). B) p62 aggregation in Mcoln1-/- hippocampus. Hippocampal sections from the CA1 region of WT (left panel) and Mcoln1-/- (right panel) mouse tissues. P62 positive structures were again not readily detected in WT hippocampus, while scattered punctate aggregates could be found in Mcoln1-/-, as indicated by white arrows. C) p62 aggregation in Mcoln1-/- mouse cerebellum. Image of WT cerebellum (left panel)) demonstrating occasional p62-positive puncta (noted by arrows), and from Mcoln1-/- mice (right panel), showing an abundant presence of small inclusions positive for p62. D) Example of typical neocortical p62+aggregate morphology. High magnification (63x) single plane images of p62 aggregates through a dorsal neocortical cell exhibiting clusters of aggregates, forming rings around diffuse, autofluorescent material. Yellow arrows denote distinguishable “rings” of p62. CD68 signal was tagged using Alexa Fluor 546 secondaries, and as autofluorescent material in MLIV fluoresces within near red wavelengths, CD68 signal overlapped with autofluorescent storage (labeled here as CD68/AutoF). Last image is a maximum intensity projection of previous slices in panel. Unless otherwise noted, all images are maximum intensity projections of acquired optical slices and collected from 6-9 month old mice. 9-11 Mcoln1-/- mice per panel were probed for p62 aggregation with matching WT counterparts.
Figure 3
Figure 3
Role of mucolipin-1 (TRPML1) during mTOR and TFEB signaling. As described in the text, in response to a low nutrient state (1) mTOR dissociates from the lysosome and lysosomal calcium efflux is triggered through mucolipin-1 (2), allowing for activation of the phosphatase calcineurin (3). Calcineurin in turn dephosphorylates TFEB (4), which promotes its translocation across the nuclear envelope to activate lysosomal biogenesis and autophagy gene expression (5). In response to a high nutrient state (6), mTORC1 scaffolding proteins are recruited to the lysosomal surface and mTORC1 docks on this lysosomal platform (7). Activated mTORC1 then phosphorylates TFEB (8), which promotes its association with 14-3-3 proteins sequestering TFEB within its cytosolic location and thereby downregulates lysosomal biogenesis/autophagy gene expression (9). In response to nutrient availability, mTORC1 also phosphorylates mucolipin-1 directly (10), which reduces its channel activity and would further promote cytosolic localization of TFEB in a phosphorylated state. Mucolipin-1's ability to respond to low nutrient states by releasing calcium (see 2, above) has been suggested to be due to relief of mTOR-mediated phosphorylation of mucolipin-1. These key roles of mucolipin-1 in the nutrient sensing pathway governed by mTORC1 have been previously highlighted in other reviews (Venkatachalam et al. 2013). (Medina et al. 2015; Wang et al. 2015; Onyenwoke et al. 2015; Roczniak-Ferguson et al. 2012; Settembre et al. 2013a).
Figure 4
Figure 4. TFEB signaling in the mucolipin-1-deficient cell
As described in the text and Fig. 3, mucolipin-1 plays an integral role in the mTORC1/TFEB nutrient response pathway – with the reported consequences of mucolipin-1's absence reviewed here. As described in the text, loss of mucolipin-1 removes the primary calcium source required to activate the phosphatase calcineurin, thus impairing TFEB dephosphorylation and its nuclear translocation (1-3). This may contribute to the mucolipin-1-deficient cell failing to properly respond to low nutrient availability via TFEB-mediated activation of the lysosomal network. (Medina et al. 2011; Medina et al. 2015; Wang et al. 2015). A full review of the role of TFEB in regulating the lysosomal network has been recently presented elsewhere (Sardiello 2016).
Figure 5
Figure 5. A proposed complex network of disease pathogenesis and potential therapeutic targets surrounding the mucolipin-1-deficient cell
This schematic summarizes the multifaceted defects that may ensue in the absence of mucolipin-1. Loss of this cation-permeable TRP channel leads to defective lysosomal cation flux (inner circle), including loss of iron and calcium efflux (Dong et al. 2008; Dong et al. 2009). This loss subsequently leads to secondary defects (yellow middle circle). The absence of mucolipin-1-mediated calcium is followed by a failure to complete fusion of organelles within the endosomal/lysosomal network and a failure to balance the mTOR/TFEB signaling axis in response to cellular nutrient states (Medina et al. 2015; Medina et al. 2011; Curcio-Morelli et al. 2010; Miedel et al. 2008; Pryor et al. 2006). These perturbations would be expected to lead to a compromise in autophagy, aberrations in endocytic trafficking, and possibly defects in mTORC1 signaling. These secondary defects may cause further dysfunction in the mucolipin-1-deficient neuron and lead to the pathologies documented within the brain (outer blue circle), including p62 aggregation, heterogenous storage, and changes to the lipid composition of cells (Micsenyi et al. 2009; Boudewyn et al. 2017; Grishchuk et al. 2014). Such chronic changes beginning early in life could contribute to developmental abnormalities, synaptic dysfunction, and even perhaps structural changes to neurons or cell death. Further still, loss of lysosomal iron efflux through mucolipin-1 (inner circle) is reported to contribute to the formation of reactive oxygen species (ROS), which leads to mitochondrial aberrations (middle yellow circle) (Zhang et al. 2016; Colletti et al. 2012; Grishchuk et al. 2015). Many of these defective pathways would be anticipated to intersect and exacerbate one another. Defects in autophagy for example may contribute to a failure to degrade damaged lysosomes or mitochondria, the buildup of which may contribute to cell death mechanisms in MLIV through release of apoptotic-triggering cathepsins (Colletti et al. 2012). Understanding the interplay between this deeply intertwined network, including in different cell types, will be important to understanding the full picture of MLIV neuropathogenesis and help us better understand how to treat this disorder. Therapeutic targets are also highlighted with yellow arrows denoting potential intervention points, including compensation for mucolipin-1's loss (inner circle, perhaps with gene therapy), targeting reactive oxygen specials for example with antioxidants (middle circle), or targeting mTOR/TFEB signaling (perhaps with mTOR and TFEB modulators), storage (perhaps with substrate reduction therapy), or cell death (perhaps with miglustat or other therapeutics when cell death mechanisms are better identified in this disease). This does not include all potential therapeutics, and additional broader therapies such as anti-inflammatories or other small molecule-based treatments remain to be explored in the context of MLIV.
Figure 6
Figure 6. Preliminary data on behavioral performance and calbindin density in senescent Mcoln1 heterozygous mice
A) Average number of slips on the narrow (i.e., most difficult), medium (intermediate difficulty, B), and widest (easiest, C) beam are plotted. While heterozygotes on the hard beam appears to have slightly more slips than WTs, no significant differences were detected for this or other beams analyzed by ANOVA (A-C). N=number of mice tested on each plot. D) Calbindin density detected via western blot of heterozygous versus WT tissues (n=4 mice aged 20-25 months per genotype, average density plotted +/- SEM, p=0.4914) suggested Purkinje cell health is comparable between the two genotypes. Statistical analyses performed using unpaired t-test with Welch's correction.
Figure 7
Figure 7. Canonical ultrastructural features of Mcoln-/- mice compared to aged heterozygous and WT mice
A) Characteristic pathology in Mcoln1-/- 3-month old tissues revealed by electron microscopy. Shown here is a neocortical neuron with the neuronal cytoplasm found congested with multiple compound, electron dense storage bodes as well as granulomembranous storage bodies and an enlarged apical dendrite (cell membrane outlined in green). B-C) Electron dense, compound storage bodies typically found in 3-month-old mutant mice, taken here from neocortex and highlighted by green arrows. E-G) Neocortical neurons from (E) WT, (F) heterozygote, and (G) mutant. E-F) WT and heterozygote neocortex exhibited neurons possessing lipofuscin accumulation (pink arrowheads) but otherwise devoid of pathological, compound storage bodies readily detectable in the mutant (G, green arrowheads). Neither heterozygous or WT neurons (both shown here at 18+ months) were found to exhibit abnormal structures, such as the swollen apical dendrites captured in 3-month old mutant neocortex (A). H-I) Hippocampal sections from WT (H) and heterozygous (I) mice. Similar to findings in cerebral cortex, hippocampal neurons displayed comparable features between WT (H) and heterozygotes (I) such as lipofuscin granulation storage with no evidence of pathological storage. Lipofuscin was not found to be quantitatively increased in the heterozygote in either cerebral cortex or hippocampus (data not shown). All tissues shown here are representative images from mice aged 18+ months, with the exception of the mutant at 3 months. 2 heterozygotes, 4 WT, and 4 Mcoln1-/- mice were probed for pilot EM data.

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