Unique molecular signature in mucolipidosis type IV microglia

Antony Cougnoux, Rebecca A Drummond, Mason Fellmeth, Fatemeh Navid, Amanda L Collar, James Iben, Ashok B Kulkarni, James Pickel, Raphael Schiffmann, Christopher A Wassif, Niamh X Cawley, Michail S Lionakis, Forbes D Porter, Antony Cougnoux, Rebecca A Drummond, Mason Fellmeth, Fatemeh Navid, Amanda L Collar, James Iben, Ashok B Kulkarni, James Pickel, Raphael Schiffmann, Christopher A Wassif, Niamh X Cawley, Michail S Lionakis, Forbes D Porter

Abstract

Background: Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease.

Methods: We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses.

Results: We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases.

Conclusions: The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders.

Trial registration: ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.

Keywords: CCL5; Fabry disease; Lysosomal disease; Microglia; Mucolipidosis type IV; Neuroinflammation.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Analysis of the microglia in Fabry disease and mucolipidosis type IV mice. Mean fluorescence intensity (MFI) of the surface expression of microglial lineage markers a CX3CR1 and CD11b and microglial activation markers. N ≥ 5; *p < 0.05, **p < 0.01 Mann-Whitney U test vs. WT. b CD86 and MHCII. WT, control; FD, 2-month-old Fabry disease; MLIV, 2-, 4-, and 8-month-old mucolipidosis type IV. N ≥ 5; *p < 0.05, **p < 0.01 Mann-Whitney U test vs. WT. c DCFDA analysis of free radical production by 10,000 microglial cells. 1 μM H2O2 treatment was used as a positive control for free radical production. N = 5, n ≥ 3; *p < 0.05, **p < 0.01 Mann-Whitney U test vs. WT. d Western blot analysis iNOS, P-ERK1/2, ERK1/2, and HIF1α and β-tubulin on 100,000 microglia lysates. N = 4, *p < 0.05 Mann-Whitney U test. e Basal extracellular acidification rate (ECAR) in mpH.minutes−1 of unbuffered seahorse media by 10,000 cells. N = 5, n ≥ 3; *p < 0.05 Mann-Whitney U test vs. WT
Fig. 2
Fig. 2
Microglia transcriptome analysis. a Heatmap of the microglia/macrophage activation markers in MLIV and FD expressed as Log2 fold change relative to control levels. b Heatmap of neurotoxic and neuroprotective markers in MLIV and FD expressed as Log2 fold change relative to control levels. The genes in this list are based upon [16, 24, 44]. c Top 50 most differentially overexpressed genes in MLIV compared to control microglia. Interferon signaling genes are boxed in red. Data underlying the heatmaps are provided in Additional file 5: Table S1 and Additional file 6: Table S2
Fig. 3
Fig. 3
Analysis of CCL5 expression in microglia. aCcl5 Log2 fold change and Log10 (pAdj) in 2-month-old Fabry disease (FD), 7-week-old Niemann-Pick type C1 (NPC1), 3-month-old amyotrophy lateral sclerosis (SOD1), 16-month-old App-Ps1 (AD), and 2-month-old mucolipidsosis type IV (MLIV) microglia isolated from mouse brain b IBA1 and CCL5 double staining in hippocampi. The scale bar is 10 μm. c CCL5 quantification by enzyme-linked immunosorbent assay (ELISA) in mouse serum. Npc1−/−N = 7, MLIV N = 8, FD N = 16, 2-month-old Gaa−/− (Pompe disease) N = 10, 3-month-old Npc2−/−N = 7, Sod1tgG93AN = 4, 2-, 3-, and 6-month-old Cln3−/−N = 4–6. HPβCD/VTS-270-treated animals were subcutaneously injected every other day from day 7 to 49 [24], N = 4 and 5 for control and Npc1−/−, respectively. d CCL5 ELISA from control and MLIV patient’s serum. Ncontrol = 14, NMLIV = 48
Fig. 4
Fig. 4
Comparison of microglia transcriptomes in diseases with neuroinflammation. a Volcano plot differentially expressed genes X = Log2 fold change vs control, Y = − Log10(p). The data are supplied in Additional file 7: Table S3. The dashed lines are set at the pAdj of 0.05 and Log2 fold change of – 1 and 1 on Y- and X-axis, respectively. b Venn diagram of the significantly differentially expressed genes (pAdj < 0.05, Log2FC > 1) genes in ALS (SOD1), AD (APP), NPC1, and MLIV disease mouse models. c Heatmap of the 100 modified genes AD, ALS, MLIV, and NPC1 diseases across aging (aged and ERCC1) and FD datasets. Yellow: DAM markers. Orange: HIF1 signaling. Blue: lysosome. The heatmap with all genes labeled is provided in Additional file 4: Figure S4a

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