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.2009 Feb;68(2):125-35.
doi: 10.1097/NEN.0b013e3181942cf0.

Neuropathology of the Mcoln1(-/-) knockout mouse model of mucolipidosis type IV

Affiliations

Neuropathology of the Mcoln1(-/-) knockout mouse model of mucolipidosis type IV

Matthew C Micsenyi et al. J Neuropathol Exp Neurol.2009 Feb.

Abstract

The recently developed Mcoln1(-/-) knockout mouse provides a novel model for analyzing mucolipin 1 function and mucolipidosis type IV disease. Here we characterize the neuropathology of Mcoln1(-/-) mouse at the end stage. Evidence of ganglioside accumulation, including increases in GM2, GM3, and GD3 and redistribution of GM1, was found throughout the central nervous system (CNS) independent of significant cholesterol accumulation. Unexpectedly, colocalization studies using immunofluorescence confocal microscopy revealed that GM1 and GM2 were present in separate vesicles within individual neurons. While GM2 was significantly colocalized with LAMP2, consistent with late-endosomal/lysosomal processing, some GM2-immunoreactivity occurred in LAMP2-negative sites, suggesting involvement of other vesicular systems. P62/Sequestosome 1 (P62/SQSTM1) inclusions were also identified in the CNS of the Mcoln1(-/-) mouse, suggesting deficiencies in protein degradation. Glial cell activation was increased in brain, and there was evidence of reduced myelination in cerebral and cerebellar white matter tracts. Autofluorescent material accumulated throughout the brains of the knockout mice. Finally, axonal spheroids were prevalent in white matter tracts and Purkinje cell axons. This neuropathological characterization of the Mcoln1(-/-) mouse provides an important step in understanding how mucolipin 1 loss of function affects the CNS and contributes to mucolipidosis type IV disease.

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Figures

Figure 1
Figure 1
Biochemical analysis of lipid profiles in cerebrum of end-stage (8-month-old) Mcoln1−/− mice. (A) Non-acidic and acidic lipid fractions of cerebrum from Mcoln1−/− mice (−/−) and Wt control mice (+/+). GalCer, galactosylceramide; PE, ethanolamine phosphoglycerides; PC, choline phosphoglycerides; Sph, sphingomyelin; BMP, bis(monoacylglycero)phosphate; PS, serine phosphoglycerides. (B) Ganglioside profiles of Mcoln1−/− mice (−/−), wild type control mice (+/+), and a reference Niemann-Pick type C1 mouse.
Figure 2
Figure 2
Accumulation of GM2 ganglioside in the CNS of the Mcoln1−/\− mouse. (A, B) Hemi-coronal brain sections stained for GM2 and counterstained with Nissl showing almost no positive immunoreactivity in a wild type (Wt) mouse (A), but strong staining throughout the neocortex (NCX) and hippocampus (HP) in the Mcoln1−/− mouse (B). (C–F) Higher magnification of Wt (C, E) cortical sections and GM2 storage in pyramidal neurons (based on size and morphology) of the Mcoln1−/− mouse (D, F). (G, H) Additionally, cerebellar sections stained for GM2 ganglioside showed no accumulation in a Wt mouse (G), but sporadic accumulation in a Mcoln1−/− mouse (H) including in Purkinje cells (denoted by *) of the Purkinje cell layer (PCL), the molecular layer (ML), and the granule cell layer (GCL). Scale bars:A = 500 μm and also pertains toB; C = 10 m and pertains toD; E = 5 μl and pertains toF; G = 20 μm and pertains toH.
Figure 3
Figure 3
GM3, GD3, and GM1 ganglioside accumulation in cerebral brain tissue of the Mcoln1−/− mouse by immunohistochemistry. (A, B) Hemi-coronal cerebral brain sections immunoperoxidase labeled for GM3 and Nissl counterstained showed no accumulation in the CA3 region of the hippocampus of a wild type (Wt) mouse (A), but significant accumulation in a Mcoln1−/− mouse (B). (C, D) Similarly, high magnification images of cortical pyramidal neurons showed no GM3 in a Wt mouse (C), but perinuclear accumulation of GM3 in neurons of the Mcoln1−/− mouse (D). (E–I) Immunoperoxidase labeling for GD3 in the amygdala showed no accumulation in a Wt mouse (E) but significant accumulation in the Mcoln1−/− mouse (F). GD3 accumulation was also absent in the CA1/CA2 region of the hippocampus of a Wt mouse (G) but is conspicuous throughout this region of a Mcoln1−/− mouse (H). Cortical pyramidal neuron of the Mcoln1−/− mouse (I) showed large amounts of GD3 accumulation throughout the perikaryon as well as in the axon hillock, apical dendrite, and other neuritic processes. (J–L) GM1 ganglioside appeared redistributed in an intracellular vesicular pattern in cortical pyramidal neurons of the Mcoln1−/− mouse (K, M) compared to a Wt mouse (J, L) by immunoperoxidase labeling. Scale bars:A andG = 50 μm and also pertain toB andH, respectively;C andL = 5 μm and pertain toD andM, respectively;E andJ = 10 μm and pertain toF andK, respectively;I = 5 μm. Nucleus, N; axon hillock, arrowhead; perikaryon, PK; apical dendrite, arrow.
Figure 4
Figure 4
Cholesterol in the brain of the Mcoln1−/− mouse. (A) Cerebral cortical sections were labeled with filipin to detect non-esterified cholesterol accumulation. There was no significant labeling in a Wt mouse, but some focal accumulation in the Mcoln1−/− mouse (arrows). Niemann-Pick C1 (NPC1) mouse tissue was used as a reference. (B) Cerebellar sections labeled with filipin similarly show minimal cholesterol in the Wt mouse but occasional accumulation in the molecular cell layer and apical dendrites of Purkinje cells. The NPC1 mouse tissue shows abundant cholesterol accumulation. Arrows indicate filipin positive cholesterol accumulation. Molecular layer, ML; Purkinje cell layer, PCL; Granule cell layer (GCL). Scale bars:A = 50 μm and also pertains to the top 3 panels;B = 50 μm and pertains to the bottom 3 panels.
Figure 5
Figure 5
Colocalization of GM1 and GM2 gangliosides, and GM2 and LAMP2 in cortical pyramidal neurons of the Mcoln1−/− mouse by confocal microscopy. (A) Cerebral cortical sections labeled for GM1 (green) and GM2 gangliosides (red) show almost no colocalization (white arrow indicates colocalization). (B) Immunofluorescence labeling for LAMP2 (green) and GM2 ganglioside (red) shows colocalization in cortical pyramidal neurons of the Mcoln1−/− mouse (small white arrows indicate GM2 independent of LAMP2 and not colocalized). The first (left-most) image inA andB shows maximum projections of confocal z-series, followed by three single XY optical plane images taken at varying intervals along the Z-axis, and finally three single YZ orthogonal optical plane images at varying intervals. The XY images inA are a 2x magnification of the maximum projection image; the XY images inB are a 1.5x magnification of the corresponding maximum projection image. Black, red, and blue arrows indicate Y-axis at which orthogonal planes were sampled and correspond to the first, second, and third YZ images respectively from left to right. Scale bars: inA andB = 10 μm.
Figure 6
Figure 6
P62/SQSTM1 inclusions in the brain of Mcoln1−/− mice. Sections of a Mcoln1−/− mouse show large P62 positive inclusions in the neocortex and brainstem; no inclusions are seen in wild type (Wt) mouse tissue. The inset in the top right panel shows a Nissl counterstained section with P62 inclusions in a glial cell, (identified based on size and morphology), directly adjacent to a cortical pyramidal neuron. Additionally, smaller punctate inclusions are seen in pyramidal neurons of the CA3 region of the hippocampus; no inclusions are present in Wt mouse tissue. Finally, the granule cell layer of the cerebellum shows numerous small punctate inclusions whereas the Wt mouse does not. Scale bars: left panels = 50 μm and also pertain to matched right panels; inset scale bar = 5 μm.
Figure 7
Figure 7
Glial involvement in the brains of Mcoln1−/− mice. (A–F) Hemi-coronal brain sections labeled by immunoperoxidase for glial fibrillary acidic protein (GFAP) show weak reactivity in the wild type (Wt) mouse (A), but strong staining throughout the cortex of the Mcoln1−/− mouse (B). Higher magnification images show GFAP staining in the neocortex of the Wt mouse (C) and the Mcoln1−/− mouse (D). GFAP staining in the cerebellum of the Wt (E) and Mcoln1−/− mouse (F) shows a similar pattern. (G–L) Microglial staining for CD11b in Wt mouse neocortex shows weak reactivity (G) compared to the Mcoln1−/− mouse (H). Similarly, there was weak staining in a Wt mouse brainstem (I) and white matter tracts of the cerebellum (K) compared to the strong immunoreactivity in brainstem (J) and cerebellum (L) of the Mcoln1−/− mouse. (M–R) Luxol fast blue staining of paraffin embedded tissue identified decreased myelination as indicated by less intense blue staining of the subcortical white matter of the Mcoln1−/− mouse (N) compared to a Wt mouse (M). There is less staining in sections of deep layer cortex and white matter tracts of the cerebellum of the Mcoln1−/− mouse (P, R) compared to a Wt mouse (O, Q). Scale bars:A andE = 500 μm and also pertain toB andF, respectively;C andG = 50 μm and pertain toD andH, respectively;I = 25 μm and pertains toJ; K, M, andQ = 100 μm and pertain toL,N, andR, respectively;O = 20 μm and pertains toP.
Figure 8
Figure 8
Accumulation of autofluorescent material throughout the cerebrum of a Mcoln1−/− mouse. Large amounts of autofluorescence are evident in cell bodies of the neocortex, the CA1 region of the hippocampus, and the cerebellum of a Mcoln1−/− mouse compared to a Wt mouse. Scale bar equals 50 μm and pertains to all panels.
Figure 9
Figure 9
Axonal spheroids in the cerebellum of the Mcoln1−/− mouse. (A, B) Calbindin immunoperoxidase labeling demonstrates a large number of axonal spheroids in Purkinje cell axons in a Mcoln1−/− mouse (ML, molecular layer; GCL, granule cell layer). Higher magnification (V) of an axonal spheroid extending from a Purkinje cell is calbindin-positive (arrow indicates spheroid; arrowheads show proximal and distal portions of the axon in relation to the spheroid and Purkinje cell body; PC, Purkinje cell). (C–E) Electron microscopy of axonal spheroids in myelinated axons of cerebellum. There is accumulation of multivesicular and dense bodies and large numbers of mitochondria (C, D) and granulomembranous storage bodies in the perikaryon of a cortical pyramidal neuron (E). Scale bars:A = 50 μm;B = 10 μm;C = 2 μm;D andE = 1 μm.
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