Neuropathology in Mouse Models of Mucopolysaccharidosis Type I, IIIA and IIIB

Mucopolysaccharide diseases (MPS) are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Neurodegenerative MPS diseases exhibit cognitive decline, behavioural problems and shortened lifespan. We have characterised neuropathological changes in mouse models of MPSI, IIIA and IIIB to provide a better understanding of these events

A Micropolymorphism Altering the Residue Triad 97/114/156 Determines the Relative Levels of Tapasin Independence and Distinct Peptide Profiles for HLA-A*24 Allotypes

While many HLA class I molecules interact directly with the peptide loading complex (PLC) for conventional loading of peptides certain class I molecules are able to present peptides in a way that circumvents the PLC components. We investigated micropolymorphisms at position 156 of HLA-A*24 allotypes and their effects on PLC dependence for assembly and peptide binding specificities. HLA-A*24:06156Trp and HLA-A*24:13156Leu showed high levels of cell surface expression while HLA-A*24:02156Gln was expressed at low levels in tapasin deficient cells. Peptides presented by these allelic variants showed distinct differences in features and repertoire. Immunoprecipitation experiments demonstrated all the HLA-A*24/156 variants to associate at similar levels with tapasin when present. Structurally, HLA-A*24:02 contains the residue triad Met97/His114/Gln156 and a Trp156 or Leu156 polymorphism provides tapasin independence by stabilizing these triad residues, thus generating an energetically stable and a more peptide receptive environment. Micropolymorphisms at position 156 can influence the generic peptide loading pathway for HLA-A*24 by altering their tapasin dependence for peptide selection. The trade-off for this tapasin independence could be the presentation of unusual ligands by these alleles, imposing significant risk following hematopoietic stem cell transplantation (HSCT)

Neuropathology in mouse models of mucopolysaccharidosis type I, IIIA and IIIB.

Mucopolysaccharide diseases (MPS) are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Neurodegenerative MPS diseases exhibit cognitive decline, behavioural problems and shortened lifespan. We have characterised neuropathological changes in mouse models of MPSI, IIIA and IIIB to provide a better understanding of these events.Wild-type (WT), MPSI, IIIA and IIIB mouse brains were analysed at 4 and 9 months of age. Quantitative immunohistochemistry showed significantly increased lysosomal compartment, GM2 ganglioside storage, neuroinflammation, decreased and mislocalised synaptic vesicle associated membrane protein, (VAMP2), and decreased post-synaptic protein, Homer-1, in layers II/III-VI of the primary motor, somatosensory and parietal cortex. Total heparan sulphate (HS), was significantly elevated, and abnormally N-, 6-O and 2-O sulphated compared to WT, potentially altering HS-dependent cellular functions. Neuroinflammation was confirmed by significantly increased MCP-1, MIP-1α, IL-1α, using cytometric bead arrays. An overall genotype effect was seen in all parameters tested except for synaptophysin staining, neuronal cell number and cortical thickness which were not significantly different from WT. MPSIIIA and IIIB showed significantly more pronounced pathology than MPSI in lysosomal storage, astrocytosis, microgliosis and the percentage of 2-O sulphation of HS. We also observed significant time progression of all genotypes from 4-9 months in lysosomal storage, astrocytosis, microgliosis and synaptic disorganisation but not GM2 gangliosidosis. Individual genotype*time differences were disparate, with significant progression from 4 to 9 months only seen for MPSIIIB with lysosomal storage, MPSI with astrocytocis and MPSIIIA with microgliosis as well as neuronal loss. Transmission electron microscopy of MPS brains revealed dystrophic axons, axonal storage, and extensive lipid and lysosomal storage. These data lend novel insight to MPS neuropathology, suggesting that MPSIIIA and IIIB have more pronounced neuropathology than MPSI, yet all are still progressive, at least in some aspects of neuropathology, from 4-9 months

Significant neuroinflammation in MPS cerebral cortex at 4 and 9 months of age.

<p>Representative sections of positively stained (A) astrocytes (GFAP; brown) and (C) microglia (Isolectin B4:brown) at 4 and 9 months of age (4 m and 9 m) that correspond to a high power view covering cortical layer IV (from section 2a, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035787#pone-0035787-g001" target="_blank">Figure 1A</a>). Boxed inserts show an enlarged single positively stained astrocyte or microglial cell. Sections were counterstained with Mayer's haematoxylin to highlight the nuclei. Bar = 100 µm. Whole fields of view used for counting are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035787#pone.0035787.s002" target="_blank">Figures S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035787#pone.0035787.s003" target="_blank">S3</a>. Four sections of brain were stained concurrently with either GFAP or Isolectin B4, and images of two low power fields of view covering cortical layers II/III–VI (boxed areas, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035787#pone-0035787-g001" target="_blank">Figure 1A</a>) were captured from each section. The number of GFAP or Isolectin B4 positive cells in these areas were counted using ImageJ and expressed as an average number of cells per section (B and D respectively; n = 3 mice per group). Error bars represent the SEM and p values are from two way ANOVA with Tukey's multiple comparisons test. Significant overall genotype differences are denoted by thick black lines. Individual genotype*time differences are shown by thin green lines. The significant individual genotype*time differences (p<0.001) between all MPSs and WT at each time point are not shown for clarity. (E) Low power plans (×2.5 objective) showing 4 representative brain sections from WT and MPS IIIA mice stained with Isolectin B4. Sections from MPSI and MPSIIIB are not shown but are virtually identical in distribution and intensity. Bar = 100 µm.</p

Lysosomal compartment size is significantly increased in MPS brain and localises to neurons and microglia.

<p>Lysosomal compartment size was measured by quantifying LAMP2 immunohistochemical staining of WT, MPSI, IIIA and IIIB mouse cerebral cortex at 4 and 9 months of age (4 m and 9 m; n = 3 mice per group). (A) Four sections of brain from Bregma 0.26, −0.46 −1.18 and −1.94 mm were stained concurrently. (B) Representative sections stained with LAMP2 that correspond to a whole field of view covering cortical layers II/III–VI (Section 2a in A). Bars = 100 µm. (C) Two fields of view (boxed areas in A; ×20 objective) from each section were quantified using ImageJ. Error bars represent the SEM and p values are from two way ANOVA with Tukey's multiple comparisons test. Significant overall genotype differences are denoted by thick black lines. Individual genotype*time differences are shown by thin green lines. The significant individual genotype*time differences (p<0.001) between all MPSs and WT at each time point are not shown for clarity. (D) LAMP2 (green) was detected in NeuN-positive neurons (red) and in ILB4-positive microglia (red) in layer II/III of WT, MPSI, IIIA and IIIB cerebral cortex. Nuclei are stained with DAPI (blue); Bar = 10 µm. Single colours and overlays are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035787#pone.0035787.s001" target="_blank">Figure S1</a>.</p

Significantly reduced levels of VAMP2 and Homer-1 and a change in the localisation of VAMP2 in MPS mouse cerebral cortex.

<p>Representative images of low (cerebral cortical layers II/III–VI) and high power sections (cerebral cortical layers II/III) of VAMP2 (A) and Homer-1 (D) stained sections at 9 months of age (9 m). WTs exhibit discrete VAMP2 punctate staining (A; white arrows) which is lost in MPSI, IIIA and IIIB mouse brain. Bar = 100 µm. Four sections of brain (Bregma 0.26, −0.46 −1.18 and −1.94 mm) were stained concurrently for VAMP2 (B), synaptophysin (C) and Homer-1 (E) and images of two low power fields of view covering cortical layers II/III–VI (boxed areas, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035787#pone-0035787-g001" target="_blank">Figure 1A</a>) were captured from each section and quantified using ImageJ (n = 3 mice per group). Error bars represent the SEM and p values are from two way ANOVA with Tukey's multiple comparisons test. Significant overall genotype differences are denoted by thick black lines and individual genotype*time differences are shown by thin green lines. The significant individual genotype*time differences (p<0.001) between all MPSs and WT at each time point are not shown for clarity.</p

Transmission electron microscopic analysis of MPS brain showing an increase in lysosomal burden in MPS cerebral cortex and large dystrophic axons in MPSI and IIIB.

<p>Images show an increase in lysosomal burden (black arrows outlined in white) in MPSI, IIIA and IIIB (B–D and F; Bars = 1 µm) compared to WT cerebral cortex (A; Bar = 1 µm). Lipid (white arrows outlined in black) is also stored in the lysosomes of MPS brain (B, C, and D) and also a small amount in WT (A). Dystrophic axons were observed in MPSI (E; Bar = 0.5 µm) and IIIB (arrow in G; Bar = 1 µm and enlarged in H; Bar = 0.5 µm) cerebral cortex but not in MPS IIIA (F; Bar = 1 µm). These structures contained organelles similar to immature and mature autophagosomes with electron dense material and some mitochondria (*; E and H). Normal axons were also observed in all MPS types (E, F and G; arrow heads).</p