To understand the effects of HS accumulation on brain pathology, changes in brain architecture were evaluated by histology.
Increased lysosomal storage in the forebrain of MPS IIIB dogs
Lysosomal membrane protein-1 (LAMP1) has been used as a surrogate marker of lysosomal storage burden and has been shown to indicate the size of the lysosomal compartment, despite only being a proxy measure10. Tissue from the forebrain of MPS IIIB dogs 15 months of age showed increased LAMP1 staining over the corresponding unaffected controls (Fig. 2). Due to the diffuse background staining within the neuropil of the oldest available MPS IIIB dogs (data not shown), increased LAMP1 staining was most evident in 15-month-old MPS IIIB dogs compared to age-matched unaffected dogs (Fig. 2). A dense accumulation of punctate LAMP1 immunoreactivity was present to different extents in the cytoplasm of individual cells. These cells predominantly had neuronal morphology, although other smaller cells, presumed to be glia, also contained intense LAMP1 immunoreactivity. This staining of individual cells also revealed a series of different regional patterns of LAMP1 immunostaining within the forebrain of 15-month-old MPS IIIB dogs.
In the cortex of 15-month-old MPS IIIB dogs, immunostaining for LAMP1 varied in intensity across the cortical laminae, and at different levels of the brain (Fig. 2). Rostrally, LAMP1 immunostaining was darker within cells in both superficial (II/III) and deeper laminae (VI), but was less intense in the other laminae. In more caudal regions of cortex, this distinction was less apparent with a more even, pan-laminar staining of cells (Fig. 2). Within the white matter, there was also a stark contrast in the distribution of LAMP1 staining; the white matter of the corona radiata that lies directly under cortical grey matter had more intense LAMP1 immunoreactivity than the adjacent white matter of the corpus callosum, where much less LAMP1 staining was evident.
Hippocampal neurons displayed subfield-specific LAMP1 staining in both unaffected and MPS IIIB dogs. This immunoreactivity was much darker in pyramidal neurons of CA3 rather than CA1 of MPS IIIB dogs and also within granule neurons of the dentate gyrus (Fig. 3). There was also staining of LAMP1 with what appeared to be glia scattered through the hippocampal neuropil of 15-month-old MPS IIIB dogs that was not present in unaffected control dogs (Fig. 3). MPS IIIB dogs also had markedly more LAMP1 immunoreactivity throughout the thalamus and hypothalamus, with much more staining within morphologically identified glia, as well as more numerous large, darkly LAMP1-stained neurons in both structures (Fig. 4). In the hypothalamus, the neurons in the paraventricular nucleus seemed to have a particularly increased intensity of LAMP1 staining, as did the nearby substantia nigra. Although unaffected control dogs displayed strong LAMP1 staining in neurons within the substantia nigra pars compacta, these nigral neurons appeared to be enlarged in MPS IIIB dogs. This morphological appearance is indicative of pronounced storage accumulation, consistent with more intense LAMP1 immunostaining in these affected dogs. In addition, other cell types (likely microglia) were also stained in this brain region of MPS IIIB dogs. In the thalamus, the dorsolateral geniculate nucleus had robust LAMP1 staining in both MPS IIIB and unaffected dogs, though staining in neurons of MPS IIIB animals was much darker, as was background staining of the neuropil.
MPS IIIB dogs do not display cortical atrophy
Previous data from MPS IIIB mice revealed no evidence of significant cortical atrophy9. Given that mice may not live long enough to develop the full extent of cortical pathology, we evaluated the presence of cortical atrophy in MPS IIIB dogs by measuring cortical thickness in the most severely affected MPS IIIB dogs ≥ 20 months of age. Measurements were obtained from the somatosensory cortex, a representative region of the sensory cortex that is most severely affected in large animal models of similar neuropathic lysosomal disorders19. There was no significant difference in the thickness of the rostral suprasylvian gyrus (Supplementary Figure 3) between this oldest cohort of MPS IIIB dogs and their age-matched unaffected counterparts indicating that even in advanced stages of the disease, cortical atrophy does not occur in this canine MPS IIIB model.
MPS IIIB dogs display qualitative increases in microglial activation in the cerebral cortex
Despite their lack of overt cortical neurodegeneration, MPS IIIB mice display pronounced and progressive microglial activation9,20. Typical markers of microglial activation, such as CD68, often do not have reactivity in canine tissue. Instead, immunostaining was performed with Iba1, a microglial marker that is present in quiescent cells and is increased with microglial activation21. Marked differences in microglial activation and morphology were observed between unaffected and MPS IIIB dogs. Such changes were evident by 3 months of age and became more pronounced over time (Fig. 5).
Surveying the distribution of Iba1 immunoreactivity across brain regions of MPS IIIB dogs revealed no particular focal area of microglial activation. For example, within the cortical mantle, Iba1 immunoreactivity was uniformly increased across the depth of all cortical regions of MPS IIIB dogs, with no greater increase in any individual lamina (Fig. 5, upper panels). Similar but less pronounced changes were evident within the underlying white matter (Fig. 5, upper panels). A notable exception was the presence of localized clusters of intensely stained Iba1-positive microglia in white and grey matter of MPS IIIB dogs of all ages (Fig. 6). Similar microglial clusters were also observed in unaffected dogs, but to a lesser extent. The distribution of these clusters did not appear to favor any specific brain region, but occurred with a similar frequency at all rostro-caudal levels of the brain in both cortical and subcortical structures. Equally, there was no evidence that the clusters associated with blood vessels and the frequency of the clusters did not appear to correlate with disease progression.
There were also clear differences in microglial morphology between unaffected and MPS IIIB dogs, which became more pronounced in MPS IIIB dogs with age. In unaffected animals, the many thin ramified Iba1-positive processes characteristic of quiescent or resting microglia gave the background of the neuropil a “lacy” appearance (Fig. 5, cortical layer inset), an appearance that was less apparent in MPS IIIB dogs. Morphological changes in microglia in MPS IIIB dogs reflected their activated state, being not only more intensely stained than cells in unaffected controls, but also with fewer thickened processes and an enlarged cell soma, typical of activated microglia.
Iba1-positive morphologically active microglia were particularly evident in the cortical mantle of MPS IIIB dogs from 3 months of age and were distributed uniformly throughout the cortex (Fig. 5, and with age morphological differences between Iba1-positive microglia became more pronounced. Twenty-month-old MPS IIIB dogs had increased incidence of distended microglial cell bodies in the cortical neuropil (Fig. 5). Measurements of the cross-sectional area of Iba1-positive microglial soma confirmed that these microglia were significantly larger in MPS IIIB dogs than their wild-type counterparts at 20 months of age or older (Supplemental Figure 2). There was also a characteristic increase in microglial activation in the white matter in MPS IIIB dogs that was not seen in unaffected controls (Fig. 5). Although the morphological changes in white matter microglia were not as pronounced in the cortical grey matter, the soma of these microglia also appeared larger at later stages of disease progression (Fig. 5, inset).
The initial characterization of the schipperke dog model of MPS IIIB reported neurologic signs consistent with cerebellar disease, evidence for cerebellar atrophy, and Purkinje neuron loss17. To broaden these observations, we assessed the degree of storage burden, glial activation, and neuronal survival in the cerebellum of MPS IIIB dogs.
Increased lysosomal storage in the cerebellum of MPS IIIB dogs
Lysosomal storage burden was greatly increased in several areas of the cerebellum in the schipperke MPS IIIB dogs17. In our study, immunostaining of LAMP1, a surrogate marker for storage burden, was increased in MPS IIIB dogs at all ages examined, as compared to age-matched unaffected dogs, although there was significant inter-sample variability in the immunostaining intensity (Fig. 7). In unaffected dogs, LAMP1 staining was very faint, and only appreciable within the characteristically large soma of Purkinje neurons, whose morphology was readily apparent. In contrast, there was dramatically more LAMP1 staining in MPS IIIB dogs. While this increase was evident at all ages examined, the difference between the diseased state and unaffected controls was more obvious by 9 months of age. In these MPS IIIB dogs, LAMP1 not only intensely stained Purkinje neurons and their projections within the molecular layer, but also stained the granule cell layer and DCN, becoming especially pronounced at 15 months of age (Fig. 7). In the oldest MPS IIIB dogs, the pattern of LAMP1 staining changed again, with far fewer stained Purkinje neurons, markedly less staining in the molecular layer, and fewer, more intensely stained cells in the granule cell layer. This change in distribution could reflect the loss of Purkinje neurons and granule cells in MPS IIIB dogs, with more LAMP1 staining in microglia (Fig. 7). These findings suggest that storage burden, as measured by LAMP1 immunostaining, reaches a peak in brains of MPS IIIB dogs before tapering off in later stages of the disease, likely due to cell death of various cell types expressing LAMP1.
Progressive gliosis in the MPS IIIB cerebellum
The activation of both astrocytes and microglia occurs as a response to cell damage in LSDs such as MPS IIIB9,10,20. Using GFAP as a marker, we assessed the extent of astrogliosis in canine MPS IIIB disease.
Progressive astrogliosis in the cerebellum of MPS IIIB dogs
Across all age groups, GFAP immunostaining was consistently elevated in MPS IIIB dogs compared to the unaffected controls. However, the high inter-animal variability precluded quantitative analyses. However, even unaffected animals at all ages displayed a substantial number of GFAP-positive Bergman glia within most folia, although these cells displayed relatively thin processes (Fig. 8, Bergman glia). This was accompanied in unaffected dogs by relatively little staining of fibrous astrocytes within the cerebellar white matter (Fig. 8, white matter). In contrast, the pattern and intensity of GFAP immunostaining was markedly different in MPS IIIB dogs at all ages examined (Fig. 8, grey), and became more complex with disease progression. Bergman glia in MPS IIIB dogs displayed much thicker processes than in corresponding age-matched unaffected controls (Fig. 8), and a distinctive band of GFAP immunostaining was evident at the interface between granule and molecular layers, marking where Purkinje cells normally sit (Fig. 8, Purkinje layer). This reached its peak intensity in MPS IIIB dogs by 15 months of age, before declining in older MPS IIIB dogs (Fig. 8). This was accompanied by progressively more GFAP immunostaining with increased age within both the granule cell layer, and perhaps most notably within the white matter and DCN of MPS IIIB dogs (Fig. 8). Increased GFAP immunostaining within the white matter was already visible in MPS IIIB dogs from 9 months of age, revealing a distinctive outlining of astrocyte processes around blood vessels within the cerebellar white matter (Fig. 8). These results suggest progressive changes in astrocyte activation in MPS IIIB dogs, specifically around the Purkinje cell layer and cerebellar white matter.
Microgliosis is most prominent in the DCN in cerebellum of MPS IIIB dogs
Iba1 immunoreactivity was much less variable than GFAP staining and revealed changes in morphology and increased Iba1 immunostaining in MPS IIIB dogs compared to the unaffected controls from 9 months of age, and were more pronounced at later stages (Fig. 9). Compared to the lacy appearance of the neuropil in unaffected dogs, Iba1-positive microglia in MPS IIIB dogs displayed fewer, thicker processes and had enlarged cell soma. A distinctive appearance of very large microglia and/or clusters of intensely stained Iba1-positive cells were seen sporadically within both granule and molecular layers of MPS IIIB dogs from 9 months of age. Compared to astrocytosis, microglial activation was less pronounced within the white matter of MPS IIIB dogs, but was still markedly higher than in unaffected dogs, with localised activation evident in the DCN (Fig. 9). Quantification of Iba1 immunoreactivity via thresholding image analysis revealed some inter-animal variability. In particular, one of the 9-month-old MPS IIIB brains had markedly increased Iba1 immunostaining than any other brain of the same age. However, there was a significant increase in Iba1 immunoreactivity in white matter of MPS IIIB dogs at > 20 months of age (Fig. 9D), with a trend starting at 15 months of age. A similar trend in relative Iba1 immunoreactivity was seen in the DCN of MPS IIIB dogs, but not in their cerebellar cortices in either the molecular or granular layers. These results indicate, similarly to the atrophy, much of the cerebellar microgliosis occurs in the white matter. While there were obvious changes in microglial morphology in the cerebellar cortical layers of MPS IIIB dogs, significant changes in Iba1 immunoreactivity were detected only in the white matter and the DCN.
Loss of neurons and white matter atrophy in the cerebellum of MPS IIIB dogs
Purkinje cells and neurons in the DCN provide the major efferent output from the cerebellum, indicating their importance for cerebellar functions. Loss of these cells was analyzed by Nissl staining and the sections were used to evaluate white matter volume loss and cerebellar cortical thickness (Fig. 10). The thickness of cerebellar molecular and granular layers was measured in the posterior lobe of the cerebellum both separately and together, but revealed no significant change in the thickness of any cerebellar cortical layer in MPS IIIB dogs at any age (data not shown). Cerebellar white matter volume changes were apparent in MPS IIIB dogs from 15 months of age, but became statistically significant at > 20 months of age when the volume was decreased by more than 50% (Fig. 10A). There was an increase in white matter volume in unaffected dogs over time, potentially due to ongoing myelination during postnatal maturation, which was not evident in MPS IIIB dogs, resulted in a significantly smaller volume in the oldest MPS IIIB dogs (Fig. 10A). A marked decrease in Purkinje cells (15–20%) was observed in the posterior lobe of the cerebellum of MPS IIIB dogs at all ages when compared to age-matched unaffected controls. Though this did not reach statistical significance, the trend in Purkinje cell loss with disease progression was evident (Fig. 10B). Optical fractionator counts of Nissl-stained DCN neurons showed a pronounced loss of these cells in MPS IIIB dogs by 9 months of age, which then remained stable at later stages of disease (Fig. 10C). Compared to unaffected controls, the loss of DCN neurons in MPS IIIB dogs became statistically significant after 15 months of age. However, compared to 3- month-old MPS IIIB dogs, the loss of DCN neurons was statistically significant from 9 months onwards (Fig. 10C). Taken together, these data indicate that cerebellar atrophy occurs primarily in the white matter, and that there is progressive loss of DCN neurons that has not been reported previously.