Development of Sensory, Motor and Behavioral Deficits in the Murine Model of Sanfilippo Syndrome Type B

BACKGROUND: Mucopolysaccharidosis (MPS) IIIB (Sanfilippo Syndrome type B) is caused by a deficiency in the lysosomal enzyme N-acetyl-glucosaminidase (Naglu). Children with MPS IIIB develop disturbances of sleep, activity levels, coordination, vision, hearing, and mental functioning culminating in early death. The murine model of MPS IIIB demonstrates lysosomal distention in multiple tissues, a shortened life span, and behavioral changes. PRINCIPAL FINDINGS: To more thoroughly assess MPS IIIB in mice, alterations in circadian rhythm, activity level, motor function, vision, and hearing were tested. The suprachiasmatic nucleus (SCN) developed pathologic changes and locomotor analysis showed that MPS IIIB mice start their daily activity later and have a lower proportion of activity during the night than wild-type controls. Rotarod assessment of motor function revealed a progressive inability to coordinate movement in a rocking paradigm. Purkinje cell counts were significantly reduced in the MPS IIIB animals compared to age matched controls. By electroretinography (ERG), MPS IIIB mice had a progressive decrease in the amplitude of the dark-adapted b-wave response. Corresponding pathology revealed shortening of the outer segments, thinning of the outer nuclear layer, and inclusions in the retinal pigmented epithelium. Auditory-evoked brainstem responses (ABR) demonstrated progressive hearing deficits consistent with the observed loss of hair cells in the inner ear and histologic abnormalities in the middle ear. CONCLUSIONS/SIGNIFICANCE: The mouse model of MPS IIIB has several quantifiable phenotypic alterations and is similar to the human disease. These physiologic and histologic changes provide insights into the progression of this disease and will serve as important parameters when evaluating various therapies

Age-related progressive loss of Purkinje cells in the cerebellar lobules in MPS IIIB mice.

<p>ANOVA of Purkinje cell count by lobe showed that old MPS IIIB mice (white bars) had significantly lower neuronal counts across cerebellar lobes compared to old heterozygous controls (black bars) although differences between groups were lobe dependent. Pairwise comparisons conducted in the older cohort of mice showed that differences between groups were greatest in lobes VI (*p = 0.001), IV/V (*p = 0.003), VIII (*p = 0.004), IX (*p = 0.011), and III (*p = 0.036). An ANOVA of the Purkinje cell count data that included both young and old cohorts of mice resulted in a significant main effect of Age (p<0.001) and a significant Age by Genotype interaction (p = 0.003). Other important effects included significant Lobe by Genotype (p = 0.010), and Lobe by Age (p<0.001) interactions. Additional comparisons showed that the older MPS IIIB mice had significantly fewer neurons across the cerebellar lobes compared to the younger MPS IIIB mice (p<0.001, (p<0.006, the Bonferroni corrected value)), while no differences were found between older and younger control mice.</p

Older MPS IIIB mice are impaired on the rocking rotarod.

<p>Mean duration (in seconds) of the time spent on the rocking rotarod as a function of increasing ages (days) for MPS IIIB (open squares) and heterozygous (filled diamonds) control mice. No performance differences were observed between groups on the rocking rotarod in the younger cohort of mice (left panel). In contrast, an ANOVA conducted on the first 4 data points from the older cohort of mice (right panel) revealed a significant main effect of Genotype (p = 0.030), thus showing, in general, that the older MPS IIIB mice, remained on the rod for a significantly shorter time compared to the heterozygous control mice. Differences between groups were greatest when the mice were tested at 244 (*p = 0.013) and 302 (†p = 0.034) days of age. Even greater deficits were observed in the MPS IIIB mice when they were tested at older ages although small sample sizes resulting from high mortality rates in the MPS IIIB mice precluded formal statistical analyses. Numbers in parentheses represent sample sizes at specific ages of testing. The dotted line indicates that the last two test sessions (327 and 372 days) were not included in the overall ANOVA since sample sizes were so small due to increased mortality.</p

Electroretinography.

<p>Mixed (cones and rods) photoreceptor b-wave amplitudes (microvolts) to a light stimulus in dark-adapted wild type (diamonds on dashed line) and MPS IIIB (squares on solid line) mice at ages 4 to 40 weeks demonstrating reduced response in MPS IIIB mice, that is statistically significant from 16 weeks onward. Cone response b-wave amplitudes after light adaptation is depicted for wild type (open diamond/dashed line) and MPS IIIB (open square/solid line) at the same ages and shows no difference in cone response.</p

Locomotor activity records of a representative wild-type and MPS IIIB mouse.

<p>Running wheel revolutions per minute (black bars) is plotted for 24 h on each line with subsequent days plotted on the line below. In a light:dark schedule (gray bars show when lights were on), the mutant mice adjusted rhythm to light similar to wild type mice. The mice experienced a series of light:dark, dark:dark, and skeleton photoperiod schedules from ages 70–275 days.</p

Inner ear pathology in MPS IIIB mice at 30 wks.

<p>A. Radial view of cochlear upper basal turn showing grossly normal features. B. Crista ampullaris of lateral semicircular canal in the same animal shows abnormal lysosomal storage in both hair cells and supporting cells of the sensory epithelium (black arrow), and in dark cells (white arrow). C. Expanded view of medial organ of Corti in A. The only cells showing storage are inner pillar cells (IP) (black arrow). Mesothelial cells lining the basilar membrane (BM) show the aberrant storage (white arrowheads), as do adherent inflammatory cells (black arrowheads). Vacuoles within inner hair cells (asterisk, IHC) are probably a processing artifact. D. Spiral ganglion cell region from A shows aberrant storage only in glial cells (white arrowheads). E. Spiral ligament and lateral organ of Corti from A shows aberrant storage in outer sulcus cells (white arrowhead, OSC), epithelial cells of spiral prominence (black arrowhead, SP), Type III fibrocytes (white arrows, T3), and Type II fibrocytes (black arrows, T2). Type IV fibrocytes (T4) also showed storage, not apparent in this view. F. Complete loss of hair cells and other differentiated cell types of the organ of Corti (black arrow), with secondary loss of neuronal processes (white arrow). This was more prevalent in MPS IIIB mice than in WTs. G. Plot of the number of hair cell profiles versus neuronal density, as seen in radial view in the lower base for 5 MPS IIIB and 5 WT mice at 30 wks. Differences in hair cell numbers by genotype are highly significant (t-test, p<.001). Because inner hair cells were less affected than outer hair cells, only numbers <1.0 indicate loss of IHCs. Neuronal density decreases only when IHCs are missing, so that neuronal loss appears secondary to hair cell loss in MPS IIIB. StV: Stria vascularis; RM: Reissner's membrane; TM: Tectorial membrane; OHC: Outer hair cells; DC: Deiters' cells; OP: Outer pillar cell; Lim: Spiral limbus.</p

Eye Pathology.

<p>Light micrographs showing pathology from normal (A, F, H) and MPS IIIB (B–D, E, G, I) mouse retinas (A–E), sclera (F, G), and ciliary bodies (H, I). (A) Normal histology from a 4-week old wild type mouse retina is shown and is indistinguishable from older wild type retinas at 45 weeks. (B) In the 8-week old MPS IIIB mouse retina, aberrant lysosomal storage can be seen in non-neuronal cells in the inner retina (arrow). Inset: Localized disruption of the retinal pigment epithelium (RPE) from a nearby region of the same retinal section is shown. (C) At about 16 weeks, the outer segments (OS) of the MPS IIIB mouse are shortened, the outer nuclear layer (ONL) is reduced by 2–4 rows of nuclei, and pyknotic nuclei are seen in the ONL (arrow). Macrophage-like cells are present in the subretinal space (arrowhead). (D) In the 30-week old MPS IIIB mouse retina, OSs are further shortened and the ONL is reduced by nearly half. Inset: higher magnification of the boxed area showing dense, round melanosome-like structures in the RPE. (E) By 34 weeks, a subpopulation of cells in the ganglion cell layer (GCL) has a dense appearance with numerous lysosomal inclusions in the cytoplasm. (F) Normal histology from a 4-week old wild type mouse sclera is shown. (G) Lysosomal storage appears as pale vesicles in the sclera of the MPS IIIB mouse (arrowheads). (H) Normal histology from a 4-week old wild type mouse ciliary body is shown. (I) By 30 weeks of age, the ciliary body of the MPS IIIB mouse appears disorganized and swollen with lysosomal storage vesicles (arrow). Numerous dense, round melanosome-like structures are seen (arrowhead). IS, photoreceptor inner segments; OPL, outer plexiform layer; INL inner nuclear layer; IPL, inner plexiform layer. Smaller bar = 10 micron for A–D, H, I. Larger bar = 10 micron for E–G.</p

Auditory-evoked Brainstem Responses (ABR).

<p>Mean (±SEM) ABR thresholds in MPS IIIB and WT mice at 5, 10, 20 and 40 kHz from age 4 to 45 weeks. Earliest differences by genotype appear at high frequencies (40 kHz). By 30 wks, high frequency hearing losses due to background deficits in C57BL/6 dominate. Threshold differences at lower test frequencies, apparent after 16 wks, probably reflect mixed cochlear and middle ear pathology. Differences by genotype were significant at all ages (p<.001, 2-way ANOVA).</p