Cerebellar Alterations and Gait Defects as Therapeutic Outcome Measures for Enzyme Replacement Therapy in α-Mannosidosis

Abstract

α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyl-linked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy (ERT) that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term ERT partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides and cholesterol. The present study demonstrates cerebellar alterations in α-mannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures.

Chronic enzyme replacement therapy ameliorates neuropathology in alpha‐mannosidosis mice

“…2E) of both genotypes. Finally, we compared neuropathological alterations such as microglia activation which appear secondary to primary substrate storage 17. A similar degree of microglia activation as indicated by increased labeling with CD68 throughout the cortex was evident (Fig.…”

“…S2C). These storage vacuoles are strongly positive for the Lysosome‐associated membrane glycoprotein 1 (LAMP‐1) 17. Morphological examination of brain sections immunologically stained for LAMP‐1 displayed a similar increase in vesicular labeling of LAMP‐1 within neurons of the hippocampus, cerebellum, and cortex (Fig.…”

“…In order to investigate the chronic impact of this ERT regimen and whether chronic treatment can compensate for a lower dose, 4‐6 weeks old mice with an established blood‐brain barrier23, 24 were biweekly injected with 125 and 500 U/kg rhLAMAN (corresponding to 4.4 and 15.6 mg/kg per bw), respectively. ERT was completed at an age of 16‐18 weeks, a time point where alpha‐mannosidosis mice display a prominent storage phenotype 16, 17. Of note, for this and the following experiments prior to harvesting brain and other organs, mice were perfused with phosphate buffer to avoid contamination with plasma‐derived rhLAMAN.…”

“…So far, several LSDs including Morbus Gaucher (Type I), mucopolysaccharidoses (I, II, VI), Morbus Fabry, and Morbus Pompe have been shown to benefit from ERT 14. We have previously demonstrated the value of high‐dose short‐term ERT on clearance of primary brain substrate storage in alpha‐mannosidase‐deficient (knockout) mice,15 a valid mouse model for the human disease, using rhLAMAN of a laboratory scale batch 16, 17, 18. However, immunological responses to the injected enzyme associated with high mortality precluded long‐term ERT of this mouse strain.…”

Distribution and Severity of Neuropathology in β-Mannosidase-Deficient Mice is Strain Dependent

“…In other lysosomal storage disorders, different, but disease-specific, patterns of neuropathology are characteristic. For example, a-mannosidosis (OMIM 248500), caused by mutations in the a-mannosidase (MAN2B1, EC 3.2.1.24, 609458) gene, has been described in cats, cattle, guinea pigs, humans, and knockout mice (Auclair and Hopwood 2007;Blanz et al 2008;Crawley and Walkley 2007;Damme et al 2011;Stinchi et al 1999;Vite et al 2001) and has somewhat different neurological manifestations and neuropathological characteristics depending on the species. Aspartylglucosaminuria (AGU, OMIM 208400) (Dunder et al 2010;Jalanko et al 1998;Kaartinen et al 1996) is caused by mutations in the gene for glycosylasparaginase (AGA, EC 3.5.1.26, 613228), the enzyme necessary for hydrolysis of the proteinoligosaccharide linkage in N-linked glycoproteins.…”