Failure of Neuroprotection Despite Microglial Suppression by Delayed-Start Myeloperoxidase Inhibition in a Model of Advanced Multiple System Atrophy: Clinical Implications

Abstract

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease. Post-mortem hallmarks of MSA neuropathology include oligodendroglial α-synuclein (αSYN) inclusions, striatonigral degeneration, olivopontocerebellar atrophy, and increased microglial activation that accompanies the wide spread neurodegeneration. Recently, we demonstrated upregulation of myeloperoxidase (MPO) in activated microglia and provided evidence for the role of microglial MPO in the mediation of MSA-like neurodegeneration (Stefanova et al. Neurotox Res 21:393-404, 2015). The aim of the current study was to assess the therapeutic potency of MPO inhibition (MPOi) in a model of advanced MSA. We replicated the advanced pathology of MSA by intoxicating transgenic PLP-α-synuclein transgenic mice with 3-nitropropionic acid (3NP). After onset of the full-blown pathology, MSA mice received either MPOi or vehicle over 3 weeks. Motor phenotype and neuropathology were analyzed to assess the therapeutic efficacy of MPOi compared to vehicle treatment in MSA mice. MPOi therapy initiated after the onset of severe MSA-like neuropathology in mice failed to attenuate motor impairments and neuronal loss within the striatum, substantia nigra pars compacta, inferior olives, pontine nuclei, and cerebellar cortex. However, we observed a significant reduction of microglial activation in degenerating brain areas. Further, nitrated αSYN accumulation was reduced in the striatonigral region. In summary, delayed-start MPOi treatment reduced microglial activation and levels of nitrated αSYN in a mouse model of advanced MSA. These effects failed to impact on motor impairments and neuronal loss in contrast to previously reported disease modifying efficacy of early-start therapy with MPOi in MSA.

Fig. 1

a The daily clinical motor score served to evaluate the time course of the motor impairment induced by 3NP treatment (day 1–day 8) and its course over the treatment period with AZD3241 or vehicle (day 9–day 30). b Mean clinical motor score per group over the total experimental time indicated lack of effect of AZD3241 treatment (MPOi) on the general motor disability in MSA mice. c Stride length was not changed under MPOi treatment of MSA mice compared to vehicle-treated ones. d, e Rearing and horizontal open field activities were not affected by the MPOi treatment of MSA mice compared to vehicle-treated MSA mice. Data are presented as mean ± SEM. MSA + vehicle group, n = 15, MSA + MPOi group, n = 14

Fig. 2

DARPP32-positive medium spiny neurons of the striatum of MSA + vehicle (n = 9) (a) and MSA + MPOi group (n = 7) (b). There was no significant effect of AZD3241 treatment on the number of striatal DARPP32 positive neurons in MSA mice (c). TH-positive dopaminergic neurons in SNc of MSA + vehicle (n = 14) (d) and MSA + MPOi group (n = 13) (e). MPOi treatment showed no significant neuroprotective effect on nigral TH neurons in MSA mice (f). Further, no neuroprotective efficacy of MPOi could be registered in the inferior olives (n _vehicle = 6, n _MPOi = 6) (g), the pontine nuclei (n _vehicle = 5, n _MPOi = 7) (h), and the Purkinje cells in the cerebellar cortex (n _vehicle = 6, n _MPOi = 8) (i). Data are presented as mean ± SEM

Fig. 3

CD11b immunohistochemistry in striatum of a MSA + vehicle and b MSA + MPOi (AZD3241) group. c Statistical analysis indicated a tendency to reduction of the microglial activation in the striatum without reaching significance (n _vehicle = 14, n _MPOi = 14). CD11b immunohistochemistry in substantia nigra (SN) of d MSA + vehicle and e MSA + MPOi group. f MPOi treatment of MSA mice resulted in significant reduction of CD11b ROD in substantia nigra (*p < 0.05 compared to MSA + vehicle; n _vehicle = 14, n _MPOi = 14). CD11b immunohistochemistry in the pontine nuclei of g MSA + vehicle and h MSA + MPOi group. i MPOi treatment of MSA mice resulted in significant reduction of CD11b ROD in the pontine nuclei of MSA mice receiving AZD3241 compared to vehicle-treated animals (**p < 0.01; n _vehicle = 12, n _MPOi = 12). CD11b immunohistochemistry in the inferior olives of j MSA + vehicle and k MSA + MPOi group. l MPOi treatment of MSA mice resulted in significant reduction of CD11b ROD in the inferior olives of MSA mice receiving AZD3241 compared to vehicle-treated animals (*p < 0.05; n _vehicle = 13, n _MPOi = 14). CD11b immunohistochemistry in the cerebellar cortex of m MSA + vehicle and n MSA + MPOi group. o MPOi treatment of MSA mice resulted in significant reduction of CD11b ROD in the pontine nuclei of MSA mice receiving AZD3241 compared to vehicle-treated animals (***p < 0.001; n _vehicle = 11, n _MPOi = 11). Data are presented as mean ± SEM

Fig. 4

Nitrated α-synuclein immunohistochemistry in striatum of a, MSA + vehicle and b MSA + MPOi (AZD3241) group. c Statistical analysis indicated significant reduction of the density of GCIs positive for nitrated α-synuclein in the striatum of MSA mice receiving AZD3241 (*p < 0.05; n _vehicle = 10, n _MPOi = 13). Nitrated α-synuclein immunohistochemistry in substantia nigra of d MSA + vehicle and e MSA + MPOi group. f MPOi treatment of MSA mice resulted in significant reduction of GCIs density in substantia nigra (**p < 0.01 compared to MSA + vehicle; n _vehicle = 10, n _MPOi = 14). Nitrated α-synuclein immunohistochemistry in the pontine nuclei of g MSA + vehicle and h MSA + MPOi group. i MPOi treatment of MSA mice resulted in no significant reduction of GCIs density in the pontine nuclei of MSA mice receiving AZD3241 compared to vehicle-treated animals (n _vehicle = 12, n _MPOi = 12). Nitrated α-synuclein immunohistochemistry in the inferior olives of j MSA + vehicle and k MSA + MPOi group. l MPOi treatment of MSA mice resulted in no change in the GCIs density in the inferior olives of MSA mice receiving AZD3241 compared to vehicle-treated animals (n _vehicle = 13, n _MPOi = 12). Nitrated α-synuclein immunohistochemistry in the cerebellar cortex of m MSA + vehicle and n MSA + MPOi group. o MPOi treatment of MSA mice resulted in no change in the GCIs density in the cerebellar cortex of MSA mice receiving AZD3241 compared to vehicle-treated animals (n _vehicle = 11, n _MPOi = 11). Data are presented as mean ± SEM