Here, α-synuclein expression was examined in various Gba1 mutated mice with or without CNS phenotypes including: Gba1 point mutated mice, and hypomorphic prosaposin mice (PS-NA), with or without 9H or 4L, and in CBE - treated Gba1 mutated mice. The 9H/PS-NA mice had the most significant α-synuclein accumulation, particularly in the deeper layers of the cortex. Brain lipid analyses showed moderately increased levels of glucosylceramide in the cortex and other regions compared with PS-NA and point mutated Gba1 models; this suggested a correlation of glucosylceramide accumulation with significant α-synuclein aggregation. In comparison with 9H/PS-NA mice, similar glucosylceramide levels and neuronopathology, but less significant α-synucleinopathy were observed in 4L/PS-NA mice. This result indicates that glucosylceramide levels are one factor in the development of α-synucleinopathy and that additional pathophysiologic mechanisms are needed, e.g., the in vivo properties of mutant GCases, (4L or 9H). Interestingly, glucosylsphingosine accumulation in 4L/PS-NA is much higher (10 times) than that in 9H/PS-NA, but with greater α-synuclein accumulations in the latter. Thus, glucosylsphingosine is not likely to play a major role in the α-synucleinopathy, but rather it contributes to neuronal and cell death. The rapid neuronal degeneration and death induced by glucosylsphingosine may not support the time needed for α-synuclein aggregate deposition. A complex and chronic mechanism is implied for α-synucleinopathy development.
The chronicity of the α-synuclein accumulation is supported by its age-dependency in the 9H/PS-NA, 4L/PS-NA, and Gba1
point-mutated mice. Low levels of α-synuclein accumulation were detected as early as 4-wks in 9H/PS-NA mice with progression to substantial aggregates by 12-20 wks. Immunoblot analysis confirmed the formation of α-synuclein oligomers in 9H/PS-NA and 4L/PS-NA cortices that contribute to the aggregates. The short-lived kn-9H mice (14 days) [34
] that were GCase null in the CNS and had glucosylceramide accumulation in the brain, did not have α-synuclein aggregates (data not shown). In CBE induced GCase deficiency in adult mice, α-synuclein aggregates only developed after long-term treatment. These observations indicate that α-synuclein aggregation is of a chronic progressive nature, consistent with the time-dependent formation of α-synuclein aggregates following an in vitro
initial nucleation event [50
]. α-Synuclein aggregates could be formed under a number of conditions including simple solutions, incubation with lipids, in cell cultures, or in animal models of disease [52
]. However, in vivo
systems are complicated by interactions among multiple proteins/biomolecules involved in the formation of inclusions [52
]. Here, ultrastructural analyses showed numerous inclusion bodies in neurons and the structure of these inclusions suggested aggregated proteins.
Various patterns of α-synuclein aggregation were observed in brains from the Gba1
mouse variants in these studies, particularly in 9H/PS-NA. Numerous large α-synuclein aggregates were present in the deep layers of cortex overlaying corpus callosum, whereas fine α-synuclein particles were in the hippocampal CA3 and dentate gyrus. The patterns of α-synuclein aggregates are similar to pathologic findings in Gaucher disease patients who had Parkinsonism. However, such patients characteristically exhibit Lewy bodylike α-synuclein inclusions with gliosis in cortical layers 3 and 5 (deeper layers) and in the hippocampal neurons (CA2-4 regions) [15
]. Similar aggregates were detected in 4L/PS-NA and 9H/PS-NA brain, but Lewy bodies were not observed in the current studies.
The relationships of α-synuclein accumulation and ubiquitin, astrocyte (GFAP), dopaminergic neuron (tyrosine hydroxylase), and microglial cells (CD68) were examined in brain sections. In 9H/PS-NA or 4L/PS-NA and PS-NA mice, ubiquitin partially colocalized with α-synuclein signals. In human samples, α-synuclein positive Lewy bodies were generally ubiquitin positive [31
]. The differences in co-localization of α-synuclein and ubiquitin between human and mouse suggest variation in the initiation mechanisms between these species. However, the end-stage nature of autopsy specimens from patients with both Gaucher disease and PD patients does not provide insights into the time course of this process. In comparison, 9H/PS-NA mice showed a progressive α-synucleinopathy that was detected as early as 4-wks, whereas ubiquitin aggregates were seen almost exclusively after 10 wks. The precise role of α-synuclein in the Lewy body formation or in the neurodegenerative processes is unclear. Lewy bodies are complex structures and contain multiple proteins [52
]. However, GCase has recently been found in Lewy bodies in patients heterozygous for GBA1
mutations suggesting a role for the GCase in their formation [55
]. In 9H/PS-NA or 4L/PS-NA brains, massive astrogliosis, and activated microglial cells were present, but signals for GFAP (astrocytes) or CD68 (microglial cells) colocalized with α-synuclein only occasionally. Interestingly, a few α-synuclein signals localize near the center of CD68 positive microglial cells (, CD68 positive), suggesting a microglial cell response to aggregated proteins in the brain. These observations indicate that α-synuclein was not expressed to a great extent in either astrocytes nor in microglial cells, but rather in neurons. Tyrosine hydroxylase expression in the mice used here was not significantly different from WT control mice.
Screening of several other LSD mouse models confirmed the presence of small α-synuclein aggregates in brains from NPC1 and MPS1 mice (data not shown), but not in LAL or individual saposin deficiency mice. NPC1 mice have a defect in a cholesterol efflux protein in the membrane of the lysosome [56
]. However, glycosphingolipids, including glucosylceramide and gangliosides, also accumulate in NPC1 brain. MPS1 mice accumulate glycosaminoglycans heparan and dermatan sulfate, as well as GM2 and GM3 [59
]. These observations support the contention that glycosphingolipids and gangoliosides contribute to the pathogenesis of these diseases and may increase the risk for developing α-synuclein accumulations. In Sandhoff mice, neuronal accumulations of α-synuclein and GM2 ganglioside were present throughout the brain [60
]. α-Synuclein aggregates are also found in cathepsin D deficient mice, sheep, and humans. This is potentially due to the role of cathepsin D in α-synuclein processing. α-Synuclein and ubiquitin signals in NPC1 and MPS1 brains were significantly less than those in 9H/or 4L/PS-NA brains, some specificity for the α-synucleinopathy. Specific saposin A, B, C or D deficiencies in mice produced no α-synuclein and ubiquitin accumulation. The accumulated GSLs in each of these models are different, e.g., galactosylceramide and galactosylsphingosine with saposin A deficiency [61
], sulfatide with saposin B deficiency [62
], lactosylceramide with saposin C deficiency [63
], and ceramide with saposin D deficiency [64
]. In total saposin deficient mice, (i.e., prosaposin knockout) moderate increases of glucosylceramide and positive anti-ubiquitin antibody staining were observed in neurons [65
]. These studies suggest that the respective sphingolipids or other saposin effects in the saposin deficient mice are not causally related to α-synuclein and ubiquitin accumulation. With the hypomorphic prosaposin-deficiency combined with low activity GCase mutations (4L/PS-NA or 9H/PS-NA), α-synuclein accumulation was observed as early as 4-wks. These studies suggest a more complicated relationship between glucosylceramide, GCase mutations, GSL accumulations, and the risk for α-synuclein/ubiquitin accumulations.
Many LSDs are associated with progressive and severe neurodegeneration. To date, the diverse biological pathways from lysosomal enzyme deficiency to neuron dysfunction and death have not been clarified [66
]. In the mouse models of multiple sulfatase deficiency (MSD) and mucopolysaccharidosis type IIIA (MPSIIIA), defective autophagosome-lysosome fusion was observed with severe neurodegeneration [67
]. In Huntington disease or familial of Parkinson disease, impaired autophagocytic pathways led to aggregation-prone proteins, which cause late-onset neurodegenerative conditions [68
]. However, knockout of selected autophagy genes caused abnormal protein accumulation in ubiquitinated inclusions and neurodegeneration in mice [69
]. These phenotypic similarities suggest the presence of common pathogenic mechanisms for LSDs with neurodegeneration [71
Clinically, earlier onset and more progressive PD patients had heterozygous mutations in GBA1
]. However, Parkinsonism manifestations [6
] and α-synuclein/ubiquitin aggregates [7
] were found in only some GD type 1 or 3 patients. Thus, the connections between GCase mutations (gain or loss of function or both) and the development of Parkinsonism or PD remain unclear. The current findings in mice provide a biological system and potential clues to the pathogenesis of neuronopathic Gaucher disease including the α-synuclein distribution patterns in Gba1
mutant mice. Ongoing studies should provide insight into the underlying neuronopathic mechanisms in Gaucher disease and the relationship between GCase deficiency and/or GBA1
heterozygosity and the development of Parkinsonian manifestations.