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Title: Viable Neuronopathic Gaucher Disease Model in Medaka (Oryzias latipes) Displays Axonal Accumulation of Alpha-Synuclein}
Article:
Abstract:
Homozygous mutations in the glucocerebrosidase( GBA) gene result in Gaucher disease( GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson’s disease( PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant( GBA-/-) medaka that are completely deficient in glucocerebrosidase( GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein( α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.
Introduction:
Gaucher disease( GD) is the most common lysosomal storage disease and is caused by homozygous mutations in glucocerebrosidase( GBA). Mutations in GBA lead to decreased enzymatic activity of glucocerebrosidase( GCase) and result in the accumulation of its substrates, glucocerebroside and glucosylsphingosine[1, 2]. GD is classically divided into three subtypes: a non-neuronopathic form( type 1), an acute neuronopathic form( type 2), and a subacute neuronopathic form( type 3). Visceral manifestations of all forms are characterized by hepatosplenomegaly, cytopenia, and skeletal disease. Pathologically, the accumulation of lipid-laden macrophages, called Gaucher cells, are observed in the affected organs. Neurological manifestations of neuronopathic forms include brainstem dysfunction, intellectual disability, seizures, and myoclonic movement. Pathological features of neuronopathic forms are neuronal loss, astrogliosis, microgliosis, and perivascular accumulation of Gaucher cells[3]. The most severe neuronopathic form, called the perinatal lethal type, has also been reported[4]. Common presentations of patients with the perinatal lethal type are hydrops fetalis and congenital ichthyosis. Almost no residual GCase enzymatic activity is found in these cases. Because currently available therapies are ineffective for neurological manifestations, a strong demand exists for elucidation of the pathological mechanisms and the development of novel therapies. Parkinson’s disease( PD) is the most common neurodegenerative movement disorder. GBA has recently drawn considerable attention because heterozygous mutations in this gene confer a high risk for sporadic PD[5, 6]. In addition, patients with type 1 GD also have an increased life-time risk of developing PD[7]. PD patients carrying GBA mutations show intraneuronal accumulation of alpha-synuclein( α-syn) called Lewy bodies and Lewy neurites, which are the pathological hallmarks of sporadic PD[3]. Several cellular, animal, and postmortem studies have indicated an association between GBA mutations and α-syn accumulation. For example, deficiency in GCase enzymatic activity causes lysosomal dysfunction and α-syn accumulation[8, 9, 10, 11, 12]. Increased α-syn in turn creates a vicious cycle by inhibiting the trafficking of GCase to lysosomes, thus leading to decreased GCase activity in lysosomes[9]. Consistent with this notion, mouse models overexpressing α-syn and postmortem tissue from patients with PD show reduced GCase activity in the brains[13, 14, 15]. Although several hypotheses have been proposed, further mechanisms of how GBA mutations contribute to the development of PD remain elusive. Medaka( Oryzias latipes) are a versatile vertebrate animal model for disease research. These fish are easy to handle, have a relatively short generation time( 2–3 months), produce a large number of progeny per generation, and have several inbred strains[16]. Importantly, medaka have an advantage as an animal model of PD due to endogenous α-syn in contrast to invertebrate models that lack α-syn. Moreover, several genetic manipulations can be performed in medaka in addition to established transgenic techniques[17, 18, 19, 20, 21]. So far, we have reported genetic PD models of medaka that develop locomotor dysfunction accompanied by the selective loss of dopaminergic and noradrenergic neurons[22, 23]. Considering these lines of evidence, medaka have the potential to be a new animal model of PD. Here, we generated GBA nonsense mutant medaka and found that homozygous GBA nonsense mutant( GBA-/-) medaka are a viable neuronopathic GD model. GBA-/- medaka developed remarkable α-syn accumulation in the brains and thus provide novel insights into the association of GBA mutations with α-syn accumulation. Furthermore, we revealed minimal contribution of endogenous α-syn to the pathogenesis of neuronopathic GD in medaka.
Discussion:
To date, several genetic animal models of neuronopathic GD including mouse, dog, and sheep have been reported[42]. Gba null mice die within 24 hours of birth due to permeability barrier defects in the skin[26, 43]. Thus, conditional knockout K14-lnl/lnl mice with normal GCase activity in their skin were generated[27]. However, use of these mice is limited because they survive only 2 weeks after birth. Mice harboring a Gba missense mutation combined with prosaposin- or saposin C-deficient mice, another neuronopathic GD model, also show neuronopathic abnormalities[29, 34] and have the advantage of longer survival than K14-lnl/lnl mice. However, the relevance of these mice to neuronopathic GD is controversial. The present study revealed that GBA-/- medaka survive long enough for pathological analysis of disease progression. As an example, GBA-/- medaka showed α-syn accumulation at 2 months, not at 1 month after fertilization. Considering these observations, GBA-/- medaka are useful as a viable neuronopathic GD model with endogenous α-syn accumulation. GBA-/- medaka showed several phenotypes different from those of mammalian neuronopathic GD model. Firstly, the skin of GBA-/- medaka looks intact whereas severe skin lesion is observed in patients with perinatal lethal type GD and Gba null mice[4, 43]. Secondly, GBA-/- medaka exhibited PAS-positive abnormal cells in spleen and kidney which presumably correspond to human Gaucher cells. In patients with GD, Gaucher cells are not found in kidney, but in liver, spleen and bone marrow. These differences could be explained by the fact that in adult teleost fish kidney has hematopoietic function instead of mammalian bone marrow[44]. Lastly, the proliferation of GFAP-positive radial glial cells was not observed in GBA-/- medaka whereas astrogliosis is observed in humans and mice with neuronopathic GD. It was reported that the reaction of GFAP-positive radial glial cells to inflammation is different from that of mammalian astrocytes[31]. Lysosomes play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their contents[45]. In general, lysosomal dysfunction results in defective digestion and accumulation of autophagic substrates such as polyubiquitinated proteins, p62, and dysfunctional mitochondria, accompanied by accumulation of autophagosomes. Axonal swellings containing autophagosome-like structures are observed in mouse models of lysosomal storage diseases such as neuronopathic GD mice, Niemann-pick type C1 mice, Cathepsin D-deficient mice, and Cathepsin B/L double-deficient mice[34, 46, 47]. A previous study reported that autophagosomes are formed in the distal part of the axon and undergo retrograde transport toward the cell body[48]. Another previous study demonstrated that inhibition of lysosomal function in primary neurons from mouse embryos disrupts the axonal transport of autophagosomes and causes their accumulation in axons[49]. Considering these lines of evidence, we propose that GCase deficiency primarily causes lysosomal dysfunction, leading to disrupted retrograde transport of autophagosomes in axons and formation of axonal swellings with α-syn accumulation( Fig. 6). Contrary to our expectations, p62-positive aggregates did not colocalize with LC3- and α-syn-positive axonal swellings, but were mainly located in the perikarya of neurons. These results suggest the possibility that presynaptic α-syn is transported proximally and degraded in a p62-independent autophagy-lysosome pathway. A previous study supports this hypothesis. Conditional knockout mice lacking ATG7 in TH-positive neurons show α-syn aggregates in swollen axons in the striatum, but not in cell bodies in the midbrain, indicating that autophagic dysfunction initially causes α-syn aggregates in the distal part of axons[50]. In addition, another previous study demonstrated that α-syn aggregation occurs earlier in axons than in neuronal cell bodies in the cardiac sympathetic nervous system in PD patients[51]. Because axonal retrograde transport may be involved in the degradative pathway of α-syn and may be a therapeutic target in PD, further studies are required to elucidate the precise mechanisms. Because GBA+/- medaka as old as 12 months did not show any apparent abnormal phenotypes, we could not directly investigate how heterozygous GBA mutations cause PD. Meanwhile, according to a recent report, induced pluripotent stem cell-derived neurons from PD patients carrying heterozygous GBA mutations show α-syn accumulation, an impaired autophagy-lysosome pathway, and dysregulation of calcium homeostasis[12]. The reason for differences in phenotypes between in vivo and in vitro models is unclear. However, our findings from GBA+/- medaka seem to be reasonable because the penetrance of PD in GBA mutation carriers is estimated to be at most 30% by the age of 80 years[52]. Thus, second hits such as environmental factors and other genetic factors are probably required for the development of PD pathology in vivo. Cellular and animal PD models overexpressing α-syn have provided evidence for the various potential toxic mechanisms of α-syn. However, few studies have demonstrated the pathological role of endogenous α-syn in vivo, which may reflect the authentic role for α-syn in PD. A previous study showed that α-syn null mice are resistant to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine( MPTP) toxicity compared with wild-type mice[53]. However, it is unclear whether this improvement is due to attenuation of α-syn toxicity or α-syn-mediated changes in the presynaptic machinery. Another previous study showed that the formation of intraneuronal inclusions and neurodegeneration in 26S proteasome-depleted mice is independent of α-syn[54]. In the present study, disruption of α-syn did not improve the life span, neuronal loss, or neuroinflammation in GBA-/- medaka. Moreover, α-syn was not involved in the accumulation of autophagosomes in axons. Our data indicate that α-syn accumulation is a downstream event, and other severe pathological factors may obscure the involvement of α-syn in the pathogenesis of neuronopathic GD in medaka. In summary, the present study showed that GBA-/- medaka are useful as a viable neuronopathic GD model with endogenous α-syn accumulation. Long-term survival of these fish allows us to observe the pathological progression. Our data revealed that GCase deficiency causes lysosomal dysfunction in neurons and α-syn accumulation in axonal swellings containing autophagosomes. Axonal transport of α-syn may play an important role in the mechanisms of GBA mutations leading to PD and may also be a therapeutic target in PD. Furthermore, we demonstrate the minimal contribution of α-syn to the pathogenesis of neuronopathic GD in medaka. GBA-/- medaka represent a valuable model for exploring the pathological mechanisms and also provide a new platform for developing treatments in PD with GBA mutations as well as neuronopathic GD.
Parkinson’s disease ( PD ) is a neurodegenerative disease characterized by intraneuronal accumulation of alpha-synuclein ( α-syn ) called Lewy bodies and Lewy neurites . Recent genetic studies have revealed that mutations in glucocerebrosidase ( GBA ) , a causative gene of Gaucher disease ( GD ) , are a strong risk for PD . However , its pathological mechanisms leading to PD remain largely unknown . Here , we generated GBA nonsense mutant ( GBA-/- ) medaka which survive long enough for pathological analysis of disease progression . These mutant medaka display not only the phenotypes resembling human neuronopathic GD but also axonal accumulation of α-syn accompanied by impairment of the autophagy-lysosome pathway . Furthermore , the present study demonstrates this α-syn accumulation has negligible contribution to the pathogenesis of neuronopathic GD in medaka . GBA-/- medaka represent a valuable model for exploring the pathological mechanisms of PD with GBA mutations as well as neuronopathic GD , and our findings have important implications for the association of GBA mutations with PD .