Propagation of pathological ?-synuclein in marmoset brain

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease, and Lewy bodies (LBs) and Lewy neurites (LNs) are characteristic features of PD. Dementia with Lewy bodies (DLB) is also a progressive neurodegenerative disease characterized by the appearance of LBs and LNs in cortex [17, 22]. The discovery of disease-associated mutation in the ?-synuclein gene SNCA and subsequent immunostaining studies with antibodies demonstrated that ?-synuclein is the major component of LBs and LNs [2, 55, 56]. It is also the major component of glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA) [54, 58]. These diseases are collectively referred to as ?-synucleinopathies. To date, six missense mutations in the SNCA gene and occurrence of gene multiplication have been identified in familial forms of PD and DLB [1, 5, 24, 28, 29, 41, 52, 62]. ?-Synuclein is a small protein of 140 amino acids, which is localized in presynaptic termini, and is involved in maintenance of synapses and synaptic plasticity. In PD, DLB, or MSA patients, it is deposited in the brain as a filamentous form with cross-? structure [51], which is abnormally phosphorylated at Ser129 and partially ubiquitinated [15, 21]. ?-Synuclein is natively unfolded, but readily assembles into amyloid-like fibrils under appropriate conditions. Pathogenic mutations affect fibril formation in vitro, either accelerating fibril formation [6, 7, 16] or resulting in formation of fibrils that are more fragile and easier to propagate than wild-type (WT) fibrils [61]. Moreover, the spreading of pathological ?-synuclein is closely correlated with disease progression; indeed, the distribution pattern and spread of the pathologies are useful for disease staging of sporadic PD [3, 48]. These results suggest that intracellular amyloid-like ?-synuclein fibrils can cause PD and DLB, and spreading of ?-synuclein pathology in the brain is considered to be the underlying mechanism of progression of these diseases. Recently, it was experimentally demonstrated that intracerebral injection of synthetic ?-synuclein fibrils and/or insoluble ?-synuclein from diseased brain converts normal ?-synuclein into an abnormal form, and the abnormal ?-synuclein propagates throughout the brain in a prion-like manner in WT mouse [30, 33, 34, 57], ?-synuclein transgenic mouse [31, 36, 60] and monkey [44].

Common marmoset (Callithrix jacchus) is a very small new world primate, about 25?–?35 cm in height and 300?–?500 g in weight, and is far more experimentally tractable than macaque monkey. Since it has high fecundity, with a short sexual maturation period of 18 months, it is attracting increasing attention as an experimental model of primates. In fact, a national project called Brain/MINDS (Brain Mapping by Integrated Neurotechnologies for Disease Studies) was started in 2014 in Japan to develop the common marmoset as a model animal for neuroscience [19, 38, 39]. The marmoset cortex is relatively smooth, but the gyrencephalic and cortical sheet is divided into functionally distinct cortical areas, as in Old World monkeys [45], and thus is suitable for studies of higher cognitive functions and social communication [11]. Therefore, marmosets are considered to be a good experimental model animal to understand the evolution of brain development and function. Moreover, transgenic marmosets have already been generated, demonstrating the feasibility of gene manipulation in this species [49].

To date, mouse models have been used to investigate brain development, circuits, and higher cognitive functions, but they have limitations for exploration of the evolution and development of the primate neocortex. In situ hybridization analysis of marmoset brain revealed that the expression patterns of the genes that regulate brain development (such as EphA6) are different, especially in brain areas that have connections to the prefrontal cortex and are presumably involved in higher cognitive functions, although similar broad regional patterns of expression were observed in both species [32].

A particular difference in brain development and structure between mouse and marmoset is that striatum of marmoset is separated into caudate nucleus and putamen, while these are not distinguishable in rodents. It has been considered that caudate nucleus and putamen were originally one structure and that they became separated by the internal capsule during evolution [25]. Thus, the marmoset has advantageous characteristics as an experimental animal to study brain networks, functions and disease conditions.

Here, we investigated whether intracerebral injection of ?-synuclein fibrils can induce PD/DLB-like pathologies in marmoset, and we present the first marmoset model of ?-synuclein propagation. We found that marmosets developed abundant phosphorylated ?-synuclein pathologies, similar to those observed in PD/DLB, in various brain regions, including striatum, cortex and substantia nigra, at only three months after injection. Remarkably, many LB-like inclusions are observed in tyrosine hydroxylase (TH)-positive dopamine neurons, and a significant decrease in TH-staining was seen in the injection hemisphere. The inclusions were also positive for fluorescent ?-sheet ligands, thioflavin-S and FSB, implying that ?-synuclein deposits in these animals should be detectable in vivo by positron emission tomography (PET) with a suitable small-molecular agent. Taking account of the advantages of marmosets over mice, we believe the current experimental model would be particularly useful to examine the relationships between PET-detectable ?-synuclein lesions and disruptions of neural networks in the absence and presence of candidate ?-synucleinopathy-modifying therapeutics.