Review: A model for seed-dependent intracellular accumulation of α-synuclein
Dr.Takashi Nonaka , Dementia Project,
Tokyo Metropolitan Institute of Medical Science
Introduction
New hypotheses have been proposed regarding the formation mechanism of intracellular abnormal protein aggregates that appear in the brains of patients with neurodegenerative diseases. In other words, intracellular aggregates due to abnormal accumulation of tau and Parkinson's disease (PD) in Alzheimer's disease (AD) and α-synuclein in dementia with Lewy bodies have the same properties as abnormal prion proteins in prion diseases, It has been suggested that it propagates between cells and functions as a seed for aggregate formation within the cells to which it propagates. This "cell-to-cell propagation of aggregated proteins" may partially explain the phenomenon in which abnormal lesions expand as the disease progresses, and is one of the hottest topics both in Japan and overseas. be. Here, we introduce our research results on intracellular accumulation models of α-synuclein using cultured cells and mice.
1. Alpha-Synuclein
In 1997, genetic analysis of a familial PD family in southern Italy identified α-synuclein as the causative gene (1) . Alpha-synuclein has been identified (2,3) . Alpha-synuclein is a polypeptide consisting of 140 amino acids and a soluble protein abundant in the brain, but its function is not very clear. In the patient's brain, α-synuclein is phosphorylated and ubiquitinated to accumulate and fibrillate, and the site of appearance of Lewy bodies is well consistent with the site of neuronal loss. Therefore, a mechanism is assumed in which accumulation of α-synuclein in cells causes some cytotoxicity, which in turn induces neuronal cell death, ultimately leading to onset.
Regarding the spread of Lewy body lesions in sporadic PD, Braak et al. proposed the hypothesis that the appearance of Lewy bodies originates in the dorsal motor nucleus of the vagus nerve and gradually spreads upward along the brainstem . 4,5) . On the other hand, Saito and Murayama et al. found a pathway that Lewy body pathology spreads from the olfactory bulb limbus to the preolfactory sense and then to the amygdala (6) . In addition, the possibility of propagation of Lewy body lesions in the brains of actual patients was first shown by analyzes of PD patient brains in which fetal mesencephalic neurons were transplanted. reportedly found (7,8) . These results suggest that α-synuclein aggregates present in host tissues from PD patients are transmitted to the neurons of the graft, leading to seed-dependent α-synuclein accumulation in the transplanted neurons, which should not have Lewy bodies. could have occurred.
2. Seed-dependent accumulation model of α-synuclein using cultured cells
In this way, it was suggested that α-synuclein accumulates in patient brains in a seed-dependent manner. was
It has already been shown that α-synuclein aggregates in vitro in a seed-dependent manner. That is, even if a solution of recombinant α-synuclein monomer is allowed to stand at 37° C., formation of amyloid fibrils hardly occurs. Formation progresses at an accelerated rate (9) . On the other hand, even if α-synuclein is transiently expressed in cultured cells, intracellular accumulation does not occur simply by expressing it. We tried to recreate seed-dependent α-synuclein accumulation in cultured cells, and succeeded in constructing it in 2005 (10,11) . That is, when cells in which an α-synuclein plasmid was transiently expressed in advance were treated with in vitro -prepared recombinant α-synuclein amyloid fibrils together with a plasmid transfection reagent, the extracellularly introduced fibrils were used as seeds for intracellular transfection. soluble α-synuclein aggregated in the cytoplasm in a seed-dependent manner. These aggregates also have the characteristics of accumulated α-synuclein, such as phosphorylation and ubiquitination, and are considered to be a cell model that reproduces the Lewy bodies seen in patient brains (Fig. 1). This method of introducing protein fibrils using this transfection reagent is highly applicable, and not only can fibrils and aggregates composed of recombinant proteins such as α-synuclein and tau be introduced into cells, but they can also be accumulated in actual patient brains. It is also possible to introduce protein fractions (detergent-insoluble fractions) into cells.
Furthermore, in this model cell, it was confirmed that necrosis-like cell death occurred 4 to 5 days after the formation of aggregates. In addition, proteasome activity is suppressed in cells with intracellular α-synuclein accumulation, suggesting that there is some relationship between this suppression and cell death induction (11) .
Figure 1. Cultured cell model for seed-dependent intracellular α-synuclein accumulation
A: Untreated cells
B: Cells transiently expressing α-synuclein (aS) plasmid
C: α-synuclein fibrils (seed) transfected with lipofectamine Cells treated with Lipofectamine D: cells
pre-expressed transiently with plasmid and cells treated with Lipofectamine for seeds
All samples were immunostained with a phosphorylated α-synuclein-specific antibody (green), and the nucleus was stained with TO-PRO-3 (blue). Since α-synuclein is hardly phosphorylated when it is only transiently expressed in cells, almost nothing is stained in (A). In addition, in (C), the introduced seeds were phosphorylated in cells and stained with dots. When seeds were treated in cells that had previously expressed an α-synuclein plasmid (D), a large number of intracellular aggregates (arrows) positive for phosphorylated α-synuclein antibodies were observed. Both are approximately 10 µm in diameter. Scale bar is 20 µm. Extracted and modified from Reference 11.
3. α-synuclein accumulation model using mice
It is easily conceivable that this method of introducing insolubilized proteins as seeds into cells can be applied not only to cultured cells but also to experimental animals. Therefore, we injected wild-type mouse brains with recombinant human α-synuclein monomers and fibrils or mouse α-synuclein fibrils as seeds, and examined whether α-synuclein accumulates in a seed-dependent manner in mouse brains. Immunohistochemical analysis of mouse brains taken 15 months after seed inoculation revealed that the accumulation and spread of aberrantly phosphorylated α-synuclein in the seed-inoculated group was observed not only in the inoculated side but also in the opposite direction. We found that it was also induced in the lateral side (Fig. 2) (12) . Biochemical analysis revealed that endogenous mouse α-synuclein accumulated after phosphorylation and ubiquitination, rather than inoculated human α-synuclein fibers accumulating in the mouse brain. This result indicates that α-synuclein accumulates and aggregates in a seed-dependent manner also in the mouse brain. Furthermore, in the group inoculated with mouse α-synuclein fibers as a seed, the incidence of pathology of α-synuclein accumulation and the frequency of propagation to the non-inoculated side were higher than in the group inoculated with human fibers. It can be said that a wall exists. On the other hand, no accumulation or propagation of α-synuclein was observed in the monomer-inoculated group. Furthermore, we found that the site of abnormal pathology changes depending on the site of α-synuclein fiber inoculation, that is, the pathology propagates to the site of inoculation and nerve communication, suggesting that part of the α-synuclein fiber is transmitted through synapses. demonstrated the potential for cell-to-cell propagation (13) .
Conclusion
In recent years, not only our group but also other research groups have suggested that abnormal proteins that accumulate in the brains of patients with neurodegenerative diseases may propagate between neurons like abnormal prion proteins. The possibility of this abnormal protein dissemination may explain the regular spread of abnormal lesions in AD and PD to some extent. It goes without saying that it is sexual. So far, it seems that basic research has been carried out with the aim of suppressing the "initial accumulation" of abnormal proteins in the central nervous system in therapeutic strategies for neurodegenerative diseases. Considering the nature of the treatment, it is not necessarily limited to suppressing the initial aggregate formation step, but suppressing the subsequent step in which aggregates propagate between cells may also be a therapeutic target.
Figure 2. Sites of abnormal pathology in the brain of mice inoculated with human α-synuclein fibers Human α-synuclein fibers were inoculated into the
substantia nigra, and 15 months later, the brain was excised and immunostained with an anti-phosphorylated α-synuclein antibody. . As a result, the spread of the pathology was observed not only around the substantia nigra of the inoculation site, but also in other areas such as the cortex and the left hemisphere on the non-inoculation side. Extracted and modified from reference 12.
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