Sachi Okabayashi

Dynein dysfunction induces endocytic pathology accompanied by an increase in Rab GTPases: a potential mechanism underlying age-dependent endocytic dysfunction.

Growing evidence suggests that endocytic dysfunction is intimately involved in early stage Alzheimer disease pathology, such as the accumulation of β-amyloid precursor protein in enlarged early endosomes. However, it remains unclear how endocytic dysfunction is induced in an age-dependent manner. Cytoplasmic dynein, a microtubule-based motor protein, interacts with another microtubule-associated protein, dynactin. The resulting dynein-dynactin complex mediates minus end-directed vesicle transport, including endosome trafficking. We have previously shown that the interaction between dynein-dynactin complexes is clearly attenuated in aged monkey brains, suggesting that dynein-mediated transport dysfunction exists in aged brains. Our immunohistochemical analyses revealed that age-dependent endocytic pathology was accompanied by an increase in Rab GTPases in aged monkey brains. Here, we demonstrated that siRNA-induced dynein dysfunction reproduced the endocytic pathology accompanied by increased Rab GTPases seen in aged monkey brains and significantly disrupted exosome release. Moreover, it also resulted in endosomal β-amyloid precursor protein accumulation characterized by increased β-site cleavage. These findings suggest that dynein dysfunction may underlie age-dependent endocytic dysfunction via the up-regulation of Rab GTPases. In addition, this vicious circle may worsen endocytic dysfunction, ultimately leading to Alzheimer disease pathology.

Leucine-rich glioma inactivated 3 is involved in amyloid β peptide uptake by astrocytes and endocytosis itself

We earlier showed that leucine-rich glioma inactivated 3 (LGI3) colocalizes with amyloid &bgr; peptide (A&bgr;) taken up by astrocytes both in vitro and in vivo, and that LGI3 accumulated with endocytosis-associated proteins in aged monkey brains. In this study, we confirmed that LGI3 localizes to the endocytic pathway and found that its accumulation is caused by endocytic perturbation. Most notably, RNA interference experiments demonstrated that the downregulation of LGI3 clearly inhibited A&bgr; uptake by cultured rat astrocytes, moreover, transferrin uptake by both astrocytes and neuronal cells. Together with our earlier findings, our results suggest that LGI3 is involved in A&bgr; uptake by astrocytes and even endocytosis itself.

Long-term persistent GBV-B infection and development of a chronic and progressive hepatitis C-like disease in marmosets

It has been shown that infection of GB virus B (GBV-B), which is closely related to hepatitis C virus, develops acute self-resolving hepatitis in tamarins. In this study we sought to examine longitudinally the dynamics of viral and immunological status following GBV-B infection of marmosets and tamarins. Surprisingly, two of four marmosets but not tamarins experimentally challenged with GBV-B developed long-term chronic infection with fluctuated viremia, recurrent increase of alanine aminotransferase and plateaued titers of the antiviral antibodies, which was comparable to chronic hepatitis C in humans. Moreover, one of the chronically infected marmosets developed an acute exacerbation of chronic hepatitis as revealed by biochemical, histological, and immunopathological analyses. Of note, periodical analyses of the viral genomes in these marmosets indicated frequent and selective non-synonymous mutations, suggesting efficient evasion of the virus from antiviral immune pressure. These results demonstrated for the first time that GBV-B could induce chronic hepatitis C-like disease in marmosets and that the outcome of the viral infection and disease progression may depend on the differences between species and individuals.

Dynein Dysfunction Disrupts Intracellular Vesicle Trafficking Bidirectionally and Perturbs Synaptic Vesicle Docking via Endocytic Disturbances: A Potential Mechanism Underlying Age-Dependent Impairment of Cognitive Function

Although genetic studies have demonstrated that β-amyloid protein (Aβ) plays a pivotal role in Alzheimers disease (AD) pathogenesis, how aging contributes to AD onset remains unclear. Moreover, growing evidence suggests that Aβ-independent mechanisms, such as altered intracellular signaling cascades and impaired neurotransmitter release, also are likely involved in this process. Cytoplasmic dynein, a microtubule-based motor protein, mediates minus end-directed vesicle transport via interactions with dynactin, another microtubule-associated protein. We previously showed that normal aging attenuates the interaction between dynein-dynactin complexes in monkey brain and that dynein dysfunction reproduces age-dependent endocytic disturbances, resulting in intracellular Aβ accumulation. In this study, we report that dynein dysfunction disrupts not only retrograde transport of neurotrophic receptors but also anterograde transport of synaptic vesicles, which occurs concomitantly with an increase in Rab3 GTPase levels. Additionally, synaptic vesicle docking was perturbed via enhanced endocytosis. Dynein dysfunction also induced neuritic swelling, which is accompanied by a significant accumulation of neurofilaments. Moreover, we also confirmed that the dynein dysfunction-related disturbances are associated with aging in monkey brains and that age-dependent endocytic disturbances precede Aβ abnormality. These findings suggest that dynein dysfunction can alter neuronal activity via endocytic disturbances and may underlie age-dependent impairment of cognitive function. Moreover, in the presence of other risk factors, such as intracellular Aβ accumulation, dynein dysfunction may contribute to the development of AD.

Diabetes Mellitus Accelerates Aβ Pathology in Brain Accompanied by Enhanced GAβ Generation in Nonhuman Primates

Growing evidence suggests that diabetes mellitus (DM) is one of the strongest risk factors for developing Alzheimer’s disease (AD). However, it remains unclear why DM accelerates AD pathology. In cynomolgus monkeys older than 25 years, senile plaques (SPs) are spontaneously and consistently observed in their brains, and neurofibrillary tangles are present at 32 years of age and older. In laboratory-housed monkeys, obesity is occasionally observed and frequently leads to development of type 2 DM. In the present study, we performed histopathological and biochemical analyses of brain tissue in cynomolgus monkeys with type 2 DM to clarify the relationship between DM and AD pathology. Here, we provide the evidence that DM accelerates Aβ pathology in vivo in nonhuman primates who had not undergone any genetic manipulation. In DM-affected monkey brains, SPs were observed in frontal and temporal lobe cortices, even in monkeys younger than 20 years. Biochemical analyses of brain revealed that the amount of GM1-ganglioside-bound Aβ (GAβ)—the endogenous seed for Aβ fibril formation in the brain—was clearly elevated in DM-affected monkeys. Furthermore, the level of Rab GTPases was also significantly increased in the brains of adult monkeys with DM, almost to the same levels as in aged monkeys. Intraneuronal accumulation of enlarged endosomes was also observed in DM-affected monkeys, suggesting that exacerbated endocytic disturbance may underlie the acceleration of Aβ pathology due to DM.

Immunohistochemical and Biochemical Analyses of LGI3 in Monkey Brain: LGI3 Accumulates in Aged Monkey Brains

Leucine-rich glioma inactivated (LGI) 3 encodes a leucine-rich repeat protein. The precise function of LGI3, however, remains unknown. We have previously shown that amyloid-β peptide (Aβ) upregulates LGI3 and that Aβ and LGI3 colocalize on plasma membranes of cultured rat astrocytes. In the present study, we performed immunohistochemical and biochemical analyses of LGI3 using various aged monkey brains. Immunohistochemistry showed that LGI3 was present in almost all neural cells and mainly localized at plasma membranes and nuclei. In aged monkey brains, we found that LGI3 accumulated on or near the plasma membranes of neurons, and colocalized with endocytosis-associated proteins and lipid raft markers. Double immunohistochemistry also showed that LGI3 colocalized with Aβ in astrocytes of aged brains. Moreover, Western blot analyses revealed that LGI3 may be cleaved in brain. Additionally, in aged monkeys LGI3 accumulated in microsomal and nuclear brain fractions.

LGI3 interacts with flotillin-1 to mediate APP trafficking and exosome formation.

We recently showed that leucine-rich glioma inactivated 3 (LGI3) mediates the internalization of β-amyloid protein and transferrin, a well-known marker for clathrin-dependent endocytosis, in neural cells. These findings strongly suggest that LGI3 is involved in the endocytosis system in the brain; however, the precise function of LGI3 remains unclear. Here, we show that LGI3 interacts with flotillin-1 (Flo1), and RNA interference analysis shows that LGI3 stabilized Flo1, and Flo1 also stabilized LGI3 vice versa. Moreover, the downregulation of the LGI3/Flo1 complex altered β-amyloid precursor protein trafficking directly to late endosomes and disrupted exosome formation, suggesting that LGI3 is involved not only in endocytosis but also in another intracellular transport system through binding with its co-factor such as Flo1.

Transthyretin amyloidosis and two other aging-related amyloidoses in an aged vervet monkey.

An aged male vervet monkey showed severe cardiac arrhythmia for more than 3 years. A multifocal amyloid consisting of transthyretin was deposited in all areas of the heart wall, especially in the extracellular stroma among muscle fibers and external tunica of arterioles. Moreover, the amyloid was deposited in the stroma and arterioles of other systemic organs except the liver and spleen. These characteristics are consistent with senile systemic amyloidosis in humans. A second amyloid consisting of amyloid β protein was in senile plaques and cerebral amyloid angiopathy in the cerebral cortex. A third amyloid consisting of islet amyloid polypeptide was deposited in islets of the pancreas. Apolipoprotein E and amyloid P component colocalized with the 3 amyloids. Thus, 3 different aging-related amyloids were found in an aged vervet monkey. In particular, to our knowledge, this is the first report on spontaneous transthyretin amyloidosis in animals.

Dynein dysfunction disrupts β-amyloid clearance in astrocytes through endocytic disturbances.

We showed previously that aging attenuates the interaction between dynein–dynactin complexes in cynomolgus monkey brain and that dynein dysfunction reproduces age-dependent endocytic disturbances, resulting in intracellular &bgr;-amyloid (A&bgr;) accumulation, synaptic vesicle transport deficits, and neuritic swelling. It remains unclear whether such endocytic disturbances also occur in glial cells. Here, we show that endocytic pathology, such as intracellular accumulation of enlarged endosomes, occurs in astrocytes of aged monkey brains. Also, A&bgr; accumulates in these enlarged endosomes. RNA interference studies have shown that dynein dysfunction reproduces astroglial endocytic pathology and disrupts A&bgr; clearance in astrocytes through endocytic disturbances. These findings suggest that endocytic disturbances can alter astroglial functions and may also be involved in age-dependent A&bgr; pathology.

Emergence of infectious malignant thrombocytopenia in Japanese macaques ( Macaca fuscata ) by SRV-4 after transmission to a novel host

We discovered a lethal hemorrhagic syndrome arising from severe thrombocytopenia in Japanese macaques kept at the Primate Research Institute, Kyoto University. Extensive investigation identified that simian retrovirus type 4 (SRV-4) was the causative agent of the disease. SRV-4 had previously been isolated only from cynomolgus macaques in which it is usually asymptomatic. We consider that the SRV-4 crossed the so-called species barrier between cynomolgus and Japanese macaques, leading to extremely severe acute symptoms in the latter. Infectious agents that cross the species barrier occasionally amplify in virulence, which is not observed in the original hosts. In such cases, the new hosts are usually distantly related to the original hosts. However, Japanese macaques are closely related to cynomolgus macaques, and can even hybridize when given the opportunity. This lethal outbreak of a novel pathogen in Japanese macaques highlights the need to modify our expectations about virulence with regards crossing species barriers.