Superparamagnetic Iron Oxide-enhanced Magnetic Resonance Imaging of Spontaneous Hepatic Neoplasia in a Cynomolgus Macaque (Macaca fascicularis).

Although the number of reports describing tumors in aged NHP has increased, spontaneous neoplasias in NHP are extremely rare, with the notable exception of prosimians, in which spontaneous hepatic neoplasms arise. In addition to radiography and ultrasonography, superparamagnetic iron oxide (SPIO)-enhanced MRI tends to be applied in human practice to non-invasively locate, identify, and size liver tumors and to define the border between neoplastic and normal tissues. Here we report a 13-y-old female cynomolgus monkey with anorexia and serologically normal liver enzymes. After fluid therapy, the condition remained in remission for several months. Later, however, a palpable mass was assessed by using ultrasonography, radiology, and SPIO-MRI; T2-weighted images revealed a clear border between a hepatocellular carcinoma and normal liver tissue. Findings at necropsy supported the imaging data. Serologic assessment after euthanasia revealed a positive reaction to an abnormal form of prothrombin (PIVKA-II). We recommend SPIO-MRI as a practical and useful for diagnosing hepatocellular neoplasias in NHP. This study is the first to demonstrate the applicability of SPIO-MRI for the identification of hepatocellular carcinoma in NHP.

Dynein Dysfunction Reproduces Age-Dependent Retromer Deficiency: Concomitant Disruption of Retrograde Trafficking Is Required for Alteration in ß-Amyloid Precursor Protein Metabolism.

It is widely accepted that ß-amyloid (Aß) protein plays a pivotal role in Alzheimer disease pathogenesis, and accumulating evidence suggests that endocytic dysfunction is involved in Aß pathology. Retromer, a conserved multisubunit complex, mediates the retrograde transport of numerous kinds of cargo from endosomes to the trans-Golgi network. Several studies have found that retromer deficiency enhances Aß pathology both in vitro and in vivo. Cytoplasmic dynein, a microtubule-based motor protein, mediates minus-end-directed vesicle transport via interactions with dynactin, another microtubule-associated protein that also interacts with retromer. Aging attenuates the dynein-dynactin interaction, and dynein dysfunction reproduces age-dependent endocytic disturbance, resulting in the intracellular accumulation of beta-amyloid precursor protein (APP) and its ß-cleavage products, including Aß. Here, we report that aging itself affects retromer trafficking in cynomolgus monkey brains. In addition, dynein dysfunction reproduces this type of age-dependent retromer deficiency (ie, the endosomal accumulation of retromer-related proteins and APP. Moreover, we found that knockdown of Rab7, Rab9, or Rab11 did not alter endogenous APP metabolism, such as that observed in aged monkey brains and in dynein-depleted cells. These findings suggest that dynein dysfunction can cause retromer deficiency and that concomitant disruption of retrograde trafficking may be the key factor underlying age-dependent Aß pathology.

Tracking cells implanted into cynomolgus monkeys (Macaca fascicularis) using MRI.

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger's disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy.

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.

Diabetes mellitus accelerates Aß pathology in brain accompanied by enhanced GAß 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 Aß 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 Aß (GAß)--the endogenous seed for Aß 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 Aß pathology due to DM.

Dynein dysfunction disrupts ß-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 ß-amyloid (Aß) 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, Aß accumulates in these enlarged endosomes. RNA interference studies have shown that dynein dysfunction reproduces astroglial endocytic pathology and disrupts Aß clearance in astrocytes through endocytic disturbances. These findings suggest that endocytic disturbances can alter astroglial functions and may also be involved in age-dependent Aß pathology.

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 ß-amyloid protein (Aß) plays a pivotal role in Alzheimer's disease (AD) pathogenesis, how aging contributes to AD onset remains unclear. Moreover, growing evidence suggests that Aß-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 Aß 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 Aß 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 Aß accumulation, dynein dysfunction may contribute to the development of AD.

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 beta-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 beta-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.

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 beta-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 beta-amyloid precursor protein accumulation characterized by increased beta-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 beta peptide uptake by astrocytes and endocytosis itself.

We earlier showed that leucine-rich glioma inactivated 3 (LGI3) colocalizes with amyloid beta peptide (Abeta) 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 Abeta 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 Abeta uptake by astrocytes and even endocytosis itself.

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-beta peptide (Abeta) upregulates LGI3 and that Abeta 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 Abeta 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.

Pterygodermatites nycticebi (Nematoda: Rictulariidae): accidental detection of encapsulated third-stage larvae in the tissue of a white-fronted marmoset.

Twin, white-fronted marmosets (Callithrix geoffroyi) born and raised in a zoo in Japan died at 7 mo of age. Several encapsulated nematode larvae were detected in the intestinal wall, as well as a few in the mesenteric lymph nodes of 1 of the twins. In the other marmoset, no encapsulated nematode larva was detected in the organs, but many adult Pterygodermatites nycticebi were found in the intestinal lumen. In the past 5 yr, 5 primates kept in the same zoo, i.e., 1 squirrel monkey (Saimiri sciureus), 2 Pygmy marmosets (Cebuella pygmaea), 1 Senegal galago (Galago senegalensis), and 1 cotton-top tamarin (Saguinus oedipus), died from heavy infestation with the same nematode. A few migrating larvae of the rictulariid were also identified histologically in the intestinal wall and liver of the cotton-top tamarin. Although no other primate currently held in the same zoo was infected with the rictulariid, German cockroaches (Blattella germanica) collected with traps near marmoset cages had encapsulated P. nycticebi larvae, indicating latent perpetuation of the life cycle of this rictulariid species in the zoo premises. Our results indicated that encapsulation or migration of third-stage larvae of P. nycticebi might occur accidentally in the organs of callithrichid primates.