Fumiko Ono

Molecular composition of drusen and possible involvement of anti-retinal autoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca fascicularis)

We have previously reported a cynomolgus monkey (Macaca fascicularis) pedigree with early onset macular degeneration that develops drusen at 2 yr after birth (1). In this study, the molecular composition of drusen in monkeys affected with late onset and early onset macular degeneration was both characterized. Involvement of anti‐retinalautoimmunity in the deposition of drusen and the pathogenesis of the disease was also evaluated. Funduscopic and histological examinations were performed on 278 adult monkeys (mean age=16.94 yr) for late onset macular degeneration. The molecular composition of drusen was analyzed by immunohistochemistry and/or direct proteome analysis using liquid chromatography tandem mass spectroscopy (LC‐MS/MS). Anti‐retinal autoantibodies in sera were screened in 20 affected and 10 age‐matched control monkeys by Western blot techniques. Immunogenic molecules were identified by 2D electrophoresis and LC‐MS/MS. Relative antibody titer against each antigen was determined by ELISA in sera from 42 affected (late onset) and 41 normal monkeys. Yellowish‐white spots in the macular region were observed in 90 (32%) of the late onset monkeys that were examined. Histological examination demonstrated that drusen or degenerative retinal pigment epithelium (RPE) cells were associated with the pigmentary abnormalities. Drusen in both late and early onset monkeys showed immunoreactivities for apolipoprotein E, amyloid P component, complement component C5, the terminal C5b‐9 complement complex, vitronectin, and membrane cofactor protein. LC‐MS/MS analyses identified 60 proteins as constituents of drusen, including a number of common components in drusen of human age‐related macular degeneration (AMD), such as annexins, crystallins, immunoglobulins, and complement components. Half of the affected monkeys had single or multiple autoantibodies against 38, 40, 50, and 60 kDa retinal proteins. The reacting antigens of 38 and 40 kDa were identified as annexin II and μ‐crystallin, respectively. Relative antibody titer against annexin II in affected monkeys was significantly higher than control animals (P<0.01). Significant difference was not observed in antibody titer against μ‐crystallin; however, several affected monkeys showed considerably elevated titer (360–610%) compared with the mean for unaffected animals. Monkey drusen both in late and early onset forms of macular degeneration had common components with drusen in human AMD patients, indicating that chronic inflammation mediated by complement activation might also be involved in the formation of drusen in these affected monkeys. The high prevalence of anti‐retinalautoantibodies in sera from affected monkeys demonstrated an autoimmune aspect of the pathogenesis of the disease. Although further analyses are required to determine whether and how autoantibodies against annexin II or μ‐crystallin relate to the pathogenesis of the disease, it could be hypothesized that immune responses directed against these antigens might trigger chronic activation of the complement cascade at the site of drusen formation.

Histopathological studies of senile plaques and cerebral amyloidosis in cynomolgus monkeys

Senile plaques (SPs) and cerebral amyloid angiopathy (CAA), pathological hallmarks of Alzheimers disease, have not been thoroughly investigated histopathologically in nonhuman primates. To determine the onset age and histopathological characteristics of SPs and CAA, we examined the brains of 64 cynomolgus monkeys (Macaca fascicularis) from 2 to 35 years old. Mature (classical and primitive) plaques appeared in 16 out of 25 monkeys that were >20 years old. Moreover, mature plaques were observed more frequently than diffuse plaques and were located in the temporal cortex of the superior or inferior gyri and amygdala. Diffuse plaques in contrast to mature plaques did not show definite tendencies in onset age and distribution. CAA appeared in more than 22‐year‐old monkeys in 10 out of 16 animals and was frequently observed in capillaries and often found adjoining mature plaques. During immunohistochemical examination, an antiserum for amyloid β protein (Aβ) 1–40 could detect all SPs, whereas a monoclonal antibody for Aβ 8–17 could not detect any diffuse plaques and only one third of the primitive plaques. As for CAA, the polyclonal antiserum was more sensitive than the monoclonal antibody. The present study describes the histopathological features of SPs and CAA in old cynomolgus monkeys.

Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.

A Rice-Based Oral Cholera Vaccine Induces Macaque-Specific Systemic Neutralizing Antibodies but Does Not Influence Pre-Existing Intestinal Immunity

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates.

Multitracer assessment of dopamine function after transplantation of embryonic stem cell-derived neural stem cells in a primate model of Parkinson's disease

The ability of primate embryonic stem (ES) cells to differentiate into dopamine (DA)‐synthesizing neurons has raised hopes of creating novel cell therapies for Parkinsons disease (PD). As the primary purpose of cell transplantation in PD is restoration of dopaminergic neurotransmission in the striatum, in vivo assessment of DA function after grafting is necessary to achieve better therapeutic effects. A chronic model of PD was produced in two cynomolgus monkeys (M‐1 and M‐2) by systemic administration of neurotoxin. Neural stem cells (NSCs) derived from cynomolgus ES cells were implanted unilaterally in the putamen. To evaluate DA‐specific functions, we used multiple [11C]‐labeled positron emission tomography (PET) tracers, including [β‐11C]L‐3,4‐dihydroxyphenylalanine (L‐[β‐11C]DOPA, DA precursor ligand), [11C]‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane ([11C]β‐CFT, DA transporter ligand) and [11C]raclopride (D2 receptor ligand). At 12 weeks after grafting NSCs, PET demonstrated significantly increased uptake of L‐[β‐11C]DOPA (M‐1:41%, M‐2:61%) and [11C]β‐CFT (M‐1:31%, M‐2:36%) uptake in the grafted putamen. In addition, methamphetamine challenge in M‐2 induced reduced [11C]raclopride binding (16%) in the transplanted putamen, suggesting release of DA. These results show that transplantation of NSCs derived from cynomolgus monkey ES cells can restore DA function in the putamen of a primate model of PD. PET with multitracers is useful for functional studies in developing cell‐based therapies against PD. Synapse 63: 541‐548, 2009.

Aging attenuates dynactin-dynein interaction: down-regulation of dynein causes accumulation of endogenous tau and amyloid precursor protein in human neuroblastoma cells.

Impaired axonal transport may promote pathogenesis in neurodegenerative disorders, such as Alzheimers disease (AD). We previously showed that tau, amyloid precursor protein (APP), and intracellular amyloid β‐protein (Aβ) accumulate in the nerve‐ending fraction of aged monkey brains, perhaps because of impaired axonal transport. In the present study, we assessed age‐related changes of axonal transport motor proteins in aged monkey brains. Western blotting showed that kinesin, dynein, and dynactin (DYN) localizations dramatically changed with aging, and dynein level in nerve‐ending fractions increased significantly. Coimmunoprecipitation analyses showed that DYN–dynein intermediate chain (DIC) interactions decreased, suggesting that age‐related attenuation of this interaction may cause the impairment of dynein function. Moreover, RNAi‐induced down‐regulation of DIC in human neuroblastoma cells caused endogenous tau and APP to accumulate, and their subcellular localizations were also affected. Our findings suggest that aging attenuates DYN–DIC interaction, representing one of the risk factors for age‐related impaired dynein function and even for accumulation of disease proteins.

Carboxyl end-specific monoclonal antibodies to amyloid β protein (Aβ) subtypes (Aβ40 and Aβ42(43)) differentiate Aβ in senile plaques and amyloid angiopathy in brains of aged cynomolgus monkeys

Abstract Senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in the brains of five aged (20–26 years old) cynomolgus monkeys were investigated immunohistochemically using two monoclonal antibodies (anti-Aβ 40 (BA27) and anti-Aβ 42(43) (BC05)) that can differentiate the carboxyl termini of amyloid β protein (Aβ) subtypes. In four of five animals, all types of SPs (i.e. diffuse, primitive, and classical plaques; DPs, PPs, and CPs, respectively) were identified by BC05. However, BA27 did not label DPs and stained only about one third of PPs and CPs, mainly labeling granular structures and cored portions, respectively. In CAA, lesions of cortical capillaries reacted to BC05 in four of five cases, but rarely and weakly to BA27 in two of five cases. On the other hand, lesions of parenchymal and meningeal arterioles were stained by both BA27 and BC05. These staining profiles of SPs in cynomolgus monkeys correspond well to those in humans, although there are two remarkable features in cynomolgus monkeys. First, BA27 stained PPs associated with granular structures. Secondly, capillary Aβ reacted intensely to BC05 but only slightly to BA27. Despite these unique features, the results suggest that aged cynomolgus monkeys can be used to investigate the pathogenesis of Aβ deposition in SPs and CAA.

White matter activated glial cells produce BDNF in a stroke model of monkeys.

Lacunar-type stroke accounts for approximately a quarter of all ischemic strokes, and is the most common cause of vascular dementia. Despite its importance, there are few specific treatments for lacunar stroke, probably due largely to a lack of animal models. In this study, we developed a stroke model in a higher primate, the Macaque monkey. This was achieved by occluding the deep subcortical penetrating arteries with agarose spheres of mean diameters around 50 microm, and the appropriateness of this model as a lacunar-type stroke was verified by MRI. We observed widespread gliosis in the ipsilateral white matter (WM) of the stroke monkey. We also analyzed the expression of neurotrophins in the activated glial cells, and found that their expression of BDNF was stimulated in the affected WM following ischemic injury. Our results support the idea that WM glial cells play an active role in protecting and promoting the regeneration of nerve fibers in the affected WM of the ischemic brain, by producing BDNF. These findings may be useful for the development of new therapeutic strategies aimed at preventing or treating stroke.

DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field.

Minimizing the inhibitory effect of neutralizing antibody for efficient gene expression in the liver with adeno-associated virus 8 vectors.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.