Kyoko Chiba

Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport.

APP associates with kinesin-1 via JIP1. In JIP1-decicient neurons, the fast velocity and high frequency of anterograde transport of APP cargo are impaired to reduced velocity and lower frequency, respectively. Interaction of JIP1 with KLC via two novel elements in JIP1 plays an important role in efficient APP axonal transport.

Simple and Direct Assembly of Kymographs from Movies Using KYMOMAKER

In tracking analysis, the movement of cargos by motor proteins in axons is often represented by a time‐space plot termed a ‘kymograph’. Manual creation of kymographs is time‐consuming and complicated for cell biologists. Therefore, we developed KYMOMAKER, a simple system that automatically creates a kymograph from a movie without generating multiple time‐dissected movie stacks. In addition, KYMOMAKER can automatically extract faint vesicle traces, and can thereby effectively analyze cargos expressed at low levels in axons. A filter can be applied to remove traces of non‐physiological movements and to extract meaningful traces of anterograde or retrograde cargo transport. For example, only cargos that move at a speed of >0.4 µm/second for a distance of >1 µm can be included. Another function of KYMOMAKER is to create a color kymograph in which the color of the trace varies according to the position of the fluorescent particle in the axis perpendicular to the long axis of the axon. Such positional information is completely lost in conventional kymographs. KYMOMAKER is an open access program that can be easily used to analyze vesicle transport in axons by cell biologists who do not have specific knowledge of bioimage informatics.

Facilitation of brain mitochondrial activity by 5-aminolevulinic acid in a mouse model of Alzheimer's disease.

The activities of mitochondrial enzymes, which are essential for neural function, decline with age and in age-related disease. In particular, the activity of cytochrome c oxidase (COX/complex IV) decreases in patients with Alzheimers disease (AD). COX, a mitochondrial inner membrane protein complex that contains heme, plays an essential role in the electron transport chain that generates ATP. Heme synthesis begins with 5-aminolevulinic acid (5-ALA) in mitochondria. 5-ALA synthetase is the rate-limiting enzyme in heme synthesis, suggesting that supplementation with 5-ALA might help preserve mitochondrial activity in the aged brain. We administered a diet containing 5-ALA to triple-transgenic AD (3xTg-AD) model mice for 6 months, starting at 3 months of age. COX activity and protein expression, as well as mitochondrial membrane potential, were significantly higher in brains of 5-ALA-fed mice than in controls. Synaptotagmin protein levels were also significantly higher in 5-ALA-fed mice, suggesting improved preservation of synapses. Although brain Aβ levels tended to decrease in 5-ALA-fed mice, we observed no other significant changes in other biochemical and pathological hallmarks of AD. Nevertheless, our study suggests that daily oral administration of 5-ALA could preserve mitochondrial enzyme activities in the brains of aged individuals, thereby contributing to the preservation of neural activity.

Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1

ETOC: Phosphorylation of KLC1 at Thr466 in kinesin-1 regulates the interaction with APP mediated by JIP1b. Substitution of Glu for Thr466 abolished this interaction and impaired the enhanced fast velocity of APP anterograde transport. This phosphorylation of KLC1 increased in aged brains, suggesting deficient APP transport in neurons after aging.

Phosphorylation of multiple sites within an acidic region of Alcadein α is required for kinesin-1 association and Golgi exit of Alcadein α cargo.

Alcadein a (Alca) is reported to function as a cargo receptor when associated with kinesin-1. Phosphorylation of three serine residues in the acidic region located between the two WD motifs of Alca is required for interaction with kinesin-1 and Golgi exit of Alca cargo.

The cytoplasmic region of the amyloid β‐protein precursor (APP) is necessary and sufficient for the enhanced fast velocity of APP transport by kinesin‐1

Amyloid β‐protein precursor (APP) is transported mainly by kinesin‐1 and at a higher velocity than other kinesin‐1 cargos, such as Alcadein α (Alcα); this is denoted by the enhanced fast velocity (EFV). Interaction of the APP cytoplasmic region with kinesin‐1, which is essential for EFV transport, is mediated by JNK‐interacting protein 1 (JIP1). To determine the roles of interactions between the APP luminal region and cargo components, we monitored transport of chimeric cargo receptors, Alcα (luminal)–APP (cytoplasmic) and APP (luminal)–Alcα (cytoplasmic). Alcα‐APP is transported at the EFV, whereas APP‐Alcα is transported at the same velocity as wild‐type Alcα. Thus, the cytoplasmic region of APP is necessary and sufficient for the EFV of APP transport by kinesin‐1.

REGULATION AND FUNCTION OF APP AS A CARGO-RECEPTOR OF KINESIN-1 IN NEURONS

Background: Type I membrane protein APP is the precursor of neurotoxic Ab peptides. Important function of APP in neuron, among many suggested functions, is a cargo-receptor of kinesin-1, which is the anterograde molecular motor first found in squid giant axon. Conventional kinesin-1 is composed of two KLC and two KHC molecules. APP associates with KLC via JNK-interacting protein 1/JIP1, and JIP1 plays an important role in the effective anterograde transport of APP in axon. Impaired APP transport by kinesin-1 increases Ab generation, however the mechanisms how APP associates with and dissociate from kinesin-1 remain unclear in detail. We analyze molecular mechanisms of APP transport by kinesin-1 in neuron. Methods: APPJIP1b-KLC interaction was analyzed biochemically. Transport of APP-EGFP in living mouse primary cultured neurons and differentiating CAD cells was observed with TIRF microscopy, and anterograde velocity and frequency of APP cargo were analyzed in detail. Results:Association of APP with kinesin-1 is largely regulated within the interaction between JIP1 and KLC. In the absence of JIP1 or deficiency of JIP1 function, the efficiency of APP anterograde transport was significantly impaired. The interaction between JIP1b and KLC1 was more complex than previously thought, and the conventional interaction of the JIP1b C11 region with TPR motifs of KLC alone is not sufficient to generate efficient transport of APP in neuron. The interaction between JIP1b and KLC1 was regulated by the phosphorylation of KLC1. Conclusions:Traffic jam of APP cargos is known to increase Ab generation, therefore, we reveled the regulatory interaction of APP with kinesin-1. APP showed an effective anterograde transport by kinesin-1 along with the enhanced fast velocity and high frequency in the presence of JIP1. Phosphorylation of KLC1 regulates the effective APP transport mediated by JIP1b, thus, alteration of protein phosphorylation system in AD neurons may impair the efficiency of APP axonal transport, inducing the increased Abgeneration. Araki et al., EMBO J. [2007] 26, 1475; Chiba et al., Mol. Biol. Cell [2014] 25, 3569; Chiba et al submitted: Sobu et al submitted; Shiraki et al., submitted.

Brain Aging as a Cause of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of senile dementia. Identification of genes causally associated with familial Alzheimer’s disease (FAD) advanced our understanding of the molecular mechanisms of AD pathogenesis. However, FAD is much less common than sporadic Alzheimer’s disease (SAD), which constitutes the majority of cases. Despite its similar pathology (albeit at a later age of onset), SAD is not linked to mutations in FAD-associated genes. In both FAD and SAD, the generation and oligomerization of amyloid β (Aβ) peptide play central roles in neurotoxicity, but it remains unclear how qualitative and quantitative alterations in Aβ occur in SAD patients in the absence of causative mutations. The predominant risk factor for SAD is aging, suggesting that some as-yet-unknown alterations in the aged brain augment the amyloidogenic metabolism of APP and promote the neural toxicity of Aβ oligomers. In this chapter, we discuss potential biochemical changes in amyloid β precursor protein (APP) and proteins related to APP metabolism and function in the aged brain. APP axonal transport, membrane microlocalization and metabolism, including generation of Aβ in neurons, are regulated by interactions with several cytoplasmic proteins and phosphorylation of the APP cytoplasmic region. Age-related decline or aberration in the regulation of APP transport, localization and metabolism may induce generation of altered Aβ. Here, we focus on APP phosphorylation at threonine 668 in the cytoplasmic domain and the roles of APP regulatory proteins, including X11-like (X11L), JIP1, kinesin-1, and Alcadein, on the regulation of APP metabolism and intracellular trafficking.