measuring-axonal-cargo-transport-mouse-primary-cortical-cultured

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient ...

Hokkaido University

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo.

Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid β-protein precursor (APP) transport into the late secretory pathway and facilitates APP cleavage.

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 Alzheimer's 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.

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

Alcadein α (Alcα) is a major cargo of kinesin-1 that is subjected to anterograde transport in neuronal axons. Two tryptophan- and aspartic acid-containing (WD) motifs located in its cytoplasmic domain directly bind the tetratricopeptide repeat (TPR) motifs of the kinesin light chain (KLC), which activate kinesin-1 and recruit kinesin-1 to Alcα cargo. We found that phosphorylation of three serine residues in the acidic region located between the two WD motifs is required for interaction with KLC. Phosphorylation of these serine residues may alter the disordered structure of the acidic region to induce direct association with KLC. Replacement of these serines with Ala results in a mutant that is unable to bind kinesin-1, which impairs exit of Alcα cargo from the Golgi. Despite this deficiency, the compromised Alcα mutant was still transported, albeit improperly by vesicles following missorting of the Alcα mutant with amyloid β-protein precursor (APP) cargo. This suggests that APP partially compensates for defective Alcα in anterograde transport by providing an alternative cargo receptor for kinesin-1.

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

In neurons, amyloid β-protein precursor (APP) is transported by binding to kinesin-1, mediated by JNK-interacting protein 1b (JIP1b), which generates the enhanced fast velocity (EFV) and efficient high frequency (EHF) of APP anterograde transport. Previously, we showed that EFV requires conventional interaction between the JIP1b C-terminal region and the kinesin light chain 1 (KLC1) tetratricopeptide repeat, whereas EHF requires a novel interaction between the central region of JIP1b and the coiled-coil domain of KLC1. We found that phosphorylatable Thr466 of KLC1 regulates the conventional interaction with JIP1b. Substitution of Glu for Thr466 abolished this interaction and EFV, but did not impair the novel interaction responsible for EHF. Phosphorylation of KLC1 at Thr466 increased in aged brains, and JIP1 binding to kinesin-1 decreased, suggesting that APP transport is impaired by aging. We conclude that phosphorylation of KLC1 at Thr466 regulates the velocity of transport of APP by kinesin-1 by modulating its interaction with JIP1b.

Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes 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.

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.

X11/Mint 1 and X11-like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11-, X11L-, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non-PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro-localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild-type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

X11 and X11-like proteins regulate the level of extrasynaptic glutamate receptors.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-β (Aβ) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aβ in mouse neurons. CLA treatment did not affect the level of β-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aβ protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aβ generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aβ in neurons.

Suppression of amyloid-β secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

γ-Secretase cleaves type I transmembrane proteins in a hydrophobic membrane environment following ectodomain shedding. Mutations in PSEN genes, encoding the catalytic subunits of γ-secretase, presenilins, are the most common cause of familial Alzheimer's disease (ad). Pathogenic mutations in PSEN genes increase production of longer and neurotoxic amyloid-β (Aβ) by intramembrane cleavage of membrane-associated amyloid-β protein precursor (APP) carboxyl-terminal fragment β, which is generated via primary cleavage of APP by β-site APP cleaving enzyme 1. The longer Aβ is prone to aggregate and accumulate in the brain; however, the accumulation of Aβ in brain is also a pathological feature of sporadic ad. Increased pathogenic Aβ generation, even in the absence of pathogenic PSEN gene mutations, is one of proposed mechanisms for sporadic ad pathogenesis. γ-Secretase digests substrates in the transmembrane region, generating Aβ peptide intermediates of various lengths. The end products, shorter Aβ40 and Aβ38 peptides, are less neurotoxic, whereas PSEN gene mutations increase the production ratio of longer, neurotoxic Aβ species such as Aβ42, an intermediate in Aβ38 production. γ-Secretase activity or structures is altered because of its aberrant membrane localization or changes in the ambient environment such as luminal acidification. Interestingly, γ-secretase has a pH sensor in presenilins.

γ-Secretase structure and activity are modified by alterations in its membrane localization and ambient environment.

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease (AD). Here, we investigated brain amyloid-β (Aβ) accumulation throughout the aging process in an amyloid precursor protein (APP) knock-in (KI) mouse model of AD that expresses human APP with or without human apoE4 or apoE3. Brain Aβ42 levels were significantly lower in 9-month-old mice that express human isoforms of apoE than in age-matched APP-KI control mice. Linear accumulation of Aβ42 began in 5-month-old apoE4 mice, and a strong increase in Aβ42 levels was observed in 21-month-old apoE3 mice. Aβ42 levels in cerebroventricular fluid were higher in apoE3 than in apoE4 mice at 6-7 months of age, suggesting that apoE3 is more efficient at clearing Aβ42 than apoE4 at these ages. However, apoE3 protein levels were lower than apoE4 protein levels in the brains of 21-month-old apoE3 and apoE4 mice, respectively, which may explain the rapid increase in brain Aβ42 burden in apoE3 mice. We identified genes that were downregulated in a human apoE-dependent (apoE4 > apoE3) and age-dependent (apoE3 = apoE4) manner, which may regulate brain Aβ burden and/or AD progression. Analysis of gene expression in AD mouse models helps identify molecular mechanisms of pleiotropy by the human APOE gene during aging.

Accumulation of amyloid-β in the brain of mouse models of Alzheimer's disease is modified by altered gene expression in the presence of human apoE isoforms during aging.

Article	Year
Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid β-protein precursor (APP) transport into the late secretory pathway and facilitates APP c... The Journal of biological chemistry\| PubMed ID: 25406318	2015
Facilitation of brain mitochondrial activity by 5-aminolevulinic acid in a mouse model of Alzheimer's disease. Nutritional neuroscience\| PubMed ID: 27329428	2017
Phosphorylation of multiple sites within an acidic region of Alcadein α is required for kinesin-1 association and Golgi exit of Alcadein α cargo. Molecular biology of the cell\| PubMed ID: 29093024	2017
Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1. Molecular biology of the cell\| PubMed ID: 29093025	2017
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. FEBS letters\| PubMed ID: 30055048	2018
X11 and X11-like proteins regulate the level of extrasynaptic glutamate receptors. Journal of neurochemistry\| PubMed ID: 30411795	2019
Suppression of amyloid-β secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid. Journal of neurochemistry\| PubMed ID: 34379812	2021
γ-Secretase structure and activity are modified by alterations in its membrane localization and ambient environment. Journal of biochemistry\| PubMed ID: 34865063	2022
Accumulation of amyloid-β in the brain of mouse models of Alzheimer's disease is modified by altered gene expression in the presence of human apoE isoforms during aging. Neurobiology of aging\| PubMed ID: 36638682	2023

Yuriko Sobu

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