Koichi Iijima

Neuron‐Specific Phosphorylation of Alzheimer's β‐Amyloid Precursor Protein by Cyclin‐Dependent Kinase 5

Abstract: The mature form of Alzheimers β‐amyloid precursor protein (APP) is phosphorylated specifically at Thr668 in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin‐dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr668. Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense‐treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.

Aβ42 Mutants with Different Aggregation Profiles Induce Distinct Pathologies in Drosophila

Aggregation of the amyloid-β-42 (Aβ42) peptide in the brain parenchyma is a pathological hallmark of Alzheimers disease (AD), and the prevention of Aβ aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Aβ can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Aβ42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Aβ42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Aβ42Arc) and an artificial mutation (Aβ42art) that is known to suppress aggregation and toxicity of Aβ42 in vitro. In the Drosophila brain, Aβ42Arc formed more oligomers and deposits than did wild type Aβ42, while Aβ42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Aβ peptides. Surprisingly, however, Aβ42art caused earlier onset of memory defects than Aβ42. More remarkably, each Aβ induced qualitatively different pathologies. Aβ42Arc caused greater neuron loss than did Aβ42, while Aβ42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Aβ aggregates: Aβ42Arc formed large deposits in the cell body, Aβ42art accumulated preferentially in the neurites, while Aβ42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Aβ42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.

JNK/FOXO-mediated Neuronal Expression of Fly Homologue of Peroxiredoxin II Reduces Oxidative Stress and Extends Life Span

Activation of c-Jun N-terminal kinase (JNK) signaling in neurons increases stress resistance and extends life span, in part through FOXO-mediated transcription in Drosophila. However, the JNK/FOXO target genes are unknown. Here, we identified Jafrac1, a Drosophila homolog of human Peroxiredoxin II (hPrxII), as a downstream effecter of JNK/FOXO signaling in neurons that enhances stress resistance and extends life span. We found that Jafrac1 was expressed in the adult brain and induced by paraquat, a reactive oxygen species-generating chemical. RNA interference-mediated neuronal knockdown of Jafrac1 enhanced, while neuronal overexpression of Jafrac1 and hPrxII suppressed, paraquat-induced lethality in flies. Neuronal expression of Jafrac1 also significantly reduced ROS levels, restored mitochondrial function, and attenuated JNK activation caused by paraquat. Activation of JNK/FOXO signaling in neurons increased the Jafrac1 expression level under both normal and oxidative stressed conditions. Moreover, neuronal knockdown of Jafrac1 shortened, while overexpression of Jafrac1 and hPrxII extended, the life span in flies. These results support the hypothesis that JNK/FOXO signaling extends life span via amelioration of oxidative damage and mitochondrial dysfunction in neurons.

Mitochondrial Mislocalization Underlies Aβ42-Induced Neuronal Dysfunction in a Drosophila Model of Alzheimer's Disease

The amyloid-β 42 (Aβ42) is thought to play a central role in the pathogenesis of Alzheimers disease (AD). However, the molecular mechanisms by which Aβ42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Aβ42 induces mitochondrial mislocalization, which contributes to Aβ42-induced neuronal dysfunction in a transgenic Drosophila model. In the Aβ42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Aβ42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Aβ42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Aβ42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Aβ42 in vivo.

Loss of axonal mitochondria promotes tau- mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1.

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimers disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi–mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD–related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.

Transgenic Drosophila models of Alzheimer's disease and tauopathies.

Alzheimer’s disease (AD) is the most common form of senile dementia. Aggregation of the amyloid-β42 peptide (Aβ42) and tau proteins are pathological hallmarks in AD brains. Accumulating evidence suggests that Aβ42 plays a central role in the pathogenesis of AD, and tau acts downstream of Aβ42 as a modulator of the disease progression. Tau pathology is also observed in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and other related diseases, so called tauopathies. Although most cases are sporadic, genes associated with familial AD and FTDP-17 have been identified, which led to the development of transgenic animal models. Drosophila has been a powerful genetic model system used in many fields of biology, and recently emerges as a model for human neurodegenerative diseases. In this review, we will summarize key features of transgenic Drosophila models of AD and tauopathies and a number of insights into disease mechanisms as well as therapeutic implications gained from these models.

Physiological Mouse Brain Aβ Levels Are Not Related to the Phosphorylation State of Threonine-668 of Alzheimer's APP

Background Amyloid-β peptide species ending at positions 40 and 42 (Aβ40, Αβ42) are generated by the proteolytic processing of the Alzheimers amyloid precursor protein (APP). Aβ peptides accumulate in the brain early in the course of Alzheimers disease (AD), especially Aβ42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFα, CTFβ). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Aβ levels, prompting the current study. Methodology In order to evaluate whether the phosphorylation state of Thr668 controlled brain Aβ levels, we studied the levels and subcellular distributions of holoAPP, sAPPα, sAPPβ, CTFα, CTFβ, Aβ40 and Aβ42 in brains from “knock-in” mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein. Conclusions The levels and subcellular distributions of holoAPP, sAPPα, sAPPβ, CTFα, CTFβ, Aβ40 and Aβ42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Aβ40 or Αβ42 in vivo.

Overexpression of Neprilysin Reduces Alzheimer Amyloid-β42 (Aβ42)-induced Neuron Loss and Intraneuronal Aβ42 Deposits but Causes a Reduction in cAMP-responsive Element-binding Protein-mediated Transcription, Age-dependent Axon Pathology, and Premature Death in Drosophila

The amyloid-β42 (Aβ42) peptide has been suggested to play a causative role in Alzheimer disease (AD). Neprilysin (NEP) is one of the rate-limiting Aβ-degrading enzymes, and its enhancement ameliorates extracellular amyloid pathology, synaptic dysfunction, and memory defects in mouse models of Aβ amyloidosis. In addition to the extracellular Aβ, intraneuronal Aβ42 may contribute to AD pathogenesis. However, the protective effects of neuronal NEP expression on intraneuronal Aβ42 accumulation and neurodegeneration remain elusive. In contrast, sustained NEP activation may be detrimental because NEP can degrade many physiological peptides, but its consequences in the brain are not fully understood. Using transgenic Drosophila expressing human NEP and Aβ42, we demonstrated that NEP efficiently suppressed the formation of intraneuronal Aβ42 deposits and Aβ42-induced neuron loss. However, neuronal NEP overexpression reduced cAMP-responsive element-binding protein-mediated transcription, caused age-dependent axon degeneration, and shortened the life span of the flies. Interestingly, the mRNA levels of endogenous fly NEP genes and phosphoramidon-sensitive NEP activity declined during aging in fly brains, as observed in mammals. Taken together, these data suggest both the protective and detrimental effects of chronically high NEP activity in the brain. Down-regulation of NEP activity in aging brains may be an evolutionarily conserved phenomenon, which could predispose humans to developing late-onset AD.

Tau Ser262 phosphorylation is critical for Aβ42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease

The amyloid-beta 42 (Abeta42) peptide has been suggested to promote tau phosphorylation and toxicity in Alzheimers disease (AD) pathogenesis; however, the underlying mechanisms are not fully understood. Using transgenic Drosophila expressing both human Abeta42 and tau, we show here that tau phosphorylation at Ser262 plays a critical role in Abeta42-induced tau toxicity. Co-expression of Abeta42 increased tau phosphorylation at AD-related sites including Ser262, and enhanced tau-induced neurodegeneration. In contrast, formation of either sarkosyl-insoluble tau or paired helical filaments was not induced by Abeta42. Co-expression of Abeta42 and tau carrying the non-phosphorylatable Ser262Ala mutation did not cause neurodegeneration, suggesting that the Ser262 phosphorylation site is required for the pathogenic interaction between Abeta42 and tau. We have recently reported that the DNA damage-activated Checkpoint kinase 2 (Chk2) phosphorylates tau at Ser262 and enhances tau toxicity in a transgenic Drosophila model. We detected that expression of Chk2, as well as a number of genes involved in DNA repair pathways, was increased in the Abeta42 fly brains. The induction of a DNA repair response is protective against Abeta42 toxicity, since blocking the function of the tumor suppressor p53, a key transcription factor for the induction of DNA repair genes, in neurons exacerbated Abeta42-induced neuronal dysfunction. Our results demonstrate that tau phosphorylation at Ser262 is crucial for Abeta42-induced tau toxicity in vivo, and suggest a new model of AD progression in which activation of DNA repair pathways is protective against Abeta42 toxicity but may trigger tau phosphorylation and toxicity in AD pathogenesis.

A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein

Abstract : Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimers disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645‐694 of APP695 and identified a protein of ~130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV‐DDB (UV‐damaged DNA‐binding protein, calculated molecular mass of 127 kDa). UV‐DDB/p127 co‐immunoprecipitated with APP using an anti‐APP antibody from PC12 cell lysates. APP also co‐immunoprecipitated with UV‐DDB/p127 using an anti‐UV‐DDB/p127 antibody. These results indicate that UV‐DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S‐transferase‐APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid β protein secretion, may be involved in the interaction between UV‐DDB/p127 and APP.