Joo-Yong Lee

Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice

Endogenous metals may contribute to the accumulation of amyloid plaques in Alzheimers disease. To specifically examine the role of synaptic zinc in the plaque accumulation, Tg2576 (also called APP2576) transgenic mice (hAPP+) expressing cerebral amyloid plaque pathology were crossed with mice lacking zinc transporter 3 (ZnT3−/−), which is required for zinc transport into synaptic vesicles. With aging, female hAPP+:ZnT3+/+ mice manifested higher levels of synaptic zinc, insoluble amyloid β, and plaques than males; these sex differences disappeared in hAPP+:ZnT3−/− mice. Both sexes of hAPP+:ZnT3−/− mice had markedly reduced plaque load and less insoluble amyloid β compared with hAPP+:ZnT3+/+ mice. Hence, of endogenous metals, synaptic zinc contributes predominantly to amyloid deposition in hAPP+ mice.

Disease-causing mutations in Parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy

Mutations in parkin, a ubiquitin ligase, cause early-onset familial Parkinsons disease (AR-JP). How parkin suppresses parkinsonism remains unknown. Parkin was recently shown to promote the clearance of impaired mitochondria by autophagy, termed mitophagy. Here, we show that parkin promotes mitophagy by catalyzing mitochondrial ubiquitination, which in turn recruits ubiquitin-binding autophagic components, HDAC6 and p62, leading to mitochondrial clearance. During the process, juxtanuclear mitochondrial aggregates resembling a protein aggregate-induced aggresome are formed. The formation of these mito-aggresome structures requires microtubule motor-dependent transport and is essential for efficient mitophagy. Importantly, we show that AR-JP-causing parkin mutations are defective in supporting mitophagy due to distinct defects at recognition, transportation, or ubiquitination of impaired mitochondria, thereby implicating mitophagy defects in the development of parkinsonism. Our results show that impaired mitochondria and protein aggregates are processed by common ubiquitin-selective autophagy machinery connected to the aggresomal pathway, thus identifying a mechanistic basis for the prevalence of these toxic entities in Parkinsons disease.

The lipophilic metal chelator DP-109 reduces amyloid pathology in brains of human β-amyloid precursor protein transgenic mice

Metals such as zinc, copper and iron contribute to aggregation of amyloid-beta (Abeta) protein and deposition of amyloid plaques in Alzheimers disease (AD). We examined whether the lipophilic metal chelator DP-109 inhibited these events in aged female hAbetaPP-transgenic Tg2576 mice. Daily gavage administration of DP-109 for 3 months markedly reduced the burden of amyloid plaques and the degree of cerebral amyloid angiopathy in brains, compared to animals receiving vehicle treatment. Moreover, DP-109 treatment appeared to facilitate the transition of Abeta from insoluble to soluble forms in the cerebrum. These results further support the hypothesis that endogenous metals are involved in the deposition of aggregated Abeta in brains of AD patients, and that metal chelators may be useful therapeutic agents in the treatment of AD.

Histone Deacetylase 6 Regulates Growth Factor-Induced Actin Remodeling and Endocytosis

ABSTRACT Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that uniquely catalyzes α-tubulin deacetylation and promotes cell motility. However, the mechanism underlying HDAC6-dependent cell migration and the role for microtubule acetylation in motility are not known. Here we show that HDAC6-induced global microtubule deacetylation was not sufficient to stimulate cell migration. Unexpectedly, in response to growth factor stimulation, HDAC6 underwent rapid translocation to actin-enriched membrane ruffles and subsequently became associated with macropinosomes, the vesicles for fluid-phase endocytosis. Supporting the importance of these associations, membrane ruffle formation, macropinocytosis, and cell migration were all impaired in HDAC6-deficient cells. Conversely, elevated HDAC6 levels promoted membrane ruffle formation with a concomitant increase in macropinocytosis and motility. In search for an HDAC6 target, we found that heat shock protein 90 (Hsp90), another prominent substrate of HDAC6, was also recruited to membrane ruffles and macropinosomes. Significantly, inhibition of Hsp90 activity suppressed membrane ruffling and cell migration, while expression of an acetylation-resistant Hsp90 mutant promoted ruffle formation. Our results uncover a surprising role for HDAC6 in actin remodeling-dependent processes and identify the actin cytoskeleton as an important target of HDAC6-regulated protein deacetylation.

Zinc released from metallothionein-iii may contribute to hippocampal CA1 and thalamic neuronal death following acute brain injury

Vesicular zinc was initially considered the sole source of toxic intraneuronal zinc accumulation in response to acute brain injury, but recent evidence suggests that additional sources also exist. Because metallothioneins (MTs) can bind and release zinc, we examined the possibility that the brain-specific form, MT-III, is such a zinc source. After kainate-induced seizures, cytoplasmic zinc accumulation and neuronal death in the hippocampal CA1 region and the thalamus were substantially lower in Mt3-null mice than in wild-type mice. Furthermore, compared with zinc transporter 3 (Znt3)-null mice, Znt3/Mt3 double-null mice exhibited further reductions in neuronal death in CA1 following kainate-induced seizures. Similar reductions in zinc accumulation and neuronal death in hippocampal CA1 and the dentate gyrus in Mt3-null mice were observed in a sodium nitroprusside model of acute brain injury. In contrast to CA1, more neuronal death occurred after kainate-induced seizures in CA3 of Mt3-null mice. These results suggest that intracellular zinc release from MT-III may contribute substantially to zinc-mediated neuronal death in certain brain areas, including the hippocampal CA1 region and the thalamus.

Neuronal Zinc Exchange with the Blood Vessel Wall Promotes Cerebral Amyloid Angiopathy in an Animal Model of Alzheimer's Disease

Cerebral amyloid angiopathy (CAA) is common in Alzheimers disease (AD) and may contribute to dementia and cerebral hemorrhage. Parenchymal β-amyloid deposition is dependent on the activity of zinc transporter 3 (ZnT3), a neocortical synaptic vesicle membrane protein that causes enrichment of exchangeable Zn2+ in the vesicle, which is externalized on neurotransmission. However, the contribution of zinc to vascular β-amyloid deposition remains unclear. Here, we identify for the first time an exchangeable pool of Zn2+ in the cerebrovascular wall of normal mice. This histochemically reactive Zn2+ is enriched in CAA in a transgenic mouse model of AD (Tg2576), and a dramatic reduction of CAA occurs after targeted disruption of the Znt3 gene in these mice. Also, in Znt3 knock-out mice, the amount of exchangeable Zn2+ [detected by N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide (TFL-Zn)] in the perivascular space was significantly decreased in the neocortex but not in peripheral organs. ZnT3 was not detected in the cerebral vessel walls or in blood components of wild-type mice. Thus, synaptic ZnT3 activity may promote CAA by indirectly raising exchangeable Zn2+ concentrations in the perivascular spaces of the brain.

Acetylation Goes Global: The Emergence of Acetylation Biology

Proteomic analysis shows protein acetylation to be more prevalent than previously appreciated. For the first 30 years since its discovery, reversible protein acetylation has been studied and understood almost exclusively in the context of histone modification and gene transcription. With the discovery of non–histone acetylated proteins and acetylation-modifying enzymes in cellular compartments outside the nucleus, the regulatory potential of reversible acetylation has slowly been recognized in the last decade. However, the scope of protein acetylation involvement in complex biological processes remains uncertain. The recent development of new technology has enabled, for the first time, the identification and quantification of the acetylome, acetylation events at the whole-proteome level. These efforts have uncovered a stunning complexity of the acetylome that potentially rivals that of the phosphoproteome. The remarkably ubiquitous and conserved nature of protein acetylation revealed by these new studies suggests the regulatory power of this dynamic modification. The establishment of comprehensive acetylomes will change the landscape of protein acetylation, where an exciting research frontier awaits.

Estrogen Decreases Zinc Transporter 3 Expression and Synaptic Vesicle Zinc Levels in Mouse Brain

Previous studies suggest that female sex hormones modulate synaptic zinc levels, which may influence amyloid plaque formation and Alzheimers disease progression. We examined the effects of ovariectomy and estrogen supplement on the levels of synaptic zinc and zinc transporter protein Znt3 in the brain. Ovariectomy was performed on 5-month-old mice, and 2 weeks later, pellets containing vehicle, low (0.18 mg/pellet), or high dose (0.72 mg) 17β-estradiol were implanted. After 4 weeks, animals were decapitated, and blood and brain were collected for analysis. Blood analysis indicated that estrogen implants altered plasma estrogen levels in a dose-dependent manner. Analysis of brain tissue showed that ovariectomy raised hippocampal synaptic vesicle zinc levels, whereas estrogen replacement lowered these zinc levels. Western blots revealed that Znt3 levels in the brain were modulated in parallel with synaptic zinc levels, whereas no change was detected in the levels of Znt3 mRNA, as determined by Northern blot and reverse transcriptase-PCR analysis. However, mRNA levels of the δ subunit of adaptor protein complex (AP)-3, which modulates the level of Znt3 levels, were altered by estrogen depletion or replacement. These data demonstrate that estrogen alters the levels of Znt3 and synaptic vesicle zinc in female mice, probably through changing AP-3 δ expression. Since synaptic zinc may play a key role in neuronal death in acute brain injury as well as in plaque formation in Alzheimers disease, and since estrogen may be beneficial in both conditions, our results may provide new insights into the effects of estrogen on the brain.

E2-25K/Hip-2 regulates caspase-12 in ER stress–mediated Aβ neurotoxicity

Amyloid-β (Aβ) neurotoxicity is believed to contribute to the pathogenesis of Alzheimers disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Aβ neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)–resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Aβ increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2–mediated cell death. Finally, we find that E2-25K/Hip-2–deficient cortical neurons are resistant to Aβ toxicity and to the induction of ER stress and caspase-12 expression by Aβ. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress–mediated Aβ neurotoxicity.

Induction of pro-apoptotic calsenilin/DREAM/KChIP3 in Alzheimer's disease and cultured neurons after amyloid-β exposure

Calsenilin/DREAM/KChIP3 was identified as a calcium‐binding protein that interacts with presenilins, serves as a transcription repressor, and binds to the A‐type potassium channel. In this study, we hypothesized that calsenilin might be involved in the neurodegeneration of Alzheimers disease and examined calsenilin expression in Alzheimers disease. Calsenilin levels were elevated in the cortex region of Alzheimers patient brains and in the neocortex and the hippocampus of Swedish mutant β‐amyloid precursor protein transgenic mice brains. Induction of calsenilin was also observed in the activated astroglia as well as in the neurons surrounding β‐amyloid (Aβ)‐ and Congo red‐positive plaques. Exposing cultured cortical and hippocampal neurons to Aβ42, an amyloid‐β peptide whose deposition in the brain is a characteristic of Alzheimers disease, induced both calsenilin protein and mRNA expression, and cell death. Moreover, blocking the calsenilin expression protected the neuronal cells from Aβ toxicity. These findings suggest that chronic up‐regulation of calsenilin may be a risk factor for developing Alzheimers disease, perhaps by facilitating calsenilin‐mediated neurodegeneration.