β‑Amyloid and α‑Synuclein Cooperate To Block SNARE-Dependent Vesicle Fusion

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are caused by β-amyloid (Aβ) and α-synuclein (αS), respectively. Ample evidence suggests that these two pathogenic proteins are closely linked and have a synergistic effect on eliciting neurodegenerative disorders. However, the pathophysiological consequences of Aβ and αS coexistence are still elusive. Here, we show that large-sized αS oligomers, which are normally difficult to form, are readily generated by Aβ₄₂-seeding and that these oligomers efficiently hamper neuronal SNARE-mediated vesicle fusion. The direct binding of the Aβ-seeded αS oligomers to the N-terminal domain of synaptobrevin-2, a vesicular SNARE protein, is responsible for the inhibition of fusion. In contrast, large-sized Aβ₄₂ oligomers (or aggregates) or the products of αS incubated without Aβ₄₂ have no effect on vesicle fusion. These results are confirmed by examining PC12 cell exocytosis. Our results suggest that Aβ and αS cooperate to escalate the production of toxic oligomers, whose main toxicity is the inhibition of vesicle fusion and consequently prompts synaptic dysfunction

Apoptotic protein expression and cellular death in both exogenous Aβ_1–42 treatment and mito Aβ_1–42–transfected cells.

<p>Western blot analysis was performed in both exogenous Aβ_1–42 treatment and mito Aβ_1–42–transfected HT22 cells to characterize the expression level of Aβ_1–42, Bcl-2, Bax using 6E10, Bcl-2 antibody and Bax antibody (A). Expression level of Bcl-2 and Bax was confirmed in mitochondrial fraction (B). Different concentrations of mito Aβ_1–42 DNA constructs were used, 2 µg and 4 µg. Cytochrome C release assay (C) and calcein cell viability assay (D) were performed in vehicle-treated, exogenous Aβ_1–42-treated, mock and mito Aβ_1–42 transfected HT22 cells, respectively (* p<0.05, ** p<0.01 compared with vehicle, # p<0.05 compared to mock).</p

Morphological alteration of mitochondria in AβPP/PS1 mice brains and Aβ_1–42-treated HT22 cell line.

<p>A. Electron microscopic (EM) image of mitochondria in wild type and AβPP/PS1 mice (10 months, cortex, scale bar: 2 µm) B. EM image of mitochondria in vehicle and Aβ-treated HT22 cells (yellow scale bar: 2 µm, white scale bar: 1 µm) C. Immunostaining of HSP60 in Aβ_1–42-treated HT22 cell line by different time period of treatment (scale bar: 20 µm).</p

Clathrin-mediated endocytosis blocker inhibited Aβ_1–42-induced mitochondrial dysfunction.

<p>A. Mitochondrial shapes were identified by immunostaining of HSP60 in vehicle, Aβ_1–42, chlorpromazine+Aβ_1–42, mouse anti-RAGE IgG+Aβ_1–42 treatment in HT22 cells, respectively (Scale bar: 20 µm). B. Altered mitochondrial shapes are quantified using form factor and aspect ratio (blue: vehicle, red: chlorpromazine+Aβ_1–42, green: Aβ_1–42 in left graph). Four functional assessments of mitochondria are shown, including MTT (C), ROS levels (D), ATP generation (E) and TMRM intensity (F). * p<0.05, ** p<0.01, *** p<0.001 compared with vehicle, # p<0.05 compared with Aβ_1–42.</p

Mitochondria-Targeting Ceria Nanoparticles as Antioxidants for Alzheimer’s Disease

Mitochondrial oxidative stress is a key pathologic factor in neurodegenerative diseases, including Alzheimer’s disease. Abnormal generation of reactive oxygen species (ROS), resulting from mitochondrial dysfunction, can lead to neuronal cell death. Ceria (CeO₂) nanoparticles are known to function as strong and recyclable ROS scavengers by shuttling between Ce³⁺ and Ce⁴⁺ oxidation states. Consequently, targeting ceria nanoparticles selectively to mitochondria might be a promising therapeutic approach for neurodegenerative diseases. Here, we report the design and synthesis of triphenylphosphonium-conjugated ceria nanoparticles that localize to mitochondria and suppress neuronal death in a 5XFAD transgenic Alzheimer’s disease mouse model. The triphenylphosphonium-conjugated ceria nanoparticles mitigate reactive gliosis and morphological mitochondria damage observed in these mice. Altogether, our data indicate that the triphenylphosphonium-conjugated ceria nanoparticles are a potential therapeutic candidate for mitochondrial oxidative stress in Alzheimer’s disease

Mito Aβ_1–42 induces not only mitochondrial morphological alteration but also functional impairments.

<p>A. Immunostaining of HSP60 and YFP in mock or mito Aβ_1–42–transfected HT22 cells (white scale bar: 20 µm). B. Quantification of alteration in mitochondrial shape is presented as form factor and aspect ratio (** p<0.01). Four functional assessments for mitochondria are shown, including MTT (C), ROS levels (D), ATP generation (E) and TMRM staining (F). G. TMRM intensity is quantified as percent of control. * p<0.05, ** p<0.01, *** p<0.001 compared to mock, white scale bar in F: 50 µm.</p

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

<p>The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.</p

Mitochondria-specific accumulation of mito Aβ_1–42.

<p>A. Western blot analysis showed the mitochondria-specific accumulation of Aβ_1–42 with the presence of TOM20, mitochondrial marker and the absence of β-actin. B. EM image of mitochondria in mock and mito Aβ_1–42-transfected HT22 cells (yellow scale bar: 2 µm, white scale bar: 1 µm).</p

Functional assays for mitochondria in AβPP/PS1 mice brains and Aβ_1–42-treated HT22 cell line.

<p>Four types of mitochondrial functional assays: MTT (A); ROS level (B); ATP generation (C) and TMRM intensity (D) were assessed in vehicle or 5 µM Aβ_1–42-treated HT22 cells. MTT assay were measured after 24 h of Aβ treatment, other assays were after 6 h of Aβ treatment (* p<0.05, ** p<0.01).</p