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

Activation of SHH signaling inhibits melanogenesis in B16F1 cells.

<p>B16F1 cells were treated with the ciliogenesis activator, Smo-agonist (SAG; 0.5 or 1 μM) for 24 h. The proportion of ciliated cells was measured (A). B16F1 cells were pre-treated with α-MSH (1 μM). After 24 h, the cells were further co-treated with α-MSH (1 μM) and SAG (5 μM) or arbutin (500 μM). After 24 h, the cellular melanin content was measured (B). The protein expression levels of tyrosinase and TRP1 were analyzed (C). Data represent the mean ± SE of 3 independent experiments (* <i>p</i> < 0.05, ** <i>p</i> < 0.01).</p

The induction of ciliogenesis reduces melanin synthesis in mouse normal melanocytes (Melan-a cells).

<p>Melan-a cells were treated with cytochalasin D (Cyto D, 0.25 μM) for 48 h. The induction of polyglutamylated-tubulin protein (PG-tubulin, green) was observed (A, scale bar = 10 μm). Ciliated cells were measured (Cyto D 0.25 and 0.5 μM, B). Melan-a cells pre-treated with α-MSH (1 μM) for 24 h were further treated with Cyto D (0.25 and 0.5 μM) for 48 h. Then, cellular melanin content was measured (C). The protein expression levels of PG-tubulin, tyrosinase, and TRP1 were analyzed (D). Data were obtained from at least 3 independent experiments and the values were presented as mean ± SE (* <i>p</i> < 0.05, ** <i>p</i> < 0.01).</p

Induction of ciliogenesis suppresses pigmentation in normal human epidermal melanocytes (NHEMs) and a human skin model.

<p>NHEMs were treated with cytochalasin D (0.5 μM) for 48 h. After immunostaining for acetylated-tubulin (green) and ARL13B (red), ciliated cells were counted (A, scale bar = 10 μm). NHEMs treated with cytochalasin D (0.5 or 1 μM) for 48 h were harvested to measure the cellular melanin content (B). Photographs of 3-dimensional human skin substitutes treated with Smo-agonist SAG (5 μM), cytochalasin D (0.5 μM), or kojic acid (5 mM) for 13 and 20 days were taken (C). The pigmentation of the skin substitutes was measured and ΔL was calculated between the control and treated groups (D). Data represent the mean ± SE of 3 experiments (* p < 0.05, ** p < 0.01).</p

GLI2 mediates the regulation of melanogenesis by primary cilia.

<p>Melan-a cells were transfected with scrambled control (siNC) or GLI2 siRNA. After 24 h, the cells were treated with cytochalasin D (0.5 μM) or serum-free media. After 48 h, the proportion of ciliated cells was counted (A). Cellular melanin content was analyzed (B). GLI2, tyrosinase, and TRP1 were analyzed (C). B16F1 cells were transfected with siNC or GLI2 siRNA. After 1 day, the cells were treated with cytochalasin D (0.5 μM) or serum-free media for 2 days. Ciliated cells and cellular melanin content were analyzed (D, E). GLI2, tyrosinase, and TRP1 were analyzed (F). Data represent the mean ± SE of 3 experiments (* p < 0.05, ** p < 0.01).</p

Inhibition of primary cilia induces melanin synthesis in Melan-a cells.

<p>Melan-a cells were incubated with serum free media and treated with ciliobrevin A1 (Cilio A, 10 μM) for 48 h. Primary cilia stained by polyglutamylated-tubulin antibody (green) were observed (A, scale bar = 10 μm) and ciliated cells were counted (Cilio A 10 and 30 μM, B). The cellular melanin content was measured (C). The expression levels of polyglutamylated-tubulin, tyrosinase, and TRP1 were analyzed (D). Data represent the mean ± SE of 3 independent experiments (* <i>p</i> < 0.05, ** <i>p</i> < 0.01).</p