Parkin Null Cortical Neuronal/Glial Cultures are Resistant to Amyloid-β1-42 Toxicity: A Role for Autophagy?

Abstract

Abstract. Dementia occurs often in late stages of Parkinson's disease (PD) but its cause is unknown. Likewise there is little information about the interaction between proteins that produce PD and those implicated in Alzheimer's disease (AD). Here we have investigated the interactions between parkin protein and the amyloid-␤ (A␤) 1-42 peptide. We examined the effects of oligomeric A␤ 1-42 peptide on the survival, differentiation, and signaling pathways in cortical cultures from wild type (WT) and parkin null (PK-KO) mice. We discovered that PK-KO cells were more resistant than WT to A␤ 1-42 . This peptide induced neuronal cell death, astrogliosis, microglial proliferation, and increased total and hyperphosphorylated tau and levels of chaperones HSP-70 and CHIP in WT, but not in A␤-treated PK-KO cultures. A␤ 1-42 decreased proteasome activities in WT and PK-KO cultures, but the ubiquitination of proteins only increased in WT cultures. A␤ 1-42 induced a short activation of ERK1/2 and AKT signaling pathways, implicated in cell survival, in PK-KO-treated cells, and a shift in the autophagy marker LC3-II/LC3-I ratio. In addition, the percentage of cells immunoreactive to both HSC70 and LAMP-2A increased in PK-KO cultures versus WT and furthermore in PK-KO cultures treated with A␤ 1-42 . Pre-treatment with inhibitors of glutathione synthesis or autophagy reverted the resistance to A␤ 1-42 of the PK-KO cultures. In conclusion, the loss of parkin protein triggers the compensatory mechanisms of cell protection against A␤ . Parkin suppression, therefore, is not a risk factor for dementia of AD type.

Trehalose Improves Human Fibroblast Deficits in a New CHIP-Mutation Related Ataxia

“…The protein concentration was assayed from the resulting supernatants by the BCA protein assay kit. The 20S proteasomal chymotrypsin-like activity was quantified [25] by monitoring the accumulation of the fluorescent cleavage product 7-amino-4-methylcoumarin (AMC) from the synthetic proteasomal substrate Suc-Leu-Leu-Val-Tyr-aminomethylcoumarin (LLVY-AMC) using the 20S proteasome activity assay kit (Chemicon, Temecula, CA, USA) according to the manufacturer's instructions.…”

“…We analyzed the percentage of cells immunoreactive to LC3, LAMP-2A, CD63 and HSC70/LAMP-2A colocalization as markers of macro-autophagy and chaperon-mediated autophagy [25], [26]. The fibroblast cultures were fixed with 4% paraformaldehyde, permeabilized with ethanol-acetic acid (19∶1), and incubated at 4°C for 24 h with primary antibodies diluted in PBS containing 10% fetal calf serum.…”

“…Poly-ubiquinated proteins were measured as previously described [25]. Human skin fibroblasts cultures were treated with epoxomicin (10 nM) or pre-treated with trehalose (50 mM) 15 min before the treatment with epoxomicin for 24 h. For detection of ubiquitinated proteins by Western blot, 15 µg of protein were used to immunoblot assay with a mouse monoclonal antibody to ubiquitin diluted 1/500 from Chemicon (Temecula, CA, USA).…”

Relationship between amyloid-beta and the ubiquitin–proteasome system in Alzheimer’s disease

“…However, adverse results were also reported. Parkin null cortical neuronal/glial cultures are more resistant to Abeta42 toxicity than wild type, 31 suggesting that parkin suppression may not be a risk factor for dementia of AD type.…”

Trehalose rescues glial cell dysfunction in striatal cultures from HD R6/1 mice at early postnatal development

“…Poly-ubiquinated proteins were measured as previously described by Solano et al (Solano et al, 2012). Glial cultures were treated with epoxomicin (10 nM) for 24 h. The cells were washed with PBS plus phenylmethylsulfonyl fluoride (PMSF), scraped in 150 μL of lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 50 mM sodium fluoride (NaF), 20 mM N-ethyl-maleimide, 100 μM sodium ortovanadate, 1 mM PMSF, and protease inhibitors cocktail (Sigma)] and immediately boiled for 5 min.…”