Effects of 4-Phenylbutyric Acid and Caprylic Acid on Proteostasis and Mitochondrial Homeostasis in an Alzheimer’s Disease Model of the Nematode Caenorhabditis elegans
Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common form of dementia. The neurodegeneration results in progressive memory loss as well as psychological changes and can ultimately lead to an almost complete loss of cognitive abilities. With age being the main risk factor of sporadic AD and the advancing demographic change, ... the disease is a growing burden of modern society. Despite its great importance and intensive research, there is currently no cure for the disease, limiting therapy to symptomatic treatment. The pathogenesis of AD is a complex irreversible process characterized by a long preclinical phase, in which accumulation of proteotoxic amyloid-β (Aβ) aggregates and mitochondrial dysfunction were identified as major features that mutually aggravate one another in a vicious cycle, eventually leading to progressive disturbance of neurotransmission and neurodegeneration. In the present work, the molecular effects of the aromatic short-chain fatty acid 4-phenylbutyric acid (4-PBA) and the medium-chain fatty acid caprylic acid (CA) on proteostasis and mitochondrial homeostasis were investigated using the transgenic Caenorhabditis elegans strain GMC101, expressing human Aβ1-42 in body wall muscle cells. Computer-based analysis of motility revealed that Aβ proteotoxicity particularly impaired the average speed of GMC101 compared to its corresponding control strain CL2122. 4-PBA and CA both increased the motility of GMC101 nematodes dose-dependently and selectively. Their selective effect on Aβ proteotoxicity was further reflected by a reduction of Aβ aggregation, as assessed using the Aβ-specific fluorescent probe NIAD 4. Given that 4-PBA, as a weak inhibitor of class I and IIb histone deacetylases (HDACs), may activate protein quality control through several mechanisms, the importance of major proteostasis transcription factors was investigated using RNA interference (RNAi). Knockdown of hsf-1, an ortholog essential for the cytosolic heat shock response, abolished the reduction of Aβ aggregation and proteotoxicity by 4-PBA incubation. Since knockdown of hda-1, an ortholog of HDAC2, also increased motility in a hsf-1-dependent manner, and application of 4-PBA under hda-1 RNAi showed no additive effect, it can be assumed that 4-PBA activates HSF 1 via inhibition of HDA-1. Next, it was tested whether the attenuation of Aβ proteotoxicity by 4-PBA was associated with improved mitochondrial function. Accordingly, incubation with 4-PBA increased adenosine triphosphate (ATP) levels, measured via luciferase assay, and the mitochondrial membrane potential (MMP), quantified using the fluorescent probe TMRE. Using RNAi against genes involved in β-oxidation (fatty acid oxidation; FAO), and complex I as well as complex II of the electron transport chain (ETC), the motility increasing effect of 4 PBA was demonstrated to be dependent on its oxidation to phenylacetic acid (PAA) and thus on its properties as an energy source. Finally, knockdown of genes involved in mitochondrial fission and receptor-mediated mitophagy resulted in a decreased protective effect of 4-PBA. This was further evidenced using genetic crossings of Aβ expressing GMC101 nematodes with transgenic fluorescent reporter strains, confirming increased mitochondrial fission and eventually induction of mitophagy. CA is a precursor for the biosynthesis of α-lipoic acid (ALA), a cellular antioxidant and coenzyme involved in energy metabolism, as well as the ketone body β-hydroxybutyric acid (BHB), which may serve as an alternative energy substrate and signaling molecule. The motility increase by CA incubation, however, was found to be independent of ALA and BHB biosynthesis. As in the case of 4-PBA, RNAi against genes involved in FAO, and complex I as well as complex II of the ETC abolished the motility increasing effect of CA. In accordance with the necessity of a functional FAO and delivery of reduction equivalents to the ETC as a requirement to enable the motility increasing effect of CA in GMC101 nematodes, ATP levels, oxygen consumption and the MMP were also elevated as a consequence of CA incubation. In conclusion, both 4-PBA as well as CA attenuate Aβ proteotoxicity in an AD model of the nematode C. elegans. They share the reduction of Aβ aggregation and improvement of mitochondrial function as common targets. Moreover, both substances serve as energy fuel via FAO, thus circumventing the steps specific for glucose oxidation that are impaired in AD. 4-PBA further activates the proteostasis network and the mitochondrial quality control, explaining its overall increased protective effect relative to CA.