Gene expression profiles of autophagy-related genes in multiple sclerosis

Highlights

• We aimed to examine the expression levels of autophagy-related genes in multiple sclerosis patients.

• The blood samples of 95 unrelated patients and 95 healthy controls were analyzed.

• mRNA levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered.

Abstract

Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17–65 years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96 × 78 (samples × primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate < 0.05). Thus, altered expression levels of several autophagy related genes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis.

Introduction

Multiple sclerosis (MS) is the most common disease of the central nervous system characterized by inflammation and neurodegeneration, affecting especially young adults (Compston and Coles, 2002).While the cause is not clear, the underlying mechanism is considered to be destruction by the immune system (T cell-mediated demyelination) or failure of the myelin-producing cells (Nakahara et al., 2012). Proposed causes involve genetic susceptibility and environmental factors such as infections (Compston and Coles, 2002, Ascherio and Munger, 2007). However, increased T cell proliferation and prolonged T cell survival have been linked to MS (Alirezaei et al., 2009). The best defining feature of the disease is formation of the sclerotic plaques, which is the end stage of several processes such as inflammation caused by recognition of myelin as a foreign antigen by T cells, demyelination and remyelination, oligodendrocyte depletion and astrocytosis, and neurodegeneration (Compston and Coles, 2002).

Macroautophagy, hereafter referred to simply as autophagy, is a conserved intracellular process which provides instruments for the toxic, aggregation-prone proteins and damaged organelles, such as mitochondria; to be degraded (Yang and Klionsky, 2010) Autophagy is initiated with a double-membrane structure formation called autophagosome. Yeast has approximately 20 different autophagy genes (Atg) that are necessary for autophagosome formation and the further stages of autophagy (Yang and Klionsky, 2010). Autophagy is greatly up-regulated by prolonged starvation or fasting (Levine and Kroemer, 2008). However, it was discovered that autophagy also has a significant role in the immune responses, and autophagy is not limited to the conditions under the stimulus of starvation (Beau et al., 2011). Autophagy is responsible for constitutive protein turnover even under nutrient-rich conditions.

In autophagy process, the cargos are packaged into autophagasomes and shipped to the lysosome for degradation. Since autophagosomes are non-degradative vacuoles, they just seclude the cytoplasmic material (Beau et al., 2011). The structure known as phagophore or the isolation membrane is derived from a single membrane and elongates around the cellular cargo (Yang and Klionsky, 2010). Autophagy begins with the construction of the complex that initiates phagophore membrane formation, including Beclin-1 (BECN1, ATG6), vacuolar protein sorting 34 (Vps34), and class III phosphoinositide 3-kinase, plus autophagy genes (Beau et al., 2011, Yordy et al., 2012). Following the formation of the phagophore membrane, construction of the autophagosome continues with the elongation and closure of the membrane. In mammalians, while Atg9, ULK1 (Unc51-Like Kinase 1) complex, and the PI3K (Phosphatidylinositol 3-Kinase) complex initiate the autophagy under certain circumstances (Beau et al., 2011), Atg 14 has the critical role in the endoplasmic reticulum (ER) targeting of the PI3K complex (Matsunaga et al., 2010). To form the autophagosome, Atg12-Atg5, Atg16 and LC3-II (Microtubule-Associated Protein 1, Light Chain 3) act to extend the phagophore membrane (Nakatogawa et al., 2009). The mitochondrial outer membrane (Hailey et al., 2010), the plasma membrane (Ravikumar et al., 2010) and also post-Golgi compartments (Mari et al., 2010) may be another sources of the phagophore membrane. After closure, except the LC3-II, the Atg proteins are reacquired from the autophagosome membrane (Yang and Klionsky, 2010).

Malfunction of autophagy contributes to the pathogenesis of certain diseases such as cancer, infectious diseases, cardiovascular and neurodegenerative disorders (Ravikumar et al., 2010). Inhibiton of autophagy significantly accelerates the development of neurodegeneration (Hara et al., 2006). In neurodegenerative disorders such as amyotrophic lateral sclerosis, Parkinson's and Huntington's diseases, the lack of autophagy is associated with neurodegeneration even in the absence of harmful proteins (Komatsu et al., 2006).

As known, the cells that play roles in the initiation and progression of inflammation are CD4 + T cells, CD8 + T cells, gamma/delta T cells, B cells, antibodies and cells of innate immune system such as microglia and macrophages (Friese and Fugger, 2005). Macrophages use autophagy to clear microbial pathogens (Pua et al., 2007). It is now known that autophagy has a role in the development and function of adaptive immune system (Pua and He, 2007).

The discovery of autophagy-related genes has highly improved our understanding and established our perspective about the mechanism responsible for the fate of autophagy from autophagosome formation to maturation and lysosomal degradation. The regulation of autophagy by protein kinases and transcription factors involving in nuclear regulation of autophagy genes is now better known. Based on the possible role of autophagy in neurodegenerative disorders and adaptive immune system, in this study, we aimed to understand whether there might be an association between autophagy and multiple sclerosis pathogenesis by monitoring the gene expression levels of several autophagy-related genes (Table 1) involving in autophagy machinery in different stages as discussed in Discussion section.