ALS-associated P56S-VAPB mutation restrains 3T3-L1 preadipocyte differentiation

Highlights

• P56S mutation leads to aberrant aggregation of VAPB in 3T3-L1 cells.

• P56S mutation impairs the lipid droplet formation in differentiating 3T3-L1 cells.

• P56S mutation restrains the expression levels of differentiated-associated genes in 3T3-L1.

• P56S mutation enhances the expression levels of ATF4 and CHOP in 3T3-L1 cells.

Abstract

Amyotrophic lateral sclerosis (ALS), which is the most common motor neuron disease in adults, is a neurodegenerative disease that involves the selective and systematic death of upper and lower motor neurons. In addition to the motor neuron death, altered metabolic functions, such as dyslipidemia, have also been reported for ALS patients; however, the underlying mechanism remains unknown. In the present study, we investigated the effects of ALS-associated P56S-vesicle-associated membrane proteinassociated protein B (VAPB), P56S-VAPB on 3T3-L1 preadipocyte differentiation and on the expression of differentiation-associated genes and unfolded protein response (UPR)-related genes. Experiments with 3T3-L1 cells transfected with wild-type (Wt)-VAPB and P56S-VAPB expression vectors showed that the size of lipid droplets was markedly smaller in P56S-VAPB-expressing cells, although fat accumulated intracellularly. In P56S-VAPB-expressing cells, increased the expression of PPARγ2, aP2, and C/EBPα, the genes deeply involved in adipocyte differentiation, was not observed. Furthermore, the expression levels of the UPR-related ATF4 and CHOP genes were found to be enhanced in the P56S-VAPB-expressing cells. From these results, P56S-VAPB was found to suppress adipocyte differentiation by promoting the activation of the ATF4-CHOP pathway. Given previous reports showing increased ATF4 and CHOP expression levels in neurons of ALS patients, results from the present study suggest that dyslipidemia is caused by enhanced ATF4–CHOP pathway in the adipose tissue of ALS patients.

Introduction

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder that affects upper and lower motor neurons [1]. Initially, pathological abnormalities in ALS were thought to be restricted to motor neurons, but descriptions of a wider dissemination of effects throughout the body have challenged this classic paradigm. ALS disease seems to be restricted not only to the CNS but also affects whole-body physiology [2]. In particular, energy metabolism is severely altered in patients with ALS, which has notable clinical implications. Patients with ALS are generally lean with a normal or low body-mass index [3], [4], [5] and typically lose weight and body fat as disease progresses [6], [7], [8]; therefore, energy stores are decreased. Hyperlipidemia has been put forward as an explanation for energy imbalance in ALS. Previous studies have demonstrated that increased blood lipid concentrations and suggested that the lipid metabolism and the nutritional status of ALS patients are important prognostic factors [9], [10]. However, the causes of hyperlipidemia are unclear in patients with ALS.

Although most cases of ALS are sporadic, 5–10% of cases occur in families with at least one other affected family member, and some families display a clear Mendelian inheritance of ALS with high penetrance of the disease [11]. A mutation in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB) causes ALS type-8 and some other related forms of motor neuron disease including late onset spinal muscular atrophy [12]. The mutation that causes ALS type-8 involves a proline to serine substitution at position-56 (P56S-VAPB). VAPB protein is ubiquitously expressed and a type II integral membrane protein that mainly locates at the endoplasmic reticulum (ER). It contains an N-terminal domain homologous to the major sperm protein of nematode worms, a central coiled-coil region and a C-terminal transmembrane domain through which it is anchored in the ER membrane; the N-terminus of VAPB projects from the ER into the cytoplasm [13], [14], [15]. VAPB has been proposed to act in the regulation of COPI-mediated protein transport within the Golgi apparatus and from the Golgi back to the ER [16]. VAPB has been implicated in a variety of processes including ER stress and the unfolded protein response (UPR), ER to Golgi transport and bouton formation at the neuromuscular junction [17], [18]. ER stress is linked to the pathogenesis of ALS [19], [20] and several studies implicate P56S-VAPB in abnormal UPR but the mechanisms are unclear.

Here, we report for the first time that P56S-VAPB impairs lipid droplet formation in 3T3-L1 adipocyte. We also found that P56S-VAPB expressing 3T3-L1 cells restrained the mRNA expression of mature adipocyte marker and enhanced the expression levels of the UPR-related ATF4 and CHOP genes. These results indicate that P56S-VAPB suppress adipocyte differentiation by promoting the activation of the ATF4-CHOP pathway. Our results thus may provide a mechanism by which the energy metabolism is severely altered in ALS.