Plasma ATG5 is increased in Alzheimer’s disease

Alzheimer’s disease (AD) is a major cause of dementia. Growing evidence suggests that dysregulation of autophagy, a cellular mechanism essential for self-digestion of damaged proteins and organelles, is involved in neurological degenerative diseases including AD. Previously, we reported that autophagosomes are increased in the brains of AD mouse model. However, the plasma levels of autophagic markers have not yet been investigated in patients with AD. In this study, we investigated the expression of autophagy-related genes 5 and 12 (ATG5 and ATG12, respectively) in cells in vitro upon amyloid-beta (Aβ) treatment and in the plasma of AD patients. ATG5-ATG12 complex levels were increased in primary rat cortical neurons and human umbilical vein endothelial cells after Aβ treatment. Furthermore, we compared plasma from 69 patients with dementia, 82 patients with mild cognitive impairment (MCI), and 127 cognitively normal control participants. Plasma levels of ATG5 were significantly elevated in patients with dementia (149.3 ± 7.5 ng/mL) or MCI (152.9 ± 6.9 ng/mL) compared with the control subjects (129.0 ± 4.1 ng/mL) (p = 0.034, p = 0.016, respectively). Our results indicate that alterations in the plasma ATG5 levels might be a potential biomarker in patients at risk for AD.

Here, we investigated the proteins of the autophagic machinery, in particular ATG5 and ATG12 in AD patients as well as in cells in vitro upon Aβ treatment in order to examine the importance of these autophagic markers as potent biomarkers for AD.

Results
ATG5-ATG12 conjugation is induced in the endothelial cell-conditioned media upon Aβ treatment. Several lines of evidence demonstrate that autophagic activation is involved in Aβ clearance and might play a role in the pathogenesis of AD. Since conjugation of ATG5-ATG12 is critical for the formation of autophagosome, we first asked whether conjugation of ATG5 and ATG12 is induced by Aβ. Western blot in primary rat cortical neurons and endothelial cells treated with Aβ, demonstrated that the conjugation between ATG5-ATG12 was increased (Fig. 1).
Given that the protein levels of ATG5 and ATG12 are not altered in the brains of AD patients 12 , we sought to examine ATG5 and ATG12 mRNA levels in human induced pluripotent stem cell (iPSC)-derived neural progenitor stem cells isolated from a patient with AD. The mRNA levels of ATG5 and ATG12 were found unchanged in iPSC-derived neurons of an AD patient compared with those in iPSC-derived neurons of a healthy control donor ( Fig. 2A,B). However, the mRNA levels of p62 and LC3A were significantly increased in iPSC-derived neurons of an AD patient compared with those in iPSC-derived neurons of a healthy control donor (Fig. 2C,D).
Furthermore, the immunoreactivity of ATG12 was increased in the cortex of APPsw/PS1ΔE9 transgenic (APP Tg) mice as shown by diaminobenzidine staining in Fig. 3. Indeed, some of the ATG12-immunopositive cells were found at higher densities near the amyloid plaques stained by Congo Red in the brains of APP Tg mice. Mice lacking ATG5 develop progressive deficits in motor function. Moreover, the autophagic flux in CA1 hippocampal neurons of AD patients was impaired with neuritic dystrophy 13,14 . Plasma ATG5 levels are elevated in AD patients. Recent studies have shown increased plasma level of autophagic markers in patients with diseases such as stroke 11 . For a more specific indication of the implication of autophagy in AD pathogenesis, we measured ATG5 and ATG12 levels in the plasma from patients with AD. Before that, we asked whether ATG5 and ATG12 were secreted into the conditioned medium from cells treated with Aβ. After treatment of Aβ in human umbilical vein endothelial cells (HUVECs) with Aβ, we found that ATG5 levels in the conditioned medium were increased (Fig. 4). This effect was dose dependent. However, we could not detect ATG12 band in the conditioned medium by western blot analysis.   . Immunostaining for ATG12 in the brain of APP transgenic mice. Brain cortex sections from 16-month-old wild type (WT) and APP transgenic (TG) mice were immunostained with anti-ATG12, and counterstained with Congo Red for amyloid plaques. Congophilic plaque was indicated by an asterisk. www.nature.com/scientificreports www.nature.com/scientificreports/ dementia were 75.3 ± 0.71 and 73.59 ± 0.54 years for patients with MCI and 72.0 ± 0.4 years for normal control participants; 75.3% of patients with dementia, 58.5% of patients with MCI, and 59% of control subjects were women.

Discussion
It has been well demonstrated that autophagy is linked to neuronal apoptosis and is impaired in neurodegenerative diseases including AD, Parkinson's disease, and Huntington's disease 3,15 . Here, we showed that plasma ATG5 levels were increased both in APP Tg mice and human patients with dementia or MCI compared to healthy control subjects. To the best of our knowledge, this is the first study to determine the autophagic markers ATG5 and ATG12 in plasma from patients with AD.
Increasing evidences have shown that autophagy dysfunction is a common feature in AD. During AD progression, one of the earliest pathological features is the accumulation of autophagosomes 3 . It is well established that in neurodegenerative diseases, abnormal protein degradation through dysregulation of autophagic flux may result in neuronal loss. Indeed, fronto-temporal dementia and amyotrophic lateral sclerosis (ALS) have been linked to mutations in the charged multivesicular body protein-2B 16 . Moreover, polyglutamine aggregates in Huntington's disease have been demonstrated to disrupt autophagy 17 and decreased Beclin-1 levels were observed in sporadic AD 18 . However, the levels of protein components of the autophagy pathway such as ATG5, ATG12 and ATG7 in the brain were not significantly different between controls and AD patients 12 . On the other hand, ATG12-immunoreactive endothelial cells were found spatially associated with Aβ-positive plaques 8 . Consistently, our previous study showed that colocalization of ATG12 and small ubiquitin-related modifier 1 (SUMO1) was increased in the brain of an AD mouse model 19 . Furthermore, Aβ treatment increased conjugation of ATG5-ATG12 in human brain neuroglioma H4 cells and microglia 5,7 . In congruence with these observations, in our current study increased conjugation of ATG5-ATG12 induced by Aβ was also observed in primary rat neurons and endothelial cells, suggesting critical roles of autophagy in AD. This is the first study to determine the levels of autophagic markers ATG5 and ATG12 in endothelial cell medium and in plasma from patients with AD. During disease progression, several proteins are released in the cerebrospinal fluid (CSF) and blood due of brain damage. In the central nervous system (CNS), autophagy can be activated by aging, nutrient deprivation and cerebral ischemia 20 . Recently, a study reported increase in autophagic markers such as LC3B and Beclin-1 in the CSF and peripheral blood in patients with acute ischemic stroke 11 . Moreover, plasma ATG5 levels were higher in the patients with coronary artery disease compared with healthy controls 9 . However, plasma ATG5 levels were lower in cerebral palsy patients compared with controls 10 . Yet, little is known about the regulation of autophagy pathways including autophagosome synthesis and autophagic flux in the blood of patients with AD. In the present study, we investigated alterations in ATG12 and ATG5 in the plasma of AD patients, triggered by the observed increased ATG5-ATG12 conjugation upon Aβ treatments in vitro (Fig. 1).
Our results showed that both LC3 and p62 mRNA levels were increased in human iPSC-derived neuronal cells (Fig. 2C,D). In addition, we confirmed that the levels of LC3 and p62 proteins in endothelial cells cultured in the presence of Aβ, pretreated with bafilomycin A 1 , were even more increased (data not shown). These results imply that Aβ treatment caused accumulation of autophagosomes and increased functional autophagic activity. In consistency with our paradoxical results, a previous study reported that the expression levels of p62 do not always inversely correlate with autophagic activity 21 . Indeed, until now there is no single "gold standard" in measuring autophagic flux. The p62 protein levels correlates with both activation and suppression of autophagy 22 . The autophagic system is highly dynamic and consists of a sequence of steps, each characterized by its own rate 23 . In this study, Aβ was demonstrated to increase autophagic activity and could also affect the autophagic steps, altering the rate of protein degradation through the entire autophagic pathway.  Table 3. Correlation between plasma biomarkers and clinical rating scales. Spearman rank correlation coefficient test was used for assessment of correlation.
www.nature.com/scientificreports www.nature.com/scientificreports/ In addition, ATG5 levels after Aβ treatment were increased in the endothelial cell medium (Fig. 4). Plasma ATG5 levels were also increased in patients with AD (Table 2). This is a very interesting observation because an autophagic biomarker, ATG5, may be a potent candidate as a blood biomarker to reflect autophagy alterations in AD.
In conclusion, our data confirmed that the autophagic marker ATG5 is associated with AD pathophysiology. Additionally, we observed that plasma ATG5 levels in patients with AD were increased and associated with CDR, suggesting ATG5 as a potential biomarker in those at risk for AD.

Methods
Subjects. The obtained subjects used in this study were selected from the population-based Ansan Geriatric (AGE) cohort, Korea [24][25][26] . Dementia and MCI diagnoses were established by a Korean version of Consortium to Establish a Registry for Alzheimer's Disease (CERAD-K) neuropsychological battery 27 . All participants were clinically assessed according to published guidelines, and each dementia patient met the criteria for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition 28 . All dementia patients met the criteria for probable AD established by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) 29 . Diagnosis of MCI was based on the Mayo Clinic criteria 24 as described previously 30,31 . In total, blood samples from 278 subjects were collected, and the distribution on each of the subjects are shown in Table 1. Global clinical dementia rating (CDR) scores are 0 for normal, 2 and 3 for moderate to severe dementia 32 . All participants provided written informed consent and the study has been approved by the Institutional Review Board (IRB) of the Korea Centers for Disease Control and Prevention (KCDC). All experiments were conducted in accordance with relevant guidelines and regulations.
Cell cultures. Human umbilical vein endothelial cells (HUVECs) purchased from Lonza (Walkersville, MD, USA) and cultured in Endothelial Growth Medium-2 (EGM-2)-MV BulletKit (Lonza). HUVECs were used at passages 5 to 9 for experimentation. Human iPSC-derived neural progenitor stem cells were purchased from Axol Bioscience (Little Chesterford, UK) and were differentiated to cerebral cortical neurons following the recommended manufacturer's protocol 26 . Primary cortical neuronal cells obtained from cortexes of embryonic rat brains were maintained in neurobasal medium supplemented with B27 (Invitrogen) as described previously 33 . Animals. APPsw/PS1ΔE9 transgenic mice were used for present study, as previously reported 19 . All animal experiments were performed in compliance with the guidelines for the care and use of laboratory animals by the Korea Centers for Disease Control and Prevention (KCDC) and approved by the Institutional Animal Care and Use Committee (IACUC) of the KCDC.

Measurement of autophagic markers by ELISA.
All the cell-free plasma samples were aliquoted and stored at −80 °C until assayed collectively for ATG5 and ATG12 by an investigator who was blinded to patient assignment. The levels of ATG5 and ATG12 were determined by the enzyme-linked immunosorbent assays kit following the manufacturer's instructions (USCN, Wuhan, China).
Immunohistochemistry and Congo Red Staining. Brains from 16-month-old APP Swedish/PS1dE9 transgenic (Tg) mice together with their wild-type controls were fixed in 4% (w/v) paraformaldehyde. Cryostat sagital sections were cut on a sliding microtome into 10 μm slices at −20 °C and placed on a microslide for immunostaining. The cortex sections were immunostained with rabbit monoclonal antibodies against ATG12 (1:100, Cell Signaling Technology, 4180). When required, immunolabeled sections were then incubated for 3 min in a