Growth Arrest Specific 6 Concentration is Increased in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease

INTRODUCTION

Alzheimer’s disease (AD) is the most common neurodegenerative disease and the main cause of dementia worldwide [1].

AD pathogenesis involves several mechanisms including protein dysfunction and deposition, immune and inflammatory reactions, synaptic impairment, and neuronal loss [2–4]. Such interrelated events likely begin years before the onset of clinical manifestations, such as memory impairment, followed by loss of acquired skills, and personality changes [5]. Senile plaques and neurofibrillary tangles, caused respectively by extracellular Aβ peptide deposition and intracellular aggregation of hyperphosphorylated and oxidized tau protein, are the neuropathological hallmarks of the disease [5]. There is convincing evidence that protein aggregation and deposition are accompanied by a complex immune and inflammatory response, involving lymphocytes, cytokines, chemokines, and especially microglia, the latter being the arm of the monocyte-macrophage system in the brain [6].

Growth arrest specific 6 (Gas6) is a multimodular secreted protein and the natural ligand of Tyro-3, Axl, and Mertk, collectively known as the tyrosine kinase receptor family TAM [7–10]. All three receptors are expressed in the central nervous system (CNS), both in progenitor and in differentiated neuronal cells [11]. Notably, the absence of both Axl and Mertk receptors determines early differentiation and migration of neural stem cells (NSC) in the subventricular zone (SVZ) [11], whereas knockout of Gas6 decreases the number of NSC in the SVZ [12]. Also Gas6 is extensively expressed in the CNS, suggesting that interactions between Gas6 and its receptors are likely to have physiologically relevant functions [13]. Accordingly, several reports have linked Gas6/TAM to both neurotrophic and neuroimmune functions [14, 15]. On the one hand, CNS Gas6/TAM expression is proportional to synaptogenesis [16, 17], and their signaling stimulates proliferation and inhibits apoptosis of hippocampal and neocortical neurons [16, 18]. On the other hand, Gas6 was shown to exert an anti-inflammatory function, which involves the microglia component [14].

Given these premises, the aim of this work was to verify if Gas6 is overexpressed in the cerebrospinal fluid (CSF) of AD patients compared to controls, and to correlate Gas6 CSF concentration to clinical parameters, such as disease stage and progression of cognitive decline.

PATIENTS AND METHODS

Sixty-three patients with a diagnosis of AD [19], and 67 controls were enrolled in the study. Clinical and demographic features are summarized in Table 1. Patients underwent a standard evaluation, including medical history, physical and neurological examination, screening laboratory tests, neuropsychological evaluation, brain MRI, and, when indicated, PET scan. Moreover, for all patients, we calculated the time between the first complaint and the diagnosis. CSF was always collected at diagnosis, and all subjects underwent both routine CSF study and AD biomarkers assay (Aβ₄₂ and tau levels). Either patients or caregivers gave informed consent to lumbar puncture and to CSF analyses for both diagnostic and research purposes. After recruitment, follow-up information confirmed the initial diagnosis of AD for all patients. Furthermore, 50 patients completed a two-year follow-up, during which their cognitive status was recorded every six-months by Mini-Mental State Examinations (MMSE) [20].

The control group was composed by 67 subjects matched for age, gender, and ethnic background, who came to our attention due to acute headache, compressive radiculopathy, or non-immune mediated peripheral neuropathy and who underwent lumbar puncture during their diagnostic workup. No control had memory complaints, signs of inflammatory, neoplastic, or neurodegenerative disorders, or family history of neurodegenerative diseases. All subjects gave informed consent to participate in the study, which was approved by the local ethical committee and was conducted in strict accordance with the principles of the Declaration of Helsinki.

CSF samples were obtained by lumbar puncture at the L4/L5 or L3/L4 interspace, centrifuged at 4°C and stored at –80°C until analysis. CSF cell counts, glucose, and proteins were determined. Albumin was measured by rate nephelometry. To evaluate the integrity of the brain-blood barrier (BBB) and the intrathecal IgG production, the albumin quotient (CSF albumin/serum albumin)×10³ and the IgG index (CSF albumin/serum albumin)/(CSF IgG/serum IgG) were calculated [21]. Aβ₄₂, tau, and P-tau CSF levels were determined with human specific ELISA kits (Innogenetics), as previously reported [22]. Cut-off values for diagnostic biomarkers were: Aβ₄₂ > 600 pg/ml; tau < 350 pg/ml (between 50 and 70 years of age),<550 pg/ml (above 70 years of age); P-tau<61 pg/ml; tau/Aβ₄₂ > 0.52 [22, 23].

CSF Gas6 concentration was measured using a validated custom-made ELISA kit, already used in human studies for plasma and CSF assay [24–26]. A 96-well plate (NUNC ImmunoPlates MaxiSorp F96, NUNC, Hereford, UK) was coated overnight with anti-Gas6 primary antibody (goat polyclonal affinity purified IgG, R&D Systems, Minneapolis, USA). The antigen was detected by a secondary biotin conjugated antibody (Biotinylated anti human Gas6 antibody, R&D Systems, Minneapolis, USA), and a streptavidin-peroxidase conjugate (Sigma, St. Louis, MO, USA) and TMB (3,3^′,5,5^′-tetramethylbenzidine, Sigma, St. Louis, MO, USA). The reaction was blocked with sulphuric acid 1.8 M and absorbance detected at 450 nm with a reference wavelength set at 570 nm. Optical density was fitted versus nominal concentration by applying a four-parameter logistic regression to the calibration curve prepared in BSA (Bovine serum albumin, further purified fraction V, ≥98%, Sigma, St. Louis, MO, USA). The method has been validated according to the Food and Drug Administration guidelines for inter- and intra-assay % coefficient of variation (% CV) for Gas6 measurement in human plasma and CSF (all % CVs were within 15% with negligible matrix effect). The lowest quantification limit was 0.26 ng/ml [24].

RESULTS

DISCUSSION

Our study provides the first evidence of Gas6 involvement in AD, demonstrating that CSF Gas6 levels are significantly increased in AD patients compared to controls. Moreover, Gas6 increase was more pronounced in the early disease phases, and patients with higher CSF Gas6 levels at diagnosis displayed a less pronounced cognitive deterioration over a two-year follow-up. Taken altogether, such findings suggest a protective function of Gas6 in AD, which may be ascribed to different yet synergistic functions.

Firstly, Gas6 is involved in neurotrophic functions [27]. TAM signaling favors neuronal stem cell (NSCs) survival and growth and prevents NSCs apoptosis. Furthermore, NSCs derived from triple TAM knock-out (TKO) mice show impaired differentiation toward neurons, being on the contrary preserved the differentiation in astrocytes; these actions are, at least partially, due to the regulation of neurotrophin expression. Accordingly, the mRNA and protein levels of nerve growth factor are significantly inhibited in the hyppocampi and cultured NSCs of TKO mice [28]. Gas6 plays also a double protective role in the context of neuronal apoptosis: i) it counteracts the detrimental intracellular increase of Ca²⁺ induced by Aβ [18, 29–31], ii) it favors apoptotic cell uptake by macrophages through a highly specialized process, in which the binding of Gas6 to TAM receptors allows the uptake of the apoptotic bodies, without eliciting a detrimental immune response [32–34].

A second important function related to Gas6 signaling is the control of inflammation. Inflammatory processes play a complex role in AD. On the one hand, activated microglia can display a pro-inflammatory M1 phenotype, exerting detrimental effects [35]. Both Aβ and tau trigger M1 microglia activation and release of pro-inflammatory/cytotoxic molecules such as nitric oxide (NO), reactive oxygen species (ROS), IL-1β, IL-6, IL-18, and TNFα, thereby causing neuronal death [36–38]. On the other hand, there is also evidence of a compensatory action of inflammation in AD. In this context, microglia assume a protective M2 phenotype, which is able to degrade extracellular Aβ plaques through the secretion of proteolytic enzymes [39] and clear the deposits through phagocytosis [40, 41]. For that reason, an interesting point was to explore the relationship between CSF Gas6 concentration and biomarkers of AD pathology, and particularly CSF Aβ₄₂ levels. Nonetheless, our findings showed no correlation between CSF Gas6 and both Aβ₄₂ and tau CSF levels, likely suggesting that Gas6 pathway has no direct influence on circulating biomarkers.

Macrophage colony-stimulating factor (M-CSF) can activate the phagocytic activity of M2 microglia [42]. This notion is supported by the finding of lower M-CSF concentrations in patients with mild cognitive impairment (MCI) converting to AD compared to stable MCI patients [43]. Microglia express TAM receptors [14] and work from our group showed that Gas6 signaling down-modulates TNF-α, IL-6, and IL-1β expression in monocyte/macrophage lineage [44]. Evidence supporting a role of Gas6/TAM signaling on microglia comes from experiments on TKO mice. Indeed, it was shown that microglia derived from TKO mice show hyper-reactivity to toll like receptor stimuli, leading to excessive pro-inflammatory cytokines production (IL-1β, IL-6, TNF, and iNOS) [40]. On the contrary, pre-treatment of wild type mice with Gas6 significantly reduces LPS-induced upregulation of pro-inflammatory cytokines, which has a detrimental impact on NSCs survival and differentiation [45]. In agreement with these observations, TKO mice develop inflammatory injury in the brain [46]. Thus, the early increase of CSF Gas6 concentrations is more likely the result of a protective reaction. Indeed, in our study, the two-year follow-up of 30 AD patients who underwent CSF study at a very early disease phase showed an inverse correlation between baseline Gas6 levels and cognitive deterioration, suggesting a protective role on disease progression. Similar findings have been reported in multiple sclerosis, where CSF Gas6 levels had an inverse correlation with relapse severity and duration, further suggesting a protective role of this pathway in neuroinflammatory conditions [47]. Whether inflammation is detrimental or protective in AD, it is likely that most inflammatory phenomena take place in the early disease stages, when neuronal loss is still limited [48–50]. Accordingly, it was shown that increased Axl CSF levels pre-date future Aβ₄₂ CSF decrease in cognitively healthy people, suggesting a potential prediction of AD development [51].

On a theoretical level, the observed increase of CSF Gas6 concentration in AD patients showing better MMSE performance might have been driven by other factors. Cognitive reserve (CR) is an important compensatory mechanism of dementia [52], and its relationship with anti-inflammatory molecules has not been explored so far. Here we tested whether a component of CR, i.e., years of formal schooling, had an influence on CSF Gas6 concentration. Our analysis showed that the two parameters were not correlated, suggesting that they are biologically independent. These findings are in line with Vemuri et al. who showed that CR and biomarkers of AD pathology are independent predictors of cognitive performance [53].

In conclusion, Gas6 increase in AD may be part of a compensatory reaction aimed at down-regulating pro-inflammatory cytokines production, favoring amyloid clearance, and promoting a regenerative response. Nonetheless, further studies with a larger population and long term follow-up are necessary to better characterize the role of Gas6 pathway in AD and to confirm its protective role on cognitive deterioration.