Modulation of RAS and PI3-AKT pathway by Stavudine (d4T) in PBMC of Alzheimer’s Disease Patients

Background: Aβ 42 -deposition plays a pivotal role in AD-pathogenesis by inducing the activation of microglial cells and neuroinammation. This process is antagonized by microglia-mediated clearance of Aβ plaques. Activation of the NLRP3 inammasome is involved in neuroinammation and in the impairments of Aβ-plaques clearance. Stavudine (d4T) on the other hand down-regulates the NLRP3 inammasome and stimulates autophagy-mediated Aβ-clearing in a TPH-1 cell line model. We explored the effect of d4T on Aβ-autophagy using PBMC of AD patients that were primed with LPS and stimulated with Aβ in the absence/presence of d4T. We analyzed the NLRP3 activity by measuring NLRP3-ASC complexes formation by AMNIS Flow-sight and pro-inammatory cytokines (IL-1β, IL-18 and Caspase-1) production by ELISA. Western blot analyses were used to measure phosphorylation and protein expression of p38, CREB, ERK and AKT, p70, LAMP 2A, Beclin-1 and Bax. Results: Data showed that d4T: 1) down regulates NLRP3 inammasome activation and the production of down-stream proinammatory cytokines even in PBMC; 2) stimulates the phosphorylation of AKT, ERK, p70 as well as LAMP2A production, but does modulate beclin-1, suggesting a selective effect of this compound on chaperone-mediated autophagy (CMA); 3) up regulates p-CREB and BAX, possibly diminishing Aβ-mediated cytotoxicity; and 4) reduces the phosphorylation of p-38, a protein involved in the production of pro-inammatory cytokines. Conclusions: d4T reduces the activation of the NLRP3 inammasome and stimulates CMA autophagy as well as molecular mechanisms that modulate cytotoxicity and reduce inammation in cells of AD patients. It might be interesting to verify the possibly benecial effects of d4T in the 1,500 for resuspended in of PBS, and examined using the AMNIS FlowSight. Results were analyzed by IDEAS analysis software The analysis of NLRP3 expression was performed by internalization feature utilizing a mask representing the whole cell, dened by the brighteld (BF) image, and an internal mask dened by eroding the whole cell mask. Apoptosis-associated speck-like protein containing CARD speck formation was analyzed using the same mask of internalization feature, differentiating diffuse or spot (speck) uorescenceinside of cells. Threshold mask was used to separate all ASC positive cells population in ASC-Speck spot cells or ASC-diffuse cells by the different


Background
Alzheimer's disease (AD), the most prevalent type of dementia, is characterized by the deposition of amyloid-β (Aβ), the formation of neuro brillary tangles, and neuroin ammation [1]. Thus, in AD patients Aβ accumulation induces the release of in ammatory mediators by the microglia; this facilitates Aβ deposition and neuroin ammation in a self-feeding pathogenic loop [2]. Microglia, on the other hand, plays an important role against AD that is mediated by the generation of Aβ-speci c antibodies, the clearance of amyloid plaques, and the recruitment of peripheral immune cells that cross the blood-brain barrier (BBB) in an attempt to remove Aβ aggregates [3][4][5]. The initiation of the in ammatory response by microglia and peripheral immune cells involves cytosolic multiprotein platforms known as in ammasomes. The NLRP3 in ammasome is the best characterized of such platforms and its formation requires multiple steps. In a priming step, transcriptionally-active signalling receptors induce the NF-kB-dependent induction of NLRP3 itself, as well as that of the caspase-1 substrates pro-IL-1β and IL-18 [6]; a second signal leads to the assembly of a multimolecular complex with ASC and Caspase-1. At this point procaspase-1 or procaspase-8 are recruited [7], ASC specks are formed [8], caspases are activated, and cytokines are produced.
Activation of the NLRP3 in ammasome is a tightly regulated process; autophagy, in particular, an adaptive response to stress, down regulates NLRP3 in ammasome activation by a complex metabolic mechanism [9] that includes different molecular pathways [10][11][12][13][14]. Notably, autophagy defects in myeloid cells results in the aberrant activation of the in ammasome [15,16], leading to development of in ammatory disorders. Different compounds can modulate the NLRP3 in ammasome activation as well Stavudine (d4T), a nucleoside reverse transcriptase inhibitor (NRTIs), in particular was shown to hamper NLRP3 in ammasome activation as well as caspase-1 and IL-18 production both in vivo [17,18] and in a TPH-1 cell line in vitro model [19]. Notably, in this model d4T did not have any effect on Aβ-phagocytosis, but it stimulated autophagy-mediated Aβ-clearing [19], as witnessed by its ability to modulate the ERK1/2 and AKT pathways and to upregulate LAMP-2A and p70-S6K, their downstream targets.
Given the fact that: 1) autophagy is necessary for the degradation of Aβ in microglia; 2) p-AKT [20] and p-ERK [21] associate with Aβ-accumulation and τ-phosphorylation in AD animal models [22,23]; and 3) autophagy is modulated by d4T [19], we explored more in details the mechanisms responsible for d4Tmediated autophagy clearing of Aβ. To this end we used peripheral blood mononuclear cells of AD patients that were stimulated with LPS + Aβ in the absence/presence of d4T.

Patient's characteristics
Epidemiological, clinical and genetic characteristics of AD patients enrolled in the study are presented in Table 1. CSF biomarkers analysis were analyzed in all the patients and included the concentration of Aβ 42 , t-t, and p-t (i.e. phosphorylated at threonine 181 or p-t 181p). Each CSF biomarker was dichotomously classi ed as positive or negative according to validated cut-off values: Cut-off thresholds of normality were: Aβ ≥ 600 pg/ml; t ≤500 pg/ml for individuals older than 70; Ptau ≤ 61 pg/ml [24]. Results are shown in Table 1. 2. d4T down-regulates NLRP3-complex activation and in ammasome derived cytokines d4T was shown to signi cantly reduce the activation of the NLRP3 in ammasome in a THP-1-derived macrophage cell line that was LPS-primed and Ab 42 -stimulated; we veri ed whether the same effect could be observed in PBMC of AD patients. Results obtained when NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC)-speck formation was investigated showed that ASC speck formation was signi cantly increased in LPS-primed and Ab 42 -stimulated compared to unstimulated cells (p< 0.05) (Fig. 1a), and indicated that ASC-speck positive cells were signi cantly decreased by d4T (p< 0.05) (Fig. 1b). d4T prevented the generation of ASC-specks, impeding the assembly of NLRP3in ammasome complexes: the percentage of cells co-localizing ASC and NLRP3 i.e. cells in whom a fully functional NLRP3 in ammasome complex is formed, was signi cantly reduced (p < 0.05) in LPS+Ab 42 -stimulated cells in the presence of d4T (Fig.1c). These results were con rmed when activated Caspase-1,IL-18 and IL-1b production was analyzed. Thus, IL-1b, IL-18 and activated Caspase-1 production was signi cantly increased in LPS+Ab 42 stimulated PBMC of AD patients (p < 0.05)(data not shown) and the production of all these proteins was reduced by d4T ( Fig. 2 a,b,c); the differences reached statistically signi cance for IL-18 (p = 0.004) ( Fig. 2. a) and activated caspase-1 (p = 0.001) ( Fig. 2.b).
3. Effect of d4T on autophagy-pathways Interaction of NLR domains with autophagy proteins provides a mechanism for direct NLR regulation of autophagy. In particular, in ammasome-forming NLRs were interact with beclin1, a pivotal protein in autophagy; we thus next investigated the ability of d4T to modulate authophagy.
Beclin-1, and phospho-p70S6K signals were examined using WB analysis in protein extract of PBMCs from AD patients that were either unstimulated (med) or LPS-primed and Ab 42 stimulated in the absence/presence of d4T. Beclin-1, an index of the activation of p-ERK1/2-and p-AKT-mediated macroautophagy was only marginally increased by d4T in LPS-primed and Ab 42-stimulated-PBMCs (Fig.3a).
Phosphorylation of p70S6Kinase, their down-stream target, on the other hand, was signi cantly increased by d4T (p < 0.005). Notably, both phospho-p70S6K isoforms: the 70 KDa cytosolic form and the 85KDa nuclear one, were signi cantly upregulated by d4T (Fig 3b).

Modulation of MEK-(ERK and p38) and AKT-Protein Kinesis pathways by d4T
The NLRP3 in ammasome can also negatively regulate autophagy by reducing the expression of phosphatases and kinases. This was analyzed by evaluating ERK, p38 and AKT phosphorylation in PBMC of AD patients that were either unstimulated (med) or had been LPS-primed and Ab 42 stimulated in the absence/presence of d4T. Results showed that all these proteins was increased in LPS+Ab 42 stimulated PBMC. Addition of d4T to cell cultures signi cantly down-modulated p38 (p = 0.0008) (Fig 4a) whereas it further up-regulated p-ERK1,2 (p = 0.025) (Fig. 4b) and p-AKT (p = 0.015) (Fig 4c). Finally, LAMP2A, a protein in the AKT-mediated pathway that plays a pivotal role in chaperon (CMA) mediated autophagy, was signi cantly increased by d4T in LPS-primed and Ab 42-stimulated-PBMCs (p=0.0007) ( Fig. 5).

Downstream MEK and AKT pathways: CREB and Bax
Protein expression of CREB and Bax, two essential downstream mediators in the ERK and AKT-mediated pathways, as well as CREB phosphorylation (p-CREB) status were analyzed next in the same cultural conditions. Also in this case, the level of these proteins was increased in LPS+Ab 42 stimulated PBMC.

Discussion
Microglia-driven neuroin ammation has been identi ed as a key player in the pathogenesis of AD; deposition of Aβ 42 in particular is an early event in the disease that induces microglia-activation and in ammation. It has been shown that this process is highly dependent on the activation of the NLRP3/ASC-in ammasome both in microglial cells [25,26] and in circulating peripheral monocytes [27].
Thus, systemic in ammation triggers a neuroin ammatory response that results in microglial activation with deleterious consequences for learning and memory in rodent models [28][29][30] and in humans [31][32][33][34][35][36][37]. Neuroin ammation has also convincingly been shown to down regulate autophagy [38][39][40] we have recently described how NLRP3/ASC-in ammasome activation hampers microglial clearance of Aβ in an in vitro experimental model of macrophages-derived THP-1 cell line [19]. Notably, in that experimental setting Stavudine (d4T), a prototypical NRTI designed used in HIV-infection prevented active Caspase-1 release but it did not restore microglial Aβ phagocytosis. Given this background, we investigated Aβstimulated in ammation and autophagy and the effects of d4T on these processes using peripheral blood immune cells of AD patients.
Results herein show that d4T is capable of signi cantly reducing NLRP3 in ammasome activation and the down-stream production of IL-18 and Caspase-1 even in PBMC of AD patients, but had only a marginal effect on IL-1β. Recent results showed that gasdermin D, a substrate for caspases that mediates the activation of pyroptosis no NLRP3-dipendent, induces the formation of pores in the plasma membrane. Because pores can serve as a gate for the release of mature IL-1β from cells [41,43], this could explain the different behavior of IL-18, Caspase-1 and IL-1β.
Results obtained when autophagy was analyzed showed that p-AKT-and p-ERK as well as the expression of Bax, p-CREB, LAMP2A, and of the cytosolic and nuclear p70S6 Kinase isoforms was increased in d4Tstimulated PBMC of AD patients, indicating that d4T up regulates the downstream AKT-and MEK-kinase pathways. In contrast Beclin-1 was modulated by d4T, suggesting that this compound mainly stimulates chaperone-mediated autophagy (CMA).
Autophagy is a complex molecular system that removes damaged organelles and proteins from the cytoplasm and is involved in programmed cell death and neurodegeneration; importantly, autophagy also represents an alternative pathway of cellular defense by removing intracellular pathogens [44]. The three main forms of autophagy are: macroautophagy-beclin-1 dependent, microautophagy endosomal mediated, and CMA the latter is associated with lysosomal degradation and is mediated by a cytoplasmic complex of chaperone proteins, including p70S6K, that interacts with LAMP2A [45,46]. The phosphorylation of p70S6K by AKT signal is also able to interact with Atg proteins, promoting phagosoma formation in mTOR-independent way [47]. All these three forms of autophagy crosstalk with each other to eliminate aberrant proteins and modulating cell apoptosis [48].
Emerging evidences show that CMA is involved in the degradation of several proteins associated with neurodegenerative diseases [46] including α-synuclein [48,50], huntingtin [51] and amyloid precursor protein [52]. Degradation by CMA involves the selective delivery of single proteins to lysosomes upon the recognition and binding of a speci c KFERQ-like pentapeptide motif to cytosolic hsp70 [53]. The chaperone-substrate complex binds the cytosolic tail of LAMP2A, triggering its assembly into a multimeric complex (protein target, KFERQ, p70S6K and LAMP2A) that mediates substrate translocation in lysosome [54,55]. CMA is modulated by the AKT and ERK signaling pathways: p-AKT-and p-ERK; both these proteins were increased by d4T in Aβ-stimulated PBMC of AD patients, as were Bax, p-CREB, p-p70S6K and LAMP2A. Because active AKT can phosphorylate a variety of downstream molecules including Bax and CREB, these results indicate that d4T can up regulate this whole signaling pathways.
d4T-induced modulation of CREB is particularly important as this protein has a well-documented role in neuronal plasticity, and long-term memory formation, and, on the other hand, CREB alterations result in oxidative stress, apoptosis, and neurodegeneration [56]. Importantly, p-CREB expression is reduced in PBMC of AD subjects [57,58]. d4T-induced Bax upregulation is potentially important as well, as Bax is a pro-apoptotic protein that promotes mitochondrial membrane permeabilization. d4T-mediated up regulation of p-CREB and Bax could thus stimulate CMA and apoptosis to counteract Aβ toxicity. Notably, behavioral symptoms and progression of AD in a murine model were shown to be alleviated by the activation of PI3K/AKT signaling pathway [59]. Increased p-AKT expression and activation of Baxmediated apoptosis were also demonstrated to improve the memory capacity of AD rats [60].
Potentially important is also the effect of d4T on p38. Thus, p38 is activated in macrophages, neutrophils, and T cells by extracellular mediators of in ammation including cytokines, chemokines, and LPS [61][62], and phospho-p38 induces Th1 immune response and the production of proin ammatory cytokines [63]. The observed d4T down-regulation of this protein thus could be bene cial in reducing neuroin ammation in AD.

Conclusions
E cient autophagic activity prevents the activation of in ammasomes [64], and stimulation of autophagy was shown to reduce soluble τ, as well as Aβ and amyloid plaques in 3xTgAD mice [65]. These results will need to be con rmed, possibly using animal models of AD. In conclusion, herein we show results obtained in peripheral immune cells of AD patients that indicate how d4T, beside reducing NLRP3 in ammasome activation, up-regulates CMA as well as CREB phosphorylation and the proapoptotic protein Bax. Taken together, these ndings could warrant the investigation of the use of D4T the clinical scenario.

Patients
Thirteen AD patients who ful lled inclusion criteria for a clinical diagnosis of AD were randomly selected within a large database of patients consecutively admitted between January 2017 and September 2019 by the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico in Milano, Italy. All patients were enrolled in a cognitive rehabilitation experimental program and underwent complete medical and neurological evaluation, laboratory analysis, CT scan or MRI, and other investigations (e.g., EEG, SPET scan, CSF examination, etc.) to exclude reversible causes of dementia. The clinical diagnosis of AD was performed according to the NINCDS-ADRDA work group criteria [66] and the DMS IV-R (American Psychiatric Association) [67]. Neuropsychological evaluation and psychometric assessment were performed with a Neuropsychological Battery that included Mini Mental State Examination (MMSE) [68].

APO e4 genotyping
Apoe genotype was determined by allelic discrimination as previously described [70]. APOE genotyping was available for all subjects and was dichotomized as being a carrier of at least one allele ε4 or carrying no ε4 allele.
CSF collection and Aβ and tau determination CSF was collected between 8 and 10 a.m. after one-night fasting by LP in the L3/L4 or L4/L5 interspace according to standardized local procedures. CSF samples were centrifuged in 1500 rpm for 10 minutes at 4C°. The supernatants were aliquoted in polypropylene tubes and stored at -80 °C. CSF cells, glucose, and proteins were determined. CSF Aβ, tau and P tau were measured using commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits (Fujirebio, Ghent, Belgium).

Blood sample collection and processing
Whole blood was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA) and washed twice in PBS at 1500 RPM for 10 min; viable leukocytes were determined using a TC20 Automated Cell Counter (Biorad Hercules,California, USA).
d4T Cellular toxicity Viability of PBMC incubated with d4T was determined using the MTT 3-(4,5-dimethylthiazol-2,5diphenyltetrazolium bromide) (Sigma-Aldrich) assay, as previously described [71].    Figure 1 In ammasome production and ASC-Speck formation. Images from a single representative experiment of Nod-like receptor protein 3 (NLRP3) expression and apoptosis-associated speck-like protein containing CARD (ASC)-speck formation in LPS-primed and A -stimulated PBMCs of 13 AD patients in the presence/absence of d4T. ASC-speck (a) and ASC-diffuse (b) images were obtained by AMNIS FlowSightmerged (Ch02/03) NLRP3-FITC (Ch02) and ASC-PE (Ch03) uorescences; analysis of ASC-specks and its co-localization with NLRP3 were performed by IDEAS software that provides tools to evaluate image regions (masks) and perform calculations (features). The percentage of ASC speck positive cells is shown in panel c. The statistical analysis were performed; signi cance was calculated by t-test (unpared) and was shown (*p <0.05).

Figure 2
Caspase-1 and in ammasome effector cytokines production. IL-18 (a), Caspase-1 (b) and IL-1 (c) production by unstimulated (medium) or by LPS+A -stimulated PBMCs of 13 AD patients in the presence/absence of d4T. Data are expressed as median of cytokines and caspase-1 concentration (pg/ml) of stimulated cells medium subtracted. The statistical analysis were performed; signi cance was calculated by t-test (unpared) and was shown (*p <0.05).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table1.pdf