Alzheimer's Disease (AD) and Type 2 Diabetes (T2D) share a common hallmark of insulin resistance. Reportedly, two non-canonical Receptor Tyrosine Kinases (RTKs), ALK and RYK, both targets of the same micro RNA miR-1271, exhibit significant and consistent functional down-regulation in post-mortem AD and T2D tissues. Incidentally, both have Grb2 as a common downstream adapter and NOX4 as a common ROS producing factor. Here we show that Grb2 and NOX4 play critical roles in reducing the severity of both the diseases. The study demonstrates that the abundance of Grb2 in degenerative conditions, in conjunction with NOX4, reverse cytoskeletal degradation by counterbalancing the network of small GTPases. PAX4, a transcription factor for both Grb2 and NOX4, emerges as the key link between the common pathways of AD and T2D. Down-regulation of both ALK and RYK through miR-1271, elevates the PAX4 level by reducing its suppressor ARX via Wnt/β-Catenin signaling. For the first time, this study brings together RTKs beyond Insulin Receptor (IR) family, transcription factor PAX4 and both AD and T2D pathologies on a common regulatory platform.

Introduction

Epidemiological studies show that type 2 diabetes (T2D) increases the risk of Alzheimer's Disease (AD) by at least 2-fold, although there are only a few mechanistic models that provide a clear pathophysiological link [1]. PET and MRI studies show marked impairment of glucose and energy metabolism in both T2D and AD [2], amyloidogenesis being a salient feature in both. Reportedly, diabetic mice overexpressing islet amyloid polypeptide (IAPP) develop oligomers and fibrils with more severe diabetic traits, akin to AD mice models overexpressing amyloid precursor protein (APP) [3]. Additionally, traits like insulin resistance, altered amyloid metabolism, synaptic dysfunction, activation of the inflammatory response pathways and impairment of autophagy have been shown as common pathological features in both the diseases [4]. With the understanding that several pathological signals are being shared by AD and T2D, AD has been suggested to be a neuroendocrine disorder that resembles T2D [5].

Insulin receptor (IR), a typical receptor tyrosine kinase (RTK), further links both of them through resistance to insulin and other metabolic imbalances. Our recent study lists the differential activities of several human RTKs in post-mortem brain tissues of AD patients and liver tissues of T2D patients and categorizes them into functional and regulatory clusters [6]. In this context, we have further shown that two RTKs, Anaplastic Lymphoma Kinase (ALK) and Receptor-like Tyrosine Kinase (RYK), functionally behave in a similar fashion in both the disease situations [6]. Both ALK and RYK are involved in the regulation of Wnt/β-Catenin signaling [7–9], which behaves aberrantly in both AD and T2D [10]. In AD, Aβ increases GSK3β activity by inhibiting the canonical Wnt Signaling [11]. GSK3β is one of the kinases that hyperphosphorylate Tau protein, leading to the formation of neurofibrillary tangles of AD [12]. In turn, hyperphosphorylated Tau dissociates from microtubule and leads to the cytoskeleton instability [10–12]. In addition, both isoforms of GSK3 (GSK3α and GSK3β) play crucial roles in the insulin signaling of T2D [13]. Also, the non-canonical Wnt/planar cell polarity (PCP) signaling is correlated with the cytoskeleton stability through Wnt/RYK/Dvl/DAAM1/RhoA pathway [14].

Besides these pathways and signaling modalities, Reactive Oxygen Species (ROS) mediated oxidative stress has also been shown to be a common trigger in both the diseases [15]. A ROS producing NADPH oxidase, NOX4, is constitutively active with its regulatory protein tyrosine kinase substrate (Tsk4/5) with multiple SH3 domains [16] and interacts with Growth factor receptor-bound protein 2 (Grb2) naturally [17,18]. Phosphorylation of Nox4 Tyr-491 after IGF-I stimulation is responsible for Nox4 binding to the SH2 domain of Grb2 [18]. Reportedly in AD post-mortem brain, in AD cell models as well as in AD mouse model (APP/PS1 mouse), Grb2 transcript is significantly up-regulated [19]. In AD brain, the NOX4 expression is significantly increased with substantial correlation between its activity and age-dependent increases of Aβ and cognitive dysfunction [20,21]. In T2D, pancreatic β-cell dysfunction is promulgated by NOX4 over-activity and is sufficient to induce insulin resistance [22]. Growing number of evidence suggest that Nox4-derived ROS contributes to oxidative stress during the initial and chronic stages of T2D [23].

A third factor of commonality are the causative oligopeptides like Amylin and Aβ. The role of human Amylin was first observed as early in 1901, when it was described as hyaline deposits in the pancreatic islets of T2D patients [24]. Amylin is a Calcitonin family peptide and it tends to interact with GPCRs of class B [25] in non-disease conditions. However, the interaction pattern of misfolded or aggregated Amylin in disease conditions is still unclear. On the other hand, Aβ oligomers interact with the extracellular domain of Metabotropic glutamate receptors (mGluR5), which is another GPCR of class C [26]. More recently Amylin has emerged as a crucial component in AD pathogenesis. Both Amylin and Aβ interact with each other and lead to the formation of cross-seeded oligomers, where Amylin acts as the seed for Aβ aggregation [27–33].

It would be intuitive to think that the microRNAs, transcription factors (TF) and adapter proteins might stitch these factors together. Chanda et al. [34] had deduced several miRNAs from the bioinformatics analysis of the small RNA sequence data of AD like cell model, where AICD was transiently transfected and Aβ was treated externally in SHSY5Y cells. The analysis showed alteration of the expression of miR-1271 [34] among others, in the AD like situation. Reportedly, miR-1271 is up-regulated in palmitate induced insulin resistance in HepG2 cell lines [35]. In recent times the therapeutic potential of a cell-lineage specific TF, Paired Box Protein 4 (PAX4), is under scanner for T2D treatment [36]. A previous study showed elevated expression of PAX4 in islets derived from T2D donors owing to high circulating blood glucose [37]. PAX4 regulates the development of islets of Langerhans by increasing the β and δ-cells population [38]. Concomitantly, another TF, Aristaless Related Homeobox (ARX), up-regulates the α-cell population while reducing the β and δ-cells proliferation [39]. Furthermore, PAX4 and ARX are not only antagonistic functionally, they negatively regulate the transcriptions of their own genes [39,40]. PAX4 has also been shown to be involved in inducing regenerative capacity in insulin-positive islet cells in mice [36,41]. PAX4 mutation have been implicated in T2D [42] and its overexpression led to β-cell proliferation and apoptosis reduction [43]. Only very recently PAX4 has been implicated in denervation [44] and neurodegeneration, especially in Parkinson's disease models [45]. Reportedly, ARX mutation is associated with neurodegenerative and neurodevelopmental disorders through the impairment of the Wnt/β-Catenin signaling pathway [46–48]. Additionally, hippocampal and select cortical neurons in AD manifest phenotypic changes indicative of neurons re-entering cell division cycle [49,50]. Pathological signals trigger the Grb2/SOS/Ras cascade [51] that initiates cell cycle re-entry and proliferation but remains incomplete due to the lack of cell division machinery. Grb2 happens to be an important adapter of the insulin receptor and interestingly, insulin resistance is a noteworthy phenotype common to AD and T2D.

In the backdrop of AD and T2D, the present study, in one hand focuses on the regulation of both Grb2 and NOX4 by TFs like PAX4, and at the same time explores the roles of miRNA regulated non-canonical RTKs. We have investigated the consequences of pathological perturbations in both the diseases, starting from RTK signaling through their downstream effectors with an aim to link them with the culminating cellular event of cytoskeleton stability and its underpinning transcriptional regulation. Specifically, the study for the first time attempts to interpret the effects of contrasting and extraneous signals, akin to AD and T2D that utilize similar signaling gateways to achieve common outcome in two apparently diverse diseases.

Materials and methods

AD and T2D human tissues

AD (NB820-59363) and Non-AD (NB820-59177) post-mortem whole brain lysates and Type 2 Diabetic (NB820-59232) and Non-Diabetic (NB820-59291) post-mortem whole liver lysates were purchased from Novus Biologicals. For statistical reasons, we procured products from different patients with different lot numbers (for patients details see Supplementary Table S1a,b).

Ethics statement

All animal experimentations using AD and T2D mouse models were conducted following the institutional guidelines for the use and care of animals and approved by the Institutional Animal and Ethics Committee of NBRC, Gurgaon (NBRC/IAEC/2012/71) and IICB, Kolkata (IICB/AEC/Meeting/2016/AUG), respectively.

APP/PS1 mice

APP/PS1 or B6C3-Tg (APPswe, PSEN1dE9/) 85Dbo/J mice were obtained from the Jackson Laboratory (U.S.A.) and maintained in the Institutes animal house facility. These transgenic mouse line for AD express human APPswe mutations (K670N and M671L) and exon 9-deleted human presenilin 1(PSEN1dE9) under the control of the mouse prion gene promoter. Animals were provided water and food /ad libitum/. The genotyping was carried out using PCR as described previously [52]. AD mice along with controls at their age of 8 and 12 months were anaesthetised with Xylazine (10 mg/kg body weight) and Ketamine (100 mg/kg body weight) and perfused transcardially with PBS followed by 4%paraformaldehyde (w/v) in PBS. Brains were collected and further placed in 4% paraformaldehyde for 24 h and then treated with 10, 20 and 30% sucrose (in PBS) followed by sectioning in freezing microtome (20 µm thickness). Sections (both control and AD) were placed on the same slides. These APP/PS1 mice were maintained in the animal house facility of NBRC, Gurgaon.

C57BL/6 mice with obesogenic western diet (OWD)

Animal experiments were performed under the approved Institutional Animal Ethics Committee (Approved by CPCSEA, India) protocol. Wild-type male C57BL/6 mice (6–8 weeks old) were kept at ambient temperature (22 ± 1°C) maintaining 12 : 12 h light-dark cycles and fed with standard chow diet (4.3% lipid and 70% carbohydrate) and/ or obesogenic western diet (45% kcal fat, MP Biomedical) for respective experiments. Weight and age-matched male C57BL/6 mice were fed with an obesogenic western diet for up to 24 weeks. These C57BL/6 mice were maintained in the animal house facility of IICB, Kolkata.

We have tested for gradual weight gain and hyperglycemia at each 4 weeks interval. All mice were fasted for 6 h and fasting blood glucose was measured using a calibrated glucometer by taking one drop of blood from the tail tip cut (Supplementary Figure S1).

AD and T2D cell models, cell culture, transfection, plasmids, siRNAs and antibodies

Clones of both Amyloid-β Protein Precursor Intracellular Domain-GFP (AICD-GFP) construct or ‘AICD’ and Grb2-DsRed construct or ‘Grb2’ were available in the lab [53–57]. ALK (siRNA ID: s1269; no. 4392420), RYK (siRNA ID: s12390; no. 4390824) and PAX4 (Assay ID s10061, 4392420) siRNAs were purchased from Ambion, life technologiesTM. Amyloid β (Aβ)-peptide (A980), Sodium Palmitate (P9767) and Insulin (I9278-5ML) were purchased from Sigma–Aldrich. Amylin (ab142398) was purchased from Abcam. Antibodies were purchased from Abcam and CST (see Supplementary Table S2 for details).

Human neuroblastoma (SHSY-5Y) and liver carcinoma (HepG2) cell-lines were obtained from National Cell Science Centre, Pune, India and were cultured in respective media of DMEM-F12 (Gibco) and DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in 5% CO2 atmosphere under humidified condition. Transfection of cells were done using Lipofectamine 2000 (Invitrogen) and as described [19]. For co-transfection, constructs were taken in equal proportions. Human neuroblastoma (SHSY-5Y) were transiently transfected with AICD and were externally treated with 0.5 µM Aβ 1–42 (Sigma A980) for 3 h after transfection and samples were collected after 48 h of addition [58]. An amount of 0.75 mM aqueous Sodium Palmitate (Sigma P9767) along with 1% fatty acid free BSA (Sigma A8806-5G) was added to 24 h serum starved media of HepG2 cells. Aqueous Amylin (Abcam ab142398) was added to the media at a final concentration of 0.5 µM after 3 h treatment of Palmitate. Samples were then collected after 16 h with or without insulin (Sigma I9278-5ML) (100 nM) shock of 10 min.

Protein from mammalian cell

Phosphate buffer saline (PBS) washed pellet from cell lines were lysed on ice in lysis buffer (1 M Tris–HCl, pH 7.5, 1 N NaCl, 0.5 M EDTA, 1 M NaF, 1 M Na3VO4, 10% SDS, 20 mM PMSF, 10% Triton X-100, 50% glycerol) for 30 min in presence of complete protease inhibitor (Roche Diagnostics) and centrifuged at 13 000g for 15 min. Protein concentration was determined by Bradford protein estimation assay. Western blots and quantification were done as per described protocol [19]. Depending on the antibodies specificity and the cross reactivity, multiple antibodies were probed on a single blot.

Fluorescence-activated cell sorting (FACS) and estimation of ROS activity

Palmitate/Amylin treated HepG2 cells were harvested and stained with CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′ dichlorodihydrofluoresceindiacetate, acetyl ester) according to manufacturer's protocol. The cells were then analyzed for ROS activity by FACS (BD FACS Calibur platform, U.S.A.).

RNA isolation, c-DNA preparations and real time PCR

RNA was isolated from cells by TRIzol Reagent (Invitrogen, U.S.A.) extraction method following manufacturer's protocol which discussed in Majumder et al. 2017 [19]. Real time RT-PCR reaction was carried out using Syber green 2X Universal PCR Master Mix (Applied Biosystems, U.S.A.) in ABI Prism 7500 sequence detection system. The absolute quantification given by the software was in terms of CT values. The relative quantification of target genes was obtained by normalizing with internal control gene (GAPDH gene). Primer sequences and PCR conditions are mentioned in Supplementary Table S3.

Chromatin immuno precipitation (ChIP)

We used High Sensitivity ChIP Kit (ab185913) to perform the ChIP assay and followed manufacturer's protocol. qRT-PCR analysis was done with the purified DNA using primers for GRB2 and NOX4 gene, more specifically around the PAX4 binding sequence. Primer details are given in Supplementary Table S4.

Validation of ALK and RYK double knockdown model

ALK and RYK double knockdown models were established in two cell lines, SHSY-5Y (human neuroblastoma cells) and HepG2 (human liver carcinoma cells). Here ALK and RYK two genes were simultaneously knocked down in cells by using siRNAs against both ALK and RYK compared with cells where two non-targeted (MOCK) siRNAs were simultaneously knocked down. The extent of knockdown was checked by measuring expression levels of both ALK and RYK by western blot in both SHSY-5Y and HepG2 cells.

Statistical and bioinformatics analysis

Unpaired ‘t’ test was carried out to compare the means of two experimental groups. The error bar represents standard error [(standard deviation/ √n); n = sample size]. To arrive at the statistically significant sample size for each experiment we did power analysis using the apriori model [59] as incorporated in the G*power 3.1 [60] software [19].

We used Transfac® MATCH1.0 (http://www.gene-regulation.com/pub/programs.html#match) online search tool to identify the TFs for the Grb2 and NOX4 gene. Additionally, miRDB [61] (http://www.mirdb.org) was used to analyze the targets of miR-1271.

Gene enhancers (genehancer) were identified using genehancer v4.8 database (downloaded from https://genecards.weizmann.ac.il/geneloc/index.shtml) [62].

Results

miRNA 1271 restricts the expressions of ALK/RYK that in turn elevate Grb2/NOX4 levels

We began with estimating the transcriptional and translational levels of Grb2 and NOX4 in both AD and T2D like situations. The expression level of NOX4 was elevated by 1.86 fold (Figure 1A,B; Supplementary Figure S2) in AD post-mortem brain as opposed to non-AD control. Grb2 and NOX4's expression levels were higher by 5.27 and 2.4 folds in human post-mortem liver tissue of T2D patients with respect to control, respectively (Figure 1D–F). Similarly, the transcript levels of Grb2 in T2D mice were raised by 3.01 fold (Figure 1G) and that of NOX4 in both AD and T2D mice showed significant up-regulation of 5.06 and 1.45 folds, respectively (Figure 1C,H). Additionally, Grb2 and NOX4 expression levels were up-regulated significantly in T2D mice (Supplementary Figure S3) and cell (Supplementary Figure S4) models.

Alteration of expression of Grb2 and NOX4 in clinical, in AD or T2D mimicking mouse.

Figure 1.
Alteration of expression of Grb2 and NOX4 in clinical, in AD or T2D mimicking mouse.

(A) Western blot showing the NOX4 and GAPDH levels in AD whole brain lysate. (B) Histogram representing the mean value of optical density of the bands, normalized against GAPDH. NOX4 was overexpressed by 1.86 fold in AD Brains. (C) Shows NOX4 transcript level being up-regulated by 5.06 fold by qRT-PCR of APP/PS1 mouse brain tissue. (D) Western blot showing Grb2, NOX4 and GAPDH levels in T2D whole liver lysate. (E and F) Histograms representing the mean values of Grb2 and NOX4 normalized against GAPDH. Grb2 and NOX4 were overexpressed in T2D liver by 5.27 fold and 2.4 fold, respectively. (G and H) show qRT-PCR results inT2D mouse model where C57BL/6 mice are maintained in OWD, Grb2 and NOX4 transcript levels are overexpressed by 3.01 fold and 1.45 fold, respectively. (I and J) the transcript levels of miR-1271 in AD and T2D cell model, respectively. All the statistical information is compiled in Supplementary Table S7.

Figure 1.
Alteration of expression of Grb2 and NOX4 in clinical, in AD or T2D mimicking mouse.

(A) Western blot showing the NOX4 and GAPDH levels in AD whole brain lysate. (B) Histogram representing the mean value of optical density of the bands, normalized against GAPDH. NOX4 was overexpressed by 1.86 fold in AD Brains. (C) Shows NOX4 transcript level being up-regulated by 5.06 fold by qRT-PCR of APP/PS1 mouse brain tissue. (D) Western blot showing Grb2, NOX4 and GAPDH levels in T2D whole liver lysate. (E and F) Histograms representing the mean values of Grb2 and NOX4 normalized against GAPDH. Grb2 and NOX4 were overexpressed in T2D liver by 5.27 fold and 2.4 fold, respectively. (G and H) show qRT-PCR results inT2D mouse model where C57BL/6 mice are maintained in OWD, Grb2 and NOX4 transcript levels are overexpressed by 3.01 fold and 1.45 fold, respectively. (I and J) the transcript levels of miR-1271 in AD and T2D cell model, respectively. All the statistical information is compiled in Supplementary Table S7.

In SHSY-5Y cells, knockdown of ALK and RYK down-regulated their expression levels by 2.42 (Supplementary Fig. S5A(i),B(i); * P = 0.0088 < 0.05; n = 3) and 1.2 folds (Supplementary Figure S5A (ii),B(iii); *P = 0.019 < 0.05; n = 2), respectively. In HepG2 cells, knockdown of ALK and RYK reduced the expression levels of ALK by 1.38 fold (Supplementary Figure S5A(iv),B(ii); * P = 0.0088 < 0.05; n = 2) whereas RYK did not show significant decrease (Supplementary Figure S5A(v),B(iv); N.S.; P = 0.22 > 0.05; n = 2). We designed double knockdown models for both ALK and RYK genes in SHSY-5Y and HepG2 cells (Supplementary Figure S5). Grb2 protein and transcript levels showed significant up-regulation for double knockdown condition of SHSY-5Y (1.22 fold in protein level, 4.08 fold in transcript level) (Supplementary Fig. S6A,B,F) and HepG2 cell lines (2.34 fold in protein level, 5.9 fold in transcript level), respectively (Supplementary Figure S6A,C,F). Similarly, protein and mRNA expression levels for NOX4 was also elevated significantly for both SHSY-5Y (1.35 fold in protein level 3.02 fold transcript level) (Supplementary Figure S6A,D,G) and HepG2 cell lines (2.62 fold in protein level 4.13 fold transcript level) (Supplementary Figure S6A,E,G). Interestingly, miRNA target analysis using miRDB revealed that both ALK and RYK were prominent target genes for miR-1271 (Supplementary Information S1). This was validated by the qRT-PCR results, which showed significant increase in miR-1271 transcript levels in both AD cell model and T2D cell model (palimitic acid and amylin treated HepG2 cells) by 3.8 and 3.7 folds, respectively (Figure 1I,J). Additionally, the bioinformatics analysis of MIR1271 gene gave us the genehancers using genehancer v4.8 database. The GH05J17641 showed the significantly high score compared with other genehancers and it had 186 transcription binding sites (Supplementary Figure S7). The SP1 transcription factor had the binding site in GH05J17641 genehancer.

PAX4 regulates the transcription of GRB2 and NOX4

To search for the molecular players behind GRB2 and NOX4 transcriptional up-regulation, we used Transfac® tool to search upto 10 Kb upstream of both the genes. In case of GRB2, 36 probable binding sites for 18 different TFs (Supplementary Table S5) were found, whereas, for NOX4, 32 probable binding sites for 16 different TFs (Supplementary Table S6) were noted. Comparing both the results, three (Nkx2-5, Foxd3 and PAX4) top hits were selected among the six common (Nkx2-5, Foxd3, PAX4, CHOP C/EBP, Oct1 and Evi-1) ones. The transcript levels of these three TFs were measured for both AD and T2D cell models (Figure 2A,B) and it was seen that whereas the mRNA levels of Nkx2-5 were down-regulated, those of PAX4 and FOXD3 were up-regulated in both the models.

Role of PAX4 in transcription of Grb2 and NOX4.

Figure 2.
Role of PAX4 in transcription of Grb2 and NOX4.

(A) shows the transcript level alterations of −3.6 fold for Nkx2-5, +4.19 fold for PAX4 and +2.89 fold for FOXD3 in AD (AICD + Aβ) cell models compared with control. Similarly, (B) shows the transcript level alterations of −2.59 fold for Nkx2-5, +4.88 fold for PAX4 and +4.39 fold for FOXD3 in T2D (+PA, +Amylin, +Insulin) cell models compared with controls. (C) shows PAX4 protein overexpression by 1.8 and 2.00 folds by Western Blot in (D) AD patients’ whole brain samples and (E) T2D patients’ whole liver samples, respectively. The alterations in transcript levels of (F) endogenous PAX4 [in SHSY5Y 2.99 fold and in HepG2 1.58 fold], (G) GRB2 [in SHSY-5Y 1.16 fold and in HepG2 1.52 fold], and (H) NOX4 [in SHSY5Y 2.83 fold and in HepG2 2.3 fold] in PAX4 knockdown situation in SHSY5Y and HepG2 cell lines. All statistical information is compiled in Supplementary Table S7.

Figure 2.
Role of PAX4 in transcription of Grb2 and NOX4.

(A) shows the transcript level alterations of −3.6 fold for Nkx2-5, +4.19 fold for PAX4 and +2.89 fold for FOXD3 in AD (AICD + Aβ) cell models compared with control. Similarly, (B) shows the transcript level alterations of −2.59 fold for Nkx2-5, +4.88 fold for PAX4 and +4.39 fold for FOXD3 in T2D (+PA, +Amylin, +Insulin) cell models compared with controls. (C) shows PAX4 protein overexpression by 1.8 and 2.00 folds by Western Blot in (D) AD patients’ whole brain samples and (E) T2D patients’ whole liver samples, respectively. The alterations in transcript levels of (F) endogenous PAX4 [in SHSY5Y 2.99 fold and in HepG2 1.58 fold], (G) GRB2 [in SHSY-5Y 1.16 fold and in HepG2 1.52 fold], and (H) NOX4 [in SHSY5Y 2.83 fold and in HepG2 2.3 fold] in PAX4 knockdown situation in SHSY5Y and HepG2 cell lines. All statistical information is compiled in Supplementary Table S7.

Specifically, perturbation of PAX4 was most significant and noticeable. PAX4 expression was measured by western blot and was found to be overexpressed in both AD and T2D cell models (Supplementary Figure S8). This was validated with clinical samples of AD and T2D (Figure 2C–E), where PAX4 expression was up-regulated by 1.8 and 2 folds, respectively. Consequences of PAX4 knockdown using Silencer® select PAX4 siRNA constructing SHSY-5Y and HepG2 (Figure 2F) have been measured. Although non-significant in SHSY-5Y, the transcript level of GRB2 decreased significantly in HepG2 (Figure 2G) consequently. Additionally, PAX4 knockdown significantly decreased the level of NOX4 in SHSY-5Y, and in HepG2, the change was insignificant (Figure 2H). We went further to measure the mRNA levels of Nkx2-5, PAX4 and FOXD3 in ALK/RYK double knockdown cells. qRT-PCR results showed +5.75, +5.84 and +3.13 folds’ alterations, respectively, in SHSY-5Y (Figure 3A) and +2.91, +4.8 and +2.46 fold changes, respectively, in HepG2 (Figure 3B). Thus, ALK/RYK double knockdown cells resembled AD and T2D cell models in terms of PAX4 up-regulation. Expectedly, both the double knockdown models showed significant up-regulation of PAX4 protein levels in SHSY-5Y and HepG2 by 1.65 and 1.98 folds, respectively (Figure 3C–E). Furthermore, Chromatin Immuno-precipitation (ChIP) assays confirmed elevated recruitment of PAX4 protein on the GRB2 and NOX4 upstream regions. For the AD cell model PAX4 recruitment increased by 27.49 and 5.35 folds (Figure 3F) and in T2D cell model it was elevated by 18.33 and 7.22 folds (Figure 3G), for GRB2 and NOX4 genes, respectively.

Effect of ALK and RYK double knockdown on PAX4 levels.

Figure 3.
Effect of ALK and RYK double knockdown on PAX4 levels.

The transcript level alterations of Nkx2-5, PAX4 and FOXD3 in ALK and RYK double knockdown (K/D) situation in (A) SHSY-5Y [+ 5.75 fold for Nkx2-5, +5.84 fold for PAX4 and +3.13 fold for FOXD3] and (B) HepG2 cell line [+2.91 fold for Nkx2-5, +4.81 fold for PAX4 and +2.46 fold for FOXD3]. (C) shows the PAX4 protein level alterations by Western Blot in ALK and RYK double knockdown (K/D) model in both SHSY-5Y and HepG2 cell lines. (D and E) histograms that graphically denotes the up-regulation of endogenous PAX4 levels in double knockdown model in both SHSY-5Y [+1.65 fold] and HepG2 cells [+1.98 folds], respectively. PAX4 binds to the upstream region of Grb2 and NOX4 gene. The ChIP data shows that PAX4 significantly up-regulates GRB2 expression in both (F) AD [+27.49 fold] and (G) T2D [+18.33 fold] cell models by binding at its upstream region and acting like an enhancer. In case of NOX4, PAX4 fails to significantly up-regulate its expression in both (F) AD [+5.35 fold] and (G) T2D [+7.22 fold] cell models. All the statistical information is available on Supplementary Table S7.

Figure 3.
Effect of ALK and RYK double knockdown on PAX4 levels.

The transcript level alterations of Nkx2-5, PAX4 and FOXD3 in ALK and RYK double knockdown (K/D) situation in (A) SHSY-5Y [+ 5.75 fold for Nkx2-5, +5.84 fold for PAX4 and +3.13 fold for FOXD3] and (B) HepG2 cell line [+2.91 fold for Nkx2-5, +4.81 fold for PAX4 and +2.46 fold for FOXD3]. (C) shows the PAX4 protein level alterations by Western Blot in ALK and RYK double knockdown (K/D) model in both SHSY-5Y and HepG2 cell lines. (D and E) histograms that graphically denotes the up-regulation of endogenous PAX4 levels in double knockdown model in both SHSY-5Y [+1.65 fold] and HepG2 cells [+1.98 folds], respectively. PAX4 binds to the upstream region of Grb2 and NOX4 gene. The ChIP data shows that PAX4 significantly up-regulates GRB2 expression in both (F) AD [+27.49 fold] and (G) T2D [+18.33 fold] cell models by binding at its upstream region and acting like an enhancer. In case of NOX4, PAX4 fails to significantly up-regulate its expression in both (F) AD [+5.35 fold] and (G) T2D [+7.22 fold] cell models. All the statistical information is available on Supplementary Table S7.

ARX couples ALK/RYK down-regulation with PAX4 via β-Catenin signaling

In the course of exploring the mechanism behind PAX4 up-regulation in the degenerative diseases, the role of ARX was evident. The significant up-regulation of ARX transcript levels by 3.3 and 3.1 folds in PAX4 knockdown conditions, in both SHSY-5Y and HepG2 cells, established the mutually repressive natures of ARX and PAX4 (Figure 4A). Moreover, the ARX transcript levels were significantly down-regulated by 2.6, 3.7, 3 and 3.7 folds in the AD and T2D cell models and ALK/RYK double knockdown in both SHSY-5Y and HepG2 cells, respectively (Figure 4B). Additionally, β-Catenin's role as ARX-regulator was also established by measuring its expression levels. Western blots showed significant down-regulation of β-Catenin by 3.5 and 1.5 folds in AD and T2D cell models, respectively (Figure 4C,D). The ALK/RYK double knockdown also showed a significant β-Catenin down-regulation by 1.15 and 6.5 folds in both SHSY-5Y and HepG2 cells, respectively (Figure 4E,F).

Role of ARX and β-Catenin in AD and T2D cell model and in ALK/RYK double knockdown model.

Figure 4.
Role of ARX and β-Catenin in AD and T2D cell model and in ALK/RYK double knockdown model.

(A) shows that ARX transcript level is being up-regulated in PAX4 knockdown condition by 3.35 and 3.13 fold by qRT-PCR in both SHSY-5Y and HepG2 cells, respectively. (B) Shows the down-regulation of ARX transcript levels by 2.6, 3.7, 3 and 3.7 folds in AD (AICD + Aβ) and T2D (+PA, +Amylin, +Insulin) cell models and also in ALK/RYK double knockdown conditions in SHSY-5Y and HepG2 cells, respectively. (C) Western blot showing the reduction in β-Catenin expression levels in (i) AD and (ii) T2D cell model. (D) Histogram representing β-Catenin being down-regulated by 3.5 and 1.5 fold in AD and T2D cell models, respectively. (E and F) shows the down-regulation of β-Catenin expression levels in ALK and RYK double knockdown situation in both (i) SHSY-5Y and (ii) HepG2 cells by 1.15 and 6.5 folds, respectively. All the statistical information is available in Supplementary Table S7.

Figure 4.
Role of ARX and β-Catenin in AD and T2D cell model and in ALK/RYK double knockdown model.

(A) shows that ARX transcript level is being up-regulated in PAX4 knockdown condition by 3.35 and 3.13 fold by qRT-PCR in both SHSY-5Y and HepG2 cells, respectively. (B) Shows the down-regulation of ARX transcript levels by 2.6, 3.7, 3 and 3.7 folds in AD (AICD + Aβ) and T2D (+PA, +Amylin, +Insulin) cell models and also in ALK/RYK double knockdown conditions in SHSY-5Y and HepG2 cells, respectively. (C) Western blot showing the reduction in β-Catenin expression levels in (i) AD and (ii) T2D cell model. (D) Histogram representing β-Catenin being down-regulated by 3.5 and 1.5 fold in AD and T2D cell models, respectively. (E and F) shows the down-regulation of β-Catenin expression levels in ALK and RYK double knockdown situation in both (i) SHSY-5Y and (ii) HepG2 cells by 1.15 and 6.5 folds, respectively. All the statistical information is available in Supplementary Table S7.

Expressions of cytoskeletal proteins change in conjunction with ALK/RYK deactivation

To explore cytoskeletal degradation, the protein and transcript levels of four cytoskeleton proteins (viz., α-Tubulin, Vimentin, α-Smooth muscle actin (α-SMA) and Stathmin1) were compared between the whole liver tissue of T2D patients and non-Diabetic whole liver samples. The relative protein levels were down-regulated by 1.16 fold for α-Tubulin, 1.83 fold for Vimentin, 1.68 fold for α-SMA, and 1.95 fold for Stathmin1, respectively (Figure 5A,B). The transcript levels also showed significant (-5.3 fold α-tubulin; -6.49 fold Vimentin; -2 fold α-SMA and -2.9 fold Stathmin1) down-regulation in T2D mice (Figure 5C). Similarly, in the T2D cell model the relative protein levels were down-regulated by 2.7 fold for α-Tubulin, 1.98 fold for Vimentin, 1.27 fold for α-SMA and 1.97 fold for Stathmin1, respectively (Figure 5D,E). Moreover, to link degeneration with ALK/RYK deactivation, we compared the protein and the transcript levels of α-Tubulin in ALK/RYK double knockdown conditions. A significant reduction in protein levels (2.64 and 1.42 folds, respectively) was observed in both SHSY-5Y and HepG2 cell lines (Figure 5F,G). Similarly, transcript levels of α-Tubulin were also down-regulated by 2.3 and 1.5 folds for both SHSY-5Y and HepG2 cell lines, respectively (Figure 5H). In both Palmitate and Amylin treated situations, the percentage recovery of cytoskeletal proteins from disassembly, upon Grb2 overexpression, up-regulated the expressions of α-Tubulin, Vimentin, α-SMA and Sthathmin1 by 98.2%, 84.39%, 508.8% and 62.2%, respectively (Figure 5I).

Participation of ALK and RYK in signaling events leading to cytoskeleton degradation and Grb2 mediated reversal in T2D: cytoskeleton degradation.

Figure 5.
Participation of ALK and RYK in signaling events leading to cytoskeleton degradation and Grb2 mediated reversal in T2D: cytoskeleton degradation.

(A) Representative western blots (n = 2) of four cytoskeletal proteins (i) α-Tubulin, (ii) Vimentin, (iii) α-SMA and (iv) Stathmin1 with (v) GAPDH used as internal control in human diabetic whole liver lysates, compared with wild type whole liver lysate. (B) Histogram representing the mean value of optical density of the protein bands, normalized against GAPDH, with a decrease in 1.16 fold for α-Tubulin, 1.83 fold for Vimentin, 1.68 fold for α-SMA and 1.95 fold for Stathmin1. Samples are derived from the same experiments and the blots are processed in parallel. (C) Shows transcript level changes in α-Tubulin (−5.3 fold), Vimentin (−6.49 fold), α-SMA (−2 fold), and Stathmin1 (−2.9 fold) by qRT-PCR in C57BL/6 mice on OWD compared with normal diet fed controls. (D) Western blots depict alteration in the expression of (i) α-Tubulin, (ii) Vimentin, (iii) α-Smooth muscle actin (α-SMA) and (iv) Stathmin1with GAPDH in T2D cell model (PA, +Amylin, +Insulin induced HepG2 cells). The samples are derived from the same experiments and the blots are processed in parallel. (E) Histograms showing changes in the four cytoskeletal proteins, normalized by GAPDH, with decrease 2.7 fold for α-Tubulin, 1.98 for Vimentin, 1.27 fold for α-SMA and 1.97 fold for Stathmin1. ALK and RYK double knockdown condition controls α-Tubulin degradation. (F and G) shows in western blot, the decrease in α-Tubulin in both SHSY-5Y [−2.64 fold] and HepG2 cell line [−1.42 fold] in ALK and RYK double knockdown (K/D) situation. (H) shows the α-Tubulin transcript level down-regulation in ALK and RYK double knockdown (K/D) model in both SHSY5Y [−2.3 fold] and HepG2 cell lines [−1.5 fold] by qRT-PCR. (I) Shows transcript level changes for the four cytoskeletal proteins in T2D (PA +Amyli +Insulin induced condition) with or without Grb2.All the statistical information is available on Supplementary Table S7.

Figure 5.
Participation of ALK and RYK in signaling events leading to cytoskeleton degradation and Grb2 mediated reversal in T2D: cytoskeleton degradation.

(A) Representative western blots (n = 2) of four cytoskeletal proteins (i) α-Tubulin, (ii) Vimentin, (iii) α-SMA and (iv) Stathmin1 with (v) GAPDH used as internal control in human diabetic whole liver lysates, compared with wild type whole liver lysate. (B) Histogram representing the mean value of optical density of the protein bands, normalized against GAPDH, with a decrease in 1.16 fold for α-Tubulin, 1.83 fold for Vimentin, 1.68 fold for α-SMA and 1.95 fold for Stathmin1. Samples are derived from the same experiments and the blots are processed in parallel. (C) Shows transcript level changes in α-Tubulin (−5.3 fold), Vimentin (−6.49 fold), α-SMA (−2 fold), and Stathmin1 (−2.9 fold) by qRT-PCR in C57BL/6 mice on OWD compared with normal diet fed controls. (D) Western blots depict alteration in the expression of (i) α-Tubulin, (ii) Vimentin, (iii) α-Smooth muscle actin (α-SMA) and (iv) Stathmin1with GAPDH in T2D cell model (PA, +Amylin, +Insulin induced HepG2 cells). The samples are derived from the same experiments and the blots are processed in parallel. (E) Histograms showing changes in the four cytoskeletal proteins, normalized by GAPDH, with decrease 2.7 fold for α-Tubulin, 1.98 for Vimentin, 1.27 fold for α-SMA and 1.97 fold for Stathmin1. ALK and RYK double knockdown condition controls α-Tubulin degradation. (F and G) shows in western blot, the decrease in α-Tubulin in both SHSY-5Y [−2.64 fold] and HepG2 cell line [−1.42 fold] in ALK and RYK double knockdown (K/D) situation. (H) shows the α-Tubulin transcript level down-regulation in ALK and RYK double knockdown (K/D) model in both SHSY5Y [−2.3 fold] and HepG2 cell lines [−1.5 fold] by qRT-PCR. (I) Shows transcript level changes for the four cytoskeletal proteins in T2D (PA +Amyli +Insulin induced condition) with or without Grb2.All the statistical information is available on Supplementary Table S7.

The signaling pathways also show a reversal of outcome through Grb2–NOX4 interaction

While investigating the pathways that could be responsible for degradation of cytoskeletal proteins, involvement of three small GTPases viz., RhoA, Rac1 and Cdc42 (Figure 6A,B) was observed. Similar to AD situations, under Palmitate and Amylin treated disease inducing conditions, the expression levels of RhoA and Rac1 decreased significantly by 2.13 (n = 3) and 1.63 folds (n = 3) respectively and the Cdc42 protein levels increased by 64.2% (n = 3). Down the line, the significantly reduced Cofilin activity bounced back by 43.2% (Figure 6C,D) upon Grb2 overexpression and Palmitate/Amylin treatment. Besides these, SSH-1's (an activator of Cofilin through dephosphorylation) distal upstream effector NOX4 was overexpressed significantly by 1.31 fold post Palmitate/Amylin treatment, that did reduce significantly by 1.26 fold (Figure 6E,F) upon Grb2 overexpression. Interestingly, Nox4, an interactor of Grb2, was found to be 1.34 fold overexpressed endogenously in T2D cell model but reduced in the presence of Grb2 (Figure 6E,F). We validated the interaction between NOX4 and Grb2 with co-immunoprecipitation (Co-IP) experiment. We pulled down the interacting complex by anti-Grb2 antibody and probed the expression of NOX4 in that complex by immunoblot. The interaction of Grb2 with NOX4 was enhanced 1.35 fold in T2D scenario (Figure 7A,B). Furthermore, the intensity of interaction of NOX4 and Grb2 was checked in T2D mouse liver lysate that increased 2.87 fold in T2D situation compared with the wild type (Figure 7C,D). Besides the reversal, we also measured the ROS activities of Palmitate/Amylin and Palmitate/Amylin/Grb2 by flow cytometry by using CMH2-DCFDA ROS indicator. Although overexpressed Grb2 could significantly reverse the effects of many T2D related metabolic changes, to our surprise in the working T2D like model, over-expression of Grb2, instead of reducing the activity of ROS, rather elevated it by 3.57 fold (Figure 7E,F). Nevertheless, the extent of this limited fate reversal upon Grb2 overexpression was estimated, and in both Palmitate and Amylin treated situations, Grb2 overexpression up-regulated the expressions of cytoskeletal proteins significantly.

Signaling molecules participate in T2D.

Figure 6.
Signaling molecules participate in T2D.

(A) Western blot showing protein level changes of small GTPases i.e. (i) RhoA [−2.13 fold and in presence of Grb2 −1.22 fold], (ii) Rac1 [−1.63 fold and in presence of Grb2 −1.26 fold] and (iii) CDC42 [+1.36 fold and in presence of Grb2 −2.05 fold] in T2D inducing (+PA, +Amylin, +Insulin in HepG2 cells) and in reversing conditions (+PA, +Amylin, +Insulin, +Grb2 in HepG2 cells). In (B) Bar diagram represents the activity alterations for the small GTPases. (C and D) Western blots depict the protein levels or activation of signaling molecules like Cofilin [−1.43 fold]. (E and F) depictNOX4 [+1.31 fold and in presence of Grb2 −1.26 fold] by Western blot. All the statistical information is available in Supplementary Table S7.

Figure 6.
Signaling molecules participate in T2D.

(A) Western blot showing protein level changes of small GTPases i.e. (i) RhoA [−2.13 fold and in presence of Grb2 −1.22 fold], (ii) Rac1 [−1.63 fold and in presence of Grb2 −1.26 fold] and (iii) CDC42 [+1.36 fold and in presence of Grb2 −2.05 fold] in T2D inducing (+PA, +Amylin, +Insulin in HepG2 cells) and in reversing conditions (+PA, +Amylin, +Insulin, +Grb2 in HepG2 cells). In (B) Bar diagram represents the activity alterations for the small GTPases. (C and D) Western blots depict the protein levels or activation of signaling molecules like Cofilin [−1.43 fold]. (E and F) depictNOX4 [+1.31 fold and in presence of Grb2 −1.26 fold] by Western blot. All the statistical information is available in Supplementary Table S7.

Grb2 and NOX4 interaction prevents cytoskeletal degradation in T2D like scenario and ROS activity.

Figure 7.
Grb2 and NOX4 interaction prevents cytoskeletal degradation in T2D like scenario and ROS activity.

(A) shows the variation of interaction of Grb2 and NOX4 in T2D inducing (+Palmitate, +Amylin and +Insulin in HepG2) conditions compared with controls (−Palmitate, −Amylin and +Insulin in HepG2). (B) graphical representation of the intensity of the interaction between NOX4 and Grb2. (C) shows the variation of interaction of Grb2 and NOX4 in type 2 diabetic mouse (OWD C57BL/6) whole liver lysate compared with WT (Normal fed C57BL/6) control. (D) graphical representation of the intensity of the interaction between NOX4 and Grb2 in T2D mouse liver lysate. ROS activity Assay. (E) (i), (ii), (iii) and (iv) show the FACS data for ROS activity of unstained, −Palmitate –Amylin +DsRed +Insulin, +Palmitate, +Amylin, +DsRed, +Insulin and +Palmitate, +Amylin, +Grb2, +Insulin, respectively, in HepG2 cells. (F) Shows graphically that ROS activity increases in T2D inducing (+Palmitate, +Amylin, +DsRed, +Insulin) condition by 1.3 fold and in presence of Grb2 ROS activity further significantly increases by 3.5 fold. All the statistical information is available on Supplementary Table S7.

Figure 7.
Grb2 and NOX4 interaction prevents cytoskeletal degradation in T2D like scenario and ROS activity.

(A) shows the variation of interaction of Grb2 and NOX4 in T2D inducing (+Palmitate, +Amylin and +Insulin in HepG2) conditions compared with controls (−Palmitate, −Amylin and +Insulin in HepG2). (B) graphical representation of the intensity of the interaction between NOX4 and Grb2. (C) shows the variation of interaction of Grb2 and NOX4 in type 2 diabetic mouse (OWD C57BL/6) whole liver lysate compared with WT (Normal fed C57BL/6) control. (D) graphical representation of the intensity of the interaction between NOX4 and Grb2 in T2D mouse liver lysate. ROS activity Assay. (E) (i), (ii), (iii) and (iv) show the FACS data for ROS activity of unstained, −Palmitate –Amylin +DsRed +Insulin, +Palmitate, +Amylin, +DsRed, +Insulin and +Palmitate, +Amylin, +Grb2, +Insulin, respectively, in HepG2 cells. (F) Shows graphically that ROS activity increases in T2D inducing (+Palmitate, +Amylin, +DsRed, +Insulin) condition by 1.3 fold and in presence of Grb2 ROS activity further significantly increases by 3.5 fold. All the statistical information is available on Supplementary Table S7.

Discussion

This work unveils the commonality of two diverse diseases, AD and T2D. Despite efforts to relate the two for more than a decade, none of the studies focused on the signaling components and their possible convergence on a transcription factor.

The precise common signaling mechanism for AD and T2D is still unclear, albeit the fact that both the diseases confer insulin resistance. Up until our recent work, Insulin Receptor (IR) family was the only RTKs known to be involved in the signaling in both the diseases [6]. Profiling the activities of other RTKs in the two diseases opened up a larger picture and the effects of both Aβ and Amylin oligomers were predicted to have impacts on other receptor tyrosine kinases [6].

Amongst all the common RTKs, ALK and RYK were the two showing similar levels of deactivation in all working models of AD and T2D [6]. ALK, a member of insulin receptor superfamily, is commonly known for its involvement in many cancer types, especially in non-small-cell lung cancer (NSCLC) [63]. RYK is a co-receptor of non-canonical Wnt signaling [64]. The involvement of Wnt signaling pathway in AD [65] and T2D [66,67] is well established. We further verified the association of classical Wnt pathway through the down-regulation of β-Catenin (Figure 4C–F) and up-regulation of GSK3β [6,19] in both diseases. This is a significant revelation considering that α-Tubulin is down-regulated in T2D conditions, and that the activity of Gsk3β, a kinase that would hyperphosphorylate Tau leading to destabilization of the microtubule network, increases under disease inducing Palmitate/Amylin treatment [6]. The activity of its upstream effector AKT1 also decreases under T2D inducing condition [6].

Recent reports and our real-time PCR data (Figure 1I,J) have revealed the active involvement of miR-1271 in both the diseases, AD [34] and T2D [35]. miR-1271 and miR-96 are known paralogs to each other [68]. The mirDB [61] target analysis of of both miR-1271 (Supplementary Information S1) and miR-96 (Supplementary Information S2) have also confirmed their roles in the ALK and RYK down-regulation. This observation is further corroborated by Kong et al., who have demonstrated ALK as the target for miR-1271 in oral squamous cell carcinoma (OSCC) [69]. RYK was observed as a target gene for miR-96, the paralog of miR-1271[68,70,71]. Additionally, the expression of miR-96 is also up-regulated in Alzheimer's disease [72]. Up-regulated expression of miR-1271, or its paralog, in both the diseases, and their targeting of both ALK and RYK are well perceived. The mirDB [61] analysis further demonstrated that mechanistic target of rapamycin (mTOR) is targeted by both miR-1271 and miR-96 [Supplementary Information 1 and 2]. Several studies confirmed that down-regulation of mTOR led to autophagy [73–75]. Concomitantly, both AD [76] and T2D [77] induced autophagy by the inhibition of mTOR protein. Thus, the elevated levels of miR-1271 could affect both AD and T2D not only by ALK/RYK, but also through the mTOR/autophagy pathway. Furthermore, the genehancer analysis revealed SP1 (specificity protein 1) as one of the putative transcription factors of miR-1271. Besides, the role of SP1 in the expression of several AD-related proteins, including amyloid-β protein precursor (APP), BACE1 and tau is well documented [78,79]. Similarly, SP1 plays a crucial role in the development of insulin resistance in T2D [80,81]. Therefore, elevated levels of SP1 could be the reason behind up-regulation of miR-1271.

Grb2 happens to be a common downstream adapter for both the RTKs [63,82]. It was shown previously that Grb2 strengthened its interaction with NOX4 to rescue the cytoskeleton degradation in AD like situations [19]. We postulate that the two RTKs, ALK and RYK, might act like non-canonical receptors with the potential to become a significant link between AD and T2D via Grb2 and NOX4.

The downstream consequences at the molecular level for both the signals were significant compromise of cytoskeleton integrity. Interestingly, ALK and RYK double knockdown cell lines also resembled similar phenotypes (Figure 5F–H). Searching for molecules that could connect the common RTKs with downstream cytoskeleton degradation, we singled out Grb2 and NOX4, both of which had elevated levels in the disease scenarios (Figure 1; Supplementary Figures S2–S4). Several reports on Grb2 strongly suggested its role in cellular survival in AD [55,57]. Transcript levels of the cytoskeletal protein components were checked and were found to increase several folds with Grb2's overexpression in both AD [19] and T2D models (Figure 5I).

It was prudent therefore to try to understand the underpinning cellular mechanisms that help in the cytoskeletal restructuring, with possibly Grb2 playing a pivotal role. The expressions of three small GTPases, RhoA, Rac1 and Cdc42, known to regulate cell morphology through rearrangement of cytoskeletal proteins [83–85] acting as molecular switches, were significantly altered and again renormalized with overexpression of Grb2. These alterations in the activities of small GTPases were sufficient to perturb the downstream signaling events and, ultimately, the cytoskeletal proteins were degraded primarily through Cofilin mediated mechanism. Cofilin is a downstream effector of RhoA, Rac1 and Cdc42 and it is one of the actin-binding proteins whose dephosphorylation enables actin depolymerization [86]. These results convincingly implies similar pathways for the rearrangement of the cytoskeleton network in AD and T2D. Grb2's intervention in both disease models significantly inactivates Cofilin by phosphorylation [19] (Figure 6C,D), a crucial regulator of actin dynamics [87]. Similar to the case in AD, in T2D also Grb2 helps reversing the signals involved in the degradation of cytoskeletal proteins.

Abnormal NOX activation is already reported in T2D [88] leading to its interaction with Grb2 and Src activation [18]. Adapter protein NOX4, from the ROS production pathway, is another Cofilin regulator in conjunction with Slingshot homolog-1 (SSH-1), the phosphatase of Cofilin. Both the disease models showed significant up-regulation of NOX4 (Figure 6E,F), which was subsequently decreased with Grb2's overexpression [19]. These small checks and balances, at different levels along the signaling cascades, culminated in a larger scale perturbation in the cytoskeleton network and Grb2's role emerged as a reversing switch of these perturbations (Figure 5I) [19]. Furthermore, while examining other effects on cytoskeleton integrity due to Grb2's overexpression, it was noted that NOX4 interacted with Grb2 in normal conditions [17,18] which increased several folds under both AD [19] and T2D disease conditions (Figure 7A–D). As NOX4 was responsible for ROS generation, we also checked whether Grb2 overexpression could put a check on the ROS, which it could not. On the contrary, the ROS activity had increased in the presence of too much of Grb2 (Figure 7E,F). This might be a hint towards the fact that even though the cells geared up the protective mechanisms, the defense was lost with the progression of the disease and beyond a threshold.

The study unraveled the relation of ALK and RYK in controlling the cytoskeleton integrity through the overexpression of Grb2 and NOX4 (Supplementary Figure S6), with miR-1271 as the master-regulator controlling ALK/RYK expression (Supplementary Information S1; Figure 1I,J). Employing bioinformatics tools and qRT-PCR, we identified PAX4 as a central regulator (Supplementary Tables S5,S6; Figures 2A,B,F–H, 3A,B), which is also crucial in regulating the gene network governing β-cell mass expansion and survival under the pathophysiological conditions of T2D [89]. Recent studies have shown association of PAX4 mutations with T2D in Japanese and Afro-Americans populations [42,90]. However, PAX4 gene dysfunction increases the susceptibility of apoptosis along with reducing cell proliferation leading to a gradual loss of β-cell and ultimately to diabetes [89]. Interestingly, the PI3K inhibitor, Wortmannin, showed potential to induce both insulin resistance and PAX4 expression [89,91]. Naturally, PAX4 emerged as a survival gene because of its role in regulating both β-cell mass expansion and Grb2 expression. On the other hand, the antagonism between PAX4 and ARX (Figure 4A) could connect the aberrant Wnt/β-Catenin signaling with the up-regulation of PAX4 via reduction in ARX in both the diseases (Figure 4B). This helps connect the dots between the deactivation of ALK and RYK with the down-regulation of β-Catenin expression (Figure 4C–F) levels, followed by a decrease in ARX. Thus, down-regulation of ALK and RYK implies an elevation of the PAX4 level (Figures 2C–E, 3C–E; Supplementary Figure S8), which in turn up-regulates Grb2 and NOX4 in both AD and T2D.

NOX4 reportedly induces mitochondrial dysfunction by inhibiting the mitochondrial chain complex 1 [92]. Alterations in the expression of genes coding for mitochondrial and cytoskeletal proteins contribute to the mitochondrial dysfunction observed in insulin-resistant conditions of both AD and T2D [93,94]. Interestingly, the majority of differentially expressed genes targeted by PAX4 are commonly enriched in both oxidative phosphorylation and neurodegenerative diseases [45]. Besides, this work establishes PAX4 as a promising candidate behind up-regulation of GRB2 and NOX4 in T2D, acting as the coveted ‘missing link’ between AD and T2D's common signaling pathways (Figures 2C–H, 3C–G; Supplementary Figure S8). Like a pivot, it links ALK, RYK as upstream receptors and Grb2, NOX4 as downstream adapters, eventually affecting the cytoskeleton. The status of all the dysregulated proteins involved in this study are compiled in Table 1.

Table 1
A summary of status of dysregulated proteins in AD and T2D disease conditions
Sl No.Name of proteinUpstream effector/effectDownstream effector/effectStatus in disease condition
ADT2D
ALK miR-1271 Grb2, NOX4 ↓[6↓[6
RYK miR-1271 Grb2, NOX4 ↓[6↓[6
Grb2 ALK, RYK, PAX4 Recover Cytoskeleton Proteins degradation after Grb2–NoX4 interaction ↑[19↑ 
NOX4 Reactive Oxygen Species (ROS) [95], Rac1 [96], ALK, RYK, PAX4 SSH-1 [18,19], Recover Cytoskeleton Proteins degradation after Grb2–NoX4 interaction ↑[19↑ 
PAX4 ARX Grb2, NOX4 ↑ ↑ 
ARX β-Catenin PAX4 ↓ ↓ 
β-Catenin GSK3β ARX ↓ ↓ 
GSK3β activity AKT1 β-Catenin ↑[6↑[6
AKT1 activity PTEN [97GSK3β ↓[6↓[6
10 Cytoskeleton Proteins (α-Tubulin, Vimentin, α-SMA, Stathmin1) RhoA, Rac1 and C dc 42 Degeneration of cell ↓[19↓ 
11 RhoA AKT1 [98Cytoskeleton proteins ↓[19↓ 
12 Rac1 AKT1 [98Cytoskeleton proteins ↓[19↓ 
13 Cdc42 AKT1 [98Cytoskeleton proteins ↑[19↑ 
14 Cofilin activity LIMK [99F-Actin ↓[19↓ 
Sl No.Name of proteinUpstream effector/effectDownstream effector/effectStatus in disease condition
ADT2D
ALK miR-1271 Grb2, NOX4 ↓[6↓[6
RYK miR-1271 Grb2, NOX4 ↓[6↓[6
Grb2 ALK, RYK, PAX4 Recover Cytoskeleton Proteins degradation after Grb2–NoX4 interaction ↑[19↑ 
NOX4 Reactive Oxygen Species (ROS) [95], Rac1 [96], ALK, RYK, PAX4 SSH-1 [18,19], Recover Cytoskeleton Proteins degradation after Grb2–NoX4 interaction ↑[19↑ 
PAX4 ARX Grb2, NOX4 ↑ ↑ 
ARX β-Catenin PAX4 ↓ ↓ 
β-Catenin GSK3β ARX ↓ ↓ 
GSK3β activity AKT1 β-Catenin ↑[6↑[6
AKT1 activity PTEN [97GSK3β ↓[6↓[6
10 Cytoskeleton Proteins (α-Tubulin, Vimentin, α-SMA, Stathmin1) RhoA, Rac1 and C dc 42 Degeneration of cell ↓[19↓ 
11 RhoA AKT1 [98Cytoskeleton proteins ↓[19↓ 
12 Rac1 AKT1 [98Cytoskeleton proteins ↓[19↓ 
13 Cdc42 AKT1 [98Cytoskeleton proteins ↑[19↑ 
14 Cofilin activity LIMK [99F-Actin ↓[19↓ 

In the context of Insulin Receptor (IR) insensitivity being the pathological hallmark of both diseases, previous findings called for understanding the roles of other members of the RTK family. For the first time two non-canonical receptors (ALK or RYK) have been shown to link disparate signals (AICD/Aβ and Palmitate/Amylin) leading to similar pathways (Grb2/PI3K/AKT/RhoA/Rac1/Cdc42/Cofilin or NOX4/SSH-1/Cofilin) that ultimately culminate in similar mechanistic consequences (degradation of cytoskeleton network) (Figure 8; Supplementary Figure S9).

Summary diagram.

Figure 8.
Summary diagram.

A cartoon representation summarizing the overlap of signaling pathways for both AD and T2D, mediated via the down-regulation of membrane bound ALK/RYK, consequent up-regulation of the transcription factor PAX4, which in turn up-regulates Grb2/NOX4. These effectors, through the signaling molecules, affect the fate of the cytoskeletal proteins.

Figure 8.
Summary diagram.

A cartoon representation summarizing the overlap of signaling pathways for both AD and T2D, mediated via the down-regulation of membrane bound ALK/RYK, consequent up-regulation of the transcription factor PAX4, which in turn up-regulates Grb2/NOX4. These effectors, through the signaling molecules, affect the fate of the cytoskeletal proteins.

Conclusions

The emerging model from this study would help understand both the diseases from each other's perspectives. The work unraveled (a) a novel avenue to study the implications of other RTKs, besides IR, for both the diseases, (b) the unique role of Grb2 and NOX4 in the management of cytoskeleton degradation under AD/T2D conditions and (c) most importantly, the emergence of PAX4 as the first Transcription Factor that actively regulates the pathways of both AD and T2D.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the IBOP project, HBNI, Department of Atomic Energy, Government of India.

CRediT Author Contribution

Debashis Mukhopadhyay: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Project administration, Writing — review and editing. Piyali Majumder: Conceptualization, Data curation, Software, Formal analysis, Investigation, Methodology, Writing — original draft, Writing — review and editing. Kaushik Chanda: Data curation, Methodology. Debajyoti Das: Data curation, Methodology. Brijesh Kumar Singh: Methodology. Partha Chakrabarti: Resources, Data curation, Methodology. Nihar Ranjan Jana: Resources, Methodology.

Abbreviations

     
  • AD

    Alzheimer's Disease

  •  
  • ALK

    anaplastic lymphoma kinase

  •  
  • APP

    amyloid precursor protein

  •  
  • ARX

    Aristaless Related Homeobox

  •  
  • IR

    insulin receptor

  •  
  • PAX4

    Paired Box Protein 4

  •  
  • ROS

    reactive oxygen species

  •  
  • RTK

    receptor tyrosine kinase

  •  
  • RYK

    Receptor-like Tyrosine Kinase

  •  
  • SMA

    smooth muscle actin

  •  
  • SSH-1

    Slingshot homolog-1

  •  
  • TF

    transcription factors

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Supplementary data