SH2B1 is Involved in the Accumulation of Amyloid-β 42 in Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is characterized by deficits in learning and memory abilities, as well as pathological changes of amyloid-β (Aβ) plaque and neurofibrillary tangle formation in the brain. Insulin has been identified as a modulator of the neuronal pathways involved in learning and memory, and is also implicated as a modulator of Aβ and tau metabolism. Disrupted insulin signaling pathways are evident in AD patients and it is understood that type 2 diabetes can increase the risk of developing AD, suggesting a possible link between metabolic disorders and neurodegeneration. SH2B1 is a key protein in the insulin signaling pathway involved in regulating the activity of the insulin receptor. To further identify the role of the insulin signaling pathway in the pathology of AD, SH2B (dSH2B homologue in flies) in neurons was partially knocked out or overexpressed in an AD Drosophila model expressing Aβ₄₂. Partial knockout of neuronal SH2B in the Aβ₄₂-expressing Drosophila had a detrimental effect on mobility and neurotransmission, and increased levels and intraneuronal accumulation of Aβ₄₂, as assessed by ELISA and immunostaining. Alternatively, partial overexpression of neuronal SH2B in the Aβ₄₂-expressing Drosophila improved lifespan, mobility, and neurotransmission, as well as decreased levels and intraneuronal accumulation of Aβ₄₂. Thus, SH2B1 may be an upstream modulator of Aβ metabolism, acting to inhibit Aβ accumulation, and has a role in the pathogenesis of AD. SH2B1 may therefore have potential as a therapeutic target for this common form of dementia.

Keywords

Alzheimer’s disease amyloid-β accumulation diabetes SH2B1 protein

INTRODUCTION

Alzheimer’s disease (AD) is the most common cause of senile dementia and characterized by cognitive decline, primarily in learning and memory. The incidence of dementia rapidly increases with age, from 2-3% at 70–75 years of age to 20–25% among individuals aged 85 years or more [1]. In 2001, more than 24 million people were diagnosed with dementia worldwide and due to an increase in global life expectancy, this number is expected to double every 20 years to 81 million by 2040 [2]. Currently, a positive diagnosis for AD can only be made by postmortem detection of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain [3]. Aβ is a cleavage product of a larger protein, the amyloid-β protein precursor (AβPP), and NFTs consist of hyperphosphorylated tau protein. The accumulation of Aβ and NFTs are considered toxic to neurons and contribute to the deterioration of the brain and onset of AD. Although strong links have been shown between Aβ, NFTs, and neurodegeneration, the mechanisms involved in the accumulation of these toxic proteins in AD are not fully understood.

In recent years, several studies have suggested metabolic disorders, such as diabetes, may contribute to the development of AD. Epidemiological studies have found individuals with type 2 diabetes have a 60% increased risk of developing cognitive decline and dementia [4]. Furthermore, higher than average blood glucose levels in non-diabetic patients have been associated with an increased risk to developing dementia, and this risk is further increased with a diagnosis of diabetes [5]. Clinically, diabetic patients show decreased executive function, information processing, planning, and visual memory; a phenotype reminiscent to that seen in dementia patients. Diabetes is a disease of high blood glucose levels caused by a lack of producing insulin (type 1) or a failure of cells to respond to insulin (type 2). In the brain, insulin is a neuromodulator and important not only as a regulator of neuronal glucose use, but also in the release and uptake of neurotransmitters [6]. Insulin receptors are evident throughout the brain, but highly abundant in the hypothalamus and hippocampus, areas of the brain that are damaged in AD. Many AD patients show evidence of insulin dysregulation. In the brain of AD patients, decreased levels of insulin and insulin receptors have been found and plasma levels of insulin are increased [7 –9].

Besides regulating glucose metabolism, insulin is also implicated as a modulator of Aβ and tau metabolism. Insulin has been shown to inhibit the degradation of Aβ₄₀ and Aβ₄₂, and may also have a role in the secretion of Aβ. In vitro, insulin has been shown to assist in the trafficking of Aβ from the Golgi and trans-Golgi network to the plasma membrane [10, 11]. Furthermore, Aβ can be directly degraded by the insulin-degrading enzyme (IDE), thus Aβ and insulin may competitively bind to IDE [12]. Postmortem hippocampal tissue from AD patients shows decreased mRNA and protein levels of IDE [13]. Insulin has also been shown to modulate the phosphorylation of tau by inhibiting glycogen synthase kinase-3 (GSK-3) in neurons [14]. It has long been known that GSK-3β is able to phosphorylate tau and its activity is increased in AD [15]. Thus, the lack of functional insulin in diabetes could contribute to an increase in the accumulation of Aβ and phosphorylation of tau, which in turn can promote the development of AD. In support of this, both Aβ and tau accumulations have been found in the pancreatic islets (the tissue in which insulin-producing cells reside) of type 2 diabetic patients [16]. GSK-3 activity is also increased, along with an overproduction of Aβ₄₀ and Aβ₄₂ and hyperphosphorylated tau, in the hippocampus of a diabetic rat model [17]. Thus, there is mounting evidence to suggest disruptions in the insulin signaling pathway may contribute to the pathology of AD.

The Src homology 2B (SH2B) family members (SH2B1, SH2B2 and SH2B3) are adaptor signaling proteins involved in obesity, insulin resistance, and glucose intolerance [18]. In mammalian cells, SH2B proteins have dual functionality during insulin signaling transduction and can activate as well as inhibit downstream intracellular signaling events [19]. SH2B1 can directly enhance insulin signaling by promoting insulin receptor phosphorylation of insulin receptor substrate (IRS) proteins [20]. This can then activate a number of downstream pathways, such as PI3K/Akt or MAPK [21]. SH2B1 can also enhance leptin signaling by stimulating JAK2 activity and assembling the JAK2 and IRS1/2 signaling complex [22]. Systemic homozygous deletion of SH2B1 results in severe obesity, glucose intolerance and insulin resistance in mice [23]. Furthermore, neuronal restoration of SH2B1 expression alone is sufficient to correct the metabolic disorders observed in null SH2B1 mice [23]. Thus, neuronal SH2B1 is a critical metabolic regulator in mammals. In neurodegenerative disease, little is known about the role of SH2B proteins, but single nucleotide polymorphisms in the SH2B3 gene have been associated with an increased risk to developing amyotrophic lateral sclerosis (ALS), a neurodegenerative movement disorder caused by deteriorating motor neurons [24]. Impaired energy metabolism has been suggested as a cause of ALS, thus SH2B proteins may have a vital role in neurodegeneration.

In the present study, to further identify the role of the insulin signaling pathway in the pathology of AD, dSH2B (homologue of human SH2B1 in Drosophila) was investigated in an AD Drosophila model. Previous studies have shown that Aβ₄₂ expression in Drosophila can cause early death, an age-dependent reduction in mobility and the intraneuronal accumulation of Aβ in the polysynaptic pathway of the giant fiber (GF) system [25 –27]. Using this Drosophila AD model, dSH2B was genetically decreased (partial gene knockout) or partially overexpressed in neurons and the effect on lifespan, mobility, and Aβ₄₂ accumulation was assessed. This study aimed to further understand the link between metabolic disorders and AD.

MATERIALS AND METHODS

Genetic and stocks

Drosophila (fruit fly) stocks were purchased from the Drosophila Bloomington Stock Center (University of Indiana, Bloomington, IN) and cultured on standard medium: ddH₂O 0.65 L/L, baker’s yeast 16 g/L, corn flour 80 g/L, agar 6.5 g/L, brown sugar 137.5 g/L, and beer yeast 7.5 g/L. The recipe for the preservative used was: methylparaben 2 g/L, alcohol 20 ml/L, ddH₂O 100 ml/L, and propionic acid 6.25 ml/L. After pupation, the adult flies were cultured on standard medium and entrained into a 12-h light/dark cycle at 25°C. The Gal4-UAS system was used to drive the over-expression of arctic Aβ₄₂ or Drosophila SH2B.

The transgenes from previous studies [25 , 29], [Gal4]A307 (expressing transcription factor, Gal4, in the neurons of GF system and other components of the nervous system), UAS-Aβ₄₂ and UAS-SH2B (driven by transcription factor Gal4 expressing arctic Aβ₄₂ and Drosophila SH2B protein, respectively) were used as well as a heterozygous deletion of the SH2B gene (partial gene knockout, SH2B^–/+). Flies containing these transgenes or mutation were back-crossed to an isogenic wild-type fly to pure genetic background, and then crossed to obtain the following groups:

Control (CTRL): contains one copy of [Gal4] A307 genetically modified wild-type flies;

SH2B^+/–: contains one copy of [Gal4]A307 with heterozygote expression of SH2B with one SH2B gene knocked out;

SH2B^OE: contains one copy of [Gal4]A307 and upstream activation system (UAS)-SH2B;

Aβ²₄₂: contains one copy of [Gal4]A307 and one copy of Aβ₄₂ inserted into the second chromosome, UAS-Aβ₄₂ transgenic flies;

Aβ²₄₂;SH2B^+/–: Aβ²₄₂-expressing Droso-phila with one copy of the SH2B gene knocked out in the second chromosome;

Aβ³₄₂: contains one copy of [Gal4]A307 and one copy inserted into the third chromosome, UAS-Aβ₄₂ transgenic flies;

Aβ³₄₂;SH2B^OE: Aβ³₄₂-expressing Drosophila with one copy of the SH2B overexpressed in the third chromosome.

To generate dSH2B transgenic flies, a full length of dSH2B cDNA was generated by RT-PCR using the primers 50-GCTGGGTAACTCGTGTGG and 50-CGGACTTAGGTGAAGCTGTAC, verified byDNA sequencing and inserted into a P-element vector (pUAST-dSH2B). The pUAST-dSH2B vectors were used to generate three independent UAS-dSH2B transgenic lines following standard germline transformation procedures [30]. The UAS-dSH2B transgenic lines were crossed with a double balancer line (CyO/Bl;TM2,Ubx/TM6B,Tb) to identify the chromosomes that contain the UAS-dSH2B transgene. A UAS-dSH2B line in which the transgene was inserted on the second chromosome was used in the current study. The UAS-dSH2B line or a coisogenic wild-type line was crossed with actin-GAL4, elav-GAL4, adh-GAL4, lsp2-GAL4, or ppl-GAL4 driver lines to generate various dSH2B transgenic lines in which dSH2B was specifically overexpressed in the whole body (actin-GAL4/UAS-dSH2B), neurons (elav-GAL4/UAS-dSH2B). The recording environment temperature was25°C.

Real time-polymerase chain reaction (RT-PCR)

To measure the expression levels of SH2B in Drosophila, head and thoracic segments from 30 female Drosophila, 25 days after eclosion, were dissected and the RNA isolated for RT-PCR. Briefly, RNA was isolated using the HiScript QRT SuperMix for qPCR kit (Vazyme). cDNA synthesis was performed using the 2xTaq PCR Mastermix (TianGen). Analyzed products were assayed in triplicate, with each replicate including 10 samples per group taken from an independent experiment. The primers used for dSH2B in flies were: forward primer 5^′-CGCCGAGACATTGCAACGAAGTGATTACC-3^′ and reverse primer 5^′-AAGGGAGGCGTTCGAAGCCCGC-3^′. The protein RLP32 was used as a control and the primers used as follows: forward primer 5^′-TAATTGTCGTTTTTGGCGGTTTC-3^′ and reverse primer 5^′-TCATCTTGAAGCAGGTTGGGC-3^′.

Longevity assay

The longevity assay was performed to measure the survival rate of each genotype of Drosophila, as previously described [25]. In summary, 100 flies from each genotype were separated equally into five vials containing standard fly food and dry yeast, and cultured at 25°C. After every 3 days, flies were transferred into vials with fresh food and dried yeast, and dead flies were counted. Survival rates were analyzed using Kaplan–Meier.

Behavioral assay

The climbing assay was performed according to the negative-geotaxis climbing assay or automatic rapid iterative negative geotaxis (aRING) assay, as previously described [31]. In summary, a vial of flies (n≥30, 25 days after eclosion) were consecutively tapped 4 times, using an automated electric motor, to cause all flies to drop to the bottom of the vial. A digital video recorder mounted on a tripod 50 cm in front of the vial was used to record the climbing behavior of the flies. Flies were assessed in 3–5 consecutive trials separated by 60-s intervals. The fly climbing video captures images at 5 s, which are then imported into an in-house program to analyze the average climbing height of the flies. All behavioral recording was done at 25°C.

Electrophysiology

At 28 days after eclosion, the GF neurons were directly stimulated in the brain of adult flies (n≥5) and recordings from the output muscles of the GF system (dorsal longitudinal muscles, DLMs) were obtained. Flies were anaesthetized with CO₂. The fly legs and wings were removed and the rest of the body was fixed into wax. A glass ground electrode was placed into the posterior end of the abdomen and two glass stimulating electrodes placed through the eyes into the brain. A glass recording electrode was placed into the left (or right) DLM muscle fiber. During 100 Hz-frequency stimulation, the stimulation intensity was 30∼60 V with the duration of 0.1 ms, 150% of the threshold stimulation intensity at 0.5 Hz. A sudden potential drop of 20–60 mV monitored intracellular penetration into the muscle. The muscle identity (DLM) was determined by electrode placement. Signals were amplified by Multiple Clamp 700B (Molecular Devices) and digitized at 20 kHz by Digidata 1440A (Molecular Devices). Data was collected and analyzed using the pClamp software (version 10.0; Molecular Devices). All glass electrodes were filled with 3 M KCl. The recording environment temperature was 25°C.

ELISA quantification of Aβ₄₂

To analyze the levels of Aβ₄₂ in the fly brain, the intact brains of flies (n≥20, 25 days after eclosion) were dissected in cold phosphate buffered saline (PBS), placed immediately into cold ELISA sample buffer supplemented with cocktail protease inhibitor (Calbiochem) and incubated on ice for 3 h. Samples were then centrifuged at 4,000 g for 5 min and the resulting supernatant collected for analysis using the Aβ₄₂ Human ELISA kit (Invitrogen), according to manufacturer’s instructions. In summary, 50 μl of supernatant was incubated with a human Aβ₄₂ detection antibody for 3 h at room temperature while shaking. Samples were then washed 4 times with wash buffer, before incubation with anti-rabbit IgG HRP for 30 min at room temperature. After washing 4 times with wash buffer, stabilized chromagen was added to each well and the ELISA plate incubated in the dark for 30 min at room temperature. 100 μL of stop solution was then added and the absorbance readings of each sample measured at 450 nm (A450). The absorbance values for the control samples were taken as background readings (wild-type flies do not produce Aβ) and the rest of the sample values calculated as absorbance readings minus the control (background) reading. Levels of Aβ₄₂ were quantified using a standard curve and values calculated in pg of Aβ per fly head.

Immunostaining and imaging

Whole mount Aβ staining was performed using the whole central nervous system (including the brain and ventral ganglion) of Drosophila, as previously described [25], 25 days after eclosion. The central nervous system of 5 flies per group, from 3 separate vials (total n≥15/group), were dissected out in PBS and fixed with 4% paraformaldehyde in PBS for 45 min. Preparations were washed twice with PBS for 10–15 min before treatment with 70% formic acid for 30 min to re-expose the epitope. Preparations were then washed three times with PBS Triton X-100 (0.5%) with BSA (5%) and incubated for 2–3 h with primary antibody (6E10, Covance #SIG-39320-1000). After incubation with primary antibody, preparations were washed with PBS, incubated for 2 h with FITC-conjugated secondary antibody and then imaged using confocal microscopy (Zeiss LSM 710 METANLO).

Western blotting

At 10 days after eclosion, the head and torso of female flies were dissected (n = 20 per group). Samples were then homogenized with a homogenization buffer (1 : 50 RIPA buffer (APPLYGEN) and protease inhibitor cocktail set III EDTA-free (Calbiochem)) and diluted in sample buffer, before separation on an 8% Tris-glycine gel in SDS-running buffer at 100 volts for 1 h. After gel electrophoresis, the proteins were transferred onto nitrocellulose membrane at 100 volts for 2 h, before incubating in blocking buffer (4% casein/PBS) at room temperature for 1 h. After blocking, membranes were then incubated with primary antibody (Phospho-Akt (Ser473) (pAkt, 1 : 1000, Cell Signaling Technology), Akt (1 : 1000, Cell Signaling Technology) or beta-actin (β-actin, 1 : 1000, Proteintech)) in 4% Casein/PBS at 4°C overnight. After primary incubation, membranes were washed three times with tris-buffered saline/0.05% (v/v) Tween-20 (TBST) and incubated with secondary antibody (goat anti-rabbit, 1 : 10000, Thermofisher) for 2 h at room temperature. Membranes were then washed three times with TBST before being imaged using the Odyssey Two-Color Infrared Imaging System (LI-COR Biosciences). The relative abundance of proteins within the sample was assessed using the ImageJ software and β-actin as a loadingcontrol.

Data analysis and statistics

All data are presented as mean±SD. A Student’s t-test was used to analyze between two groups (e.g., Aβ²₄₂ versus Aβ²₄₂-SH2B^+/–), with significant difference taken as p < 0.05. The statistical software GraphPad Prism was used to represent and analyze the data.

RESULTS

Behavioral analysis of neuronal dSH2B partial gene knockout or overexpression in Aβ₄₂-expressing Drosophila

Levels of dSH2B were analyzed in control, Aβ-expressing, dSH2B partial gene knockout and partial overexpression of dSH2B Drosophila to confirm the SH2B mutants. RT-PCR results showed Aβ-expressing Drosophila with a partial gene knockout of dSH2B did have a decrease in dSH2B mRNA (^*p < 0.05) and partial overexpression of dSH2B Drosophila did have an increase in dSH2B mRNA (^*p < 0.05) (Fig. 1A). The longevity assay was used to assess the survival rate of Aβ₄₂-expressing Drosophila with partial gene knockout or overexpression of neuronal dSH2B compared with wild-type (CTRL) flies. Consistent with previous data from our laboratory [25], the Aβ-expressing Drosophila showed decreased lifespan compared to the CTRL Drosophila (^*p < 0.05, Fig. 1Bi and Ci). Partial gene knockout (SH2B^+/–) or overexpression of dSH2B (SH2B^OE) in the CTRL Drosophila did not significantly affect the lifespan of the female and male flies (Fig. 1Bii and Cii, respectively). Compared to the Aβ²₄₂-expressing Drosophila, partial gene knockout of dSH2B in the Aβ²₄₂-expressing Drosophila (Aβ²₄₂-SH2B^+/–) did not significantly affect lifespan of the female and male flies (Fig. 1Bi p = 0.11 and Ci p = 0.75, respectively). Compared to the Aβ³₄₂-expressing Drosophila, partial overexpression of dSH2B in the Aβ³₄₂-expressing Drosophila (Aβ³₄₂-SH2B^OE) significantly increased lifespan of the female and male flies (Fig. 1Bi and Ci ^***p < 0.001, respectively). When genders of flies were combined, the data also showed the same trends between groups (data not shown). Thus, the following experiments used only female flies, as the female fly anatomy is larger and easier to conductanalyses.

To assess the effect of genetically altering levels of SH2B on behavior in the Aβ₄₂-expressing Drosophila, the climbing assay was used to analyze the mobility of the flies. The landing height of flies from each genotype was assessed. Partial gene knockout or overexpression of dSH2B in the CTRL Drosophila did not significantly affect climbing ability and mobility of the flies (p = 0.21 and p = 0.33, respectively; Fig. 1D). The Aβ₄₂-expressing Drosophila had a significantly lower landing height compared to control flies, indicating impaired climbing ability and mobility (1.90 cm in Aβ²₄₂- and 1.24 cm in Aβ³₄₂-expressing flies compared to 2.83 cm in CTRL flies, Student’s t-test ^*p < 0.05, Fig. 1D). Compared to the Aβ₄₂-expressing Drosophila, partial gene knockout of dSH2B in the Aβ²₄₂-expressing Drosophila (Aβ²₄₂-SH2B^+/–) significantly decreased the landing height (1.46 cm, Student’s t-test ^*p < 0.05, Fig. 1D), but partial overexpression of dSH2B in the Aβ³₄₂-expressing Drosophila (Aβ³₄₂-SH2B^OE) increased the landing height similar to wild-type levels (2.06 cm, Student’s t-test ^*p < 0.05, Fig. 1D).

Electrophysiology of Aβ₄₂-expressing Drosophila is altered with partial knockout or overexpression of neuronal dSH2B

To investigate more directly the effect of genetically altering levels of SH2B on Aβ₄₂-induced neuronal changes, the neural transmission through the GF system in vivo was examined. Repetitive brain stimulation at 100 Hz (total of 100 stimuli) was used to activate the GF system and evoked excitatory junction potentials (EJPs) were intracellularly recorded in the DLM (representative images of recordings from each group are shown in Fig. 2A). At 28 days after eclosion, expression of Aβ₄₂ in Drosophila altered neurotransmission by decreasing EJP in the GF system (43.33% success rate in Aβ²₄₂- and 36.80% success rate in Aβ³₄₂-expressing flies compared to 93.25% success rate in CTRL flies, Student’s t-test ^*p < 0.05, Fig. 2B). Partial gene knockout of dSH2B in Aβ₄₂-expressing Drosophila further decreased EJP (18.00% success rate in Aβ²₄₂-SH2B^+/– flies, Student’s t-test ^***p < 0.001, Fig. 2B), but overexpression of dSH2B improved neurotransmission by increasing EJP (68.80% success rate in Aβ³₄₂-SH2B^OE flies, Student’s t-test ^***p < 0.001, Fig. 2B) in the GF system compared to Aβ₄₂-expressingDrosophila.

Aβ₄₂ levels and intraneuronal accumulations are increased with partial knockout, but decreased with partial overexpression of neuronal dSH2B in Aβ₄₂-expressing Drosophila

To determine the effect of genetically altering levels of SH2B1 in neurons on levels of Aβ₄₂, the heads of flies were collected, homogenized and the supernatant collected for ELISA. Partial gene knockout of dSH2B in the Aβ₄₂-expressing Drosophila increased levels of Aβ₄₂ (13.49 pg/head in Aβ²₄₂-SH2B^+/– flies compared to 7.97 pg/head in Aβ²₄₂-expressing flies, Student’s t-test ^*p < 0.05, Fig. 3A), but overexpression of dSH2B decreased levels (8.28 pg/head in Aβ³₄₂-SH2B^OE flies compared to 15.05 pg/head in Aβ³₄₂-expressing flies, Student’s t-test ^*p < 0.05, Fig. 3A).

To analyze the intraneuronal accumulation of Aβ₄₂ in the flies, the central nervous system tissue (including the brain and ventral ganglion) were whole mounted for immunostaining. As expected, the control flies showed no Aβ₄₂ accumulations (Fig. 3Bi), as Drosophila do not produce Aβ₄₂, but intraneuronal accumulation of Aβ₄₂ was observed in the Aβ²₄₂- and Aβ³₄₂-expressing flies (Fig. 3Bii and 3Biv, respectively). Partial gene knockout of dSH2B in the Aβ₄₂-expressing Drosophila increased the amount of intraneuronal Aβ₄₂ accumulations observed (Aβ²₄₂-SH2B^+/–, Fig. 3Biii), but overexpression of dSH2B decreased the amount of intraneuronal Aβ₄₂ accumulations observed (Aβ²₄₂-SH2B^OE, Fig. 3Bv).

Partial overexpression of neuronal dSH2B in Aβ₄₂-expressing Drosophila can increase phosphorylated Akt levels

To identify if the partial knockout or overexpression of neuronal dSH2B in Aβ₄₂-expressing Drosophila could affect downstream signaling pathways known to be involved in AD and diabetes, levels of protein kinase B (Akt) and its activated phosphorylated form were investigated. Western blotting analysis showed levels of pAkt were not significantly different between Aβ²₄₂-expressing and partial gene knockout dSH2B Drosophila (Aβ²₄₂-SH2B^+/–, Student’s t-test p = 0.53, Fig. 4), but overexpression of dSH2B increased levels of pAkt (Aβ²₄₂-SH2B^OE, Student’s t-test ^**p < 0.01, Fig. 4). Levels of pAkt were also greater in the Drosophila with an overexpression of dSH2B compared to control (CTRL) flies (Student’s t-test ^*p < 0.05, Fig. 4).

DISCUSSION

A recent meta-analysis found diabetes and coronary heart disease, a complication associated with diabetes, increased the risk of mortality in AD patients [32]. Many AD patients present with symptoms of diabetes, such as high blood glucose levels and high plasma and low cerebrospinal fluid insulin levels [7 –9]. Insulin, a key modulator of glucose levels and dysregulated in diabetes, has also been implicated as a modulator of Aβ and tau metabolism. Thus, metabolic disorders may be involved in the development of AD, and diabetes comorbid with AD could accelerate disease progression and exacerbate disease pathology. SH2B1 is a key protein in the insulin signaling pathway and involved in regulating the activity of the insulin receptor. To further identify the role of the insulin signaling pathway in the pathology of AD, this study genetically decreased or overexpressed SH2B in neurons of the GF system in an AD Drosophila model expressing Aβ₄₂. Partial gene knockout of neuronal dSH2B (SH2B1 homologue in flies) in Aβ₄₂-expressing Drosophila showed no significant effect on lifespan, but had a detrimental effect on mobility and neurotransmission, and increased the levels and intraneuronal accumulation of Aβ₄₂. Partial overexpression of neuronal dSH2B in Aβ₄₂-expressing Drosophila significantly increased lifespan, improved mobility and neurotransmission, and decreased the levels and intraneuronal accumulation of Aβ₄₂. Furthermore, partial overexpression of dSH2B in Aβ₄₂-expressing Drosophila increased levels of phosphorylated protein kinase B (pAkt). Thus, neuronal SH2B1 may be an upstream modulator of Aβ metabolism, acting to inhibit Aβ accumulation.

This is the first study to investigate SH2B1 in an animal model of AD and further supports the hypothesis that dysregulated insulin signaling can impact the pathology of AD. Systemic knockout of SH2B1 results in severe obesity, glucose intolerance, and insulin resistance in mice, but neuronal restoration of SH2B1 expression alone can sufficiently correct the metabolic disorders observed in null SH2B1 mice [23]. Consistent with this, partial knockout of neuronal dSH2B had a detrimental affect on the phenotype of Aβ₄₂-expressing Drosophila, whereas overexpression of neuronal dSH2B led to an improvement in phenotype. Thus, neuronal SH2B1 is a key metabolic regulator. Previous study in our laboratory has shown the expression of Aβ₄₂ in Drosophila is able to cause the accumulation of Aβ in the cell body and axons of neurons [25]. This intraneuronal accumulation of Aβ causes the age-dependent decline in mobility and early death in flies. Consistent with this, in this study, the Aβ-expressing Drosophila showed decreased lifespan compared to control Drosophila. Furthermore, lifespan was significantly improved with partial overexpression of neuronal dSH2B compared to the Aβ-expressing Drosophila, but not significantly affected with apartial knockout of dSH2B. These discrepancies on the effect of dSH2B expression on lifespan may be due to the expression of dSH2B restricted to only the GF system and not the entire neuronal or peripheral system in the fly. To further identify the effect of SH2B on lifespan in Drosophila, null dSH2B and overexpression of dSH2B in the whole central nervous system will need to be done. Of interest, null SH2B mice have been studied and are viable [23]. Analysis of the neural transmission through the GF system showed deficits consistent with an AD pathology and partial gene knockout of dSH2B worsened neural transmission, but partial overexpression of dSH2B was sufficient to improve these deficits. Thus, these results may indicate that synaptic pathology is sensitive to alterations related to insulin signaling. In recent years, the intraneuronal accumulation of Aβ has gained in significance as a crucial part of AD pathology and is associated with abnormal synaptic pathology, development of cognitive decline and formation of Aβ plaques [33, 34]. Thus, the results from this study, showing genetic manipulation of SH2B can affect the intraneuronal accumulation of Aβ, suggests targeting SH2B could modulate an integral part of ADpathology.

While Drosophila are valuable animals for investigating the effect of genetic manipulation on cellular pathways, further analysis using higher organisms relatable to humans will be necessary to fully examine the role of SH2B1 on AD pathology. Thus, it would be of interest to see if similar genetic manipulation of SH2B1 levels can improve the behavior of other animal models of AD, such as in rodents. While there are cognitive (learning and memory) tests which can be performed using fly models [35], it would be more relatable to humans if the effect of partial deletion or overexpression of SH2B1 on the cognitive symptoms of AD were analyzed in rodent (mouse or rat) models of disease. Furthermore, in this study, the expression of Aβ and dSH2B in Drosophila were restricted to only the GF nervous system, which is primarily used in flies for mobility to direct flight muscles and not in learning and memory [36]. Thus, to test if the partial knockout of SH2B1 worsens cognitive decline or if overexpression of SH2B1 improves the cognitive deficits represented in AD mouse models, our laboratory is currently conducting these studies. Nonetheless, this study does show significant initial data that SH2B1 may be involved in the intraneuronal accumulation of Aβ and development of an AD phenotype, further implicating the insulin signaling pathway is involved in the pathogenesis of AD.

In the body, insulin binding to the insulin receptor causes autophosphorylation and the recruitment of adaptor proteins such as insulin receptor substrate (IRS) and SH2B proteins [37]. This causes downstream signaling, such as an activation of PI3K/Akt or MAPK pathways, which can involve numerous cellular processes including apoptosis, Aβ secretion, or tau phosphorylation. Thus, SH2B directly enhances insulin signaling. To preliminarily determine if altering levels of SH2B can affect downstream pathways, levels of Akt were analyzed. Levels of activated phosphorylated Akt (pAkt) were not significantly different in the control, Aβ₄₂-expressing, and partial gene knockout of neuronal dSH2B flies, but levels of pAkt were increased in the flies expressing a partial overexpression of neuronal dSH2B. This suggests the partial overexpression of neuronal SH2B can activate downstream signaling proteins such as Akt in the insulin signaling pathway. This may have contributed to the beneficial outcomes found with the partial overexpression of neuronal dSH2B in Drosophila. However, other proteins involved in the insulin signaling pathway,such as IRS1/2, will need to be investigated in all groups to further conclude this. Other pathways involved with SH2B1, such as an interaction with tyrosine kinase receptors (Trks) to promote neuronal survival [38], could also be affected and involved in causing the effects seen in this study. This will also need to be investigated in further studies. However, these preliminary results show promise that targeting SH2B in AD can increase the insulin signaling pathway and improve disease symptoms. There is very little data on a direct relationship between SH2B proteins and neurodegeneration, but the SH2B3 gene has been associated with an increased risk to developing ALS, a neurodegenerative movement disorder caused by deteriorating motor neurons [24]. Impaired energy metabolism has been implicated as a cause of ALS, further suggesting metabolic disorders have a role in the development of neurodegenerative disease. Alternatively, insulin has been investigated heavily in AD research due to multiple lines of evidence showing insulin expression and levels in the CNS are altered in AD patients [8]. Further linking diabetes and AD, elderly diabetic patients often present in the clinic with poorer memory scores compared to healthy elderly patients, and short trials delivering intranasal insulin to healthy or AD patients improve cognitive performance [39, 40]. Healthy, elderly individuals with high, but non-diabetic, blood glucose levels are also associated with poorer memory performance and decreased hippocampal volume [41]. The intranasal delivery of insulin has effectively been shown to pass the blood-brain barrier, directly increasing insulin levels in the brain and improving neural metabolic processes [42]. However, these effects are short-lived as patients became desensitized to the delivered insulin [43]. Thus, targeting insulin as a therapeutic strategy for treating AD is a viable option, but further study is needed to identify the best method to target this pathway and to improve the method of delivery to patients. SH2B1 may be one such target to boost insulin signaling in the AD brain to better treat thisdisease.

In summary, the results from this study suggest that dysregulation of the insulin signaling pathway is involved in the pathogenesis of AD. This study is the first to show that neuronal SH2B1, a key protein in insulin signaling, may have a role in the accumulation of Aβ₄₂ in an animal model of AD. Thus, the presence of a metabolic disorder in AD patients may have a detrimental effect on disease progression and treatment targeting insulin dysregulation may improve the quality of life for these patients.

Footnotes

ACKNOWLEDGMENTS

We thank the Drosophila Bloomington Stock Center (University of Indiana, Bloomington, IN) for providing stocks. This work was supported by the National Natural Science Foundation of China (81371400 and 81071026), the National Basic Research Development Program of China (2013CB530900) and the support for key discipline of Chongming County.

Authors’ disclosures available online ().

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SH2B1 is Involved in the Accumulation of Amyloid-β 42 in Alzheimer’s Disease

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Genetic and stocks

Real time-polymerase chain reaction (RT-PCR)

Longevity assay

Behavioral assay

Electrophysiology

ELISA quantification of Aβ42

Immunostaining and imaging

Western blotting

Data analysis and statistics

RESULTS

Behavioral analysis of neuronal dSH2B partial gene knockout or overexpression in Aβ42-expressing Drosophila

Electrophysiology of Aβ42-expressing Drosophila is altered with partial knockout or overexpression of neuronal dSH2B

Aβ42 levels and intraneuronal accumulations are increased with partial knockout, but decreased with partial overexpression of neuronal dSH2B in Aβ42-expressing Drosophila

Partial overexpression of neuronal dSH2B in Aβ42-expressing Drosophila can increase phosphorylated Akt levels

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

ELISA quantification of Aβ₄₂

Behavioral analysis of neuronal dSH2B partial gene knockout or overexpression in Aβ₄₂-expressing Drosophila

Electrophysiology of Aβ₄₂-expressing Drosophila is altered with partial knockout or overexpression of neuronal dSH2B

Aβ₄₂ levels and intraneuronal accumulations are increased with partial knockout, but decreased with partial overexpression of neuronal dSH2B in Aβ₄₂-expressing Drosophila

Partial overexpression of neuronal dSH2B in Aβ₄₂-expressing Drosophila can increase phosphorylated Akt levels