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Insulin Promotes Neuronal Survival via the Alternatively Spliced Protein Kinase CδII Isoform*

      Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.

      Introduction

      Strong links between insulin and cognitive function are supported by epidemiological data, studies of laboratory animals, and in vitro research (
      • Benedict C.
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      Intranasal insulin improves memory in humans. Superiority of insulin aspart.
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      Intranasal insulin to improve developmental delay in children with 22q13 deletion syndrome. An exploratory clinical trial.
      ,
      • Park C.R.
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      • Craft S.
      • Woods S.C.
      Intracerebroventricular insulin enhances memory in a passive-avoidance task.
      ). Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration caused by apoptosis (
      • Francis G.J.
      • Martinez J.A.
      • Liu W.Q.
      • Xu K.
      • Ayer A.
      • Fine J.
      • Tuor U.I.
      • Glazner G.
      • Hanson L.R.
      • Frey 2nd, W.H.
      • Toth C.
      Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.
      ,
      • Francis G.J.
      • Martinez J.A.
      • Liu W.Q.
      • Xu K.
      • Ayer A.
      • Fine J.
      • Tuor U.I.
      • Glazner G.
      • Hanson L.R.
      • Frey 2nd, W.H.
      • Toth C.
      Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.
      ,
      • Reger M.A.
      • Watson G.S.
      • Green P.S.
      • Baker L.D.
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      • Plymate S.R.
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      • Schellenberg G.D.
      • Frey 2nd, W.H.
      • Craft S.
      Intranasal insulin administration dose-dependently modulates verbal memory and plasma amyloid-β in memory-impaired older adults.
      ,
      • Benedict C.
      • Hallschmid M.
      • Hatke A.
      • Schultes B.
      • Fehm H.L.
      • Born J.
      • Kern W.
      Intranasal insulin improves memory in humans.
      ). However, the neuronal molecular mechanisms of these effects have not been studied in depth. Insulin signaling in the CNS is important in learning, memory, and synaptogenesis and activates survival pathways in neurons. Protein kinase C (PKC) isoforms are serine/threonine kinases, which are involved in the regulation of cellular differentiation, growth, and apoptosis. The insulin and PKC pathways are integrated and function closely together. PKC activation in the brain is essential for learning, synaptogenesis, and neuronal repair (
      • Alkon D.L.
      • Epstein H.
      • Kuzirian A.
      • Bennett M.C.
      • Nelson T.J.
      Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning.
      ,
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      • Kerr D.S.
      • Bevilaqua L.R.
      • Izquierdo I.
      Inhibition of PKC in basolateral amygdala and posterior parietal cortex impairs consolidation of inhibitory avoidance memory.
      ,
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      • Nelson T.J.
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      • Van Leuven F.
      • Alkon D.L.
      Therapeutic effects of PKC activators in Alzheimer's disease transgenic mice.
      ). Insulin signaling pathways activate PKC and its substrates, many of which are essential components of learning, memory, and cognition. In particular, PKCδ (PRKCD) has been implicated in memory, neuronal survival, and proliferation (
      • Conboy L.
      • Foley A.G.
      • O'Boyle N.M.
      • Lawlor M.
      • Gallagher H.C.
      • Murphy K.J.
      • Regan C.M.
      Curcumin-induced degradation of PKC δ is associated with enhanced dentate NCAM PSA expression and spatial learning in adult and aged Wistar rats.
      ,
      • Nelson T.J.
      • Alkon D.L.
      Neuroprotective versus tumorigenic protein kinase C activators.
      ,
      • Gallagher H.C.
      • Murphy K.J.
      • Foley A.G.
      • Regan C.M.
      Protein kinase C δ regulates neural cell adhesion molecule polysialylation state in the rat brain.
      ). PKCδ improves learning and memory by preventing neuronal loss and maintaining the synapses (
      • Nelson T.J.
      • Sun M.K.
      • Hongpaisan J.
      • Alkon D.L.
      Insulin, PKC signaling pathways and synaptic remodeling during memory storage and neuronal repair.
      ). PKCδ plays a central role in apoptosis and has dual effects: as a mediator of apoptosis and as a pro-survival effector (
      • Peluso J.J.
      • Pappalardo A.
      • Fernandez G.
      Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKC δ-dependent pathway.
      ,
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      • Lee J.Y.
      • Haines K.M.
      • Campbell D.E.
      • Sullivan K.E.
      • Korchak H.M.
      A role for PKC-δ and PI 3-kinase in TNF-α-mediated antiapoptotic signaling in the human neutrophil.
      ,
      • McCracken M.A.
      • Miraglia L.J.
      • McKay R.A.
      • Strobl J.S.
      Protein kinase Cδ is a prosurvival factor in human breast tumor cell lines.
      ,
      • Zhang J.
      • Hung A.C.
      • Ng P.Y.
      • Nakayama K.
      • Hu Y.
      • Li B.
      • Porter A.G.
      • Dhakshinamoorthy S.
      PKCδ mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis.
      ,
      • Liu S.
      • Yuan Q.
      • Zhao S.
      • Wang J.
      • Guo Y.
      • Wang F.
      • Zhang Y.
      • Liu Q.
      • Zhang S.
      • Ling E.A.
      • Hao A.
      High glucose induces apoptosis in embryonic neural progenitor cells by a pathway involving protein PKCδ.
      ). Our previous work demonstrated that the mouse splice variants, PKCδI and PKCδII, are a switch that determines cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. PKCδII is the mouse homolog of human PKCδVIII; both are generated by alternative 5′ splice site usage, and their transcripts share >94% sequence homology. We have shown that PKCδII and PKCδVIII function as pro-survival proteins (
      • Patel N.A.
      • Song S.S.
      • Cooper D.R.
      PKCδ alternatively spliced isoforms modulate cellular apoptosis in retinoic acid-induced differentiation of human NT2 cells and mouse embryonic stem cells.
      ,
      • Jiang K.
      • Apostolatos A.H.
      • Ghansah T.
      • Watson J.E.
      • Vickers T.
      • Cooper D.R.
      • Epling-Burnette P.K.
      • Patel N.A.
      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ); the functions of the other PKCδ splice variants (PKCδIII, -IV, -V, -VI, or -VII) are not yet established. A recently reported mouse splice variant, PKCδIX, is described as a dominant-negative inhibitor of the apoptotic property of PKCδ in vivo (
      • Kim J.D.
      • Seo K.W.
      • Lee E.A.
      • Quang N.N.
      • Cho H.R.
      • Kwon B.
      A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis.
      ). PKCδII is generated by the insertion of 78 bp (26 amino acids) via utilization of an alternative downstream 5′ splice site of PKCδ pre-mRNA exon 9 in its caspase-3 recognition sequence (DILD). Previously, we showed that overexpression of PKCδII decreased apoptosis and promoted survival (
      • Patel N.A.
      • Song S.S.
      • Cooper D.R.
      PKCδ alternatively spliced isoforms modulate cellular apoptosis in retinoic acid-induced differentiation of human NT2 cells and mouse embryonic stem cells.
      ). It is established that insulin improves cognition and that neuronal survival is critical for promoting cognition and memory formation. However, the molecular determinants of insulin-mediated neuronal survival remain unknown. Here, we established the link between insulin-mediated alternative splicing of a key signaling kinase, PKCδ, and increased neuronal survival, which ultimately improves cognitive function. We also demonstrate that PKCδII is upstream of the Bcl2-mediated survival cascade.

      EXPERIMENTAL PROCEDURES

       Cell Culture

      The studies were carried out using an immortalized clonal mouse hippocampal cell line (HT22) obtained from Dr. D. R. Schubert (Salk Institute). HT22 cells were cultured in 75-cm2 flasks in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (50 units/ml), and 2 mm glutamine. Cells were maintained at 37 °C in a humidified incubator containing 5% CO2. HT22 cells were sub-cultured into either 25-cm2 flasks or 100-mm2 dishes and used for experiments at 60–80% confluence.

       Western Blot Analysis

      Cell lysates (60 mg) were separated on 10% PAGE-SDS. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with Tris-buffered saline/0.1% Tween 20 containing 5% nonfat dried milk, washed, and incubated with a polyclonal antibody against either anti-PKCδ (BioSource) or PKCδII-specific polyclonal antibody (Patel laboratory). PKCδII-specific antibody was raised against the amino acids of the extended hinge region of mouse PKCδII. This region is absent in PKCδI. PKCδII-specific polyclonal antibody was raised in rabbits by Bio-Synthesis, Inc. (Lewisville, TX) to the synthetic peptide NH2-HISLKSFPSRAKEKDSSET-COOH (corresponding to the V3-hinge domain of PKCδII). The antibody specificity was confirmed by ELISA with the above epitope region peptide and was characterized alongside unreactive pre-immune antisera and shown to recognize PKCδII in mouse HT22 cells. A peptide/antigen assay was also performed to confirm its specificity. Other antibodies, anti-Bcl2, anti-Bcl-xL, anti-pBAD, anti-PARP, anti-XIAP, and anti-GAPDH, were purchased from Cell Signaling. Following incubation with anti-rabbit IgG-HRP, enhanced chemiluminescence (Pierce) was used for detection.

       Transient Transfection of Plasmid DNA

      2 μg of PKCδI-pTracer/CMV and PKCδII-pTracer/CMV plasmids (from Dr. Harutoshi Kizaki, Tokyo Dental College, Japan) were transfected into HT22 cells using a Nucleofector kit from Amaxa. 106 HT22 cells were combined with 100 μl of Nucleofector solution L (Amaxa) and 2 μg of plasmid DNA. The cell/DNA suspension was transferred into a cuvette and nucleofected using program A-020. The cells were then incubated in 100-mm plates for 24–48 h in HT22 growth media at 37 °C.

       siRNA Transfection

      Custom-designed, validated siRNA for PKCδII were purchased from Ambion. These are validated for specificity to eliminate off-target gene effects. The efficiency and conditions of transfections were validated by using silencer GAPDH siRNA from Ambion. The negative control siRNA (scrambled) was simultaneously transfected into HT22 cells. The Ambion siRNA ID, 444054, worked most efficiently for PKCδII, and it was used for further studies. 25–50 nm siRNAs were transfected in serum-free medium with siPORTTM (Ambion) as per the manufacturer's protocol or nucleofected as described above. The cells were then incubated in the growth medium at 37 °C for 72 h.

       RT-PCR

      Total RNA was isolated from cells using RNA-BeeTM (Tel Test, Inc.) as per the manufacturer's instructions. 2 μg of RNA was used to synthesize first strand cDNA using an Oligo(dT) primer and OmniscriptTM kit (Qiagen). PCR was performed using 2 μl of cDNA and Takara Taq polymerase. The primers are listed: PKCδ sense primer (5′-CACCATCTTCCAGAAAGAACG-3′) and antisense primer (5′-CAACAACGGGACCTATGGCAAG-3′); PKCδII-specific antisense primer (5′-TCGCAGGTCTCACTACTGTCCTTTTCC-3′); Bcl-x sense primer (5′-GAGGCAGGCGACGAGTTTGAA-3′) and antisense primer (5′-TGGGAGGGTAGAGTGGATGGT-3′); GAPDH sense primer (5′-CTTCATTGACCTCAACTCATG-3′) and antisense primer (5′-TGTCATGGATGACCTTGGCCAG-3′). The PCR products were as follows: using PKCδ primers, PKCδI and PKCδII were detected simultaneously: PKCδI was 351 bp and PKCδII was 429 bp. Using the PKCδ sense primer and PKCδII-specific antisense primer, PKCδII was 408 bp, Bcl-xL was 460 bp, and Bcl-xS was 261 bp; GAPDH was 391 bp. Following PCR, 5% of products were resolved on 6% PAGE gels and detected by silver staining. The PCR reaction was optimized for linear range amplification to allow for quantification of products. Densitometric analyses of the bands were done using the Un-Scan ITTM Analysis Software (Silk Scientific).

       Animals

      We studied 12 male C57BL6 mice (Harlan). Mice were procured, raised, and studied in pathogen-free environments. Mice were housed in plastic, sawdust-covered cages with a normal light-dark cycle and free access to chow and water. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee at J. A. Haley Veterans Hospital and the University of South Florida, Division of Comparative Medicine. Whole blood glucose measurements were performed weekly with puncture of the tail vein and a hand-held freestyle glucometer. Intranasal insulin was administered as described by previous studies (
      • Francis G.J.
      • Martinez J.A.
      • Liu W.Q.
      • Xu K.
      • Ayer A.
      • Fine J.
      • Tuor U.I.
      • Glazner G.
      • Hanson L.R.
      • Frey 2nd, W.H.
      • Toth C.
      Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.
      ,
      • Francis G.
      • Martinez J.
      • Liu W.
      • Nguyen T.
      • Ayer A.
      • Fine J.
      • Zochodne D.
      • Hanson L.R.
      • Frey 2nd, W.H.
      • Toth C.
      Intranasal insulin ameliorates experimental diabetic neuropathy.
      ), which established pharmacokinetic measurements, optimum concentrations, and uptake and demonstrated that intranasal insulin directly improves learning and memory and has no effect on circulating blood glucose levels. Briefly, mice are administered intranasal insulin (Humulin R, Eli Lilly, 100 units/ml) at final concentrations of 1.0 units/ml or 1% intranasal saline (control). A total of 24 μl of containing the insulin (or saline) was administered as four 6-μl drops in alternating nostrils every minute while each mouse was held in supine position with its neck in extension. Mice were administered intranasal insulin once daily for 4 weeks. Mice were tested in a radial arm water maze to assess their cognitive functions. At the end of the trials, the hippocampi of the mice were collected for RNA and protein analysis.

       Radial Arm Water Maze Testing

      An aluminum insert was extended 5 cm above the surface of the water, allowing the mice to easily view surrounding visual cues, which were generously placed outside of the pool. Visual/spatial cues consisted of large, brightly colored objects, including a beach ball, poster, and inflatable pool toys. In one arm, a transparent 9-cm escape platform was placed 1.5 cm below the surface of the water near the wall end. Each mouse was given thirteen 1-min trials per day. The last of 12 consecutive acquisition trials (Trial 12) and a 60-min delayed retention trial (Trial 13) are indices of working memory. The next day (24 h later), the mice were given the trial once again to measure the long term memory retention. The escape platform location was placed at the end of one of the six arms and was moved to a different arm in a semi-random fashion for each day of testing. Moving the escape platform forces the mouse to learn a new platform location daily and therefore evaluates working memory. On each day, different start arms for each of the daily trials were selected from the remaining five swim arms in a semi-random sequence that involved all five arms. For any given trial, the mouse was placed into the start arm, facing the center swim area, and given 60 s to find the platform. When the mouse made an incorrect choice, it was gently pulled back to the start arm, and an error was recorded. An error was also recorded if the mouse failed to make a choice in 20 s (in which case it was returned to the start arm) or entered the platform-containing arm but failed to locate the platform. After finding the platform, the mouse was allowed to stay on it for 30 s. If the mouse did not find the platform within 60 s, it was guided to the platform, allowed to stay for 30 s, and assigned a latency of 60 s. Errors (incorrect arm choices) and escape latency were recorded for each daily trial.

       Cell Viability and Proliferation Assays

      HT22 cells were assessed for cell viability and proliferation. HT22 cells were either treated with insulin or transfected with PKCδII plasmid or PKCδII siRNA as described above. The treatments were performed in triplicate in a 48-well plate. The 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay kit was purchased from Millipore, catalogue no. 2750, and used as per manufacturer's instructions to quantitatively evaluate the number of actively proliferating cells. Briefly, 100 μl of BrdU was added per well of the 48-well plate and incubated overnight. BrdU incorporation was detected using peroxidase conjugate. The plate was read using a spectrophotometer microplate reader set at a dual wavelength of 450 nm/550 nm. The results were normalized against the blank and background readings. The cell proliferation kit was purchased from Chemicon International, catalogue no. 2210, and uses WST-1/ECS as per the manufacturer's instructions. The formazon dye produced by viable cells is quantified using a spectrophotometer set at a wavelength of 440 nm.

       Statistical Analysis

      The significance for the animal studies was assessed by the matched Student's t test in which the mice cohort was n = 6 per treatment for every time trial. A level of p < 0.05 was considered statistically significant. The gels were densitometrically analyzed using UN-SCAN-ITTM software (Silk Scientific, Inc.). PRISMTM software was used for statistical analysis. The results are expressed as mean ± S.E. of densitometric units or as a percentage of exon inclusion.

      RESULTS

       Insulin Increases PKCδII Expression

      Because the hippocampus is critical for memory and cognition, we used the immortalized mouse hippocampal cell line HT22 for our studies. These cells have been used to study neuronal survival and are established as an in vitro model for mechanistic studies for neurodegenerative diseases such as Alzheimer disease (
      • Liu J.
      • Li L.
      • Suo W.Z.
      HT22 hippocampal neuronal cell line possesses functional cholinergic properties.
      ,
      • Fukui M.
      • Song J.H.
      • Choi J.
      • Choi H.J.
      • Zhu B.T.
      Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells.
      ,
      • Adamczyk A.
      • Kamierczak A.
      • Czapski G.A.
      • Strosznajder J.B.
      α-Synuclein induced cell death in mouse hippocampal (HT22) cells is mediated by nitric oxide-dependent activation of caspase-3.
      ). PKCδI and PKCδII are alternatively spliced variants of PKCδ pre-mRNA; PKCδI is pro-apoptotic, while PKCδII promotes survival. Insulin was added in increasing doses (10 to 100 nm) to HT22 cells for 24 h. Total protein lysates were harvested, and Western blot analysis was performed with a PKCδII-specific antibody raised by our laboratory (see “Experimental Procedures”) or a COOH-terminal PKCδ antibody, which recognizes both variants. In separate experiments, total RNA was extracted and analyzed by RT-PCR with primers that detect PKCδI and PKCδII simultaneously. Insulin increased PKCδII expression, whereas PKCδI levels remained constant (Fig. 1, a and b). In subsequent experiments, the lowest dose (10 nm) of insulin was used. This is the first report demonstrating that insulin regulates PKCδ pre-mRNA alternative splicing in neuronal cells.
      Figure thumbnail gr1
      FIGURE 1Insulin increases PKCδII expression. HT22 cells were treated with increasing doses (10 and 100 nm) of insulin for 24 h. a, whole protein lysates were separated and analyzed by Western blot analysis using PKCδ antibody and anti-GAPDH. b, total RNA was extracted and RT-PCR performed with primers that simultaneously detect PKCδI and PKCδII mRNA. The gels are representative of four experiments performed with similar results. The graph represents the percentage of exon inclusion calculated as SS II/(SS II + SS I) × 100 and is representative of four experiments performed separately.

       Insulin Mediates Its Effect via a PI3K Pathway

      Tyrosine phosphorylation of the insulin receptor and its downstream substrates initiates a series of events through tyrosine and serine/threonine kinases that mediate the effects of insulin. Insulin may mediate its effects by activating kinase pathways such as the mitogen-activated protein kinase pathway, the Janus-activated kinase/signal transducers and activators of transcription pathway, or the phosphatidylinositol 3-kinase (PI3K) pathway. To determine which pathway is involved in insulin-mediated PKCδ alternative splicing in neuronal cells, we used inhibitors to these pathways. Inhibitors were added 30 min before insulin (10 nm) treatment. The PI3K inhibitor LY294002 (1 μm) blocked the insulin-mediated increase in PKCδII, whereas the mitogen-activated protein kinase inhibitor PD98059 (10 μm) did not (Fig. 2a). Rapamycin, a p70/85 S6 kinase inhibitor, had no effect (data not shown), and the Janus-activated kinase inhibitor AG490 decreased PKCδII by 5%.
      Figure thumbnail gr2
      FIGURE 2a, PI3K inhibitor LY294002 blocked insulin-mediated increase of PKCδII expression. HT22 cells were treated with signaling cascade inhibitors: PI3K inhibitor, LY294002 (1 μm), Janus-activated kinase inhibitor, AG490 (5 μm), or MEK inhibitor, PD98059 (10 μm) for 30 min prior to the addition of 10 nm insulin. Whole cell lysates were analyzed by Western blot analysis using antibodies as indicated. The graph shows PKCδII densitometric units normalized to GAPDH and represents three separate experiments. The results were analyzed with a two-tailed Student's t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of p < 0.05 was considered statistically significant. ***, p < 0.0001. Significance was determined after three or more experiments. b, LY294002 blocked insulin-mediated increase of PKCδII expression and phosphorylation of Akt kinase. HT22 cells were treated with the PI3K inhibitor, LY294002 (1 μm), for 30 min prior to the addition of 10 nm insulin for 24 h. Whole cell lysates were analyzed by Western blot analysis using antibodies against PKCδII, p-AKT, AKT, and GAPDH as indicated. The gels represent three experiments performed separately with similar results. The graph shows PKCδII densitometric units normalized to GAPDH and represents three separate experiments. The results were analyzed with a two-tailed Student's t test using PRISM4 statistical analysis software (GraphPad). A level of p < 0.05 was considered statistically significant. ***, p < 0.0001. Significance was determined after three or more experiments.
      Because Akt kinase is a key downstream mediator of the PI3K pathway, we designed experiments to assess its phosphorylation state. HT22 cells were treated with LY294002 before addition of insulin. Insulin increased the phosphorylation of Akt kinase (Ser-473) and simultaneously increased PKCδII levels; the latter increase was inhibited by LY294002, and total Akt levels remained the same (Fig. 2b). These findings indicate that insulin promotes alternative splicing of PKCδII via the PI3K-Akt pathway.

       Insulin Increases Alternative Splicing of PKCδII Pre-mRNA via Phosphorylation of Splicing Factor SC35

      In pre-mRNA, sequences exist as auxiliary cis-elements that recruit trans-acting factors to promote alternative splicing. Exonic or intronic splicing enhancers often bind serine/arginine-rich nuclear factors (SR proteins)
      The abbreviations used are: SR
      serine/arginine-rich
      RS
      arginine/serine-rich
      CA
      constitutively active
      PARP
      poly-(ADP-ribose)polymerase.
      to promote the choice of splice sites in the pre-mRNA. The binding of SR proteins to exonic or intronic sites determines splice site choice. The mouse splice variant PKCδII is generated by use of an alternative downstream 5′ splice site in exon 9 of PKCδ pre-mRNA. Therefore, we sought to identify the trans-factors that bind to the PKCδ pre-mRNA upon insulin treatment and that influence splice site use.
      HT22 cells were treated with insulin (10 nm) for 24 h, and lysates were analyzed by Western blotting with mAb104. Used extensively by RNA biologists, this antibody recognizes the common epitope of all SR proteins and can detect them simultaneously. Insulin increased the expression of a protein of ∼30 kDa, either SRp30a or SRp30b. Using specific antibodies to individual SR proteins, we observed an increase in SRp30b (SC35) expression concurrent with increased PKCδII levels in response to insulin; SRp30a (SF2/ASF) levels were unaffected (Fig. 3a). Hence, SC35 was the logical factor to pursue.
      Figure thumbnail gr3
      FIGURE 3a, insulin increases alternative splicing of PKCδII mRNA via SC35. HT22 cells were treated with increasing doses (10, 50, and 100 nm) of insulin for 24 h or left untreated (control). Whole protein lysates were separated and analyzed by Western blot analysis using either mAb104, PKCδII, SF2/ASF, or SC35antibody as indicated. The gels represent three experiments performed separately with similar results. b, SC35 protein sequence showing Akt kinase consensus sequences RXRXX(S/T) in its RS domain. c, HT22 cells were treated with the Akt inhibitor (124005, 1 μm) for 30 min prior to the addition of 10 nm insulin for 24 h. Whole cell lysates were analyzed by Western blot analysis using antibodies as indicated. The gels represent three experiments performed separately with similar results. The graph represents the percentage of exon inclusion calculated as SS II/(SS II + SS I) × 100 and is representative of four experiments performed separately. The results were analyzed with a two-tailed Student's t test using PRISM4 statistical analysis software (GraphPad). A level of p < 0.05 was considered statistically significant. ***, p < 0.0001. Significance was determined after three or more experiments. d, HT22 cells were treated with increasing doses (10, 50, and 100 nm) of insulin for 24 h or transiently transfected with CA-Akt2 or WT-Akt2. Whole protein lysates were separated and analyzed by Western blot analysis using either PKCδII, phospho-SC35, or GAPDH antibody as indicated. The gels represent three experiments performed separately with similar results.
      SC35 has an NH2-terminal RNA recognition motif domain and a COOH-terminal arginine/serine-rich (RS) domain. The RNA recognition motif domain interacts and binds to the target pre-mRNA, whereas the RS domain is highly phosphorylated and is the protein interaction region. Phosphorylation of SR proteins may result in their differential distribution to alternate sites. Splicing factors are phosphorylated on their RS domain, and the phosphorylation state (hypo- or hyper-) also affects splice site selection. SC35 has several AKT motifs (RXRXX(S/T)) in its RS domain (Fig. 3b). In separate experiments, HT22 cells were treated with insulin and an AKT inhibitor (124005, Calbiochem), which inhibits all Akt isoforms. Inhibition of AKT prevented insulin-mediated increase of alternative splicing of PKCδII mRNA (Fig. 3c). A slight decrease of PKCδI was also observed, and the graph reflects the percent PKCδII exon inclusion for each lane and may be attributed to Akt being the central kinase mediating splicing events. Our previous publications and several other laboratories have shown that Akt2 kinase is central to insulin signaling and phosphorylation of SR proteins in insulin-mediated alternative splicing. Akt2 kinase phosphorylates splicing factors such as SRp40 (
      • Patel N.A.
      • Kaneko S.
      • Apostolatos H.S.
      • Bae S.S.
      • Watson J.E.
      • Davidowitz K.
      • Chappell D.S.
      • Birnbaum M.J.
      • Cheng J.Q.
      • Cooper D.R.
      Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CβII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40.
      ) and SRp75, SC35, and SRp55 (
      • Jiang K.
      • Patel N.A.
      • Watson J.E.
      • Apostolatos H.
      • Kleiman E.
      • Hanson O.
      • Hagiwara M.
      • Cooper D.R.
      Akt2 regulation of Cdc2-like kinases (Clk/Sty), serine/arginine-rich (SR) protein phosphorylation, and insulin-induced alternative splicing of PKCβII messenger ribonucleic acid.
      ). To determine whether Akt2 kinase phosphorylates SC35 thereby increasing PKCδII expression, we transfected constitutively active (CA) or wild-type (WT) Akt2 kinase into HT22 cells compared with HT22 cells treated with increasing amounts of insulin. Akt1 or Akt3 did not have a significant effect on splicing (data not shown). Whole cell lysates were analyzed by Western blotting with antibodies against PKCδII and phospho-SC35, a monoclonal antibody (S4045, Sigma) that recognizes the phospho-epitope of SC35. Phosphorylation of SC35 increased in cells transfected with CA-Akt2 kinase, thereby mimicking the effects of insulin (Fig. 3d).

       Insulin Increases Expression of Pro-survival Proteins Bcl2 and bcl-xL with a Concomitant Increase in PKCδII

      PKCδII is a pro-survival protein. We showed that its overexpression decreases PARP cleavage and DNA fragmentation, both of which are indicators of apoptosis (
      • Patel N.A.
      • Song S.S.
      • Cooper D.R.
      PKCδ alternatively spliced isoforms modulate cellular apoptosis in retinoic acid-induced differentiation of human NT2 cells and mouse embryonic stem cells.
      ). Because our data showed an increase in PKCδII expression with insulin treatment, we used an apoptosis micro-array (SuperArray, catalogue no. PAHS-012A) to determine the profiles of proteins associated with the apoptotic cascade. RNA was isolated from control and insulin-treated HT22 cells. Real-time RT-PCR was performed according to the manufacturers' protocol, and data were analyzed by SuperArray software. Two genes, Bcl2 and bcl-xL, were increased 5-fold in response to insulin (data not shown), which were concurrent with an increase in PKCδII levels with insulin treatment. We observed a moderate increase (0.25-fold) in Mcl-1. To confirm this finding, we treated HT22 cells with insulin (10 nm) for 24 h and analyzed whole cell lysates by Western blot. As shown (Fig. 4), expression of Bcl2 and bcl-xL increased along with PKCδII expression following insulin treatment.
      Figure thumbnail gr4
      FIGURE 4Insulin increases expression of the pro-survival proteins. HT22 cells were treated with 10 nm insulin for 24 h. Whole cell lysates were analyzed by Western blot analysis using antibodies against PKCδII, Bcl2, bcl-xL, p-BAD, BAD, and GAPDH as indicated. The gels represent four experiments performed separately with similar results. The graph shows PKCδII densitometric units normalized to GAPDH and represents three separate experiments. The results were analyzed with a two-tailed Student's t test using PRISM4 statistical analysis software (GraphPad). A level of p < 0.05 was considered statistically significant. ***, p < 0.0001. Significance was determined after three or more experiments.
      When Bad is in a complex with Bcl2, the mitochondria-mediated survival pathway is inhibited. Upon phosphorylation, p-BAD dissociates thereby allowing Bcl2-Bcl-xL complex to promote survival. p-BAD is then sequestered by 14-3-3 further inhibiting apoptosis. Other studies have shown that p-AKT phosphorylates BAD in several cell types (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
      ,
      • Yip W.K.
      • Leong V.C.
      • Abdullah M.A.
      • Yusoff S.
      • Seow H.F.
      Overexpression of phospho-Akt correlates with phosphorylation of EGF receptor, FKHR and BAD in nasopharyngeal carcinoma.
      ). Because our data (Fig. 4) indicated that insulin mediated its effects via AKT kinase and that pAKT levels were increased with insulin treatment, we immunoblotted with p-BAD antibody. Our results (Fig. 4) show an increase in phosphorylation of BAD at Ser-136 (and not Ser-122 or Ser-115, data not shown) with insulin treatment.

       PKCδII Overexpression Mimics Insulin Treatment in HT22 Cells

      Next, we determined whether overexpression of PKCδII directly affects the levels of these proteins. PKCδI or PKCδII pTracer/CMV expression plasmids were transiently transfected into HT22 cells. Overexpression of PKCδII increased expression of Bcl2 and bcl-xL thereby mimicking insulin effects (Fig. 5a). Interestingly, overexpression of PKCδII increased phosphorylation of BAD. This finding suggests that insulin promotes cell survival via the splice variant PKCδII by increasing the expression of the pro-survival proteins Bcl2 and Bcl-xL. PKCδI overexpression did not promote these effects of insulin.
      Figure thumbnail gr5
      FIGURE 5a, overexpression of PKCδII increased expression levels of Bcl2 and BclxL. HT22 cells were transfected with either PKCδI-pTracer or PKCδII-pTracer vector overnight. The cells were then treated with 10 nm insulin or left untreated for 24 h. Whole cell lysates were analyzed by Western blot analysis using antibodies against PKCδII, Bcl2, bcl-xL, p-BAD, and GAPDH as indicated. The gels represent three experiments performed separately with similar results. b, overexpression of PKCδII promotes Bcl-xL alternative splicing. HT22 cells were transfected with PKCδII-pTracer vector and RT-PCR performed with Bcl-x primers that detect Bcl-xS and Bcl-xL levels simultaneously. The graph represents the percentage of exon inclusion calculated as Bcl-xL/(Bcl-xS + Bcl-xL) × 100 and is representative of four experiments performed separately.
      There are two splice variants of bcl-x (BCL2L1): the short form is Bcl-xS, which is apoptotic, and the long form is Bcl-xL, which is pro-survival. Bcl-x splicing is shown to be regulated by ceramide in lung adenocarcinoma cells where Bcl-xS is increased (
      • Chalfant C.E.
      • Rathman K.
      • Pinkerman R.L.
      • Wood R.E.
      • Obeid L.M.
      • Ogretmen B.
      • Hannun Y.A.
      De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.
      ,
      • Massiello A.
      • Salas A.
      • Pinkerman R.L.
      • Roddy P.
      • Roesser J.R.
      • Chalfant C.E.
      Identification of two RNA cis-elements that function to regulate the 5′ splice site selection of Bcl-x pre-mRNA in response to ceramide.
      ). Another regulatory mechanism affecting the expression of Bcl-x splice variants is shown to be coupled with PKC signaling (
      • Revil T.
      • Toutant J.
      • Shkreta L.
      • Garneau D.
      • Cloutier P.
      • Chabot B.
      Protein kinase C-dependent control of Bcl-x alternative splicing.
      ). Our data (Fig. 5a) demonstrated that PKCδII increased the endogenous expression Bcl-xL isoform. We hypothesized that PKCδII can regulate the alternative splicing of Bcl-x isoforms such that the expression is switched to the Bcl-xL splice variant. HT22 cells were transfected with PKCδII-pTracer/CMV vector. Total RNA was isolated, and RT-PCR was carried out with primers that detect both Bcl-xL and Bcl-xS. PKCδII increased expression of the pro-survival protein Bcl-xL and decreased expression of the apoptotic protein Bcl-xS (Fig. 5b).

       Knockdown of PKCδII Expression Decreases Bcl2 and bcl-xL Expression

      Next, we determined whether knockdown of PKCδII affects Bcl2 and bcl-xL levels after insulin treatment. These experiments provide insights into the extent to which PKCδII affects the expression of these pro-survival proteins after insulin treatment, because there are other mechanisms by which Bcl2 and bcl-xL may be expressed in neuronal cells. siRNAs have been used to specifically knock down PKCδ isoforms and have been used successfully by our laboratory previously (
      • Jiang K.
      • Apostolatos A.H.
      • Ghansah T.
      • Watson J.E.
      • Vickers T.
      • Cooper D.R.
      • Epling-Burnette P.K.
      • Patel N.A.
      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ). HT22 cells were transfected with specific siRNA for PKCδII (increasing doses) or scrambled siRNA (control) in the presence or absence of insulin. Bcl2 and bcl-xL expression levels were determined in control and PKCδII-siRNA-transfected HT22 cells by Western blot analysis with antibodies against COOH-terminal PKCδ, which detects PKCδI and PKCδII simultaneously, anti-Bcl2, anti-pBAD, or anti-Bcl-xL. Our results (Fig. 6) show that knockdown of PKCδII decreased Bcl2 and bcl-xL expressions, whereas the expression of PKCδI and GAPDH remained unaffected. Further, insulin treatment of cells with PKCδII siRNA failed to increase Bcl2 and bcl-xL expression. This indicates that PKCδII is a crucial signaling kinase mediating neuronal survival cascade.
      Figure thumbnail gr6
      FIGURE 6Knockdown of PKCδII decreased expression levels of Bcl2 and Bcl-xL. HT22 cells were transfected with increasing doses (25 and 50 nm) of PKCδII siRNA (indicated by the triangle above the lanes) or scrambled siRNA for 48 h. The cells were then treated with 10 nm insulin for 24 h. Whole cell lysates were analyzed by Western blot analysis using COOH-terminal PKCδ antibody which recognizes both PKCδI and -δII isoforms, anti-Bcl2, anti-Bcl-xL, anti-phosphoBAD, or anti-GAPDH as indicated. The gels represent three experiments performed separately with similar results. The graph shows the percentage of densitometric units normalized to GAPDH for each antibody and represents three separate experiments.

       Insulin and PKCδII Decrease Neuronal Apoptosis

      We have previously shown that PKCδII is a pro-survival protein (
      • Patel N.A.
      • Song S.S.
      • Cooper D.R.
      PKCδ alternatively spliced isoforms modulate cellular apoptosis in retinoic acid-induced differentiation of human NT2 cells and mouse embryonic stem cells.
      ). XIAP is bound to caspase-3 (p20/p12, intermediate caspase-3 form) in the cytosol and prevents the onset of apoptosis by activated caspase-3. We have shown that PKCδII overexpression increases the expression of XIAP (
      • Jiang K.
      • Apostolatos A.H.
      • Ghansah T.
      • Watson J.E.
      • Vickers T.
      • Cooper D.R.
      • Epling-Burnette P.K.
      • Patel N.A.
      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ). Here we sought to establish that insulin treatment decreases apoptosis via PKCδII. HT22 cells were transfected with PKCδII pTracer/CMV expression plasmid or PKCδII-specific siRNA as described above and then treated with insulin. Whole cell lysates were separated, and Western blot analysis was performed with antibodies against PKCδII or poly-(ADP-ribose)polymerase (PARP), an indicator of apoptosis. In apoptotic cells, PARP is cleaved by caspase-3 into an 85-kDa fragment, which is detected, along with the 116-kDa fragment, by Western blot analysis with anti-PARP antibody. PARP is differentially processed in apoptosis and necrosis, and hence its activity can be used to distinguish the two forms of cell death (
      • Putt K.S.
      • Beilman G.J.
      • Hergenrother P.J.
      Direct quantitation of poly(ADP-ribose) polymerase (PARP) activity as a means to distinguish necrotic and apoptotic death in cell and tissue samples.
      ). Insulin increased PKCδII levels with concurrent decrease in PARP cleavage and increase in XIAP expression. Further, knockdown of PKCδII prevented insulin-mediated decrease in PARP cleavage and inhibited XIAP expression (Fig. 7). These results indicate that insulin decreases neuronal apoptosis via PKCδII.
      Figure thumbnail gr7
      FIGURE 7Insulin decreases neuronal apoptosis via PKCδII. HT22 cells were transfected with either 2 μg of PKCδII-pTracer vector or 50 nm PKCδII siRNA for 48 h and then were treated with 10 nm insulin overnight as indicated. Whole cell lysates were analyzed by Western blot analysis using antibodies against PKCδII, XIAP, or PARP. PARP_F, full-length PARP; PARP_C, cleaved fragment of PARP. The gels represent three experiments performed separately with similar results. The graph shows the percentage of densitometric units normalized to GAPDH for each antibody and represents three separate experiments.

       PKCδII Overexpression Promotes Neurogenesis

      Increased Bcl2 and bcl-xL expression is shown to enhance neurogenesis. Further, hippocampal neurogenesis is linked to the formation of memories and cognition. To test whether PKCδII-mediated increases in Bcl2 and Bcl-xL expression ultimately result in new cell birth, we performed a BrdU-coupled enzyme-linked immunosorbent assay in HT22 cells overexpressing PKCδII or PKCδII siRNA-treated cells in the presence of insulin. The incorporation of BrdU into replicating DNA was used to label proliferating cells. BrdU is incorporated into S-phase cells, serves as a proliferation marker, and can be quantitatively assayed to determine cell proliferation. BrdU is detected immunochemically allowing for the assessment of neuronal cells synthesizing DNA. Our data (Fig. 8a) demonstrated that PKCδII overexpression increased the amount of BrdU concentration in HT22 cells thereby indicating that PKCδII overexpression promotes neurogenesis and proliferation thereby mimicking insulin action. PKCδII siRNA prevented the effects of insulin on neurogenesis and proliferation.
      Figure thumbnail gr8
      FIGURE 8Overexpression of PKCδII increases HT22 proliferation and viability. HT22 cells were transfected with either 2 μg of PKCδII-pTracer vector or 50 nm PKCδII siRNA for 48 h and then were treated with 10 nm insulin overnight as indicated. The BrdU assay and cell viability assay were performed. The graphs represent BrdU incorporation in PKCδII-overexpressing cells as a percentage of control cells (a) and cell viability in PKCδII-overexpressing cells as a percentage of control cells (b). The measurements were made in triplicate in three separate experiments. The results were analyzed with a two-tailed Student's t test using PRISM4 statistical analysis software (GraphPad). A level of p < 0.05 was considered statistically significant. ***, p < 0.0001. Experiments were performed in triplicate, and significance was determined after three experiments.
      To further verify that PKCδII overexpression increases cell viability and proliferation, we performed a cell proliferation assay based on WST1 (a tetrazolium salt) cleavage to formazan by mitochondrial dehydrogenases. Increased viability of cells results in increased activity of the mitochondrial dehydrogenases in the sample, which can be measured quantitatively by increases in formazan dye production. Data from the assay (Fig. 8b) demonstrated that PKCδII overexpression increased HT22 cell viability and proliferation and knockdown of PKCδII inhibited the effect of insulin on HT22 cell viability and proliferation.

       Intranasal Insulin Treatment in Mice Improved Cognitive Function with a Concomitant Increase in PKCδII Levels

      Administration of intranasal insulin significantly improves cognitive performance in humans and rodent models (
      • Benedict C.
      • Hallschmid M.
      • Schmitz K.
      • Schultes B.
      • Ratter F.
      • Fehm H.L.
      • Born J.
      • Kern W.
      Intranasal insulin improves memory in humans. Superiority of insulin aspart.
      ,
      • Schmidt H.
      • Kern W.
      • Giese R.
      • Hallschmid M.
      • Enders A.
      Intranasal insulin to improve developmental delay in children with 22q13 deletion syndrome. An exploratory clinical trial.
      ,
      • Park C.R.
      • Seeley R.J.
      • Craft S.
      • Woods S.C.
      Intracerebroventricular insulin enhances memory in a passive-avoidance task.
      ). Insulin also prevents neuronal damage, thereby promoting healthy synapses, which are critical for cognition. It also improves memory, reduces cerebral atrophy, and reverses neurodegeneration due to apoptosis (
      • Francis G.J.
      • Martinez J.A.
      • Liu W.Q.
      • Xu K.
      • Ayer A.
      • Fine J.
      • Tuor U.I.
      • Glazner G.
      • Hanson L.R.
      • Frey 2nd, W.H.
      • Toth C.
      Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.
      ,
      • Francis G.J.
      • Martinez J.A.
      • Liu W.Q.
      • Xu K.
      • Ayer A.
      • Fine J.
      • Tuor U.I.
      • Glazner G.
      • Hanson L.R.
      • Frey 2nd, W.H.
      • Toth C.
      Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.
      ,
      • Reger M.A.
      • Watson G.S.
      • Green P.S.
      • Baker L.D.
      • Cholerton B.
      • Fishel M.A.
      • Plymate S.R.
      • Cherrier M.M.
      • Schellenberg G.D.
      • Frey 2nd, W.H.
      • Craft S.
      Intranasal insulin administration dose-dependently modulates verbal memory and plasma amyloid-β in memory-impaired older adults.
      ,
      • Benedict C.
      • Hallschmid M.
      • Hatke A.
      • Schultes B.
      • Fehm H.L.
      • Born J.
      • Kern W.
      Intranasal insulin improves memory in humans.
      ). Intranasal delivery is a noninvasive method that bypasses the blood-brain barrier and delivers insulin to the brain and the spinal cord, thereby avoiding systemic side effects (e.g. hypoglycemia and increased serum insulin levels) (
      • Hallschmid M.
      • Benedict C.
      • Schultes B.
      • Fehm H.L.
      • Born J.
      • Kern W.
      Intranasal insulin reduces body fat in men but not in women.
      ,
      • Kern W.
      • Born J.
      • Schreiber H.
      • Fehm H.L.
      Central nervous system effects of intranasally administered insulin during euglycemia in men.
      ,
      • Stockhorst U.
      • de Fries D.
      • Steingrueber H.J.
      • Scherbaum W.A.
      Insulin and the CNS: effects on food intake, memory, and endocrine parameters and the role of intranasal insulin administration in humans.
      ). Here we sought to measure the effects of insulin on cognitive function in mice and simultaneously evaluate PKCδII expression in the hippocampus. We tested six older mice (C57 BL/6, 18 months) treated with intranasal insulin (1 unit/ml, daily for 4 weeks) and matched controls in the radial arm water maze. The mice treated with intranasal insulin demonstrated improved cognition as shown by decrease in number of errors and decreased time to find the platform (Fig. 9a). At the end of the trials, the hippocampi of the mice were collected for RNA and protein analysis. Our data showed that intranasal insulin treatment in mice increased the expression of PKCδII, Bcl2, and bcl-xL in the hippocampus. These results taken together indicated that intranasal insulin improved performance in memory tests, and this was accompanied by increased expression of PKCδII in the hippocampus (Fig. 9, b and c). Hence, neuronal survival is a contributing factor in increasing cognition by intranasal insulin treatment.
      Figure thumbnail gr9
      FIGURE 9Intranasal insulin improved cognitive functions in vivo with concurrent increase in PKCδII expression. a, experimental setup and behavior tests after intranasal insulin treatment. Performance during the last block of pre-testing in the radial arm water maze task during working memory trials T11 and/or T12 is shown; retention trial (short term memory) and long term memory retention for both errors and latency measurements are shown. Results revealed insulin treatment improved performance in memory tests. The hippocampus was harvested and analyzed with Western blot analysis using PKCδII-specific antibody (b) and RT-PCR analysis using primers that simultaneously detect PKCδI and PKCδII mRNA levels (c). The gels are representative of six mice per treatment in experiments performed with similar results. The graph represents the percentage of exon inclusion calculated as SS II/(SS II + SS I) × 100 and is representative of six experiments performed separately. The results were analyzed with a two-tailed Student's t test using PRISM4 statistical analysis software (GraphPad). A level of p < 0.05 was considered statistically significant. ***, p < 0.0001.

      DISCUSSION

      Neuronal insulin signaling is critical in mediating learning and memory and functions as a neuroprotectant in the CNS. Insulin receptor signaling in the CNS is important in learning, memory, synaptogenesis, and regulation of neurodegeneration. Insulin also promotes the recovery of neurons after injury by activating survival pathways. Deficiencies in insulin and insulin receptor signaling are detrimental to learning and memory. Injected insulin lowers blood glucose, but the effects on cognition are not evident. Although it is not synthesized in the brain, insulin crosses the blood-brain barrier and is taken up by insulin receptors in the hippocampus. Reduced levels of insulin and insulin receptors in the brain are seen in the elderly and in patients with dementia and Alzheimer disease (
      • Arvanitakis Z.
      • Wilson R.S.
      • Bienias J.L.
      • Evans D.A.
      • Bennett D.A.
      Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function.
      ). Even in states of hyperinsulinemia, insulin levels are much lower in cerebrospinal fluid than in serum (
      • Clegg D.J.
      • Riedy C.A.
      • Smith K.A.
      • Benoit S.C.
      • Woods S.C.
      Differential sensitivity to central leptin and insulin in male and female rats.
      ), and both type I and II diabetic patients often have larger declines in cognition at an earlier age. Human studies and epidemiological data suggest a strong link between insulin and cognition (
      • Lu F.P.
      • Lin K.P.
      • Kuo H.K.
      Diabetes and the risk of multi-system aging phenotypes. A systematic review and meta-analysis.
      ). Intranasal insulin treatment in humans and rodent models significantly improves cognition (
      • Benedict C.
      • Hallschmid M.
      • Hatke A.
      • Schultes B.
      • Fehm H.L.
      • Born J.
      • Kern W.
      Intranasal insulin improves memory in humans.
      ). Our in vivo data are in agreement with previous studies, and it further establishes that insulin affects cognition with concurrent increases in PKCδII expression in the hippocampus, the seat of memory and cognition. This knowledge will bridge the gap between insulin signaling, splicing, and cognition.
      Insulin and PKC pathways are integrated and function closely together. PKCs, serine/threonine kinases, are involved in the regulation of cellular differentiation, growth, and apoptosis. PKC activation in the brain is essential for learning, synaptogenesis, and neuronal repair (
      • Alkon D.L.
      • Epstein H.
      • Kuzirian A.
      • Bennett M.C.
      • Nelson T.J.
      Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning.
      ,
      • Bonini J.S.
      • Cammarota M.
      • Kerr D.S.
      • Bevilaqua L.R.
      • Izquierdo I.
      Inhibition of PKC in basolateral amygdala and posterior parietal cortex impairs consolidation of inhibitory avoidance memory.
      ,
      • Etcheberrigaray R.
      • Tan M.
      • Dewachter I.
      • Kuipéri C.
      • Van der Auwera I.
      • Wera S.
      • Qiao L.
      • Bank B.
      • Nelson T.J.
      • Kozikowski A.P.
      • Van Leuven F.
      • Alkon D.L.
      Therapeutic effects of PKC activators in Alzheimer's disease transgenic mice.
      ). In particular, PKCδ has been implicated in memory, neuronal survival, and proliferation and activates survival pathways in neurons (
      • Conboy L.
      • Foley A.G.
      • O'Boyle N.M.
      • Lawlor M.
      • Gallagher H.C.
      • Murphy K.J.
      • Regan C.M.
      Curcumin-induced degradation of PKC δ is associated with enhanced dentate NCAM PSA expression and spatial learning in adult and aged Wistar rats.
      ,
      • Ferri P.
      • Cecchini T.
      • Ambrogini P.
      • Betti M.
      • Cuppini R.
      • Del Grande P.
      • Ciaroni S.
      α-Tocopherol affects neuronal plasticity in adult rat dentate gyrus. The possible role of PKCδ.
      ,
      • Fujiki M.
      • Hikawa T.
      • Abe T.
      • Uchida S.
      • Morishige M.
      • Sugita K.
      • Kobayashi H.
      Role of protein kinase C in neuroprotective effect of geranylgeranylacetone, a noninvasive inducing agent of heat shock protein, on delayed neuronal death caused by transient ischemia in rats.
      ). The two alternatively spliced variants of PKCδ expressed in mouse neuronal cells are PKCδI and PKCδII. We have previously demonstrated that PKCδI is apoptotic, whereas PKCδII promotes survival. Recent reports of clinical trials have emphasized the role of intranasal insulin treatment for improving memory and cognition. However, the cellular target of insulin and its neuronal molecular mechanisms are less studied. Because insulin improves cognition and neuronal survival is critical for promoting cognitive function and memory formation, we sought to determine whether there is a link between insulin and PKCδ. This is the first report showing that insulin regulates alternative splicing of PKCδ, a key signaling kinase in the neuronal cells.
      Alternative splicing events in the brain resulting in diverse proteins are now shown to promote specific functionality and to be the cause of several neurodegenerative diseases. Alternative splicing is a means by which multiple proteins can be produced from a single gene. The large, diverse proteome present at any given time in the human body is the direct evidence of the genome's power to generate functional alternatively spliced variants of the gene. Alternative splicing in neurons is now considered to be a central phenomenon in development, evolution, and neuronal survival. We have demonstrated that insulin mediates PKCδ alternative splicing in neuronal cells. Insulin regulates alternative splicing of PKCβ pre-mRNA in other tissues where it regulates glucose uptake and cellular differentiation (
      • Patel N.A.
      • Apostolatos H.S.
      • Mebert K.
      • Chalfant C.E.
      • Watson J.E.
      • Pillay T.S.
      • Sparks J.
      • Cooper D.R.
      Insulin regulates protein kinase CβII alternative splicing in multiple target tissues: Development of a hormonally responsive heterologous minigene.
      ). However, its role in neuronal alternative splicing mediating the apoptotic pathway was not yet determined.
      The human homolog PKCδVIII has a >94% sequence similarity with the mouse PKCδII. Both PKCδII and PKCδVIII splice variants are generated via utilization of an alternative 5′ splice site. Computational analysis showed a conserved SC35 binding site on the 5′ intron of PKCδII exon 9 pre-mRNA. However, the sequence of exon 9 and its flanking introns, which is the region of alternative splicing of PKCδII pre-mRNA, is different from the sequence comprising of PKCδVIII exon 10. We are cloning a PKCδII splicing minigene to further study the molecular mechanisms of alternative splicing of PKCδII mRNA regulated by insulin, thereby evaluating the interplay of PKCδII cis-elements with their trans-factors.
      Here we demonstrated that insulin acts through PKCδII to increase expression of the pro-survival proteins Bcl2 and bcl-xL, which increase neurogenesis and neuronal survival (
      • Gal A.
      • Szilagyi G.
      • Wappler E.
      • Safrany G.
      • Nagy Z.
      Bcl-2 or Bcl-XL gene therapy reduces apoptosis and increases plasticity protein GAP-43 in PC12 cells.
      ,
      • Sasaki T.
      • Kitagawa K.
      • Yagita Y.
      • Sugiura S.
      • Omura-Matsuoka E.
      • Tanaka S.
      • Matsushita K.
      • Okano H.
      • Tsujimoto Y.
      • Hori M.
      Bcl2 enhances survival of newborn neurons in the normal and ischemic hippocampus.
      ,
      • Zhang K.Z.
      • Westberg J.A.
      • Hölttä E.
      • Andersson L.C.
      BCL2 regulates neural differentiation.
      ,
      • Abe-Dohmae S.
      • Harada N.
      • Yamada K.
      • Tanaka R.
      Bcl-2 gene is highly expressed during neurogenesis in the central nervous system.
      ,
      • Adams J.M.
      Ways of dying. Multiple pathways to apoptosis.
      ,
      • Adams J.M.
      • Cory S.
      The Bcl-2 apoptotic switch in cancer development and therapy.
      ,
      • Chang M.Y.
      • Sun W.
      • Ochiai W.
      • Nakashima K.
      • Kim S.Y.
      • Park C.H.
      • Kang J.S.
      • Shim J.W.
      • Jo A.Y.
      • Kang C.S.
      • Lee Y.S.
      • Kim J.S.
      • Lee S.H.
      Bcl-XL/Bax proteins direct the fate of embryonic cortical precursor cells.
      ). Our results with insulin treatment and in cells overexpressing PKCδII show an increase in phosphorylation of BAD at Ser-136, which indicates that this increase occurs via the AKT pathway and not the ERK or PKA pathways, because we did not see increases at Ser-122 or Ser-115. It may also be possible that PKCδII is an additional kinase phosphorylating BAD such that it dissociates from the Bcl2-BAD complex thereby initiating the survival pathway. We are currently expanding the study to evaluate this in our laboratory. Knockdown of PKCδII further prevented insulin-mediated increases in Bcl2, Bcl-xL, and pBAD levels.
      Currently, there is a lack of a good inhibitor for PKCδ. Rottlerin, the widely used PKC inhibitor, is shown to also inhibit other kinases such as PKA, calmodulin kinase, and other additional PKC isoforms (
      • Soltoff S.P.
      Rottlerin. An inappropriate and ineffective inhibitor of PKCδ.
      ). Hence we used siRNA in our studies, which also offers the additional benefit of selecting for a specific splice variant. Our data indicated that PKCδII knockdown affected Bcl2 and bcl-xL levels, and the expression of PKCδI, which remained constant in the PKCδII-siRNA cells, did not affect their levels. These results suggest that PKCδII is an important kinase in the Bcl2-mediated survival pathway.
      In conclusion, our studies demonstrate that insulin regulates alternative splicing of a key signaling kinase, PKCδ, in neuronal cells. Further, we have shown that PKCδII is a critical signaling component that modulates neuronal survival, which is essential to improve memory and learning. These findings contribute to the therapeutic potential of PKCδII and elucidate molecular mechanisms underlying intranasal insulin treatment, which ultimately contribute to improved cognition and memory.

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