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. 2016 Nov 14;158(1):183–195. doi: 10.1210/en.2016-1819

MALAT1 in Human Adipose Stem Cells Modulates Survival and Alternative Splicing of PKCδII in HT22 Cells

Ghattas El Bassit 3,*, Rekha S Patel 3,*, Gay Carter 1, Vyshakh Shibu 2, Achintya A Patel 2, Shijie Song 1, Michel Murr 4, Denise R Cooper 1,3, Paula C Bickford 1,5, Niketa A Patel 1,3,✉
PMCID: PMC5412980  PMID: 27841943

Abstract

Brain injury may be caused by trauma or may occur in stroke and neurodegenerative diseases. Because the central nervous system is unable to regenerate efficiently, there is utmost interest in the use of stem cells to promote neuronal survival. Of interest here are human adipose-derived stem cells (hASCs), which secrete factors that enhance regeneration and survival of neurons in sites of injury. We evaluated the effect of hASC secretome on immortalized mouse hippocampal cell line (HT22) after injury. Protein kinase C δ (PKCδ) activates survival and proliferation in neurons and is implicated in memory. We previously showed that alternatively spliced PKCδII enhances neuronal survival via B-cell lymphoma 2 Bcl2 in HT22 neuronal cells. Our results demonstrate that following injury, treatment with exosomes from the hASC secretome increases expression of PKCδII in HT22 cells and increases neuronal survival and proliferation. Specifically, we demonstrate that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA contained in the hASC exosomes mediates PKCδII splicing, thereby increasing neuronal survival. Using antisense oligonucleotides for MALAT1 and RNA immunoprecipitation assays, we demonstrate that MALAT1 recruits splice factor serine-arginine-rich splice factor 2 (SRSF2) to promote alternative splicing of PKCδII. Finally, we evaluated the role of insulin in enhancing hASC-mediated neuronal survival and demonstrated that insulin treatment dramatically increases the association of MALAT1 and SRSF2 and substantially increases survival and proliferation after injury in HT22 cells. In conclusion, we demonstrate the mechanism of action of hASC exosomes in increasing neuronal survival. This effect of hASC exosomes to promote wound healing can be further enhanced by insulin treatment in HT22 cells.

Protein kinase C (PKC) is a family of serine/threonine kinases with 11 isoforms. The PKC family is subdivided into 3 groups based upon their activation by calcium, phosphatidyl serine, diacyl glycerol, or phorbol esters: classical or conventional PKCs (α, βI, βII, and γ), unique PKCs (δ, ε, η, and θ), and atypical PKCs (ζ, λ/ι). Activation of these proteins in the brain is essential for learning, synaptogenesis, and neuronal survival (1–3). In particular, PKCδ, a unique PKC, has been implicated in memory, neuronal proliferation, and activation of survival pathways (4–6). PKCδ is alternatively spliced to PKCδI (ubiquitous expression in human, mouse, and rat), PKCδII, PKCδIV, PKCδV, PKCδVI, and PKCδVII (mouse-specific splice variants), PKCδIII (rat-specific splice variant), and PKCδVIII (human-specific splice variant). In mouse, PKCδII is generated via alternative 5′ splice site usage of exon 9 and functions as a prosurvival kinase (7, 8).

Adult stem cells have the potential for healing dermal wounds, pressure sores as well as promoting survival of neurons after injury. Stem cells residing in the adipose tissue have substantial advantage with its ease of isolation, ability to obtain large quantities, and potential of personalized regenerative medicine. Our study (9) and others (10, 11) have demonstrated that human adipose-derived stem cells (hASC) present surface antigens similar to mesenchymal stem cells and self-renewal capacity. The hASC secrete factors (in their secretome) that function in a paracrine manner to enhance cell survival and proliferation. The secretome content, rich in proteins and RNA, is transported in vesicles and smaller exosomes and transferred to the recipient cells where they exert their effect. Secretome (collected as conditioned media [CM]) from hASC is known for its regenerative properties including neuroprotection (12, 13).

We previously demonstrated that CM from hASC are promising therapy for mild traumatic brain injury. Our in vivo study in rats with mild traumatic brain injury showed substantial amelioration of motor and cognitive functions following treatment with CM from hASC (14). Separately, we demonstrated the role of alternatively spliced variant PKCδII in increasing neuronal survival in HT22 cells and mouse models for cognition (7). Here, we sought to determine the molecular mechanisms involved in increased neuronal proliferation and survival with treatment with exosomes derived from hASC.

Exosomes isolated from hASC CM contain many noncoding RNAs. Of specific interest are the long noncoding RNAs (lncRNAs) that have varied functions, which include signaling, molecular decoys, scaffolding, and guiding ribonucleoprotein complexes. Multiple lines of evidence link regulatory lncRNAs to human diseases (15–18). The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was first reported in tumors (19, 20) and is shown to regulate alternative splicing of genes and function as transcriptional regulator as well as play a role in cell cycle and cellular mitosis (21–24). MALAT1 is referred to as NEAT2 (noncoding nuclear-enriched abundant transcript 2) in some reports (25). MALAT1 is shown to regulate cellular function, gene expression, and splicing in several systems (26–28). In this study, we elucidate a role for lncRNA MALAT1 in increasing neuronal proliferation and survival via alternative splicing of PKCδII in HT22 cells. Additionally, we evaluated insulin treatment in combination with exosomes from hASC to improve and accelerate neuronal survival.

Methods

Cell culture

The studies were carried out using immortalized clonal mouse hippocampal cell line (HT22) obtained from Dr. D.R. Schubert (Salk Institute). HT22 cells were cultured in 75 cm2 flasks in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin (50 U/mL), and 2 mM glutamine. Cells were maintained at 37°C in a humidified incubator containing 5% CO2. HT22 cells were subcultured into either 25-cm2 flasks or 100-mm2 dishes and used for experiments at 60% to 80% confluence.

Western blot analysis

Cell lysates (40 μg) were separated on 10% polyacrylamide gel electrophoresis-sodium dodecyl sulfate. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with tris(hydroxymethyl)aminomethane-buffered saline/0.1% Tween 20 containing 5% nonfat dried milk, washed, and incubated with a polyclonal antibody against either anti-B-cell lymphoma 2 (anti-Bcl2; Cell Signaling) or PKCδII-specific polyclonal antibody [described in (7)]. Anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) was purchased from Cell Signaling. Following incubation with anti-rabbit immunoglobulin G (IgG)-HRP, enhanced chemiluminesence (Pierce) was used for detection. Images were digitally captured by ProteinSimple FluorChem, and densitometric analysis was performed using AlphaView Software.

Transient transfection of plasmid DNA

hASC was trypsinized and cell pellets were collected in 100 μL Nucleofector solution (Lonza) and combined with pMAX green fluorescent protein (GFP; 2 µg). The cell/DNA solution was transferred to a cuvette, and the program was initiated (0.34 kV, 960 microfarads). Medium (500 μL) was added immediately, and cells were gently transferred to 60-mm plates and allowed to grow for 24 hours or more according to the experiment.

Silencer RNA transfection

Two silencer RNAs (siRNAs) that target separate areas were used to knockdown expression of serine-arginine-rich splice factor 2 (SRSF2). SRSF2 siRNAs (IDs: 12628 and 12444) along with its scrambled control were purchased from Ambion and transfected using Ambion’s siRNA transfection kit. These were validated for specificity to eliminate off-target gene effects. Ambion’s PARIS kit (Thermo Fisher Scientific, Waltham, MA, cat. no. AM1921) was used to simultaneously isolate proteins and RNA to verify knockdown by siRNA transfection.

Real-time quantitative polymerase chain reaction

Total RNA was isolated from cells using RNA-Bee (Tel Test, Inc.) as per manufacturer’s instructions. Two micrograms of RNA was used to synthesize first-strand complementary DNA (cDNA) using random hexamer primers and the Omniscript kit (Qiagen). One microliter of cDNA was amplified by real-time quantitative polymerase chain reaction (qPCR) using Maxima SYBR Green/Rox qPCR master mix (Thermo Fisher Scientific) in an ABI ViiA7 sequence detection system (PE Applied Biosystems) to quantify the relative levels of the transcripts in the samples. The primers are: PKCδI sense primer 5′-ACATCCTAGACAACAACGGGAC-3′ and antisense 5′-ACCACGTCCTTCTTCAGACAC-3′; PKCδII sense primer 5′-CACCATCTTCCAGAAAGAACG-3′ and antisense 5′-TCGCAGGTCTCACTACTGCCTTTTCC-3′; GAPDH sense primer 5′-TGACGTGCCGCCTGGAGAAAC-3′ and antisense 5′-CCGGCATCGAAGGTGGAAGAG-3′; human MALAT1 sense: 5′-GAGTTCTAATTCTTTTTACTGCTCAATC-3′ and antisense 5′-TCAAGTGCCAGCAGACAGCA-3′; mouse MALAT1 sense: 5′-TGCAGTGTGCCAATGTTTCG-3′ and antisense 5′-GGCCAGCTGCAAACATTCAA-3′; and U1 small nuclear RNA (U1snRNA) sense: 5′-TCCCAGGGCGAGGCTTATCCATT-3′ and antisense 5′-GAACGCAGTCCCCCACTACCACAAAT-3′. Amplification was performed on the ViiaA 7 (Applied Biosystems). Real-time polymerase chain reaction (PCR) was then performed in triplicate on samples and standards. The plate setup included a standard series, no template control, no RNA control, no reverse transcription control, and no amplification control. After primer concentrations were optimized to give the desired standard curve and a single melt curve, relative quotient was determined using the comparative method (∆∆CT) with U6snRNA or GAPDH as the endogenous control and HT22 control samples as the calibrator sample. Experiments were repeated 4 times.

For absolute quantification, a standard curve was generated for each gene in every assay. To do so, 100 to 0.4 ng of RNA were reverse transcribed as described previously. The resulting cDNA was used to obtain a standard curve correlating the amounts with the threshold cycle number. A linear relationship (r2 > 0.96) was obtained for each gene. Real-time PCR was then performed on samples and standards in triplicates. The plate setup also included a standard series, no template control, no RNA control, no reverse transcription control and no amplification control. The dissociation curve was analyzed for each sample. Absolute quantification of mRNA expression levels for PKCδI and PKCδII was calculated by normalizing the values to GAPDH.

Adipose-derived stem cells

hASCs were isolated as previously described by our laboratory (9). Briefly, white adipose tissue was obtained as discarded tissue from surgeries performed at Tampa General Hospital by Dr. Michel Murr. Donors consented to their waste tissue to be used in research. The lean adipose tissue samples were obtained from subcutaneous depot of a female donor with body mass index of 21.3. Subject was nondiabetic, a nonsmoker, and did not have any form of cancer. The de-identified samples were obtained under an Institutional Review Board–approved protocol (University of South Florida Institutional Review Board number 20295) with a “not human research activities determination” and were transported to the laboratory and processed within 24 hours of collection. Adipose tissue was cut up into small pieces and digested with 0.075% collagenase type 1 (Worthington) in modified phosphate-buffered saline (PBS) for 2 hours at 37°C. The suspension was filtered and centrifuged at 400g at room temperature. The pellet contains the stromal vascular fraction. The pellet was resuspended in 1 mL of the erythrocyte lysis buffer (Stem Cell Technologies) for 10 minutes and washed in 20 mL PBS with 2% penicillin/streptomycin before centrifugation (300 g to 500 g, 5 minutes). The supernatant was aspirated, and the cell pellet resuspended in a 3-mL stromal medium (α-MEM; Mediatech) with 20% fetal bovine serum, 1% l-glutamine (Mediatech), and 1% penicillin/streptomycin. Following 3 rinses in the stromal medium, stromal vascular fraction cells were plated for initial cell culture at 37°C with 5% CO2 in hASC medium from ZenBio (catalong number PM-1). Subconfluent cells were passaged by trypsinization. CM was collected after 24 to 48 hours. Experiments were conducted within passages 2 and 3.

Exosome isolation

Exosomes were isolated as previously described by our laboratory (29). CM derived from hASC was collected after 48 hours and centrifuged at 3,000g for 15 minutes to remove dead cells. ExoQuick (SBI) reagent was added to the CM and incubated overnight at 4°C. Following centrifugation at 1,500g for 30 minutes, the pellet was further processed. ExoCap (JSR Life Sciences) composite reagent containing magnetic beads for cluster of differentiation 9 (CD9), CD63, and CD81 was used to purify exosomes. Exosomes were eluted from beads using the manufacturer’s elution buffer and used in experiments as described.

RIP assay

The RNA-immunoprecipitation (RIP) kit was purchased from Sigma and protocol followed as per manufacturer’s instruction. SRSF2 antibody and SNRNP70 antibody were purchased from Millipore, and IgG antibody was included in the kit (Sigma). Cell lysate (10%) was removed for input sample. Immunoprecipitation was performed with 2 μg SRSF2 antibody, snRNP70 antibody (positive control), or IgG antibody (as negative control). RNA was purified and treated with DNAse to remove genomic DNA. SYBR Green Real-Time qPCR was performed as described earlier using MALAT1 primer sets and primers for U1 RNA, the binding partner for the positive control SNRNP70. The yield (percentage input) and specificity (fold enrichment) were calculated using the Microsoft Excel template for RIP from Sigma.

Cell survival assay

WST-1 (Roche Molecular Biochemicals, IN) was added to HT22 cells (in triplicate) in the presence of hASC exosomes (10 μg) to a final concentration of 10% (v/v). Cells were incubated for 2 hours at 37°C. The formazon dye produced by viable cells is quantified using a spectrophotometer set at a wavelength of 440 nm, and absorbance was recorded for each well (reference wavelength, 690 nm).

Cell proliferation assay

HT22 cells were treated with hASC exosomes (10 μg). The treatments were performed in triplicate in a 48-well plate. The BrdU cell proliferation assay kit was purchased from Millipore (catalog number 2750) and used as per manufacturer’s instructions to quantitatively evaluate the number of actively proliferating cells. Briefly, 100 μL 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 dual wavelength of 450 nm/550 nm. The results were normalized against the blank and background readings.

Cell migration assay

Scratch assay is an established method to measure cell migration and wound healing in vitro (30). HT22 cells were plated in 35-mm dishes. After 24 hours, the cell monolayer was scraped with a P100 pipette tip, creating a scratch. Cell debris was removed by washing with culture medium. Parallel lines on the outside surface of the dish were made to mark boundaries and create reference points. The cells were treated with hASC exosomes (10 μg) or insulin (10 nM) as indicated in the experiments. A Nikon microscope was used to capture phase contrast images at 24 hours at 20× magnification. Five separate fields of 1μm2 were counted for each plate for migration distances and averaged to determine overall scratch width after 24 hours post treatments compared with control. Experiments were repeated thrice.

Immunochemistry

HT22 cells were plated in 8-well chamber plates and were either treated with exosomes from hASC and with or without 10 nM insulin treatment. After 24 hours, medium was removed, and cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 30 minutes. Cells were rinsed with PBS and blocked with 1% bovine serum albumin for 30 minutes. Primary antibodies for either Ki-67 or doublecortin were incubated overnight at 4°C. Cells were washed 3 times with PBS and were incubated with secondary fluorescent antibody for 1 hour at room temperature. To visualize nucleus, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 minutes at room temperature.

Statistical analysis

The gels were densitometrically analyzed using AlphaView software (ProteinSimple). PRISM software was used for statistical analysis. A level of P < 0.05 was considered statistically significant. The results are expressed as mean ± standard error of mean (SEM) or as percentage of exon inclusion.

Results

Exosomes secreted by hASC increase expression of PKCδII in HT22 cells

The immortalized mouse hippocampal cell line HT22 is widely used to study neuronal survival and is established as an in vitro model for mechanistic studies for neuronal diseases. PKCδ is alternatively spliced to PKCδI and PKCδII in mouse HT22 cells. We previously showed that PKCδI is proapoptotic, while PKCδII promotes survival. Additionally, we showed that PKCδII increases neuronal survival via Bcl2 in HT22 cells (7). We have also separately shown that CM from hASCs increased neuroprotection and improved cognition in vivo in mice with mild traumatic brain injury (14). Hence, we sought to examine the effect of hASC secretome (collected as CM) on PKCδ alternative splicing in HT22 cells. The secretome of hASC contains microvesicles and exosomes, which are taken up by target cells. The contents of the exosomes (50 to 100 nM in size) often influence the cellular functions of the target cells. hASC cells were grown to confluency, and CM was collected after 48 hours. Exosomes were isolated from CM (see “Methods” section). HT22 cells were treated with 10 μg hASC exosomes for 24 hours. Our western blot analysis and PCR results indicate that treatment with hASC exosomes increased PKCδII and Bcl2 levels in HT22 cells [Fig. 1(A–C)].

Figure 1.

Figure 1.

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Exosomes from hASC increase PKCδII expression in HT22 cells. HT22 cells were treated with 10 μg of hASC exosomes for 24 hours. Whole-cell lysates were collected, and western blot analysis was performed with antibodies as indicated. (A) The gels represent 3 experiments performed separately with similar results. (B) The graph shows percentage of densitometric units normalized to GAPDH for each antibody and represents 3 separate experiments. The results were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. ***P < 0.0001, highly significant between control and hASC exosome–treated cells for PKCδII and Bcl2. (C) SYBR Green Real-Time qPCR using PKCδI and PKCδII primers was performed for absolute quantification. GAPDH served as control. For absolute quantification, a standard curve was generated for each gene in every assay. Absolute quantification of mRNA expression levels for PKCδI and PKCδII was calculated by normalizing the values to GAPDH. Experiments were repeated 4 times with similar results. The results were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. ***P < 0.0001, highly significant between control and hASC exosome–treated cells for PKCδII. AQ, absolute quantification; Con, control; IB, immunoblot.

Exosomes from HASC are taken up by HT22 cells

We sought to verify that exosomes from hASC are taken up by HT22 cells. hASC was transiently transfected with pMAX-GFP using nucleofection. The cells were allowed to recover for 48 hours, and fresh dye-free media was added to the hASC. After 48 hours, the CM was collected and exosomes were isolated. Ten micrograms of exosomes derived from the GFP-transfected hASC was added to HT22 cells for 24 hours. The cells were harvested, and using qPCR, we show GFP in recipient HT22 cells [Fig. 2(A) and 2(B)]. Additionally, western blot analysis showed presence of human exosome cell surface markers CD9, CD63, and CD81 in HT22 cells treated with hASC exosomes [Fig. 2(C)].

Figure 2.

Figure 2.

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Exosomes from hASC are taken up by HT22 cells. hASC was transfected with pMAX-GFP for 24 hours. Exosomes were isolated, and HT22 cells were treated with 10 μg of exosomes isolated from GFP-transfected hASC (GFP-hASC Ex). (A) Western blot analysis was performed with the antibodies as indicated in the figure. (B) SYBR Green Real-Time qPCR was performed using GFP-hASC Ex as the reference sample. Experiments were repeated thrice with similar results. (C) HT22 whole-cell lysates were collected, and western blot analysis was performed with antibodies CD9, CD63, and CD81, which are human exosome cell surface markers. Blots represent 3 experiments performed separately with similar results. Con, Control; IB, immunoblot; RQ, relative quantification.

Exosomes from hASC enhance neuronal survival and proliferation

Because exosomes from hASC increased PKCδII, a prosurvival kinase, we sought to determine whether hASC exosomes increased survival and proliferation in HT22 cells. We performed BrdU-coupled enzyme-linked immunosorbent assay in HT22 cells treated with hASC exosomes (10 μg). The incorporation of 5-bromo-2′-deoxyuridine (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. 3(A)] demonstrated treatment with hASC increased the amount of BrdU concentration in HT22 cells.

Figure 3.

Figure 3.

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Exosomes from hASC increase proliferation and survival in HT22 cells. HT22 cells were treated with 10 μg of hASC exosomes for 24 hours. The BrdU assay and cell viability assay were performed. The graphs represent (A) BrdU incorporation in hASC exosome (hASC Ex)–treated cells as a percentage of control cells and (B) cell survival in hASC exosome–treated cells as a percentage of control cells. The measurements were made in triplicate in 3 separate experiments. The results were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. ***P < 0.0001. Experiments were performed in triplicate, and significance is determined after 3 experiments. Con, control.

Next, to verify that hASC exosomes increase cell survival and proliferation, we performed a cell proliferation assay based on WST1 (a tetrazolium salt) cleavage to formazan by mitochondrial dehydrogenases. Increased proliferation 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. 3(B)] demonstrated that hASC exosomes increased HT22 cell survival and proliferation.

hASC exosomes contain lncRNA MALAT1

lncRNAs are emerging as important modulators of gene expression. We screened for lncRNAs that were enriched within exosomes derived from hASC. Our recent publication demonstrated that the lncRNAs in exosomes from hASC had distinct expression patterns. Comparing the levels between hASC, CM, and its exosomes, we demonstrated that exosomes secreted from subcutaneous hASC were significantly enriched in MALAT1, GAS5, and lncRNA-VLDLR (29). Of particular interest is the lncRNA MALAT, which predominantly regulates alternative splicing of genes by modulating the activity of splice factors. MALAT1 has been extensively studied in several cancers (19, 22, 31), where it is shown to regulate gene expression either by promoting alternative splicing or regulating assembly of complexes mediating gene expression. HT22 cells were treated with 10 μg of exosomes from hASC. To delineate the amount of MALAT1 taken up from exosomes as compared with endogenous MALAT1 in HT22 cells, we used primers specific for either mouse or human MALAT1 in SYBR Green qPCR. Our results demonstrate that human MALAT1 levels are increased in HT22 cells treated with hASC exosomes [Fig. 4(A)].

Figure 4.

Figure 4.

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Exosomes from hASC contain the lncRNA MALAT1. (A) RNA was isolated from HT22 cells treated with hASC exosomes (hASC Ex). Absolute SYBR Green Real-Time qPCR was performed using human or mouse MALAT1 primers. Experiments were repeated 5 times with similar results. The results were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. *** P < 0.0001, highly significant between hASC exosomes and control for MALAT1. (B) Exosomes from hASC were treated with ribonuclease cocktail (hASC-Ex ribonuclease). Total RNA was isolated from hASC-Ex ribonuclease, and absolute SYBR Green Real-Time qPCR was performed using human MALAT1 primers. Experiments were repeated 3 times with similar results. (C) HT22 cells were either untreated (control) or treated with 10 μg of hASC Ex or 10 μg of ribonuclease cocktail–treated hASC-Ex (hASC-Ex ribonuclease). Total RNA was isolated from HT22 cells, and SYBR Green Real-Time qPCR was performed for PKCδI and PKCδII. Experiments were repeated 3 times with similar results. The results were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. ***P < 0.0001, highly significant between HT22 control and hASC exosome–treated cells for MALAT1. AQ, absolute quantification; Con, control; RQ, relative quantification.

hASC secretome contains a milieu of protein and RNA factors. The RNA component is encapsulated in vesicles such as exosomes to prevent its degradation. To determine if it was an RNA component of the exosome that increased PKCδII expression, 10 μg hASC exosomes was incubated with a cocktail of ribonucleases (1 μL RiboShredder RNase Blend Epicenter, catalog number RS12100) at 37°C for 1 hour. We evaluated the levels of human MALAT1 in the exosomes. Our results [Fig. 4(B)] show that ribonuclease cocktail–treated hASC exosomes were depleted of MALAT1. HT22 cells were either treated with 10 μg hASC exosomes or 10 μg ribonuclease cocktail–treated exosomes. Our qPCR results indicated that ribonuclease cocktail–treated hASC exosomes did not increase PKCδII expression in HT22 cells [Fig. 4(C)].

Depletion of MALAT1 impacts hASC-mediated PKCδII splicing

Because our results demonstrated that hASC exosomes had significantly higher levels of MALAT1 and that PKCδII levels were increased in HT22 following treatment with hASC exosomes, we sought to determine whether depletion of MALAT1 in hASC exosomes affected PKCδII levels in HT22 cells. We evaluated 2 antisense oligonucleotides (ASOs) to human MALAT1 (IDs 395254 and 395240) along with the scrambled ASOs (control; ASOs from Ionis Pharmaceuticals [formerly known as ISIS Pharm]; validated for specificity and designed for efficient uptake by cells). The ASOs were added to hASC for 48 hours. Exosomes were isolated from CM. The expression levels of MALAT1 were determined in the exosomes using human MALAT1 primers in qPCR. Our results show exosomes from hASC treated with ASOs 395254 and 395240 (abbreviated as M1ASO-254 Ex or M1ASO-240 Ex) had depleted MALAT1 levels [Fig. 5(A)].

Figure 5.

Figure 5.

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Depletion of MALAT1 inhibits hASC exosome-mediated increase of PKCδII expression. hASC was treated with 50 nM MALAT1 ASOs 395254 and 395240 or scrambled ASO control (ASOscr) for 48 hours. Exosomes were isolated from the CM (abbreviated as M1ASO-254 Ex, M1ASO-240 Ex, or ScrASO Ex). (A) Total RNA was isolated from exosomes, and SYBR Green Real-Time qPCR was performed for human MALAT1. Results indicated 80% decline in M1ASO-254 Ex or M1ASO-240 Ex samples. MALAT1 levels remained constant in ScrASO Ex. Experiments were repeated 5 times with similar results. (B) ScrASO Ex, M1ASO-254 Ex, or M1ASO-240 Ex (10 μg each) was added to HT22 cells for 24 hours. Total RNA was extracted from HT22 cells, and SYBR Green Real-Time qPCR was performed using PKCδI and PKCδII primers. Experiments were repeated 5 times with similar results. (C) Total RNA was extracted from HT22 cells, and SYBR Green Real-Time qPCR was performed using mouse MALAT1 primers. Experiments were repeated 5 times with similar results. The results for all experiments were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. ***P < 0.0001 highly significant between HT22 control and ScrASO Ex–treated cells for PKCδII. ***P < 0.0001 highly significant between ScrASO Ex–treated cells and M1ASO-254 Ex–treated cells or M1ASO-240 Ex–treated cells for PKCδII. AQ, absolute quantification; RQ, relative quantification.

Ten micrograms scrambledASO exosomes, M1ASO-254 exosomes, or M1ASO-240 exosomes was added to HT22 cells for 24 hours. Total RNA was isolated from HT22 cells, and real-time absolute qPCR was performed to determine levels of PKCδ splicing variants. Results demonstrated M1ASO-254 exosomes or M1ASO-240 exosomes did not increase PKCδII splicing in HT22 cells. The scrambledASO exosomes increased PKCδII splicing in HT22 cells, mimicking control hASC exosomes treated with HT22 cells. PKCδI splicing was not affected significantly with hASC exosomes, M1ASO-254 exosomes, or M1ASO-240 exosomes [Fig. 5(B)]. As a control, we measured the levels of endogenous MALAT1 levels in HT22 cells using mouse MALAT1 primers in qPCR. Our results [Fig. 5(C)] indicated that addition of hASC exosomes did not significantly alter mouse MALAT1 levels in HT22 cells.

MALAT1 promotes PKCδII alternatively splicing via SRSF2

MALAT1 interacts with several pre-mRNA splice factors and modulates alternative splicing. We previously demonstrated that in HT22 cells, PKCδII alternative splicing is regulated by SRSF2, also known as SC35 (7). We evaluated whether MALAT1 in hASC exosomes could promote PKCδII splicing in the absence of SRSF2. SRSF2 was depleted in HT22 cells using siRNA (50 nM for 48 hours; SRSF2 siRNA from Ambion 12444, previously validated by our laboratory) (32). Ten micrograms of hASC exosomes was added for 24 hours, and RNA was isolated for qPCR analysis. Our results using SYBR Green Absolute qPCR indicate that HT22 cells treated with MALAT1 containing hASC exosomes increased PKCδII, while the SRSF2-depleted HT22 cells did not show an increase in PKCδII with hASC exosome treatment [Fig. 6(A)].

Figure 6.

Figure 6.

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MALAT1 promotes splicing of PKCδII via SRSF2. (A) SRSF2 siRNA was transfected into HT22 cells for 48 hours. Fresh medium was replaced, and HT22 cells were treated with 10 μg of hASC exosomes. Absolute SYBR Green qPCR was performed with GAPDH as internal control. A standard curve was generated for each gene. Absolute quantification of mRNA expression levels for PKCδI and PKCδII was calculated by normalizing the values to GAPDH. The experiments were repeated 4 times with similar results. ***P < 0.0001 highly significant. (B) HT22 cells were treated with 10 μg of hASC exosomes. RIP assay was performed, and immunoprecipitated RNA was analyzed by qPCR for PKCδ using primers flanking the SRSF2 binding site on PKCδ pre-mRNA. Graph is plotted as fold enrichment of SRSF2 using qPCR in the RIP assay with SRSF2 IP and fold enrichment of U1 RNA using SNRP70 IP (positive control). Results show SRSF2 binding to PKCδ pre-mRNA. Inset shows immunoblot of SRSF2 IP and IgG IP (negative control) using antibody against SRSF2. Experiments were repeated 4 times with similar results. (C) HT22 cells were transfected with 2 μg PKCδ splicing minigene and treated with or without 10 μg hASC exosomes for 24 hours. Total RNA was isolated and reverse transcribed, and 2 μL cDNA was used in PCR. The primers used were on the SD and SA exons of the pSPL3 splicing vector as indicated in the schematic of the PKCδ splicing minigene. Five percent of the products were separated by polyacrylamide gel electrophoresis and silver stained for visualization. Graphs represent percentage of exon inclusion calculated as SS II/(SS II + SSI) x 100 in the samples and are representative of 4 experiments performed separately. (D) HT22 cells were treated with 10 μg hASC exosomes. The nuclear and cytoplasmic fractions were separated from the HT22 cells followed by RIP assay. Immunoprecipitated RNA was analyzed by real-time qPCR for human MALAT1. Graph is plotted as fold enrichment of MALAT1 using qPCR in the RIP assay with SRSF2 IP and fold enrichment of U1 RNA using SNRP70 IP (positive control). Results show SRSF2 binding to lncRNA MALAT1. Inset shows immunoblot of SRSF2 IP and IgG IP (negative control) using antibody against SRSF2. Experiments were repeated 4 times with similar results. AQ, absolute quantification; Con, Control; IB, immunoblot; IP, immunoprecipitation; SA, splice site acceptor exon; SD, splice site donor exon.

We previously demonstrated that SRSF2 regulated PKCδII alternative splicing (7). We sought to evaluate whether MALAT1 influenced the binding of SRSF2 to PKCδ pre-mRNA. HT22 cells were treated with hASC exosomes containing MALAT1, and RNA coimmunoprecipitation (RIP) assay was performed using primers to PKCδ pre-mRNA. Our results [Fig. 6(B)] demonstrate that SRSF2 binds to PKCδ pre-mRNA. HT22 cells treated with hASC exosomes showed an enrichment of SRSF2 on PKCδ pre-mRNA. Inset shows western blot analysis of SRSF2 in input sample of RIP.

Separately, HT22 cells were transfected with 2 μg of PKCδ heterologous splicing minigene (8) and treated with or without hASC exosomes (10 μg) for 24 hours. PKCδ splicing minigene was previously described by our laboratory. Briefly, mouse exon 9 of PKCδ along with its flanking 3′ and 5′ introns were cloned into the pSPL3 splicing vector. Utilization of 5′ splice site I results in PKCδI, while utilization of 5′ splice site II results in PKCδII (8). Our results demonstrate that treatment with hASC exosomes increased utilization of 5′ splice site II in the pSPL3-PKCδ minigene [Fig. 6(C)].

Cells use lncRNAs to maximize the ability of splice factors to regulate splicing. Our results suggested that MALAT1 recruits SRSF2 and stabilizes its association with PKCδ pre-mRNA to promote PKCδII splicing. We previously demonstrated that SRSF2 regulated PKCδII alternative splicing (7). To evaluate whether MALAT1 bound to SRSF2 in HT22 cells, we performed the RNA coimmunoprecipitation (RIP) assay. MALAT1 is shown to colocalize in the nuclear speckles with SRSF2 (25, 33, 34). Hence, we sought to evaluate whether MALAT1 and SRSF2 associated in the nucleus of HT22 cells. HT22 cells were treated with hASC exosomes (10 μg) containing MALAT1 for 24 hours. The nuclear and cytoplasmic RNA from HT22 cells were separated and isolated (RNA subcellular isolation kit, ActiveMotif). RNA coimmunoprecipitation (RIP) assay was performed using SRSF2 antibody and primers in real-time qPCR specific for human MALAT1. Our results demonstrated that human MALAT1 from the hASC exosomes bound to SRSF2 in HT22 cells with a twofold higher affinity when compared with binding of the positive control snRNP70 and U1 RNA in the nuclear fraction, while no association was observed in the cytoplasmic fraction [Fig. 6(C)].

Insulin enhances the ability of hASC secretome to increase neuronal proliferation and survival

We previously demonstrated that alternative splicing of PKCδII in HT22 cells was increased with insulin treatment and that intranasal insulin treatment increased cognition and neuronal survival via PKCδII in vivo in a mouse model (7). We demonstrated that insulin increased phosphorylation of SRSF2, which is essential for its function to promote splicing. Here, we sought to see the effect of combining insulin treatment with hASC exosomes treatment in enhancing neuronal proliferation and survival.

To determine if hASC exosomes and insulin may be neuroprotective, we induced oxidative stress with hydrogen peroxide. Oxidative stress–induced cell damage is common in etiology of several neurobiological disorders, including traumatic brain injury. HT22 cells were treated with 100 μm H2O2 for 24 hours. The cells were then treated with either 10 μg hASC exosomes, 10 μg MALAT1-depleted exosomes (M1ASO-254 exosomes), or insulin (10 nM) alone or in combination with exosomes treatment. Ki67 staining was used to evaluate neuronal proliferation following oxidative stress. DAPI images show nuclear staining. The merged images show the amount of proliferating cells compared with total HT22 cells. Our results show that H2O2 treatment significantly decreased the number of proliferating HT22 cells. Treatment of H2O2-treated HT22 cells with hASC exosomes significantly increased HT22 proliferation, while MALAT1-depleted exosomes (M1ASO-254 exosomes) had lower ability to increase HT22 proliferation post H2O2 treatment. Additionally, our results demonstrate that addition of insulin post H2O2 treatment along with hASC exosomes in HT22 cells significantly increased proliferation in HT22 cells [Fig. 7(A)].

Figure 7.

Figure 7.

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hASC exosomes and insulin treatment promotes neuronal proliferation and survival. HT22 cells were treated with 100 μm H2O2 for 24 hours, followed by treating the cells with either hASC exosomes or MALAT1-depleted exosomes M1ASO-254 Ex, 10 nM insulin, or a combination thereof (as indicated in the figure) for 24 hours. (A) Immunochemistry was performed with either Ki67 (for proliferating cells) or DAPI (for nuclei). The merged images are shown on right and show proliferating cells with the different treatments compared with control cells. White bar in images is 50 μm. Inset (left of B) shows HT22 cells stained with doublecortin, confirming neuronal cells. Experiments were performed 5 times with similar results. Images were captured on a Nikon microscope. (B) Scratch assay for cell migration and wound healing: HT22 cells were scratched using a P100 pipette tip to mimic injury, and boundary of scratch on each side was marked on the outside bottom of the plate. The injured HT22 cells were treated with either hASC exosomes or MALAT1-depleted exosomes M1ASO-254 Ex, 10 nM insulin, or a combination thereof for 24 hours. Cell images were captured using a Nikon microscope (20× magnification) at 24 hours post treatment. (C) Five separate fields within the marked boundaries were measured for each plate in the scratch assay after 24 hours. The average width of wound was measured and calculated as percentage of control wound width. Experiments were repeated thrice. (D) In the previous experiments, total RNA was isolated separately and SYBR Green Real-Time qPCR was performed using PKCδI and PKCδII primers for absolute quantification. GAPDH served as control. For absolute quantification, a standard curve was generated for each gene in every assay. Absolute quantification of mRNA expression levels for PKCδI and PKCδII was calculated by normalizing the values to GAPDH. Experiments were repeated 4 times with similar results. The results were analyzed with a 2-tailed Student t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of P < 0.05 was considered statistically significant. ***P < 0.0001, highly significant between hASC exosomes treated with or without insulin. AQ, absolute quantification.

Next, we used an in vitro scratch assay to study injury to the neurons and its wound healing in response to treatment with exosomes (30). HT22 cells were plated in a 35-mm plate, and cells were scratched using a pipette tip to mimic injury. Ten micrograms of hASC exosomes, 10 μg MALAT1-depleted exosomes (M1ASO-254 exosomes), or insulin (10 nM) were added to the HT22 cells for 24 hours. Using a Nikon microscope, cell images were captured for 5 different fields of 1-μm2 area, and wound width was measured 24 hours posttreatments. Our results show that hASC exosomes and insulin treatment either separately or in combination enhance wound healing, as measured by decrease in wound width compared with the control wound width. MALAT1-depleted exosomes increased wound healing but to a smaller extent compared with hASC exosomes [Fig. 7(B) and 7(C)].

In the preceding proliferation and migration assays, we sought to verify that the increase in neuronal proliferation and survival due to hASC exosomes and insulin treatment was due to increased splicing of PKCδII. Our SYBR Green qPCR results show PKCδII alternative splicing was increased by hASC exosomes and hASC exosomes with insulin treatment [Fig. 7(D)]; PKCδI levels were not significantly affected. These results demonstrate that insulin enhances hASC and its secretome’s ability to increase neuronal survival via PKCδII.

Insulin increases association of MALAT1 with SRSF2

We previously showed that insulin treatment increases phosphorylation of SRSF2 by AKT serine/threonine kinase 2, which results in increased alternative splicing of PKCδII. Our results cited previously (Fig. 6) demonstrate that MALAT1 sequesters SRSF2 in HT22 cells. Hence, we sought to determine the effect of insulin treatment on association of MALAT1 and SRSF2 in HT22 cells. Our results demonstrate that insulin dramatically increased the association of MALAT1 and SRSF2 in HT22 cells in the RIP assay (Fig. 8) compared with HT22 cells without insulin treatment. These results suggest that MALAT1 sequesters SRSF2 to enhance the ability of kinases (such as AKT serine/threonine kinase 2) to phosphorylate SRSF2, which is essential for the function of SRSF2 to promote alternative splicing of PKCδII.

Figure 8.

Figure 8.

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Insulin increases association of MALAT1 with SRSF2. HT22 cells were treated with or without 10 nM insulin for 24 hours. RIP assay was performed on HT22 cells treated with or without 10 nM insulin. Immunoprecipitated (IP) RNA was analyzed with primers for MALAT1 flanking the SRSF2 putative binding site. Graph is plotted as fold enrichment of MALAT1 using qPCR in the RIP assay with SRSF2 IP and fold enrichment of U1 RNA using SNRP70 IP (positive control). Results show that SRSF2 binding to lncRNA MALAT1 increases with insulin treatment. Experiments were repeated 4 times with similar results.

Discussion

Regenerative medicine is exploding with the availability of stem cells and their potential use in treating neuronal injuries. hASCs provide additional benefits with their ease of isolation and the ability to obtain them in large quantities. We have previously published that hASCs present cell surface antigens similar to mesenchymal stem cells (9). hASC may influence other target organs via its protein and RNA-rich secretome. The secretome contains microvesicles, among which exosomes are the smallest vesicles at 50 to 100 nm in diameter. We recently published that hASC and its secretome differ in their genetic composition and cargo based on the adipose depot from which it is isolated. We demonstrated that the hASC and its secretome from the subcutaneous depot (used in the current study) has significantly higher levels of MALAT1 in the exosomes compared with the omental depot (29). MALAT1 regulates alternative splicing of several genes, including CAMK2B, CDK7, SAT1, HMG2L1, B-MYB, and MGEA6 (35). In addition, MALAT1 associates with SRSF2 splicing domains in multiple mammalian species (25). The lncRNA NEAT1 is also shown to regulate alternative splicing of genes. NEAT1 is present in the CM of hASC; however, it is not present within the exosomes (29). It may be possible that NEAT1 is packaged in other vesicles. Our data using GFP demonstrates that HT22 cells take up exosomes and that exosomes from hASC increases neuronal survival and proliferation. These studies demonstrate that exosomes from hASC can be used in therapeutic applications in neuronal repair. An advantage to identifying exosomes as the important component of the secretome that increases neuronal survival is that directly injecting hASC to the neuronal site of injury may result in growth and integration of stem cells into neurons, which may be detrimental to the brain. Our results identifying MALATI as an important component of hASC exosomes promise the development of therapeutic regiments targeted to specific areas of neuronal injury and degeneration. The usage of MALAT1 in a systemic manner would be discouraged to avoid possible malignancy-promoting effects of lncRNAs.

Other lncRNAs such as Pinky are shown to have a role in neurons (36). We focused on MALAT1 in this study for several reasons: Its expression was higher in exosomes from subcutaneous hASC; subcutaneous hASC is predominantly used in applications toward regenerative medicine; and MALAT1 was shown to have a prominent role in alternative splicing of genes. MALAT1 locates to the nuclear speckles, where it interacts with the pre-mRNA to regulate splicing (34). Our data using RIP assays show that MALAT1 associates with SRSF2 in the nuclear fraction. Earlier reports have shown that MALAT1 sequesters SRSF2 and promotes its phosphorylation in the serine-arginine domain (35). Our previous data showed that SRSF2 regulates PKCδII expression (7). Our results here demonstrate that MALAT1 from hASC exosomes recruits SRSF2 to regulate PKCδII alternative splicing in HT22 neuronal cells. Our previous data demonstrated that insulin increased phosphorylation of SRSF2. Interestingly, our results here show that insulin treatment dramatically increases association of SRSF2 with MALAT1. These studies demonstrate that MALAT1 binds tightly to SRSF2 such that kinases may efficiently phosphorylate SRSF2 to promote alternative splicing of PKCδII in HT22 cells.

There are several interesting studies about the potential of insulin treatment in neuronal survival in trauma as well as neurodegenerative diseases. We showed earlier that intranasal insulin treatment in vivo increased the cognition and memory in mice (7). Hence, it was of interest to evaluate the combination therapy of hASC with insulin in neuronal survival and proliferation. Our results indicated that hASC exosomes improved neuronal survival and proliferation to a higher extent than insulin alone; combination treatment of hASC exosomes and insulin further enhanced the ability of hASC to increase neuronal survival. These results show a promise of using hASC exosomes with insulin in models of traumatic brain injury or other neurodegenerative diseases. Our study provides the mechanism of action of hASC and its secretome in neuronal survival and identifies MALAT1 as a central lncRNA in hASC exosomes and neuronal regenerative medicine. It is also important to note that MALAT1 may have additional targets that play a role in neuronal survival and proliferation. Additionally, apart from MALAT1, other components of the exosomes may contribute to increased neuronal survival. Future studies are needed to identify all neuronal targets of MALAT1 contained in hASC exosomes.

Acknowledgments

This work was supported by the Department of Veterans Affairs Medical Research Grant 821-MR-EN-20606 (to N.A.P.).

Acknowledgments

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
ASO
antisense oligonucleotide
Bcl2
B-cell lymphoma 2
CD
cluster of differentiation
cDNA
complementary DNA
CM
conditioned media
DAPI
4′,6-diamidino-2-phenylindole
GAPDH
glyceraldehyde 3-phosphate dehydrogenase
GFP
green fluorescent protein
hASC
human adipose-derived stem cell
HT22
immortalized mouse hippocampal cell line
IgG
immunoglobulin G
lncRNA
long noncoding RNA
MALAT1
metastasis-associated lung adenocarcinoma transcript 1
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
PKC
protein kinase C
qPCR
quantitative polymerase chain reaction
RIP
RNA-immunoprecipitation
siRNA
silencer RNA

Author contributions: N.A.P. conceived, designed, and analyzed data, coordinated the study, and wrote the paper. G.E.B. and R.S.P. designed, performed, and analyzed the experiments shown in Figures 1–8. G.C. designed, performed, and analyzed the experiments for MALAT1 qPCR shown in Figure 4. S.S. contributed in design and analysis of Figure 7. V.S. and A.A.P. provided technical support in all experiments. M.M. provided the adipose tissues for hASC and discussed and contributed to hASC application. D.R.C. and P.C.B. discussed and contributed to hASC exosome application. All authors reviewed the results and approved the final version of the manuscript.

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