Jo-Wen Liu

Activation and reversal of lipotoxicity in PC12 and rat cortical cells following exposure to palmitic acid

Lipotoxicity involves a series of pathological cellular responses after exposure to elevated levels of fatty acids. This process may be detrimental to normal cellular homeostasis and cell viability. The present study shows that nerve growth factor‐differentiated PC12 cells (NGFDPC12) and rat cortical cells (RCC) exposed to high levels of palmitic acid (PA) exhibit significant lipotoxicity and death linked to an “augmented state of cellular oxidative stress” (ASCOS). The ASCOS response includes generation of reactive oxygen species (ROS), alterations in the mitochondrial transmembrane potential, and increase in the mRNA levels of key cell death/survival regulatory genes. The observed cell death was apoptotic based on nuclear morphology, caspase‐3 activation, and cleavage of lamin B and PARP. Quantitative real‐time PCR measurements showed that cells undergoing lipotoxicity exhibited an increase in the expression of the mRNAs encoding the cell death‐associated proteins BNIP3 and FAS receptor. Cotreatment of NGFDPC12 and RCC cells undergoing lipotoxicity with docosahexaenoic acid (DHA) and bovine serum albumin (BSA) significantly reduced cell death within the first 2 hr following the initial exposure to PA. The data suggest that lipotoxicity in NGFDPC12 and cortical neurons triggers a strong cell death apoptotic response. Results with NGFDPC12 cells suggest a linkage between induction of ASCOS and the apoptotic process and exhibit a temporal window that is sensitive to DHA and BSA interventions.

Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids.

Epidermal fatty acid‐binding protein (E‐FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E‐FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E‐FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n‐3 docosahexaenoic acid (DHA), C20:5n‐3 eicosapentaenoic acid (EPA), and C20:4n‐6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E‐FABP expression that is blocked by mitogen‐activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor‐differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12‐AS) which exhibit low E‐FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12‐S). Replenishing NGFDPC12‐AS cells with biotinylated recombinant E‐FABP (biotin‐E‐FABP) protein restores normal neurite outgrowth. Cellular localization of biotin‐E‐FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E‐FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12‐S than in NGFDPC12‐AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E‐FABP.

Induction and axonal localization of epithelial/epidermal fatty acid‐binding protein in retinal ganglion cells are associated with axon development and regeneration

Epithelial/epidermal fatty acid‐binding protein (E‐FABP) is induced in peripheral neurons during nerve regeneration and is found at high levels in central neurons during neuronal migration and development. Furthermore, E‐FABP expression is required for normal neurite outgrowth in PC12 cells treated with nerve growth factor (NGF). The present study examined whether E‐FABP plays a role in retinal ganglion cell (RGC) differentiation and axon growth. Rat retinal tissues from embryonic (E) and postnatal (P) development through adulthood were examined using immunocytochemical labeling with E‐FABP and growth‐associated protein 43 (GAP‐43) antibodies. E‐FABP colocalized with GAP‐43 at E14 through P10. At E14, E‐FABP immunoreactivity was confined to the somas of GAP‐43‐positive cells in the ganglion cell layer, but it was localized to their axons by E15. The axons in the optic nerve were GAP‐43‐positive and E‐FABP‐negative on E15, but the two proteins were colocalized by E18. Retinal cultures at E15 confirmed that E‐FABP and GAP‐43 colocalize in RGCs. Postnatally, labeling was present between P1 and P10 but decreased at older ages and was minimally present or absent in adult animals. Western immunoblotting revealed that at E18, P1, and P10 E‐FABP levels were at least fourfold greater than those in the adult. By P15, protein levels were only twofold greater, with adult levels reached by P31. Furthermore, E‐FABP could be reinduced during axon regeneration. Dissociated P15 retinal cells cultured in the presence of brain‐derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor exhibited sixfold more GAP‐43 and E‐FABP double‐positive RGCs (cell body and axons) than controls. Moreover, all GAP‐43‐immunoreactive RGCs were also positive for E‐FABP. Taken together, these results indicate the following: 1) E‐FABP is expressed in RGCs as they reached the ganglion cell layer and 2) E‐FABP plays a functional role in the elaboration of RGC axons in both development and regeneration. J. Neurosci. Res. 66:396–405, 2001.

Depletion of a fatty acid-binding protein impairs neurite outgrowth in PC12 cells.

Epithelial fatty acid-binding protein (E-FABP) is up-regulated in rat dorsal root ganglia after sciatic nerve crush and in differentiating neurons during development. The present study investigates the role of E-FABP during nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells. Undifferentiated PC12 cells express low levels of E-FABP, while NGF triggers a 6- and 8-fold induction of E-FABP mRNA and protein, respectively. Up-regulation of E-FABP mRNA occurs as early as 24 h after NGF treatment and remains highly expressed over the course of several days, corresponding to NGF-mediated neurite outgrowth. Withdrawal of NGF leads to down-regulation of E-FABP mRNA and retraction of neurites. Immunofluorescence microscopy reveals E-FABP immunoreactivity in the perinuclear cytoplasm, neurites and growth cones of NGF-differentiated cells. To examine the role of E-FABP during neurite outgrowth, PC12 cells were transfected with a constitutive antisense E-FABP vector to create the E-FABP-deficient line PC12-AS. By morphometric analysis, PC12-AS cells treated for 2, 4, and 7 days with NGF exhibited significantly decreased neurite expression relative to control (mock-transfected) cells. Taken together, these data indicate that E-FABP is important in normal NGF-mediated neurite outgrowth in PC12 cells, a finding that is consistent with a potential role in axonal development and regeneration.

Lipotoxicity-mediated cell dysfunction and death involve lysosomal membrane permeabilization and cathepsin L activity.

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity.

Epidermal fatty acid-binding protein protects nerve growth factor-differentiated PC12 cells from lipotoxic injury

Epidermal fatty acid‐binding protein (E‐FABP/FABP5/DA11) binds and transport long‐chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E‐FABP protects nerve growth factor‐differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM‐induced lipotoxicity (PAM‐LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E‐FABP. Antioxidants MCI‐186 and N‐acetyl cysteine prevented E‐FABPs induction in expression by PAM‐LTx, while tert‐butyl hydroperoxide increased ROS and E‐FABP expression. Non‐metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E‐FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE‐FABP showed reduced E‐FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E‐FABP cellular levels by pre‐loading the cells with recombinant E‐FABP diminished the PAM‐induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E‐FABP expression and enhanced the resistance of NGFDPC12 cells to PAM‐LTx. We conclude that E‐FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.

SONIC HEDGEHOG ESTABLISHES THE FIBROBLAST GROWTH FACTORS RESPONSIVENESS IN THE LIMB BUD SHH-FGF FEEDBACK LOOP.: 176

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), whereas progressive limb pattern formation is regulated by SHH emanating from the zone of polarizing activity (ZPA). SHH also up-regulates factors that stimulate posteriorly accentuated FGFs in the AER. In turn, these FGFs maintain SHH expression in the adjacent distal mesoderm to complete the SHH-FGF feedback loop. Interestingly, application of FGF to the anterior mesoderm fails to up-regulate SHH, indicating a requirement for additional factors. We hypothesized that SHH induces this FGF responsiveness in limb mesoderm. To test this hypothesis, we ectopically expressed a SHH-GFP construct in the distal-anterior mesoderm by confined microelectroporation (CMEP) at HH 18-19. We demonstrated subsequent expression of SHH and its downstream target, the patched receptor (PTCH), by whole mount in situ hybridization (WISH). After 18 hours (HH 23-24) of exposure to SHH, we applied an FGF-soaked bead to the anterior margin. After an additional 24 hours, chicks were harvested and the SHH expression was determined by WISH. SHH and PTCH expression were up-regulated 24 hours after CMEP. The initial ectopic expression of SHH correlates with the site of transfection indicated by GFP fluorescence. Interestingly, by 42 hours the expression of SHH has shifted to lie immediately underneath the distal anterior AER and does not correlate with GFP fluorescence. These data signify that endogenous expression and FGF-regulated maintenance of SHH has been established. Furthermore, anteriorly placed FGF-soaked beads locally up-regulate SHH expression. Collectively, these data indicate that exposure of limb mesoderm to SHH activates a unique mechanism to produce SHH in response to FGF and thus link SHH expression tightly to the AER during limb outgrowth.

THE ROLE OF SONIC HEDGEHOG IN THE INDUCTION OF CHICK LIMB REGENERATION/REDEVELOPMENT.: 178

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), tightly linked to progressive limb pattern formation regulated by SHH emanating from the posteriorly positioned zone of polarizing activity (ZPA). The reciprocal induction of SHH and FGF during limb development has been termed the SHH-FGF loop. Amputation of a chick wing bud during early development results in removal of the AER and the ZPA with truncation of the distal limb. Addition of FGF to the posterior, but not the anterior, stump immediately after amputation induces tissue regeneration and reactivates development of the distal limb structures. SHH expression is up-regulated adjacent to posteriorly applied FGF-soaked beads but not anterior applied FGF and correlates with regenereative capacity. We wondered whether SHH was sufficient to induce regenerative capacity. To determine the role of SHH in limb regenerative capacity, we amputated developing wings (HH stage 23) and ectopically expressed SHH by confined microeclectroporation (CMEP) in the anterior and posterior stump margins in the presence or absence of FGF-soaked beads. Embryos were harvested at day 10 (6 days after amputation) and the limb morphology analyzed by alcian green staining of the skeletal elements. In other experiments, chicks were harvested 30 to 40 hours after electroporation, and whole-mount in situ hybridization (WMISH) to FGF4 was used to identify any ectopic ectodermal FGF expression. Ectopic expression of SHH in the posterior stump alone could not reestablish segmented limb development. In contrast, expression of ectopic SHH in the anterior stump induced segmented regeneration/redevelopment in the absence of FGF (9/20 ≈ 40%). However, WMISH for FGF4 identified ectopic FGF4 expression in the ectoderm overlying ectopic anterior SHH expression, indicating the local presence of both factors. Thus, it appears that both SHH and FGF are required to regenerate lost tissue and initiate redevelopment. Our data suggest that SHH induces both positive and negative regulators of FGF expression. Initial SHH expression in naive limb mesoderm (anterior stump cells) up-regulates FGFs in the overlying ectoderm, while cells with prior exposure to SHH (posterior stump cells) no longer have the ability to up-regulate FGF in response to SHH. This shutdown of the SHH-FGF-loop, overcome in amphibian regeneration, may be a critical step in promoting mammalian limb regeneration.

230 PALMITIC ACID TRIGGERS CELL DEATH IN PC12 CELLS THROUGH THE ACTIVATION OF MITOCHONDRIAL PROAPOPTOTIC GENES.

The present study uses PC12 cells to investigate the mechanism by which pathological concentrations of saturated free fatty acids (FFAs) trigger cell death. Fatty acid-induced apoptotic cell death has been proposed to play a role in the neuronal loss observed following traumatic injury in the CNS and PNS. To further study this observation, we performed a series of c-DNA array experiments using mRNA of PC12 cells exposed to palmitic acid complexed with bovine serum albumin (BSA) (2:1 ration). Our data show that palmitic acid changes the levels of expression of several mRNAs of proteins that have been associated with apoptosis such as BNIP3 and Bax. Quantitative real-time RT-PCR (QRTPCR) experiments revealed an up-regulation of the proapoptotic mRNA BNIP3 that reached a maximum of 4-fold induction and Bax that reached a maximum of 3.5 induction during the course of the exposure of PC12 cells to palmitic acid. Western blots results also show an induction in BNIP3 and Bax protein. Additional activation of BNIP3/Bax can induce cell death by heterodimerization with antiapoptotic Bcl-2/Bcl-xl or by opening of the permeability transition pore resulting in mitochondrial dysfunction. Our results suggest that saturated FFAs induced cell death through the activation of proapoptotic mitochondrial genes like BNIP3 and Bax.

181 PALMITIC ACID-INDUCED CELL DEATH IN NERVE GROWTH FACTOR DIFFERENTIATED PC12 CELLS INVOLVES THE UP-REGULATION OF HYPOXIA-INDUCING FACTOR 1 AND βNIP-3.

Hypoxic-ischemic injury in the nervous system is associated with the degradation of membrane phospholipids with subsequent release of free fatty acids (FFAs). It has been proposed that this increase in the release of FFA can cause fatty acid-induced apoptotic neuronal cell loss and would be at least in part responsible for the observed secondary injury seen following hypoxia/ischemia and nerve injury insult. Purpose The present study uses NGF differentiated PC12 (NGFDPC12) cells to study neuronal apoptosis and to investigate the mechanism by which pathological concentrations of saturated FFA trigger neuronal cell death. We previously reported that NGFDPC12 cells exposed to palmitic acid (PA) show a dramatic loss of viability as early as 12 hours after treatment and exhibit an increase in the expression levels of several mRNAs of apoptosis-associated proteins such as BAK and βNIP-3 and hypoxic-inducing factor 1 alpha (HIF-1). Methods To further study this observation, we performed a series of Western blots experiments using cell extract of NGFDPC12 cells exposed to PA bound to bovine serum albumin (BSA) (2:1 ratio) to determine the levels of βNIP-3 and its activator HIF-1. Cells were treated with the PA for 6, 9, 12, and 24 hours. Cells were then harvested and lysed in an extraction buffer with protease inhibitors. Next, 20 μg of total cellular protein were electrophoresed on 4-12% gradient SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and blocked with 7.5% nonfat milk in TTBS for 1 hour. Blots were then probed with antibodies for HIF-1 or βNIP-3 followed by a secondary antibody conjugated to horseradish peroxidase. Antigen-antibody complexes were visualized with enhanced chemiluminescence. Results Our Western blot results show an increase in both proteins HIF-1 and βNIP-3. HIF-1 and βNIP-3 have a maximum expression at 6 hours after PA treatment and gradually decreased thereafter. Conclusion These findings suggest that these two apoptosis-associated proteins may play a role in the apoptotic cell damage mediated by saturated FFA in NGFDPC12 cells. This work is supported in part by NIGMS award 2R25GM060507-05 to M.D.L.