Loss in PKC Epsilon Causes Downregulation of MnSOD and BDNF Expression in Neurons of Alzheimer’s Disease Hippocampus 1

Abstract

Oxidative stress and amyloid-β (Aβ) oligomers have been implicated in Alzheimer’s disease (AD). The growth and maintenance of neuronal networks are influenced by brain derived neurotrophic factor (BDNF) expression, which is promoted by protein kinase C epsilon (PKCɛ). We investigated the reciprocal interaction among oxidative stress, Aβ, and PKCɛ levels and subsequent PKCɛ-dependent MnSOD and BDNF expression in hippocampal pyramidal neurons. Reduced levels of PKCɛ, MnSOD, and BDNF and an increased level of Aβ were also found in hippocampal neurons from autopsy-confirmed AD patients. In cultured human primary hippocampal neurons, spherical aggregation of Aβ (amylospheroids) decreased PKCɛ and MnSOD. Treatment with t-butyl hydroperoxide (TBHP) increased superoxide, the oxidative DNA/RNA damage marker, 8-OHG, and Aβ levels, but reduced PKCɛ, MnSOD, BDNF, and cultured neuron density. These changes were reversed with the PKCɛ activators, bryostatin and DCPLA-ME. PKCɛ knockdown suppressed PKCɛ, MnSOD, and BDNF but increased Aβ. In cultured neurons, the increase in reactive oxygen species (ROS) associated with reduced PKCɛ during neurodegeneration was inhibited by the SOD mimetic MnTMPyP and the ROS scavenger NAc, indicating that strong oxidative stress suppresses PKCɛ level. Reduction of PKCɛ and MnSOD was prevented with the PKCɛ activator bryostatin in 5–6-month-old Tg2576 AD transgenic mice. In conclusion, oxidative stress and Aβ decrease PKCɛ expression. Reciprocally, a depression of PKCɛ reduces BDNF and MnSOD, resulting in oxidative stress. These changes can be prevented with the PKCɛ-specific activators.

Keywords

Alzheimer’s disease BDNF hippocampus MnSOD oxidative stress

INTRODUCTION

Protein kinase C (PKC) and its downstream signaling pathways have been implicated in memory enhancement and synaptogenesis [1, 2]. PKCɛ can reduce amyloid-β (Aβ) level by activating α-secretase and anti-amyloidogenic pathways [3, 4], by increasing the degradation of Aβ by endothelin-converting enzyme [5, 6], and by inhibiting β-secretase, which cleaves amyloid-β protein precursor (AβPP) to Aβ [7]. Treatment of transgenic Alzheimer’s disease (AD) mice with the PKCɛ activators, given before memory loss occurs, prevents a decrease in neuronal PKCɛ level, reduces the levels of soluble Aβ and amyloid plaque deposition, and also prevents memory impairment and the loss of hippocampal synapses [8]. Decreases in PKCɛ were recently found in AD autopsy samples and in animal models of AD [8, 9]. PKC may act by preventing the reduction of brain-derived neurotrophic factor (BDNF) expression. However, the effects of decreased PKCɛ and its downstream signals have not been clearly investigated in AD. One common factor in many neurodegenerative diseases is mitochondrial dysfunction. It has previously been shown that neurons in the AD brain exhibit damaged mitochondria and oxidative stress [10]. Chronic oxidative stress can both be a cause and a consequence of chronic neuroinflammation [11 –14]. Inflammation increases the expression of AβPP and the APP-cleaving enzyme 1 (BACE1 or β-secretase), but decreases ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10), an enzyme with α-secretase activity, resulting in increased Aβ production [15 –17]. Aβ oligomers induce prolonged inflammation and oxidative stress, which can inhibit neurogenesis and synaptogenesis [18 –20].

PKCɛ can prevent oxidative stress by inhibiting mitochondrial superoxide (O₂^–) generation [19]. PKCɛ activation can enhance anti-inflammatory and anti-apoptotic gene expression via CREB and ERK1/2 [20 –23]. Learning can stimulate the PKC-activated nuclear export of Hu proteins (or ELAV mRNA-stabilizing proteins) into the dendritic shaft, resulting in prolonged mRNA accumulation [1, 24]. These processes are regulated by the interaction of three RNA recognition motifs in Hu with adenine- and uridine-rich instability-conferring elements (ARE) in the 3^′-untranslated regions of target mRNAs [25, 26], including BDNF, nerve growth factor, and neurotrophin-3 [27]. PKCɛ stimulates HuD-mediated mRNA stability and protein expression of BDNF and enhances dendritic maturation of hippocampal neurons in culture [8, 27]. MnSOD mRNA also contains AREs [28]. However, the effect of PKCɛ on MnSOD has not been clearlydemonstrated.

We investigated the reciprocal interaction among oxidative stress, Aβ, and PKCɛ level and subsequent PKCɛ-dependent MnSOD and BDNF expression in pyramidal neurons in the CA1 stratum radiatum of AD human hippocampus using AD-confirmed human autopsy samples. We then used primary cultures of human hippocampal neurons and Tg2576 AD transgenic mice to define the underlying molecular pathophysiology.

MATERIAL AND METHODS

Preparation of human autopsy hippocampal sections

Autopsy confirmed age-matched control (n = 9) and AD (n = 9) brains at 0.5–0.8 cm thickness and fixed in 4% paraformaldehyde were kindly provided by the Harvard Brain Tissue Resource Center, McLean Hospital, Boston, MA. The pathological diagnosis of AD was conducted according to the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Approval for the study was obtained from Dr. Francine M. Benes at the Harvard Brain Tissue Resource Center. All patients (or relatives/representatives who had the power of attorney) signed informed consent forms. Autopsy was conducted between 6.83 and 26.18 h postmortem. All samples were matched for age (AD 78.3 ± 10.5 years and control 78.9 ± 10.4 years) and gender (50% male and female); demographic details of each individual brain are reported in Table 1. The present work was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans (https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/).

Table 1

Demographic data

Patient ID	Diagnosis	Braak	Age (y)	Gender	PMI (h)	Vascular risk factor
AN15589	AD	II	84	F	19.25	CAA
AN06468	AD	IV	98	M	22.70	Atherosclerosis
AN01667	AD	V	68	M	15.67	Hypertension, atherosclerosis, CAA
AN02773	AD	V	81	F	20.75	Hypertension, atherosclerosis
AN14554	AD	V	61	F	21.42	Hypertension, atherosclerosis, CAA
AN10254	AD	VI	87	M	15.25	Atherosclerosis, CAA
AN16195	AD	V	73	F	17.67	Hypertension, atherosclerosis, CAA
AN17726	AD	II	72	M	6.83	Atherosclerosis, CAA
AN02930	AD	II/III	80	M	23.17	Hypertension, atherosclerosis
AN10329	Control	I	81	F	26.18	Hypertension, atherosclerosis
AN08277	Control	0	61	F	16.80	Hypertension
AN00704	Control	I	82	F	15.70	Atherosclerosis
AN15515	Control	I	73	M	20.88	Hypertension, atherosclerosis
AN17896	Control	I	69	M	18.43	Hypertension, atherosclerosis
AN00316	Control	I	75	F	20.08	(Hypertension), atherosclerosis, CAA
AN08396	Control	I	76	M	12.25	Atherosclerosis
AN12667	Control	II	86	M	25.28	Hypertension, atherosclerosis, CAA
AN03324	Control	II	97	M	17.00	(Hypertension), atherosclerosis,

AD, Alzheimer’s disease, CAA, cerebral amyloid angiography. Note: (Hypertension) is classified with brain arteriolosclerosis that is the previous sign of hypertension. AD brains were from subject diagnosed with dementia and confirmed with the amyloid plaques and neurofibrillary tangles, while control brains were from subjects without dementia.

Human hippocampi were sectioned into 0.4 cm slices with a vibratome (Leica VT1200S) and processed for paraffin embedding. Paraffin-embedded sections (10μm) were mounted on glass slides and deparaffinized with xylene (2 times; 5 min each) and 100% ethanol for 5 min. Tissue sections were then rehydrated with graded alcohol (100%, 95%, 85%, 70%, and 50%; at 3 min each step) and washed with distilled water (2 times; 5 min each).

Culturing of primary fetal hippocampal neurons

Human primary hippocampal neurons (ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in an 8-well chamber mounted on a glass slide (Lab-Tek II CC² chamber slide, Nunc, Rochester, NY, USA), coated with poly-L-lysine. Cultured neurons were maintained in neuronal medium (ScienCell) supplemented with neuronal growth supplement (NGS, ScienCell) for 21 days before being used for experiments. For maintenance of neurons, half of the media was changed every 3 days.

PKCɛ knockdown in cultured human neurons

PKCɛ knockdown was performed using 100 nM of three target-specific 19–25 nucleotide PKCɛ si-RNA constructs (sc-36251), purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). For negative control, a scrambled si-RNA (sc-37007) was used. Transfection was performed using Lipofectamine 3000, per the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Medium was changed 6 h after addition of lipofectamine. PKC downregulation was assessed 72 h after transfection, as mentioned earlier [2, 29] and in the Lipofectamine 3000 protocol.

Increased superoxide production and PKCɛ activator treatment in cultured human neurons

Cultured neurons were treated with 200μM tert-butyl hydroperoxide (TBHP) for 1 h. After treatment, the media was changed with fresh media and maintained in culture without TBHP for 4 days [30]. For (sham) controls, cultured neurons were treated similarly without PKCɛ si-RNA transfection and TBHP, and cells were used at the same age. For PKCɛ activator treatment, bryostatin was purchased from Biomol International (Farmingdale, NY, USA) and methyl ester of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCPLA-ME) was synthesized in our laboratory following the method described earlier [6, 31] and shown to be specific for PKCɛ. Primary human neurons were treated with bryostatin (0.27 nM) [6, 32], DCPLA-ME (100 nM) [32], or drug solvent control (ethanol) for at least 4 days. Cultured neurons were either fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min at room temperature or lysed and prepared for immunoblot.

Measurement of increased superoxide or reactive oxygen species in cultured neurons

For O₂^– measurement, the specific probe hydroethidine (Thermo Fisher Scientific/Life Technologies) was used to measure changes in cellular O₂^– production [33]. The reaction between O₂^– and hydroethidine generates a highly specific red fluorescent product, 2-hydroxyethidium. In biological systems, another red fluorescent product (ethidium) is also formed, usually at a much higher concentration than 2-hydroxyethidium [34]. Cultured human neurons, grown on glass slides, were incubated with hydroethidine (2μg/ml) at room temperature for 15–20 min and then fixed with 4% paraformaldehyde. Increased O₂^– production was qualitatively determined with a confocal microscope at 488 nm/>510 nm (excitation/emission).

Effect of ROS scavenger drugs

Primary cultures of human hippocampal neurons were incubated with the ROS scavenger N-acetylcysteine (NAc, 5 mM for 15 h) or the SOD mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 25μM for 45 min). Following incubation, NAc and MnTMPyP were removed and the neurons were treated with TBHP at 200μM for 1 h. After 3 days of recovery without TBHP, NAc and MnTMPyP cultured neurons were used for western blot analysis.

Cell lysis and western blot analysis

Human neuron cultures were harvested in homogenizing buffer (HB) containing 10 mM Tris-Cl (pH 7.4), 1 mM PMSF (phenylmethylsulfonylfluoride), 1 mM EGTA, 1 mM EDTA, 50 mM NaF, and 20μM leupeptin, and were lysed by sonication. The homogenate was centrifuged at 100,000×g for 15 min at 4°C to obtain the cytosolic fraction (soluble) and membrane (particulate). The pellet was resuspended in the HB by sonication. HB used for whole cell protein isolation from primary neurons also contained 1% Triton X-100. Protein concentration was measured using the Coomassie Plus (Bradford) Protein Assay kit (Pierce, Rockford, IL, USA). Following quantification, 20μg of protein from each sample was subjected to SDS-PAGE analysis in a 4–20% gradient Tris-glycine polyacrylamide gel (Invitrogen, Carlsbad, CA, USA). The separated protein was then transferred to a nitrocellulose membrane. The membrane was blocked with 1.5% BSA and incubated with primary antibody (the same antibody as used in immunohistochemistry) overnight at 4°C. After incubation, the blot was washed 3×with TBS-T (Tris-buffered saline-0.1% Tween 20) and further incubated with alkaline phosphatase-conjugated secondary antibody at 1:10000 dilution for 45 min. The membrane was finally washed 3×with TBS-T and developed using the 1-step NBT-BCIP substrate (Pierce, Rockford, IL, USA). The blot was imaged in ImageQuant RT-ECL (GE Life Sciences, Piscataway, NJ), and densitometric quantification was performed using IMAL software. For quantifying expression of a protein, the densitometric value for the protein of interest was normalized against β-actin (loadingcontrol).

Preparation of Aβ oligomers

Amylospheroids-enriched Aβ-oligomers (ASPDs) were prepared as previously described [32, 35]. Briefly, Aβ_1–42 was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol and incubated overnight at 4°C and then for 3 h at 37°C. The dissolved Aβ_1–42 was lyophilized to 40 nmol/tube. The lyophilized Aβ was dissolved in PBS without Ca^2 + or Mg^2 + to less than 50μM concentration and rotated for 14 h at 4°C. The resulting ASPD enriched preparation was purified using a 100-kDa molecular weight cutoff filter (Amicon Ultra,Millipore).

Native gel analysis of ASPD enriched preparation

Native gel analysis was performed using 4–20% gradient Tris-Glycine gel (Invitrogen, USA) and Novex Tris-Glycine native running buffer (Invitrogen, USA) at 100V and 4°C. Gels were stained with Sypro Ruby Red stain (Molecular Probes) [32].

Viability assay

Viability of cultured neurons treated with ASPD enriched preparation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [36]. Dissolved MTT is a tetrazolium salt that is cleaved to insoluble formazan by succinate dehydrogenase that is only active in viable cells [37]. After treatment, the cells were washed with 1×PBS and were incubated with 200μl of 1 mg/ml MTT solution (Sigma, USA) at 37°C for 2 h. Then the MTT-solution was removed, and the cells were lysed with 200μl isopropanol containing 0.04 M HCl and 160 mM NaOH for 10 min. Finally, the absorbance was measured at 570 nm and 630 nm. All the samples were done in triplicate, and the data were represented as the percentage of control.

Preparation of hippocampal sections from transgenic mice

Littermate Tg2576 and control wild type mice (Genotype: Hemizygous for 129S6.Cg-Tg(APPSWE)2576Kha N20+?; Taconic, Hudson, NY, USA) were used. Tg2576 mice at 8 weeks of age were treated with bryostatin (from the National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 30μg/kg, i.p.) for a 12-week period. The dose of 30μg/kg was selected because it is comparable to the doses used clinically (20, 25, and 40μg/kg). We have found that this dose results in a brain concentration of 0.1–0.3 nM, which is within the optimum range for PKCɛ activation. Mice at almost 6 months of age were then used for histology studies. Under anesthesia (pentobarbital, 80 mg/kg body weight, i.p.), mice were perfused through the left cardiac ventricle with PBS at room temperature by gravity to wash out the blood, and then fixed with 4% paraformaldehyde in PBS at room temperature. Brains were removed, post-fixed at 4°C overnight, and stored in PBS at 4°C. Right dorsal hippocampi were dissected and sectioned with a vibratome at 300μm thickness. The hippocampal sections were re-sectioned to 5-μm thickness. Serial sections at one section at every 600μm of each hippocampus (4 sections per hippocampus) were processed free floating for immunohistochemistry and blood vessel density studies.

Immunohistochemistry

Due to long-term storage in paraformaldehyde, we treated the human autopsy brain sections for antigen retrieval before immunohistochemistry. Deparaffinized hippocampal sections were incubated in 10 mM citrate and 0.05% Tween 20 in distilled water (adjusted to pH 6.0 with 1 N NaOH), for 30 min at 95°C. Tissue sections were then washed with PBS (3 times; 5 min each). For immunohistochemistry of hippocampal sections and cultured neuron samples, the samples were treated with Image-iT^® FX signal enhancer (Thermo Fisher Scientific, Grand Island, NY, USA) for 30 min at room temperature to block non-specific protein binding sites. The samples were incubated with primary antibodies against Aβ peptide (mouse monoclonal MOAB-2 antibody; 1:400; EMD Millipore, Billerica, MA, USA), Aβ oligomers (rabbit polyclonal A11 antibody, 1:100, EMD Millipore), BDNF (mouse monoclonal antibody, 1:50, Santa Cruz Biotechnology), MnSOD (rabbit polyclonal antibody, 1:200; EMD Millipore), PKCɛ (rabbit polyclonal antibody, 1:75, EMD Millipore), neuron-specific enolase (NSE, chicken polyclonal IgG; 1:50; Thermo Fisher Scientific), and 8-hydroxyguanosine (8-OHG; goat IgG; 1:100; Alpha Diagnostic International, San Antonio, TX, USA). Samples for cell culture were incubated overnight at room temperature for mouse tissue samples, or 48 h at 4°C and then overnight at room temperature for fixed human tissue samples.

The secondary antibody was 1) Alexa Fluor 488 or 568 goat anti-rabbit, anti-mouse, or anti-goat antibodies (1:200, Thermo Fisher Scientific) for 3 h at room temperature, or 2) biotinylated horse anti-mouse or anti-rabbit IgG (1:20, Vector laboratories, Burlingame, CA) for 3 h at room temperature, followed by streptavidin conjugated with Alexa Fluor 488 (1:100, Thermo Fisher Scientific) for 3 h at room temperature. After antibody incubation the samples were washed with PBS 3 times (each time for a period of 5 min). The samples mounted on glass slides were sealed with cover glasses, using Vectashield mounting medium with DAPI (Vector Laboratories) to counter stain nuclei and were processed for confocal microscopy.

Confocal microscopy

The samples were oriented with a Zeiss Axio Observer Z1 microscope equipped with a 710 confocal scanning system using the 10x objective lens in the DAPI channel (for staining nuclei). The random area that appeared immediately after switching to the higher magnification lens, either 63x or 100x Plan-APOChromat oil immersion objectives (1.4 NA), was imaged for appropriate fluorescence (e.g., Alexa 488 and/or 568). Confocal images (512×512 pixels) were acquired in line scan mode with a pinhole of approximately 1.00 Airy unit. Averaged data from several (8x) images were reported. Confocal images, obtained with a Zeiss confocal microscope, were stored and quantified offline. Immunofluorescence intensity was evaluated with the ImageJ program (http://rsb.info.nih.gov/ij/). All experiment and control tissue sections were processed for staining and microscopy on the same day. Results were divided by DAPI (4’,6-diamidino-2-phenylindole) staining, which stains A-T rich regions in DNA in nuclei, to correct for effects of fluorescence decay and/or different postmortem interval.

Statistical analyses

Briefly, data with a significant overall difference among the groups as demonstrated by ANOVA were further analyzed with Tukey’s multiple comparison test. For data with only 2-group comparison (e.g., AD versus age-matched control in postmortem human brains, PKCɛ knockdown in cultured neurons), paired two-tailed t-test comparisons were used.

RESULTS

Changes in pyramidal neurons in human autopsy-confirmed AD hippocampus

In the present study (Table 1) human brains were collected at autopsy between 6.83 and 26.18 h postmortem. No significant difference between postmortem interval and immunohistochemistry with any primary antibody or neuron density was seen using linear regression analysis (not shown), suggesting that our results were not affected by postmortem change within 26.18 h. The Braak staging of AD brains was 4.2 ± 0.47 (mean ± S.E.; range = 2 – 6). For the factors of age and sex, paired two-tailed t tests were used to compare equal numbers of AD and age/sex-matched controls (AC).

Biochemical changes in the hippocampal CA1 pyramidal neurons were quantified using immunohistochemistry and imaged with confocal microscopy (Fig. 1). PKCɛ, BDNF, and MnSOD were decreased, while the oxidative DNA/RNA damage marker 8-OHG and Aβ were increased in the cell bodies of the hippocampal CA1 neurons (stratum pyramidale) (p < 0.005, Fig. 1A-E). We used the Aβ-specific antibody MOAB-2, which recognizes both soluble and insoluble Aβ. A decrease in neuron number was found in the AD hippocampus (p < 0.005,Fig. 1F).

Fig.1

A decrease in PKCɛ, BDNF, and mitochondrial MnSOD associated with an increase in Aβ, oxidative DNA/RNA damage, and neuron loss in autopsy-confirmed AD hippocampus. Immunohistochemistry and confocal microscopy were used. Change in neuronal (A) PKCɛ, (B) BDNF, (C) mitochondrial MnSOD, (D) the oxidative stress marker 8-OHG, (E) Aβ, and (F) the number of neurons marked with neuron specific enolase (NSE) per 100μm² CA1 area were compared between human AD and age-matched control (AC) hippocampal neurons. Results (except neuron density) were divided by DAPI (4’,6-diamidino-2-phenylindole) staining, which stained A-T rich regions in DNA in nuclei, to correct for effects of fluorescence decay and/or different postmortem interval. Data (mean ± SEM) were from n = 9 AC or 9 AD brains, 13–25 random CA1 neurons/human subject or 13–17 CA1 areas/human subject. ***p < 0.005; Student’s t-test.

Neurotoxic effect of amylospheroid-enriched Aβ-oligomers (ASPDs) in human primary hippocampal neurons in culture

Earlier we showed that ASPDs are the most toxic form of Aβ oligomers, causing a 50% death rate of primary rat hippocampal neurons at 100 nM [32]. To determine the neurotoxic effect of ASPD enriched preparation (Fig. 2A), primary cultures were prepared from human hippocampal neurons (ScienCell Research Laboratories) that were isolated (>90% purity) from hippocampal region of human fetal brain tissues of both sexes. The age of the donor was between 19–22 weeks gestation. All control and experiment tissue cultures were from the same donor and processed for experiment at the same time. We assessed the viability of neurons by MTT-assay after treatment of neurons with 100 nM ASPD enriched preparation for 24 h. The treatment of ASPD enriched preparation reduced the viability of human neurons by 26.7 ± 1.7 % (p < 0.0001) (Fig. 2B) compared to untreated control neurons. Western blot analysis (Fig. 2C) showed that after 24 h the ASPD-treated neurons expressed lower amounts of PKCɛ (p = 0.0008) (Fig. 2D) and MnSOD (p = 0.02) (Fig. 2E).

Fig.2

Neurotoxic effect of Aβ on PKCɛ and MnSOD in cultured human hippocampal neurons. Primary cultures of hippocampal neurons were treated with vehicle (Control) or 100 nM amylospheroids (ASPD) enriched preparation for 24 h. A) Native gel analysis showed that the ASPD enriched preparation (Aβ monomers and oligomers) was in the range of 150–220 kDa. B) Viability of neurons was measured by MTT assay. The treatment of ASPD enriched preparation reduced neuronal viability. C) Immunoblot showing the expression of PKCɛ, MnSOD, and β-actin in control and ASPD treated cells. ASPD enriched preparation reduced (D) the expression of PKCɛ by 36.0 ± 4.0 % and (E) MnSOD by 25.6 ± 6.9 %. M = molecular weight marker. Data are represented as mean ± SEM of three independent experiments (Student’s t-test, *p < 0.05; ***p < 0.005; ****p < 0.0005).

Effects of PKCɛ knockdown in human primary hippocampal neurons in culture

PKCɛ knockdown (KD) was achieved using human specific PKCɛ si-RNA. After 72 h of transfection, PKCɛ, BDNF, and MnSOD levels were measured by immunoblot (Fig. 3A). PKCɛ KD cells expressed significantly lower amounts of PKCɛ (F_2,6 = 8.000, p = 0.02), BDNF (F_2,6 = 8.500, p = 0.02), and MnSOD (F_2,6 = 16.300, p = 0.004). For negative controls, cells were transfected with scrambled si-RNA. Scrambled si-RNA did not affect the expression of PKCɛ, BDNF, and MnSOD (Fig. 3B-D). Primary cultures were then processed with immunocytochemistry and confocal microscopy (Fig. 4). Tissue cultures were oriented with the 10x objective lens in the DAPI channel (for staining nuclei). The random area that appeared immediately after switching to the higher magnification lens (63x or 100x) was imaged for appropriate fluorescence. PKCɛ KD decreased PKCɛ (p < 0.005), BDNF (p < 0.005), and MnSOD (p < 0.01) levels (Fig. 4A-C), confirming the western blot results (Fig. 3B-D). Immunohistochemistry additionally showed that PKCɛ KD increased Aβ (p < 0.005) but decreased (p < 0.01) neuron density (Fig. 4D, E).

Fig.3

PKCɛ knockdown decreases PKCɛ, BDNF, and MnSOD expression levels in the cultures of primary human hippocampal neurons. Effect of PKCɛ reduction was studied in cultured neurons transfected with small double-stranded interfering RNA (si-RNA), which targeted and interfered with mRNA to prevent protein translation. A) Immunoblot showing expression of PKCɛ, BDNF, MnSOD, and β-actin in untreated (Control), scrambled si-RNA (negative control), and PKCɛ si-RNA treated human neurons after 72 h. M = molecular weight marker. PKCɛ knockdown reduces the expression of (B) PKCɛ by 50%, (C) BDNF by 16%, and (D) MnSOD by 40%. Scrambled si-RNA produced no significant change in expression. Data are reported as mean ± SEM of three independent experiments. (*p < 0.05; **p < 0.01; one-way ANOVA, post hoc Tukey’s multiple comparison test).

Fig.4

PKCɛ knockdown increased Aβ staining and neuronal loss associated with the reduction in PKCɛ, BDNF, and MnSOD staining in primary human hippocampal neurons. Cultured neurons were transfected without (Control) or with small double-stranded interfering RNA (si-RNA), which targeted and interfered mRNA to prevent protein translation. After transfection of PKCɛ si-RNA for 72 h, immunohistochemistry and confocal microscopy were used. PKCɛ knockdown decreased (A) PKCɛ, (B) BDNF, and (C) MnSOD, but increased (D) Aβ and (E) neuronal death (a reduction of neuron number per 1 mm² culture area) using light microscopy. Data are reported as mean ± SEM, n = 3–4 cultures (98–197 random neurons or 56–75 random areas) per experiment. **p < 0.01; ***p < 0.005; one-way ANOVA and paired two-tailed t-test comparison test.

Effects of tert-butyl hydroperoxide treatment on oxidative damage in human primary hippocampal neurons in culture

The stable organic peroxide tert-butyl hydroperoxide (TBHP) was used to induce oxidative modification of enzymatic complexes of the respiratory chain and mitochondrial matrix, mitochondrial reduced glutathione depletion, protein glutathionylation, membrane lipid peroxidation, and mitochondrial Ca^2 + overload [38], resulting in an increase in reactive oxygen species (ROS) including O₂^– and oxidative stress [39, 40]. In Fig. 5, primary cultures of human hippocampal neurons were treated with TBHP at 200μM for 1 h and then cultured with fresh culture medium without TBHP for 4 more days. A fluorescent indicator for O₂^– (added 15–20 min before fixing the neurons with formaldehyde) revealed that O₂^– was increased (p < 0.005, Fig. 5A, B).

Fig.5

Oxidative DNA/RNA damage is associated with a decrease in PKCɛ, BDNF, and MnSOD staining in primary human hippocampal neurons. Treatment of tert-butyl hydroperoxide (TBHP) was used to induce mitochondrial dysfunction and ROS increase. Cultured neurons were treated without (Control) or with 200μM TBHP for 1 h and maintained in culture medium without TBHP for 4 days. A) The hydroethidine indicator showed (B) an increase in superoxide (O₂^–) in cultured human neurons. C) Immunohistochemistry and confocal microscopy. TBHP treatment increased (D) the oxidative DNA/RNA damage marker 8-OHG alongside with a reduction in (E) PKCɛ, (F) BDNF, and (G) MnSOD, but the increase in (H) Aβ and (I, J) neuronal loss (decreased neuron density). The effect of TBHP treatment was normalized with the PKCɛ activators bryostatin (Bry, 0.27nM) and DCPLA-ME (DCP, 100n M) continuously presented in the culture medium after 1-h TBHP treatment. Data are reported as mean ± SEM, n = 3 cultures (37–152 random neurons or 54–65 random areas) per experiment; *p < 0.05; **p < 0.01; ***p < 0.005; one-way ANOVA, post hoc Tukey’s multiple comparison test.

With immunohistochemistry and confocal microscopy (Fig. 5C), ANOVA revealed significant differences among experimental groups for 8-OHG (F_3,563 = 17.805, p < 0.001), PKCɛ (F_3,365 = 8.786, p < 0.001), BDNF (F_3,284 = 9.508, p < 0.001), MnSOD (F_3,298 = 26.914, p < 0.001), Aβ (F_3,276 = 11.771, p < 0.001), and neuron density (F_3,209 = 5.646, p < 0.001). The TBHP treatment increased the oxidative damage marker 8-OHG (Fig. 5D) that was associated with decreases in PKCɛ (p < 0.01), BDNF (p < 0.005), MnSOD (p < 0.005), and neuron density (p < 0.01), but an increase in Aβ (p < 0.01) (Fig. 5E-I). Both PKCɛ activators bryostatin and DCPLA-ME prevented the loss of PKCɛ compared to TBHP without the PKCɛ activator (p < 0.05, Fig. 5E). Bryostatin, but not DCPLA-ME, even enhanced PKCɛ level compared to the control level (p < 0.05, Fig. 5E). PKCɛ activators also prevented the decrease in MnSOD (p < 0.01) and abolished the increases in oxidative damage and Aβ (p < 0.005, Fig. 5D, G, H). Both bryostatin and DCPLA-ME also prevented the loss of BDNF (p < 0.005, Fig. 5F) and the decrease in neuron density (p < 0.05, Fig. 5I, J).

Effects of tert-butyl hydroperoxide treatment on membrane translocation and activation of PKCɛ and expression of PKCɛ, MnSOD, and BDNF in human primary hippocampal neurons

To assess whether TBHP treatment affects PKCɛ activity we treated the human neurons with TBHP for 1 h. We also determined the effect of PKCɛ activators (bryostatin and DCPLA-ME) in preventing TBHP induced toxicity. After 1 h, we measured the PKCɛ activation by immunoblot. Untreated and TBHP-treated neurons were lysed and separated into soluble (S) and particulate (P) fractions. PKC activity is calculated and represented as the percentage of total protein in the membrane or particulate fraction. TBHP treatment reduced the membrane associated PKCɛ by 20.2 ± 1.8 % (p < 0.01) (Fig. 6A, B). This effect was prevented with bryostain1 (p < 0.01) or DCPLA-ME (p < 0.05).

Fig.6

Oxidative stress promoted a decrease in PKCɛ translocation to the plasma membrane and consequent PKCɛ activation alongside with a decrease in the expression levels of PKCɛ, BDNF, and MnSOD in primary human hippocampal neurons. Treatment oftert-butyl hydroperoxide (TBHP) was used to induce mitochondrial dysfunction and ROS increase. Cultured neurons were treated without (Control) or with 200μM TBHP for 1 h and maintained in culture medium without TBHP for 4 days. A, B) Immunoblot showing TBHP treatment reduces membrane associated PKCɛ in the particulate (P) fraction. S = soluble fraction, M = molecular weight marker. C) Immunoblot showing expression of PKCɛ, BDNF, MnSOD, and β-actin after 72 h. TBHP reduced the expression of (D) PKCɛ, (E) BDNF, and (F) MnSOD. The effect of TBHP treatment was normalized with the PKCɛ activators bryostatin (Bry, 0.27nM) and DCPLA-ME (DCP, 100nM) continuously presented in the culture medium after 1-h TBHP treatment. Data are reported as mean ± SEM of three independent experiments, *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0005; (B) Student’s t-test or (D-F) one-way ANOVA, post hoc Tukey’s multiple comparison test.

Next, we used immunoblot to measure the expression of PKCɛ, MnSOD, and BDNF in the 1 h TBHP-treated cells after a 3-day survival time (Fig. 6C). TBHP treatment inhibited the expression of PKCɛ (F_5,12 = 16.400, p < 0.0001) (Fig. 6D), BDNF (F_5,12 = 23.400, p < 0.0001) (Fig. 6E), and MnSOD (F_5,12 = 8.300, p = 0.001) (Fig. 6F). Bryostatin and DCPLA-ME protected against the TBHP-induced loss in protein expression. Western blot analysis (Fig. 6D-F) confirmed the results from the immunohistochemistry (Fig. 5E-G). These results suggest that TBHP treatment increases excessive O₂^– production and oxidative damage and suppresses PKCɛ levels, thereby decreasing PKCɛ-activated BDNF and MnSOD expression and PKCɛ-dependent inhibition of Aβ synthesis.

Effect of ROS scavenger drugs on oxidant-mediated reduction of PKCɛ, BDNF, and MnSOD

Significant differences among experimental groups were found for PKCɛ (F_5,12 = 4.236, p < 0.05), BDNF (F_5,12 = 8.743, p < 0.001), and MnSOD (F_5,12 = 9.787, p < 0.0001). The ROS scavenger NAc suppressed (p < 0.05) the TBHP-induced reduction of PKCɛ and MnSOD (Fig. 7B, D). NAc had no effect on the reduction of BDNF after TBHP treatment (Fig. 7C). The cell-permeable SOD mimetic MnTMPyP effectively protected the TBHP-induced decrease in PKCɛ (p < 0.05), BDNF (p < 0.05), and MnSOD (p < 0.01) (Fig. 7B-D).

Fig.7

Neurodegeneration in primary cultures of human hippocampal neurons is associated with a high level of reactive oxygen species and with a decrease in PKCɛ staining. Effect of the ROS scavengers NAc and MnTMPyP on oxidant-mediated reduction of PKCɛ, BDNF, and MnSOD was investigated in cultured human neurons treated with TBHP. (A) Immunoblot was used to determine the levels of (B) PKCɛ, (C) BDNF, and (D) MnSOD. NAc prevented the TBHP-induced reduction of PKCɛ and MnSOD, while MnTMPyP protected the TBHP-induced decrease in of PKCɛ, BDNF, and MnSOD. Data are reported as mean ± SEM of three independent experiments, *p < 0.05; **p < 0.01; one-way ANOVA, post hoc Tukey’s multiple comparison test.

Effects of PKCɛ on Aβ and MnSOD levels in the hippocampal neurons from Tg2576 transgenic AD mice

We previously demonstrated that the A11 antibody detects only Aβ oligomers (not monomers or fibrils), AβPP, and α-synuclein in the CA1 hippocampal neurons [8]. Target-specific antibodies showed a decrease in AβPP but no change in α-synuclein. Therefore, these earlier results showed that an increase in A11 antibody staining indicates an increase in Aβ oligomers in the hippocampal CA1 pyramidal neurons.

In the present study, AD transgenic mice Tg2576 mice at 2 months of age were treated with bryostatin (30μg/kg, i.p., twice a week) for a 3-month period. We imaged the mossy fibers, which are composed of axons of granule cells in the dentate gyrus that form synapses with dendritic spines of CA3 pyramidal neurons, using the Aβ-specific antibody MOAB-2 that recognizes both soluble and insoluble Aβ (Fig. 8A). An inverse relationship between PKCɛ and Aβ was found in the mossy fibers of wild type mice, AD transgenic mice, and AD transgenic mice treated with the PKCɛ activator (Fig. 8B).

Fig.8

An inverse correlation between PKCɛ and Aβ and effect of PKCɛ activation on MnSOD level in hippocampal neurons from Tg2576 transgenic AD mice. Tg2576 (Tg) and wild-type (Wild) control mice at 8 weeks of age were treated with or without the PKCɛ activator bryostatin (Bry, 30μg/kg, ip, 2 times per week) for a 12-week period. A) Immunohistochemistry and confocal microscopy demonstrated (B) an inverse correlation between Aβ and PKCɛ in mossy fibers. Data are represented as mean, n = 9 mice per condition and 3–4 measurements per mouse (C) Immunohistochemistry showed that (D) the reduction of MnSOD was prevented with bryostatin 1 in the cell bodies of the CA1 hippocampal pyramidal neurons. Data are represented as mean ± SEM, n = 3 mice (59–70 neurons per mouse). Data were divided by DAPI (4’,6-diamidino-2-phenylindole) staining, ***p < 0.005; one-way ANOVA, post hoc Tukey’s multiple comparison test.

Immunohistochemistry and confocal microscopy of the CA1 pyramidal neurons revealed a significant difference among experimental groups for MnSOD (F_2,529 = 13.227, p < 0.001) (Fig. 8C). MnSOD was reduced (p < 0.005) in the cell bodies of hippocampal CA1 pyramidal neurons; this was prevented with bryostatin (p < 0.005) (Fig. 8D). These data indicate that increased Aβ is associated with the reduction in PKCɛ and MnSOD expression in hippocampal CA1 pyramidal neurons.

DISCUSSION

The present study demonstrated the role of PKCɛ in regulating Aβ level and the production of reactive oxygen species, as summarized in Fig. 9. Changing the balance among these three factors may contribute to or enhance AD pathogenesis. During aging, decrements in gene expression, impairments in intracellular signaling, and cytoskeletal transport may mediate a decline in cholinergic function [41], leading to the reduction of PKCɛ translocation and activation [42].

Fig.9

Diagram showing imbalance of reciprocal interaction among PKCɛ, Aβ, and oxidative stress during AD pathogenesis. Reduction of PKCɛ decreases α-secretase activity (non-amyloidogenic pathway) [3, 4] decreases endothelin converting enzyme (ECE) activity (Aβ degradation) [5 , 27], and increases β-secretase activity (amyloidogenic pathway) [7], resulting in a rise in Aβ production. Aβ directly binds and inhibits PKCɛ [45, 46]. Reduction of PKCɛ level and activity decreases the activation of ELAV-like Hu RNA-binding proteins (Hu), increasing degradation of mRNAs, and decreasing expression of BDNF and MnSOD proteins. The suppression of PKCɛ-dependent MnSOD expression stimulates a prolonged and sustained increase of O₂^– that reacts with the free radical nitric oxide (NO) to form peroxynitrite (ONO₂^–), a powerful oxidant that damages DNA, RNA, and proteins, including PKC [47]. Reduced MnSOD also decreases the conversion of O₂^– to hydrogen peroxide (H₂O₂) that can oxidize and increase PKC activity [33, 47].

We previously demonstrated that PKCɛ reduction is associated with increased Aβ in autopsy-confirmed human neurons and fibroblasts and transgenic AD mice [8, 9]. In the present study, we provided direct evidence that PKCɛ KD increases Aβ in cultured neurons. The likely mechanism is a decrease in PKCɛ level and its downstream effector activities (Fig. 9), including PKCɛ-activated α-secretase [3, 4], PKCɛ-inhibited β-secretase [7], and PKCɛ-activated degradation of Aβ via endothelin-converting enzyme (ECE) [5 , 45].

The present study also showed that Aβ (ASPD enriched preparation) reciprocally decreased PKCɛ levels. This could be mediated by the well-documented effects of Aβ on cholinergic neurons [42 –44]. Aβ directly binds and inhibits PKCɛ [45, 46]. Under non-disease conditions, O₂^– is converted to hydrogen peroxide (H₂O₂) that oxidizes and promotes PKC activity [33, 47] (Fig. 9). Age-related reduction in MnSOD expression may decrease the conversion of O₂^– to H₂O₂ that can oxidize and increase PKC activity [33, 47].

We found that the levels of PKCɛ, BDNF, and MnSOD decreased in autopsy-confirmed human sporadic AD hippocampus. We therefore demonstrated that PKCɛ KD reduces cell viability, decreases MnSOD and BDNF, and elevates Aβ in primary cultures of human hippocampal neurons, suggesting that PKCɛ protects against AD pathogenesis in the human hippocampus. Activating PKCɛ restores BDNF and MnSOD to normal levels. Therefore, the expression of MnSOD and BDNF is downstream of PKCɛ in hippocampal neurons. The present study showing that the expression of MnSOD and BDNF in pyramidal neurons in the hippocampal CA1 area is PKCɛ-dependent suggests the involvement of Hu-promoted mRNA stabilization and transcription of MnSOD and BDNF [27 , 49] (Fig. 9). PKCɛ can also induce PP2A-mediated dephosphorylation of Akt at Ser-473 that in turn leads to Forkhead box class O (FOXO) 3a dephosphorylation at Ser-253 and its activation of MnSOD expression [48].

The present study also showed that the TBHP-induced ROS increase reciprocally suppressed PKCɛ level and its molecular targets that was prevented by NAc and MnTMPyP. These results indicate a direct effect of oxidative stress on PKCɛ expression. One possible explanation is that sustained and significant increases in O₂^–, which might occur under pathological conditions, favor reaction with the free radical nitric oxide (NO) to form peroxynitrite (ONO₂^–), a powerful oxidant that damages DNA, RNA, and proteins, including PKC [50] (Fig. 9).

We found that MnTMPyP completely rescued, while NAc partially protected the reduction of PKCɛ, BDNF, and MNSOD levels. The reason may be that MnTMPyP is a membrane– permeable SOD mimetic and may have direct anti-oxidant effect. In contrast, NAc (N-acetylcysteine) is an aminothiol and synthetic precursor of intracellular cysteine and reduced glutathione (GSH), an important antioxidant [51]. Although NAc also possesses a reducing property through its thiol-disulfide exchange activity, in the present study, cultured human neurons had been pretreated with either NAc or MnTMPyP, and these compounds were not present during the 1 h TBHP treatment and subsequent 4-day survival. This was to avoid the effect of long-term NAc incubation; for example, NAc has been shown to induce cell cycle arrest in hepatic stellate cells by modulating the mitogen-activating protein (MAPK) pathway [51].

We also investigated Tg2576 mice that overexpress a mutant form of human AβPP. The result in Tg2576 mice confirms our previous study showing an inverse relationship between PKCɛ and Aβ oligomers in human hippocampal neurons [9]. The present studies showed that neuronal PKCɛ and MnSOD levels were decreased in Tg 2576 mice at 5–6 months of age when an increase in Aβ oligomers, but not amyloid plaque formation, was found [8 , 52–54]. We then confirmed that the PKCɛ activators prevented MnSOD in Tg2576 mice and cultured human neurons.

Conclusions

In summary, our findings demonstrate that an imbalance among PKCɛ, Aβ, and ROS may contribute to neuronal dysfunction in AD. Oxidative stress and Aβ oligomers decrease PKCɛ expression. On the other hand, depression of PKCɛ reduces BDNF and MnSOD synthesis, resulting in an increase in oxidative stress. The PKCɛ-specific activators can prevent the loss of PKCɛ, BDNF, and MnSOD and the increase in oxidative stress.

Footnotes

ACKNOWLEDGMENTS

We are grateful to the families of the patients for providing brain tissue for research and the pathologists who referred case material to Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA. We are also indebted to all staff at Harvard Brain Tissue Resource Center for providing tissue for this work.

Authors’ disclosures available online ().

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