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. 2009 Feb 13;29(5):635–641. doi: 10.1007/s10571-009-9356-8

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Ornella Spadoni 1, Alessio Crestini 1, Paola Piscopo 1, Lorenzo Malvezzi-Campeggi 1, Irene Carunchio 2, Massimo Pieri 2, Cristina Zona 2, Annamaria Confaloni 1,✉
PMCID: PMC11506110  PMID: 19214738

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.

Keywords: APP, BACE1, Tg SOD1G93A mice, Amyotrophic lateral sclerosis, Alzheimer

Introduction

The neurodegenerative disorders, a heterogeneous group of chronic progressive diseases, are among the most puzzling and devastating illnesses in medicine. They affect over five million people of the aging population in the US and Europe alone. Yet, it is only during the past 20 years that the molecular events that precede and cause the disease process have begun to be understood. This has led to the recognition that many of the underlying pathogenic processes linked to neurodegenerative disorders are common molecular themes in many neurodegenerative diseases, including protein misfolding, oxidative stress, cytoskeletal abnormalities, disruption of calcium homeostasis, and inflammation, all of which increase during aging (Bossy-Wetzel et al. 2004; Forman et al. 2004; Selkoe 2004).

Some of these disorders such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), can occur sporadically and, in some instances, are caused by inheritance of gene mutations. Transgenic mice that express disease-causing genes recapitulate many features of these pathologies and the emerging insights are relevant to the comprehension of the underlying molecular mechanisms of these diseases.

Amyotrophic lateral sclerosis has long been considered a disease of both upper and lower motor neurons. However, there has been increased interest, recently, in the relationship between ALS and altered cognition (Strong et al. 1999; Lomen-Hoerth et al. 2003). Estimates suggest that from one-third to a half of all ALS patients have cognitive impairment (Ringholz et al. 2005) and many studies have pointed to an overlap between ALS associated with cognitive impairment and frontotemporal dementias (Abrahams et al. 2005; Hamilton and Bowser 2004).

The overlap between cognitive impairment and motor neuron disease is far more extensive than previously recognized. In fact, a recent study suggests that the number of patients, with clinically defined ALS, who also have neuropathological changes indicative of AD, is much larger than previously thought (Wheaton et al. 2007). In particular, an hypothetic link between AD and ALS was suggested by finding of both an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons (Sasaki and Iwata 1999) and Aβ peptides in ALS patients skin (Tamaoka et al. 2000). Moreover, a recent study reported an increase of APP in the hind limb muscles of SOD1G93A transgenic mice (Koistinen et al. 2006), a well-known model for ALS.

The APP is synthesized by neurons as a 100 kDa glycosylated transmembrane protein with a single membrane-spanning domain. The use of cellular models has clearly identified two catabolic pathways for APP: a non-amyloidogenic pathway, in which APP is cleaved by α-secretase within the sequence of the amyloid peptide. This cleavage precludes the formation of the full-length Aβ found in the amyloid core of senile plaques. A second catabolic pathway of APP leads to the production of Aβ from its precursor. In this amyloidogenic pathway, APP is cleaved by β-secretase at the N-terminus of Aβ. The C-terminal fragment of APP thus formed is in turn cleaved by γ-secretase to release the full-length amyloid peptide (Octave 2005). An altered proteolysis of APP, due for example to missense mutations or overexpression, induces an increased production of Aβ with the correlated accumulation and deposition as diffuse plaques (Selkoe 2001). APP processing could be altered by oxidative stress, facilitating the cellular accumulation of Aβ (Kolecki, et al. 2008; Misonou et al. 2000; Ohyagi et al. 2000) or, conversely, Aβ could trigger oxidative stress (Butterfield and Bush 2004; Hensley et al. 1994).

β-Site APP cleaving enzyme 1 (BACE1) is the primary transmembrane aspartyl protease responsible for β-secretase activity in the brain and carries out the first cleavage step leading to Aβ production. Moreover, BACE1 protein and its activity levels are elevated in AD brains compared to controls, further suggesting its involvement in AD pathogenesis (Roßner et al. 2006).

The aim of this study was to unravels eventual common molecular pathways underlying pathogenic processes by analyzing some genes involved in the amyloidogenic pathway of AD, in an animal model of ALS. In particular, we analyzed beyond APP and BACE1 also some genes involved in the γ-secretase multi-protein complex as Presenilin 1 (PSEN1), Presenilin 2 (PSEN2), nicastrin (NCSTN), and anterior pharynx defective 1 (APH-1a).

Methods

Transgenic Animal Model

B6SJL-TgN (SOD1G93A) 1 Gur mice expressing the G93A mutant human SOD1 (G93A) and B6SJL-TgN (SOD1) 2 Gur mice expressing wild-type human SOD1 (SOD1), constructed by Gurney ( 1994), were originally obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and then housed in our animal facilities. Screening for the presence of the human transgene was performed on tail tips from adult mice and on the body from each embryo after removal of the brain (Pieri et al. 2003). Control cells were obtained from non-transgenic mice. Procedures involving animals and their care were conducted in strict accordance with the Policy on Ethics approved by the Society for Neuroscience and with the European Communities Council Directive for Experimental Procedures. Every effort was made to minimize the number of animals used and their suffering.

Cortical Neuron Cultures

Cortical cultures were prepared as previously described (Carunchio et al. 2007). Briefly, cortical neurons were prepared from 15-day-old embryos. Cortical tissue was dissected and individually incubated for 10 min in 0.025% trypsin and then dissociated by gentle trituration. Cells were plated in 2 ml MEM (Minimum Essential Medium, Gibco, Invitrogen, Milan, Italy) supplemented with 5% foetal bovine serum (FBS, Gibco), 5% horse serum (HS, Gibco), 25 mM d-glucose (Sigma-Aldrich, Milan, Italy), 2 mM glutamine (Gibco) and 0.1 mg/ml gentamicine (Sigma-Aldrich), in petri dishes (Falcon, 4 cm2 growth area, 35-mm well diameter), previously coated with poly-l-lysine (Sigma). Cells were plated at a constant density of 7.5 × 105 cells/well and kept in a 5% CO2 humified incubator at 37°C. After 24 h, the medium was replaced with Neurobasal supplemented with B-27 (2%, Gibco), 0.5 mM glutamine and 0.1 mg/ml gentamicine (Sigma-Aldrich). Every 3 days Neurobasal medium supplemented only with B27 was replaced. Cortical cells were collected for following experiments at 8 DIV.

SOD1 Activity

Transgenic and non-transgenic control cultures were homogenized in RIPA buffer containing 50 mM Tris, 150 mM NaCl, 10% NP-40, 10% Na-deoxycholate plus a cocktail of protease inhibitors (Sigma-Aldrich, Milan, Italy). After total protein quantification the samples were electrophoresed on a 10% polyacrylamide gel, and SOD1 activities were determined as previously reported by the method of Beauchamp and Fridovich (1971). The gels were soaked in 2.5 mM NBT for 45 min followed by immersion for 15 min in phosphate buffer, containing 28 μM riboflavin. After lighting, gels become uniformly blue except at the positions containing SOD1. The SOD1 activity gel images were acquired with a FX-Imager densitometer and analyzed by the Quantity One software (Biorad Laboratories).

Quantitative RT-PCR

Total RNA was collected as previously described (Piscopo et al. 2008). Briefly, total RNA from tissue samples (10 mg) was extracted (RNeasy Lipid Tissue, Qiagen) and retrotranscribed in cDNA using SuperScript III first-strand cDNA synthesis kit (Invitrogen Inc., Carlsbad, CA, USA) with random primers, according to the manufacturer’s protocol. Each cDNA synthesis reaction was performed with specific primers and probes (Applied Biosystems) and using ABI PRISM 7000 sequencer. In particular, a 20 μl reaction mixture containing 2 μl of cDNA template, 10 μl TaqMan Universal PCR Master Mix and 1 μl primer probe mixture was amplified as follows: after incubation at 50°C for 2 min and denaturing at 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. The relative quantification was performed, using comparative Ct method and 18s gene, as endogenous reference.

Immunocitochemistry

Cortical cells (8 DIV) were washed and fixed in 0.4% phosphate-buffered formaldehyde for 10 min at room temperature. After fixation, cells were washed, treated with 4,6-diamidino-2-phenylindole (DAPI; Sigma; 0.1 mg/ml final concentration in PBS) for nuclear counterstaining, and permeabilized by dipping in Triton X-100 (0.2% in PBS) for 5 min at room temperature. Subsequently, cell preparations were incubated for 2 h at room temperature with primary monoclonal antibodies diluted in PBS. In the present study, the following primary antibodies were used: (1) a monoclonal mouse Anti-Alzheimer precursor protein A4, raised against an intracellular N-terminal epitope (residues 66–81; Chemicon International, diluted 1:50); (2) a polyclonal rabbit anti-BACE1 raised against an intracellular C-terminal epitope (residues 485–501; Calbiochem, diluted 1:100). Wells were carefully washed with buffer before incubating for 1 h at room temperature with Cy3-conjugated secondary antibodies that were diluted 1:100 in PBS. Donkey anti-mouse and anti-rabbit secondary antibodies (Chemicon International) were used to react against the primary monoclonal antibodies. Immunolabeled cells were washed a final time, and the coverslip was mounted with Fluoromount G (EMS, Rome, Italy). Nuclei were identified by DAPI staining. The cells were examined with a Zeiss fluorescence microscope (Axioplan), and digital images acquired by an AxioCam (Zeiss).

Cell Lysis and Protein Amount Quantification

Cell pellets were detergent-extracted on ice using RIPA buffer (50 mM Tris, 150 mM NaCl, 10% NP-40, 10% Na-deoxycholate) plus a cocktail of protease inhibitors (Sigma). The lysates were collected, sonicated, and quantified for total proteins by the Quick Start Bradford Protein Assay kit (Biorad Laboratories).

Western Blot Analysis

Twenty micrograms of total protein of each sample was subjected to SDS-PAGE using 10% Tris/glycine gels under reducing conditions and proteins were then transferred to PVDF membranes in SDS-free transfer buffer. Nonspecific proteins were blocked using 5% non-fat dry milk in TBST (Tris-buffered saline/Tween) for 1 h. Blots were then incubated with a primary antibody followed by a secondary antibody (horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies 1:10,000; Chemicon). Monoclonal antibody APP at a ratio of 1:1000 (catalog number MAB348; Chemicon) was used to recognize APP (110 and 120 kDa); polyclonal antibody BACE1 at a ratio of 1:1000 (catalog number ab2077; Abcam) was used to detect BACE1 (70 kDa). Western blot signals were acquired and analyzed by a FX-Imager densitometer and the Quantity One software (Biorad Laboratories). We used the levels of β-actin to normalize the levels of APP and BACE1 to control for loading differences in total protein amounts.

Statistical Analysis

Data are expressed as mean ± SEM. Comparisons among groups were made using Student’s t test, and by one-way ANOVA followed by Bonferroni’s multiple comparison test, with significance set at P < 0.05.

Results

SOD Activity

To check the presence of the human transgenic SOD1 (hSOD1) form in primary cortical cultures, we performed an activity gel assay. In non-transgenic derived cell culture a single weak band corresponding to the murine SOD1 homodimer was observed (Fig. 1, lane 1). Both in hSOD1 and ALS (Tg SOD1G93A) mice derived primary cultures, the wild-type and mutated SOD1 form were observed as an intense band (corresponding to the mSOD/hSOD1 heterodimer and human homodimer unresolved bands), which migrates, as attended, to lower molecular weights respect to the murine SOD1.

Fig. 1.

Fig. 1

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SOD1 activity gel. Total protein extracts (30 μg of protein) from murine control (lane 1), Tg hSOD1 (lane 2) and Tg SOD1G93A (lane 3) cell cultures were analyzed. The position of the homodimeric murine SOD1 detectable exclusively in murine control derived cells is shown by “m”

Gene Expression Analysis

For gene expression analysis, we carried out a quantitative RT-PCR on wild-type hSOD1 and ALS mice. We found that APP mRNA levels were increased 1.5-fold (P < 0.001) in cortical cells of SOD1G93A mice respect to hSOD1 mice. However, comparing the transgenic models with murine cells, the APP transcripts decreased in both hSOD1 and in ALS mice relative to non-transgenic mice cells (P < 0.001 and P < 0.05, respectively) (Fig. 2a).

Fig. 2.

Fig. 2

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Real-Time PCR analysis of APP and BACE1 mRNA in murine controls, Tg hSOD1 mice, and in Tg SOD1G93A (a, b). We found that either APP or BACE1 mRNA levels of G93A mice had an increase of 50% respect hSOD1 mice, in cortical cells. The histograms show, on the ordinate axis, the relative amount of specific cDNA normalized with 18s cDNA levels. Data, expressed as fold changes relative to each own control, are means ± SEM of at least eight different preparations. Data were analyzed by Student’s t test and ANOVA (***P < 0.001, **P < 0.01)

As regard to genes involved into the processing of APP, we analyzed BACE1 and some genes of γ-secretase multi-protein complex as PSEN1, PSEN2, nicastrin, and APH1a. The quantitative analysis showed a significant decrease of BACE1 transcripts only in hSOD1 cortical cells relative to the murine control, while an increase of BACE1 of about 1.5-fold (P < 0.01) in ALS mice respect to hSOD1 was observed similar to APP data (Fig. 2b). The expression of γ-secretase genes, PSEN1, PSEN2, nicastrin, and APH-1a, showed no statistical differences among murine control, Tg hSOD1, and ALS mice (data not shown).

Immunocytochemical Analysis

To determine whether the increase of APP and BACE1 mRNA levels observed in ALS mice corresponded to an elevation of the protein levels, we set out to assess a qualitative analysis by immunostaining on cortical cultures from non-transgenic, Tg hSOD1, and ALS mice.

Cellular immunoreactivity for APP protein, revealed by an antibody recognizing the N-terminal fragment, showed the cortical cell immunoreactivity of Tg hSOD1 mice and Tg SOD1G93A quite similar to the non-transgenic mice (Fig. 3a–c). The same result was observed for BACE1 protein, by an antibody specific for the C-terminal fragment (Fig. 3d–f).

Fig. 3.

Fig. 3

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Immunostaining of APP and BACE1 in cortical neurons of murine control, Tg hSOD1 and Tg SOD1G93A mice. a–c cell immunoreactivity of Tg hSOD1 mice and Tg SOD1G93A results quite similar to the non-transgenic mice. d–f Also BACE1 signal appeared comparable in all tested cell cultures. Scale bars in a–f = 20 μm

Biochemical Analysis

To quantify the protein levels in cortical cells, we performed a western blot analysis. We used an antibody recognizing 66–81 amino acids of the N-terminus on the APP and an antibody that recognizes 485-501 amino acids of BACE1.

Signal normalization, obtained from β-actin protein, allowed us to perform a semi-quantitative analysis that showed no statistical differences (P > 0.05) for both APP and BACE1 among Tg hSOD1, Tg SOD1G93A, and non transgenic cell cultures (Fig. 4). In addition, we investigated the protein levels of γ-secretase components, but the analysis did not show any change in their protein expression (data not shown).

Fig. 4.

Fig. 4

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Semi-quantitative protein analysis in cortical cells of murine control, Tg SOD1G93A and Tg hSOD1 mice. a, b Representative immunoblots of APP, BACE1, and β-Actin. Panels c and d present the quantitative analyses data of the immunoblots. The optical density was evaluated for each band and values for Tg SOD1G93A and Tg hSOD1 were normalized to murine control after correction for protein loading with β-actin. No statistical differences were found for both APP and BACE1 in all tested cortical cells. Each histogram shows the mean ± SEM of at least three culture preparations. Data were analysed by Student’s t test and ANOVA

Discussion

The molecular bases underlying the pathogenesis of neurodegenerative diseases are gradually disclosed. One problem that investigators face is distinguishing primary from secondary events. Rare, inherited mutations causing familial forms of these disorders have provided important insights into the molecular networks implicated in disease pathogenesis. Evidence is accumulating to suggest that such chronic neurodegenerative disorders as AD and ALS are caused by a combination of events that impair normal neuronal function. At this regard, Koistinen and colleagues showed higher APP mRNA levels in ALS mice muscle, in which a transcript increase of 1.7-fold was observed in ALS respect to wild-type mice (Koistinen et al. 2006). Our results seem to confirm the potential link between ALS-associated mutant SOD1 expression and the modulation of some gene involved in AD pathogenesis. In fact, we revealed an APP and BACE1 over-expression in transgenic Tg SOD1G93A mice, respect to Tg hSOD1 mice.

As regards to BACE1, it was observed that gene expression is tightly regulated at both transcriptional and translational levels (Roßner et al. 2006). The BACE1 promoter contains a number of putative binding sites for transcription factors that become activated in response to cellular stress. For instance, acute hypoxia modulates the expression and the enzymatic activity of BACE1 by up regulating its level, which triggers the Aβ generation (Zhang et al. 2007).

Given the apparent importance of metabolic dysfunction and amyloidosis in AD, it is noteworthy that BACE1 up-regulation has been observed under various experimental conditions likely involving energy disruption and/or mitochondrial stress (Xiong et al. 2007). In our animal model, the presence of mutation in Tg hSOD1 gene could be responsible of a metabolic alteration that induces transcriptional activation of BACE1. For instance, Velliquette and collegues showed that a reduced energy metabolism increased cerebral BACE1 levels in APP transgenic mice, compared with controls (Velliquette et al. 2005).

Our data did not show any difference in the protein expression between transgenic models, suggesting the existence of further post-translational mechanisms that regulate APP and BACE1 levels. For instance, recent evidences have shown that macroautophagy is an active pathway for turning over APP and generating Aβ peptide, which is then delivered principally to lysosomes and degraded by cathepsins (Yu et al. 2005). In addition, recent studies on Tg SOD1G93A mice found an increased autophagy at symptomatic stage compared with non-transgenic or human wild-type SOD1 transgenic animals (Morimoto et al. 2007).

On the other hand, it is well known that studies using transgenic rodent models of ALS disease found that there is an induction of proteasome activity within the CNS as the animals developed neurological disease. In fact, Puttaparthi and colleagues have found that there is induction of immuno-proteasomes in the spinal cords of mutant SOD1 transgenic mice as they develop neurological dysfunction, suggesting a potential compensatory role for immuno-proteasomes as a response to the increasing accumulation of SOD1 aggregates (Puttaparthi et al. 2007).

Our data indicate that in our model some post-translational mechanisms, probably triggered by the presence of mutated SOD1, are able to modify the levels of APP and BACE1. In fact, some important mechanisms exist like autophagy, or the ubiquitin-proteasome system, which are eligible to avoid the accumulation of proteins known to be toxic. However, further analysis has to be made in our model to clarify the process that plays a protective role in the maintenance of the brain homeostasis.

Acknowledgement

This work was supported by MIUR Italy (FIRB: contract RBAU01A7T4 to C.Z.)

Abbreviations

APP

Amyloid precursor protein

BACE1

β-Site APP cleaving enzyme

ALS

Amyotrophic lateral sclerosis

CNS

Central nervous system

PSEN1

Presenilin 1

PSEN2

Presenilin 2

NCSTN

Nicastrin

APH1

Anterior pharynx defective 1

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