Alterations in G₁ to S Phase Cell-Cycle Regulators during Amyotrophic Lateral Sclerosis

Source of Tissue Samples

The lumbar spinal cord and pre-/postcentral gyrus (motor/sensory cortex) region from 18 cases of clinically diagnosed sporadic ALS, and 9 non-ALS age-matched controls, were used to examine protein expression and distribution. All tissues were obtained from the University of Pittsburgh ALS tissue bank. The average age at death was 60.05 ± 12.22 years for ALS cases (range, 40 to 76 years) and was not significantly different from control cases (65.33 ± 15.37 years; range, 51 to 95 years; P = 0.34). The average postmortem interval times for ALS and control cases were 6.02 ± 3.35 hours (range, 2.5 to 14 hours) and 9.88 ± 5.68 hours (range, 5 to 20 hours), respectively. The difference in the postmortem interval time was statistically significant (P = 0.039). Some of the controls and ALS cases (indicated with an asterisk in Table 1 ▶ ) were neuropathologically diagnosed as cases with possible Alzheimer disease (Braak II or III/VI). Approval for use of human tissues was obtained from the University of Pittsburgh Interval Review Board. For immunohistochemistry, all tissues were fixed in 10% buffered formalin for 1 week and 8-μm paraffin-embedded sections were examined as follows.

Antibodies

Monoclonal antibodies were used to detect hypophosphorylated pRb (Pharmingen, La Jolla, CA), E2F-1 (clones KH95 and C-20; Santa Cruz Biotechnology, Santa Cruz, CA), and cyclin D1 (Santa Cruz Biotechnology). Polyclonal antibodies were used to detect hyperphosphorylated pRb (ser-795, NEN Biolabs, Boston, MA), active form of CDK4 (Santa Cruz Biotechnology), actin (Chemicon, Temecula, CA), and glial fibrillary acidic protein (GFAP) (DAKO, Carpinteria, CA). Western gels were probed at dilutions of 1:750 (pRb, ppRb, E2F-1), 1:200 (cyclin D1, CDK4), or 1:15000 (actin). For immunohistochemistry, these antibodies were used at dilutions of 1:150 (pRb, ppRb, E2F-1) and 1:200 (CDK4, cyclin D1, GFAP).

Immunohistochemistry

Paraffin-embedded tissue sections were microwave treated for 4 minutes at full power followed by 7 minutes at 40% power in 1× citra antigen retrieval (Biogenex, San Ramon, CA), cooled to room temperature for 90 minutes, and then incubated in 3% H₂O₂ and 0.25% Triton X-100 in phosphate-buffered saline (PBS) for 30 minutes. The sections were then blocked in 5% milk/PBS for 1 hour. Primary antibodies were added in 1× PBS and incubated overnight at 4°C. After four 15-minute washes in PBS, sections were incubated in biotinylated goat anti-rabbit or anti-mouse IgG secondary antibody (1:1000 dilution; Southern Biotechnology Labs., Birmingham, AL) for 1.5 hours. The signal was further amplified using biotinylated tyramide according to the manufacturer’s protocol (TSA Biotin System, NEN Biolabs). On washing, the sections were incubated in streptavidin-horseradish peroxidase (1:1000 dilution) for 1 hour and the reaction product visualized using 3-amino-9-ethylcarbazole (AEC) (for 3 to 5 minutes) (Biogenex, San Ramon, CA). This reaction results in a red end product and all sections were then counterstained with hematoxylin.

Protein Extraction

Spinal cord and motor cortical frozen tissue samples from controls cases and ALS cases (Table 1) ▶ were used for immunoblotting and DNA-binding assays. For total cell lysates, tissue samples were homogenized using polytron homogenizer (PGC Scientific, Gaithersburg, MD) set at 15,000 rpm for 45 seconds. It was performed in lysis buffer containing 25 mmol/L HEPES (pH 7.4), 50 mmol/L NaCl, protease inhibitor cocktail II (Sigma Chemical Co., St. Louis, MO), and 1% Triton X-100. The homogenized product was spun at 14,000 rpm in a cold microfuge and the supernatant saved as the total cell lysate. Nuclear and postnuclear extracts were extracted as described previously. 54 Briefly, protein extracts were prepared by detergent lysis on ice (0.1% Nonidet P-40, 10 mmol/L Tris, pH 8.0, 10 mmol/L MgCl₂, 15 mmol/L NaCl, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 μg/ml pepstatin A, and 1 μg/ml leupeptin). The nuclei were collected by low-speed centrifugation at 800 × g for 5 minutes. The supernatant was saved as the postnuclear supernatant and the pellet containing the nuclei was further extracted with high-salt buffer (0.42 mol/L NaCl, 20 mmol/L HEPES, pH 7.9, 20% glycerol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 μg/ml pepstatin A, and 1 μg/ml leupeptin) on ice for 10 minutes. Residual insoluble material was removed by centrifugation at 14,000 × g for 5 minutes. The resulting supernatant fraction was collected and termed the nuclear extract. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad, Richmond, CA).

Immunoblotting

Total cell lysates, nuclear extracts and postnuclear supernatants were fractionated by electrophoresis on an 8, 10, or 12% sodium dodecyl sulfate-polyacrylamide gels. The proteins were transferred to polyvinylidene difluoride nylon membranes (NEN Biolabs) and blocked in 5% nonfat milk/1× PBS or 0.5% bovine serum albumin/0.15% glycine in 1× PBS overnight at 4°C. The blots were probed individually with the antibodies and concentrations as aforementioned, overnight at 4°C in 0.5% milk/PBS. The blots were washed three times in PBS/0.1% Tween-20 for 15 minutes. Isotype-specific horseradish peroxidase-conjugated secondary antibodies (Chemicon) specific for each primary antibody were added for 2 hours at room temperature. The secondary antibodies were washed extensively in PBS/0.1% Tween-20 (three times for 20 minutes). The final reaction products were visualized using enhanced chemiluminescence (Pierce, Rockford, IL) and the band intensities were within the linear range of detection. The density of bands was measured using the NIH Image software version 1.58 (National Institutes of Health, Atlanta, GA). Actin was used to normalize protein levels within each sample.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts (20 to 25 μg) were preincubated with salmon sperm DNA (120 ng) as a nonspecific competitor in 12 to 15 μl of EMSA buffer (20% glycerol, 150 mmol/L KCl) before addition of ³²P-labeled oligo to reduce nonspecific DNA-protein interactions. Wild-type and mutant oligonucleotides were synthesized and gel purified (Oligos ETC.). The sequences were: wild type E2F-1 5′-ATTTAAGTTTCGCGCCCTTTCTCAA-3′; mutant E2F-1 5′-ATTTAAGTTTCGATCCCTTTCTCAA-3′. For competition reactions, unlabeled E2F-1 competitor (3, 30, and 100 ng) or unlabeled unrelated competitor (5′-GATCATTCAGGTCATGACCTGA-3′; 100 and 300 ng) oligos were preincubated with the protein for 5 minutes on ice before addition of labeled probe. For positive controls, nuclear extracts were prepared from NIH3T3 cells. The reaction mixture was loaded onto a 6% nondenaturing polyacrylamide gel and electrophoresed at 100 V in 1× Tris-borate-ethylenediaminetetraacetic acid. The polyacrylamide gel was removed from the apparatus, dried, and exposed to autoradiography film. The density of the complexes was measured using the NIH Image software version 1.58 (National Institutes of Health).

Statistical Analysis

Comparisons between any two groups of data were done using the single-factorial analysis of variance. A P value of ≤0.05 was considered statistically significant. Numerical data were expressed as means ± the SD with n = number of experiments or cases.

Increased Immunoreactivity of G₁ to S Phase Cell-Cycle Proteins in ALS Spinal Cord Motor Neurons

Sections of lumbar spinal cord and pre-/postcentral gyri were immunostained using commercially available antibodies (see Materials and Methods). For light microscopy, the antigen-antibody complex was visualized with AEC and counterstained with hematoxylin (see Materials and Methods). Cyclin D1, a D-type cyclin functional in the hyperphosphorylation of pRb and G₁ to S phase transition of the cell cycle, exhibited increased cytoplasmic distribution in the spinal motor neurons of ALS patients (Figure 1, A and B) ▶ . Sections were also stained for the active form of CDK4. Although active CDK4 immunoreactivity was primarily negligible in the ventral horn of control cases (Figure 1D) ▶ , increased and punctate CDK4 immunoreactivity was apparent in the cytoplasm and occasionally in the nucleus of ventral horn motor neurons of ALS patients (Figure 1E) ▶ . CDK4 and cyclin D1 immunoreactivities were negligible in the dorsal horn sensory neurons and surrounding glia in the ALS patients (Figure 1, C and F) ▶ .

One downstream target of active cyclin D/CDK4 complex is the retinoblastoma protein (pRb), with cyclin D-specific phosphorylation of serine at position 795. Therefore, we examined the phosphorylation state of pRb in ALS using a phospho-specific anti-pRb antibody to serine-795 that has been shown to detect increased pRb phosphorylation in Alzheimer’s disease. 24,25,55 We detected abundant nuclear and punctate cytoplasmic staining of Ser-795 phosphorylated pRb (ppRb) in the motor neurons of ALS spinal cord but not in the age-matched control tissues (Figure 1, G and H) ▶ . When morphologically distinct motor neurons from multiple lumbar spinal cord sections of 10 ALS cases and 5 control cases were counted (total of 450 motor neurons per condition) the percentage of ppRb-positive lower motoneurons in ALS (85.5%) was significantly greater than controls (2.8%). The retinoblastoma protein regulates the transactivation activity of the E2F family of transcription factors. We noted punctate E2F-1 immunoreactivity specific to the cytoplasm of ALS spinal motoneurons but not in control motor neurons (Figure 1, J and K) ▶ . The percentage of E2F-1-positive motor neurons in ALS cases was significantly greater (80.8%) than in control cases (4.5%). There were negligible levels of nuclear E2F-1 irrespective of the disease state. These results were reproduced using a second E2F-1 antibody that recognizes a distinct epitope not encompassing the pRb-binding domain (data not shown). In contrast to the ALS ventral horn motor neurons, the dorsal horn sensory neurons were ppRb- and E2F-1-negative (Figure 1, I and L) ▶ .

Cellular Distribution of Cell-Cycle Proteins in Motor Cortex

We next examined the expression patterns of cell-cycle proteins in the motor and sensory cortex (pre- and postcentral gyrus) of control and ALS patients. All ALS cases except one (ALS 10, Table 1 ▶ ) exhibited substantial loss of Betz cells in the motor cortex and the presence of gliosis was used to identify the motor cortex when all Betz cells were absent. Although cyclin D1 expression was primarily cytoplasmic in any remaining large pyramidal neurons (Betz cells) of the motor cortex of ALS patients (Figure 2, A and B) ▶ , CDK4 immunoreactivity was both nuclear and cytoplasmic in these neurons (Figure 2, D and E) ▶ . There was variation in the level of CDK4 immunoreactivity within the motor cortex of ALS patients (Table 2) ▶ , although this did not correlate to any reported clinical information for the patients. The cortical neurons in the postcentral gyrus of these patients exhibited little immunoreactivity for cyclin D1 or active CDK4 (Figure 2, C and F) ▶ . Hyperphosphorylated pRb (ppRb) was detected predominantly in the nucleus of many cortical neurons in the precentral gyrus of ALS patients but not in control cases (Figure 2, G and H) ▶ . The reactivity of ppRb in the cortical sensory neurons was low or negligible (Figure 2I) ▶ . E2F-1 immunoreactivity was observed in the cytoplasm of neurons in the ALS motor cortex with no protein detected within control tissues (Figure 2, J and K) ▶ . E2F-1 immunoreactivity was absent in the postcentral gyrus (Figure 2L) ▶ . It is interesting to note that one ALS case lacking loss of upper motor neurons (ALS case 13, Table 1 ▶ ) exhibited little immunoreactivity for these G₁ to S phase cell-cycle regulators (Table 2) ▶ .

Distribution of Cell-Cycle Proteins in White Matter of ALS Spinal Cord and Motor Cortex

Subsequently we investigated the presence of these proteins in cells with a glial morphology. We observed moderate immunoreactivity for cyclin D1 and CDK4 in cells with morphological characteristics of astrocytes in the white matter of ALS lumbar spinal cord (data not shown). Hyperphosphorylated pRb (ppRb) immunoreactivity appeared in the nucleus of cells in the spinal cord white matter of ALS patients but not in control cases (Figure 3A) ▶ . Furthermore, glial cells with a morphology of activated astrocytes in the white matter of ALS lumbar spinal cord tissues was E2F-1 immunoreactive (Figure 3B) ▶ . Within the motor cortex, ppRb immunoreactivity was present in the cytoplasm of cells in the white matter of ALS patients but not in control cases (Figure 3C) ▶ . Using consecutive sections, we demonstrated the presence of GFAP-positive astrocytes in the white matter that were ppRb- and E2F-1-positive (asterisk in Figure 3; C to E ▶ ). Additional GFAP-labeled astrocytes were E2F-1-positive but ppRb-negative (arrowheads in Figure 3, D and E ▶ ).

Analysis of Protein Levels by Immunoblot

To determine whether altered immunostaining correlates to increased protein levels in control and ALS spinal cords, we performed immunoblot analysis using total cell lysates from lumbar spinal cord tissues for 18 ALS and 9 control cases. We show representative data from 10 ALS and 6 control cases (Figure 4) ▶ . The level of ppRb was significantly increased in the spinal cord of ALS patients, although the predominant pRb family member phosphorylated is p130. (Figure 4, A and B) ▶ . There was also an increase in phosphorylated Rb in nuclear extracts whereas it was undetected in postnuclear supernatants (data not shown). We failed to detect altered levels of total pRb in nuclear extracts from control or ALS spinal cord, suggesting that phospho-pRb results from phosphorylation of pre-existing pRb (data not shown). Cyclin D1, CDK4, and E2F-1 expression exhibited increased levels in the total cell lysates (Figure 4, A and B) ▶ . Increased levels of cyclin D1, ppRb, and E2F-1 in ALS extracts were statistically significant (P ≤ 0.05). E2F-1 was increased specifically in soluble postnuclear supernatants but undetectable in the nuclear fraction from the spinal cord of ALS patients (data not shown). All protein levels were normalized to levels of actin and quantitated to demonstrate statistical significance of these findings (Figure 4) ▶ .

We further examined protein expression in the motor cortex by immunoblot analysis. There was a decrease in levels of cyclin D1, CDK4, and E2F-1 in total cellular extracts (Figure 4C) ▶ . This decrease was statistically significant for cyclin D1 (P ≤ 0.05). Hyperphosphorylated pRb (ppRb) was undetectable in the total cell extracts. However, we did notice a significant accumulation of ppRb in nuclear extracts of ALS patients but not in the postnuclear supernatants (data not shown). Also, increased levels of cyclin D1 and E2F-1 were present in postnuclear supernatants (data not shown). There was a significant twofold increase in the levels of active CDK4 in the nuclear fraction by immunoblot correlating with nuclear CDK4 immunoreactivity as noticed in many of the ALS cases (data not shown).

DNA-Binding Activity of E2F-1

We next examined the DNA-binding activity of E2F-1 in control and ALS patients as a measure of E2F-1 functional activity. To evaluate DNA-binding activity, gel mobility shift assays (EMSA) were performed using nuclear extracts from spinal cord and motor cortex, with extracts from proliferating NIH3T3 cells as positive control. [γ-³²P]-labeled double-stranded oligonucleotides containing the consensus E2F-1-binding site were incubated with 25 μg of 3T3 cells, spinal cord, or motor cortex nuclear extracts. Retardation in protein mobility because of DNA-protein complexes was visualized by autoradiography. We first demonstrated that the very sensitive EMSA could detect nuclear E2F-1 that was below the limits of detection by immunoblot. The spinal and cortical nuclear extracts contained E2F-1 protein that bound the recognized E2F-1-binding element with specificity as demonstrated by competition with excess wild-type cold oligos but not with excess mutant or unrelated oligos (Figure 5A) ▶ . In Figure 5A ▶ , the slowest migrating complex is competed away by excess unlabeled oligos but not by mutant or unrelated oligos and were interpreted to contain E2F-1. We next used nuclear extracts from proliferative 3T3 cell nuclear extracts as positive control (lane 2 in left panel of Figure 5B ▶ ) and competed the E2F-1 complex with increasing levels of excess cold oligo (Figure 5B ▶ , lanes 3 and 4). We next examined nuclear extracts of control and ALS spinal cord and motor cortex for E2F-1 DNA-binding activity (Figure 5B) ▶ . Densitometric measurement of the protein:DNA complex indicates no significant change in the intensity of DNA binding in spinal cord (P = 0.189) or motor cortex (P = 0.724) of ALS patients when compared to age-matched controls (Figure 5, B and C) ▶ .