Does apoptosis occur in amyotrophic lateral sclerosis? TUNEL experience from human Amyotrophic Lateral Sclerosis (ALS) tissues

The role that apoptosis plays in the pathogenesis of amyotrophic lateral sclerosis (ALS) is still unclear. From our autopsy samples, we have undertaken an effort to verify if apoptosis in ALS really occurs or if can at least be detected. The study was performed using TUNEL method for screening the apoptotic changes in the autopsy samples from 8 ALS cases compared with 16 control cases. No features of apoptosis (DNA cleavages) were noted in any of the investigated regions of the central nervous system in ALS cases as well as in controls. These preliminary results seem to support the reports, which deny the role of apoptosis in human ALS. The following investigations using additional methods will be performed for detection the apoptotic signals in ALS

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma

Analysis of TUNEL assay and protein biomarkers in cortical and basal ganglia regions of untreated 14-day-old organotypic brain slice cultures (BOCSC).

<p>Statistical analyses of control <b>CRE3</b><i>vs</i><b>N</b><b><i>casp8</i></b><b>^−/−</b> mouse specimens were performed using Student's t-test. The resulting average percentages of positive/immunopositive cells and standard error of mean (SEM) values are provided. <i>P</i> values<0.05 are reported as statistically significant. “NS” stands for nonsignificant (<i>p</i>≥0.05).</p

Caspase 8 deficiency affects functional and histopathological outcome following TBI and facilitates learning and memory retention in aged mice.

<p>(<b>A</b>) Sensorimotor function was measured as the number of foot slips while traversing the length of the beam three times in the beam walking trial. Training was followed by probe 1 (one week interval) before CCI procedure and repeated 7 and 21 days after the procedure (probe 2 and 3). <i>P</i> values result from Student's t-test (*<i>p</i> = 0.009; **<i>p</i> = 0.04). (<b>B</b>) Wire grip scores were quantified using a 5-point scale <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024341#pone.0024341-Hall1" target="_blank">[33]</a> to assess motor function. Tests were conducted 1 day before CCI (probe 1) and 7 (probe 2; *<i>p</i> = 0.03) and 21 days (probe 3) after brain trauma. (<b>C</b>) Morris water maze (MWM) performance was tested in the control (Ctrl; n = 12) and N<i>casp8</i>^−/− (n = 15) mice. Average latency to reach and climb the platform or enter the platform zone before and after CCI is shown (mean±SEM) (*<i>p</i> = 0.005; **<i>p</i> = 0.001; ***<i>p</i> = 0.009). Beam walking (<b>D</b>) and wire grip (<b>E</b>) tests were conducted in uninjured 18 month old <i>casp8^fl/fl</i> (Ctrl; n = 19) and N<i>casp8</i>^−/− (n = 20) mice (3 testing sessions, spaced one week apart). <i>P</i> values result from Student's t-test (*<i>p</i> = 0.01). (<b>F</b>) MWM task was performed in the same aged cohort to test memory retention up to 60 days after completion of the learning sessions. Average latency to reach and climb the platform is shown (mean±SEM) (*<i>p</i> = 0.04). (<b>G–I</b>) Histopathological outcomes were compared between the control (Ctrl) and N<i>casp8</i>^−/− mice 2 h, 6 h, 24 h, 48 h, and 3 weeks after the trauma (8–10 mice per time point in each experimental group). Using Aperio scanning system, lesion (<b>G</b>) and cavity (<b>H</b>) volumes were determined by summing each lesion or cavity area multiplied by the distance between each coronal slice (mean±SEM). Plots depict average lesion volume (<b>G</b>: *<i>p</i> = 0.03; **<i>p</i> = 0.002; ***<i>p</i> = 0.003), cavity volume (<b>H</b>: *<i>p</i> = 0.02; **<i>p</i> = 0.03), and neuropathological scores (<b>I</b>: *<i>p</i> = 0.002; **<i>p</i> = 0.02; ***<i>p</i> = 0.004). (<b>J</b>) Time course for density (n/mm²) of NeuN-immunopositive neurons in traumatic brain lesions is depicted for the control (Ctrl) and N<i>casp8</i>^−/− mice. (<b>K</b>) The NeuN immunostaining results are compared among the six experimental groups [control (Ctrl) and N<i>casp8</i>^−/− naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice] in impact lesions (CCI-treated) or corresponding regions (naïve or sham-operated) at 21 day after procedure. Density (n/mm²) of NeuN-immunopositive neurons in impact lesions or matching ipsilateral regions (IL) and corresponding contralateral (CL) regions of the control (Ctrl) (<b>L</b>) and N<i>casp8</i>^−/− (<b>M</b>) naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice is presented in a time-course manner (<b>L, M</b>) or at 21 day after procedure (<b>N</b>) (<b>J</b>: *<i>p</i> = 0.02; **<i>p</i> = 0.01; ***<i>p</i> = 0.0004; <b>L</b>: *<i>p</i> = 0.0005; **<i>p</i><.0001; ***<i>p</i> = 0.0008; <b>M</b>: *<i>p</i> = 0.0002; **<i>p</i> = 0.006; ***<i>p</i> = 0.002; ****<i>p</i> = 0.0007). (<b>O–P</b>) Percentages of MAP2 immunopositive neurons were compared in impact lesions or corresponding ipsilateral regions of the control (Ctrl) and N<i>casp8</i>^−/− naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice (8–10 mice/time point/group) and presented as time-course data (<b>O</b>) at 5 time points post CCI (mean±SEM) (<b>L</b>: *<i>p</i> = 0.002) and at 21 day after CCI (bar graph)(<b>P</b>). (<b>Q</b>) Phospho-tau immunoreactivity was assessed in ipsilateral semihemispheres of the control and N<i>casp8</i>^−/− mice 48 h and 3 weeks after CCI (n = 8–10 per group) (mean ± SEM *<i>p</i> = 0.001).</p

Generation of mouse line (Ncasp8^−/−) with neuron-specific ablation of caspase8.

<p>(<b>A</b>) PCR was performed using 1 µg tail genomic DNA for transgenic CRE3 and caspase 8<i>^flox</i> animals. PCR products were generated using amplification primers specific to either the <i>cre</i> or <i>casp8</i> genes. PCR products were analyzed by agarose gel-electrophoresis and ethidium bromide staining. Arrows indicate the <i>cre</i> PCR product of 750 bp and <i>casp8</i> PCR products of 800 bp for floxed genes and 650 bp for wild-type <i>casp8</i>. (<b>B</b>) Brain-specific recombination of floxed <i>casp8</i> genes in CRE3 mice. Genomic DNA was isolated from various tissues of CRE3 mice containing either wild-type <i>casp8</i> genes or homozygous floxed <i>casp8</i> genes (N<i>casp8</i>^−/−). PCR was performed using primers specific for <i>casp8</i> (top) or the <i>cre</i> transgene (bottom). Brain tissues included cortex (lanes #1), thalamus/basal ganglia (lanes #2), and brain stem (lanes #3). Arrows indicate the expected PCR products for unrecombined (fl/fl) at 650 bp, recombined <i>caspase8</i> gene knockout at ∼200 bp (−/−), and wild-type unfloxed (+/+) c<i>asp8</i> genes at ∼800 bp. (<b>C</b>) SDS-PAGE immunoblot analysis of caspase 8 protein levels in mouse brain tissues. Brain samples from cortex (C), cerebellum (CB) or basal ganglia (BG) of <i>casp8^fl/fl</i>-CRE3 mice were compared with CRE3 and <i>casp8^fl/fl</i> animals. Lysates were normalized for total protein content (30 µg/lane) and analyzed by SDS-PAGE/immunoblotting using antibodies specific for mouse caspase 8 (top), or β-actin (bottom), using a multiple antigen detection method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024341#pone.0024341-Krajewski1" target="_blank">[39]</a>. (<b>D–K</b>) Examples are provided of immunostaining for mouse caspase 8 in the brains of CRE3 (<b>D–G</b>) and N<i>casp8</i>^−/− mice (<b>H–K</b>). Antibody detection was accomplished using diaminobenzidine (DAB) chromogen (brown); nuclei were counterstained with hematoxylin (blue). Magnification scale bar = ∼100 µm.</p

Analysis of TUNEL assay and protein biomarkers in cortical and basal ganglia regions of untreated and TRAIL-treated coronal brain slices (3-day-old BOCSC).

<p>Statistical analyses of control (<b>CRE3</b>, <b><i>casp8^fl/fl</i></b>) <i>vs</i><b>N</b><b><i>casp8</i></b><b>^−/−</b> mouse specimens were performed using Student's t-test. The resulting average percentages of positive/immunopositive cells and standard error of mean (SEM) values are provided. <i>P</i> values<0.05 are reported as statistically significant. “NS” stands for nonsignificant (<i>p</i>≥0.05).</p

Caspase 8 gene ablation provides neuroprotection in vitro in primary neuronal cultures and brain organotypic cultures.

<p>(<b>A–B</b>) 3-day-old PNC derived from embryos (E16.5) of CRE3 (<b>A</b>) and N<i>casp8</i>^−/− homozygous mice (<b>B</b>) were treated with recombinant murine TNFα and cycloheximide (CHX), then fixed and stained with Hoechst dye and immunostained with an antibody to NeuN followed by application of Alexa Fluor 594 conjugated anti-mouse antibody. (<b>C1–J4</b>). Untreated (UNT <b>C1–F4</b>), STS-treated, (<b>G1–H4</b>) and recombinant murine TNFα and CHX-treated (<b>I1–J4</b>) 14-day-old neuronal cultures derived from CRE3 and N<i>casp8</i>^−/− mice were preincubated with pSIVA reagent (green) for 15 min prior to Z-fix fixation, then immunostained for MAP2 (red) and stained with DAPI (blue). To assess neuronal phenotype, the transmission light, single (<b>C1, D1, E4, G1, H1, H3, I1, I2, I4, J1</b>), double (<b>C2, D2, E1, E2, F1, F3, G4, H2, H4, I3, J2</b>), and triple (<b>E3, F2, F4, G2, G3, J3, J4</b>) channel images are provided. Colocalization of neuronal marker MAP2 (red) with pSIVA signal (green) resulted in yellow color denoting degenerating neurons whose number was counted and plotted as ratio to the total number of neurons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024341#pone-0024341-g002" target="_blank"><b>Figure 2B</b></a>). Single and multichannel panels confirm specificity of the double labeling. PSIVA was visualized from the early (membranous staining) to later stages (staining in the cell body and nucleus, the latter in cells with loss of plasma cell integrity) of neuronal degeneration.</p

Casp8 ablation provides neuroprotection in organotypic brain slice cultures.

<p>The coronal brain slices derived from the CRE3 control line and N<i>casp8</i>^−/− homozygous mice were maintained in <i>in vitro</i> conditions for 3 (<b>A1–M</b>) and 14 days (<b>N–X</b>). The presented results were obtained from untreated (<b>A1–D3, I, K, N–X</b>) and TRAIL-treated (<b>E–H, J, L</b>) cultures. Immunohistochemical stainings are depicted in cortical and subcortical regions of CRE3 (<b>A1, A2, E, F, Q, S, W</b>) and N<i>casp8</i>^−/− (<b>B1–D3, G–P, R, T, U, X</b>) brain slices. Examples of single marker (<b>M–W</b>) and double (<b>A1–L, X</b>) immunostainings are presented for S100 (<b>M, N</b>), NeuN (<b>O–T</b>), and β3-tubulin (<b>U, W</b>) as well as for double labeled MAP2 (DAB-brown) and cleaved caspase 3 (SG-black; <b>A1–B2</b>) or GFAP (DAB; <b>D1–D3</b>), TUNEL (DAB; <b>E–H</b>) with MAP2/NeuN antibody cocktail (SG) and S100 (DAB; <b>I–J</b>); IbA1 (DAB; <b>C1–C2</b>) or GFAP (DAB; <b>D1–D3</b>) with NeuN (SG) immunostaining. Scale bar indicates ∼1000 µm on macro images and ∼100 µm on high magnification images of the slices. Abbreviations: <b>DG</b> = Dental Gyrus; <b>CA2; CA4</b> = sectors of hippocampus; <b>TH</b> = Thalamus; <b>AMG</b> = Amygdala; <b>PC</b> = Piriformis Cortex; <b>WM</b> = White Matter.</p