Histone H4 deacetylation plays a critical role in early gene silencing during neuronal apoptosis

<p>Abstract</p> <p>Background</p> <p>Silencing of normal gene expression occurs early in the apoptosis of neurons, well before the cell is committed to the death pathway, and has been extensively characterized in injured retinal ganglion cells. The causative mechanism of this widespread change in gene expression is unknown. We investigated whether an epigenetic change in active chromatin, specifically histone H4 deacetylation, was an underlying mechanism of gene silencing in apoptotic retinal ganglion cells (RGCs) following an acute injury to the optic nerve.</p> <p>Results</p> <p>Histone deacetylase 3 (HDAC3) translocates to the nuclei of dying cells shortly after lesion of the optic nerve and is associated with an increase in nuclear HDAC activity and widespread histone deacetylation. H4 in promoters of representative genes was rapidly and indiscriminately deacetylated, regardless of the gene examined. As apoptosis progressed, H4 of silenced genes remained deacetylated, while H4 of newly activated genes regained, or even increased, its acetylated state. Inhibition of retinal HDAC activity with trichostatin A (TSA) was able to both preserve the expression of a representative RGC-specific gene and attenuate cell loss in response to optic nerve damage.</p> <p>Conclusions</p> <p>These data indicate that histone deacetylation plays a central role in transcriptional dysregulation in dying RGCs. The data also suggests that HDAC3, in particular, may feature heavily in apoptotic gene silencing.</p

Dominant inheritance of retinal ganglion cell resistance to optic nerve crush in mice

BACKGROUND: Several neurodegenerative diseases are influenced by complex genetics that affect an individual's susceptibility, disease severity, and rate of progression. One such disease is glaucoma, a chronic neurodegenerative condition of the eye that targets and stimulates apoptosis of CNS neurons called retinal ganglion cells. Since ganglion cell death is intrinsic, it is reasonable that the genes that control this process may contribute to the complex genetics that affect ganglion cell susceptibility to disease. To determine if genetic background influences susceptibility to optic nerve damage, leading to ganglion cell death, we performed optic nerve crush on 15 different inbred lines of mice and measured ganglion cell loss. Resistant and susceptible strains were used in a reciprocal breeding strategy to examine the inheritance pattern of the resistance phenotype. Because earlier studies had implicated Bax as a susceptibility allele for ganglion cell death in the chronic neurodegenerative disease glaucoma, we conducted allelic segregation analysis and mRNA quantification to assess this gene as a candidate for the cell death phenotype. RESULTS: Inbred lines showed varying levels of susceptibility to optic nerve crush. DBA/2J mice were most resistant and BALB/cByJ mice were most susceptible. F1 mice from these lines inherited the DBA/2J phenotype, while N2 backcross mice exhibited the BALB/cByJ phenotype. F2 mice exhibited an intermediate phenotype. A Wright Formula calculation suggested as few as 2 dominant loci were linked to the resistance phenotype, which was corroborated by a Punnett Square analysis of the distribution of the mean phenotype in each cross. The levels of latent Bax mRNA were the same in both lines, and Bax alleles did not segregate with phenotype in N2 and F2 mice. CONCLUSION: Inbred mice show different levels of resistance to optic nerve crush. The resistance phenotype is heritable in a dominant fashion involving relatively few loci. Bax was excluded as a candidate gene for this phenotype

Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric

BACKGROUND: Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the βGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice. RESULTS: Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of βGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal. CONCLUSION: A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma

Distribution of guanylate cyclase within photoreceptor outer segments

Guanylate cyclases play an essential role in the recovery of vertebrate photoreceptor cells after light activation. Here, we have investigated how one such guanylate cyclase, RetGC-1, is distributed within light- and dark-adapted rod photoreceptor cells. Guanylate cyclase activity partitioned with the photoreceptor outer segment (OS) cytoskeleton in a light-sensitive manner. RetGC-1 was found to bind actin filaments in actin blot overlays, suggesting a mechanism for its association with the OS cytoskeleton. In retinal sections, this enzyme was immunodetected only in the OSs, where it appeared to be distributed throughout the disk membranes

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of ApcMin/+ ROSA11 Gene Trap Mice

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in ApcMin/+ mice. The insertion site of the β-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis

<div><p>The pro-apoptotic <i>BCL2</i> gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous <i>BAK</i> or <i>BAX</i> genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.</p></div

Cytochrome c, SMAC and AIF are released at different times during BAX recruitment.

<p>Time-lapse videos were collected from D407 cells expressing mCherry-BAX, mito-BFP and one of three GFP-tagged apoptotic molecules that are released from mitochondria after being challenged with 1 μM staurosporine. Representative graphs indicate the release of apoptotic molecules (A) cytochrome c-GFP, (E) SMAC-GFP, and (I) AIF-GFP, and the recruitment of mCherry-BAX at one mitochondrial focus. (B-D) Representative images show (B) cytochrome c-GFP localized at the mitochondria (pre-initiation), and (C) diffuse cytochrome c-GFP four minutes after BAX recruitment initiation and (D) 30 minutes after BAX recruitment initiation. (F-H) Images from a time-lapse video of SMAC-GFP and mCherry-BAX (F) pre-initiaton, (G) four minutes and (H) 30 minutes after BAX recruitment initiation, show the same localization patterns as cytochrome c-GFP. (J-L) Images from a time-lapse video of AIF-GFP and mCherry-BAX show (J) AIF-GFP localized at the mitochondria (pre-initiation), (K) AIF-GFP partially retained in the mitochondria four minutes after BAX recruitment initiation and (L) diffuse AIF-GFP 30 mins after BAX recruitment initiation. For all three datasets, the time of molecule release was compared to the time of BAX recruitment initiation. (M) A box and whisker plot shows that cytochrome c-GFP and SMAC-GFP are released -2.5 ± 3 mins and -1.3 ± 1.6 mins before mathematical assignment of the initiation of BAX recruitment, respectively, while AIF is released at 10.3 ± 11 mins after recruitment initiation. Each molecule’s relative release time was significantly different from the others by one-way ANOVA (p** < 0.01; n = 53, 52, 46 mitochondria foci for cytochrome c, SMAC, and AIF, respectively). Cytochrome c and SMAC release times were also significantly different (p* < 0.05). (N) A bar graph showing the rate of apoptotic molecule release for each molecule. Cytochrome c was released at a rate of -0.78 ± 0.46 RFU/min, SMAC at -1.36 ± 0.72 RFU/min and AIF at -0.04 ± 0.07 RFU/min (n = 54, 52, 51 mitochondria foci respectively). All were significantly different (p < 0.001), while SMAC exhibited a significantly faster release rate when compared to the other molecules (*p< 0.001).</p

BAX fusion proteins parallel spatial and temporal localization patterns of endogenous BAX.

<p>D407 cells were nucleofected with mito-BFP alone or with GFP-BAX or mCherry-BAX. Cells were treated 24 hours later with 1μM staurosporine (STS) for 1, 2, or 3 hours or DMSO control. (A-C) Endogenous activated BAX was monitored using the BAX 6A7 antibody. In DMSO only treated cells (shown after 3 hour incubation) the 6A7 antibody does not cross-react with BAX. Punctate labeling, co-localized to mitochondria, is detected within 1 hour of STS addition. (D-F) Exogenous GFP-BAX and (G-I) mCherry-BAX demonstrate similar localization changes. Size bar = 5 μm. Three independent experiments were quantified. (J) The percentage of punctate cells (number of punctate cells over the total number of nucleofected cells for cells expressing BAX fusion proteins) increased similarly for each time point among all BAX conditions (p > 0.05).</p