Endoplasmic reticulum pathology and stress response in neurons precede programmed necrosis after neonatal hypoxia-ischemia

Abstract

The endoplasmic reticulum (ER) is tasked, among many other functions, with preventing excitotoxicity from killing neurons following neonatal hypoxia-ischemia (HI). With the search for delayed therapies to treat neonatal HI, the study of delayed ER responses becomes relevant. We hypothesized that ER stress is a prominent feature of delayed neuronal death via programmed necrosis after neonatal HI. Since necrostatin-1 (Nec-1), an inhibitor of programmed necrosis, provides delayed neuroprotection against neonatal HI in male mice, Nec-1 is an ideal tool to study delayed ER responses. C57B6 male mice were exposed to right carotid ligation followed by exposure to FiO2=0.08 for 45 min at p7. Mice were treated with vehicle or Nec-1 (0.1 μl of 8 μmol) intracerebroventricularly with age-matched littermates as controls. Biochemistry assays at 3 and 24h and electron microscopy (EM) and immunohistochemistry at 96 h after HI were performed. EM showed ER dilation and mitochondrial swelling as apparent early changes in neurons. With advanced neurodegeneration, large cytoplasmic fragments containing dilated ER "shed" into the surrounding neuropil and calreticulin immunoreactivity was lost concurrent with nuclear features suggestive of programmed necrosis. Nec-1 attenuated biochemical markers of ER stress after neonatal HI, including PERK and eIF2α phosphorylation, and unconventional XBP-1 splicing, consistent with the mitigation of later ER pathology. ER pathology may be an indicator of severity of neuronal injury and potential for recovery characterized by cytoplasmic shedding, distinct from apoptotic blebbing, that we term neuronal macrozeiosis. Therapies to attenuate ER stress applied at delayed stages may rescue stressed neurons after neonatal HI.

Keywords: Cell death; Cytoplasmic shedding; Endoplasmic reticulum stress; Macrozeiosis; Neonatal hypoxia-ischemia; Programmed necrosis; Unfolded protein response.

Figure 1

Representative endoplasmic reticulum (ER) pathology following neonatal HI (n=4 pups). (A) Neuron with normal appearing ER (er), mitochondria (m) and nuclear structure (n) were present in neuropil with evidence of very mild dendritic swelling (d). (B) Elongation and marginalization of ER (er) was found in a neuron with one darkened mitochondria and very minimal chromatin condensation appearing as a slight peppering of the nucleus (boxed area). Surrounding neuropil (np) was significantly disrupted with a shed cytoplasmic fragment (asterisks) containing much more dilated ER (er) at the top edge of the photomicrograph. (C) The earliest signs of swelling were appreciated in elongated and marginalized ER (er) in a neuron with progressive random condensation of nuclear chromatin (boxed area). Surrounding neuropil (np) was significantly disrupted. (D) Dilation of ER (er) along the nuclear membrane (nm) was present. (E) Dilation throughout the full complement of cytoplasmic ER (er) occurred with swollen appearing mitochondria (m) and still random small condensations of nuclear chromatin. (F) Advancement to larger random chromatin fragments (boxed area) was encountered with advanced ER (er) dilation and mitochondrial (m) swelling and disruption. (G) Progressive stages of massive ER swelling (er) with pseudo-inclusions («) were encountered within neurons with a similar degree of chromatin breakdown. (H) A neuron with complete detachment of the ER from nuclear membrane (nm) including a large bleb, more advanced, “clock-face” nuclear chromatin condensation (boxed area) appeared to be shedding a large fragment of cytoplasm with hugely dilated ER (er) and completely disrupted mitochondria (m) included. (I) Neurons with very small amounts of remaining cytoplasm, loss of continuity of the cellular membrane (∞) and advanced pathology of ER (er) may had already shed portions of their cytoplasmic contents including swollen ER into the neuropil. Larger but still random chromatin condensation occurred in the nucleus. Magnification noted with measure bar in right lower corner of each panel.

Figure 2

High resolution and close approximation of endoplasmic reticulum ER fragments with mitochondria within cytoplasmic compartment of neurons following neonatal HI (panels A–C). (n=4 pups). (A) Minimal ER (er) dilation. (B) Progression of ER (er) dilation and multiple contact points (white dots) with mitochondria (m). (C) Massively dilated ER (er) encircling clumps of cytoplasm. These massively dilated sections of ER (er) are still found abutting (white dots) swollen mitochondria (m). (D and E) Representative examples of shed cytoplasmic fragments (***) containing swollen ER (er) with pseudo-inclusions of cytoplasm («). These shed fragments also included large and swollen mitochondria (m). Smaller shed fragments with the same contents as in D (*** in E) within a highly disrupted neuropil containing swollen dendrites (^^^) with swollen and disrupted mitochondria (m). (F) Microglia (μg) were attracted to the area of neurons and shed cytoplasmic fragments (***). Neuron and shed cytoplasmic fragments had marked ER (er) pathology including blebbing from the nuclear membrane (nm) in neurons and swelling. Magnification noted with measure bar in right lower corner of each panel. (G–L) Calreticulin immunostaining as a marker of ER pathology after neonatal HI. Calreticulin staining in neurons mimicked some of the ultrastructural pathology seen following neonatal HI. (G) Healthy pyramidal neurons within layer IV of the cortex display fine, reticular pattern of calreticulin staining (***) at p11. (H) Neurons in areas with mild HI injury had increased calreticulin staining in pyramidal neurons (***). (I and J) In areas with more advanced injury, increasing numbers of neurons display massive accumulation of calreticulin within the cytoplasm (***). (K) In areas with the most severe injury, some neurons had massive accumulation of calreticulin within the cytoplasm, including punctate areas of immunoreactivity suggesting especially high concentrations (***). Other neurons within those areas had lost staining for calreticulin and appeared to be shedding fragments that were still weakly immunoreative for calreticulin (^^^). (L) At most advanced stages of injury, minimal calreticulin staining was identified within pyramidal neurons, and large numbers of microglia occupy most of the field (*). Magnification noted with measure bar in right lower corner of each panel.

Figure 3

Early αII-fodrin cleavage as a marker of injury and neuroprotection with necrostatin-1 (Nec-1) treatment in experimental neonatal HI. Bar graphs showing relative cleavage of αII-fodrin (240 kDa) protein into calpain-dependent (150 kDa, A) and caspase-dependent (120 kDa, B) fragments. Bars represent the mean ± SEM (adjusted OD) measured in forebrain of naive control (white), vehicle (black), and necrostatin (grey) treated male mice. Analysis by one-way ANOVA. *, p < 0.05 (Tukey’s post-hoc); n= 4 mice/treatment/time. Representative immunoblots with corresponding loading controls (β-actin, 42 kDa) are shown at 3 and 24h.

Figure 4

Necrostatin-1 (Nec-1) protects against endoplasmic reticulum (ER) pathology after neonatal HI (n=4 pups/treatment). Neurons in the ipsilateral cortex at 96 hours after neonatal HI and vehicle treatment (panels A–D) display progressive stages of ER dilation and disintegration. (A) ER(er) were mildly dilated throughout the cell soma initially, then (B) in the moderate stage, more significantly enlarged showing pseudo-inclusions («) of cytoplasm as dilation progresses with mitochondrial swelling and nuclear chromatin randomly condensed. (C) Severe ER swelling including that in continuity with the nuclear membrane (nm), accompanies severely swollen and disrupted mitochondria (m) and more, but still random and incomplete chromatin condensation especially around the nuclear margin. (D) Shed pieces of neuronal cytoplasm (***) containing swollen ER (er) with the pseudo-inclusions («) of cytoplasm are frequently found in the disrupted neuropil in the cortex of vehicle treated HI injured mice. Not the apparent lack of cell membrane around the shed fragment of cytoplasm (∞). Prominent neuroprotection after neonatal HI and Nec-1 treatment occurs with preservation of ER ultrastructure (E–H). (E) Normal appearing neuron with intact ER and mitochondria exists within slightly rarefied neuropil. (F) Abundant, intact ER are somewhat marginalized in an otherwise appearing neuron. (G) Mild dilation of ER (er) in the cell soma periphery occurs in a neuron with some darkened and rounded mitochondria (m). (H) Neuropil in cortex after HI and Nec-1 treatment is largely intact without evidence of shed fragments of cytoplasm containing dilated ER. Magnification noted with measure bar in right lower corner of each panel.

Figure 5

Endoplasmic reticulum (ER) unfolded protein response (UPR) signaling intermediates at 3 and 24 hours following experimental HI in mice treated with vehicle (black) or necrostatin-1 (Nec-1, grey) vs. naive control (white). ER stress regulator, GRP78 protein (A) and mRNA (B) levels; phosphorylation of ER sensor PERK (C) and downstream eIF2α (D) relative to total PERK or eIF2α, respectively; expression of XBP-1 mRNA (E) and its unconventional spliced product (F) downstream to eIF2α, as well as the negative feedback effector, GADD34 mRNA (G) and protein (H) are shown. CHOP is a final arbiter of apoptotic cell death in response to ER stress and its changes in mRNA (I) and protein (J) levels are also shown. Normally distributed data are shown as bar graphs representing mean ± SEM and analyzed one-way ANOVA, while not normally distributed data are shown as box and whiskers plot and analyzed using Mann-Whitney U test (vs. naive control set at 1). Boxes in box and whiskers plots represent the interquartile range (IQR) between 25 to 75 percentiles, the solid line within the box represents the median, and the whiskers extends to the last data point within 1.5 times the IQR. Protein levels were measured by western blot, while mRNA levels were measured by real time qRT-PCR. Figures present data obtained from forebrain homogenates of naive control (white), vehicle (black), and necrostatin (grey) treated mice. *, p < 0.05; n= 4 mice/treatment/time per technique. Representative immunoblots (A, C, D, H, J) and melting curves (B, G, I) are shown.

Figure 6

Simplified schematic representation of the early biochemical markers of ER stress and delayed ER ultrastructural pathology in neurons following neonatal HI. At baseline GRP78, a Ca²⁺ dependent master cell stress regulator found in the ER lumen, binds to ER sensors (PERK, Ire1 and ATF6) (1). Following neonatal HI, reperfusion induces free radical production (ROS, NO•) and Ca²⁺ dysregulation (2) and consequently misfolded, damaged, or truncated proteins accumulate and bind to GRP78, releasing PERK and other ER stress sensors (3). These steps initiate the unfolded protein response (UPR) that follows ER stress. After release, PERK oligomerizes, autophosphorylates and induces early phosphorylation of eIF2α producing global translation inhibition with preferential translation of ATF4 (4). ATF4 facilitates GADD34 transcription, which provides negative feedback to limit eIF2α phosphorylation (5), and via Xbox protein (Xbp) 1, increases CHOP transcription (6). Ire1 endonuclease activity also produces splicing of Xbp1mRNA allowing translation of an active protein that becomes part of the transcription complex for CHOP (7). The characterization of individual selectively vulnerable cortical neurons at delayed stages after HI in the mouse permitted the reconstruction of a possible evolution of ER stress related neurodegeneration. Schematic morphology of a cortical neuron showing the rough ER (er) bound to ribosomes (r) surrounding the nucleus (n) and with intact mitochondria (m) (8). Mild ER pathology is characterized by distended vermiform ER strands sometimes marginated to the periphery of the cortical neuron and associated in some instances with dark appearing nuclear speckles (peppering) (9). Moderate ER pathology includes the formation of large swollen ER cisterns. As ER pathology advances, these fragments of cytoplasm containing dilated ER and in some instances including disrupted mitochondria (m) appear to be shed from the neuron in a macrozeiotic-like process. The nucleus may show larger speckles with thickening and blebbling of the nuclear envelope (10). The most severe ER pathology includes significant shedding of hugely dilated ER cisterns within cytoplasmic fragments (cf), along with neuronal volume shrinkage and microglia (μg) infiltration (11). While ER stress response with UPR is initiated quickly following neonatal HI, a delayed cell death proceeds with significant intracellular ER pathology proceeds via a form of programmed cell necrosis once ER compensatory and autophagy mechanisms are overwhelmed.