Hippocampal region-specific endogenous neuroprotection as an approach in the search for new neuroprotective strategies in ischemic stroke. Fiction or fact?

Ischemic stroke is the leading cause of death and long-term disability worldwide, and, while considerable progress has been made in understanding its pathophysiology, the lack of effective treatments remains a major concern. In that context, receiving more and more consideration as a promising therapeutic method is the activation of natural adaptive mechanisms (endogenous neuroprotection) - an approach that seeks to enhance and/or stimulate the endogenous processes of plasticity and protection of the neuronal system that trigger the brain's intrinsic capacity for self-defence. Ischemic preconditioning is a classic example of endogenous neuroprotection, being the process by which one or more brief, non-damaging episodes of ischemia-reperfusion (I/R) induce tissue resistance to subsequent prolonged, damaging ischemia. Another less-known example is resistance to an I/R episode mounted by the hippocampal region consisting of CA2, CA3, CA4 and the dentate gyrus (here abbreviated to CA2-4, DG). This can be contrasted with the ischemia-vulnerable CA1 region. There is not yet a good understanding of these different sensitivities of the hippocampal regions, and hence of the endogenous neuroprotection characteristic of CA2-4, DG. However, this region is widely reported to have properties distinct from CA1, and capable of generating resistance to an I/R episode. These include activation of neurotrophic and neuroprotective factors, greater activation of anti-excitotoxic and anti-oxidant mechanisms, increased plasticity potential, a greater energy reserve and improved mitochondrial function. This review seeks to summarize properties of CA2-4, DG in the context of endogenous neuroprotection, and then to assess the potential utility of these properties to therapeutic approaches. In so doing, it appears to represent the first such addressing of the issue of ischemia resistance attributable to CA2-4, DG.

Is Nrf2 Behind Endogenous Neuroprotection of the Hippocampal CA2-4,DG Region?

The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is the master regulator of genes known to be involved in antioxidant, and anti-inflammatory processes, metabolic regulation, and other cellular functions. Here, we also hypothesize a core role for it in endogenous neuroprotection, i.e., the natural adaptive mechanisms protecting the brain from ischemia-reperfusion (I/R) episode. An example of endogenous neuroprotection is ischemia-resistance of the hippocampal regions comprising the CA2, CA3, CA4 and dentate gyrus subfields (here abbreviated to CA2-4,DG) which can be contrasted with the ischemia-vulnerable CA1 region. In the work detailed here, we used a gerbil model of transient cerebral ischemia to examined Nrf2 activation in CA1 and CA2-4,DG, in a control group, and post I/R episode. Data obtained indicate enhanced Nrf2 activity in CA2-4,DG as compared with CA1 in the control, with this difference seen to persist even after I/R. While I/R does indeed cause further activation of Nrf2 in CA2-4,DG, it is associated with slight and transient activation in CA1. Sub-regional differences in Nrf2 activity correlate with immunoreactivity of Keap1 (an Nrf2 suppressor) and Nrf2 target proteins, including heme oxygenase 1, the catalytic and modulatory sub-units of glutamate-cysteine ligase, and glutathione peroxidase 1. Pharmacological Nrf2 activation by sulforaphane results in protection of CA1 after I/R episode. Our results therefore suggest that high Nrf2 activity in CA2-4,DG may guarantee resistance of this region to I/R, potentially explaining the differential sensitivities of the hippocampal regions.

Mitochondrial dynamics, elimination and biogenesis during post-ischemic recovery in ischemia-resistant and ischemia-vulnerable gerbil hippocampal regions.

Transient ischemic attacks (TIA) result from a temporary blockage in blood circulation in the brain. As TIAs cause disabilities and often precede full-scale strokes, the effects of TIA are investigated to develop neuroprotective therapies. We analyzed changes in mitochondrial network dynamics, mitophagy and biogenesis in sections of gerbil hippocampus characterized by a different neuronal survival rate after 5-minute ischemia-reperfusion (I/R) insult. Our research revealed a significantly greater mtDNA/nDNA ratio in CA2-3, DG hippocampal regions (5.8 ± 1.4 vs 3.6 ± 0.8 in CA1) that corresponded to a neuronal resistance to I/R. During reperfusion, an increase of pro-fission (phospho-Ser616-Drp1/Drp1) and pro-fusion proteins (1.6 ± 0.5 and 1.4 ± 0.3 for Mfn2 and Opa1, respectively) was observed in CA2-3, DG. Selective autophagy markers, PINK1 and SQSTM1/p62, were elevated 24-96 h after I/R and accompanied by significant elevation of transcription factors proteins PGC-1α and Nrf1 (1.2 ± 0.4, 1.78 ± 0.6, respectively) and increased respiratory chain proteins (e.g., 1.5 ± 0.3 for complex IV at I/R 96 h). Contrastingly, decreased enzymatic activity of citrate synthase, reduced Hsp60 protein level and electron transport chain subunits (0.88 ± 0.03, 0.74 ± 0.1 and 0.71 ± 0.1 for complex IV at I/R 96 h, respectively) were observed in I/R-vulnerable CA1. The phospho-Ser616-Drp1/Drp1 was increased while Mfn2 and total Opa1 reduced to 0.88 ± 0.1 and 0.77 ± 0.17, respectively. General autophagy, measured as LC3-II/I ratio, was activated 3 h after reperfusion reaching 2.37 ± 0.9 of control. This study demonstrated that enhanced mitochondrial fusion, followed by late and selective mitophagy and mitochondrial biogenesis might together contribute to reduced susceptibility to TIA.

Mitochondrial Metabolism behind Region-Specific Resistance to Ischemia-Reperfusion Injury in Gerbil Hippocampus. Role of PKCßII and Phosphate-Activated Glutaminase.

Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCßII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCßII specific inhibitor peptide ßIIV5-3-treated IR. Vehicle or ßIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCßII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 µM) to evaluate the inhibition of GLS1 on neuronal viability. PKCßII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by ßIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCßII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.

Hippocampal Sector-Specific Metabolic Profiles Reflect Endogenous Strategy for Ischemia-Reperfusion Insult Resistance.

The gerbil is a well-known model for studying cerebral ischemia. The CA1 of the hippocampus is vulnerable to 5 min of ischemia, while the CA2-4 and dentate gyrus (DG) are resistant to it. Short-lasting ischemia, a model of transient ischemic attacks in men, results in CA1 neuron death within 2-4 days of reperfusion. Untargeted metabolomics, using LC-QTOF-MS, was used to enrich the knowledge about intrinsic vulnerability and resistance of hippocampal regions and their early post-ischemic response (IR). In total, 30 significant metabolites were detected. In controls, taurine was significantly lower and guanosine monophosphate was higher in CA1, as compared to that in CA2-4,DG. LysoPG and LysoPE were more abundant in CA1, while LysoPI 18:0 was detected only in CA2-4,DG. After IR, a substantial decrease in the citric acid level in CA1, an accumulation of pipecolic acid in both regions, and opposite changes in the amount of PE and LysoPE were observed. The following metabolic pathways were identified as being differentially active in control CA1 vs. CA2-4,DG: metabolism of taurine and hypotaurine, glycerophospholipid, and purine. These results may indicate that a regulation of cell volume, altered structure of cell membranes, and energy metabolism differentiate hippocampal regions. Early post-ischemia, spatial differences in the metabolism of aminoacyl-tRNA biosynthesis, and amino acids and their metabolites with a predominance of those which upkeep their well-being in CA2-4,DG are shown. Presented results are consistent with genetic, morphological, and functional data, which may be useful in further study on endogenous mechanisms of neuroprotection and search for new targets for therapeutic interventions.