The generation of LDHB KO mouse

Whole body LDHB gene knockout mouse is generated by inactivation of LDHB gene by cre/loxP system (Figure 4A). The LDHB gene which has flanked by two loxP sites can be excised and inactivated by Cre recombinase recognizing two loxP sites.

Two mouse lines were required for conditional gene-LDHB deletion. First, a conventional transgenic mouse line with Cre targeted to protamine. Secondly a mouse strain that has a target gene–LDHB flanked by two loxP sites. Recombination occurs only in those cells expressing Cre recombinase. Thus, the LDHB remains active in all cells and tissues which do not express Cre recombinase. We successfully generated whole body LDHB knockout mouse. The whole body LDHB knockout mouse (WLDHB KO) showed 403 bp size band, while the whole body LDHB wild type mouse (WLDHB WT) showed 660 bp size band by PCR (Figure 4B).

Figure 4. The generation of whole body LDHB knockout mouse A. The scheme of WLDHB Knockout mouse model by loxP/cre system B. The genotype of WLDHB WT and KO by PCR

2. Ischemic cell death was attenuated in LDHB KO mice by PcomA size in vivo

In brain, cells consume large amounts of metabolite and energy substrate, including glucose, lactate, and ketone. Especially lactate is the main energy source in neuron cells and it should transfer to pyruvate before oxidized for ATP production. For this reason, LDHB may play a crucial role on neuronal metabolism. To investigate whether LDHB contributes to neuron cell survival, histological staining using cresyl violet was performed. Interestingly, there was no phenotypical change by LDHB KO, including neuronal cell number, in normal condition (Figure 5A, upper panel). It is assumed that LDHB KO neurons may metabolically adapted. So, I further examined in metabolic stress condition like ischemic condition for observing dramatical change by LDHB. For this purpose, bilateral common carotid occlusion (BCCO) in the mouse is utilized as a common ischemic model, in terms of mortality and susceptible neuronal degeneration of CA1 region (Barone,1993). As shown in Figure 5, LDHB KO mice was resistant to neuronal cell death in ischemic condition. The results of histological staining using cresyl violet showed that ischemic stress effectively induced cell death in neuron cells of LDHB WT mice, but not those of KO mice (Figure 5A). In addition, ischemic damage in neuron cells was evaluated using the following scale: no damaged, and every quarters are expressed to grade 0 (0%), grade 1 (up to 24%), grade 2 (25% - 50%), grade 3 (51% - 75%), and grade 4 (upper than 75%), respectively (Figure 5B). Furthermore, when survival neuron cells of CA1 region were counted and described to graph, these results showed that neuronal cell survival was markedly increased in LDHB deficient mice (Figure 5C). To confirm preceding data, TUNEL assay was performed in ischemic condition. Ischemic condition outstandingly induced TUNEL fluorescence in neuronal cells of LDHB WT mice, whereas TUNEL-positive cells were decreased in those of LDHB KO mice (Figure 6A, B). I drew TUNEL-positive cells graphically by counting cells (Figure 6B). Collectively, I found that deficiency of LDHB contributes to cell survival ability

of neuronal cells and resistance to ischemic cell death.

The patency of the PcomA is regarded as a crucial factor in ischemia after bilateral common carotid artery occlusion (BCCO) (Kitagawa et al., 1998). Commonly, neuron cell death is induced at less than 30% PcomA/BA (basilar artery) (Cho, 2006). While there is no difference of the PcomA size between WLDHB WT and WLDHB KO in normal condition, it markedly distinguished in ischemic condition (Figure 7). LDHB KO mice having over 30% PcomA/BA was more than WLDHB KO mice after tBCCAO. These in vivo results suggest that the increase of PcomA size in WLDHB KO mice affects neuron cell survival in ischemic condition.

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Figure 5. Increased neuronal cell survival of LDHB KO mice under ischemic condition

A. Cresyl violet staining of the fixed hippocampus isolated from the mouse after ischemic stress

B. Neuronal damage grades of the hippocampus C. Survival neuron cell count in CA1 region

Figure 6. Decreased TUNEL-positive fluorescence in LDHB KO neuronal cells, compared with those of WT

A. TUNEL staining of the fixed hippocampus isolated from the mouse after ischemic stress

B. The graphical description of TUNEL positive cells

Figure 7. The difference of PcomA/BA (%) between LDHB WT and KO under ischemic stress

A. Representative images of latex dye casting show PcomA B. The graphical description of PcomA/BA (%)

3. Hypoxia response was more upregulated in LDHB KO neuron cells after treatment with CoCl2 than LDHB expressing neuron cells

For neuron primary culture in vitro, first I mated mice WLDHB WT female/male and KO female/male, respectively. Fourteen days after plug checked, I cultured primary neuron cells using cortex and hippocampus isolated from embryos. Additionally, 2 days after culture, cytosine arabinoside (AraC) was treated for 3 days to decrease glial cell contamination, which was known as anti-mitotic agent reducing the population of non-neuronal cells capable of DNA synthesis (Seibenhener et al. 2012). After treatment, the neuronal cells were used to perform following experiments (Figure 8 A). For confirming neuron cells, immunocytochemistry was performed using specific antibody targeting NeuN proteins, neuron marker protein (Figure 8 B). Similar to in vivo data, in vitro cells, phase-contrast microscopy showed that there is no difference between primary neuron cells from WLDHB WT mice and those of WLDHB KO mice (Figure 8 B).

To confirm genetic type of primary neuron cells, protein was extracted from each cultured primary neuron cells and then western blot was performed (Figure 8 C). The reason of increasing LDHA in hypoxia condition is that LDHA is the downstream target of HIF-1α. Furthermore, since LDH is homo- or hetero-tetrameric proteins, isoenzyme pattern analysis was performed. To confirm isoenzyme patterns, protein lysates were isolated from both WT and KO neuron cells using specific protein extraction solution which is not contain detergent and reducing agent and separated by Native-PAGE. LDH isozyme is separated by their charge. LDHB carry more negative charges than LDHA. So, LDHA is appeared more upper than LDHB. When isoenzyme patterns were detected by activity staining using NBT/PMS under dark, only LDH5 was expressed in KO neuron cells, which is LDHA only homo-tetrameric enzyme (Figure 8 D).

Hypoxia inducible factor-1 (HIF-1) is composed of HIF-1α and HIF-1β subunit as a heterodimeric transcription factor (Zhou J et al, 2004). HIF-1β is constitutively present and its mRNA and protein level is sustained constantly regardless of oxygen availability, while HIF-1α has a short half-life and its stability is controlled by oxygen (Ke et al. 2006). In normoxia, HIF-1α exists low level du to continuous degradation by the 26S proteasome, whereas under hypoxia, HIF-1α becomes stabilized by blocking prolyl hydroxylases (HIF-PH). In this way, many chemicals as transition metals such as CoCl2, or the iron chelator desferroxamine (DFX) are widely used by blocking HIF-PH and in turn inducing HIF-1α stabilization (Zhou J et al, 2004).

So, to mimic ischemic condition in vitro, I treated cobalt chloride (CoCl2), which enhance the stability of hypoxia-inducible factor (HIF)-1α, in the neuron cell (Zhang et al. 2014). After treating CoCl2 for 12 hour, MTT assay is used for identification of neuron cell viability (Figure 9 A). The decreasing viability of LDHB KO neuron cells after treatment with CoCl2 was similar to that of LDHB WT neuron cells. These results suggest that resistance of LDHB KO mice to neuronal cell death under ischemic condition might be irrelevant to cellular level. Rather, PcomA size might play crucial rules on resistance to ischemic stress. To identify the reason of resistance to ischemic stress, I further tested whether proteins associated with hypoxia response were activated by CoCl2 in LDHB KO neuron cells. Even though HIF-1α protein level was dramatically induced by CoCl2 in protein extracts of neuron cells isolated from LDHB WT and KO (Figure 9 B), its target gene, VEGFA, was significantly increased in neuronal cells of LDHB KO, more dramatically than those of WT (Figure 10). On the other hand, brain-specific angiogenesis inhibitor 1 (BAI 1) displayed a tendency to decrease in LDHB KO cells compared to LDHB WT cells (Figure 10). Increase of the VEGF expression by HIF-1 and decrease of the BAI 1 could induce the development of new blood vessels of the target area in the brain. The production of new blood vessels leads to provide an increased blood flow and oxygen supply and that results in reducing ischemic damage (KE et al., 2006; Tulsulkar et al., 2013).

Taken together, these results suggest that resistance of LDHB KO to neuronal cell death under ischemic condition is derived from upregulated response to hypoxia prior to PcomA size increase.

Figure 8. Culture of primary neuron cells A. The schedule of primary cell culture

B. Morphology of neuronal cells and the confirmation of neuron cell by ICC C. The western blot of the extracted protein comparing LDHB KO with WT D. LDH isoenzyme pattern comparing LDHB KO with WT

Figure 9. No difference of cell viability in vitro under hypoxia mimic condition &

the conformation of hypoxia condition induced by CoCl2

A. Cellular viability using MTT assay after treatment of primary neuronal cells with CoCl2

B. Western blotting of HIF1a, LDHA and LDHB was performed in LDHB WT, KO neuron cells. Tubulin was used as a loading control in western blots

Figure 10. mRNA levels associated with angiogenesis after treatment with CoCl2

VEGF-A, BAI 1 mRNA levels through Real-Time PCR after extracting cDNA of WT, KO neuron cells.

IV. DISCUSSION

In this study, I found that LDHB deficiency increase neuronal cell survival under ischemic condition in vivo. LDHB null mice did not show any different phenotype compared with wild type mice (Figure 5 A). In parallel, no morphological difference also was observed in primary neuronal cells isolated from cortex and hippocampus of mice having LDHB mice (Figure 8 B). But in metabolic stress condition, absence of LDHB increased the cell survival ability via increase of PcomA size. When I performed the histological staining using a cresyl violet to detect survival cells in ischemic condition, decrease of live cells was observed in only WT mice, but not KO mice (Figure 5 A). In addition, when I further examined TUNEL-assay to detect dead cells, TUNEL-fluorescence positive cells were outstandingly induced in only WT mice (Figure 6). These results indicated that loss of LDHB positively affects cell viability and increase of PcomA size (Figure 7) might be reasonable explain. When I treated of primary neuronal cells with CoCl2 to mimic ischemic condition in cell culture system, the cell viability is no different between LDHB WT and KO (Figure 9). But, in ischemic condition, VEGFA was effectively increased in LDHB KO neuron cell (Figure 10). Taken together, in ischemic stress condition, deficiency of LDHB protects ischemic neuronal cell death by increasing PcomA size and VEGFA level.

The relationship of VEGFA and PcomA is unclear. But, VEGFA may be also relevant to vasodilation, although VEGFA is well known as the role of mediating angiogenesis and vasculogenesis. I will further investigate the mechanism of vessel dilation in LDHB KO brain. In ischemic condition, HIF-1α expression is increased and in turn, increased HIF-1α induce production of VEGFA. In addition, VEGFA stimulates endothelial nitric oxide synthase (eNOS) which produces nitric oxide (NO). NO is a crucial mediator of blood vessel reactivity, angiogenesis and vasodilation (Johanna et al., 2012). So, it is expected that VEGFA may be involve in increasing PcomA size.

And then, further study is needed to confirm why PcomA size is increased in LDHB deficiency under ischemic condition and how PcomA size is regulated.

Metabolic adaptation also is in close connection with ischemic cell death (Michelle et al., 2009). LDHB is most important enzyme for energy metabolism of neuronal cells, according to astrocyte-to-neuron lactate-shuttle theory. Therefore, LDHB deficiency may induce metabolic adaptation, such as increased capillary surface area that results in increased oxygen delivery, decreased energy demand and increased energy production efficiency, and this metabolic adaptation might contribute to resistance of neuronal cells to cell death under ischemic stress. LDHB deficiency mice also may be involved in the ischemic precondition which is resistance to the loss of blood supply and oxygen. To confirm whether ischemic precondition is induced by LDHB deficiency, further studies are needed.

In the ischemic condition, mitochondrial dysfunction is considered as key cause of neuronal cell death (Baxter et al., 2014; Niizuma et al., 2009). Therefore, mitochondrial oxygen consumption rates, ROS levels, ATP levels, and mitochondrial membrane potential should be examined using mice and its primary cultured neuron cells, having or not LDHB gene, under ischemic stress.

In brain, increasing activity is accompanied by changes in local blood flow and glucose utilization being referred to as neurovascular and neurometabolic coupling (Bélanger et al., 2011). So, brain energy metabolism has relevance to vascular and metabolic sources.

Here, I found that ischemic neurons of LDHB deficiency mice survive well by the increased PcomA size. Although more studies are need for the mechanism of increasing PcomA size, LDHB may be related to vasodilation. So, I suggest that LDHB has the potential of remedial agent as vasodilator for treating the diseasesthe blood vessels get blocked such as stroke and cerebral infarction. And I also expect that LDHB is helpful for surviving neurons under ischemic condition. So, LDHB may be possible to be a good candidate for treating neurodegenerative disease such as

epilepsy, stroke, Alzheimer’s disease, aging, ischemia and dementia (Sada et al., 2015;

Rho, 2015).

V. CONCLUSION

In this study, I demonstrated that LDHB KO mouse ameliorate ischemic neuronal cell death through the increase of PcomA size which was supported by the fact that VEGFA level is upregulated I cells. LDHB is an important enzyme in neuron for using lactate as energy source by converting lactate to pyruvate, since neuron uses lactate more than glucose as energy source. In normal condition, LDHB KO neuron cell appeared no difference, but in ischemic condition, the decreased neuronal cell death is shown. That means LDHB KO neuron cell may be less vulnerable to ischemia by preconditioning compared to LDHB WT. To elucidate preconditioning in LDHB KO neuron cells, further studies are needed.

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핵심어 : 젖산탈수소효소, 허혈성 스트레스, 신경세포 사멸, 혈관 표피 성장인자