Oxidative stress

Increase of oxidative stress was evaluated by various oxidative stress markers. Each marker was observed to have an increased positive signal in the hippocampus pyramidal layer of LDHB KO mice compared to WT mice. In the hippocampus CA1 of the LDHB KO mice, the intensity of neuronal 3-nitrotyrosine tended to be higher compared to WT mice, but it was statistically marginal (p = 0.059; Fig. 8A-C).

Methylglyoxal was often observed in the dark neuronal cells in the hippocampus CA1 of the LDHB KO mice. The intensity of neuronal methylglyoxal tended to be higher compared to WT mice, but it was statistically insignificant between groups (p

= 0.245; Fig. 8D–F). For 8-OhdG, which is associated with DNA oxidation in neuronal cells, the intensity of neuronal 8-OHdG at the hippocampal CA1 of the LDHB KO mice tended to be higher compared to WT mice (p = 0.051; Fig. 8G–I).

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Figure 8. Immunohistochemistry detection of oxidative stress makers in LDHB KO mice. A-C. The intensity of 3-nitrotyrosine tends to increase in the hippocampal pyramidal neurons of LDHB KO mice compared to WT mice (p = 0.059). D-F. A tendency of increase in methylglyoxal is shown in the neurons of LDHB KO mice;

however, there was no statistical significance (p = 0.245). G-I. 8-OHdG level tended to increase in LDHB KO mice compared to WT mice (p = 0.051). Data are expressed as the mean ± S.E.M. (n = 10). Magnification = 100X. White box of left lower of figure = magnification of arrow-indicated area.

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5. Glucose transporter 3 (GLUT3)

Expression of GLUT3 was confirmed to show glucose utilization. GLUT3 was mostly expressed in neurons in the hippocampal CA1 region (Fig. 9A, B); however, the intensity of GLUT3 did not show difference between the two groups (p = 0.735;

Fig. 9C). Nevertheless, the higher intensities in GLUT3 appeared to be more prominent in the dark neurons.

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Figure 9. Immunohistochemistry detection of glucose transporter 3 (GLUT3) in hippocampal pyramidal cell layer in LDHB KO mice. A-B. Cellular density of GLUT3 appeared to increase in the smaller neurons of LDHB KO. This finding suggests that higher density might be more prominent in dark neurons C.

Quantification of GLUT3 densities, however, did not differ between the neurons of LDHB KO and WT mice. Data are expressed as the mean ± S.E.M. (n = 10).

Magnification = 400X

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6. MR spectroscopic analysis

To evaluate changes in brain lactate levels, ¹H-MRS was performed in the hippocampus of aging mice (Fig. 10A). Lactate peak was identified at 1.33 ppm with higher lactate peaks in LDHB KO mice (Fig. 10B). When its levels were quantified as a ratio to N-acetyl aspartate, the lactate ratio in the hippocampus of the LDHB KO mice showed about 3 times higher levels in the hippocampus of LDHB KO mice than in WT mice (p < 0.005; Fig. 10C).

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Figure 10. Lactate levels was increased in the LDHB KO mice brain. A. Position of the volume of interest (2 mm x 2 mm x 2 mm) in the hippocampus in the sagittal and coronal plane. B. ¹H-magnetic resonance spectroscopy obtained from the hippocampus. The lactate peak is located at 1.3 ppm chemical shift whereas the N-acetyl aspartate (NAA) peak is located at 2.0 ppm chemical shift (red arrow = lactate;

brown arrow = NAA). C. Quantification as a ratio of lactate to total NAA. LDHB KO mice show significantly elevated lactate ratio compared to WT mice. 20~24-month-old WT, n = 6; LDHB KO, n = 7 mice. Independent t-test, *p = 0.009.

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IV. DISCUSSION

In the current study, we have examined whether a deficiency of the LDHB enzyme related to energy metabolism affects the behavior and pathologic findings of mice brain. We observed that locomotor activity decreased, and learning and memory were deteriorated in LDHB KO mice. These changes were accompanied by neuronal change with dark staining and small size but without loss of neuron cells, and decrease in astrocytic cell count. There was a tendency for elevation of oxidative stress markers, and a significant elevation of lactate/NAA ratio in the LDHB KO group. In conclusion, LDHB deficiency seems to aggravate neuronal and behavioral changes with a lack of lactate utilization in neurons. Overall, it is regarded that these changes related to one depletion of several neuronal energy metabolisms seem to be similar as a cross section of neuronal aging process.

Morphological changes in neuron and insignificant decrease in behavior Many behavioral studies have shown that behavior is worsened by aging in normal animals (Hamezah, Durani et al. 2017). The cell morphologic changes underlying the behavioral changes seen in LDHB KO mice may be interpreted as findings that can be seen in aged mice. In our study, cognitive functions just tended to be decreased in LDHB KO mice without statistical significance. This finding is consistent with histological findings in that neuronal cell loss was not evident and total neuronal cell count did not differ between KO and WT mice. Nevertheless, following histological changes might influence the subtle behavioral changes. The increase of small dark cells without decrease in cell number in hippocampus and cortex, decrease of astrocytes, and increase of ROS is a common pathologic change in the LDHB KO mice. As animals age, cell atrophy occurs naturally, and is followed by cell death via apoptosis or necrosis. However, it has been reported that age is associated with changes in cell morphology but not overall number, and our study results are

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consistent with these findings (Villena, Diaz et al. 1997, Kim, Kim et al. 2011, Stranahan, Jiam et al. 2012, Ojo, Rezaie et al. 2015).

ROS change

Changes in neuronal morphology may also result from the production of reactive oxygen species (ROS), which was generally increased in LDHB KO mice.

Nitrotyrosine and 8-OHdG were increased in LDHB KO mice compared to WT mice.

In both neurodegenerative disease as well as aging, a decline of normal antioxidant defense mechanisms has a harmful effect on cells and increases oxidative damage (Harman 1956, Dias, Lourenco et al. 2016, Garaschuk, Semchyshyn et al. 2018).

The level of methylglyoxal, however, did not significantly differ between KO and WT mice. Methylglyoxal, a byproduct of glycolysis, is the most powerful glycation agent in the cell, and its accumulation is harmful to the body. It is also a precursor to advanced glycation end (AGE) products, which are related to severe neurodegenerative disorders, diabetes complications, and aging. The glyoxalase system is of primary importance in protecting the brain against the accumulation of AGEs. Normal neurons have few glycolytic processes to protect them from oxidative stress, and glycolysis through astrocytes is predominant. In the process of glycolysis, methylglyoxal is produced and then its detoxification should be achieved through the glyoxalase system (Belanger, Yang et al. 2011, Allaman, Belanger et al. 2015).

Metabolism change

When GLUT-3 was used to examine the glucose utilization of neurons, we observed no significant difference between WT mice and LDHB KO mice. Although the neuronal glucose utilization is increased in aging, this finding is not sufficient to account for the increase in glycolysis. In relation to this, GLUT3 is initially decreased in normal aging according to age, and has been reported to increase at over 15 months.

In addition, 3xTg mice were found to be more decline in glucose transporter than

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normal mice and metabolism could be changed to ketogenic system (Ding, Yao et al.

2013).

Given that when glucose enters the cell, it is converted into glucose-6-p, it is used as a source for glycolysis, the pentose phosphate pathway (PPP), and glycogen synthesis. Glycolysis is controlled by catalysis by the enzymes hexokinase (HEX), phosphofructokinase (PFK) and pyruvate kinase (PK). The catalytic activity of PFK in cells is inhibited by increasing the proton concentration and the intracellular pH is the direct control mechanism. Neuron has a low concentration of PFK, so the process is inevitable. However, there is a possibility that the change in catalytic activity of PFK due to the increase of lactate (acidification) may have occurred.

The brain produces glycogen and lactate in astrocyte, whereas in neuron it is metabolized mainly through pentose phosphate pathway and consumes lactate.

Pentose phosphate pathway is involved in biosynthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) with glucose-6-p. This is also related to the GSH regeneration tube, which is an antioxidant mechanism in neurons (Fernandez-Fernandez, Almeida et al. 2012), (Deitmer, Theparambil et al.

2017). We can identify NADPH or GSH levels in the following experiments and identify them as changes in LDHB KO mice.

Another possibility is that LDHB KO mice use metabolic pathways that utilize forms of energy other than glucose. For example, when the body's glucose levels are low, ketone bodies produce energy by introducing lipids to be directly used in the TCA cycle in neuronal mitochondria (Owen. 2005), (Yao, Irwin et al. 2009, Yao, Hamilton et al. 2010). In LDHB KO mice, it can be explained that the neurons may utilize the more convenient ketone bodies as a source of energy.

Direct toxicity of lactate

1H-magnetic resonance spectroscopy results showed that lactate levels were increased in the brain of LDHB KO mice. While, lactate produced by aerobic

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glycolysis in astrocytes is an essential metabolite for memory consolidation (Newman, Korol et al. 2011, Steinman, Gao et al. 2016), pathologically increased lactate production in transgenic rodent models such as APP/PS1 contributes to cognitive dysfunction associated with Alzheimer disease and promotes neuronal death (Harris, Tindale et al. 2016). The highly elevated lactate levels may also have toxic effects in our study.

It was also reported that the damage to the mitochondria slowly increases with age in brains of mice and causes changed expression in certain genes that are responsible for the accumulation of lactate (Ross, Oberg et al. 2010). An increase in intracerebral lactate, due to an abnormal rather than normal aging process, can lead to the production of reactive oxidative stress as well as the impairment of neuronal function, resulting in cognitive dysfunction (Ross, Oberg et al. 2010).

In conclusion, LDHB deficiency results in aging-like changes in behavior and cognitive function, suggesting the possibility of accelerating cerebral degenerative change by some abnormal energy metabolism (Figure. 11).

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Figure 11. Abnormal energy metabolism of the brain in aged LDHB KO mice.

In the normal state, glucose from astrocyte produces lactate through glycolysis and goes to neuron and used as a mitochondrial energy (ANLS). By LDHB depletion, lactate is not utilized in neuron after transferred from astrocyte, and lactate accumulates in brain. In turn, this environment increases reactive oxygen species production, thereby promoting aging-like process of the animal.

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aspartate (NAA) 비율의 비율이 증가함을 나타냈다.

결론을 말하여 나이든 LDHB KO 마우스에서는 lactate의 증가와 활성 산소 종의 증가로 인해 신경세포의 형태변화 및 행동 저하를 야기하는 것으로 나타났다.

핵심어: 젖산 탈수소 효소 B, knock-out 마우스, 젖산, 활성 산소 종, 신경세 포, 성상세포