Mitochondrial Dysfunction and Changes in High-Energy Compounds in Different Cellular Models Associated to Hypoxia: Implication to Schizophrenia

Schizophrenia (SZ) is a multifactorial mental disorder, which has been associated with a number of environmental factors, such as hypoxia. Considering that numerous neural mechanisms depends on energetic supply (ATP synthesis), the maintenance of mitochondrial metabolism is essential to keep cellular balance and survival. Therefore, in the present work, we evaluated functional parameters related to mitochondrial function, namely calcium levels, mitochondrial membrane potential, redox homeostasis, high-energy compounds levels and oxygen consumption, in astrocytes from control (Wistar) and Spontaneously Hypertensive Rats (SHR) animals exposed both to chemical and gaseous hypoxia. We show that astrocytes after hypoxia presented depolarized mitochondria, disturbances in Ca2+ handling, destabilization in redox system and alterations in ATP, ADP, Pyruvate and Lactate levels, in addition to modification in NAD+/NADH ratio, and Nfe2l2 and Nrf1 expression. Interestingly, intrauterine hypoxia also induced augmentation in mitochondrial biogenesis and content. Altogether, our data suggest that hypoxia can induce mitochondrial deregulation and a decrease in energy metabolism in the most prevalent cell type in the brain, astrocytes. Since SHR are also considered an animal model of SZ, our results can likewise be related to their phenotypic alterations and, therefore, our work also allow an increase in the knowledge of this burdensome disorder.


Spontaneusly hypertensive rats (SHR) and Schizophrenia
Today, numerous tasks are performed in rodents in order to identify changes in their behavioral that can be referred to human disorders; this definitely helps researchers to understand the pathophysiology of several diseases [1][2][3][4][5] , including SZ 6 .
In fact, in Spontaneously Hypertensive Rats (SHRs) were identified and characterized various phenotypic alterations resembling SZ. SHRs, when submitted to various investigations, present hyperlocomotion, social interaction deficit, decreased contextual fear conditioning and decreased performance in prepulse inhibition of startle (PPI) [7][8][9][10][11][12][13][14][15][16] . Specifically, regarding the augmentation in rearing phenotype in social context, it is also considered an antisocial behaviour, indicating that the animal prefers to explore the environment to have to interact with an unfamiliar rodent 7,17,18 . The augmentation of exploratory behaviour may still be a reflection of working memory deficits, a cognitive symptom observed in SZ 6 .
Interestingly, the rise in rearing phenotype is correlated to dopamine release in the striatum 6,19 , a biochemical characteristic also observed in patients with SZ and linked to positive symptoms 20 .
Despite such evidences, there are still manuscripts in the literature that use SHR as a model of Attention Deficit Disorder (ADHD). However, it is important noting that such studies use young SHR animals as well as Wistar Kyoto (WKY) as control; the use of WKY animals is not indicated, since they present depressive-like behaviour 21 . In addition, several studies report that the phenotype of SHR do not respond to psych stimulant drugs, which are used to treat ADHD; they even make the picture worse 17,15,18 . All these data reinforce the SHR as model for SZ.

Lipid peroxidation investigation
To evaluated lipid peroxidation status in Wistar and SHR astrocytes, we On the other hand, to analyze HNE levels, we used the western blot technique and we evaluated the levels of alpha-HNE protein (68 kDa) (JAICA-MHN020P; 1:3.000) 23 . Data were normalized to ACTIN levels and results were expressed as percentage of control group.
Cells were then transferred to an O 2 electrode chamber (DW1, Clark type electrode, Hansatech Instruments, Norfolk, UK) with a constant temperature (37°C) coupled to an analogue/computer system, after calibration for gas phase (following manufacturer's protocol). Afterwards, we measured basal O 2 consumption and the respiration rate after the addition of FCCP (10 µM) [24][25][26][27] . The rate of O 2 consumption was acquired as nmol/mL/min. After readings, protein content was obtained for normalization (cells were lysed using 1 M NaOH) and we performed Bradford assay.
O 2 consumption is shown as nmol/mL/min/mg of protein normalized to control group (in percentage) 24 .

Increased lipid peroxidation in astrocytes after neonatal hypoxia
As we observed alterations in mitochondrial metabolism in SHR astrocytes when compared to control group, we decided to further investigate lipid peroxidation in both groups ( Figure 1SS). As a side note, lipid peroxidation refers to the oxidative degradation of lipids, in which free radicals take electrons from the lipids (generally from cell membranes), resulting in cellular damage and even cell death.
To verify lipid peroxidation in our system, we evaluated two peroxidation byproducts, malondialdehyde (MDA) and 4-hydroxynonenal (HNE). As we can observe on Figure 1SS, there is a significant increase in MDA (µM/ml) levels in SHR astrocytes in relation to Wistar cells (*p<0.01). In addition, our results also demonstrated a significant augmentation in αHNE levels in SHR astrocytes (*p<0.01) ( Figure 1SS).

Increase in oxygen consumption after intense chemical hypoxia
The analysis revealed that there was no change in oxygen consumption between untreated Wistar and SHR astrocytes ( Figure 2SS), indicating that despite changes in calcium levels, mitochondrial membrane potential and oxidative stress, the total respiration was not affected.

TOM-40 levels augmentation in neonatal hypoxic astrocytes
As shown in Figure 7D and E, we detect a significant increment in TOM-40 levels in SHR astrocytes. All data presented reinforce that an increased mitochondrial content in SHR astrocytes could be related to changes in mitochondrial metabolism.
The uncropped blots of ACTIN and TOM-40 are shown below ( Figure 3SS and 4SS).