ABSTRACT

Conclusion:

These findings suggest that the excess of thyroid hormone during neuronal plasticity partially alters the expression of genes related to neurodegeneration.

Results:

After HFS, LTP was induced in the control group at the EPSP slope, while LTP was not induced in the hyperthyroid group. However, LTP was induced at the PS amplitude in both groups. In hyperthyroid rats, Gsk3β, P35(Anxa) Calpn1 and Bace1, Psen2-mRNA levels were not significantly different compared to the control group, while Akt1, Cdk5 and Mapt-mRNA levels were found to be increased significantly compared to the control group (p<0.05).

Materials and Methods: 12 male Wistar-albino rats were used in the study. The rats were divided into two groups as control and hyperthyroidism groups. To induce hyperthyroidism, L-tyroxine was administered intraperitoneally at a dose of 400 μg/kg/day for 21 days, starting on the 39^th day. To investigate LTP, when rats were 60-day-old, LTP was induced by giving HFS at 100 Hz for 1 second at 5-minute intervals after 15 minutes of basal recording to the perforating pathway and the excitatory postsynaptic potential (EPSP) slope and population spike (PS) amplitude were recorded in the granule cell layer of the dentate gyrus. The mRNA levels of genes related to neurodegeneration in stimulated hippocampus were measured by reverse transcription-polymerase chain reaction method.

Aim:

Imbalances in plasma thyroid hormone levels cause changes in synaptic response, resulting in impairments in learning and memory. It is not well understood how the excess of these hormones (T3 and T4 hormones), which modulate gene transcription, affects gene expression after high-frequency stimulation (HFS). Therefore, in this study, it was aimed to investigate the transcriptional changes accompanying long-term potentiation (LTP) in genes related to neurodegeneration in rats with hyperthyroidism.

INTRODUCTION

Thyroid hormones have genomic and non-genomic effects. Thyroid hormones cause the activation of intracellular PI3K-Akt/Gsk3β and Mapk signaling pathways by interacting with the specialized membrane proteins monocarboxylate transporter 8 and integrin αVβ3, which are involved in the communication between the extracellular and intracellular environment, with their non-genomic effect. The non-genomic effects of thyroid hormones are short term^1-4. The main effect of thyroid hormone is genomic, it binds to its receptors in the nucleus and initiates various transcription events and directly affects the biological functions of the cell¹. Changes in hormone level can affect the expression levels of genes associated with synaptic plasticity and neurodegenerative diseases. The underlying cause of the majority of neurodegenerative diseases is based on defects in gene expression. In recent years, studies performed to explain the causes of neurodegenerative diseases have focused on changes in gene expression^5,6. The hippocampus is considered one of the most important brain regions affected by these diseases. In the previous studies conducted in our laboratory, it was determined that high or low thyroid hormone levels caused significant changes on hippocampal synaptic plasticity forms^7,8.

Long-term potentiation (LTP) means permanently increasing the strength and number of synaptic connections, depending on the increase in the frequency of neural activity from a presynaptic neuron or nerve pathway, and is a form of synaptic plasticity that is the most studied and well-known. A growing number of studies on synaptic plasticity have shown that LTP not only strengthens synaptic connections during learning, but also promotes the formation of new neurons in the hippocampus^9,10. LTP requires the phosphorylation of some critical kinases¹¹. At the molecular level, the effect of an increase in kinase or phosphatase activity is relatively short term, and longer maintenance of these changes requires gene transcription and synthesis of neuronal proteins¹².

Changes in the expression of certain genes following the induction of the forms of synaptic plasticity that underlie learning and memory allow these forms of plasticity to persist through protein synthesis¹³. On the other hand, the over- or under-synthesized protein products formed by genes with changed expression in thyroid hormone deficiency or excess may cause tau and amyloid beta proteins, which are associated with some neurodegenerative diseases, to be synthesized and phosphorylated at different levels. As a result, the incidence of neurodegenerative diseases such as Alzheimer’s disease, characterized by dementia, may increase.

In our study, it is aimed to investigate the change in mRNA level of amyloid precursor protein (APP)-associated (Bace1 and Psen2) and tau phosphorylation-related proteins (Gsk3β, Cdk5, Akt1ve Mapt, P35(Anxa), Capn1), associated with neurodegenerative proteins and accompanying the induction of LTP in the hippocampus of hyperthyroid induced rats.

GİRİŞ

Tiroid hormonlarının genomik ve genomik olmayan etkileri vardır. Tiroid hormonları genomik olmayan etkisiyle hücre dışı ile hücre içi ortam arası iletişimde görev alan özelleşmiş membran proteinleri monokarboksilat transporter 8 ve integrin αVβ3 ile etkileşime geçerek, hücre içi PI3K-Akt/Gsk3β ve Mapk sinyal yolaklarının aktivasyonuna neden olur. Tiroid hormonlarının genomik olmayan etkileri kısa sürelidir^1-4, tiroid hormonunun esas etkisi genomik olup, nukleusta yer alan reseptörlerine bağlanır ve çeşitli transkripsiyon olaylarını başlatır ve hücrenin biyolojik fonksiyonlarını doğrudan etkiler¹. Hormon seviyesindeki değişiklikler sinaptik plastisite ilişkili ve nörodejenatif hastalıklarla ilişkili genlerin ekspresyon düzeyini etkileyebilir. Nörodejeneretif hastalıkların çoğunluğunun altında yatan neden gen ifadesinde meydana gelen bozukluklara dayanmaktadır. Son yıllarda nörodejeneratif hastalıkların gelişme nedenlerini açıklama amaçlı yapılan çalışmalar, gen ifadesindeki değişimler üzerine yoğunlaşmıştır^5,6. Hipokampüs bu hastalıklardan etkilenen en önemli beyin bölgelerinden biri olarak kabul edilir. Laboratuvarımızda yapılan daha önceki çalışmalarda tiroid hormon yüksekliği ya da azlığının hipokampal sinaptik plastisite formları üzerinde önemli değişiklikler meydana getirdiği belirlenmiştir^7,8.

Uzun dönemli güçlenme (UDG), presinaptik bir nöron veya sinir yoluna gelen nöral aktivite frekansının artışına bağlı olarak sinaptik bağlantı gücünün ve sayısının kalıcı olarak artırılması anlamına gelmektedir ve üzerinde en çok çalışılan ve bilgi sahibi olunan sinaptik plastisitenin bir formudur. Sinaptik plastisite konusunda giderek artan sayıdaki çalışmalarda, öğrenme sürecinde UDG’nin sinaptik bağlantıları güçlendirmekle birlikte hipokampüsteki yeni nöronların oluşumunu da teşvik ettiği gösterilmiştir^9,10. UDG, kritik bazı kinazların fosforilasyonunu gerektirir¹¹. Moleküler düzeyde kinaz veya fosfataz aktivitesindeki artışın etkisi nispeten kısa sürelidir ve bu değişikliklerin daha uzun idame ettirilmesi, gen transkripsiyonunu ve nöronal proteinlerin sentezini gerektirir¹².

Öğrenme ve belleğin altında yatan sinaptik plastisite formlarının indüklenmesini takiben belirli genlerin ekspresyonundaki değişiklikler, bu plastisite formlarının protein sentezi üzerinden kalıcı hale gelmesini sağlar¹³. Bununla birlikte, ekspresyonu değişen genlerin oluşturduğu protein ürünlerinin tiroid hormonu azlığı ya da fazlalığında az ya da çok sentezlenmesi bazı nörodejeneratif hastalıklarla ilişkili olan tau ve amiloid beta proteinlerinin farklı düzeylerde sentezlenmesine ve farklı düzeylerde fosforillenmesine neden olabilir. Bunun sonucunda demansla karakterize Alzheimer hastalığı benzeri nörodejeneratif hastalıkların görülme sıklığı artabilir.

Çalışmamızda, hipertiroidi oluşturulan sıçanların hipokampüsünde UDG’nin indüklenmesi ile birlikte plastisite indüksiyonuna eşlik eden, nörodejenearatif proteinlerle bağlantılı olan, amiloid prekürsör protein (APP) ilişkili (Bace1 ve Psen2) ve tau fosforilasyonuyla ilişkili proteinlerin (Gsk3β, Cdk5, Akt1ve Mapt, P35(Anxa), Capn1) mRNA düzeyindeki değişiminin araştırılması amaçlanmıştır.

METARIALS AND METHODS

Experimental Animals

This study was carried out within the scope of the project numbered TDK-2019-9405 supported by Erciyes University Scientific Research Projects Unit and with the approval of Erciyes University Animal Experiments Ethics Committee dated 17.07.2019 and numbered 19/133. For the study, 12 Wistar albino male rats, weighing 180-200 gr, produced in Erciyes University Hakan Çetinsaya Experimental and Clinical Research Center, were used. Ethical principles were taken into account in the study in order not to use unnecessary experimental animals and not to cause pain on experimental animals. Rats were divided into two groups, as the control group (n=6) and the hyperthyroidism group (n=6).

Creating Hyperthyroidism

To induce hyperthyroidism (n=6), 39-day-old animals were given 400 µg/kg/day L-thyroxine (Sigma, catalog number: T2501-5G, 0.5 mL) intraperitoneally (ip) for 21 days¹⁴. The control group (n=6) rats were given saline ip in the volumes given to the experimental group rats for 21 days starting from the 39^th day.

Electrophysiological Recording

After rats were anesthetized with urethane (1.2 g/kg), they were placed in stereotaxis using ear and mouth sticks. The stimulating electrode was lowered into the hippocampus according to the coordinates of anteroposterior (AP): 6.5 and mediolateral (ML): 3.8 mm, and the recording electrode according to the coordinates of AP: 3 mm ML: 2.13 mm, and the two-channel glass micropipette filled with 3 M NaCl was lowered into the hippocampus until a typical response was observed.

The perforating pathway was stimulated with stimuli varying in the range of 0.1-1.5 mA with the stimulus electrode, and the stimulus intensity (test stimulus intensity), which constituted half of the maximum intensity of the responses received from the dentate gyrus granule cells, was determined by input/output experiments. The basal record of the experiment was recorded for 15 minutes. During the basal recording, the perforating pathway was stimulated with this determined stimulus intensity every 30 seconds, and the excitatory postsynaptic potential (EPSP) slope, field potentials in the dentate gyrus granule cell layer, and population spike (PS) amplitude, were recorded for 15 minutes. For the LTP response, a stimulus was given every 30 seconds, followed by a 15-minute basal recording, and by induction with high-frequency stimulation (HFS) at 100 Hz for 1 second at 5 minutes intervals (15 minutes in total). EPSP slope and PS amplitude were recorded by continuing the stimulation with a test stimulus every 30 seconds for a total of 60 minutes following HFS stimulation.

After the electrophysiological recordings were taken, the blood of the rats were taken intracardiacly, then they were decapitated, and their brains removed, and their hippocampi were isolated. The collected blood was centrifuged and the plasma was stored at -20 °C until the study day. Isolated hippocampi were stored at -80 °C until real-time PCR (rt-PCR) analysis. The level of fT4 in plasma and mRNA levels of APP-related (Bace1 and Psen2) and tau phosphorylation-related proteins (Gsk3β, Cdk5, Akt1 P35(Anxa), Calpn1 and Mapt) in hippocampus tissue were measured.

Measuring Plasma Free T₄ Levels

After collecting plasma samples from all groups, plasma fT4 levels were measured in a plate reader (Multiskan^TM FC Microplate Photometer) with a commercial ELISA kit (Cloud Cone Corporation, USA).

Real-Time-PCR Measurements

RNA Isolation from Hippocampus Tissue

Hippocampus tissues taken into tubes each containing 1000 µL of RNA isolation solution (PureZOL™: BioRad, USA) were homogenized with the help of a homogenizer. They were then incubated at room temperature for 5 min. 400 µL of chloroform was added to them and vortexed for 15 seconds. They were incubated for 15 min at room temperature and centrifuged at 14000 g for 20 min at +4 °C. The aqueous phase formed at the end of centrifugation was taken into a new eppendorf tube. 300 µL of isopropanol was added to it (depending on the amount of aqueous phase) and inverted several times. It was incubated at room temperature for 10 minutes and centrifuged at 14000 g for 30 minutes at +4 °C. The supernatant obtained at the end of centrifugation was discarded. 1 mL of 75% ethanol was added to the pellet portion at the bottom and vortexed. Afterwards, it was centrifuged at 7500 g for 5 min at +4 °C. The supernatant formed at the end of centrifugation was discarded and the ethanol around the pellet was pipetted and cleaned. The pellet was dried at room temperature for 10-15 minutes until the ethanol evaporated. After drying, 20 µL of NFW (Nuclease Free Water) was added and the pellet was resuspended. It was then placed in ice and kept in a refrigerator at +4 °C for 10-15 minutes. At the end of this period, RNA concentration (ng/µL) was measured in the nanodrop.

cDNA Synthesis

Before starting the study, all RNA samples from rats were numbered and these numbers were written on the PCR tubes. The iScrip^TMcDNA synthesis Kit (1708890, BioRad, USA) was used for cDNA synthesis. cDNA synthesis was performed according to the iScrip^TMcDNA Kit protocol. In order to create a reaction with equal amount of RNA in each sample, the amounts of RNA and water to be added were calculated according to the RNA quantification results. Desired amount of NFW and RNA samples were added to the PCR tubes with a total volume of 15 µL. Afterwards, 5 µL of 5X reaction buffer (4 µL) and Reverse Transcriptase (1 µL) mixture was dispensed into each PCR tube and the tubes were placed in the PCR device (CFX Connect rt-PCR Detection System BioRad). At the end of PCR, 20 µL of cDNA product was formed. In the PCR device (CFX Connect rt-PCR detection System, BioRad), it was kept for the steps of the incubation, denaturation and extension and annealing for 5 minutes at 25 °C, 30 minutes at 42 °C and 5 minutes at 85 °C, respectively.

mRNA Expression Study

The mRNA expressions of Gsk3β, Akt1, Cdk5, Mapt, P35(Anxa), Calpn1, Bace1, and Psen2 genes were studied on the BIORAD CFX Connect rt-PCR instrument (BIORAD, USA), using the SsO Advanced Universal IT SYBR Green Supermix kit (10000076382, BIORAD, USA). A reaction mixture consisting of SYBR Green Supermix (10 µL), Nuclease Free Water (8 µL), Primer F (0.5 µL) and Primer R (0.5 µL) included in the kit was prepared and dispensed into the wells in on a 96-well plate, and cDNA (1 µL) was added to each well. Then, it was placed in the CFX Connect rt-PCR Detection System PCR device.

PCR was held at 95 °C for 3 min, at 95 °C for 5 sec, and at 60 °C for 15 sec (45 cycles) and at 40 °C for 30 sec for pre-incubation, amplification, and cooling conditions, respectively. All samples were duplicated in order to avoid errors that might arise due to manipulation. β-Actin gene was used as house-keeping gene. Ct (thresholdcycle) values obtained at the end of the process were calculated using the 2^-∆Ct method and normalized.

Statistical Analysis

The slope of the EPSP wave was calculated as 20-80% of the voltage difference between the beginning of the wave and the beginning of the PS wave. PS amplitude was calculated as the mean of the first negative spike and the next positive spike. The mean slope and amplitude values of EPSP and PS of 30 field potentials triggered during the initial 15-minute baseline recording time were accepted as 100. The averages of EPSP slope and PS amplitude values were calculated from 10 field potentials recorded in the first 5-minute period after HFS and in the last 5-minute period at the end of the experiment.

A single sample t-test was used to compare the values obtained within a group with the baseline values. The non-parametric Mann-Whitney U test was used to determine whether there was significance between the groups. Significance level was accepted as p<0.05.

Non-parametric Mann-Whitney U test was employed to determine whether there was significance between groups in fT4 level analysis. Significance level was accepted as p<0.05.

The conformity of the data obtained with rt-PCR to normal distribution was evaluated with histogram, q-q graphs and Shapiro-Wilk test, and homogeneity of variance was evaluated with the Levene’s test. Analysis of the data was performed using GraphPad Prism software. Statistical significance level was accepted as p<0.05. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (SPSS, Chicago, IL).

RESULTS

Plasma Free T₄ Level

Plasma fT4 levels of rats belonging to control and hyperthyroidism groups were measured after electrophysiological recording (Figure 1). In the L-thyroxine group, higher plasma fT4 level was measured than in the control group (p<0.004). This result is an indication of the development of hyperthyroidism in rats.

Analysis of Input/Output Curves

The effect of L-thyroxine administration on the basal activity of synapses and neurons before plasticity induction was evaluated with input-output curves. The mean and standard error values of EPSP slope and PS amplitude measured against stimulus intensity were given graphically (Figure 2D; Figure 3D).

Baseline efficacy, ANOVA test results, with repeated measurements of input/output curves obtained by applying pulses of varying intensity between 0.1 mA-0.5 mA before HFS, revealed a significant increase in EPSP slopes (F_7.70=6.017; p<0.001 Figure 2D) and PS amplitudes (F_7.70=13.460; p<0.001 Figure 3D) as the stimulus intensity increased, as expected. Insignificant interaction between EPSP slope (F_7.70=1.32; p=0.27) and PS amplitude (F_7.70=0.46; p=0.515 after lower-bound correction) and stimulus intensity showed that this increase was similar in all groups. The group effect was not found to be significant for EPSP slope (F_1.10=0.33; p=0.577) and PS amplitude (F_1.10=2.65; p=0.134). These findings indicate that the efficacy of the perforating pathway-dentate gyrus synapses before the triggering of LTP with HFS was similar in the control and hyperthyroid groups.

The Effect of HFS on EPSP and PS

LTP responses were measured using EPSP slope and PS amplitude. The 5-minute period (15^th min-20^th min) after induction was called the post-HFS period, and the 5-minute period between 70^th and 75^th minutes at the end of the experiment was called the maintenance period.

With single-sample t-test, EPSP slopes of the control (154.2±16.0%, t₅=8.261, p=0.000; Figure 2B) and hyperthyroidism (148.5±18.2%, t₅=5.993, p=0.002; Figure 2B) groups were detected to be statistically higher after HFS compared to the recording of baseline period. During the maintenance period, EPSP slopes were found to be statistically significantly higher in the control group (119.3±9.0%, t₅=5.202 p=0.003, Figure 2C) compared to baseline recording. No statistically significant difference was found in the hyperthyroidism (109.0±12.5, t₅=1.765 p=0.138, Figure 2C) groups according to baseline recording. This finding shows us that LTP response did not occur in the synaptic component in the hyperthyroidism group.

When the differences between the groups were examined with the Mann-Whitney U test for EPSP slope, there was no statistically significant difference in EPSP slope between the groups in the post-HFS period (Z=-0.801 p=0.4) and the maintenance period (Z=-0.601 p=0.1).

When PS amplitude was evaluated considering baseline values, with single sample t-test, PS amplitudes of the control (271.0±62.4%, t₅=6.716, p=0.01; Figure 3B) and hyperthyroidism (213.47±23.2%, t₅=11.967, p=0.00; Figure 3B) groups in the post-HFS period were found to be statistically higher compared to the baseline recording. In the maintenance period, PS amplitudes of the control (212.3±21.9%, t₅=12.536, p=0.00; Figure 3C) and hyperthyroidism (182.5±35.3, t₅=5.723, p=0.02 Figure 3D) groups increased significantly compared to the baseline recording.

When the differences between the groups were examined for PS amplitude with the Mann-Whitney U test, no statistically significant difference was found between the groups in PS amplitude in the post-HFS period (Z=-0.922 p=0.055) and in the maintenance period (Z=-0.281 p=0.2).

Analysis of Real-Time PCR Outcomes

The mRNA expression levels of tau protein and amyloid beta precursor protein-related genes in the hippocampus, which were removed at least 60 minutes after HFS, obtained by rt-PCR method are graphically shown in Figure 4.

According to the Mann-Whitney U test, there was a statistically significant difference between the groups in the expression levels of tau-associated Akt1-mRNA (p=0.02), Cdk5-mRNA (p=0.03), and Mapt1-mRNA (p=0.006), while no statistically significant difference was found in the expression levels of tau-associated Gsk3β - P35(Anxa)-mRNA and Capn1-mRNA expression and amyloid beta precursor protein-associated Bace1-mRNA and Psen2-mRNA (p>0.05).

DISCUSSION

Changes in LTP and Gene Expression in Thyroid Hormone Excess

Tetanic stimulation with a frequency of one hundred Hz and a duration of 1 second can be applied as a series or as four repetitive sequences as in our study. Powerful four-repeat induction protocols are often preferred because they cause increased transcription factors and gene expression, and thus have greater potential for longer persistence of LTP. In our study, the fact that both the EPSP slope and PS amplitude were significantly higher in the control (euthyroid) group 1 hour after the application of the induction protocol indicates that this protocol is sufficient to induce a permanent LTP (Figures 2, 3). However, this protocol, which was able to induce LTP lasting at least 1 hour in rats in the control group, was able to induce a non-permanent synaptic LTP in the hyperthyroid group. There was an increase in PS amplitude 1 hour after induction in both control and hyperthyroid groups. These findings, which are consistent with the previous findings of our group^7,15,16, can be interpreted as the effect of hyperthyroidism is limited to the synapse level.

While mRNA expression levels of Gsk3β, P35 (Anxa), Capn1, Bace1 and Psen2 genes evaluated in the hippocampus tissue after LTP recording did not differ significantly between the groups, Akt1, Cdk5 and Mapt-mRNA expression levels were increased in the hyperthyroidism group compared to the control group.

The absence of a difference between the groups in the mRNA levels of Gsk3β, P35 (Anxa), Calpn1, Bace1 and Psen2 genes in hyperthyroid rats that did not show a permanent synaptic LTP 1 hour after HFS indicates that the products of these genes cannot be responsible for the deterioration in LTP.

Gsk3β is an important kinase that exhibits persistent activity under basal conditions and is inhibited by phosphorylation from the ser9 position by many kinases, including Akt¹⁷. Gsk3β ser9 phosphorylation, which occurs with the increased activity of the PI3K-Akt pathway during HFS, inhibits this kinase and promotes the formation of LTP, while preventing the formation of UDB¹⁸. In other words, the formation of LTP (with the effect of Akt1) requires Gsk3β to remain inactive, while the formation of UDB (with the activity of protein phosphatases) requires it to remain active¹⁹.

Akt and Cdk5 proteins, which are the target products of Akt1 and Cdk5 gene expression, are proteins with strong tau kinase properties^20-23. Serum tau levels of hyperthyroid cases are significantly increased compared to the controls²⁴. Therefore, the increase in Akt1 and Cdk5 mRNA in hyperthyroid rats is consistent with the hyperthyroid condition having a higher risk of dementia than the euthyroid condition²⁵. The increase in LTP-related tau expression in the hyperthyroid condition may have a function that prevents the persistence of synaptic LTP and triggers neurodegenerative changes. The fact that Cdk5 down-regulation potentiated UDB by causing BDNF/CREB activation in hippocampal sections²⁶ supports the role of increased Cdk5 expression in the synaptic LTP disorder we observed in hyperthyroid rats.

Cdk5 is a neuron-specific kinase that plays important roles in many cellular functions and is mainly involved in many neurodevelopmental processes such as synaptic plasticity, learning, and memory²⁷. For Cdk5 activation, it requires the binding of p35, p39, or p25 (a proteolytic part of p35) to Cdk5²⁸. In Alzheimer’s disease, p35, which is Cdk5 activator protein, undergoes N-terminal cleavage by calpain (calcium-dependent protease) to form p25. p25 forms a stable complex with Cdk5, which retains its active state longer, leading to hyperphosphorylation of tau. p25-bound Cdk5 also acts as a priming kinase and promotes Gsk3β-mediated Tau phosphorylation²³. Although the monomeric form of Cdk5 is enzymatically inactive, it has been reported that it may cause an increase in Cdk5 tau phosphorylation and neurodegeneration in Alzheimer’s disease²⁹. Cdk5 inhibitors have been shown to protect hippocampal neurons against both abnormal tau phosphorylation and neuronal death²⁷. In addition, some study findings show that Cdk5 activity facilitates LTP by increasing the number of NMDA receptors NR2A and NR2B³⁰. Therefore, apart from the expression level of Cdk5, how the effect of thyroid hormones will change the activity of this kinase should also be investigated.

β-secretase, known as Bace1, is one of the first enzymes involved in the APP cleaving, and this is a neurotoxic abnormal cleaving. At the end of this cleaving, amyloid beta peptide production occurs and contributes to the development of AH³¹. However, it has been reported that Bace1 deficiency impairs the formation of LTP by causing a decrease in synaptic proteins (mGluR1 and postsynaptic PSD-95 proteins). In other words, it has been reported that inhibition or deficiency of Bace1 causes a decrease in glutamate (Glu) release with a decrease in the distribution of synaptic vesicles to the active site, and thus a weaker activation of NMDA and AMPA receptors on the postsynaptic membranes of a small number of glutamate, thereby impairing the formation of LTP³². On the other hand, it has been reported that Gsk3β activation increases Bace1 gene expression and increases amyloid peptide formation¹⁷. In our study, while there was no significant difference in Gsk3β-mRNA expression level between the control group and the hyperthyroidism group, there was no difference between both Bace1 and Psen2 genes.

Study Limitations

The following limitations should be taken into account when interpreting the results of our study, which examined the changes in the expression of genes related to neurodegeneration after LTP in the hippocampus of hyperthyroid rats: compared to the control group, measurement of plasma free T4 level in the L-thyroxine group as higher confirms that hyperthyroidism has occurred. Although changes in free T3 and TSH levels, consistent with the change in T4 level, are expected, plasma fT3 and TSH levels could not be measured (due to limited financial support). The real-time quantitative PCR method used in the study only gives information about the mRNA levels of the measured proteins. It does not provide information about the loss, destruction or activity of protein that may occur while being synthesized. For this reason, there is a need for further studies in which protein analyses will be performed.

TARTIŞMA

Tiroid Hormon Fazlalığında UDG ve Gen Anlatımındaki Değişiklikler

Yüz Hz frekanslı 1 sn süreli tetanik uyarım bir dizi halinde veya çalışmamızda olduğu gibi tekrarlayan dört dizi halinde uygulanabilmektedir. Dört tekrarlı güçlü indüksiyon protokolleri, transkripsiyon faktörlerinin ve gen anlatımının artışına neden olmasından ve böylece UDG’nin daha uzun süre kalıcı olmasını sağlama potansiyeli daha fazla olduğundan dolayı sıklıkla tercih edilmektedir. Çalışmamızda indüksiyon protokolü uygulamasından 1 saat sonra hem EPSP eğiminin hem de PS genliğinin kontrol (ötiroid) grubunda indüksiyondan önceki değere göre anlamlı derecede daha yüksek olması, bu protokolün kalıcı bir UDG indüklemek için yeterli olduğunu göstermektedir (Şekil 2, 3). Ancak kontrol grubu sıçanlarda en az 1 saat süren UDG indükleyebilen bu protokol hipertiroidi grubunda kalıcı olmayan bir sinaptik UDG indükleyebilmiştir. PS genliğinde indüksiyondan 1 saat sonra hem kontrol hem de hipertiroidi grubunda artış görülmüştür. Grubumuzun önceki bulguları^7,15,16 ile uyumlu olan bu bulgular, hipertiroidinin etkisinin sinaps seviyesi ile sınırlı kaldığı şeklinde yorumlanabilir.

UDG kaydı sonrasında hipokampüs dokusunda değerlendirilen Gsk3β, P35 (Anxa), Capn1, Bace1 ve Psen2 genlerinin mRNA ekspresyon düzeyleri gruplar arasında anlamlı farklılık göstermezken Akt1, Cdk5 ve Mapt-mRNA ekspresyon düzeylerinde kontrol grubuna göre hipertiroidi grubunda artış bulunmuştur.

YFU’dan 1 saat sonra kalıcı bir sinaptik UDG göstermeyen hipertiroidili sıçanlarda Gsk3β, P35 (Anxa), Calpn1, Bace1 ve Psen2 genlerinin mRNA düzeylerinde gruplar arası farklılık bulunmaması bu genlerin ürünlerinin UDG’deki bozulmadan sorumlu olamayacağına işaret etmektedir.

Gsk3β, bazal koşullarda süre-giden bir aktivite gösteren ve aralarında Akt’nin de olduğu pek çok kinaz tarafından ser9 pozisyonundan fosforillenerek inhibe edilen önemli bir kinazdır¹⁷. YFU sırasında PI3K-Akt yolağının artmış aktivitesi ile oluşan Gsk3β ser9 fosforilasyonu, bu kinazı inhibe ederek UDG oluşumunu teşvik ederken UDB oluşmasını engellemektedir¹⁸. Bir başka ifade ile UDG oluşumu (Akt1’in etkisi ile) Gsk3β’nın inaktif durumda kalmasını, UDB oluşumu ise (protein fosfatazların aktivitesi ile) aktif durumunu sürdürmesini gerektirmektedir¹⁹.

Akt1 ve Cdk5 gen anlatımının hedef ürünleri olan Akt ve Cdk5 proteinleri güçlü tau kinaz özelliği gösteren proteinlerdir^20-23. Hipertiroidi olgularının serum tau düzeyleri kontrole göre önemli derecede artış göstermektedir²⁴. Dolayısıyla hipertiroidili sıçanlarda Akt1 ve Cdk5 mRNA’nın artışı hipertiroidi koşulun, ötiroid koşula göre daha yüksek bir demans riskine sahip olması ile uyumludur²⁵. UDG ilişkili tau anlatımının hipertiroidi koşulunda artması, sinaptik UDG’nin kalıcı olmasını önleyen ve nörodejeneratif değişiklikleri tetikleyen bir işleve sahip olabilir. Cdk5 down-regülasyonunun hipokampal kesitlerde BDNF/CREB aktivasyonuna neden olarak UDB’yi güçlendirmiş olması²⁶, hipertiroid sıçanlarda gözlediğimiz sinaptik UDG bozukluğunda artmış Cdk5 anlatımının rolünü desteklemektedir.

Cdk5, birçok hücresel fonksiyonda önemli roller oynayan nörona özgü bir kinaz olduğu gibi esas olarak sinaptik plastisite, öğrenme ve bellek gibi birçok nörogelişimsel süreçte yer alır²⁷. Cdk5 aktivasyonu için p35, p39 veya p25’in (p35’in proteolitik bir parçası) Cdk5’e bağlanmasını gerektirir²⁸. Cdk5 Alzheimer hastalığında, Cdk5 aktivatör proteini p35, p25 oluşturmak için kalpain (kalsiyum bağımlı proteaz) ile N-terminal kesilmesine uğrar. p25, aktif durumunu daha uzun süre koruyan Cdk5 ile stabil bir kompleks oluşturur ve bu taunun hiperfosforilasyonuna yol açar. p25 bağlı Cdk5 ayrıca priming kinaz görevi görür ve Gsk3β aracılı Tau fosforilasyonunu destekler²³. Cdk5’in monomerik formu enzimatik olarak inaktif olmasına rağmen, Alzheimer hastalığında, Cdk5 tau fosforilasyonu ve nörodejenerasyonda bir artışa neden olabileceği bildirilmiştir²⁹. Cdk5 inhibitörlerinin, hipokampal nöronları hem tau anormal fosforilasyonlarına hem de nöronal ölüme karşı koruduğu gösterilmiştir²⁷. Ayrıca bazı çalışma bulguları Cdk5 aktivitesinin NMDA reseptörleri NR2A ve NR2B sayısını artırarak UDG’yi kolaylaştırdığını göstermektedir³⁰. Bu nedenle Cdk5’in ifadelenme seviyesinden ayrı olarak tiroid hormonlarının etkisinin bu kinazın aktivitesini nasıl değiştireceği de araştırılmalıdır.

Bace1 olarak bilinen β-sekretaz, APP kesiminde yer alan ilk enzimden bir tanesidir ve bu kesim nörotoksik anormal kesimdir. Bu kesim sonunda amiloid beta peptid üretimi oluşarak AH’nın gelişimine katkıda bulunur³¹. Fakat Bace1 eksikliğinin ise sinaptik proteinlerde (mGluR1 ve postsinaptik PSD-95 proteinleri) azalmaya neden olarak UDG’nin oluşumunu bozduğu bildirilmiştir. Yani Bace1 inhibisyonu veya eksikliğinin, sinaptik veziküllerin aktif bölgeye dağılımında azalmayla birlikte glutamat (Glu) salınımda azalmaya ve böylelikle az sayıdaki glutamatın postsinaptik zarlarda NMDA ve AMPA reseptörlerinin daha zayıf aktivasyonuna neden olarak UDG’nin oluşumunu bozduğu bildirilmiştir³². Bununla birlikte Gsk3β aktivasyonunun Bace1 gen ekspresyonunu artırarak amiloid peptid oluşumunu artırdığı bildirilmiştir¹⁷. Çalışmamızda Gsk3β-mRNA ekspresyon düzeyinde kontrol grubu ile hipertiroidi grubu arasında anlamlı farklılık bulunmamakla birlikte hem Bace1 hem de Psen2 genleri arasında da farklılık bulunmamıştır.

Çalışmanın Kısıtlılıkları

Hipertiroidili sıçanların hipokampüsünde UDG sonrası nörodejenerasyonla ilgili genlerin anlatımındaki değişikliklerin incelendiği çalışmamızın sonuçları yorumlanırken şu kısıtlılıklar göz önünde bulundurulmalıdır: Plazma serbest T4 seviyesinin, kontrol grubuna göre L-tiroksin verilen grupta yükselmiş olarak ölçülmesi hipertiroidinin oluştuğunu doğrulamaktadır. Her ne kadar, serbest T3 ve TSH seviyesinde, T4 seviyesindeki değişmeye uygun değişmeler beklense de plazma sT3 ve TSH düzeyleri (sınırlı maddi destek nedeni ile) ölçülememiştir. Çalışmada kullanılan gerçek zamanlı kantitatif PZR yöntemi sadece ölçümü yapılan proteinlerin mRNA düzeyleri hakkında bilgi verir; bu proteinin sentezlenirken oluşabilecek kaybı, yıkımı veya aktivitesi hakkında bilgi vermez. Bu nedenle ilerde yapılacak olan çalışmalar için protein analizlerinin de yapılacağı çalışmalara ihtiyaç vardır.

CONCLUSION

Physiological levels of thyroid hormones are important for the expression of some genes accompanying the induction of plasticity to remain within physiological limits. Changes in hormone level can alter mRNA expression of genes related to neuronal plasticity, depending on the induction protocol.