Effect of mPFC KLF4 on autism-like behaviors and possible mechanisms

doi:10.21203/rs.3.rs-1728882/v1

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Effect of mPFC KLF4 on autism-like behaviors and possible mechanisms

https://doi.org/10.21203/rs.3.rs-1728882/v1

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Autism spectrum disorder (ASD) is a series of neurodevelopmental disorders with two core symptoms, repetitive behavioral phenotype, and social impairments, yet the underlying cellular and molecular mechanisms are still obscure. KLF4 (Kruppel-like factor 4) is a zinc finger protein transcription factor that has been widely reported in the regulation of cell proliferation, differentiation, survival, and apoptosis. Recently, increasing evidence demonstrated its critical role in neurodevelopment, microglia activating, and neuroinflammation in the central nervous system (CNS). Little is known about the effect of KLF4 in the medial prefrontal cortex (mPFC) on autism and autism-like behaviors. Here, we found that high and low expression levels of KLF4 in mPFC induced significant social novelty impairments. The molecular mechanisms might be associated with overexpression of KLF4 in mPFC promote activation of microglia and altered synaptic E/I ratio by affecting the expression of GluA1 and GABA-related proteins, the specific molecular mechanisms still need more investigations.

KLF4

autism spectrum disorder

neuroglia

social behavior

synapse

GABA

Autism spectrum disorder(ASD) is a series of neurodevelopmental disorders with two core symptoms, repetitive behavioral phenotype and social impairments[1–3]. KLF4 (Kruppel-like factor 4) is a eukaryotic zinc finger protein transcription factor, a member of the Kruppel-like transcription factor protein family, which was first reported in 1996[4, 5]. It is widely reported that KLF4 plays a critical role in cell proliferation[6–9], differentiation[10–13], survival, and apoptosis[14–16] Significantly, Kazutoshi et al. firstly reported that KLF4 combined with three transcription factors Sox2, Oct4, and c-Myc together can induce mouse embryonic and adult fibroblasts into pluripotent stem cells and named them induced pluripotent stem cells (IPS)[17]. These results suggest a critical role of KLF4 might play in the neurodevelopmental process. Accumulating results have shown that KLF4 regulation in the central nervous system (CNS), including axon regeneration of adult retinal ganglion cells (RGCs)[18, 19], microglia activation mediated neuroinflammation process[20–22], and neurophysiology of neurodegenerative disorders[20]. So far, no evidence investigated the relationship between KLF4 and ASD.

Human and animal evidence have shown that aberrant activation of microglia and subsequent neuroinflammation process might be implicated in the ASD pathogenesis[23–25]. Dysfunction of microglia affects synaptic structure and function. Another hypothesis of ASD is that aberrant neurotransmission, especially the increased ratio of excitatory to inhibitory transmission (E/I ratio)in CNS, is the basis of hyperexcitability and excess spiking[26]. Our previous study demonstrated that KLF4 significantly prolonged the duration of sedation induced by acute pentobarbital administration, indicating a potential promotion of KLF4 in the GABA system[27]. Therefore, KLF4 is thought to be involved in ASD and autism-like behaviors. Thus, in this study, we aimed to investigate the effect of medial prefrontal cortex (mPFC) KLF4 on autistic behaviors.

Animal models were constructed by stereotactic mPFC of mice and injection of lentivirus-mediated interference/overexpression KLF4 virus. Autism-like behavioral phenotypes of mice were confirmed by a series of behavioral pharmacological experiments. The expression levels of KLF4 and downstream pstat3/stat3 signalings were verified by immunoblotting and immunofluorescence staining. We also investigated the altered expression of markers of neurons and neuroglia, excitatory and inhibitory synapse-related proteins by immunofluorescence and western blotting. Our results showed that KLF4 in mPFC plays a critical role in social novelty behaviors. Overexpression of KLF4 in mPFC promotes activation of microglia and altered synaptic E/I ratio by affecting the expression of GluA1 and GABA-related proteins. However, the specific molecular mechanisms of KLF4 involved in ASD pathogenesis need to be further investigated.

Animals

Healthy 4-week-old C57BL/6J mice (18 - 22 g) were used in the experiment and were purchased from Changchun Yis Biological Company. All animals were housed in the Experimental Animal Center of the School of Basic Medicine, Jilin University, in a standard experimental animal environment with a light/dark cycle of 12: 12 - h and constant temperature (22 ± 2 °C). All animals have free access to food and water. The experiments were approved by the Animal Ethics Committee of Jilin University.

Stereotaxic virus microinfusion

Referring to previous literature[28, 29], mice were anesthetized with pentobarbital (dissolved with saline) at a dose of 65 mg/kg and were administrated intraperitoneally at 0.01 ml per g according to the body weight. The coordinates of mPFC administration were determined by referring to the brain atlas[30] with bregma as the coordinate origin (AP: ± 1.8 mm; ML: ± 0.4 mm; DV: -1.6 mm). Overexpressed and interfered RNA virus (Genechem, China) were slowly injected at a rate of 0.5 ul/min, both sides of the mPFC were administered with 1.5 ul volume of virus. After surgery, the mice were kept in a single cage, and subsequent behavioral and molecular biology experiments were performed after three weeks of recovery.

Behavioral tests

Open-field test (OFT)

Animals were placed in a rectangular open field box of size (60 cm × 90 cm × 16 cm), divided equally into 9 sections (3 × 3). The mouse was placed in the central part, after 30 min of adaptation, the exploration, and locomotor activity were recorded with a camera for the next 10 min. The time spent in the central area was analyzed to evaluate the anxiety. The experimental data were collected and analyzed by Ethovision XT 13(Noldus, Wageningen, The Netherlands).

Light-dark box test (LDBT)

The experimental method was referred to in a previous study[31]. A chamber (20 cm × 40 cm × 20 cm) was divided equally into two parts of the same size (20 cm × 20 cm), where the dark box was covered and the light box was kept bright, and there was a 7.5 cm × 7.5 cm channel in both boxes, which allowed the mice to travel freely. The time spent in the light box and the number of entries into a lightbox of mice were recorded by a camera for 10 min.

Three-chamber sociability test (TCST)

The TCST was used to detect social exploration behaviors, social recognition, and social novelty of mice. The experimental method is based on published literature[32]. Animals were placed in the room for behavioral experiments one day before tests for adaptation. The three-box apparatus (60 cm × 90 cm × 35 cm) was divided equally into three chambers (30 cm × 60 cm × 35 cm), each chamber has a small door that could allow the animal to pass through freely. The experimental approach has three stages. The animal was put in the middle chamber and allowed to freely explore for 10 min with the doors opened in the first stage. In the second stage (social interaction), two cylindrical cages (15 cm × 10 cm) were placed in each side of the chamber, a sex and age-matched companion was placed in one cage and the other was empty. In the third phase (social novelty), a sex and age-matched stranger was put in the empty cage and the mouse in the stage 2 was used as a familiar companion. The animal was put in the middle chamber and allowed to freely explore for 10 min. The time spent in the two chambers, the sniffing time to two cages and the difference index (difference/sum) in the sniffing time were recorded and analyzed at each stage. Difference Index (DI) was defined as the difference between sniffing time/ the sum of sniffing time. The experimental data were collected and analyzed by Ethovision XT 13(Noldus, Wageningen, The Netherlands).

Marble burying test (MBT)

MBT was performed to measure the repetitive behaviors and compulsive phenotype of subject rats. 12 glass marbles (1.4 cm diameter) were placed on 5cm-depth fresh bedding in a new cage. Each mouse was placed on the bedding and allowed to freely explore for 30 min. A marble covered over more than 70% by the bedding was defined as buried marble and was counted as a buried marble.

Western blot

Western blots procedures were performed as previous studies[33]. All tissues were extracted homogenized with lysis buffer (137 mM NaCl, 20 mM TRIS, 1% NP40, 10% glycerol, 1 phenylmethylsulphonyl fluoride (PMSF), 10 μg/ml aprotinin, 1 μg/ml leupeptin, 0.5 mM sodium vanadate, 0.5mM sodium fluoride) on ice. The proteins in different samples were separated by running on 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS- PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The blots were visualized by gel imaging system (Tanon, China). The membranes were exposed to chemiluminescent detection reagents and imaged for calculation of optical density by Fiji/ImageJ. Primary antibodies used (KLF4, Abcam, ab129473, 1:1000; pstat3, CST9131s, 1:1000; stat3, CST4904s, 1:1000; GAD65, Abcam, ab26113, 1:1000; GAD67, Abcam, ab26116, 1:1000; GABAα1, Abcam, ab33299, 1:1000; GluA1, Abcam, ab109450, 1:1000; PSD95, Abcam, ab2723, 1:1000; SYN, Abcam, ab64581, 1:1000; GAPDH, tansgen, HC301-01, 1:5000). Second antibodies used (goat-anti rabbt, ZSGB-BIO, ZB2301, 1:5000; goat-anti mouse, ZSGB-BIO, ZB2305, 1:5000)

Immunofluorescence

The entire brains were carefully peeled and dehydrated after a saline and 4 % PFA (dissolved with phosphate-buffered saline) perfusion. The perfused brains were cut into 25 μm slices with Leica CM1860. Sections were washed 3 times and then blocked with 5 % goat serum (diluted with 0.1 M PBS solution) for 1h at room temperature. Sections were incubated in primary antibodies overnight at 4 ℃ and the next day incubated in secondary antibodies at room temperature after 3 times wash and then counterstained with DAPI (rocche236276, 1ug/ml) for 5 min. After sealed under glycerin, sections were detected with a fluorescence microscope (IX73/BX51W1, Olympus). Two mice from each group and 2-3 sections from each brain were selected for statistics. Primary antibodies used (Neun, Abcam, ab104224, 1:500; Iba1, Arigobio, ARG63338, 1:200; GFAP, ABclonal, A0237, 1:200). Second antibodies used (IFKineTM Green donkey anti-goat, Abbkine, A24231, 1:500; IFKineTM Red donkey-anti rabbit, Abbkine, A24421, 1:500)

Statistical analysis

All data were are represented as the mean ± SEM; and the comparison of behavioral and immunoblotting results between the two groups was performed by unpaired t t-test, the staying and sniffing time of the TCST were compared by one-way ANOVA, and the post hoc Tukey test was used for post hoc comparative analysis; All data were compared using SPSS 22.0. *P < 0.05, **P < 0.01 and ***P < 0.001.

Effect of mPFC KLF4 on stat3 signaling in mice

The experimental approach and timeline of our study were showed in Fig 1A. We stereolocalized mPFC (AP: ± 1.8 mm; ML: ± 0.4 mm; DV: -1.6 mm) for targeted injection of interference RNA and overexpression RNA virus. After three weeks of recovery, the KLF4 expression of mPFC was verified by direct observation of viral immunofluorescence (Fig 1B, C). Western blotting results showed the expression of KLF4 decreased in the interference virus injection group(Ad - shKLF4) (p<0.01, Fig 1D, E) and increased in the overexpression KLF4 group(Ad - KLF4)(p＜0.05, Fig 1F, G). Due to the evidence that KLF4 exerts action in CNS mainly by regulating stat3 and the downstream transcription, we detect the expression levels of pstat3, stat3, and the ratio of pstat3/stat3 expression after virus-mediated KLF4 expression alteration. Our results showed that the mPFC of Ad - shKLF4 group mice showed an increased pstat3/stat3 ratio than the control group(t₉ = 2.640，p＜0.05, Fig 1D, E). Inversely, overexpression of KLF4 induces a reduction of the pstat3/stat3 ratio in the mPFC of mice(t₄ = 185，p＜0.05, Fig 1F, G).

【Figure 1】

Effect of mPFC KLF4 on sociability and anxiety-like behavior in mice

To explore the relationship of mPFC KLF4 and autistic-like behaviors, mainly embodied in social impairments and repetitive phenotype. We performed OFT, and LDBT to explore the activities and the anxiety-like behaviors. We found no significant difference in exploratory behaviors and time-spent in the center of mice between Ad - shKLF4 and controls in OFT (Fig 2A). Also, no significant difference in time spent in a light box and the number of light entries of mice in Ad - shKLF4 and controls in LDBT (Fig 2C). Our results showed that mice in the Ad - KLF4 group spent less time in the center part of OFT than the control group (t₁₇ = 3.162, p＜0.01, Fig 2B). Consistent with those findings, mice in the Ad - KLF4 group showed decreased time spent in the light box in LDBT comparing with the control group (t₁₈ = 2.383, p＜0.05, Fig 2D). Our results indicate overexpression of mPFC KLF4 might increase anxiety-like behaviors in mice.

【Figure 2】

Effect of mPFC KLF4 on repetitive behavior in mice

Increased repetitive behaviors are another core symptom of ASD. Thus, we performed MBT to explore the repetitive behaviors. We found no significant difference in number of buried marbles between the Ad - shKLF4 and the control group. Intriguingly, our results showed that overexpression of mPFC KLF4 reduced the number of buried marbles in MBT, indicating a reduced repetitive phenotype.

【Figure 2】

Effect of mPFC KLF4 on social behavior in mice

ASD is characterized by significant sociability impairments. TCST is a widely used experimental test for testing sociability in mice. In the social interaction stage, we found that mice in the Ad - shKLF4 group displayed a significant difference in distinguishing empty cage from a stranger mouse and that the difference index was comparable with the control group (Fig 3A). Although mice in the Ad - KLF4 group appeared to be hard to distinguish the empty cage from stranger mice, the difference index did not show statistical difference (Fig 3B). In the social novelty stage, we observed mice in the Ad - shKLF4 group did not distinguish familiar and stranger mice, thus displaying a significantly decreased difference index comparing with the controls (Fig 3C). Similarly, we observed overexpression of mPFC KLF4 also induce impairments in distinguishing familiar mouse from the stranger mouse (t₁₀ = 2.772， p＜0.05， Fig 3D). These results indicate KLF4 expression levels might be strongly implicated in the regulation of social ability, especially social novelty.

【Figure 3】

Effect of mPFC KLF4 on Neun, GFAP, and Iba1 expression in mice

Several evidence has demonstrated that KLF4 is implicated in the activation of microglia, thus we further investigate whether mPFC KLF4 is involved in sociability by affecting neuroglia. We detect the expression levels of Neun, GFAP, and Iba1 both by immunofluorescence and western blotting. Our results showed mice with interfered KLF4 expression in mPFC showed significantly reduced Neun+ cell number in mPFC (t₃₄ = 3.067，p＜0.01, Fig 4A), comparable numbers of GFAP and Iba1-positive cells in mPFC (Fig 4B, C). Western blotting results showed that Ad - shKLF4 mice have significant increment on Neun expression (t₁₁ = 2.6077, p＜0.5, Fig 4D) and reduction on Iba1 (t₁₁ = 2.878, p＜0.5, Fig 4D) and GFAP expression (t₁₁ = 2.414, p＜0.5, Fig 4D).

Immunofluorescence results showed that mice with mPFC KLF4 overexpression displayed decreased number of Neun+ cells (t₁₀ = 2.411, p＜0.05, Fig 5A), relatively increased number of Iba1+ cells (t₁₀ = 2.411, p＜0.05, Fig 5B) and comparable number of GFAP+ cells. We observed that mice with KLF4 overexpression in mPFC have significantly reduced expression of Neun (t₂₇= 2.861, p＜0.01, Fig 5D) and increased expression of Iba1 (t₂₇ = 2.861, p＜0.01, Fig 5D), while no significant changes in GFAP levels were detected (Fig 5D). These results indicated that altered KLF4 expression levels affect the number of microglia and GFAP in mPFC and whether the consequently altered ratio of Neun to neuroglia implicated in regulation of KLF4 in sociability needs to be further investigated.

【Figure 4, 5】

Effect of mPFC KLF4 on the expression of synapse-related and GABA-related protein in mice

Some evidence has demonstrated that KLF4 regulates microglial activation and polarization. KLF4 is highly expressed in neural precursor cells and immature neurons and decreased with differentiation. Whether KLF4 can exert a regulatory effect on a mature neuron or indirectly affect neuron function by neuroglia is obscure. Our results suggest that mPFC KLF4 affects the expression of specific microglia marker, iba1, and mature neuron marker, Neun. Immunofluorescence histochemical double-stain results showed that mPFC KLF4 of overexpressed KLF4 mouse can label with both iba1(Fig 6A) and Neun(Fig 6B), indicating KLF4 might direct regulate neurons. Previous studies have shown that synaptic-related proteins are closely related to cognitive function, thus we measured the expression of GluA1, SynapsinΙ, and PSD95 after interference with mPFC KLF4 expression. The results showed that GluA1 expression was significantly increased after interference with mPFC KLF4 expression (t₁₀ = 2.249, p<0.05, Fig 6C), and the expression levels of SynapsinΙ and PSD95 were not significantly changed. We next measured the expression of mPFC GluA1, SynapsinΙ, and PSD95 in mice with KLF4 overexpression in mPFC. Our results showed that the expression of mPFC GluA1 was significantly lower in the Ad - KLF4 group than in the control group (t₁₀ = 2.24958, p<0.001, Fig 6D). The results were similar to those obtained by interfering with the expression of mPFC KLF4, the expression of synapsin Ι was not significantly changed in Ad - KLF4 comparing with the control group. To further investigate the effect of KLF4 on excitatory and inhibitory synaptic transmission, we next compared the expression of two groups of GABA-related proteins, glutamic acid decarboxylase (GAD), a key enzyme in GABA synthesis, and γ-aminobutyric acid A receptor α1 isoform (GABAARα1), a subunit type that constitutes the GABA-A receptor. Our results showed that interference with mPFC KLF4 expression resulted in no significant changes in GAD65, GAD67, and GABAAR α1(Fig 6C). The expression of GAD65 of mice with mPFC KLF4 overexpression was upregulated with statistically significant differences (t₁₀ = 2.333, p＜0.05, Fig 6D), while the GAD67 expression was not significantly altered, the GABAα1 expression was also significantly upregulated (t₁₀ = 2.48, p＜0.05, Fig 6D), these results suggest that mPFC KLF4 overexpression may promote inhibitory neurotransmission by promoting GABA synthesis and upregulating GABA receptor expression.

【Figure 6】

KLF4 (Kruppel-like factor 4) is a eukaryotic zinc finger protein transcription factor, a member of the Kruppel-like transcription factor protein family, which was first reported in 1996[4, 5]. KLF4 is reported expressed in peripheral tissues, including epithelial cells of oral, respiratory, and gastrointestinal tracts[34, 35], vascular endothelial cells, and mucosal cells of conjunctiva and cornea[36]. It is widely reported that KLF4 plays a critical role in cell proliferation[6–9], differentiation[10–13], survival, and apoptosis[14–16] Recently, several evidence have shown that KLF4 is involved in axon regeneration of adult retinal ganglion cells (RGCs) [18, 19]and microglia activation mediated development of neuroinflammation[20–22], and neurophysiology of neurodegenerative disorders[20]. Significantly, Kazutoshi et al. firstly reported that KLF4 combined with three transcription factors Sox2, Oct4, and c-Myc together can induce mouse embryonic and adult fibroblasts into pluripotent stem cells and named them IPS[17]. These results suggest a critical role of KLF4 might play in the neurodevelopmental process. ASD is a series of developmental disorders characterized by repetitive behaviors and social impairments. So far, no evidence has investigated the relationship between KLF4 and ASD. Thus, we aimed to investigate the effect of mPFC KLF4 on autistic behaviors. In our study, we found that both overexpressed or interfered with KLF4 expression in mPFC can induce social novelty impairments. Consistent with earlier studies, mPFC KLF4 appears to positively regulate markers of neuroglia, including microglia and astrocytes. Altered expression levels of KLF4 also affect GluA1 expression, KLF4 overexpression upregulated GAD65 and GABAA α1, these might be involved in the molecular mechanisms of KLF4 regulation in social impairments.

In our study, we found both overexpressed and interfered KLF4 expression of mPFC induce significant social novelty impairments in TCST. Only overexpression of KLF4 in mPFC induces anxiety-like phenotype in OFT and LDBT. The number of buried marbles in MBT could also be used as a measurement of anxiety behaviors[37]. However, our results showed a significant reduction of buried marbles of mice with mPFC KLF4 overexpression in MBT. Some evidence suggests that the marble-burying behavior in mice is a stereotypical repetitive behavior and cannot reflect novelty-induced anxiety[38]. Thus, our results showed a reduced repetitive behavior in overexpressed KLF4 mice, this is inconsistent with the social impairments observed in overexpressed KLF4 mice. We presume that mPFC KLF4 might be involved in the molecular mechanism of social regulation, especially social novelty behaviors, due to the evidence showing that mPFC might be involved in the regulation of social novelty by an independent molecular mechanism[39, 40].

In consideration of that KLF4 play a critical role in microglia activation in CNS, we further investigate the effect of altered KLF4 expression in mPFC on neurons and neuroglia using immunofluorescence and western blot. Our results showed that both immunofluorescence and western blotting results showed interfered expressed mPFC KLF4 increased Neun expression and overexpression KLF4 in mPFC decreased Neun levels. Conversely, we observed a significant increment of iba1 expression in mPFC of KLF4 overexpressed mice, significantly reduced protein expression levels in mPFC of KLF4 interfered-expressed mice while no significant changes in immunofluorescence. These results suggest that altered KLF4 expression in mPFC causes an imbalance in the ratio of mature neurons to neuroglia in adult mice. Although we obtained a significant difference in Neun expression when mPFC KLF4 expression levels were upregulated or downregulated, considering that most mature prefrontal neurons exclusively originated from the early developmental period, only about 3% of precursor cells of adult mouse mPFC can differentiate into neurons, and about 15% of precursor cells can differentiate into neuroglia[41], thus the significantly altered Neun levels in adult mice might be the subsequent result of altered expression of markers of neuroglia. The decrement of Neun expression with KLF4 overexpression in mPFC might be due to KLF4 activating apoptosis signaling in neurons, further investigation is needed.

Several evidence from in vivo and in vitro studies have shown that cytokines and LPS stimulate KLF4 expression[42–45]. IL1β induces KLF4 expression by acting on specific IL1β-R to activate PI3K / Akt pathway [44]. LPS increased KLF4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of KLF4 blockade the LPS stimulation in pro-inflammatory cytokines TNF-α, MCP-1, IL-6, iNOS, and Cox-2 expression[42]. These results suggest a critical role of KLF4 in positively regulating the activation of microglia and subsequent release of pro-inflammatory factors. It was widely acknowledged that microglia activation is accompanied by increased iba1 expression and morphological alterations, such as increased soma area and reduced branching[46, 47]. In our study, we observed significantly increased iba1 expression levels in mice with mPFC KLF4 overexpression, indicating an activated state of microglia. However, we did not focus on the morphological alterations in immunofluorescence results. In addition, it was found that Aβ stimulates KLF4 expression and KLF4 silencing in BV2 cells attenuates the release of tumor necrosis factor-a (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)[20]. These results indicate that KLF4 acts as a critical modulator involved in neurological disorders based on neuroinflammation regulation. Whether the regulation of KLF4 in social behavior depends on microglia-mediated neuroinflammation need to be further investigated. KLF4 is highly expressed in neural precursor cells and immature neurons and decreased with differentiation. Whether KLF4 can exert a regulatory effect on a mature neuron or indirectly affect neuron function by neuroglia is obscure. Our results suggest that mPFC KLF4 of the adult brain can be labeled with both iba1 and Neun, indicating KLF4 might directly regulate neurons.

Accumulating evidence has shown that the imbalance of excitatory/inhibitory neurotransmission plays a key role in the pathogenesis of psychiatric and neurological disorders, including schizophrenia[48, 49], autism[50, 51], and epilepsy [52, 53]. Cognitive and social-behavioral regulation is also closely related to excitatory and inhibitory transmission[49, 54]. PSD95 is a postsynaptic scaffolding protein in excitatory neurons and affects synaptic strength and stability[55]. Synapsins encode neuronal phosphoproteins and are mainly related to synaptic vesicle transport and neurotransmitter release, synapsinⅠis a member of the synapsins family [56]. GluA1, AMPAR receptor subunit 1, has been shown to underlie the molecular mechanism of LTP, a process depicting intracellular transport of AMPAR to the postsynaptic membrane based on GluA1 rapidly accumulation at the postsynaptic membrane[57, 58]. It was found that dysfunction of trafficking of GluA1 during LTP directly relates to psychiatric symptoms. Our result showed overexpressed KLF4 decreased GluA1 protein levels in mPFC, conversely increased when knockdown of KLF4, while no significant changes were observed in the expression of synapsinⅠ and PSD95. Some evidence showed that FMRP promotes synaptic GluA1 membrane delivery and subsequent excitatory neuronal maturation[59]. Restoration of synapse expression of AMPA GluA1 alleviated cognitive and spine abnormalities in Fmr1 KO mouse[60]. These results indicate dysfunction of synaptic GluA1-mediated synaptic transmission related to the neuropathology of ASD which mPFC KLF4 might be implicated in.

With regard to GABA-related signaling proteins, knockdown with KLF4 in mFPC did not alter the expression of GAD65, GAD67, and GABAA α1, overexpression of KLF4 promote GAD65 and GABAA α1 levels. Our previous study demonstrated that hypothalamic overexpression of KLF4 significantly prolonged the duration of sedation induced by acute pentobarbital administration, with increased c-Fos expression in the sleep-related nuclei, the lateral hypothalamic nucleus(LH), and the ventral preoptic nucleus (VLPO), and decreased c-Fos expression in the wakefulness-related nucleus TMN[27], indicating KLF4 overexpression can enhance the activation of inhibitory neurotransmission by pentobarbital. Our results indicate KLF4 might promote the GABA system and alter the synaptic E/I ratio by affecting the secretion of GABA and expression levels of GABA receptors and this also further confirmed our previous findings.

In summary, we found that KLF4 in mPFC plays a critical role in social novelty behaviors. Both high and low expression levels of KLF4 induce significant behavior impairments in social novelty. The molecular mechanisms might be associated with overexpression of KLF4 in mPFC promote activation of microglia and altered synaptic E/I ratio by affecting the expression of GluA1 and GABA-related proteins. Specific molecular mechanisms need more investigations.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All samples were obtained under Institutional Review Board approved protocols. The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Jilin university.

Consent for publication

Not applicable

Availability of data and materials

All data are available from corresponding author by request.

Competing interests

The authors declare no competing financial or non-financial interests.

FUNDING INFORMATION

National Natural Science Foundation of China (Grant Numbers: 31971078).

Author Contributions

T.T.G performed biochemical experiments and wrote the draft. H.S.Z, M.M.D performed the animal experiments. W.Y, B.J.L, and R.J.C provide the final revision.

Acknowledgements

The author(s) confirm that this article content has no conflict of interest.

Baieli S, Pavone L, Meli C, Fiumara A, Coleman M (2003) Autism and phenylketonuria. J Autism Dev Disord 33(2):201–204. https://doi.org/10.1023/a:1022999712639
Folstein SE (1985) Genetic aspects of infantile autism. Annu Rev Med 36:415–419. https://doi.org/10.1146/annurev.me.36.020185.002215
Khemir S, Halayem S, Azzouz H, Siala H, Ferchichi M, Guedria A, Bedoui A, Abdelhak S, Messaoud T, Tebib N, Belhaj A, Kaabachi N (2016) Autism in Phenylketonuria Patients: From Clinical Presentation to Molecular Defects. J Child Neurol 31(7):843–849. https://doi.org/10.1177/0883073815623636
Riverso M, Montagnani V, Stecca B (2017) KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth. Oncogene 36(23):3322–3333. https://doi.org/10.1038/onc.2016.481
Yet SF, McA'Nulty MM, Folta SC, Yen HW, Yoshizumi M, Hsieh CM, Layne MD, Chin MT, Wang H, Perrella MA, Jain MK, Lee ME (1998) Human EZF, a Krüppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains. J Biol Chem 273(2):1026–1031
Chew YC, Adhikary G, Wilson GM, Reece EA, Eckert RL (2011) Protein kinase C (PKC) delta suppresses keratinocyte proliferation by increasing p21(Cip1) level by a KLF4 transcription factor-dependent mechanism. J Biol Chem 286(33):28772–28782. https://doi.org/10.1074/jbc.M110.205245
Katz JP, Perreault N, Goldstein BG, Actman L, McNally SR, Silberg DG, Furth EE, Kaestner KH (2005) Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach. Gastroenterology 128(4):935–945. https://doi.org/10.1053/j.gastro.2005.02.022
Rowland BD, Peeper DS (2006) KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer 6(1):11–23. https://doi.org/10.1038/nrc1780
Wei D, Kanai M, Jia Z, Le X, Xie K (2008) Kruppel-like factor 4 induces p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells. Cancer Res 68(12):4631–4639. https://doi.org/10.1158/0008-5472.CAN-07-5953
Murgai M, Ju W, Eason M, Kline J, Beury DW, Kaczanowska S, Miettinen MM, Kruhlak M, Lei H, Shern JF, Cherepanova OA, Owens GK, Kaplan RN (2017) KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis. Nat Med 23(10):1176–1190. https://doi.org/10.1038/nm.4400
Stenmark KR, Mecham RP (1997) Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59:89–144. https://doi.org/10.1146/annurev.physiol.59.1.89
Swamynathan SK, Katz JP, Kaestner KH, Ashery-Padan R, Crawford MA, Piatigorsky J (2007) Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells. Mol Cell Biol 27(1):182–194. https://doi.org/10.1128/MCB.00846-06
Zheng B, Han M, Wen JK (2010) Role of Krüppel-like factor 4 in phenotypic switching and proliferation of vascular smooth muscle cells. IUBMB Life 62(2):132–139. https://doi.org/10.1002/iub.298
Li Z, Zhao J, Li Q, Yang W, Song Q, Li W, Liu J (2010) KLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involving the bcl-2/bax pathway. Cell Stress Chaperones 15(6):905–912. https://doi.org/10.1007/s12192-010-0199-5
Mohan N, Ai W, Chakrabarti M, Banik NL, Ray SK (2013) KLF4 overexpression and apigenin treatment down regulated anti-apoptotic Bcl-2 proteins and matrix metalloproteinases to control growth of human malignant neuroblastoma SK-N-DZ and IMR-32 cells. Mol Oncol 7(3):464–474. https://doi.org/10.1016/j.molonc.2012.12.002
Wang GC, He QY, Tong DK, Wang CF, Liu K, Ding C, Ji F, Zhang H (2016) MiR-367 negatively regulates apoptosis induced by adriamycin in osteosarcoma cells by targeting KLF4. J Bone Oncol 5(2):51–56. https://doi.org/10.1016/j.jbo.2016.02.002
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676. https://doi.org/10.1016/j.cell.2006.07.024
Moore DL, Blackmore MG, Hu Y, Kaestner KH, Bixby JL, Lemmon VP, Goldberg JL (2009) KLF family members regulate intrinsic axon regeneration ability. Science 326(5950):298–301. https://doi.org/10.1126/science.1175737
Qin S, Zou Y, Zhang CL (2013) Cross-talk between KLF4 and STAT3 regulates axon regeneration. Nat Commun 4:2633. https://doi.org/10.1038/ncomms3633
Li L, Zi X, Hou D, Tu Q (2017) Krüppel-like factor 4 regulates amyloid-β (Aβ)-induced neuroinflammation in Alzheimer's disease. Neurosci Lett 643:131–137. https://doi.org/10.1016/j.neulet.2017.02.017
Li T, Luo Y, Zhang P, Guo S, Sun H, Yan D, Liu X, Yang B (2020) LncRNA MEG3 regulates microglial polarization through KLF4 to affect cerebral ischemia-reperfusion injury. J Appl Physiol (1985). https://doi.org/10.1152/japplphysiol.00433.2020
Wen M, Ye J, Han Y, Huang L, Yang H, Jiang W, Chen S, Zhong W, Zeng H, Li DY (2018) Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 in cerebral edema treatment. Biochem Biophys Res Commun 499(2):345–353. https://doi.org/10.1016/j.bbrc.2018.03.161
Andoh M, Ikegaya Y, Koyama R (2020) Microglia in animal models of autism spectrum disorders. Prog Mol Biol Transl Sci 173:239–273. https://doi.org/10.1016/bs.pmbts.2020.04.012
Blaylock RL, Strunecka A (2009) Immune-glutamatergic dysfunction as a central mechanism of the autism spectrum disorders. Curr Med Chem 16(2):157–170. https://doi.org/10.2174/092986709787002745
Liao X, Yang J, Wang H, Li Y (2020) Microglia mediated neuroinflammation in autism spectrum disorder. J Psychiatr Res 130:167–176. https://doi.org/10.1016/j.jpsychires.2020.07.013
Nelson SB, Valakh V (2015) Excitatory/Inhibitory Balance and Circuit Homeostasis in Autism Spectrum Disorders. Neuron 87(4):684–698. https://doi.org/10.1016/j.neuron.2015.07.033
Cheng Z, Yang W, Li B, Cui R (2020) KLF4 Exerts Sedative Effects in Pentobarbital-Treated Mice. J Mol Neurosci. https://doi.org/10.1007/s12031-020-01680-y
Banks WA, Kastin AJ, Trentman TL, Haynes HS, Johnson BG, Galina ZH (1988) Mediation of serotonin-induced analgesia by the 5HT2 receptor in the pentobarbital anesthetized mouse model. Brain Res Bull 21(6):887–891. https://doi.org/10.1016/0361-9230(88)90022-6
Horn T, Klein J (2013) Neuroprotective effects of lactate in brain ischemia: dependence on anesthetic drugs. Neurochem Int 62(3):251–257. https://doi.org/10.1016/j.neuint.2012.12.017
Messier C, Emond S, Ethier K (1999) New techniques in stereotaxic surgery and anesthesia in the mouse. Pharmacol Biochem Behav 63(2):313–318. https://doi.org/10.1016/s0091-3057(98)00247-0
Bourin M, Hascoët M (2003) The mouse light/dark box test. Eur J Pharmacol 463(1–3):55–65. https://doi.org/10.1016/s0014-2999(03)01274-3
Servadio M, Melancia F, Manduca A, di Masi A, Schiavi S, Cartocci V, Pallottini V, Campolongo P, Ascenzi P, Trezza V (2016) Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid. Transl Psychiatry 6(9):e902. https://doi.org/10.1038/tp.2016.182
Ge TT, Yao XX, Zhao FL, Zou XH, Yang W, Cui RJ, Li BJ (2020) Role of leptin in the regulation of food intake in fasted mice. J Cell Mol Med 24(8):4524–4532. https://doi.org/10.1111/jcmm.15110
Shields JM, Christy RJ, Yang VW (1996) Identification and characterization of a gene encoding a gut-enriched Krüppel-like factor expressed during growth arrest. J Biol Chem 271(33):20009–20017. https://doi.org/10.1074/jbc.271.33.20009
Yang Y, Goldstein BG, Chao HH, Katz JP (2005) KLF4 and KLF5 regulate proliferation, apoptosis and invasion in esophageal cancer cells. Cancer Biol Ther 4(11):1216–1221. https://doi.org/10.4161/cbt.4.11.2090
Norman B, Davis J, Piatigorsky J (2004) Postnatal gene expression in the normal mouse cornea by SAGE. Invest Ophthalmol Vis Sci 45(2):429–440. https://doi.org/10.1167/iovs.03-0449
Njung'e K, Handley SL (1991) Evaluation of marble-burying behavior as a model of anxiety. Pharmacol Biochem Behav 38(1):63–67. https://doi.org/10.1016/0091-3057(91)90590-x
Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R (2009) Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology 204(2):361–373. https://doi.org/10.1007/s00213-009-1466-y
Cao W, Lin S, Xia QQ, Du YL, Yang Q, Zhang MY, Lu YQ, Xu J, Duan SM, Xia J, Feng G, Xu J, Luo JH (2018) Gamma Oscillation Dysfunction in mPFC Leads to Social Deficits in Neuroligin 3 R451C Knockin Mice. Neuron 97(6):1394. https://doi.org/10.1016/j.neuron.2018.03.006
Donegan ML, Stefanini F, Meira T, Gordon JA, Fusi S, Siegelbaum SA (2020) Coding of social novelty in the hippocampal CA2 region and its disruption and rescue in a 22q11.2 microdeletion mouse model. Nat Neurosci 23(11):1365–1375. https://doi.org/10.1038/s41593-020-00720-5
Mandyam CD, Koob GF (2012) The addicted brain craves new neurons: putative role for adult-born progenitors in promoting recovery. Trends Neurosci 35(4):250–260. https://doi.org/10.1016/j.tins.2011.12.005
Kaushik DK, Gupta M, Das S, Basu A (2010) Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation. J Neuroinflammation 7:68. https://doi.org/10.1186/1742-2094-7-68
Kaushik DK, Mukhopadhyay R, Kumawat KL, Gupta M, Basu A (2012) Therapeutic targeting of Krüppel-like factor 4 abrogates microglial activation. J Neuroinflammation 9:57. https://doi.org/10.1186/1742-2094-9-57
Kaushik DK, Thounaojam MC, Kumawat KL, Gupta M, Basu A (2013) Interleukin-1β orchestrates underlying inflammatory responses in microglia via Krüppel-like factor 4. J Neurochem 127(2):233–244. https://doi.org/10.1111/jnc.12382
Rickert U, Cossais F, Heimke M, Arnold P, Preuße-Prange A, Wilms H, Lucius R (2018) Anti-inflammatory properties of Honokiol in activated primary microglia and astrocytes. J Neuroimmunol 323:78–86. https://doi.org/10.1016/j.jneuroim.2018.07.013
Aldana BI (2019) Microglia-Specific Metabolic Changes in Neurodegeneration. J Mol Biol 431(9):1830–1842. https://doi.org/10.1016/j.jmb.2019.03.006
Inoue K, Tsuda M (2018) Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential. Nat Rev Neurosci 19(3):138–152. https://doi.org/10.1038/nrn.2018.2
Grent-'t-Jong T, Gross J, Goense J, Wibral M, Gajwani R, Gumley AI, Lawrie SM, Schwannauer M, Schultze-Lutter F, Navarro Schröder T, Koethe D, Leweke FM, Singer W, Uhlhaas PJ (2018) Resting-state gamma-band power alterations in schizophrenia reveal E/I-balance abnormalities across illness-stages. Elife 7. https://doi.org/10.7554/eLife.37799
Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, O'Shea DJ, Sohal VS, Goshen I, Finkelstein J, Paz JT, Stehfest K, Fudim R, Ramakrishnan C, Huguenard JR, Hegemann P, Deisseroth K (2011) Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477(7363):171–178. https://doi.org/10.1038/nature10360
Gogolla N, LeBlanc JJ, Quast KB, Südhof TC, Fagiolini M, Hensch TK (2009) Common circuit defect of excitatory-inhibitory balance in mouse models of autism. J Neurodev Disord 1(Pt 1)
Nelson SB, Valakh V (2015) Excitatory/Inhibitory Balance and Circuit Homeostasis in Autism Spectrum Disorders.Neuron87(4)
Esclapez M, Hirsch JC, Ben-Ari Y, Bernard C (1999) Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy.J Comp Neurol408(4)
Massimo A, de Marco C, Vadym G, Jean G, Rüdiger K, Maxime L, Frédéric M, Zahra S, Sylvain W (2016) Specific imbalance of excitatory/inhibitory signaling establishes seizure onset pattern in temporal lobe epilepsy.J Neurophysiol115(6)
Yamamuro K, Bicks LK, Leventhal MB, Kato D, Im S, Flanigan ME, Garkun Y, Norman KJ, Caro K, Sadahiro M, Kullander K, Akbarian S, Russo SJ, Morishita H (2020) A prefrontal-paraventricular thalamus circuit requires juvenile social experience to regulate adult sociability in mice. Nat Neurosci 23(10):1240–1252. https://doi.org/10.1038/s41593-020-0695-6
El-Husseini AE, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-Campbell C, Aguilera-Moreno A, Nicoll RA, Bredt DS (2002) Synaptic Strength Regulated by Palmitate Cycling on PSD-95.Cell108(6)
Chi P, Greengard P, Ryan TA (2001) Synapsin dispersion and reclustering during synaptic activity.Nat Neurosci4(12)
Henley JM, Wilkinson KA (2016) Synaptic AMPA receptor composition in development, plasticity and disease. Nat Rev Neurosci 17(6):337–350. https://doi.org/10.1038/nrn.2016.37
Selcher JC, Xu W, Hanson JE, Malenka RC, Madison DV (2012) Glutamate receptor subunit GluA1 is necessary for long-term potentiation and synapse unsilencing, but not long-term depression in mouse hippocampus.Brain Res1435
Guo W, Polich ED, Su J, Gao Y, Christopher DM, Allan AM, Wang M, Wang F, Wang G, Zhao X (2015) Fragile X Proteins FMRP and FXR2P Control Synaptic GluA1 Expression and Neuronal Maturation via Distinct Mechanisms. Cell Rep 11(10):1651–1666. https://doi.org/10.1016/j.celrep.2015.05.013
Tian M, Zeng Y, Hu Y, Yuan X, Liu S, Li J, Lu P, Sun Y, Gao L, Fu D, Li Y, Wang S, McClintock SM (2015) 7, 8-Dihydroxyflavone induces synapse expression of AMPA GluA1 and ameliorates cognitive and spine abnormalities in a mouse model of fragile X syndrome. Neuropharmacology 89:43–53. https://doi.org/10.1016/j.neuropharm.2014.09.006

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Effect of mPFC KLF4 on autism-like behaviors and possible mechanisms

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