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ISSN: 2637-6628

Online Journal of Neurology and Brain Disorders

Research Article(ISSN: 2637-6628)

Overexpression of Neuregulin-1 Type III has Impact on Visual Function in Mice Volume 5 - Issue 3

Nan Su1,3, Weiqi Zhang2, Nicole Eter1, Peter Heiduschka1* and Mingyue Zhang2*

  • 1Department of Ophthalmology, Research laboratory, University Hospital Muenster, Germany
  • 2Lab for Molecular Neuroscience, Clinic for mental Health, University Hospital Muenster, Germany
  • 3Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, People’s Republic of China, China

Received:March 10, 2021   Published:March 17, 2021

Corresponding author: Mingyue Zhang, Lab for molecular neuroscience, Clinic for Mental Health, University Hospital Muenster, Muenster, Germany

DOI: 10.32474/OJNBD.2021.05.000215

Abstract PDF

Abstract

Schizophrenia is associated with several brain deficits, including abnormalities in visual processes. Neuregulin-1 (Nrg1) is a family of trophic factors containing an Epidermal Growth Factor (EGF) like domain. It is thought to play a role in neural development and has been linked to neuropsychiatric disorder. Abnormal Nrg1 expression has been observed in schizophrenia. Moreover, there is more and more evidence found about pathological changes of the retina regarding structural, neurochemical and physiological parameters. However, mechanisms of these changes are not well known. To investigate this, we analyzed function of the visual system using Electroretinography (ERG) and measurement of Visual Evoked Potentials (VEP) in a transgenic mouse overexpressing Nrg1 type III. ERG amplitudes tended to be higher in transgenic mice in younger animals, whereas the amplitudes were almost similar in older mice. VEP amplitudes are larger in transgenic mice in both younger and older animals, with significance in older animals. Nonetheless, latencies did not differ considerably in both age groups (wt & tg). Our data show for the first time that overexpression of Nrg1 type III changed visual function in transgenic mice. Overall, this Investigation of visual function in transgenic mice is helpful for understanding corresponding changes that occur in schizophrenia, as they may find use as biomarkers for psychiatric disorders as well as a potential.

Introduction

Schizophrenia is a complex disorder that affects 0.5-1% of the adult population throughout different ethnics in the world. According to findings reported in literature, several genes have been associated with the neuropathology in diverse populations [1]. Among these candidates, genes encoding the proteins neuregulin (Nrg1) and its receptor ErbB4 have been shown to be promising susceptibility genes of schizophrenia [1]. Some patients show abnormal levels of expression of Nrg1 and ErbB4 isoforms in different brain regions [2]. Schizophrenic patients are impaired in cognitive abnormalities, including executive control and working memory [3]. In addition, elevated levels of Nrg1 and ErbB4 proteins have been found studies on post-mortem schizophrenic patients studies [4]. Further, our early work on mice has shown that elevated Nrg1 expression (Nrg1-III-tg) showed ventricular enlargement and symptoms similar to schizophrenia [5]. Similarly, in 2018 Olaya and colleagues showed that overexpression of Nrg 1 type III in mice confers schizophrenia-like behaviors [6]. Importantly, another study it has confirmed that Nrg1/ErbB4 regulates visual cortical plasticity [7].

Researchers have long been aware of the link between schizophrenia and visual processing impairments, which are accompanied by multiple structural and functional disturbances in patients. Further, the retina may be particularly affected, as it is an extension of the central nervous system and shows similarities to the brain and spinal cord in terms of structure, functionality, response to insult, and immunology [8]. In addition, from embryonic point of view, the retina and optic nerve, which have a neuroectodermal origin, emerge from diencephalon and can be seen with the naked eye in its natural state in living organism [9]. Visual processing impairments are well established in schizophrenia, including multiple structure and functional disturbances in patients. In addition, even when studies have controlled for factors such as psychotic symptoms and auditory distortions, visual distortions (including those of the retina) have been associated with suicidal ideation [10]. These visual alterations include dopaminergic abnormalities, abnormal output, maculopathies and retinopathies, cataracts, poor visual acuity, and thinning of the Retinal Fibre Layer (RNFL) [9]. Looking at this from another angle, some classic ocular pathologies have been found to occur in the context of several majorneurodegenerative disorders and RNFL thinning is related to brain volume loss in aging and illness progression, as well as cognitive decline in multiple sclerosis and Alzheimer’s disease [11-13].

The visual functions can be assessed with the flash Electroretinography (ERG) and measurement of Visual Evoked Potentials (VEP). While several ERG anomalies have been identified in patients with psychiatric disorders [14], the underlying mechanisms and visual processing abnormalities in schizophrenia are still unknown. To investigate this, we used transgenic mice overexpressing Nrg1 type III, as this protein is linked with both schizophrenia and visual processing and analysed the mice’s retinal function using Electroretinogram (ERG) and Visual Evoked Potentials (VEP).

Materials and Methodse

Animals

The experiments were performed in accordance with European Communities Council Directive (86/EEC) and were approved by the Federal State Office for Consumer Protection and Food Safety of North Rhine-Westphalia, Germany. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments to a minimum necessary for reliable statistical analyses. The generation and genotyping of transgenic Nrg1-III-tg mice have been described in detail previously [15-16].

Visual Electrophysiology

Electroretinography was performed as described previously in Schubert et al., 2015 [17] Briefly, the animals were anaesthetised using a standard intraperitoneal ketamine/xylazine injection. The Sleeping animals were placed on a heating pad, with pupils dilated by tropicamide and neosynephrine eye drops. Desensitisation of the cornea was achieved by a drop of proparacaine. For the ERG and VEP measurement, the commercial measuring device RetiPort from Roland Consult (Brandenburg) was used. During the measurement, the animals were placed on a heated plate at 37°C to prevent cooling of the animals. For the ERG measurement, a gold ring electrode was placed on the cornea of the left eye without damaging the cornea. VEP was recorded simultaneously by inserting a stainless-steel needle electrode subcutaneously above the visual centre of the mice, on top the skull between the ears. Another gold electrode that was moistened with saline and placed into the mouth of the animals served as the reference. Measurement was performed in the scotopic mode, with animals that were dark adapted for at least 12 hours. Visual stimulation was performed by application of flashes of six different light intensities, ranging from 0.0003 to 30 cd∙s/m². Responses of the visual system were recorded, averaged and stored by the RetiPort device. After the measurement, the still sleeping animal were kept in a separate box and given back into the cage after awakening.

Immunohistochemistry

Eyes of euthanised mice were isolated and fixed in 4% paraformaldehyde for 1hour, washed 2x in PBS pH 7.4 for 5 minutes and frozen in NEG-50TM. Cryo sections (thickness 10 μm) were cut using a Cryostar NX70 cryostat (Thermo Fisher Scientific), placed on Starfrost Advanced Adhesive glass slides (Engelbrecht) and stored at -20 °C until used for immunohistochemistry. For immunohistochemistry, sections were blocked with Power Block reagent (HK085-5K, BioGenex) at room temperature for 6 minutes, then washed 3 x with 0.1 M PBS and incubated overnight with primary antibodies at 4°C. The sections were then washed 3 x with 0.1 M PBS and incubated with appropriate secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (4′6′-diamidino-2-phenylindole dihydrochloride), diluted with pure water 1:300, for 7 minutes at room temperature. The primary antibody (Neuregulin-1; Gene Tex; 1:1000) was diluted in 1% bovine serum albumin containing 0.1% Triton X-100, and the secondary antibody goat anti rabbit (Alexa fluor 568; Abcam; 1:800) were diluted with 1% bovine serum albumin. Finally, sections were washed 3 with 0.1 M PBS and mounted under glass coverslips using mounting medium (ImmuMount TM Thermo Scientific).

Data Analysis

Data are shown as mean ± SEM. Evaluation of the data was performed by separately comparing the means of each parameter obtained in wildtype (wt) and Nrg1-III-tg mice. Parametric unpaired Student’s t-tests were used to determine difference between wt and Nrg1-III-tg mice. The level of statistical significance was set at p=0.05, statistical significance is indicated as an *p<0.05, **p<0.01.

Results

Investigation of the Function of the Visual System Measured by ERG and VEP

ERG and VEP measurements were carried out in younger (12 weeks old) and older (55 weeks old) mice; in these age groups, transgenic (tg) and wt mice were compared. In each age group (younger and older), the latencies in scotopic ERG had similar ranges when comparing tg and wt mice. Because of the inevitable spread of results, most error bars overlap, making it difficult to offer stringent statements about differences between the wt and transgenic age-matched mice in the younger and elder groups. In contrast, the amplitudes of ERG a-waves and b-waves tended to be higher in younger Nrg1-III-tg mice than wt mice over the whole range of light intensities, whereas the amplitudes of a-waves and b-waves in older mice are quite similar for the tg and wt mice at all light intensities (Figure 1).

Similar to the situation for the scotopic ERG, the latencies in scotopic VEP were similar for tg and wt mice in each age group. However, the amplitudes of scotopic VEP in the Nrg1-III-tg mice were larger than those of wt mice in both young and old control mice. In the group of younger mice (12 weeks old), there was a significant difference in VEP amplitude of tg and wt mice at the highest light intensity (30 cd·s/m²), whereas the difference in the older group (55 weeks old) was significant at all light intensities except of 0.0003 cd·s/m² (Figure 2).

Figure 1: Scotopic Amplitudes and Latencies in 12- and 55- Week Mice
Data are shown as mean values and standard deviations. Number of animals in the control younger (n=3), Nrg1-III-tg younger (n=2); older control (n=8) and older Nrg1-III-tg mice (n=7).

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Figure 2: Scotopic VEP Amplitudes and Latencies in 12 -and 55-Week-old Mice
Data are shown as mean values and standard deviations. Number of animals in the control younger (n=3), Nrg1-III-tg younger (n=2); older control (n=8) and older Nrg1-III-tg mice (n=7).

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Immunohistochemical Staining

Immunohistochemical staining was carried out against Nrg1. In the sections stained against neuregulin, it can be seen that Immunoreactivity (IR) of Nrg1 was clearly stronger in the 24 weeks old Nrg1-III-tg mouse than in the age-matched wt mice (Figure 3).

Figure 3: Immunohistochemical Staining Against Neuregulin. The trophic factor NRG1 (red) in frozen sections of the retina of the mouse eye at 24-week-old, and 55-week-old of the control and Nrg1-III-tg mice as indicated. The cell nuclei were stained with DAPI (blue).

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Discussion

In the current study, we investigated the influence of neuronal overexpression of Nrg1-Type III-tg mice on the visual function evaluated by electroretinography and VEP measurement. The above data demonstrate that ERG responses are larger in Nrg1 III tg mice compared to WT mice in younger mice, but not in elderly mice. VEP amplitudes were larger in Nrg1 III tg mice at both ages, with significance in elderly mice. Latencies were always similar between wild-type and transgenic mice. The human Neuregulin1 (Nrg1) gene is a major schizophrenia susceptibility gene, and its association with the illness has been found in different populations [18]. In addition, dysregulated expression of Nrg1 increases disease susceptibility and has been found in studies of post-mortem brain tissue from schizophrenia patients, inclusive elevation of expression of Nrg1 [19]. In our previous mouse studies, gene disruption that increased expression of Nrg1-Type III can confer distinct schizophrenia-like behavioural and brain biological phenotypes [5]. More and more studies are showing that schizophrenia is associated with several brain deficits, including visual processing deficits [20, 21]. As key player in visual processing, the retina is part of the central nervous system and is composed of several layers. To evaluate the function of specific layers or neurons of the retina, ERG can be used, as it records the light-evoked electrical potential [22]. The cornea-negative a-wave indicates the electrical activity of the photoreceptors, and ON-bipolar cells are the source of the b-wave in the retina of mice [23, 24].

In the present study, we examined the effects of the overexpression of Nrg1 on visual function. We found that electroretinographic a-waves and b-waves were larger in transgenic mice compared to the controls in the younger mice. Yet, as the sample size was relatively small in the young mice group, these findings needs to be confirmed further. These data suggest that ERG abnormalities are seen only in younger mice, in accordance with the neurodevelopmental theory that schizophrenia arises at the early stages of brain formation. Transmission and processing of nerve signals in the retina depend on different neurotransmitters, such as glutamate and dopamine [25,26]. As has been found in the brain, Nrg1 signaling pathway has effect on neurotransmission and synaptic plasticity [5]. Hence, to explore what type of changes in the neural signaling occur in the retina of transgenic mice requires further studies. The VEP approach, which is an electrical potential recorded from the visual cortex in response to a visual stimulus, can be used to evaluate post retinal function [27]. Our measurements of flash evoked VEP showed that the amplitudes were larger in the young transgenic mice compared to the age-matched wt mice, but this difference was statistically significant for the older transgenic mice vs. their age-matched wt control mice. Nonetheless, in each age group the latencies were in a similar range (Figure 3). The observed increase in the VEP amplitude in transgenic mice may be caused by the presence of abnormal ganglion cells, abnormal either myelination of the optic nerve or abnormal number of ganglion cells. This has to be evaluated in further studies.

From our immunofluorescence results, we observed that there was more immunoreactivity for Nrg1 in the retina of young transgenic mice than in wt mice. As overexpression of Nrg1 is performed under the control of the Thy-1 promoter, it could be anticipated that mainly retinal ganglion cells showed immunoreactivity for Nrg1, because especially these cells produce Thy-1 at young ages [28]. Given that the expression of Nrg1 seems to change with age, this may suggest that Nrg/ErbB signaling plays a role during the early development of the retina. Our previous work showed that dysregulation of Nrg1 by cortical pyramidal neurons disrupts GABAergic and glutamatergic neurotransmission in cortex as well as synaptic plasticity [5]. All these changes in transgenic mice have the potential to alter the visual system and further to eventually impact to ERG and VEP. Given the findings of the current study, further investigations should more deeply explore the mechanisms by which of these visual anomalies occur, as they will be helpful for understanding the biological basis of the psychiatric disorder.

Acknowledgmentse

The authors would like to thank Christiane Schettler, Kathrin Schwarte, Helen Haupt and Mechthild Wissing for their support to this study.

References

  1. PJ Harrison, AJ Law (2006) Neuregulin 1 and Schizophrenia: Genetics, Gene Expression, and Neurobiolog. Biological Psychiatry 60(2): 132-140.
  2. AJ Law, JE Kleinman, DR Weinberger, CS Weickert (2007) Disease-associated intronic variants in the ErbB4 gene are related to altered ErbB4 splice-variant expression in the brain in schizophrenia. Hum Mol Genet 16(2): 129-141.
  3. DA Lewis, RA Sweet (2009) Schizophrenia from a neural circuitry perspective: Advancing toward rational pharmacological therapies. Journal of Clinical Investigation 119(4):706-716.
  4. VZ Chong, M Thompson, S Beltaifa, MJ Webster, AJ Law, et al. (2008) Elevated neuregulin-1 and ErbB4 protein in the prefrontal cortex of schizophrenic patients. Schizophr Res 100(1-3): 270-280.
  5. A Agarwal, Mingyue Zhang, Irina Trembak-Duff, Tilmann Unterbarnscheidt, Konstantin Radyushkin, et al. (2014) Dysregulated expression of neuregulin-1 by cortical pyramidal neurons disrupts synaptic plasticity. Cell Rep 8(4): 1130-1145.
  6. JC Olaya, Carrie L Heusner, Mitsuyuki Matsumoto, Duncan Sinclair, Mari A Kondo, et al (2018) Overexpression of Neuregulin 1 Type III Confers Hippocampal mRNA Alterations and Schizophrenia-Like Behaviors in Mice. Schizophr Bull 44(4): 865-875.
  7. Y Sun, Taruna Ikrar, Melissa F Davis, Nian Gong, Xiaoting Zheng, et al. (2016) Neuregulin-1/ErbB4 Signaling Regulates Visual Cortical Plasticity. Neuron 92(1): 160-173.
  8. A London, I Benhar, M Schwartz (2013) The retina as a window to the brain - From eye research to CNS disorders. Nature Reviews Neurology 9(1): 44-53.
  9. SM Silverstein, R Rosen (2015) Schizophrenia and the eye. Schizophrenia Research: Cognition 2(2): 46-55.
  10. N Granö, L Salmijärvi, M Karjalainen, S Kallionpää, M Roine, et al. (2015) Early signs of worry: Psychosis risk symptom visual distortions are independently associated with suicidal ideation. Psychiatry Res 225(3): 263-267.
  11. E Gordon Lipkin, B Chodkowski, D S Reich, S A Smith, M Pulicken, et al. (2007) Retinal nerve fiber layer is associated with brain atrophy in multiple sclerosis. Neurology 69(16): 1603-1609.
  12. YT Ong, Saima Hilal, Carol Y Cheung, Narayanaswamy Venketasubramanian, Wiro J Niessen, et al. (2015) Retinal neurodegeneration on optical coherence tomography and cerebral atrophy. Neurosci Lett 584: 12-16.
  13. L Ferrari, SC Huang, G Magnani, A Ambrosi, G Comi, et al. (2017) Optical Coherence Tomography Reveals Retinal Neuroaxonal Thinning in Frontotemporal Dementia as in Alzheimer’s Disease. J Alzheimer’s Dis 56(3): 1101-1107.
  14. M Hébert, Chantal Mérette, Anne Marie Gagné, Thomas Paccalet, Isabel Moreau, et al. (2020) The Electroretinogram May Differentiate Schizophrenia from Bipolar Disorder. Biol Psychiatry 87(3): 263-270.
  15. V Velanac, Tilmann Unterbarnscheidt, Wilko Hinrichs, Maike N Gummert, Tobias M Fischer, et al. (2012) Bace1 processing of NRG1 type III produces a myelin-inducing signal but is not essential for the stimulation of myelination. Glia 60(2): 203-217.
  16. GV Michailov, Michael W Sereda, Bastian G Brinkmann, Tobias M Fischer, Bernhard Haug, et al. (2004) Axonal Neuregulin-1 Regulates Myelin Sheath Thickness. Science 304(5671): 700-703.
  17. T Schubert, Corinna Gleiser, Peter Heiduschka, Christoph Franz, Kerstin Nagel-Wolfrum, et al. (2015) Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype. Cell Mol Life Sci 72(20): 3953-3969.
  18. H Stefansson, V Steinthorsdottir, TE Thorgeirsson, JR Gulcher, K Stefansson (2004) Neuregulin 1 and schizophrenia. Annals of Medicine 36(1): 62-71.
  19. CS Weickert, Y Tiwari, PR Schofield, BJ Mowry, JM Fullerton (2012) Schizophrenia-associated HapICE haplotype is associated with increased NRG1 type III expression and high nucleotide diversity. Transl Psychiatry 2(4): 104.
  20. NN Samani, Frank A Proudlock, Vasantha Siram, Chathurie Suraweera, Claire Hutchinson, et al. (2018) Retinal Layer Abnormalities as Biomarkers of Schizophrenia. Schizophr Bull 44(4): 876-885.
  21. SA Adams, HA Nasrallah (2018) Multiple retinal anomalies in schizophrenia. Schizophrenia Research 195: 3-12.
  22. LH Pinto, B Invergo, K Shimomura, JS Takahashi, JB Troy (2007) Interpretation of the mouse electroretinogram. Doc Ophthalmol 115(3): 127-136.
  23. RD Penn, WA Hagins (1969) Signal transmission along retinal rods and the origin of the electroretinographic a-Wave. Nature 223(5202): 201-205.
  24. JG Robson, H Maeda, SM Saszik, LJ Frishman (2004) In vivo studies of signaling in rod pathways of the mouse using the electroretinogram. Vision Research 44(28): 3253-3268.
  25. E Popova (2014) Role of dopamine in distal retina. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 200(5): 333-358.
  26. V Connaughton (1995) Glutamate and Glutamate Receptors in the Vertebrate Retina. University of Utah Health Sciences Center, US.
  27. WH Ridder, S Nusinowitz (2006) The visual evoked potential in the mouse-Origins and response characteristics. Vision Res 46(6-7): 902-913.
  28. CJ Barnstable, UC Drager (1984) Thy-1 antigen: A ganglion cell specific marker in rodent retina. Neuroscience 11(4): 847-855.
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