Imaging the Cytoskeleton to Understand Schizophrenia
Introduction

Imaging the Cytoskeleton to Understand Schizophrenia

Scientists demonstrate that schizophrenia is a disease of the cytoskeleton using confocal and scanning electron microscopy.

Schizophrenia is a mental disorder in which patients often suffer hallucinations, delusions and disordered thinking that can severely impair their daily lives. Approximately 20 million people worldwide are affected with schizophrenia. Despite its prevalence, very little is understood about the molecular mechanisms causing this disease.

A collaborative group of scientists, directed by Prof. Nobutaka Hirokawa of the University of Tokyo, and supported by Prof. Takeo Yoshikawa, a molecular psychiatrist from the RIKEN Center for Brain Science, have combined their expertise and demonstrated in S. Yoshihara et al. that schizophrenia is a disease of the cytoskeleton. Their teams used both confocal microscopy and scanning electron microscopy data to support this.

Scientific Discovery

Schizophrenia is a Disease of the Cytoskeleton

Lifeact-mRuby- and EB1-YFP-expressing primary hippocampal neurons at DIV1 (one day in vitro) treated with or without 500 µM betaine. Imaged using ZEISS confocal microscope with Airyscan detector.

Understanding the Pathogenic Mechanisms of Schizophrenia

In S. Yoshihara et al., the team demonstrates that schizophrenia is a disease of the cytoskeleton. Imaging actin dynamics in neurons using a ZEISS confocal microscopy with Airyscan as well as a ZEISS scanning electron microscope contributed to their findings.

They also identified that both elevated carbonyl stress in the body and the KIF3 kinesin motor dysfunction in cells are synergistically leading to neuronal cytoskeletal disorders and dendritic hyperbranching. These can be reversed by the carbonyl stress scavenger, betaine. Based on these data, they propose betaine as a new remedy for schizophrenia. 

Dr. Yosuke Tanaka, University of Tokyo, Japan

Cytoskeletal structures are very dynamic and small, which makes them difficult to image because they require high resolution and high speed. We mainly use a ZEISS confocal microscope with an Airyscan detector as it beautifully depicts the actin bundle alignment and the gaps between the actin bundles. In order to observe individual actin fibers, we utilized a ZEISS scanning electron microscope, which is able to very clearly image not only the actin bundles but also individual actin fibers.

Dr. Yosuke Tanaka

Researcher and Contributing Author, University of Tokyo, Japan

Marked Abnormalities in Neuron Lamellipodia

Imaged using ZEISS Confocal with Airyscan Detector

Time-lapse recording of microtubule invasion from the C- into the P-domains of lamellipodia. Arrows show movements of typical puncta of microtubule plus ends. Imaged using a ZEISS confocal microscope with Airyscan detector.
Time-lapse recording of microtubule invasion from the C- into the P-domains of lamellipodia. Arrows show movements of typical puncta of microtubule plus ends. Imaged using a ZEISS confocal microscope with Airyscan detector.

Time-lapse recording of microtubule invasion from the C- into the P-domains of lamellipodia. Arrows show movements of typical puncta of microtubule plus ends. Imaged using a ZEISS confocal microscope with Airyscan detector.

Time-lapse recording of microtubule invasion from the C- into the P-domains of lamellipodia. Arrows show movements of typical puncta of microtubule plus ends. Imaged using a ZEISS confocal microscope with Airyscan detector.

Observing Lamellopodia in Neurons from Mice with Schizophrenia-like Behavioral Phenotypes

Imaged using ZEISS Confocal with Airyscan Detector

Kinesin-like protein KIF3B is a protein that complexes with two other kinesin proteins to form motors that assist with movement of proteins along microtubules. Kif3b+/− mice exhibit schizophrenia-like behavioral phenotypes.

By observing immature Kif3b+/- neurons with confocal microscopy, the team reports marked abnormalities in lamellipodia, which are the peripheral membrane-like structure of developing neurons. Normal lamellipodia continuously showed erratic movements due to the folding of actin bundles, but these movements were significantly impaired in Kif3b+/- neurons. Furthermore, they found that this hyperstabilization of the lamellipodial movement lead to abnormal invasion of microtubules into the peripheral actin-rich domain of lamellipodia, by simultaneously imaging F-actin and microtubules.

Imaging of actin cytoskeleton in the lamellipodia of primary hippocampal neurons of the indicated conditions at DIV1 (day 1 in vitro) either by Airyscan superresolution microscopy labelled with fluorescent phalloidin or by scanning electron microscopy. Scale bars, 2 μm (confocal microscopy) and 1 μm (electron microscopy).
Imaging of actin cytoskeleton in the lamellipodia of primary hippocampal neurons of the indicated conditions at DIV1 (day 1 in vitro) either by Airyscan superresolution microscopy labelled with fluorescent phalloidin or by scanning electron microscopy. Scale bars, 2 μm (confocal microscopy) and 1 μm (electron microscopy).

Imaging of actin cytoskeleton in the lamellipodia of primary hippocampal neurons of the indicated conditions at DIV1 (day 1 in vitro) either by Airyscan superresolution microscopy labelled with fluorescent phalloidin or by scanning electron microscopy. Scale bars, 2 μm (confocal microscopy) and 1 μm (electron microscopy).

Imaging of actin cytoskeleton in the lamellipodia of primary hippocampal neurons of the indicated conditions at DIV1 (day 1 in vitro) either by Airyscan superresolution microscopy labelled with fluorescent phalloidin or by scanning electron microscopy. Scale bars, 2 μm (confocal microscopy) and 1 μm (electron microscopy).

Dendrite Hyperbranching Due to Loss of Actin Bundling

Data from Confocal and Scanning Electron Microscopy

The team observed that the microtubule plus ends tend to move horizontally in Kif3b+/+ neuron lamellipodia, but rather perpendicularly in Kif3b+/- ones toward the periphery of the lamellipodia. These excessive peripheral microtubules hyperstabilized the processes on the Kif3b+/- lamellipodia, which was considered to be the basis of dendritic hyperbranching.

Very intriguingly, a significant loss of actin bundling was found there. According to both confocal microscopy with Airyscan and scanning electron microscopy, the density of actin bundles in Kif3b+/- lamellipodia was significantly lower than that of Kif3b+/+ lamellipodia. These data suggested that the continuous actin bundle dynamics may be essential for excluding microtubules from the peripheral region of the lamellipodia and for suppressing the dendrite hyperbranching. These cytoskeletal phenotypes could be reversed by betaine administration to the culture medium.

Kif3b+/+ primary hippocampal neuron at DIV1 (day 1 in vitro) labeled with an antibody against CRMP2 (magenta), an antibody against KIF3A (yellow), fluorescent phalloidin (cyan), and an antibody against α-tubulin (green). Imaged with confocal microscopy with Airyscan.
Kif3b+/+ primary hippocampal neuron at DIV1 (day 1 in vitro) labeled with an antibody against CRMP2 (magenta), an antibody against KIF3A (yellow), fluorescent phalloidin (cyan), and an antibody against α-tubulin (green). Imaged with confocal microscopy with Airyscan.

Kif3b+/+ primary hippocampal neuron at DIV1 (day 1 in vitro) labeled with an antibody against CRMP2 (magenta), an antibody against KIF3A (yellow), fluorescent phalloidin (cyan), and an antibody against α-tubulin (green). Imaged with confocal microscopy with Airyscan.

Kif3b+/+ primary hippocampal neuron at DIV1 (day 1 in vitro) labeled with an antibody against CRMP2 (magenta), an antibody against KIF3A (yellow), fluorescent phalloidin (cyan), and an antibody against α-tubulin (green). Imaged with confocal microscopy with Airyscan.

Possible Mechanism of Betaine

The team had previously identified the neuronal regulator protein, collapsin response mediator protein 2 (CRMP2), as a major target of carbonylation in the brain (M. Toyoshima et al.). Using confocal microscopy, they show that KIF3 binds to CRMP2, and the KIF3-CRMP2 complex localizes to microtubule and actin cytoskeleton. This CRMP2 distribution was significantly reduced in the periphery of Kif3b+/- neurons.

To find the mechanistic link between betaine and CRMP2 activity, the team also conducted biochemical assays. Unmodified CRMP2, but not the carbonylated CRMP2 had strong actin bundling activity. This suggests that decarbonylation of CRMP2 by betaine could functionally compensate the CRMP2 deficiency in Kif3b+/- lamellipodia by improving its actin bundling activity. (see S. Yoshihara et al. for data).

Prof. Nobutaka Hirokawa, University of Tokyo, Japan

Since our discovery of KIFs, we have studied the molecular mechanism of intracellular transports. Among our approaches we especially like to visualize the molecular mechanisms using various kinds of microscopy. I’m very honored and glad to see that we finally got a nice approach to the fundamental remedy to schizophrenia in this study as one of the most fruitful outcomes of our KIF research. I hope this remedy will be further appreciated by the clinicians, because there have been still few good medications for schizophrenia.

Prof. Nobutaka Hirokawa

Corresponding Author, University of Tokyo, Japan


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