KR102437852B1 - Method for restoring brainwave impairments in alzheimer's brain using optogenetical activation - Google Patents

Abstract

The present invention relates to a method for recovering EEG damage in Alzheimer's disease brain using an optogenetic technique.
The present invention is a method of recovering EEG that has been damaged in Alzheimer's disease brain. It does not directly damage nerve cells through photostimulation rather than electrical stimulation. After specifying the inhibitory neurons, the damaged brain waves in the Alzheimer's disease brain can be restored to a normal level by selectively controlling the activity of the damaged neurons using light through an optogenetic technique.

Description

EEG damage recovery method in Alzheimer's disease brain using optogenetic technique

The present invention relates to a method for recovering EEG damage in the brain of Alzheimer's disease using an optogenetics technique, and more particularly, to a method for recovering EEG that has been damaged in an Alzheimer's disease brain. By using optogenetic technique without directly damaging It relates to a method for recovering EEG damage in an Alzheimer's disease brain using an optogenetic technique, which selectively recovers damaged theta and gamma waves in the Alzheimer's disease brain by transmitting a stimulus to control the activity of the corresponding neuron.

Alzheimer's disease is a representative degenerative brain disease, and it is known that beta-amyloid, a toxic substance, is abnormally deposited in the brain to cause learning and memory deterioration.

In the normal brain, for memory formation, delta waves (0.1-3 Hz), theta waves (3-12 Hz), alpha waves (8-15 Hz), beta waves (16-30 Hz) and gamma waves due to the activity of various inhibitory neurons (20-120Hz), etc., occur in various frequency bands. In Alzheimer's disease brain, EEG production is inhibited due to the decrease in inhibitory nerve cell function due to beta-amyloid deposition, which is a major factor in learning and memory deterioration in Alzheimer's disease. cause

In order to recover the damaged EEG in the conventional Alzheimer's disease brain, a technique for recovering the damaged EEG by inserting an electrode and giving electrical stimulation of a high frequency band has been proposed.

However, this electrical stimulation not only has the risk of inflicting direct damage to the nerve cells, but also reflects the various functions and activity patterns of the inhibitory nerve cells in the actual brain because the target of the stimulation is non-selective in the electrical stimulation technique. Therefore, there is a disadvantage in that it is not possible to selectively control the activity.

Therefore, a technique for treating neurological diseases through optogenetics rather than electrical stimulation has been proposed. As an example, Patent Document 1 discloses the steps of: transfecting a target tissue with a photosensitive channel protein sensitive to light of a specific wavelength; delivering light in a wavelength range to a target tissue through an optical stimulation device; detecting a biosignal at substantially the same time as delivering light to a target tissue; determining a patient treatment status based on the biosignal; Based on the sensed patient's treatment status, light stimulation therapy performed as a step of adjusting the transmission of light to a target tissue is disclosed, and it is described that it is applied to neurological disorders such as dysfunction, dementia, Parkinson's disease, spasticity, and epilepsy.

In addition, according to Patent Document 2, recently, a method for treating depression through optogenetic stimulation of the left brain medial prefrontal cortex is disclosed.

Specifically, it was confirmed that social interaction according to stress is closely related to the left medial prefrontal cortex, and based on the above results, social interaction can be regulated through photostimulation in the left medial prefrontal cortex. It is described as being useful in the treatment of depression by expressing channelrhodopsin in the prefrontal cortex and providing control information of the interaction (social interaction) using means of photostimulation.

Although the possibility of optogenetic technology as a treatment method for neurological diseases has been raised through the prior inventions, no Alzheimer's disease treatment method using optogenetic technology has been reported. There is no technology available

Accordingly, as a result of the present inventors' efforts to improve the problems of the prior art, it is a method of recovering brain waves that have been damaged in Alzheimer's disease brain. Alzheimer's disease brain by optogenetic activity of SST inhibitory neurons by selectively regulating the activity by expressing photoreceptors only in PV and SST inhibitory neurons, which are inhibitory neurons known to play the most direct role in EEG generation By providing a technology that can selectively induce the recovery of damaged gamma waves in the Alzheimer's disease brain by optogenetic activity of PV inhibitory neurons in the theta wave damaged in was completed.

US Patent Publication No. 2011-0125078 (published on May 26, 2011) Republic of Korea Patent Publication No. 2014-0125659 (published on October 29, 2014)

It is an object of the present invention to provide a method for recovering EEG damage in Alzheimer's disease brain using an optogenetic technique.

In order to achieve the above object, the present invention is an animal model production step in which the CRE gene is expressed in inhibitory neurons,

Injecting a viral vector containing a photoreceptor gene designed to target the inhibitory neurons in which the CRE gene is expressed into the animal model and expressing the viral vector;

Inserting an electrode and an optical fiber into the animal model injected with the viral vector;

EEG damage recovery in Alzheimer's disease brain using optogenetic technique comprising the step of selectively recovering damaged EEG in Alzheimer's disease brain by delivering photostimulation that responds to each nerve cell designed to the photoreceptor gene through the electrode and optical fiber provide a way

In the present invention, after the step of recovering the damaged EEG in the Alzheimer's disease brain, the step of quantifying the damage EEG recovery effect may be further performed.

In the above, the inhibitory neurons are neurons expressing PV or SST, and can selectively restore damaged gamma waves or damaged theta waves by the photostimulation.

In the present invention, as the photoreceptor, it is preferable to use a photoreceptor that responds to light in the wavelength range of 450, 470, 565 and 590 nm.

The method of recovering EEG damage in Alzheimer's disease brain using the optogenetic technique of the present invention is different from the conventional EEG treatment technology through electrical stimulation that directly damages nerve cells, without using photostimulation to damage nerve cells. EEG recovery in Alzheimer's disease brain is possible through the regulation of nerve cell activity.

In addition, unlike the prior art, which non-selectively applied electrical stimulation to a large number of unspecified neurons, the method of recovering EEG damage in the Alzheimer's disease brain using the optogenetic technique of the present invention uses a viral vector to activate photoreceptors of specific neurons. By using genetic manipulation technology to insert genes, it is possible to selectively control the activity of only one type of cell among various inhibitory neurons in the brain.

In addition, the present invention can induce a neuronal activity pattern similar to the activity pattern of inhibitory neurons that appears when generating an actual normal brain EEG in the Alzheimer's disease brain, so that the EEG generation mechanism of a normal brain can be induced by using various photostimulation patterns. It can be simulated to treat Alzheimer's disease brain wave damage.

1 shows the Aß polymer synthesis process,
2 shows a viral vector and Aß injection technique using an optogenetic genetic manipulation technique and a stereotaxic method;
3 is a photograph of Aß expression and SST/PV neurons in the hippocampus of Alzheimer's disease mice;
Figure 4 shows the damage theta wave recovery results in Alzheimer's disease hippocampus through optogenetic SST neuronal activity regulation,
5 shows the damage gamma wave recovery results in the hippocampus of Alzheimer's disease through optogenetic PV neuronal activity regulation.

Hereinafter, the present invention will be described in detail.

The present invention uses various photostimulation patterns to induce a neuronal activity pattern similar to that of inhibitory neurons that appears during actual normal brain EEG generation in the Alzheimer's disease brain, thereby mimicking the EEG generation mechanism of a normal brain and Alzheimer's disease. It relates to a method of recovering brain wave damage.

Specifically, the present invention provides a step for preparing an animal model in which the CRE gene is expressed in 1) inhibitory neurons;

2) injecting a viral vector containing a photoreceptor gene designed to target the inhibitory neurons expressing the CRE gene into the animal model and expressing the viral vector;

3) inserting an electrode and an optical fiber into the animal model injected with the viral vector; and

4) EEG in Alzheimer's disease brain using optogenetic technique comprising the step of selectively recovering damaged EEG in Alzheimer's disease brain by transmitting photostimulation that responds to each nerve cell designed to the photoreceptor gene through the electrode and optical fiber Provides a way to recover from damage.

Hereinafter, each step will be described in detail with reference to the drawings.

In the method of recovering EEG damage in the brain of Alzheimer's disease using the optogenetic technique of the present invention, step 1) is the production step of an animal model for Alzheimer's disease. A method of inducing Alzheimer's disease by directly injecting an Aß polymer (oligomer) into the brain or an animal model of Alzheimer's disease previously invented through genetic manipulation technology is used.

FIG. 1 shows the Aß polymer synthesis process. After treating the Aß monomer with HFIP (Hexafluoro-2-propanol), an organic solvent, and floating it for 5 to 10 minutes to form a monomer, Aß using a vacuum concentrator Remove the HFIP from the suspension and film it. The filmed Aß is treated with DMSO (dimethyl sulfoxide) to slow the aggregation of Aß and suspend it to prepare an Aß suspension. At this time, the Aß suspension to be used in the experiment is treated with artificial cerebrospinal fluid, diluted to 200 nM, and then refrigerated for about 12 hours to synthesize and use a soluble Aß polymer. The Aß polymer synthesized as above is directly injected into the animal brain through the stereotaxic method to induce Alzheimer's disease.

The previously invented Alzheimer's disease animal model of step 1) includes Alzheimer's disease animal models manufactured through genetic manipulation, such as 5xFAD mice, APP/PS1 mice, and the like.

In step 1), the inhibitory neurons use PV or SST neurons, and the CRE gene is expressed in the specific inhibitory neurons.

The CRE is a CRE recombination enzyme, which recognizes DNA of a specific sequence and cuts and splices it. , CRE enzyme is expressed only in neurons expressing PV.

In the method of the present invention, step 2) prepares a viral vector containing a photoreceptor designed to target the inhibitory neurons expressing the CRE gene, and in the brain of the CRE transgenic animal model of step 1), the photoreceptor This is the step of injecting the viral vector containing the gene.

The viral vector refers to an adeno-associated virus having promoters such as EF1a-DIO, CAG-Flex, and Syn-Flex, and targets only specific neurons expressing the CRE gene to deliver photoreceptors. can

The photoreceptor includes all kinds of photoreceptors that increase the activity of nerve cells expressing the photoreceptor in response to light. Specific examples include ChIEF, which responds maximally to light with a wavelength of 450 nm; Crimson et al., which respond maximally to light with a wavelength of nm. The photoreceptors of step 2) include all kinds of photoreceptors capable of activating nerve cells in addition to the photoreceptors presented in the above example.

Figure 2 shows the viral vector and Aß injection technology using the optogenetic genetic manipulation technique and the stereotaxic method. CRE transgenic animal models, more preferably SST-CRE and PV-CRE animal models, which are preferred embodiments of the present invention. If the viral vector encoding the photoreceptor is injected through a micro-injector after setting the location of the brain to be injected using the stereotaxic method, the photoreceptor can be selectively expressed only in SST and PV neurons. can

After the viral vector injection, the injected viral vector is expressed in specific inhibitory neurons containing the CRE gene to cause genetic modification, a step of expressing the viral vector for 2 to 3 weeks is performed.

Then, in the method of the present invention, step 3) is a step of inserting an electrode and an optical fiber into the animal model injected with the viral vector, and an electrode and optical fiber for EEG measurement and optical stimulation delivery in the brain of an animal model of Alzheimer's disease. insert

Specifically, in the case of an in vitro situation, it is possible to measure EEG by inserting a microglass electrode into a brain section of an animal model of Alzheimer's disease, and the photoelectrode can be delivered through a light source and a microscope lens.

In addition, in the case of an in vivo situation, a photoelectrode (electrode + optical fiber) is injected after a hole is drilled in the skull of an anesthetized animal and the location of the brain where the electrode is to be inserted through the stereotaxic method is set, and then dental cement Cover the surgical site through

Thereafter, the present invention comprises a step of selectively recovering damaged brain waves in Alzheimer's disease brain by transmitting photostimulation through the electrode and optical fiber through step 4).

Step 4) is a photostimulation step in which light in a wavelength band such as 450, 470, 565 and 590 nm is irradiated to the brain of Alzheimer's disease. Regulates the activity of sexual neurons.

3 shows photographs of Aβ expression and SST/PV neurons taken from the hippocampus of Alzheimer's disease mice. That is, when photostimulation with a wavelength of 470 nm is given to the hippocampus of SST-CRE or PV-CRE mice injected with Aß and virus, it is possible to selectively activate ChR2-expressing SST/PV neurons in the brain of Alzheimer's disease. can restore damaged theta and gamma waves by irradiating the brain with Alzheimer's disease.

The photostimulation varies depending on the type of photoreceptor, and in the case of ChR2, a wavelength of 470 nm is used, and in the case of C1V1, light of a wavelength of 565 nm is used, and is given through a photoelectrode inserted into the brain.

Figure 4 shows the damage theta wave recovery results in the hippocampus of Alzheimer's disease through optogenetic regulation of SST nerve cell activity. The damaged theta wave recovery is achieved by activating photoreceptor-expressing SST nerve cells through photostimulation.

In addition, FIG. 5 shows the results of recovery of damaged gamma waves in the hippocampus of Alzheimer's disease through optogenetic control of PV nerve cell activity. it has been done

The intensity and pattern of photostimulation are provided in a direction to simulate various activity patterns for each type of inhibitory neuron occurring in the normal brain. By irradiating the neuronal cells, it is possible to mimic the activity pattern of par albumin neurons, which show activity in the gamma wave band in the normal brain, and to restore gamma wave damage in the brain with Alzheimer's disease. In this case, it will be understood that the intensity of the photostimulation may be given in proportion to the degree of EEG damage in the Alzheimer's disease brain.

In addition, the pattern of the photostimulation may be given as a constant and continuous stimulation pattern, a sine wave pattern, a specific frequency pattern, or the like.

Thereafter, the EEG damage recovery method in the Alzheimer's disease brain using the optogenetic technique of the present invention is to quantify the EEG recovery effect by verifying 5) whether the damaged EEG is recovered after the 4) step of recovering the damaged EEG in the Alzheimer's disease brain. More steps may be performed.

That is, the recovery of EEG damage in the brain with Alzheimer's disease is determined by band-pass filtering the EEG to the wavelength band to be observed, and then using a power analysis technique using Fast Fourier Transform, etc. can be quantified.

The EEG damage recovery method in the Alzheimer's disease brain using the above optogenetic technique is capable of EEG recovery in the Alzheimer's disease brain by controlling the activity of nerve cells without damaging nerve cells using photostimulation.

In addition, by the method of recovering EEG damage in the brain of Alzheimer's disease using the optogenetic technique of the present invention, the photoreceptor gene of a specific nerve cell is not selectively applied to a large number of non-specific nerve cells in the prior art, unlike the prior art. By using a genetic manipulation technique that inserts, it is possible to selectively control the activity of only one type of inhibitory neuron in the brain.

In particular, the present invention is capable of recovering theta wave and gamma wave damage, which are known to be closely related to memory damage in the actual Alzheimer's disease brain.

In the above, the present invention has been described in detail only with respect to the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical spirit of the present invention, and it is natural that such variations and modifications belong to the appended claims.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Example 1> Theta wave damage recovery method in Alzheimer's disease brain

1. Establishment of Alzheimer's disease animal model through amyloid beta polymer injection

The Aß monomer was treated with HFIP (Hexafluoro-2-propanol) solvent, suspended at a concentration of 1 mM for monomerization, and then HFIP was removed from the Aß suspension using a vacuum concentrator to form a film. The filmed Aß was subjected to DMSO solvent treatment to suspend the aggregation of Aß while suspending to prepare a 5 mM Aß suspension. The Aß suspension to be used in the experiment was treated with physiological saline, diluted to 200 nM, and then refrigerated (4° C.) for about 12 hours to synthesize a soluble amyloid beta polymer (Aß oligomer).

3 μl of the synthesized Aβ polymer was injected into the hippocampus CA1 of SST-CRE mice using the stereotaxic method. At this time, the Aß polymer was injected using a micro-injector at a rate of 100 nl/min, and the micro-injector was fixed for 3 minutes to spread the Aß well.

2. Virus Injection for Optogenetics Techniques

In order to express photoreceptors in SST neurons, ChR2 was used among photoreceptors. -p2A-EYFP-WPRE virus (1 μl) was injected.

Virus injection was performed using a micro-injector at a rate of 100 nl/min. After injection, the micro-injector was fixed for 3 minutes so that the virus could spread well, and then a recovery period of 3 weeks was maintained.

3. Inserting the electrode and optical fiber into the animal model injected with the viral vector

A photoelectrode was inserted into the anesthetized rat brain through stereotactic method to measure EEG in the hippocampus CA1 of an Alzheimer's mimic rat.

The photoelectrode includes an electrode for EEG measurement and an optical fiber for photostimulation transmission, and applies a band-pass filter to the local field potential measured through the electrode with a band-pass filter in the 3-12Hz band to generate theta wave. was obtained, and the power value and frequency band value of theta wave were obtained through fast Fourier transform.

4. Verification of EEG recovery of theta waves through photostimulation in the hippocampus of an animal model of Alzheimer's disease

For the EEG recovery of theta wave, the Aß and virus-injected SST-CRE rat hippocampus was irradiated with a wavelength of 470 nm through a photoelectrode. At this time, the photostimulation was continuously delivered for 3 seconds at a constant intensity. As a result, when SST inhibitory neurons were activated using optogenetic photostimulation, the theta waves, which were damaged in the hippocampus of mice mimicking Alzheimer's disease, were restored to normal levels [Fig. 4].

<Example 2> Gamma wave damage recovery method in Alzheimer's disease brain

An amyloid beta polymer (Aß oligomer) was synthesized in the same manner as in step 1 of Example 1, and 3 μl of the synthesized Aß polymer was injected into the hippocampus CA1 of PV-CRE mice using a stereotaxic method. At this time, the Aß polymer was injected using a micro-injector at a rate of 100 nl/min, and the micro-injector was fixed for 3 minutes to spread the Aß well.

To express photoreceptors in PV neurons, ChR2 among photoreceptors was used. -p2A-EYFP-WPRE virus (1 μl) was injected. Virus injection was performed using a micro-injector at a rate of 100 nl/min. After injection, the micro-injector was fixed for 3 minutes so that the virus could spread well, and then a recovery period of 3 weeks was maintained.

A photoelectrode was inserted into the anesthetized rat brain through the stereotactic method to measure EEG in the hippocampus CA1 of an Alzheimer's mimic rat, and a band pass in the 20-120Hz band to the local field potential measured through the electrode. -pass) filter was applied to obtain a gamma wave, and the power value and frequency band value of the gamma wave were obtained through fast Fourier transform.

For EEG recovery of gamma waves, the Aß and virus-injected PV-CRE mouse hippocampus were irradiated with a wavelength of 470 nm through a photoelectrode. At this time, the photostimulation was continuously delivered for 3 seconds at a constant intensity. As a result, when PV inhibitory neurons were activated using optogenetic photostimulation, the theta wave, which was damaged in the hippocampus of mice mimicking Alzheimer's disease, was restored to a normal level again [FIG. 5].

In the above, the present invention has been described in detail only with respect to the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical spirit of the present invention, and it is natural that such variations and modifications belong to the appended claims.

Claims (5)

Animal model production step in which the CRE gene is expressed in inhibitory neurons,
Injecting a viral vector containing a photoreceptor gene designed to target the inhibitory neurons in which the CRE gene is expressed into the animal model and expressing the viral vector;
Inserting an electrode and an optical fiber into the animal model injected with the viral vector;
Transmitting photostimulation to a photoreceptor gene designed to selectively express only target neurons through the electrode and optical fiber to restore damaged brain waves in Alzheimer's disease brain,
The photoreceptor is
A photoreceptor that responds to light in the wavelength region of 450, 565 and 590 nm, comprising at least one of ChIEF, C1V1, and Crimson,
The pattern of photostimulation is
EEG damage recovery in Alzheimer's disease brain using optogenetic technique, which is a pattern that simulates the activity pattern for each inhibitory neuron type occurring in the normal brain, including a constant and continuous stimulation pattern, a sine wave pattern, and a specific frequency pattern Way. The method of claim 1, wherein after the step of recovering the damaged EEG in the Alzheimer's disease brain, the step of quantifying the damage EEG recovery effect is further performed. The method of claim 1, wherein the inhibitory nerve cells are nerve cells expressing parvalbumin or somatostatin. delete The method of claim 1, wherein the damaged gamma wave or the damaged theta wave is selectively damaged by the photostimulation.

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