CN112585270A

CN112585270A - Compositions and methods for assessing or improving brain function, learning ability or memory

Info

Publication number: CN112585270A
Application number: CN201880096673.8A
Authority: CN
Inventors: 王秀杰; 张泽宇; 王猛; 谢冬芳; 黄增辉
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2018-08-15
Filing date: 2018-08-15
Publication date: 2021-03-30
Anticipated expiration: 2038-08-15
Also published as: WO2020034127A1; CN112585270B

Abstract

Compositions and methods for improving brain function or enhancing learning or memory are provided. The composition may comprise increasing METTL3 (N) in at least one body part of a subject⁶-adenosine methyltransferase 70kDa subunit). The methods can include administering to a subject a composition comprising one or more agents that increase the efficacy of METTL3 in at least one body part of the subject. Methods for assessing learning or memory ability of a subject are also provided. The method may comprise assessing N in at least one body part of the subject⁶-methyladenosine (m)⁶A) The level of the protein of interest is,and m is⁶A or said m⁶The level of the a-related protein is compared to a standard level. The method may further comprise basing the m⁶A or said m⁶Comparing the level of the a-related protein to a standard level to determine the learning or memory ability of the subject.

Description

Compositions and methods for assessing or improving brain function, learning ability or memory

Technical Field

The present application relates generally to assessment or improvement of physiological and/or psychological functions, and more particularly to compositions and methods for improving brain function, learning ability or memory, and methods for assessing brain function, learning ability or memory.

Background

Memory and learning play an important role in people's daily life. Many people suffer from memory and learning disorders or deficits such as agnosia, alzheimer's disease, amnesia and dementia. These people often have difficulty remembering information and learning new skills, and thus suffer from pain and a reduction in quality of life. Furthermore, persons with normal memory and learning ability often desire to enhance such ability, or to prevent the memory and learning ability from gradually declining with age. Memory and learning ability depend to a large extent on the function of the brain to receive, store, consolidate and retrieve information. Brain function may be associated with neuronal plasticity and expression of certain genes (e.g., early response genes). Accordingly, it is desirable to develop compositions and methods for assessing and/or improving brain function, learning ability, or memory in healthy subjects, as well as subjects suffering from disorders or deficiencies.

Disclosure of Invention

According to an aspect of the present application, a method is provided. The methods may include administering to a subject a composition for improving brain function or enhancing learning or memory in the subject, the composition comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

In some embodiments, the subject may be a human or an animal.

In some embodiments, the subject may have a learning or memory disorder.

In some embodiments, the subject may have a learning or memory deficit.

In some embodiments, the subject may have agnosia, alzheimer's disease, amnesia, brain trauma, or dementia.

In some embodiments, the subject may be mentally healthy.

In some embodiments, the at least one body part of the subject may comprise the brain of the subject.

In some embodiments, the at least one body part of the subject may comprise the hippocampus of the subject.

In some embodiments, the one or more agents may be configured to increase the amount of METTL3 in the at least one body part.

In some embodiments, the one or more agents may include a METTL3 peptide.

In some embodiments, the one or more reagents can include a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

In some embodiments, the one or more reagents can include an engineered vector comprising a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

In some embodiments, the one or more reagents can include an engineered virus comprising a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

In some embodiments, the virus may comprise an adeno-associated virus, an adenovirus, a lentivirus, or a sendai virus.

In some embodiments, the one or more agents may be configured to increase METTL3 expression by stimulating a positive factor that enhances METTL3 expression.

In some embodiments, the one or more agents may be configured to increase METTL3 expression by inhibiting a negative factor that decreases METTL3 expression.

In some embodiments, the one or more reagents may comprise an antibody.

In some embodiments, the one or more agents may be configured to stimulate METTL3 activity in the at least one body part.

In some embodiments, the one or more agents may include a METTL3 agonist.

In some embodiments, the one or more agents may be configured to increase METTL3 activity by stimulating a positive factor that enhances METTL3 activity.

In some embodiments, the one or more agents may be configured to increase METTL3 activity by inhibiting a negative factor that decreases METTL3 activity.

In some embodiments, administering the composition to the subject may comprise administering the composition to the skin of the subject.

In some embodiments, administering a composition to a subject may comprise injecting the composition into the subject.

In some embodiments, administering a composition to a subject may comprise orally administering the composition to the subject.

In some embodiments, the composition may be configured as a suppository.

According to another aspect of the present application, a method is provided. The methods can include administering to a subject a composition to enhance learning ability of the subject, the composition comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit) One or more agents of potency.

According to another aspect of the present application, a method is provided. The methods may include administering to a subject a composition to enhance memory in the subject, the composition comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

According to another aspect of the present application, a method is provided. The methods can include administering to a subject a composition for enhancing long term memory consolidation in the subject, the composition comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

According to another aspect of the present application, a method is provided. The methods can include administering to a subject a composition for enhancing long term memory consolidation in the subject, the composition comprising increasing METTL3 (N) in the subject's hippocampus⁶-adenosine methyltransferase 70kDa subunit).

According to another aspect of the present application, a method is provided. The methods can include administering to a subject a composition for improving brain function in the subject with a mental disorder, the composition comprising increasing METTL3 (N) in the hippocampus of the subject⁶-adenosine methyltransferase 70kDa subunit).

According to another aspect of the present application, a method is provided. The methods can include administering to a subject a composition for enhancing long-term memory consolidation in the subject having a memory deficit, the composition including increasing METTL3 (N) in the hippocampus of the subject⁶-adenosine methyltransferase 70kDa subunit).

According to another aspect of the present application, a method is provided. The method can include administering to a subject a composition for enhancing long-term memory consolidation in the subject who does not suffer from a memory deficit, the composition including in the subject's seaIncreasing METTL3 (N) in horses⁶-adenosine methyltransferase 70kDa subunit).

According to another aspect of the present application, a method is provided. The method may comprise administering to a subject a composition for improving brain function or enhancing learning or memory in the subject, the composition comprising increasing N in at least one body part of the subject⁶-methyladenosine (m)⁶A) An abundance of one or more reagents.

According to another aspect of the present application, a method for assessing learning or memory of a subject is provided. The method may comprise assessing N in at least one body part of the subject⁶-methyladenosine (m)⁶A) The level of the protein of interest, and⁶a or said m⁶The level of the a-related protein is compared to a standard level. The method may further comprise basing the m⁶A or said m⁶Comparing the level of the a-related protein to a standard level to determine learning or memory of the subject.

In some embodiments, the m⁶The a-related protein may be METTL3 protein.

In some embodiments, the standard level is determined by evaluating the m of at least one body part of a control subject⁶Levels of a-related protein.

According to yet another aspect of the present application, a composition is provided. The composition may be configured to improve brain function or enhance learning or memory of the subject. The composition may comprise increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

In some embodiments, the subject may be a human or an animal.

In some embodiments, the subject may have a learning or memory disorder.

In some embodiments, the subject may have a learning or memory deficit.

In some embodiments, the subject may be mentally healthy.

In some embodiments, the one or more agents may include a METTL3 peptide.

In some embodiments, the one or more reagents may comprise an antibody.

In some embodiments, the one or more agents may include a METTL3 agonist.

Additional features of the present application will be set forth in part in the description which follows. Additional features of some aspects of the present application will be apparent to those of ordinary skill in the art in view of the following description and accompanying drawings, or in view of the production or operation of the embodiments. The features of the present application may be realized and obtained by means of the instruments and methods and by means of the methods and combinations set forth in the detailed examples discussed below.

Drawings

The present application will be further described by way of exemplary embodiments. These exemplary embodiments will be described in detail by means of the accompanying drawings. It should be noted that the drawings are not to scale. These embodiments are non-limiting exemplary embodiments in which like reference numerals represent similar structures throughout the several views of the drawings, and wherein:

FIGS. 1A-1F show exemplary results of a postnatal knockout of Mettl3 in the hippocampus resulting in a prolonged Long Term Memory (LTM) consolidation process, according to some embodiments of the present application;

2A-2D show exemplary results of electrophysiological testing of a hippocampus knockout Mettl3 according to some embodiments of the present application;

FIGS. 3A-3D illustrate METTL3 passing through its m according to some embodiments of the present application⁶Exemplary results of modulation of long-term memory formation by A methyltransferase function;

FIGS. 4A-4D illustrate dynamically adjusting m during memory consolidation according to some embodiments of the present application⁶Exemplary results of A methylation;

FIGS. 5A-5F illustrate m according to some embodiments of the present application⁶Exemplary results of a promoting translation of early response genes following activity induction;

6A-6E illustrate exemplary results of overexpression of METTL3 enhancing the development of long term memory according to some embodiments of the present application;

FIG. 7 illustrates an exemplary proposed model according to some embodiments of the present application;

figures 8A-8H show exemplary results of brain gross morphology characterization of Mettl3 cKO mice according to some embodiments of the present application;

figures 9A-9F show exemplary results of Mettl3 cKO mice showing no differences in locomotion, exploration, and anxiety compared to the control group according to some embodiments of the present application;

10A-10C show exemplary results of the electrophysiological properties of cKO mice, according to some embodiments of the present application;

11A-11B illustrate exemplary results of an analysis of transcriptome changes during early time points after training according to some embodiments of the present application;

FIG. 12 shows exemplary results of MeRIP-qPCR validation according to some embodiments of the present application; and

fig. 13A-13B show exemplary results of overexpression of Mettl3 in hippocampus or primary neurons according to some embodiments of the present application.

Detailed Description

The following description is presented to enable any person skilled in the art to make and use the application, and is provided in the context of a particular application and its requirements. It will be apparent to those skilled in the art that various modifications to the disclosed embodiments are possible, and that the general principles defined in this application may be applied to other embodiments and applications without departing from the spirit and scope of the application. Thus, the present application is not limited to the described embodiments, but should be accorded the widest scope consistent with the claims.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes" and/or "including," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

The combination of these features of the present application, as well as the methods of operation and functions of the structure of the related elements, and the parts and economies of manufacture, will become more apparent upon consideration of the following description with reference to the accompanying drawings, all of which form a part of this specification. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the application. It should be understood that the drawings are not to scale.

Compositions and methods for improving brain function or enhancing learning or memory, and methods for assessing brain function, learning or memory are provided. In some embodiments, the composition can comprise one or more agents that increase METTL3 (N) in at least one body part of the subject (e.g., the brain of the subject)⁶Adenosine methyltransferase 70kDa subunit). In some embodiments, the one or more agents may be configured to increase the amount of METTL3 or METTL3 activity in at least one body part of the subject. In some embodiments, the subject may be mentally healthy, or the subject may have a learning disorder, a memory disorder, a learning deficit, a memory deficit, or the like, or any combination thereof. In some embodiments, a method for improving brain function or enhancing learning or memory may comprise administering to a subject a composition as described above. In some embodiments, the present disclosure also includes compositions disclosed hereinUse for the manufacture of a medicament or food supplement for the treatment of a disorder or defect in brain function, learning ability or memory in a subject. In some embodiments, the present disclosure also includes use of a composition disclosed herein in the manufacture of a medicament or food supplement for improving a disorder or deficiency in brain function, learning ability, or memory in a subject. In some embodiments, the composition can be administered to the subject by oral administration, injection administration, topical administration, and the like. In some embodiments, the composition may be configured as a suppository. In some embodiments, a method for assessing brain function, learning ability, or memory of a subject may include assessing N of at least one body part of the subject⁶-methyladenosine (m)⁶A) Or m is⁶Levels of a-related proteins (e.g., METTL3, METTL 14). In some embodiments, m may be⁶A or m⁶The estimated level of the a-related protein is compared to a standard level. In some embodiments, may be based on m⁶A (or m)⁶A-related protein) to determine brain function, learning ability, or memory of the subject. In response to determining m⁶A (or m)⁶A-related protein) below the corresponding standard level, it can be determined that the subject has a relatively low brain function, learning ability, or memory.

As used herein, the term "potency" refers to the total catalytic ability of an enzyme (e.g., RNA, protein, peptide, or fragment thereof) in a subject or in certain regions of a subject (e.g., a body part, tissue, or organ). The region may include, but is not limited to, the entire body or a portion of the body of the subject. In some embodiments, the subject may be a human or non-human animal. In some embodiments, a portion of a body may include an organ, tissue, blood vessel, or portion thereof, or a combination thereof. By way of example only, the region may include the head, brain, hippocampus, cortex, prefrontal cortex, neocortex, amygdala, striatum, cerebellum, and the like, or any combination thereof. In certain embodiments, the area includes a human hippocampus. In some embodiments, the effectiveness of an enzyme may depend on the total amount (or concentration) of the enzyme in the region and/or the activity of the enzyme in the region. The term "activity" of an enzyme refers to the catalytic ability of a certain number of enzymes in that region.

As used herein, the term "brain function" refers to the ability of the brain to receive, process, and/or store various information, and/or to control and/or modulate various biological activities. In some embodiments, brain function may be affected or impaired by one or more factors such as aging, brain trauma, tumors, physiological disorders, and the like, or any combination thereof. If the brain function is abnormal, one or more abilities related to the subject's brain function (e.g., understanding, memory, reading, spoken language, hearing, vision, learning, etc.) may be affected. In some embodiments, brain function may be improved by the compositions and methods described herein.

As used herein, the term "memory" refers to the ability to acquire information stored in the brain that can be retrieved, or encode, store, register, access and/or retrieve the acquired information. In some embodiments, the terms "memory" and "memorability" are used interchangeably herein to refer to the ability to encode, store, register, access and/or retrieve information. In some embodiments, the memory may include short-term memory and/or long-term memory. In some embodiments, short-term memory may refer to the ability to save a small amount of information in the brain over a short period of time (e.g., 1s, 2s, 3s, 4s, 5s, 10s, 15s, 20s, 25s, 30s, etc.). In some embodiments, the short-term memory can develop rapidly and can last for a relatively short period of time (e.g., one or more seconds, one or more minutes, one or more hours, one or more days, etc.). In some embodiments, long-term memory may be a stage in the Atkinson-Shiffrin memory model where knowledge of information may be maintained indefinitely. In some embodiments, the rate of development of long-term memory may be slower and may last for a relatively longer period of time (e.g., one or more days, one or more weeks, one or more months, one or more years, etc.) than the short-term memory. In some embodiments, the newly acquired information may be initially stored in the brain in a vulnerable state and may tend to be gradually forgotten by the subject. The fragile state of the acquired information may transition to a relatively stable state in the brain during memory integration, and thus, the acquired information is less likely to be forgotten by the subject. In some embodiments, the memory consolidation process may occur naturally over time, or as the same acquired information (or related information) is retrieved.

As used herein, the term "learning capabilities" refers to the ability to obtain new information, knowledge and/or skills through a process that includes experiencing, learning, or receiving training, etc. In some embodiments, learning capabilities may depend at least in part on the development of memory. For example, during learning, known information in existing memory may be retrieved and used to generate new information, such as new understandings, knowledge, skills, and the like, or any combination thereof. In some embodiments, the new information may be stored in the brain for further learning processes. Thus, in some embodiments, if a subject suffers from a memory disorder, it may be indicated that the subject also suffers from a learning disorder. In some embodiments, a memory impairment and/or a learning impairment may be induced or indicated if the brain function used to receive, process and/or store information is abnormal.

According to an aspect of the present application, there is provided a composition configured to improve brain function or enhance learning or memory of a subject. In some embodiments, the composition may comprise a peptide configured to increase m in at least one body part of the subject⁶A level of one or more agents. In some embodiments, the composition may include a METTL3 (N) configured to increase in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

In some embodiments, the subject may be a human or an animal. For example, the subject may be an infant, child, adolescent, young adult, middle aged or elderly human. In some embodiments, the subject may be a vertebrate or an invertebrate. Exemplary animals can be monkeys, orangutans, tigers, cats, dogs, rabbits, ferrets, pigs, gerbils, hamsters, yellow rats, mice, guinea pigs, hedgehogs, pouzoles, yellow squirrels, marmot, squirrels, fish, turtles, and the like, or any combination thereof. In some embodiments, the subject may be a mammal. In some embodiments, the subject may include a companion animal (also referred to as a "pet"), an animal of a protected species, an animal used for scientific research, an animal assisting police (e.g., a police dog), and the like, or any combination thereof. In some embodiments, animals used for scientific research can include normal animal models (e.g., animals that have not received any experimental treatment) and/or test animal models (e.g., animals that have received one or more experimental treatments). In some embodiments, the test animal model may have received one or more experimental treatments relating to toxicology testing, disease treatment testing, xenograft testing, drug testing, defense testing, behavioral testing (e.g., long term memory testing, short term memory testing, learning testing). In some embodiments, the one or more experimental treatments may include, but are not limited to, administering a test composition (e.g., a drug, a supplement or an active ingredient thereof, or a food, a beverage, etc.) to the test animal model, altering the conditions of growth and development of the test animal model, modifying one or more genes of the test animal model, etc., or any combination thereof.

In some embodiments, the subject may suffer from a learning deficit and/or a memory deficit. As used herein, the term "learning deficit" (also referred to as learning disability) may refer to a subject having an unusually difficult to learn information, knowledge and/or skills, or having symptoms of a disease associated with learning disability or reduced learning ability. As used herein, the term "memory deficit" may refer to a symptom of a subject having exceptional difficulty encoding, storing, registering, accessing, and/or obtaining learned information, or having a disease associated with memory disability or memory loss. In some embodiments, the subject may have a learning disorder and/or a memory disorder. As used herein, the term "learning disorder" may refer to a neurological development, neural structure, or neurochemical problem that causes a person with normal intellectual potential to encounter, in their learning function, an abnormal difficulty that cannot be explained with insufficient educational opportunities or emotional or mental disorders. As used herein, the term "memory disorder" may refer to damage to one or more neuro-anatomical structures or the result of a neurochemical problem that has impeded the storage, retention, and/or recovery of memory. In some embodiments, the subject may be impaired in brain function (i.e., brain dysfunction) in receiving, processing, and/or storing information, and this may lead to memory impairment and/or learning impairment. In some embodiments, the learning disorder, learning deficit, memory disorder, memory deficit, and/or brain dysfunction may be caused by one or more factors, such as developmental problems, aging, brain trauma, tumors, physiological disorders, and the like, or any combination thereof. For example, the subject may have traumatic brain injury, agnosia, alzheimer's disease, amnesia, dementia, huntington's disease, parkinson's disease, weirnike-koxsackov syndrome, or the like, or any combination thereof. As another example, a subject may have a tumor located in or near the central nervous system (e.g., the brain), which may affect the normal biological functions of the brain.

In some embodiments, the composition can be administered to a subject to treat or alleviate a learning disorder, learning deficit, memory disorder, memory deficit, and/or brain dysfunction. For example, the composition may be configured to reduce, alleviate or eliminate one or more symptoms of a learning disorder, learning deficit, memory disorder, memory deficit, and/or brain dysfunction, and/or reduce or slow further progression. In some embodiments, the compositions may be administered to a subject to improve the brain's ability to receive, process, and store various information, and/or the ability to control and/or modulate various biological activities; enhancing the ability of the subject to encode, store, register, access and/or retrieve the acquired information, and/or enhancing the ability of the subject to obtain new information, knowledge and/or skills.

In some embodiments, the subject may be mentally healthy. As used herein, the term "mental health" refers to the absence of learning disorders, learning deficits, memory disorders, memory deficits, and/or brain dysfunction as described above. A mentally healthy subject may have normal learning, memory and/or brain function. In some embodiments, the composition can be administered to a mental healthy subject to improve brain function, enhance learning and/or memory in the subject, or prevent a decline in learning and/or memory (e.g., with age). In some embodiments, the compositions may be administered to a subject at risk of learning disorders, learning deficits, memory disorders, memory deficits, and/or brain dysfunction to prevent these and other disorders. In some embodiments, the subject may be an elderly human. In some embodiments, the subject may be an infant, child, juvenile, or adult from a family with a genetic history associated with a learning deficit, memory deficit, and/or brain dysfunction.

In some embodiments, the composition may include a peptide configured to increase m⁶A methyltransferase activity of a. m is⁶The a methyltransferase may include, but is not limited to, METTL3, METTL14, WTAP, KIAA1429, or the like, or a fragment thereof, or any combination thereof. In some embodiments, the composition may comprise a peptide configured to reduce m⁶A Demethiylase potency to increase m⁶One or more agents of A abundance. m is⁶A demethylases may include, but are not limited to, FTO, ALKBH5, and the like, or fragments thereof, or any combination thereof. In the following description, the compositions and methods for increasing the efficacy of METTL3 are provided for illustrative purposes only and are not intended to limit the scope of the present application. It should be noted that similar strategies may also be applied to add one or more other m⁶Compositions and methods for reducing the potency of A methyltransferases, or reducing the potency of one or more m⁶A demethylase or other m⁶Compositions and methods for the efficacy of A-related enzymes or any combination thereof.

In some embodiments, the composition may include one or more agents that increase the efficacy of METTL3 in at least one body part of the subject. METTL3 is N⁶-adenosine methyltransferase 70kDa subunit. As used herein, the term "METTL 3 potency" may refer to METTL3 catalyzing in at least one body part of a subjectTotal capacity for methylation of RNA (e.g., mRNA) of (a). In some embodiments, METTL3 can catalyze methylation of mRNA to facilitate translation of mRNA. In some embodiments, METTL3 can recognize and/or facilitate translation of methylated mRNA. In some embodiments, METTL3 can bind chromatin. For example, METTL3 can bind DNA at the transcriptional initiation region (e.g., promoter). In some embodiments, METTL3 may be determined by dependence on m⁶A or independently of m⁶The mechanism of a promotes translation of genes (e.g., neuronal early response genes) to regulate memory formation and/or memory consolidation. In some embodiments, METTL3 (or other m)⁶A methyltransferase) may enhance long-term potentiation (LTP). As used herein, the term "long-term potentiation" refers to the persistent enhancement of synapses based on recent patterns of synaptic activity. In some embodiments, the recent pattern of synaptic activity may produce a persistent increase in signaling between two neurons. In some embodiments, enhancement of LTP may result in improvement in brain function, learning ability, and memory (e.g., long-term memory). In some embodiments, the agent that increases the efficacy of METTL3 may be configured to increase the amount of METTL3 in at least one body part of the subject and/or increase the activity of METTL3 in at least one body part unit number of the subject. In some embodiments, the at least one body part may comprise an organ, tissue or part thereof. By way of example only, the at least one body part may include, but is not limited to, the head, brain, hippocampus, cortex, prefrontal cortex, neocortex, amygdala, striatum, cerebellum, and the like, or any combination thereof.

In some embodiments, the efficacy of METTL3 may be increased by increasing the amount of METTL3 peptide or increasing the activity of METTL3 peptide in at least one body site. As used herein, the term "METTL 3 peptide" refers to a full-length METTL3 protein, or a fragment of METTL3 protein that retains the efficacy of METTL3 (i.e., the ability of the METTL3 peptide to catalyze methylation of RNA (e.g., mRNA)).

In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site may comprise a METTL3 peptide. In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site can include a METTL3cDNA sequence and/or a fragment of METTL3cDNA sequence. As used herein, Mettl3cDNA sequence and/or fragments thereof can refer to DNA sequences encoding Mettl3 peptides. In some embodiments, the agent that increases the efficacy of METTL3 in at least one body part may include a METTL3mRNA sequence and/or a fragment of a METTL3mRNA sequence. As used herein, a METTL3mRNA sequence and/or a fragment of METTL3mRNA sequence may refer to mRNA transcribed from a fragment of METTL3cDNA sequence and/or METTL3cDNA sequence. In some embodiments, a Mettl3cDNA sequence, a fragment of a Mettl3cDNA sequence, a Mettl3mRNA sequence, and/or a fragment of a Mettl3mRNA sequence can increase expression of a Mettl3 peptide in at least one body site, thereby increasing the amount of expression of a Mettl3 peptide in at least one body site.

In some embodiments, an agent that increases the efficacy of METTL3 in at least one body site may include an engineered METTL3 peptide, an engineered METTL3 peptide refers to a mutant form of full-length METTL3 or a METTL3 fragment that retains catalytic activity, and a peptide comprising a wild-type or mutant METTL3 peptide coupled or linked to a carrier agent. For example, the carrier agent may be a carrier nanoparticle or a carrier peptide configured to facilitate passage of the METTL3 peptide across the Blood Brain Barrier (BBB). In certain embodiments, liposomes can be loaded onto carrier nanoparticles, thereby facilitating the transfer of the engineered METTL3 peptide across the BBB. In certain embodiments, the vector peptide may be a peptide having a sequence obtained from the brainpps database and capable of passing through the BBB, e.g., by active transport.

In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site can include an engineered vector comprising a METTL3cDNA sequence or a fragment of METTL3cDNA sequence. In some embodiments, the agent that increases the efficacy of METTL3 in at least one body part may comprise an engineered vector comprising a METTL3mRNA sequence or a fragment of a METTL3mRNA sequence. Vectors may include, but are not limited to, plasmids, cosmids, synthetic nucleic acids, artificial chromosomes, and the like. By way of example only, a Mettl3cDNA sequence, a fragment of Mettl3cDNA sequence, a Mettl3mRNA sequence, and/or a fragment of Mettl3mRNA sequence can be incorporated into nucleic acids, respectively, to produce engineered nucleic acids. In some embodiments, the engineered nucleic acid can be used directly to transfect cells in at least one body part of a subject.

In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site may comprise an engineered virus. The engineered virus can include a Mettl3cDNA sequence, a fragment of Mettl3cDNA sequence, a Mettl3mRNA sequence, and/or a fragment of Mettl3mRNA sequence. In some embodiments, the engineered nucleic acid can be introduced into a virus to obtain an engineered virus. For example, the virus may include adenovirus, adeno-associated virus, sendai virus (SeV), retrovirus, polyoma virus, epstein-barr virus, and the like, or any combination thereof. In some embodiments, the retrovirus may include one or more viruses from the alpha retrovirus genus (e.g., avian leukemia virus), the bertaro retrovirus genus (e.g., mouse mammary tumor virus), the gamma retrovirus genus (e.g., murine leukemia virus), the delta retrovirus genus (e.g., bovine leukemia virus), the epsilon retrovirus genus (e.g., Walleye skin sarcoma virus), the lentivirus genus (e.g., human immunodeficiency virus), the foamy virus genus (e.g., simian foamy virus), and the like, or any combination thereof. In some embodiments, the engineered virus may infect one or more cells in at least one body site, and expression of the METTL3 peptide may be increased in the at least one body site.

In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site may comprise a positive factor that enhances METTL3 expression. In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site may be configured to increase METTL3 expression by stimulating a positive factor that enhances METTL3 expression. For example, the agent may include a peptide, a lipid, a polysaccharide, a nucleic acid, a steroid, or any combination thereof.

In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site may be configured to increase METTL3 expression by inhibiting a negative factor that decreases METTL3 expression. In some embodiments, the negative factor that reduces METTL3 expression may include microrna (mirna), antisense DNA, small interfering rna (sirna), or the like, or any combination thereof. In some embodiments, the complementary strand of the miRNA, antisense DNA, and/or siRNA can bind to the METTL3mRNA sequence or a fragment of the METTL3mRNA sequence through a base pairing mechanism, such that the METTL3mRNA sequence can be silenced, and expression of the METTL3 peptide can be inhibited. In some embodiments, an agent that inhibits a negative factor may include an inhibitor of miRNA, antisense DNA, and/or siRNA, an enzyme that degrades miRNA, antisense DNA, and/or siRNA, a nucleic acid encoding an enzyme that degrades miRNA, antisense DNA, and/or siRNA, a vector comprising a nucleic acid encoding an enzyme that degrades miRNA, antisense DNA, and/or siRNA, an engineered virus including an enzyme that encodes an enzyme that degrades miRNA, antisense DNA, and/or siRNA, and the like, or any combination thereof. By way of example only, mirnas that reduce METTL3 expression may include let-7g mirnas that target the 3 '-UTR (3' untranslated region) of METTL3 mRNA. Exemplary agents that inhibit let-7g mirnas and increase METTL3 expression may include, but are not limited to, mammalian hepatitis b virus X protein binding protein (HBXIP) or fragments thereof. In some embodiments, the agent that increases the efficacy of METTL3 in at least one body site may comprise an antibody. In some embodiments, the antibody can specifically bind to a negative factor that reduces METTL3 expression. For example, an antibody can include an IgG, IgM, IgA, IgD, or IgE molecule or an antigen-specific fragment thereof (e.g., Fab fragment, Fv fragment, scFv fragment, single domain antibody, disulfide-linked scFv fragment, etc.).

In some embodiments, the composition may include one or more agents configured to stimulate METTL3 activity in at least one body part. In some embodiments, the one or more agents may include a METTL3 agonist. As used herein, the term "METTL 3 agonist" refers to a substance that can increase the activity of the METTL3 peptide. The activity of METTL3 may refer to the ability of a unit number of METRT3 peptides in at least one body site to catalyze the methylation of RNA. In some embodiments, the METTL3 agonist may comprise a peptide, a lipid, a polysaccharide, a nucleic acid, a steroid, or any combination thereof. For example, a METTL3 agonist may include a miRNA that promotes METTL3 binding to mRNA for methylation. Such miRNAs may include, but are not limited to, miR-423-3p, miR-1226-3p, miR-330-5p, miR-668-3p, miR-1224-5p, miR-1981 and the like, or any combination thereof.

In some embodiments, the agent may be configured to increase METTL3 activity by stimulating a positive factor that enhances METTL3 activity. In some embodiments, the agent may be configured to increase the activity of one or more mirnas that promote binding of the METTL3 peptide to mRNA. In some embodiments, the agent may be configured to increase the expression of one or more mirnas that promote binding of the METTL3 peptide to mRNA. For example, the agent may comprise an enzyme that promotes the production of one or more mirnas, such as a Dicer protein and/or a fragment of a Dicer protein. As another example, an agent can include a nucleic acid and/or fragment of a nucleic acid encoding a Dicer protein or fragment of a Dicer protein. As yet another example, an agent can comprise a vector or engineered virus comprising a nucleic acid and/or fragment of a nucleic acid encoding a Dicer protein and/or fragment of a Dicer protein.

In some embodiments, the agent may be configured to increase METTL3 activity by inhibiting a negative factor that decreases METTL3 activity. By way of example only, a negative factor may include one or more small ubiquitin-like modifying (SUMO) proteins. SUMO proteins may be covalently linked to the METTL3 peptide to inhibit the catalytic ability of the METTL3 peptide. For example, SUMO proteins may include SUMO-1, SUMO-2, SUMO-3, SUMO-4, and the like, or any combination thereof. In some embodiments, the agent may inhibit the activity of one or more SUMO proteins. For example, the agent may be an antibody that binds to one or more SUMO proteins. In some embodiments, the agent may inhibit the expression of SUMO protein.

In some embodiments, the agent can increase the amount of METTL3 peptide and/or the activity of METTL3 peptide by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 150%, 200%, 300% or more.

In some embodiments, a composition for improving brain function and/or enhancing learning and/or memory may be administered orally to a subject. In some embodiments, the composition for oral administration may be formulated as a medicament, dietary supplement, food additive, beverage additive, or the like, or any combination thereof. In some embodiments, the composition may be formulated as a tablet, granule, powder, micelle, liquid, suspension, emulsion, or the like, or any combination thereof. In some embodiments, the composition may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may need to be non-toxic and may not negatively impact the activity of the agent that increases the efficacy of METTL3 in the composition. For example, a pharmaceutically acceptable carrier may include excipients, diluents, auxiliary ingredients, and the like, or any combination thereof. Exemplary excipients can include, but are not limited to, emulsifiers, flow agents, flavoring agents, coloring agents, and the like, or any combination thereof. In some embodiments, the pharmaceutically acceptable carrier can protect the agent from oxidation and/or degradation by enzymes and low/high pH values, thereby maintaining the efficacy of the composition. For example, the pharmaceutically acceptable carrier may include a coating layer, a capsule, a microcapsule, a nanocapsule, etc., or any combination thereof. In some embodiments, the pharmaceutically acceptable carrier may have the ability to control the release of an agent that may increase the efficacy of METTL 3. Controlled release may include, but is not limited to, slow release, sustained release, targeted release, and the like, or any combination thereof. By way of example only, pharmaceutically acceptable carriers may include hydrogel capsules, microcapsules, or nanocapsules made of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polylactic acid, and the like, or any combination thereof.

In some embodiments, the composition for improving brain function and/or enhancing learning and/or memory may be a parenteral formulation. For example, the composition may be in the form of a solution, suspension, emulsion, powder, or the like for injection. In some embodiments, the composition in powder form may be dissolved or dispersed in a solution, suspension, or emulsion prior to injection. In some embodiments, the injectable formulation may further comprise other pharmaceutically injectable ingredients, such as glucose, sodium chloride, potassium chloride, and the like, or any combination thereof. In some embodiments, the composition can be administered to the subject by intravenous injection or intraperitoneal injection. In some embodiments, the composition can be stereotactically injected into at least one body part or an area proximate to the at least one body part to increase METTL3 efficacy in the at least one body part. For example, the composition can be administered to the head, brain, hippocampus, cortex, prefrontal cortex, neocortex, amygdala, striatum, cerebellum, and the like, or any combination thereof.

In some embodiments, the composition may be formulated (or configured) as a suppository. As used herein, "suppository" refers to a dosage form that is inserted into the rectum, vagina, urethra, etc., or any combination thereof. In some embodiments, the suppository may include a pharmaceutically acceptable carrier that contains the active ingredients of the composition (e.g., one or more agents that increase the efficacy of METTL 3). In some embodiments, the pharmaceutically acceptable carrier may gradually dissolve, melt, or degrade (e.g., in the rectum, vagina, urethra) to release the active ingredient to produce a local or systemic effect. In some embodiments, the composition can be administered vaginally, rectally, nasally (e.g., in the form of nasal drops), otically (e.g., in the form of ear drops), intramedullary, intraarticular, intrapleurally, etc., or any combination thereof. In some embodiments, the composition may be administered to the skin of a subject. For example, the composition may be formulated as a powder, granules, nanoparticles, cream, lotion, ointment, suspension, solution, and the like. By way of example only, the composition may be applied or sprayed onto the skin of the subject (e.g., skin of at least one body part, skin of a nearby area of at least one body part).

According to another aspect of the present application, a method for improving brain function and/or enhancing learning and/or memory of a subject is provided. In some embodiments, the method may comprise administering an effective amount of a composition comprising one or more agents that increase the efficacy of METTL3 (e.g., as previously described herein) in at least one body part of the subject.

In some embodiments, the subject may be a human or an animal. In some embodiments, the subject may have a learning or memory disorder. In some embodiments, the subject may have a learning or memory deficit. In some embodiments, the subject may be afflicted with agnosia, alzheimer's disease, amnesia, traumatic brain injury, dementia, huntington's disease, parkinson's disease, west-korsakoff syndrome, or the like, or any combination thereof. In some embodiments, the subject may be mentally healthy. In some embodiments, the at least one body part of the subject may include the head, brain, hippocampus, cortex, prefrontal cortex, neocortex, amygdala, striatum, cerebellum, and the like, or any combination thereof.

In some embodiments, the composition may include one or more agents configured to increase the efficacy of METTL3 in at least one body part. In some embodiments, the agent may include a METTL3 peptide. In some embodiments, reagents can include a fragment of the Mettl3cDNA sequence and/or the Mettl3cDNA sequence. In some embodiments, reagents can include engineered vectors that include a Mettl3cDNA sequence and/or a fragment of Mettl3cDNA sequence. In some embodiments, the agent can include an engineered vector comprising a METTL3mRNA sequence and/or a fragment of a METTL3mRNA sequence. In some embodiments, reagents can include engineered viruses that include a Mettl3cDNA sequence and/or a fragment of Mettl3cDNA sequence. In some embodiments, the agent can include an engineered virus that includes a METTL3mRNA sequence and/or a fragment of a METTL3mRNA sequence. More description of compositions including agents that increase the efficacy of METTL3 can be found elsewhere in the application.

In some embodiments, a composition for improving brain function and/or enhancing learning and/or enhancing memory may be administered orally to a subject. In some embodiments, the composition for improving brain function and/or enhancing learning and/or enhancing memory may be administered to the subject by parenteral administration, e.g., intravenous injection, intraperitoneal injection. In some embodiments, the composition can be stereotactically injected into at least one body part or an area proximate to the at least one body part to increase METTL3 efficacy in the at least one body part. For example, the composition can be administered to the head, brain, hippocampus, cortex, prefrontal cortex, neocortex, amygdala, striatum, cerebellum, and the like, or any combination thereof. In some embodiments, the composition can be administered to a subject via vaginal, rectal, nasal, otic, intramedullary, intra-articular, intra-pleural, and the like, or any combination thereof. In some embodiments, the composition may be administered to the skin of a subject. For example, the composition may be formulated as a powder, granules, nanoparticles, cream, lotion, ointment, suspension, solution, and the like. By way of example only, the composition may be applied or sprayed onto the skin.

In some embodiments, the composition may be administered to the subject once a day, twice a day, three times a day, four times a day, etc. In some embodiments, the composition can be administered to the subject every two days, every three days, weekly, biweekly, monthly, etc.

In some embodiments, the composition may be used in combination with other drugs and/or dietary supplements to improve brain function and/or enhance learning and/or enhance memory. Exemplary drugs for improving brain function and/or enhancing learning and/or enhancing memory may include, but are not limited to, donepezil, rivastigmine, galantamine, maytansine, and the like, or any combination thereof. Exemplary core ingredients of the dietary supplement may include acetyl-l-carnitine (acetyl-l-carnitine), Bacopa monnieri (Bacopa monnieri), citicoline (citicoline), curcumin (curcumin), ginseng, huperzine A (huperzine A), and the like, or any combination thereof.

According to yet another aspect of the present application, a method for assessing learning and/or memory ability of a subject is provided. The method can comprise the following steps: (a) assessing N in at least one body part of a subject⁶-methyladenosine (m)⁶A) And/or one or more of m⁶The level of a-related protein; (b) m is to be⁶Level of A and/or m⁶Comparing the level of the a-related protein to a standard level; (c) determining (or evaluating) the learning ability and/or memory ability of the subject based on the comparison of (b).

In some embodiments, the at least one body part may comprise an organ, tissue, blood vessel, etc. or a portion thereof or any combination thereof. In some embodiments, the at least one body part may comprise the entire body of the subject. In some embodiments, the at least one body part may comprise one or more regions of the subject. Exemplary regions may include the head, brain, hippocampus, cortex, prefrontal cortex, neocortex, amygdala, striatum, cerebellum, and the like, or any combination thereof.

In some embodiments, to evaluate m⁶Level of A and/or m⁶Levels of a-related protein, one or more biological samples may be obtained from the subject. In some embodiments, the biological sample may include, but is not limited to, a tissue sample, a cell sample, a bodily fluid sample, and the like, or any combination thereof. Exemplary bodily fluid samples may include blood samples, mucus samples, semen samples, saliva samples, urine samples, breast milk samples, interstitial fluid, cerebrospinal fluid, lymph fluid, and the like, or any combination thereof. In some embodiments, the biological sample may be obtained from at least one body part (e.g., the hippocampus). In some embodiments, a biological sample may be obtained from one or more other body parts of a subject to assess m in at least one body part⁶Level of A and/or m⁶Levels of a-related protein. For example, the biological sample may comprise a venous blood sample obtained from a vein of the subject. In some embodiments, the biological sample may be obtained from at least one body part at any point in time. In some embodiments, the biological sample may be obtained from at least one body part after the learning test and/or the memory test. In some embodiments, the biological sample may be obtained from at least one body part after the subject has received a certain amount of training (i.e., the subject has learning and/or memory activity).

In some embodiments, a biological sample can be pretreated to obtain a pretreated biological sample. In some embodiments, a biological sample (e.g., a tissue sample) may be mashed in the pre-treatment. In some embodiments, in the pretreatment, the biological sample may contain cells and may be subjected to a cell membrane disruption processTo release intracellular RNA or protein. Exemplary operations for disrupting cell membranes may include homogenization treatment, sonication treatment, organic solvent treatment, acid treatment, base treatment, and the like, or any combination thereof. In some embodiments, total protein can be extracted from a biological sample to obtain a pretreated biological sample. In some embodiments, total RNA can be extracted from the biological sample. In some embodiments, a portion of total RNA that satisfies a predetermined condition can be isolated to obtain a measurement for m⁶A level of pretreated biological sample. In some embodiments, the predetermined condition may relate to the integrity of the RNA. For example, the integrity of RNA can be tested by agarose gel electrophoresis. In some embodiments, the separation may be of about 2: 1 ratio of 28S and 18S ribosomal RNA gel bands to obtain a pretreated biological sample. In some embodiments, the pretreated biological sample can be used to measure m quantitatively or qualitatively⁶The level of A.

In some embodiments, m⁶The level of A may refer to m for RNA (e.g., mRNA) in at least one body part⁶Degree of modification of a. In some embodiments, m of all RNAs in a biological sample may be pretreated⁶Intensity of A methylation site (i.e., m)⁶Intensity of A peak) to evaluate m⁶The level of A. In some embodiments, m of an mRNA that can be transcribed from a set of target genes⁶Evaluation of m by the intensity of A methylation sites⁶The level of A. In some embodiments, the set of target genes may be selected from a set of genes associated with memory and/or learning. For example, the set of target genes may include an Immediate Early Gene (IEG). In some embodiments, IEGs may include, but are not limited to, Arc, Btg2, Egr1, c-Fos, Npas4, Nr4a1, and the like, or any combination thereof. In some embodiments, the IEG may be activated quickly after learning. In some embodiments, IEGs may be essential for long-term memory development.

Evaluation of m⁶Exemplary methods for level a may include liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), colorimetry, methylated single nucleotide-resolved cross-linking and immunoprecipitation sequencing (miCLIP-seq) methods, methylated RNA co-immunoprecipitation-real-time quantitative PCR (MeRIP-qPCR) method, or the like, or any combination thereof. In some embodiments, for evaluating m⁶The method of level A may comprise contacting the pretreated biological sample with a specifically bindable moiety m⁶Contacting the reagent A. In some embodiments, the agent may be labeled with a detectable label. For example, the reagent may comprise a specific binding m⁶An antibody or fragment thereof of A. As another example, the label may include a fluorescent label, an isotopic label, or the like, or any combination thereof. m is⁶The level of a can be assessed on the basis of a detectable marker.

In some embodiments, m⁶The A-related protein may include m⁶A methyltransferase, m⁶A demethylase, and the like, or any combination thereof. E.g. m⁶The A methyltransferase may include, but is not limited to, METTL3, METTL14, WTAP, KIAA1429, and the like, or a fragment thereof, or any combination thereof. As another example, m⁶A demethylases may include, but are not limited to, FTO, ALKBH5, and the like, or fragments thereof, or any combination thereof. In certain embodiments, m⁶The a-related protein is METTL 3.

In some embodiments, for evaluating m⁶The method of the level of the A-related protein may comprise assessing m⁶Expression level of A-related protein and/or evaluation of m⁶Activity level of a related protein. In some embodiments, m can be measured in a unit volume or unit mass of the biological sample (or pretreated biological sample)⁶mRNA level and/or m of A-related protein⁶Amount of A-related protein to evaluate m⁶Expression level of a related protein. Evaluation of m⁶Exemplary methods of mRNA levels of a-related proteins can include, but are not limited to, Polymerase Chain Reaction (PCR), reverse transcription PCR (RT-PCR), in situ hybridization, knose hybridization, sequence analysis, microarray analysis, reporter gene detection, and the like, or any combination thereof. For evaluating m in a biological sample per unit volume or unit mass⁶Exemplary methods of measuring the amount of A-related protein may include, but are not limited to, gel electrophoresis, mass spectrometry, Quantitative Dot Blot (QDB) analysis, spectrophotometry (e.g., bicinchoninic acid assay (BCA assay), etc.,or any combination thereof. Evaluation of m⁶Exemplary methods of activity level of a-related protein may include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), western blot, dot blot, and the like, or any combination thereof.

In some embodiments, m in at least one body part of each reference subject in the control group can be assessed⁶A and/or m⁶The level of the A-related protein is used to obtain a standard level, for example, by using the methods described above. In some embodiments, the reference subject in the control group can be mental healthy. In some embodiments, the reference subject in the control group can have normal learning and/or memory abilities. In some embodiments, the standard level may be based on m in at least one body part of a reference subject in a control group⁶A and/or m⁶A standard threshold or standard level range determined by statistical analysis of a-related protein levels.

In some embodiments, to assess the learning and/or memory abilities of a subject, the same assessment method as the control group may be used to assess m of at least one body part of the subject⁶A and/or m⁶Levels of a-related protein. In some embodiments, m of the subject may be⁶A and/or m⁶Evaluation of level of A-related protein and m⁶Standard level of A and/or m⁶Standard levels of a-related protein were compared. For example, if the assessment is above or equal to a standard threshold, or falls within a standard level range, it may be determined that the learning and/or memory ability of the subject is normal. If the assessment is below the corresponding standard threshold, or is not within a standard level range, it may be determined that the subject may have a disorder or deficit in learning and/or memory. In some embodiments, the learning and/or memory of a subject can be assessed based on a comprehensive analysis. For example, other factors to be considered in the integrated analysis may include the presence or absence of symptoms of learning and/or memory impairment or insufficiency, neuropsychological test results, Magnetoencephalogram (MEG) test results, brain imaging assessment (e.g., computed tomography: (a)CT), Magnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET)) results, or the like, or any combination thereof.

The present application is further described in terms of the following examples, which should not be construed as limiting the scope of the application.

Examples of the invention

Materials and methods

Animal(s) production

All mice used in the examples were born and housed in temperature-controlled (22 + -1 deg.C) rooms in ventilated cages within specific pathogen-free barriers, with a 12h light/dark illumination cycle (7:30 to 19:30 illumination times) and a humidity of approximately 50%. After birth, young mice were housed together with their mother mice, weaned on day 21 after birth, and then divided into groups by sex, and 4 to 5 mice were housed in each cage, and food and water were taken ad libitum. All wild type mice used in this procedure had a C56BL/6J genetic background and were purchased from vita River Laboratory (Beijing). To generate Mettl3 conditional knockout mice, Mettl3 was first introduced^flox/floxMice were mated with CaMKII α -Cre mice (Jax #005359) to produce heterozygous mice (Mettl 3)^flox/+(ii) a CamKII α -Cre), and then, the hybrid mouse is mixed with other hybrid mice or Mettl3^flox/floxMice were mated to produce cKO mice (Mettl 3)^flox/flox(ii) a CamKII α -Cre) and litter control mice (Mettl 3)^Flox/flox). The genotype of each mouse was determined by genomic DNA extracted from the tail tip tissue. All experiments followed the guidelines of the institutional animal care and use committee of genetics and developmental biology of the chinese academy of sciences.

General conditions for behavioral testing

All behavioral tests were performed on male mice 8 to 12 weeks old. Each test was performed at a fixed time (between 8:30m and 18:30 pm) per training day. Animals were treated for 2min 3 days before behavioral testing and transferred to the testing room 24h before behavioral testing, allowing animals participating in multiple tests to rest for at least 3 days between tests. Unless otherwise stated, after testing each mouse, the device should be cleaned with 75% ethanol to prevent any deviation from olfactory cues. All behavioral tests were performed in the presence of two non-genotyped researchers.

Elevated plus maze test

The elevated plus labyrinth consists of two opposing open arms (10X 35cm) and two opposing closed arms (10X 35X 15 cm). The arms were attached to a central square (10 x 10cm) and mounted on an overhead (60 cm from the ground). The maze is made of opaque blue plastic. Each mouse was gently placed in the central area of the maze facing one open arm and then recorded with a hanging camera for 10 min. The time spent on different arms by each mouse was analyzed using EthoVision XT13 (Noduls).

Open field testing

The open field box (40X 35cm) was made of white opaque plastic. Each mouse was gently placed in the central area of the box, left to explore freely for 10min, and recorded with a hanging camera. The total travel distance and time spent by each mouse in the central region of the box was calculated using EthoVisionXT13 (Noduls).

Rotation test

The mice (4 per group) were gently placed on a spinning bar (UGO Basile) at an initial speed of 4rpm for 30s and then accelerated from 4rpm to 50rpm over 5 min. The latency of each mouse falling off the rod was recorded by a relay under the rod. Each mouse was tested 3 times with 3h intervals between each test.

New object identification testing

The new object identification test was performed using the same equipment as the open field test. On the first day, mice were placed in an open field box for 10 min. On the following day, each mouse was gently placed in the center of the box, two objects (a T-25 flask with red ink and a 50ml centrifuge tube with water) were placed in the central area, allowed to explore freely for 10min, and then the mice were returned to their home cages. After 30min, the mice were again placed back in the box (one object was replaced with a 10cm tall vase) for 5min for memory retention testing. (ii) a The behaviour of each mouse was recorded using the EhtoVisionXT 13. Frequency of investigation (f) for each subject was scored manually by two experienced researchers. The discrimination index is calculated as (f)_novel-f_famliar)/(f_novel+f_familiar)×100％。

Moris water maze test

The Morris water maze test was performed in a room in a fixed environment in a round water basin (120 cm diameter, 30cm depth, opaque by the addition of titanium dioxide, held at 21. + -. 1 ℃ C.) filled with water. A circular platform (diameter 9cm) was submerged below the water level in the center of the target quadrant. For each test, mice were gently placed in the water from one of the four quadrants, facing the pool wall, and allowed to swim for a maximum of 60s to position the hidden platform. The release quadrant is randomly altered for each test. Mice that did not find a platform were guided to the platform with long metal rods and left on the platform for 5s (test correlation between METTL3 protein abundance and learning) or 30s (all other experiments). Mice were trained twice daily, 6h apart (starting at 8:00am and 14:00pm, respectively). During the exploratory test, the platform was removed and the mice were left to swim for 60 s. The first heuristic test was performed on day 4, prior to training on day 4 (16 h after the last training on day 3), and the second heuristic test was performed on

day

6, 24h after the last training on day 5. The swim path and time for each quadrant were recorded and analyzed using EthoVisionXT13 (Noduls).

Contextual fear conditioned reflex test

Mice were gently placed into conditioning chambers (Panlab, Harvard Apparatus), respectively, and left to explore for 2min, followed by one light shock (0.8mA for 2s) or 3 light shocks (0.8mA for 2s with 60s intervals). The mice were allowed to stand in the conditioning cabinet for a further 60s and then returned to the rearing cage. After 30min (short-term test) or 24h (long-term test), the mice were returned to the conditioning box for 5 min. The freezing behaviour was recorded and analysed with PACKWIN2.0.5 software (Panlab, Harvard Apparatus).

Immunohistochemistry

Fresh or perfused brain samples were instilled in 4% Paraformaldehyde (PFA) at 4 ℃ in 1xPBS for 48h fixation, then washed twice with 1xPBS, cryo-protected with 30% sucrose, frozen in tissue cryo-medium (TFM, general data) and sectioned (25-35 μm thick) with a cryostat (Leica). Sections were infiltrated and blocked for 30min at room temperature by blocking buffer, formulated in 1xPBS, containing 0.2% Triton X-100 and 2% bovine serum. The sections were then incubated overnight at 4 ℃ with primary antibody diluted with blocking buffer and secondary antibody diluted with blocking buffer for 2h at room temperature. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) with a blocking agent. Antibodies used for immunofluorescence labeling were as follows:

anti-METTL 3 (1: 200, Abcam, ab195352), anti-CUX 1 (10. mu.g/ml, Abcam, ab54583), goat anti-rabbit Alexa Fluor 594 (1: 500, Abcam, ab150080) and goat anti-mouse Alexa Fluor 488 (1: 500, Abcam, ab 150113). Images were acquired using a Leica SP8 confocal microscope.

Hematoxylin-eosin (HE) staining

Brain Tissue (fixed in 4% PFA at 4 ℃ for at least 48h) was automatically dehydrated by Tissue-TekVIP5Jr (Sakura) and embedded in paraffin (56-58 ℃) by Tissue-TekTEC5 Tissue embedding machine (Sakura). Brain paraffin blocks were cut into sections 8 μm thick with a manual rotary microtome (Leica, RM2235) and these sections were mounted on poly-D-lysine coated microscope slides (CITOGLAS, 10127105P). For hematoxylin-eosin staining, sections were first dewaxed in xylene (2 times 10min each), rehydrated in alcohol (100% alcohol 5min, 95% alcohol 3min, 70% alcohol 3min, then rinsed with distilled water) and then automatically stained by Tissue-Tek Tissue section staining machine (Sukara, DRS 2000). Stained sections were dehydrated in 100% alcohol (3 times 5min each), washed in xylene (3 times 5min each), and placed in neutral balsam (Solarbio, G8590). The slide was scanned using a Nanozomer RS scanner (Hamamatsu) to obtain a full slide image.

Nie's dyeing

Nisshin staining was performed using the staining kit (Coolabler, DL0135) according to the manufacturer's instructions. Briefly, paraffin-embedded sections were deparaffinized and hydrated following the same protocol as in HE staining. The sections were then stained in cresyl violet staining solution at 56 ℃ (in 56 ℃ incubator) for 1h, washed with distilled water, and then dipped into the differentiation solution provided in the kit until the background became clear. Stained sections were dehydrated in 100% alcohol (3 times 5min each), washed in xylene (2 times 5min each), and fixed in neutral balsam (Solarbio, G8590). Slide images were obtained by scanning the slides using a Nanozomer RS scanner (Hamamatsu).

TUNEL staining

TUNEL staining was performed using the in situ cell death detection kit (Roche, 11684809910) according to the manufacturer's instructions. Briefly, cryopreserved tissue sections (prepared according to the procedures described in immunohistochemical experiments) were fixed in 4% PFA in 1XPBS for 20min at room temperature, then washed with 1XPBS for 30min and permeabilized with 0.1% sodium citrate with 0.1% Triton X-100 for 2min at 4 ℃. Each tissue section was stained with 50. mu.l of the labeling solution plus 50. mu.l of the enzyme solution in a humidified 37 ℃ incubator for 1h, rinsed 3 times in 1xPBS, and then counterstained with DAPI following the procedure described in the immunochemical experiment. Slides were imaged using a Leica fluorescence microscope (DMI 3000B). Positive and negative control staining samples were performed according to the manufacturer's instructions to confirm the validity of the staining results (data not shown).

Electrophysiological recording

Mice were deeply anesthetized (8 weeks) with sodium pentobarbital (2%, 0.3ml/100g) and decapitated. The brains were quickly removed and stored in ice-cold artificial cerebrospinal fluid (ACSF, in mM: 124NaCl, 2.5KCl, 1.2 NaH)₂PO₄、24NaHCO₃12.5D-glucose, 2CaCl₂And 1.5MgSO₄With 95% O₂And 5% CO₂Saturation, pH adjustment to 7.3, osmotic pressure adjustment to sucrose

). Acute brain slices (300 μm thick) of hippocampus were prepared with a vibrating microtome (Leica) perfused with ice-cold ACSF and incubated with oxygen-containing ACSF for 1h at room temperature. The individual brain slices were then transferred to recording chambers filled with oxygen-containing ACSF (2ml/min perfusion rate) at 31. + -. 1 ℃. Visual localization of CA1 pyramidal neurons was performed using an Olympus microscope (Olympus BX 50-WI). From 110mm boronA pipette having a resistance of 4-6 M.OMEGA.was withdrawn from a silica glass capillary (Sutter Instrument). The internal solutions used were (in mM): 140K-gluconate, 2MgCl₂、8KCl、10HEPES、0.2Na-GTP、2Na₂-ATP (pH 7.3). Recording and analysis was performed by Axomatch 700B Amplifier (AXON), Digidata 1440A (Molecular Devices), and pCLAMP 10.6 software (Molecular Devices). Cells were monitored for series resistance and input resistance in each experiment while meeting the following requirements (high seal resistance)>1G Ω, series resistance below 25M Ω, series resistance and input resistance varied by less than 15%) for further analysis.

Minimal excitatory postsynaptic current (MEPSC) was recorded at-70 mV by keeping the cells in whole-cell voltage clamp mode. To isolate AMPA receptor-mediated mEPSC, recordings were made in ACSF containing 1. mu.M TTX and 100. mu.M PTX. Record each cell for at least 5 min. The action potential was recorded in whole-cell current-clamp mode, and was caused by a series of depolarizing current pulses between 60 and 500pA in step increments of 20 pA. A single 500pA current (500ms) was injected to measure the inter-peak interval and 3 repeated-20 pA currents (800ms) were injected to measure the fast/slow post-hyperpolarization potential (f/sAHPs).

Short-term plasticity and long-term potentiation measurements

Acute brain sections (380 μm) were prepared as described above and incubated for 1.5h at room temperature in oxygenated ACSF. Individual sections were transferred to a recording chamber (perfusion rate of 6 ml/min) at 31. + -. 1 ℃ into oxygen-containing ACSF. The extracellular recording electrode is filled with ACSF and is located in the radiation layer of the dorsal hippocampus CA1 region. A concentric stimulating electrode was placed on the radiation of CA 3. Each recording began with the measurement of the input/output ratio by adjusting the stimulation intensity from 0 to 80 μ a in 5 μ a increments. The Paired Pulse Ratio (PPR) was evaluated by applying a series of paired pulses with time intervals of 20ms, 50ms, 100ms and 200 ms. Wait 0.5-1h and further record the Long Term Potentiation (LTP) of the same brain slice. The stimulation intensity was set by stimulating 40% of the maximal response as baseline level. Stable baseline was reached at least 30min before Theta Burst Stimulation (TBS). LTP 1h was then recorded.

Virus preparation

The cDNA of Mettl3 gene was amplified from mice and cloned into T vector (TransGen, CB 101). The DPPW motif (residue 395-399) of Mettl3 (wild-type Mettl3), which is important for the binding of ademetionine, was mutated to APPA (mutant Mettl3) by PCR site-directed mutagenesis. Wild-type Mettl3 and mutant Mettl3 were subcloned into pAAV2/DJ-CMVMSC-RFP vector (HANBIO). pAAV-RC and pHelper were co-transfected into AAV-293 cells by using LipoFiter transfection reagent (HANBIO) with pAAV2/DJCMV-wildtype-Mettl3-RFP (AAV2/DJ-WT-Mettl3), pAAV2/DJ-CMV-mutated-Mettl3-RFP (AAV2/DJ-Mut-Mettl3) or pAAV2/DJ-CMV-MSC-RFP (AAV2/DJ-RFP) to produce adeno-associated virus (AAV). AAV2/DJ propagated in AAV-293 cells was purified and virus titer was measured by plaque assay. The stock solutions of AAV2/DJ-WT-Mettl3, AAV2/DJ-Mut-Mettl3 and AAV2/DJ-RFP were 1.0-1.2X 10, respectively¹²Plaque Forming Units (PFU)/ml. The primers used in the examples are listed in table 1.

TABLE 1 primers for vector construction and qRT-PCR

Three-dimensional positioning injection

Adult mice were anesthetized with isoflurane (8 weeks) and placed in a stereotaxic apparatus (RWD). The virus was delivered by hamilton syringe at 0.1ul per minute and the needle was allowed to stand for an additional 1min before withdrawal. Mu.l AAV2/DJ virus carrying wild-type Mettl3, mutated Mettl3 or RFP, respectively (all 1.0-1.2X 10)¹²PFU/ml) was injected bilaterally into the dorsal aspect of the hippocampus (relative to the chimney: AP ═ 1.9mm, ML ═ 1.2mm, DV ═ 1.3 mm). After surgery, the mice were placed on a warm pad for a short recovery time and then returned to the cage and monitored for 24 h. Mice were placed 2 weeks post-operatively and then behavioral testing was performed.

RNA isolation

Total RNA was extracted from cells or hippocampal tissue using TRNzol Universal (TIANGEN, DP 424). RNA concentration was measured using a NanoPhotometer P330(Implen), onlyOD 260/280nm ratio of

The samples of (2) were used in subsequent experiments. RNA integrity was tested by agarose gel electrophoresis of total RNA with only 28S and 18S ribosomal RNA gel bands at a ratio of about 2: 1 was used for further studies.

Methylated RNA immunoprecipitation

Using Epimark N⁶Methyladenosine enrichment kit (NEB, E1610S) for methylated RNA immunoprecipitation (MeRIP). Briefly, 2. mu. l m⁶The a antibody was linked to protein G magnetic beads (NEB, S1430). Then, will carry m ⁶100. mu.g total RNA of A control RNA (Gaussia luciferase, GLuc) and unmodified control RNA (Cypridina luciferase, CLuc) were incubated with magnetic beads at 4 ℃ for 1 h. The magnetic beads were washed with reaction buffer (150mM NaCl, 10mM Tris-HCl, pH 7.5, nuclease-free H)₂0.1% NP-40 in O), low salt reaction buffer (50mM NaCl, 10mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease free water) and high salt reaction buffer (500mM NaCl, 10mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease free water) were washed twice. Purification of enriched m by phenol-chloroform extraction⁶A RNA。

qRT-PCR

The extracted RNA was treated with DNaseI (ThermoFisher Scientific, EN0525) and reverse transcribed to cDNA by reverse transcriptase (ThermoFisher Scientific, EP 0441). The qRT-PCR experiment was performed using SYBR Green PCR Master Mix (Toyobo, QPK-201). By using 2^-ΔΔCtThe relative expression was calculated. The primers are listed in Table 1.

m⁶Dot blot analysis

M as described above was performed on a Bio-Dot apparatus (Bio-Rad)⁶Dot blot analysis, with minor modifications. Briefly, total RNA isolated from rapidly frozen (by liquid nitrogen) hippocampal tissue (male 8 weeks) was quantified using NanoPhotometer P330(Implen) and spotted onto positively charged nylon-based membranes (GE Healthcare, RPN 303B). Then 5% skim milk (Amres) dissolved in blocking buffer (LI-COR, 927-50000) was used at room temperatureco, M203) blocking the RNA sample for 2h, and mixing with anti-M⁶A primary antibody (1: 3000, Abcam, ab151230) was incubated overnight at 4 ℃. The RNA samples were then washed with 1 XTSST (3X 5min, CWBIO, CW00435), incubated with IRDye 800CW secondary antibody (1: 5000, Odyssey, 926-doped 32211) at room temperature for 2h, and then washed again with 1 XTSST (2X 5 min). Images were obtained from ODYSSEY CLx (LI-COR) and analyzed by ImageJ (v1.51K).

miCLIP-SMARTer-m⁶A-seq

Small scale single base resolution m⁶The A methylation group detection was performed according to the previously reported modification procedure. Briefly, 100ng of mRNA was isolated from the hippocampus of 5 mice (male 8 weeks) using the Dynabeads mRNA purification kit (Life Technologies, 61006) and fragmented using a fragmentation reagent (Life Technologies, AM8740)

This was then mixed with 5. mu.g of anti-m⁶Antibody A was incubated in 450. mu.l immunoprecipitation buffer (50mM Tris, 100mM NaCl, 0.05% NP-40, adjusted to pH7.4) for 2h at 4 ℃ with slow rotation. The mixture was then transferred to clear flat-bottomed 96-well plates (Corning) on ice and subjected to 254nm, 0.15J/cm in a CL-1000 ultraviolet crosslinking apparatus (UVP)^-2Irradiation was performed 3 times. After irradiation, the mixture was collected and incubated with 50. mu.l of prewashed protein A magnetic beads (Life Technologies, 1001D) for 2h at 4 ℃. After extensive washing (2 times) with high salt buffer (2 times, 50mM Tris, 1M NaCl, 1mM EDTA, 1% NP-40, 0.1% SDS, adjusted to pH7.4) and immunoprecipitation buffer, the microbeads were dephosphorylated with T4PNK (NEB, M0201L) for 20min at 37 ℃. RNA was eluted from the beads by proteinase K (Sigma, P2308) treatment at 55 ℃ for 1h, followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA was library-constructed using the SMARTer smRNA-Seq kit for Illumina (Clontech Laboratories, 635030) according to the manufacturer's instructions and sequenced on the Illumina HiSeq X Ten platform.

RNA sequencing

Preparation of RNA according to the instructions of TruSeq RNA sample preparation kit (Illumina, FC-122-1001)And (5) sequencing the sample. Briefly, total RNA was extracted from the hippocampal tissue of the snap-frozen mice

And used to generate a cDNA library. All samples were sequenced on the Illumina HiSeq X Ten platform. Two replicates (replicates) were sequenced for each condition (each replicate represents one mouse).

Protein extraction and western blotting

Total proteins from mouse hippocampus or primary cortical neurons were extracted by N-PER neuronal protein extraction reagent (Thermo Fisher Scientific, 87792). Protein concentrations were calculated using the Pierce Coomassie protein detection kit (Thermo Fisher Scientific, 23200). Separation of protein fractions by 10% SDS-PAGE

And immunoblot analysis was performed with the corresponding antibodies, anti-METTL 3 (1: 1000, Abcam, ab195352), anti-beta TUBULIN (1: 2000, Abcam, ab108342), anti-GAPDH (1: 7500, Protein tech, 60004-1-AP), anti-c-FOS (1: 1000, Abcam, ab214672), anti-EGR 1 (1: 1000, Thermo Fisher Scientific, MA5-15008), anti-NPAS 4 (1: 500, Abcam, ab109984), anti-ARC (1: 1000, Abcam, ab183183) and anti-NR 4A1 (1: 1000, Abcam, ab 109180). Protein detection was performed by LI-COR Odyssey imaging system (ODYSSEY CLx, LI-COR) using IRDye secondary antibody. Relative protein levels were analyzed by ImageJ (v1.51k).

miCLIP-m⁶A-seq data analysis

The miCLIP-seq data were analyzed according to a preset protocol (paired end sequencing). Briefly, the linker sequence was trimmed by Cutadapt (v1.7.1) using the parameters: q 5-O5-m 20. Forward reads (forward reads) were demultiplexed (demultiplexed) using fastq2collapse. pl (CTKToolKit, v1.0.9), reverse reads (reverse reads) were first converted to reverse complement using fastx _ reverse _ completion (FASTXToolkit, v0.0.13) and then processed in the same manner. Next, the random barcode was striped using stripbarcode. pl (CTK tool kit, v1.0.9)Processing and appending it to the read headings to facilitate downstream CIMS analysis, then pulling the forward reads and converted reverse reads of each sample into a single file and aligning them with a reference Genome (mm10, USCS Genome Browser) using BWA (v0.7.12-r1039) using the parameters: n0.06-q 35. Cross-linking induced mutation sites (CIMS) were identified using the CTK tool kit (v 1.0.9): the uniquely aligned reads were selected using part alignment.pl (- -map-qual 1- -min-len 18) and PCR copies (PCR products) were folded using tag2collapse.pl (- -EM 30- -seq-error-model alignment). Py was then used to identify the mutation sites (insertions, deletions and substitutions) and CIMSC → T transition was designated using cims.pl (-n 10). Only CIMS sites with a transition number of > 2(m > 2) and a total coverage of the transition of between 1% and 50% (0.01. ltoreq. m/k. ltoreq.0.5) were selected for further analysis. Adenosine located 5' to the CIMS site was identified as m⁶Position A, and is labeled by bed2annode.pl (-dbkeymm 10). Meta plotR was used to analyze meta gene distribution, and fine Motifs. pl (Homerv4.8) was used to perform motif enrichment analysis (motif enrichment analysis).

RNA-seq data analysis

Paired-end, jointless reads were first aligned to a reference Genome (mm10, UCSC Genome Browser) using Tophat2(v2.1.1) with default parameters. The uniquely mapped reads were assembled into transcripts using Cufflinks (v2.2.1) and the respective abundances (FPKM) were estimated using default parameters. Cuffdiff (2.2.1) was used to identify differentially expressed genes between samples with fold change ≥ 2 and q-value ≤ 0.05 as the threshold. Gene ontology (bioprocess) enrichment analysis was performed using the metascap online service (metascap. org/gp/index. html #/main/step 1).

Cell culture

From E18.5 Mettl3 for sex Distinguishing^f/fPrimary neurons were isolated from cortical tissue of embryos. Briefly, cerebral cortical tissue was isolated using a neural tissue dissociation kit (Miltenyi, 130092628) according to the manufacturer's instructions. Make the neuron at 5 × 10⁵cells/cm²Was seeded on matrigel (Corning, 354227) precoat (Coring) and supplemented with 2% B-27(Gibco, 175040)44) 1% GlutaMAX (Gibco, 35050061), 1% non-essential amino acid solution (Gibco, 11140050) and 1% penicillin-streptomycin-neomycin antibiotic mixture (Gibco, 15640055) in a neurobasal medium (Gibco, 21103049). At 37 ℃ and 5% CO₂The cells were maintained and the medium was half-changed every two days. For Mettl3 knockout and rescue experiments, neurons cultured in vitro on day 3 (DIV3) were first infected with adenoviruses expressing Cre-GFP (HANBIO, HBAD1016) or GFP (HANBIO, HBAD1009) to achieve Mettl3 conditional knockouts, and neurons cultured in vitro on day 5 (DIV5) were infected with AAV2/DJ expressing wild-type Mettl3, mutant Mettl3 or RFP. For Mettl3 overexpression experiments, neurons cultured in vitro at day 5 were infected directly with AAV2/DJ expressing wild type Mettl3, mutant Mettl3 or RFP of DIV 5. Half of the medium was collected prior to infection and neurons were incubated overnight with the corresponding virus. The next day, cells were washed and replaced with media consisting of media collected before viral infection (50%) and fresh media (50%). At days 8 to 10 of in vitro culture, neurons were stimulated with 25mM KCl (Sigma, P5405) in an incubator for 1h, and then collected for downstream analysis.

Statistical test

The results are shown as box plots (median, 25 th and 75 th percentiles) with data points representing mean ± standard error (mean ± SEM) plotted in boxes or in bar and dot plots. Statistical analysis and mapping was performed using Prism GraphPad5 or R (v3.1.3). The data distribution between groups is assumed to be normal and homovariance, but this has not been formally examined. Unless otherwise stated, comparisons between two groups were analyzed by two-tailed Student t-test (two-tailed Student's t-test), and comparisons between three or more groups were analyzed by one-way ANOVA (one-way ANOVA) and Tukey's HSD (post hoc test). The statistical test, exact P-value, sample size (n) for each experiment are specified in the legend.

The development of Long Term Memory (LTM) is crucial for learning ability and social behavior in humans and animals, but the underlying mechanisms are largely unknown. Long Term Memory (LTM) accumulated in learning or experience of the immortal memories is crucial for behavioral adaptation and intellectual development in mammals. Transformation of short-term memory into long-term memory requires synthesis of nascent proteins for synaptic consolidation, and long-term potentiation (LTP) of neurons is considered to be one of the major contributors. Although very important, the molecular mechanisms that regulate LTM formation, especially at the post-transcriptional level, may remain largely unclear.

The following examples show that the efficacy of hippocampus-dependent memory consolidation is regulated by METTL 3. Depletion of METTL3 in the hippocampus of mice reduces memory consolidation, but if given sufficient training or recovery of m of METTL3⁶A methyltransferase function can improve learning outcome. The abundance of METTL3 in wild type mouse hippocampus was positively correlated with learning potency, whereas overexpression of METTL3 significantly enhanced long-term memory consolidation.

Example 1 Mettl3 in the hippocampus of knockout adult mice did not alter the anatomical features of the brain

Mettl3^flox/floxMice were mated with CaMKII α -Cre mice to generate forebrain excitatory neuron-specific Mettl3 conditional gene knockout mice (Mettl 3)^flox/flox(ii) a CaMKII α -Cre, hereinafter cKO) as shown in fig. 1A and 5A. cKO mice were alive, fertile and developed normally to adulthood, with littermate control mice (Mettl 3)^flox/floxHereinafter CTRL) equivalent body and brain weight (fig. 5B and 5C). After 8 weeks, cKO mice had normal brain architecture with respect to brain morphology and the number and distribution of neurons, astrocytes and microglia, as shown in fig. 5E-5I. After the spin test, open field test, elevated plus maze test, and morris water maze test, the CTRL and cKO mice detected no differences in motor coordination, exploratory behavior, anxiety level, and swimming ability (see fig. 6A-6F). In the new object recognition test, cKO mice showed complete short-term memory (see FIG. 1B).

Example 2 depletion of METTL3 in hippocampus leads to reduced LTM forming ability and prolonged learning time

The morris water maze test was performed to examine the formation of hippocampus-dependent long-term memory (LTM) in cKO mice. In the initial training, cKO mice spent more time than the CTRL group to find hidden platforms, they could still learn gradually. On day 5, cKO mice reached the plateau as fast as CTRL (fig. 1C). Consistently, cKO mice subjected to the first challenge test after day 3 training spent significantly less time in the target quadrant than CTRL, but showed no difference compared to cKO mice subjected to the second challenge test after day 5 training (fig. 1D-1E). Fear conditioning reflex tests were performed. The cKO mice behaved identically to the CTRL mice in the contextual freeze assessment within 30min before and after a mild shock (FIG. 1F), indicating that cKO mice had normal peripheral pain and short-term memory. However, in the contextual test 24h after the first shock, the cKO mice had a freezing time of only half that of the CTRL mice, indicating that LTM formation was insufficient (fig. 1F). In conjunction with the water maze test, three mild shock trains over time increased the duration of episodic freezing behavior of CTRL and cKO mice to similar levels (fig. 1F). Overall, these data indicate that depletion of the hippocampus METTL3 results in reduced LTM formation and extended learning time, but that the final training results are unchanged after adequate training is provided.

Example 3 Long Term Potentiation (LTP) reduction leads to LTM deficiency

To investigate the physiological causes of this phenotype, whole cell patch clamping was performed on CA1 pyramidal neurons from CTRL and cKO mice. cKO mice exhibited normal resting membrane potential, membrane resistance, discharge rate, amplitude and duration, and response to injected current in CA1 neurons (fig. 2A and 10A). The synaptic transmission ability of CA1 pyramidal neurons was tested by calculating the input-output relationship (I/O) by measuring the mini excitatory post-synaptic current (mepscs) and synaptic strength. No significant change was observed for cKO neurons (fig. 2B and 10B). The contribution of paired pulses was also normal in cKO neurons, indicating that these cells have normal short-term synaptic plasticity (fig. 2C). However, LTP on the hippocampus schafer collateral pathway showed a significant decrease in the slope of the field excitatory postsynaptic potential (fEPSP) (fig. 2D and 10C), which was sufficient to cause LTM deficiency.

Example 4 formation of LTM associated with METTL3 was dependent on m of METTL3⁶A methyltransferase function

Rescue experiments were performed by stereotactic injection of adeno-associated virus 2/DJ (AAV2/DJ) carrying the methyltransferase domain mutation (DPPW motif mutated to APPA, M3-Mut) of the Mettl3cDNA sequence (M3) or Mettl3cDNA sequence into the dorsal side of the hippocampus of 7 week old cKO mice. CTRL and cKO mice of the same age injected with AAV2/DJ bearing red fluorescent protein were used as positive and negative controls (CTRL + RFP, cKO + RFP), respectively. After 2 weeks of recovery, the four groups of mice were examined using the morris water maze test and the conditioned fear test. As expected, cKO + M3 and cKO + Mut mice synthesized more METTL3 protein in hippocampus than in cKO + RFP group, but M was detected only in cKO + M3 mice⁶A abundance increased (fig. 3A). Thus, cKO + M3 mice performed as well as the CTRL + RFP group in the water maze and conditioned fear test, but cKO + Mut mice did not show any improvement compared to the cKO + RFP group (fig. 3B-3D), indicating that METTL 3-associated LTM formation is dependent on M of METTL3⁶A methyltransferase function. Again, after 3 shocks of 5 days of water maze and conditioned fear training, all four groups of mice reached the same level of performance (fig. 3B-3D), demonstrating the ability of cKO + Mut mice to form LTMs after full training.

Example 5m⁶Role of A in memory consolidation

Collected for miCLIP-m 0min (initial), 30min, 1h and 4h after conditioned situational fear training by using one shock⁶CTRL mice of A-seq Single base m in Hippocampus tissue⁶A methylation detection (FIG. 4A). At the above time points, expressed genes (hereinafter referred to as m) corresponding to 4424, 3440, 3643 and 5063, respectively, were determined⁶A-tag gene) 8941, 5995, 6367 and 10853 m⁶The A site (with the RRACU motif and distributed near the stop codon) (FIG. 4B). Wherein about 15-20% of the expressed genes are represented by m⁶A was specifically modified and resulted in modification of the 1184 gene at all time points. The gene ontology analysis shows that the gene is continuously repairedIn decorated genes, synaptic signaling and neural development functions were abundant, in genes specifically modified within 30min and 1h, membrane-associated protein deposition was abundant, and in 4h specific genes (FIG. 4C), axon projection function was abundant by 35, consistent with the mechanism of memory consolidation. RNA-seq detected m between 0min (initial), 30min, 1h and 4h collection of CTRL and cKO mouse hippocampal tissue following contextual fear conditioning training⁶There were no significant differences in expression of the a-tag gene (fig. 4D, 11A and 11B), indicating that transcriptional level regulation of cells in cKO mouse hippocampus remained intact.

To investigate the seemingly contradictory phenomena between functional deficits and mRNA expression consistency between CTRL and cKO mice, western blotting was used to examine the protein abundance of 6 well-studied early response genes (IEGs), which should be activated rapidly after learning, and which are essential for LTM formation. M was detected on transcripts of all 6 genes by MeRIP-qPCR of CTRL samples (FIGS. 5A and 12)⁶And (C) modifying. However, although all of these genes showed similar learning-induced changes in expression in CTRL and cKO samples at RNA level (fig. 5B), they all produced less protein in cKO mice than in CTRL mice (fig. 5C-5F), suggesting that the prolongation of the learning process in cKO mice may be due to the lack of m⁶A is caused by insufficient protein synthesis. For further study, expression of Arc and c-Fos was induced in cultured primary cortical neurons by KCl treatment. Consistent with the in vivo data, Mettl3-KO neurons produced less ARC and C-FOS proteins than CTRL, but this difference could be rescued by introducing wild-type METTL3 into cKO neurons (FIGS. 5C-5E). Likewise, METTL3 with a mutated methyltransferase domain failed to rescue protein translation defects, suggesting m⁶The important role of a in such regulation.

Example 6 RNA m in hippocampus⁶A-induced differences are the cause of individual differences in memory forming potency

The above findings indicate that the abundance of the hippocampus METTL3 in individuals may explain the changes in its spatial learning potency. Indeed, in the Morris water maze test, genes were found in wild type mice (8 weeks, males)Moderate positive correlation between METTL3 protein abundance and learning potency (r ═ 0.378) in the hippocampus (fig. 6A). In the first probing test, mice with more METTL3 tended to spend more time on the target quadrant, but after sufficient training (probing test II), this correlation disappeared (fig. 6A). To further characterize the relationship between METTL3 abundance and learning potency, AAV2/DJ virus carrying wild-type METTL3, methyltransferase domain mutated METTL3 or RFP was injected bilaterally into the dorsal hippocampus of wild-type mice (WT + M3, WT + Mut and WT + RFP, respectively) (fig. 13A). As expected, the over-expressed Mettl3 significantly improved the learning potency of the mice in both the morris water maze test and the one-shock conditioned contextual fear test, but the over-expression of Mettl3 with a mutated methyltransferase domain had no effect (fig. 6B-6D), indicating that Mettl3 modulates m by⁶The formation of a plays a role. Also, with appropriate training (three shock fear conditioning training), no behavioral differences were detected between mettl3 overexpression and the other groups (fig. 6D). Mettl3 overexpressed in KCl-treated primary cortical neurons also significantly enhanced the translation of IEGsArc and c-Fos compared to wild-type controls (FIGS. 6E and 13B).

As shown above, METTL3 was demonstrated to enhance hippocampus-dependent LTM by promoting activity-induced translational potency of IEG. In this application, METTL3 was demonstrated to mediate LTM formation. In this application, Mettl3 in knockout adult mouse hippocampus does not alter brain anatomical features or short-term memory-related electrophysiological activity, a phenomenon that should be distinguished from developmental stage studies in which depletion of Mettl3 causes severe defects in the whole brain. Thus, m⁶The dynamic functional preference of the A-modified genes at different post-training points (FIG. 4B) also indicates that m⁶The A modified gene responds rapidly to the stimulus, suggesting that other stimulus-related physiological responses may also be related to m⁶A is related. Interestingly, despite the lack of Mettl3/m⁶A resulted in a reduction in learning performance, but the same training effect was achieved after prolonged water maze training or excessive shock, indicating Mettl3/m⁶A is useful in regulating memory consolidation but is not necessarily requiredHas little effect. Evidence of expression of IEG may further support this conclusion, since induction of IEG after training can still be detected at the mRNA and protein levels of cKO mice, but protein levels of cKO mice are much weaker than those of CTRL mice. Therefore, repeated induction of IEG proteins in reduced abundance may be able to achieve the desired synaptic consolidation effect. The correlation of METTL3 abundance with mouse learning capacity suggests that this molecule in hippocampus may be responsible, in part, for individual differences in memory-forming potency, and that drugs that enhance METTL3 expression may improve learning capacity and slow memory loss associated with age or disease.

Fig. 1A-1F show exemplary results of processes by which the acquired absence of Mettl3 in the hippocampus may prolong LTM consolidation, according to some embodiments of the present application. FIG. 1A shows the characteristics of Mettl3 conditional gene knockout in the CA1 region of 8-week male mice brains (scale bar, 100 μm). FIG. 1B shows a control group (CTRL, Mettl3)^f/f) And Mettl3 conditional knockouts (cKO, Mettl3)^f/f(ii) a CaMKII α -Cre) mice were tested in a new object recognition test (CTRL, n 13 mic; cKO, n is 10 mic). Figure 1C shows the results of the morris water maze test for five consecutive training days. FIG. 1D shows quadrant occupancy of CTRL and cKO mice in two probing tests ("a" for CTRL and "b" for cKO). Fig. 1E shows a corresponding representative swim path. Fig. 1F shows the freezing behavior before (initial) and 30min (short term) after one shock fear condition, and during contextual testing 24h after one or three shock exercises. In fig. 1D-1F, CTRL, n ═ 14 mice; cKO, n is 13 mice. Student's t test, P<0.05，**P<0.01，***P<0.001, N.S, no significance.

Figures 2A-2D show exemplary results of electrophysiological testing of a hippocampus knockout Mettl3 according to some embodiments of the present application. Fig. 2A shows representative traces and numbers of action potentials in CTRL and cKOCA1 pyramidal neurons (3 mice per group, n ═ 10 hippocampus slices) in response to stimuli of different intensities. Figure 2B shows a representative trace of mESPC and the distribution of cumulative probability of mepscs amplitude and frequency. The inset shows a comparison of the mean values. Fig. 2C shows the paired pulse ratio for different stimulation intervals in CTRL and cKO groups (3 mice per group, n ═ 9 hippocampus slices). Fig. 2D shows the field EPSP slope change in the CTRL and cKO groups after a single Theta Burst Stimulation (TBS). Inset shows representative traces at baseline and 1h post-TBS induction (3 mice per group, n ═ 9 hippocampal sections). Student t test, n.s., no significance.

FIGS. 3A-3D illustrate indicating METTL3 via m thereof according to some embodiments of the present application⁶A methyltransferase function regulates exemplary consequences of long-term memory formation. FIG. 3A shows cKO METTL3 and m in the hippocampus of mice⁶And (4) recovering the A. #1 and #2 represent two biological replications. FIG. 3B shows that wild-type Mettl3(M3) was tested in the Moris water maze test instead of having a lack of M⁶Mettl3(Mut) for a methyltransferase function was reintroduced into hippocampus, saving cKO mice for learning delay. FIG. 3C shows probing tests of the panel of FIG. 3B ("a" for CTRL + RFP, "B" for cKO + M3, "C" for cKO + Mut, "d" for cKO + RFP). Upper plate, target and non-target quadrant occupancy frequencies; lower panel, representative swimming path of the upper panel. In fig. 3B and 3C, RFP: controlling injection; CTRL + RFP, n ═ 10 mice; cKO + M3, n ═ 10 mice; cKO + Mut, n-8 mice; cKO + RFP, n ═ 9 mice. Figure 3D shows that reintroduction of wild-type Mettl3 rescued the learning deficit of cKO mice in fear conditioning testing (CTRL + RFP, n-10 mice; cKO + M3, n-9 mice; cKO + Mut, n-10 mice; cKO + RFP, n-10 mice). FIGS. 3B-3D, ANOVA and Tukey's HSD post hoc testing, P<0.05，**P<0.01，***P<0.001, n.s., no significance.

FIGS. 4A-4D illustrate dynamically adjusting m during memory consolidation according to some embodiments of the present application⁶Exemplary results of A methylation. Fig. 4A shows an experimental design of a sampling strategy. FIG. 4B shows m before and after fear conditioning reflex training⁶A distribution (upper panel), motif (middle panel) and m⁶Number of genes marked A (lower panel). FIG. 4C shows common and time-point specific m⁶And (3) enriching and analyzing a Gene Ontology (GO) of the A-tag gene. Nodule size proportional to associated basis factor, tone scale representationThe phrase enhances meaning. FIG. 4D shows m between CTRL and cKO hippocampus at different points in time⁶And (4) comparing the expression of the A marker gene. r is the Pearson correlation coefficient.

FIGS. 5A-5F illustrate m according to some embodiments of the present application⁶A promotes translation of early response genes upon activity induction. FIG. 5A shows early response genes (IEG) by m⁶A labeled and were induced by comparative fear conditioning training in the hippocampus of CTRL and cKO mice (fig. 5B). FIGS. 5C-5E show that cKO mouse IEG is impaired in translation after training and can pass Mettl3^f/fM of METTL3 in primary neurons⁶A methyltransferase activity was used for rescue. Fig. 5C shows western blots and fig. 5D-5E show relative quantitation. Fig. 5F shows immunofluorescence images of EGR1 in the CA1 region before and after fear conditioning reflex training (1h and 4 h). Student's t test, P<0.05、**P<0.01、***P<0.001, n.s., without significance, in fig. 5C-5E, n-3 repeats, and in fig. 5B, n-2 repeats.

Fig. 6A-6E show exemplary results of overexpression of METTL3 enhancing the development of long term memory according to some embodiments of the present application. Figure 6A shows the correlation between hippocampus METTL3 levels and mouse performance in two probing tests. n-20 mice. r, representing the pearson correlation coefficient. 6B-6C show the Morris water maze test. Figure 6D shows a fear conditioned reflex test, which indicates that the learning efficacy of WT + M3 mice is superior to other mice. WT + Mut and WT + RFP mice were shock trained once, but not three times. Fig. 6E shows that overexpression of Mettl3 enhances translation of IEGs in primary neurons after KCl treatment. In fig. 6B-6E, student t-test, P <0.01, P <0.001, n.s., was not significant. In fig. 6B-6D, n is 10 mice/group. In fig. 6E, n is 3 repeats.

FIG. 7 illustrates an exemplary proposed model according to some embodiments of the present application. In FIG. 7, METTL3 mediated m⁶A modification can modulate long-term memory consolidation by increasing the translation efficiency of early response genes in the mouse hippocampus. As shown, long training may offset m⁶Learning deficit due to A deficiency, whereas of METTL3Overexpression may enhance learning performance.

Figures 8A-8H show exemplary results of brain gross morphology characterization of Mettl3 cKO mice according to some embodiments of the present application. FIG. 8A shows that CaMKII α -Cre-mediated KO of Mettl3 reduces m in hippocampus⁶A abundance (n ═ 3 repeats). cKO mice developed normally to adulthood, normal body weight (fig. 8B), normal brain weight (fig. 8C), normal brain morphology (fig. 8D-8G) and no apoptosis was observed in the hippocampus (8 weeks) (fig. 8H). Student's t test, P<0.05，**P<0.01, n.s., no significance; in fig. 8B-8C, n-8 mice/group.

Figures 9A-9F show exemplary results of Mettl3 cKO mice showing no differences in locomotion, exploration, and anxiety compared to CTRL, according to some embodiments of the present application. Fig. 9A shows the rotation test measuring the motor coordination of the animals (CTRL, n-14 mice; cKO, n-11 mice). Fig. 9B shows the total movement distance within 10min on an open field. Fig. 9C shows the duration and representative motion trajectory of mice staying in the central area (n-10 mice/group). Figure 9D shows the duration and representative traces of mice in either the open or closed arms (CTRL, n-12 mice; cKO, n-11 mice). Fig. 9E shows a representative heat map showing that the preference of the animal for new objects (filled circles) is higher than the preference for old objects (dashed circles). Fig. 9F shows the total swimming distance of the mice (CTRL, n ═ 11 mice; cKO, n ═ 10 mice). Student t test, n.s., no significance.

Fig. 10A-10C show exemplary results of characterization of electrophysiological properties of cKO mice, according to some embodiments of the present application. Fig. 10A shows measurements from whole-cell recordings of CA1 pyramidal neurons. fhhp, fast hyperpolarized post-potential; sAHP, potential after slow post-hyperpolarization. Fig. 10B shows the input-output curves of the fEPSP in the CA1 region in response to different stimuli. Fig. 10C shows the slope of fEPSP over the last 10min of LTP recording. Baseline was measured during the last 10min before the theta burst stimulation. In fig. 10A, 10 brain slices were taken from 3mice per group. In fig. 10B-10C, 9 brain sections were taken from 3mice per group. Student t test, P <0.001, n.s., no significance.

11A-11B show exemplary results of transcriptome change analysis at early time points after training according to some embodiments of the present application. Figure 11A shows a volcano plot with no significant transcriptome abundance changes (FPKM) between CTRL and cKO mice at all time points. m is⁶The genes of the A-tag are each color-coded. FIG. 11B shows a heatmap of cKO and CTRL mice expression of key genes involved in synaptic function at different time points (except not from miCLIP-m)⁶All genes listed, except Cdk5 identified in A-seq, were found to be m⁶A modification). Both CTRL and cKO groups contained two RNA-seq replicates.

Figure 12 shows exemplary results of MeRIP-qPCR validation according to some embodiments of the present application. Positive and negative controls for the MeRIP-qPCR experiment (relevant to figure 5A). n-3 replicates.

Fig. 13A-13B show exemplary results of overexpression of Mettl3 in hippocampus or primary neurons according to some embodiments of the present application. FIG. 13A shows that overexpression of Mettl3 in mouse hippocampus increases m⁶And (4) abundance of A. Student's t test, P<0.01，***P<0.001, n.s., no significance. Figure 13B shows that overexpression of Mettl3 in primary neurons enhanced translation of IEG (induced by KCl treatment). n-3 replicates.

It should be noted that the above examples are provided for illustrative purposes only and are not intended to limit the scope of the present application. Many variations and modifications may be made to the teachings of the present application by those of ordinary skill in the art. However, such changes and modifications do not depart from the scope of the present application.

Having thus described the basic concepts, it will be apparent to those of ordinary skill in the art having read this application that the foregoing disclosure is to be construed as illustrative only and is not limiting of the application. Various modifications, improvements and adaptations of the present application may occur to those skilled in the art, although they are not explicitly described herein. Such modifications, improvements and adaptations are proposed in the present application and thus fall within the spirit and scope of the exemplary embodiments of the present application.

Also, this application uses specific language to describe embodiments of the application. For example, "one embodiment," "an embodiment," and "some embodiments" mean a particular feature, structure, or characteristic described in connection with at least one embodiment of the present application. Therefore, it is emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of the application may be combined as appropriate.

Additionally, the order in which elements and sequences of the processes described herein are processed, the use of alphanumeric characters, or the use of other designations, is not intended to limit the order of the processes and methods described herein, unless explicitly claimed. While various presently contemplated embodiments of the invention have been discussed in the foregoing disclosure by way of example, it is to be understood that such detail is solely for that purpose and that the appended claims are not limited to the disclosed embodiments, but, on the contrary, are intended to cover all modifications and equivalent arrangements that are within the spirit and scope of the embodiments herein. For example, although the system components described above may be implemented by hardware devices, they may also be implemented by software-only solutions, such as installing the described system on an existing server or mobile device.

Similarly, it should be appreciated that in the foregoing description of embodiments of the application, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the application aiding in the understanding of one or more of the various embodiments. This method of application, however, is not to be interpreted as reflecting an intention that the claimed subject matter to be scanned requires more features than are expressly recited in each claim. Indeed, the embodiments may be characterized as having less than all of the features of a single embodiment disclosed above.

Claims

1. A method, comprising:

administering to a subject to improve brain function or enhance learning or memory of the subjectThe composition of (a), comprising enhancing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

2. The method of claim 1, wherein the subject is a human or an animal.

3. The method of any one of claims 1-2, wherein the subject has a learning or memory disorder.

4. The method of any one of claims 1-2, wherein the subject has a learning or memory deficit.

5. The method of any one of claims 1-2, wherein the subject has agnosia, alzheimer's disease, amnesia, brain trauma, or dementia.

6. The method of any one of claims 1-2, wherein the subject is psychologically healthy.

7. The method of any one of claims 1-6, wherein the at least one body part of the subject comprises the brain of the subject.

8. The method of any one of claims 1-6, wherein the at least one body part of the subject comprises a hippocampus of the subject.

9. The method of any one of claims 1-8, wherein the one or more agents are configured to increase the amount of METTL3 in the at least one body part.

10. The method of claim 9, wherein the one or more agents comprise a METTL3 peptide.

11. The method of claim 9, wherein the one or more reagents comprise a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

12. The method of claim 9, wherein the one or more reagents comprise an engineered vector comprising a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

13. The method of claim 9, wherein the one or more reagents comprise an engineered virus comprising a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

14. The method of claim 13, wherein the virus comprises an adeno-associated virus, an adenovirus, a lentivirus, or a sendai virus.

15. The method of claim 9, wherein the one or more agents are configured to increase METTL3 expression by stimulating a positive factor that enhances METTL3 expression.

16. The method of claim 9, wherein the one or more agents are configured to increase METTL3 expression by inhibiting a negative factor that decreases METTL3 expression.

17. The method of claim 16, wherein the one or more reagents comprise an antibody.

18. The method of any one of claims 1-8, wherein the one or more agents are configured to stimulate METTL3 activity in the at least one body part.

19. The method of claim 18, wherein the one or more agents comprise a METTL3 agonist.

20. The method of claim 18, wherein the one or more agents are configured to increase METTL3 activity by stimulating a positive factor that enhances METTL3 activity.

21. The method of claim 9, wherein the one or more agents are configured to increase METTL3 activity by inhibiting a negative factor that decreases METTL3 activity.

22. The method of any one of claims 1-21, wherein administering a composition to a subject comprises applying the composition to the skin of the subject.

23. The method of any one of claims 1-21, wherein administering a composition to a subject comprises injecting the composition into the subject.

24. The method of any one of claims 1-21, wherein administering a composition to a subject comprises orally administering the composition to the subject.

25. The method of any one of claims 1-21, wherein the composition is configured as a suppository.

26. A method, comprising:

a composition for administration to a subject to enhance learning ability of the subject, the composition comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

27. A method, comprising:

a composition for administration to a subject to enhance memory in the subject, the composition comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

28. A method, comprising:

a composition for administration to a subject to enhance long term memory consolidation in the subject, the composition comprising an increase of METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

29. A method, comprising:

a composition for administration to a subject to enhance long term memory consolidation in said subject, said composition comprising an increase in METTL3 (N) in the hippocampus of said subject⁶-adenosine methyltransferase 70kDa subunit).

30. A method, comprising:

a composition for administration to a subject to improve brain function in the subject suffering from a mental disorder, the composition comprising increasing METTL3 (N) in the hippocampus of the subject⁶-adenosine methyltransferase 70kDa subunit).

31. A method, comprising:

a composition for administration to a subject to enhance long term memory consolidation in said subject having a memory deficit, said composition comprising an increase in METTL3 (N) in the hippocampus of said subject⁶-adenosine methyltransferase 70kDa subunit).

32. A method, comprising:

a composition for administration to a subject to enhance long term memory consolidation in said subject who does not suffer from a memory deficit, said composition comprising an increase in METTL3 (N) in the hippocampus of said subject⁶-adenosine methyltransferase 70kDa subunit).

33. A method, comprising:

a composition for administration to a subject to improve brain function or to enhance learning or memory in said subject, said composition comprising an increase in N in at least one body part of said subject⁶-methyladenosine (m)⁶A) An abundance of one or more reagents.

34. A method of assessing learning or memory ability of a subject, comprising:

(a) assessing N in at least one body part of the subject⁶-methyladenosine (m)⁶A) The level of the protein of interest;

(b) m is to be⁶A or said m⁶Comparing the level of the a-related protein to a standard level; and

(c) determining the learning or memory ability of the subject from the comparison of (b).

35. The method of claim 34, wherein m is⁶The A-related protein is METTL3 protein.

36. The method of claim 34, wherein the at least one body part of the subject comprises a hippocampus of the subject.

37. The method of claim 34, wherein said standard level is determined by evaluating said m for at least one body part of a control subject⁶Levels of a-related protein.

38. A composition configured to improve brain function or enhance learning or memory in a subject, comprising increasing METTL3 (N) in at least one body part of the subject⁶-adenosine methyltransferase 70kDa subunit).

39. The composition of claim 38, wherein the subject is a human or an animal.

40. The composition of any one of claims 38-39, wherein the subject has a learning or memory disorder.

41. The composition of any one of claims 38-39, wherein the subject has a learning or memory deficit.

42. The composition of any one of claims 38-39, wherein the subject has agnosia, Alzheimer's disease, amnesia, brain trauma, or dementia.

43. The composition of any one of claims 38-39, wherein the subject is mental healthy.

44. The composition of any one of claims 38-43, wherein the at least one body part of the subject comprises the brain of the subject.

45. The composition of any one of claims 38-43, wherein the at least one body part of the subject comprises the hippocampus of the subject.

46. The composition of any one of claims 38-45, wherein the one or more agents are configured to increase the amount of METTL3 in the at least one body part.

47. The composition of claim 46, wherein the one or more agents comprise a METTL3 peptide.

48. The composition of claim 46, wherein the one or more reagents comprise a Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

49. The composition of claim 46, wherein the one or more reagents comprise an engineered vector comprising the Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

50. The composition of claim 46, wherein the one or more reagents comprise an engineered virus comprising the Mettl3cDNA sequence or a fragment of the Mettl3cDNA sequence.

51. The composition of claim 50, wherein the virus comprises an adeno-associated virus, an adenovirus, a lentivirus, or a Sendai virus.

52. The composition of claim 46, wherein the one or more agents are configured to increase METTL3 expression by stimulating a positive factor that enhances METTL3 expression.

53. The composition of claim 46, wherein the one or more agents are configured to increase METTL3 expression by inhibiting a negative factor that decreases METTL3 expression.

54. The composition of claim 53, wherein the one or more agents comprise an antibody.

55. The composition of any one of claims 38-45, wherein the one or more agents are configured to stimulate METTL3 activity in the at least one body part.

56. The composition of claim 55, wherein the one or more agents comprise a METTL3 agonist.

57. The composition of claim 55, wherein the one or more agents are configured to increase METTL3 activity by stimulating a positive factor that enhances METTL3 activity.

58. The composition of claim 46, wherein the one or more agents are configured to increase METTL3 activity by inhibiting a negative factor that decreases METTL3 activity.