WO2022033529A1 - Utilisation d'un antagoniste de sema3d dans la prévention ou le traitement de maladies neurodégénératives et prolongeant la durée de vie - Google Patents

Utilisation d'un antagoniste de sema3d dans la prévention ou le traitement de maladies neurodégénératives et prolongeant la durée de vie Download PDF

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WO2022033529A1
WO2022033529A1 PCT/CN2021/112108 CN2021112108W WO2022033529A1 WO 2022033529 A1 WO2022033529 A1 WO 2022033529A1 CN 2021112108 W CN2021112108 W CN 2021112108W WO 2022033529 A1 WO2022033529 A1 WO 2022033529A1
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sema3d
mice
antagonist
mir
disease
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PCT/CN2021/112108
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Chinese (zh)
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卓夙航
卓庆昌
陈建元
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洪明奇
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to the use of a composition in the preparation of a medicament for preventing or treating neurodegeneration and prolonging lifespan, wherein the composition comprises a Sema3D antagonist.
  • AD Alzheimer's disease
  • FTLD frontotemporal Frontotemporal lobar degeneration
  • NSCs neural stem cells
  • miR-195 could exert neuroprotective effects and improve functional recovery in acute stroke rats.
  • Other studies have also demonstrated that miR-195 downregulates amyloid-beta (amyloid-beta), a core component of Alzheimer's plaques, and that miR-195 alleviates hypoperfusion-induced dementia. Therefore, it is reasonable to speculate that miR-195 may play a role in regulating neuronal function in the aging brain.
  • Semaphorins 3 Class III Semaphorins, Sema3 A-G have been previously reported as axon guidance molecules.
  • Sema3 semaphorin 3A
  • semaphorin 3D semaphorin 3D
  • Sema3D semaphorin 3D
  • Sema3A is a secreted protein that mainly binds to class A plexin receptors (PlexinA1–PlexinA4).
  • PlexinA1–PlexinA4 class A plexin receptors
  • Sema3 family members are also involved in other functions related to the pathogenesis of various diseases, such as neurodegenerative diseases and diabetic retinopathy.
  • Sema3A is a direct target of miR-195 and that Sema3A is involved in neuronal damage caused by acute stroke.
  • the present invention provides a method of preventing or treating a neurodegenerative disease in an individual comprising administering to the individual suffering from the neurodegenerative disease a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • the method according to the present invention is characterized by providing a method for prolonging lifespan in an individual, comprising administering to said individual in need of lifespan extension a medicament comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist combination.
  • a medicament comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist combination.
  • Another feature of the method according to the present invention is that there is also provided a method for promoting nerve regeneration in an individual, comprising administering to the individual in need of nerve regeneration a therapeutically effective amount of semaphorin 3D (Sema3D) Antagonist pharmaceutical compositions.
  • a method for promoting nerve regeneration in an individual comprising administering to the individual in need of nerve regeneration a therapeutically effective amount of semaphorin 3D (Sema3D) Antagonist pharmaceutical compositions.
  • Sema3D semaphorin 3D
  • another feature is that there is also provided a method for preventing or treating retinal neurodegenerative diseases in an individual, comprising administering to the individual suffering from retinal neurodegenerative diseases a method comprising a therapeutically effective A pharmaceutical composition for an amount of a semaphorin 3D (Sema3D) antagonist.
  • a method for preventing or treating retinal neurodegenerative diseases in an individual comprising administering to the individual suffering from retinal neurodegenerative diseases a method comprising a therapeutically effective A pharmaceutical composition for an amount of a semaphorin 3D (Sema3D) antagonist.
  • Sema3D semaphorin 3D
  • the term “subject” refers to an animal, especially a mammal. In a preferred embodiment, the term “individual” refers to a human being.
  • the present invention provides a method of preventing or treating a neurodegenerative disease in an individual comprising administering to the individual suffering from the neurodegenerative disease a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist .
  • a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist .
  • the present invention also provides the use of a composition in the preparation of a medicament for preventing or treating neurodegenerative diseases, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a semaphorin 3D Sema3D
  • prevention refers to preventing the onset, recurrence, or spread of a disease or disorder or one or more symptoms thereof. In certain embodiments, this term refers to the use of the drugs provided herein to treat a particular disorder described herein, with or without one or more other additional active agents, prior to the onset of symptoms or an individual at risk of a disorder, or to which a drug provided herein is administered.
  • treating means alleviating symptoms or complications; delaying progression of a disease, disorder or condition; alleviating or alleviating symptoms and complications; and/or curing or eliminating a disease, disorder or condition.
  • the individual suffers from a neurodegenerative disease of the central nervous system.
  • neurodegenerative diseases as used herein is also used to describe an acute, progressive or chronic disease caused by damage to the central nervous system, which damage can be caused by the Sema3D antagonist treatment to reduce and/or alleviate.
  • the neurodegenerative disease comprises Alzheimer's disease (AD), Parkinson's disease (Parkinson's disease), frontotemporal dementia (FTLD), frontotemporal dementia with ubiquitin inclusion bodies ( frontotemporal lobar degeneration with ubiquitinated inclusions, FTLD-U), age-associated cognitive decline (age-associated cognitive decline), vascular dementia (Vascular dementia), cortico-basal ganglionic degeneration (CBD), progressive Progressive supranuclear palsy (PSP), Lewy body dementia (Lewy body dementia), Huntington's chorea, Alzheimer's disease, Pick's disease (PiD), argyrophilic grain dementia), Guam parkinsonism-dementia complex (Guam parkinsonism-dementia complex), Lytico-Bodig disease (Lytico-Bodig disease), Amyotrophic lateral sclerosis (ALS), spinocerebellar atrophy , Spinal and bulbar muscular atrophy (SBMA), Motor Neuron Disease (AD), Parkinson's
  • the individual is an elderly individual.
  • the drug or pharmaceutical composition comprising the Sema3D antagonist can prevent/inhibit neurodegeneration by increasing the dendritic spine of the hippocampus and promoting nerve regeneration. Therefore, the Sema3D antagonist can be used to prevent or treat neurodegeneration. In a specific embodiment, the Sema3D antagonist prevents or treats the neurodegenerative disease by increasing dendritic spines in the hippocampus and promoting nerve regeneration.
  • the Sema3D antagonist increases dendritic spine density in CA1 pyramidal neurons in the hippocampus. In a preferred embodiment, the Sema3D antagonist prevents or treats neurodegeneration by increasing the density of dendritic spines in CA1 pyramidal neurons in the hippocampus. In a more preferred embodiment, the Sema3D antagonist prevents or treats the neurodegenerative disease by increasing the density of dendritic spines of CA1 pyramidal neurons in the hippocampus.
  • the effect of the Sema3D antagonist in promoting nerve regeneration is achieved by increasing neural stem cells, up-regulating autophagy and promoting neuron proliferation.
  • the promoting nerve regeneration comprises increasing neural stem cells, upregulating autophagy, and promoting neuronal proliferation.
  • the Sema3D antagonist can increase neural stem cells in the hippocampal dentate gyrus and subventricular zone (SVZ) to promote nerve regeneration.
  • the Sema3D antagonist increases neural stem cells in the dentate gyrus and subventricular zone of the hippocampus to promote nerve regeneration to prevent or treat neurodegeneration.
  • the Sema3D antagonist increases neural stem cells in the dentate gyrus and subventricular zone of the hippocampus to promote nerve regeneration to prevent or treat the neurodegenerative disease.
  • an “antagonist” includes, but is not limited to, a molecule that disrupts, prevents, inhibits, reduces or neutralizes a target activity or expression.
  • “Sema3D antagonist” refers to any molecule that blocks, inhibits or reduces (including significantly affects) the biological activity or expression of Sema3D, the biological activity of which includes Sema3D downstream signaling pathways.
  • the term “antagonism” does not imply a specific biological mechanism of activity, and its expression encompasses all possible direct or indirect pharmacological, physiological and biochemical effects on Sema3D.
  • the Sema3D antagonist comprises an aganist Sema3D conjugate, wherein the conjugate comprises a compound, polypeptide, antibody, antibody fragment or oligonucleotide.
  • the Sema3D antagonists include anti-Sema3D antibodies or fragments thereof, anti-sense molecules corresponding to Sema3D (including antisense molecules encoded by nucleic acids corresponding to Sema3D), and small interfering RNAs (small interfering RNAs) corresponding to Sema3D.
  • siRNA short hairpin RNA
  • miRNA microRNA
  • aptamer nucleic acid aptamer
  • Sema3D inhibitory compounds but the Sema3D of the present invention Antagonists are not limited to this.
  • the Sema3D antagonist comprises an anti-Sema3D antibody, an anti-Sema3D antibody fragment, an antisense nucleic acid that inhibits the expression of Sema3D, an siRNA that inhibits the expression of Sema3D, an shRNA that inhibits the expression of Sema3D, and an antisense nucleic acid that inhibits the expression of Sema3D. miRNA or nucleic acid aptamer that inhibits Sema3D expression.
  • the compound comprises a molecular compound.
  • the molecular compounds include small molecular compounds and macromolecular compounds.
  • the antibody or antibody fragment comprises a polyclonal antibody, monoclonal antibody, humanized antibody, diabody, antibody fragment Fab, Fv, F(ab') 2 , single chain Fv (scFv ), Fv fragments or peptoids of antibodies.
  • the oligonucleotide comprises an antisense strand produced by the principle of nucleic acid complementation.
  • the oligonucleotide comprises a nucleotide sequence with sufficient complementarity to the Sema3D gene to directly bind Sema3D RNA to interfere with Sema3D RNA function.
  • the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer.
  • the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises microRNA-195 (miRNA-195), modified miRNA-195 or mimics of miRNA-195.
  • the Sema3D antagonists described in the present invention do not contain molecules such as miRNA-195, modified miRNA-195 or mimetics of miRNA-195 to prevent or treat neurodegenerative diseases.
  • the present invention provides a method of extending lifespan in an individual comprising administering to said individual in need of lifespan extension a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • the present invention also provides the use of a composition in the manufacture of a life-extending medicament, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a semaphorin 3D Sema3D
  • the Sema3D antagonist extends lifespan by delaying aging.
  • the Sema3D antagonist has the effect of delaying aging.
  • the Sema3D antagonist extends lifespan by delaying aging.
  • the Sema3D antagonist is used to delay aging and prolong lifespan.
  • the Sema3D antagonist delays aging by preventing/inhibiting neurodegeneration, thereby prolonging lifespan.
  • the Sema3D antagonist delays aging by preventing/inhibiting neurodegeneration, thereby extending lifespan.
  • the Sema3D antagonist prevents/inhibits neurodegeneration by increasing dendritic spines in the hippocampus and promoting nerve regeneration.
  • the Sema3D antagonist inhibits the expression of aging-related biomarkers.
  • the aging-related biomarkers comprise senescence-associated ⁇ -galactosidase (senescence-associated ⁇ -galactosidase, SA ⁇ -gal), p16 Ink4a and p19 Arf .
  • the Sema3D antagonist can delay brain aging to prolong lifespan.
  • the Sema3D antagonist delays brain aging to prolong lifespan by promoting neurogenesis.
  • the Sema3D antagonist increases neural stem cells in the dentate gyrus and subventricular zone of the hippocampus to promote neurogenesis. Therefore, the Sema3D antagonist has the effect of promoting neurogenesis, thereby prolonging lifespan.
  • the individual is an elderly individual.
  • the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a molecular compound, polypeptide, antibody, antibody fragment or oligonucleotide.
  • the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer.
  • the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises microRNA-195 (miRNA-195), modified miRNA-195 or a mimetic of miRNA-195.
  • the present invention provides a method of promoting nerve regeneration in an individual comprising administering to said individual in need of nerve regeneration a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • the present invention also provides the use of a composition in the manufacture of a medicament for promoting nerve regeneration, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a semaphorin 3D Sema3D
  • the Sema3D antagonist promotes nerve regeneration by increasing neural stem cells, upregulating autophagy, and promoting neuronal proliferation.
  • the Sema3D antagonist can upregulate the PI3K/AKT/mTOR pathway to upregulate autophagy to promote nerve regeneration to inhibit neurodegeneration.
  • the up-regulation of autophagy comprises up-regulation of the PI3K/Akt/mTOR signaling pathway.
  • the individual is an elderly individual.
  • the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a molecular compound, polypeptide, antibody, antibody fragment or oligonucleotide.
  • the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer.
  • the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises microRNA-195 (miRNA-195), modified miRNA-195 or a mimetic of miRNA-195.
  • the Sema3D antagonists described in the present invention do not comprise molecules such as miRNA-195, modified miRNA-195 or mimetics of miRNA-195 to promote nerve regeneration.
  • the present invention provides a method of preventing or treating retinal neurodegenerative disease in an individual comprising administering to said individual suffering from retinal neurodegenerative disease a medicament comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist combination.
  • a semaphorin 3D Sema3D
  • the present invention also provides use of a composition in the preparation of a medicament for preventing or treating retinal neurodegenerative diseases, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.
  • a semaphorin 3D Sema3D
  • the retinal neurodegenerative disease comprises diabetic retinopathy (diabetic retinopathy), age-related macular degeneration (age-related macular degeneration), optic neuritis, myopia-induced retinopathy or Glaucoma-associated retinal disorders.
  • the retinal neurodegenerative disease comprises diabetic retinopathy.
  • the individual is an elderly individual.
  • the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a molecular compound, polypeptide, antibody, antibody fragment or oligonucleotide.
  • the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer.
  • the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  • the medicament or the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier, adjuvant or excipient.
  • the medicament or the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is determined by a particular combination of administration and a particular method of administration of the composition.
  • carrier includes, but is not limited to, any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents such as osmotic and absorption delaying agents, buffers, carrier solutions, suspensions or colloid, etc. Such media and agents for pharmaceutically active substances are well known in the art.
  • compositions Unless any conventional medium or agent is incompatible with the active ingredient, its therapeutic combination needs to be considered. Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a subject.
  • the preparation of aqueous compositions with proteins as active substances is common knowledge in the art. Typically, such compositions are prepared as liquid solutions, troches, capsules, or injections as suspensions; solid forms that can be dissolved or suspended for injections can also be prepared.
  • the aforementioned pharmaceutically acceptable salts include, but are not limited to: inorganic cation salts, for example, alkali metal salts such as sodium, potassium or amine salts; alkaline earth metal salts such as magnesium or calcium salts; and salts containing divalent or tetravalent cations, such as zinc, aluminium or zirconium salts.
  • organic salts such as dicyclohexylamine salt, methyl-D-glucamine, and amino acid salts such as arginine, lysine, histidine or glutamic acid amide.
  • the medicine or pharmaceutical composition of the present invention can be prepared into a dosage form suitable for the present invention by using the techniques well known to those skilled in the art to prepare the above-mentioned Sema3D antagonist and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers thus include, but are not limited to, liposomes, water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and white petrolatum (white petrolatum), waxes (such as paraffin and yellow wax), preserving agents, antioxidants, solvents, emulsifiers, suspending agents (suspending agent), decomposer (decomposer), binder (binding agent), excipient (excipient), stabilizer (stabilizing agent), chelating agent (chelating agent), diluent (diluent), gelling agent ( gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emolli
  • the oligonucleotides or microRNA-195 (miRNA-195) of the present invention can be administered in combination with the following pharmaceutically acceptable carriers, including but not limited to: liposomes, micelles , metal particles, polymer particles, solvents, dispersion media, coatings, antibacterial and antifungal agents or isotonic and absorption delaying agents, etc.
  • pharmaceutically acceptable carriers including but not limited to: liposomes, micelles , metal particles, polymer particles, solvents, dispersion media, coatings, antibacterial and antifungal agents or isotonic and absorption delaying agents, etc.
  • pharmaceutical compositions can be formulated into different dosage forms using general methods.
  • the above-mentioned pharmaceutically acceptable carrier may be liposomes, micelles, metal particles or polymer particles.
  • the particles described above can be prepared from various materials such as lipids, proteins, polysaccharides and synthetic polymers. Depending on the preparation method, nanoparticles, nanospheres, nanocapsules and the like can be obtained
  • administering refers to providing a drug or pharmaceutical composition to an individual.
  • the medicament or pharmaceutical composition comprising the Sema3D antagonist can be administered to the individual by any suitable route of enteral or parenteral administration.
  • Suitable routes of enteral administration in the present invention include, for example, oral, rectal, or intranasal administration.
  • Suitable routes of parenteral administration include, for example, intravascular administration (eg, intravenous bolus injection, intravenous infusion, arterial bolus, arterial infusion, and catheter instillation into blood vessels); peri- and intra-tissue injection (eg, intramuscular).
  • injection peritumoral and intratumoral injection, intravitreal injection or subretinal injection
  • subcutaneous injection or deposition including subcutaneous infusion (eg, by osmotic pump); direct administration to the tissue of interest, such as by catheter or other placement Devices (eg, retinal pellets or suppositories or implants containing porous, non-porous, or gel-like substances); and inhalation. More preferred routes of administration are injection, infusion and direct injection to the target.
  • One of skill in the art can determine the amount of Sema3D to administer to a given individual by considering the following factors: the individual's size and weight; the severity of the disease; the individual's age, health, and gender; the route of administration; effective amount of antagonist.
  • One of skill in the art can also readily determine the appropriate dosage regimen in administering the isolated said Sema3D antagonist to a given individual.
  • the Sema3D antagonist can be administered to the individual once or twice.
  • the effective amount of the Sema3D antagonist administered to the individual may comprise the total amount of drug administered during the entire dosage regimen.
  • the Sema3D antagonist in the form of an oligonucleotide can be delivered to the individual using a recombinant vector.
  • the recombinant vector comprises plastid DNA or a viral vector.
  • the viral vector comprises an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a poliovirus vector, a herpes simplex virus (HSV) vector or a A vector for the murine Maloney virus.
  • the Sema3D antagonist can inhibit the expression of Sema3D in vivo to achieve the above effect.
  • the present invention confirms that the expression level of Sema3D is related to neurodegeneration and regeneration of the brain, reduction of life span and retinal degenerative diseases. Therefore, by inhibiting Sema3D and its related signaling pathways, symptoms such as neurodegeneration, shortened lifespan, retinal degenerative diseases, etc. in the brain can be improved, and nerve regeneration can be promoted.
  • Figure 1 shows the original mice with two miR-195 genes, one of which was knocked out (Knockout, KO) called miR-195a KO mice.
  • Figure 1A and Figure 1B show that miR-195a KO mice have lower amounts of miR-195 in the central nervous system and other organs compared to age-matched wild type (WT) mice, the number of per organ is 3.
  • Figure 1C shows an age-dependent decrease in the amount of miR-195 in the total brain of wild-type (WT) mice (3/group). *p ⁇ 0.05; **p ⁇ 0.01 compared to 4-month-old mice.
  • Figure 2A shows the scheme of learning trials and memory trials.
  • Figure 2B shows that longer escape latency to the hidden platform indicates lower learning ability.
  • Figure 2C shows that longer escape latencies to reach the platform indicate poorer memory.
  • Two-way analysis of variance was used to assess overall differences in multiple days between knockout (KO) and wild-type (WT) mice.
  • Figure 2D shows memory trials (the frequency in the platform quadrant) of miR-195a KO and WT mice.
  • Figure 3A shows the Y-maze test to assess working memory by measuring the time to reach the arm of a new toy; quantitative data are shown in the right panel.
  • Figure 3B shows that locomotor function was measured by an open field test. Representative images (left panel) show the walking trajectories of WT and miR-195a KO mice in the open field; quantitative data are shown in the right panel.
  • 3M 3-5 month old mice; 6M: 6-9 month old mice; 12M: 10-12 month old mice; 18M: 15-18 month old mice; and 24M : 21-24 month old mice.
  • Figure 4A shows the analysis of the lifespan of miR-195a KO mice (number of 28). Historical data of WT mice served as a reference. Results for mean lifespan, median lifespan and percent reduction are shown in Table 3.
  • Figure 4C shows the use of X-gal-based staining to measure senescence-associated beta-galactosidase (SA[beta]-gal) activity.
  • the intensity of the green signal represents the activity of the SA ⁇ -gal enzyme.
  • Representative images show an age-dependent increase in SA ⁇ -gal activity in the cortex and hippocampus of WT mice. Green signal intensity was similar between 4-month-old miR-195a KO mice and 12-month-old WT mice. Scale bar: 200 ⁇ m.
  • Figure 4D shows p16 Ink4a (left panel) and p19 Arf (right panel) expression in the whole brain of 4-month-old miR-195a KO mice and age-matched WT mice (number 3/group) quantitative PCR analysis. Data are presented as mean ⁇ standard error of the mean (SEM). *p ⁇ 0.05;**p ⁇ 0.01. 4M WT: 4 month old WT mouse; 12M WT: 12 month old WT mouse; 24M WT: 24 month old WT mouse; and 4M miR -195a KO: 4-month-old miR-195a KO mice.
  • FIG. 5A shows that miR-195a KO mice have fewer neural stem cells (NSCs) populations compared to age-matched WT mice.
  • Figure 5B shows reduced dendritic spine density in miR-195a KO mice.
  • Figure 6A shows reporter plasmids carrying wild-type or mutant Sema3D 3'-UTRs were transfected into HEK293 cells. In cells transfected with a plasmid carrying the wild-type Sema3D 3'-UTR, miR-195 dose-dependently reduced luciferase activity.
  • Figure 6B shows western blot and quantitative data (3/group) of Sema3A and Sema3D protein levels in the hippocampus of 4-month-old WT and miR-195a KO mice. Sema3A in the hippocampus of WT mice served as a reference group.
  • Figure 6C shows the mRNA expression of Sema3A and Sema3D in the hippocampus of 4-month-old miR-195a KO mice and age-matched WT mice (number 3/group). WT mice served as the reference group.
  • Figure 6D shows the mRNA expression of Sema3D and Sema3A in the human hippocampus according to RNA sequencing data from the Allen Brain Atlas (number 95). Data are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001. NC: negative control group.
  • Figure 7A shows immunohistochemistry (IHC) staining of Sema3D protein in brain. According to the degree of expression of Sema3D, it showed an age-dependent increase in whole brain and hippocampus. Top panel: slice of whole brain; bottom panel: hippocampus.
  • Figure 7B shows a flowchart for selection and analysis of neurodegenerative disease transcriptomes. Computational analyses consist of four main steps shown in oval boxes. 4M WT: 4 month old WT mouse; 13M WT: 13 month old WT mouse; and 21M WT: 21 month old WT mouse.
  • Figure 8A shows at day 7, Western blot data showing that after bilateral injection of a Sema3D-expressing lentivirus (Lv. Sema3D) into the hippocampus of 4-month-old WT mice, the Sema3D protein was present at higher levels, but not in the cortex.
  • Lentivirus for control (Lv.Ctrl) was injected as a control group (the number was 3/group). Quantitative data from western blotting are shown in the right panel.
  • Figure 8B shows the hippocampus from the same mouse shown in Figure 8A, stained with Golgi-cox stain. Dendritic spine density in the hippocampus was measured using Golgi-Cox staining.
  • FIG. 8C shows administration of Lv.Sema3D or Lv.Ctrl to WT mice; learning and memory tests were performed according to the protocol.
  • Figures 8D to 8F show control and Sema3D overexpressing 4-month-old mice (4/group) tested for learning and spatial memory performance by Morris Water Maze test (MWM).
  • Figure 8D shows a learning assay showing that mice overexpressing Sema3D took longer to reach the hidden platform, suggesting lower learning ability.
  • Figures 8E and 8F show escape latency and frequency, respectively, as measured in memory trials. Mice overexpressing Sema3D had longer avoidance latencies and lower frequencies in the target quadrant.
  • Figure 8G shows the effect of Sema3D on short-term memory assessed by the novel object recognition test (number of 4/group). Quantitative data are shown in the right panel. Data in Figures 8B to 8E are presented as mean ⁇ SEM. *p ⁇ 0.05 compared to Lv.Ctrl mice on the same experimental day. Two-way ANOVA was used to assess overall differences in multiple days between Sema3D-overexpressing and control mice. **p ⁇ 0.01 and ***p ⁇ 0.001.
  • Figure 9A shows representative images of immunofluorescence staining in the dentate gyrus (DG) of the hippocampus showing the efficiency of Sema3D silencing (knockdown).
  • siRNA-Sema3D si-Sema3D
  • siRNA-Ctrl siRNA-Ctrl
  • Figures 9B to 9D show spatial working memory and motor function of miR-195a KO mice (12 months old) (number 7/group) receiving siRNA-Ctrl or siRNA-Sema3 injections measured by the Y-maze test.
  • Figure 9B shows the protocol showing details of the date of administration of siRNA to miR-195a KO mice, testing and brain sample collection.
  • Figure 9C shows that mice were allowed to freely explore the Y-maze for 15 minutes. A higher percentage of alternations indicates better spatial working memory.
  • Figure 9D shows the use of total walking distance to determine motor function, with longer distance indicating better motor function. Testing of siRNA-Sema3D and siRNA-Ctrl mice was performed on the same experimental day.
  • FIG. 9E shows representative images of dendritic spine density of CA1 pyramidal neurons in siRNA-Sema3D injected mice (number of 3 per group).
  • siRNA-Sema3D or siRNA-Ctrl were injected into the bilateral hippocampus of 12-month-old miR-195a KO mice and sacrificed on day 15 after injection.
  • Scale bar 5 ⁇ m.
  • the right panel shows quantification of dendritic spine density.
  • Figure 10A shows survival curves of Sema2A-overexpressing flies and control flies (300 per group).
  • Drosophila Sema2A is the homologous gene of human and mouse Sema3D.
  • the Gehan-Breslow-Wilcoxon test was used to compare the lifespan of Drosophila. Average lifespan, median lifespan, longest survival time and percentage of reduced lifespan are shown in Table 6.
  • Figure 1OC shows representative images of human neural stem cells forming neurospheres 48 hours after Sema3D treatment.
  • Figure 11A shows injection of Lv.Sema3D or Lv.Ctrl into the bilateral hippocampus of 4-month-old WT mice. On day 14, Sema3D and autophagy-associated proteins in the hippocampus were measured by western blotting (number 3/group). Right panel shows quantitative western blot data.
  • Figure 11B shows Western blotting of autophagy-related proteins p62, Beclin-1 and LC3-II/I in human neurons 72 hours after Sema3D treatment. Right panel shows quantitative western blot data.
  • Figure 11C shows Western blot of the effect of Sema3D on phosphorylation of the PI3K/Akt/mTOR signaling pathway 24 hours after Sema3D treatment.
  • FIG. 1 shows quantitative western blot data. All quantitative data are presented as mean ⁇ SEM from three independent experiments, *p ⁇ 0.05 and **p ⁇ 0.01.
  • Figure 1 ID shows that Rapamycin reverses the inhibitory effect of Sema3D on cell proliferation.
  • Human neuronal cells SY5Y
  • SY5Y Human neuronal cells
  • Proliferation of neuronal cells was determined using Ki67 staining.
  • Scale bar 50 ⁇ m.
  • Quantitative data are presented as mean ⁇ SEM from three independent experiments. *p ⁇ 0.05 and **p ⁇ 0.01 based on SY5Y cell data without Sema3D and rapamycin treatment.
  • Figure 12 shows that Sema3D is highly expressed in the eyes of diabetic (Diabetes Mellitus, DM) rats and mice.
  • Figure 12A shows that Sema3D mRNA expression is increased 4 weeks after the onset of diabetes, and quantitative data show that Sema3D mRNA is expressed to a higher degree than Sema3A mRNA. *p ⁇ 0.05 compared to the level of Sema3A of the rats in the control group; ##p ⁇ 0.01 compared with the level of Sema3D of the rats of the control group, the number is 2/group. Data are presented as mean ⁇ SEM from three independent experiments.
  • Figure 12B shows representative IHC images showing that Sema3D protein in the retina increases with time to diabetes onset (brown; number 3/group).
  • MiR-195 and negative control microRNA were purchased from Ambion Inc. (Austin, TX, USA). The sequence information is:
  • miR-195 mimic: 5'-UAGCAGCACAAGAAAUAUUGGC-3' (SEQ ID NO: 1);
  • anti-miR-195 5'-GCCAATATTTCTGTGCTGCTA-3' (SEQ ID NO: 2);
  • Negative control sequence 5'-AGUACUGCUUACGAUACGG-3' (SEQ ID NO: 3).
  • Green PCR Master Mix Reverse Transcriptase Kit was purchased from Applied Biosystems (Foster City, CA, USA). Rapamycin, a chemical mTOR inhibitor, was purchased from Sigma Aldrich (St. Louis, MO, USA). Recombinant mouse Sema3D and human Sema3D proteins were purchased from R&D Systems (Minneapolis, MN, USA). All other reagents were analytical grade unless otherwise stated.
  • anti-SOX2 (ab97959, Abcam; Cambridge, MA, USA), anti-Sema3D (ab180147, Abcam), anti-GAPDH (5174, Cell Signaling; Beverly, MA, USA), anti-Ki67 (ab16667, Abcam), anti-Beclin -1 (ab207612, Abcam), anti-LC3 (ab48394, Abcam), anti-p62/SQSTM1 (ab56416, Abcam), anti-PI3K (E-AB-32575, Elabscience; Houston, Texas, USA), anti-phospho-PI3K (E-AB-32575, Elabscience; Houston, Texas, USA) - AB-20966, Elabscience), anti-Akt (9272S, Cell Signaling), anti-phospho-Akt (9271S, Cell Signaling), anti-mTOR (E-AB-32128, Elabscience) and anti-phospho-mTOR (E-AB-20929 , Elabscience) for western blot, immunohistochemical staining
  • Oligonucleotide pools of siRNA targeting human Sema 3D and non-targeting (control) RNA were purchased from Dharmacon (Lafayette, CO, USA).
  • the sequences of siRNA-Sema3D are 5'-CUGUGAUGUAUAAGUCCGU-3' (SEQ ID NO: 4), 5'-GCAAUAUGAUGGAAGGAUA-3 (SEQ ID NO: 5), 5'-CUGCCAACUUAUAAUGUUU-3' (SEQ ID NO: 6) and 5'-GCUAUGUGCUUAAUGUUUC-3' (SEQ ID NO: 7).
  • siRNA-Ctrl The sequences of siRNA-Ctrl are 5'-UGGUUUACAUGUCGACUAA-3' (SEQ ID NO: 8), 5'-UGGUUUACAUGUUUUCUGA-3' (SEQ ID NO: 9), 5'-UGGUUUACAUGUUUUCCUA-3' (SEQ ID NO: 10) and 5'-UGGUUUACAUGUUGUGUGA-3' (SEQ ID NO: 11).
  • the human neuronal cell line SY5Y was obtained from the American Type Culture Collection.
  • HEK293 cells BCRC90016) were obtained from Bioresource Collection and Research Center. SY5Y cells and HEK293 cells were maintained in a humidified incubator with 37°C and 5% CO supplemented with 10% FBS (Invitrogen, Waltham, MA, USA), 1% penicillin and streptomycin (Biowest) , Loire Valley, France) and 1% L-glutamine (Invitrogen) in DMEM.
  • Human neural stem cells (NSCs) are derived from human induced pluripotent stem cells (iPSCs).
  • human iPSCs were first cultured into embryoid bodies (EB) medium on matrigel-coated dishes (BD Biosciences; Franklin Lakes, NJ, USA), supplemented with With recombinant human Noggin protein (250ng/ml, R&D).
  • the medium was changed to supplemented with Sonic Hedgehog (SHH, 20ng/ml, R&D) and fibroblast growth factor 8 (FGF8) (100ng/ml, R&D) EB medium.
  • SHH Sonic Hedgehog
  • FGF8 fibroblast growth factor 8
  • Sema3D induces neurodegeneration via the PI3K/Akt/mTOR/autophagy pathway
  • SY5Y cells were treated with recombinant Sema3D protein for 72 hours, and cell lysates were collected for western blot analysis.
  • 1 ⁇ M rapamycin and Sema3D were co-administered into cultured SY5Y cells for 72 hours, and cell viability was detected by Ki67 staining at 72 hours.
  • CHOV20191024 a novel Sema3D antagonist
  • Sema3D were co-treated with SY5Y cells to demonstrate the biological activity of CHOV20191024.
  • MTT analysis, Western blot and cell survival analysis were used to assess the rescue ability of CHOV20191024.
  • Sema3D affects neural stem cells
  • Sema3D was added to the culture medium of human neural stem cells. To assess whether Sema3D can affect neural stem cell properties (stemness). Sphere formation assay and counting of sphere numbers were performed on day 5 after Sema3D treatment.
  • Sema3D overexpressing mice 4 month old C57BL/6 mice were used to generate Sema3D overexpressing mice. Mice were anesthetized by using a mixture of Zoletil and Rompun (ratio: 3:1, 1 mg kg -1 , intraperitoneal) for stereotaxic injection procedures. A solution containing Sema3D lentivirus (Lv. Sema3D; 4.5 x 105 TU/ml) was injected into the bilateral hippocampus (-1.2 mm anterior-posterior, 1 mm medial-lateral, and -2 mm dorsal-ventral Relative to bregma; -3.6 mm anterior-posterior, 3.2 mm medial-lateral and -4 mm dorsal-ventral relative to bregma). On day 7 post-injection, Sema3D overexpressing mice were subjected to behavioral testing, Golgi-cox stain or signaling pathway analysis. Mice receiving control lentivirus (Lv.Ctrl
  • miR-195a KO and WT mice were used to investigate the role of miR-195 in cognitive function, neurogenesis and brain senescence.
  • the cognitive function of miR-195a KO and WT mice was assessed by Morris Water Maze (MWM) test, Y-maze test (Y-maze test) and open field test (OFT).
  • Neurogenesis capacity was assessed by quantifying SOX2 + neural stem cells in the dentate gyrus (DG) and subventricular zone (SVZ).
  • DG dentate gyrus
  • SVZ subventricular zone
  • SA ⁇ -gal Senescence-associated ⁇ -galactosidase
  • p16 INK4a /p19 Arf expression were used to assess brain aging.
  • Sema3D the role of Sema3D in cognitive function, neurodegeneration and neurogenesis was explored by using Sema3D overexpressing mice.
  • Cognitive function was assessed through the MWM test, the novel object recognition test, the Y-maze test, and the open field test. Cognitive testing was performed on day 7 after lentivirus administration.
  • Golgi-Cox staining was used to detect neurodegeneration in the same mice after behavioral testing. If Golgi-Cox staining shows a decrease in the density of the dendritic spines of neurons, there is neurodegeneration in the brain.
  • Sema3D neurogenesis
  • recombinant Sema3D protein was injected intracerebroventricularly (ICV) into 4-month-old WT mice.
  • Neurogenesis was assessed by quantifying SOX2 + neural stem cells in the dentate gyrus (DG) and subventricular zone (SVZ).
  • DG dentate gyrus
  • SVZ subventricular zone
  • rapamycin was delivered into the hippocampus of Sema3D-overexpressing mice to rescue autophagy efficiency. Rapamycin and Lv.Sema3D were injected into the bilateral hippocampus of mice. Golgi-Cox staining was performed to visualize dendritic spine density to assess the severity of Sema3D-induced neurodegeneration.
  • Sema3D siRNA or control siRNA was delivered into the hippocampus of 12-month-old miR-195a KO mice. Spatial working memory and motor function obtained by the Y-maze test were used to evaluate the effect of Sema3D siRNA on cognitive function. Rescue was assessed again using Golgi-Cox staining.
  • the Y maze is composed of three closed arms, which are 50 cm long, 11 cm wide, and 10 cm high, and are made of black plexiglass (Plexiglas); and the three arms are 120° to each other in a Y shape.
  • the first protocol consisted of two separate experiments performed at 15 min intervals in miR-195a KO mice and Sema3D overexpressing mice. Briefly, in the first trial (acquisition trial), mice were placed at the end of a selected arm (initial arm) and allowed to close in one of the arms (noted as the new arm) Exploring the maze for 5 minutes. The mice were then returned to their home cages away from the testing room for 15 minutes. In the second trial (the retention trial), mice were allowed to freely explore all three arms of the maze for 5 minutes and the time taken to reach the new arm (previously closed in the first trial) was recorded. The longer it takes to reach the new arm, the worse the working memory performance.
  • mice The second protocol was performed with Sema3D/control siRNA-treated miR-195a KO mice. Briefly, each animal was placed in the center of the Y-maze and was free to explore the arena for 8 minutes. Mice tended to explore the least recently visited arm and therefore tended to alternate visits among the three arms. In order to alternate effectively, mice need to use working memory, keep a constant record of recently visited arms, and continually update those records. Access to the arm was scored when the mouse placed its four paws inside the arm. Spatial working memory was assessed by alternation percentage. Alternation is defined as successive selection into three different arms. Alternation percentage is calculated as the ratio of the actual number of alternations to the maximum number of alternations. The maximum number of possible alternations is defined as the total number of entries into the arm minus 2. A low percentage of alternation indicates impaired spatial working memory, as the mouse does not remember which arm it has just visited, and thus shows reduced spontaneous alternation.
  • mice were trained to find a hidden platform in opaque water for 5 days, with 4 acquisition trials per day from a pseudorandomized starting position. During a 5-day acquisition trial, the latency to find the hidden platform was recorded as an indicator of spatial learning ability. Next, to assess spatial memory ability, probe trials (in which the hidden platform was removed) were performed on days 1, 7, and 14 after the acquisition trial, recording the total time spent finding the hidden platform. The latency to find a hidden platform and the frequency of reaching the platform quadrant were recorded as indices of spatial memory capacity. All MWM experiments were recorded and analyzed using the ANY-Maze Animal Behavior Analysis System (Stoelting, Chicago, IL, USA).
  • Recognition memory was assessed by a novel object recognition test. The mice were first placed in the center of the arena with two identical objects for 10 minutes. The mice were then returned to their home cages away from the testing room for another 15 minutes. Next, a 5-minute recognition memory test in which a familiar object was replaced by a new one. The time each animal spent exploring each object during the test was recorded with a video tracking system (ViewPoint Behavior Technology; Lyon, France). Object memory ability is shown as the ratio of the time spent exploring new objects to the time spent exploring all objects (discrimination index).
  • Dendritic spine density of hippocampal neurons in the CA1 area is shown by Golgi-Cox staining. Brains were immersed in Golgi staining solution according to the manufacturer's protocol (FD Rapid GolgiStainTM Kit, FD Neuro Technologies Inc., MD, USA). The stained brains were then sectioned using a vibratome (Leica VT1000S) to obtain 100 ⁇ m thick coronal sections from the dorsal hippocampus. Sections were embedded on gelatin-coated glass slides, then placed in Kodak Film Fixer for 15 minutes and dehydrated in xylene-based media.
  • Golgi-Cox staining Brains were immersed in Golgi staining solution according to the manufacturer's protocol (FD Rapid GolgiStainTM Kit, FD Neuro Technologies Inc., MD, USA). The stained brains were then sectioned using a vibratome (Leica VT1000S) to obtain 100 ⁇ m thick coronal sections from the dors
  • NSCs neural stem cells
  • neural stem cells were detected using immunofluorescence staining. Neural stem cells were confirmed and quantified by SOX2-positive signal with clearly distinguishable nuclei (DAPI-positive cells). Briefly, brains were fixed with 4% paraformaldehyde (PFA), cryopreserved in 30% sucrose solution at 4°C for 24 hours, and then embedded in OCT. 15 ⁇ m thick cryosections were collected and stored at ⁇ 20 °C until use. For immunostaining, brain sections were incubated with SOX2 antibody in 5% BSA in PBS overnight at 4°C with secondary antibody conjugated to Alexa Fluor 647 (Invitrogen). Images were acquired by immunofluorescence confocal microscopy (Leica SP2/SP8X). The number of SOX2-positive cells located in the dentate gyrus (DG) and subventricular zone (SVZ) was quantified by Image J software.
  • PFA paraformaldehyde
  • SVZ subventricular zone
  • the spheroid formation assay was used to determine the effect of Sema3D on the properties of neural stem cells. Briefly, human neural stem cells were seeded in ultra-low attachment 24-well plates (Corning; NY, USA). The number of spheroids (>50 ⁇ m in diameter) was counted on day 5 of culture.
  • the Gene Expression Omnibus (GEO) database (as of December 2019) was queried for human hippocampal microarray gene expression datasets related to neurodegenerative diseases and aging.
  • the specific search terms used were: "neurodegeneration”, “dementia”, “cognitive impairment” and "postmortem brain”.
  • the retrieved datasets were filtered according to the following criteria: (1) derived from available raw data from human hippocampal tissue; (2) for neurodegenerative disease datasets, there was at least one control group (normal individuals) and one disease group; and (3) Sema3D should be detected in the microarray results. Table 1 summarizes all retrieved datasets and the reasons for their inclusion or exclusion in the present analysis.
  • Raw gene expression data and disease severity classifications obtained from the GEO database.
  • the retrieved datasets were microarray datasets obtained from two platforms: the Affymetrix human gene body U133 and the Affymetrix human gene 1.0ST array (Table 1). After filtering, retention of six human hippocampal microarray datasets from dementia, aging, frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U), and Alzheimer's disease (AD) (GSE84422, GSE11882, GSE13162, GSE1297, GSE48350 and GSE36980) for further analysis.
  • FTLD-U frontotemporal lobar degeneration with ubiquitinated inclusions
  • AD Alzheimer's disease
  • RNA-sequencing data were downloaded from the Allen Brain Atlas (https://portal.brain-map.org/).
  • RNA sequencing data of 94 donors aged 77 to 100+ years were analyzed for Sema3A and Sema3D gene expression.
  • SA ⁇ -gal Aging-Associated ⁇ -Galactosidase
  • GAPDH internal control group
  • miR-195 or negative control microRNA was transfected into HEK-293 cells by HiPerFect transfection reagent (Invitrogen) to investigate whether miR-195 could directly interact with the target 3'-untranslated region ( 3'-untranslated region, 3'-UTR) sequence binding. Luciferase activity was compared between cells transfected with normal or mutant plasmids. If Sema3D is the target of miR-195, the luciferase activity should be higher in cells transfected with the mutant plasmid, since miR-195 is unable to exert its knockdown effect.
  • HiPerFect transfection reagent Invitrogen
  • Drosophila carrying elav-Gal4 or UAS-Sema2a-GFP were obtained from the Bloomington Drosophila stock center (Indiana University, Bloomington, IN, USA) and incubated at 25°C with a 12-hour light-dark cycle and grown on standard cornmeal medium at 60% relative humidity.
  • To overexpress Sema2A in the nervous system virgin female flies carrying the neuron-specific driver gene elav-Gal4 were crossed with male flies carrying UAS-Sema2a-GFP, and their F1 offspring would be Sema2A-overexpressing flies .
  • F1 progeny of virgin female flies and male wild-type flies carrying elav-Gal4 served as control flies.
  • Sema2A-overexpressing flies and control flies 300/group were used, and surviving flies were counted every 7 days. The number of dead flies was recorded and a survival curve was drawn. The Gehan-Breslow-Wilcoxon test was used to determine statistical differences between Sema2A-overexpressing flies and control flies.
  • the locomotor activity of flies overexpressing Sema2 was determined using a negative Geotaxis assay. Briefly, groups of approximately 15 flies were placed in vertical cylinders (25 cm in length, 1.5 cm in diameter) with tapered bottom ends. On a light tap, the flies startle and climb up. After 30 s, flies above the midline were counted, and climbing scores were determined by calculating the ratio of flies above the midline to the total number of flies. The Mann-Whitney test was used to determine statistical differences between Sema2A-overexpressing flies and control flies.
  • the structure of human Sema3D is not available in the RCSB Protein Data Bank (http://www.rcsb.org/). BLASTP was used to identify homologs in the RCSB protein database.
  • Systemic miR-195a knockout (KO) mice have been produced. Notably, one mouse had two miR-195 genes (miR-195a and miR-195b), whereas only the miR-195a gene was knocked out in the mouse model of the present invention.
  • the level of miR-195 expression in the brain is higher than in several internal organs.
  • the level of miR-195 expression was significantly reduced by 25-50% in the brain of the miR-195a KO mice of the present invention, and by 50-75% in other organs (FIGS. 1A-1B).
  • the level of miR-195 in the brain of WT mice decreased with age (Fig. 1C).
  • MWM testing was performed according to the protocol described in Figure 2A.
  • Another memory parameter in the MWM test was the entry frequency of the target quadrant, but did not show any difference between KO and WT mice regardless of age (Fig. 2D). Compared with WT mice aged 6-12 months, miR-195a KO mice exhibited poor motor activity (Fig. 3B). Similar to memory and spatial learning, aged KO and WT mice did not differ in motor activity. The above results suggest that miR-195a KO mice may accelerate the aging process of the brain, resulting in poorer cognitive function.
  • miR-195 deficiency is also associated with other aging phenotypes, including lifespan, molecular biomarkers, neural stem cells (NSCs), and dendritic spines. Therefore, the results of the present invention comparing the lifespan, mean lifespan, median lifespan and percentage reduction of WT mice and miR-195a KO mice are shown in Table 3. The mean and median lifespan of miR-195a KO mice was reduced by approximately 25% compared to WT mice (Fig. 4A). Furthermore, the present invention examines two biomarkers of aging in the brain, aging-associated beta-galactosidase (SAbeta-gal) activity and p16 Ink4a /p19 Arf expression.
  • SAbeta-gal aging-associated beta-galactosidase
  • the present invention compares the number of Sox2 + neural stem cells in the dentate gyrus (DG) and subventricular zone (SVZ) between miR-195a KO and WT mice.
  • DG dentate gyrus
  • SVZ subventricular zone
  • neural stem cell numbers decreased with age in the DG (Fig. 5A) and SVZ (Fig. 5C) of WT mice.
  • 4-month-old miR-195a KO mice the number of neural stem cells in the DG and SVZ was reduced by 40% and 50%, respectively, which was the same result observed in 12-month-old WT mice.
  • histological analysis of Golgi-Cox staining revealed a significant 50% decrease in dendritic spine density in the hippocampus of miR-195a KO mice compared with age-matched WT mice (Fig. 5B).
  • Sema3A is a direct target of miR-195 and that neuronal cells are overexpressed under stress to promote apoptosis.
  • bio-informatics algorithms miRanda and TargetScan
  • Sema3A and Sema3D were shown to be direct targets of miR-195. Therefore, the corresponding seed region in the Sema3D 3'-UTR was mutated to disrupt the base pairing between Sema3D and miR-195.
  • the present invention mutates at positions 2772-2792 of Sema3D 3'-UTR, and the mutation positions are shown in Table 4.
  • the underlined part is the mutation position.
  • Reporter plasmids carrying wild-type or mutant Sema3D 3'-UTR were transfected into HEK293 cells. Luciferase reporter assay confirmed that miR-195 binds directly to the Sema3D RNA 3'-UTR (Fig. 6A). The amounts of both Sema3A and Sema3D were increased in the hippocampus of miR-195a KO mice (Fig. 6B-6C), and the amount of Sema3D protein was 3-fold higher than that of Sema3A in WT mice (Fig. 6B).
  • RNA-seq analysis of the Allen Human Brain Atlas revealed that the amount of Sema3D mRNA in the human hippocampus was 50% higher than that of Sema3A (Fig. 6D). Since it has been reported that Sema3A is related to neurodegenerative diseases, and the role of Sema3D in brain function is still unclear, the present inventors decided to further study the role of Sema3D in this aspect.
  • Sema3D degree correlates with aging characteristics and neurodegenerative diseases
  • datasets include one dataset of individuals with dementia according to the Clinical Dementia Rating, three datasets of Alzheimer's disease (AD) patients, one patient with ubiquitin inclusion bodies A dataset of patients with temporal lobe dementia (FTLD-U) as well as a dataset of a normal elderly individual.
  • FTLD-U temporal lobe dementia
  • Sema3A data were also analyzed and presented in Table 5.
  • Sema3D is a direct cause of neurodegeneration and cognitive impairment
  • Sema3D-expressing lentivirus (Lv. Sema3D) was administered to the bilateral hippocampus of 4-month-old WT mice. This injection avoids overexpression of Sema3D in the cortex. The efficiency of viral transfection was confirmed by measuring the degree of expression of Sema3D protein on day 7. Immunoblot data showed a 3-fold increase in Sema3D expression in the hippocampus, while only a slight increase in Sema3D expression in the cortex (Fig. 8A).
  • Sema3D-overexpressing mice were tested to examine the deleterious effects of Sema3D on cognitive function in Sema3D-overexpressing mice.
  • a novel object recognition test was also used in Sema3D-overexpressing mice to perform recognition memory.
  • Sema3D in the hippocampus was knockdowned by a single injection of siRNA into 12-month-old miR-195a KO mice.
  • the silencing efficiency of Sema3D was assessed by immunofluorescence analysis ( Figure 9A), and spatial working memory and motor function were assessed by the Y-maze test (see protocol described in Figure 9B).
  • the results show that mice that received siRNA-Sema3D injections exhibited better spatial working memory than mice that received siRNA-Ctrl, as demonstrated by a higher percentage of alterations and an optimal fitness with a p-value of 0.042.
  • the Drosophila model was used to test the association between Sema3D extent and lifespan. Since the Sema2A gene in Drosophila is the homolog of the Sema3D gene in human and mouse, Sema2A is overexpressed in the nervous system of Drosophila (annotated as Sema2A overexpressed Drosophila). As shown in Figure 10A, the lifespan of flies overexpressing Sema2A was significantly shorter than that of control flies (300 per group, p ⁇ 0.0001). As shown in Table 6, the mean lifespan of the Sema2A group was reduced by 26%, and the maximum lifespan was also significantly reduced from 75 days to 63 days.
  • the present invention explores two possible mechanisms for the deleterious effects of Sema3D, the function of neural stem cells and autophagy.
  • the function of neural stem cells is related to nerve regeneration.
  • the present invention speculates whether Sema3D can disrupt neural stem cell function to explain its effect on neurodegeneration, and partially explain the low neural stem cell population density in miR-195a KO mice ( Figure 5A and Figure 5C).
  • Neurosphere formation assays were performed and the number of neural stem cells was quantified. The results showed that Sema3D dose-dependently inhibited neurosphere formation of human neural stem cells, suggesting that Sema3D impairs the stem cell properties of neural stem cells (Fig. 10C).
  • Sema3D When recombinant Sema3D was injected intraventricularly (ICV) into mice, Sema3D dose-dependently reduced neural stem cell population size in the DG and SVZ (DG results in Figure 10D and SVZ results in Figure 10E). Therefore, Sema3D reduces neural stem cell properties and causes neural stem cell loss, which may lead to neurodegeneration.
  • Sema3D may induce neurodegeneration by regulating autophagy and the PI3K/AKT/mTOR pathway.
  • the present invention first explored the effect of Sema3D on the efficiency of autophagy by overexpressing Sema3D in the hippocampus of mice and treating SY5Y cells with Sema3D.
  • Lv.Sema3D-treated mice had higher degrees of p-mTOR/mTOR and p62, and lower degrees of Beclin-1 and LC3-II/I ratios in the hippocampus (FIG. 11A). Consistently, Sema3D dose-dependently increased p62 and decreased the ratio of Beclin-1 and LC3-II/I in Sema3D-treated SY5Y cells (FIG. 11B). Thus, both in vitro and in vivo models suggest that Sema3D significantly disrupts autophagy.
  • the present invention assessed whether the PI3K/AKT/mTOR pathway could be modulated by Sema3D and whether Sema3D-induced neurodegeneration could be rescued by rapamycin, an mTOR inhibitor.
  • the results of the present invention show that Sema3D dose-dependently increased the phosphorylation of PI3K, Akt and mTOR in SY5Y cells (Fig. 11C).
  • Sema3D inhibited the proliferation of SY5Y cells, which was reversed by rapamycin (Fig. 11D).
  • a single dose of rapamycin was injected into the bilateral hippocampus 5 minutes after the injection of Lv.
  • Sema3D into the bilateral hippocampus. Histological images showed that rapamycin reversed Sema3D-induced neurodegeneration as evidenced by increased density of dendritic spines (FIG. 11E). Taken together, these data suggest that Sema3D impairs neuronal autophagy through an mTOR-dependent pathway that is rescued by rapamycin.
  • Sema3D is highly expressed in diabetic rats
  • Sema3D expression was also increased in diabetic retina. Furthermore, Sema3D was expressed to a higher degree than Sema3A in the retina of diabetic animals.

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Abstract

La présente invention concerne une utilisation d'une composition dans la préparation d'un médicament pour prévenir ou traiter des maladies neurodégénératives, la composition comprenant une quantité thérapeutiquement efficace d'un antagoniste de Sema3D. La présente invention concerne également une utilisation d'une composition dans la préparation d'un médicament pour prolonger la durée de vie ou favoriser la régénération nerveuse, la composition comprenant une quantité thérapeutiquement efficace d'un antagoniste de Sema3D.
PCT/CN2021/112108 2020-08-12 2021-08-11 Utilisation d'un antagoniste de sema3d dans la prévention ou le traitement de maladies neurodégénératives et prolongeant la durée de vie WO2022033529A1 (fr)

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