Use of atdr and atdra in treating leukodystrophy

Abstract

The present disclosure provides the use of ATDR and ATDRA in treating white matter diseases, in particular, provides the use of all-trans-13,14-dihydroretinol (ATDR) and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a pharmaceutically acceptable salt thereof, or a composition comprising all-trans-13,14-dihydroretinol (ATDR) and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a pharmaceutically acceptable salt thereof, retinol saturase (Retsat) or a variant thereof, a nucleic acid encoding Retsat or a variant thereof, an expression cassette comprising a nucleic acid encoding Retsat or a variant thereof, a vector comprising a nucleic acid encoding Retsat or a variant, a cell expressing Retsat or a variant thereof in the preparation of a medicament for preventing and / or treating white matter diseases in a subject in need thereof, which can promote the differentiation of oligodendrocytes and myelination to treat white matter diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of ATDR and ATDRA in the treatment of white matter lesions. Background Technology

[0002] The white matter of the brain is mainly composed of nerve fibers, axons, myelin sheaths, and glial cells. White matter lesions (WMLs) are defined as a clinical syndrome characterized by scattered punctate or confluent patchy changes widely distributed in the subcortical white matter, periventricular region, and centrum semiovale of the brain, which can be observed on imaging. Based on different etiologies and pathophysiological processes, white matter lesions can be classified into leukoaraiosis, age-related leukoencephalopathy, toxic leukoencephalopathy, and subcortical arteriosclerotic encephalopathy. Its main pathological feature is the destruction, loss, or impaired formation of myelin sheaths in central nervous fibers. Demyelination changes are present in the common pathological stages of various diseases, including multiple sclerosis (MS), Alzheimer's disease (AD), vascular cognitive impairment, and dementia (VCID), and are closely related to glial cells (microglia, oligodendrocytes, and astrocytes). The pathogenesis of white matter injury (WML) mainly involves hypoperfusion damage, blood-brain barrier injury, immune inflammatory response, endothelial dysfunction, and oxidative stress. In recent years, with the development of medical imaging technology, the detection rate of WML has been increasing, with surveys showing a prevalence exceeding 30% in people over 60 years of age. The intracranial distribution of white matter varies, leading to significant differences in clinical manifestations: periventricular white matter lesions often present as cap-shaped, linear, or crescent-shaped lesions. Smaller cap-shaped or punctate lesions may be asymptomatic and progress slowly; deep white matter lesions often present as punctate, patchy, or large confluent areas. These lesions progress rapidly, leading to cognitive impairment, abnormal emotional fluctuations, gait instability, urinary incontinence, and other clinical manifestations, causing serious socioeconomic and family problems in daily life. Summary of the Invention

[0003] On the one hand, this disclosure provides the use of all-trans-13,14-dihydroretinol (ATDR) or a salt thereof, all-trans-13,14-dihydroretinoic acid (ATDRA) or a salt thereof, or a composition comprising all-trans-13,14-dihydroretinol (ATDR) or a salt thereof and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a salt thereof in the preparation of a medicament for the prevention and / or treatment of leukoencephalopathy in a subject in need.

[0004] In some embodiments, the composition may optionally contain a pharmaceutically acceptable carrier or excipient, and may optionally further contain a second drug for treating leukoencephalopathy.

[0005] On the other hand, this disclosure provides the use of retinol saturase or variant thereof, nucleic acid encoding retinol saturase or variant thereof, expression cassette containing nucleic acid encoding retinol saturase or variant thereof, vector containing nucleic acid encoding retinol saturase or variant thereof, and cells expressing retinol saturase or variant thereof in the preparation of a medicament for the prevention and / or treatment of leukoencephalopathy in subjects in need.

[0006] In some embodiments, the retinol saturase has the biological activity of converting all-trans retinol to all-trans-13,14-dihydroretinol (ATDR), and the retinol saturase variant contains one or more amino acid substitutions, deletions, insertions and / or additions compared to the retinol saturase, and has enhanced retinol saturase activity. Preferably, the retinol saturase has a Q247R substitution relative to SEQ ID NO: 1 or a Q246R substitution relative to SEQ ID NO: 5.

[0007] In some embodiments, the retinol saturase or a variant thereof comprises a sequence selected from SEQ ID NO: 1-2, 5-6 or having at least 85%, 90%, 95%, or 100% identity with SEQ ID NO: 1-2, 5-6.

[0008] In some embodiments, the nucleic acid encoding retinol saturase or a variant thereof is DNA, cDNA, or mRNA.

[0009] In some embodiments, the nucleic acid encoding retinol saturase or a variant thereof comprises a sequence selected from SEQ ID NO: 3-4, 7-8 or having at least 85%, 90%, 95%, or 100% identity with SEQ ID NO: 3-4, 7-8.

[0010] In some embodiments, the expression cassette containing a nucleic acid encoding a retinol saturase or a variant thereof includes: a nucleic acid encoding a retinol saturase or a variant thereof and a transcriptional regulatory element operatively linked to the nucleic acid encoding a retinol saturase or a variant thereof.

[0011] In some embodiments, the vector containing nucleic acid encoding retinol saturase or a variant thereof is selected from plasmids, viral vectors, and non-viral vectors. Preferably, the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentiviral vector, more preferably an AAV vector, wherein the AAV vector is selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-PHP.B, AAV-PHP.eB, or variants thereof. The non-viral vector is selected from nanoparticles, nanobodies, liposomes, biodegradable polymer complexes, or combinations thereof. The nanoparticles include liposomes, lipid nanoparticles, polymer nanoparticles, dendritic polymers, cyclodextrins, silica nanoparticles, polymer complexes, magnetic nanoparticles, gold nanoparticles, quantum dots, carbon nanotubes, or combinations thereof.

[0012] In some specific embodiments, the retinol saturase or a variant thereof is specifically expressed in neurons.

[0013] In some specific implementations, the leukoencephalopathy is a myelin sheath injury-related disease, including hypoxic-ischemic encephalopathy, demyelinating diseases, Alexander's disease, leukoencephalopathy, metachromatic leukodystrophy (MLD), adrenoleukodystrophy, and myeloleukoencephalopathy. Preferably, the hypoxic-ischemic encephalopathy is neonatal hypoxic-ischemic encephalopathy. The demyelinating diseases include multiple sclerosis, autoimmune encephalomyelitis (EAE), progressive multifocal leukoencephalopathy, acute sporadic encephalomyelitis, spinal cord injury, stroke, neuromyelitis optica, Guillain-Barré syndrome, diffuse sporadic encephalomyelitis, and acute sporadic encephalomyelitis. Multiple encephalomyelitis, diffuse sclerosis, central pontine myelinolysis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, subacute combined degeneration caused by nutritional deficiencies, subacute sclerosing panencephalitis caused by viral infection, diabetic neuropathy, and systemic lupus erythematosus neuropathy; preferably, the demyelinating disease is an acute or chronic demyelinating-related disease, more preferably, the demyelinating disease is selected from one or more of multiple sclerosis, autoimmune encephalomyelitis (EAE), acute inflammatory demyelinating polyneuropathy, or chronic inflammatory demyelinating polyneuropathy.

[0014] In some specific implementations, the subject is a mammal, preferably a human.

[0015] In some specific embodiments, the drug is administered via intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, intrathecal injection, or intraperitoneal injection, preferably via intraperitoneal injection.

[0016] Beneficial effects This disclosure provides the use of all-trans-13,14-dihydroretinol (ATDR) and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a pharmaceutically acceptable salt thereof, retinol saturase (Retsat) and / or an enhanced variant thereof, nucleic acids encoding Retsat and / or an enhanced variant thereof, expression cassettes containing nucleic acids encoding Retsat and / or an enhanced variant thereof, vectors containing nucleic acids encoding Retsat and / or an enhanced variant thereof, and cells expressing Retsat and / or an enhanced variant thereof in the preparation of medicaments for the prevention and / or treatment of white matter injury in subjects of need. The Q246R mutation corresponding to the murine sequence increases Retsat enzyme activity, promotes ATDR synthesis, and ATDR and / or ATDRA improve spatial memory deficits and motor coordination in patients with white matter injury by enhancing oligodendrocyte differentiation and myelination, alleviating inhibition of myelination, and maintaining myelin structure. Attached Figure Description

[0017] This disclosure can be more fully understood with reference to the following figures.

[0018] Figure 1 The diagram shows that the Retsat-Q246R mutation can prevent developmental hypoxia-induced myelin degeneration. (A) Schematic diagram of the chronic hypoxia experimental design. (B) As shown in Figure A, after hypoxia, wild-type and Retsat-Q246R mutations... Q / R and Retsat R / R Route map of mice reaching the hidden platform at 40 days of age. (Retsat) Q / R This refers to a Q246R mutation on a chromosome, Retsat R / R (Refers to a Q246R mutation occurring on two chromosomes) (C, D) P40 normoxic or hypoxic wild type, Retsat Q / R and Retsat R / R Morris water maze (MWM) spatial learning and memory test in mice. Escape latency (C) was measured as the average time during the training trial. On the second day, the time mice spent in the target quadrant (D) was analyzed (number of normoxic and hypoxic mice, n = 5). (E) Wild-type and Retsat mice were assessed using a three-box social test. Q / R and Retsat R / R Social behavior of mice on day 45 after normoxic or hypoxic conditions (n≥5 represents the number of normoxic or hypoxic mice). (F and G) Social behavior of wild-type and Retsat mice after normoxic or hypoxic conditions. Q / R and Retsat R / RImmunostaining (F) and quantification (G) of NeuN in the cortex of mice at P10 (n = 3 mice per experiment). Scale bar, 300 μm. (H and I) Wild-type and Retsat mice at P10 after normoxic or hypoxic conditions. Q / R and Retsat R / R Immunostaining (H) and quantification (I) of NF200 in mouse corpus callosum (n = 3 mice per experiment). Scale bar, 100 μm. (J and K) Wild-type and Retsat at P10 after normoxic or hypoxic conditions. Q / R and Retsat R / R Immunostaining (J) and quantification (K) of mouse corpus callosum (CC) MBP (n = 3 mice per experiment). Scale bar, 120 μm. (L and M) Wild-type and Retsat mice at P10 after normoxic or hypoxic conditions. Q / R and Retsat R / R In situ hybridization (L) and quantification (M) of mouse corpus callosum Plp1+ cells (n = 3 mice in each experiment). Scale bar, 200 μm. (N) After normoxic or hypoxic conditions, wild-type, Retsat Q / R and Retsat R / R Real-time PCR analysis of cortical myelin-related genes in mice at P10 (N = 3 mice per case). (O) Wild-type and Retsat mice after normoxic or hypoxic conditions. R / R Electron microscopic analysis of the corpus callosum in mice at P14. Scale bar, 2 μm. (P) Wild-type and Retsat mice under normoxic or hypoxic conditions. R / R Quantitative analysis of myelinated axons in the corpus callosum at P14 in mice (n = 3 mice in each experiment). (Q) Wild-type and Retsat mice under normoxic or hypoxic conditions. R / R Mouse corpus callosum myelin sheath thickness (g-ratio) (n = 3 mice per experiment). g-ratio is the ratio of inner axon diameter to total outer diameter. Data are expressed as mean ± standard deviation; ns indicates no statistical significance. p<0.05, p<0.01, p<0.001, p<0.0001 (based on one-way ANOVA and Tukey's multiple comparison test).

[0019] Figure 2 The study demonstrates that the Retsat-Q246R mutation has a protective effect against developmental disorders caused by hypoxia-induced motor coordination deficits. (A) After normoxic or hypoxic conditions, wild-type and Retsat... R / RAt 40 days of age, the motor function of mice was assessed using a rotating bar test (n = 5 normoxic and hypoxic mice). (B) Digital PCR (dPCR) was used to detect wild-type and Retsat mice at 26 days of age. Q / R and Retsat R / R Retsat expression levels in the mouse cortex (n = 3 mice per experiment). Data are expressed as mean ± standard deviation. This indicates p < 0.001 (using one-way ANOVA and Tukey multiple comparison test).

[0020] Figure 3 The study showed that Retsat promotes oligodendrocyte differentiation and myelination, and the Q246R mutation enhances this function. (A, B) After normoxic or hypoxic conditions, in wild-type and Retsat-positive cells... Q / R and Retsat R / R Immunostaining (A) and quantification (B) of CC1 in the corpus callosum of mice at 10 days of age (n = 3 mice in each experiment). Scale bar, 30 μm. (C and D) Immunostaining (A) and quantification (B) in wild-type and Retsat mice after normoxic or hypoxic conditions. Q / R and Retsat R / R Immunostaining (C) and quantification (D) of oligodendrocytes of the corpus callosum at 10 days of age in mice (n = 3 mice in each experiment). Scale bar, 40 μm. (E, F) After hypoxia, in wild-type and Retsat mice... Q / R and Retsat R / R Immunostaining (E) and quantification (F) of oligodendrocyte proliferation in the corpus callosum of mice at 10 days of age (n = 3 mice per experiment). Scale bar, 50 μm. (G and H) in wild-type, Retsat Q / R and Retsat R / RApoptotic cell detection (G) and quantitative statistics (H) in the corpus callosum of mice at 10 days of age (n = 3 mice per experiment). Scale bar, 30 μm. (I and J) Immunostaining (I) and quantification (J) of myelin basic protein in the corpus callosum of wild-type and Retsat knockout mice at 14 days of age (n = 3 mice per experiment). Scale bar, 300 μm. (K and L) Immunostaining (K) and quantification (L) of mature oligodendrocytes in the corpus callosum of wild-type and Retsat knockout mice at 14 days of age (n = 3 mice per experiment). Scale bar, 150 μm. (M and N) Immunostaining (M) and quantification (N) of oligodendrocyte precursor cells in the corpus callosum of wild-type and Retsat knockout mice at 14 days of age (n = 3 mice per experiment). Scale bar, 150 μm. (O and P) Immunostaining (O) and quantification (P) of myelin basic protein in the corpus callosum of wild-type and Retsat knockout mice at 14 days after birth under normoxic or hypoxic conditions (n ​​= 3 mice per experiment). Scale bar, 100 μm. (QS) Immunostaining (Q) and quantification (R, S) of mature oligodendrocytes, oligodendrocyte precursor cells, and oligodendrocyte lineage cells in the corpus callosum of wild-type and Retsat knockout mice at 10 days after birth under normoxic or hypoxic conditions (n ​​= 3 mice per experiment). Scale bar, 50 μm. Data are expressed as mean ± standard deviation; ns indicates no statistical significance. p<0.05, p<0.01, p<0.001, p < 0.0001. Among them, Figures J, L, N, P, R, and S were tested using two-tailed unpaired Student's t-test; Figures B, D, F, and H were tested using one-way ANOVA and Tukey's multiple comparison test.

[0021] Figure 4This study demonstrates that Retsat deletion does not affect OPC proliferation, neurons, astrocytes, or microglia. (A) Immunoblotting verification of Retsat gene knockout efficiency in the liver of wild-type or Retsat knockout mice at 80 days of age. (B) Real-time PCR analysis of Retsat gene expression in the cortex of wild-type and Retsat knockout mice at 10 days of age (n = 3 mice per experiment). (C and D) Immunostaining (C) and quantification (D) of proliferating oligodendrocyte lineage cells in the corpus callosum region of wild-type and Retsat knockout mice at 10 days of age after normoxic or hypoxic conditions (n ​​= 3 mice per experiment). Scale bar, 50 μm. (E) Immunostaining of neurons, astrocytes, and microglia in wild-type and Retsat knockout mice at 10 days of age after normoxic or hypoxic conditions. Scale bar, 100 μm. (F to H) Quantitative statistical analysis of immunostaining of neurons, astrocytes, and microglia in wild-type and Retsat knockout mice at 10 days of age after normoxic or hypoxic conditions (n ​​= 3 mice per experiment). (I and J) Immunostaining (I) and quantification (J) of NF200 in the corpus callosum of wild-type and Retsat knockout mice at 10 days of age after normoxic or hypoxic conditions (n ​​= 3 mice per experiment). Scale bar, 150 μm. Data are expressed as mean ± standard deviation; ns indicates no statistical significance. This indicates p < 0.001 (using a two-tailed unpaired Student's t test).

[0022] Figure 5This study demonstrates how the Retsat Q246R mutation promotes myelin repair in the central nervous system of adult mice. (A, B) EdU and CC1 immunostaining (A) and CC1+ & EdU+ cell quantification analysis (B) were performed on control and mutant mice at 60 days of age after continuous administration of EdU for 30 days (n=3 mice). Scale bar = 90 μm. (C, D) Representative images of fluorescent myelin staining (C) and myelin volume quantification (D) of corpus callosum lesions in control and mutant mice (8 weeks old) 14 days after LPC injection. Scale bar = 250 μm. (E, F) MBP and Iba1 immunostaining (E) and MBP volume quantification (F) of corpus callosum lesions in control and mutant mice 14 days after LPC injury. Scale bar = 80 μm. (G, H) Plp1 in situ hybridization (G) and Plp1+ cell quantification (H) in corpus callosum lesions of control and mutant mice 14 days after injury. Scale bar = 500 μm (left) and 250 μm (right). (IL) Immunostaining (I, K) and quantitative analysis (J, L) of specified proteins in corpus callosum lesions of control and mutant mice 14 days after LPC injury. Scale bar = 50 μm (I), 100 μm (K). (MO) Electron microscopic image analysis (M), myelinated nerve fibers (N), and myelin g-value quantification (O) of the corpus callosum of control and mutant mice 10 days after LPC injury. Scale bar = 5 μm. (P, Q) Representative images (P) and myelin volume quantification (Q) of the corpus callosum of control and mutant mice after 5 weeks of 0.2% copper plasmid treatment and 1 week of recovery. Scale bar = 300 μm. (R, S) Immunostaining (R) and quantification (S) of mature oligodendrocytes and oligodendrocytes in the corpus callosum of control and mutant mice. Scale bar = 40 micrometers. Data are expressed as mean ± standard deviation. Statistical analysis was performed using a two-tailed unpaired Student's t-test; p < 0.01, p < 0.001, p<0.0001.

[0023] Figure 6This study illustrates the regenerative capacity of adult mice impaired by Retsat deficiency. (A, B) Representative images of fluorescent myelin staining in the corpus callosum of control and mutant mice (8 weeks old) after 5 weeks of treatment with 0.2% copper sulfate and 1 week of recovery (A) and quantitative analysis of myelin volume (B). Scale bar = 150 μm. (CF) Immunostaining (C, E) and quantitative analysis (D, F) of specified proteins in the corpus callosum of control and knockout mice at 65 days of age (65 days). Scale bar = 250 μm (C), 100 μm (E). (G, H) Representative images of fluorescent myelin staining in the corpus callosum lesions of control and knockout mice (8 weeks old) 14 days after LPC injection (G) and quantitative analysis of myelin volume (H). Scale bar = 350 μm. (I, J) Immunostaining (I) and quantitative analysis of myelin area (J) in the corpus callosum lesions of control and knockout mice 14 days after injury. Scale bar = 100 micrometers. (K, L) Immunostaining (K) and quantitative analysis (L) of mature oligodendrocytes in corpus callosum lesions of control and knockout mice. Scale bar = 50 micrometers. Data are expressed as mean ± standard deviation. ns indicates no statistical difference. p<0.05, p<0.01 (two-tailed unpaired Student t test).

[0024] Figure 7This study demonstrates that Retsat Q246R promotes myelination in a neuron-specific rather than oligodendrocyte-specific manner. (A) Representative images of whole-brain Retsat staining at 7, 14, and 60 days postnatal in wild-type mice (left) and high-magnification images of the cortical / corpus callosum region (right). Scale bar = 2 mm (left), 100 μm (right). (B, C) Co-immunostained Retsat with specific cellular markers in the brain of wild-type mice at 10 days postnatal (B) and quantitative analysis of the positive proportions of various cellular markers in Retsat+ cells (C). Arrows indicate Retsat-positive cells. Scale bar = 20 μm. (D, E) Immunostaining of myelin basic protein and oligodendrocyte markers in oligodendrocyte precursor cells of control and mutant mice under differentiation induction and normoxic or hypoxic conditions (D) and quantitative analysis of mature oligodendrocytes (E) (n = 3 independent experiments). Scale bar = 50 μm. (F) Real-time quantitative PCR analysis of myelination-related genes in oligodendrocyte precursor cells of control and mutant mice under differentiation induction and normoxic or hypoxic conditions (n=3 independent experiments). (G) Schematic diagram of constructing Rosa26-Retsat Q246R knock-in mice to achieve Cre-mediated cell-specific expression. EGFP: Enhanced green fluorescent protein; IRES: Internal ribosome entry site; HA: Tag protein. (HL) Immunostaining (H, K) and quantitative analysis (I, J, L) of specified proteins in the corpus callosum and cortex of control and O-Retsat R mice at time point P14. Scale bar = 150 μm (H), 50 μm (K). O-Retsat R: Specific overexpression of Retsat Q246R in oligodendrocytes. (M) Immunostaining of myelin basic protein and oligodendrocyte markers in oligodendrocyte precursor cells of control and knockout mice under differentiation conditions (left panel) and quantitative analysis of mature oligodendrocytes (right panel) (n=3 independent experiments). Scale bar = 100 μm. (N, O) Immunostaining of myelin and axonal tissue in cerebellar sections of control and mutant mice 3 days after birth (N) and quantitative analysis (O) (n=3 independent experiments) (n=3 independent experiments). Scale bar = 50 μm. (P, Q) Immunostaining of myelin basic protein in the corpus callosum and cortex of control and N-RetsatiR mice at 14 days after birth (P) and quantitative analysis (Q). Scale bar = 100 μm. (R, S) Immunostaining of myelin basic protein and mature oligodendrocyte markers in the corpus callosum of control and N-RetsatiR mice treated with normoxic or hypoxic methods from 3 to 10 days after birth (R) and quantitative analysis (S) of cells. Scale bar = 50 micrometers. N-RetsatiR: Specific overexpression of Retsat Q246R in neurons. (T, U) Immunostaining (T) and quantitative analysis (U) of mature oligodendrocytes in primary oligodendrocyte precursor cells treated with conditioned medium from control or mutant mice.Scale bar = 100 micrometers. (V) Real-time quantitative PCR analysis of myelin formation-related genes in oligodendrocytes treated with conditioned medium from primary neurons of control or mutant mice. Data are expressed as mean ± standard deviation; ns indicates no statistical difference, p < 0.05, p < 0.01, p < 0.001. p<0.0001; Statistical methods used included two-tailed unpaired Student t-test (L, M, Q) or Tukey multiple comparisons one-way ANOVA (E, F, I, J, O, S, U, V).

[0025] Figure 8 This demonstrates that Retsat-Q246R does not promote myelination through an oligodendrocyte-autonomous mechanism. (AC) Analysis of Retsat expression and cellular localization in the brains of wild-type mice under normoxic or hypoxic conditions. (A) Representative images of Retsat immunohistochemical staining. (B) Quantitative cellular analysis. (C) Colocalization analysis of Retsat with various neural cell markers. Scale bar = 20 μm. (D) Real-time quantitative PCR analysis of gene expression in primary oligodendrocyte precursor cells of control and mutant mice treated with or untreated with retinol under differentiation induction and normoxic or hypoxic conditions. (E, F) Immunostaining (E) and quantitative analysis (F) of proliferating cells in primary oligodendrocyte precursor cells of control and mutant mice. Scale bar = 50 μm. (G, H) Immunostaining (G) and quantitative analysis (H) of cell death in primary oligodendrocyte precursor cells of control and mutant mice. Scale bar = 50 μm. (I, J) Real-time quantitative PCR analysis of myelin formation-related genes in primary oligodendrocyte precursor cells of mice (I) and rats (J) transfected with control, mouse Retsat, and Q246R under differentiation induction and normoxic or hypoxic conditions (n ​​= 3 independent experiments). (K) Co-immunostained green fluorescent protein and specific cell markers in the brain of P9 O-RetsatiR mice. Arrows indicate GFP+ cells. Scale bar = 30 μm. (L) Immunostaining of HA and oligodendrocyte markers in the brain of 14-day-old control and O-RetsatiR mice. Scale bar = 50 μm. (M, N) Immunostaining of specified proteins in the corpus callosum of 9-day-old control and O-RetsatiR mice. Scale bar = 100 μm (M), 50 μm (N). (O) Real-time quantitative PCR analysis of gene expression in primary oligodendrocyte precursor cells of control and knockout mice under differentiation induction and normoxic or hypoxic conditions (n ​​= 3 independent experiments). (P) Real-time quantitative PCR analysis of myelin formation-related genes in primary oligodendrocyte precursor cells of Retsat knockdown rats under differentiation conditions (n ​​= 3 independent experiments). Data are expressed as mean ± standard deviation; ns indicates no statistical difference. p<0.01, p<0.0001; statistical methods used included two-tailed unpaired Student t-test (B, F, H, P) or Tukey multiple comparisons one-way ANOVA (D, I, J, O).

[0026] Figure 9 The following images show that neuron-specific expression of Retsat Q246R does not affect oligodendrocyte progenitor cell proliferation (A, B). Immunostaining of green fluorescent protein (GFP) and specific cell markers in the brains of 14-day-old N-RetsatiR mice (A) and quantitative analysis of the positive proportion of each cell marker in Retsat-positive cells (B). Arrows indicate GFP-positive cells. Scale bar = 20 μm. (C) Immunostaining of HA and neurons in the brains of 14-day-old control and N-RetsatiR mice. Arrows indicate HA-positive cells. Scale bar = 20 μm. (D, E) Immunostaining of oligodendrocyte progenitor cells and oligodendrocyte markers in the corpus callosum of control and N-RetsatiR mice treated with normoxic or hypoxic methods from 3-10 days of age (D) and quantitative analysis of oligodendrocyte progenitor cells (E). Scale bar = 150 μm. Data are presented as mean ± standard deviation. One-way ANOVA with multiple Tukey comparisons showed no statistically significant differences between groups (ns).

[0027] Figure 10This study demonstrates how point mutations in retinol saturase promote oligodendrocyte differentiation via the neuronal dihydroretinol-retinoid X receptor signaling axis. (A) Immunoblot analysis of 293T cells transfected with mouse retinol saturase or point mutations. (B) Immunostained COS7 cells after transfection with retinol saturase or point mutations, respectively. Scale bar, 10 μm. (C) HPLC analysis of 293T cells transfected with Retsat, Retsat (Q246R), Retsat (E96A), or Retsat (Q246R; E96A), showing peaks at approximately 12 min (retinol) and approximately 14 min (ATDR). Quantification is based on peak area. (D) ATDR concentration in primary wild-type or point-mutant neurons detected by HPLC-MS (n = 3 independent experiments). (E) HPLC-MS detection of retinol and ATDR concentrations in the brains of wild-type or point-mutant mice at P75 (n = 3 mice per condition). (F) Schematic diagram of the experimental procedure: Conditioned medium from primary mouse neurons was collected and then centrifuged and filtered at a 3-kDa threshold. The filtrate was applied to OPCs. (G) Real-time PCR analysis of myelination-related gene expression in primary rat oligodendrocyte precursors after differentiation induction and treatment with <3 kDa filtrate from wild-type or point-mutant neuronal conditioned medium (CM) as described in (F) (n = 3 independent experiments). (H) Real-time PCR analysis of myelination-related genes in primary mouse oligodendrocyte precursors after treatment with retinol or dihydroretinol under normoxic or hypoxic conditions (n ​​= 3 independent experiments). (I and J) Immunostaining (I) and quantification (J) of myelin and axonal tissue in cerebellar slice cultures prepared from P3 mice and cultured in vitro for 12 days (DIV 12). Sections were exposed to normoxic or hypoxic conditions and treated with either control or dihydroretinol (n = 3 independent experiments). Scale bar, 50 μm. (K) Schematic diagram of the experimental procedure: Wild-type neurons were incubated with dihydroretinol, and conditioned medium was collected and passed through a 3-kDa cutoff filter. The <3 kDa filtrate was then applied to oligodendrocyte precursor cultures to induce differentiation. (L) Real-time PCR analysis of myelin basic protein expression in primary rat oligodendrocyte precursors after differentiation induction and treatment with the specified <3 kDa conditioned medium filtrate (as described in (K)) (n = 3 independent experiments). (M) Real-time PCR analysis of retinol dehydrogenase-related genes in primary mouse neurons and oligodendrocyte precursors (n = 3 independent experiments). (N) Immunostaining of alcohol dehydrogenase 1, aldehyde dehydrogenase 1A2, neurons, and oligodendrocytes in the cortex adjacent to the corpus callosum of P14 mice. Scale bar, 20 μm. (O and P) Immunostaining (O) and quantification (P) of MBP, CNP enzymes and oligodendrocytes in primary rat oligodendrocyte precursors under differentiation conditions and after treatment with specified conditions (n ​​= 3 independent experiments).Scale bar, 100 μm. (Q) Real-time PCR analysis of myelination-related gene expression in primary rat oligodendrocyte precursors after specified condition treatment under differentiation conditions (n ​​= 3 independent experiments). (R) Detection of dihydroretinoic acid concentration in primary wild-type or point mutant neurons by HPLC-MS (n = 3 independent experiments). (S) Detection of dihydroretinoic acid concentration in the brains of wild-type and point mutant mice at P75 by HPLC-MS (n = 3 mice for each condition). (T) Real-time PCR analysis of retinoid X receptor pathway-related genes in primary rat oligodendrocyte precursors after specified condition treatment (n = 3 independent experiments). (U) Real-time PCR analysis of retinoid X receptor target genes in the cortex of wild-type and point mutant mice at P10 (n = 3 mice for each condition). (V and W) Immunostaining (V) and quantification (W) of myelin sheath in the corpus callosum of wild-type, point-mutant, and point-mutant + retinoid X receptor- / - mice at P14 (n = 3 mice per condition). Scale bar, 100 μm. (X and Y) Immunostaining (X) and quantification (Y) of mature oligodendrocytes and oligodendrocytes in the corpus callosum of wild-type, point-mutant, and point-mutant + RXRγ- / - mice at P14 (n = 3 mice per condition). Scale bar, 40 μm. Data are presented as mean ± SD; ns = not significant. p<0.05， p<0.01, p<0.001, p<0.0001, by two-tailed unpaired Student's t test (D, E, L, M, R, S), or by one-way ANOVA with Tukey multiple comparisons (G, H, J, P, Q, T, W, Y).

[0028] Figure 11 Immunostaining of designated proteins in the cortex of P17 mice is shown in (A) Immunostaining of retinol saturase point mutations acting through the retinoid X receptor pathway. Scale bar, 5 μm. (B) Real-time quantitative PCR analysis of myelination-related genes in primary rat oligodendrocyte precursors after treatment with retinol or dihydroretinol under normoxic or hypoxic conditions (n ​​= 3 independent experiments). (C to F) Immunostaining (C) and quantification of neurons (D), astrocytes (E), and microglia (F) in the corpus callosum of wild-type, point-mutant, and point-mutant + retinoid X receptor - / - mice at P14 (n = 3 mice per condition). Scale bar, 50 μm. Data are presented as mean ± SD; ns = no significance, analyzed by one-way ANOVA with Tukey multiple comparisons.

[0029] Figure 12This study demonstrates the therapeutic potential of dihydroretinol and dihydroretinoic acid in myelination disorders. (A and B) Immunostaining (A) and quantification (B) of myelin and axonal tissue cultures prepared from P3 mice and maintained in vitro for 14 days (DIV14). At DIV14, sections underwent lysophosphatidylcholine-induced demyelination, simultaneously treated with either control or dihydroretinol (n = 3 independent experiments). Scale bar, 50 μm. (C) Following intraperitoneal administration of a dose of (g, body weight), the concentrations of dihydroretinol and dihydroretinoic acid in the brains of P14 wild-type mice were determined by HPLC-MS (n = 3 mice for each condition). (D) Following intraperitoneal administration of a dose of (g, body weight), the concentration of retinol in the brains of P14WT mice was detected by HPLC-MS (n = 3 mice per condition). (E) Schematic diagram showing wild-type mice treated with hypoxia and dihydroretinol and dihydroretinoic acid at specified time points. (F and G) Immunostaining (F) and quantification (G) of myelin sheath in the corpus callosum of P14 wild-type mice after hypoxia and dihydroretinol and dihydroretinoic acid treatment (n = 3 mice per condition). Scale bar, 100 μm. (H) Immunostaining of mature oligodendrocytes and oligodendrocyte precursor cells in the corpus callosum of P14 wild-type mice after hypoxia and dihydroretinol and dihydroretinoic acid treatment (n = 3 mice per condition). Scale bar, 50 μm. (I) Quantification of mature oligodendrocytes in the corpus callosum of P14 wild-type mice after hypoxia and treatment with dihydroretinol and dihydroretinic acid (n = 3 mice per condition). (J) Schematic diagram of the dihydroretinol administration and lysophosphatidylcholine injection schedule. (K and L) Immunostaining (K) and quantification (L) of myelin in corpus callosum lesions of control and dihydroretinol-treated mice at 10 days post-injury (10 dpl). Scale bar, 400 μm. (M) Immunostaining of myelin and microglia in corpus callosum lesions of control and dihydroretinol-treated mice at 10 dpl. Scale bar, 50 μm. (N) Quantification of myelin in corpus callosum lesions of control and dihydroretinol-treated mice at 10 dpl (n = 3 mice per condition). (O) Immunostaining of mature oligodendrocytes and oligodendrocytes in corpus callosum lesions of mice in the control and dihydroretinol-treated groups at 10 dpl. Scale bar, 50 μm. (P) Quantification of mature oligodendrocytes in corpus callosum lesions of mice in the control and dihydroretinol-treated groups at 10 dpl (n = 3 mice per condition). (Q) Daily clinical scores of 8-week-old female WT mice induced with EAE, starting daily treatment with oil (control) or ATDR (10 mg / kg / day) after disease onset (10 days post-immunization) (n = 3 independent experiments). (R and S) Immunostaining (R) and quantification (S) of MBP in the spinal cord of P90WT mice after EAE and ATDR treatment (n = 3 mice per condition). Scale bar, 50 μm. (T) Electron microscopic analysis of the spinal cord of P90 wild-type mice after autoimmune encephalomyelitis model and dihydroretinol treatment (n = 3 mice per condition). Scale bar, 2 μm. (U) Quantification of myelin axons in the spinal cord of P90 wild-type mice after autoimmune encephalomyelitis model and dihydroretinol treatment (n = 3 mice per condition). (V) Myelin sheath thickness (g ratio) in the spinal cord of P90 wild-type mice after autoimmune encephalomyelitis model and dihydroretinol treatment (n = 3 mice per condition). Data are expressed as mean ± SD; ns = not significant. p<0.05， p<0.01, p<0.001, p < 0.0001, by two-tailed unpaired Student's test (C, D, L, N, P, S, U), or one-way ANOVA with Tukey multiple comparisons (B, G, I).

[0030] Figure 13 The following HPLC-MS chromatograms (A) show no adverse effects on body weight or cell death after administration of dihydroretinol and dihydroretinoic acid, as indicated by intraperitoneal injection of corn oil (control) or... Peak intensities of 9-cis-retinoic acid (9CRA) and RA (m / z = 301.2162) in the brains of P14 wild-type mice after administration of retinol at a dose of (g, body weight). (B) Bar graph shows the peak intensities of 9-cis-retinoic acid (9CRA) and RA (m / z = 301.2162) in the brains of P14 wild-type mice after administration of oil (control) or by gavage. (g, body weight) Change in body weight of mice over 3 days after administration of dihydroretinol (n = 3 mice for each condition). (C) Administration of oil (control), dihydroretinol, or dihydroretinoic acid (dose of) by gavage. The results of the TUNEL assay were analyzed after the mice were weighed (g, body weight). Detailed Implementation

[0031] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0032] This disclosure provides the use of all-trans-13,14-dihydroretinol (ATDR) and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a pharmaceutically acceptable salt thereof, or a composition comprising all-trans-13,14-dihydroretinol (ATDR) and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention and / or treatment of leukoencephalopathy in a subject of need.

[0033] In some specific embodiments, the composition may optionally include a second drug for treating leukoencephalopathy, and a pharmaceutically acceptable carrier or excipient.

[0034] In some specific embodiments, the composition may be a liquid or a solid, such as a powder, gel, or paste. Preferably, the pharmaceutical composition is a liquid, and more preferably an injectable liquid. Suitable carriers will be known to those skilled in the art.

[0035] In some specific implementations, some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0036] In some specific embodiments, the composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension, or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0037] This disclosure provides the use of retinol saturase or variant thereof, nucleic acid encoding retinol saturase or variant thereof, expression cassette containing nucleic acid encoding retinol saturase or variant thereof, vector containing nucleic acid encoding retinol saturase or variant thereof, and cells expressing retinol saturase or variant thereof in the preparation of a medicament for the prevention and / or treatment of leukoencephalopathy in subjects of need.

[0038] In some specific embodiments, the retinol saturase has the biological activity of converting all-trans retinol to all-trans-13,14-dihydroretinol (ATDR), and the retinol saturase variant contains one or more amino acid substitutions, deletions, insertions and / or additions compared to the retinol saturase, and has enhanced retinol saturase activity.

[0039] In some specific embodiments, the retinol saturase now has a Q247R substitution relative to SEQ ID NO: 1 or a Q246R substitution relative to SEQ ID NO: 5, wherein the retinol saturase or a variant thereof comprises an amino acid sequence selected from SEQ ID NO: 1-2, 5-6 or having at least 85%, 90%, 95%, or 100% identity with SEQ ID NO: 1-2, 5-6.

[0040] In some specific embodiments, the nucleic acid encoding retinol saturase or a variant thereof is DNA, cDNA, or mRNA.

[0041] In some specific embodiments, the nucleic acid encoding retinol saturase or a variant thereof comprises a sequence selected from SEQ ID NO: 3-4, 7-8 or having at least 85%, 90%, 95%, or 100% identity with SEQ ID NO: 3-4, 7-8.

[0042] In some specific embodiments, the expression cassette containing a nucleic acid encoding a retinol saturase or a variant thereof includes: a nucleic acid encoding a retinol saturase or a variant thereof, and a transcriptional regulatory element operatively linked to the nucleic acid encoding a retinol saturase or a variant thereof.

[0043] In some specific embodiments, the vector containing nucleic acid encoding retinol saturase or a variant thereof is selected from plasmids, viral vectors, and non-viral vectors. Preferably, the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentiviral vector, more preferably an AAV vector, wherein the AAV vector is selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-PHP.B, AAV-PHP.eB, or variants thereof; the non-viral vector is selected from nanoparticles, nanobodies, liposomes, biodegradable polymer complexes, or combinations thereof; the nanoparticles include liposomes, lipid nanoparticles, polymer nanoparticles, dendritic polymers, cyclodextrins, silica nanoparticles, polymer complexes, magnetic nanoparticles, gold nanoparticles, quantum dots, carbon nanotubes, or combinations thereof.

[0044] In some specific embodiments, the transcriptional regulatory element directs the expression of a nucleic acid sequence in target cells of the nervous system. The transcriptional regulatory element may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, nucleic acid restriction site, and homologous recombination site operatively linked to the nucleic acid sequence.

[0045] The phrase "pharmaceutically acceptable" is recognized in the art and refers to compositions, polymers, and other materials and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. For example, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, relate to carrying or transporting any supplement or composition or its components from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of compatibility with the other components of the supplement and harmlessness to the patient. Optionally, pharmaceutically acceptable carriers are pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and Soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0046] Vectors can be viral or non-viral (e.g., plasmids). Viral vectors include those derived from: adenoviruses, including mutant forms of adeno-associated virus (AAV), retroviruses, lentiviruses, herpesviruses, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxviruses, and SV40. Preferably, the viral vector is replication-defective, although it is envisioned to be replication-deficient, capable of replication, or conditionally replicating. Viral vectors can generally remain in an extrachromosomal state without integrating into the genome of the target cell. Preferred viral vectors encoding nucleic acid sequences of retinol saturase or variants thereof are AAV vectors, such as self-complementary adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotypes 2 to 12, AAV-DJ) or modified versions of any of these serotypes (including AAV 4YF, AAV 7m8, AAV-PHP.B, and AAV-PHP.eB).

[0047] Viral vectors can be modified to delete any non-essential sequences. For example, in AAV, the virus can be modified to delete all or part of the IX gene, Ela, and / or Elb gene. For wild-type AAV, the absence of helper viruses such as adenovirus makes replication very inefficient. For recombinant adeno-associated viruses, preferably, the replication gene and capsid gene are provided in trans form (in the pRep / Cap plasmid), and only the ITR of the AAV genome is preserved and packaged into the virion, while the required adenovirus genes are provided by adenovirus or another plasmid. Similar modifications can be made to lentiviral vectors.

[0048] Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids can be conjugated with agents to facilitate the uptake of the viral vector by target cells. Such agents include polycationic agents. Alternatively, delivery systems such as liposome-based delivery systems may be used. The vectors used in this disclosure are preferably suitable for in vivo or in vitro use, and are preferably suitable for human use. The plasmids are preferably AAV vector plasmids pAAV, pAAV-MCS, pAAV-MCS2, or pAAV-2Aneo.

[0049] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / RNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a “collapsed” structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0050] Suitable lipids can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Mo.; dihexadecanyl phosphate (“DCP”) is available from K&K Laboratories (Plainview, NY); and cholesterol (“Choi”) is available from Calbiochem. Behring is available; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of the lipids in chloroform or chloroform / methanol can be stored at approximately [time missing]. 20°C. Chloroform was used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of monolayer and multilayer lipid mediators formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions having structures in solution different from normal vesicular structures are also included. For example, lipids may exhibit a micellar structure or exist simply as heterogeneous aggregates of lipid molecules. Liposomes are also considered. Nucleic acid complex.

[0051] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0052] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0053] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions where identical residues occur. This is typically expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0054] As used herein, the terms “subject” and “required subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, and goats.

[0055] The terms “leukoencephalopathy,” “white matter lesion,” or “white matter injury” used in this article refer to a class of central nervous system demyelinating diseases characterized by demyelinating changes. Based on whether the myelin sheath is mature at the time of onset, these diseases can be divided into two main categories: demyelinating diseases with normal myelin development, such as multiple sclerosis, progressive multifocal leukodystrophy, acute sporadic encephalomyelitis, spinal cord injury, stroke, neuromyelitis optica, Guillain-Barré syndrome, diffuse sporadic encephalomyelitis, acute sporadic encephalomyelitis, diffuse sclerosis, central pontine myelinolysis, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, subacute combined degeneration caused by nutritional deficiencies, subacute sclerosing panencephalitis caused by viral infection, diabetic neuropathy, systemic lupus erythematosus neuropathy, leukoazotosis, adrenoleukodystrophy, and myeloleukoencephalopathy; and diseases with impaired myelin formation, such as globoid cell leukodystrophy, metachromatic leukodystrophy, and spongiform encephalopathy.

[0056] The term "retinol saturase" or "Retsat" as used herein catalyzes the conversion of all-trans retinol to all-trans-13,14-dihydroretinol (ATDR). Retsat "Gen locus" and " RetsatA "gene" refers to a coding sequence, introns, and regulatory elements that regulate transcription and / or translation. The term "gene" is used in this context. Retsat "Gen locus" and " Retsat The term "gene" includes all allelic variations of RETSAT. Retsat "Nucleic acid" refers to nucleic acid derived from Retsat Polynucleotides at gene loci, such as those encoding Retsat Messenger RNA, DNA, complementary DNA, genomic DNA, and antisense nucleic acids and polynucleotides encoding their fragments, derivatives, and analogs. Useful fragments and derivatives include sequences based on all possible codon selections of the same amino acid, as well as codon selections based on conserved amino acid substitutions. Useful derivatives also include those related to... Retsat Nucleic acids are polynucleotides with at least 50% or at least 70% (usually 80%, more typically 90%) sequence identity.

[0057] Table 1. Sequence List

[0058] Experimental Materials and Methods mice All mice were housed at the core animal facility of Shanghai Jiao Tong University School of Medicine, and all experiments were approved by the Institutional Animal Ethics and Use Committee of Shanghai Jiao Tong University. Mice were housed in an environment with a 12-hour light / dark cycle and free access to food and water. Both male and female mice were included in the study. The retinol saturase (Retsat) heterozygous Q246R was knocked into the mice (…). Retsat Q / R They mate to produce Retsat R / R Homozygous mutant mice and wild-type mice; mice with Retsat heterozygous gene knockout ( Retsat + / - They mate to produce Retsat - / - Homozygous knockout mice and wild-type mice. The Retsat-Q246R mutation was introduced into Rosa26-LSL (…). loxP -Stop- loxP The system (pmid: 26772810) was then compared with... Olig1 - Cre + / – mice (Xin, Mei et al. "Myelinogenesis and axonal recognition by oligodendrocytes in brain are uncoupled in Olig1-null mice.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 25,6 (2005): 1354-65. doi:10.1523 / JNEUROSCI.3034-04.2005) or Syn1 - Cre + / – mice (Zhu, Y) et al. "Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.” Genes&development vol. 15,7 (2001): 859-76. doi:10.1101 / gad.862101) Mating. All mice were bred on a mixed genetic background of C57BL / 6 and 129Sv.

[0059] Labeling and Detection of 5-ethynyl-2'-deoxyuridine (EdU) In the cumulative labeling assay, EdU (5-ethynyl-2'-deoxyuridine) was dissolved in drinking water at a concentration of 0.2 mg / mL. The solution was changed every 48 hours, and mice were continuously ingested with EdU for up to 30 days. EdU staining was performed using an EdU cell proliferation assay kit.

[0060] Cell culture and transfection The isolation of primary mouse oligodendrocyte precursor cells (OPCs) was performed according to previous literature. (Liu, Z. et al. Sec13) promotes oligodendrocyte differentiation and myelin repair through autocrine pleiotrophin signaling. J Clin Invest 132, doi:10.1172 / JCI155096 (2022).)The method is briefly described as follows: Cells are obtained from the cortex of mice aged 4-7 years (P4-P7) by sequentially screening culture plates coated with anti-GalC and anti-O4 antibodies using an immune disc. The method for isolating rat OPCs is similar: mixed cortical cells are obtained from the cortex of young mice aged 2-4 years (P2-P4) by sequentially screening culture plates coated with anti-GalC and anti-A2B5 antibodies using an immune disc. The isolated OPCs were cultured in OPC growth medium (OGM) based on DMEM / F-12 (Gibco, Thermo Fisher Scientific) supplemented with the following components: 2% B-27 (Gibco, Thermo Fisher Scientific); 1% N2 (Gibco, Thermo Fisher Scientific); 20 ng / mL PDGF-AA (Peprotech, 100-13A); 10 ng / mL CNTF (Peprotech, 450-13); 20 ng / mL bFGF (SinoBiological, 10014-HNAE); 5 μM forskolin (Sigma, F6886); 10 ng / mL biotin (Sigma, B4639); 5 μg / mL insulin (Sigma, I-6634); trace element B (Corning, 25-022-Cl) (diluted 1000 times); 1 mM sodium pyruvate (Thermo Fisher Scientific, ...). 11360-070); 100 U / ml penicillin-streptomycin (Thermo Fisher Scientific, 15140-122); and 1 ng / mL NT3 (Peprotech, 450-03). OPC differentiation culture was performed using OPC differentiation medium (ODM), which differs from OGM in that it contains 60 nM T3 (Sigma, T6397) and removes PDGF-AA, bFGF, and NT3.

[0061] The agreement One rat oligodendrocyte precursor cell (OPC) was resuspended in 100 mL of the mammalian glial cell transfection reagent from the Basic Nucleofectors kit (Lonza, O-017), with 5 μg of the corresponding plasmid or 5 μL of 20 mM siRNA (siRetsat sequence: GAAGAAAGTTCTCAAACAA). The mixture was transferred to an electroporation cuvette provided with the Nucleofectors kit and electroporated using a Lonza Nucleofector 2b device (LONZA) according to the manufacturer's recommended procedure O-017. The cells were then resuspended in culture medium and incubated at 37°C, 5% CO2 for 30 minutes, with the medium replaced to remove dead cells. Plasmid transfection of primary mouse OPCs was performed using the FuGENE® HD transfection reagent (Promega, E2311) under growth conditions according to the manufacturer's instructions. 293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1× penicillin-streptomycin, and transfection was performed using polyethyleneimine (Fushen Biotechnology, FSF0001).

[0062] Digital PCR Total RNA was extracted from the mouse cerebral cortex using the TRIzol method. 1000 ng of RNA was then reverse transcribed into cDNA using a 5× All-In-One RT Master Mix (Abm, G592). The obtained cDNA was diluted 5-fold as a template. A 25 μL reaction mixture was prepared as follows: 5 μL template, 2.5 μL 10 μM primer 1, 2.5 μL 10 μM primer 2, 1.25 μL 20 μM probe 1, 1.25 μL 20 μM probe 2, 5 μL 5× Perfecta Multiplex qScript ToughMix (Pexbio, 95147), and 2.5 μL 1 μM sodium fluorescein. Water was added to bring the total volume to 25 μL. The reaction mixture was added to the wells of a Sapphire microarray and amplified using the Naica Geode droplet generation and amplification system. Finally, the reaction was analyzed using the Naica™ Prism droplet reading system.

[0063] Primers: Mouse-F: 5'-GGCTACTGACTCGTTTCTCT-3' Mouse-R: 5'-TGGAGCTCACGGGAAG-3' Probe: Mouse-WT: 5'-FAM-CAAGTCCTGCAGCAGCTTGG-BHQ1-3' RNA extraction and real-time quantitative PCR Total RNA was extracted using the TRIzol method, and cDNA was synthesized using the All-In-One RT MasterMix reverse transcription reagent. Real-time quantitative PCR was performed using the Bio-Rad real-time quantitative PCR system. The primer sequences used for mouse gene amplification are as follows: Table 2. Primer sequences used for mouse gene amplification

[0064] The primer sequences used for rat gene amplification are as follows: Table 3. Primer sequences used for rat gene amplification

[0065] Tissue preparation and immunohistochemistry After anesthetizing the animals, the heart was perfused with ice-cold phosphate-buffered saline (PBS), followed by perfusion with ice-cold 2% paraformaldehyde (PFA). Brain tissue was fixed in the same fixative at 4°C for 6–8 hours, and spinal cord samples were fixed under the same conditions for 2 hours. After cryoprotection with 30% sucrose solution, the tissues were embedded in OCT embedding medium (SAKURA) and cut into 12 μm thick sections using a cryostat (Leica).

[0066] In immunohistochemical experiments, frozen sections were permeabilized and blocked for 30 minutes at room temperature in PBS containing 0.4% Triton X-100 and 3% bovine serum albumin (BSA). The sections were then incubated overnight at 4°C with an appropriately diluted primary antibody. After thorough washing with PBS, the sections were incubated for 1 hour at room temperature with Alexa Fluor-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:500) and DAPI (1:50). Before imaging, slides were mounted with anti-fluorescence quenching mounting medium.

[0067] In immunocytochemistry experiments, cultured cells were fixed with 4% PFA at room temperature for 10 minutes, permeabilized with 0.5% Triton X-100, and blocked with 3% BSA for 1 hour. The cells were incubated with primary antibody overnight at 4°C. After washing with PBS, the cells were co-incubated with fluorescent secondary antibody and DAPI at room temperature for 1 hour before mounting.

[0068] In situ hybridization was performed according to previous methods. A simplified procedure is as follows: frozen sections were hybridized with a digoxigenin-labeled RNA antisense probe targeting the mouse Plp1 gene. The hybridization probe was detected by alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche, 11093274910), and a colorimetric reaction was performed using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indole phosphate (BCIP) (Sangon, A600116).

[0069] plasmid preparation The mouse Retsat gene (gene ID: 67442) was cloned into the pcDNA3.3 vector. Retsat mutants were constructed using PrimeSTAR® HS DNA polymerase (Takara, R040A) via PCR-based site-directed mutagenesis.

[0070] Conditioned culture medium Primary neurons were isolated from the cerebral cortex of P1-P2 wild-type (WT) and gene knock-in (KI) mice and cultured in DMEM / F-12 medium supplemented with 2% B27, 1 mM L-alanyl-glutamine, 1 mM cytarabine, and 1× penicillin-streptomycin. To maintain the culture system, half of the medium was replaced with fresh medium every two days.

[0071] On day 6 of in vitro culture (DIV 6), the culture medium was replaced with ODM-T3 differentiation medium. After 72 hours of culture (DIV 9), the conditioned medium (CM) was collected. This conditioned medium was used in one of two ways: (1) directly on oligodendrocyte precursor cells (OPCs) for a 3-day differentiation experiment; (2) using a <3 kDa molecular cutoff filter membrane (Millipore, UFC5003) for component separation, replacing DMEM / F-12 with the filtrate to prepare OPC growth medium, and then using it to culture OPCs for 3 days. Cell differentiation was analyzed after treatment.

[0072] To collect secreted proteins, neurons were first washed three times with PBS, then replaced with DMEM / F-12 medium and subjected to 48 hours of hypoxia (10% oxygen concentration). The collected medium was centrifuged at 1200 rpm for 5 minutes and cell debris was removed by passing through a 0.45 μm filter membrane. Each 10 mL of DMEM / F-12 medium from neurons was mixed with 10 mL of fresh DMEM / F-12 and concentrated to 250 μL using Amicon Ultra-15 ultrafiltration centrifuge tubes (Merck).

[0073] Brain slice culture Cerebellar tissue sections were prepared from mice on day 3 (P3). The whole brain was dissected and placed in chilled artificial cerebrospinal fluid (ACSF). 300 μm thick cerebellar sections were obtained using a vibratory microtome (Leica) and transferred to Millipore (0.4 μm) organ culture inserts. The section culture system used MEM section medium supplemented with the following components: 20% heat-inactivated horse serum, 0.5% D-glucose, 500 μg / mL sodium bicarbonate, 2 mM calcium chloride, 2 mM magnesium sulfate, 25 mM HEPES (pH 7.4), 12 μg / mL ascorbic acid, 1 μg / mL insulin, and 1× penicillin-streptomycin.

[0074] All cultures were cultured at 37°C and 5% CO2. On day 2 of in vitro culture (DIV 2), sections were subjected to 24 hours of hypoxia (2% O2), followed by restoration to normoxic conditions. Cultures were then continued for 10 days in media with or without ATDR.

[0075] On day 14, demyelination was induced by adding 0.05% lysophosphatidylcholine (LPC; Sigma) to the culture medium for 16 hours. After demyelination, the sections were transferred to new culture plates containing fresh culture medium (with or without ATDR) and cultured for another 10 days to observe the remyelination process.

[0076] Chronic hypoxia model Newborn mice and their mothers were placed in a chronic sublethal hypoxic environment: a continuous supply of 10% inhaled oxygen was provided from day 3 to day 10 after birth (P3-P10). Oxygen concentration was continuously monitored and maintained using an oxygen regulator (AIPUINS, XBX-03S). Cellular hypoxia was treated by culturing oligodendrocytes or neurons at 1% oxygen concentration for 2-3 days using an oxygen regulator (ESCO, CCL-050T-8).

[0077] LPC-induced demyelinating injury model Refer to the methods in previous literature ( Sec13 promotes oligodendrocyte differentiation and myelin repair through autocrine pleiotrophin signaling. J Clin Invest 132, doi:10.1172 / JCI155096 (2022))LPC-induced demyelination was induced in the corpus callosum of 10-week-old mice. The injection cannula was fixed to the vertical axis of a stereotactic operating table, and the injection area was precisely located using a stereotactic coordinate system (RWD Life Science, China). The procedure was briefly as follows: 1.5 μL of 1% LPC was microinjected into the corpus callosum region to induce demyelinating lesions. The location coordinates were: 1.0 mm posterior to the anterior fontanelle, 1.0 mm lateral to the anterior fontanelle, and 1.5 mm below the skull surface. Mice were sacrificed at different time points after injection. The injection center point was marked with a gel pen for accurate localization. Tissue was cut into 4 mm thick sections along the injection mark, and 12 μm thick sections were collected for histological examination. Sections from the central region or from the same relative position as the control group were selected for analysis to obtain a focal demyelination model.

[0078] Copper-induced demyelination model In a copper-induced demyelination model, 8-week-old male C57BL / 6J mice were fed a standard rodent powder diet containing 0.2% (w / w) copper (bis(cyclohexanone)oxalyl dihydrazone; Sigma-Aldrich, C9012) for 5 consecutive weeks. The copper-containing diet was changed every 48 hours to ensure compound activity. Tissue samples were then collected one week after resuming a normal diet.

[0079] Autoimmune encephalomyelitis (EAE) model Experimental autoimmune encephalomyelitis (EAE) was induced in 8-10 week old female C57BL / 6 mice. Complete Freund's adjuvant (CFA) was prepared by adding 10 mg / ml of heat-inactivated Mycobacterium tuberculosis H37Ra (BD, #231141) to incomplete Freund's adjuvant (BD, #263910). CFA was then mixed with an equal volume of MOG. 35-55 The polypeptide (GL Biochem) solution (prepared with 2 mg / ml sterile distilled water) was mixed and emulsified at a 1:1 ratio to prepare an antigen emulsion. 200 μl of the antigen emulsion was subcutaneously injected into each mouse. Pertussis toxin (400 ng / dose; List Biological Laboratories, #181) in PBS solution was injected intraperitoneally immediately after immunization and again 48 hours later. Mice were observed daily and EAE clinical symptoms were scored according to the following criteria: 0 points, no abnormalities; 1 point, tail flaccidity; 2 points, hind limb weakness; 3 points, hind limb paralysis; 4 points, hind limb paralysis with forelimb weakness; 5 points, near-death state or death.

[0080] Metabolite extraction and LC-MS analysis Optimized procedures were employed to extract metabolites from biological samples. For adherent cells, a cell suspension was formed in the original culture medium by direct pipetting, followed immediately by the addition of methanol and acetonitrile to achieve a final extraction solvent ratio of 2:2:1 (v / v / v). After thorough vortexing, the mixture was incubated on ice for further processing. For tissue samples, approximately 60 mg of tissue was homogenized in ultrapure water (5 μL / mg tissue). 300 μL of the homogenate was mixed with 1.2 mL of extraction solvent (methanol:acetonitrile = 1:1, v / v) for subsequent processing. All samples were vortexed for 30 seconds, followed by sonication on ice for 10 minutes, and then subjected to three liquid nitrogen freeze-thaw cycles (sonication on ice for 10 minutes after each thaw). After incubation at -20°C for 1 hour, the samples were centrifuged at 13,000 rpm for 15 minutes at 4°C. The supernatant was collected, concentrated by vacuum centrifugation, and then analyzed.

[0081] Chromatographic separation was performed using a 5 μm C18 reversed-phase column at 40℃ and a flow rate of 1.0 mL / min. The mobile phase consisted of solvent A (ultrapure water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid), with the following gradient program: 0–20 min, phase A 20%–0%; 20–25 min, phase A 0%. Detection was performed using UV and mass spectrometry: UV detection wavelengths were set to retinol 325 nm, ATDR 290 nm, and RA / 9CRA 354 nm; mass spectrometry was performed in positive ion mode, with the following mass-to-charge ratios (m / z): retinol… 269.2260, RA 301.2162, 9CRA 301.2162, ATDR 289.2526, ATDRA 303.2324.

[0082] ADTRA Synthesis Synthesis Strategy The synthesis of ATDRA begins with compound 1 and proceeds through two steps: first, it is oxidized to aldehyde intermediate 2, and then silver-mediated oxidation is performed to obtain the final carboxylic acid product.

[0083] Step 1: Synthesis of Aldehyde Intermediate 2 Under nitrogen protection, Dess-Martin periodane (DMP, 30.8 mg, 72.8 μmol) was added to a 0.50 mL solution of anhydrous DCM (dichloromethane) containing compound 1 (7.00 mg, 24.2 μmol) and pyridine (3.84 mg, 48.5 μmol, 3.92 μL). The reaction mixture was stirred at 25°C for 1 hour. TLC analysis (hexane / ethyl acetate = 10:1 (volume ratio)) showed complete consumption of the starting material and the formation of a new active UV spot. The reaction solution was directly subjected to preparative TLC (…). Purification was performed using hexane / ethyl acetate (10:1) to give colorless oily aldehyde intermediate 2 (4.00 mg, yield 57.5%).

[0084]

[0085] Step 2: Synthesis of ATDRA A sodium hydroxide (2.79 mg, 69.8 μmol) aqueous solution (0.40 mL) was cooled to 0 °C, followed by the addition of a silver nitrate (5.93 mg, 34.9 μmol) aqueous solution (0.40 mL). The mixture was stirred at 0 °C in the dark for 30 minutes to form an active silver oxide (I) reagent. Then, a THF (tetrahydrofuran) solution (0.20 mL) of aldehyde intermediate 2 (4.00 mg, 13.9 μmol) was slowly added dropwise at 0 °C. The reaction system was allowed to warm naturally to 25 °C and then stirred in the dark for 2 hours. The main peak at the target mass number was detected by LC-MS, and the formation of new spots was confirmed by TLC (dichloromethane / methanol = 20:1), indicating complete reaction. The reaction solution was quenched in 0.5 M hydrochloric acid (1.00 mL) and extracted with ethyl acetate (2 × 1.00 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was analyzed by preparative TLC (…). The mixture was purified with dichloromethane / methanol (20:1) to obtain a white solid ATDRA (1.60 mg, yield 37.8%).

[0086]

[0087] Features of ADTRA LCMS (EB18998-4-P1A1): m / z = 303.5 , R t = 2.521 min. LCMS (EB18998-4-P1M1): m / z = 303.3 Rt = 3.283 min. H NMR (400 MHz, CDCl3) δ 6.46 (dd, J = 15.2, 11.2 Hz, 1H), 6.06-6.12(m, 1H), 5.96-6.04 (m, 1H), 5.66 (dd, J = 15.2, 8.0 Hz, 1H), 5.35-5.37 (m,1H), 2.79 (br d, J = 7.2 Hz, 1H), 2.35-2.43 (m, 2H), 2.23 (t, J = 7.6 Hz,1H), 2.02 (br d, J = 6.0 Hz, 3H), 1.87-1.94 (m, 3H), 1.70 (s, 3H), 1.42-1.44(m, 2H), 1.13 (d, J = 6.8 Hz, 3H), 1.01 (s, 6H). electron microscope The simplified steps are as follows: Mice were deeply anesthetized and perfused with pre-cooled sodium dimethylarsenate buffer. The corpus callosum or spinal cord tissue was immediately separated, fixed overnight in 2.5% glutaraldehyde at 4°C, then treated with 1% osmium tetroxide, dehydrated, and embedded in PolyBed resin. Subsequently, 70 nm ultrathin sections were prepared, stained with lead citrate, and images were acquired using a JEM-2100HC electron microscope with a Hitachi HT-7800 camera. The g-ratio of myelinated fibers, i.e., the ratio of axonal diameter to the total diameter (axon + myelin sheath), was calculated using ImageJ software.

[0088] Behavioral testing Morris Water Maze Experiment A circular plastic pool with a diameter of 1.2 meters was used, and the water temperature was maintained at 22±2℃. Mice were placed into the maze from one of four random starting points, and their movement was recorded by a top camera. The mice were given 60 seconds to freely search for the hidden platforms. If they could not locate a platform, they were gently guided to it and stayed there for 10 seconds. After 4 days of training, the platforms were removed, and a detection test was conducted. The following parameters were recorded: percentage of time spent in each quadrant, number of times the target (platform) area was traversed, average movement speed, and total movement distance.

[0089] Three-room social behavior test First, the mice were allowed to acclimatize in the empty apparatus for 5 minutes. Then, a social skills test was conducted: the mice were allowed 10 minutes to freely explore the chamber containing the unfamiliar mouse (unfamiliar mouse 1) and the empty chamber. Next, a social novelty preference test was performed: a second unfamiliar mouse (unfamiliar mouse 2) was introduced and observed for 10 minutes. Interaction time and chamber dwell time were quantitatively analyzed using a Noldus EthoVision XT video tracking system. The apparatus was cleaned with 70% ethanol between each test round, and all experimental procedures were approved by the institution's Animal Care and Use Committee.

[0090] Quantitative and Data Analysis All data in this paper are expressed as arithmetic mean ± standard deviation. Specific sample sizes (n-values) are detailed in the Results section and figure captions. All statistical analyses were performed using GraphPad Prism 8.0 software. The criterion for statistical significance between two groups was a two-tailed unpaired Student's t-test with a p-value less than 0.05. All quantitative analyses were based on at least three independent experiments and were evaluated using a blinded approach. Sample size was determined without statistical methods but was consistent with the standard size for this field. Data collection was not randomized, but all data were quantitatively analyzed using a blinded approach.

[0091] Example To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0092] Example 1: The Retsat Q246R mutation confers resistance to hypoxic-ischemic white matter damage in newborns. Chronic hypoxia is widely considered a major factor leading to neurodevelopmental disorders (especially white matter damage) and causing long-term neurological deficits. Wild-type (WT) and heterozygous (carrying a heterozygous mutation in retinol saturase Q246R) mice were compared. Retsat Q / R ) and homozygous (mice carrying the Q246R homozygous mutation, Retsat R / R Gene knock-in mice were placed in a hypoxic environment (10% oxygen) from day 3 (P3) to P10 after birth, and then returned to a normal oxygen concentration environment to obtain a neonatal hypoxic-ischemic encephalopathy model. Analysis was performed at different time points. Figure 1 A). In the Morris water maze test, hypoxic WT mice showed impaired escape latency learning; in the probe test, they spent significantly less time in the target quadrant, indicating spatial memory deficits. Conversely, mice carrying the Q246R point mutation showed significantly improved function in all test phases. Figure 1 B-1D). Furthermore, in the swivel bar test, Q246R point mutant mice were less likely to fall off the swivel bar, indicating improved motor coordination. Figure 2 A). In social interaction tests, although chronic hypoxia impaired the social abilities of WT mice, this deficiency was largely absent in Q246R point mutant mice. Figure 1 E). Given the intermediate level of phenotypic improvement observed in heterozygous (KI / +) mice, we used digital PCR to quantify allele-specific expression. The results showed that the levels of wild-type and Q246R mutant mRNA expressed in heterozygous mice were approximately equal, each about half the total Retsat mRNA level in WT mice; while the level of Q246R mutant mRNA expressed in homozygous (KI / KI) mice was comparable to the total mRNA level in the WT control group. Figure 2 B). This dose-dependent gene expression pattern is consistent with the observed phenotypic severity grading.

[0093] To systematically assess the impact of chronic hypoxia on brain tissue integrity, we first performed histological analysis. NeuN staining, a neuronal marker, showed that the number of neurons was comparable between wild-type and Q246R point mutant mice, indicating that neuronal survival was not significantly affected by gene mutation or hypoxic stress. Figure 1 F, 1G). Neurofilament 200 (NF200) immunostaining also showed no obvious abnormalities in the axonal structure of each group. Figure 1 H, 1I). Given the known susceptibility of myelin formation to hypoxia-induced injury, we then focused on white matter integrity studies. Analysis of key myelin components showed that chronic hypoxia significantly inhibited myelin formation in wild-type mice, but this inhibition was significantly alleviated in Q246R mutant mice. Specifically, immunohistochemistry, in situ hybridization, and quantitative PCR revealed that under hypoxic conditions, mutant mice exhibited higher expression levels of myelin basic protein (MBP), myelin lipoprotein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) compared to hypoxic wild-type mice. Figure 1 J-1N). We further verified the above findings at the ultrastructural level using electron microscopy: In the brains of P14 mice, chronic hypoxia led to severe myelination defects in wild-type mice, manifested as significant loss of myelinated axons; in contrast, Q246R point mutant mice were able to significantly maintain myelin structure after hypoxic stress, with significantly better myelinated axon proportion and myelin thickness than wild-type mice. Figure 1 O-1Q).

[0094] In summary, these results indicate that the Retsat Q246R mutation can specifically and potently protect developing white matter from hypoxic damage, thereby improving myelin formation.

[0095] Example 2: Retsat can promote oligodendrocyte differentiation and myelination, while the Q246R mutation can enhance this function.

[0096] In the central nervous system, myelin is generated by mature oligodendrocytes. The improved myelin formation observed in Q246R point mutant mice under chronic hypoxia suggests that the Retsat mutant protein may regulate oligodendrocyte differentiation and maturation. To test this hypothesis, we quantitatively analyzed mature oligodendrocytes and oligodendrocyte precursor cell (OPC) populations in wild-type and Q246R mutant mice. Chronic hypoxia significantly reduced the number of CC1 (a monoclonal antibody targeting APCs)-positive mature oligodendrocytes in wild-type mice; in contrast, Q246R mutant mice showed more mature oligodendrocytes under both normoxic and hypoxic conditions than wild-type mice. Figure 3 A, 3B). Conversely, the density of platelet-derived growth factor receptor α (PDGFRα) positive OPCs did not change due to hypoxic conditions or Retsat genotype. Figure 3 C, 3D). Furthermore, OPC proliferation was assessed by Ki67 staining, and cell viability was assessed by TUNEL assay. Results showed no significant differences between wild-type and mutant mice. Figure 3 (E-3H) indicates that this mutation does not affect the proliferation or survival of OPCs.

[0097] We then constructed a Retsat systemic knockout (KO) mouse model ( Figure 4 (A, 4B) This study aimed to investigate the necessity of Retsat protein for myelination and oligodendrocyte differentiation. On day 14 after birth (P14), compared with the wild-type control group, Retsat knockout mice exhibited moderate but significant impaired myelination and oligodendrocyte maturation. Figure 3 I-3L), while the density of oligodendrocyte precursor cells (OPCs) remained unchanged. Figure 3 M, 3N). Retsat deficiency significantly exacerbates damage during chronic hypoxic stress, leading to a greater reduction in myelin protein MBP expression and the number of CC1-positive mature oligodendrocytes. Figure 3 O-3R). However, the number of PDGFRα-positive OPCs was not affected by Retsat deletion (O-3R). Figure 3 Q, 3S). Ki67 staining confirmed that the proliferation capacity of OPCs was not affected by either hypoxia or Retsat deficiency. Figure 4C, 4D). We also examined other major neuronal cell types (including NeuN-positive neurons, GFAP-positive astrocytes, IBA1-positive microglia, and NF200-positive axons) and found that their number or structural integrity was not impaired by Retsat loss. Figure 4 E-4J).

[0098] In summary, these findings confirm that Retsat proteins are crucial for myelination and oligodendrocyte differentiation during development, especially under hypoxic stress, and that the Q246R point mutation can enhance this function.

[0099] Example 3: The Retsat Q246R mutation promotes more effective myelin repair in the adult central nervous system.

[0100] Oligodendrocyte precursor cells (OPCs) persist in the adult brain, continuously proliferating and differentiating—a process crucial for remyelination following white matter injury. Given the promoting effect of the Retsat Q246R mutation on OPC differentiation during development, we further investigated its role in adult oligodendrocyte formation and remyelination. We first assessed adult oligodendrocyte formation by adding EdU to the drinking water of P30 mice to label newly generated cells. Analysis at P60 showed that, compared to the wild-type control group, Q246R mutant mice had significantly more EdU and CC1 double-positive cells (representing newly generated mature oligodendrocytes). Figure 5 (A, 5B) indicates that this mutation enhances persistent oligodendrocyte production in adulthood. This finding prompted us to further evaluate the functional remyelination capacity of Q246R mutant mice using a lysophosphatidylcholine (LPC)-induced focal demyelination model. This model rapidly induces focal myelin disintegration, subsequently initiating the remyelination process. On day 14 post-injury (14dpl), FluoroMyelin staining and immunostaining analysis of myelin proteins (MBP, PLP) revealed that the remyelination extent in the corpus callosum region of Q246R mutant mice was more extensive than that in wild-type mice. Figure 5 C-5H). Similarly, the lesion areas of mutant mice contained more CC1-positive mature oligodendrocytes, while the density of PDGFRα-positive OPCs did not differ significantly between the two groups, indicating that the Q246R mutation mainly enhances repair capacity by promoting OPC differentiation rather than recruitment. Figure 5 I-5L). Electron microscopy ultrastructural analysis further confirmed that the mutant mice had higher remyelination efficiency, specifically manifested in a higher proportion of myelinated axons and thicker myelin sheaths (reflected in a lower g-ratio). Figure 5 M-5O).

[0101] We further investigated whether the enhanced remyelination effect of the Q246R mutation was applicable to a copper phosphate model simulating the complex demyelination / remyelination dynamics of multiple sclerosis. After feeding mice a 0.2% copper phosphate diet for five weeks, followed by a one-week recovery period with a normal diet, significantly enhanced remyelination was observed in the Q246R mutant mice using TrueGold and FluoroMyelin staining. Figure 5 P-5Q and 6A, 6B). Consistent with this, co-staining of the mature oligodendrocyte marker aspartate acyltransferase (ASPA) and the oligodendrocyte lineage pan-marker Olig2 showed that, after copper-induced demyelination and the recovery period, the corpus callosum of Q246R mutant mice had a greater number of mature oligodendrocytes that were double-positive for both ASPA and Olig2. Figure 5 These data collectively suggest that the Retsat Q246R mutation promotes oligodendrocyte differentiation, thereby improving developmental myelination under hypoxic stress and conferring stronger remyelination capacity in the adult brain after experiencing two distinct types of demyelinating injury.

[0102] We further investigated the necessity of this gene in adult remyelination using Retsat knockout (KO) mice. Although Retsat KO mice exhibited insufficient myelination in early development, by adulthood, assessments of myelin protein expression levels and the number of mature oligodendrocytes revealed that their myelination level had recovered to wild-type levels. Figure 6 C-6F). However, after LPC-induced demyelination (in a focal demyelination model), FluoroMyelin and MBP staining showed that Retsat KO mice exhibited impaired remyelination on day 14 post-injection. Figure 6 G-6J). In KO mice, the number of CC1-positive mature oligodendrocytes was reduced in the lesion area, while the recruitment of IBA1-positive microglia was unaffected. Figure 6 I, 6K, 6L).

[0103] In summary, these results indicate that Retsat is a necessary condition for effective remyelination in the adult brain, and that gain-of-function mutations in Q246R can significantly enhance this repair process.

[0104] Example 4: The Retsat Q246R mutation in neurons (non-oligodendrocytes) mediates the promyelination effect.

[0105] To elucidate the cellular mechanism by which the Retsat Q246R point mutation, a product of evolutionary selection, promotes oligodendrocyte differentiation, we first characterized its spatiotemporal expression pattern. Immunofluorescence analysis of brain slices from P4, P14, and P65 mice showed that Retsat was significantly expressed in the cortex, hippocampus, thalamus, and hypothalamus. Figure 7 A). Co-staining with multiple cell markers indicated that this protein is mainly localized in neurons, moderately expressed in oligodendrocytes, and detected at extremely low levels in astrocytes, microglia, or vascular endothelial cells. Figure 7 B, 7C). Furthermore, hypoxic exposure did not alter its expression levels, cell distribution, or distribution pattern across different cell types (B, 7C). Figure 8 A-8C). To verify whether the enhanced oligodendrocyte differentiation in Q246R mice stemmed from cell autonomy within the oligodendrocyte lineage, we isolated oligodendrocyte precursor cells (OPCs) from wild-type and Q246R mutant mice. In vitro differentiation experiments showed that, under normoxic or hypoxic conditions, the differentiation capacity of mutant OPCs did not exhibit intrinsic differences, as assessed by the percentage of MBP and Olig2 double-positive cells or myelin gene expression. Figure 7 D-7F). Even after adding retinol, the substrate of Retsat, to the culture medium, no differences were observed between groups. Figure 8 D). Furthermore, the proliferation (Ki67 marker) and survival (TUNEL assay) of OPCs remained unchanged. Figure 8 E-8H). Overexpression of wild-type or Q246R-type Retsat in mouse or rat OPCs did not affect their differentiation ability. Figure 8 I, 8J).

[0106] To definitively verify whether the Q246R mutation plays a role in cell autonomy within oligodendrocyte lineages, we constructed the Rosa26-LSL ( loxP -Stop- loxP )-Retsat(Q246R)-HA-IRES-GFP knock-in mice (abbreviated as) Retsat iR Where "i" represents inducible and "R" represents the Retsat-Q246R mutant), and compare it with... Olig1 -Cre mice were mated to specifically express the mutant protein (O-) in oligodendrocyte lineages. Retsat iR (where "O" represents oligodendrocytes) Figure 7 G). Although GFP and HA tags confirmed the specific expression of this protein in Olig2-positive cells ( Figure 8 K, 8L), but compared with the control group from the same litter, this oligodendrocyte-specific Q246R mutant did not increase the expression level of MBP or the number of CC1-positive mature oligodendrocytes in the corpus callosum or cortex at the P9 or P14 stage ( Figure 7 H-7J, 8M). The density of PDGFRα-positive OPCs also remained unchanged. Figure 7Consistent with these findings, from K, 7L, 8N). Retsat OPCs isolated from KO mice, or wild-type OPCs transfected with Retsat-targeting siRNA, all exhibited normal differentiation capacity in vitro. Figure 7 (M, 8O, 8P). These data collectively indicate that Retsat and its Q246R mutation do not promote myelination through oligodendrocyte autonomy mechanisms.

[0107] Therefore, we switched to an in vitro brain slice culture system for our study. Compared to wild-type mice, organoid cortical sections from Q246R mutant mice showed enhanced myelination under both normal oxygen concentration and hypoxic conditions. Figure 7 This result contrasts sharply with the negative findings in the purified OPC culture system, strongly suggesting that Retsat must function within the complex cellular environment of the brain. Given that Retsat is primarily expressed in neurons, we then investigated whether this mutation specifically acts on neurons. By comparing Rosa26-LSL-Retsat(Q246R) mice with... Syn1 By mating Cre mice, we achieved neuron-specific expression (N-) Retsat iR (where "N" represents a neuron). Immunostaining with GFP and HA confirmed the specific expression of this protein in neurons. Figure 9 A-9C). The neuron-specific Q246R mutant increased MBP expression in P14 mice under normoxic conditions and significantly alleviated myelin insufficiency after chronic hypoxia, accompanied by an increase in the number of CC1-positive mature oligodendrocytes. Figure 7 P-7S), while the density of OPCs was unaffected (P-7S). Figure 9 D, 9E).

[0108] To directly verify whether neurons carrying the Q246R mutation promote OPC differentiation, we collected conditioned medium from primary cortical neurons isolated from wild-type and Q246R mutant mice and applied it to a wild-type OPC culture system. The conditioned medium from Q246R mutant neurons significantly enhanced the differentiation of wild-type OPCs, specifically manifested in an increased proportion of MBP and CNP-positive cells and upregulated expression of myelin-related genes (…). Figure 7 T-7V).

[0109] In summary, these data demonstrate that the promyelination effect of the Retsat Q246R mutation is achieved through a non-cellular autonomous mechanism—the mutant protein functions in neurons, thereby enhancing oligodendrocyte differentiation and myelin formation.

[0110] Example 5: The Q246R mutation enhances the enzymatic activity of Retsat, prompting neurons to produce all-trans-13,14-dihydroretinoic acid (ATDRA). After confirming that the Retsat Q246R mutation acts on neurons, we further investigated its molecular mechanism. First, we found that Q246R substitution did not affect Retsat protein expression or its subcellular localization: Western blotting of HA-tagged Retsat protein showed comparable protein expression levels. Figure 10 A); Immunofluorescence staining showed that HA signals in both wild-type and Q246R variants co-localized with the endoplasmic reticulum marker PDI (A). Figure 11 A, 5B). Retsat is known to act as a retinol saturase, catalyzing the conversion of all-trans-retinol to all-trans-13,14-dihydroretinol (ATDR). Through HPLC analysis of cell lysates after transfecting 293T cells with retinol, we found that the Q246R mutation significantly enhanced Retsat enzyme activity. Figure 10 C). The E96A mutation located in the cofactor-binding domain completely eliminates enzyme activity. Figure 10 C), the double mutant Q246R / E96A also lost activity, indicating that the enhanced enzyme activity of the Q246R mutation still depends on cofactor binding ( Figure 10 C). Consistent with this, primary neurons isolated from Q246R mutant mice produced more ATDR from retinol substrates than wild-type neurons. Figure 10 D, 11B). Furthermore, ATDR levels are elevated in the brain tissue of Q246R mutant mice (D, 11B). Figure 10 E, 11C), while retinol levels remained unchanged (E, 11C). Figure 10 E, 11D). These results suggest that the Q246R mutant enhances myelin formation through its catalytic products.

[0111] We then collected conditioned medium from wild-type and mutant neurons, and removed molecules larger than 3 kDa by centrifugation and filtration to eliminate interference from secreted proteins. Figure 10 F). Culture medium filtered from Q246R neurons significantly enhanced OPC differentiation, while culture medium filtered from wild-type neurons did not have this effect. Figure 10 G). This result led us to hypothesize that the catalytic product ATDR mediates this effect. However, unexpectedly, direct addition of ATDR to OPCs—whether under normal oxygen concentrations or hypoxic conditions, or in mouse or rat culture systems—failed to promote their differentiation. Figure 10 H, 11E). Interestingly, myelination under hypoxic conditions was enhanced after wild-type brain slices were treated with ATDR (H, 11E). Figure 10I, 10J). More importantly, after co-culturing ATDR with wild-type neurons, applying the conditioned medium containing molecules larger than 3 kDa filtered out to OPCs can effectively promote OPC differentiation. Figure 10 These findings suggest that ATDR must be metabolized into a certain derivative in neurons in order to activate the differentiation of oligodendrocyte precursor cells.

[0112] We hypothesize that ATDR, as an alcohol compound, needs to be successively oxidized to aldehydes and then converted to carboxylic acids to acquire biological activity. Expression profiling analysis of related metabolic enzymes showed that neurons highly express the enzyme responsible for oxidizing alcohols to acids (…). Figure 10 M, 10N), while oligodendrocytes highly express enzymes involved in the degradation of acidic compounds (M, 10N), Figure 10 M). Therefore, we examined the oxidized form of ATDR—all-trans-13,14-dihydroretinoic acid (ATDRA). Assessment of myelin gene expression revealed that direct addition of ATDRA significantly promoted OPC differentiation, while ATDR had no such effect. Figure 10 O-10Q). Importantly, the primary neurons of Q246R mutant mice have a stronger ability to generate ATDRA from retinol than those of wild-type mice (O-10Q). Figure 10 R, 11F). Consistent with this, higher levels of ATDRA were detected in the brain tissue of Q246R mutant mice. Figure 10 S).

[0113] Example 6: The therapeutic potential of ATDR and ATDRA in myelination disorders Given that ATDR and ATDRA significantly promote oligodendrocyte (OPC) differentiation, we further investigated their physiological functions in myelin repair. In an ex vivo brain slice culture model of LPC-induced demyelination, ATDR treatment enhanced remyelination (…). Figure 12 A, 12B). We then administered retinol, ATDR, or ATDRA to mice via intraperitoneal injection and found that both ATDR and ATDRA significantly increased their respective levels in the cerebellum (A, 12B). Figure 12 C). Surprisingly, administration of retinol did not significantly increase the levels of retinol itself and its derivatives (retinoic acid and 9-cis-retinoic acid) in the mouse brain. Figure 12 D). 9-cis-retinoic acid was almost undetectable in the mouse brain ( Figure 13 A). Furthermore, no adverse effects on body weight or cell death (TUNEL staining) were observed after administration of ATDR or ATDRA. Figure 13 B, 13C), indicating good safety. Therefore, we tested their efficacy in a neonatal chronic hypoxia model (B, 13C). Figure 12E). Both ATDR and ATDRA significantly improved myelin deficiency and increased the number of CC1-positive mature oligodendrocytes (E). Figure 12 F-6I). Due to the limited availability of synthetic ATDRA, we only tested ATDR in an adult demyelination model. In an LPC-induced focal demyelination model ( Figure 12 J), TrueGold and MBP staining showed that ATDR treatment significantly enhanced remyelination and the generation of new oligodendrocytes, manifested as an increase in CC1-positive cells in the lesion area (J). Figure 12 K-12P). Finally, we evaluated the therapeutic effect of ATDR in an experimental autoimmune encephalomyelitis (EAE) model, which reproduces key features of multiple sclerosis. ATDR treatment significantly reduced clinical symptom scores in EAE and improved motor function (K-12P). Figure 12 Q). This was accompanied by significant preservation of MBP expression and marked improvement in axonal myelination as observed by electron microscopy. Figure 12 R-12V).

[0114] By incorporating via reference The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0115] Equivalence This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. Use of all-trans-13,14-dihydroretinol (ATDR) or a salt thereof, all-trans-13,14-dihydroretinoic acid (ATDRA) or a salt thereof, or a composition comprising all-trans-13,14-dihydroretinol (ATDR) or a salt thereof and / or all-trans-13,14-dihydroretinoic acid (ATDRA) or a salt thereof in the preparation of a medicament for the prevention and / or treatment of multiple sclerosis and / or autoimmune encephalomyelitis in subjects of need.

2. The use according to claim 1, wherein the composition comprises a pharmaceutically acceptable excipient.

3. The use according to claim 1 or 2, further comprising a second drug for treating multiple sclerosis and / or autoimmune encephalomyelitis.

4. The use according to claim 1, wherein the subject is a mammal.

5. The use according to claim 1, wherein the subject is a human.

6. The use according to any one of claims 1 to 5, wherein the drug is administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, intrathecal injection or intraperitoneal injection.

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