Methods and molecules for reducing axonal tau protein accumulation via blockade of HNRNP R-mediated Mapt mRNA transport for the treatment of Alzheimer's disease

Abstract

The present invention includes methods for reducing axonal tau protein. These methods include inhibiting the binding between MAPT mRNA and hnRNP R.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to (i) U.S. Provisional Patent Application No. 63 / 364,629, filed May 13, 2022, and (ii) U.S. Provisional Patent Application No. 63 / 382,536, filed Nov. 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Field of the Invention The present invention generally relates to a method for selective reduction of tau in axons by preventing the transport of microtubule - associated protein tau (hereinafter, MAPT) mRNA (Mapt mRNA), which encodes tau protein, from the cell body to the axon by blocking the interaction between Mapt mRNA and hnRNP R or by reducing the hnRNP R level. hnRNP R is an RNA - binding protein that interacts with the 3’UTR of Mapt mRNA. Molecules that inhibit such interaction between Mapt mRNA and hnRNP R or reduce the hnRNP R level reduce axonal tau protein.

Background Art

[0003] Alzheimer's disease (hereinafter, AD) is a neurodegenerative disorder and the most common form of late - onset dementia, affecting a significant proportion of individuals over 65 years of age. It is characterized by progressive memory loss and is expected to increase dramatically over the next few decades as aging is the major risk factor. AD is caused by the accumulation of insoluble protein aggregates in the brain, including the formation of tau fibrils in neuronal axons, leading to neuronal dysfunction and loss, which in turn results in progressive memory loss with a decline in the ability to perform daily functions.

[0004] The treatment of AD is difficult due to the complex etiology of the disease. In pre-symptomatic AD patients, the following two types of protein aggregates are present in the brain: extracellular accumulation of amyloid-β (hereinafter referred to as Aβ) protein (senile plaques: hereinafter referred to as SP) and intracellular protofibrils of hyperphosphorylated tau protein (neurofibrillary tangles: hereinafter referred to as NFT). SPs are widely present in the brains of AD patients, but the temporal and spatial formation of NFTs is more closely correlated with cognitive impairment and disease progression. Most treatment approaches have focused on removing or delaying the formation of SPs, but it has become clear today that preventing the formation of NFTs or halting their spread are promising treatment options. Furthermore, current treatment strategies targeting the prevention or deceleration of plaque and tangle formation through antibody-based targeting of Aβ or tau can induce unwanted side effects.

[0005] NFTs are first formed in the entorhinal cortex and hippocampus, and in AD, the memory formation sites are first affected. On the other hand, SPs occur more diffusely throughout the brain. Furthermore, axonal dysfunction is an early event in AD that induces neuronal degeneration via a "dying back" mechanism that spreads from damaged axons to neuronal cell bodies (Salvadores, N., et al., Axonal Degeneration in AD: The Contribution of A beta and Tau. Front Aging Neurosci, 2020.12: p.581767).

[0006] Tau is required in the brain for axon maintenance by stabilizing the cytoskeleton through microtubule assembly. This function is impaired by hyperphosphorylation of tau, leading to its fibrillation and toxic accumulation as NFTs in axons. As a result, axonal tau aggregates occur, which develop early in AD and disrupt the transport of RNA and proteins necessary for axon and synapse maintenance (Robbins, M., et al., Synaptic tau: A pathological or physiological phenomenon Acta Neuropathol Commun, 2021.9(1):p.149). This suggests that NFT formation is an important step in the etiology underlying AD. Therefore, in a mouse model of AD, genetic deletion of tau improved cognitive ability without affecting the deposition of SP, so reducing tau and consequently reducing tau aggregates seems to be a promising therapeutic approach (Roberson, E.D., et al., Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science, 2007.316(5825):p.750-4).

[0007] In the hippocampus of AD patients, axonal tau pathology precedes tau deposition in the somatodendritic compartment, suggesting that dysregulation of axonal tau alteration is an early event in the abnormal AD cascade (Christensen, K.R., et al., Pathogenic tau modifications occur in axons before the somatodendritic compartment in mossy fiber and Schaffer collateral pathways. Acta Neuropathol Commun, 2019.7(1):p.29). Beyond this early event, tau is secreted upon neuronal activation via its interaction with synaptic vesicle proteins and promotes the spread of abnormal tau pathology between brain regions in AD (de Calignon, A., et al., Propagation of tau pathology in a model of early Alzheimer's disease. Neuron, 2012.73(4):p.685-97). Decrease of tau shows a causal role of tau as an AD etiology since it improves the phenotypic deficits of AD mouse models and restores axonal transport deficits induced by Aβ (Leroy, K., et al., Lack of tau proteins rescues neuronal cell death and decreases amyloidogenic processing of APP in APP / PS1 mice. Am J Pathol, 2012.181(6):p.1928-40).

[0008] Strategies for tau removal can be broadly classified into two approaches. In the first approach, tau is globally reduced: the Mapt mRNA level is decreased in a targeted manner through the delivery of short double-stranded RNAs in the form of short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), or through the delivery of antisense oligonucleotides (ASOs) that induce RNase H-mediated mRNA degradation. However, since tau is involved in the regulation of gene expression, its non-specific loss affects many other pathways (Montalbano, M., et al., Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes. Front Mol Neurosci, 2021.14:p.742790). Furthermore, depletion of tau throughout neurons is detrimental to its function as it is involved in several processes related to synaptic plasticity. Thus, tau knockout mice exhibit cognitive and motor deficits (Lei, P., et al., Motor and cognitive deficits in aged tau knockout mice in two background trains. Mol Neurodegener, 2014.9:p.29). Additionally, bilateral acute knockdown of tau in the mouse hippocampus caused deficits in motor coordination and spatial memory (Velazquez, R., et al., Acute tau knockdown in the hippocampus of adult mice causes learning and memory deficits. Aging Cell, 2018.17(4):p.e12775).Therefore, the overall reduction of tau protein is not an effective therapeutic strategy because the removal of NFTs that can be achieved by this approach is accompanied by undesirable changes in these cellular functions normally performed by tau in the cell body of neurons.

[0009] In a second approach, tau-specific antibodies that neutralize and / or remove abnormal tau are used (Jadhav, S., et al., A walk through tau therapeutic strategies. Acta Neuropathol Commun, 2019.7(1):p.22). To that end, various antibodies have been developed that are specific for the hyperphosphorylated form of tau and target various regions of tau. However, antibody-based strategies have shown some success by preventing tau seeding and NFT formation, but this strategy is limited by the occurrence of multiple tau isoforms and abnormal fragments that may not be targeted simultaneously by individual antibodies. Furthermore, antibody delivery to the brain is inefficient and may require repeated dosing. Additionally, immunization against targets in the brain, whether active or passive, can induce an inflammatory cascade, cause further complications, and lead to an acute disease state.

[0010] Furthermore, adverse effects often occur in connection with currently available treatment responses. For example, immunotherapies that utilize antibodies targeting Aβ or tau to induce clearance of SPs and NFTs by the immune system can induce inflammatory responses, brain edema, or hemorrhage as side effects. Other approaches target reducing the level of tau protein via antisense oligonucleotide (ASO)-mediated degradation of the MAPT transcript encoding tau (De Vos, S.L., et al., Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy. Sci Transl Med, 2017.9(374):p.eaag0481; Easton, A., et al., Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies. Mol Ther Nucleic Acids, 2022.29:p.625-42).

[0011] Additional challenges for Aβ and tau immunotherapies are to identify the isoforms and aggregated species that need to be targeted to achieve treatment outcomes (Song, C., et al., Immuno therapy for Alzheimer's disease:targeting beta-amyloid and beyond. Transl Neurodegener, 2022.11(1):p.18). Both Aβ and tau exist as fragments of different lengths or splice isoforms, and their aggregation progresses from the oligomeric state to fibrillary deposits. Therefore, in addition to currently available therapeutic strategies that target selected Aβ and tau deposits, it is necessary and desirable to identify additional factors that can modulate the abundance of SP and NFT and for which such manipulation may be tolerated.

[0012] Therefore, there is a need for new therapeutic methods that selectively reduce tau concentration from neuronal axons. One such method can involve inhibition, reduction and / or depletion of the RNA-binding protein hnRNP R, which can lead to a reduction in plaques and tangles. SUMMARY OF THE INVENTION

[0013] The present invention discloses a method for preventing the transport of microtubule-associated protein tau (MAPT) mRNA (Mapt mRNA), which encodes tau protein, from the cell body to the axon. By blocking the interaction between the RNA-binding protein "hnRNP R", which interacts with the 3’UTR of Mapt mRNA and promotes its axonal localization, or by reducing the level of "hnRNP R", decreased Mapt mRNA transport occurs. As a result of the decrease in mRNA transport, local tau protein synthesis in the axon is reduced, and selectively lower tau protein levels are achieved in the axon while maintaining tau levels in the somato-dendritic compartment. The consequence (possibility) of the decrease in newly synthesized axonal tau protein is to limit the level of tau protein, which can be hyperphosphorylated and transmitted synaptically, thereby reducing the formation of NFTs and the spread of abnormal tau. This should prevent the progression of AD. This method allows tau to continue to function in the neuronal cell body while blocking axonal NFT formation. Preventing local accumulation of tau in the axon achieves more specific removal of tau aggregates with fewer side effects than prior art methods.

Brief Description of the Drawings

[0014] This patent application or application documents include at least one drawing created in color. Copies of this patent application or patent application publication with color drawings are provided by the Patent and Trademark Office upon payment of the claims and the necessary fees.

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[0017] Figure 1C shows compartmentalized cultures of mouse motor neurons.

[0018] Figure 1D shows the results of quantitative PCR (qPCR).

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DETAILED DESCRIPTION OF THE INVENTION

[0041] In the present invention, the method characterized, in part, is based on the confirmation that the RNA-binding protein hnRNP R interacts with the 3’UTR of MAPT mRNA in motor neurons and regulates its axonal localization (see FIGS. 1A and 1B).

[0042] To facilitate understanding of the present invention, a number of terms enclosed in quotation marks are defined below. Note that the drawings of the present application are provided for illustrative purposes only, and thus, it should be noted that the drawings are not drawn to scale. It should also be noted that similar and corresponding elements are referred to by similar reference numerals.

[0043] In the following description, numerous specific details such as specific structures, components, materials, dimensions, processing steps, and techniques are described in order to provide an understanding of various embodiments of the present application. However, it will be understood by those skilled in the art that various embodiments of the present application may be practiced without these specific details. In other instances, well-known structures or processing steps are not described in detail to avoid obscuring the present application.

[0044] As used herein, the terms "substantially" or "substantial" are equally applicable when used in a negative sense to denote the lack of being complete or nearly complete of an action, characteristic, property, state, structure, item, or result. For example, a "surface that is substantially flat" is either completely flat or nearly flat, and as a result, the effect is the same as when it is completely flat.

[0045] As used herein, terms defined in the singular are intended to include terms defined in the plural and vice versa.

[0046] As used in this specification and the appended claims, terms such as "a", "an", and "the" are not intended to refer to only a single entity, but include a general class of which a particular example may be used for illustration, unless the context otherwise indicates. The terms in this specification are used to describe particular embodiments of the invention, but their use does not limit the invention except as described in the claims.

[0047] Unless otherwise specified, all numbers representing characteristics such as amounts of components, molecular weights, reaction conditions, etc. used in this specification and the claims should be understood to be modified in all cases by the term "about". Accordingly, unless otherwise specified, the numerical parameters in this specification and the claims are approximate values that may vary depending on the desired characteristics to be obtained by the present invention. At a minimum, without limiting the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters describing the broad scope of the invention are approximations, the numerical values in specific examples are reported as accurately as possible. However, any numerical value inherently includes a standard deviation that necessarily results from the error found in the numerical test measurements.

[0048] Accordingly, any reference in this specification to a numerical range includes explicitly each numerical value (including fractions and integers) subsumed by that range. By way of illustration, a reference in this specification to a range of "at least 50" or "at least about 50" includes integers such as 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers such as 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a further illustration, a reference in this specification to a range of "less than 50" or "less than about 50" includes integers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc. In yet another example, a reference in this specification to a range of "5 to 10" includes integers 5, 6, 7, 8, 9, and 10, and fractional numbers such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, etc.

[0049] In the detailed description and claims of this specification, the term "about" indicates that the indicated value may vary somewhat, provided that such variation does not result in an inappropriate process or structure for the indicated embodiment. For example, for some elements, the term "about" may refer to a variation of ±0.1%, and for other elements, the term "about" may refer to a variation of ±1% or ±10%, or any point therebetween.

[0050] According to the method of the present invention, the disclosed inhibitory compounds can be administered alone or in combination with other immunosuppressive or anti-inflammatory agents for either prophylactic or therapeutic purposes. When provided prophylactically, the immunosuppressive compound is provided prior to any inflammatory response or symptoms (e.g., prior to, at the time of, or immediately following an organ or tissue transplant, but prior to any symptoms of organ rejection).

[0051] The inhibitory compounds disclosed can, according to the present invention, be administered in single or divided doses by oral, parenteral or topical routes. (Formula I) Suitable oral dosages of the compounds of the present invention range from about 0.001 mg to about 10 g per day. In parenteral formulations, suitable dosage units may contain from about 0.001 mg to about 250 mg of said compound, while in topical administration, formulations containing from about 0.001% to about 1% of the active ingredient are contemplated. However, it should be understood that the dosage varies from patient to patient and that the dosage for any particular patient depends on the judgment of the clinician using it as a criterion for fixing the appropriate dosage in view of the patient's size and condition as well as the patient's response to the drug.

[0052] When the compounds of the present invention are administered by the oral route, they can be administered as a medicament in the form of a pharmaceutical preparation containing them together with a compatible pharmaceutical carrier and / or excipient material. Such carriers and / or excipient materials can be inert organic or inorganic carriers and / or excipient materials suitable for oral administration. Examples of such carriers and / or excipient materials are water, gelatin, talc, starch, magnesium stearate, gum arabic, vegetable oils, polyalkylene glycols, petrolatum, etc.

[0053] Pharmaceutical preparations can be prepared by conventional methods and the finished dosage form can be a solid dosage form, such as tablets, coated tablets, capsules, etc., or a liquid dosage form, such as solutions, suspensions, emulsions, etc. Pharmaceutical preparations can be subjected to conventional pharmaceutical operations such as sterilization. Furthermore, pharmaceutical preparations can contain conventional adjuvants such as preservatives, stabilizers, emulsifiers, flavor improvers, wetting agents, buffers, salts for changing osmotic pressure, etc. Examples of solid carriers and / or excipient materials that can be used are starch, lactose, mannitol, methylcellulose, microcrystalline cellulose, talc, silica, dibasic calcium phosphate, and high molecular weight polymers (such as polyethylene glycol, etc.).

[0054] For parenteral use, the inhibitory compound may contain bacteriostatic agents, antioxidants, preservatives, buffers, or other solutes that make the solution isotonic with blood, thickeners, suspending agents, or other pharmaceutically acceptable additives, and can be administered as an aqueous or non-aqueous solution, suspension, or emulsion in a pharmaceutically acceptable oily or liquid mixture. Examples of this type of additive include, for example, tartrate, citrate, and acetate buffers, ethanol, propylene glycol, polyethylene glycol, complexing agents (e.g., EDTA), antioxidants (e.g., sodium bisulfite, sodium metabisulfite, and ascorbic acid), high molecular weight polymers for viscosity adjustment (e.g., liquid polyethylene oxide), and polyethylene derivatives of sorbitol anhydride. Optionally, preservatives such as benzoic acid, methyl or propyl paraben, benzalkonium chloride, and other quaternary ammonium compounds can also be added.

[0055] The compounds of the present invention may also be administered as a solution for nasal application, and in addition to the compounds of the present invention, appropriate buffers, tonicity adjusters, microbial preservatives, antioxidants, and thickeners can be contained in an aqueous vehicle. Examples of agents used to increase viscosity are polyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone, polysorbate, or glycerin. Examples of microbial preservatives that may be added include benzalkonium chloride, thimerosal, chlorobutanol, or phenylethyl alcohol.

[0056] Furthermore, the compounds provided in the present invention can be administered topically or by suppository.

Examples

[0057] The functional significance of the interaction between the RNA-binding protein hnRNP R and the 3’UTR of Mapt mRNA in motor neurons was investigated. HnRNP R knockout mice (Hnrnpr- / -) were generated, and their motor neurons were cultured in microfluidic chambers (Figure 1C). Such compartmentalization of motor neurons enables the separate extraction of RNA from their somatic dendritic and axonal regions for transcript level analysis by quantitative PCR (qPCR). This showed that loss of hnRNP R depletes Mapt mRNA from axons but not from the cell body including dendrites (Figure 1D). This result suggests that hnRNP R mediates the transport of Mapt mRNA to axons.

[0058] Figure 1A shows hnRNP R binding sites along the Mapt pre-mRNA revealed by individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP). Figure 1B shows co-immunoprecipitation of MAPT mRNA with anti-hnRNP R antibody from mouse motor neurons. Figure 1C is a schematic of a microfluidic chamber for compartmentalized neuron culture. Figure 1D is quantitative PCR of Mapt from somatic dendritic RNA and axonal RNA of Hnrnpr+ / + and - / - mouse motor neurons. Statistical analysis was performed using two-way ANOVA with Sidak's multiple comparison test. ***p < 0.001.

[0059] Next, fluorescence in situ hybridization (FISH) of Mapt mRNA was performed in cultured motor neurons from hnRNP R knockout mice and wild-type control mice. Consistent with the qPCR results from the compartmentalized chambers, FISH revealed a decrease in the amount of Mapt mRNA in the axons of motor neurons depleted of hnRNP R.

[0060] Figure 2A shows the results of fluorescence in situ hybridization (FISH) of Mapt mRNA in motor neurons cultured from Hnrnpr+ / + and - / - mice. Figure 2B shows the quantification of FISH signals. Statistical analysis was performed using the Wilcoxon test. ****p < 0.0001.

[0061] Finally, immunostaining showed that the loss of hnRNP R decreased the levels of tau protein in axons in both their proximal and distal regions, while its levels in soma remained unchanged. Figure 3A shows the tau immunostaining of motor neurons cultured from Hnrnpr+ / + and - / - mice, with the proximal (P) and distal (D) regions of the axons marked. Figure 3B is the quantification of tau immunostaining signals by immunostaining. Statistical analysis was performed using the Wilcoxon test. **p < 0.01; n.s., not significant.

[0062] Figure 4 (left panel: upper figure) shows that in the diseased state, Mapt mRNA is transported to axons by hnRNP R, where it is locally translated into tau protein, resulting in NFTs.

[0063] However, considering that hnRNP R interacts with hundreds of RNAs including the abundant non-coding RNA 7SK, its extensive depletion may have harmful side effects. Therefore, the use of small molecules, peptides, ASOs, or combinations thereof can be used to inhibit the association between MAPT 3’UTR and hnRNP R. These small molecules, peptides, ASOs, or combinations thereof anneal to regions of the MAPT 3’UTR where hnRNP R binds in other ways to block the interaction between hnRNP R and MAPT. As a result of these treatments, the transport of MAPT to axons and the local synthesis of tau protein should be reduced, thereby alleviating abnormal tau.

[0064] Figure 4 (right panel: lower figure) shows the proposed mechanism of the present invention, where ASOs are used to block the binding of hnRNP R to Mapt mRNA, thereby preventing its axonal localization and local synthesis of tau. As a result, the formation of tau fibrils is reduced.

[0065] ASOs have been successfully delivered to the central nervous system and are therapeutically used to correct splicing defects underlying the motor neuron disorder in spinal muscular atrophy. Jablonka, S. and M. Sendtner, Developmental regulation of SMN expression: pathophysiological implications and perspectives for therapy development in spinal muscular atrophy. Gene Ther, 2017. 24(9): p. 06 - 513.

[0066] When either of the two binding sites on Mapt mRNA is bound by a small molecule, peptide, or ASO, mRNA binding is blocked. The two mRNA sites related to the hnRNP R binding protein are: MAPT - S1: 5'-TTTGGCTCGGGACTTCAAAA - 3', and MAPT - S2: 5'-ATTTCATCTTTCCAAATTGA - 3'.

[0067] ASOs can be prepared that bind to these two mRNA sites and prevent association with the hnRNP R binding protein. Figure 5 shows the design of ASOs that inhibit hnRNP R binding to MAPT.

[0068] Two exemplary ASOs that bind to the Mapt mRNA site and prevent association with the hnRNP R binding protein are: MAPT - ASO1: 5'-TTTTGAAGTCCCGAGCCAAA - 3', and MAPT-ASO2: 5'-TCAATTTGGAAAGATGAAAT-3'.

Example

[0069] The blockade of axonal Mapt transport was investigated for a decrease in axonal tau protein production, providing a therapeutic strategy for the protection of neurons against NFT formation and spread.

[0070] In this example, the 3’UTR of Mapt contains a binding site for the RNA-binding protein hnRNP R, and it has been demonstrated that the loss of hnRNP R selectively abolishes axonal Mapt translocation without affecting the total levels of Mapt and tau, reducing axonal tau. Antisense oligonucleotides (ASOs) were designed to disrupt the association between hnRNP R and Mapt, and a similar decrease in axonal Mapt and tau was observed in neurons treated with these ASOs. Thus, ASO-mediated depletion of axonal tau could be a therapeutic option for the treatment of AD and other tauopathies.

[0071] In this example, individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) for hnRNP R in primary mouse embryonic motor neurons was performed. Visual inspection of the Mapt transcript identified hnRNP R iCLIP hits in the 3’UTR (Figure 6A). RNA immunoprecipitation using an antibody against hnRNP R confirmed its association with Mapt (Figure 6B). It was also investigated whether hnRNP R regulates the intracellular distribution of Mapt. In motor neurons derived from hnRNP R knockout mice (Hnrnpr- / -), a decrease in axonal Mapt levels was detectable by fluorescence in situ hybridization (FISH) compared to + / + motor neurons (FIG. 7A-7B). The axonal level of MAPT mRNA decreased in Hnrnpr- / - compared to + / + motor neurons cultured in vitro for 6 days (DIV) in a microfluidic chamber, but the axonal dendrite level did not decrease (Figure 7C-7D). Immunostaining for tau revealed lower levels of tau protein in the axons, but not in the cell bodies of Hnrnpr- / - motor neurons (Figure 8A-8C). Thus, hnRNP R regulates axonal tau levels via translocation of Mapt.

[0072] Continuing with this example, two 2'-O-methyl- and phosphorothioate-modified ASOs (MAPT-ASO1 and 2) were designed to be complementary to the Mapt 3’UTR region with hnRNP R iCLIP hits to block the association between hnRNP R and Mapt (Figure 9). Binding regions conserved or substantially conserved between mouse and human were selected. Cy3-labeled sense oligonucleotides were used for optimization of uptake. Efficient uptake was observed by incubating motor neurons (Figure 10A) or hippocampal neurons (Figure 10B) with 10 μM oligonucleotide.

[0073] In this example, in Example 2, the motor neurons treated with MAPT-ASO1 or MAPT-ASO2 and cultured for 6 DIV showed a decrease in axonal Mapt mRNA levels as detected by FISH compared to untreated motor neurons (Figs. 11A-11C). A similar decrease in axonal Mapt was also detectable in cultured hippocampal neurons (Figs. 11D-11F). Importantly, the Mapt levels in the cell bodies of MAPT-ASO-treated neurons did not change (Figs. 11B, 11E).

[0074] Next, it was investigated whether depletion of axonal Mapt via MAPT-ASO could also downregulate tau protein in the axons. Considering the relatively long half-life of tau, tau was evaluated by immunostaining in MAPT-ASO-treated and untreated motor neurons cultured for 11 DIV. After MAPT-ASO treatment, tau decreased in the axons but not in the cell bodies of motor neurons (Figs. 12A-12D). The axonal tau decrease was stronger for MAPT-ASO2 compared to MAPT-ASO1 (Fig. 12D). Collectively, these data indicate that tau protein levels can be selectively reduced in axons by ASOs that block the hnRNP R binding site in the Mapt 3’UTR.

[0075] Since MAPT-ASO2 was demonstrated to more efficiently reduce axonal tau compared to MAPT-ASO1, MAPT-ASO2 was used and compared to its scrambled version (MAPT-ASO2Scr) as an additional control. At 10 μM, Cy3-labeled MAPT-ASO2 and Scr were efficiently taken up by motor neurons (Figure 13A) and hippocampal neurons (Figure 13B). Compared to Scr, MAPT-ASO2 significantly downregulated axonal Mapt levels in motor neurons (Figures 14A-14C) and hippocampal neurons (Figures 14D-14F). Subsequently, axonal translation of tau was evaluated using Puro-PLA. Motor neurons treated with MAPT-ASO2 revealed a decreased Puro-PLA signal for tau in the axons compared to motor neurons treated with Scr and untreated motor neurons (Figures 15A-15C). Tau synthesis in the cell body was not affected by MAPT-ASO2 treatment (Figure 15B).

[0076] Similarly, tau protein levels were decreased in the axons of MAPT-ASO2-treated motor neurons (Figures 16A-16D) and hippocampal neurons (Figures 16E-16G) compared to Scr-treated neurons. Consistent with the function of tau in axonal growth, the axonal length of motor neurons treated with MAPT-ASO2 was decreased compared to Scr treatment, but survival was not affected (Figures 17A-17C). A decrease in axonal growth was also detected for hippocampal neurons exposed to MAPT-ASO2 (Figures 17D-17E). Thus, MAPT-ASO2 treatment can reduce the axonal level of Mapt, resulting in less axonal tau due to decreased local translation.

[0077] Additional MAPT-ASOs were designed along the Mapt 3’UTR in regions containing hnRNP RiCLIP hits and conserved between mouse and human (Figs. 18A - 18B). These ASOs were screened for their potential to reduce axonal Mapt mRNA levels by fluorescence in situ hybridization (FISH) in hippocampal neurons. Several candidates were identified that reduced axonal Mapt levels by >50% in MAPT-ASO treatment compared to untreated hippocampal neurons (Figs. 18B - 18C). Two of these MAPT-ASOs (19 and 20) were truncated forms of MAPT-ASO2 with lengths of 18 and 16 nucleotides, respectively.

[0078] Tau reduction has emerged as a promising therapeutic strategy for the treatment of AD and other tauopathies. However, long-term depletion of tau can be harmful, justifying the development of more targeted approaches to selectively prevent tau elevation and NFT formation in axons. The strategy demonstrated here utilizes the hnRNP R-dependent axonal transport of Mapt mRNA and its local translation into tau protein. By blocking the interaction between Mapt and hnRNP R, a reduction in axonal tau levels can be achieved without affecting tau levels in somatic cells (Fig. 4). When administered sufficiently early in the disease course, the MAPT-ASOs described herein may be useful for limiting the initiation and spread of abnormal tau in AD.

Example

[0079] This example is directed to a method of reducing the number of SPs and NFTs by depleting the RNA-binding protein hnRNP R. This method is based on the observation that in the case of hnRNP R deficiency, an AD mouse model overexpressing the brains of 5xFAD mice, mutant human amyloid precursor protein (APP) and presenilin 1 (PS1) shows a reduced number of SPs.

[0080] As shown in the image of Figure 19, at 9 months of age, 5xFAD mice show extensive deposition of SPs in the cortex and hippocampus, with activated microglia revealed by 1ba1 immunostaining. However, 5xFAD mice homozygous for the hnRNP R knockout allele (5xFAD;Hnrnpr- / -) have reduced SP deposition and less microglial activation.

[0081] As shown in the image of Figure 20, the absence of hnRNP R also reduced the amount of hyperphosphorylated tau, the main component of NFTs, in these brain regions.

[0082] These results support that depletion of hnRNP R by degradation of its mRNA via antisense oligonucleotides or inhibition of hnRNP R via small molecules, peptides and oligonucleotides can be used to reduce the amount of SPs and NFTs and represent a therapeutic option for the treatment of AD. This mechanism is shown in the form illustrated in Figure 21.

[0083] This detailed description is to be understood as illustrative and exemplary in every respect and not restrictive, and the scope of the inventive concept disclosed herein should be construed in accordance with the full breadth permitted by patent law. The embodiments shown and described herein are merely illustrative of the principles of the inventive concept, and it should be understood that various modifications can be made by those skilled in the art without departing from the scope and spirit of the inventive concept. Those skilled in the art can implement various other combinations of features without departing from the scope and spirit of the inventive concept.

Claims

1. A method for reducing axonal tau protein involves inhibiting the binding between MAPT mRNA and hnRNP R.

2. The method of claim 1, wherein the binding molecule binds to the binding site of the MAPT mRNA.

3. The method of claim 2 , wherein the binding molecule comprises a small molecule.

4. The method of claim 2 or claim 3, wherein the binding molecule comprises a peptide.

5. The method of claim 2 , wherein the binding molecule comprises an antisense oligonucleotide.

6. 6. The method of claim 5, wherein one of the antisense oligonucleotides is 5'-TTTTGAAGTCCCGAGCCAAA-3'.

7. 6. The method of claim 5, wherein one of the antisense oligonucleotides is 5'-TCAATTTGGAAAGATGAAAT-3'.

8. 6. The method of claim 5, wherein one of the antisense oligonucleotides is selected from the group consisting of MAPT-ASO1 and MAPT-ASO2.

9. 3. The method of claim 2, wherein one of the binding sites of the MAPT mRNA is 5'-TTTGGCTCGGGACTTCAAAA-3'.

10. 3. The method of claim 2, wherein one of the binding sites of the MAPT mRNA is 5'-ATTTCATCTTTCCAAATTGA-3'.

11. 2. The method of claim 1, comprising administering at least one of MAPT-AS01 and MAPT-AS02.

12. 1. A medicament for reducing hnRNP R protein levels in a subject, the medicament comprising: administering to said subject an antisense oligonucleotide, wherein said antisense oligonucleotide is configured to reduce hnRNP R mRNA levels.

13. 1. A medicament for reducing hnRNP R protein activity in a subject, the medicament comprising: Administered to one or more of the following: small molecules designed to inhibit hnRNP R; A peptide configured to inhibit hnRNP R; and oligonucleotides configured to inhibit hnRNP R,

14. 1. A medicament for treating a subject having Alzheimer's disease, the medicament comprising administering to the subject one or more of the following: antisense oligonucleotides, wherein the antisense oligonucleotides are configured to reduce hnRNP R mRNA levels; small molecules designed to inhibit hnRNP R; Peptides designed to inhibit hnRNP R; oligonucleotides configured to inhibit hnRNP R; a small molecule that inhibits the interaction between MAPT mRNA and hnRNP R; A peptide that inhibits the interaction between MAPT mRNA and hnRNP R; and Antisense oligonucleotides that inhibit the interaction between MAPT mRNA and hnRNP R.

15. 1. A medicament for treating Alzheimer's disease, comprising a pharmaceutical composition comprising: 5'-TTTTGAAGTCCCGAGCCAAA-3' or a derivative thereof; and A pharmaceutically acceptable carrier or excipient.

16. 1. A medicament for treating Alzheimer's disease, comprising a pharmaceutical composition comprising: 5′-TCAATTTGGAAAGATGAAAT-3′ or a derivative thereof; and Pharmaceutically acceptable carriers and / or excipients.

17. A medicament for treating Alzheimer's disease, comprising a pharmaceutical composition comprising: at least one of MAPT-ASO1 and MAPT-ASO2; and Pharmaceutically acceptable carriers and / or excipients.

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