Methods and materials for treating proteinopathies

Abstract

This document provides methods and materials for treating a mammal (e.g., a human) having or at risk of developing a proteinopathy. For example, one or more importin polypeptides (and / or nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy to treat the mammal.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 193,927, filed May 27, 2021, and U.S. Patent Application No. 63 / 256,318, filed October 15, 2021. The disclosures of the prior applications are considered part of (and are incorporated by reference into) the present disclosure of this application.

[0002] Array List This document contains a Sequence Listing that has been submitted electronically as an ASCII text file named 07039-2043WO1_SL_ST25.txt. The ASCII text file was created on May 16, 2022 and is 362 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.

[0003] This document relates to methods and materials for treating a mammal (e.g., a human) having or at risk of developing a proteinopathy. For example, one or more importin polypeptides (and / or nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy to treat the mammal. [Background technology]

[0004] Common age-related proteinopathies, such as Alzheimer's disease and frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) disease spectrum, are characterized by defects in protein homeostasis and the propagation and spread of pathological protein aggregates (Jucker et al., Nature, 501(7465):45-51 (2013)). Currently, there is a lack of understanding of what causes these pathologies and how they can be reversed by effective therapeutic agents (Ciechanover et al., Exp. Mol. Med., 47:e147 2015; Klaips et al., J. Cell. Biol., 217(1):51-63 (2018)). Summary of the Invention

[0005] This document provides methods and materials for treating a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a proteinopathy associated with aggregation of TAR DNA binding protein 43 (TDP-43) polypeptide. A proteinopathy associated with aggregation of TDP-43 polypeptide may also be referred to as a TDP-43 proteinopathy. For example, one or more importin polypeptides and / or fragments thereof (and / or nucleic acids designed to express importin polypeptides and / or fragments thereof) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) to treat the mammal.

[0006] As demonstrated herein, importin polypeptides and fragments of importin polypeptides can reduce neurodegeneration in TDP-43 proteinopathies. For example, importin polypeptides and fragments of importin polypeptides can prevent TDP-43 from undergoing pathological phase transitions, reverse the formation of insoluble aggregates, restore TDP-43 nuclear localization, and restore TDP-43 function in the nucleus. In some cases, importin polypeptides and fragments of importin polypeptides can slow, delay, or prevent the progression of neurodegeneration in the central nervous system (CNS; e.g., brain) of a mammal. In some cases, importin polypeptides and fragments of importin polypeptides can slow, delay, or prevent the onset of neurodegeneration in the CNS of a mammal. The ability to reduce neurodegeneration in TDP-43 proteinopathies as described herein (e.g., by administering one or more importin polypeptides and / or fragments thereof, and / or nucleic acids designed to express importin polypeptides and / or fragments thereof) provides a unique and unrealized opportunity to treat mammals having or at risk of developing a TDP-43 proteinopathies.

[0007] In general, one aspect of this document features a method of treating a mammal having a TDP-43 proteinopathy. The method can include or consist essentially of administering an Importin 3 (IPO3) polypeptide or a fragment of an IPO3 polypeptide to a mammal having a TDP-43 proteinopathy. The IPO3 polypeptide or fragment can be effective in reducing symptoms of the TDP-43 proteinopathy. The symptoms may be depression, apathy, social withdrawal, mood swings, irritability, aggression, changes in sleep patterns, wandering, loss of inhibitions, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, blunted emotions, compulsive or ritualistic behavior, changes in eating habits or diet, deficits in executive function, lack of insight, agitation, emotional lability, difficulty producing or understanding spoken or written language, memory loss, muscle weakness, muscle atrophy, fasciculations, spasticity, dysarthria, dysphagia, behavioral variant frontotemporal dementia (bvFTD), or primary progressive aphasia (PPA). The method may include administering an IPO3 polypeptide to the mammal. The method may include administering a fragment of the IPO3 polypeptide to the mammal. The fragment of the IPO3 polypeptide may consist of an amino acid sequence set forth in any one of SEQ ID NOs: 26-55. The mammal may be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, traumatic brain injury (TBI), chronic traumatic brain injury (CTE), limbic-predominant senile TDP-43 encephalopathy (LATE), dementia with Lewy bodies (DLB), Parkinson's disease, Huntington's disease, argyrophilic grain disease (AGD), hippocampal sclerosis (HS), Guam ALS, Guam Parkinson-Dementia Complex (G-PDC), Perry's disease, facial onset sensorimotor neuronopathy (FOSMN), inclusion body myositis (IBM), hereditary inclusion body myopathy, oculopharyngeal muscular dystrophy (OPMD), or distal myopathy with rimmed vacuoles. Administering may include intracerebral injection. Administering may include intrathecal injection.

[0008] In another aspect, this document features a method of reducing aggregation of a TDP-43 polypeptide in the CNS of a mammal having a TDP-43 proteinopathy. The method can include or consist essentially of administering an IPO3 polypeptide or a fragment of an IPO3 polypeptide to a mammal having a TDP-43 proteinopathy. The method can include administering an IPO3 polypeptide to the mammal. The method can include administering a fragment of an IPO3 polypeptide to the mammal. The fragment of an IPO3 polypeptide can consist of an amino acid sequence set forth in any one of SEQ ID NOs: 26-55. The mammal can be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, traumatic brain injury (TBI), chronic traumatic brain injury (CTE), limbic-predominant senile TDP-43 encephalopathy (LATE), dementia with Lewy bodies (DLB), Parkinson's disease, Huntington's disease, argyrophilic grain disease (AGD), hippocampal sclerosis (HS), Guam ALS, Guam Parkinson-Dementia Complex (G-PDC), Perry's disease, facial onset sensorimotor neuronopathy (FOSMN), inclusion body myositis (IBM), hereditary inclusion body myopathy, oculopharyngeal muscular dystrophy (OPMD), or distal myopathy with rimmed vacuoles. Administering may include intracerebral injection. Administering may include intrathecal injection.

[0009] In another aspect, this document features a method of treating a mammal having a TDP-43 proteinopathy. The method can include or consist essentially of administering a nucleic acid encoding an IPO3 polypeptide or a fragment of an IPO3 polypeptide to a mammal having a TDP-43 proteinopathy, where the IPO3 polypeptide or fragment is expressed by cells within the CNS of the mammal. The IPO3 polypeptide or fragment can be effective in reducing symptoms of the TDP-43 proteinopathy. The symptoms may be depression, emotional blunting, social withdrawal, mood swings, irritability, aggression, changes in sleep habits, wandering, loss of inhibitions, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, emotional blunting, compulsive or ritualistic behavior, changes in eating habits or diet, deficits in executive function, lack of insight, agitation, emotional lability, difficulty producing or understanding spoken or written language, memory loss, muscle weakness, muscle atrophy, fasciculations, spasticity, dysarthria, dysphagia, bvFTD, or PPA. The nucleic acid may encode an IPO3 polypeptide. The nucleic acid may encode a fragment of an IPO3 polypeptide. The fragment of an IPO3 polypeptide may consist of an amino acid sequence set forth in any one of SEQ ID NOs: 26-55. The nucleic acid may be in the form of a vector. The vector may be an adeno-associated virus (AAV) vector. The vector may be an expression plasmid. The nucleic acid may be contained within a nanoparticle. The mammal may be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, traumatic brain injury (TBI), chronic traumatic brain injury (CTE), limbic-predominant senile TDP-43 encephalopathy (LATE), dementia with Lewy bodies (DLB), Parkinson's disease, Huntington's disease, argyrophilic grain disease (AGD), hippocampal sclerosis (HS), Guam ALS, Guam Parkinson-Dementia Complex (G-PDC), Perry's disease, facial onset sensorimotor neuronopathy (FOSMN), inclusion body myositis (IBM), hereditary inclusion body myopathy, oculopharyngeal muscular dystrophy (OPMD), or distal myopathy with rimmed vacuoles. Administering may include intracerebral injection. Administering may include intrathecal injection.

[0010] In another aspect, this document features a method of reducing aggregation of a TDP-43 polypeptide in the CNS of a mammal. The method can include or consist essentially of administering to the mammal a nucleic acid encoding an IPO3 polypeptide or a fragment of an IPO3 polypeptide, where the IPO3 polypeptide or fragment is expressed by a cell in the CNS of the mammal. The nucleic acid can encode an IPO3 polypeptide. The nucleic acid can encode a fragment of an IPO3 polypeptide. The fragment of an IPO3 polypeptide can consist of an amino acid sequence set forth in any one of SEQ ID NOs: 26-55. The nucleic acid can be in the form of a vector. The vector can be an adeno-associated virus (AAV) vector. The vector can be an expression plasmid. The nucleic acid can be contained within a nanoparticle. The mammal can be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, traumatic brain injury (TBI), chronic traumatic brain injury (CTE), limbic-predominant senile TDP-43 encephalopathy (LATE), dementia with Lewy bodies (DLB), Parkinson's disease, Huntington's disease, argyrophilic grain disease (AGD), hippocampal sclerosis (HS), Guam ALS, Guam Parkinson-Dementia Complex (G-PDC), Perry's disease, facial onset sensorimotor neuronopathy (FOSMN), inclusion body myositis (IBM), hereditary inclusion body myopathy, oculopharyngeal muscular dystrophy (OPMD), or distal myopathy with rimmed vacuoles. Administering may include intracerebral injection. Administering may include intrathecal injection.

[0011] In another aspect, this document features a method of treating a mammal having a TDP-43 proteinopathy. The method can include or consist essentially of administering an Importin 13 (IPO13) polypeptide or a fragment of an IPO13 polypeptide to a mammal having a TDP-43 proteinopathy. The IPO13 polypeptide or fragment can be effective in reducing a symptom of a TDP-43 proteinopathy. The symptom can be depression, apathy, social withdrawal, mood swings, irritability, aggression, changes in sleep habits, wandering, loss of inhibitions, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, emotional blunting, compulsive or ritualistic behavior, changes in eating habits or diet, deficits in executive function, lack of insight, agitation, emotional lability, difficulty producing or understanding spoken or written language, memory loss, muscle weakness, muscle atrophy, fasciculations, spasticity, dysarthria, dysphagia, bvFTD, or PPA. The method may include administering an IPO13 polypeptide to the mammal. The method may include administering a fragment of an IPO13 polypeptide to the mammal. The fragment of an IPO13 polypeptide may consist of an amino acid sequence set forth in any one of SEQ ID NOs: 56-77. The mammal may be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, TBI, CTE, LATE, DLB, Parkinson's disease, Huntington's disease, AGD, HS, Guam ALS, G-PDC, Perry's disease, FOSMN, IBM, hereditary inclusion body myopathy, OPMD, or distal myopathy with rimmed vacuoles. The administering may include intracerebral injection. The administering may include intrathecal injection.

[0012] In another aspect, this document features a method of reducing aggregation of a TDP-43 polypeptide in the CNS of a mammal having a TDP-43 proteinopathy. The method can include or consist essentially of administering an IPO13 polypeptide or a fragment of an IPO13 polypeptide to a mammal having a TDP-43 proteinopathy. The method can include administering an IPO13 polypeptide to the mammal. The method can include administering a fragment of an IPO13 polypeptide to the mammal. The fragment of an IPO13 polypeptide can consist of an amino acid sequence set forth in any one of SEQ ID NOs: 56-77. The mammal can be a human. The TDP-43 proteinopathy can be Alzheimer's disease, FTD, ALS, TBI, CTE, LATE, DLB, Parkinson's disease, Huntington's disease, AGD, HS, Guam ALS, G-PDC, Perry's disease, FOSMN, IBM, hereditary inclusion body myopathy, OPMD, or distal myopathy with rimmed vacuoles. The administering can include intracerebral injection. Administering can include intrathecal injection.

[0013] In another aspect, this document features a method of treating a mammal having a TDP-43 proteinopathy. The method can include or consist essentially of administering a nucleic acid encoding an IPO13 polypeptide or a fragment of an IPO13 polypeptide to a mammal having a TDP-43 proteinopathy, where the IPO13 polypeptide or fragment is expressed by cells within the CNS of the mammal. The IPO13 polypeptide or fragment can be effective in reducing symptoms of the TDP-43 proteinopathy. The symptoms may be depression, apathy, social withdrawal, mood swings, irritability, aggression, changes in sleep habits, wandering, loss of inhibitions, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, blunted emotions, compulsive or ritualistic behavior, changes in eating habits or diet, deficits in executive function, lack of insight, agitation, emotional lability, difficulty producing or understanding spoken or written language, memory loss, muscle weakness, muscle atrophy, fasciculations, spasticity, dysarthria, dysphagia, bvFTD, or PPA. The nucleic acid may encode an IPO13 polypeptide. The nucleic acid may encode a fragment of an IPO13 polypeptide. The fragment of an IPO13 polypeptide may consist of an amino acid sequence set forth in any one of SEQ ID NOs: 56-77. The nucleic acid may be in the form of a vector. The vector may be an AAV vector. The vector may be an expression plasmid. The nucleic acid may be contained within a nanoparticle. The mammal may be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, TBI, CTE, LATE, DLB, Parkinson's disease, Huntington's disease, AGD, HS, Guam ALS, G-PDC, Perry's disease, FOSMN, IBM, hereditary inclusion body myopathy, OPMD, or distal myopathy with rimmed vacuoles. Administering may include intracerebral injection. Administering may include intrathecal injection.

[0014] In another aspect, this document features a method of reducing aggregation of a TDP-43 polypeptide in the CNS of a mammal. The method can include or consist essentially of administering a nucleic acid encoding an IPO13 polypeptide or a fragment of an IPO13 polypeptide to a mammal having a TDP-43 proteinopathy, where the IPO13 polypeptide or fragment is expressed by cells in the CNS of the mammal. The nucleic acid can encode an IPO13 polypeptide. The nucleic acid can encode a fragment of an IPO13 polypeptide. The fragment of an IPO13 polypeptide can consist of an amino acid sequence set forth in any one of SEQ ID NOs: 56-77. The nucleic acid can be in the form of a vector. The vector can be an AAV vector. The vector can be an expression plasmid. The nucleic acid can be contained within a nanoparticle. The mammal can be a human. The TDP-43 proteinopathy may be Alzheimer's disease, FTD, ALS, TBI, CTE, LATE, DLB, Parkinson's disease, Huntington's disease, AGD, HS, Guam ALS, G-PDC, Perry's disease, FOSMN, IBM, hereditary inclusion body myopathy, OPMD, or distal myopathy with rimmed vacuoles. Administering may include intracerebral injection. Administering may include intrathecal injection.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0017] [Figure 1A] Expression of KPNB1 reduces the aggregation and toxicity of the C-terminal fragment of TDP. Figure 1A) Representative images of SH-SY5Y human neuroblastoma cells expressing an mCherry-tagged C-terminal fragment of human TDP-43 (TDP-CTF) derived from amino acids 207-414 together with either EGFP or EGFP-KPNB1. Expression of KPNB1 reduces pathological TDP-CTF aggregates. [Figure 1B] Figure 1B) Western blot analysis of soluble and insoluble fractions of SH-SY5Y cells co-expressing TDP-CTFs with EGFP or EGFP-KPNB1. KPNB1 significantly reduces both RIPA buffer soluble and insoluble levels of TDP-CTFs without affecting endogenous TDP-43 protein levels. N=6; ***p<0.005. [Figure 2A-1] KPNB1 and other β-importin nuclear import receptors (NIRs) reduce TDP-CTF aggregation. Figure 2A) SH-SY5Y human neuroblastoma cells were co-transfected with mCherry or mCherry-TDP-CTF along with GFP-tagged karyopherin α and karyopherin β nuclear transport receptor proteins. Expression of KPNB1 and other specific NIRs reduces the formation of TDP-CTF aggregates. Scale bar: 5 μm. [Figure 2A-2] Same as Figure 2A-1. [Figure 2B] Figure 2B) Western blot analysis and quantification of insoluble levels of mCherry-TDP-CTF co-transfected with expression plasmids for different karyopherin α and karyopherin β transport receptors in SH-SY5Y cells shows that KPNB1 and other β-importins significantly reduce TDP-CTF aggregation. N=4; *p<0.5, ***p<0.005. [Figure 3A]N-terminal fragments of KPNB1 are necessary and sufficient to reduce TDP-CTF aggregates. Figure 3A) Top: Schematic of the HEAT repeat domain in KPNB1 (H1-19). Bottom left: Representative images of SH-SY5Y cells expressing mCherry-TDP-CTF together with either EGFP, EGFP-KPNB1 full-length (FL), the N-terminal fragment of KPNB1 containing HEAT repeats 1-9 (H1-9), or the C-terminal fragment of KPNB1 (H10-19). Both full-length KPNB1 and its N-terminal fragment significantly reduce the appearance of cytoplasmic TDP-CTF aggregates, while the C-terminal half of KPNB1 coaggregates with TDP-CTF (arrows). Bottom right: Both full-length KPNB1 and its N-terminal fragment, but not its C-terminal fragment, significantly reduce the amount of RIPA buffer-insoluble TDP-CTF. [Figure 3B] Figure 3B) Different truncated constructs consisting of the N-terminal half of KPNB1 were co-transfected with either mCherry or mCherry-TDP-CTF. Only constructs H1-9 and H1-8 completely suppressed TDP-CTF aggregation and caused a diffuse nucleocytoplasmic distribution of mCherry-TDP-CTF. Scale bar = 5 μm. [Figure 4A] Figure 4: Selected NIRs are involved in the nuclear localization of TDP-43 variants even if they lack a functional nuclear localization sequence / signal (NLS). Representative images of SH-SY5Y human neuroblastoma cells expressing full-length TDP-43 with a mutated inactive NLS (TDP-43mNLS) together with GFP-tagged TDP-CTFs or mCherry-tagged NIRs. (Figure 4A) KPNB1, a splicing isoform of TNPO2 / IPO3 (IPO3A and IPO3B and IPO13), can import TDP-CTFs into the nucleus. [Figure 4B](Figure 4B) KPNB1 and IPO13 can import the full-length TDP-43 mNLS into the nucleus. The truncated N-terminal spliced ​​isoform of TDP-43 (sTDP-43) is partially disaggregated but remains largely cytoplasmic, likely due to the presence of a nuclear export signal. [Figure 5A] C-terminal fragments of IPO3B are necessary and sufficient to mediate nuclear localization of TDP-CTFs and TDP-43 mNLS. Figure 5A) Top: Schematic of the HEAT repeat domains within IPO3B (H1-20). Bottom: Representative images of SH-SY5Y cells expressing mCherry-TDP-CTFs together with either EGFP or EGFP-IPO3B constructs. Deletion of more N-terminal HEAT repeats than H13 (H14-20) results in reduced nuclear localization, but still reduced TDP-43 aggregation. The strongest nuclear import activity was observed in IPO3b H8-H20. Bottom: Full-length IPO3 and IPO13, as well as truncated IPO3 constructs (H2-20, H3-20, and H4-20) reduce the levels of RIPA buffer-insoluble GFP-TDP-CTF and GFP-TDP-43 mNLS protein levels in transfected HEK293 cells. [Figure 5B] Figure 5B) C-terminal deletion of IPO3B H20 (H8-19) and beyond causes prominent cytoplasmic coaggregation. These data demonstrate that H13-20 are required for nuclear localization of the TDP-43 mNLS. H20 is required for both nuclear transport and disaggregation. [Figure 5C] FIG. 5C) IPO3B H8-H20 induce nuclear localization of TDP-CTF, reduce its insoluble aggregation, and ameliorate TDP-CTF-dependent toxicity in SH-SY5Y cells. [Figure 6A]IPO13 fragments are involved in the nuclear localization of TDP-CTFs. Figure 6A) TDP-CTFs become nuclear but appear to aggregate when co-expressed with IPO13 deletion constructs (H1-19, H2-20, and H1-10), but less so with the H1-11 construct. Western blot analysis of the insoluble proteome reveals that only the full-length and IPO13 constructs with C-terminal deletions after H20 (Δ934-964) significantly reduce detergent-insoluble GFP-TDP-43mNLS protein levels. Other IPO13 constructs tested make TDP-CTFs appear aggregated but more nuclear, explaining why no effect on insoluble TDP-43 levels is observed. [Figure 6B] Figure 6B. N-terminal deletions indicate that IPO13 H4-19 is strongly involved in the nuclear localization of TDP-CTFs but not in the disaggregation of TDP-CTFs and TDP-43mNLS. [Figure 7] Figure 1: KPNB1 interacts with the C-terminal domain of TDP-43 in the absence of an NLS. Co-IP of mCherry-tagged TDP-43 deletion constructs with GFP-KPNB1 reveals that the intrinsically disordered, aggregation-prone prion-like domain (PrLD) is not required for the association. [Figure 8A] NIRs that block TDP-CTF aggregation also reduce TDP-CTF-induced cell death in neuronal SH-SY5Y cells. Figure 8A) Representative images of SH-SY5Y cells co-expressing EGFP-tagged NIRs with mCherry-TDP-CTF show that KPNB1, IPO13, and to a lesser extent KPNB2 / TNPO1 reduce aggregation. Scale bar: 5 μm. [Figure 8B] Figure 8B) Expression of KPNB1 and IPO13 significantly reduced TDP-CTF-dependent cell death, while KPNB2 / TNPO1 had a weaker effect. N=3; *p<0.5, ***p<0.005. [Figure 9]Figure 1: KPNB1 fragments that reduce TDP-CTF aggregation also block TDP-CTF-induced cell death. Co-expression of mCherry-TDP-CTF with GFP-tagged full-length KPNB1, KPNB1 (H1-9), and KPNB1 (H10-19) in neuronal SH-SY5Y cells reveals that only disaggregating full-length KPNB1 and its N-terminal fragments (H1-9) reduce TDP-CTF-induced cell death. [Figure 10A] FIG. 10A) The amino acid sequence of an exemplary KPNB1 polypeptide (SEQ ID NO:1). [Figure 10B] FIG. 10B) An exemplary nucleic acid encoding a KPNB1 polypeptide (SEQ ID NO:2). [Figure 11A] FIG. 11A) The amino acid sequence of an exemplary IPO3 polypeptide (SEQ ID NO:3). [Figure 11B] FIG. 11B) An exemplary nucleic acid encoding an IPO3 polypeptide (SEQ ID NO:4). [Figure 12A] FIG. 12A) The amino acid sequence of an exemplary IPO13 polypeptide (SEQ ID NO:5). [Figure 12B] FIG. 12B) An exemplary nucleic acid encoding an IPO13 polypeptide (SEQ ID NO:6). [Figure 13A] KPNB1 reduces TDP-43 aggregation in a cellular model of ALS / FTD. Figure 13A) Cells co-transfected with mCherry-TDP-CTF and either EGFP or EGFP-KPNB1 demonstrate through fluorescence microscopy that KPNB1 reduces TDP-43 aggregation. Western blot analysis of the insoluble fraction further demonstrates this reduction in TDP-43 aggregation. [Figure 13B] FIG. 13B) Western blot analysis of insoluble fractions of co-transfected cells demonstrates that the disaggregation activity of KPNB1 against TDP-43 known to carry the familial mutations Q331K, M337V, or A382T is similar to that of wild-type TDP-43. [Figure 13C] FIG. 13C) Fluorescence microscopy reveals that KPNB1 has disaggregation activity against wild-type GFP-tagged TDP-CTFs, as well as splicing isoforms lacking the C-terminal prion-like domain (sTDP) and TDP-43 with a mutated nuclear localization signal (mNLS). [Figure 13D] FIG. 13D) Western blot analysis reveals that KPNB1 has disaggregation activity against wild-type GFP-tagged TDP-CTF, TDP-43 sTDP, and TDP-43 mNLS. [Figure 13E] Figure 13E) Live cell fluorescence microscopy analysis of primary cortical neurons co-transfected with expression constructs for TDP-CTF-mApple, mTagBFP2, and either mEGFP or KPNB1-2A-mEGFP demonstrated that the time-dependent increase in TDP-CTF-mEGFP fluorescence was blocked by KPNB1 expression. [Figure 13F] FIG. 13F) Inducible expression of KPNB1 blocks the time-dependent increase in fluorescence of the TDP-CTF construct (CTF). [Figure 13G] FIG. 13G) Inducible expression of KPNB1 blocks the time-dependent increase in fluorescence of the TDP-dNLS construct (CTF). [Figure 14A] KPNB1 interacts with and reduces TDP-CT aggregation. Figure 14A) Western blot analysis of immunoprecipitation reveals that both GFP-TDP-43 and GFP-TDP-CTF interact with KPNB1, even though GFP-TDP-CTF lacks a nuclear localization signal. [Figure 14B] FIG. 14B) Western blot analysis of cells co-transfected with expression constructs for GFP-TDP-CTF and either mCherry-tagged or untagged KPNB1 demonstrates a similar reduction in RIPA-insoluble TDP-CTF levels compared to untreated, with or without the mCherry tag. [Figure 15A]The N-terminal domain of KPNB1 interacts with phenylalanine- and glycine-rich nucleoporins. FIG. 15A) Western blot analysis of co-immunoprecipitation experiments with GFP-tagged KPNB1 truncations demonstrates that KPNB1 FL, KPNB1 H1-8, and KPNB1 H1-9, but not the smaller truncations, bind to the phenylalanine- and glycine-rich nucleoporins (FG-Nups) NUP62, NUP50, RAN, and importin-a1. [Figure 15B] FIG. 15B) Co-immunoprecipitation of FG-Nup Nup62 by KPNB1 is abolished by mutations within the nucleoporin interaction sequence (I178A, F217A, Y255A, and I263R; mNIS). [Figure 15C] FIG. 15C) Fluorescence microscopy of cells co-expressing TDP-43 and either KPNB1 H1-8 or KPNB1 H1-8 mNIS demonstrates reduced disaggregation activity in the mNIS variants. [Figure 15D] FIG. 15D) Western blot analysis of insoluble fractions from cells co-expressing TDP-43 and either KPNB1 H1-8 or KPNB1 H1-8 mNIS demonstrates reduced disaggregation activity in the mNIS variants. [Figure 16A] TDP-CTFs bind to KPNB1 via their RRM2 and PrLD domains. Figure 16A) Western blot analysis of co-immunoprecipitation experiments reveals that the mCherry-KPNB1 interaction of TDP-CTF 208-239 is stronger than that of TDP-CTF 208-229. [Figure 16B] Figure 16B) Western blot analysis of co-immunoprecipitation experiments reveals that a TDP-CTF fragment lacking aa230-240 (D230~240) has reduced interaction with mCherry-KPNB1 compared to TDP-CTF. [Figure 16C](Figure 16C) Western blot analysis of the insoluble fractions of co-transfected cells reveals that both GFP-TDP-CTF and GFP-TDP-CTF del(aa230-240) are decondensed at similar levels to mCherry-KPNB1. [Figure 17A] FG-Nups are involved in the interaction and disaggregation activity of KPNB1 towards TDP-43. Figure 17A) Fluorescence microscopy of co-transfected cells reveals that GFP-tagged TDP-CTFs, TDP-43mutNLS, and TDP PrLD, but not the N-terminal fragment TDP-43mutNLS aa1-265 lacking PrLD and sTDP. [Figure 17B] Figure 17B) Western blot analysis of insoluble fractions of cells expressing mutant variants of TDP-CTFs in which residues in PrLD were substituted with other residues as indicated demonstrates that mutation of V, L, I, and M residues to F reduced disaggregation by KPNB1. [Figure 17C] FIG. 17C) Co-immunoprecipitation experiments reveal that VLIM-F variants of TDP-CTFs have reduced interaction with mCherry-tagged KPNB1. [Figure 17D] FIG. 17D) Immunocytochemistry of cells expressing GFP-tagged TDP-CTFs and a TDP-43 (mutNLS) variant in which the V, L, I, and M amino acid residues in the PrLD are all mutated to F (VLIM-F) reveals reduced co-localization with both Nup62 and KPNB1. [Figure 17E] FIG. 17E) Western blot analysis of insoluble fractions from co-transfected cells demonstrates that the VLM-F variants of TDP-CTFs are not decondensed by KPNB1 to the same extent as TDP-CTFs. [Figure 18A]KPNB1 reduces endogenous TDP-43 aggregation in a polyGR model of C9orf72-associated ALS (C9ALS). Figure 18A) Cells transfected to express GFP-GRx100 C9orf72 to induce C9ALS show coaggregation of TDP-43 with GRx100 C9orf72 and Nup62. [Figure 18B] FIG. 18B) Full-length KPNB1 and its truncations H1-8 reduced the number of GR C9orf72 aggregates containing mislocalized TDP-43, but H1-8 with a mutated NIS was inactive. [Figure 19A-1] KPNB1 and other β-type importins reduce pathological aggregation of TDP-CTFs. Fig. 19A) Immunofluorescence of SH-SY5Y human neuroblastoma cells co-expressing mCherry or mCherry-TDP-CTF with 27 GFP-tagged transport receptors. Screen results were subdivided into 4 categories based on their activity: "no effect", "coaggregation", "reduce aggregation" (=transport proteins only reduce the size of TDP-CTF aggregates) and "no aggregation" for most β-importins. Arrowheads indicate coaggregation. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 19A-2] Same as FIG. 19A-1. [Figure 19B-1] FIG. 19B) Western blot analysis and quantification of insoluble mCherry-TDP-CTF protein levels in SH-SY5Y cells expressing GFP or one of each of the 27 GFP-tagged transport receptors. Multiple β-importins significantly reduced insoluble TDP-CTF levels. KPNB1 (bold) was used as a reference. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, n=3). [Figure 19B-2] Same as Figure 19B-1. [Figure 20A]Figure 20: The N-terminal half of KPNB1 is necessary and sufficient to reduce TDP-CTF aggregation and toxicity. Figure 20A) (Top) Schematic domain structure of the N-terminal half (HEAT repeats 1-9, H1-9) and C-terminal half (HEAT repeats 10-19, H10-19) of KPNB1. (Bottom) Immunofluorescence (IF) of SH-SY5Y cells co-expressing mCherry or mCherry-TDP-CTF with GFP, full-length (FL) GFP-KPNB1, H1-9, or H10-19. Both full-length and the N-terminal half of KPNB1 reduce TDP-CTF aggregation, while the C-terminal half of KPNB1 co-aggregates with TDP-CTF in the cytoplasm. Arrowheads indicate co-aggregation. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 20B] FIG. 20B) Western blot analysis and quantification of insoluble mCherry-TDP-CTF protein levels in SH-SY5Y cells expressing GFP, full-length (FL) GFP-KPNB1, H1-9, or H10-19. Both full-length and the N-terminal half of KPNB1 significantly reduce insoluble TDP-CTF levels. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (**p<0.01, ***p<0.001, n=4). [Figure 20C]FIG. 20C) Quantification of TDP-CTF-induced cytotoxicity upon KPNB1 overexpression. mCherry or mCherry-TDP-CTF was co-expressed with GFP, full-length (FL) GFP-KPNB1, H1-9, or H10-19 in SH-SY5Y cells. 48 hours after transfection, cells were labeled with Live-or-Dye™ 640 / 662 dyes and dead cells were quantified per group. Both full-length and the N-terminal half of KPNB1 significantly ameliorated TDP-CTF-mediated toxicity. Statistical analysis was performed using two-way ANOVA and Bonferroni post-hoc test (4 independent experiments; **p<0.001. mCherry: GFP (n=400), GFP-KPNB1FL (n=405), GFP-KPNB1H1–9 (n=426), GFP-KPNB1H10–19 (n=406); mCherry-TDP-CTF: GFP (n=405), GFP-KPNB1FL (n=401), GFP-KPNB1H1–9 (n=405), GFP-KPNB1H10–19 (n=400)). [Figure 20D] FIG. 20D) IF of SH-SY5Y cells co-expressing mCherry or mCherry-TDP-CTF with GFP-KPNB1 H1-9, or C-terminal deletion constructs. TDP-CTF appeared nuclear-cytoplasmic for KPNB1 H1-9 and H1-8, while it formed small aggregates when KPNB1 H1-7 or H1-6 were present. Arrowheads indicate coaggregations. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 20E] FIG. 20E) Western blot analysis and quantification of insoluble mCherry-TDP-CTF protein levels in SH-SY5Y cells expressing GFP, full-length (FL) GFP-KPNB1, H1-9 or C-terminal deletion constructs. KPNB1 H1-8 was the most active fragment in reducing insoluble TDP-CTF levels. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, n=4). [Figure 21A]KPNB1-mediated reduction of TDP-CTF and TDP-43 aggregates depends on its FG-Nup interaction domain. Figure 21A) Schematic domain structure of KPNB1. KPNB1 is composed of 19 HEAT repeats (H1-19). RanGTP and importin-α interact with H1-8 and H8-19, respectively. FG-Nup binds to two regions of KPNB1 (H5-7 and 14-16). [Figure 21B] (B) Lysates from HEK293T cells expressing GFP, full-length (FL) GFP-KPNB1, N-terminal fragments of KPNB1 (H1-9...H1), or C-terminal fragments of KPNB1 (H10-19) were immunoprecipitated with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. KPNB1 H1-8, the smallest KPNB1 fragment fully active in reducing TDP-43 aggregation, interacts more strongly with FG-Nup and Ran and less strongly with importin-α1 compared to full-length KPNB1. [Figure 21C] FIG. 21C) (Top) Schematic domain structure of KPNB1 H1-8mNIS harboring four missense mutations (I178A, F217A, Y255A, I263R) within the nucleoporin interaction site (NIS). (Bottom) Lysates from HEK293T cells expressing GFP, GFP-KPNB1 H1-8WT, or H1-8mNIS were subjected to immunoprecipitation with GFP-Trap magnetic beads. Western blot analysis was performed on whole cell lysates (input) and immunoprecipitates (IP) using the indicated antibodies. KPNB1 H1-8mNIS shows a significant reduction in interaction with FG-Nups. [Figure 21D]FIG. 21D) Immunofluorescence (IF) of SH-SY5Y cells co-expressing mCherry or mCherry-TDP-CTF with GFP-KPNB1 H1-8WT or H1-8mNIS. KPNB1 H1-8mNIS does not reduce cytoplasmic TDP-CTF aggregates. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 21E] FIG. 21E) Western blot analysis and quantification of insoluble mCherry-TDP-CTF in SH-SY5Y cells expressing GFP, GFP-KPNB1 H1-8WT or H1-8mNIS. KPNB1 H1-8WT significantly reduced insoluble TDP-CTF levels, whereas KPNB1 H1-8mNIS did not. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (**p<0.01, ***p<0.001, n=4). [Figure 21F] FIG. 21F) IF of HEK293T cells co-expressing GFP-(GR)100 with empty plasmid (ctrl), FLAG-tagged KPNB1 H1-8WT or H1-8mNIS and stained for endogenous TDP-43. Unlike KPNB1 H1-8mNIS, KPNB1 H1-8WT prevents sequestration of endogenous TDP-43 into cytoplasmic GR aggregates. Arrowheads point to cytoplasmic GR aggregates. Hoechst staining was used to outline the nucleus. Scale bar: 5 μm. [Figure 21G] FIG. 21G) Quantification of the percentage of cells with cytoplasmic GR aggregates positive for endogenous TDP-43. KPNB1 H1-8WT significantly reduced the number of TDP-43-positive GR aggregates, while KPNB1 H1-8mNIS had no effect. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (3 independent experiments; *p<0.05, ***p<0.001. n=50-52 cells per group). [Fig. 21H]FIG. 21H) IF of HEK293T cells co-expressing GFP-(GR)100 with empty plasmid (ctrl), FLAG-tagged KPNB1 H1-8WT or H1-8mNIS and stained for endogenous Nup62. Unlike KPNB1 H1-8mNIS, KPNB1 H1-8WT prevents sequestration of endogenous Nup62 into cytoplasmic GR aggregates. Arrowheads point to cytoplasmic GR aggregates. Hoechst staining was used to outline the nucleus. Scale bar: 5 μm. [Figure 21I] FIG. 21I) Quantification of the percentage of cells with cytoplasmic GR aggregates positive for endogenous Nup62. KPNB1 H1-8WT significantly reduced the number of Nup62-positive GR aggregates, while KPNB1 H1-8mNIS had no effect. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (3 independent experiments; ***p<0.001. n=50-51 cells per group). [Figure 22A] Nup62 coaggregates with TDP-43 PrLDs and introduction of KPNB1 promotes the reduction of aggregation. FIG. 22A) Immunofluorescence (IF) of HEK293T cells co-expressing GFP-tagged TDP-CTFs, sTDP, or TDP-43mNLS with mCherry or mCherry-KPNB1. KPNB1 abolished TDP-CTF and TDP-43mNLS aggregates, while sTDP aggregates were only reduced in size. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 22B] FIG. 22B) Western blot analysis and quantification of insoluble GFP-tagged TDP-CTF, sTDP, and TDP-43mNLS in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 significantly reduced insoluble TDP-CTF and TDP-43mNLS protein levels, and to a lesser extent sTDP levels. β-actin was used as a loading control. Statistical analysis was performed using two-way ANOVA with Bonferroni's post-hoc test (*p<0.05, ***p<0.001, n=3). [Figure 22C]FIG. 22C) IF of HEK293T cells co-expressing mCherry or Nup62-mCherry with GFP, or GFP-tagged TDP-CTFWT, TDP-43mNLS, TDP-43mNLS 1-265, TDP-PrLD, or sTDP. TDP-CTFWT and TDP-43mNLS significantly co-localize with Nup62 and its PrLD, whereas sTDP, which lacks this domain, does not. Arrowheads indicate coaggregation. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 22D] (Figure 22D) Lysates from HEK293T cells expressing GFP, GFP-TDP-CTF, or GFP-sTDP were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. Unlike sTDP, TDP-CTFs strongly interact with endogenous Nup62 and KPNB1. [Figure 22E] FIG. 22E) IF of HEK293T cells co-expressing mCherry / Nup62-mCherry / mCherry-Nup85, GFP-TDP-CTF / GFP-sTDP, and FLAG-KPNB1. Nup62 recruits KPNB1 to TDP-CTFs but not to sTDP aggregates. Nup85, which lacks FG repeats, does not recruit KPNB1 to either TDP-CTFs or sTDP, although it co-localizes with both aggregates. Arrowheads indicate coaggregations. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 22F] FIG. 22F) IF of HEK293T cells co-expressing Nup62-mCherry with GFP or GFP-tagged KPNB1WT, KPNB1mNIS, KPNB1 H1-8WT or H1-8mNIS. KPNB1WT associated with Nup62 aggregates and significantly reduced their size, whereas KPNB1 H1-8WT rendered Nup62 completely soluble. Arrowheads indicate coaggregations. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 22G] FIG. 22G) IF of HEK293T cells co-expressing Nup62-mCherry with GFP, or GFP-tagged IPO13, TNPO1, XPO1, XPO7, or RANBP17. IPO13 associated with Nup62 aggregates and significantly reduced their size. TNPO1 and exportin coaggregated with Nup62, but only TNPO1 slightly reduced the size of individual aggregates. Arrowheads indicate coaggregation. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 23A] KPNB1 promotes nuclear localization of TDP-43mNLS in primary neurons and mouse brain tissue. FIG. 23A) Immunofluorescence (IF) of primary cortical neurons co-expressing NLS-mTagBFP (nuclear marker), HaloTag (cellular marker), GFP, or GFP-tagged TDP-43WT / TDP-43mNLS / TDP-CTF, and mScarlet / mScarlet-KPNB1. KPNB1 increased nuclear localization of TDP-43mNLS and TDP-CTF. Scale bar: 25 μm. [Figure 23B] FIG. 23B) Quantitative analysis of the N-to-C ratios of GFP or GFP-tagged TDP-43WT, TDP-43mNLS, or TDP-CTF in primary neurons expressing mScarlet or mScarlet-KPNB1 72 hours after transfection. Statistical analysis was performed using one-way ANOVA with Bonferroni's post-hoc test (**p<0.01, ***p<0.001, n=52-264 neurons per group). [Figure 23C] FIG. 23C) IF of HEK293T cells co-expressing GFP-TDP-43mNLS with mCherry, or mCherry-tagged full-length KPNB1 (FL), or fragments thereof. KPNB1 FL, H1-9, and H1-8 constructs increase nuclear localization of TDP-43mNLS. Arrowheads indicate coaggregation. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 23D]FIG. 23D) Quantification of the percentage of HEK293T cells exhibiting nuclear (N), nucleocytoplasmic (NC), or cytoplasmic (C) GFP-TDP-43mNLS distribution upon expression of different mCherry-KPNB1 constructs. Statistical analysis was performed using two-way ANOVA with Bonferroni's post-hoc test (3 independent experiments; n=66-81 cells per group; statistics summarized in Table 8). [Figure 23E] FIG. 23E) IF of mouse brain slice cultures (DIV12) co-expressing AAV-GFP-TDP-43mNLS with AAV-FLAG-mCherry, or AAV-FLAG-KPNB1 H1-9WT, H1-9mNIS, H1-8WT, H1-8mNIS, or H10-19. KPNB1 H1-9WT and H1-8WT increase nuclear localization of TDP-43mNLS, while the NIS mutation abolishes this effect. Hoechst staining was used to outline the nuclei. Scale bar: 50 μm. [Figure 23F] FIG. 23F) Quantification of the percentage of neurons showing nuclear (N), nucleocytoplasmic (NC), or cytoplasmic (C) GFP-TDP-43mNLS distribution upon expression of different AAV-KPNB1 constructs. Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc test (3 independent experiments; n=150-154 neurons per group; statistics summarized in Table 8). [Figure 24A] Figure 24: Nup62 and KPNB1 co-localize with pathological pTDP-43 aggregates in human postmortem brain tissue. Simultaneous immunofluorescence (IF) staining was performed on fixed spinal cord or posterior hippocampus of neuropathologically diagnosed ALS and FTLD cases, respectively, including non-affected control tissue. Figure 24A and Figure 24B) High-resolution simultaneous IF staining of pTDP-43 and Nup62 or KPNB1 with Hoechst. Each image is represented as a panel of projected optical sections from each z-series for red and green channels, merged with Hoechst DNA stain. Volume-rendered z-series of all channels are shown as 3D insets. Scale bar: 5 μm. [Figure 24B]Same as Figure 24A. [Figure 24C] (C and D) Hippocampal tile scan images of simultaneous IF of pTDP-43 aggregates with Nup62 or KPNB1 in case sFTLD-B#1. Scale bar: 50 μm. [Figure 24D] Same as Figure 24C. [Figure 25A] Figure 25. Model for NIR as a molecular chaperone for FG-Nup containing assemblies: Figure 25A) NIR facilitates nuclear transport by releasing the hydrophobic interactions between FG repeats that form a hydrogel barrier within the nuclear pore complex. [Figure 25B] FIG. 25B) NIR blocks deleterious phase transitions of FG-Nups and other aggregation-prone proteins that form cytoplasmic foci under cell crowding conditions. [Figure 25C] FIG. 25C) NIR is introduced into pathological FG-Nup and TDP-43 coaggregates, independent of the NLS, opposes their aberrant phase transition and relocates TDP-43 into the nucleus. [Figure 26A] KPNB1 specifically reduces insoluble protein levels of TDP-CTF. FIG. 26A) (Top) Western blot analysis and quantification of insoluble mCherry-TDP-CTF and endogenous TDP-43 protein levels in SH-SY5Y cells expressing GFP or GFP-KPNB1. KPNB1 significantly reduces insoluble TDP-CTF levels, while endogenous TDP-43 levels were unaffected. β-tubulin was used as a loading control. Statistical analysis was performed using Student's t-test (***p<0.001, n=10). (Bottom) Immunofluorescence of SH-SY5Y cells co-expressing mCherry-TDP-CTF with GFP or GFP-KPNB1. mCherry-TDP-CTF forms cytoplasmic aggregates but shows diffuse localization in the presence of GFP-KPNB1. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 26B]FIG. 26B) Western blot analysis of soluble and insoluble GFP-TDP-CTFs co-expressed with mCherry, mCherry-KPNB1, or non-tagged KPNB1 in HEK293T cells. Only KPNB1 reduces insoluble TDP-CTF levels. β-Tubulin was used as a loading control. [Figure 26C] FIG. 26C) Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT, TDP-CTFQ331K, TDP-CTFM337V, and TDP-CTFA382T in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 similarly reduced insoluble wild-type and ALS-derived mutant TDP-CTF protein levels. β-tubulin was used as a loading control. Statistical analysis was performed using Student's t-test (***p<0.001, n=3). [Figure 26D] FIG. 26D) Western blot analysis of total protein levels of GFP-tagged TDP-CTFs or TDP-43WT in HEK293T cells expressing mCherry, mCherry-KPNB1, or non-tagged KPNB1. Cells were lysed in 7M urea buffer to collect total protein. KPNB1 reduces total protein levels of TDP-CTFs, but not TDP-43WT. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (*p<0.05, ***p<0.001, n=4). [Figure 26E] e, TDP-CTF and TDP-43 transcript levels were quantified in HEK293T cells co-expressing GFP-tagged TDP-CTF or TDP-43 with mCherry, mCherry-KPNB1, or untagged KPNB1. GAPDH was used as a reference to normalize transcript levels. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (n=3). [Figure 27]Figure 1 shows that expression of full-length KPNB1 and its N-terminal half does not reduce endogenous TDP-43 levels. Quantification of endogenous insoluble TDP-43 protein levels in SH-SY5Y cells co-expressing mCherry-TDP-CTF with GFP, GFP-KPNB1 full-length (FL), H1-9, or H10-19. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (*p<0.05, n=4). [Figure 28A] Cytoplasmic GFP-(GR)100 aggregates sequester endogenous TDP-43 and Nup62. FIG. 28A) Immunofluorescence of HEK293T cells expressing GFP-(GR)100 and stained for endogenous TDP-43 and Nup62. Only cytoplasmic GFP-(GR)100 aggregates are immunopositive for TDP-43 and Nup62. Nuclear GFP-(GR)100 aggregates cause diffuse TDP-43 staining (purple arrowheads), while Nup62 accumulates as puncta in the cytoplasm (red arrowheads). White arrowheads point to cytoplasmic GR aggregates. Hoechst staining was used to outline the nucleus. Scale bar: 5 μm. [Figure 28B] FIG. 28B) Quantification of the percentage of cytoplasmic GFP-(GR)100 aggregates positive for TDP-43 or Nup62 in HEK293T cells. Nearly all cells are positive for both proteins. Statistical analysis was performed using Student's t-test (3 independent experiments; n=239-249 cells per group). [Figure 29A]Untagged KPNB1 interacts with both TDP-CTFs RRM2 and PrLD. FIG. 29A) (Left) Lysates from HEK293T cells expressing untagged KPNB1 together with GFP alone or GFP, GFP-TDP-CTF, or GFP-TDP43WT were immunoprecipitated with GFP-Trap magnetic beads. Western blot analysis was performed on whole cell lysates (input) and immunoprecipitates (IP) using the indicated antibodies. (Right) Quantification of relative KPNB1 levels in IP normalized by KPNB1 levels in input. Statistical analysis was performed using one-way ANOVA with Bonferroni's post-hoc test (**p<0.01, ***p<0.001, n=3). [Figure 29B] Figure 29B) Schematic domain structure of full-length TDP-CTF (208-414). TDP-CTF contains a portion of RRM2 (non-PrLD, 208-274) and the C-terminal PrLD (275-414). [Figure 29C] FIG. 29C) (Left) Lysates from HEK293T cells expressing GFP alone or untagged KPNB1 with GFP, GFP-TDP-CTF, GFP-TDP-CTFnon-PrLD, or GFP-TDP-PrLD were immunoprecipitated with GFP-Trap magnetic beads. Western blot analysis was performed on whole cell lysates (input) and immunoprecipitates (IP) using the indicated antibodies. (Right) Quantification of relative KPNB1 levels in IP normalized by KPNB1 levels in input. Statistical analysis was performed using one-way ANOVA with Bonferroni's post-hoc test (***p<0.001, n=3). [Figure 30A] KPNB1 reduces TDP-CTF aggregation independent of its interaction with the RRM2 region. Figure 30A) Schematic domain structure of full-length TDP-CTF(208-414) and its C- and N-terminal deletion constructs used in co-IP experiments. [Figure 30B](B) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTF, or C-terminal deletion constructs of GFP-TDP-CTF were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. Deletion of amino acids 230-240 within TDP-CTF reduced its binding to KPNB1. [Figure 30C] (Figure 30C) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTF, or N-terminal deletion constructs of GFP-TDP-CTF were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. Deletion of amino acids 230-240 in TDP-CTF reduces its binding to KPNB1. TDP-PrLD weakly interacts with KPNB1. [Figure 30D] FIG. 30D) (Left) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTFWT, or GFP-TDP-CTFΔ230-240 were immunoprecipitated with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. KPNB1 interacts weakly with TDP-CTFΔ230-240. (Right) Quantification of relative GFP levels in IP normalized by GFP levels in input confirms that GFP-TDP-CTFΔ230-240 interacts weakly with mCherry-KPNB1. [Figure 30E]FIG. 30E) Immunofluorescence of HEK293T cells co-expressing GFP-tagged TDP-CTFWT or TDP-CTFΔ230-240 with mCherry or mCherry-KPNB1. TDP-CTFΔ230-240 forms small nuclear and large cytoplasmic aggregates. KPNB1 reduces both TDP-CTFWT and TDP-CTFΔ230-240 aggregates and also makes TDP-CTFΔ230-240 more nuclear. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 30F] FIG. 30F) Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT or TDP-CTFΔ230-240 in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 reduced insoluble TDP-CTFWT and TDP-CTFΔ230-240 protein levels in a similar manner. β-tubulin was used as a loading control. Statistical analysis was performed using two-way ANOVA with Bonferroni's post-hoc test (***p<0.001, n=3). [Figure 31A] Figure 31. TDP-43 PrLD interacts with FG-Nup and KPNB1. Figure 31A) Immunofluorescence (IF) of HEK293T cells expressing GFP or GFP-TDP-PrLD and stained for endogenous Nup98 and Nup62. TDP-PrLD forms small cytoplasmic foci positive for Nup62. [Figure 31B] FIG. 31B) IF of HEK293T cells expressing GFP or GFP-TDP-PrLD and stained for endogenous KPNB1. KPNB1 co-localizes with TDP-PrLD foci in the cytoplasm. Hoechst staining was used to outline the nucleus. Scale bar: 5 μm. [Figure 31C](C) Lysates from HEK293T cells expressing GFP or GFP-TDP-PrLD were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. TDP-43 PrLD interacts with Nup62, Nup98, and KPNB1, but not Ran. [Diagram 32] Figure 1: Colocalization of Nup62 with TDP-43 constructs is dependent on PrLD. Colocalization between Nup62-mCherry and GFP or GFP-tagged TDP-43 constructs was measured using Mander's overlap coefficient. Values ​​close to 0 and 1 indicate weak and strong colocalization, respectively. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (***p<0.001. n=21-23 cells per group). [Diagram 33] Figure 1: The NES of sTDP does not prevent it from binding to Nup62 aggregates. Immunofluorescence of HEK293T cells co-expressing Nup62-mCherry with either sTDP or sTDPmNLS ΔNES. Nup62 aggregates do not co-localize with sTDP even after its NLS and NES are mutated. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 34A] TDP-CTF mutations that reduce association with Nup62 abolished the interaction and reduced aggregation by KPNB1. FIG. 34A) Immunofluorescence (IF) of HEK293T cells co-expressing Nup62-mCherry with either GFP-tagged TDP-CTFWT or TDP-CTF constructs harboring different mutations in the PrLD. All TDP-CTF mutant variants co-localize with Nup62 aggregates, except for TDP-CTFVLIM-F (green arrowhead), which accumulates near Nup62 (red arrowhead). Scale bar: 5 μm. [Figure 34B]Figure 34B) Co-localization between Nup62-mCherry and GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F was measured using Mander's overlap coefficient. Values ​​close to 0 and 1 indicate weak and strong co-localization, respectively. Statistical analysis was performed using Student's t-test (***p<0.001, n=21-22 cells per group). [Figure 34C] (C) IF of HEK293T cells co-expressing Nup62-mCherry with either GFP-tagged wild-type or mutant TDP-43mNLS constructs. RNA-binding defective TDP-43mNLS (TDP-43mNLS 5F-L) can still co-localize with Nup62. Only TDP-43mNLS VLIM-F aggregates (green arrowheads) do not contain Nup62 (red arrowheads). [Fig. 34D] FIG. 34D) IF of HEK293T cells co-expressing Nup62-mCherry with GFP-FUSΔ14. Cytoplasmic FUS aggregates (green arrowheads) do not co-localize with Nup62 (red arrowheads). Scale bar: 5 μm. [Figure 34E] Figure 34E) Protein sequences of TDP-CTFWT (SEQ ID NO: 78) and TDP-CTFVLIM-F (SEQ ID NO: 79) showing the FG repeats (underlined) and VLIM-F mutations (red) within the PrLD. [Fig. 34F]FIG. 34F) (Left) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-tagged TDP-CTFWT, or TDP-CTFVLIM-F were immunoprecipitated with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. Introduction of the VLIM-F mutation in the TDP-CTF significantly reduces its binding to KPNB1. (Right) Relative GFP levels in IP normalized by GFP levels in input confirm that GFP-TDP-CTFVLIM-F interacts weakly with mCherry-KPNB1. Statistical analysis was performed using Student's t-test (*p<0.05, n=3). [Figure 34G] FIG. 34G) IF of HEK293T cells co-expressing GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F with mCherry or mCherry-KPNB1. KPNB1 only reduces the size of TDP-CTFVLIM-F aggregates. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Fig. 34H] FIG. 34H) Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 significantly reduced the protein levels of insoluble TDP-CTFWT, but not TDP-CTFVLIM-F. β-tubulin was used as a loading control. Statistical analysis was performed using two-way ANOVA with Bonferroni's post-hoc test (***p<0.001, n=4). [Diagram 35]Nup98 strongly associates with TDP-CTFWT, but not with TDP-CTFVLIM-F and sTDP. Immunofluorescence of HEK293T cells co-expressing GFP-Nup98 with mCherry or mCherry-tagged TDP-43mNLS, TDP-43mNLS 1-265, TDP-PrLD, sTDP, TDP-CTFWT, or TDP-CTFVLIM-F. Arrowheads indicate coaggregation. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Diagram 36] KPNB1 H1-8 reduces cytoplasmic Nup62 aggregates. Immunofluorescence of HEK293T cells co-expressing Nup62-GFP with mCherry, mCherry-tagged KPNB1 H1-8WT or H1-8mNIS. KPNB1 H1-8WT suppresses Nup62 aggregates, while the NIS mutation abolishes this activity. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 37A] KPNB1 increases the N-to-C ratio of different TDP-43 constructs in rodent primary neurons. Quantitative analysis of the N-to-C ratio of GFP or GFP-tagged TDP-43WT, TDP-43mNLS, or TDP-CTF in primary neurons expressing mScarlet or mScarlet-KPNB1 24 hours (FIG. 37A) or 48 hours (FIG. 37B) after transfection. KPNB1 only increases the nuclear levels of TDP-43WT at this stage. Statistical analysis was performed using one-way ANOVA and Bonferroni's post-hoc test (***p<0.001, n=52-264 neurons per group). [Figure 37B] Same as Figure 37A. [Figure 38A]KPNB1 reduces cytoplasmic aggregation of TDP-CTF and promotes its nuclear localization in BSCs. FIG. 38A) Immunofluorescence of mouse BSCs (DIV12) co-expressing AAV-mScarlet-TDP-CTF with AAV-GFP, AAV-GFP-KPNB1 H1-9WT, AAV-FLAG-KPNB1 H1-9WT, H1-9mNIS, H1-8WT, H1-8mNIS, or H10-19. KPNB1 H1-9WT and H1-8WT reduce TDP-CTF cytoplasmic aggregates (arrows) and make them highly nuclear (arrowheads), whereas NIS mutations significantly reduce this function. Hoechst staining was used to outline the nuclei. Scale bar: 50 μm. [Figure 38B] FIG. 38B) Quantification of the percentage of neurons showing nuclear (N), nucleocytoplasmic (NC), or cytoplasmic (C) distribution of mScarlet-TDP-CTF upon expression of different AAV-KPNB1 constructs. Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc test (3 independent experiments; n=150-178 neurons per group; statistics summarized in Table 8). [Figure 39] Figure 1: Nup62 co-localizes with pTDP-43 aggregates in the spinal cord of TARDBP ALS patients. High-resolution simultaneous fluorescent immunostaining of pTDP-43 (magenta) and Nup62 (green) with Hoechst (blue) was performed on fixed spinal cords of neuropathologically diagnosed TARDBP ALS cases. pTDP-43 was stained with 647 / Cy5 secondary antibody and Nup62 with 488 / FITC antibody to ensure there was no bleed through between channels. Images are presented as panels of projected optical sections from each z-series for the magenta and green channels and merged with Hoechst DNA staining. Volume-rendered z-series of all channels are shown as 3D insets. Scale bar: 5 μm. [Figure 40A]KPNB1 expression does not deregulate nucleocytoplasmic transport. Figure 40A) Immunofluorescence (IF) of SH-SY5Y cells co-expressing the transport reporter NES-tdTomato-NLS with GFP or GFP-tagged KPNB1 full-length (FL), H1-9, or H1-8. [Figure 40B] FIG. 40B) Quantification of the N-to-C ratio of the NES-tdTomato-NLS reporter. Statistical analysis was performed using one-way ANOVA with Bonferroni's post-hoc test (*p<0.05, n=21-29 cells per group). [Figure 40C] FIG. 40C) IF of SH-SY5Y cells expressing GFP, or GFP-tagged full-length KPNB1 (FL), H1-9, or H1-8, and stained for endogenous Ran and Nup98. Both proteins remain primarily nuclear in the presence of KPNB1. Hoechst staining was used to outline the nucleus. Scale bar: 5 μm. [Figure 41A] Expression of the N-terminal half of KPNB1 does not affect the nuclear localization of endogenous TDP-43 in cells and BSCs. FIG. 41A) Immunofluorescence (IF) of HEK293T cells co-expressing GFP with empty plasmid (ctrl), FLAG-tagged KPNB1 H1-8WT or H1-8mNIS and stained for endogenous TDP-43. TDP-43 remains nuclear in the presence of KPNB1 H1-8. Scale bar: 5 μm. [Figure 41B] FIG. 41B) IF of mouse BSCs (DIV15) expressing AAV-FLAG-KPNB1 H1-9 and stained for endogenous TDP-43. TDP-43 remains nuclear in the presence of KPNB1 H1-9. Scale bar: 25 μm. [Figure 42A]Nup62 and TDP-CTF aggregates are positive for amyloid staining and their co-expression promotes TDP-CTF aggregation. FIG. 42A) Immunofluorescence of HEK293T cells expressing mCherry-TDP-CTF or Nup62-mCherry forming Amylo-Glo-positive aggregates. Hoechst staining was used to outline the nuclei. Scale bar: 5 μm. [Figure 42B] (B) Western blot analysis of insoluble mCherry-TDP-CTFs in HEK293T cells expressing GFP or Nup62-GFP. Nup62 significantly increases the insoluble levels of TDP-CTFs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] This document provides methods and materials for treating a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy). For example, one or more importin polypeptides and / or fragments thereof (and / or nucleic acids designed to express an importin polypeptide and / or fragments thereof) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) to treat the mammal.

[0019] In some cases, the methods and materials described herein can be used to treat a proteinopathy (e.g., TDP-43 proteinopathy). For example, one or more importin polypeptides (and / or one or more nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a proteinopathy, such as a TDP-43 proteinopathy, etc.) to slow, delay, or prevent the progression of a proteinopathy (e.g., TDP-43 proteinopathy). In some cases, one or more importin polypeptides (and / or one or more nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human having or at risk of developing a proteinopathy, such as a TDP-43 proteinopathy, etc.) in need thereof to slow, delay, or prevent the onset of a proteinopathy (e.g., TDP-43 proteinopathy, etc.).

[0020] In some cases, the methods and materials described herein can be used to reduce or eliminate one or more symptoms of a proteinopathy (e.g., TDP-43 proteinopathy). For example, one or more importin polypeptides (and / or one or more nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a proteinopathy, such as a TDP-43 proteinopathy) to reduce or eliminate one or more symptoms of a proteinopathy (e.g., TDP-43 proteinopathy). Examples of symptoms of proteinopathy (e.g., TDP-43 proteinopathy) include, but are not limited to, depression, blunted affect, social withdrawal, mood swings, irritability, aggression, changes in sleep habits, wandering, loss of inhibitions, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, blunted affect, compulsive or ritualistic behavior, changes in eating habits or diet, deficits in executive function, lack of insight, agitation, emotional lability, difficulty producing or understanding spoken or written language, memory loss, and symptoms of motor neuron disease (e.g., muscle weakness, muscle atrophy, fasciculations, spasticity, dysarthria, and dysphagia), behavioral frontotemporal dementia (bvFTD), and primary progressive aphasia (PPA). In some cases, the materials and methods described herein can be used to reduce the severity of one or more symptoms of a proteinopathy (e.g., TDP-43 proteinopathy) in a mammal (e.g., a human) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.

[0021] In some cases, the methods and materials described herein can be used to reduce or eliminate neurodegeneration associated with a proteinopathy (e.g., TDP-43 proteinopathy). For example, one or more importin polypeptides (and / or one or more nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a proteinopathy, such as TDP-43 proteinopathy, etc.) to slow, delay, or prevent the progression of neurodegeneration associated with a proteinopathy (e.g., TDP-43 proteinopathy, etc.). In some cases, one or more importin polypeptides (and / or one or more nucleic acids designed to express an importin polypeptide) can be administered to a mammal (e.g., a human having or at risk of developing a proteinopathy, such as TDP-43 proteinopathy, etc.) in need thereof to slow, delay, or prevent the onset of neurodegeneration associated with a proteinopathy (e.g., TDP-43 proteinopathy, etc.).

[0022] In some cases, the methods and materials described herein can be used to reduce or eliminate aggregation of TDP-43 polypeptides. For example, the materials and methods described herein can be used to reduce the number of TDP-43 polypeptide aggregates present in the CNS (e.g., brain) of a mammal having a proteinopathy (e.g., TDP-43 proteinopathy) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more. For example, the materials and methods described herein can be used to reduce the size (e.g., volume) of one or more TDP-43 polypeptide aggregates present in a mammal having a proteinopathy (e.g., TDP-43 proteinopathy) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more.

[0023] In some cases, the methods and materials described herein can be used to reduce or eliminate detergent-insoluble TDP-43 polypeptides. For example, the materials and methods described herein can be used to reduce the number of detergent-insoluble TDP-43 polypeptides present in the CNS (e.g., brain) of a mammal having a proteinopathy (e.g., TDP-43 proteinopathy) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more. For example, the materials and methods described herein can be used to reduce the proportion or percentage of detergent-insoluble TDP-43 polypeptides within one or more TDP-43 polypeptide aggregates present in a mammal having a proteinopathy (e.g., TDP-43 proteinopathy) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more.

[0024] In some cases, the methods and materials described herein can be used to reduce or eliminate cell death associated with a proteinopathy (e.g., TDP-43 proteinopathy). For example, the materials and methods described herein can be used to reduce the number of apoptotic cells present in the CNS (e.g., brain) of a mammal having a proteinopathy (e.g., TDP-43 proteinopathy) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.

[0025] In some cases, the methods and materials described herein can be used to restore nuclear localization of TDP-43 polypeptides. For example, the materials and methods described herein can be used to increase the number of TDP-43 polypeptides that can be, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more hyperlocalized to the nucleus of cells present in the CNS (e.g., brain) of a mammal having a proteinopathy (e.g., a TDP-43 proteinopathy).

[0026] In some cases, the methods and materials described herein can be used to restore function of a TDP-43 polypeptide. For example, the materials and methods described herein can be used to increase the level of nuclear TDP-43 polypeptide function (e.g., increasing expression, splicing, and polyadenylation of transcripts derived from stathmin-2 and / or UNC13A) of cells present in the CNS (e.g., brain) of a mammal having a proteinopathy (e.g., TDP-43 proteinopathy) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more.

[0027] Any suitable mammal having or at risk of developing a proteinopathy (e.g., TDP-43 proteinopathy) can be treated as described herein (e.g., by administering one or more importin polypeptides and / or fragments thereof, and / or nucleic acids designed to express an importin polypeptide and / or fragments thereof). Examples of mammals that can be treated as described herein include, but are not limited to, humans, non-human primates, such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, rabbits, hamsters, guinea pigs, and ferrets.

[0028] As described herein, when treating a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) (e.g., by administering one or more importin polypeptides and / or fragments thereof, and / or nucleic acids designed to express an importin polypeptide and / or fragments thereof), the proteinopathy can be any type of proteinopathy. In some cases, the proteinopathy is a TDP-43 proteinopathy (e.g., can include aggregation of TDP-43 polypeptides). In some cases, the proteinopathy can be a neurodegenerative disease. Examples of types of proteinopathies treatable as described herein include, but are not limited to, Alzheimer's disease, FTD, ALS, traumatic brain injury (TBI), chronic traumatic brain injury (CTE), limbic-predominant senile TDP-43 encephalopathy (LATE), dementia with Lewy bodies (DLB), Parkinson's disease, Huntington's disease, argyrophilic grain disease (AGD), hippocampal sclerosis (HS), Guam ALS, Guam Parkinson-Dementia Complex (G-PDC), Perry's disease, facial onset sensorimotor neuronopathy (FOSMN), and rimmed vacuolar myopathy (e.g., inclusion body myositis (IBM), hereditary inclusion body myopathy, oculopharyngeal muscular dystrophy (OPMD), and distal myopathy with rimmed vacuoles). When the proteinopathies treated as described herein are ALS, the ALS can be familial ALS. Familial ALS may be associated with one or more mutations (e.g., insertions, deletions, indels, substitutions, and / or expansions) in any relevant nucleic acid (e.g., any relevant gene). Examples of familial ALS include, but are not limited to, C9ALS (associated with one or more mutations in the C9orf72 gene), ALS1 (associated with one or more mutations in the SOD1 gene), ALS4 (associated with one or more mutations in the SETX gene), ALS6 (associated with one or more mutations in the FUS gene), ALS8 (associated with one or more mutations in the VABP gene), ALS9 (associated with one or more mutations in the ANG gene), ALS10 (associated with one or more mutations in the TARDBP gene), ALS11 (associated with one or more mutations in the FIG4 gene), ALS12 (associated with one or more mutations in the OPTN gene), ALS13 (associated with one or more mutations in the OPTN gene), ALS14 (associated with one or more mutations in the OPTN gene), ALS15 (associated with one or more mutations in the OPTN gene), ALS16 (associated with one or more mutations in the OPTN gene), ALS17 (associated with one or more mutations in the OPTN gene), ALS18 (associated with one or more mutations in the OPTN gene), ALS19 (associated with one or more mutations in the OPTN gene), ALS20 (associated with one or more mutations in the OPTN gene), ALS21 (associated with one or more mutations in the OPTN gene), ALS22 (associated with one or more mutations in the OPTN gene), ALS23 (associated with one or more mutations in the OPTN gene), ALS24 (associated with one or more mutations in the OPTN gene), ALS25 (as ALS13 (associated with one or more mutations in the ATXN2 gene), ALS14 (associated with one or more mutations in the VCP gene), ALS15 (associated with one or more mutations in the UBQLN2 gene), ALS17 (associated with one or more mutations in the CHMP2B gene), ALS18 (associated with one or more mutations in the PFN1 gene), ALS19 (associated with one or more mutations in the ERBB4 gene), ALS20 (associated with one or more mutations in the HNRNPA1 / A2B1 gene), ALS21 (associated with one or more mutations in the MATR3 gene), ALS22 (associated with one or more mutations in the TUBB4A gene), ALS23 (associated with one or more mutations in the ANXA11 gene), ALS24 (associated with one or more mutations in the NEK1 gene), and ALS25 (associated with one or more mutations in the KIF5A gene).

[0029] In some cases, a mammal (e.g., a human) may be identified as having or at risk for developing a proteinopathy (e.g., a TDP-43 proteinopathy). For example, genetic testing, imaging techniques (e.g., CNS scanning techniques such as magnetic resonance imaging (MRI), computed tomography (CT) scanning, fluorodeoxyglucose positron emission tomography (FDG-PET) scanning, and SPECT (single proton emission CT) scanning), and / or biomarker detection techniques using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), cerebrospinal fluid real-time quaking-induced conversion (RT-QuIC), single molecule array (Simoa), Western blot analysis, and / or mass spectrometry can be used to diagnose a mammal as having or at risk for developing a proteinopathy (e.g., a TDP-43 proteinopathy).

[0030] Once identified as having or at risk for developing a proteinopathy (e.g., a TDP-43 proteinopathy), a mammal (e.g., a human) can be administered or instructed to self-administer one or more importin polypeptides and / or fragments thereof (and / or nucleic acids designed to express importin polypeptides and / or fragments thereof) as described herein.

[0031] Any suitable importin polypeptide or fragment thereof (and / or a nucleic acid designed to express an importin polypeptide or fragment thereof) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy as described herein (e.g., a TDP-43 proteinopathy). In some cases, the importin polypeptide can be a beta-karyopherin-type importin polypeptide. Examples of importin polypeptides include, but are not limited to, KPNB1 polypeptides, IPO3 polypeptides (e.g., IPO3a polypeptides and IPO3b polypeptides), importin 4 (IPO4) polypeptides, importin 9 (IPO9) polypeptides, importin 12 (IPO12) polypeptides, and IPO13 polypeptides.

[0032] When the importin polypeptide is a KPNB1 polypeptide, any suitable KPNB1 polypeptide (and / or a nucleic acid designed to express a KPNB1 polypeptide) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) as described herein. Examples of KPNB1 polypeptides and nucleic acids encoding KPNB1 polypeptides include, but are not limited to, those set forth in the National Center for Biotechnology Information (NCBI) database, e.g., accession numbers NP_002256 (version NP_002256.2), NP_001263382 (version NP_001263382.1), and NP_032405 (version NP_032405.3).

[0033] In some cases, the KPNB1 polypeptide can have the amino acid sequence shown in SEQ ID NO: 1 (see, e.g., FIG. 10A). In some cases, the nucleic acid encoding the KPNB1 polypeptide can have the nucleotide sequence shown in SEQ ID NO: 2 (see, e.g., FIG. 10B).

[0034] Where the importin polypeptide fragment is a KPNB1 polypeptide fragment, any suitable KPNB1 polypeptide fragment (and / or a nucleic acid designed to express a KPNB1 polypeptide fragment) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy as described herein (e.g., a TDP-43 proteinopathy). In some cases, the KPNB1 polypeptide fragment can be derived from the amino acid sequence set forth in SEQ ID NO:1.

[0035] KPNB1 polypeptide fragments can be of any suitable length (e.g., can contain any number of amino acids), provided that they maintain at least some function of a naturally occurring KPNB1 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation and / or restore nuclear localization of at least some TDP-43 polypeptides). For example, KPNB1 polypeptide fragments can be from about 30 amino acids to about 900 amino acids in length (e.g., from about 30 amino acids to about 850 amino acids, from about 30 amino acids to about 800 amino acids, from about 30 amino acids to about 750 amino acids, from about 30 amino acids to about 700 amino acids, from about 30 amino acids to about 650 amino acids, from about 30 amino acids to about 600 amino acids, from about 30 amino acids to about 550 amino acids, from about 30 amino acids to about 5 ...00 amino acids, from about 30 amino acids to about 700 amino acids, from about 30 amino acids to about 800 amino acids, from about 30 amino acids to about 850 amino acids, from about 30 amino acids to about 900 amino acids, from about 30 amino acids to about 900 amino acids, from about 30 amino acids to about 1000 amino acids, from about 30 amino acids to about 1500 amino acids, from about 30 amino acids to about 1600 amino acids, from about 30 amino acids to about 1700 amino acids, from about 30 amino acids to about 1800 amino acids, from about 30 amino acids to about 1900 amino acids, from about 3 00 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 250 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 100 amino acids, about 50 amino acids to about 900 amino acids, about 100 amino acids to about 900 amino acids, about 150 amino acids to about 900 amino acids, about 200 amino acids to about 900 amino acids, about 250 amino acids to about 900 amino acids, about 300 amino acids to about 900 amino acids, about 350 amino acids to about 900 amino acids, about 400 amino acids to about 900 amino acids, about 450 amino acids to about 900 amino acids, about 500 amino acids to about 900 amino acids, about 550 amino acids to about 900 amino acids, about 6 00 amino acids to about 900 amino acids, about 650 amino acids to about 900 amino acids, about 700 amino acids to about 900 amino acids, about 750 amino acids to about 900 amino acids, about 800 amino acids to about 900 amino acids, about 50 amino acids to about 800 amino acids, about 100 amino acids to about 700 amino acids, about 200 amino acids to about 600 amino acids, about 300 amino acids to about 500 amino acids, about 100 amino acids to about 300 The fragment may be about 100 to about 400 amino acids, about 200 to about 400 amino acids, about 400 to about 600 amino acids, about 500 to about 700 amino acids, or about 600 to about 800 amino acids), provided that the fragment maintains at least some of the function of a naturally occurring KPNB1 polypeptide (e.g., the ability to reduce at least some of the TDP-43 aggregation).

[0036] In some cases, the KPNB1 polypeptide fragment may comprise, consist essentially of, or consist of an amino acid sequence shown in Table 1.

[0037] [Table 1] TIFF2024520414000002.tif204165

[0038] In some cases, the KPNB1 polypeptide fragments provided herein have 0, 1, 2, 3, or more (e.g., 5, 8, 12, 15, 18, 20, 25, 30, 35, 40, or more) amino acid substitutions (e.g., one or more K→R substitutions) within the sequence identifier (e.g., any one of SEQ ID NOs: 7-16), 0, 1, 2, 3, 4, or 5 amino acid residues preceding the sequence identifier (e.g., any one of SEQ ID NOs: 7-16), and / or may include an amino acid sequence set forth in any one of SEQ ID NOs: 7-16, which has 0, 1, 2, 3, 4, or 5 amino acid residues following the designated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 7-16), provided that the KPNB1 polypeptide fragment retains at least some activity elicited by a naturally occurring KPNB1 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation and / or restore nuclear localization of at least some TDP-43 polypeptides). Examples of such KPNB1 polypeptide fragments may be those set forth in Table 2.

[0039] [Table 2] TIFF2024520414000004.tif76166

[0040] Where the importin polypeptide is an IPO3 polypeptide (also referred to as a transportin 2 (TNPO2) polypeptide), any suitable IPO3 polypeptide (and / or nucleic acid designed to express an IPO3 polypeptide) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) as described herein. Examples of IPO3 polypeptides and nucleic acids encoding IPO3 polypeptides include, but are not limited to, those set forth in the NCBI database, e.g., accession numbers NP_001369170 (version NP_001369170.1), NP_038461 (version NP_038461.2), and NP_001369172 (version NP_001369172.1).

[0041] In some cases, an IPO3 polypeptide can have the amino acid sequence set forth in SEQ ID NO:3 (see, e.g., FIG. 11A). In some cases, a nucleic acid encoding an IPO3 polypeptide can have the nucleotide sequence set forth in SEQ ID NO:4 (see, e.g., FIG. 11B).

[0042] Where the fragment of an importin polypeptide is an IPO3 polypeptide fragment, any suitable IPO3 polypeptide fragment (and / or a nucleic acid designed to express an IPO3 polypeptide fragment) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy as described herein (e.g., a TDP-43 proteinopathy). In some cases, the IPO3 polypeptide fragment can be derived from the amino acid sequence set forth in SEQ ID NO:3.

[0043] An IPO3 polypeptide fragment can be of any suitable length (e.g., can contain any number of amino acids), provided that it maintains at least some function of a naturally occurring IPO3 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation and / or restore nuclear localization of at least some TDP-43 polypeptides). For example, an IPO3 polypeptide fragment can be from about 100 amino acids in length to about 850 amino acids in length (e.g., from about 100 amino acids in length to about 800 amino acids, from about 100 amino acids in length to about 750 amino acids, from about 100 amino acids in length to about 700 amino acids, from about 100 amino acids in length to about 650 amino acids, from about 100 amino acids in length to about 600 amino acids, from about 100 amino acids in length to about 550 amino acids, from about 100 amino acids in length to about 500 amino acids, from about 100 amino acids in length to about 600 amino acids, from about 100 amino acids in length to about 750 amino acids, from about 100 amino acids in length to about 800 amino acids, from about 100 amino acids in length to about 900 amino acids, from about 100 amino acids in length to about 15 ... about 100 to about 450 amino acids, about 100 to about 400 amino acids, about 100 to about 350 amino acids, about 100 to about 300 amino acids, about 100 to about 250 amino acids, about 100 to about 200 amino acids, about 150 to about 850 amino acids, about 200 to about 850 amino acids, about 250 to about 850 amino acids, about 300 to about 850 amino acids Amino acids, about 350 amino acids to about 850 amino acids, about 400 amino acids to about 850 amino acids, about 450 amino acids to about 850 amino acids, about 500 amino acids to about 850 amino acids, about 550 amino acids to about 850 amino acids, about 600 amino acids to about 850 amino acids, about 650 amino acids to about 850 amino acids, about 700 amino acids to about 850 amino acids, about 750 amino acids to about 850 amino acids, about 200 The IPO3 polypeptide may be a polypeptide of at least about 800 amino acids, at least about 300 amino acids, at least about 700 amino acids, at least about 400 amino acids, at least about 600 amino acids, at least about 200 amino acids, at least about 400 amino acids, at least about 300 amino acids, at least about 500 amino acids, at least about 500 amino acids, at least about 700 amino acids, or at least about 600 amino acids, provided that it retains at least a portion of the function of a naturally occurring IPO3 polypeptide (e.g., at least a portion of the TDP-43 aggregates). The assumption is that the objective of the proposed method is to maintain the ability to reduce the concentration of

[0044] In some cases, an IPO3 polypeptide fragment may comprise, consist essentially of, or consist of an amino acid sequence shown in Table 3.

[0045] [Table 3] TIFF2024520414000006.tif175166TIFF2024520414000007.tif161166TIFF2024520414000008.tif19116 6TIFF2024520414000009.tif175166TIFF2024520414000010.tif187166TIFF2024520414000011.tif59166

[0046] In some cases, the IPO3 polypeptide fragments provided herein have 0, 1, 2, 3, or more (e.g., 5, 8, 12, 15, 18, 20, 25, 30, 35, 40, or more) amino acid substitutions (e.g., one or more K→R substitutions) within the designated sequence of the sequence identifier (e.g., any one of SEQ ID NOs:26-42), have 0, 1, 2, 3, 4, or 5 amino acid residues preceding the designated sequence of the sequence identifier (e.g., any one of SEQ ID NOs:26-42), and / or have a sequence identifier. The IPO3 polypeptide fragments may include any one of the amino acid sequences set forth in SEQ ID NOs: 26-42, which have 0, 1, 2, 3, 4, or 5 amino acid residues following the specified sequence (e.g., any one of SEQ ID NOs: 26-42), provided that the IPO3 polypeptide fragments provided herein retain at least some activity of a naturally occurring IPO3 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation and / or restore at least some nuclear localization of a TDP-43 polypeptide). Examples of such IPO3 polypeptide fragments may be those set forth in Table 4.

[0047] [Table 4] TIFF2024520414000013.tif207166TIFF2024520414000014.tif211166TIFF2024520414000015.tif141166

[0048] Where the importin polypeptide is an IPO13 polypeptide, any suitable IPO13 polypeptide (and / or nucleic acid designed to express an IPO13 polypeptide) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) as described herein. Examples of IPO13 polypeptides and nucleic acids encoding IPO13 polypeptides include, but are not limited to, those set forth in the NCBI database, e.g., accession number NP_055467 (version NP_055467.3), accession number XP_024306837 (version XP_024306837.1), and accession number NP_666264 (version NP_666264.1).

[0049] In some cases, an IPO13 polypeptide can have the amino acid sequence set forth in SEQ ID NO:5 (see, e.g., FIG. 12A). In some cases, a nucleic acid encoding an IPO13 polypeptide can have the nucleotide sequence set forth in SEQ ID NO:6 (see, e.g., FIG. 12B).

[0050] Where the importin polypeptide fragment is an IPO13 polypeptide fragment, any suitable IPO13 polypeptide fragment (and / or a nucleic acid designed to express a KPNB1 polypeptide fragment) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy as described herein (e.g., a TDP-43 proteinopathy). In some cases, the IPO13 polypeptide fragment can be derived from the amino acid sequence set forth in SEQ ID NO:5.

[0051] An IPO13 polypeptide fragment can be of any suitable length (e.g., can contain any number of amino acids), provided that it maintains at least some function of a naturally occurring IPO13 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation and / or restore nuclear localization of at least some TDP-43 polypeptides). For example, an IPO13 polypeptide fragment can be from about 100 amino acids in length to about 950 amino acids in length (e.g., from about 100 amino acids in length to about 800 amino acids, from about 100 amino acids in length to about 750 amino acids, from about 100 amino acids in length to about 700 amino acids, from about 100 amino acids in length to about 650 amino acids, from about 100 amino acids in length to about 600 amino acids, from about 100 amino acids in length to about 550 amino acids, from about 100 amino acids in length to about 500 amino acids, from about 100 amino acids in length to about 600 amino acids, from about 100 amino acids in length to about 750 amino acids, from about 100 amino acids in length to about 800 amino acids, from about 100 amino acids in length to about 950 amino acids, from about 100 amino acids in length to about 950 amino acids, from about 100 amino acids in length to about 950 amino acids, from about 100 amino acids in length to about 150 ... 0 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 200 amino acids to about 950 amino acids, about 250 amino acids to about 950 amino acids, about 300 amino acids to about 950 amino acids, about 350 amino acids to about 950 amino acids, about 400 amino acids to about 95 0 amino acids, about 450 amino acids to about 950 amino acids, about 500 amino acids to about 950 amino acids, about 550 amino acids to about 950 amino acids, about 600 amino acids to about 950 amino acids, about 650 amino acids to about 950 amino acids, about 700 amino acids to about 950 amino acids, about 750 amino acids to about 950 amino acids, about 800 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 30 0 amino acids to about 800 amino acids, about 400 amino acids to about 700 amino acids, about 500 amino acids to about 600 amino acids, about 200 amino acids to about 400 amino acids, about 300 amino acids to about 500 amino acids, about 400 amino acids to about 600 amino acids, about 500 amino acids to about 700 amino acids, about 600 amino acids to about 800 amino acids, or about 700 amino acids to about 900 amino acids), provided that maintains at least some function of a naturally occurring IPO13 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation).

[0052] In some cases, the IPO13 polypeptide fragment may comprise, consist essentially of, or consist of an amino acid sequence shown in Table 5.

[0053] [Table 5] TIFF2024520414000017.tif213166TIFF2024520414000018.tif192166TIFF20245204140 00019.tif147166TIFF2024520414000020.tif212166TIFF2024520414000021.tif121166

[0054] In some cases, the IPO13 polypeptide fragments provided herein have 0, 1, 2, 3, or more (e.g., 5, 8, 12, 15, 18, 20, 25, 30, 35, 40, or more) amino acid substitutions (e.g., one or more K→R substitutions) within the sequence identifier (e.g., any one of SEQ ID NOs: 56-66), 0, 1, 2, 3, 4, or 5 amino acid residues preceding the sequence identifier (e.g., any one of SEQ ID NOs: 56-66), and / or has 0, 1, 2, 3, 4, or 5 amino acid residues following the designated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 56-66), with the proviso that an IPO13 polypeptide fragment can include an amino acid sequence set forth in any one of SEQ ID NOs: 56-66 that retains at least some activity of a naturally occurring IPO13 polypeptide (e.g., the ability to reduce at least some TDP-43 aggregation and / or restore nuclear localization of at least some TDP-43 polypeptides). Examples of such IPO13 polypeptide fragments can be those set forth in Table 6.

[0055] [Table 6] TIFF2024520414000023.tif162167TIFF2024520414000024.tif193168TIFF2024520414000025.tif217168TIFF2024520414000026.tif55168

[0056] Any suitable method can be used to deliver one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) to a mammal. When one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) are administered to a mammal (e.g., a human), the one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) can be administered to the CNS (e.g., the brain) of the mammal (e.g., a human). In some cases, the one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) can be administered to the CNS (e.g., the brain) of the mammal (e.g., a human) by direct injection into the CNS (e.g., into the brain and / or spinal cord).

[0057] Any suitable method can be used to obtain the importin polypeptide or fragment thereof provided herein (e.g., a polypeptide comprising, substantially consisting of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 7-77). For example, the importin polypeptide or fragment thereof provided herein (e.g., a polypeptide comprising, substantially consisting of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 7-77) can be obtained by synthesizing the polypeptide of interest using a suitable polypeptide synthesis technique, such as those described elsewhere (see, for example, Fields et al., Curr. Protoc. Protein Sci., Chapter 18:Unit 18.1 (2002); Hartrampf et al., Science, 368(6494):980-987 (2020); and Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)).

[0058] When one or more nucleic acids designed to express an importin polypeptide or fragment thereof are administered to a mammal (e.g., a human), the nucleic acid may be in the form of a vector (e.g., a viral or non-viral vector).

[0059] When a nucleic acid encoding an importin polypeptide or a fragment thereof is administered to a mammal, the nucleic acid can be used for transient expression of the importin polypeptide or a fragment thereof, or for stable expression of the importin polypeptide or a fragment thereof. When a nucleic acid encoding an importin polypeptide or a fragment thereof is used for stable expression of the importin polypeptide or a fragment thereof, the nucleic acid encoding the importin polypeptide or a fragment thereof can be engineered to be integrated into the genome of the cell. The nucleic acid can be engineered to be integrated into the genome of the cell using any suitable method. For example, gene editing techniques (e.g., CRISPR or TALEN gene editing) can be used to integrate a nucleic acid designed to express an importin polypeptide or a fragment thereof into the genome of the cell.

[0060] When the vector used to deliver the nucleic acid encoding the importin polypeptide or a fragment thereof to a mammal (e.g., human) is a viral vector, any suitable viral vector can be used. The viral vector can be derived from a positive strand virus or a negative strand virus. The viral vector can be derived from a virus having a DNA genome or an RNA genome. In some cases, the viral vector can be a chimeric viral vector. In some cases, the viral vector can infect dividing cells. In some cases, the viral vector can infect non-dividing cells. Examples of viral-based vectors that can be used to deliver the nucleic acid encoding the importin polypeptide to a mammal (e.g., human) include, but are not limited to, viral-based vectors based on adenovirus, AAV, Sendai virus, retrovirus, lentivirus, herpes simplex virus (HSV), vaccinia virus, and baculovirus.

[0061] When the vector used to deliver the nucleic acid encoding the importin polypeptide or a fragment thereof to a mammal (e.g., a human) is a non-viral vector, any suitable non-viral vector can be used. In some cases, the non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

[0062] When one or more nucleic acids designed to express an importin polypeptide or fragments thereof are administered to a mammal (eg, a human), the nucleic acid may be contained within a nanoparticle (eg, a liposome).

[0063] In addition to the nucleic acid encoding an importin polypeptide or a fragment thereof, a vector (e.g., a viral vector or a non-viral vector) may contain one or more control elements operably linked to the nucleic acid encoding an importin polypeptide or a fragment thereof. Such control elements may include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that regulate the expression (e.g., transcription or translation) of the nucleic acid. The selection of control elements that can be included in a vector depends on several factors, including, but not limited to, inducibility, target target, and desired expression level. For example, a promoter can be included in the vector to promote transcription of the nucleic acid encoding the importin polypeptide. The promoter may be a naturally occurring promoter or a recombinant promoter. Promoters can be ubiquitous or inducible (e.g., in the presence of tetracycline), but can also affect expression of a nucleic acid encoding a polypeptide in a general or tissue-specific manner (e.g., the prion protein (Prp) promoter, the synapsin promoter, the methyl-CpG-binding protein 2 (MeCP2) promoter, the neuron-specific enolase (NSE) promoter, the promoter of the vesicular glutamate transporter promoter (vGLUT), and the Hb9 promoter). Examples of promoters that can be used to drive expression of an importin polypeptide in a cell include, but are not limited to, the cytomegalovirus / chicken β-actin (CBA) promoter, the cytomegalovirus (CMV) promoter, the ubiquitin C (UbC) promoter, the Prp promoter, the synapsin promoter, the MeCP2 promoter, the NSE promoter, the vGLUT promoter, and the Hb9 promoter. As used herein, "operably linked" refers to the positioning of control elements within a vector in relation to a nucleic acid encoding a polypeptide in such a manner as to allow or facilitate expression of the encoded polypeptide.For example, a vector can contain a promoter and a nucleic acid encoding an importin polypeptide or fragment thereof, where the promoter is operably linked to the nucleic acid encoding the importin polypeptide or fragment thereof such that it drives expression of the importin polypeptide or fragment thereof in the cell.

[0064] In some cases, the nucleic acid encoding the importin polypeptide or a fragment thereof may contain a nucleic acid encoding a detectable label. For example, a vector may contain a nucleic acid encoding an importin polypeptide or a fragment thereof and a nucleic acid encoding a detectable label (arranged such that the encoded polypeptide is a fusion polypeptide comprising an importin polypeptide or a fragment thereof fused to a detectable polypeptide). In some cases, the detectable label may be a peptide tag. In some cases, the detectable label may be a fluorescent molecule (e.g., a fluorescent dye and a fluorescent polypeptide). Examples of detectable labels that can be used as described herein include, but are not limited to, HA tag, Myc tag, FLAG tag, green fluorescent polypeptide (GFP; e.g., enhanced GFP), red fluorescent polypeptide (e.g., mCherry) polypeptide, Halo Tag, SNAP tag, 6xHis tag, GST tag, MBP tag, Strep-Tag, and V5 tag.

[0065] Nucleic acids encoding importin polypeptides or fragments thereof can be produced by techniques including, but not limited to, standard molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques. For example, PCR or RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acid (e.g., genomic DNA or RNA) encoding an importin polypeptide.

[0066] In some cases, one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) may be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a human). For example, one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) may be formulated into a pharma- ceutically acceptable composition for administration to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy). In some cases, one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) may be formulated with one or more pharma- ceutically acceptable carriers (additives), excipients, and / or diluents.Examples of pharma- ceutically acceptable carriers, excipients, and diluents that can be used in the compositions described herein include, but are not limited to, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses, such as microcrystalline cellulose, and cellulose ethers, such as hydroxypropyl cellulose (HPC) and the cellulose ether hydroxypropylmethylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cross-linked polyvinylpyrrolidone (crospovidone), carboxymethylcellulose, polyethylene-polyoxypropylene-block polymers, and cross-linked sodium carboxymethylcellulose (croscarmellose sodium)), titanium dioxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methylparaben and propylparaben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts, or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, sodium acetate, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, SM-102, dimyristoyl glycerol, cholesterol, tromethamine, and lecithin.

[0067] Compositions (e.g., pharmaceutical compositions) containing one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragment thereof) can be formulated into any suitable dosage form, including solid or liquid forms, including but not limited to gels, liquids, suspensions, solutions (e.g., sterile solutions), sustained release formulations, and delayed release formulations.

[0068] Compositions (e.g., pharmaceutical compositions) containing one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express importin polypeptides or fragments thereof) may be designed for parenteral (e.g., intravenous, intracerebral, intraventricular, and intrathecal) administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules or vials, and may be stored in a freeze-dried (vacuum lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0069] Compositions (e.g., pharmaceutical compositions) containing one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) can be administered locally or systemically. For example, compositions containing one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) can be administered locally by direct injection (e.g., intracerebral injection) into the CNS (e.g., brain) of a mammal (e.g., human).

[0070] An effective amount of a composition (e.g., a pharmaceutical composition) containing one or more importin polypeptides or fragments thereof (and / or a nucleic acid designed to express an importin polypeptide or fragment thereof) can be any amount capable of treating a mammal without producing significant toxicity to the mammal. For example, an effective amount of one or more importin polypeptides or fragments thereof can range from about 0.1 milligrams of polypeptide per kilogram of body weight (mg / kg) per dose to about 100 mg / kg per dose of the mammal. (For example, per dose, about 0.1 mg / kg to about 75 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 100 mg / kg, about 10 mg / kg to about 100 mg / kg, about 20 mg / kg to about 100 mg / kg, about 30 mg / kg to about 100 mg / kg, about 50 mg / kg to about 10 The effective amount may be about 0 mg / kg, about 75 mg / kg to about 100 mg / kg, about 1 mg / kg to about 75 mg / kg, about 10 mg / kg to about 50 mg / kg, about 20 mg / kg to about 40 mg / kg, about 1 mg / kg to about 10 mg / kg, about 10 mg / kg to about 30 mg / kg, about 30 mg / kg to about 50 mg / kg, about 40 mg / kg to about 60 mg / kg, about 50 mg / kg to about 70 mg / kg, about 60 mg / kg to about 80 mg / kg, or about 70 mg / kg to about 90 mg / kg). The effective amount may remain constant or may be adjustable, such as by sliding the scale or modifying the dose, depending on the mammal's response to treatment. Various factors may affect the actual effective amount used in a particular application. For example, frequency of administration, duration of treatment, use of multiple treatments, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.

[0071] The frequency of administration of a composition (e.g., a pharmaceutical composition) containing one or more importin polypeptides or fragments thereof (and / or a nucleic acid designed to express an importin polypeptide or fragment thereof) can be any frequency that can treat a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) without producing significant toxicity to the mammal. For example, the frequency of administration can be about twice a day to about once a week, about twice a day to about twice a week, or about once a day to about twice a week. The frequency of administration can remain constant or can be changed during the treatment period. The course of treatment with a composition containing one or more chimeric vesiculoviruses provided herein can include a drug holiday. As with the effective amount, various factors can affect the actual frequency of administration used in a particular application. For example, the effective amount, the duration of treatment, the use of multiple treatment agents, the route of administration, and the severity of the condition may require an increase or decrease in the frequency of administration.

[0072] The effective period of administering a composition (e.g., a pharmaceutical composition) containing one or more importin polypeptides or fragments thereof (and / or a nucleic acid designed to express an importin polypeptide or fragment thereof) can be any period of time that treats a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) without producing significant toxicity to the mammal. For example, the effective period can vary from days to weeks, months, or even years. In some cases, the effective period for treating a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) can be for a period of about one month to about ten years. Several factors can affect the actual effective period used in a particular treatment. For example, the effective period can vary with the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the condition being treated.

[0073] In some cases, one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) can be used as the sole active agent used to treat a mammal (e.g., a human) having or at risk for developing a proteinopathy (e.g., a TDP-43 proteinopathy).

[0074] In some cases, the methods and materials described herein may include one or more (e.g., 1, 2, 3, 4, 5, or more) additional therapeutic agents used to treat a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy). Examples of therapeutic agents used to treat a proteinopathy (e.g., a TDP-43 proteinopathy) that can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) in conjunction with one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragment thereof) include, but are not limited to, antidepressants, antipsychotics, riluzole (e.g., RILUTEK®), edaravone (e.g., RADICAVA®), molecules that can target C9orf72 repeat expansions (e.g., antisense oligonucleotides), molecules that can target amyloid beta (Aβ) polypeptides (e.g., Aβ polypeptides present in amyloid plaques) (e.g., antibodies), and molecules that can target tau polypeptides (e.g., tau polypeptides present in neurofibrillary tangles) (e.g., antibodies). In some cases, the one or more additional therapeutic agents can be administered together with one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof; e.g., in the same composition). In some cases, the one or more additional therapeutic agents can be administered independently from the one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof). When the one or more additional therapeutic agents are administered independently from the one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof), the one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express an importin polypeptide or fragments thereof) can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa.

[0075] In some cases, the methods and materials described herein may include subjecting a mammal having or at risk of developing a proteinopathy (e.g., TDP-43 proteinopathy) to one or more (e.g., 1, 2, 3, 4, 5, or more) additional treatments (e.g., therapeutic interventions) that are effective in treating the proteinopathy (e.g., TDP-43 proteinopathy). Examples of therapies that can be used to treat a proteinopathy include, but are not limited to, physical therapy, occupational therapy, speech disorder therapy, and any combination thereof. In some cases, the one or more additional treatments that are effective in treating a proteinopathy (e.g., TDP-43 proteinopathy) can be administered simultaneously with the administration of one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express importin polypeptides or fragments thereof). In some cases, the one or more additional treatments that are effective in treating a proteinopathy (e.g., TDP-43 proteinopathy) can be administered before and / or after the administration of one or more importin polypeptides or fragments thereof (and / or nucleic acids designed to express importin polypeptides or fragments thereof).

[0076] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0077] [Example] [Example 1] Nuclear import receptors (NIRs) reduce TDP-CTF aggregates

[0078] This example describes the discovery that NIR polypeptides can reduce TDP-43 aggregation.

[0079] method Cell culture and transfection Human HEK293 cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293 and SH-S5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0080] Immunofluorescence and image acquisition Cells were seeded on coverslips in 12-well plates, transfected for 24 h, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. For high-resolution imaging, z-series image stacks were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera (Zyla sCMOS, Andor). In each experiment, all groups were imaged using identical acquisition settings. Image stacks were deconvolved using a 3D blind constrained iterative algorithm (Nikon NIS Elements).

[0081] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). Samples were left at room temperature for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction.

[0082] For immunoblotting, samples were boiled in Laemmli sample buffer at 98°C for 5 min and separated in Bolt™ 4-12% Bis-Tris gels (Fisher). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Fisher). Membranes were blocked with Odyssey blocking buffer (LiCor) for 1 h, then incubated with primary antibodies (anti-TDP-43 (1:1000, 12892-1-AP, Proteintech) and anti-β-tubulin (1:1000, DHSB)) at 4°C overnight and incubated with secondary antibodies (1:10000, LiCor) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Blots were imaged using an Odyssey scanner (LiCor).

[0083] Immunoprecipitation HEK293 cells were seeded in 6-well plates, transfected for 48 h, then lysed in IP lysis buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. A small aliquot of the collected supernatant was set aside (input). Pulldown was performed using GFP- and RFP-TRAP magnetic beads (Chromotek) according to the manufacturer's protocol. The beads were briefly washed twice in wash buffer (10 mM Tris / Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The lysate was then incubated with the equilibrated beads overnight at 4°C with end-over-end rotation. The next day, the beads were magnetically separated from the supernatant (flow-through) and washed three times with wash buffer. Bound proteins were released from the beads in elution buffer (200 mM glycine, pH 2.5). Samples were boiled in Laemmli buffer at 95°C for 10 min and used for immunoblotting.

[0084] Cell death assay Cell death in SH-SY5Y cells was assessed by uptake of membrane-impermeable Live-or-Dye™ Fixable Dead Cell Dye 640 / 662 (Live-or-Dye™ Fixable Viability Staining Kit, 32007, Biotium). The dye can penetrate dead cells with compromised membrane integrity and label amines on intracellular proteins. Cells were co-transfected with mCherry / mCherry-TDP-CTF and EGFP / EGFP-tagged transportin constructs using FuGene6 (Promega). 48 hours after transfection, cells were incubated with the dye for 30 minutes at 37°C in the dark, then fixed in 4% paraformaldehyde and imaged using an epifluorescence microscope (Nikon Eclipse Ti2). Cell death was assessed by counting the number of dead cells that had taken up the dye.

[0085] result The import receptor KPNB1 reduces TDP-CTF aggregates. We found that expression of importin β1 / karyopherin β1 (KPNB1) reduced the aggregation of insoluble TDP-CTF proteins in the cytoplasm, resulting in a significant decrease in TDP-CTF protein levels, while endogenous TDP-43 levels were unaffected (Figure 1).

[0086] Specific nuclear import receptors (NIRs) reduce TDP-CTF aggregates To clarify whether this effect was specific to KPNB1, all seven alpha-type and 20 beta-type karyopherins were obtained or cloned as expression constructs and tested for their ability to reduce TDP-CTF aggregation in SH-SY5Y cells. KPNB1 / IPO1, IPO3, 4, 9, and 13 had the strongest effect on reducing TDP-CTF aggregation (Figure 2), whereas exportin and the seven α-karyopherins had no significant effect.

[0087] The N-terminal 112-end fragment of KPNB1 reduces TDP-CTF aggregation To determine the minimal domain of KPNB1 that is necessary and sufficient for activity against TDP-43, we generated an extensive series of truncated KPNB1 constructs based on sequential deletion of α-helical HEAT repeats (H1-19). These constructs were used to map the most active domain within the N-terminal half of KPNB1 H1-8 (Figure 3). Shorter fragments become unstable or progressively lose their activity against TDP-CTFs.

[0088] Specific NIRs restore nuclear localization of TDP-43 variants lacking a functional NLS The specific NIR is expressed by the C-terminal fragment TDP-CTF (TDP-43 208~414 ) and full-length TDP-43 with a mutated NLS (TDP-43 ΔNLS), as well as restored nuclear localization of TDP-43 constructs lacking a functional NLS (nuclear localization signal). The NIRs KPNB1, IPO3A and 3B, and to a lesser extent IPO13, caused nuclear localization of both normally cytoplasmic TDP-43 constructs (Figure 4). The localization of a truncated TDP-43 (short TDP; sTDP) splicing isoform that has an active NES (nuclear export sequence) at its C-terminus was not significantly affected. To determine which domains of IPO3 are necessary and sufficient for its activity on TDP-43 constructs, a series of truncation mutants were generated and tested. Results showed that the IPO3b H8~20 fragment significantly inhibited the TDP-43 mNLS It is clear that TDP-43 has the strongest activity in restoring the nuclear localization of TDP-43 (Figures 5A and 5C). mNLS C-terminal deletions cause the IPO3b fragment to become increasingly cytoplasmic. mNLS The active IPO3b H8-20 fragment also reduces insoluble TDP-CTF protein levels and TDP-CTF-induced cell death.

[0089] N- and C-terminal truncated fragments of IPO13 restore nuclear localization of TDP-CTFs. In the case of IPO13, the full-length protein is expressed by the TDP-CTF (Figure 2B) and TDP-43 ΔNLS The TDP-CTFs have strong aggregation-reducing activity (Figure 6A) and are involved in the nuclear localization of TDP-CTFs in some cells (Figure 4A). This activity was not observed in the truncated constructs (N-terminal fragment H1-11 and C-terminal fragment H4-19) which are sufficient to strongly localize TDP-CTFs in the nucleus, but in the TDP-43 ΔNLS This is different from the results of the previous study (Figures 6A and 6B), in that the strongest activity involved in the nuclear localization of TDP-CTFs resides in H4 to H11, whereas TDP-43 ΔNLS We demonstrate that the strongest activity in reducing aggregation of required all 20 HEAT repeats and is present in amino acid residues 1 to 934 (Figures 6A and 6B).

[0090] TDP-43 associates with KPNB1 via its RRM2 domain To determine which domains are necessary and sufficient to participate in the association with KPNB1, a series of TDP-43 constructs were tested (Figure 7). In pull-down experiments from lysates, the strongest association was found with a sequence from aa 229 to 259 within the RRM2 domain. A weaker association involved its C-terminal prion-like domain. This demonstrates that the NLS is not required for the association of KPNB1 with the TDP-43 fragment.

[0091] Specific NIR reduces TDP-43-mediated toxicity To clarify whether restoring the solubility of pathological TDP-43 would be beneficial for cell survival, we performed cell death assays of SH-SY5Y cells expressing toxic TDP-43 constructs and selected NIRs. Experiments with TDP-CTFs revealed that co-expressed KPNB1, and even more so IPO13, reduced cell death, whereas the non-disaggregating NIRs KPNA1 and XPO1 showed no such activity (Figure 8), demonstrating that NIRs that reduce TDP-43 aggregation also reduce TDP-43 toxicity in vitro.

[0092] Comparing the activities of KPNB1 constructs against TDP-CTF-induced cell death, consistent with their corresponding activities against reducing TDP-CTF aggregates (Fig. 3A), it is clear that only full-length KPNB1 and its N-terminal fragment, but not the C-terminal fragment, reduced cell death (Fig. 9).

[0093] Collectively, these results demonstrate that NIR polypeptides can be used to treat proteinopathies associated with TDP-43 polypeptide aggregation, for example, NIR polypeptides can be used to prevent TDP-43 polypeptides from undergoing pathological phase transitions, reverse the formation of insoluble aggregates, restore TDP-43 nuclear localization, and / or restore TDP-43 function in the nucleus.

[0094] [Example 2] KPNB1 reduces TDP-43 aggregation in multiple cell models containing disease-associated mutations.

[0095] method Cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0096] Immunofluorescence and image acquisition Cells were seeded on coverslips in 12-well plates, transfected for 24 h, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. For high-resolution imaging, z-series image stacks were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera (Zyla sCMOS, Andor). In each experiment, all groups were imaged using identical acquisition settings. Image stacks were deconvolved using a 3D blind constrained sequential algorithm (Nikon NIS Elements).

[0097] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). Samples were left at room temperature for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction. For immunoblotting, samples were boiled at 98 °C for 5 min in Laemmli sample buffer and separated in BoltTM 4-12% Bis-Tris gels (Fisher). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Fisher). Membranes were blocked with Odyssey blocking buffer (LiCor) for 1 h, then incubated with primary antibodies (anti-TDP-43 (1:1000, 12892-1-AP, Proteintech), anti-GFP (1:2000, Aves Labs), anti-β-tubulin (1:1000, DHSB)) at 4°C overnight, and incubated with secondary antibodies (1:10000, LiCor) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Blots were imaged using an Odyssey scanner (LiCor).

[0098] result Full-length KPNB1 successfully blocks the aggregation of TDP-43-CTF in co-transfected cells (Figure 13A). TDP-43 containing various disease-associated mutations (Q331K, M337V, or A382T) is similarly affected in transfected cell models (Figure 13B). Aggregation of TDP-43-CTF engineered to lack the C-terminal prion-like domain (sTDP) or to contain a mutated nuclear localization signal (mNLS) is also reduced by expression of KPNB1 (Figures 13C and 13D). The inhibitory activity of KPNB1 is also maintained in more physiologically relevant primary cortical neurons (Figures 13E-G). KPNB1 exhibits robust activity against a range of disease-associated forms of TDP-43 in multiple cellular environments.

[0099] [Example 3] KPNB1 can be modified to maintain its functional activity.

[0100] method Cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0101] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). Samples were left at room temperature for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction. For immunoblotting, samples were boiled at 98 °C for 5 min in Laemmli sample buffer and separated in BoltTM 4-12% Bis-Tris gels (Fisher). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Fisher). Membranes were blocked with Odyssey blocking buffer (LiCor) for 1 h, then incubated with primary antibodies (anti-TDP-43 (1:1000, 12892-1-AP, Proteintech), anti-GFP (1:2000, Aves Labs), anti-β-tubulin (1:1000, DHSB)) at 4°C overnight, and incubated with secondary antibodies (1:10000, LiCor) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Blots were imaged using an Odyssey scanner (LiCor).

[0102] Immunoprecipitation HEK293T cells were seeded in 6-well plates and transfected for 48 h. Cells were then lysed in IP lysis buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. A small aliquot of the collected supernatant was set aside (input). Pulldown was performed using GFP- and RFP-TRAP magnetic beads (Chromotek) according to the manufacturer's protocol. The beads were briefly washed twice in wash buffer (10 mM Tris / Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The lysate was then incubated with the equilibrated beads overnight at 4°C with end-over-end rotation. The next day, beads were magnetically separated from the supernatant (flow-through) and washed three times with wash buffer. Bound proteins were released from the beads in elution buffer (200 mM glycine, pH 2.5). Samples were boiled in Laemmli buffer at 95°C for 10 min and used for immunoblotting. The following primary antibodies were used: anti-Mab414 (1:1000, Abcam), anti-mCherry (1:1000, Novus Biologicals), anti-GFP (1:2000, Aves Labs), anti-Nup62 (1:1000, BD labs), anti-Nup50 (1:1000, Abcam), anti-Ran (1:2000, Sigma), and anti-importin α / KPNA2 (1:1000, Novus Biologicals).

[0103] result Wild-type KPNB1 has the ability to directly bind to both wild-type TDP-43 and TDP-43-CTF (the C-terminal fragment of the aggregation-prone protein) (Figure 14A). KPNB1 maintains its activity toward GFP-TDP-43-CTF regardless of the presence or absence of N-terminal mCherry fluorescent protein modification (Figure 14B). The function of KPNB1 may be enhanced through its interaction with the C-terminal fragment of TDP-43.

[0104] [Example 4] The FG-Nup interaction sequence is required for the reduction of TDP-43 aggregation by KPNB1.

[0105] method Cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0106] Immunofluorescence and image acquisition Cells were seeded on coverslips in 12-well plates, transfected for 24 h, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. For high-resolution imaging, z-series image stacks were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera (Zyla sCMOS, Andor). In each experiment, all groups were imaged using identical acquisition settings. Image stacks were deconvolved using a 3D blind constrained sequential algorithm (Nikon NIS Elements).

[0107] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). Samples were left at room temperature for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction. For immunoblotting, samples were boiled at 98 °C for 5 min in Laemmli sample buffer and separated in BoltTM 4-12% Bis-Tris gels (Fisher). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Fisher). Membranes were blocked with Odyssey blocking buffer (LiCor) for 1 h, then incubated with primary antibodies (anti-TDP-43 (1:1000, 12892-1-AP, Proteintech), anti-GFP (1:2000, Aves Labs), anti-β-tubulin (1:1000, DHSB)) at 4°C overnight, and incubated with secondary antibodies (1:10000, LiCor) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Blots were imaged using an Odyssey scanner (LiCor).

[0108] Immunoprecipitation HEK293T cells were seeded in 6-well plates and transfected for 48 h. Cells were then lysed in IP lysis buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. A small aliquot of the collected supernatant was set aside (input). Pulldown was performed using GFP- and RFP-TRAP magnetic beads (Chromotek) according to the manufacturer's protocol. The beads were briefly washed twice in wash buffer (10 mM Tris / Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The lysate was then incubated with the equilibrated beads overnight at 4°C with end-over-end rotation. The next day, beads were magnetically separated from the supernatant (flow-through) and washed three times with wash buffer. Bound proteins were released from the beads in elution buffer (200 mM glycine, pH 2.5). Samples were boiled in Laemmli buffer at 95°C for 10 min and used for immunoblotting. The following primary antibodies were used: anti-Mab414 (1:1000, Abcam), anti-mCherry (1:1000, Novus Biologicals), anti-GFP (1:2000, Aves Labs), anti-Nup62 (1:1000, BD labs), anti-Nup50 (1:1000, Abcam), anti-Ran (1:2000, Sigma), and anti-importin α / KPNA2 (1:1000, Novus Biologicals).

[0109] result KPNB1 has the ability to bind to phenylalanine- and glycine-rich nucleoporins (FG-Nups). This interaction is maintained even when truncated C-terminally to the ninth HEAT domain (Figure 15A). Mutations in the nucleoporin-interacting sequence (I178A, F217A, Y255A, and I263R; mNIS) disrupt this interaction (Figure 15B). KPNB1-mNIS also shows a reduction in its TDP-43-CTF disaggregation ability (Figures 15C and 15D). This suggests that the disaggregation activity of KPNB1 against TDP-43 depends on its interaction with FG-Nups such as NUP62.

[0110] [Example 5] TDP-CTF binds to KPNB1 via its RRM2 domain.

[0111] method Cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0112] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). Samples were left at room temperature for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction. For immunoblotting, samples were boiled at 98 °C for 5 min in Laemmli sample buffer and separated in BoltTM 4-12% Bis-Tris gels (Fisher). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Fisher). Membranes were blocked with Odyssey blocking buffer (LiCor) for 1 h, then incubated with primary antibodies (anti-TDP-43 (1:1000, 12892-1-AP, Proteintech), anti-GFP (1:2000, Aves Labs), anti-β-tubulin (1:1000, DHSB)) at 4°C overnight, and incubated with secondary antibodies (1:10000, LiCor) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Blots were imaged using an Odyssey scanner (LiCor).

[0113] Immunoprecipitation HEK293T cells were seeded in 6-well plates and transfected for 48 h. Cells were then lysed in IP lysis buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. A small aliquot of the collected supernatant was set aside (input). Pulldown was performed using GFP- and RFP-TRAP magnetic beads (Chromotek) according to the manufacturer's protocol. The beads were briefly washed twice in wash buffer (10 mM Tris / Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The lysate was then incubated with the equilibrated beads overnight at 4°C with end-over-end rotation. The next day, beads were magnetically separated from the supernatant (flow-through) and washed three times with wash buffer. Bound proteins were released from the beads in elution buffer (200 mM glycine, pH 2.5). Samples were boiled in Laemmli buffer at 95°C for 10 min and used for immunoblotting. The following primary antibodies were used: anti-Mab414 (1:1000, Abcam), anti-mCherry (1:1000, Novus Biologicals), anti-GFP (1:2000, Aves Labs), anti-Nup62 (1:1000, BD labs), anti-Nup50 (1:1000, Abcam), anti-Ran (1:2000, Sigma), and anti-importin α / KPNA2 (1:1000, Novus Biologicals).

[0114] result Truncating TDP-43-CTF at its C-terminus demonstrates that removing the PrLD does not inhibit KPNB1 interaction. The interaction is maintained as long as residues 208-239 are present, which includes the entire RRM2 domain (Figure 16A). In the longer form of TDP-43-CTF that contains the PrLD, deletion of residues 230-240 within the RRM2 domain reduces binding to KPNB1 (Figure 16B). This reduced binding has no significant effect on the ability of KPNB1 to disaggregate TDP-43 (Figure 16C).

[0115] [Example 6] FG-Nups are involved in the activity of KPNB1 against TDP-43

[0116] method Cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0117] Immunofluorescence and image acquisition Cells were seeded on coverslips in 12-well plates, transfected for 24 h, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. For high-resolution imaging, z-series image stacks were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera (Zyla sCMOS, Andor). In each experiment, all groups were imaged using identical acquisition settings. Image stacks were deconvolved using a 3D blind constrained sequential algorithm (Nikon NIS Elements).

[0118] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). Samples were left at room temperature for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction. For immunoblotting, samples were boiled at 98 °C for 5 min in Laemmli sample buffer and separated in BoltTM 4-12% Bis-Tris gels (Fisher). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Fisher). Membranes were blocked with Odyssey blocking buffer (LiCor) for 1 h, then incubated with primary antibodies (anti-TDP-43 (1:1000, 12892-1-AP, Proteintech), anti-GFP (1:2000, Aves Labs), anti-β-tubulin (1:1000, DHSB)) at 4°C overnight, and incubated with secondary antibodies (1:10000, LiCor) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at room temperature. Blots were imaged using an Odyssey scanner (LiCor).

[0119] Immunoprecipitation HEK293T cells were seeded in 6-well plates and transfected for 48 h. Cells were then lysed in IP lysis buffer (Fisher) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. A small aliquot of the collected supernatant was set aside (input). Pulldown was performed using GFP- and RFP-TRAP magnetic beads (Chromotek) according to the manufacturer's protocol. The beads were briefly washed twice in wash buffer (10 mM Tris / Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The lysate was then incubated with the equilibrated beads overnight at 4°C with end-over-end rotation. The next day, beads were magnetically separated from the supernatant (flow-through) and washed three times with wash buffer. Bound proteins were released from the beads in elution buffer (200 mM glycine, pH 2.5). Samples were boiled in Laemmli buffer at 95°C for 10 min and used for immunoblotting. The following primary antibodies were used: anti-Mab414 (1:1000, Abcam), anti-mCherry (1:1000, Novus Biologicals), anti-GFP (1:2000, Aves Labs), anti-Nup62 (1:1000, BD labs), anti-Nup50 (1:1000, Abcam), anti-Ran (1:2000, Sigma), and anti-importin α / KPNA2 (1:1000, Novus Biologicals).

[0120] result Along with interacting with KPNB1, Nup62 colocalizes with TDP-43 aggregates as long as the PrLD is maintained (Figure 17A). KPNB1 showed reduced disaggregation activity when the V, L, I, and / or M residues in the PrLD were mutated to F (VLIM-F) (Figure 17B). These same mutations abolished the interaction between KPNB1 and TDP-43 (Figure 17C). Colocalization between TDP-43 and Nup62 was also reduced by VLIM-F mutation (Figure 17D). TDP-43 containing VLIM-F mutation also showed resistance to KPNB1 disaggregation (Figure 17E). This suggests that the disaggregation activity of KPNB1 against TDP-43 depends on the tripartite interaction with FG-Nup.

[0121] [Example 7] KPNB1 reduces TDP-43 aggregation in a familial model of ALS (C9ALS)

[0122] method Cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS and transfected with PolyMag Neo (Oz Biosciences) according to the manufacturer's protocol. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS and transfected with NeuroMag Neo (Oz Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were obtained from ATCC (CRL-1573 and CRL-2266, respectively).

[0123] Immunofluorescence and image acquisition Cells were seeded on coverslips in 12-well plates, transfected for 24 h, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. For high-resolution imaging, z-series image stacks were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera (Zyla sCMOS, Andor). In each experiment, all groups were imaged using identical acquisition settings. Image stacks were deconvolved using a 3D blind constrained sequential algorithm (Nikon NIS Elements).

[0124] result Expression of C9orf72 with a large GR extension is associated with the familial ALS variant C9ALS. When polyGR C9orf72 is present, it coaggregates with both TDP-43 and Nup62 (Figure 18A). Introduction of KPNB1 successfully reduced aggregation. Truncated KPNB1 (H1-H8) shows increased efficacy in this family model of aggregation, but mutation of NIS blocks this inhibitory activity (Figure 18B). This suggests that active truncation of KPNB1 maintains the interaction with both FG-Nups and TDP-43, allowing KPNB1 activity to be exerted.

[0125] [Example 8] Nuclear import receptors are recruited by FG-nucleoporins and reduce hallmarks of TDP-43 proteinopathy

[0126] This example describes the discovery that one or more importin polypeptides and / or fragments thereof (and / or nucleic acids designed to express importin polypeptides and / or fragments thereof) can rescue all hallmarks of TDP-43 pathology by restoring TDP-43 polypeptide solubility and TDP-43 polypeptide subnuclear localization, thereby reducing neurodegeneration in cellular and animal models of ALS / FTD.

[0127] The results in this example reproduce and expand on at least some of the results provided in other examples.

[0128] method DNA constructs Expression plasmids used in this study were obtained or generated from multiple sources as summarized in Table 9. Flexible Linker (GGGS) 3 (SEQ ID NO: 80) was inserted between the fluorescent protein fusion proteins to ensure proper protein folding.

[0129] [Table 7] TIFF2024520414000028.tif250160TIFF2024520414000029.tif247160

[0130] Mammalian cell culture and transfection Human HEK293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS. SH-SY5Y neuroblastoma cells were cultured in DMEM / F12 medium (Life Technologies) containing 10% FBS. Cells were transfected with NeuroMag Neo (OZ Biosciences) according to the manufacturer's protocol. HEK293T and SH-SY5Y cells were purchased from ATCC (CRL-1573 and CRL-2266, respectively).

[0131] Immunostaining and image acquisition and analysis Cells were seeded on cover slips in 12-well plates. 24 hours after transfection, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature (RT). GFP-(GR) 100For experiments with , cells were fixed 48 h after transfection, permeabilized with 0.2% Triton X-100 in PBS for 10 min, blocked with 5% bovine serum albumin (BSA) for 45 min, and incubated overnight at 4°C with the following primary antibodies: anti-FLAG (1:500), anti-TDP-43 (1:500), anti-Nup62 (1:250), anti-Nup98 (1:250), anti-Ran (1:200), and anti-KPNB1 (1:100), followed by incubation with fluorophore-coupled secondary antibodies for 1 h at RT.

[0132] For high-resolution fluorescence microscopy, single and multiple z-plane images were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera (Zyla sCMOS, Andor). In each experiment, all groups were imaged using identical acquisition settings. Image stacks were deconvolved using a 3D blind constrained sequential algorithm (Nikon NIS Elements).

[0133] To quantify the spatial overlap between Nup62-mCherry and GFP-tagged TDP-43 constructs, Mander's colocalization coefficient was calculated on ImageJ using the JACoP plugin (Bolte and Cordelieres, 2006). Values ​​close to 0 and 1 indicate weak and complete colocalization, respectively.

[0134] As a reporter for protein nucleocytoplasmic transport, NES-tdTomato-NLS was co-transfected with GFP or GFP-tagged KPNB1 constructs in SH-SY5Y cells. The average pixel intensity of NES-tdTomato-NLS in the nucleus and cytoplasm was measured, and the nucleus-to-cytoplasm (N-to-C) ratio was calculated.

[0135] GFP-tagged TDP-43 mNLS To semiquantitatively determine the nuclear-cytoplasmic localization of TDP-43, HEK293T cells were transfected with GFP-TDP-43 mNLSand different mCherry-KPNB1 constructs, fixed, and z-stacks acquired as described above. Images were blinded across conditions and were mNLS Fluorescence signals were characterized as nuclear / nucleocytoplasmic / cytoplasmic in triplicates for at least 20 cells per condition.

[0136] Cell lysis, subcellular fractionation, and immunoblotting Cells were seeded in 12-well plates and transfected for 48 h. Cells were lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as detergent soluble fraction and the insoluble pellet was recovered in urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM Tris, pH 8, Roche complete protease inhibitor cocktail). For experiments with total protein lysates, cells were collected in urea buffer. Samples were left at RT for 20 min, then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as detergent insoluble fraction. For immunoblotting, samples were boiled in Laemmli sample buffer at 98°C for 5 min and separated in Bolt™ 4-12% Bis-Tris gels (Thermo Fisher Scientific). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 gel transfer device (Thermo Fisher Scientific). Membranes were blocked with Odyssey blocking buffer (LI-COR) for 1 h and then incubated with the following primary antibodies at 4°C overnight: anti-mCherry (1:1,000), anti-GFP (1:2,000), anti-β-tubulin (1:1,000), anti-β-actin (1:1,000), and anti-KPNB1 (1:1,000), and then incubated with secondary antibodies (1:10,000) in PBS blocking buffer containing 0.05% Tween 20 for 1 h at RT. Blots were imaged using an Odyssey scanner (LI-COR).

[0137] Immunoprecipitation (IP) HEK293T cells were seeded in 6-well plates. 48 hours after transfection, cells were lysed in IP lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche) and centrifuged at 15000 rpm for 20 min at 4°C. A small amount of the collected supernatant was collected as the input extract and the rest was used for IP. Pulldown was performed using GFP- and RFP-Trap magnetic beads (Chromotek) according to the manufacturer's protocol. The beads were briefly washed twice in wash buffer (10 mM Tris / Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The lysate was then incubated with the equilibrated beads overnight at 4°C with end-over-end rotation. The beads were magnetically separated from the supernatant (flow-through) and washed three times with wash buffer. Bound proteins were released from the beads in elution buffer (200 mM glycine, pH 2.5). Samples were boiled for 10 min at 95°C in Laemmli buffer and used for immunoblotting. The following primary antibodies were used: anti-mCherry (1:1,000), anti-GFP (1:2,000), anti-mAb414 (1:1,000), anti-Nup62 (1:1,000), anti-Nup50 (1:1,000), anti-importin α1 (1:1,000), and anti-Ran (1:2,000).

[0138] RNA isolation and real-time RT-PCR RNA from cultured HEK293T cells was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. The isolated RNA was treated with RQ1 DNase (Promega) and used for cDNA synthesis with the SuperScript First-Strand Synthesis System (Invitrogen) using oligo-dT and random hexamer primers. Real-time RT-PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan Gene Expression Assays for EGFP (Mr04097229_mr, Thermo Fisher Scientific) and human GAPDH (Hs02758991_g1, Thermo Fisher Scientific) on a QuantStudio 7 Flex Real-Time PCR system (Applieded Biosystems).

[0139] Cell death assay Cell death in SH-SY5Y cells was assessed by uptake of membrane-impermeable Live-or-Dye™ Fixable Dead Cell Dye 640 / 662 (Live-or-Dye™ Fixable Viability Staining Kit, 32007, Biotium). The dye penetrates dead cells with compromised membrane integrity and labels amines on intracellular proteins. Cells were co-transfected with mCherry / mCherry-TDP-CTF and GFP / GFP-tagged KPNB1 constructs using FuGene6 (Promega). 48 hours after transfection, cells were incubated with the dye for 30 minutes at 37°C in the dark, then fixed in 4% PFA and imaged using an epifluorescence microscope (Nikon Eclipse Ti2). Cell death was assessed by scoring the percentage of dead cells that had taken up the dye.

[0140] Simultaneous immunofluorescence on human postmortem tissues In addition to tissues from neuropathologically normal control tissue blocks, tissues from the spinal cord and hippocampus were obtained from patients with ALS or FTLD, respectively. Neuropathological curation of cases consisted of sporadic and familial disease, and screened cases were available for patients with ALS or FTLD due to genetic mutations in C9orf72, SOD1, or TARDBP. Demographics of postmortem cases are presented in Table 7. After brains were processed in 10% neutral buffered formalin, tissue blocks were selected during gross neuroanatomical examination and embedded in paraffin for preservation and sectioning. Double fluorescent immunostaining was performed on 5 μm formalin-fixed paraffin-embedded (FFPE) tissue sections obtained from the spinal cord or posterior hippocampus. FFPE tissue sections were deparaffinized by immersion in xylene for a short period and rehydrated through a graded series of ethanol solutions (100%, 90%, and 70% ethanol). Tissues were then transferred to dHO for 3 min. 2 Antigen retrieval was performed by rinsing in 2020 and steaming the tissue sections in citrate buffer, pH 6 (Dako Target Retrieval Solution, S2369) for 30 min. The tissue sections were cooled to RT and then eluted in dHO. 2The sections were then rinsed with O for 10 min. Sections were permeabilized and blocked with serum-free protein block containing 0.3% Triton X100 for 1 h at RT. Tissues were immunostained with antibodies against pTDP-43 (1:200), Nup62 (1:100), or KPNB1 (1:2000), diluted in antibody diluent (Dako, S0809) and incubated overnight at 4°C in a humidified chamber. Tissue sections were washed three times for 10 min each in a solution of Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T). Alexa Fluor-conjugated secondary antibodies (1:1000, F'(ab) anti-mouse 488, or anti-rat 555, or anti-rat 647, Thermo Fisher Scientific) were diluted in antibody diluent (Dako, S0809) and incubated for 2 h at RT. Tissues were washed with TBS-T and incubated with Hoechst 33342 (1:1000, Thermo Fisher Scientific) for 20 min at RT, then rinsed with TBS. To quench autofluorescence, tissues were stained with 1× True Black lipofuscin autofluorescence quencher (Biotium, 23007) for 30 s and resuspended in dH 2The tissue sections were mounted on glass slides using ProLong Glass Antifade Mountant (Invitrogen). Optical sections were acquired based on tissue depth to perform high-resolution imaging and capture the complete contours of pTDP-43 aggregates. Z-series were acquired using an ECLIPSE Ti2 fluorescence microscope (Nikon) equipped with a Spectra X multi-LED light engine (Lumencor), single bandpass filter cubes for DAPI, EGFP / FITC, and TRITC (Chroma), and a ZYLA 4.2 PLUS sCMOS camera (Andor) using NIS Elements HC V5.30 software (Nikon). Immunofluorescence staining of all cases and CNS regions was performed simultaneously, and all pathological subgroups were imaged using the same acquisition environment. Out-of-focus light from each z-series was removed using the Clarify.ai module of the NIS-Elements Advanced Research (V5.30) deconvolution package. To demonstrate co-localization of KPNB1 and Nup62 with pTDP-43 inclusions, the cleared z-series are presented as optical projections and 3D volume-rendered views. Fluorescent tile images of the same tissue section were captured using a 40× objective on an inverted fluorescent microscope (Keyence BZ-X800), and high-resolution image stitching was completed using the Keyence analyzer package (BZ-X series).

[0141] [Table 8]

[0142] Organotypic brain slice cultures, transduction, and immunohistochemistry Brain slice cultures (BSCs) were prepared from postnatal day 8–9 (P8–9) C57BL / 6 mice. Pups were placed in a chamber containing isoflurane and decapitated after a toe-pinch response. Hemibrains were dissected and the cortex and hippocampus were collected in sterile filtered ice-cold dissection buffer (Hank's Balanced Salt Solution (HBSS), calcium, magnesium, phenol red free (Thermo Fisher Scientific), 2 mM ascorbic acid (Sigma Aldrich), 39.4 μM ATP (Sigma Aldrich), and 1% (v / v) penicillin / streptomycin (Thermo Fisher Scientific)). Each hemibrain was placed on filter paper and cut into 350 μm slices using a McIllwain™ tissue chopper (Stoelting Co.). Slices were placed in 6-well tissue culture plates to accommodate 3 slices per semi-porous membrane insert per well (pore diameter 0.4 μm, MilliporeSigma) in sterile filtered culture medium (Basal Medium Eagle (Thermo Fisher Scientific), containing 26.6 mM HEPES (pH 7.1, Thermo Fisher Scientific), 511 μM ascorbic acid, 1% (v / v) GlutaMAX (Thermo Fisher Scientific), 0.033% (v / v) insulin (Sigma Aldrich), 1% (v / v) penicillin / streptomycin (Thermo Fisher Scientific), and 25% (v / v) heat-inactivated horse serum (Sigma Aldrich)) at 37°C and 5% CO. 2 The BSCs were maintained at 4 °C for 1 h. The culture medium was changed every 3 to 4 days.

[0143] For AAV transduction of BSCs, AAV plasmids were first transfected in HEK293T using polyethyleneimine hydrochloride (Polysciences). Three to four days after transfection, the cell medium was collected and centrifuged at 800rcf for 5 min to recover the supernatant containing the secreted AAV. AAV was applied directly into the culture medium on day 0 (DIV0) of BSCs.

[0144] For immunostaining of BSCs, slices were fixed 12–15 days after transduction (DIV12–15) with 4% PFA for 1 h at RT, permeabilized with 0.5% Triton X-100 overnight at 4°C, and blocked with 20% BSA for 4 h. BSCs were incubated with the following primary antibodies in 5% BSA at 4°C for 48 h, followed by incubation with fluorophore-coupled secondary antibodies for 4 h at RT. To stain nuclei, slices were incubated with Hoechst dye (1:5000) for 30 min at RT and then coverslipped with ProLong Glass Antifade Mountant (Thermo Fisher Scientific). Nuclear-cytoplasmic localization of TDP-43 was determined semiquantitatively as described above for HEK293T cells.

[0145] statistical analysis Data obtained from different experiments were analyzed by analysis of means (Student's t-test) or analysis of variance (one-way or two-way ANOVA) using GraphPad Prism software. Bonferroni's post-hoc test was used. Differences were considered statistically significant when p < 0.05. All values ​​are means and SEM.

[0146] [Table 9] TIFF2024520414000032.tif235164TIFF2024520414000033.tif82164

[0147] result A subset of β-importin family nuclear cytoplasmic import receptors (NIRs) reduces cytoplasmic aggregation of TDP-CTFs TDP-CTF is a 25 kDa C-terminal fragment of TDP-43 that lacks the NLS but contains a portion of the RRM2 and the complete PrLD, and is a major component of insoluble cytoplasmic aggregates in the cortical and hippocampal regions of ALS and FTLD-TDP patient brain tissue. Its expression is related to phospho-TDP-43 S409 / 410 KPNB1 recapitulates the histopathological features of TDP-43 pathology by forming detergent-insoluble inclusions that are positive for TDP-43, p62 / SQSTM1, and ubiquitin, and induces high levels of toxicity in neuronal cell lines and primary neurons. KPNB1 reduces the cytoplasmic aggregation of TDP-CTFs in human neuroblastoma SH-SY5Y cells, making their localization more nuclear-cytoplasmic (Figure 26A). In complementary biochemical fractionation experiments, expression of KPNB1 significantly reduces detergent-insoluble protein levels of TDP-CTFs and variants carrying ALS-causing point mutations without affecting endogenous nuclear TDP-43 levels (Figures 26A and 26C). Expression of mCherry-KPNB1 or untagged KPNB1 significantly reduced insoluble TDP-CTF protein levels with only minor effects on soluble TDP-CTFs (Figure 26B). In addition, both constructs significantly reduced the total protein levels of TDP-CTFs, but not TDP-43. WT This was not the case for TDP-43, suggesting that KPNB1 specifically targets insoluble TDP-CTFs and reduces their aggregation in a tag-independent manner (Figure 26D). KPNB1 did not affect the transcript levels of either TDP-CTFs or TDP-43 (Figure 26E).

[0148] Mammalian cells express several members of the α- and β-karyopherin families of nuclear transport receptors (which share a similar curved α-solenoid structure composed of stacks of related armadillo or HEAT repeats, respectively). To determine whether KPNB1 is the only transport factor capable of reducing TDP-43 pathology, all seven α-importin, twenty β-importin, and exportin protein family members were tested for their effects on TDP-CTF aggregates in SH-SY5Y cells using fluorescence microscopy (Figure 19A) and quantitative Western blot analysis of insoluble proteins (Figure 19B). Based on the microscopy results, transport receptors were subdivided into four categories: "no effect," "coaggregation," "aggregation reduction" (including proteins that only reduced the size of individual TDP-CTF aggregates), and "no aggregation." Several β-importins, including IPO3 / TNPO2, IPO4, IPO9, and IPO13, were able to reduce insoluble TDP-CTF levels, similar to KPNB1. TNPO1 / KPNB2 did not reduce insoluble TDP-CTF protein levels, but reduced the size of cytoplasmic TDP-CTF inclusions (Figures 19A and 19B). No such effect was found for α-importins and exportins, and some exportins showed coaggregation (Figures 19A and 19B). These results suggest that although KPNB1 and other β-importins differ in their substrate specificity for NLS recognition and nuclear import, they share a common mechanism in reducing TDP-CTF aggregation.

[0149] The N-terminal half of KPNB1 is necessary and sufficient to reduce TDP-CTF aggregation and toxicity Our focus was on gaining a deeper mechanistic understanding of how NIRs reduce TDP-CTF aggregation in KPNB1, the best-characterized member of the NIR family, and on determining the regions necessary and critical for this activity. Since KPNB1 is composed of a highly flexible array of 19 short amphipathic tandem α-helical HEAT repeats, we generated a series of constructs based on this repeat. First, we tested the N-terminal half (HEAT repeats 1–9, H1–9) and the C-terminal half (H10–19) of KPNB1. Just like the full-length protein, KPNB1 H1–9 showed diffuse localization enriched within the nuclear envelope, whereas KPNB1 H10–19 was predominantly located in the nucleocytoplasm (Figure 20A). Expression of full-length KPNB1 and H1-9, except H10-19, reduced TDP-CTF aggregate formation (Figure 20A) and also reduced detergent-insoluble levels of TDP-CTF (Figure 20B) without affecting endogenous nuclear TDP-43 protein levels (Figure 27). Co-expression of full-length KPNB1 or H1-9 significantly reduced TDP-CTF-induced cell death by approximately 58%, while KPNB1 H10-19 had no effect (Figure 20C), demonstrating that the N-terminal portion of KPNB1 is necessary and sufficient to reduce cytoplasmic TDP-43 aggregation and toxicity.

[0150] To further elucidate the active region within the KPNB1 H1-9 fragment, a series of constructs with deletions of the C-terminal HEAT repeats were generated, ranging from H1-8 to H1. Only KPNB1 H1-8 was significantly enriched on the nuclear envelope and identified as the smallest fragment with full activity for reducing TDP-CTF aggregation, conferring TDP-CTF diffusibility, and significantly reducing insoluble TDP-CTF protein levels (approximately 77%) (Figures 20D and 20E). KPNB1 H1-7 and KPNB1 H1-6 were less active, only slightly reducing insoluble TDP-CTF protein levels and aggregate size, whereas the smaller KPNB1 fragments colocalized with TDP-CTF aggregates and did not exhibit any significant chaperone activity to reduce aggregation and solubility (Figures 20E and 20F).

[0151] Reduced TDP-CTF aggregation depends on the FG-Nup interacting domain of KPNB1 Ran GTP We mapped the binding sites for KPNB1 interactors that control its nuclear import activity, including importin-α, importin-α, and FG-Nups that can bind KPNB1 at two distinct Nup-interaction sites (NIS1 and 2) (Figure 21A). Co-immunoprecipitation (co-IP) experiments with KPNB1 constructs were used to map high affinity interactions with FG-Nups, including Nup62 and Nup50, as well as with importin-α1 and Ran. KPNB1 H1-8 and H1-9 interacted with high molecular weight FG-Nups with even higher affinity than full-length KPNB1 (Figure 21B). Although structural studies have revealed that importin-α contacts KPNB1 H7-19 (Cingolani et al., Nature, 399:221-229 (1999)), these results suggest that H1-8 may be necessary and sufficient for the high-affinity interaction, possibly via an acidic loop in H8.

[0152] The KPNB1 construct with the greatest effect on TDP-CTF aggregation is also the construct that binds FG-Nups; this raises the question of whether the proposed chaperone activity of KPNB1 depends on its interaction with FG-Nups. Therefore, to reduce Nup binding, missense mutations (I178A, F217A, Y255A, I263R) were introduced into KPNB1 H1-8 (H1-8 mNIS ) was introduced into NIS1 (Figure 21C). WT Expression of KPNB1 caused a diffuse nuclear-cytoplasmic distribution of TDP-CTF, reduced the formation of TDP-CTF cytoplasmic inclusions, and reduced insoluble TDP-CTF, whereas expression of KPNB1 H1-8 mNIS lost most of its activity (Fig. 21D and Fig. 21E).

[0153] To clarify whether KPNB1 can also reduce the aggregation of cytoplasmically mislocalized endogenous TDP-43, we used a C9ALS / FTD disease model based on the expression of a poly(GR) dipeptide repeat protein derived from the C9orf72 repeat expansion, which has been shown to induce mislocalization of endogenous TDP-43 in mice. 100 KPNB1 H1-8 was found to form both nuclear and cytoplasmic aggregates in cell culture, but only the cytoplasmic aggregates were positive for endogenous TDP-43 and Nup62. Cells with nuclear GR aggregates showed irregular nuclear envelopes, nuclear-cytoplasmic distribution of TDP-43, and abnormal aggregation of Nup62 in the cytoplasm (Figures 28A and 28B). WT KPNB1 H1-8 did not affect GR inclusion formation, but abolished the accumulation of TDP-43 and Nup62 within these aggregates in approximately 60% of cells (Figures 21F-I). Most GR aggregates were mNIS This effect was dependent on the FG-Nup interaction domain, as KPNB1-mediated TDP-43 aggregation downregulation remained positive for TDP-43 and Nup62 in the presence of KPNB1 (Figures 21F-I). Collectively, these results suggest a role for FG-Nups in facilitating KPNB1-mediated TDP-43 aggregation downregulation.

[0154] KPNB1 associates with TDP-CTFs via both RRM2 and PrLD To identify the TDP-43 region required for KPNB1 transduction, a series of interaction domain mapping experiments were performed. Co-IP experiments in HEK293T cell lysates revealed that GFP-TDP-CTF strongly interacted with untagged KPNB1 through both the remaining RRM2 domain fragment (aa208-274) present in the TDP-CTF and, to a lesser extent, its PrLD (aa275-414) (Figure 29).

[0155] To elucidate the interaction domain within the RRM2 region (non-PrLD) of TDP-CTF, a series of TDP-CTF deletion constructs (Figure 30A) were tested in co-IP experiments with KPNB1, and the major interaction domain was mapped to TDP-CTF amino acids 230-240 (Figure 30V and Figure 30C). This indicates that these 10 amino acids (TDP-CTF Δ230~240 ) and TDP-CTF WT This was confirmed by the DP-CTF deletion constructs, which interacted with KPNB1 significantly weaker than that of TDP-CTF (Figure 30D). WT Similarly, TDP-CTF Δ230~240 We were still able to reduce aggregates and insoluble protein levels (Figures 30E and 30F), leading to the conclusion that the region 230-240 within TDP-CTF may be involved in the interaction with KPNB1 but is dispensable for its TDP-CTF aggregation reducing activity.

[0156] These findings suggest that KPNB1 may be a TDP-CTF, TDP-43, mNLS This led to experiments investigating whether PrLD is required for reducing TDP-43 aggregation by co-expressing KPNB1 with TDP-CTFs and TDP-43 (full-length TDP-43 containing a mutant NLS) or short TDP (an alternatively spliced ​​isoform of TDP-43 in which PrLD replaces the nuclear export signal (NES)). mNLS Not only did it reduce aggregation, but it also reduced TDP-43 mNLS KPNB1 also increased the nuclear localization of PrLD in the expression of sTDP-43 (Figure 22A). In contrast, sTDP aggregates were reduced in size but still present when KPNB1 was expressed. Western blot analysis of insoluble TDP-43 protein revealed that KPNB1 reduced the levels of all three TDP-43 constructs, but to a much lesser extent, sTDP (Figure 22B), suggesting an NLS-independent mode of interaction and an important role for PrLD in enabling KPNB1 to reduce protein aggregation.

[0157] Nup62 associates with TDP-43 PrLDs and recruits KPNB1 into the aggregates The TDP-PrLD fragment (aa275-414) is mostly soluble in cells, but can form small cytoplasmic granules that are positive for Nup62 and KPNB1 (Figure 31A and Figure 31B). Since the aggregation-reducing activity of KPNB1 was found to depend on its FG-Nup interaction domain (Figure 21), we investigated whether FG-Nup or Ran could introduce KPNB1 into TDP-43 aggregates. Pull-down experiments in HEK293T cell lysates confirmed that TDP-PrLD interacts with FG-Nups Nup62 and Nup98, as well as KPNB1, but not Ran (Figure 31C), suggesting that Ran is dispensable for the association between TDP-CTFs and KPNB1. To investigate the role of FG-Nups in introducing KPNB1 into TDP-43 aggregates, a series of GFP-tagged TDP-43 constructs were tested for colocalization with mCherry-tagged Nup62, itself previously shown to form large cytoplasmic aggregates in cell culture and to colocalize with TDP-43 aggregates. mNLS was found to significantly co-localize with Nup62-mCherry in HEK293T cells (Figures 22C and 32). mNLS The N-terminal half of TDP-43 mNLS 1-265) and TDP-PrLD are soluble when co-expressed with mCherry, but only TDP-PrLD coaggregates with Nup62-mCherry. Notably, sTDP aggregates lacking PrLD do not colocalize with Nup62, but accumulate around Nup62 inclusions (Figures 22C and 32). Like Nup62, Nup98 also binds to the TDP-CTF, TDP-43. mNLS, and TDP-PrLD, but not sTDP (Figure 35). sTDP also showed reduced interactions with endogenous Nup62, Nup98, and KPNB1 compared to TDP-CTF (Figure 22D). These findings may explain why KPNB1 cannot reduce sTDP aggregation as effectively as TDP-CTF (Figures 22A and 22B). TDP-43 mNLS In comparison, sTDP lacks the PrLD but also has an intact NLS and an additional C-terminal NES that may interfere with the interaction with TDP-43-Nup62. mNLS ΔNES) was co-expressed with Nup62-mCherry and found to still form aggregates around Nup62 foci (Figure 33), confirming that it is the TDP-43 PrLD that is required for its association with Nup62.

[0158] We next tested whether expression of Nup62 could increase the association between TDP-CTF aggregates and KPNB1 in HEK293T cells. KPNB1 significantly reduced the spreading of TDP-CTF aggregates; rare residual TDP-CTF aggregates weakly overlapped with KPNB1, and co-expression of Nup62 dramatically increased the recruitment of KPNB1 into TDP-CTF aggregates (Figure 22E). Nup62 and KPNB1 did not co-localize with sTDP aggregates. The non-FG nucleoporin Nup85 showed significant co-localization with TDP-CTFs but did not recruit KPNB1 into the aggregates (Figure 22E). These findings raise the question of whether this FG-Nup-dependent recruitment of NIR into TDP-43 aggregates might contribute to its aggregation-reducing activity.

[0159] Association of TDP-43 PrLD with Nup62 is required for KPNB1-mediated aggregation reduction To test whether specific mutations occurring within the TDP-43 PrLD that disrupt the association of TDP-43 with Nup62 also inhibit KPNB1-dependent aggregation reduction, we generated an extensive series of PrLD mutation variants (Figure 34). FUSΔ14 was used as a negative control because FUS aggregates do not colocalize with Nup62 (Figure 34D). Deletion of any of the three segments that make up the PrLD (Δ274-313, Δ314-353, or Δ354-393) did not disrupt coaggregation with Nup62, suggesting multiple interactions across the PrLD instead of one specific interaction domain (Figure 34A). Because the TDP-43 PrLD harbors six FG motifs, including four GXFG repeats (Figure 34E), we attempted to clarify whether TDP-CTFs and Nup62 could associate through their respective FG repeats. However, TDP-CTF mutants in which phenylalanine residues in PrLD were replaced with alanine, glycine, or tyrosine residues (FA, FG, or FY) still strongly coaggregated with Nup62 (Figure 34A). The role of charged (KRED), aliphatic (VLIM), and aromatic (FYW) amino acid residues was also examined for the association of Nup62 with PrLD. Only one of these mutations had a strong effect on TDP-CTF colocalization with Nup62: Substitution of nine aliphatic residues with phenylalanine (VLIM-F) reduced the TDP-CTF colocalization with Nup62. VLIM-F Not only did it render TDP-43 more aggregation-prone, but it also prevented its co-localization with Nup62 (Figures 34A and 34B) and Nup98 (Figure 35). Similarly, TDP-43 carrying a VLIM-F mutation in the PrLD mNLS The aggregates were close to, but did not overlap, Nup62 granules (Figure 34E). This allowed us to investigate whether reduced association with Nup62 affected the KPNB1-dependent reduction in aggregation. WT TDP-CTF with KPNB1 compared to VLIM-FThe interaction was reduced in co-IP experiments (Figure 34F), and the insoluble protein levels were only slightly affected by KPNB1 (Figures 34G and 34H). VLIM-F Although it is possible to hypothesize that the introduction of FG-Nups and KPNB1 into inclusion bodies is hindered by their dense nature, other TDP-CTF mutations that promote their aggregation did not affect the association with Nup62. Collectively, these results strongly suggest that FG-Nup interactions play an important role in introducing KPNB1 to reduce protein aggregation.

[0160] KPNB1 reduces TDP-CTF and Nup62 aggregates in a similar manner One plausible model for the canonical activity of NIR in nuclear import is based on its structural flexibility, which allows NIR to form multiple interactions with FG motifs. We investigated whether KPNB1 could dissolve or prevent the formation of Nup62-TDP-43 coaggregates through a similar mechanism. WT Coexpression of Nup62 with KPNB1 led to the formation of smaller Nup62 aggregates that significantly colocalized with KPNB1, whereas KPNB1 mNIS KPNB1 H1-8 weakly associated with Nup62 aggregates and had no effect on their size (Fig. 22F). WT KPNB1 H1~8 abolishes Nup62 aggregates mNIS This was not the case (Figure 22F and Figure 36).

[0161] Although importins and exportins interact with Nup62 and other FG-Nups during nuclear transport, we observed that only a subset of β-importins can reduce TDP-CTF aggregates. IPO13 reduces TDP-CTF aggregation more potently than KPNB1, while TNPO1 partially disaggregates TDP-CTFs into smaller aggregates, and exportins XPO1, XPO7, and RANBP17 coaggregate with TDP-CTFs (Fig. S19A). These transport receptors were coexpressed with Nup62-mCherry, and a very similar effect was also found for Nup62 (Fig. S2G). All three exportins strongly associate with Nup62 aggregates but do not affect their morphology, while IPO13 causes significant dissociation and TNPO1 partially disaggregates TDP-CTFs into smaller aggregates (Fig. S2G). These results indicate that specific transport receptors have similar effects on the formation of both TDP-43 and Nup62 aggregates, suggesting a common mechanism.

[0162] Expression of KPNB1 restores the nuclear localization of mislocalized TDP-43 in primary neurons and mouse brain tissue, independent of its NLS Expression of KPNB1 is related to TDP-43 mNLS Given our unexpected finding that KPNB1 increases the nuclear localization of TDP-43 (Figure 22A), we measured the nuclear-cytoplasmic (N-to-C) ratio of TDP-43 constructs over time upon expression in primary neuronal cultures. WT KPNB1 further increased the N-to-C ratio of cytoplasmic TDP-43, even though it is mainly nuclear, at an early stage 24 hours after transfection (Figures 23A and 23B and 37). However, KPNB1 did not increase the N-to-C ratio of cytoplasmic TDP-43. mNLS and nuclear localization of TDP-CTFs was also significantly increased at 48 and 72 hours post-transfection, demonstrating that KPNB1 can increase the nuclear import of TDP-43 independent of its NLS (Figures 23A and 23B and Figure 37).

[0163] To clarify whether both the aggregation-reducing activity and the nuclear import activity exist within the same domain of KPNB1, we investigated the mechanism of action of TDP-43. mNLS The effect of truncated KPNB1 constructs on the localization of TDP-43 in the nucleus was examined in HEK293T cells. Full-length KPNB1, as well as KPNB1 H1-9 and H1-8, were found to mediate the localization of TDP-43 in the nucleus. mNLS The levels of KPNB1 were significantly increased by KPNB1 H1-7, whereas the nuclear import activity of KPNB1 H1-7 was weak (Fig. 23C and D). mNIS is KPNB1 H1~8 WT Compared to TDP-43 mNLS This finding suggests that the nuclear import activity of KPNB1 also depends on the FG-Nup interaction domain.

[0164] To clarify the effect of KPNB1 on TDP-43 localization in the context of intact CNS tissue, we utilized AAV-transduction of organotypic mouse brain slice cultures (BSCs) (Figure 23E). AAV-GFP-TDP-43 mNLS BSCs were co-transduced with various AAV-FLAG-KPNB1 constructs or AAV-FLAG-mCherry as a control. mNLS were cytoplasmic and showed no obvious signs of aggregation (Figure 23E). WT and H1~8 WT Expression of TDP-43 mNLS KPNB1 constructs carrying NIS mutations that block FG-Nup-binding had no effect (Figures 23E and 23F). We tested the effect of these constructs on AAV-mScarlet-TDP-CTF (which forms small cytoplasmic aggregates in this model) (Figure 38A, arrow). TDP-CTF significantly increased the nuclear localization of KPNB1 H1-9 (arrowheads). WT and KPNB1 H1~8 WT This effect was observed in the presence of KPNB1 H1~9 mNISIt is not so obvious for KPNB1 H1~8 mNIS KPNB1 H10-19 is completely absent from TDP-43 (Figure 38). mNLS and has no effect on TDP-CTF localization (Figures 23E and 23F and 38). Collectively, these data demonstrate that KPNB1 reduces TDP-43 cytoplasmic aggregation and mislocalization in a FG-Nup-dependent, but NLS-independent manner in both primary neurons and the BSC model of TDP-43 proteinopathy.

[0165] Nup62 and KPNB1 are sequestered into cytoplasmic pTDP-43 aggregates in postmortem tissues of ALS / FTD To clarify that Nup62 and pTDP-43 are mislocalized within pTDP-43 inclusions in ALS / FTLD, co-immunostaining for Nup62 and KPNB1 was performed in patient tissues with phospho-TDP-43 (pTDP-43) pathology (case demographics are summarized in Table 7). pTDP-43 inclusions were positive for both Nup62 and KPNB1 in the spinal cord of subjects diagnosed with sporadic ALS, C9-ALS, and mutant TARDBP-ALS (Figures 24A and 24B and 39). Nup62 was almost completely absent from the nuclear rim in these cells, while KPNB1 was still partially present in the nucleus. Similar observations were noted in the hippocampus of sporadic and C9-FTLD-TDP patients, although Nup62 was still weakly detected on the nuclear envelope (Figures 24A-D). No cytoplasmic Nup62 or KPNB1 aggregates were observed in spinal cord tissue from subjects diagnosed with SOD1-ALS or FUS-ALS who did not show TDP-43 pathology (Figures 24A and 24B). Neuropathologically normal control spinal cord and hippocampus showed clear localization of Nup62 on the nuclear rim of most cells and nuclear cytoplasmic localization of KPNB1 in the absence of pTDP-43 pathology. The finding that both Nup62 and KPNB1 are incorporated into TDP-43 inclusions in ALS / FTD patients strongly supports the relevance of our data from disease models for human TDP-43 proteinopathy.

[0166] Collectively, these results demonstrate that one or more importin polypeptides and / or fragments thereof (and / or nucleic acids designed to express importin polypeptides and / or fragments thereof) can be administered to a mammal (e.g., a human) having or at risk of developing a proteinopathy (e.g., a TDP-43 proteinopathy) to treat the mammal.

[0167] [Example 9] Treatment of ALS

[0168] A human identified as having or at risk for developing ALS is administered one or more IPO3 polypeptides or fragments thereof that can slow, delay, or prevent the progression of neurodegeneration in the human brain and / or spinal cord.

[0169] [Example 10] Treatment of ALS

[0170] A nucleic acid encoding an IPO3 polypeptide or a fragment thereof is administered to a human identified as having or at risk of developing ALS under conditions such that the IPO3 polypeptide or a fragment thereof is expressed in the human's brain and / or spinal cord to slow, delay or prevent the progression of neurodegeneration in the human's brain and / or spinal cord.

[0171] [Example 11] Treatment of ALS

[0172] A human identified as having or at risk for developing ALS is administered one or more IPO13 polypeptides or fragments thereof that can slow, delay, or prevent the progression of neurodegeneration in the human brain and / or spinal cord.

[0173] [Example 12] Treatment of ALS

[0174] A human identified as having or at risk for developing ALS is administered a nucleic acid encoding an IPO13 polypeptide or a fragment thereof under conditions such that the IPO13 polypeptide or a fragment thereof is expressed in the human's brain and / or spinal cord to slow, delay or prevent the progression of neurodegeneration in the human's brain and / or spinal cord.

[0175] Other embodiments While the present invention has been described in connection with its detailed description, it is understood that the above description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A pharmaceutical composition for treating a mammal having TAR DNA-binding protein 43 (TDP-43) proteinopathy, comprising an importin-13 (IPO13) polypeptide or a fragment of said IPO13 polypeptide.

2. The pharmaceutical composition according to claim 1, wherein said IPO13 polypeptide or said fragment is effective for reducing the symptoms of said TDP-43 proteinopathy.

3. The symptoms are selected from the group consisting of depression, emotional blunting, social withdrawal, mood swings, irritability, aggression, changes in sleep habits, wandering, loss of inhibition, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, blunting of affect, compulsive or ritualistic behavior, changes in eating habits or diet, defects in executive function, lack of insight, excitation, emotional instability, difficulty generating or understanding spoken or written language, memory loss, muscle weakness, muscle atrophy, fasciculation, spasticity, dysarthria, dysphagia, behavioral variant frontotemporal dementia (bvFTD), and primary progressive aphasia (PPA). The pharmaceutical composition according to claim 2.

4. A pharmaceutical composition for reducing the aggregation of TDP-43 polypeptide in the central nervous system (CNS) of a mammal having TDP-43 proteinopathy, comprising an IPO13 polypeptide or a fragment of said IPO13 polypeptide.

5. The pharmaceutical composition according to any one of claims 1 to 4, comprising said IPO13 polypeptide.

6. The pharmaceutical composition according to any one of claims 1 to 4, comprising said fragment of the IPO13 polypeptide.

7. The pharmaceutical composition according to claim 6, wherein said fragment of the IPO13 polypeptide consists of an amino acid sequence shown in any one of SEQ ID NOs: 56 to 77.

8. A pharmaceutical composition for treating a mammal having TDP-43 proteinopathy, comprising a nucleic acid encoding an IPO13 polypeptide or a fragment of said IPO13 polypeptide, wherein said IPO13 polypeptide or said fragment is expressed by cells in the CNS of said mammal.

9. The pharmaceutical composition according to claim 8, wherein said IPO13 polypeptide or said fragment is effective for reducing the symptoms of said TDP-43 proteinopathy.

10. The pharmaceutical composition according to claim 9, wherein the symptoms are selected from the group consisting of depression, emotional blunting, social withdrawal, mood swings, irritability, aggression, changes in sleep habits, wandering, loss of inhibition, delusions, deterioration in behavior and personality affecting conduct, judgment, empathy and insight, emotional blunting, compulsive or ritualistic behavior, changes in eating habits or diet, defects in executive function, lack of common sense, excitability, emotional instability, difficulty in generating or understanding spoken or written language, memory loss, muscle weakness, muscle atrophy, fasciculation, spasm, dysarthria, dysphagia, bvFTD, and PPA.

11. A pharmaceutical composition for reducing the aggregation of TDP-43 polypeptide in the central nervous system (CNS) of a mammal, comprising a nucleic acid encoding an IPO13 polypeptide or a fragment of said IPO13 polypeptide, wherein the IPO13 polypeptide or the fragment is expressed by cells within the CNS of the mammal.

12. The pharmaceutical composition according to any one of claims 8 to 11, wherein the nucleic acid encodes the IPO13 polypeptide.

13. The pharmaceutical composition according to any one of claims 8 to 11, wherein the nucleic acid encodes the fragment of the IPO13 polypeptide.

14. The pharmaceutical composition according to claim 13, wherein the fragment of the IPO13 polypeptide consists of an amino acid sequence shown in any one of SEQ ID NOs: 56 to 77.

15. The pharmaceutical composition according to any one of claims 8 to 11, wherein the nucleic acid is in the form of a vector.

16. The pharmaceutical composition according to claim 15, wherein the vector is an AAV vector or an expression plasmid.

17. The pharmaceutical composition according to any one of claims 8 to 11, wherein the nucleic acid is contained within nanoparticles.

18. The pharmaceutical composition according to any one of claims 1 to 4 and 8 to 11, wherein the mammal is a human. **Claim 19**: The pharmaceutical composition according to any one of claims 1 to 4 and 8 to 11, wherein the TDP-43 proteinopathy is selected from the group consisting of Alzheimer's disease, FTD, ALS, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE), dementia with Lewy bodies (DLB), Parkinson's disease, Huntington's disease, argyrophilic grain disease (AGD), hippocampal sclerosis (HS), Guam ALS, Guam Parkinson's dementia complex (G-PDC), Perry disease, facial-onset sensory and motor neuronopathy (FOSMN), inclusion body myositis (IBM), hereditary inclusion body myopathy, oculopharyngeal muscular dystrophy (OPMD), and distal myopathy with rimmed vacuoles. **Claim 20**: The pharmaceutical composition according to any one of claims 1 to 4 and 8 to 11, for intracerebral injection. **Claim 21**: The pharmaceutical composition according to any one of claims 1 to 4 and 8 to 11, for intrathecal injection.

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