Compositions and methods for treatment
A technology for neurodegenerative, disease, applied in the field of compositions and methods for treatment, which can solve the problems of poor prognosis and limited treatment
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Embodiment 1
[0123] Example 1 - Identification of new interaction partners of TDP-43
[0124] To identify novel interacting partners of the C-terminal glycine-rich domain (GRD) of TDP-43, the inventors performed a bacterial two-hybrid screen as described above. The best candidate identified (11 of 65 hits) was 14-3-3Θ (encoded by the YWHAQ gene), a member of the 14-3-3 scaffold protein family. Co-immunoprecipitation from murine N2a cells and mouse brain confirmed the interaction between endogenous 14-3-3θ and TDP-43 ( figure 1 ).
[0125] To test whether the 14-3-3θ / TDP-43 interaction is associated with disease, the inventors expressed 14-3-3θ and TDP-43 mutants in 293T HEK cells. Surprisingly, 14-3-3θ interacted significantly more with TDP-43 variants carrying pathogenic mutations, including the A315T mutation, and such TDP-43 variants were associated with the family Pathogenic variants associated with ALS and FTD (see figure 2 ). Coexpression of 14-3-3θ with TDP-43-A315T resulted in ...
Embodiment 2
[0128] Example 2 – Interaction motifs in 14-3-3θ that mediate binding to TDP-43
[0129] The 14-3-3 dimer normally interacts with the phosphorylated form of its interacting partner. In contrast, however, in the case of TDP-43, the inventors found that phosphorylated mimetic variants of TDP-43 interact less with 14-3-3θ, supporting a non-canonical type of interaction (data not shown) .
[0130] Structurally, 14-3-3θ contains nine α-helices (see Figure 4 ), where helices αC, αE, αG, and αI contribute to the canonical partner binding of the 14-3-3θ dimer. To identify the interaction motif in 14-3-3θ that mediates TDP-43 binding, the inventors truncated stepwise 14-3-3θ, revealing that the interaction is initiated by the sixth α-helix of 14-3-3θ (αF )Mediated( Figure 4 ).
[0131] The inventors generated α-helix 6 (αF) comprising only 14-3-3θ (30 amino acid sequence shown in SEQ ID NO: 2; corresponding to amino acids 135-164 of the wild-type human TDP-43 sequence) , and th...
Embodiment 3
[0132] Example 3 - Effect of increased 14-3-3θ expression in vivo
[0133] To study the effect of increased 14-3-3θ levels in vivo, the inventors used adeno-associated virus (AAV) in three-month-old non-transgenic mice and iTDP-43 A315T 14-3-3θ is expressed in the hippocampus of mice. Increased neuronal 14-3-3θ levels lead to accumulation of insoluble TDP-43 fragments in non-transgenic mice and in iTDP-43 A315T This is especially true in mice ( Figure 7 A). Furthermore, AAV-14-3-3θ-injected iTDP-43 A315T Mice showed massive loss of hTDP-43 expressing hippocampal neurons ( Figure 7 B). Thus, increased 14-3-3θ levels in vivo lead to disease-like insolubility and fragmentation of endogenous and transgenic TDP-43, further exacerbating iTDP-43 A315T Neuropathological phenotypes in mice.
[0134] The inventors also tested the naive Does chronic AAV-mediated 14-3-3θ overexpression in the spinal cord of C57Bl / 6 mice lead to altered endogenous TDP-43 and functional deficits....
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