TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad-resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance-associated mutation (G275E) is predicted to lie at the top of the third α-helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate-gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance-associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action.

Wiseman et al. show that triplication of genes other than APP is sufficient to exacerbate Aβ deposition and associated cognitive changes in a mouse model of Down syndrome – Alzheimer’s disease. This occurs independently of changes to γ-secretase but results from a novel mechanism that lowers the soluble Aβ40/42 ratio.

RIC‐3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). In this study, we report the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC‐3 (DmRIC‐3). Heterologous expression studies of alternatively spliced DmRIC‐3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled‐coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline‐rich N‐terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC‐3 to influence nAChR maturation was examined in co‐expression studies with human α7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC‐3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC‐3. In contrast, when expressed in a human cell line, human RIC‐3 enhanced nAChR maturation more efficiently than DmRIC‐3. The cloning and characterisation of 11 alternatively spliced DmRIC‐3 isoforms has helped to identify domains influencing RIC‐3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC‐3.

Ivermectin is a macrocyclic lactone that acts as a positive allosteric modulator of ␣7 nicotinic acetylcholine receptors (nAChRs) but has no modulatory activity on 5-hydroxytryptamine (5-HT) type 3 (5-HT 3 ) receptors. By examining the influence of ivermectin on subunit chimeras containing domains from the nAChR ␣7 subunit and the 5-HT3A subunit, we have concluded that the transmembrane domains play a critical role in influencing allosteric modulation by ivermectin. A series of mutations located within the ␣-helical transmembrane domains of the ␣7 subunit were examined, and seven were found to have significant effects on allosteric modulation by ivermectin. Four mutations (A225D, Q272V, T456Y, and C459Y) caused a significant reduction in the potency of ivermectin as an allosteric potentiator. Compared with wild-type ␣7 nAChRs, potentiation by ivermectin was reduced dramatically (by 89 -97%) by these mutations. Somewhat unexpectedly, three mutations (S222M, M253L, and S276V located in TM1, TM2, and TM3) converted ivermectin from a positive allosteric modulator into an antagonist. Levels of inhibition of 56, 84, and 89% were observed on M253L, S276V, and S222M, respectively. Antagonism by ivermectin was insurmountable and had no effect on EC 50 of acetylcholine, indicating that it is acting noncompetitively. The seven mutations that influence allosteric modulation by ivermectin are located near a predicted intrasubunit transmembrane cavity. Computer docking simulations provide support for the hypothesis that ivermectin binds in close proximity to this cavity. We conclude that transmembrane mutations in ␣7 nAChRs are able to convert ivermectin from a positive to a negative allosteric modulator.