Figure 4.

The amino-terminus S44L mutation exacerbates R388 catalytic domain mutant phenotypes but is itself only mildly deleterious. (A) L44 expressed heterozygously (H^WT,H^L44) or alone (H^L44,Ø) rescues eclosion as effectively as one or two copies of the wild-type transgene, H^WT (P > 0.1). Co-expression of R388 and L44 slightly but significantly lowers eclosion compared with the H^WT,H^R388 genotype, consistent with L44 being largely silent in combination with wild-type Spastin, but deleterious when in trans with a catalytic domain mutant in Spastin (P < 0.02). (B, C) Post-eclosion, survival of adult flies is dramatically affected by Spastin genotype. Compared with controls, H^WT,H^R388 heterozygotes survive on average only about half as long, and H^L44,H^R388 compound heterozygotes survive less than half as well as those (P < 9 × 10⁻⁶). No deleterious effect of the L44 allele is seen in H^WT,H^L44 heterozygotes compared with wild-type, however. (D, E) H^WT,H^L44 larvae show moderate but significant increases in bouton number and the percentage of terminal boutons lacking MAP1B compared with H^WT,H^WT controls, indicating that L44 is somewhat deleterious at the cellular level (P < 0.01 for both parameters). When in trans with R388, however, L44 strongly enhances the R388 loss of function phenotype, to levels equivalent to spastin nulls (P > 0.3). Bouton number is significantly greater in the compound versus single mutants (P < 6 × 10⁻⁷), although both genotypes are deficient in terminal bouton microtubules, similar to nulls. (F) Representative synaptic bouton arbors with their MAP1B-positive microtubule distribution are shown for the different ‘AD-HSP’ genotypes. Presynaptic arbors in H^WT,H^L44 animals resemble wild-type, with large, round terminal boutons (e.g. arrows) sometimes penetrated by microtubules. H^WT,H^R388 larvae exhibit smaller, more clustered boutons often devoid of microtubule staining, and H^L44,H^R388 synapses are similarly, but much more severely, affected.