Modeling Clinically Heterogeneous Presenilin Mutations with Transgenic Drosophila

SummaryTo assess the potential of Drosophila to analyze clinically graded aspects of human disease, we developed a transgenic fly model to characterize Presenilin (PS) gene mutations that cause early-onset familial Alzheimer's disease (FAD). FAD exhibits a wide range in severity defined by ages of onset from 24 to 65 years [1]. PS FAD mutants have been analyzed in mammalian cell culture, but conflicting data emerged concerning correlations between age of onset and PS biochemical activity [2–4]. Choosing from over 130 FAD mutations in Presenilin-1, we introduced 14 corresponding mutations at conserved residues in Drosophila Presenilin (Psn) and assessed their biological activity in transgenic flies by using genetic, molecular, and statistical methods. Psn FAD mutant activities were tightly linked to their age-of-onset values, providing evidence that disease severity in humans primarily reflects differences in PS mutant lesions rather than contributions from unlinked genetic or environmental modifiers. Our study establishes a precedent for using transgenic Drosophila to study clinical heterogeneity in human disease

<i>Nmf9</i> Encodes a Highly Conserved Protein Important to Neurological Function in Mice and Flies

<div><p>Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, <i>Nmf9</i>, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with <i>Nmf9</i> expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian <i>Nmf9</i>. Intriguingly, homozygotes for null mutations in the <i>Drosophila</i> homolog, <i>CG45058</i>, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.</p></div

Mouse <i>nmf9</i> alleles generated by CRISPR/Cas9-mediated genome editing.

<p>Phenotypes are noted as 2 for strong, 1 for mild, or 0 for not evident.</p><p>Mouse <i>nmf9</i> alleles generated by CRISPR/Cas9-mediated genome editing.</p

Genome editing of <i>Drosophila CG45058</i> exons results in severe locomotor deficits and early death.

<p>(A) Target sites and recovered alleles are shown for separately targeted exons encoding the first ANK repeat, FN3 domain, and the GLYLGYLK peptide. (B) Posture of a heterozygous control fly. (C) Posture of a mutant fly homozygous for a frame-shifting mutation in conserved domain 2. Note wings held up at an angle with respect to the body. (D) The same mutant fly subsequently fell over and moved legs in an uncoordinated fashion. (E) Survival frequencies at eclosion and 7 days post-eclosion. (F) Total waking activity (beam breaks per minutes awake) for males (blue) and females (pink) of the indicated genotypes. Heterozygotes for framshifting mutations in <i>CG45058</i> ANK, FN3 or conserved domain 2 consistently showed reduced activity relative to +/+ control, similar in magnitude to <i>wake</i> (EY02219) homozygotes. *** p<2.2x10^-16.</p

Mouse <i>nmf9</i> alleles generated by CRISPR/Cas9-mediated genome editing.

<p>Phenotypes are noted as 2 for strong, 1 for mild, or 0 for not evident.</p><p>Mouse <i>nmf9</i> alleles generated by CRISPR/Cas9-mediated genome editing.</p

<i>CG45058</i> null heterozygotes have abnormal sleep patterns.

<p>Male heterozygotes (blue graphs) recorded less total sleep per 24 hr. period and slightly longer latency to first sleep bout after lights on (daytime sleep latency) than +/+ controls, but no significant difference in latency after lights off (nighttime sleep latency). By contrast female heterozygotes (pink graphs) recorded slightly more overall sleep than +/+ controls and slightly longer daytime and nighttime sleep latencies. Heterozygous <i>wake</i> P-element alleles (EY02219, KG02188, KG08407 and MI02905 insertion lines) and a homozygous <i>wake</i> allele (EY02219) were recorded as a comparison. * p<0.025, *** p<5x10^-4, ns p = 0.78, Wilcoxon rank sum test.</p

Genome editing of <i>Drosophila CG45058</i> exons results in severe locomotor deficits and early death.

<p>(A) Target sites and recovered alleles are shown for separately targeted exons encoding the first ANK repeat, FN3 domain, and the GLYLGYLK peptide. (B) Posture of a heterozygous control fly. (C) Posture of a mutant fly homozygous for a frame-shifting mutation in conserved domain 2. Note wings held up at an angle with respect to the body. (D) The same mutant fly subsequently fell over and moved legs in an uncoordinated fashion. (E) Survival frequencies at eclosion and 7 days post-eclosion. (F) Total waking activity (beam breaks per minutes awake) for males (blue) and females (pink) of the indicated genotypes. Heterozygotes for framshifting mutations in <i>CG45058</i> ANK, FN3 or conserved domain 2 consistently showed reduced activity relative to +/+ control, similar in magnitude to <i>wake</i> (EY02219) homozygotes. *** p<2.2x10^-16.</p

<i>Nmf9</i> is important to fear learning and circadian rhythm.

<p>(A) Mutant animals showed decreased freezing behavior in both tone cued and contextual fear conditioning. (2-factor ANOVA p = 0.0018 for genotypes in contextual conditioning, 0.002 in tone-cued conditioning. Sex effects were non-significant, p = 0.29 in contextual, p = 0.09 in tone-cued, N = 14 +/+ female, 8 mutant female, 11 +/+ male, 8 mutant male, although the interaction between genotype and sex was supported for contextual fear conditioning, p = 0.026.) (B) Double-plotted actograms of running-wheel counts of mutant and wildtype from the same litter housed in 12 hour light: 12 hour dark (LD) cycle and continuous dark (DD) illustrates less robust circadian rhythm in a more severely affected animal. Arrowheads indicate transfer to DD. (C) Mutants showed a small but significant decrease in period length (p = 0.014) and an increase in period onset error (p = 0.034) during DD (2-factor ANOVA; effects of sex p = 0.14 for period length, p = 0.012 for onset error, with larger deviation in females. N = 14 wildtype females, 12 mutant females, 9 wildtype males, 10 mutant males).</p

Tests of LS, PC/olfactory pathway, and VMH.

<p>Behaviors that depend on <i>Nmf9</i>-expressing circuits were assayed and assessed by 2-factor ANOVA or MANOVA for sex and genotype. Number tested for each group (N) is indicated in each bar or a legend to line graphs. (A) Elevated Plus Maze behaviors were not significantly different between control (+/+) and mutant (n/n) littermates (Percent time in open arm p = 0.27 for genotype, p = 0.22 for sex; Number of transitions p = 0.12 for genotype, p = 0.23 for sex). (B) In the Open Field Test, mutant animals showed increased time spent in center, but potentially confounded by increased number of line crosses due to hyperactivity (Time in center p = 0.0043 for genotype, p = 0.82 for sex; number of transitions, 3.4x10^-7 for genotype, p = 0.0004 for sex). (C) No significant difference between genotypes was detected for the Marble Burying Test (p = 0.15 and 0.68 for genotype and sex, respectively). (D) While mutant females took somewhat longer in olfactory-dependent Buried Food Finding Test, this was did not reach conventional significance (p = 0.11 for genotype, 0.09 for sex). (E) Similarly, Odor Habituation / Dishabituation Tests did not support a significant difference (MANOVA, p = 0.69 for genotype, p = 0.30 for sex). (F) Weight was significantly different by genotype at 2 months (ANOVA, p = 0.014) and remained significant at 6 months (p = 0.0088), including well-known sex differences, while food consumption (G) was not significantly different (p = 0.11 for genotype, 0.75 for sex).</p