Lissencephaly-1 mutations enhance traumatic brain injury outcomes in Drosophila

Abstract

Traumatic brain injury (TBI) outcomes vary greatly among individuals, but most of the variation remains unexplained. Using a Drosophila melanogaster TBI model and 178 genetically diverse lines from the Drosophila Genetic Reference Panel (DGRP), we investigated the role that genetic variation plays in determining TBI outcomes. Following injury at 20-27 days old, DGRP lines varied considerably in mortality within 24 h ("early mortality"). Additionally, the disparity in early mortality resulting from injury at 20-27 vs 0-7 days old differed among DGRP lines. These data support a polygenic basis for differences in TBI outcomes, where some gene variants elicit their effects by acting on aging-related processes. Our genome-wide association study of DGRP lines identified associations between single nucleotide polymorphisms in Lissencephaly-1 (Lis-1) and Patronin and early mortality following injury at 20-27 days old. Lis-1 regulates dynein, a microtubule motor required for retrograde transport of many cargoes, and Patronin protects microtubule minus ends against depolymerization. While Patronin mutants did not affect early mortality, Lis-1 compound heterozygotes (Lis-1x/Lis-1y) had increased early mortality following injury at 20-27 or 0-7 days old compared with Lis-1 heterozygotes (Lis-1x/+), and flies that survived 24 h after injury had increased neurodegeneration but an unaltered lifespan, indicating that Lis-1 affects TBI outcomes independently of effects on aging. These data suggest that Lis-1 activity is required in the brain to ameliorate TBI outcomes through effects on axonal transport, microtubule stability, and other microtubule proteins, such as tau, implicated in chronic traumatic encephalopathy, a TBI-associated neurodegenerative disease in humans.

Keywords: Drosophila melanogaster; Lissencephaly-1; Patronin; nudE; genome-wide association study; microtubule; neurodegeneration; traumatic brain injury.

Fig. 1.

Genotype affects the MI₂₄ of flies injured at 20–27 days old and the difference in MI₂₄ between flies injured at 20–27 vs 0–7 days old. a) Mean and standard error of the mean (SEM) of the MI₂₄ for 178 DGRP lines injured using the standard TBI protocol at 20–27 days old (n = 3). Supplementary Table 1 lists MI₂₄ values for each DGRP line. b) Mean and SEM of the MI₂₄ for 178 DGRP lines injured at 0–7 days old (n = 3). Supplementary Table 1 and Katzenberger et al. (2015a) list MI₂₄ values for each DGRP line. c) The difference in MI₂₄ values between flies injured at 20–27 and 0–7 days old. DGRP lines are ordered the same as in panels A and B.

Fig. 2.

Maps of patronin and lis-1 alleles. Schematic diagrams of (A) Patronin and B) Lis-1 alleles (http://flybase.org). Colored and white boxes indicate coding and non-coding exons, respectively, and intervening lines indicate introns. Locations and orientations of transposons are indicated by triangles and arrows, respectively. The orientation of Lis-1^E415 is not known (Swan et al. 1999), Lis-1^G10.14 contains a nonsense mutation (R239stop) (see references in Kudumala et al. 2017), and deficiencies Df(2R)BSC355 and Df(2R)Jp8, respectively, delete Patronin and Lis-1 (http://flybase.org). Transcription start sites are indicated by horizontal arrows followed by the gene name. Supplementary Table 2 provides genotypes of Patronin and Lis-1 mutant lines.

Fig. 3.

Lis-1 mutations increase the MI₂₄ of flies injured at 20–27 or 0–7 days old. The MI₂₄ of mixed-sex Lis-1 mutants of the indicated genotypes injured using the standard TBI protocol at (A) 20–27 days old (n = 6) and (B) 0–7 days old (n = 10). Chromosomes wild type for Lis-1 are designated +(w¹¹¹⁸), +(DGRP774), and +(DGRP892), based on their parental origin. Symbols indicate the following: box, second and third quartiles of data; +, mean; horizonal bar, median; and whiskers, minimum and maximum data points. Note that data for DGRP774 and DGRP892 lines are the same in Figs. 4a and b, and data for w¹¹¹⁸ flies are the same in Fig. 4a.

Fig. 4.

Patronin mutations do not affect the MI₂₄ of flies injured at 20–27 or 0–7 days old. The MI₂₄ of mixed-sex Patronin mutants of the indicated genotypes injured using the standard TBI protocol at (A) 20–27 days old (n = 6) and (B) 0–7 days old (n = 10). Chromosomes wild type for Patronin are designated +(w¹¹¹⁸), +(DGRP774), and +(DGRP892), based on their parental origin. Symbols indicate the following: box, second and third quartiles of data; +, mean; horizonal bar, median; and whiskers, minimum and maximum data points. Note that data for DGRP774 and DGRP892 lines are the same in Figs. 3a and b, and data for w¹¹¹⁸ flies are the same in Fig. 3a.

Fig. 5.

Overexpression of Patronin increases the MI₂₄ of flies injured at 0–7 day old. a) A representative Western blot and (b) quantitation of Western blots for Lis-1 and α-tubulin protein expression in head extracts of 0–7 day old male flies of the indicated genotypes (n = 4). e) A representative Western blot and (f) quantitation of Western blots for Patronin and α-tubulin protein expression in head extracts of 0–7 day old male flies of the indicated genotypes (n = 4). In panels A and E, protein size markers are indicated on the left in kDa. (c, d, g, and h) The Mi₂₄ of mixed-sex flies of the indicated genotypes injured using the standard TBI protocol at the indicated age (n = 12). Symbols indicate the following: box, second and third quartiles of data; +, mean; horizonal bar, median; and whiskers, minimum and maximum data points.

Fig. 6.

Lis-1 mutations do not affect lifespan following TBI. Percent survival of mixed-sex Lis-1 mutants either uninjured or injured using the standard TBI protocol. Data are averages and SEM of four Lis-1^x/+ heterozygous and four Lis-1^x/Lis-1^y compound heterozygous genotypes shown in Table 2. Heterozygous Lis-1 mutants were generated by crossing Lis-1 mutants to the w¹¹¹⁸ line. For each genotype, at least 100 flies were examined in groups of 20. Lifespan analysis of individual Lis-1 mutant genotypes is shown in Supplementary Fig. 6.

Fig. 7.

Effects of minor SNPs identified by GWAS on expression of Lis-1 and Patronin mRNAs and proteins in uninjured flies. qRT-PCR analysis of Lis-1 and Patronin mRNAs relative to Rpl32 mRNA in uninjured, whole, male DGRP lines at (a) 20–27 days old and (d) 0–7 days old (n = 3). Dots and whiskers indicate the average and SEM, respectively (n = 3). (c and d) Representative Western blots and (e and f) quantitation of Western blots (n = 3) for Lis-1 and α-tubulin protein expression in head extracts from (c and e) 20–27 day old and (d and f) 0–7 day old male flies of the indicated DGRP line. (g and h) Representative Western blots and (i and j) quantitation of Western blots (n = 3) for Patronin and α-tubulin protein expression in head extracts from (g and i) 20–27 day old and (H and J) 0–7 day old male flies of the indicated genotypes. (c, d, g, and h) Protein size markers are indicated on the left in kDa. (e, f, i, and j) Symbols indicate the following: box, second and third quartiles of data; +, mean; horizonal bar, median; and whiskers, minimum and maximum data points.

Fig. 8.

TBI does not markedly alter expression of Lis-1 and Patronin mRNAs in w¹¹¹⁸ flies. qRT-PCR analysis of the amount of Lis-1 and Patronin mRNAs relative to Rpl32 mRNA in uninjured (U) flies at time zero and at indicated time points after injury using the standard TBI protocol. a) Male w¹¹¹⁸ flies were injured at 20–27 or 0–7 days old and mRNA level was determined in whole flies at the indicated time points over 24 h following injury (n = 8). mRNA level in (b) whole, male w¹¹¹⁸ flies and (c) heads of male w¹¹¹⁸ flies was determined for flies every 7 days over 49 days following injury or no injury (uninjured) at 0–7 days old (n = 6). Each data point represents the mean and SEM.

Fig. 9.

Lis-1 mutations enhance neurodegeneration in the brain following TBI. a) The number of large (>5 μM) holes in the brain of female Lis-1 mutants of the indicated genotypes at two weeks after injury at 0–7 days old (injured) or at the same age in the absence of injury (uninjured) (n = 4). Lis-1 heterozygotes were generated by crossing Lis-1 mutants to the w¹¹¹⁸ line. Brain sections chosen for analysis were at equivalent depths in the brain. Genotypes are ordered from low to high, based on the mean number of holes per brain in injured flies. Symbols indicate the following: box, second and third quartiles of data; +, mean; horizonal bar, median; and whiskers, minimum and maximum data points. (b–d) Representative images of sections of fly brains from flies of the indicated genotypes and injury status. (e–g) High-magnification images of boxed regions in b–d, respectively. Arrows indicate large holes.