Sleep/Wake Disruption in a Mouse Model of BLOC-1 Deficiency

Mice lacking a functional Biogenesis of Lysosome-related Organelles Complex 1 (BLOC-1), such as those of the pallid line, display cognitive and behavioural impairments reminiscent of those presented by individuals with intellectual and developmental disabilities. Although disturbances in the sleep/wake cycle are commonly lamented by these individuals, the underlying mechanisms, including the possible role of the circadian timing system, are still unknown. In this paper, we have explored sleep/circadian malfunctions and underlying mechanisms in BLOC-1-deficient pallid mice. These mutants exhibited less sleep behaviour in the beginning of the resting phase than wild-type mice with a more broken sleeping pattern in normal light-dark conditions. Furthermore, the strength of the activity rhythms in the mutants were reduced with significantly more fragmentation and lower precision than in age-matched controls. These symptoms were accompanied by an abnormal preference for the open arm in the elevated plus maze in the day and poor performance in the novel object recognition at night. At the level of the central circadian clock (the suprachiasmatic nucleus, SCN), loss of BLOC-1 caused subtle morphological changes including a larger SCN and increased expression of the relative levels of the clock gene Per2 product during the day but did not affect the neuronal activity rhythms. In the hippocampus, the pallid mice presented with anomalies in the cytoarchitecture of the Dentate Gyrus granule cells, but not in CA1 pyramidal neurones, along with altered PER2 protein levels as well as reduced pCREB/tCREB ratio during the day. Our findings suggest that lack of BLOC-1 in mice disrupts the sleep/wake cycle and performance in behavioural tests associated with specific alterations in cytoarchitecture and protein expression

Strategy for the genetic modifier screening carried out in this study.

<p>The primary screening involved parental (P₀) crosses between male flies carrying a deficiency (<i>Df</i>) over a balancer in an autosomal chromosome (Chr. 2, 3 or 4) and female flies homozygous for the hypomorphic <i>g</i>^<i>2</i> allele of the X-linked gene encoding the δ subunit of AP-3. The eye color of male progeny (F₁) carrying one copy of each deficiency (without the balancer) was compared with that of control <i>g</i>^<i>2</i> males and, if deemed different, the corresponding deficiency was selected for a secondary screening involving the same P₀ cross followed by quantification of red and brown pigments in the F₁ males carrying the deficiency. In cases in which differences in both red and brown pigment content were statistically significant, further validation and fine mapping was attempted using independent deficiency lines in which the deleted genomic regions partially overlapped with that of the deficiency identified through screening. When successful, theses steps allowed identification of a relatively small genomic region (rectangle) containing a modifier gene of interest (red arrow).</p

Red pigment content in eyes of <i>g</i>^<i>2</i> mutant flies carrying hemizygous deletions selected through primary screening.

<p>Red pigments were extracted from the heads of adult <i>g</i>^<i>2</i> mutant males carrying no deletions (—) or a single copy of the indicated deficiencies, quantified as described under Materials and Methods, and expressed as percentages of the red pigment content of male flies of the wild-type line (Canton-S). Bars represent means + SD of 2–28 biological replicates. Grey bars denote values obtained for flies carrying the marker allele <i>w</i>^<i>+mC</i> linked to the deficiency. Dashed lines indicate threshold values corresponding to 66.7% (2/3) and 150% (3/2) of the pigment content of control <i>g</i>^<i>2</i> flies carrying no deletion (black bar). One-way ANOVA followed by Dunnett’s test of each group versus <i>g</i>^<i>2</i> flies carrying no deletion: ***p<0.001.</p

<i>ArfGAP1</i> as a modifier of the AP-3 eye pigmentation phenotype.

<p>(A-C) Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds carrying wild-type (+) or mutant (<i>G3-85</i>) alleles of the <i>ArfGAP1</i> gene on chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 3–17 biological replicates. Statistical analyses were performed by means of Student’s t-test (A and B) or one-way ANOVA followed by Dunnett’s test of each group versus <i>g</i>^<i>2</i> flies carrying no deletion (C): *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of <i>ArfGAP1</i>^<i>G3-85</i> mutant allele over wild-type <i>ArfGAP1</i> was sufficient to ameliorate the pigmentation defects of both <i>g</i>^<i>2</i> (A) and <i>rb</i>^<i>1</i> (B) AP-3-subunit mutants. Notice in (C) that such partial suppressor effect was not exacerbated in flies homozygous for the <i>ArfGAP1</i>^<i>G3-85</i> allele or heterozygous for this allele over any of two deficiencies in which the deleted genomic regions include the entire <i>ArfGAP1</i> gene, namely <i>Df(3L)eyg</i>^<i>C1</i> (Df1) and <i>Df(3L)BSC380</i> (Df2). The deficiency <i>Df(3L)BSC413</i>, in which the deleting genomic region excludes the <i>ArfGAP1</i> gene, was used as a control (Df3).</p

Homozygous ArfGAP1-null mutants display pleiotropic effects depending on genetic background.

<p>(A-C) Genetic crosses were set up to obtain flies carrying wild-type (+) or null mutant (<i>G3-85</i>) alleles of the <i>ArfGAP1</i> gene in the genetic backgrounds of the wild-type line Canton-S (A), the BLOC-1-subunit mutant <i>blos1</i>^<i>ex2</i> (B) and the Lightoid GTPase mutant <i>ltd</i>^<i>1</i> (C). Notice in (B) that males that were double homozygous for <i>ArfGAP1</i>^<i>G3-85</i> and <i>blos1</i>^<i>ex2</i> did not survive to adulthood (indicated with X). In those cases in which viable adult males were obtained, red pigments were extracted and quantified as described under Materials and Methods. Values were expressed as percentages of the pigment content of wild-type flies. Bars represent means + SD of 3–9 biological replicates. Statistical analyses were performed by means of Student’s t-test (B) or one-way ANOVA followed by Dunnett’s test of each group versus that of flies homozygous for the wild-type <i>ArfGAP1</i> allele in the corresponding genetic background (A and C): ns, not significant; *p<0.05, ***p<0.001. (D and E) eye morphology of adult flies homozygous for <i>ltd</i>^<i>1</i> (D) and for both <i>ltd</i>^<i>1</i> and <i>ArfGAP1</i>^<i>G3-85</i> (E), with the latter displaying a mild interommatidial facets phenotype.</p

Effects of the <i>Atg2</i>^{<i>EP3697</i>} allele on red pigment content in various genetic backgrounds.

<p>Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds lacking (open bars) or carrying (filled bars) one copy of the <i>Atg2</i>^{<i>EP3697</i>} allele over a normal chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 7–15 biological replicates. One-way ANOVA followed by Bonferroni comparison of selected group pairs: *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of <i>Atg2</i>^{<i>EP3697</i>} over a normal <i>Atg2</i> allele increased pigmentation of two AP-3-subunit mutants (<i>g</i>^<i>2</i> and <i>rb</i>^<i>1</i>), a BLOC-1-subunit mutant (<i>blos1</i>^<i>ex2</i>), and a mutant in the Rab-GTPase Lightoid (<i>ltd</i>^<i>1</i>), though it also increased the red pigment content of wild-type flies. Although the transposon inserted in <i>Atg2</i>^{<i>EP3697</i>} carries the <i>w</i>^<i>+mC</i> marker, its weak activity led to barely detectable red pigments in a White-null background (<i>w</i>^<i>1118</i>).</p

Validation and fine mapping of the critical region responsible for the modifier effect observed for <i>Df(3L)eyg</i>^<i>C1</i>.

<p>Red pigments were extracted from the heads of adult <i>g</i>^<i>2</i> mutant males carrying no deletions (—) or a single copy of the indicated deficiencies, quantified as described under Materials and Methods, and expressed as percentages of the red pigment content of male flies of the wild-type (Canton-S) line. Bars represent means + SD of 6–10 biological replicates. One-way ANOVA followed by Dunnett’s test of each group versus <i>g</i>^<i>2</i> flies carrying no deletion (black bar): ***p<0.001. Shown on the left is a schematic representation of the extent of overlap between the chromosomal region deleted in the deficiency that had been identified through screening (blue) and those deleted in independent deficiencies that elicited (red) or failed to elicit (grey) a similar modifier effect on the <i>g</i>^<i>2</i> eye color phenotype. The critical genomic region responsible for the observed modifier effect is highlighted with black dashed lines, and the relative location of genes found within this region (adapted from the FlyBase database) is depicted at the bottom.</p

Attempts to validate selected deficiencies carrying the <i>w</i>^<i>+mC</i> marker as genetic modifiers of the <i>g</i>^<i>2</i> mutation.

<p>(A) Red pigments were extracted from the heads of AP-3-deficient (<i>g</i>^<i>2</i>) or White-negative (<i>w</i>^<i>1118</i>) adult males carrying single copies of the indicated deficiencies with their associated <i>w</i>^<i>+mC</i> marker. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 2–10 biological replicates. Notice that the activity of the <i>w</i>^<i>+mC</i> marker associated with deficiency <i>Df(3L)ED4978</i> resulted in a red pigment content (arrow) higher than that of <i>g</i>^<i>2</i> flies (black bar). (B-D) Analyses of red pigment content in the eyes of adult <i>g</i>^<i>2</i> mutant males carrying no deletions (—), single copies of the deficiencies <i>Df(3L)ED4978</i> (B), <i>Df(3R)Exel6195</i> (C) and <i>Df(2L)XE-3801</i> (D) that had been identified through screening, or single copies of deficiencies with partially overlapping deletions and devoid of the <i>w</i>^<i>+mC</i> marker. Schematic representations of the extent of overlap between the chromosomal regions deleted in the deficiencies identified through screening (blue) and the others (grey) are included in each figure panel. Notice in (C) that a small portion of the chromosomal region deleted in <i>Df(3R)Exel6195</i> (dashed box) did not overlap with any of those deleted in other available deficiencies. One-way ANOVA followed by Dunnett’s test of each group versus control <i>g</i>^<i>2</i> flies carrying no deletion (black bars): **p<0.01; ***p<0.001.</p