10.5068/D1R67M
Ghiani, Cristina
0000-0002-9867-6185
David Geffen School of Medicine at UCLA
Dell'Angelica, Esteban
University of California Los Angeles
Lee, Frank Y.
University of California Los Angeles
Larimore, Jennifer
Agnes Scott College
Faundez, Victor
0000-0002-2114-5271
Emory University
Dell’Angelica, Esteban C.
0000-0002-6363-6845
University of California Los Angeles
Ghiani, Cristina A.
0000-0002-9867-6185
University of California Los Angeles
Sex‐dimorphic effects of biogenesis of lysosome‐related organelles
complex‐1 deficiency on mouse perinatal brain development
Dryad
dataset
2020
Biogenesis of Lysosome-related Organelles Complex 1 (BLOC-1)
Brain development
Cellular and Molecular Neuroscience
National Cancer Institute
https://ror.org/040gcmg81
R01GM112942S1
National Institutes of Health
https://ror.org/01cwqze88
1R56MH111459
National Institutes of Health
https://ror.org/01cwqze88
1RF1AG060285
National Institutes of Health
https://ror.org/01cwqze88
R01GM112942
National Institutes of Health
https://ror.org/01cwqze88
R01GM112942S1
2020-02-12T00:00:00Z
2020-02-12T00:00:00Z
en
https://doi.org/10.1002/jnr.24620
26343354 bytes
5
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
The function(s) of the Biogenesis of Lysosome-related Organelles Complex 1
(BLOC-1) during brain development is hitherto largely unknown. Here, we
investigated how its absence alters the trajectory of postnatal brain
development using as model the pallid mouse. Most of the defects observed
early postnatally in the mutant mice were more prominent in males than
in females and in the hippocampus. Male mutant mice, but not females, had
smaller brains as compared to sex-matching wild-types at postnatal day 1
(P1), this deficit was largely recovered by P14 and P45. An
abnormal cytoarchitecture of the pyramidal cell layer of the hippocampus
was observed in P1 pallid male, but not female, or juvenile mice (P45),
along with severely decreased expression levels of the radial glial marker
GLAST. Transcriptomic analyses showed that the overall response to the
lack of functional BLOC-1 was more pronounced in hippocampi at P1 than
at P45 or in the cerebral cortex. These observations suggest that absence
of BLOC-1 renders males more susceptible to perinatal brain maldevelopment
and although most abnormalities appear to have been resolved in juvenile
animals, still permanent defects may be present, resulting in faulty
neuronal circuits, and contribute to previously reported cognitive and
behavioural phenotypes in adult BLOC-1-deficient mice.
Histomorphometrical and Immunohistochemical Analyses P1 male and female,
and P45 male, wild-type and pallid mice were anesthetized with
isoflurane (30-32%) and transcardially perfused with phosphate-buffered
saline (PBS, 0.1 M, pH 7.4) containing 4% (w/v) paraformaldehyde (Electron
Microscopy Sciences, Hatfield, PA). The brains were rapidly dissected out,
post-fixed overnight in 4% (w/v) paraformaldehyde at 4°C, and
cryoprotected in 15% (w/v) sucrose. Coronal sections were cut on a
cryostat (Leica, Buffalo Grove, IL) collected sequentially, and paired
along the anterior-posterior axis before further processing. For Nissl
Staining, coronal brain sections (20 μm) were stained with a 1% (w/v)
cresyl violet (Sigma-Aldrich Corp., St. Louis, MO) solution as previously
reported (Lee et al., 2018). Photographs were acquired on a Zeiss Axioskop
equipped with a Zeiss colour or monochrome Axiocam using the AxioVision
software (Zeiss, Pleasanton, CA) and used to determine the thickness of
the cerebral cortex (layers I-VI) in rostral and caudal sections, the
outer border and total sectional area of the hippocampus, and the cell
density and cytoarchitecture in the Cornu Ammonis (CA)1 subfield of the
hippocampus. Measurements were performed by two observers masked to the
genotype, sex and age of the animals, from which each histological section
has been generated, with the aid of the Zeiss Axiovision or the NIH Image
Software (ImageJ, http://rsb.info.nih.gov/ij/). To control and reduce
variations due to staining intensity, for the analysis of CA1 cell
density, images were acquired using a monochromatic camera (AxioCam;
Zeiss). For image analysis of the CA1 cell layer, the “vertical profile
plot analysis” feature of ImageJ was used as follows: a grid set at 16,000
square pixels was over-imposed onto each image, and three identical
rectangles of height equal to 800 pixels and width equal to 400 pixels
(156 µm x 88 µm) were set at a fixed distance from the lateral ventricle
(third, fifth, and seventh columns of the grid). The rectangles were
positioned such that the long axis would cross completely the pyramidal
cell layer, and used to automatically calculate a profile plot as the
average pixel intensity per row (i.e., across the short horizontal axis)
represented as a function of the position on the long vertical axis. The
plots (three plots for each of the left and right hippocampi in each
section) were then averaged to obtain one profile per section. To average
the profiles of all the sections per animal (8-12 consecutive sections per
animal), sixth-order polynomial curves were fitted to each profile per
image and used to automatically estimate the mid-point of the cell layer,
which in turn was used to align and average the profiles from the
different sections to yield one average profile per animal. Data are shown
as the mean ± SEM of 4-5 animals per sex, genotype and age. total RNA
extractions for Biochemical Analyses Hippocampi from P1 and P45 wild-type
and pallid mice were rapidly dissected, and the two halves frozen
separately to be used for protein or total RNA extraction (Ghiani et al.,
2010; Lee et al., 2018). Total RNA was extracted using the Invitrogen™
TRIzol™ reagent (ThermoFisher; Carlsbad, CA) following the manufacturer’s
protocol from P1 wild-type, pallid and sandy, as well as P45 wild-type and
pallid hippocampi. Samples were further purified by treatment with Ambion®
TURBO DNA-free™ (Life Technologies; Waltham, MA), followed by a second
extraction with phenol/chloroform. Sample concentrations and purity were
assessed using a ThermoScientific™ NanoDrop™ One Microvolume UV-Vis
Spectrophotometer (Canoga Park, CA). Microarray Analysis Microarray
hybridization was performed at the Southern California Genotyping
Consortium. Prior to hybridization, the quality of RNA was further
monitored using micro-capillary electrophoresis (Bioanalizer 2100, Agilent
Technologies; Santa Clara, CA). Total RNA (10 ng/ml) was amplified,
labelled, and hybridized to the Illumina MouseRef-8 v2.0 expression array
(Illumina; San Diego, CA) overnight at 58°C. The microarrays were then
scanned using an iScan reader, and the signal compiled using BeadStudio
software (Illumina). Raw microarray data were analysed using Bioconductor
packages and online tools [Database for Annotation, Visualization and
Integrated Discovery (DAVID) Bioinformatics Resources,
http://david.abcc.ncifcrf.gov/]. Quality assessment was performed by
examining the inter-array Pearson correlation and clustering based on the
top variant genes. Contrast analysis of differential expression was
performed by using the LIMMA (Linear Models for Microarray and RNA-Seq
Data) package (Ritchie et al., 2015). After linear model fitting, a
Bayesian estimate of differential expression was calculated at a false
discovery rate (FDR) of 5% or less. To identify genes with differential
expression in P1 hippocampus potentially due to BLOC-1 deficiency, as
opposed to strain-specific effects, average mutant-to-wild-type signal
ratios (in logarithmic scale base 2) were calculated for each probe
yielding a strong signal in at least one genotype (quartile >0.66)
in both BLOC-1-deficient mutants pallid and sandy (relative to their
matched wild-type controls), and only those probes resulting in a greater
than 50% signal increase (logarithm base 2 > 0.6) in both mutants
were selected for further analyses. Top enriched biological functions were
inferred by means of the Gene Set Enrichment Analysis (GSEA), using the
online GEne SeT AnaLysis Toolkit (WebGestalt, http://www.webgestalt.org)
and the datasets comprising the relative signals obtained in each
BLOC-1-deficient mutant and its wild-type control for all microarray
probes yielding relatively strong signals (defined as quartile
>0.66 in at least one sample), using standard parameters (5-2000
genes per category, Benjamini-Hochberg procedure to control for multiple
comparison).