BIOLOGY 199

Date: September 7, 2015

Speaker: Christian Jirard Z. Custodio
TITLE: Profiling
Transcriptomics

of

Neurological

Behavior

and

Disorders

through

OBJECTIVES:
Specifically, the objectives of this seminar are
1. to explain how important the role of transcriptomics is in profiling
delicate
biological processes specifically neurological phenomena,
2. to discuss new methodologies in transcriptome analysis to identify
the molecular
mechanism of certain neurological phenomena and
3. to describe the key genes involved in aggression, brain ischemia,
and Alzheimers
disease in different organisms and trace the possible mechanism
of gene regulation.
DISCUSSION:
A. Transcriptomics in Analyzing Brain Processes
(Munro and Perreau, 2009).
o
o

o
o

The CNS is an extremely complex structure in animal anatomy with an

equally complex transcriptome.
The application of gene expression data to elucidate functional
information from such a complex system requires elegant experimental
design, generation of vast datasets and analysis tools capable of
identifying biologically relevant genes or patterns from within the data.
High-throughput gene expression analysis platforms, such as
microarrays, have proven to be powerful tools for generating large
amounts of transcriptomic data.
Complexity of the Central Nervous System is attributed to the
heterogeneity of the transcriptome across structures, regions, and cell
types.

Structural and Regional Heterogeneity

Transcriptional profiling of 20 different anatomically distinct sites across the human
CNS identified:
1. Distinct expression signatures between CNS and non CNS
structures
2. Distinct expression signatures within each CNS region
Cellular Heterogeneity
Three main neural cell types are commonly discussed in the literature,
neurons, oligodendrocytes and astrocytes. Thus, it is important to characterise
the cell-specific transcriptome and response to injury.

B.

Comparison of Tame
Transcriptome

and

Aggressive

Foxes

Based

on

Brain

(Kukekova et al, 2011).

Red/Silver Fox
The red fox/ silver fox (Vulpes vulpes) is one of the most widely distributed
mammalian species across the globe and are close relatives of the domestic dog
(Canis lupus familiaris).
Methodology
Breeding
o Selectively bred tame and aggressive strains.
o The behavioural phenotypes of these strains were genetically verified.
o

Due to their intermediate nature compared to rodent and primate

complexity, these foxes can serve as models for studying variations of
aggressive behaviours.
Brain Samples

Pre-frontal cortex tissues were collected from 7 month old

male foxes Total RNA was extracted using TRIZOL reagent
cDNA library was prepared and sequenced using 454 GS FLX
Titanium
sequencing
Reads were assembled and bioinformatics analyses were
performed
Gene expression between the two strains were compared
through transcriptome sequencing (RefSeq) and candidate
genes were validated through RT-qPCr

o
o
o

Results
Comparison of gene expression between the two fox samples
1.) A total of 27 genes had a tenfold difference of expression between the
tame and aggressive
individuals.
2.) 335 genes had at least a twofold difference of expression (280 up in tame,
55 up in
aggressive).
Biofunctional Classification of involved genes
1.Behavior
2. Nervous System Development and Function
3. Cardiovascular Development and Function
o
o

Overexpression of genes involved in neurological diseases was

observed in the tame sample.
Genes involved in cardiovascular diseases were overexpressed
in the aggressive sample.

Validation of gene expression by RT-qPCR

HTR2C: sertonergic and dopaminergic signalling

C. Gene Expression in Response to Brain Ischemia

(Hori et al, 2011)
Background
o Brain ischemia is a common cause of death and its molecular basis is
therefore widely essential to medicine.
o Brain ischemia, also termed cerebral ischemia, is a condition in which
there is insufficient blood flow to the brain leading to tissue death due
to poor oxygen supply
Methodology
o
o

Use of a 32 mouse permanent middle cerebral artery occlusion

(PMCAO) model
33 high-throughput DNA microarray analysis on an Agilent microarray
platform.

Results (Genomic response to ischemia)

Changes in gene expressions in the ischemic brain and their
functional categorization
At 6 and 24 hours respectively:
o 1237 and 2759 cases of gene upregulation (> 1.5-fold)
o 620 and 2102 cases of downregulation
Categorization of involved genes based on their function under gene
ontology terms
Through analysis of brain transcriptomes post-ischemia, several differentially
expressed genes have been identified leading to the possible identification of target
genes for reversal.
1. Biological process: 3440 and 6826 induced genes, and 1398 and 3531
suppressed genes at 6
and 24 hours, respectively
2. Molecular function: 875 and 1732 induced genes, and 394 and 1021
suppressed genes at 6
and 24 hours, respectively
3. Cellular component: 1788 and 3522 induced genes, and 762 and 2015
suppressed genes at 6
and 24 hours, respectively
Specific Genes
1. S100 calcium-binding protein family
2. matrix metalloproteinases
3. Chemokines and Interleukins
Validation with RT-PCR
D. Profile of Possible Genes Involved in Alzheimers Disease by Analyzing
Divergence
of Mouse and Human Brain Transcriptome
(Miller et al, 2010)

Background
o Comparison of mouse and human gene transcription has highlighted
divergence of brain transcriptomes
o Between species, alteration of gene network positioning has a role in
the expression of human-specific disease phenotypes
o Two factors were analysed in networks between the species, and these
are expression levels and connectivity.
Methodology and Results
Gene coexpression assessment through module-by-module basis
Differences in Mouse and Human Modules Provide Insight into AD.
Variation of network organization between species could explain why
Alzheimers Disease is enriched in the human population.
Modules Invloved
M9h (FBXW12, LOC152719/ZNF721, FLJ12151, and ZNF160)
M7h (GSK3 and tau)
Divergence between mouse and human bran networks
o 67 validated, human- specific marker genes for cell type
o PSEN1
o Divergence between mouse and human AD models may be due
to evolutionary changes in expression patterns of PSEN1 in the
context of neuronoligodendrocyte interactions.
(Ray et al, 2008)
o
o

1,663 differentially expressed genes between AD samples and controls

Construction co-expression networks by clustering 1,663 genes into 6
modules

Modules Involved
Module 1 (associated with cardiovascular diseases and diabetes)
contained 18 disease-associated genes
evidence of strong association between CVD and the incidence
of AD
SUMMARY AND CONCLUSION:
Transcriptomics is an essential method for understanding neurological
phenomena. Different approaches can be used, but the most popular methods are
through DNA microarrays and Validation through RT-PCR of randomly selected
differentially expressed genes. Through the identification of key genes involved in
these neurological phenomena, certain risk factors to and possible targets for
control and reversal of crucial neurological phenomena may also be identified and
addressed.
REFERENCES:

Hori, M. et al. (2011). Unraveling the ischemic brain transcriptome in a permanent

middle cerebral artery occlusion model by DNA microarray analysis. DMM doi:
10.1242/dmm.008276.
Kukekova, A. et al. (2011). Sequence comparison of prefronatal cortical brain
transcriptome from a tame and an aggressive silver fox (Vulpes vulpes). BMC
Genomics, 12(482): 1471-2164.
Miller, J., Horvath, S., and Geschwind, D. (2010). Divergence of human and mouse
brain transcriptome highlights Alzheimer disease pathways. PNAS, 107(28):
12698-12703.
Munro, K. and Perreau, V. (2009). Current and future applications of transciptomics
for discover in CNS disease and inury. Neurosignals, 17: 311-327.
Ray, M., Ruan, J., and Zhang, W. (2008). Variations in the transcriptome of
Alzheimers disease reveal molecular networks involved in cardiovascular
disease. Genome Biology, 9(10): R148.