Abstract #0022
BRAIN GENE EXPRESSION SIGNATURES FROM
CEREBROSPINAL FLUID EXOSOME RNA PROFILING
S. B. Zanello1, L. Hu3, I. Sinclare3, B. S. Carter2, G. Brock3 and J. Skog3
1Universities Space Research Association, Houston, TX; 2University of California San Diego Medical
3
School, San Diego, CA; and Exosome Diagnostics, Cambridge, MA
1. BACKGROUND
2. Materials and Methods
VIIP and IIH
Table 1. CSF samples from 10 patients with elevated ICP and control
• Visual symptoms reported in astronauts returning from long
duration missions in low Earth orbit are thought to be related
to fluid shifts within the body due to microgravity exposure,
leading to increased intracranial pressure (ICP) and visual
impairment and intracranial pressure (VIIP) syndrome.
• Idiopathic intracranial hypertension (IIH) is a condition
characterized by increased ICP without clinical, laboratory, or
radiologic evidence of an intracranial space-occupying lesion,
meningeal inflammation or venous outflow obstruction.
• While the described VIIP syndrome focuses on ocular
symptoms, spaceflight has been also associated with a number
of other performance and neurologic signs, such as headaches,
cognitive changes, vertigo, nausea, sleep/circadian disruption
and mood alterations, which, albeit likely multifactorial, can
also result from elevation of ICP.
• We hypothesize that these various symptoms are caused by
disturbances in the neurophysiology of the brain structures
and correlated with molecular markers in the cerebrospinal
fluid (CSF) as indicators of neurophysiological changes.
• The purpose of this study is to investigate changes in brain
gene expression via exosome analysis in patients suffering
from ICP elevation of varied severity as a first step toward
obtaining evidence suggesting that cognitive function and ICP
levels can be correlated with biomarkers in the CSF.
Exosomes
• Exosomes are 30-200 nm microvesicles that are actively
released from cells into all biofluids such as blood, urine, and
CSF. They carry a highly rich source of intact protein and RNA
cargo (Figure 1).
• Exosomes have been isolated from CSF and measured for brain
associated genes (Figure 2).
A
B
ID
Description
BRKpool1
BRKpool2
CCC002
CCC003
CCC004
CCC005 (*)
CCC007 (*)
CCC008
CCC009
CCC010
CCC014
CCC015
Pooled 5 bio-reclamation samples, replicate 1
Pooled 5 bio-reclamation samples, replicate 2
SAH due to ruptured aneurysm
ACA aneurysm rupture
SAH due to ruptured aneurysm
Colloid cyst of Third ventricle
ACA aneurysm rupture
Gunshot injury
SAH
AVM
SAH due to ruptured aneurysm
Trauma - Fall from train/bus
CSF from 10 patients with
elevated ICP
Exosome isolation from
2ml CSF per patient using
membrane affinity spin
columns (Figure 4)
RNA profiling on
OpenArray® Human
Inflammation Panel
APP
F
F
M
F
F
M
M
F
F
M
15
18
12
13
15
7
16
10
3
6
Figure 5. RNA profiling raw data
OpenArray® is a TaqMan qPCR array. Human Inflammation Panel
consists of 586 target and 21 endogenous control assays.
Numbers in red color indicate # of assays with readout. (A) All
607 assays. (B) 21 endogenous control assays only.
UC San Diego
Exosome Diagnostics
CLIA facility
Figure 3. Workflow diagram
• QIAzol lysis and release of RNA
Figure 1. Exosome/microvesicle biogenesis
Exosomes and other vesicles can be released by (A) multivesicular
body pathway or through (B) direct budding at the plasma
membrane. (C) Transmission electron microscopy of microvesciles.
BACE1
B
• Bind vesicles to membrane & wash
Spin column
HIF1A
A
Gender ICP (mmHG)
SAH: subarachnoid hemorrhage
ACA: anterior communicating artery
AVM: arteriovenous malformation
*: excluded downstream due to low quality of exosome RNA
C
Gene
3. Data and Results
•
•
•
•
Phenol/Chloroform extraction
Ethanol conditioning
Bind to RNeasy column and wash
Elute RNA
Figure 4. Exosome RNA extraction
Spin column to purify exosomes and the extraction workflow
4. Conclusions
Figure 6. Differentially expressed genes
Raw Crt values were normalized using the geometric mean of 6
globally expressed endogenous control assays. Two-group
contrast between 2 control samples and 8 patient samples was
performed. 84 genes here were significant (p-value<0.05). Red
color means higher Crt, blue color lower Crt.
Description
Hypoxia-inducible factor 1
β-site amyloid precursor protein
cleaving enzyme 1
Amyloid precursor protein
MAPT/Tau Microtubule associated protein
SOD1
Superoxide dismutase 1
ACTB
Beta-actin
18S
18S ribosomal RNA
Figure 2. Expression of brain associated genes in CSF exosomes
CSF from 36 individual donors were pooled. Exosome RNA was
extracted 3 times from the same CSF batch. All 7 genes were
reproducibly detected.
• Exosomes from CSF can be efficiently used for RNA profiling.
• Exosomes contain brain derived RNA transcripts as well as
markers of inflammation.
• The expression trend of some of the inflammation markers
examined suggest a concerted inflammatory response in
response to the injuries.
• No correlation analysis with ICP was possible, since these
samples corresponded to patients that underwent treatment
for ICP reduction in the trauma unit.
• The next step will include samples from IIH patients with
known ICP values.
This work is funded by the Human Research Program (Omnibus grant NNJ12ZSA002N)
Figure 7. Expression profile
of 5 inflammation markers