1. J of Proteomics | April 20, 2007 | vol. 103 | no.10
Development and implementation of a novel interactome
platform in studies on neurodegeneration.
Ewelina Maliszewska-Cyna, Kelly Markham, Gerold Schmitt-Ulms
Centre for Research in Neurodegenerative Diseases (CRND),
Proteomics Unit
Tanz Neuroscience Building
University of Toronto
_________________________________________________________
Background
Neurodegenerative disorders
Neurodegenerative diseases are among the
major scourges of modern society. To a certain
degree, these diseases represent deficits
frequently associated with the experience of
senescence, such as forgetfulness, loss of
dexterity and deterioration in strength and
locomotor functions (1). These disorders have
been termed a “silent epidemic”, because they
have been inadequately recognized by the
public and, more importantly, because of their
late onset leading to predominant involvement
of the elderly, who have low public visibility
because of their social isolation. The most
common disorders in this category are
Alzheimer’s disease, Parkinson’s disease and
amyotrophic lateral sclerosis (2). In addition,
there exists a large collection of less common
neurodegenerative disorders. Together, they
represent an enormous burden on both patients
and their families. These diseases also
constitute an increasing economic burden on
society.
In spite of the increasing prevalence of
neurodegeneration, their nature remains an
enigma. Where is the line to be drawn between
a disease and the normal progression of
senescence associated with the natural
degradation of neurons? What is the
mechanism by which functionally related
neurons die surrounded by other regions that
are spared? Is there more than one cause for
each of the relatively clear defined syndromes?
Is there a shared pathogenesis or a clinical
overlap among the neurodegenerative
disorders?
These are only some of the many
issues that face researchers today. Substantial
advances have taken place in the field of
neuroscience that have had direct relevance to
neurodegeneration. Molecular biology,
immunohistochemistry, clinical physiology,
and pharmacology have all provided an insight
into our understanding of these degenerative
processes (3). In some areas, these
developments generated practical benefits
leading to the establishment of treatment
methods, whereas in other instances the
knowledge has remained frustratingly
theoretical.
Amyloid hypothesis of Alzheimer’s disease
One of best known neurodegenerative
disorders is Alzheimer’s disease (AD) and the
proposed amyloid hypothesis has become the
main focus of AD research (4). Amyloid β-
peptide (Aβ) is recognized as the primary
component of the neuritic plaques of AD
patient’s brain tissue (Fig.1). Consequently,
identification of mutations in the gene coding
for the Aβ precursor protein (APP) illustrated
that APP mutations contribute to the formation
of Aβ deposits, also called the amyloid fibrils.
It is well known that most of the mutations
occur within or in close proximity to the APP
gene, that is normally cleaved by proteases
called the α-, β- and γ-secretases (5). In the
absence of mutations in the APP gene, the
function of α-secretase is being favored,
whereas in the mutated APP gene it is the β-
and γ-secretases that are predominantly active.
Furthermore, APP mutations internal to the Aβ
sequence elevate the self-aggregation of Aβ
into amyloid fibrils – the hallmark of
Alzheimer’s disease (2).
In addition to the mutations in the APP
gene, there are other genes associated with
AD, the presenilin family being the most
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prominent one. Mutations in the PSs interfere
with the cleavage of APP causing the
overproduction of the most destructive
amyloidogenic peptide, the Aβ-42 (5).
Presenilin 1 (PS1) and presenilin 2 (PS2) are
incorporated into protein complexes destined
for maturation within the ER and Golgi
apparatus. The maturation involves
endoproteolysis of PSs into N- and C-terminal
fragments, and the recruitment of additional
components into the complex, all of these
processes being essential for normal activity of
secretases. Consequently, mutation within PS1
or PS2 causes the abnormal maturation and
results in the disruption of the protein complex
function.
The formation of tau protein
neurofibrillary tangles constitutes another
hallmark of AD pathology. Studies have
shown that tau tangles are being deposited only
after changes in Aβ metabolism and initial
plaque formation has occurred. Therefore,
cerebral Aβ accumulation is the primary
influence in AD patients and the rest of the
disease process, including tau protein
neurofibrillary tangles formation - an
imbalance between Aβ production and Aβ
clearance.
Knowledge of molecular and
biochemical processes behind Alzheimer’s
disease allows our research team to pursue
interactome studies. Our objective is to
carefully analyze AD bait interactomes, which
involves co-immunoprecipitation (Co-IP)
studies. Co-IP has its disadvantages, mostly
associated with the interference from antibody
bands in gel analysis. In those cases, where
several proteins may be co-precipitated with
the target, presence of the co-eluted antibody
heavy and light chains (25 and 50 kDa bands
in reducing SDS-PAGE gel) in the preparation
can obscure the results. The ideal situation
would be to conduct the Co-IP without
contamination of the eluted antigen with the
antibody. With this potential interference
eliminated, only the co-precipitated proteins
will be present and detected on a gel. One such
method has been reported by Burckstummer et
al., who suggest tandem affinity purification
(TAP), a generic two-step affinity purification
protocol that enables the isolation of protein
complexes under close-to-physiological
conditions for subsequent analysis by mass
spectrometry (6). It is because of the above
reasons that protein tagging with the TAP
system would become a method of choice in
our laboratory for these types of experiments.
Cell culture models of neurodegeneration.
Neurospheres
Cultures of purified cell populations obtained
from animal live tissue are an invaluable
model for the study of the nervous system. The
goal of such a culture is to establish a cell
population that retains its cytoarchitecture,
maintains maturation and differentiation
patterns and preserves its original function.
Cell cultures provide the following advantages:
1.Control over the environment, such as
nutrients, ions, temperature, gas phases and
cofactors. 2. Isolation from modulators, such
as hormonal, humoral and metabolic
influences, normally present in the body. 3.
Direct accessibility to the cells allowing for
visual observation of morphology and cell
dynamics. 4. Rapid preparation for
morphological techniques, immunocyto-
chemistry assays and biochemical analyses (7).
In addition, protein tagging in an entire
organism poses great difficulties, hence a cell
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culture is a preferred model to study
manipulated cell systems.
All of these characteristics make
neural cell culture one of the most efficient and
versatile experimental models available for
studying the possible role of various physical
and chemical agents involved in neuronal loss
associated with neurodegenerative disorders.
Cancerous cells and neuroblastoma cells in
particular, are used by many research centres
to study neurodegeneration, as those cells have
the capacity to proliferate indefinitely (8).
Nevertheless, there are limitations to this
model associated with little control over their
cell cycle and proliferation rates. As a result,
our lab explores other possible models.
One such model is the neuronal stem
cells system (NSCs). This is the
undifferentiated pool of cells of the nervous
system with the ability to proliferate, self-
renew and generate a large number of
differentiated progeny cells (9). Data also
indicate that stem cells are not only
functioning during embryonic development,
but also throughout our lives. Reynolds and
Weiss (1992) were the first to isolate NSCs
(10). In vitro, NSCs form aggregates, or
neurospheres (NS), in the presence of
epidermal growth factor (EGF) or basic
fibroblast growth factor (bFGF) (Fig. 2).
Neurospheres are in fact a heterogeneous
mixture of stem and progenitor cells, however,
over time, there are only nestin-positive NSCs
that retain the ability to form neurospheres and,
more importantly, retain their multipotentiality
over extended periods of time.
Although many researchers report that
NSCs are indeed a useful tool, there are certain
caveats associated with this model. One of
them comes into play when one desires to
deliver a gene of interest into neurospheres (9,
10). It appears that widely used transfection
methods, with the use of such reagents as
Lipofectamine 2000, are not applicable when
handling NS. Because of the relatively slow
growth rate of NS, a considerable amount of
transfected signal disappears before cells
achieve the desired titer. Moreover, because
NSCs aggregate into NS and later on grow in
suspension, LF2000 must be constantly present
in the medium, which proves to be toxic and
leads to cell death. Davidson et al. succeeded
in adeno-viral mediated gene transfer into
nestin-positive NSCs while preserving their
potential for self-renewal and proliferation (10)
It is for this reason that adenoviral transfection
is used instead of the standard LF2000 method
when delivering the gene of interest into
neurospheres (10, 11).
In view of the above evidences, it
seems plausible to create a stable pool of
neuronal stem cells that would serve as a
model to study neurodegeneration involved in
Alzheimer’s disease. The objective of my
project consisted of two parts. Firstly, the
development and maintenance of a stable
culture of neurospheres was to be achieved.
Secondly, optimization of a transfection
method using lentivirus was necessary in order
to establish a stable line of transfected cells
expressing a gene of interest. An application of
this tool to study the expression of PS1 and
PS2 proteins was also explored.
Materials and Methods
Neurospheres culture in T75 flasks
Procedure for tissue extraction was adapted
from Weiss et al. with few modifications (8).
Whole brains were removed and cleared of
meninges from mouse embryos at embryonic
day 14 (Charles River Laboratories, CD-1
strain) (Fig.3). Tissue wastriturated with fire
polished pipette in 3 ml cold PBS and
centrifuged at 1500 rpm for 10 min (Napco
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2028R). Supernatant was removed and cells
were resuspended in 1 ml DMEM/F-12
medium supplemented with 6% glucose G-
7021), 2mM L-glutamine (Sigma, G-3202),
3mM sodium bicarbonate (Sigma, S-5761),
5mM HEPES (Sigma, H-4034), 30 nM sodium
selenite (Sigma, S-9133) and hormone mix
containing 25ug/ml insulin (Sigma, I-5500),
100 ug/ml transferring (Sigma, T-2252), 20nM
progesterone (Sigma, P-6149) and 60uM
putrescine (Sigma, P-7505). 20 ng/ml of hEGF
(PeproTech, 315-09), 100 units of penicillin
and 100ug/ml of streptomycin (Gibco, 15140-
122) was also added to the medium. Finally,
cells were plated at a density of 0.75x105
cells/ml in T75 flasks containing 10ml of
medium. Cells were cultured in the incubator
at 37o
C and 5% CO2 and passaged every 7
days. To test for the presence of NS, an aliquot
of cells was collected during each passage and
tested for the presence of nestin using
monoclonal antibody and 12.5% SDS PAGE
gel. Primary and secondary antibodies and
their respective dilutions are given in Table 1.
Cell count and viability
Cell density was determined before each
passage using a hemocytometer and viability
was determined using the standard trypan blue
test (Sigma, cat # T8154). Cell counts were
performed in duplicate as necessary. Spherical
aggregates were gently dissociated
mechanically and a sample was taken for cell
count.
Lentiviral transfection and transduction of
HEK 293T cells
One day before the experiment, cells were
plated to achieve a cell density of
2x106
/100mm dish. Calcium-phosphate
precipitate, or CPP (CalphosPM
Mammalian
Transfection Kit, Clontech, 631312) was added
to a plasmid mixture containing the envelope
(pMd2G plasmid) and packaging (pPAX2
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plasmid) fractions of lentivirus as well as
pWPI transfer insert containing the GFP
sequence (Table 2). Plasmid mixture of 700ul
was combined with 700ul of 2 x HEPES
buffered saline (HBS) and incubated for 20
min. at room temp. The mixture was added
dropwise to the cells for overnight incubation
in 37o
C and 5%CO2. After the media change,
cells were incubated overnight. The media
containing viral supernatant was collected the
following day. A new series of HEK 293T
cells was plated in two 12-well plates to
achieve 50% confluency the following day. On
the next day, a second sample of viral
supernatant was collected, combined with the
previous one in the quick seal tubes and
centrifuged at 121000 x g for 2 hours at 4o
C
(Beckman SW32ti) to concentrate the viral
sample. Viral pellet was then resuspended in
300ul of growth media. Fresh HEK 293T cells
were transduced at different dilutions of viral
supernatant (10ul – undiluted as well as 1:10
and 1:100 dilutions) and left for a 48 hour
incubation period. Finally, the cells were
examined under the ultraviolet microscope for
determination of transfection and transduction
efficiency.
Results
Neurospheres
We isolated neural stem cells from an
embryonic day 14 mouse brain and maintained
them in EGF supplemented medium. Earlier
work showed that cells propagated in EGF or
bFGF displayed characteristics of stem cells,
that is, they were self-renewable and
multipotent (7, 8). Over a period of 7 days,
single cells formed multicellular aggregates
with a morphology consistent with that of
neurospheres grown in suspension (Fig. 2).
After optimizing the medium components,
there was an almost 10-fold increase in cell
density over the period of three weeks (Table
3). After the fourth week, the cell density and
viability started to decrease rapidly. Cells from
collected neurospheres were tested for the
expression of the progenitor cell marker,
nestin, (Fig. 4).
Lentiviral transduction
After successful transfection, HEK 293T cells
were transduced at different dilutions of viral
supernatant. The results are outlined in Figure
5. Transduction achieved the highest yield
when no dilution was made and cells were
transduced with 10ul of viral suspension per
each well (Fig. 5 B, D). Conversely,
transducing cells with diluted viral material
(1:100) resulted in a very low transduction
rate. At the same time, the transduction rate
remained unchanged when used cells were
grown attached to the plates (Fig. 5A, B) or in
suspension culture (Fig. 5C, D).
Discussion
The objective of this study was to design a
robust and efficient tool for the purpose of
interactome studies. One aspect of such studies
is a requirement of a large quantity of protein-
starting material, and it is for this reason that
the neurosphere culture system requires a
substantial expansion. Many researchers
reported high yields of NS when these were
cultured in spinner flasks, also called
bioreactors (7, 12, 13). Because NS grow in
suspension, upscaling the cell system into
spinner flasks of size up to 1L does not pose
any disadvantages. When placed on the
Thermolyne Cell-Gro magnetic stirrer (speed
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at 100rpm), cells achieve high density while
retaining their characteristics of stem cells, as
reported by Sen et al. (12). Relatively slow
growth rate and a requirement of sustained low
cell density are other reasons why large
volume of starting material is plausible in
order to obtain enough material for interactome
studies coupled with mass spectrometry
analyses.
Even though the period of robust NS
culture achieved in our laboratory is shorter
than that reported in the literature (3, 6, 9), it is
sufficient for the transfection to be coupled
along with the interactome studies.
Nevertheless, if one would like to preserve this
culture system for prolonged periods of time,
one would have to consider adding a second
growth factor. It has been reported that
although initially NS cells grow well in EGF-
alone, at later stages, they change their
requirements in such a way that
supplementation with both EGF and bFGF is
required (14, 15).
Even though Lentiviral experiments
have been conducted on the HEK 293T cell
line, we infer that the same technique and
conditions can be applied to a NS system as
well. What is of concern to this system is to
what degree the biology of neurospheres be
influenced by the viral transduction and by the
presence of a foreign genome in the cell
system. Because of these reasons and in order
to avoid overexpressing the protein of interest,
fairly low levels of transduction is desirable.
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Moreover, the influence of the virus on the
activity and functioning of examined protein
remains unknown. More studies involving
protein cleavage patterns that would further
decide on choice of the protein tagging system
are necessary.
As this new interactome platform is
being developed, one can implement this tool
to study bait interactomes of Alzheimer’s
disease. The protein of interest to our lab is the
TAP-tagged PS1 and PS2. It has been reported
by Burckstummer et al. that the application of
TAP procedure resulted in a tenfold increase in
protein-complex yield and improved the
specificity of the procedure. Therefore, the
TAP purification system coupled with the
Lentiviral transduction tool has a large
potential to obtain large amounts of the intact
protein complexes as well as generate enough
material for mass spectrometry analysis. Figure
6 outlines the details of the TAP-tagged
experimental design. If successful, this system
may prove to be of more use than currently
used, yet not always reliable, antibodies.
Future perspectives
It would be worthwhile to implement this
novel interactome platform into research on
TAP-PS1 and TAP-PS2 inserts. In addition,
the research would take full advantage of many
transgenic animal models that are currently
available at CRND. Insights gained from
interactome analyses of Tg systems might lead
to novel therapeutic and diagnostic methods of
Alzheimer’s disease or simply to a better
understanding of processes governing
neurodegeneration.
Acknowledgements
The authors thank Yu Bai for technical
assistance with Lentiviral work as well as
Rasanjala Weerasekera for valuable
suggestions regarding molecular biological
techniques. This study was supported by a
grant from the Canadian Institutes of Health
Research (CIHR), operating since Sept. 15,
2003.
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