You are on page 1of 10

Analysis of enriched insoluble proteins in frontotemporal dementia and Alzheimer’s disease

Yifan “Ronnie” Li

Advisor: Dr. Chad Hales

_________________________________________ 08/17/2017
__________________
Student signature Date

As rotation advisor, I concur with the content of the rotation report.

__________________________________________ 08/17/2017
__________________
Advisor signature Date
Abstract
Frontotemporal dementia (FTD) is a neurodegenerative disorder that can be characterized
by the intracellular accumulation of hyperphosphorylated tau due to mutations in the MAPT gene.
While the sequence of cellular disease-promoting events is not yet fully understood, several
experimental therapies are currently available in the clinic. However, most of these therapies have
undesirable off-target effects. Based on quantitative data from the enriched insoluble proteome,
we used immunohistochemistry to assess for aggregates of the proteins ACTR5, COL6A1,
COL6A3, and STRN4 to elucidate the mechanisms behind FTD and AD pathology. We found no
aggregates of ACTR5, COL6A1, COL6A3, or STRN4 in both Alzheimer’s disease (AD) and FTD
cases. Western blotting results showed increased STRN4 expression in the whole brain proteome
in sporadic AD cases compared to control. Moreover, Western blotting qualitatively supported the
hypothesis that STRN4 is enriched in the insoluble fraction of FTD cases compared to control.
Together, these data confirm the efficacy of the STRN4 antibody and provide some insight into a
possible role for STRN4 in neurodegeneration.
Introduction
Frontotemporal dementia (FTD) is a neurodegenerative disorder that can cause behavioral
and language difficulties (Elahi and Miller, 2017). One of the main causes of FTD is autosomal
dominant mutations in the MAPT gene (Irwin et al., 2014). These mutations lead to the abnormal
accumulation of hyperphosphorylated tau that likely contributes to disease symptomology.
However, we do not yet fully understand the sequence of cellular events that promotes the
underlying degenerative process. One approach is to examine the insoluble brain proteome for
proteins that may be aggregating and/or dysfunctional. We and others have used this approach to
study Alzheimer’s disease (AD) and other neurodegenerative disorders (Diner et al., 2014; Hales
et al., 2016).
We previously performed quantitive proteomic sequencing on the brain insoluble proteome
from FTD-tau cases. Several enriched proteins were identified, including actin-related protein 5
(ACTR5), the A1 and A3 chains of collagen type VI (COL6A1 and COL6A3, respectively), and
striatin 4 (STRN4). Specifically, the ACTR5 protein is involved in DNA repair and possibly
transcriptional regulation (Kitayama et al., 2009), so loss of this protein might lead to deficits in
neuronal survival. Collagen type VI has been implicated in neuronal survival as well, as loss of
this protein facilitates apoptosis and promotes neurodegeneration during aging (Cescon et al.,

1
2016). Finally, a recent study of tauopathy in mice found that the Strn4 gene was upregulated in
Mapt knockout mice (Maphis et al., 2017). Interestingly, STRN4 also interacts with protein
phosphatase 2A (PP2A; Goudreault et al., 2009), which could further implicate its role in
regulating tau phosphorylation.
Since insoluble proteins may form intracellular aggregates, we performed
immunohistochemistry (IHC) on post-mortem human FTD-tau brains to assess for aggregates of
ACTR5, COL6A1, COL6A3, and STRN4. Control and AD cases were also included to determine
specificity of immunostaining for FTD-tau. We also performed Western blotting to compare
expression levels of STRN4 among FTD, AD, and control cases. Elucidation of the aggregation
properties of these proteins would provide further insights into AD and FTD pathology as well as
ideas for novel therapies, since current experimental therapies for tauopathies – which include
inhibiting tau phosphorylation and tau immunotherapy – lack specificity and have untoward effects
in humans (Khanna et al., 2016).
Materials and Methods
Antibodies
Primary antibodies included ACTR5 (PA5-26274, Fischer, Waltham, MA, USA),
COL6A1 (PA5-29068, Fischer, Waltham, MA, USA), COL6A3 (PA5-52689, Fischer, Waltham,
MA, USA), STRN4 (PA5-63343, Fischer, Waltham, MA, USA), and PHF-tau (MN1020, Fischer,
Waltham, MA, USA). Secondary antibodies included biotinylated goat anti-rabbit (BA-1000,
Vector Laboratories, Burlingame, CA, USA) and biotinylated goat anti-mouse (BA-9200, Vector
Laboratories, Burlingame, CA, USA).
Immunohistochemistry
A cryostat was used to cut formalin- and paraformaldehyde-fixed post-mortem human
frontal cortex into 50 µm sections. Sections were washed in 0.1 M phosphate buffer (PB),
incubated with 3% hydrogen peroxide, washed again with PB, and then incubated with blocking
solution [Tris-buffered saline (TBS), 8% goat serum, 0.1% Triton X-100 (Fischer, Waltham, MA,
USA) and Avidin D (Vector) (10 µg/mL final concentration; Vector Laboratories, A-2000)] for 1
hour at 40C. Sections were washed with TBS and then incubated with primary antibody solution
[primary antibody, TBS, 2% goat serum, and biotin (50 µg/mL; Sigma, St. Louis, MO, USA,
B0301) overnight at 4oC. Sections were washed with TBS the following day and incubated with
biotinylated secondary antibody solution (secondary antibody, TBS, 2% goat serum) for 1 hour at

2
4oC. Sections were washed with TBS, incubated with Vectastain Elite ABC amplification reagent
(Vector Laboratories, PK-6200) for 1 hour at 4oC, washed again with TBS, incubated with 3,3-
diaminobenzidine solution (DAB; Sigma, D4418) for 3 minutes at room temperature and finally
washed with TBS. Sections were mounted in standard fashion on glass slides, dried overnight at
room temperature, dehydrated through alcohol rinses and Histo-Clear (National Diagnostics,
Atlanta, GA, USA; HS-200) and finally coverslipped with DPX mounting medium (VWR,
Atlanta, GA, USA; 360294H).
Protein blotting
Protein samples from control, FTD, and AD whole brain homogenates were run on a
gradient 4-12% polyacrylamide gel, then transferred onto a nitrocellulose membrane. The
membrane was washed in TBS with Tween 20 (TBST), incubated in TBS with blocking buffer for
30 minutes at room temperature and washed again in TBST. Primary antibody solutions against
STRN4 and tau were applied to the membranes [primary antibody, 1.5 mL TBST, 1.5 TBS with
blocking buffer], and the membranes were incubated overnight at 40 C. Membranes were washed
in TBS, fluorophore-conjugated secondary antibody solutions [secondary antibody, 10 mL TBST,
10 mL TBS with blocking buffer] were added to the membranes, and the membranes were
incubated for 1 hour at room temperature. Membranes were finally washed in TBS and analyzed
on Odyssey Reader.
Results
Selection of candidate proteins
Among more than 200 enriched potential targets in the insoluble proteome data set, the
proteins ACTR5, COL6A1, COL6A3, and STRN4 were chosen for immunohistochemical
analysis. These proteins were selected based on extensive review of literature that could propose
a relationship between their loss of function and neurodegeneration. Kitayama et al. (2009)
suggested a role for human ACTR5 in DNA repair and transcriptional regulation, two essential
functions for cell survival. Therefore, loss of this protein could ultimately translate to neuronal
death. Furthermore, loss of collagen type VI promotes neurodegeneration during aging (Cescon et
al., 2016). Lastly, loss of STRN4 could impair the regulation of tau phosphorylation through the
interaction between STRN4 and PP2A (Goudreault et al., 2009).
Immunostaining

3
Four candidate proteins from the insoluble proteome were selected based on literature
evidence that could provide a link between their loss of function and the neurodegenerative
process.
Immunostaining using the ACTR5, COL6A1, and COL6A3 antibodies yielded mostly
background, nonspecific staining (Figure 1). We observed staining of neuropil and blood vessels,

Control AD FTD

ACTR5

COL6A1

COL6A3

STRN4

Figure 1. Antibodies against ACTR5, COL6A1, COL6A3, and STRN4 yielded mostly background, nonspecific
staining in control, AD, and FTD cases. Staining of neuropil and blood vessels was also observed.

4
and nonspecific staining of cell bodies in the gray matter. We anticipated seeing pathological
aggregates of STRN4 in FTD cases, but we observed light cytoplasmic staining instead.
Qualitatively, the cytoplasmic STRN4 staining in the FTD case is more prominent than in the
control and AD cases, but quantitative analyses are needed to validate this result. We did not
observe pathological aggregates of STRN4 in control, AD, or FTD cases.
AD, FTD, and corticobasal degeneration (CBD) sections were immunostained with
antibody against PHF-tau as positive controls to demonstrate abundance of hyperphosphorylated
tau in these disease cases (Figure 2).

Western blotting
Since we observed enrichment of STRN4 in the insoluble proteome, we wanted to better
understand the transition of STRN4 into the insoluble proteome to suggest a possible mechanism
behind FTD pathology. Analysis of the whole brain total homogenate in control, AD, and FTD
cases revealed an increased expression of STRN4 in all tested sporadic AD cases and an AD case
with an amyloid precursor protein (APP) mutation compared to MAPT P301L FTD cases and a
presenilin 1 (PS1) mutant AD case (Figure 3). Counterintuitively, we observed enrichment of
STRN4 in sporadic AD but not FTD-tau cases. We did observe bands of hyperphosphorylated tau
in the positive control as expected.
Because the first Western blot used the total brain homogenate, we wished to determine
whether STRN4 is more highly expressed in just the insoluble fraction of FTD cases. Figure 4
shows a second Western blot in which only control and FTD cases were used, and the samples
were split into total brain homogenate (TH), soluble, and insoluble fractions. GADPH was used as
a normalization marker, although it was not a reliable marker for the insoluble fractions. Upon
qualitative analysis, we discovered that the STRN4 band is more intense compared to the GADPH

5
band in the insoluble fractions in two FTD cases (highlighted in yellow) compared to control. This
is consistent with our hypothesis that STRN4 is enriched in the insoluble proteome of FTD cases.

Figure 3. Protein blotting revealed the highest expression of STRN4 in sporadic AD cases and
an APP mutant AD case compared to other AD and FTD cases.

6
Figure 4. A second Western blot showed that the relative intensities of the STRN4 bands in
the insoluble fraction of FTD cases (highlighted yellow) were more intense than the
GADPH bands compared to control cases.

Discussion
Prior experimentation has identified many enriched targets in the insoluble proteome in
FTD-tau cases. We chose four candidate proteins based on compelling literature evidence that they
might play roles in neurodegeneration. We performed immunohistochemistry to assess
aggregation properties, and we utilized Western blotting with anti-STRN4 antibody to determine
enrichment of STRN4 in FTD cases. Immunohistochemical analysis using antibodies targeted to
all four candidate proteins produced mostly background and nonspecific staining in control, AD,
and FTD cases, with the exception of STRN4 displaying more cytoplasmic staining in the FTD
case than in control and AD cases. The positive control cases demonstrated neurofibrillary tangles
(NFTs) as expected. Western blotting, however, showed that STRN4 was more highly expressed
in sporadic AD cases compared to FTD cases, which was counterintuitive to our rationale.
However, a second blot comparing FTD cases and controls, separating the total brain homogenate,

7
soluble, and insoluble fractions, demonstrated qualitatively that STRN4 was enriched in the
insoluble fraction in FTD cases compared to controls, a finding that supports our hypothesis.
Western blotting also showed that the anti-STRN4 primary antibody was effective and reliable.
Taken together, these data provide intriguing insights into a possible role of STRN4 in FTD and
AD pathogenesis.
STRN4 is an interesting target primarily because of its interaction with PP2A. If STRN4
were to form aggregates and demonstrate loss of function, this could result in disruption of the
regulation of tau phosphorylation. One exciting future direction is to use shRNA to knockdown
STRN4 gene expression in cultured neurons that carry a MAPT mutation and then examine the
downstream effects on tau. This would provide additional clues into what role, if any, STRN4
plays in the neurodegenerative process.
Despite the interesting observations, there were two limitations to the first Western blot.
The first limitation was that the masses of the protein samples were not equalized prior to loading
the first blot, which could have skewed the results. Secondly, because we looked at the total brain
homogenate instead of just the insoluble fraction, there is the possibility that the cases that showed
low levels of STRN4 expression simply saw a shift of STRN4 to the insoluble proteome. However,
we performed a second blot that corrected for these limitations by normalizing the masses of
samples and by separating the total homogenate, soluble, and insoluble fractions. Our results are
still qualitative nonetheless, so more meticulous analyses are needed to quantify whether STRN4
expression is truly upregulated in the insoluble fraction.
Finally, as mentioned earlier, there are over 200 previously identified enriched protein
targets in the insoluble proteome. There could very well be other proteins we have not analyzed
that play a role in the neurodegenerative process. Further consideration must be given to each
target in order to resolve the pathogenic cascade. Nevertheless, these preliminary experiments
provide an exciting glimpse into new mechanisms behind tauopathies, and therefore new potential
treatments to target cellular processes gone awry.
References
Cescon M, Chen P, Castagnaro S, Gregorio I, Bonaldo P (2016) Lack of collagen VI promotes
neurodegeneration by impairing autophagy and inducing apoptosis during aging. Aging
8:1083-1098.

8
Diner I, Hales CM, Bishof I, Rabenold L, Duong DM, Yi H, Laur O, Gearing M, Troncoso JC,
Thambisetty M, Lah JJ, Levey AI, Seyfried NT (2014) Aggregation properties of the small
nuclear ribonucleoprotein U1-70K in Alzheimer disease. J Biol Chem 289:35296-35313.
Elahi FM, Miller BL (2017) A clinicopathological approach to the diagnosis of dementia. Nat Rev
Neurol 13:457-476.
Goudreault M, D’Ambrosio LM, Kean MJ, Mullin MJ, Larsen BG, Sanchez A, Chaudhry S, Chen
GI, Sicheri F, Nesvizhskii AI, Aebersold R, Raught B, Gingras AC (2009) A PP2A
phosphatase high density interaction network identifies a novel striatin-interacting
phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3)
protein. Mol Cell Proteomics 8:157-171.
Hales CM, Dammer EB, Deng Q, Duong DM, Gearing M, Troncoso JC, Thambisetty M, Lah JJ,
Shulman JM, Levey AI, Seyfried NT (2016) Changes in the detergent-insoluble brain
proteome linked to amyloid and tau in Alzheimer’s Disease progression. Proteomics
16:3042-3053.
Irwin DJ, Cairns NJ, Grossman M, McMillan CT, Lee EB, Van Deerlin VM, Lee VM,
Trojanowski JQ (2015) Frontotemporal lobar degeneration: defining phenotypic diversity
through personalized medicine. Acta Neuropathol 129:469-491.
Khanna MR, Kovalevich J, Lee VM, Trojanoswki JQ, Brunden KR (2016) Therapeutic strategies
for the treatment of tauopathies: hopes and challenges. Alzheimers Dement 12:1051-1065.
Kitayama K, Kamo M, Oma Y, Matsuda R, Uchida T, Ikura T, Tashiro S, Ohyama T, Winsor B,
Harata M (2009) The human actin-related protein hArp 5: nucleo-cytoplasmic shuttling
and involvement in DNA repair. Exp Cell Res 315:206-217.
Maphis NM, Jiang S, Binder J, Wright C, Gopalan B, Lamb BT, Bhaskar K (2017) Whole genome
expression analysis in a mouse model of tauopathy identifies MECP2 as a possible
regulator of tau pathology. Front Mol Neurosci 10:1-13.

9