Screen for kinases affecting amyloidogenic cleavage by BACE1

233 Pages·2011·5.56 MB·English

by  Stephan Penzkofer

Screen for kinases affecting
amyloidogenic cleavage by BACE1

Dissertation

zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Universität Konstanz
Mathematisch-Naturwissenschaftliche Sektion
Fachbereich Biologie
vorgelegt von
Stephan Penzkofer
Konstanz, Juli 2011

Tag der mündlichen Prüfung: 24.10.2011
1. Referent: Professor Dr. Marcel Leist
2. Referent: Professor Dr. Daniel Dietrich
Summary: The Amyloid β peptide (Aβ) is suspected to be a causal agent for Alzheimer’s disease (AD).
Therefore a screen for kinases downregulating the initial step of its production, the cleavage of the
Amyloid Precursor Protein (APP) by Beta-site of APP Cleaving Enzyme 1 (BACE1), was conducted in
this study. Briefly, HEK293 cells were colipofected with one of in total 1357 siRNAs against 60% of the
human kinome and either an APP construct with only the β-cleavage site left or normally cleavable
APP as control. Remaining β-cleavage was for logistic reasons firstly measured with an activity-test for
secreted alkaline phosphatase (SEAP) fused to both types of APP and subjected to Aβ-ELISA when
interesting. Before the screen, the APP-constructs were characterized in the cell types HEK293 and
CGCs with regards to cleavage, especially by BACE1. The screen resulted in 38 hits of which one,
Testis Specific Serine Kinase 3, was confirmed once more. In a second, bioinformatic project, an
initially suspected APLP-like pseudogenic-like sequence in C3orf52 was refuted. Further, analysis of
C3orf52 gene expression data hints on a role in myeloid leukemia. Lastly, the phylogenetic
relationship of the APP family paralogs was examined, also in comparison to neighboring gene
families, and found in the topology (APLP1)(APLP2/APP).

Zusammenfassung: Das Amyloid β-Peptid (Aβ) steht im Verdacht, ursächlich für die Alzheimer-
Demenz (AD) zu sein. Daher wurden in dieser Studie Kinasen gesucht, die den ersten Schritt seiner
Entstehung, Schnitt des Amyloid Precursor Proteins durch Beta-site of APP Cleaving Enzyme 1
(BACE1), herabregulieren. Dafür wurden HEK293 colipofiziert mit einer von insgesamt 1357 siRNAs
gegen 60% des menschlichen Kinoms und entweder einem ausschliesslich β-spaltbarem APP-
Konstrukt oder normal spaltbarem APP als Kontrolle. Aus logistischen Gründen wurde die
übriggebliebene β-Spaltung zunächst mit einem Aktivitätstest für sekretierbare alkaline Phosphatase
(SEAP), das an beide APPs fusioniert war, gemessen und falls interessant noch mit Aβ-ELISA. Vor
dem Screen wurden die APP-Konstrukte in den Zelltypen HEK293 und CGC mit Hinblick auf Spaltung,
v. a. durch BACE1, charakterisiert. Der Screen resultierte in 38 Hits, von denen einer, Testis Specific
Serine Kinase 3, nochmals bestätigt wurde. In einem zweiten, bioinformatischen Projekt wurde eine
zunächst vermeintlich APLP-ähnliche pseudogenartige Sequenz in C3orf52 widerlegt. Weiter deutet
die Analyse von C3orf52-Genexpressionsdaten auf eine Rolle in myeloider Leukämie hin. Zuletzt
wurden die phylogenetischen Beziehungen der APP-Familie-Paraloge untersucht, auch im Vergleich
mit benachbarten Genfamilien, und in der Topologie (APLP1)(APLP2/APP) gefunden.
Abbreviations

aa amino acids LINE1 Long Interspersed Nuclear Element1
Aβ Amyloid β peptide LOAD Late-Onset AD
AβK16V Aβ with K to V exchange, not mbp mega base pairs
cleavable by α-secretases MCA Middle Cerebral Artery
ACh Acetyl-Choline MCI Mild Cognitive Impairment
ACoA Anterior Communicating Artery MRI Magnetic Resonance Imaging
AD Alzheimer's Disease my, mya million years, - ago
AICD APP Intracellular Domain µm 10-6 m
Alu primate specific repeat class NbMc Nucleus basalis of Meynert complex
ANNE APLP-Near Notes Extant NCBI National Center for Biotechnology
APLP1 APP-Like Protein 1 Information
APLP2 APP-Like Protein 2 NCT nicastrin
APP Amyloid Precursor Protein nm 10-9 m
APPswe APP with Swedish double mutation NSAID Non-Steroidal Anti-Inflammatory Drug
AQD Amino-Quinazoline-Derivative O-GlcNAcylation O-linked-β-N-acetyl-
ATCC American Tissue type Culture glucosaminylation
Collection PBS Phosphate Buffered Saline
AU Absorption Units PFK Phosphofructokinase
BACE1 Beta-site of APP Cleaving Enzyme 1 pg picogram
BBB Blood Brain Barrier pNPP para-Nitro-Phenyl-Phosphate
bp base pairs PS1 preseniline 1
C3orf52 chr 3 open reading frame 52 rev reverse
CAA Cerebral Amyloid Angiopathy SD Standard Deviation
CArG CC-AT-rich(6)-GG box SEAP Secreted Alkaline Phosphatase
CGC Cerebellar Granule Cell siRNA small interfering RNA
ChAT choline acetyl transferase SNP Single Nucleotide Polymorphism
chr chromosome TFA Tierforschungsanlage
CSNK1D Casein Kinase 1 isoform D tko triple knockout
ct cycle of threshold TMD Transmembrane Domain
CTF C-Terminal Fragment TTMP TPA-induced Transmembrane Protein
dko double knockout us upstream
DMSO dimethylsulphoxide WHO World Health Organization
DS Down's Syndrome 3'UTR 3' Untranslated Region
ds downstream
Embry.s.c. Embryonic stem cells
ENSEMBL joint project between EMBL - EBI Bestiarium
and Wellcome Trust Sanger Institute
EOAD Early-Onset AD Ac Anolis carolinensis green anole
ER Endoplasmic Reticulum Bt Bos taurus cattle
e-value expected value Ce Caenorhabditis elegans worm
fwd forward Cf Canis familiaris dog
gbp giga base pairs Cm Callorhinchus milii elephant shark
GFP Green Fluorescent Protein Dm Drosophila melanogaster fruitfly
HBP Hexosamine Biosynthesis Pathway Dr Danio rerio zebrafish
HEK293 Human Embryonic Kidney293 cell line Ec Equus caballus horse
HIV Human Immunodeficiency Virus Ga Gasterosteus aculeatus stickleback
ICD-10 International Classification of Diseases Gg Gallus gallus chicken
10th revision Hs Homo sapiens human
inh inhibitor Mm Mus musculus mouse
IPAD Iso-Phthal-Amide-Derivative Oa Ornithorhynchus anatinus platypus
IQ Intelligence Quotient Oc Oryctolagus cuniculus rabbit
kbp kilo base pairs Ol Oryzias latipes medaka
kd knockdown Pt Pan troglodytes chimpanzee
ko knockout St Silurana tropicalis clawed frog
Tn Tetraodon nigroviridis
Tr Takifugu rubripes pufferfish
Some parts of this thesis have already been published:

Poster: Identification of BACE1- and APP-regulating kinases
Stephan Penzkofer*, Christiane Volbracht&, Karina Fog&, Kenneth Vielsted& and Marcel Leist*!
*:Doerenkamp-Zbinden Chair for alternative in vitro methods, University of Konstanz, Konstanz,
Germany; !:corresponding author &:H. Lundbeck A/S, Valby, Denmark
Presented at PENS Summer School: “Novel molecular strategies to treat neurodegenerative
diseases”, Ofir, Portugal, 7. July 2007

Christiane Volbracht, Stephan Penzkofer, David Mansson, Kenneth Vielsted Christensen, Karina Fog,
Stefan Schildknecht, Marcel Leist, Jacob Nielsen, Measurement of cellular β-site of APP cleaving
enzyme 1 activity and its modulation in neuronal assay systems, Analytical Biochemistry 387 (2009)
208-220.

Curriculum vitae of Stephan Penzkofer
06.06.1978 born in Munich, Germany
09/1984 – 06/1998 attendance of schools in Munich and Erding
07/1998 – 04/1999 compulsory military service
05/1999 – 09/1999 newspaper catering and car rental at the Munich Airport
10/1999 – 09/2000 studies in civil engineering at the Technical University of Munich

10/2000 – 09/2002 basic study period in biology at the University of Regensburg, Germany, with
intermediate examinations in physics (oral), chemistry (oral) and biology
(written; best result of the semester)
10/2002 – 03/2004 laboratory research courses in genetics, organic chemistry, biophysics and
biochemistry followed by diploma examinations in organic chemistry,
biochemistry and biophysics
04/2004 – 04/2005 diploma thesis with the Institute of Biophysics and physical Biochemistry at the
University of Regensburg: “Characterisation of the PDZ2-PIP-Interaction with
NMR-titration studies”

10/2005 – 05/2006 research attachment in cell culture, proteomics with mass spectrometry,
capillary electrophoresis and flow cytometry with the Institute of
Bioengineering and Nanotechnology, Biopolis, Singapore

09/2006 – 09/2011 doctoral thesis with the Doerenkamp-Zbinden-Chair of alternative in vitro
methods at the University of Konstanz, Germany: “Screen for kinases affecting
amyloidogenic cleavage by BACE1”
eidesstattliche Erklärung:
"Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und
ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus
anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter
Angabe der Quelle gekennzeichnet."
Table of contents

1 Alzheimer’s disease (AD) and screening for BACE1-affecting kinases 1

1.1 History of AD research and AD in Down’s syndrome 1
I
1.2 Plaques and Aβ 4
N
1.3 APP and the discovery of its cleavage by proteases in HEK293 6
T
1.4 The families of APP and BACE1 11
R
1.5 Diabetes type 2 and the families of APP and BACE1 12
O
1.6 Atrophy in AD and the cholinergic hypothesis 13
D
1.7 Tau modifications and BACE1 transcriptional regulation 17
U
1.8 Influence of kinases on Aβ-generation 23
C
1.9 Difficulties with direct inhibition of BACE1 30
T
1.10 Aims of the study 33
I
2 Evolution of the APP family and characteristics of processed pseudogenes 34
O
2.1 Evolution of the APP family 34
N
2.2 Characteristics of processed pseudogenes 37
3 Screen preparation and execution 42
M
3.1 Cells 42
A
3.2 Molecular material 45
T
3.2.1 APP-constructs 45
E
3.2.2 Inhibitors and other compounds 48
R
3.2.3 Antibodies 49
I
3.2.4 Primers 50
A
3.2.5 small interfering RNA (siRNA) 50
L
3.3 Experimental setup 69
S
3.4 Data collection and analysis 71

3.4.1 SEAP activity measurement 72
&
3.4.2 A40 quantification 72

3.4.3 Cell viability measurements 72
M
3.4.4 Western blot analysis 73
E
3.4.5 Immunocytochemistry 73
T
3.4.6 Determination of mRNA knockdown 74
H
3.4.7 Statistics 75
O
3.4.8 Screen data analysis 75
D
3.5 Laboratory equipment 76
S
4 Sequences of C3orf52, orthologs and C3orf52 primers 78

5 Characterization of cells and APP-constructs 80

5.1 Characterization of CGCs, HEK293 and SHSY5Y cells 80

5.2 Optimization of transfection for APP-construct characterization 85
R
5.3 Characterization of the APP-constructs 89
E
6 Kinome screen for BACE1-affecting kinases 114
S
6.1 Optimization of cotransfection with SEAP-APPswe-EpoR and siRNAs 114
U
6.2 Analysis and validation of screen results 123
L
7 Identification of potential APLP-like pseudogenic fragments in C3orf52 137
T
7.1 Examination of potential APLP-like pseudogenic fragments 137
S
7.2 Gene expression data analysis of C3orf52 141
8 Second attempt to identify a fourth APP family member on chromosome 3 143
9 Examination of the regions containing the APP family 144
10 Additional figures and tables from chapters 7-9 148
11 Interpretation of 185
D
11.1 cell- and APP-construct-characterization 185
I
11.2 the kinome screen for BACE1-affecting kinases 191
S
12 Interpretation of 198
C
12.1 potential APLP-like pseudogenic fragments 198
U
12.2 gene expression data analysis of C3orf52 202
S
13 The search for the 4. APP family member and the examination of the APP family regions206
S

I

O

N

14 References 208
15 Acknowledgements 226
Introduction
1 Alzheimer‟s disease (AD) and
screening for BACE1-affecting kinases
1.1 History of AD research and AD in Down‟s syndrome
AD was named in memory of Alois Alzheimer and originally the term was coined by
Emil Kraepelin in 1910 to describe the type of presenile dementia which was reported
histologically by Alzheimer in 1906 [1]. The age of its onset was earlier than in most
cases of dementia and silver staining revealed round non-cellular structures which
have earlier been called plaques [2, 3], with an average diameter of 50 µm [4]. Based
on clinical (behavioral) symptoms, the disease was earlier identified as distinct from
others by Kraepelin in whose laboratory Alzheimer searched on the histological level
for (biological) pathologies. These pathologies were postulated by Kraepelin as
underlying causes of the major psychiatric disorders.
Already in 1904, Alzheimer had published findings of plaques in cases of senile
dementia [5, 6] which were corroborated in 1907 by Oskar Fischer in 16 cases [3, 7],
but only in the early 1950s, Meta Neumann realized that most senile dementia cases
can be classified as AD [8]. Nowadays, they comprise probably around 60% of all
dementia patients or 24 Mio worldwide. The WHO labeled this senile dementia in
ICD-10 with G30.1: “Alzheimer‟s disease with age of onset 65 or older”, more
commonly called late-onset AD (LOAD) as opposed to early-onset AD (EOAD).
One of the first EOAD cases after Auguste Deter was Johann Feigl who died at age
57 [9] and was examined by Alzheimer post-mortem in 1910 [10]. In 1992, the brain
samples were rediscovered and revaluated and no genetic cause for the disease
could be found [11], as in most cases of EOAD. However dementia and mental
illness also affected his mother and three siblings, so his dementia might be called
familial [9].
Familial AD (FAD) cases require for confirmation two first-degree-relatives diagnosed
with AD and account for 7% of all EOAD cases. Around 20% of familial EOAD are
passed on in an autosomal-dominant pattern with high penetrance and 25% to 50%
of them are caused by mutations in three genes. They are directly linked to the
above-mentioned plaques.
Its main constituent, amyloid-β (Aβ), a peptide of mostly 40 or 42 amino acids, was
isolated in 1984 by Glenner [12]. Since it was noticed in 1929 that Down‟s syndrome
1
Introduction
(DS) patients develop plaques similar to those of AD patients [13, 14], the plaques of
DS patients were also examined and also demonstrated to be made up mainly of Aβ
[15, 16]. DS is a chromosomal disorder which is caused by an extra chromosome 21
in all cells (95% of DS patients) or in a fraction of the cells (=mosaicism; 1-2%). Also,
familial DS exists (=Robertsonian translocation; 2-3%) and the rare event of
duplication of parts or all of chromosome 21 [Wikipedia]. Thus it was supposed, that
Aβ is encoded on chromosome 21.
Indeed it was identified there in 1987 by the groups around Goldgaber, Tanzi and
Kang [17-19] as part of a larger protein, the Aβ Precursor Protein (APP), launching
an unprecedented boom in AD research, dealing with each and every facet of APP.
The search for genetic causes in EOAD cases resulted in a total of 25 non-silent
mutations in APP which have been detected between 1990 and 2009 [from
www.alzforum.org, APP Mutations Table]. The best known, the Swedish mutation, is
a double mutation found in a Swedish family and leading to higher Aβ production [20-
22], presumably because it leads to more cleavage of APP by a protease, called
Beta-site of APP Cleaving Enzyme 1 (BACE1). BACE1 has been identified in 1999
independently by Hussain, Lin, Sinha, Vassar and Yan and their colleagues [23-27].
For the liberation of Aβ a second protease has to cleave the truncated APP, which
was called the γ-secretase complex. It consists of the 4 proteins Nicastrin, Aph-1,
and the Presenilins-1 and -2 [28, 29] for which 92 and 11 mutations have been linked
to AD, respectively [from www.alzforum.org, Presenilin-1 or Presenilin-2 Mutations
Table]. The ratio of Aβ40 to Aβ42 is regulated by the γ-secretase complex, and many
mutations shift the ratio in favor of Aβ42 [30] which is more prone to aggregation.
These and additional facts were the basis for a nowadays widely accepted theory on
the cause of AD: the Amyloid Cascade Hypothesis by Selkoe and Hardy in 1991 [31,
32]. Additional facts were the finding that the major genetic risk factor for LOAD,
apolipoprotein E ε allel 4 (ApoEε4) [33], leads to a strong buildup of amyloid and the
knowledge of amyloid-induced diseases in other organs like kidney which can be
successfully treated by prevention of the responsible amyloid protein. Also, it was
observed that Aβ oligomers can be toxic to cells and that brain inflammatory cells,
the microglia, are activated by plaques. According to the hypothesis, Aβ42
accumulates and oligomerizes in limbic and association cortices and exerts subtle
effects on synapses as oligomers. These form fibrils and fibers and then deposit as
not matured, diffuse plaques, thereby activating microglia and astrocytes, the key
2
Introduction
players of inflammatory response in brain. Neurons in the vicinity can thus be directly
injured by oxidative stress or through altered ionic homeostasis. Kinase and
phosphatase activities could be changed and lead to a common hallmark of many
types of dementia, neurofibrillary tangles. Finally, the widespread neuronal
dysfunction and neuronal loss is clinically perceived as AD-typical dementia with
memory loss and death occurs in average nine years after disease onset.
However, the Amyloid Cascade Hypothesis was questioned in the past years, partly
because diffuse plaques as mentioned above are also found in non-demented
elderly, although not as many as in AD patients. Interestingly, Alois Alzheimer himself
interpreted plaques as signs of brain aging and did not accept Oskar Fischer‟s
suggestion that they might correlate with severity of dementia and memory loss [34].
AD in DS patients is one of the strongest arguments for the hypothesis as Aβ being
causative for dementia. On a closer look, plaques in DS patients are found as early
as 12 years and in 16% from 20-29 years and 80% from 30-39 years [35], whereas
estimates for dementia range from 6%-75% [36]. A more recent study determined the
prevalence of dementia with 13% and a mean age of onset of 55 years [37], an
earlier one with 15%-45% and 52 years [38]. Reasons for difficulties with
determination of dementia prevalence in DS patients can have its origins in the
overall physical changes coming along with trisomy 21, e.g. hypothyroidism in 40%
which leads to cognitive decline when untreated [39], mitral valve prolapse in 50%
which increases the risk for cerebrovascular accident [39] and a lifestyle with less
physical activity leading to high obesity rates [39]. Insulin resistance, also called
diabetes type 2, which is linked to obesity in DS patients affects 10% of patients [40,
41]. Also, the liver isozyme of phosphofructokinases (PFKL) resides on chromosome
21 [42].
However, regarding the facts, that at most half of DS patients with extensive plaque
generation develop AD, the existence of differences like a higher prevalence of
epilepsy and the different overall DS constitution, one might think of not totally
identical etiologies of dementia in DS and sporadic AD patients. For further
unraveling the role of Aβ for the development of AD in DS patients it would be of
great interest to find DS patients with mosaicism, such that neurons would be spared
of trisomy 21 and hence APP not overexpressed, and see whether dementia will still
be found in the usual prevalence or not.
3
Introduction
At least it could be possible to find these DS patients by testing their skin cells for
trisomy 21, since skin cells like neurons are derived from the ectodermic lineage.
They could be of DS phenotype due to mesoderm/endodermic trisomy 21 and with
normal IQ, as an inverse relationship of the fraction of skin cells with trisomy 21 and
measured IQ demonstrated [43]. Closest to the condition of mosaic DS with only two
copies of APP (also in neurons) was a 78 year old woman with partial trisomy 21 in
all cells and a phenotype suggestive of DS, although not typical. Her brain was very
similar to a DS brain with its weight of 950 g, the round shape and the small
cerebellum but lacking extensive amyloid plaques, below 1/mm2, and also atrophy.
She was not demented [44], lending support to the Amyloid Cascade Hypothesis.
Finally, a direct link exists between AD and DS patients: mothers giving birth to DS
children before age 35 have a fivefold higher rate of AD [45]. A DS child mother who
developed presenile dementia with mild cerebral atrophy at age 41 showed a
connection: at routine karyotyping she was diagnosed with trisomy 21 in 10% of white
blood cells, yet had no DS phenotypical features [46]. The lymphocyte trisomy 21
mosaicism affects only 2% of mothers with DS children but occult mosaicism may be
more common in brain and germ cells and thus in part explain the association of DS
and AD in family histories [47] or also some of the EOAD cases. So, trisomy 21
without DS phenotype leads to a higher risk for AD, thus also strengthening the
Amyloid Cascade Hypothesis.
1.2 Plaques and Aβ
Since their discovery, the plaques have been examined for their morphological
structure and chemical constitution.
Morphologically, mainly two different types of plaques in brain parenchyma are
distinguished. The earlier stages are so-called diffuse plaques which are found in
non-demented elderly and young DS patients, characterized by diameters of 10-200
µm and blurred boundaries but not containing dystrophic neurites [48]. Also, no
microglia or activated astrocytes are found in their vicinity [49]. Diffuse plaques
cannot be stained with congo-red or thioflavin-S [50] in contrast to later stages which
are called neuritic plaques and typically display a dense core in middle of a corona of
degenerating neurites [51] and the final stage of a “burnt-out” plaque with only the
core left over [50]. Whereas neuritic plaques are surrounded by microglia and
astrocytes, “burnt-out” plaques are not [52]. Neuritic and burnt-out plaques are
stainable with iodine, congo-red and thioflavin-S because they consist of amyloid
4