Jonny Daborg
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Recent animal studies have shown that synaptic elimination is dependent on
the complement system. Interestingly, knockout of C1q in transgenic mice
expressing human APP with FAD mutations produces a minor
neuropathological phenotype (Fonseca et al. 2004). In line with this
mechanism of elimination, investigation of post mortem brain tissue from AD
patients have shown increased levels of complement mRNA (Yasojima et al.
1999), this finding has been further supported by studies showing increased
levels of complement proteins in the CSF from AD patients (Finehout et al.
2005; Wang et al. 2011). In addition, it has been shown that the genes
encoding complement receptor 1 (CR1) and complement factor H (CFH) are
associated with AD diagnosis (Harold et al. 2009; Lambert et al. 2009; Naj et
al. 2011; Seshadri et al. 2010; Zetterberg et al. 2008).
In conclusion, these findings warrant a closer investigation of the
involvement of the complement system and synaptic elimination in AD.
Synaptic elimination and the complement system in Alzheimer’s disease
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2
AIM
2.1
The general aim
The general aim of this thesis was to examine the hypothesis that AD is
primarily a synaptic disease – with an emphasis on complement-mediated
elimination of synapses.
2.2
The specific aims
I.
To examine if genetic variation in the gene encoding the
RAGE receptor is associated with AD.
II.
To investigate if the complement system is involved in
elimination of synapses in the hippocampus of mice.
III.
To measure the levels of C3, C4, and CR1 in CSF from
patients with MCI, probable AD, MCI patients who later
developed probable AD, and compare them with the
levels in healthy controls, with the purpose of evaluating
these proteins as potential biomarkers for AD, and to
gain evidence for complement involvement in AD.
IV.
To evaluate the complement genes CR1, C3, C2 and
CFB as possible susceptibility genes for AD.
Jonny Daborg
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3
METHODOLOGICAL
CONSIDERATIONS
3.1
Subjects
The subjects of the different studies were chosen to best provide an answer to
the questions posed in the specific aims. In general I have preferred to work
with human subjects for both scientific and ethical reasons - mice do not
become demented nor can they provide consent.
3.1.1
Patients
For details on diagnostic procedures and other information on the patients
please refer to the different papers. The patients in our studies were
diagnosed with MCI, AD or other neurodegenerative conditions using
standardised clinical methods and test batteries performed by psychiatrists
and psychologists at specialised memory clinics. Controls were cognitively
normal according to these tests. A clinical diagnosis of AD is not perfect and
it has been estimated that 10-16% misdiagnoses occur in relation to
neuropathology (Brunnstrom and Englund 2009; Galasko et al. 1994;
Victoroff et al. 1995), which often is considered the gold standard diagnostic
method. Biomarkers for AD neuropathology, e.g., CSF levels of Aβ42 and
tau proteins, may help to increase the diagnostic accuracy compared with
clinical criteria (Blennow et al. 2010), but studies showing this for a fact are
still lacking. Clinical AD diagnoses in papers I and IV were confirmed either
by a typical CSF tau and Aβ biomarker profile or at autopsy according to the
neuropathological CERAD criteria for definitive AD (Mirra et al. 1991).
3.1.2
Mice
To study the role of the complement system in elimination of synapses in the
hippocampus, mice deficient in C3 were used (Pekna et al. 1998). These mice
do not lack the whole gene, but the critical exon 24, which renders the gene
product incomplete and thus non-functioning (Pekna et al. 1998). To ensure
almost identical genetic backgrounds between WT controls (C57Bl/6) and C3
knockout mice (C3 KO), the C3 KO mice were backcrossed for 13
generations.
Synaptic elimination and the complement system in Alzheimer’s disease
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3.2
Genotyping
Genotyping of single nucleotide polymorphisms (SNPs) was made by use of
TaqMan allelic discrimination, a polymerase chain reaction (PCR)-based
assay. PCR is a method of DNA amplification, based on the molecular
mechanisms of DNA replication (Mullis and Faloona 1987). A sample of
DNA is heated to approximately 95ºC to make the two DNA strands separate,
subsequently the temperature is lowered to 50-60ºC in order to enable
hybridisation of two oligonucleotides (predesigned DNA molecules that bind
to a specific region of the genome) that flank the desired sequence of the
sample DNA and work as primers for the DNA polymerase. Then the
temperature is raised again, to 72ºC, and the heat-stable DNA polymerase,
Taq polymerase, will commence with the work of replicating the DNA
sequence singled out by the primers. Thus, the sequence of interest is doubled
for each cycle.
The TaqMan allelic discrimination is accomplished by cleavage of a
fluorescent dye from the TaqMan probe by the exonuclease activity of the
Taq polymerase as it replicates the SNP-containing sequence. Two probes are
used in the reaction, one for each allele, thus enabling identification of
heterozygotes and both kinds of homozygotes (Livak et al. 1995).
PCR is easy in theory but difficult to optimize in practice. In the present
thesis all genotyping experiments were performed by certified lab technicians
specialised in molecular genetics, using commercially available kits.
TaqMan allelic discrimination is an excellent method for genotyping a
limited number SNP since no post-PCR handling is required, thus,
minimizing the risk of contamination.
3.3
Electrophysiology
Electrophysiology was chosen as the primary method for quantifying
synapses for the reason that the assessment only concerns functional
synapses.
3.3.1
Hippocampal slice preparation
The hippocampus (fig. 1 in paper II) has been extensively studied in the past
40 years. It is a brain formation of fundamental importance for learning and
memory, it is easily recognised, and the cell layers are arranged in a laminar
way which makes the structure very suitable to use when recording
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