Chromosome 9p21.2 and Amyotrophic Lateral Sclerosis: A
causal pathogenic link?
Author: R. van Dorland
Student number: 3157288
Daily supervisor: M. Koppers
Examiner: J. Pasterkamp
0
Table of contents
 Introduction
o Amyotrophic Lateral Sclerosis
 Amyotrophic lateral sclerosis
 Amyotrophic lateral sclerosis and frontotemporal
dementia
o Pathogenesis of ALS/FTD




SOD1
TDP-43
FUS
Genes involved in ALS/FTD
 ALS and Chromosome 9: Introduction to the
main research question
Pg. 3-8
Pg. 3
Pg. 3
Pg. 3
Pg. 4-8
Pg. 4-5
Pg. 5-6
Pg. 6-7
Pg. 7-8
Pg. 9-13
o Chromosome 9p21.2
Pg. 9
o Chromosome 9p21.2 and genome wide
association studies
Pg. 9-10
o Chromosome 9p21.2 and linkage studies
Pg. 10-13
 IFT-74
Pg. 14-15
 Tie2
Pg. 16-19
 C9orf14
Pg. 20
 C9orf11
Pg. 21-22
 MOBKL2B
Pg. 23-25
 IFNK
Pg. 26-27
 C9orf72
Pg. 28
 LINGO2
Pg. 29-30
1
 Conclusion
Pg. 31-32
 References
Pg. 33-49
2
Introduction
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is a neurological disorder that is characterized by
progressive muscle atrophy due to a loss of motor neurons in the motor cortex, brain
stem and spinal cord. Death due to respiratory failure usually occurs within 3-5
years. Functioning of the bladder, bowel sphincters and movement of the eye is
usually intact. The incidence of ALS is 1-2.5 per 100.000 population. Approximately
5% of ALS patients have a family history of ALS and the lifetime risk of developing
ALS has been found to be 1 in 2 in some families. 1, 2 This form of ALS is called
familial ALS (fALS). The remaining 95% of ALS cases are considered to be sporadic
ALS (sALS) cases. Distinguishing between fALS and sALS can sometimes be
difficult. The mean age of onset for fALS is 10 years lower than for sALS but apart
from that, genetics and family history are the main factors contributing to diagnosing
patients. Diagnosing the sporadic form of ALS in an early stage is not very common
because early symptoms such as cramps, muscle twitching, weakness or stiffness
are easily ignored. Symptoms originating from the sensory tract have also been
shown in fALS. 1, 2 The possible occurrence of cognitive problems in ALS, varying
from a minor degree of behavioral/cognitive impairment to overt dementia, generated
the concept of a possible spectrum of disorders that might be similar in their
pathogenesis. 3
Amyotrophic lateral sclerosis and frontotemporal dementia
Frontotemporal dementia (FTD) is characterized by a change in personality which
can be accompanied by socially inappropriate behavior. The disease is thought to be
caused by degeneration of neurons in the anterior temporal and frontal lobes. 3
Cognitive functioning and memory are relatively spared and language defects are
commonly seen. There are striking similarities between FTD and ALS such as the
age of onset and an average life time expectancy after disease onset of 4 years. 4, 5
Also, neuropsychological examination of ALS patients showed a cognitive or
behavioral impairment in 10-50% of the affected individuals. 6 The pattern in which
the cognitive and motor symptoms develop is not precisely defined. However, it is
known that FTD/ALS cases develop signs of motor neuron degeneration after they
have been diagnosed with FTD more frequently than the other way around.
Development of both symptoms simultaneously is rarely seen. 3 So far studies did
not prove whether the cognitive problems in FTD/ALS patients progress over time.
Genetics are also an important tool in characterizing ALS and FTD. Several genes
have been found to be involved in the pathogenesis of both diseases. For example,
an accumulation of TAR DNA binding protein 43 (TDP-43) is found in most cases of
ALS and in a common form of FTD. Also, the fused in sarcoma protein (FUS) has
been identified as a novel pathological protein in ALS and FTD. 7 The pathogenesis
of ALS/FTD and the known genes that are thought to be involved will be discussed
next.
Pathogenesis of ALS/FTD
The molecular mechanism behind ALS remained a mystery for a long time. The first
pathological mutation was found in the superoxide dismutase [Cu-Zn] (SOD1)
protein. 8 Mutations found in this gene are thought to account for 20% of the familial
3
form of ALS. Identification of more mutated and causal genes lead to a molecular
classification of the different ALS forms in which patients with SOD1 mutations are
classified as ALS-SOD. FTLD subtypes were already classified depending on the
molecular composition of their inclusions. FTLD-tau patients had Tau protein
containing inclusions whereas FTLD-U patients were named after the ubiquitinpositive inclusions. A new pathological protein, TDP-43, was discovered in 2006 and
this protein was held responsible for most FTLD-U (renamed to FTLD-TDP) and
SOD1-negative cases (ALS-TDP). 9 Next, mutations were found in the protein FUS
and this protein was found to be one of the genetic causes of TDP-43-negative and
SOD1-negative ALS (ALS-FUS). Mutations in FUS were also found in most of the
remaining tau-negative FTLD patients and these were named FTLD-FUS. The
discovery of these pathogenic genes strengthened the idea that ALS and FTD
belong to the same spectrum of diseases. 7
SOD1
The first successful linkage study that linked SOD1 to FALS was performed in 1991.
The first pathological mutations were reported two years later. The SOD1 gene
consists of 5 small exons that are translated in a metallo-enzyme containing one
copper and one zinc ion. Its function is to catalyze the reaction in which toxic
superoxide radicals are converted to hydrogen peroxide and oxygen. Mutations have
been observed in both familial and sporadic ALS patients (not in FTD patients), in
respectively 20% and 3-7%, and are scattered across all five exons. 10, 11 These
mutations lead to single amino acid substitutions, generation of truncated proteins,
insertions and deletions. It is thought that the mutated protein becomes toxic and
that the loss of enzymatic activity has less effect on the pathogenesis. Several
hypotheses have been formed that try to describe the mechanism through which the
SOD1 mutants cause ALS. One study showed a two- to four-fold increase in the use
of hydrogen peroxide by the mutated protein in vitro. 12 This change in activity
increases the production of toxic radicals. Also, conformational changes resulting
from the mutations can expose the toxic copper ion that is present in SOD1. This
may contribute to the pathogenesis of ALS. 13 The discovery of SOD1 aggregates in
ALS patients lead to the idea that mutated and aggregated SOD1 itself is toxic to
cells. It was shown that expression of mutated SOD1 caused aggregation and
toxicity of the protein specifically in motor neurons. 14 Capture of for example
chaperone proteins in the aggregates of SOD1 are also thought to play a role in the
pathogenic effect of SOD1 mutations on motor neurons. 15 The most common
mutation in the SOD1 gene is the D90A (aspartate to alanine substitution in codon
90) mutation. The mutation is especially common in Scandinavia, where the gene is
found to be mutated in 0.5-5% of the population. 16 The D90A SOD1 protein is
slightly less active (95% of activity left) and the stability is thought to be decreased.
Patients that are homozygous for this mutation develop a characteristic form of ALS
that usually starts in one lower extremity, the lower back and the hip. 2 The use of
transgenic mice has been very important in gaining insight in the role of SOD1 in
ALS. To date, a wide range of transgenic SOD1 mice have been generated. These
mouse lines can differ in the way the protein is mutated or how well the gene is
expressed, allowing investigation of different aspects of the mutated gene. SOD1
knockout mice did not develop ALS like symptoms, however, transgenic mice
expressing the mutants SOD1-G93A or SOD1-G37R did develop motor neuron
disease. 17, 18 This suggests that mutations in SOD1 cause the protein to obtain toxic
properties which are responsible for the observed disease phenotype rather than a
4
loss of SOD1 function. A downside of transgenic mice is the amount of mutant
overexpression needed to induce ALS-like phenotypes. In some mouse lines
expression can go up to 40 times the basal level. 19 Interestingly, the life span of the
transgenic mice appears to be inversely proportional to the mutant gene dosage.
These data support the idea that long-term exposure to mutated and misfolded
SOD1 can have a toxic effect. Several other SOD1 transgenic mouse models
(including SOD1-G85R, SOD1-L126Z and SOD1-G86R) showed that viability of the
animals is decreased when wild type SOD1 overexpression is combined with a
mutated SOD1 gene. It is thought that a direct interaction between the mutant and
wild-type SOD1 is needed to exacerbate ALS disease. 20, 21 The involvement of other
cell types in the development of ALS was investigated in an elegant way by
expressing SOD1-G37R flanked by LoxP sequences. This allows expression of the
mutant in specific cell types. All motor neurons and oligodendrocytes expressed
sufficient mutant SOD1 to cause early-onset, fatal motor neurons disease when
expressed ubiquitously. However, an environment that contained non-mutant, non
motor neurons increased disease-free life by 50% suggesting that not only motor
neurons are involved in the pathogenesis of ALS but that mutant SOD1 toxicity in
glial cells can influence the disease after onset. 22
TDP-43
TDP-43 is encoded by the TARDBP gene and is located on chromosome 1p36.2.
The protein has five coding regions and is localized predominantly in the nucleus. 23
Specific RNA recognition motifs allow it to bind to RNA, single stranded DNA and
proteins and this is crucial for its role in transcription and splice regulation. 23, 24 The
neuropathology of most sALS cases are characterized by a cytoplasmic
accumulation of TDP-43. These neuronal cytoplasmic inclusions can be found in
neurons and glia of the primary motor cortex, spinal cord, brainstem motor nuclei
and white matter tracts. 25 Most mutations in TARDBP result in missense changes in
the Glycine-rich region and C-terminus of TDP-43. (Figure 1) These changes can
cause an increased aggregation or altered phosphorylation of TDP-43 which might
lead to exon skipping, impaired nuclear import or transcriptional repression activity. 26
Aggregation of TDP-43 is also thought to be caused by mutations or changes in
expression of other proteins. For example, mutations in the valosin containing
protein (VCP) gene can lead to accumulation of TDP-43 and increased expression of
TMEM106B might be a risk factor for developing FTLD-TDP. 27 It is not exactly
known how the accumulations of TDP-43 are involved in the pathology of
neurodegeneration but a loss of normal protein function is suggested to be
pathogenic.
Figure 1: Most known mutations of the TDP-43 gene are located in the Gly-rich and
C-terminal area of the protein. 7
5
This idea was supported by an observation that showed how reduced TDP-43
expression in mouse models resulted in motor deficits and structural abnormalities of
motor neurons. 28 The C-terminal fragments of TDP-43 are more likely to form
ubiquitinated insoluble cytoplasmic aggregates. These aggregates might also bind to
the full length protein, thereby depleting the nucleus of TDP-43. The aggregates are
thought to have a direct toxic effect on the affected motor neurons. 29, 30 However,
the real pathogenic mechanism in TDP-43 proteinopathy is still not solved and
multiple disease mechanisms might be involved. The use of transgenic mice in
understanding the role of TDP-43 in ALS pathogenesis has been of great use.
Homozygous knockouts for TDP-43 were found not to be viable, suggesting an
essential role in development for TDP-43. 31, 32 Heterozygous TDP-43 knockout mice
only develop small muscle weakness and do not show motor neuron pathology. The
expression of mutated human TDP-43 (A315T mutation) in mice did lead to a
progressive and fatal neurodegenerative disease. However, these mice did not have
cytoplasmic TDP-43 aggregates, probably due to premature cell death caused by a
high expression level of mutated TDP-43. 33 In another study expression of human
wild type TDP-43 and a mutated form of TDP-43 with a defective nuclear localization
signal both led to ALS like symptoms. 34, 35 Again, no TDP-43 containing cytoplasmic
aggregates were found. The problem with the TDP-43 animal models is that they all
depend on high levels of expression and this can interfere with slow developing
pathogenic pathways. Also, the absence of TDP-43 positive inclusions is something
to take into account when using these models.
FUS
FUS is encoded by 15 exons and the translated protein contains 526 aminoacids.
The structural characteristics of the protein are shown in figure 2. It shows
similarities to TDP-43 because of its RRM domain and Gly-rich domain. The protein
is expressed in both the cytoplasm and the nucleus and it shuttles continuously
between these two compartments. The precise function is not well understood but
involvement in cell proliferation, transcription regulation, DNA repair and RNA and
microRNA processing is suggested. Furthermore, FUS might be involved in
transporting mRNA to dendrites and neuronal plasticity. 36, 37 Mutations in FUS were
found in patients with a familial form of ALS that was linked to chromosome 16. In
these patients researchers observed motor neuron loss in the spinal cord and
brainstem, demyelination of corticospinal tracts and FUS-positive inclusions in lower
motor neurons. 38 Later, other regions such as motor nuclei in the brainstem,
striatum, substantia nigra and the thalamus were also found to be affected. The
mechanism underlying the accumulation of FUS and the FUS-mediated
neurodegeneration is still largely unknown. Disruption of the nuclear localization
signal will impair nuclear import and alter the cytoplasmic concentration of FUS. This
might be a key mechanism by which FUS contributes to the pathogenesis of ALS. 39
6
Figure 2: Overview of the FUS protein. The protein contains a Gly-rich domain, a
RNA recognition motif, a N-terminal Gln-Gly-Ser-Tyr-rich domain, a zinc finger motif,
Arg-Gly-Gly repeats and a C-terminus that contains the nuclear localization signal.
Many mutations are found in this C-terminal area of the protein that is involved in
transport to the nucleus. 7
Genes involved in ALS/FTD
The genes that we discussed do not explain all the ALS/FTD cases and many more
genes are thought to be involved in the pathogenesis of this disease. However, in
depth discussion of these genes goes beyond the scope of this thesis. Therefore we
will shortly summarize most of the genes that are thought to be involved in the
pathogenesis of ALS.
Several candidate genes involved in axonal transport and cytoskeletal structure have
been identified. These include alsin (ALS2), dynein, dynactin (DCTN1),
neurofilament heavy subunit (NF-H) and peripherin. Alsin is located on
chromosome 2q33 and encodes an enzyme that consists of 1657 amino acids. The
enzyme can activate small GTPases by acting as a guanine nucleotide exchange
factor. Disruption of alsin functioning can affect actin dynamics, membrane trafficking
and the proper development of neurons leading to an increased susceptibility to toxic
exposure. Several point mutations have been found causing premature termination
and missense mutations of the protein. 40-42 Dynein and DCTN1 form a complex that
has a critical role in the transport of materials within cells via microtubules. Three
missense mutations have been found in DCTN1 and a decrease in function of this
protein may slow the transport of essential proteins or materials in axons of motor
neurons. 43 NF-H is a structural protein that helps to define the size and shape of
nerve cells. Deposits of neurofilaments have been found in ALS patients but it is still
unknown whether this is caused by NF-H or if it is a byproduct of dying motor
neurons. Mutations have been found in both ALS patients and healthy relatives and
therefore mutated NF-H is only considered to be a risk factor for ALS. 44
Neurovascular molecules are involved in the supply of oxygen and mutations in
these proteins can lead to hypoxia and oxidative stress in neurons. Several genes
involved in this process appear to increase the risk for developing ALS including
vascular endothelial cell growth factor (VEGF), angiogenin (ANG), progranulin
(PGRN) and paraoxonase (PON). 45 VEGF has various effects and can induce
angiogenesis, increase vascular permeability, promote cell migration, inhibit
apoptosis and promote vasculogenesis and endothelial cell growth. Carriers of a
single nucleotide polymorphism (SNP 2578AA) are more susceptible for the
7
development of ALS. Furthermore, deletion of the hypoxic response element of
VEGF in mice led to the development of an ALS-like neuron disease. 46 Angiogenin
can be found throughout the body and activation of the protein will lead to the
formation of new blood vessels. Missense mutations in ANG have been found in 20
ALS patients and it is suggested that a decrease in function of the protein can
increase the risk of developing ALS. 47 The function of PGRN in the brain is still
largely unknown although it appears to be crucial for the survival of neurons. The
protein was first identified as a gene responsible for some cases of FTLD-U but later
mutations were also found in ALS patients. PGRN might influence the disease
course of ALS by changing the age of onset and survival of patients. 48
Senataxin (SETX) is a protein consisting of 2688 amino acids and is located on
chromosome 9q34. The protein is involved in DNA repair, recombination,
transcription and replication but can also regulate RNA processing, initiation of
translation and transcript stabilization. Therefore, SETX might be essential in
synthesizing mature RNA. 49, 50 Several missense mutations have been found in
three different families and the incapacity to produce error-free mature mRNA might
be responsible for the motor neuron damage found in these patients. 51 The vesicleassociated membrane protein/synaptobrevin-associated membrane protein B
(VAPB) has been found to be mutated in several Brazilian families. This mutation is
located in exon 2 and leads to the switch of a highly conserved proline to serine at
codon 56. 52 Mutated VABP can form aggregates both in and outside the
endoplasmic reticulum resulting in deregulation of cellular secretion, intracellular
membrane trafficking and the subsequent loss of neurons. 52
The knowledge of ALS genetics has been largely broadened in the last few years.
For a long time research has been limited to SOD1 induced motor neuron
degeneration but the generation of animal models for other associated genes allows
for a much broader approach. Interestingly, the newly discovered genes appear to
functionally cluster in specific themes such as intracellular axonal transport that uses
the cytoskeleton, hypoxia, ER stress and RNA processing. More research is needed
to find out how these different pathways contribute to the development of ALS.
8
ALS and Chromosome 9: Introduction to the main research
question
Chromosome 9p21.2
Mutations in SOD1 account for up to 20% of all familial ALS cases. Also, several
other genes that contribute to the development of familial ALS have been found
including FUS, TARDBP, VAPB and VCP. However, the cause of many sporadic ALS
cases remains unknown and candidate gene association studies are used to identify
these causal genes. Recently, genome wide association studies have been
performed in sporadic ALS patients and several candidate genes have been found,
including FGGY carbohydrate kinase domain containing (FGGY), dipeptidylpeptidase 6 (DPP6) and inositol 1,4,5-triphosphate receptor 2 (ITPR2). However,
consistent replication of these findings in other studies appears to be a problem.
More recently obtained data showed a possible role of chromosome 9p21.2 in the
pathogenesis of ALS. This finding was replicated within the same study as well as in
two other genome wide association studies in different populations. 53-55 The same
area on chromosome 9 was also found in several independent studies linking
fALS/FTD to chromosome 9. (Figure 3)
Figure 3: Area of chromosome 9 associated with ALS/FTD. This region has been
found in several GWA and linkage studies.56
Chromosome 9p21.2 and genome wide association studies
A genome wide association study that included 19838 individuals from nine countries
associated the 9p.21.2 region with ALS. Also, a single nucleotide polymorphism
within the UNC13A gene was found to be associated with ALS. This SNP was also
found in the replication phase of the study. Combined analysis of both stages
showed a strong association of this SNP with ALS (P = 2.53 × 10−14). The other
region on chromosome 9 (9p21.2) was only found to be significant in the combined
analysis. 55 A genome wide association analysis in Finland was performed on a
smaller group of patients and control individuals. 54 Here, the incidence of ALS is
much higher compared to other parts of the world and the genetic homogeneity of
the Finnish population makes it easier to detect risk loci. This study included 442
patients and 521 control individuals and analysis of the data showed two significant
SNPs, namely, rs13048019 which is located on chromosome 21q22 and
corresponds with the D90A allele of the well known SOD1 gene and rs3849942. The
latter SNP is located on chromosome 9p21 and this finding corresponds with data
from van Es et al.2009. More evidence for a role of the 9p21 region of chromosome
in ALS was provided by Shatunov et al. 53 They conducted a GWAS with 599
patients and 4144 control individuals from the UK. The dataset was increased by
adding 4312 patient and 8425 control samples from eight other countries from
9
previously conducted GWAS. The authors found two SNPs that showed a significant
association with sporadic ALS. These SNPs, rs3849942 and rs2814707, were
significant in both the independent UK sample set as well as in the combined
analysis that included the samples from previously published GWAS.
Figure 4: Overview of the genes located on the associated region of chromosome 9
53
Chromosome 9p21.2 and linkage studies
Thus far, to our knowledge, a total of ten linkage studies have linked ALS/FTD to the
9p21 region of chromosome 9. 56-65 A nearby locus was first found to be associated
with ALS/FTD in 2000 by Hosler et al. 64 This study analyzed 16 families with both
ALS and ALS/FTD patients. They found linkage to region 9q21-q22 that was specific
for ALS/FTD patients. In 2006, Morita et al. showed linkage of chromosome 9p21.3p13.3 with both ALS and FTD. This study characterized 50 members of one family
that contained five ALS and nine FTD patients. 57 These results were replicated in
the same year by Vance et al. 58 Screening of 3 selected genes (Dynactin 3, KIF24
and BCL2-associated athanogene 1) in this region did not reveal any possible causal
mutations. Momeni et al. also found linkage of chromosome 9p21with fALS. They
sequenced 14 candidate genes of 12 fALS/FTD and fALS cases from two families
and found a mutation in the IFT-74 gene. This mutation was found in two cases of
the same family. (Table 1) However, sequencing of the IFT-74 gene in 420 other
ALS, ALS/FTD and FTD samples did not replicate this finding. 65 One year later three
more families with ALS, FTD and ALS/FTD patients were linked to chromosome 9p.
10
This study downsized the linked area to between the markers D9S2154 and
D9S1791. Aprataxin, BCL2-associated athanogene 1 and the tyrosine kinase
endothelial genes were selected for mutation screening but no mutations were found
in these genes. 60 The next evidence came from a study performed in a multigenerational Australian family with FTLD-MND (motor neuron disease). The family
consisted of 16 members of which 11 individuals were diagnosed with either FTD,
MND or FTD/MND. Haplotype analysis identified a critical region between markers
D9S169 and D9S1845 located on chromosome 9p21. Again, no mutations were
found in the selected genes (IFNK, LINGO2, MOBKL2B, C9orf11and C9orf72) of this
region. 62 Six additional FTLD/MN families with linkage to 9p21.3-p13.3 were
identified by Ber et al. in 2009. 63 Patients suffered either from FTD (32%), MND
(29%), or both disorders (39%). The associated area on chromosome 9 was strongly
reduced by findings of Boxer et al. 59 They linked a family with FTD, ALS and
FTD/ALS patients to a 3.7 Mb interval on chromosome 9p. This region only contains
five protein coding genes (c9orf11, MOBKL2B, IFNK, c9orf72, LINGO2), two microRNA genes (miR-873, miR-876), two predicted genes (AK074231 and RBMXP2) and
a large non-protein coding RNA gene (NCRNA00032). Direct sequencing of coding
exons, flanking intronic sequences and 3’ UTR’s of these 10 genes did not reveal
any mutations. The cDNA of MOBKL2B and C9orf72 was also analyzed but did not
identify small deletions, duplications or altered splicing in the RT-PCR products. Two
additional articles were published in 2010 regarding the linkage of chromosome 9 to
ALS/FTD. 56, 61 Gijselinck et al. linked chromosome 9p23-q21 to ALS/FTD and
Pearson et al. further refined the well known locus on chromosome 9p21 that is
linked to ALS/FTD. An overview of these studies and the genes that have been
sequenced can be found in table 1.
Study
Linked region
Families
and cases
fALS
cases
FTD
cases
fALS
/FTD
cases
Sequenced
genes
Mutated
Hosler 64
9q21-q22
NK
NK
NK
-
-
Yan 66
NK
NK
NK
27 genes sequenced
No mutations
Momeni 65
9p13.3 to 9p22.1
between D9S1684
and D9S1678
9p21.3-p13.3
16 families
93 cases
15 families
2 families
12 cases
1
-
9
IFT-74
*5
Present in 2
cases
Morita 57
9p21.3-p13.3
5
9
-
Vance 58
9p13.2–21.3
1 family
14 cases
1 family
12 cases
7
2
3
Boxer 59
3.7 Mb interval on
chromsome 9p
1 family
10 cases
2
5
3
C9orf82 (12 cases)
PLAA
IFT-74
LRRC19
Tie2
C9orf14
C9orf11
MOBKL2B
IFNK
C9orf72
LOC392298
LRRN6C
LOC653777
LOC646700
VCP (1 case)
UBQLN1
Dynactin 3 (10cases)
KIF24
BAG1
C9orf11 (10 cases)
MOBKL2B *2-3
IFNK
C9orf72 *2-3
LINGO2
miR-837
No mutations
*1
No mutations
*1
No mutations
*2
11
Valdmanis 60
chromosome 9p
between the markers
D9S2154 and
D9S1791
9p23-q21
3 families
21 cases
14
3
4
1 family
9 cases
1
8
-
Luty 62
chromosome 9p21.1q21.3 between the
markers D9S169 and
D9S1845
1 family
11 patients
2
5 (2 cases
with
dementia
)
2
Ber 63
Chromosome 9p
linkage between
markers D9S1121 and
D9S301
6 families
31 cases
9
10
12
Pearson 56
chromosome 9p21,
4.8-megabase
haplotype area
1 family
9 cases
3
-
6
Gijselinck 61
miR-876
AK074231
RBMXP2
NCRNA00032
APTX (21 cases)
BAG1
CNTFR
Tie2
MOBKL2B (2 cases)
C9orf72
ACO1
DDX58
TOPORS
NDUFB6
DNAJA1
SMU1
B4GALT1
BAG1
CHMP5
AQP3
NOL6
UBE2R2
UBAP2
WDR40A
UBAP1
IFNK (7 cases) *3
LINGO2
MOBKL2B *3
C9orf11
C9orf72 *3
ELAVL2 (31 cases)
C9orf82
PLAA
IFT-74
LRRC19
Tie2
C9orf14
C9orf11
MOBKL2B
C9orf35
IFNK
C9orf072
LRRN6C
TOPORS C9orf133
Q8N710
NP_997723.2
DNAJA1
CHMP5
SUGT1P
UBE2R2
UBAP2
WDR40A UBAP1
DNAJB5
VCP
UNC13B
TLN1
C9orf134
hsa-mir876
hsamir873
hsa-mir491
hsa-mir31
TUSC1 (9 cases)
PLAA
IFT-74
LRRC19
Tie2
MOBKL2B
IFNK
and LINGO2
No mutations
*4
No mutations
*1
No mutations
*5
No mutations
*6
No mutations
*1
*1: coding sequence analyzed
*2: genomic (gDNA) sequencing of coding exons, flanking intronic sequences and 3’ UTR’s
*3: cDNA analyzes for small deletions, duplications or altered splicing in the RT-PCR
*4: Exons and 50 base pairs past intron or exon junction were analyzed
*5: coding and non-coding exons and flanking intronic sequences
12
*6: Coding exons and intron/exon junctions were sequenced on both strands, copy number variations (CNVs) were also
investigated
Table 1: Overview of the genes that are screened for mutations in chromosome 9
linked ALS/FTD families.
Together these studies provide strong evidence of a genetic association of
chromosome 9p21 with sporadic/familial ALS and FTD. This region is depicted in
figure 4 and shows the presence of multiple genes. Thus far, only one amino acid
altering variant has been found in these genes and this suggests that the factor
underlying chromosome 9p21-related ALS might be causing the disease by altered
gene expression or splicing of genes located on chromosome 9. In the following
chapter we will discuss the possible role of IFT-74, Tie2, C9orf14, C9orf11,
MOBKL2B, IFNK, C9orf72 and LINGO2 in the pathogenesis of ALS.
13
Intraflagellar transport 74
Intraflagellar transport 74 (IFT-74) is composed of 600 amino acids and has a
molecular weight of 69-kDa. The protein contains a coiled-coil domain and localizes
to the intracellular vesicle compartment. 65 The exact function of the protein is
unknown but involvement in the intraflagellar transport (IFT) system has been
shown. 67 IFT is the bidirectional movement of multi-subunit protein particles along
the length of axonemal microtubules and requires the action of retrograde IFT-dynein
motors and anterograde kinesin-II motors. IFT is thought to play a role in many
processes including the assembly and maintenance of cilia/flagella, exocytosis,
photoreception, sensory perception and the transport of proteins to the axons and
dendrites of neurons. 65, 68 Two distinct protein complexes have been identified in the
IFT-particles that are responsible for the growth of axoneme, namely complex A and
B. These complexes are essential building blocks in the process of axonemal
growth. 69 The role of IFT-74 in this process of IFT is illustrated in figure 5. It appears
that IFT-74 is essential in functioning of this protein complex. 67, 69
Figure 5: Complex B contains at least 13 proteins including IFT-74. However, the
exact role of IFT-74 in this complex is still unknown.69
IFT is not restricted to cells with cilia and roles in targeting endosomal vesicles
containing T-cell receptors to the immune-synapse have been shown. 70 This data
led to the hypothesis that IFT-particles will probably be present and functional in
many systems that involve the transport of proteins and exocytosis. These include
neuronal synapses and the exocytosis of signaling receptors and ion channels to
specific membrane locations. 71
The possible involvement of IFT-74 in ALS was first suggested by Yan et al. 2006. 66
Shortly after this publication, the first sequence variations of this gene were reported
by Momeni et al. 2006. 65 They found an amino acid change (a C to T sequence
variant) at nucleotide 1024 in exon 13 that leads to a premature stop codon in the
peptide truncating the last 258 residues. The mutation was present in two brothers
diagnosed with ALS-FTD but absent in 1,000 chromosomes from North American
controls and four healthy individuals within the kindred. Also, additional sequence
14
variants (I55L and G58D) were found in 4 individuals diagnosed with sporadic
semantic dementia, FTD or sporadic ALS. 65 Additional evidence of a possible role of
IFT-74 in the pathogenesis of ALS is not provided since the mutations found in IFT74 are not replicated in other studies. For example, a linkage study performed in
2009 by Ber et al. did not identify any mutations or copy number variations in the
IFT-74 gene. 63 Similar results were obtained in a case-control study using a UK
dataset. 72 They evaluated the known genetic variability in IFT-74 and found that they
are not strong risk factors for ALS.
The importance of axonal transport, membrane traffic and vesicle synthesis in the
pathogenesis of ALS is becoming more clear. We already discussed the role of
several proteins involved in these processes that were previously found to be
mutated (VAPB in fALS, dynactin in sALS/fALS and alsin in juvenile ALS and fALS).
The role of IFT-74 in vesicle transport and exocytosis makes it a plausible biological
candidate gene because the transport of proteins and proper membrane trafficking is
essential in the development of neurons. Disruption of these processes can partly
explain the neurodegeneration that is characteristic of ALS. Furthermore, an 64-year
old Japanese female who had earlier suffered from Kartagener syndrome was
diagnosed with ALS. Kartagener syndrome is characterized by a disrupted axonemal
dynein function and a relationship between this syndrome and ALS is therefore very
interesting. 73 However, more research is needed to establish the precise link
between these two diseases. Although the present data about the mutations found in
IFT-74 does not provide clear evidence for a causal relationship, it may well be that
genetic variation in this gene increases the risk of developing ALS.
15
Tie2
The tyrosine kinase endothelial (Tie2) protein contains 1124 amino acids and several
domains including EGF-like domains, fibronectin type-III domains, Ig-like C2-type
(immunoglobulin-like) domains and a protein kinase domain have been identified
(Figure 6). 74
Figure 6: Schematic overview
of the Tie2 receptor domains.
The receptor contains three
EGF-like domains, three
fibrinogen-like domains, three
immunoglobulin (Ig)-like
domains and a tyrosine
kinase domain. 75
Tie2 is a single-pass type 1 membrane protein and acts as a receptor for
angiopoietin 1 (Ang-1). The Ang-1/TIE2 ligand/receptor complex is part of the
Angiopoietin/TIE system that controls endothelial cell survival and vascular
maturation. This family includes two tyrosine kinase receptors (Tie1 and Tie2) and
four corresponding ligands (Ang-1, Ang-2, Ang-3 and Ang-4). Activated Tie2 is
thought to recruit mural cells (cells involved in the formation of normal vasculature)
and to mediate survival signals for endothelial cells leading to the assembly and
maturation of vessels. 75, 76 Tie2 is expressed by endothelial cells, endothelial
precursor cells, hematopoietic cells, tumor cells and has been directly found in the
lungs, placenta, brain and kidney. 74, 75 Neuronal expression of Tie2 has also been
shown in primary neuronal cultures. 77 Tie2 functioning is crucial during the
development of the embryo where it plays a key role in stabilization, maturation and
remodeling of the cardiovascular system. Ang-1 and Ang-2 can both bind the Tie2
receptor, however, Ang-2 acts as an antagonistic ligand and can thus inhibit the
activity of the Tie2 receptor by competing with Ang-1. 78 Yet, Ang-2 was also found to
act as an agonist of Tie2 by Kim et al. 79
Tie2 is part of an extensive signal transduction cascade. Ligand binding is followed
by dimerization of the receptor and autophosphorylation of the intracellular tyrosine
kinase domain which activates intracellular signaling pathways. First, the p85 subunit
of phosphatidylinositol 3-kinase (PI3-K) is phosphorylated which in turn activates Akt.
Activated Akt phosphorylates the forkhead transcription factor FOXO-1 which is a
strong stimulant of Ang-2 expression. Also, survival is promoted by activation of antiapoptotic signals such as Survivin, eNOS, Bad and Caspase-9. Endothelial cell
permeability is reduced by activation of the Rho-GTPases. This is accomplished
16
through Src by inhibition of VE-cadherin internalization. An overview of this signaling
pathway is depicted in figure 7. 75
Figure 7: Overview of the Ang-1/Tie2 signaling pathway. 75
A more recent finding by showed a possible new role of Tie2 in endoplasmic
reticulum (ER) stress. 80 They suggested that Tie2 expression is decreased in
response to severe ER stress and that this results in cell death. This interesting
finding will be discussed further on in this chapter where we will look at a possible
role of Tie2 in the pathogenesis of ALS. Another promising finding was made by
Valable et al. 77 This study showed a protective effect of the PI3-K pathway in
primary neuronal cultures. This is an important finding since Tie2 is predominantly
expressed in endothelial cells and it was unsure whether the protective effect via the
PI3-K pathway had a protective effect in neurons. A similar finding was made by Bai
et al. They showed that activation of the Ang1-Tie2-PI3-K pathway in neural
progenitor cells in response to glucose and oxygen deprivation initiate cell survival. 81
The regulation of the vasculature does not depend on Tie receptor signaling alone.
Vascular endothelial growth factors (VEGFs) and their receptors also play a role in
this process. VEGF is thought to be more involved in the initial phase of vascular
development whereas the Ang-Tie system is essential in later stages. 82 The VEGF
promoter contains a hypoxia response element (HRE) that can bind hypoxia
inducible factors. Binding of these factors is crucial in regulating the expression of
VEGF in response to hypoxic conditions. Deletion of the HRE in neural tissue of
mice reduced hypoxic VEGF expression and caused adult-onset progressive motor
neuron degeneration possibly due to reduced neural vascular perfusion. This
17
phenotype is very similar to the degeneration of neurons found in ALS patients. 83 An
interesting finding about VEGF functioning in relation to Tie2 was done by Singh et
al. 84 They showed that VEGF can activate Tie2 by causing a four-fold increase in
tyrosine phosphorylation. Furthermore they showed that the mechanism behind this
increase involves the proteolytic cleavage of the associated tyrosine kinase Tie1
which leads to trans-phosphorylation of Tie2.
The linkage of Tie2 with fALS and FTD was first found in 2007 by Valdmanis et al.
They analyzed three families with a total of 21 cases. 60 No mutations were found in
exons or in 50 base pairs past intron or exon junction. Later, two more studies linked
Tie2 with fALS and fALS/FTD. 56, 63 Together, these studies analyzed a total of 7
families and 40 cases. No mutations were found in the analyses of the gene. (See
table 1 for sequence details)
Tie2 is an interesting candidate gene in our search for causal genes in ALS. A study
in 2003 showed that Ang-1 and its receptor Tie2 are both functionally expressed in
primary cultures of mouse cortical neurons. 77 The activation of anti-apoptotic signals
due to Tie2 activation led to a protective effect in neurons. This effect was seen
during serum deprivation induced apoptosis. Inhibition of the PI3-K pathway reduced
the protective effect suggesting the use of the same pathway as observed in
endothelial cells. Disruption of the neuroprotective role of Tie2 can have a negative
effect on the survival of neurons and thus increase the susceptibility to the
development of neurodegenerative diseases such as ALS. No mutations have been
found in fALS, sALS and FTD patients so far although a decrease in expression of
the receptor might be enough to abolish its protecting effect. Another mechanism by
which Tie2 might influence cell survival was recently discovered by Hosoi et al. 80
They found that inhibition of the PI3-K/Akt pathway is important in ER-stress induced
cell death. It appears that the expression of Tie2 is decreased in response to ERstress which leads to downregulation of the PI3K/Akt pathway and less production of
anti-apoptotic signals which results in cell death. Thus Tie2 appears to be a mediator
of the ER stress induced PI3K-Akt signal. Also, ER-stress might disrupt the normal
protein folding function of the ER which can cause an increase in the misfolding and
aggregation of proteins. Aggregation of misfolded proteins is a well known
characteristic of ALS. ER stress has already been implicated in the pathogenesis of
Parkinson’s and Alzheimer’s disease. ALS might share pathogenic pathways with
these neurodegenerative disorders suggesting a role of the ER and Tie2 in ALS
pathogenesis. A recent study directly linked VEGF expression with the amount of
tyrosine phosphorylation of Tie2. 84 It is known that a reduced expression of VEGF
during hypoxia induces an ALS-like phenotype in mice. It could be that this
phenotype is partly caused by a lower level of Tie2 phosphorylation mediated by
VEGF. Therefore it is important to find out whether a lower level of expression or
phosphorylation of Tie2 can be found in ALS/FTD patients.
We hypothesize that misfolding and aggregation of proteins in the ER and cytoplasm
leads to ER stress. Subsequently, the expression of Tie2 is decreased and less antiapoptotic signals are produced leading to neuronal cell death and the development
of ALS. A decrease in the basal level of expression of Tie2 in ALS patients could
make neurons more susceptible to degeneration. This effect becomes stronger when
ER stress due to for example misfolded SOD1 lowers the expression of Tie2 even
more and causes the degeneration of neurons. Also, a lower level of Tie2
18
phosphorylation/expression due to for example VEGF might lead to a reduced neural
vascular perfusion and an ALS-like phenotype.
19
C9orf14
Chromosome 9 open reading frame 14 (C9orf14) is located on chromosome
9 between Tie2 and C9orf11. Its official full name is non-protein coding RNA 32
(NCRNA00032). Transcription of the gene can produce 5 alternatively spliced
mRNA’s. The protein is found to be mostly expressed in the dermal system.
Expression was also found in the kidney, fetal heart and pregnant uterus.
85, 86 The exact in vivo functions of this gene are still unknown. However, a possible
role as tumor-suppressor gene has been suggested by Pujana et al. 87
Pujana et al. performed cytogenetic analysis in a family with individuals with large
number of nevi (LNN) and cutaneous malignant melanoma (CMM). They found a
t(9;12) (p21;q13) balanced translocation in three family members. Further
investigation of this translocation breakpoint led to the fine mapping of its location
between Tie2 and C9orf11. Subsequently, the C9orf14 gene was identified by
complete sequencing of public domain EST clones, RT-PCR reactions using cDNA
preparations and normal human melanocyte cDNA library screening. Identification of
differential expression levels of C9orf14 in human cancer cell lines provided new
evidence for its possible role as a tumor-suppressor gene. The gene was found to be
downregulated in three out of seven melanoma cell lines (SK-MEL-5, SK-MEL-28
and MALME-3M). 87 Downregulation of C9orf14 was also observed in ovarian cancer
cell lines as well as in the epithelium of patients with serous carcinomas, suggesting
a possible role in ovarian tumorigenesis. 88, 89 Next, Pujana et al. determined the
cellular localization of the C9orf14 protein by an immunofluorescent staining with
EmGFP-C9orf14, y-tubulin and DAPI in normal human mammary epithelial cells.
They found C9orf14 to be specifically localized in the centrosome of these cells. The
centrosome plays an important role in the organization of microtubules and is thus
also involved in many other processes including the formation of primary cilia, the
organization of mitotic spindles and the proper progression through cytokinesis.
Taken together, these data suggest that C9orf14 might be able to act as a tumor
suppressor gene by influencing the centrosome. 90
Association of C9orf14 with ALS and ALS/FTD has been found in the linkage studies
that were discussed in the introduction. Two of these studies selected the C9orf14
gene for mutational screening. 59, 63 Le Ber et al. identified six families with linkage to
chromosome 9p and they analyzed the coding exons and intron/exon junctions on
both strands as well as CNVs of 31 cases. Boxer et al. sequenced the gDNA of
coding exons, flanking intronic sequences and 3’ UTR’s of 10 patients from one
family. Both studies did not identify any mutations in the C9orf14 gene (see table 1
for a more detailed specification of the cases).
The available data about C9orf14 is limited and therefore only assumptions can and
have been made about its possible function. Thus far, no expression has been found
in the brain or neurons from the peripheral nervous system and a role in neuronal
cell death is therefore unlikely. Indirect effects of C9orf14 on the degeneration of
neurons via for example the dermal system is not very plausible since the protein is
localized intracellular near the centrosome. Regulation of cell division as a tumor
suppressor gene does not play a role in the degenerating neurons of ALS patients
since adult neurogenesis is restricted to the subventricular zone and the subgranular
zone. It is therefore highly unlikely that C9orf14 is involved in the pathogenesis of
ALS.
20
C9orf11
Chromosome 9 open reading frame 11 (C9orf11), also known as Acrosome
formation-associated factor (AFAF), is organized in eight exons that encode a total of
294 amino acids. The protein has a transmembrane domain and a leucine zipper
pattern at its C-terminal end, suggesting the possibility to bind vesicles and dimerise.
The 3’-side contains an untranslated region that possibly codes for a secretion signal
containing polypeptide. 91 Expression of the gene, determined by Northern blot
hybridization, was found to be very specific. High expression levels were found in the
testis but only minor expression was present in the amygdala, heart, kidney, pituitary
gland, placenta, jejunum, salivary gland and thymus. 91 C9orf11 appears to have
multiple transcript variants. For example, a truncated version of the protein that
misses exon 7 and 8 is expressed in the testis. Also, expression of an altered
transcript that misses exon 4 was found in the blood, skin, pancreas and the brain
(Figure 8). 91 It is assumed that this form is expressed at a low level.
Figure 8: Schematic representation of the C9orf11 protein. The protein contains eight
exons and has three known transcript variants. 91
Characterization of C9orf11 was performed because of its location on a chromosome
region (9p21) that is frequently deleted in human cancer. Researchers expected the
presence of a tumor suppressor gene but found no disease causing mutations in the
C9orf11 gene in 17 melanoma families. 92 C9orf11 might also be involved in the
formation of a specific membrane organelle that is formed during spermiogenesis,
namely the acrosome.
Figure 9: The acrosome is formed from
vesicles coming from several pathways.
C9orf11 might be involved in the formation
of this organelle. 93
21
The acrosome is formed from internalized vesicles and C9orf11 appears to have a
role in the recruitment or transport of these vesicles (Figure 9). 93
The extensive linkage of the chromosome on which this gene is located (discussed
in introduction) with ALS suggested a possible role for C9orf11 in the pathogenesis
of this disease. A linkage study by Luty et al. tried to identify mutations in this gene in
a group of patients suffering from FTLD. 62 However, no mutations were found in the
exons or flanking intronic sequences suggesting that mutations in this protein are not
likely to induce the ALS/FTLD like phenotype.
It is highly unlikely that C9orf11 is involved in the pathogenesis of ALS when we
consider its expression pattern (mainly in the testis) and involvement in acrosome
formation. The protein is hardly expressed in the brain and this makes it difficult for
the protein to regulate for instance motor neuron cell death. However, the expression
of an altered transcript variant (missing exon 4) in the brain might change the
function of the protein. Its transmembrane domain that allows it to bind to vesicles
might then be used in the intracellular transport of cargo-vesicles. This process is
thought to be disrupted in patients that suffer from ALS. 45
It is known that leucine zippers are critical for the DNA binding properties of
some transcription factors such as c-Jun and c-Fos. 91 C9orf11 contains a leucine
zipper pattern but does not possess a DNA-binding domain. It is therefore also
unlikely that regulation of transcription, a process thought to be deregulated in ALS,
is influenced by this protein.
22
MOBKL2B
Mps one binder kinase activator-like 2B (MOBKL2B) is located on chromosome
9p21.2 and the coding gene contains 216 amino acids. Two alternative names for
the protein are Mob1 homolog 2b and protein Mob3B. The gene has 4 exons and its
flanking start and stop codons have been identified. The hypothetical 3D-structure of
the gene shows the presence of several α-helices (Figure 10). Furthermore,
sequence analysis has suggested the presence of 4 metal binding sites that can bind
zinc. The functional properties of these binding sites still need to be confirmed by
experimental research. 94, 95
Figure 10: A hypothetical 3D
representation of the MOBKL2B
protein. The protein is thought to
contain several α-helices and zinc
binding sites. 94
MOBKL2B has sequence similarities with members of the MOB1/phocein family and
is thought to be a member of this family. Shared sequence similarities between the
proteins of this family can go up to 95%. The human form of MOBKL2B shares 54%
identity and 69% homology with the well studied Drosophila-MOB1 protein. The
entire MOB family is widely expressed throughout the body including the brain,
spinal cord and many other tissues/organs. 96 Experimental data about the exact
function of MOBKL2B is not available and the possible function of MOBKL2B will
thus be based on MOB1 protein data. MOB1 was first identified in yeast where it is
required for maintenance of ploidy and completion of mitosis. 97 Expression patterns
of MOB1 during cell proliferation showed that MOB1 expression begins in the G2
phase where it is located at the forming spindle poles and kinetochores. Expression
was maintained throughout cytokinesis. 98 They also suggested that MOB1 can
regulate mitotic progression by influencing the timely mobilization of the
chromosomal passenger complex. This complex is involved in coordinating the
cytoskeletal and chromosomal events of mitosis. 99
MOB1 is also involved in the ‘Hippo pathway’. This pathway has a role in cell contact
inhibition, organ size control and apoptosis. The pathway was first discovered in
Drosophila but was found to be preserved in mammals. 100 The Hippo pathway can
be divided in three parts namely the upstream regulators, the core machinery and
the downstream regulators. The upstream regulators, Crb1-3/FT1-4 and DCHS1-2,
are mainly located on the cell surface where they sense cell-cell contacts and
regulate pathway activity (Figure 11). These surface molecules can recruit the
FRMD6-NF2-Kibra complex which will then recruit the core machinery of the Hippo
pathway containing Mst1-2, WW45, LATS1/2 and MOB1. Subsequently, LATS1-2
protein kinase will be activated leading to the phosphorylation of YAP/TAZ.
23
Figure 11: Overview of the molecules
involved in the Hippo pathway. Nonphosphorylated YAP/TAZ can enter the
nucleus and interact with TEAD1-4.
This will lead to the recruitment of
transcriptional machinery that will target
genes involved in cell proliferation and
suppression of apoptosis. 100
Phosphorylated YAP/TAZ interacts with 14-3-3 proteins preventing it from moving to
the nucleus. Non-phosphorylated YAP/TAZ can move to the nucleus where it binds
to TEAD1-4 which leads to the formation of transcriptional machinery necessary for
the expression of genes that suppress apoptosis and promote cell proliferation. 100
This regulatory role in cell proliferation suggests a possible role of participants of this
pathway in cancer development. This has been confirmed by several other studies
as reviewed by Chan et al. 100 The involvement of MOBKL2B in the Hippo pathway is
firmly stated by Hartmann et al. 2010, however direct evidence of this statement
could not be found in literature. This study does provide some circumstantial
evidence by showing that a decreased expression of MOBKL2B leads to a
decreased survival in patients with mantle cell lymphoma (MCL) suggesting a
potential role as a tumor suppressor. 101 On the contrary, strong interaction of
MOBKL2B with the binding partners of MOB1, LATS1 and LATS2, was not found. 96
These data suggest that MOBKL2B is not the main binding partner of LATS1/2 and
might influence the Hippo pathway in a different way. We are especially interested in
the role of the Hippo-pathway in neurons and how this might influence neuronal cell
death. It appears that the Hippo pathway is involved in both the development of the
brain and the protection of adult neurons. 102-104 Cao et al. showed that the Hippo
pathway is involved in the development of the vertebrate neural tube. Gain of
function of YAP and TEAD, which are both directly or indirectly influenced by the
Hippo pathway, promoted cell cycle progression and caused an expansion of the
neural progenitor population. Loss of function increased apoptosis and premature
neuronal differentiation. 102 Van Hateren et al. also showed that the Hippo pathway
can regulate the number of neural progenitor cells. 103 Calamita et al. proposed a
new idea that links the Hippo pathway with autophagy in neurons. 104 They
hypothesize that the blockage of autophagy in fat/hippo mutants reveals an
unexpected role of the Hippo pathway in postmitotic neuronal homeostasis and
neurodegeneration.
MOBKL2B has been associated with sporadic ALS in 3 genome wide association
studies. 53-55 Shatunov et al. and van Es et al. showed that SNPs rs2814707 and
rs3849942, both located near MOBKL2B, are associated with sporadic ALS.
Furthermore, Laaksovirta et al. also identified rs3849942 in a 232 kb region on
24
chromosome 9 that contains the following three genes: IFNK, C9orf72 and
MOBKL2B. No causal mutations were found in these genes. We previously
discussed linkage studies that associated familial ALS/FTD with our region of interest
on chromosome 9. Four of these studies screened the coding region of the
MOBKL2B gene for mutations but no mutations were found. 56, 59, 61, 62 A total of 39
patients from 4 different families suffering from fALS, FTD, fALS/FTD and MND were
sequenced in these studies.
So far, the function of MOBKL2B has mainly been determined by comparing it with
its homolog MOB1. Also a role as a tumor suppressor gene has been proposed. 101
The ability to regulate cell proliferation and cell death are both interesting processes
that could be involved in the development of ALS/FTD. These diseases are
characterized by a loss of motor neurons. The mechanism behind this loss of
neurons is not fully understood and activation of apoptotic signals might contribute to
this cell death. So far, no mutations in MOBKL2B were found in 4 different families
and 1 genome wide association study. However, a down-regulation of this gene has
been associated with increased tumor growth and poor survival in patients with MCL.
An up-regulation in the expression of this gene could therefore lead to an apoptotic
signal in neurons, leading to the development of ALS. In addition, the Hippo pathway
has been shown to mediate crosstalk with other pathways such as the Wnt pathway,
TGFβ and BMP pathways. 105 The Wnt pathway is well known and has several
functions including neuronal tube development and axon guidance. Although farfetched, MOBKL2B might be able to influence Wnt signaling through the Hippo
pathway. This can then lead to a disturbed neuronal development causing the
system to be more susceptible for cell death. Another interesting idea is the
involvement of the Hippo pathway in autophagy. Autophagy is important in the
maintenance of neuronal homeostasis and blockage or deregulation of this process
by for instance a mutation in MOBKL2B could contribute to an increase in neuronal
cell death. The available data on the function of MOB1 does provide some clues
regarding the functioning of MOBKL2B, however, no firm evidence is provided.
Therefore, more research is needed to determine the possible role of MOBKL2B in
neuronal degeneration.
25
IFNK
Interferon kappa (IFNK) is member of a large family of type 1 IFNs. This family
contains a large number of homologous cytokines that are conserved in many
species. The gene consists of 207 amino acids, including a series of cysteines, a 27amino acid long signal peptide and a coiled coil domain. 106, 107 Interferons are best
known for their anti-viral and immunomodulatory activities but are also involved in
anti-proliferative and antitumor processes. IFNK is selectively expressed in
epidermal keratinocytes, monocytes and resting dendritic cells. The level of
expression in keratinocytes is enhanced upon exposure to viral infections or doublestranded RNA. 108
Figure 12: IFNK can bind the IFNAR receptor complex and activate an antiviral
signaling cascade. The STAT1/2 complex will bind the promoters of several genes
and initiate transcription. 109
It has been shown previously that all type 1 IFN receptors exert their effect via a
common receptor complex. 109 This receptor has two major subunits, IFNAR1 and
IFNAR2c, as well as several preassociated proteins (Figure 12). The signal
transduction cascade is started upon binding of the ligand, in our case IFNK. The
binding of IFNK is followed by phosphorylation of TYK2 by Janus activated kinase
(JAK) 1 which can then further activate JAK1 by cross-phosphorylation.
Subsequently, signal transducer and activator of transcription (STAT) 1 and 2 are
activated and form homo- or heterodimers. These complexes can translocate to the
nucleus and bind to the promoters of genes that are regulated by IFN. 109, 110 Binding
to the promoters will initiate the transcription of antiviral genes such as OAS, MxA
and PKR. These proteins can activate three well defined anti-viral pathways. Also,
STAT1 and IRF1, which are important transcription factors in the IFN response, are
up-regulated by IFNK. These up-regulated proteins are able to inhibit viral mRNA
translation, apoptosis of infected cells and degradation of viral RNA. 108 IFNK also
has a direct effect on the release of cytokines by cells of the innate immune system.
26
For example, monocytes incubated with IFNK increased their release of IL-10, MCP1 and MIP-1α but decreased IL-12 release.
IFNK has been linked and associated to fALS, sALS and fALS/FTD in several
studies. Thus far, five linkage studies sequenced the IFNK gene for mutations. 56, 59,
62, 63, 65 A total of 69 patients diagnosed with fALS, FTD and fALS/FTD from multiple
families have been sequenced but no mutations were found in the IFNK gene.
Furthermore, a genome wide association study that included 442 patients with sALS
did not find any mutations in the IFNK gene. 54
It is known that type 1 IFNs are involved in several autoimmune diseases. 111 The
development of some of these diseases is characterized by an IFN type 1 induced
gene expression profile. 112 A possible role of the immune system in the development
of ALS has also been suggested. 113 This study analyzed blood samples of 54 ALS
patients and found elevated levels of potassium channel antibodies compared to
control individuals. However, this finding does not provide any clear evidence for a
direct link between the increased level of antibodies and the development of ALS.
IFNK, a more recently discovered type 1 IFN, might be involved in a similar way in
the onset of autoimmune diseases as the other well known type 1 IFNs. It could be
that an unknown mutation or increase in expression of IFNK disturbs the balance in
the expression of antiviral proteins and lead to an autoimmune response.
IFNs are also involved in the induction of apoptosis. They can activate a proapoptotic pathway that involves the production of TNF-related apoptosis-inducing
ligand (TRAIL). 111 Overactivation of this pathway due to for example an increased
expression of IFNK can lead to the induction of apoptosis. A possible role of this
apoptosis pathway in the development of ALS is only thinkable if it also functions in a
similar way in neurons. Wang et al. investigated the responsiveness of neuronal cells
to direct exposure of type 1 IFNs and observed very responsive target cells.
However, at the present little is known about type 1 IFN signaling in neurons and
their ability to regulate cell survival and apoptosis. 114 An important finding was made
by Dedoni et al. as they reported that an abnormal prolonged exposure of neurons to
a type 1 IFN (IFN-β) caused apoptotic death. Several pathways were activated which
led to increased signaling of JAK/STAT, enhanced activation of double stranded
RNA-activated protein kinase (PKR) and inhibition of the PI3K/AKT pathway. 115
JAK/STAT and PKR are both involved in apoptotic death whereas the PI3/AKT
pathway promotes cell survival. These data clearly show how a deregulation of the
expression of type 1 IFNs can influence neuronal cell death which can then lead to
the development of ALS.
27
C9orf72
The C9orf72 gene encodes 481 amino acids and contains 11 exons. Alternative
splicing of the gene is thought to produce five isoforms. Two of these transcripts,
isoform 1 and 2, have been isolated in-vivo. Isoform 1 contains the entire sequence
whereas Isoform 2 is characterized by an Asparagine to Lysine change at amino acid
222 and the missing of amino acids 223-481. Thus far, existence of the protein has
only been shown at transcript level. 116-118 The function of C9orf72 is currently
unknown although its region on chromosome 9 has been associated with sporadic
ALS and a differential response to anti-TNF treatments for rheumatoid arthritis in 4
genome wide association studies. 53-55, 119
Funcbase is a large database of genes that uses computational gene function
prediction to determine gene function. 120 This database states that C9orf72 might be
involved in cell development and spermatogenesis although no real experimental
evidence supporting this hypothesis is available. Another possible role was
suggested by Liu et al. in 2008. They identified 16 SNPs that associated a region on
chromosome 9 with a differential response to anti-TNF treatment in Rheumatoid
Arthritis (RA). Five of these SNPs, rs774359, rs3849942, rs7046653, rs868856 and
rs2814707 are located near or in the C9orf72 gene. 119 However, these results were
not replicated by Suarez-Gestal et al. who analyzed the same SNPs in a different
patient group. 121
A new possible function was discovered in 2006 when Morita et al. linked
chromosomal region 9p21.3-p13.3 with fALS/FTD. (10) Later, three more linkage
studies that found this association looked for mutations in C9orf72. 59, 61, 62 Luty et al.
screened the coding and non-coding exonic sequence and flanking intronic regions
from the genomic template of C9orf72 of 11 fALS and FTD patients. No mutations
were found. Boxer et al. analyzed the C9orf72 gene in 10 patients of 1 family
suffering from fALS and FTD. One family member also had signs of Parkinsonism.
Screening of the genomic and complementary DNA for mutations was performed as
well as the search for complete genomic deletions or duplications. No irregulations
were found in the C9orf72 gene. Furthermore, Gijselinck et al. analyzed the entire
sequence of the C9orf72 cDNA of 9 patients that suffered from FTLD-ALS but found
no mutations. More evidence of the association of ALS with C9orf72 was provided by
three genome wide association studies. 53-55 These studies found three SNPs near
C9orf72 to be significantly associated with sporadic ALS. One of these studies 54
looked for aminoacid-changing mutations in C9orf72 but did not find any mutations.
Thus far the knowledge about C9orf72 functioning and its possible role in RA, f/sALS
and FTD pathogenesis is still limited. Association of this region of chromosome 9
with ALS/FTD has been shown several times so the gene is certainly worth looking
into. More research is needed to elucidate its possible role in the pathogenesis of
ALS.
28
LINGO2
The leucine-rich repeat and Ig domain containing 2 gene (LINGO2) contains several
domains including 13 leucine-rich repeats, an immunoglobulin I-set, a leucine-rich
repeat-containing N-terminal, a cysteine-rich flanking region of the C-terminal
domain and a transmembrane domain. LINGO2 encodes 606 amino acids and
contains 7 exons. 122, 123 The protein is an important member of the LRR gene family
and expression increases during development and seems to be limited to neuronal
tissue (Figure 13). The function of LINGO2 has not been well investigated and is
currently unknown. 124 However, a high degree of homology exists between LINGO1,
another member of the LRR gene family, and LINGO2. 125 The big overlap in gene
domains (61%) suggests that LINGO2 has a role that is comparable with the better
characterized LINGO1.
Figure 13: Expression levels of the proteins from the LRR gene family increase
during development of the embryo but is low in the adult stage. LINGO2 is mainly
expressed in a population of cells near the olfactory pit. 126
LINGO1 is a component of the nogo-66 receptor (NgR1)/p75/LINGO1 signaling
complex implicated in axonal myelination and regeneration, inhibition of
oligodendrocyte differentiation and neuronal survival. 125, 127, 128 LINGO1 expression
is increased after neuronal damage and inhibition of expression is thought to
increase axonal sprouting and to promote functional recovery. 129 These data
suggest that LINGO1 is a regulator of neuronal death and disruption of this gene
might have great implications in the pathogenesis of several neurodegenerative
diseases.
Further evidence supporting the idea that LINGO1 and LINGO2 have similar
functions came in two recently published studies. 124, 125 First, a LINGO1 SNP
(rs9652490) was associated with essential tremor (ET) and Parkinsons’s disease
(PD). PD is a neurodegenerative disease with a prevalence of 1-2% among the
population over the age of 60. The disease is caused by a loss of dopaminergic
neurons and the formation of Lewy bodies mainly in the substantia nigra pars
compacta. 130 The high level of homology between LINGO1 and LINGO2 led to the
selection of both genes as candidates for ET and PD. Wen et al. and Vilarino-Guell
et al. sequenced groups of patients that suffered from ET and PD. Wen et al. found
sequence variants in both LINGO1 and LINGO2 that were associated to ET and PD
and Vilarino-Guell et al. associated LINGO2 with ET and PD. A direct link between
the survival of dopamine neurons in PD and LINGO-1 was found by Inoue et al. This
29
study contained preliminary data that showed an increase in LINGO-1 mRNA levels
in the substantia nigra of PD patients. Furthermore they showed that inhibition of
endogenous brain LINGO-1 protects midbrain dopamine neurons against
Parkinsonism-inducing agents in vivo and in vitro. They further hypothesize that
LINGO-1 acts via the EGFR/PI3-K/Akt pathway which is involved in cell survival and
can increase or decrease its activity by regulating EGFR activity. Tie2, a different
upstream regulator of the PI3-K/Akt pathway, is also thought to influence neuronal
cell death via this pathway. This finding supports the hypothesis of Inoue et al.
Together, these data suggest a potential role of LINGO1 in the neurodegeneration
found in PD. 131 The association of LINGO2 with PD and its possible function as a
regulator of neuronal death makes LINGO2 a good candidate gene for ALS. This is
strengthened by similarities that can be found between the disease mechanisms of
PD and ALS such as damage to the mitochondria caused by mutations in
mitochondrial proteins. 132
LINGO2 has been associated with ALS in three linkage and three GWA studies. 53-56,
59, 62 The three linkage studies analyzed a total of 3 families with 30 cases that
showed clinical signs of fALS, FTD or fALS/FTD. However, causal mutations in
LINGO2 have not been found in any of these studies so sequencing of bigger patient
groups will be needed to increase chances of detecting possible mutations. (See
table 1 for detailed information about the mutational screening) LINGO2 could also
contribute to the pathogenesis of ALS by changes in the level of expression.
Assuming LINGO2 functions in a similar way as LINGO1, changes in the expression
of LINGO2 can stimulate or inhibit the growth and survival of neurons. An increase in
expression can then lead to enhanced neuronal death which is characteristic for
ALS. Inoue et al. propose a possible hypothesis in which LINGO-1 can downregulate
the activity of EGFR and subsequently the PI3-K/Akt pathway causing an increase in
neuronal cell death. However, speculation about the function of LINGO2 alone will
not be enough in proving its role in the pathogenesis of ALS. Therefore, further
functional studies regarding LINGO2 will be needed in order to prove its involvement
in ALS.
30
Conclusion
The knowledge of ALS genetics has been largely broadened in the last few years.
Interestingly, the newly discovered genes appear to functionally cluster in specific
themes such as RNA processing, ER stress, intracellular axonal transport that uses
the cytoskeleton and hypoxia. Recently, genome wide association studies have been
performed in sporadic ALS patients and showed strong associations of chromosome
9p21.2. with sALS. This region of chromosome 9 has also been linked with fALS,
FTD and fALS-FTD patients in 10 linkage studies and contains 8 genes. The data
provided by these studies suggest a possible role of IFT-74, Tie2, C9orf14, C9orf11,
MOBKL2B, IFNK, C9orf72 and LINGO2 in the pathogenesis of ALS.
IFT-74 is involved in the intraflagellar transport system which is responsible for
the bidirectional movement of multi-subunit protein particles along the length of
axonemal microtubules. An amino acid change that leads to a premature stop codon
was found by Momeni et al. Thus far this mutation has only been found in two
brothers diagnosed with fALS-FTD. The role of IFT-74 in vesicle transport and
exocytosis makes it a plausible biological candidate gene because the transport of
proteins and proper membrane trafficking is essential in the development of neurons.
Although the present data about the mutations found in IFT-74 does not provide
clear evidence for a causal relationship, it may well be that genetic variations in this
gene increase the risk of developing ALS.
The Ang-1/TIE2 ligand/receptor couple is part of the Angiopoietin/TIE system
that activates the PI3-K/AKT pathway. This pathway was found to have a protective
effect in neurons. A decrease in the basal level of expression of Tie2 in ALS patients
could thus make neurons more susceptible to degeneration. This effect becomes
stronger when ER stress due to for example misfolded SOD1 lowers the expression
of Tie2 even more and causes the degeneration of neurons. Also, a lower level of
Tie2 phosphorylation/expression due to for example VEGF might lead to a reduced
neural vascular perfusion and an ALS-like phenotype.
The available data about C9orf14 is limited and therefore only assumptions
can be made about its possible function. Thus far, no expression has been found in
the brain or neurons from the peripheral nervous system and a role in neuronal cell
death is therefore unlikely. Indirect effects of C9orf14 on the degeneration of neurons
via for example the dermal system is not very plausible since the protein is localized
intracellular near the centrosome. It is therefore highly unlikely that C9orf14 is
involved in the pathogenesis of ALS.
It is also highly unlikely that C9orf11 is involved in the pathogenesis of ALS
when we consider its expression pattern (mainly in the testis) and involvement in
acrosome formation. The protein is hardly expressed in the brain and this makes it
difficult for the protein to regulate for instance motor neuron cell death. However, the
expression of an altered transcript variant (missing exon 4) in the brain might change
the function of the protein. Its transmembrane domain that allows it to bind to
vesicles might then be used in the intracellular transport of cargo-vesicles. This
process is thought to be disrupted in patients that suffer from ALS.
MOBKL2B is thought to be play a role in the Hippo pathway which is involved
in the regulation of cell proliferation and cell death. These are both interesting
processes that could be involved in the development of ALS/FTD. So far, no
mutations in MOBKL2B were found in 4 different families and a genome wide
association study. However, an up-regulation in the expression of this gene could
lead to an apoptotic signal in neurons, leading to the development of ALS. It should
be noted that most data presented here is based on the high level of homology with
31
MOB1 and that more research is needed that focuses on MOBKL2B itself.
IFNK is a type 1 IFN involved in the immune response against viruses. Type 1
IFNs can also alter the level of activation of apoptotic pathways that involve the
activation of TRAIL, JAK/STAT, PKR and the inhibition of the PI3K/AKT pathway.
Activation of these pathways has been shown in neurons after a prolonged exposure
to a type 1 IFN. These data show how an increase in the expression of type 1 IFNs
can influence neuronal cell death which can then lead to the development of ALS.
Thus far the knowledge about C9orf72 functioning and its possible role in RA,
ALS and FTD pathogenesis is still limited. A database that uses a computational
gene function prediction method states that C9orf72 might be involved in cell
development and spermatogenesis although no real experimental evidence
supporting this hypothesis is available. Association of this region of chromosome 9
with ALS/FTD has been shown several times so the gene is certainly worth looking
into. More research is needed to elucidate its possible role in the pathogenesis of
ALS.
Assuming LINGO2 functions in a similar way as LINGO1, changes in the
expression of LINGO2 can stimulate or inhibit the growth and survival of neurons. An
increase in expression can then lead to enhanced neuronal death which is
characteristic for ALS. Inoue et al. propose a possible hypothesis in which LINGO-1
can downregulate the activity of EGFR and subsequently the PI3-K/Akt pathway
causing an increase in neuronal cell death.
In this thesis we discussed the function and possible role of IFT-74, Tie2, C9orf14,
C9orf11, MOBKL2B, IFNK, C9orf72 and LINGO2 in the pathogenesis of ALS. Some
of these genes (IFT-74, Tie2, MOBKL2B, IFNK and LINGO2) have great potential in
playing a causal role in the development of ALS. Thus far, IFT-74 is the only gene in
this region in which mutations in fALS patients have been found. Its function in
axonal transport, a crucial process for neurons, strengthens the idea of this protein
being causally linked to the development of ALS even more. Tie2, IFNK and LINGO2
all have one common trait in that they are thought to influence neuronal survival via
the PI3K/Akt pathway. This pathway is an important regulator of neuronal cell death
and this suggests a possible role of these proteins in the pathogenesis of ALS.
Future research should focus on determining the level of expression of these
promising genes in sALS and fALS patients. Also, mutational screening in sALS
patients can contribute to a better understanding of the possible role of these
candidate genes in the pathogenesis of ALS.
32
References
1. Valdmanis PN, Rouleau GA. Genetics of familial amyotrophic lateral sclerosis.
Neurology. 2008;70:144-152.
2. Shaw CE, Arechavala-Gomeza V, Al-Chalabi A. Chapter 14 familial amyotrophic
lateral sclerosis. Handb Clin Neurol. 2007;82:279-300.
3. Giordana MT, Ferrero P, Grifoni S, Pellerino A, Naldi A, Montuschi A. Dementia
and cognitive impairment in amyotrophic lateral sclerosis: A review. Neurol Sci.
2011;32:9-16.
4. Chow TW, Miller BL, Hayashi VN, Geschwind DH. Inheritance of frontotemporal
dementia. Arch Neurol. 1999;56:817-822.
5. Rascovsky K, Salmon DP, Lipton AM, et al. Rate of progression differs in
frontotemporal dementia and alzheimer disease. Neurology. 2005;65:397-403.
6. Raaphorst J, de Visser M, Linssen WH, de Haan RJ, Schmand B. The cognitive
profile of amyotrophic lateral sclerosis: A meta-analysis. Amyotroph Lateral Scler.
2010;11:27-37.
7. Mackenzie IR, Rademakers R, Neumann M. TDP-43 and FUS in amyotrophic
lateral sclerosis and frontotemporal dementia. Lancet Neurol. 2010;9:995-1007.
8. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide
dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature.
1993;362:59-62.
33
9. Neumann M, Sampathu DM, Kwong LK, et al. Ubiquitinated TDP-43 in
frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science.
2006;314:130-133.
10. McCord JM, Fridovich I. Superoxide dismutase. an enzymic function for
erythrocuprein (hemocuprein). J Biol Chem. 1969;244:6049-6055.
11. Rosen DR. Mutations in Cu/Zn superoxide dismutase gene are associated with
familial amyotrophic lateral sclerosis. Nature. 1993;364:362.
12. Wiedau-Pazos M, Goto JJ, Rabizadeh S, et al. Altered reactivity of superoxide
dismutase in familial amyotrophic lateral sclerosis. Science. 1996;271:515-518.
13. Carroll MC, Girouard JB, Ulloa JL, et al. Mechanisms for activating cu- and zncontaining superoxide dismutase in the absence of the CCS cu chaperone. Proc Natl
Acad Sci U S A. 2004;101:5964-5969.
14. Durham HD, Roy J, Dong L, Figlewicz DA. Aggregation of mutant Cu/Zn
superoxide dismutase proteins in a culture model of ALS. J Neuropathol Exp Neurol.
1997;56:523-530.
15. Bruening W, Roy J, Giasson B, Figlewicz DA, Mushynski WE, Durham HD. Upregulation of protein chaperones preserves viability of cells expressing toxic Cu/Znsuperoxide dismutase mutants associated with amyotrophic lateral sclerosis. J
Neurochem. 1999;72:693-699.
16. Andersen PM, Forsgren L, Binzer M, et al. Autosomal recessive adult-onset
amyotrophic lateral sclerosis associated with homozygosity for Asp90Ala CuZn-
34
superoxide dismutase mutation. A clinical and genealogical study of 36 patients.
Brain. 1996;119 ( Pt 4):1153-1172.
17. Reaume AG, Elliott JL, Hoffman EK, et al. Motor neurons in Cu/Zn superoxide
dismutase-deficient mice develop normally but exhibit enhanced cell death after
axonal injury. Nat Genet. 1996;13:43-47.
18. Cleveland DW. From charcot to SOD1: Mechanisms of selective motor neuron
death in ALS. Neuron. 1999;24:515-520.
19. Gurney ME, Pu H, Chiu AY, et al. Motor neuron degeneration in mice that
express a human cu,zn superoxide dismutase mutation. Science. 1994;264:17721775.
20. Deng HX, Jiang H, Fu R, et al. Molecular dissection of ALS-associated toxicity of
SOD1 in transgenic mice using an exon-fusion approach. Hum Mol Genet.
2008;17:2310-2319.
21. Swarup V, Julien JP. ALS pathogenesis: Recent insights from genetics and
mouse models. Prog Neuropsychopharmacol Biol Psychiatry. 2011;35:363-369.
22. Yamanaka K, Boillee S, Roberts EA, et al. Mutant SOD1 in cell types other than
motor neurons and oligodendrocytes accelerates onset of disease in ALS mice. Proc
Natl Acad Sci U S A. 2008;105:7594-7599.
23. Buratti E, Dork T, Zuccato E, Pagani F, Romano M, Baralle FE. Nuclear factor
TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO
J. 2001;20:1774-1784.
35
24. Buratti E, Baralle FE. Multiple roles of TDP-43 in gene expression, splicing
regulation, and human disease. Front Biosci. 2008;13:867-878.
25. Mackenzie IR, Bigio EH, Ince PG, et al. Pathological TDP-43 distinguishes
sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1
mutations. Ann Neurol. 2007;61:427-434.
26. Pesiridis GS, Lee VM, Trojanowski JQ. Mutations in TDP-43 link glycine-rich
domain functions to amyotrophic lateral sclerosis. Hum Mol Genet. 2009;18:R15662.
27. Van Deerlin VM, Sleiman PM, Martinez-Lage M, et al. Common variants at 7p21
are associated with frontotemporal lobar degeneration with TDP-43 inclusions. Nat
Genet. 2010;42:234-239.
28. Kabashi E, Lin L, Tradewell ML, et al. Gain and loss of function of ALS-related
mutations of TARDBP (TDP-43) cause motor deficits in vivo. Hum Mol Genet.
2010;19:671-683.
29. Nonaka T, Kametani F, Arai T, Akiyama H, Hasegawa M. Truncation and
pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43.
Hum Mol Genet. 2009;18:3353-3364.
30. Zhang YJ, Xu YF, Cook C, et al. Aberrant cleavage of TDP-43 enhances
aggregation and cellular toxicity. Proc Natl Acad Sci U S A. 2009;106:7607-7612.
31. Kraemer BC, Schuck T, Wheeler JM, et al. Loss of murine TDP-43 disrupts
motor function and plays an essential role in embryogenesis. Acta Neuropathol.
2010;119:409-419.
36
32. Wu LS, Cheng WC, Hou SC, Yan YT, Jiang ST, Shen CK. TDP-43, a neuropathosignature factor, is essential for early mouse embryogenesis. Genesis.
2010;48:56-62.
33. Wegorzewska I, Bell S, Cairns NJ, Miller TM, Baloh RH. TDP-43 mutant
transgenic mice develop features of ALS and frontotemporal lobar degeneration.
Proc Natl Acad Sci U S A. 2009;106:18809-18814.
34. Wils H, Kleinberger G, Janssens J, et al. TDP-43 transgenic mice develop
spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal
lobar degeneration. Proc Natl Acad Sci U S A. 2010;107:3858-3863.
35. Igaz LM, Kwong LK, Lee EB, et al. Dysregulation of the ALS-associated gene
TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest.
2011;121:726-738.
36. Lagier-Tourenne C, Polymenidou M, Cleveland DW. TDP-43 and FUS/TLS:
Emerging roles in RNA processing and neurodegeneration. Hum Mol Genet.
2010;19:R46-64.
37. Fujii R, Takumi T. TLS facilitates transport of mRNA encoding an actin-stabilizing
protein to dendritic spines. J Cell Sci. 2005;118:5755-5765.
38. Vance C, Rogelj B, Hortobagyi T, et al. Mutations in FUS, an RNA processing
protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;323:12081211.
37
39. Zakaryan RP, Gehring H. Identification and characterization of the nuclear
localization/retention signal in the EWS proto-oncoprotein. J Mol Biol. 2006;363:2738.
40. Gros-Louis F, Gaspar C, Rouleau GA. Genetics of familial and sporadic
amyotrophic lateral sclerosis. Biochim Biophys Acta. 2006;1762:956-972.
41. Cudkowicz ME, McKenna-Yasek D, Sapp PE, et al. Epidemiology of mutations in
superoxide dismutase in amyotrophic lateral sclerosis. Ann Neurol. 1997;41:210221.
42. Hadano S, Hand CK, Osuga H, et al. A gene encoding a putative GTPase
regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat Genet.
2001;29:166-173.
43. Munch C, Sedlmeier R, Meyer T, et al. Point mutations of the p150 subunit of
dynactin (DCTN1) gene in ALS. Neurology. 2004;63:724-726.
44. Al-Chalabi A, Andersen PM, Nilsson P, et al. Deletions of the heavy
neurofilament subunit tail in amyotrophic lateral sclerosis. Hum Mol Genet.
1999;8:157-164.
45. Kuzma-Kozakiewicz M, Kwiecinski H. The genetics of amyotrophic lateral
sclerosis. Neurol Neurochir Pol. 2009;43:538-549.
46. Lambrechts D, Poesen K, Fernandez-Santiago R, et al. Meta-analysis of
vascular endothelial growth factor variations in amyotrophic lateral sclerosis:
Increased susceptibility in male carriers of the -2578AA genotype. J Med Genet.
2009;46:840-846.
38
47. Fernandez-Santiago R, Hoenig S, Lichtner P, et al. Identification of novel
angiogenin (ANG) gene missense variants in german patients with amyotrophic
lateral sclerosis. J Neurol. 2009;256:1337-1342.
48. Sleegers K, Brouwers N, Maurer-Stroh S, et al. Progranulin genetic variability
contributes to amyotrophic lateral sclerosis. Neurology. 2008;71:253-259.
49. Tanner NK, Linder P. DExD/H box RNA helicases: From generic motors to
specific dissociation functions. Mol Cell. 2001;8:251-262.
50. Chen YZ, Bennett CL, Huynh HM, et al. DNA/RNA helicase gene mutations in a
form of juvenile amyotrophic lateral sclerosis (ALS4). Am J Hum Genet.
2004;74:1128-1135.
51. Chance PF, Rabin BA, Ryan SG, et al. Linkage of the gene for an autosomal
dominant form of juvenile amyotrophic lateral sclerosis to chromosome 9q34. Am J
Hum Genet. 1998;62:633-640.
52. Nishimura AL, Mitne-Neto M, Silva HC, et al. A mutation in the vesicle-trafficking
protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral
sclerosis. Am J Hum Genet. 2004;75:822-831.
53. Shatunov A, Mok K, Newhouse S, et al. Chromosome 9p21 in sporadic
amyotrophic lateral sclerosis in the UK and seven other countries: A genome-wide
association study. Lancet Neurol. 2010;9:986-994.
54. Laaksovirta H, Peuralinna T, Schymick JC, et al. Chromosome 9p21 in
amyotrophic lateral sclerosis in finland: A genome-wide association study. Lancet
Neurol. 2010;9:978-985.
39
55. van Es MA, Veldink JH, Saris CG, et al. Genome-wide association study
identifies 19p13.3 (UNC13A) and 9p21.2 as susceptibility loci for sporadic
amyotrophic lateral sclerosis. Nat Genet. 2009;41:1083-1087.
56. Pearson JP, Williams NM, Majounie E, et al. Familial frontotemporal dementia
with amyotrophic lateral sclerosis and a shared haplotype on chromosome 9p. J
Neurol. 2011;258:647-655.
57. Morita M, Al-Chalabi A, Andersen PM, et al. A locus on chromosome 9p confers
susceptibility to ALS and frontotemporal dementia. Neurology. 2006;66:839-844.
58. Vance C, Al-Chalabi A, Ruddy D, et al. Familial amyotrophic lateral sclerosis with
frontotemporal dementia is linked to a locus on chromosome 9p13.2-21.3. Brain.
2006;129:868-876.
59. Boxer AL, Mackenzie IR, Boeve BF, et al. Clinical, neuroimaging and
neuropathological features of a new chromosome 9p-linked FTD-ALS family. J
Neurol Neurosurg Psychiatry. 2011;82:196-203.
60. Valdmanis PN, Dupre N, Bouchard JP, et al. Three families with amyotrophic
lateral sclerosis and frontotemporal dementia with evidence of linkage to
chromosome 9p. Arch Neurol. 2007;64:240-245.
61. Gijselinck I, Engelborghs S, Maes G, et al. Identification of 2 loci at
chromosomes 9 and 14 in a multiplex family with frontotemporal lobar degeneration
and amyotrophic lateral sclerosis. Arch Neurol. 2010;67:606-616.
40
62. Luty AA, Kwok JB, Dobson-Stone C, et al. Sigma nonopioid intracellular receptor
1 mutations cause frontotemporal lobar degeneration-motor neuron disease. Ann
Neurol. 2010;68:639-649.
63. Le Ber I, Camuzat A, Berger E, et al. Chromosome 9p-linked families with
frontotemporal dementia associated with motor neuron disease. Neurology.
2009;72:1669-1676.
64. Hosler BA, Siddique T, Sapp PC, et al. Linkage of familial amyotrophic lateral
sclerosis with frontotemporal dementia to chromosome 9q21-q22. JAMA.
2000;284:1664-1669.
65. Momeni P, Schymick J, Jain S, et al. Analysis of IFT74 as a candidate gene for
chromosome 9p-linked ALS-FTD. BMC Neurol. 2006;6:44.
66. Yan J, Siddique T. A major novel locus for ALS/FTD on chromosome 9p21 and
its pathological correlates. April 2006;American Academy of Neurology, 58th Annual
Meeting.
67. Lucker BF, Behal RH, Qin H, et al. Characterization of the intraflagellar transport
complex B core: Direct interaction of the IFT81 and IFT74/72 subunits. J Biol Chem.
2005;280:27688-27696.
68. Scholey JM. Intraflagellar transport. Annu Rev Cell Dev Biol. 2003;19:423-443.
69. Lucker BF, Miller MS, Dziedzic SA, Blackmarr PT, Cole DG. Direct interactions of
intraflagellar transport complex B proteins IFT88, IFT52, and IFT46. J Biol Chem.
2010;285:21508-21518.
41
70. Das V, Nal B, Dujeancourt A, et al. Activation-induced polarized recycling targets
T cell antigen receptors to the immunological synapse; involvement of SNARE
complexes. Immunity. 2004;20:577-588.
71. Baldari CT, Rosenbaum J. Intraflagellar transport: It's not just for cilia anymore.
Curr Opin Cell Biol. 2010;22:75-80.
72. Xiao S, Sato C, Kawarai T, et al. Genetic studies of GRN and IFT74 in
amyotrophic lateral sclerosis. Neurobiol Aging. 2008;29:1279-1282.
73. Yamashita S, Migita A, Hayashi K, et al. Amyotrophic lateral sclerosis in a patient
with kartagener syndrome. Amyotroph Lateral Scler. 2010;11:402-404.
74. Protein information. Available at:
http://www.uniprot.org/uniprot/Q02763#section_terms.
75. Thomas M, Augustin HG. The role of the angiopoietins in vascular
morphogenesis. Angiogenesis. 2009;12:125-137.
76. Peters KG, Kontos CD, Lin PC, et al. Functional significance of Tie2 signaling in
the adult vasculature. Recent Prog Horm Res. 2004;59:51-71.
77. Valable S, Bellail A, Lesne S, et al. Angiopoietin-1-induced PI3-kinase activation
prevents neuronal apoptosis. FASEB J. 2003;17:443-445.
78. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for
Tie2 that disrupts in vivo angiogenesis. Science. 1997;277:55-60.
42
79. Kim I, Kim JH, Moon SO, Kwak HJ, Kim NG, Koh GY. Angiopoietin-2 at high
concentration can enhance endothelial cell survival through the phosphatidylinositol
3'-kinase/Akt signal transduction pathway. Oncogene. 2000;19:4549-4552.
80. Hosoi T, Tamubo T, Horie N, Okuma Y, Nomura Y, Ozawa K. TEK/Tie2 is a
novel gene involved in endoplasmic reticulum stress. J Pharmacol Sci.
2010;114:230-233.
81. Bai Y, Meng Z, Cui M, et al. An Ang1-Tie2-PI3K axis in neural progenitor cells
initiates survival responses against oxygen and glucose deprivation. Neuroscience.
2009;160:371-381.
82. Saharinen P, Bry M, Alitalo K. How do angiopoietins tie in with vascular
endothelial growth factors? Curr Opin Hematol. 2010;17:198-205.
83. Oosthuyse B, Moons L, Storkebaum E, et al. Deletion of the hypoxia-response
element in the vascular endothelial growth factor promoter causes motor neuron
degeneration. Nat Genet. 2001;28:131-138.
84. Singh H, Milner CS, Aguilar Hernandez MM, Patel N, Brindle NP. Vascular
endothelial growth factor activates the tie family of receptor tyrosine kinases. Cell
Signal. 2009;21:1346-1350.
85. NCBI Aceview. Homo sapiens gene NCRNA00032, encoding non-protein
coding RNA 32. Available at:
http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=human&l=NCRNA00
032.
43
86. Nextprot Beta. NCRNA00032 » Putative uncharacterized protein encoded by
NCRNA00032 . Available at:
http://beta.nextprot.org/db/entry/NX_P0C843/expression.
87. Pujana MA, Ruiz A, Badenas C, et al. Molecular characterization of a
t(9;12)(p21;q13) balanced chromosome translocation in combination with integrative
genomics analysis identifies C9orf14 as a candidate tumor-suppressor. Genes
Chromosomes Cancer. 2007;46:155-162.
88. Lu KH, Patterson AP, Wang L, et al. Selection of potential markers for epithelial
ovarian cancer with gene expression arrays and recursive descent partition analysis.
Clin Cancer Res. 2004;10:3291-3300.
89. Dent J, Hall GD, Wilkinson N, et al. Cytogenetic alterations in ovarian clear cell
carcinoma detected by comparative genomic hybridisation. Br J Cancer.
2003;88:1578-1583.
90. Doxsey S, McCollum D, Theurkauf W. Centrosomes in cellular regulation. Annu
Rev Cell Dev Biol. 2005;21:411-434.
91. Ruiz A, Pujana MA, Estivill X. Isolation and characterisation of a novel human
gene (C9orf11) on chromosome 9p21, a region frequently deleted in human cancer.
Biochim Biophys Acta. 2000;1517:128-134.
92. Parris CN, Harris JD, Griffin DK, Cuthbert AP, Silver AJ, Newbold RF. Functional
evidence of novel tumor suppressor genes for cutaneous malignant melanoma.
Cancer Res. 1999;59:516-520.
44
93. Li YC, Hu XQ, Zhang KY, et al. Afaf, a novel vesicle membrane protein, is related
to acrosome formation in murine testis. FEBS Lett. 2006;580:4266-4273.
94. Protein database. Available at: http://genome.ucsc.edu/cgibin/hgGene?hgg_gene=uc003zqn.2&hgg_prot=Q86TA1&hgg_chrom=chr9&hgg_sta
rt=27325207&hgg_end=27529850&hgg_type=knownGene&db=hg19&hgsid=190601
305.
95. Protein database. Available at: http://www.uniprot.org/uniprot/Q86TA1.
96. Chow A, Hao Y, Yang X. Molecular characterization of human homologs of yeast
MOB1. Int J Cancer. 2010;126:2079-2089.
97. Luca FC, Winey M. MOB1, an essential yeast gene required for completion of
mitosis and maintenance of ploidy. Mol Biol Cell. 1998;9:29-46.
98. Wilmeth LJ, Shrestha S, Montano G, Rashe J, Shuster CB. Mutual dependence
of Mob1 and the chromosomal passenger complex for localization during mitosis.
Mol Biol Cell. 2010;21:380-392.
99. Vagnarelli P, Earnshaw WC. Chromosomal passengers: The four-dimensional
regulation of mitotic events. Chromosoma. 2004;113:211-222.
100. Chan SW, Lim CJ, Chen L, et al. The hippo pathway in biological control and
cancer development. J Cell Physiol. 2011;226:928-939.
101. Hartmann EM, Campo E, Wright G, et al. Pathway discovery in mantle cell
lymphoma by integrated analysis of high-resolution gene expression and copy
number profiling. Blood. 2010;116:953-961.
45
102. Cao X, Pfaff SL, Gage FH. YAP regulates neural progenitor cell number via the
TEA domain transcription factor. Genes Dev. 2008;22:3320-3334.
103. Van Hateren NJ, Das RM, Hautbergue GM, Borycki AG, Placzek M, Wilson SA.
FatJ acts via the hippo mediator Yap1 to restrict the size of neural progenitor cell
pools. Development. 2011;138:1893-1902.
104. Calamita P, Fanto M. Slimming down fat makes neuropathic hippo: The
Fat/Hippo tumor suppressor pathway protects adult neurons through regulation of
autophagy. Autophagy. 2011;7.
105. Halder G, Johnson RL. Hippo signaling: Growth control and beyond.
Development. 2011;138:9-22.
106. Nardelli B, Zaritskaya L, Semenuk M, et al. Regulatory effect of IFN-kappa, a
novel type I IFN, on cytokine production by cells of the innate immune system. J
Immunol. 2002;169:4822-4830.
107. Protein database. Available at: http://www.uniprot.org/uniprot/Q9P0W0.
108. LaFleur DW, Nardelli B, Tsareva T, et al. Interferon-kappa, a novel type I
interferon expressed in human keratinocytes. J Biol Chem. 2001;276:39765-39771.
109. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells
respond to interferons. Annu Rev Biochem. 1998;67:227-264.
110. Kirou KA, Mavragani CP, Crow MK. Activation of type I interferon in systemic
lupus erythematosus. Expert Rev Clin Immunol. 2007;3:579-588.
46
111. Meyer O. Interferons and autoimmune disorders. Joint Bone Spine.
2009;76:464-473.
112. Trinchieri G. Type I interferon: Friend or foe? J Exp Med. 2010;207:2053-2063.
113. Nwosu VK, Royer JA, Stickler DE. Voltage gated potassium channel antibodies
in amyotrophic lateral sclerosis. Amyotroph Lateral Scler. 2010;11:392-394.
114. Wang J, Campbell IL. Innate STAT1-dependent genomic response of neurons
to the antiviral cytokine alpha interferon. J Virol. 2005;79:8295-8302.
115. Dedoni S, Olianas MC, Onali P. Interferon-beta induces apoptosis in human
SH-SY5Y neuroblastoma cells through activation of JAK-STAT signaling and downregulation of PI3K/Akt pathway. J Neurochem. 2010;115:1421-1433.
116. Protein database. Available at:
http://www.uniprot.org/uniprot/Q96LT7#section_seq.
117. Protein database. Available at: http://genome.ucsc.edu/cgibin/hgGene?hgg_gene=uc003zqq.2&hgg_prot=A8K5W0&hgg_chrom=chr9&hgg_sta
rt=27546543&hgg_end=27573481&hgg_type=knownGene&db=hg19&hgsid=190559
043.
118. AceView: a comprehensive cDNA-supported gene and transcripts annotation.
Available at:
http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?db=human&q=C9orf72.
47
119. Liu C, Batliwalla F, Li W, et al. Genome-wide association scan identifies
candidate polymorphisms associated with differential response to anti-TNF treatment
in rheumatoid arthritis. Mol Med. 2008;14:575-581.
120. Funcbase: The human gene C9orf72, computational function prediction.
Available at:
http://func.mshri.on.ca/human/genes/list_functional_scores/ENTREZ_GENE:203228.
121. Suarez-Gestal M, Perez-Pampin E, Calaza M, Gomez-Reino JJ, Gonzalez A.
Lack of replication of genetic predictors for the rheumatoid arthritis response to antiTNF treatments: A prospective case-only study. Arthritis Res Ther. 2010;12:R72.
122. Protein database. Available at:
http://www.ebi.ac.uk/interpro/ISpy?mode=single&ac=Q7L985.
123. Protein database. Available at: http://www.uniprot.org/uniprot/Q7L985.
124. Wu YW, Prakash KM, Rong TY, et al. Lingo2 variants associated with essential
tremor and parkinson's disease. Hum Genet. 2011;129:611-615.
125. Vilarino-Guell C, Wider C, Ross OA, et al. LINGO1 and LINGO2 variants are
associated with essential tremor and parkinson disease. Neurogenetics.
2010;11:401-408.
126. Haines BP, Rigby PW. Expression of the Lingo/LERN gene family during
mouse embryogenesis. Gene Expr Patterns. 2008;8:79-86.
127. Mi S, Lee X, Shao Z, et al. LINGO-1 is a component of the nogo-66
receptor/p75 signaling complex. Nat Neurosci. 2004;7:221-228.
48
128. Shao Z, Browning JL, Lee X, et al. TAJ/TROY, an orphan TNF receptor family
member, binds nogo-66 receptor 1 and regulates axonal regeneration. Neuron.
2005;45:353-359.
129. Ji B, Li M, Wu WT, et al. LINGO-1 antagonist promotes functional recovery and
axonal sprouting after spinal cord injury. Mol Cell Neurosci. 2006;33:311-320.
130. Lorenzo-Betancor O, Samaranch L, Garcia-Martin E, et al. LINGO1 gene
analysis in parkinson's disease phenotypes. Mov Disord. 2011;26:722-727.
131. Inoue H, Lin L, Lee X, et al. Inhibition of the leucine-rich repeat protein LINGO-1
enhances survival, structure, and function of dopaminergic neurons in parkinson's
disease models. Proc Natl Acad Sci U S A. 2007;104:14430-14435.
132. Martin LJ. Mitochondrial pathobiology in parkinson's disease and amyotrophic
lateral sclerosis. J Alzheimers Dis. 2010;20 Suppl 2:S335-56.
49