Joshua Hughes

Valproic Acid and Lithium Treatment May Offer Neuroprotection Against Hypoxic
Insults in HIF-1 deficient Caenorhabditis elegans
Abstract
Behavioural studies of the model organism, Caenorhabditis elegans, have long been used
as a means of characterising the biological signaling pathways associated with sensory
response. In this study, we investigate the effect of hypoxia and the associated HIF-1
pathway on chemotactic behaviour and the extent to which this behaviour is modified by
treatment with valproic acid and lithium, agents postulated to regulate HIF-expression. We
show that HIF-1-deficient organisms exhibit impaired chemotactic response following
hypoxic insult, not shared by wild-type organisms. We also demonstrate that the loss of
HIF-1 function can be abrogated by valproate and/or lithium treatment, restoring
chemotactic index to a level comparable to that of wild-type organisms. This indicates that
valproate/lithium possess neuroprotective properties, independent of their effects on HIF
expression.
1. Introduction
Hypoxia is a pathological condition whereby a region of an organism, or organism as a
whole, is deprived of oxygen to the point where the metabolic demand cannot be
sufficiently met. Brief periods of hypoxia may be tolerated by means of anaerobic
respiration, however prolonged periods result in lactic acidosis, eventually resulting in cell
death. As a result, most species, including C. elegans (the focus of this study), have
evolved a biological alarm system of sorts, known as the hypoxia signaling pathway. The
chief regulators of this pathway are the transcription factor family HIF (Hypoxia-Inducible
Factors). The most prevalent and well
characterised of these is HIF-1, a heterodimer
consisting of an oxygen-sensitive -subunit and
a constitutively expressed -subunit.
In normoxic conditions, HIF-1 is hydroxylated
at conserved proline residues (P402 and P564)
within its O2-dependent degradation domain
(ODD) by HIF prolyl-hydrolases, allowing for
recognition and binding by von Hippel-Lindau
tumour suppressor protein (pVHL). pVHL has
E3 ubiquitin ligase activity, and thus targets
HIF-1 for proteasomal degradation (Maxwell et
al., 1999). Importantly, hydroxylation does not
occur under hypoxic conditions, as HIF prolylhydrolases require oxygen as a co-substrate
(Semenza et al., 2004). In this way HIF-1 is
regulated so it is only stabilised in conditions of
hypoxia. Under this state, it undergoes nuclear
translocation and is free to dimerise with HIF-1
(ARNT) and co-activator p300 triggering
activation and subsequent DNA binding (Fig. 1).
The pathway is conserved in C. elegans,
mammalian HIF-1, HIF-1 and pVHL being
orthologous to C. elegans HIF-1, AHA-1 and
VHL-1 respectively.

Figure 1: Fate of HIF-1 under normoxia

and hypoxia. HIF-1 is hydroxylated under
normoxia, resulting in proteasome
degradation. Under hypoxia it is stabilised,
and acts (in complex with HIF-1) and coactivator, c300, as a transcription factor.

Joshua Hughes

Active HIF-1 operates to up-regulate a suite of genes which provide an adaptive response
to low oxygen, via the binding of hypoxia response elements (HRE), present in promoter/
enhancer regions. HIF-1s downstream targets promote cell survival and the restoration of
oxygen homeostasis through various cellular and systemic mechanisms, including upregulation of glycolytic enzymes (Shen et al, 2005), repression of mitochondrial activity
(Papandreou et al., 2006), inhibition of apoptotic pathways (Sendoel et al., 2010) as well
as neurone-specific protective responses (Haines et al., 2012). Neurones are particularly
hypoxia-sensitive due to to their high metabolic oxygen requirement, a demand which
cannot be satisfied through anaerobic respiration alone.
In this study, the effect of the HIF-1
pathway is observed from a
functional perspective, through
analysing changes in the chemotactic
behaviour of C. elegans, a free-living
nematode. C. elegans is capable of
detecting a broad range of volatile
and water-soluble molecules via
chemosensory neurones located
principally in the anterior amphid
organs (Fig.2). The AWC and AWA
neurones are known to mediate
Figure 2: Diagram of olfactory and gustatory
chemotaxis towards volatile
neuroanatomy in C. elegans.
Diagram shows major chemosensory neurones only.
compounds, whereas ASE is
Neurones with minor roles in chemotaxis include ASJ,
responsible for attraction towards
ASI, ASF, ASG and ASK (not shown).
water-soluble compounds (Bargmann
et al., 1991).
It can be hypothesised that neuronal cells of HIF-1 deficient mutants are significantly more
vulnerable to hypoxia, due to the absence of HIF-1-mediated protection. It follows that a
relative increase in neuronal damage/death is likely to be reflected in the neurosensory
capability of an organism, in this case the ability to chemotax towards an attractant point
source. This behaviour-centric analysis also will be used to observe the effect of extrinsic
chemical factors, valproic acid (VPA) and lithium chloride (LiCl), on hypoxia adaptation.
Valproic acid is a well characterised histone deacetylase (HDAC) inhibitor, which has been
reported to indirectly inhibit HIF-1 in mammalian cell lines (Qian et al., 2006). Conversely,
lithium (Li+) has been demonstrated to up-regulate HIF-1 through inhibition of glycogen
synthase kinase-3 (GSK-3) (Kast, 2008), which in the absence of Li+ primes HIF-1 for
ubiquitin-proteasomal degradation (Flgel et al., 2007). However, these reported effects
have not yet been substantiated in C. elegans, and it remains to be observed how
inhibition of GSK-3 and/or HDAC effects hypoxic adaptation. It is conceivable that GSK-3
and/or HDAC inhibition may also alter hypoxia adaption in a HIF-1-independent manner.
The aim of this study is to determine if and how extrinsic exposure to VPA and LiCl effect
the hypoxic adaptation of C. elegans, as represented by the ability to chemotax towards
an identified chemoattractant. To determine whether VPA/LiCl act in a HIF-dependent or
independent manner, experiments will be repeated using HIF-1 knockout organisms, in
addition to wild-type. C. elegans is almost ideally suited for this purpose; possessing a
basic, but highly characterised and invariant neurosensory system, for which a wide
variety of assays have been developed and optimised.

Joshua Hughes

Materials & Methods

Equipment provided by the University of Bath, all chemicals obtained from Sigma-Aldrich.
2.1. Strains and Culture
Nematodes were originally obtained from Caenorhabditis Genetics Centre, University of
Minnesota. C. elegans (Wild Type [N2 Bristol] and HIF-1 knockout) were cultivated on
90mm NGM petri plates seeded with a 200l suspension of OP50 e. coli (in Lysogeny
Broth). Strains were grown at 18C, under a low light environment. Passages of a small
number (~10) of nematodes to new seeded NGM plates were conducted every 2-3 days,
in order to prevent OP50 depletion and/or overcrowding (both of which induce dauer
formation). Samples designated for hypoxic assays were cultured for 48 hours in a sealed
Plexiglass chamber, under a constant 1% O2/5% CO2 gas flow. Oxygen level was
monitored using an O2 analyser.
2.2. Exposure of Extrinsic Compounds (VPA & LiCl)
Pharmacological compounds LiCl and VPA were introduced into the growth media, at
10mM and 6mM respectively. Nematodes were propagated in this adulterated media for
48 hours prior to assay.
2.3. Chemotaxis Assay Preparation
Nematodes were washed from NGM maintenance plates using 2ml M9 buffer (see
appendix). The M9-worm suspension was transferred to an eppendorf tube and
centrifuged at low speed (3200rpm). The resulting supernatant was subsequently replaced
with fresh M9, and the pellet resuspended by briefly vortexing. To ensure OP50 removal,
this process was repeated twice. Assay plates (50mm) were poured with chemotaxis agar
(see appendix), and marked into equal quadrants. A 5l drop of the washed preparation
was pipetted onto each of the two quadrants, labelled W (see Fig. 1). The attractant was
placed in the centre of quadrant A, with the opposite quadrant, C, an unaltered control.
Attractant preparation varied slightly depending upon the nature of the chemical used;
water-soluble attractants (biotin) necessitating the use of an agar plug, left to diffuse
overnight (as per Saeki et al., 2001), whereas volatile attractants were simply spotted onto
the agar surface.
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Figure 2: Configuration of chemotaxis assay plates. Petri plate

base divided into quadrants, using marker pen. Suspended
nematodes were placed in quadrants labelled W, equidistant from the
attractant and control positions. Volatile attractants were spotted (7l,
10% in M9 buffer solution) in the centre of quadrant A, water-soluble
attractants were added to the same location in the form of an agar
plug - left to diffuse overnight before initiating assays. Quadrant C was
used as a negative control, containing 7l M9 solution while
conducting volatile attractant assays, or an unadulterated agar plug in
the case of water soluble attractants.
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Joshua Hughes

2.4. Chemotaxis Assay Scoring

Numbers of organisms in each quadrant were recorded over a 120 minute time period, at
intervals of 15 minutes. Assays were scored by means of a chemotaxis index (CI), as
originally described by Bargmann et al., 1993). The equation is as follows:CI=

(Number of organisms at attractant location - Number of organisms at control location)

Total number of organisms on assay plate

To avoid measurement bias, the assays were recorded blind, i.e. quadrants were
prepared and labelled (in code) independently of data collection.
3. Results & Discussion
3.1. Selection of Chemoattractant: Butanone Selected
Various compounds, both volatile and water-soluble, were screened for chemoattractant/
repellent properties. A strong attractant, which elicited consistent response was required
so as to enable clear and meaningful comparisons. Of those tested, the most effective
water-soluble attractant was found to be biotin, while butanone was the most effective
volatile attractant. Butanone was selected as the attractant henceforth, as the assays
reached completion earlier (~60min), thus allowing for the collection of more data in a
limited time frame. Biotin could be explored as an attractant in future experiments, to
observe whether differences exist in the regulation of gustatory compared to olfactory
response.
______________________________________________________________________________________
Figure 3. Chemotactic response of Wild
Type C. elegans, under normoxia. Values
of chemotaxis index obtained from
chemotaxis assays, as prepared in 2.2. Each
data point represents two independent
assays, error bars displaying standard error
of the mean. All volatile attractants used at
7l, 10% dilution. Water-soluble attractants
were presented in agar plug form, each
containing 100mM attractant.
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3.2. HIF Knockout Organisms Exhibit Reduced Chemotaxic Response after Hypoxia
It was necessary to evaluate the phenotypic differences between the two strains under
both normoxic and hypoxic conditions, in order to confirm the initial hypothesis made
regarding the relationship between HIF-1 activation and chemotactic response. As the
HIF-1 pathway is only expressed under hypoxia, there ought to be no significant
phenotypic difference between the two strains under normoxic conditions. This was
reflected in the obtained CI data, shown in Figure 4a. Under hypoxic conditions however,
HIF-1 and its related pathway are activated (WT strain only), resulting in a significant
difference in chemotactic response compared to the HIF-1 deficient strain (Figure 4b). This
confirms that the HIF-1 pathway, or rather its downstream products, play a positive role in
regulating chemosensory behaviour after hypoxia.

Joshua Hughes

a) Normoxic

b) Hypoxic

Figure 4. Chemotactic response of Wild Type and HIF mutant C. elegans, under normoxia and
hypoxia. Values of chemotaxis index obtained from chemotaxis assays, as prepared in 2.2. Each data point
represents three independent assays, error bars displaying standard error of the mean. Experimental groups
analysed through unpaired students t-test (two-tailed). a) Difference between the two strains under normoxia
found to be insignificant (p > 0.05). b) Difference between the two strains under hypoxia found to be
significant (p < 0.05).
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3.3. Evaluation of VPA and LiCl Treatment on Chemotactic Behaviour

VPA and LiCl were introduced to the agar media (NGM) in which nematodes were
maintained for 48 hours, at 6mM and 10mM concentrations respectively (see 2.1). These
concentrations have previously been observed to be pharmacologically relevant in prior
study, hence their selection in this instance. This does not mean to imply, however, that the
concentrations used were optimal in the context of chemotaxis/neuronal modulation. In
future study it would be of interest to optimise the concentrations used for maximal
pharmacological effect, via dose-response analysis. A similar response analysis could be
used with regard to exposure time. We ourselves compared a 48 hour exposure period, as
described in 2.1, with a very brief exposure (~30mins), whereby chemical agents were
simply introduced to M9 washing buffer.
Exposure for a 30 minute duration was observed to be ineffective. This was unsurprising
given that VPA and LiCl are both postulated to act (in part) at the level of gene expression,
through regulation of transcription factor activity. In contrast, 48 hour exposure would
conceivably be sufficient to allow regulation of gene expression to become phenotypically
apparent. Indeed, this duration was observed to elicit significant alterations in chemotaxic
response (dependent on strain), as shown in Figure 6 (below).
______________________________________________________________________________________

6a) VPA Exposure

6b) LiCl Exposure

Joshua Hughes

6c) Concomitant VPA & LiCl Exposure

Figure 6. Comparison between hypoxic and normoxic chemotactic response, w/wo 48 hour treatment
of VPA and/or LiCl. Nematodes were treated for 48 hours with 6mM VPA and/or 10mM LiCl, through
incorporation into NGM (see 2.1). Hypoxia (1% O2), where appropriate, was also maintained for 48 hours
prior to experimentation. Values of chemotaxis index obtained from chemotaxis assays, as prepared in 2.2.
Each data point represents three independent assays, error bars displaying standard error of the mean.
Each assay plate contained between 9 and 70 worms. Experimental groups analysed through 2-way
ANOVA with Dunnetts post-hoc test. Significance is denoted by the asterisk system, whereby: * = P0.05, **
= P0.01, *** = P0.001 and **** = P0.0001. Differences not denoted on the graph are insignificant. Error
bars display standard error of the mean (where n= 3).
______________________________________________________________________________________

Exposure of wild-type organisms to VPA/LiCl did not elicit any significant alterations in
chemotactic response. This was initially surprising, as preexisting evidence from in-vitro
models have suggested that lithium indirectly activates HIF-1, while VPA has been shown
to indirectly inhibit HIF-1. If this is indeed the case, the extent of HIF-1 regulation
induced by VPA and lithium (in the concentrations used) do not appear to be sufficient to
significantly alter the adaptive response it provides (as represented by the CI).
Alternatively, the phenotypic effect of HIF-1 regulation may be masked by other
nonspecific responses induced by VPA/LiCl. It must also be considered that the
mechanisms by which HIF-1 has been shown to be regulated by VPA/lithium may not be
conserved in C. elegans.
In contrast, exposure of hypoxic HIF-1-deficient organisms to VPA resulted in a
pronounced increase in chemotactic behaviour with respect to HIF-1 in absence of VPA,
restoring the chemotaxic index to a level insignificantly different to that of wild-type
organisms. A similar effect was elicited with LiCl exposure, as well as concomitant VPA
and LiCl exposure. As exposure of VPA or LiCl alone restores the chemotaxis index of
HIF-1 organisms to a level comparable to that of the WT group, it is likely that concomitant
exposure is redundant; having no additional adaptive benefit.
The results indicate that VPA and LiCl can compensate for the absence of HIF-1-mediated
hypoxic response, by inducing an independent mechanism of hypoxia adaptation. The two
compounds have been reported to participate in a complex network of interactions
(summarised in Fig. 7), not limited to those mediated by HIF-1.

Joshua Hughes

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Figure 7: Schematic illustration of biochemical pathways modulated by VPA and Li+. Diagram is limited
to pathways deemed relevant in the context of chemotaxis/neuronal survival. VPA activates HSP (Marinova
et al., 2009) and inhibits HIF-1 via HDAC inhibition (Qian et al., 2006), activates AP-1 (Chen et al., 1999) and
CREB via ERK pathway activation (Creson et al., 2009). Li+ activates HIF-1 (Kast, 2008) and activates AP-1
via GSK-3 inhibition (Yuan et al., 1998). Large black arrows represent overall transcriptional effect due to
lithium. Large grey arrows represent overall transcriptional effect due to VPA. Note: not all gene products are
shown.
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However, most of the interactions in Figure 7 have been observed in mammalian or in-vitro
models, and have not yet been substantiated in C. elegans, thus limiting their usefulness.
Due to the current paucity of literature detailing the role of GSK-3 and HDAC in C.
elegans, it is challenging to propose a definitive mechanism for their role in hypoxia
adaptation. Nevertheless, there have been a few recent studies which investigate the
effect of VPA and, to a lesser extent, lithium on C. elegans in other contexts (such as
lifespan), with findings that may be tentatively applied in this instance. It was recently
demonstrated that VPA promotes DAF-16 nuclear localisation (Evason et al., 2008), a
property which commonly indicates a decrease in activity of the daf-2 signaling pathway
(Henderson et al., 2001). The daf-2 pathway has been shown in an unrelated study to
potentiate hypoxic cell death, with daf-2-deficient mutants being profoundly hypoxiaresistant (Scott et al., 2003). However the mechanism by which daf-2 induces cell death is
unclear, and there have been no studies observing chemotaxis in organisms with
concomitant HIF-1 and daf-2 mutations. Lithium has no effect on DAF-16 nuclear
localisation (McColl et al., 2008), indicating that it acts via a different mechanism.
Beyond this, the evidence linking VPA/Li and C. elegans hypoxia adaptation becomes
more tenuous, necessitating the use of in-vitro data. An alternative mechanism which
could account for apoptotic inhibition is the induction of stress response gene products
e.g. HSP70, which are known to be transcriptionally activated by VPA and possibly lithium.
VPA has been demonstrated, in-vitro, to induce HSP70 up-regulation via inhibition of
HDAC-1, leading to histone H3 hyperacetylation in the HSP70 promoter (Zhang et al.,
2012). Acetylation neutralises positively charged histone lysine residues, attenuating their

Joshua Hughes

interaction between the negative phosphate groups of DNA. This relaxes the chromatin
structure to a form more conducive to DNA transcription (Grunstein, 1997). The
mechanism by which lithium may activate HSP70 expression is unclear, but is thought to
be mediated via GSK-3 inhibition (Bijur et al., 2008). HSP70 has multiple anti-apoptotic
mechanisms, a main candidate of which being inhibitory binding to apoptotic protease
activating factor 1 (APAF1, or CED-4 in C. elegans), preventing formation of the
apoptosome (Saleh et al., 2000). It also serves to activate the anti-apoptotic factor Bcl-2
(Jiang et al., 2011) and inhibit pro-apoptotic factors e.g. Bax (Li et al., 2010). In order to
ascertain the role of HSP70 induction in response to VPA/LiCl treatment, nematodes could
be cultured in the presence of a specific HSP70 inhibitor, such as the small-molecule 2phenylethyenesulfonamide (PES), in conjunction with VPA/Li treatments. The effect on
apoptosis could be quantified using an assay such as TUNEL (terminal deoxynucleotidyl
transferase dUTP nick end labeling), which detects endonucleolytic cleavage of chromatin,
a hallmark of apoptosis.
4. Conclusions & Future Work
In summary, it has been confirmed that HIF-1-knockout mutants exhibit decreased
chemotactic response following prolonged hypoxia, relative to wild-type organisms.
Furthermore, we demonstrate that treatment with VPA and/or LiCl is capable of
compensating for HIF-1 inactivity, through the induction of an independent adaptive
response. The mechanism by which this occurs is unclear, due to current absence of
literature regarding VPA/Li mediated regulation of C. elegans gene/protein expression.
These must be profiled (e.g. through microarray analysis) before a definitive mechanism
can be elucidated.
In future study, it would also be of benefit to age-synchronise the nematode cultures. This
is because chemotactic response varies with developmental stage, young adults exhibiting
a significantly stronger response than infants (Matsuura et al., 2007). Care must be taken
when using non-synchronised cultures in assays, in order to discount infants which may
ultimately skew the data.

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Joshua Hughes

6. Appendix
C. Elegans Maintainance Agar (NGM) Formulation
16g Bacto-agar
3.0g NaCl
2.5 Bacto-peptone
In 1L dH2O
~AUTOCLAVE~
1ml 1M CaCl2
1ml 1M MgSO4
1ml Cholesterol
25ml 1M KPi
Lysogeny Broth (LB) Formulation
10g Bacto-tryptone 15g Agar
5g Bacto-yeast 1L dH2O
5g NaCl
Chemotaxis Agar Formulation
20g Agar
1mM CaCl2
1mM MgSO4
5mM KPO4
1L dH2O
M9 Buffer Formulation
3g 1M K2HPO4
3g 1M Na2PO4
5g NaCl
1ml MgSO4
1L milliQ H2O