The role of the CBS/H2S pathway in the brain during

hibernation

Report written by Jojanneke Bruintjes (s1765671) as part of her Science Rotation of the

Master study in Medicine under supervision of prof. dr. R.H. Henning, pharmacologist and

prof.dr. E.A. van der Zee, neuro- and behavioural scientist at the University of Groningen and

University Medical Center Groningen (UMCG).

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Summary

Small endothermic mammals use hibernation as a survival strategy in periods with

unreliable food availability and low ambient temperature. Hibernation consists of torpor

phases, during which metabolism is severely depressed and body temperature (Tb) extremely

reduced. The torpor phases are alternated with brief periods of restoration of metabolism and

euthermia, called arousals. Remarkably, hibernating animals do not develop organ damage

during the repetitive cooling-rewarming cycles. The CBS/H2S pathway is coined as one of

the underlying mechanisms, as H2S protects cells from damage and can also induce artificial

torpor (also known as "suspended animation"). Because of lack of information about the

regulation of the CBS/H2S pathway in the brain, this is an interesting subject to study,

particularly in face of the possible protective effects of H2S.

To this end, brain CBS expression was studied in natural hibernators (Syrian hamsters and

Djungarian hamsters) and in pharmacologically induced torpor (by 5' adenosine

monophosphate, in mice). Syrian hamsters were sacrificed in different stages of natural

hibernation (euthermic, torpor early, torpor late and arousal late). The Djungarian hamsters

and the mice were sacrificed in either torpor or euthermic conditions. CBS expression was

studied by immunohistochemistry (IHC) and western blot (WB). In all animal species a

distinctive expression pattern of CBS was found, mainly in the (pre-frontal) cortex,

hippocampus and thalamus. In Syrian hamster IHC stainings showed increased expression

levels of CBS in the retrosplenial cortex of arousal late animals, compared to animals in other

stages of hibernation. No distinctive differences in expression levels or expression patterns of

CBS were found in the Djungarian hamsters or in the mice.

From these data we conclude:

- CBS has a distinctive and similar distribution pattern in brain of both hamster species

and in mouse brain. Expression was mainly found in the (pre-frontal) cortex,

hippocampus and thalamus.

- There is a clear difference between regulation in the arousal phase in natural hibernation

and in 5'AMP induced "hibernation". This particularly concerns the retrosplenial cortex.

The main role of the retrosplenial cortex is spatial navigation and episodic memory. The

upregulation of CBS in this area during the arousal late phase can implicate higher necessity

for its product, H2S. H2S can function as a protective agent, making sure the ability of these

animals to navigate through the surroundings after arousal is maintained. Alternatively, H2S

may act as a gasotransmitter, again helping arousing animals navigate through their

environment to find the nearest food source.

Kleinere endotherme zoogdieren gebruiken winterslaap als een overlevingsstrategie in

periodes met onbetrouwbare beschikbaarheid van voedsel en een lage omgevingstemperatuur.

Winterslaap bestaat onder andere uit torpor fases, waarin de lichaamstemperatuur en het

metabolisme sterk dalen. Deze torpor fases worden onderbroken door korte periodes van

herstel van het metabolisme en een eutherme lichaamstemperatuur, de arousal fases. Tijdens

de herhaaldelijke koeling-opwarming cycli die de winterslapende dieren doorlopen,

ontwikkelen ze opmerkelijk genoeg geen orgaan schade. De CBS/H2S pathway is mogelijk

een van de onderliggende beschermende mechanismes. Het gebrek aan informatie over de

regulatie van deze pathway in het brein, maakt het een interessant onderwerp om te

bestuderen, vooral vanwege de mogelijke beschermende effecten van H2S.

Om dit te onderzoeken hebben we gekeken naar CBS expressie in het brein van natuurlijke

winterslapers (Syrische hamsters en Russische dwerghamsters) en muizen in farmacologisch

geïnduceerde (door 5' adenosine monofosfaat) torpor. De Syrische hamsters werden

opgeofferd in verschillende winterslaap fases (eutherm, torpor early, torpor late, arousal late),

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de Djungarian hamsters en de muizen in torpor of eutherme condities. Expressie van CBS

werd bestudeerd met behulp van immunohistochemie (IHC) of western blot (WB). In alle

diersoorten werd een specifiek expressie patroon van CBS gevonden, met de voornaamste

expressie in de (pre-frontale) cortex, de hippocampus en de thalamus. In de Syrische hamsters

werd een verhoogde expressie gevonden in de retrospleniale cortex van dieren in de arousal

late fase, in tegenstelling tot dieren in andere hibernatie fases. In de Djungarian hamsters en in

de muizen werden geen duidelijke verschillen gevonden in expressie patroon of expressie

sterkte van CBS.

Uit bovenstaande data kunnen we het volgende concluderen:

- CBS heeft een specifieke en gelijke distributie van expressie in de breinen van zowel

hamsters als muizen. De expressie wordt voornamelijk gevonden in de (pre-frontale)

cortex, hippocampus en thalamus.

- Er is een duidelijk verschil tussen de regulatie van CBS in de arousal fase in natuurlijke

en 5'AMP geïnduceerde winterslaap. Hierbij gaat het specifiek om de retrospleniale

cortex.

De hoofdzakelijke rol van de retrospleniale cortex is ruimtelijke navigatie en episodisch

geheugen. De hogere expressie van CBS in dit gebied tijdens de arousal fase, kan impliceren

dat er een grotere hoeveelheid van het product van CBS, H2S, nodig is. H2S kan functioneren

als een beschermer, waardoor de mogelijkheid van deze dieren om zich te navigeren door hun

directe omgeving gewaarborgd blijft. Een andere mogelijkheid is dat H2S functioneert als

gasotransmitter en daarmee de ruimtelijke navigatie ondersteund, een hersenfunctie die

essentieel kan zijn bij het zoeken naar voedsel na het ontwaken uit de winterslaap.

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Contents list

Summary 2

1. Introduction 5

2. Methods 8

3. Results 12

4. Discussion 20

5. Conclusion 23

Acknowledgements 23

References 24

Supplements 26

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1. Introduction

Mammalian hibernation

Endothermic mammals are able to maintain a constant body temperature (Tb) during

fluctuating ambient temperatures. Small endotherms have a higher surface area/volume ratio,

which means they lose heat more easily during cold exposure than large endotherms. The

small endotherms thus have to compensate with high metabolic heat production, which

requires high food intake. Hibernation provides small mammals with the most optimal

survival strategy during seasons wherein food availability is unreliable [1].

Small mammals use two different hibernation strategies: deep and daily hibernation. Deep

hibernation is found in animals such as the Syrian hamster (Mesocricetus auratus), the 13-

lined ground squirrel (Ictidomys tridecemlineatus) and the arctic ground squirrel (Urocitellus

parryii) [1]. Deep hibernation consists of prolonged torpor bouts, where metabolism is

severely depressed and Tb extremely reduced, which are alternated with brief periods of

euthermia, called arousals. In deep torpor, -

C, and lasting for 6-40 days. The arousals usually do not last longer than

24h [2]. Daily torpor is found in animals such as the Djungarian hamster (Phodopus

sungorus) and in many bird species. Mammals that use the daily torpor hibernation pattern do

not have the deep, prolonged torpor as the deep hibernators do. Instead, their torpor bout only

lasts a few hours and is alternated by shorter arousals than in deep hibernation, wherein the

animals forage and feed [1].

The changes in physiology during hibernation may be used to our advantage in clinical

settings. One example of these changes is the regulation of the immune system during

hibernation. Hibernation affects the innate as well as the adaptive immune system. The main

effect is a reduction in numbers of leukocytes [2]. The influence of hibernation on the

immune system might be beneficial by prevention of tissue destruction because of

suppression of the inflammatory responses. Another example of physiological change during

hibernation is the lowering of cardiac output, which can be useful e.g. for patients who are at

risk for hypovolemic shock due to heavy bleeding. The resistance to ischemia/reperfusion and

hypothermia/rewarming injury is another interesting physiological property of hibernators,

one that could be used to prevent these types of damage in patients [3]. Should the molecular

mechanisms of hibernation be disclosed, induction of a state of suspended animation,

resembling the physiology of natural torpor, or usage of the protective cellular mechanisms of

hibernation, may be used to reduce tissue injury in patients. This may eventually lead to a

reduction in the incidence of organ injury and optimize the outcome following surgery,

trauma or transplantation [4].

There are two promising compounds that can be used to induce suspended animation, 5'-

adenosine monophosphate (5'AMP), a metabolite of the hydrolyses of ATP, and hydrogen

sulfide (H2S), a toxic gas. There are two hypotheses about the underlying mechanisms of

5'AMP induced torpor. The first attributes its effects to the dephosphorylation of 5'AMP to

adenosine, which in turn activates adenosine receptors and thereby reduces cardiac output and

Tb. The second hypothesis states that 5'AMP uptake activates AMP-activated protein kinase

(AMPK), which plays a major role in the regulation of metabolism [5]. H2S induces torpor by

a reversible reduction in metabolism through inhibition of oxidative phosphorylation complex

IV (cytochrome c oxidase), leading to a decrease in cellular oxygen consumption. Indeed,

H2S is highly toxic in concentrations >600 ppm, however in lower concentrations it has been

described to have cytoprotective effects [6]. When mammals are exposed to moderate

amounts (80 ppm) of H2S, a reversible state of hypothermia is induced. In addition to the

effect of H2S on mitochondrial function, H2S has been suggested to activate anti-apoptotic

signaling pathways and anti-oxidant mechanisms [5].

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Gaseous H2S may be difficult to use clinically due to its toxicity at high concentrations, e.g.

locally in the long, and its foul smell. H2S donor compounds such as Na2S and NaHS have

been used, but have a major disadvantage in the form of a short half life and unpredictable

pharmacokinetics. To sustain physiological levels of H2S slow-releasing H2S donor

compounds, among others ACS48, have been developed. Very recently a novel H2S-releasing

NMDA receptor antagonist has proven to prevent ischemic neuronal death [7].

Endogenously produced H2S

As described before, H2S has an effect on mitochondrial function, resulting in suspended

animation, and can activate anti-apoptotic signaling pathways and anti-oxidant mechanisms.

Therefore it is interesting to examine the exact role of H2S in hibernation and its function in

anti-apoptotic pathways and anti-oxidant mechanisms.

The low concentrations wherein H2S seems to have physiological functions can be found in

several animals, where H2S is endogenously produced. H2S functions as a gaseous

neurotransmitter, which is synthesized in mammals from the sulfide containing amino acid L-

cysteine by the enzymes cysthathione betasynthase (CBS) and cysthathione gamma lyase

(CSE), using pyridoxal 5'phosphate (vitamin B6) as a cofactor [8].

The protective effects of hibernation, specifically H2S related, make for a new subfield of

research. Other than our finding that H2S is implicated in lung remodeling during hibernation

[9], information is lacking on its involvement in other organs or the immune system and on

regulation in hibernation. As is described below, H2S seems to have an important function in

the brain, but information is scarce. Thus, it is interesting to examine the exact role of H2S in

the hibernating brain.

The CBS/H2S pathway and the brain

CBS is the main contributor to the production of H2S in the brain. Recently another H2S

producing enzyme was found in the brain: 3 mercaptopyruvate sulfurtransferase (3MST). It

produces H2S from 3-mercaptopyruvate, which is produced by cysteine aminotransferase

(CAT), from cysteine and α-ketoglutarate. The H2S production is mainly supported by CBS

due to its function as catalyst of the β-replacement reaction of cysteine. Cysteine is an amino

acid, produced by hydrolysis of cystathionine, catalyzed by CSE. Cystathionine is a thiol

ether produced by condensation of homocysteine with serine, executed by CBS [10].

Dysregulation of CBS can be linked to different diseases, which all have mental retardation

as a similar symptom. CBS deficiency leads to homocysteinuria, a autosomal recessive

metabolic disorder caused by the a mutation in the gene that encodes CBS. Homocysteinuria

is mainly characterized by mental retardation, seizures, ectopia lentis, skeletal deformities and

occlusive vascular disease. The disease was first detected in 1962 by screening patients with

mental retardation. Another disease which links mental retardation to a disturbance in CBS

regulation is Down syndrome (DS). DS is mainly caused by a trisomy of chromosome 21, the

chromosome on which the CBS gene is located. Almost all adults with DS develop

Alzheimer's disease (AD), and this development is usually at a much younger age than in non-

DS adults. Levels of CBS in brains of DS patients are approximately three times higher than

those in healthy individuals. Thus, strangely, both diseases featuring mental retardation

appear to root in an opposite dysregulation of CBS, which likely underscores the importance

of a proper CBS balance in the brain [11,12]. However, the exact role of CBS in the mental

retardation in homocysteinuria and DS and in the development of AD in DS patients remains

unclear [13].

Information on the expression of CBS in the brain is lacking. Only a few research groups

have studied the expression of CBS, and their results are conflicting. Enokido et al. found

CBS to be preferentially expressed in the glia/astrocyte lineage, whereas Robert et al. found

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CBS to be solely localized to neurons. Linden et al. found CBS expression in many areas of

the brain but are not clear about the cell type. They state that "some cells immunoreactive for

CBS had the stellate morphology of astrocytes or oligodendrocytes", which is somewhat

vague and does not help to identify the exact location of CBS in the brain. Different

antibodies for CBS were used amongst these research groups and no one has repeated their

studies. For now we can conclude that there is no certainty about the expression and

localization of CBS in the brain [14,15,16].

Functions of H2S in the brain

Besides the lack of information about the expression of CBS in the brain, there is even less

knowledge about CBS in the hibernating brain. Previous studies in lung, kidney and smooth

muscle cells indicate that the CBS/H2S pathway plays an important role in hibernation and in

the protection of organs from ischemia/reperfusion or hypothermia/rewarming damage

[17,9,18]. It is interesting to examine if this pathway also plays an important role in

hibernators' brains, possibly by functioning as a brain protector.

Multiple research teams have shown that H2S protects neurons against hypoxic injury and

ischemia [19,20,21]. One of the reasons might be that H2S has an antioxidant effect, either

directly or indirectly by increasing the production of reduced glutathione. H2S can also

protect against LPS-induced inflammation. Neuro-inflammation is involved in almost all

major neuropathological states, such as AD, Parkinson's disease (PD) and Huntington's

disease (HD) [22] Interestingly, a protective effect of H2S against amlyoid-β induced cell

toxicity was found in murine microglial cells, which supports the possible link with AD [23].

Another study confirms this possibility, by proving that H2S attenuates spatial memory

impairment and neuro-inflammation in a beta-amyloid rat model of AD. In addition, an

interaction between CBS and huntingtin, the gene that is mutated in HD, was found [24].

Thus, there is compelling evidence that H2S may govern protective effects against

neurodegeneration. The protective effect of H2S against hypothermia/rewarming damage in

the brain however, has not been studied yet.

H2S has also been found to modulate physiological brain functions, in addition to its

attenuation of cell damage. The previously found roles of H2S in the brain include facilitation

of long term potentiation (LTP) and regulation of neurotransmission and calcium

homeostasis. The facilitation of long term potentiation (LTP) in the hippocampus is

accomplished by selectively stimulating NMDA receptor-mediated currents [25]. The

underlying mechanism of this potentiation of the NMDA receptor function remains unknown.

A more recently disclosed function of H2S is the upregulation of the γ-aminobutyric acid

(GABA) B receptor. GABA is the main inhibitory neurotransmitter in the brain and

stimulation of the post-synaptic receptors can help regulate inhibitory neurotransmission.

Thus, H2S may play a role in the maintenance of the excitatory/inhibitory neurotransmission

balance in the brain. H2S as a regulator of calcium levels was found in astrocytes and

microglia. Astrocytes communicate with each other via calcium signaling, in contrast to the

action potential signaling used in neurons, which possibly leads to modulation of neuronal

and vascular function. Microglial brain cells have a similar function as macrophages, both cell

types can be activated upon foreign challenge. Interestingly, they supposedly play a role in the

progression of AD and PD [26].

When contemplating neurodegenerative diseases, ageing is a word that comes to mind.

Healthy ageing has become a popular topic in the past decade and prevention of neuro-

degeneration is considered to be quite important. There is not much known about the effect of

CBS on ageing. A very recent article already states that H2S can possibly help prevent

physiological ageing and age-associated diseases, by its antioxidant function and the influence

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on two age-related genes, sir2 (silent information regulator 2) and Klotho [27]. However, this

link is still highly hypothetical and not much is known about the exact mechanisms.

Together, the protection and modulation of brain functions by H2S strongly support the idea

that CBS represents an important enzyme in the brain and provide a link between the

CBS/H2S pathway, neuro-inflammation, neuropathology and ageing.

Current study

The overall shortage of information about the specifics of the CBS/H2S pathway in the brain

provides us with a very interesting question that has yet to be answered: what is the exact role

of CBS in the brain? This question, however, is one that needs to be answered by doing more

research than can be done in 20 weeks. Thus, we have focused on the role of CBS in the

hibernating brain. Other pilot studies have been performed, but are not included in this report.

These pilot studies focused more on ageing and neurodegenerative diseases such as AD. Extra

results can be found on the included CD. To study the role of CBS in the hibernating brain we

determined CBS expression in brain tissues of Syrian hamsters (deep hibernator) and

Djungarian hamsters (daily hibernator) during natural hibernation. In addition to natural

hibernation, we also examined CBS expression in 5'AMP induced artifical hibernation in

mice. To study the protective effects of the CBS/H2S pathway, neuroblastoma 2A (N2A) cells

were subjected to a cooling-rewarming cycle with addition of an agent which maintains (high)

H2S levels (Sul-121).

2. Methods

Tissue processing of samples

Brain samples from different experiments were used to analyze the expression of CBS .

Syrian hamsters (Mesocricetus auratus) (n=111) were obtained from our former breeding

colony (Zoological laboratory, Haren, the Netherlands), where they were raised under

summer conditions (14h:10h light:dark at 21±1°C). The hamsters were housed in Macrolon

type 3 cages on sawdust bedding, with ad libitum water, food and hay as nesting material.

Entry into hibernation was induced by transferring the animals to a climate-controlled room.

First, the climate conditions were changes to a short-day photoperiod (8h:16h light:dark at

21±1°C) and after six to eight weeks the conditions changed to a temperature of 5±1°C with

continuous dim red light (<0.5lux). These conditions triggered the majority of the animals to

go into hibernation.

Hamsters were sacrificed at different time points and thereby divided into different groups:

summer euthermic animals (n=13), animals in entry to torpor with either Tmouth > 30°C (n=6)

or Tmouth < 30°C (n=22), animals which were 24-30h in torpor ("torpor early" or "torpor

middle", n=3) or >84h in torpor ("torpor late", n=15), animals who where rewarming from

torpor with either Tmouth < 30°C (n=23) or Tmouth > 30°C (n=11) and animals with euthermic

body temperature which where >8.5h after induction of arousal ("arousal late, n=18).

Sacrification was executed by an overdose of intraperitoneally (i.p.) injected 6% Sodium

Pentobarbital. Brains from a part of the hamsters (n=83) were collected and immediately

frozen on dry ice and stored at -80°C for further protein analyses by western blotting. The

other animals (n=23) were transcardially perfused with 0.1M phosphate buffered saline (PBS)

followed by 4% paraformaldehyde (PFA) in 0.1M PBS. The brains were dissected, post-fixed

in PFA for 24h at 5°C and stored in 0.1M PBS, containing azide, at 5°C. For cutting slices the

brains were cryoprotected with a 30% sucrose solution and cut by cryostat (25µM) to be

stored in PBSA at 4°C. The brains of transcardially perfused hamsters were used for cutting

slices.

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Djungarian hamsters were obtained from the breeding colony at the Zoological Institute,

University of Veterinary Medicine, Hannover, Germany. The animals were exposed to natural

photoperiod and temperature. Daily torpor was confirmed by regular inspection of the

animals. Daily torpor started around 07:00 hours and ended around 16:00 hours.

Hamsters were sacrificed at two different timepoints and thereby divided into two groups:

14:02 hours (+/- 19 min), the torpor group, and 22:08 hours (+/- 13min), the euthermic group.

Four animals of both groups were transcardially perfused with PBS, followed by 4% PFA in

0.1M PBS at 6°C. Brains were stored in PBS with 1% sodium-azide at 6°C, followed by

cryoprotection and cutting of the brains by cryostat (20µM), to be stored again in PBS with

1% sodium azide.

Male C57bl/6J mice (mus musculus) (n=28) were purchased from Harlan (Horst, the

Netherlands) at an age of 4 weeks, to be used for the 5'AMP induced hibernation experiments.

Prior to the experiment animals were kept under a light:dark cycle of 12h:12h at an ambient

temperature of 21±1°C. The mice were housed individually on sawdust bedding with ad

libitum water, food an nesting material. Torpor-like hypothermia was induced by an i.p.

injection of 7.5µmol/g adenosine 5'-monophosphate disodium salt, obtained from Sigma

Aldrich (Sigma, St Louis, USA) and dissolved in saline (with a final concentration of 0.75M).

The injection was given approximately 1h after lights-on (inactive phase) and resulted in

torpor bouts of approximately 4h at am ambient temperature of 21±1°C.

Animals were sacrificed during torpor-like hypothermia at an ambient temperature of

21±1°C ("torpor", n=11) or during euthermia following torpor-like hypothermia ("euthermic",

n=11). The animals in the torpor group were sacrificed on average 240 min (SD 6.9) after the

5'AMP injection. The animals in the euthermic group were sacrificed on average 576 min (SD

13.2) after the 5'AMP injection, these animals had been rewarming for 112 min (SD 47.8).

The mice were sacrificed by an overdose of 6% Sodium Pentobarbital. For each groups six

brains were immediately frozen on dry ace after collection and stored at -80°C for further

protein analyses by western blotting. Five animals of each group were transcardially perfused

with 0.1M PBS, followed by 4% PFA in 0.1M PBS. After dissection, brains were post-fixed

in PFA for 22h at 4°C and stored in 0.1M PBS, containing sodium-azide, at 4°C. For cutting

slices the brains were cryoprotected with a 30% sucrose solution and cut by cryostat (25µM)

to be stored in PBSA at 4°C. The brains of transcardially perfused hamsters were used for

cutting slices.

Young C57bl/6J mice (mus musculus) were purchased from Harlan (Horst, the Netherlands)

at an age of 2 months. Middle-aged C57bl/6J mice were purchased from Harlan at an age of

22 months. The young and middle-aged mice were individually housed on sawdust bedding

with ad libitum water, food and nesting material. The young mice were sacrificed at an age of

4 months, the middle-aged mice at an age of 28 months The sacrification protocol was similar

to that of the C57bl/6J mice used for the 5'AMP experiment, as described above.

For the Alzheimer model transgenic mice (mus musculus) with a C57BL/6J background

over-expressing the haPP695swe and presenilin-1M146V mutation, generated at

GlaxoSmithKline (Harlow, UK), were used. All animals had access to ad libitum rodent chow

(Harlan Teklad), water, nesting material and a play tube. The ambient temperature was

maintained at 21±1°C under a controlled light-dark photocycle (12:12 h; lights on 07:00).

The mice were randomly designed to 5 age groups (3 months, 8 months, 13 months, 18

months and 21 months). Two BrdU injections were given and mice were sacrificed with a

pentobarbital overdose either 2 hours (per age group: n=7, n=7, n=8, n=10, n=8) or 28 days

(per age group: n=12, n=12, n=16, n=16, n=18) after the second injection. After sacrification

a transcardial perfusion with 4% PFA was performed, followed by removing and post-fixating

the brains to be stored in 0.1% sodium-azide PBS at 4°C. Brains were then cut with cryostat

to be used for immunohistochemistry.

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All protocols and experimental procedures were approved by the Animal Ethical

Committees.

Immunohistochemistry pilots (protocol optimization)

Due to absence of a protocol for CBS staining on brain slices, one had to be developed. A

basic staining protocol for free floating brain slices was used as a starter. This protocol

consisted of washing the slices with 0.01M phosphate buffered saline (PBS), followed by

blocking of endogenous peroxidase by incubation with 0.3% H2O2 for 30 minutes at room

temperature, followed by a second washing. The primary antibody was diluted 1:300 in a

0.1% Triton X, 3% bovine albumin serum (BSA) 0.01M PBS solution. After incubation with

the primary antibody for 4 days at 4°C, the slices were washed with 0.01M PBS and

incubated with the secondary antibody, diluted 1:500 in 0.01M PBS, for 2 hours at room

temperature. Following the secondary antibody and washing again with 0.01M PBS, the slices

were incubated with Avidin Biotin complex, diluted 1:500 in 0.01M PBS, for 2 hours at room

temperature. Again the brain slices were washed with 0.01M PBS, followed by adding

3,3'diaminobenzine (DAB) solution, which was activated by adding 100µL 0.1% H2O2. After

activation the samples had to be shaken for a maximum of 1 minute to help with the activation

and equalizing of the staining. After using this protocol to stain the slices, slices could be

fixated on glass slides with a 1% gelatin solution with 50mg kaliumchrom(III) sulfate (Merck,

Germany) per 100ml. This was followed by the following set of treatments: 2x5 minutes in

100% alcohol, 5 minutes in a solution of 70% alcohol and 30% xylol, 5 minutes in a solution

of 30% alcohol and 70% xylol, 3x5 minutes in 100% xylol. The slides could then be covered

with a cover slide using a DPX mountant.

The basic staining protocol was used for a pilot staining where Syrian hamster brain slices

from animals in euthermic (n=1), torpor late (n=2) and arousal late (n=1) condition together

with 5'AMP induced hibernating mice in torpor (n=1) and euthermic (n=1) condition were

stained.

In the second pilot eleven grains of ammonium nickel sulfate were added to the DAB

solution. This was used to stain Syrian hamster brain slices from animals in euthermic (n=1),

torpor early (n=1), torpor late (n=1) and arousal late (n=1) conditions as well as animals in the

cooling (n=1) and rewarming (n=1) period between hibernating stages.

The amount of nickel ammonium sulfate grains was reduced in the third pilot from eleven to

six. Three different dilutions; 1:200, 1:300, 1:400 were used to stain hamster brain slices from

hamsters in the rewarming period (n=1 per dilution) and torpor late phase (n=1 per dilution).

The incubation of the primary antibody was shortened in the fourth pilot from four days at

4°C to one day at 4°C. The amount of nickel ammonium sulfate remained the same. DAB

time was increased to 30 minutes instead of 5 minutes and 200µl instead of 100µl of 0.1%

H2O2 was used to activate the DAB staining. This protocol was used to stain hamster slices

from hamsters in the rewarming period (n=1) and torpor late phase (n=1).

The primary antibody was incubated 2.5 hours at 37°C before incubating 4 days at 4°C in

the fifth pilot. The incubation time for the secondary antibody was increased from 2 hours to 4

hours. This last pilot was used to stain hamster brain slices from hamsters in the euthermic

(n=1), torpor early (n=1), torpor late (n=1) and arousal phase (n=1) as well as the cooling

(n=1) and rewarming (n=1) period.

The used antibodies were CBS (G-1) sc-271886 (Santa Cruz Biotechnology) and Biotin-SP-

conjugated AffiniPure Goat-Anti-Mouse IgG (H+L) (Jackson ImmunoResearch).

Immunohistochemistry sets

After optimization of immunohistochemical staining of CBS on brain slices, slices from

different experiments were processed. The first set consisted of Syrian hamsters in different

11

hibernation stages; summer euthermic (n=2), early torpor (n=2), late torpor (n=3) and late

arousal (n=3). The brains of the other transcardially perfused animals (n=13) were not used

for this study, as these animals were not in a specific hibernation phase. The second set

consisted of C57bl/6J mice where hibernation was induced by 5'AMP (n=5) and euthermic

(n=5) animals. The third set consisted of Djungarian hamsters in torpid (n=4) or euthermic

(n=4) phases. Measurements of optical densities were executed with a Leica DM IRB

microscope system and Leica Qwin Pro software.

Another set that was executed but is not included in this report, is a set of young (n=6)

versus middle-aged (n=6) mice. In a last pilot staining, slices from AD model mice in the age

groups 3 months (n=2) and 8 months (n=2) were stained. This ageing and neuro-degeneration

part of the study is still going on.

Westernblot

Western blotting was used to quantify the expression of CBS. Lysates from cortex samples

of Syrian hamsters (5mg protein per ml) were received from previous experiments and used

for a westernblot pilot. For each received lysate, containing loading buffer and protein, 25µl

was loaded onto pre-made gradient gels 4-20% (Thermoscientific, Waltham MA, USA) for

electrophoresis at 100V (80min). Following electrophoresis, the gels were blotted on

nitrocellulose membranes. To check if blotting had been executed correctly, proteins were

detected with panceau. The nitrocellulose membranes were incubated with a solution of the

primary antibody, diluted 1:100 in TBS + Tween 20 with 3% BSA. Membranes were

developed using super signal West Dura Substrate (Thermoscientific), by incubating the

membrane with the solution for 3 minute in the dark. Images were acquired with GeneSnap

(version 6.0.7., Syngene, Cambridge, UK) and analysed using GeneTools (version 3.08,

Syngene).

The used antibodies were CBS (G-1) sc-271886 (Santa Cruz Biotechnology) and Polyclonal

Rabbit Anti Mouse Ig/HRP P0260 (DAKO).

Cell culture

Neuroblastoma 2A cells (N2A) were a kind gift from Dr. Jan-Willem Kok (UMCG). N2A is

a mouse neural-crest derived cell line, mainly used for studying neuronal differentiation,

axonal growth and signaling pathways. Cells were maintained at 37°C in Dulbecco's modified

Eagly Medium (DMEM) with high glucose, L-glutamine, Phenol Red and Sodium Pyruvate

(41966-029, Gibco). Medium was supplemented with 10% fetal calf serum (FCS) and 1%

penicillin/streptomycin (PS).

The N2A cell line was new to our department, which means the first step was to analyze the

characteristics of this cell line, mainly the growth speed, for logistical reasons. 10000

cells/cm2 were added to 10 25cm

2 flasks and the cells were counted by using a Bürker

chamber, each time in 2 flasks, on day 1,2,3,4 and 7. The average cell count on each day was

used to determine a growth curve.

The cells were then subjected to a cooling-rewarming cycle to see whether, when and to

what extend damage occurs. Cells were cooled for 24 hours at 4°C and then rewarmed for

either 2h or 2.5 hours at 37°C

After determining the point of damage occurrence, cells were again subjected to a cooling-

rewarming cycle of 24h cooling and 2.5h of rewarming, but with treatment of with Sul-121

directly before cooling or 1h before rewarming. Sul-121 is a new, experimental compound

from Sulfateq, which is supposed to maintain or increase H2S in cells. We added Sul-121 to

examine if the compound protects against hypothermia/rewarming damage by increasing H2S

production. As Sul-121 is dissolved in dimethyl sulfoxide (DMSO), cells were also treated

with DMSO alone, at the same time points as Sul-121, to discriminate between the effect of

12

Sul-121 and the effect of DMSO. For both compounds a dilution of 1.10-5

was used. Control

cells were untreated but were subjected to the cooling-rewarming cycle.

Statistical analysis

Statistical data analyses were performed using the one-way ANOVA. Values of P < 0.05

were considered statistically significant.

3. Results

Immunohistochemistry pilots (protocol optimization)

For a total overview of pictures from every executed staining see included CD.

The goal of the pilot stainings was to obtain a proper CBS staining. Criteria that needed to

be met were: an equal staining result throughout the slices (no dark or light patches within one

slice), a clear specific result when compared to the negative control and a good representation

of the structures within the brain (neuron cell bodies, dendrites, molecular vs. granular layers

etcetera). The first pilot showed specific staining in different areas such as the hippocampus,

the cortex and the retrosplenial cortex. The staining itself wasn’t completely uniform. The

slices appeared to have darker areas, mostly in a stripe pattern, that were most likely caused

by an unequal spreading of the antibodies and/or staining solution. To make the staining result

from the second pilot more uniform, extra attention was paid to shaking the samples in the

first 30-40 seconds of the DAB activation. Nickel ammonium sulfate grains were added to

increase contrast and visibility of the specific staining in the found areas.

In the second pilot the uniformity of the staining was clearly increased. However, the

specific staining shown was not evaluated as the most optimal. Because the start dilution of

1:300 was from a basic protocol and not completely based on the antibody and protein,

different dilutions were used in the third pilot. The amount of nickel ammonium sulfate was

reduced to avoid extremely dark coloring in the stronger dilution.

The results of the third pilot showed a clear difference amongst the different dilutions (Fig.1,

panel B,C and D). The primary antibody worked best when diluted 1:200. The neuronal cell

bodies were stained nicely and especially the dendrites were very clear.

For logistical reasons the incubation time of the primary antibody was reduced from 4 days

to 1 day in the fourth pilot (Fig.1, panel A). However, the shorter incubation time resulted in

the loss of the nicely represented brain structures. Neuronal cells were not clearly visible and

dendrites could not be found.

To explore if the specific result could be even more improved than the good result of the

third pilot, the primary antibody was incubated 2.5 hours at 37°C before incubating 4 days at

4°C and the secondary antibody was incubated for 4 hours instead of 2 hours. The specific

staining was indeed stronger, but so was the background (Fig.2). The ratio specific

staining/background was the same. Because of the strong background caused by this protocol

option, this was not the preferable protocol.

Taken together, the optimal protocol was the protocol achieved by using the basic IHC

staining protocol with a change in the primary antibody dilution from 1:300 to 1:200. The

differences between the used protocols for pilot stainings can be seen in the figure below (Fig.

1), where the result seen in panel D is clearly the result from the most optimal protocol. This

can be concluded when looking at the specificity of staining, together with the good

representation of brain structures and an equal spreading of the staining throughout the slices.

During the optimization process specific staining was found in many different brain areas,

such as the hippocampus, cortex and the retrosplenial cortex. These areas were then compared

in the total sets.

13

Figure 1. Differences in the protocol clearly give a difference in the result of the IHC. Panel A: basic protocol, primary

antibody incubated for 1 day ; panel B: basic protocol, primary antibody diluted 1:400; panel C: basic protocol; panel D:

basic protocol, primary antibody diluted 1:200. Pictures are of the hippocampus and were made at a magnification of 2000x

Figure 2.

C for four days increased the intensity of the specific staining, as well as the intensity of the background. Panel A: Syrian

hamster in torpor late phase; panel B: Syrian hamster in the rewarming period; panel C: young mouse; panel D: middle-aged

mouse. Pictures are of the hippocampus and were made at a magnification of 1000x

14

Immunohistochemistry, CBS localization

To examine whether CBS is as omnipresent in the brain as it seems to be, as suggested by

the pilot stainings, both brain stem and prefrontal cortex were included in the analysis, in

addition to the cortex, hippocampus, hypothalamus, thalamus and retrosplenial cortex. In

Syrian hamsters, Djungarian hamsters and 5'AMP mice, specific staining was found in the

prefrontal cortex (primary motor cortex and cingulate cortex), cortex, retrosplenial cortex and

hippocampus. CBS was located in the cytosol of neuronal cell bodies and in their dendrites

(Fig.1, panel D). The retrosplenial cortex of arousal late phase Syrian hamsters showed a

different staining pattern than the retrosplenial cortex of animals in other phases of

hibernation (Fig.3). This pattern seems to be consistent with the staining of interneurons. This

specific staining pattern was absent in the retrosplenial cortices of (aroused) Djungarian

hamsters and 5'AMP mice. Other areas such as the thalamus and secondary motor cortex also

showed some specific staining in all animals, though not enough to use these areas for

comparison. No specific staining could be found in the brain stem.

The areas and the expression pattern within these areas were similar between Syrian

hamsters, Djungarian hamsters and 5'AMP mice in all conditions, with the exception of the

retrosplenial cortex in arousal late phase Syrian hamsters. CBS seems to be constitutively

present in multiple brain areas.

Figure 3. The staining pattern in the retrosplenial cortex of the animals in the arousal late phase differs from the staining

pattern in the retrosplenial cortex of animals in other hibernation phases. Panel A: summer euthermic; panel B: torpor early;

panel C: torpor late; panel D: arousal late. Pictures are of the retrosplenial cortex and were made at a magnification of 400x.

Immunohistochemistry, CBS regulation

To examine the regulation of CBS in natural hibernation, the results from the Syrian hamster

set were quantified (Fig.4). Unfortunately, the brain slices from one of the euthermic animals

had such a bad quality that it could not be properly analyzed. Almost all of the analyzed areas

show a similar pattern: an gradual increase of CBS expression during the torpor bout,

followed by a decrease in the arousal phase. The only area that showed a different pattern is

15

the retrosplenial cortex. In contrast to the other areas, CBS expression seems to increase in the

retrosprenial cortex during the arousal phase.

The staining results of the Djungarian hamster set were not uniform and qualitatively good

enough to use for quantification. The irregularity of the staining (darker and lighter patches)

precludes us from obtaining a representative result. Thus, this set was not used for

quantification.

The regulation of CBS in induced hibernation was examined by quantifying the results from

the 5'AMP mice set (Fig.5), using the same regions as those in the Syrian hamster set. All of

the analyzed areas show a similar pattern: no clear difference between euthermic and torpor

was observed. However, we found a substantial variation of staining intensity within each

slice of this set. This seriously hampered analysis and hence the results may not represent the

actual situation.

Taken together, the regulation of CBS seems to differ between natural and induced

hibernation, especially with respect to the retrosplenial cortex in the arousal phase.

16

Figure 4. Measured optical densities for Syrian hamster set. Optical densities of different areas were corrected with

corresponding background. PoDG: polymorphic layer of the dentate gyrus; MoDG: molecular layer of the dentate gyrus;

hippocampus total: includes CA1, CA3, PoDG and MoDG. *p<0.05. EU: summer euthermic; TE: torpor early; TL: torpor

late; AL: arousal late

17

Figure 5. Measured optical densities for 5'AMP induced hibernation set. Optical densities of different areas were corrected

with corresponding background. PoDG: polymorphic layer of the dentate gyrus; MoDG: molecular layer of the dentate

gyrus; hippocampus total: includes CA1, CA3, PoDG and MoDG. EU: euthermic; T: torpor.

Western blot

Results of the western blot analysis for CBS expression are shown in the figure below

(Fig.6). Syrian hamsters showed a very large variation within each group. Unfortunately the

set was not a complete set, as material of some of the groups was absent due to their use in

other western blot analyses. Figure 6A shows, again, that the antibody does work on hamster

material and clear bands for CBS could be found. The pattern shown in figure 6B suggests

that the amount of CBS protein is higher in the arousal phase than in the torpor late phase.

However, the large variation precludes this difference to reach statistical significance.

18

Figure 6. Results of the westernblot analysis on the Syrian hamster set. 6A shows the CBS upper and lower bands found in a

TL (a) and AL (b) animal. 6B shows the quantification of Syrian hamster cortex lysates in different stages of hibernation.

EU: euthermic; TM: torpor middle; TL: torpor late; AL: arousal late.

Cell culture

Neuroblastoma 2A (N2A) cells were used to study the protective effects of the CSB/H2S

pathway. Cell characteristics such as growth rate were determined, followed by subjection of

the cells to a cooling-rewarming cycle with addition of an agent which maintains (high) H2S

levels (Sul-121).

To evaluate growth characteristics of the N2A cell line, a growth curve was constructed

(Fig.7). The curve shows that the cell count increases until it reaches its highest point at day 4,

followed by a decline at day 7. At each time point pictures were made, these can be seen in

supplement 1.

Figure 7. Growth curve of the N2A cell line.

Next, we examined the effect of cooling-rewarming on the viability of the N2A cells (Fig.8)

Cooling the cells for 24h resulted in some shrinkage of the cells, however no floating cells

could be found and no cells were lost (Fig.8, panel B), suggesting that minor damage has

occurred. After 24h of cooling and 2h of rewarming cells seem to have their old size again,

but some floating cells could be found (Fig.8, panel C). However, again no cells were lost and

thus still only minor damage could be found. Prolonging the rewarming by half an hour lead

to loss of a large part of the cells, floating of another large part of the cells and shrinkage of

the remaining cells (Fig.8, panel D). These morphological changes strongly suggest the

occurrence of cooling-rewarming injury, which has lead to cell death. This type of injury

seems to occur mostly and more extensively after a longer rewarming period (longer than 2

hours).

19

Figure 8. Results of subjecting cells to a cooling-rewarming cycle to determine the timepoint of cell damage. Panel A: N2A

cells kept at 37°C; panel B: N2A cells after 24h of cooling at 4°C; panel C: N2A cells after 24h of cooling at 4°C and 2h of

rewarming at 37°C; panel D: N2A cells after 24h of cooling at 4°C and 2.5h of rewarming at 37°C

After examining the cooling-rewarming injury, we examined the potential positive effect of

induction of the CBS/H2S pathway on the N2A cells, by examining the effects of Sul-121 on

the cooling-rewarming cycle. The results of subjecting cells to a cooling-rewarming cycle

during treatment with Sul-121 at t=0 or t=23h, or treatment with DMSO at t=0 or t=23h, are

shown in the figure below (Fig.9). Sul-121 did not affect the morphology of non-cooled cells

(Fig.9. panel A). After 24h of cooling and 2.5h of rewarming, cell death occurs due to

cooling-rewarming injury (Fig.9, panel B), as observed before (Fig.8). In contrast,

pretreatment with Sul-121 substantially attenuated cooling-rewarming induced cell death, as

demonstrated by the almost complete prevention of cell shrinkage, floating of cells and cell

loss (Fig. 9, panel D). In contrast, damage prevention by Sul-121 did not occur when

administering Sul-121 to cells at the end of the cooling cycle, 1h before rewarming (Fig.9,

panel F). DMSO seems to give some sort of cell clustering in damaged cell groups (Fig. 9,

panel C and E)

20

Figure 9. Sul-121 protects N2A cells from cooling/rewarming injury. Panel A: N2A cells kept at 37°C; panel B: N2A cells

after 24h of cooling at 4°C and 2.5h of rewarming at 37°C; panel C: N2A cells after the cooling-rewarming cycle with

DMSO added at t=0; panel D: N2A cells after the cooling-rewarming cycle with Sul-121 added at t=0; panel E: N2A cells

after the cooling-rewarming cycle with DMSO added at t=23h; panel F: N2A cells after the cooling-rewarming cycle with

Sul-121 added at t=23h

4. Discussion We analyzed the expression pattern of CBS in the hibernating brain and assessed if the

CBS/H2S pathway may protect brain (N2A) cells from cooling/rewarming injury.

Immunohistochemistry analyses showed CBS to be ubiquitously expressed in the brains of

Syrian hamsters and Djungarian hamsters in different stages of natural (daily torpor and deep

hibernation) hibernation and in the brains of C57bl/6J mice in an induced torpor-like

hypothermia or euthermic condition. These findings were confirmed by protein quantification

analysis by western blot. Cell experiments with the N2A cell line showed a protective effect

of Sul-121, a compound which is supposed to maintain or increase H2S in the cells.

CBS has a very distinctive and similar expression pattern in all animal species examined. It

is located in the cytosol of neurons and in their dendrites. The main areas wherein CBS

expression was found are: the pre-frontal cortex (mainly the primary motor cortex and

cingulate cortex), cortex (specifically the retrosplenial cortex), hippocampus, hypothalamus

and the thalamus.

Furthermore, we found a very specific enhancement of the expression of CBS in the

retrosplenial cortex of the Syrian hamsters in the arousal late phase. The arousal phase is the

hibernation stage wherein the animals rewarm to their euthermic body temperature. Enhanced

expression in this specific area was absent in the brains of Syrian hamsters in other

hibernation phases, or in the other examined animals.

21

Unfortunately, no quantification could be performed on the results of the Djungarian

hamster stainings. The irregularity of the staining, due to the bad quality of the brain slices,

precludes us from obtaining a representative result in quantification. The material from these

animals was more than five years old, which explains the non-optimal staining result. To

complete the information about CBS expression in natural hibernation, it is still very

interesting to look at the CBS expression in these daily torpor animals. Therefore, it is

recommended to repeat the staining in fresh material.

However, by eye we could not find an increase in CBS expression in the retrosplenial cortex

of Djungarian hamsters. One of the differences in hibernation between the Syrian hamsters

and the Djungarian hamsters is the Tb. The Tb of Syrian hamsters drops to as low as 5°C,

whereas the Tb of the Djungarian hamsters does not drop lower than 22°C. This implicates

that the increased expression of CBS in the Syrian hamsters may be a temperature driven

process. Thus, it would be interesting to repeat the CBS staining on Djungarian hamster

brains and combine the results with a CBS staining on brains of 5'AMP mice with a Tb of

5°C. This could help determine if the increase in CBS expression is a temperature driven

process or a hibernation driven process.

Our findings in 5'AMP induced hibernation in mice were not conclusive. No clear difference

could be found between the torpor and euthermic phase. It is difficult to draw any conclusions

from these data because of the large variety of staining intensity in each slice, even though

every value was corrected for its corresponding background. Repeating the staining is

recommended in this case. What we can see, is that CBS is omnipresent in the brains of the

mice in torpor as well as in euthermic conditions, with a similar expression pattern as seen in

the Syrian hamsters.

During rewarming from hypothermic conditions many mammalian cell types are vulnerable

to damage, caused by a burst of reactive oxygen species (ROS), low ATP production and Ca2+

overload. This so called "rewarming injury" has evident apoptotic features such as cellular

shrinkage, formation of organelle-containing blebs and apoptotic bodies, and condensation of

chromatin [17,27]. In hibernating animals however, this rewarming injury does not occur to

an extent in which it causes organ damage. It seems that hibernating animals protect their

organs from hypothermia/rewarming related damage. As this damage would occur primarily

during the arousal phase it is interesting to see that CBS has a higher expression in this phase,

as well as a very distinctive expression pattern in the retrosplenial cortex.

There are two reasons why a higher CBS expression is necessary: the clearance of a higher

concentration of its substrate, homocysteine, or the production of higher amounts of its

product, H2S. There is no literature or known data about a possibly higher concentration of

homocysteine during rewarming from hypothermic conditions. Thus, higher clearance of the

substrate for CBS does not seem likely. The second possibility, necessity of higher

concentrations of H2S, seems more logical when considering the functions of H2S in the

brain. H2S has a high antioxidant potency, plays a role in the calcium regulation in the brain

and can protect against hypoxic injury and ischemia [22]. These functions could be very

helpful in the arousal phase, as this is the phase wherein the rewarming damage would occur.

An increase in the amount of H2S may help hibernators protect their brains from the

rewarming damage and might as well be one of the reasons why hibernators do not show the

organ damage, particularly in the brain).

When comparing the possibly protective functions of H2S with the damage causing

mechanisms of rewarming injury, one could say that H2S is an ideal candidate for protection

against rewarming injury. Supportive of this hypothesis is our finding that CBS has a higher

expression in the retrosplenial cortex of animals in the specific phase where the rewarming

injury might occur. Another finding in this study that supports this hypothesis is the

protection by Sul-121 from hypothermia/rewarming injury in N2A cells. The protection only

22

occurred when administered in advance of the entire cooling-rewarming cycle. Possibly,

providing Sul-121 in still normothermic conditions fires off a protective mechanism lasting

throughout the entire cooling and rewarming period. When Sul-121 is given before the

rewarming phase, the cells probably are unable to build up enough protective capacity,

possibly because of contracted cell damage during cooling.

A recent hypothesis that could help explain our findings about the location of CBS

expression is the function of H2S as a gaseous neurotransmitter [30]. H2S appears to signal

by S-sulfhydrating cysteines in its target proteins, in a similar way the gasotransmitter NO

causes S-nitrosylation. The S-nitrosylation caused by NO typically inhibits enzymes, whereas

H2S induced S-sulfhydrating activates enzymes. The physiological gasotransmitter-like effect

of H2S is comparable to that of NO and carbon monoxide. The gasotransmitter role of H2S is

relatively new and has to be investigated further. Another established role of H2S in

neurotransmission is the regulation of inhibitory neurotransmission by upregulation of the

GABA B receptor, as previously mentioned. H2S does not only help neurons communicate, it

also assists astrocyte communication, through its role in the regulation of cellular calcium

levels. The possible function of H2S as a gasotransmitter can help explain the omnipresent

expression of CBS in the brain.

Both hypotheses about the function of H2S, protective agent or gaseous neurotransmitter,

can be combined. The fact that CBS is omnipresent in the brain, in different conditions, gives

the impression that H2S needs to be continuously produced. Neurotransmitting and protection

against hypoxia/reperfusion (or hypothermia/rewarming damage) damage are two processes

that are also continuously present in the brain. The specific expression pattern and the higher

expression in the retrosplenial cortex can also combine both hypothesis. The retrosplenial

cortex is an area that could be very important during and after arousing from hibernation,

which needs to be protected from the possible rewarming damage during the arousal phase.

H2S can give this protection, as well as help with the neurotransmission in this area and

thereby supporting its function in spatial navigation and episodic memory. The CBS

expression found in the other areas implicate that H2S also assists neurotransmission in many

other parts of the brains, as well as protect them from different types of damage.

Not only the amount of CBS expression but also the very specific location of the distinctive

CBS expression pattern, the retrosplenial cortex, is a striking feature of natural deep

hibernators. In humans, the retrosplenial cortex is important for spatial navigation and it plays

a role in episodic memory, imagination and thinking about the future. In rodents, the

retrosplenial cortex plays a role in spatial memory and navigation. Spatial navigation can be

explained as a type of wayfinding, the ability to find one's way through a city centre, an

airport, a national historical park or other large-scale surroundings. When combined with

episodic memory (the memory of autobiographical events) the wayfinding becomes more

specific to one's own environment: the route from home to work, finding one's way around the

house or finding one's favorite table in a frequently visited large restaurant [28,29]. When

thinking about hibernating animals, the spatial navigation is definitively important upon

arousal. The animal needs to recognize the environment in which it has been hibernating and

navigation to the closest hoard (food supply) is highly important. Possibly, this function of the

retrosplenial cortex explains why CBS expression is increased during arousal. Increased

expression of CBS in the retrosplenial cortex can implicate a higher necessity for its product,

H2S.

Other brain areas with connections to the retrosplenial cortex are: the hippocampal

formation, the parahippocampal region and specific thalamic nuclei. In this study CBS

expression was also very clearly present in the hippocampus. The hippocampus is another

area that plays an important role in spatial navigation by retaining spatial memories.

Hippocampus is mostly known for its role in learning and memory, a process in which

23

synaptic plasticity is essential. One of the mechanisms underlying synaptic plasticity is long-

term potentiation (LTP). Thus, LTP is very important for learning and memory. The specific

type of memory as supported by the retrosplenial cortex together with the hippocampus is

spatial memory. Facilitation of LTP by H2S can therefore assist the learning and memory

processes, especially when it comes to spatial memory. Again, the CBS/H2S pathway can be

linked to spatial navigation and may well play an important role in spatial memory [22,28].

Still, the most important area in this study is the retrosplenial cortex, which interestingly is

also an important area in the development of cognitive impairment and Alzheimer's disease

(AD). There is evidence that the retrosplenial cortex is the earliest area of cortical

hypometabolism in mild cognitive impairment (MCI), the amnesic prodrome of AD [31]. This

correlates with one of the first symptoms in AD, topographical disorientation. This implicates

that the severe impairment in spatial navigation seen in AD dementia, is caused by

retrosplenial and hippocampal hypometabolism, two areas where we found a prominent

expression of CBS. The hypometabolism would cause a lower expression of CBS and

therefore a lower H2S production. There is already some evidence of a decreased brain H2S

in AD. There seems to be a role of the CBS/H2S pathway in the development of cognitive

impairment, which needs to be further investigated [32].

All things considered, H2S, in low concentrations, has a positive effect on the brain. When

combining the information known in literature with the results of this study one can say that

the CBS/H2S pathway has many important functions in the brain. The immunohistochemical

staining results and western blot results show CBS to be ubiquitously expressed, underscoring

the wide variety of functions of its product, H2S, in the brain. The cell culture experiments

with Sul-121 suggest a protective effect of H2S in brain cells. Taken together, the CBS/H2S

pathway and Sul-121 as a stimulating compound may represent a novel therapeutic direction

in limiting brain injury.

5. Conclusion Taken together, the CBS/H2S pathway seems to constitute a well conserved and important

pathway in the brain, which possibly plays a role in the protection of the brain from different

damage inducing mechanisms (neuro-inflammation, hypoxia/reperfusion and hypothermia

rewarming) and the (regulation of) neurotransmission. These functions are important to

investigate further when examining the link with clinical diseases such as homocysteinuria,

ageing and neurodegenerative diseases. Because of the lack of information about this link and

the role of the CBS/H2S pathway in these diseases, it is important to look at the specific

mechanisms by which H2S executes these functions. For example, the type of neurons in

which CBS is present and their main neurotransmitter will help to explore the pathway

further. A previous study by our group already linked the mono-anime neurotransmitters

dopamine and serotonin to the activation of the CBS/H2S pathway [17].

When studied more extensively, the CBS/H2S pathway could possibly help understanding

mental retardation, ageing of the brain and neurodegenerative diseases. If H2S proves to be

the protective agent that it seems to be, it might even help to protect against some of the

damaging mechanisms in the diseased and/or ageing brain.

Acknowledgements I thank prof. dr R.H. Henning and prof.dr. E.A. van der Zee for their support by sharing their

intellectual knowledge and for their supervision of this project, dr. A.S. Boerema for

providing (a lot of) brain material from his previous experiments to be used for this study and

24

his intellectual knowledge about this very specific subfield (the hibernating brain), M.Goris,

M.Duin and J.N. Keijser for their (bio)technical support and knowledge about sacrification,

processing brain tissue and processing brain cells, and the (PhD) students of the department of

clinical pharmacology and the department of molecular neurobiology (centre for behaviour

and neurosciences) for their intellectual knowledge of different parts of my study and for

helping me with performing and planning of my experiments.

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Supplement 1

Figure 1. Growth of N2A cells after enting 10000 cells/cm

2 on t=0, for t=1d: 5860 cells/cm

2(panel A); t=2d:

37850 cells/cm2(panel B); t=3d: 213600 cells/cm

2(panel C); t=4d: 291000 cells/cm

2(panel D); t=7d: 119700

cells/cm2(panel D)