REVIEWS

Osteocalcin in the brain: from

embryonic development to age-related
decline in cognition
Arnaud Obri1, Lori Khrimian1, Gerard Karsenty1 and Franck Oury2
Abstract | A remarkable, unexpected aspect of the bone-derived hormone osteocalcin is that it is
necessary for both brain development and brain function in the mouse, as its absence results in a
profound deficit in spatial learning and memory and an exacerbation of anxiety-like behaviour.
The regulation of cognitive function by osteocalcin, together with the fact that its circulating
levels decrease in midlife compared with adolescence in all species tested, raised the prospect
that osteocalcin might be an anti-geronic hormone that could prevent age-related cognitive
decline. As presented in this Review, recent data indicate that this is indeed the case and that
osteocalcin is necessary for the anti-geronic activity recently ascribed to the plasma of young
wild-type mice. The diversity and amplitude of the functions of osteocalcin in the brain, during
development and postnatally, had long called for the identification of its receptor in the brain,
which was also recently achieved. This Review presents our current understanding of the biology
of osteocalcin in the brain, highlighting the bony vertebrate specificity of the regulation of
cognitive function and pointing toward where therapeutic opportunities might exist.

1
Department of Genetics and
Development, Columbia
University Medical Center,
701 W 168th St. Rm 1602,
New York City,
New York 10032, USA.
2
Institut Necker-Enfants
Malades, CS 61431, Paris,
France Institut National de la
Santé et de la Recherche
Médicale, U1151, F-75014
Paris, France Université Paris
Descartes, Sorbonne Paris
Cité, 75006 Paris, France.
Correspondence
to G.K. and F.O.
gk2172@columbia.edu;
franck.oury@inserm.fr
doi:10.1038/nrendo.2017.181
Published online 29 Jan 2018
The bone-derived hormone osteocalcin has become syn-
onymous with the skeletal regulation of energy metabo-
lism, in part because this was the first endocrine function
ascribed to this hormone1. Although energy metabolism is
certainly a critical aspect of osteocalcin biology (whereby
in mice2, rats and humans3–12, osteocalcin affects the
functions of β-cells13, myoblasts and probably other cell
types), it by no means tells the entire story of osteo­calcin.
A hallmark of this hormone is that it regulates a large
and continually growing number of physiological func-
tions and developmental processes14 (FIG. 1). This Review
focuses exclusively on one aspect of osteocalcin biology
— its regulation of brain development and function,
which is sometimes overlooked. However, this function of
osteo­calcin is of fundamental importance to link this hor-
mone to the biology of ageing. The first reason to write a
review on this topic at the present time is that the biomed-
ical importance of the regulation of several behaviours
by osteocalcin was underscored and linked to the ageing
of the brain in a paper published in 2017 (REF. 15). A sec-
ond reason is that major progress has been made over
the past few years in elucidating the molecular basis of
the functions of osteocalcin in the brain15. These advances
illustrate the growing reach of this hormone as well as the
similarities and differences between the peripheral and
central functions of osteocalcin.
The function of osteocalcin in the brain
Demonstrating that anxiety-like behaviour and cog-
nition are affected by a molecule made in a peripheral
organ has never been an easy or straightforward task.
What led to this suspicion in the case of osteocalcin
(which is encoded by Bglap, also known as Osteocalcin)
was simply the fact that the extreme passivity of
Osteocalcin–/– mice16 could not be explained by any other
means. What was already known about osteocalcin
when we started to investigate this functionality was
that when under­carboxylated, this molecule promotes
insulin secretion and glucose homeostasis in animals
fed a normal diet 1,13,17. Undercarboxylated osteocalcin
also promotes testosterone synthesis in Leydig cells of
the testis but not in follicular cells of the ovary because
its receptor is expressed in the male but not in the female
gonads18–21. Although low circulating levels of testoster-
one affect several behaviours in male mice22,23, a poorly
defined yet evident passivity was observed in both male
and female Osteocalcin–/– mice. Hence, this finding
excluded the possibility that this phenotype was due to
a defect in sex steroid hormone synthesis, as this process
is not affected by osteocalcin in female mice18.
Likewise, it became clear that the passivity of
Osteocalcin–/– mice was not secondary to their abnormal
glucose metabolism, as this passivity was not seen in

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Key points each hemisphere28, is often missing in Osteocalcin–/–

mice24. Biochemically, the levels of all monoamine
• Undercarboxylated osteocalcin regulates anxiety and cognition in adult mice neuro­transmitters29, that is, serotonin, dopamine and
• Osteocalcin is necessary and sufficient to correct age-related decline in cognitive noradrenaline, are 20−50% lower in the midbrain
function in mice and brainstem of Osteocalcin–/– mice than in wild-type
• Mouse maternal osteocalcin contributes to brain development and the acquisition of mice. At the same time, the accumulation of GABA, an
cognitive function in the fetus starting at embryonic day (E) 14.5 inhibiting neurotransmitter 30, in the same areas of the
• Probable G protein-coupled receptor 158 (GPR158) regulates the role of osteocalcin brain was increased by 15−30% in Osteocalcin–/– mice.
in anxiety and cognition in the mouse brain These abnormalities are secondary, at least in part, to
transcriptional events, as the expression of the genes
needed for the synthesis of monoamine neurotransmit-
mice lacking Gprc6a, the receptor used by osteocalcin ters31–33 is decreased by 15−50%, whereas the expression
to increase insulin synthesis in pancreatic β-cells13,24–26. of two genes necessary for GABA synthesis, Gad1 and
The fact that Gprc6a–/– mice had none of the behavioural Gad2 (REF. 34), is increased by almost twofold in the brain-
abnormalities, such as increased passivity, increased anx- stem of Osteocalcin–/– mice. Even though the receptor for
iety and impaired memory, further explained below, has osteo­calcin in these neuronal cells was not identified at
been important throughout our investigation of osteo­ the time that these experiments were performed in 2013,
calcin for several reasons. It enabled us to uncover a we observed that osteocalcin binds to neurons of the
decrease in muscle function during exercise caused by CA3 region of the hippocampus, the ventral tegmental
the absence of osteocalcin signalling through G protein-­ area (a nucleus located in the midbrain that is enriched
coupled receptor family C group 6 member A (GPRC6A) in dopaminergic neurons35) and the dorsal and medial
in myoblasts2 and to distinguish this effect from the raphe, which contain serotonergic neurons36 (FIG. 2).
passivity caused by the absence of osteo­calcin signal- Accordingly, in electrophysiological assays, osteocalcin
ling through a different receptor present in the brain. increased the activation of the action potential frequency
At the onset of the study, the brain-­specific receptor was of neurons by almost twofold in the brainstem and
not known. This lack of explanation for the abnormal locus coeruleus24.
passivity of Osteocalcin–/– mice led to the suspicion that Two experiments led to the conclusion that all these
the phenotypes of these mice might indicate that osteo­ described abnormalities reflect the absence of osteo-
calcin normally signals and functions in the brain. What calcin signalling in the brains of adult mice. The first
added considerable credence to this notion is that osteo­ experiment found that the delivery of uncarboxylated
calcin had been shown in the original study of its central osteocalcin to the brain, through intracerebroventricu-
function to cross the blood–brain barrier, as peripheral lar infusion and at a dose that does not result in any
delivery of uncarboxylated osteo­calcin in Osteocalcin–/– measurable leakage of osteocalcin in the general circu-
mice resulted in an accumulation of osteocalcin in spe- lation, fully corrects anxiety-like behaviour and memory
cific regions of the brain, mainly in the midbrain and phenotypes, as well as gene expression abnormalities,
brainstem24. By contrast, when delivered peripherally, in mice24. Second, an osteoblast-specific deletion of
carbo­x ylated osteocalcin crossed the blood–brain Osteocalcin performed in adult mice resulted in the
barrier far less efficiently than uncarboxylated osteo­ same increased anxiety-like behaviour phenotype and
calcin24. This observation led us to a remarkable string memory defect, albeit in less severe forms than those
of findings. observed in Osteocalcin–/– mice24. Neurotransmitter lev-
els and gene expression were also considerably affected
Regulation of anxiety and cognition in mice with a postnatal and osteoblast-specific dele-
Moving from the entire animal to the molecular level, tion of Osteocalcin24. As expected, the only phenotype
we observed that from a young age (3 months old), adult that could not be recapitulated in mice lacking osteo­
mice lacking Osteocalcin displayed a substantial increase calcin only during postnatal stages was a developmental
in anxiety-like behaviour, including increased aversion change, that is, their hypomorphic hippocampi24.
to lit and open spaces and decreased exploratory activity,
compared with wild-type littermates and had a major The role of maternal osteocalcin
deficit in learning and memory 24. In the Morris water Two reasons that it was suspected that osteocalcin
maze test, Osteocalcin–/– mice were simply unable to influenced the brain during embryonic development
learn the location of the platform and find it in a timely were the normal brain development observed in mice
fashion27. This finding identifies osteocalcin as a periph- lacking Osteocalcin in only osteoblasts during adult-
eral hormone exerting one of the strongest influences on hood and their more modest deficit in spatial learning
spatial learning and memory. and memory. As osteocalcin expression is not detected
Anatomically, the brains of Osteocalcin–/– mice are in the mouse embryo before embryonic day (E) 17.5
consistently smaller than those of their wild-type litter- (REFS 37,38), this hypothesis implied that maternal osteo­
mates24. The most overt abnormality, however, is seen calcin might cross the placenta and the blood–brain
in the hippocampal region, where the area covered barrier to execute this putative function.
by the dentate gyrus is 30% smaller in Osteocalcin–/– This hypothesis turned out to be correct. Maternal
mice than in wild-type mice, and the corpus callosum, undercarboxylated osteocalcin begins to cross the
which serves as a bridge between the hippocampi of placenta between E14.5 and E15.5 (REF. 24), at a time

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Undercarboxylated organs, most of which are still unknown. Underscoring

osteocalcin
Testis
↑ Leydig cell
Pancreas proliferation
↑ Testosterone
↑ β-cell proliferation production
↑ Insulin secretion
Muscle
↑ IL-6 production
↑ Nutrient uptake and catabolism
↑ Adaptation to exercise
Figure 1 | Osteocalcin functions in peripheral organs.
Nature Reviews | Endocrinology
A schematic representation of the physiological functions
regulated by undercarboxylated osteocalcin in peripheral
organs is shown. In the pancreas, undercarboxylated
osteocalcin increases β‑cell proliferation and insulin
secretion. In the testis, it increases testosterone production
and prevents the apoptosis of Leydig cells. In the muscle, it
increases IL‑6 production and nutrient uptake and
utilization for the production of ATP to promote adaptation
to exercise.
when the development of brain structures, such as the
hippo­campus, is in full speed39 (FIG. 3). A complicated
but efficient breeding strategy could establish unam-
biguously that almost all the undercarboxylated osteo-
calcin found in an embryo before E18.5 is of maternal
origin. Moreover, we showed that maternally derived
osteocalcin is needed to ensure the proper progression
of fetal neurogenesis and prevent neuronal apoptosis24.
Maternal undercarboxylated osteocalcin is also neces-
sary for the establishment of spatial learning and mem-
ory in adult mice24. Indeed, delivering uncarboxylated
osteocalcin or vehicle to Osteocalcin–/– mothers crossed
with Osteocalcin–/– fathers throughout pregnancy and
testing the behavioural performance of their progeny at
3 months of age showed that maternal osteocalcin con-
tributes to a substantial extent, but does not completely
explain, the deficit in spatial learning and memory that
is observed in adult Osteocalcin–/– mice24.
What did we learn?
Taken together, this series of observations revealed sev-
eral important and novel notions about brain physiology
and brain development. Moreover, given where osteo­
calcin is synthesized, when taken at face value, these
observations also enrich bone physiology. If one looks
at brain physiology, these findings show unambiguously
that peripheral hormones, and certainly one made in
bone, osteocalcin, exert a rather powerful influence on
neuronal activity, gene expression, neurotransmitter syn-
thesis and, ultimately, behaviour, including anxiety-like
behaviour, spatial learning and memory. This notion is
of fundamental importance, as too often we conceive
and study functions of a given organ while omitting
the fact that this organ receives many inputs from other
the potential medical influence of this work, two retro­
spective studies performed in human participants40,41
after the initial paper of Oury et al.24 was published have
suggested a correlation between decreased circulating
levels of osteocalcin and poor cognitive performance in
healthy middle-aged and aged individuals (40–78 years
of age). We are fully aware that these studies, because of
their retrospective nature, can be seen as only encourag-
ing and that prospective studies are now needed. In terms
of brain development and physiology, this work shows
that one molecular underpinning of the so‑called Barker
effect 42,43 (the maternal influence on neuro­psychological
functions of the offspring) might be a lack of maternal
osteocalcin. This observation is important if we consider
that the Barker effect also includes the maternal influence
on energy metabolism in the offspring, as energy metab-
olism is one of the main physiological processes regulated
by osteocalcin44–48.
How does one place these data in a broader context of
bony-vertebrate-specific biology? Two aspects of osteo­
calcin biology are particularly helpful in that respect.
The first is that in pre-pubescent mice and in humans,
circulating levels of undercarboxylated osteocalcin
decrease steeply before 9 months of age in the mouse
and before age 30 and age 45 years in women and men,
respectively, without reverting to levels observed dur-
ing puberty 2. In other words, circulating levels of osteo­
calcin are high during life stages when most skills are
being learnt. Importantly for our purpose, this feature
is specific to osteocalcin, as circulating levels of other
bone-specific hormones such as sclerostin49 increase
with age. A second feature of osteocalcin that is worth
mentioning is that circulating levels of undercarboxy-
lated osteocalcin surge during exercise, and exercise is
known to improve cognitive function2,50–54. These find-
ings raise the following interconnected questions: could
the decrease in cognitive function that occurs during
ageing be due, at least in part, to the reduction in cir-
culating levels of osteocalcin, and could osteocalcin be
sufficient to correct age-related cognitive defects?
In addition to these biomedical questions, there is
also a molecular question that needs to be answered —
how does osteocalcin signal in the brain? Indeed, the fact
that Gprc6a is not expressed anywhere that osteocalcin
signals in the brain and does not mediate osteocalcin
signalling in the brain excludes the possibility that the
central functions of osteocalcin are secondary, even in
part, to the peripheral ones. Once this point was made,
the task remained to understand how osteocalcin signals
in the brain by identifying its receptor in the brain. This
advance could serve several purposes of varying nature.
One purpose is to achieve a better understanding of how
extracellular cues coming from the periphery can affect
brain development and function. A second purpose is,
depending on the pattern of expression of this receptor, to
possibly broaden the portfolio of the central functions of
osteocalcin. A third purpose, dependent on the nature
of this receptor and of its pattern of expression, could
be to harness the biology of the signalling axis between
osteocalcin and its receptor for therapeutic purposes.

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Blood–
brain Hippocampus
↑ Serotonin
barrier ↑ Dopamine
CA3
↑ Noradrenaline
↓ GABA

↑ Spatial learning
VTA DRN and memory
MRN ↓ Anxiety-like
behaviour
Undercarboxylated
osteocalcin

Figure 2 | Undercarboxylated osteocalcin promotes spatial learning and memory andNature Reviews
prevents | Endocrinology
anxiety-like
behaviour. Undercarboxylated osteocalcin is made in bone and uses the general circulation to cross the blood–brain
barrier, where it binds to neurons from the CA3 region of the hippocampus, the ventral tegmental area (VTA) of the
midbrain and dorsal raphe nuclei (DRN) and medial raphe nuclei (MRN) of the brainstem. Osteocalcin acts on these
neurons through transcriptional events to increase the synthesis of all monoamine neurotransmitters (serotonin,
dopamine and noradrenaline) and decrease the synthesis of GABA24. Osteocalcin promotes spatial learning and memory
and prevents anxiety-like behaviour24.

Osteocalcin and memory in ageing
The most fundamental question raised by the first
study in the mouse that established bone as a regula-
tor of brain development and function via osteocalcin24
was to determine how useful these findings could be or
could become from a medical point of view, especially
in the ageing population. This possibility stems from the
severe behavioural phenotype of the Osteocalcin–/– mice
and from the following observations. For instance, with
the progressive ageing of the general population55–57,
new ways to prevent age-related cognitive decline and
other age-related diseases affecting brain functions
are urgently needed58–62. Moreover, it has been shown
that for every function regulated by undercarboxy-
lated osteocalcin, delivery of exogenous uncarboxy-
lated osteocalcin can improve the age-related decline
of these functions2,24. One reason for this finding could
be that circulating levels of undercarboxylated osteo­
calcin plummet beyond midlife in all species tested2,63,64.
Finally, the aforementioned clinical studies40,41, which
suggested an association between circulating levels of
undercarboxylated osteocalcin and cognitive perfor-
mance, were another reason to ask whether osteocalcin
could be sufficient to restore cognitive function in older
mice. We have begun to address this question in animal
models in several ways.
The first method used the assay popularized by
Villeda et al.65, which was published in 2014, show-
ing that repeated injections of plasma obtained from
3‑month-old mice could improve cognitive function in
older mice (16‑month-old mice; FIG. 4a). We need to add
a precision here: we, as well as many other laboratories,
are able to make mice live beyond 24 months of age in
the confined environment of a modern animal facility,
and 24 months of age could be used as a definition of
ageing. After reproducing the results of Villeda et al.,
we showed that by contrast, injections of plasma from
3‑month-old Osteocalcin–/– mice into 16‑month-old
wild-type mice did not improve cognitive function or
anxiety-like behaviour, but improvements were seen
with plasma from 3‑month-old wild-type mice15 (FIG. 4a).
Many possible factors could explain these results, yet
they established, on genetic grounds, that osteocalcin is
necessary for the beneficial effect of plasma from young
mice on several brain functions in older mice15. To rule
out the possibility that this inability of plasma from
young Osteocalcin–/– mice to improve the behavioural
performance of older mice represented a developmental
effect and to link osteocalcin itself to the beneficial effect
of plasma from young wild-type mice, older mice were
injected with plasma from young Osteocalcin–/– mice
with the addition of recombinant uncarboxylated osteo-
calcin (‘spiked plasma’)15 (FIG. 4a). The fact that this spiked
plasma could improve cognitive function and decrease
anxiety-like behaviour in older wild-type mice to the
same extent as plasma from young wild-type mice was a
strong indication that osteocalcin was not only necessary
but also sufficient to improve cognitive function and to
hamper anxiety-like behaviour in adult mice. In a similar
experiment, depleting every form of osteocalcin from
the plasma of young wild-type mice through immuno-
logical means removed the ability of plasma from wild-
type mice to improve cognitive function and to decrease
anxiety-like behaviour 15 (FIG. 4b).
Another experiment has provided the most direct
evidence that osteocalcin is sufficient to rescue age-­
related cognitive decline and to decrease anxiety-like
behaviour and functions independent of any develop-
mental effect. As osteocalcin crosses the blood–brain
barrier 24, we subcutaneously implanted mini osmotic
pumps delivering uncarboxylated osteocalcin or vehicle
in wild-type mice that were either 10 or 14 months old
for 60 days15. The mice were analysed at 12 or 16 months
old. Long-term delivery of uncarboxylated osteocalcin
in mice aged 10 or 14 months fully rescued their deficit
in cognitive function and decreased their anxiety-like
behaviour 15.
In exploring the molecular bases of this remarka-
ble ability of osteocalcin to rescue age-related cognitive
decline, several lines of experimental evidence pointed
towards brain-derived neurotrophic factor (BDNF), a mol-
ecule well known to promote hippocampal-dependent

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a G protein-coupled receptor (GPCR) that belongs to

↑ Neurogenesis
↓ Neuronal
apoptosis
Undercarboxylated
osteocalcin
Placenta
Bone
Starting at E14.5
Figure 3 | Maternal undercarboxylated osteocalcin acts directly
Nature in the| Endocrinology
Reviews offspring to
regulate embryonic brain development. Maternal undercarboxylated osteocalcin
crosses the placenta starting at embryonic day (E) 14.5, promotes de novo neurogenesis
and at the same time prevents neuronal death in the hippocampal region of the offspring
where it signals.
memory 54,66–69, as a critical mediator of the regulation of
cognitive function by osteocalcin. The first indication
of this functionality was observed when monitoring mul-
tiple parameters of the transport of BDNF-containing
dense-core vesicles in embryonic rat neurons in micro-
chambers70. In this setting, uncarboxylated osteocalcin
was able to stimulate the transport of BDNF-containing
vesicles to synapses in rat neurons15. This translated to an
increase in velocity and in the number of BDNF vesicles
moving to the synapses. In in vitro studies, osteocalcin
treatment of primary hippocampal neurons stimulated
Bdnf expression. A second piece of evidence is sup-
plied by in vivo studies showing that BDNF is a medi-
ator of osteocalcin in the brain. Stereotaxic injection of
uncarboxylated osteocalcin in the anterior hippocampi
increased the accumulation of BDNF in the hippo­
campus. Lastly, peripheral injections of uncarboxylated
osteocalcin increased Bdnf expression in the hippocampi
of 16‑month-old mice15. Similarly, injection of plasma
from 3‑month-old mice to 16‑month-old mice increased
BDNF accumulation in the hippocampi of 16‑month-
old mice15. This beneficial effect of young plasma on
BDNF accumulation was abolished when plasma from
3‑month-old Osteocalcin –/– mice was injected into
16‑month-old wild-type mice15.
The receptor for osteocalcin in the brain
The fact that osteocalcin was not only necessary but
sufficient to improve cognitive decline at any age,
though it is more effective in aged mice, and to reduce
anxiety-­like behaviour made it imperative to identify
and characterize a receptor that mediates the functions
of this hormone in the brain. To tackle this problem,
three criteria were used to identify what could be a
candidate receptor of osteocalcin in the brain. First, on
the basis of what has been observed in many fields, we
posited that a new receptor for osteocalcin should be
the same class as GPRC6A, the class C family 71,72. This
criterion, because the class C orphan GPCR list is rather
small73, considerably reduced the number of options.
A second criterion was that this orphan GPCR should
be expressed in an area where it had been previously
shown that osteo­calcin binds. The third criterion was
that this orphan GPCR should not be expressed in any
of the cell types where GPRC6A mediates osteocalcin
signalling 2,13,17,18. When these three criteria were used as
a filter, only one orphan class C GPCR fulfilled all three:
probable G protein-­coupled receptor 158 (GPR158).
It remained then to demonstrate that GPR158 was the
actual receptor of osteocalcin.
The pattern of expression for Gpr158 in the mouse
brain was certainly consistent with this notion, as it
is expressed in the CA3 region of the hippocampus,
which is where osteocalcin binds; Gpr158 is expressed
in pyramidal neurons and not in astrocytes in this area of
the brain15. The fact that GPR158 was more abundant in
hippocampi from Osteocalcin–/– mice than in those from
wild-type mice was a further suggestion that it could be
a receptor of osteocalcin in the hippocampus15. Like
GPRC6A, GPR158 is expressed in humans in the same
structures and regions as it is in the mouse74,75. All the
other data that support this hypothesis were of genetic
nature and were obtained either via ex vivo or in vivo
experiments15.
For instance, local delivery of uncarboxylated osteo­
calcin in the anterior hippocampus induced proto-­
oncogene c‑FOS accumulation, a marker of neuronal
activity 76,77, in neurons of the CA3 region in wild-type
mice but not in Gpr158–/– mice15. Osteocalcin could bind
a complex containing GPR158 and guanine nucleotide-­
binding protein α-q (Gαq, also known as GANQ) in
solubilized membranes obtained from hippocampi from
Osteocalcin–/– mice, whereas the quantity bound by osteo­
calcin was considerably reduced when using solubilized
membranes obtained from hippocampi from Gpr158–/–
mice. In agreement with this observation, osteo­calcin
increased the accumulation of inositol triphosphate
(IP3), a second messenger used by GPCRs78,79, in hippo­
campal neurons from wild-type but not Gpr158 –/–
mice, where it consistently failed to increase cAMP
accumulation. In whole-cell current clamp recording
(an electrophysiological assay), osteocalcin increased
the action potential frequency in pyramidal neurons
of the CA3 region of the hippocampus of wild-type but
not of Gpr158–/– mice15; this effect of osteocalcin was
nearly abolished when using an IP3 receptor antagonist
(FIG. 5). An analysis of long-term potentiation80,81 verified
that osteocalcin signals were in hippocampal slices from
wild-type but not Gpr158–/– mice15.
Many genetic in vivo experiments also indicated that
GPR158 is a receptor for osteocalcin in the brain15. For
instance, whether we analysed mice lacking Gpr158 in
only the forebrain or in all cells, we observed the same
severe deficit in hippocampal-dependent memory 82 in
mice lacking this receptor 15. Likewise, Gpr158–/– mice
manifested more severe anxiety-like behaviour than
wild-type littermates. That neither cognitive deficit nor

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Plasma from young Plasma from young

Plasma from young Osteocalcin–/– mouse Osteocalcin–/– mouse
wild-type mouse (3 months) (3 months) + osteocalcin (3 months)

Recombinant
Undercarboxylated osteocalcin
osteocalcin (uncarboxylated)

Aged wild-type Aged wild-type Aged wild-type

mouse (16 months) mouse (16 months) mouse (16 months)

↑ Memory ↓ Memory ↑ Memory

↓ Anxiety-like behaviour ↑ Anxiety-like behaviour ↓ Anxiety-like behaviour

Plasma from young Anti-osteocalcin IgG

IgG-treated mouse (3 months) (3 months)

Aged wild-type Aged wild-type

mouse (16 months) mouse (16 months)

↑ Memory ↓ Memory
↓ Anxiety-like behaviour ↑ Anxiety-like behaviour

Figure 4 | Osteocalcin is sufficient to improve cognition in aged mice. a | Injections of plasma from young (3 months)
wild-type mice, which naturally contains undercarboxylated osteocalcin, into aged (16 months)
Naturewild-type
Reviews |mice improve
Endocrinology
memory and prevent anxiety-like behaviour. The improvement on memory and prevention of anxiety-like behaviour is not
seen when plasma from young (3 months) Osteocalcin–/– mice is used instead of plasma from young wild-type mice. When
Osteocalcin–/– plasma is spiked with mouse recombinant uncarboxylated osteocalcin, the ability of this plasma to improve
cognitive functions is restored to the same extent as when using plasma from young wild-type mice15. b | Injection of
control plasma from young mice (3 months) treated with an immunoglobulin G (IgG) antibody that does not recognize
osteocalcin into aged (16 months) wild-type mice led to improved cognition. Injections of osteocalcin-depleted (by the
use of an anti-osteocalcin IgG antibody) plasma from young (3 months) wild-type mice into aged (16 months) wild-type
mice do not improve memory performance and do not prevent anxiety-like behaviour15

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GPR158 Gαq
↑ IP3
Blood–
brain CA3
barrier ↑ BDNF
Neuron

Undercarboxylated
osteocalcin

Nature Reviews | Endocrinology

Figure 5 | Probable G protein-coupled receptor 158 mediates the functions of osteocalcin in the mouse brain.
Undercarboxylated osteocalcin binds probable G protein-coupled receptor 158 (GPR158) in pyramidal neurons of the
CA3 region of the hippocampus. This interaction promotes an increase in the accumulation of inositol triphosphate (IP3) as
a second messenger and eventually results in an increase in the expression and synthesis of brain-derived neurotrophic
factor (BDNF), a well-known mediator of cognition, in neurons54,66–69. Gαq, guanine nucleotide-binding protein α-q.

anxiety-like behaviour could be corrected by chronic Conclusions

delivery of uncarboxylated osteocalcin in Gpr158–/– mice
established that GPR158 is necessary to mediate the reg-
ulation of cognitive function and anxiety-like behaviour
by osteocalcin15. Two additional lines of evidence, of
genetic nature, demonstrated that this is indeed the case.
The first showed that stereotaxic injection of uncarbox-
ylated osteocalcin in the anterior hippocampi improved
hippocampal-dependent memory in wild-type mice but
not in mice in which GPR158 accumulation had been
decreased by a small hairpin RNA interference strat-
egy 83. Second, compound heterozygous mice lacking one
allele of Gpr158 and one allele of Osteocalcin (Gpr158+/–
Osteocalcin+/–) demonstrated the same cognitive deficits
observed in Gpr158–/– or in Osteocalcin–/– mice, whereas
single heterozygous mice behaved like wild-type mice15.
Independent of this body of work that identified
GPR158 as a receptor mediating the regulation of cogni-
tive function and anxiety-like behaviour by osteocalcin,
several other important observations were made in the
course of these investigations. One is that, as we had long
surmised, osteocalcin function in the brain might not
be limited to the hippocampus and the ventral tegmen-
tal area of the midbrain24. Indeed, a systematic analysis
of Gpr158 expression showed that it is also expressed in
the somatosensory area, the motor and auditory areas of
the cortex, the piriform cortex and the retrosplenial area,
where it was not known that osteocalcin binds24. A second
more surprising observation that needs to be interpreted
cautiously is that Gpr158 is not expressed in serotonergic
neurons of the dorsal and medial raphe of the brainstem,
where osteocalcin binds15. This observation suggests the
possibility that a third receptor for osteocalcin exists.
What was illustrated through this work is a classic and
fundamental principle of physiology; no organ, as noble
as it might be, is an island that develops and functions
at the exclusion of other organs. In our current era of
biology, we have developed the tools to explore molec-
ular events very effectively, and yet the study of mouse
genetics has enabled us to uncover broader physiological
phenomena that have remained hidden for a long time.
What we did not know and could not have anticipated
is that the skeleton, of all organs, would secrete a hor-
mone that exerts such a powerful influence on the brain
in the mouse and, it seems from retrospective studies,
probably in humans as well.
Here, one should focus first and foremost on the
salient features of osteocalcin and how they could help
medicine. It is impossible to address these questions
without considering the natural history of osteocalcin,
whose circulating levels decrease around midlife in all
species tested. This is a fundamental feature of osteo­
calcin in view of the fact that this hormone is necessary
to maintain cognitive function at an optimal level and to
decrease anxiety-like behaviour. Maybe more importantly
from a medical point of view is the notion that osteo-
calcin is necessary and sufficient to correct age-­related
cognitive defects and to hamper anxiety-like behaviour.
Together, these features identify osteocalcin as an anti-
geronic hormone that joins metalloproteinase inhibitor 2
(TIMP2)84 on the list of anti-geronic molecules. The
identification of a receptor for this hormone in the brain
now raises the prospect that this interaction between
osteocalcin and GPR158 could be used to treat various
age-­related degenerative conditions that affect the brain.

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This possibility might even be more important going for-
ward, as the pattern of expression of Gpr158 suggests that
we are not yet aware of all the functions of osteocalcin
in the brain.
The physiology and ageing of the brain are only one
side of the coin. The other aspect one has to recognize
is that osteocalcin is one of the first, if not the first,
molecular substratum for the long-suspected maternal
influence on brain development. How important the role
of osteocalcin is in brain development is not yet fully
known, in part because we do not know all the functions
of osteocalcin in the brain and in part because we also
do not know the pattern of expression of GPR158 in the
developing brain. These notions and the possibility that
a third receptor for osteocalcin might exist represent the
most concrete challenges going forward.

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Acknowledgements
This work was supported by 2P01 AG032959‑06A1 and the
Columbia Aging Center (G.K.), a 5T32DK007328‑38
Endocrinology Training Grant (L.K.) from the NIH, Fondation
pour la Recherche Medicale grant AJE20130928594, the
Human Frontier Scientific Program–Grant ATIP-AVENIR
INSERM - R14080KS - RSE15007KSA Program–INSERM,
Grant AGEMED-INSERM (F.O.) and the Philippe Foundation
(A.O.).
Author contributions
A.O., L.K., G.K. and F.O. researched data for the article, con-
tributed to discussion of the content, wrote the article and
reviewed and/or edited the manuscript before submission.
Competing interests statement
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

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