Novel CDNF/MANF Family of Neurotrophic Factors
Päivi Lindholm, Mart Saarma
Institute of Biotechnology, Viikinkaari 9, Viikki Biocenter, University of Helsinki, 00014 Helsinki, Finland
Received 16 July 2009; revised 14 September 2009; accepted 21 September 2009
ABSTRACT: Current therapeutic interventions
for neurodegenerative diseases alleviate only disease
symptoms, while treatments that could stop or reverse
actual degenerative processes are not available. Parkin-
son’s disease (PD) is a movement disorder with charac-
teristic degeneration of dopaminergic neurons in the
midbrain. Few neurotrophic factors (NTFs) that pro-
mote survival, maintenance, and differentiation of
affected brain neurons are considered as potential ther-
apeutic agents for the treatment of neurodegenerative
diseases. Thus, it is important to search and study new
NTFs that could also be used in therapy. In this review,
we discuss novel evolutionary conserved family of NTFs
consisting of two members in the vertebrates, cerebral
dopamine neurotrophic factor (CDNF) and mesence-
phalic astrocyte-derived neurotrophic factor (MANF).
Invertebrates, including Drosophila and Caenorhabditis
have a single protein homologous to vertebrate CDNF/
MANF. Characteristic feature of these proteins is eight
INTRODUCTION
Neurotrophic factors (NTFs) are an important group
of secreted proteins regulating the life and death of
neurons during development. Different NTFs affect
specific sets of neuronal subpopulations, which are
genetically competent to respond, i.e., express cog-
nate receptors on their surface, and have appropriate
transcriptional programs. During developmental peri-
ods of programmed cell death (PCD), NTFs prevent
apoptosis and regulate the number of neurons inner-
vating the target tissue. According to the classical
Correspondence to: M. Saarma (mart.saarma@helsinki.fi).
' 2010 Wiley Periodicals, Inc.
Published online 22 February 2010 in Wiley InterScience (www.
interscience.wiley.com).
DOI 10.1002/dneu.20760
360
structurally conserved cysteine residues, which deter-
mine the protein fold. The crystal structure analysis
revealed that CDNF and MANF consist of two domains;
an amino-terminal saposin-like domain that may inter-
act with lipids or membranes, and a presumably
unfolded carboxy-terminal domain that may protect
cells against endoplasmic reticulum stress. CDNF and
MANF protect midbrain dopaminergic neurons and
restore motor function in 6-hydroxydopamine rat model
of PD in vivo. In line, Drosophila MANF is needed for
the maintenance of dopaminergic neurites and dopa-
mine levels in the fly, suggesting that the function of
CDNF/MANF proteins is evolutionary conserved.
Future studies will reveal the receptors and mode of
action of these novel factors, which are potential thera-
peutic proteins for the treatment of PD. ' 2010 Wiley Peri-
odicals, Inc. Develop Neurobiol 70: 360–371, 2010
Keywords: neurotrophic factors; receptors; dopamine
neurons; Parkinson’s disease; lipids; saposins
NTF theory that is largely based on the studies on
nerve growth factor (NGF), the first growth factor
and NTF identified (Levi-Montalcini, 1987), excess
neurons compete of NTFs that are secreted by the tar-
get tissue in limited amounts. Only neurons that
receive sufficient trophic support survive and main-
tain synaptic contacts with the target tissue, whereas
other neurons die by apoptosis. NTFs also regulate
migration, differentiation, and maturation of postmi-
totic neuronal precursors and promote the regenera-
tion of neurons from injury (Huang and Reichardt,
2001; Airaksinen and Saarma, 2002). The role of
NTFs is well-established in the development of the
peripheral nervous system (PNS). However, the func-
tion of NTFs in the development and maintenance of
the central nervous system (CNS) is less clear. NTFs,
e.g., brain-derived neurotrophic factor (BDNF) are

known to regulate synaptic plasticity in adult CNS
(Lu et al., 2005). NTFs have important roles also in
non-neuronal tissues, and regulate, e.g., angiogenesis
(Zacchigna et al., 2008), kidney development, and
spermatogenesis (Airaksinen and Saarma, 2002;
Andressoo and Saarma, 2008).
In this review, we focus on the novel CDNF/
MANF family of NTFs (Petrova et al., 2003; Lind-
holm et al., 2007). Traditionally, there are three fami-
lies of NTFs: Neurotrophins NGF, BDNF, neurotro-
phin-3 (NT-3) and NT-4 (Huang and Reichardt,
2001; Lu et al., 2005); glial cell line-derived neuro-
trophic factor (GDNF) family ligands (GFLs) GDNF,
neurturin (NRTN), artemin (ARTN), and persephin
(PSPN) (Airaksinen and Saarma, 2002; Bespalov and
Saarma, 2007); and neuropoietic cytokines (also
referred as interleukin-6 (IL-6) family) (Heinrich
et al., 2003; Bauer et al., 2007). Members of these
families either signal through transmembrane recep-
tor tyrosine kinases or via kinases that interact with
their receptors (Bespalov and Saarma, 2007). Neuro-
trophic activities are also shown by other trophic and
growth factors, e.g., members of transforming growth
factor b, vascular endothelial growth factor (VEGF),
insulin-like growth factor, and fibroblast growth
factor (FGF) families (Grothe and Timmer, 2007;
Zacchigna et al., 2008). Recently, a novel evolution-
ary conserved protein Meteorin with neurotrophic
activities was characterized (Nishino et al., 2004;
Jørgensen et al., 2009).
The question whether NTFs function in the devel-
opment of invertebrate nervous system has been
uncertain. However, a recent study described Dro-
sophila Neurotrophin (DNT1; spätzle 2) a small part
of which is structurally homologous to all known
neurotrophins (Zhu et al., 2008). Although GFL
homologs have not been found in insect genomes
(Airaksinen et al., 2006), an orphan homolog for
GDNF transmembrane receptor tyrosine kinase RET
(rearranged during transfection) is present in Dro-
sophila (Sugaya et al., 1994). As we will discuss later
in more detail, Drosophila MANF (DmMANF) func-
tions as a NTF for dopaminergic neurons in the fly
(Palgi et al., 2009). Thus, it seems now evident that
NTFs have important roles also in the invertebrate
nervous system.
Since NTFs have many beneficial effects on neu-
rons, they are potential factors not only for the treat-
ment of neurodegenerative diseases, including Par-
kinson’s disease (PD), Alzheimer’s disease (AD),
amyotrophic lateral sclerosis (ALS), but also for the
treatment of neuronal trauma, such as spinal
cord injury. Shortage of NTFs is also seen in
neurodegenerative diseases, e.g., decreased levels of
Novel CDNF/MANF Family of NTFs 361
GDNF and BDNF have been detected in the substan-
tia nigra (SN) of PD patients (Mogi et al., 1999;
Parain et al., 1999; Howells et al., 2000; Chauhan et
al., 2001). Thus, delivery of NTFs may help to restore
the NTF deficiency in the disease.
None of the existing therapeutic strategies for PD
can stop or even slow down the degeneration of dopa-
minergic neurons or induce neuronal recovery. The
available therapies are based on dopamine-replace-
ment therapy using either L-DOPA (l-3,4-dihydroxy-
phenylalanine) or dopamine agonists. These treat-
ments only alleviate the disease symptoms, usually
by correcting striatal dopamine neurotransmission.
Two highly potent NTFs for dopaminergic neurons,
GDNF and NRTN have been taken to clinical trials
on PD patients, but the results from these trials have
been quite modest so far. Although direct infusion of
GDNF protein into the putamen of PD patients led to
significant clinical improvement (Gill et al., 2003;
Patel et al., 2005; Slevin et al., 2005), a double
blinded phase II study showed no clinical efficacy of
GDNF (Lang et al., 2006). Since several patients gen-
erated antisera against GDNF (Lang et al., 2006) and
at high doses GDNF induced cerebellar lesions in pri-
mates (Hovland et al., 2007), clinical trials with
GDNF protein have currently been interrupted.
Recent NRTN gene therapy trials have given
slightly more positive outcome. A press release on
May 2009 by Ceregene on phase II study reported
small positive effects on PD patients after 18 months
of treatment with CERE-120, the adeno-associated
virus serotype 2 vector carrying the gene for NRTN.
In any case, the development of NTF therapy is still
at a very early stage. Therefore, in addition to the
development of more efficient NTF delivery systems
it is of great importance to search for and study novel
NTFs that could offer new therapeutic approaches for
the treatment of neurodegenerative diseases.
The focus of this review is on the novel CDNF/
MANF family of NTFs that are structurally unrelated
to the classical NTF families, suggesting a
completely new mechanism of action and a novel
therapeutic potential for the treatment of neurodege-
nerative diseases.
CDNF AND MANF LIGANDS
Cerebral dopamine neurotrophic factor (CDNF; offi-
cial name by HUGO gene nomenclature committee;
previous name conserved dopamine NTF) and
mesencephalic astrocyte-derived neurotrophic factor
(MANF) form a novel evolutionary conserved protein
Developmental Neurobiology
362 Lindholm and Saarma
family showing neurotrophic activities (Petrova et al.,
2003; Lindholm et al., 2007, 2008; Airavaara et al.,
2009; Palgi et al., 2009; Voutilainen et al., 2009).
The founding member of the family, MANF, was
identified from the culture medium of rat Type-1
astrocyte ventral mesencephalic cell line 1 (VMCL1)
based on its survival promoting activities on cultured
embryonic dopaminergic neurons (Petrova et al.,
2003). The active rat protein was isolated and found
homologous to a predicted human arginine-rich pro-
tein (ARP) of 234 amino acids (aa; GenBank Acc.
No. NP_006001) (Shridhar et al., 1996b; Petrova et
al., 2003), which had never been expressed or purified
in vitro. Human ARP (ARMET; arginine-rich,
mutated in early stage tumors) gene contains an
amino-terminal arginine-rich region and mutations
within were associated with different cancer types
(Shridhar et al., 1996a,b, 1997), although contrasting
studies showed that variations in the arginine-rich
region of ARP represent normal polymorphisms
(Evron et al., 1997; Tanaka et al., 2000; Piepoli et al.,
2006). In line with that, the arginine-rich region of
ARP is apparently not translated in vivo (Petrova et
al., 2003). Thus, the protein encoded by ARP was
renamed MANF to better indicate its neurotrophic
functions (Petrova et al., 2003). Human MANF to-
gether with the 21 amino acids long signal sequence
consists of 179 aa that equal amino acids 56–234 of
human ARP (Petrova et al., 2003) [Fig. 1(A)]. The
signal sequence is cleaved off cotranslationally
resulting into the mature MANF protein of 158 amino
acids.
Mature CDNF (ARMETL1; GenBank Acc. No.
NM_177647) protein consists of 161 amino acid resi-
dues (preCDNF is 187 amino acids long), and is a
vertebrate specific paralog of MANF. CDNF was first
identified by bioinformatics and then biochemically
characterized (Lindholm et al., 2007). CDNF and
MANF encoding genes are highly conserved in evo-
lution. Based on sequence analyses, vertebrates have
CDNF and MANF genes (Lindholm et al., 2007),
whereas invertebrates, including C. elegans and
D. melanogaster, have a single homologous gene
more closely related to MANF than to CDNF (Palgi
et al., 2009). Characteristic feature of CDNF and
MANF proteins is eight cysteine residues, spacing of
which is conserved from vertebrates to invertebrates
(Shridhar et al., 1996b; Petrova et al., 2003; Lind-
holm et al., 2007). DmMANF of 151 aa residues (pre-
DmMANF is 173 aa) has been recently characterized
(Palgi et al., 2009). Human CDNF shows 59% amino
acid identity with human MANF, 49% identity with
D. melanogaster, and 46% identity with C. elegans
MANF proteins. Human MANF shows 53% amino
Developmental Neurobiology
acid identity with D. melanogaster and 50% identity
with C. elegans MANF proteins, respectively. Differ-
ently from neurotrophins and GDNF family ligands,
CDNF and MANF proteins seem not to contain a pro-
sequence, suggesting that enzymatic cleavage is not
necessary for their activity (Fig. 1A).
Consistently with the roles as extracellular NTFs,
CDNF and MANF are secreted from transiently trans-
fected cells (Lindholm et al., 2007, 2008; Palgi et al.,
2009). Also the secretion of endogenous MANF has
been demonstrated in vitro (Apostolou et al., 2008).
Human CDNF contains one potential N-linked glyco-
sylation site, and both unglycosylated (Lindholm et
al., 2007) and glycosylated (Apostolou et al., 2008)
form of human CDNF is secreted from transiently
overexpressing cells. The nonglycosylated CDNF is
biologically active (Lindholm et al., 2007), indicating
that glycosylation is not crucial for its neurotrophic
activity. Differently from human CDNF, mouse
CDNF, or human and mouse MANF do not have
potential N- or O-linked glycosylation sites. In con-
trast to the original report (Petrova et al., 2003),
secreted glycosylated MANF has not been detected in
later studies (Apostolou et al., 2008; Lindholm et al.,
2008). Both CDNF and MANF are highly soluble and
monomeric at neutral pH 7 (Lindholm et al., 2007;
Mizobuchi et al., 2007). Whether the secretion of en-
dogenous CDNF or MANF is regulated by physiolog-
ical stimuli or injury in vivo is currently unclear.
Although secreted, MANF proteins are also par-
tially retained in the endoplasmic reticulum (ER)
(Mizobuchi et al., 2007; Apostolou et al., 2008).
MANF and CDNF have a C-terminal sequence
(RTDL and KTEL, respectively) closely resembling
the classical ER retention signal (KDEL), which may
function as a partial ER retention signal (Raykhel et
al., 2007; Apostolou et al., 2008). Interestingly,
CDNF and MANF may have important functions also
in the ER, as discussed below.
TWO DOMAINS—TWO ACTIVITIES?
The structure of mammalian MANF and CDNF pro-
teins is unique among NTFs and growth factors, and
defines a novel type of proteins [Fig. 1(B)]. The crys-
tal structure of the mature human MANF protein and
the amino (N)-terminal domain of human CDNF
have been resolved (Parkash et al., 2009). The struc-
ture of CDNF and MANF consists of two domains, a
saposin-like N-terminal domain and a presumably
unstructured carboxy (C)-terminal domain with an
intradomain cysteine bridge in a CXXC motif. Inter-
estingly, saposin-like proteins interact with lipids

Figure 1 (A) Schematic presentation of the primary struc-
tures of human CDNF and MANF, and selected invertebrate
MANF proteins. Pre represents the signal sequence and the
numbers the length of the polypeptides in amino acids.
Yellow vertical bars show the conserved cysteine residues.
(B) Crystal structure of mature human MANF. Alpha-helices
(a) are numbered starting from the N-terminus. Below the
structural image is a schematic picture of the primary struc-
ture of mature MANF. The formation of cysteine bridges is
shown as connecting lines. (C) Structure of the N-terminal
domain of human MANF (blue) is superimposed on human
granulysin saposin (pink).
and/or membranes (Bruhn, 2005) suggesting that the
N-terminal domain of CDNF and MANF may also
interact with lipids. Furthermore, the C-terminal cys-
teine bridge of MANF is similar to the active site
motif of thiol/disulfide oxidoreductases that catalyze
the formation of intramolecular disulphide bonds,
suggesting that CDNF and MANF maybe involved in
protein folding in the ER.
The closest structural homologs for CDNF and
MANF N-terminal domain (Parkash et al., 2009)
Novel CDNF/MANF Family of NTFs 363
are saposin-like proteins (SAPLIPs) granulysin
(Anderson et al., 2003) and NK-lysin (Liepinsh et al.,
1997) [Fig. 1(C)], which function as defense proteins
against bacterial cells and are able to disrupt target
cell membranes (Jongstra et al., 1987; Andersson et
al., 1995; Pena et al., 1997). Granulysin and NK-lysin
are proteolytically processed from propolypeptides in
cytolytic granules. In contrast, CDNF and MANF are
likely functional upon synthesis and secretion since
they apparently do not contain prosequences for
enzymatic activation. However, it is possible to
speculate the N-terminal domain represents the pro-
domain of CDNF/MANF family proteins and then
proteolytic cleavage in the linker region produces the
\mature" protein, i.e., the C-terminal domain.
Characteristic features of the membrane interac-
tions of SAPLIPs are oligomerization and conforma-
tional changes. Also the composition of membrane
lipids and pH value are known to affect membrane
binding. Interestingly, the saposin-like domain of
Figure 2 Hypothetical modes of CDNF and MANF
action. (A) Change in pH or lipid binding may induce dime-
rization of CDNF or MANF. (B) C-terminal domain of
CDNF or MANF may facilitate the formation of disulfide
bridges on secretory proteins in the ER. (C) CDNF or
MANF may reduce disulfide bond on a transmembrane
receptor protein on the cell surface. (D) CDNF or MANF
may interact with the cell surface membrane and subse-
quently activate transmembrane receptor protein.
Developmental Neurobiology
364 Lindholm and Saarma
CDNF crystallized as a dimer at pH 4.6, whereas the
full-length, mature MANF crystallized as a monomer
at neutral pH (Parkash et al., 2009). Saposin C and
Saposin A form oligomers in the presence of a deter-
gent at low pH 4.8 (Ahn et al., 2006). Acidic pH also
increases the ability of Saposin C and Saposin D to
bind lipid vesicles and destabilize phospholipid-con-
taining membranes (Vaccaro et al., 1995). Differently
from acidic saposins with pI values 4.6–4.8, CDNF
and MANF SAPLIPs are slightly basic with pI values
7.7 and 8.5, respectively. It is likely that pH affects
the putative oligomerization [Fig. 2(A)] and mem-
brane interactions of CDNF and MANF.
Hydrophobic residues located at conserved posi-
tions of SAPLIPs form a hydrophobic core and may
have a role in the lipid interactions (Bruhn, 2005).
Based on the structural analysis, the saposin-like
domain of CDNF and MANF is in the closed confor-
mation and stabilized by three intramolecular disul-
fide bonds, which connect a1 and C-terminal 310
helix, a1 and a5 helix, a2 and a3 helix, respectively
(Parkash et al., 2009) [Fig. 1(B)]. Saposin B, a pro-
tein with lipid transfer activity (Vogel et al., 1991),
crystallized in an open conformation as a homodimer
with bound phospholipid (Ahn et al., 2003), whereas
Saposin C crystallized in the presence of detergent as
a monomer in an open conformation with hydropho-
bic pocket exposed to solvent (Hawkins et al., 2005).
Also at pH 4, Saposin C crystallized in an open con-
formation, but as a domain-swapped dimer that may
facilitate vesicle fusion (Rossmann et al., 2008).
Charge distribution on the surface of SAPLIPs
may have an important role in the membrane target-
ing. NK-lysin (Liepinsh et al., 1997) and granulysin
(Anderson et al., 2003) have a ring of positively
charged residues around the protein, which may
direct their interaction toward the negatively charged
lipid head groups in the membrane. The saposin-like
domain of CDNF and MANF has conserved posi-
tively charged residues on the surface in two patches
that may contribute to the membrane interactions
(Parkash et al., 2009).
We have recently confirmed the lipid interaction
of CDNF and MANF (Hongxia Zhao, Pekka Lappa-
lainen and Mart Saarma, unpublished observations).
The role of lipids and membrane interactions in the
biology of CDNF and MANF are completely
unknown. Does lipid binding induce dimerization of
CDNF and MANF [Fig. 2(A)] and subsequent activa-
tion? It does not seem likely that CDNF and MANF
disrupt the target membranes like granulysin and
NK-lysin, since cell death has not been observed
when cells were treated with CDNF or MANF.
Whether CDNF and MANF have lipid transfer prop-
Developmental Neurobiology
erties similar with those of saposins is also not
known. Hypothetical modes of CDNF and MANF
dimerization and membrane interaction are presented
in Figure 2.
PROTECTION AGAINST ER STRESS
Accumulating evidence suggests that MANF is an
ER stress response protein (Lee et al., 2003; Mizobu-
chi et al., 2007; Apostolou et al., 2008; Tadimalla et
al., 2008) and is able to protect cells against ER
stress-induced cell death in vitro (Apostolou et al.,
2008). In line, the crystal structure suggests that
MANF and CDNF may help protein folding in the
ER. In the MANF C-terminal domain, the two cys-
teines in 127CKGC130 motif (132CRAC135 in CDNF)
form a C-terminal disulfide bridge. Interestingly, a
CXXC active site motif is found in thiol/disulfide
oxidoreductases that catalyze the formation of intra-
molecular disulfide bonds. These proteins include thi-
oredoxins, like protein disulphide isomerases (PDIs),
which function in the ER (Ellgaard and Ruddock,
2005). Interestingly, nitrosylated PDI is found in
brain samples derived from PD patients, indicating a
link between PDI dysfunction and protein misfolding
in neurodegenerative disorders (Uehara et al., 2006).
MANF and CDNF may facilitate the formation of
cysteine bridges and protein folding in the ER
[Fig. 2(B)], thus reducing the ER stress caused by
unfolded or incorrectly folded proteins.
Accumulation of unfolded proteins in the ER
causes ER stress and the induction of unfolded pro-
tein response (UPR). This is a signal-transduction
pathway that counteracts ER stress (Szegezdi et al.,
2006b) by increasing protein folding capacity and
degradation of misfolded proteins in the cell. Also
protein translocation across the ER membrane is
reduced. Proteins are refolded by molecular chaper-
ones and misfolded proteins are removed by ER asso-
ciated protein degradation. ER transmembrane recep-
tors mediate the UPR pathways, including receptors
activating transcription factor 6 (ATF6), inositol-
requiring enzyme 1 (IRE1), and pancreatic ER kinase
(PKR)-like ER kinase (PERK) (Szegezdi et al.,
2006b). The expression of UPR target genes is also
regulated by the transcription factor X-box binding
protein 1 (XBP1) (Szegezdi et al., 2006b).
Upregulation of Manf by ER stress conditions has
been shown by microarray analyses (Lee et al., 2003;
Mizobuchi et al., 2007; Belmont et al., 2008), sug-
gesting that Manf has a role in the quality control of
proteins during ER stress (Lee et al., 2003). Manf was
also upregulated in cell lines by different UPR

inducers (Apostolou et al., 2008). Ischemia is known
to induce ER stress response (Szegezdi et al., 2006a).
In line, hypoxia induced upregulation of Manf in cell
lines in vitro (Romero-Ramirez et al., 2004), and
myocardial infarction upregulated Manf in vivo
(Harpster et al., 2006; Tadimalla et al., 2008). Knock-
down of Armet expression by small interfering RNA
(siRNA) increased the susceptibility of cultured cells
to tunicamycin-induced death, whereas overexpres-
sion of Armet was protective supporting the role for
ARMET/MANF as a protective protein against ER
stress-induced cell death (Apostolou et al., 2008).
Furthermore, recombinant MANF protein protected
cardiac myocytes from ischemia-induced death in
vitro (Tadimalla et al., 2008). In line, MANF protein
protects against ischemic brain injury induced by
middle cerebral artery occlusion (MCAO) in rats in
vivo (Airavaara et al., 2009). The ER stress response
element-II (ERSE-II) is located in the Armet pro-
moter and is shown to regulate Armet expression
(Mizobuchi et al., 2007). In contrast to Manf, Cdnf
(Armetl1) expression was not upregulated by ER-
stressors in vitro, suggesting that CDNF acts constitu-
tively in the ER (Apostolou et al., 2008). ER stress
had only a minor effect on the secretion of MANF
and CDNF proteins in vitro (Apostolou et al., 2008),
suggesting that they have a cell-autonomous role in
the ER stress.
Interestingly, the reduction and rearrangement of
disulfide bonds is a mechanism of controlling protein
function on the cell surface (Hogg, 2003). Cell adhe-
sion, uptake of bacterial toxins and viral fusion with a
host cell membrane may depend on thiol-disulfide
exchange reactions (Hogg, 2003). Oxidoreductases
are secreted and may associate on cell surfaces
(Jordan and Gibbins, 2006). Extracellular redox cata-
lysts have been shown to regulate the functional
forms of cell surface proteins as well as receptor–
ligand interactions. For example, thioredoxin 1
(TRX1) mediates disulfide reduction in CD30 (tumor
necrosis factor receptor superfamily member 8;
TNFRS8), which induces a subsequent conforma-
tional change and affects functional properties of the
CD30 ectodomain thus regulating inflammatory
response (Schwertassek et al., 2007). Extracellular
PDI is known to mediate entry of human immunode-
ficiency virus 1 (HIV-1) into lymphoid cells by cata-
lyzing redox changes of the HIV-1 envelope glyco-
protein gp120 (Hogg, 2003). Whether extracellular or
cell-surface bound CDNF and MANF proteins regu-
late reduction or oxidation of disulfide bonds on cell
surface proteins, e.g., transmembrane receptor
[Fig. 2(C)] is not known. The first studies did not
detect oxidoreductase activity or bound metal ions in
Novel CDNF/MANF Family of NTFs 365
MANF protein (Mizobuchi et al., 2007), but further
analyses are definitely needed.
CDNF AND MANF ACTIVITIES IN VIVO
In addition to the highly potent factors GDNF and
NRTN, which have been extensively studied using
toxin-induced rodent and primate models of PD, few
trophic factors and growth factors, e.g., EPO (Pus-
kovic et al., 2006; Kadota et al., 2009), VEGF family
members (Yasuhara et al., 2005), and FGF family
members (Fontan et al., 2002) induce the recovery of
dopaminergic function in vivo. MANF was discov-
ered based on its survival promoting activity on mid-
brain dopaminergic neurons in vitro (Petrova et al.,
2003). Since the activity of MANF or CDNF in vivo
on dopaminergic neurons had not been studied, we
used a rat 6-OHDA model of PD to test their activ-
ities on dopaminergic neurons (Lindholm et al., 2007,
Voutilainen et al., 2009). The unilateral 6-hydroxydo-
pamine (6-OHDA) rat model of PD is commonly
used for preclinical studies of novel potential thera-
peutic agents. Striatal 6-OHDA injection leads to pro-
tracted retrograde degeneration of the nigrostriatal
pathway (Sauer and Oertel, 1994) [Fig. 3(A)]. When
CDNF (Lindholm et al., 2007) or MANF (Vouti-
lainen et al., 2009) were injected into the striatum
before striatal 6-OHDA, both factors were able to
significantly reduce amphetamine-induced rotational
behavior in rats and protect tyrosine hydroxylase
(TH) positive dopaminergic cell bodies in the SN and
TH-positive fibers in the striatum [Fig. 3(B)]. Impor-
tantly, CDNF and MANF also showed prominent
neurorestorative effects. When either CDNF or
MANF was given into the striatum 4 weeks after a
striatal 6-OHDA lesion, we observed recovery of
motor function in the rats, suggesting that CDNF and
MANF prevented further degeneration of dopaminer-
gic neurons and maintained or increased the function-
ality of remaining neurons [Fig. 3(C,D)]. Thus, we
found that CDNF and MANF very efficiently stopped
the further death of degenerating dopamine neurons.
Presumably they also induced dopaminergic neurite
growth and axonal sprouting. GDNF induces sprout-
ing of striatal dopaminergic fibers and upregulation
of TH expression (Hoffer et al., 1994; Hudson et al.,
1995). Further studies are needed to reveal the effects
of CDNF and MANF on dopaminergic neurites and
TH levels in vivo.
Effects of recombinant MANF protein have also
been studied in an experimental model of stroke in
rats (Airavaara et al., 2009). Administration of
MANF into the cerebral cortex before MCAO was
Developmental Neurobiology
366 Lindholm and Saarma

Figure 3 CDNF and MANF activities in vivo. (A) Unilateral 6-OHDA model of PD. 6-OHDA is
injected (arrow) into the striatum (STR) resulting in a gradual degeneration of nigro-striatal dopaminer-
gic pathway with cell bodies located in the substantia nigra (SN). (B) GDNF and CDNF were able to
protect striatal TH+ fibers (in brown) against unilateral 6-OHDA injection (arrow). Note that the right
hemisphere served as the control. (C, D) CDNF (C) and MANF (D) can restore the motor function in
6-OHDA-lesioned rats. Animals were first injected with 6-OHDA and 4 weeks later with CDNF or
MANF, and GDNF as the control. Striatal injection of CDNF, MANF, and GDNF reduced ampheta-
mine-induced ipsilateral rotational behavior as compared with the control group with maximum effect
measured at 12 weeks postlesion. (E) MANF protein diffuses in the brain more efficiently than GDNF.
(B) and (C) were reproduced with permission of Nature Publishing Group from Lindholm et al. (2007).
(D) was reproduced with permission of the Society for Neuroscience from Voutilainen et al. (2009).
(E) was kindly provided by Merja H. Voutilainen, Susanne Bäck, and Raimo K. Tuominen.

able to significantly reduce the infarction volume and MANF protein added to the culture medium of car-
reduce markers of apoptotic cell death in the ischemic diac myocytes prevented cell death induced by simu-
cortex (Airavaara et al., 2009). In support to that lated ischemia in vitro (Tadimalla et al., 2008). Manf
Developmental Neurobiology

was transiently upregulated in adult rat brain after
status epilepticus (SE) and global forebrain ischemia
in vivo (Lindholm et al., 2008), suggesting that
MANF may regulate neuronal survival and synaptic
plasticity in the cortex and hippocampus.
Studies with Drosophila MANF-deficient flies
indicate that DmMANF, which is expressed in the
glia, functions as a NTF in the fly (Palgi et al., 2009).
The genomic-null flies die in early larval stage, and
mutant embryos show axonal degeneration, cuticular
defects, and nonapoptotic cell death. Importantly,
DmMANF is needed for the maintenance of dopami-
nergic neurites and dopamine levels, whereas moto-
neurons and serotonergic (5-HT) neurons remained
unaffected (Palgi et al., 2009). Human MANF is able
to rescue the fly DmMANF knockout phenotype
(Palgi et al., 2009), suggesting evolutionary con-
served function for MANF protein in the vertebrates
and invertebrates.
NOVEL NEUROTROPHIC FACTORS FOR
DOPAMINERGIC NEURONS
Although extensively studied, the role of NTFs in the
target-innervation of dopaminergic neurons has
remained unclear (Andressoo and Saarma, 2008).
Potential target-derived factors for midbrain dopami-
nergic neurons include GDNF, NRTN, BDNF, NT-4,
and FGF2 (Krieglstein, 2004).
Cdnf and Manf are expressed in the developing
nigro-striatal system of the mouse at P1 and P10
(Lindholm et al., 2007, 2008), that is around the two
postnatal PCD peaks of midbrain dopaminergic neu-
rons at P2 and P14 (Burke, 2004), suggesting that
CDNF and MANF may have a role in the develop-
ment of midbrain dopaminergic system. In the adult
mouse, MANF protein was also localized in TH-
positive dopaminergic neurons in the SN (Lindholm
et al., 2008). Whether MANF or CDNF function as
target-derived NTFs for midbrain dopaminergic neu-
rons is currently unknown. Future studies on CDNF-
and MANF-deficient mice should reveal the develop-
mental role of these new NTFs. The widespread
expression of CDNF and MANF (Lindholm et al.,
2007, 2008) suggests that endogenous CDNF and
MANF secreted by other sources than the striatal tar-
get may affect maturation of rodent dopaminergic
system in vivo.
Whether CDNF and MANF affect PNS neurons or
other neuronal types in addition to dopaminergic neu-
rons and cortical neurons in the CNS in vivo is
unknown. In vitro, CDNF protein was unable to
promote the survival of embryonic motoneurons or
Novel CDNF/MANF Family of NTFs 367
dorsal root ganglion (DRG) neurons, or early post-
natal sympathetic cervical ganglion (SCG) neurons
(Lindholm et al., 2007) differently from GDNF that
supports the survival of different neuronal popula-
tions including motoneurons and enteric, sensory,
parasympathetic, and sympathetic neurons (Airak-
sinen and Saarma, 2002). Similar to the results
obtained with CDNF were also reported for the
MANF protein, although MANF showed a small sur-
vival promoting effect on DRG neurons (Petrova et
al., 2003). Additional studies using, e.g., in vitro
organotypic cultures or in vivo models of neurode-
generation, including models of motoneuron degener-
ation (e.g., ALS models), models of Huntington’s dis-
ease with striatal neurodegeneration, or models of
AD are essential to reveal potential neuronal targets
of CDNF and MANF.
RECEPTOR IS UNKNOWN
Differently from GDNF and NRTN, the target mole-
cules, i.e., putative receptor(s) and signaling path-
ways activated by CDNF and MANF are still
unknown. Whether CDNF and MANF use the same
signaling receptors is also unclear. 6-OHDA is known
to induce oxidative stress, inhibition of mitochondrial
respiratory chain complex I, apoptotic cell death, and
inflammation (Schober, 2004). CDNF and MANF
presumably activate signaling pathways in cells that
counteract some of these phenomena. Interestingly,
in vitro studies have shown that ER stress and UPR
are also related to 6-OHDA toxicity (Silva et al.,
2005). Could extracellularly delivered CDNF and
MANF inhibit ER stress induced by 6-OHDA in the
brain tissue in vivo? 6-OHDA was shown to induce
upregulation of Manf expression in dopaminergic
MN9D cells in culture (Holtz et al., 2005). Whether
6-OHDA treatment affects to endogenous levels of
CDNF or MANF expression in rat brain in vivo is
under investigation.
Whether the neurotrophic activities of CDNF and
MANF are mediated by the saposin-like domain, the
C-terminal domain or whether both domains are
essential is also unknown. Neurotrophic activities of
SAPLIPs, e.g., prosaposin, saposin C, and a peptide
derived from them have been reported in vitro
(O’Brien et al., 1994, 1995) and in vivo (Kotani et al.,
1996; Liu et al., 2001), suggesting that the neurotro-
phic activity may reside in the N-terminal domain of
CDNF and MANF (Parkash et al., 2009). It is also
possible that the neurotrophic activities and cytopro-
tection against ER stress are mediated by the same
structural motif. Whether the extracellular CDNF and
Developmental Neurobiology
368 Lindholm and Saarma
MANF is able to activate a transmembrane protein
receptor, e.g., through interaction with lipids, and
induce intracellular survival promoting signaling
[Fig. 2(D)] is unclear. Another possibility is that
extracellular CDNF and MANF proteins are endocy-
tosed and subsequently able to induce neurotrophic/
cytoprotective effects inside the cell. We definitely
need additional studies to reveal the mechanisms of
CDNF and MANF neurotrophic and cytoprotective
activities at molecular level and to understand how or
if these activities are related.
POTENTIAL THERAPEUTIC PROTEINS
The development of NTF therapy for neurodegenera-
tive diseases is at an early stage. Several growth fac-
tors have been reported as the survival factors for
midbrain dopaminergic neurons, but only GDNF and
NRTN have well-established neurorestorative effects
in preclinical models of PD. Direct infusion of GDNF
into the putamen of PD patients led to significant
clinical improvement without serious side-effects
(Gill et al., 2003; Patel et al., 2005; Slevin et al.,
2005) and induced sprouting of dopaminergic fibers
(Love et al., 2005). Although the results of these
phase I studies were encouraging, a double blinded
phase II study on PD patients showed low clinical ef-
ficacy of GDNF, and the formation of GDNF function
blocking antibodies (Lang et al., 2006). Thus, GDNF
was withdrawn from clinic by Amgen.
Recent press release on May 27, 2009 by Ceregene
reported clinically modest positive effects of gene
therapy by NRTN (CERE-120) on PD patients after
18 months of treatment. Neurturin expression by
CERE-120 was detected in the targeted putamen, but
no evidence was provided for the retrograde axonal
transport of NRTN to the dopaminergic cell bodies in
the SN, evidently reducing bioactivity of the factor.
Diffusion of GDNF and NRTN are known to be lim-
ited in brain tissue due to the binding to the heparan
sulfate proteoglycans (Bespalov and Saarma, 2007).
It is important to search and study novel NTFs for
midbrain dopaminergic neurons that might give new
therapeutic approaches in the future for the treatment
of neurodegenerative diseases. CDNF and MANF are
definitely potential novel proteins for the treatment of
PD, since in animal models of PD they can protect
and repair very efficiently the nigro-striatal dopami-
nergic neurons. An important additional aspect is that
MANF readily diffuses in the brain (Voutilainen et
al., 2009), and even more efficiently than GDNF
[data obtained by Merja H. Voutilainen, Susanne
Bäck, and Raimo K. Tuominen; Fig. 3(E)]. Since
Developmental Neurobiology
MANF is slightly more basic than CDNF, we assume
that CDNF should diffuse even better than MANF in
the brain tissue. The efficient diffusion of MANF may
be of critical importance for its possible clinical use,
since growth factors delivered to human brain have to
diffuse significant distances to reach all target cells.
Indeed, the low benefit of GDNF and NRTN observed
in the recent clinical trials of PD may at least partially
result from their limited tissue distribution.
Although CDNF and MANF have potent effects
on adult midbrain dopaminergic system (Lindholm et
al., 2007, Voutilainen et al., 2009) and cortical neu-
rons in the ischemia model (Airavaara et al., 2009), it
is currently unknown which other CNS or PNS neuro-
nal cell types or non-neuronal cells CDNF and
MANF may affect in vivo. GDNF was originally
introduced as a specific NTF for midbrain dopaminer-
gic neurons (Lin et al., 1993), but it is now clear that
the claim of specificity is not valid. Further preclini-
cal studies are needed to assess the potency of CDNF
and MANF as therapeutic agents for PD and other
neurodegenerative diseases, and their possible side
effects.
The biology of novel CDNF/MANF proteins is
starting to be revealed. Gene ablation studies will pre-
sumably elucidate the roles of CDNF and MANF
in vivo. It will be exciting to see what are the molecu-
lar interactions and signaling pathways related to the
potential neurotrophic and cytoprotective activities of
CDNF and MANF.
The authors thank Dr. Veli-Matti Leppänen and Vimal
Parkash for providing the original structural images. They
are indebted to Dr. Raimo K. Tuominen, Susanne Bäck,
and Merja H. Voutilainen for making available unpublished
data.
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