Neuropsychobiology
Research Article
Neuropsychobiology
DOI: 10.1159/000540673
Received: March 7, 2024
Accepted: July 29, 2024
Published online: September 6, 2024
Lower Plasma Levels of Selective VGF
(Non-Acronymic) Peptides in Bipolar Disorder:
Comparative Analysis Reveals Distinct Patterns
across Mood Disorders and Healthy Controls
Cristina Coccoa Barbara Nolia Barbara Manconib Cristina Continic
Elias Mancad Claudia Pisanua Anna Melonia Mirko Manchiac, e, f
Pasquale Paribelloc, e Caterina Chillottig Raffaella Ardaug
Giovanni Severinoa Alessio Squassinaa, h
aDepartment of Biomedical Sciences, University of Cagliari, Cagliari, Italy; bDepartment of Life and
Environmental Sciences, University of Cagliari, Cagliari, Italy; cDepartment of Medical Sciences and Public Health,
University of Cagliari, Cagliari, Italy; dTurku Bioscience Centre, University of Turku and Åbo Akademi University,
Turku, Finland; eClinical Psychiatry Unit, University Hospital Agency of Cagliari, Cagliari, Italy; fDepartment of
Pharmacology, Dalhousie University, Halifax, NS, Canada; gClinical Pharmacology Unit, University Hospital Agency
of Cagliari, Cagliari, Italy; hDepartment of Psychiatry, Dalhousie University, Halifax, NS, Canada
Keywords
AQEE peptide · Bipolar disorder · Depression · NAPP
peptide · TLQP peptide · Biomarkers · VGF
Abstract
Introduction: Discriminating bipolar disorder (BD) from major
depressive disorder (MDD) remains a challenging clinical task.
Identifying specific peripheral biosignatures that can differen-
tiate between BD and MDD would significantly increase diag-
nostic accuracy. Dysregulated neuroplasticity is implicated in BD
and MDD, and psychotropic medications restore specific dis-
rupted processes by increasing neurotrophic signalling. The
nerve growth factor inducible vgf gene (non-acronymic) en-
codes a precursor protein named proVGF, which undergoes
proteolytic processing to produce several VGF peptides, some of
which were suggested to be implicated in mood disorders and
have antidepressant effects. Since the presence of VGF peptides
in humans has been exclusively investigated in brain and ce-
rebrospinal fluid, we aimed to identify which VGF peptides are
present in the plasma and to investigate whether their levels
could differentiate BD from MDD as well as responders from
non-responders to pharmacological interventions. Methods:
VGF peptides were investigated in plasma from patients diag-
nosedwithMDD (n= 37) or BD (n= 40 under lithiumplus n= 29
never exposed to lithium), as well as healthy controls (HC;
n = 36). Results: Three VGF peptides (TLQP-11, AQEE-14, and
NAPP-19) were identified using spectrometry analysis of plasma
from HC. These peptides were then measured in the entire
sample using ELISA, which showed significantly lower levels of
AQEE and NAPP in BD than in HC and MDD (p = 5.0 × 10−5, p =
0.001, respectively). Conclusion:Our findings suggest that lower
plasma levels of NAPP and AQEE are specifically associated with
BD, thus possibly representing a diagnostic biomarker in mood
disorders. © 2024 The Author(s).
Published by S. Karger AG, Basel
karger@karger.com
www.karger.com/nps
© 2024 The Author(s).
Published by S. Karger AG, Basel
Correspondence to:
Cristina Cocco, crcocco @ unica.it
Alessio Squassina, squassina @ unica.it
This article is licensed under the Creative Commons Attribution-
NonCommercial 4.0 International License (CCBY-NC) (http://www.
karger.com/Services/OpenAccessLicense).Usage anddistribution for
commercial purposes requires written permission.
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Introduction
Mood disorders, including bipolar disorder (BD) and
major depressive disorder (MDD), are characterised by
longitudinal occurrence of mood episodes, alternating from
depression to hypo/mania in BD or exclusively of de-
pressive polarity in MDD. According to the Global Burden
of Diseases, MDD and BD are among the leading causes of
disease burden worldwide [1]. The combined 12-month
prevalence estimate of these two disorders is higher than
9% [2]. Accurate differential diagnosis of MDD and BD
remains difficult in clinical practice because, in two-thirds
of the patients with BD, the onset is of depressive polarity;
episodes of opposite polarity occur later in the course of the
illness andmight not be recognised, especially if hypomanic
[3]. Thus, misdiagnosis betweenMDD and BD is common,
leading to ineffective treatment, which in turn might
further worsen long-term outcomes [4]. However, no
validated markers, including peripheral biomarkers, can
discriminate BD from MDD with accuracy.
Evidence suggests that altered synaptic plasticity might
be implicated in mood disorders [5]. Indeed, it has been
shown that depression is correlated with neuronal at-
rophy in limbic and cortical regions responsible for mood
and emotion control [6]. Some studies have shown that
antidepressant therapy can increase neuroplasticity and
reverse neuroanatomical changes observed in depression
[7], while lithium, the first-line treatment in BD, has been
proven to exert neuroprotection and increase neuro-
plasticity [8]. This effect seems to be mediated by in-
creased levels of neurotrophic factors, particularly brain-
derived neurotrophic factor (BDNF) [7, 8]. Interestingly,
the vgf gene (non-acronymic) has been shown to be one
of the BDNF targets [9], and its encoded polypeptide
protein (VGF non-acronymic) has been found to induce
antidepressant-like effects in rats through the enhance-
ment of proliferation in the hippocampus [10]. VGF is a
neuroprotein precursor of 615 amino acids in humans
[11], secreted in different brain regions, including the
hippocampus [10]. The non-acronymic name used for
VGF originates from the procedures through which it was
identified since the clone was selected from plate “V” of a
rat pheochromocytoma PC12 cDNA library [12, 13].
The VGF neuroprotein is also named proVGF or VGF
precursor because it contains cleavage sites for neuro-
endocrine prohormone convertases leading to a variety of
peptides (named by their 4 N-terminal amino acids and
length) some of these with reported bioactivity each with
specific tissue expression and modulations, including
IEHV [14–16], TLQP [17, 18], NAPP [19, 20], and AQEE
peptides [20]. Different modifications of the VGF, its
mRNA or even its cleaved peptides have been implicated
in pathways connected to mood disorders. For instance,
overexpression of the VGF protein in mice causes the
opposite effects from vgf gene ablation, resulting in pro-
depressant or antidepressant behaviour in the dorsal
hippocampus or nucleus accumbens, respectively [21].
Another study showed that vgf mRNA levels were sig-
nificantly reduced in human leukocytes from drug-free
depressed patients compared to controls, while exposure
to antidepressants restored its expression [22]. Further-
more, vgf mRNA was found to be downregulated in BD
patients in the CA region of the hippocampus and
Brodmann’s area 9 of the prefrontal cortex [23].
The fact that the proVGF is related to mechanisms
involved in mood disorders has stimulated the investi-
gation of its potential role as a blood biomarker for MDD
and BD. In this regard, a recent study showed that VGF
levels were found to be lower in drug-free MDD and
higher in BD patients compared with controls [24].
Similarly, another study showed lower serum VGF levels
in MDD patients with an increase following therapy [25,
26] and an inverse link to suicide risk [27]. These results
were obtained using ELISA with antibodies against the
C-terminus [24] and N-terminus [25–27] of the proVGF-
, which were non-specific to any known VGF peptide.
Interestingly, two antibodies, anti-TLQP-62 and AQEE-
30, have been reported to have antidepressant activity in
previous investigations [21] and the peptides used for
their production have been well characterised by pro-
teomic analyses in human cells [28] and CSF [29, 30].
However, despite these findings, whether these or other
VGF peptides are detectable in peripheral blood has yet to
be elucidated. Based on the findings reported above, we
hypothesise that VGF peptides might indirectly or di-
rectly play a role in mood regulation and that specific
VGF peptides might be implicated in BD and MDD as
well as in modulating treatment response.
To test this hypothesis we performed a study to
characterise all naturally occurring VGF peptides in
human plasma from a cohort of healthy controls (HCs)
and patients with a diagnosis of mood disorders (BD and
MDD) characterised for response to pharmacological
treatments.
Material and Methods
Sample
The cohort comprised 69 patients with BD and 37 patients with
MDD recruited at the community mental health centre at the
Psychiatry Unit of the Department of Medical Science and Public
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Health, University of Cagliari and University Hospital Agency of
Cagliari, and the Unit of Clinical Pharmacology, University
Hospital Agency, Cagliari. The recruitment process was based on
the inclusion and exclusion criteria described elsewhere [31].
Briefly, the diagnosis was made according to the Diagnostic and
Statistical Manual of Mental Disorders IV (DSM-IV) criteria and
Schedule for Affective Disorders and Schizophrenia Lifetime
(SADS-L) (BD patients), and Structured Clinical Interview for
DSM-IV-TR Axis I Disorders (SCID) (MDD patients). Exclusion
criteria comprised the presence of acute infections, diagnosis of
eating disorders, post-traumatic stress disorder, substance use
disorders, neurological disorders, traumatic brain injury, or severe
medical conditions. In the MDD cohort, treatment resistance was
defined according to the criteria of Souery et al. [32] based on the
clinical course and the assessment of treatment response patterns.
The BD cohort included patients treated with lithium (n = 40) and
patients who were never exposed to lithium but treated with either
valproate or lamotrigine (n = 29). Response to lithium or valproic
was characterised using the Retrospective Criteria of Long-Term
Treatment Response in Research Subjects with BD scale (or ALDA
scale) [33–35]. The scale quantifies the degree of improvement
during treatment with a score from 0 to 10, adjusted for potential
confounders. Patients with a total score of 7 were considered
responders [34, 35]. All BD patients enrolled in the study were
under mood-stabilising treatment at the time of recruitment, while
12 out of the 26 BD patients receiving lithium treatment were
responders. Among the BD patients treated with valproate, 3 were
responders, 21 were non responders, and 4 were not classified. The
cohort under study also included 38 non-psychiatric, HC subjects
with no personal or familial history of psychiatric disorders in first-
degree relatives. HCs were recruited based on the same exclusion
criteria of the patients and were administered the Italian version of
the SCID-I/NP 26 to rule out the presence of Axis I psychiatric
disorders. Patients and controls were all recruited from Sardinia
(Italy) and were Caucasian and of Italian origin. Characteristics of
the patients and controls are reported in Table 1. Physical activity
was defined as any sports activity performed at least once a week;
cardio-metabolic disorders included cardiovascular diseases,
obesity and diabetes mellitus type 2 while smoking status was
defined as smoking regularly. Fasting blood was collected in the
morning using tubes containing ethylenediamine tetra-acetic acid
(EDTA, 1.5 mg/mL). Blood samples were centrifuged (3,000 g,
10 min), and, after addiction to extraction solution (PBS plus 5 μL/
mL protease inhibitor cocktail: Sigma P8340), were frozen. At
blood drawn, patients with BD andMDDwere in euthymic phases
with an interval of at least 6 months from the last mood episode
meeting diagnostic criteria.
Nano-RP-HPLC-High Resolution ESI-MS/MS Analysis
The characterisation of the VGF peptides was obtained by
nano-reverse-phase high-performance liquid chromatography
(nano RP-HPLC) with Ultimate 3,000 Nano System HPLC (Di-
onex-Thermo Fisher Scientific) coupled with a high-resolution
mass spectrometer (ESI-HR-MS) LTQ Orbitrap Elite (Thermo
Fisher Scientific) on a pooled plasma sample obtained by mixing
2 μL from 5 HC samples (4 males, 1 female, age ± standard de-
viation 70 ± 8, 60 μg/μL of total protein concentration each).
Following the manufacturer’s instructions, the pooled plasma
sample was then depleted of albumin and IgG using the High
Select™ Depletion Spin Columns (Thermo Fisher Scientific,
Waltham, MA, USA). After the depletion, the total protein con-
centration was determined by the bicinchoninic acid assay kit
(Sigma-Aldrich/Merck, Darmstadt, Germany), following the
manufacturer’s instructions. Finally, the sample was dried and
resuspended in aqueous formic acid (FA) 0.1% v/v with a final
protein concentration of 0.1 μg/μL prior the RP-nanoHPLC-ESI-
HR-MS/MS analysis. The injection volume was 10 μL, and the
column was a C18 Easy Spray reverse-phase nano-column
(250 mm × 75 μm inner diameter I.D., Thermo Fisher Scien-
tific) with 2 μm beads. The elution of proteins and peptides was
achieved with aqueous solvent A (0.1% FA) and aqueous solvent B
(0.1% FA, 80% Acetonitrile v/v) in 50 min at a flow rate of 0.3 μL/
min with the following gradient: 0–3min at 4% B, 3–30min 4–80%
B, 30–31 min 80–90% B, 31–41 min 90% B, 41–45 min 4% B,
45–50 4% B. The mass spectrometer operated at 1.6 kV in the data-
dependent acquisition mode, with the capillary temperature set at
275°C and S-Lens RF level 68.0%. Complete MS experiments were
performed in positive ion mode from 350 to 2,000 m/z with a
resolution of 60,000 (at 400 m/z). The 10 most intense ions were
subjected to Collision Induces Dissociation (CID) or Higher-
Energy Collisional Dissociation (HCD) fragmentation by setting
35% of normalised collision energy for 20 ms, isolation width of
2 m/z and activation q of 0.25. Only single-charged ions were
excluded from the fragmentation process, and when required, a
Full MS window in a range of 100m/z centred on the ion of interest
was set to improve the quality of fragmentation by applying the
same parameters described above. The synthetic TLQP, AQEE,
and NAPP peptides (from Pepmic Co., Suzhou, China) corre-
sponding to the aminoacidic sequence of TLQP-62 from rats and
AQEE-30 and NAPP-19 from humans were analysed (200 fmol) as
standard reference under the same analytical conditions. (HR)-MS
and MS/MS data were generated by Xcalibur 2.2 SP 1.48 (Thermo
Fisher Scientific, CA, USA) and deconvoluted by using the Xtract
algorithm available in FreeStyle (version 1.8.63.0, Thermo Fisher
Scientific, CA, USA) with the following settings: 44% fit factor, 25%
remainder threshold, minimum intensity set to 1, expected in-
tensity error set to 3, and S/N threshold set to 2. The charac-
terisation of TLQP, AQEE, and NAPP peptides in plasma from
controls was achieved by comparing the theoretical MS/MS
fragmentation generated in silico by the MS-Product tool available
at the Protein Prospector website (http://prospector.ucsf.edu/
prospector/mshome.htm), setting a fragment tolerance of 300
ppm, ESI-FT-ICR-CID as MS/MS fragmentation instrument,
search of b and y.
Fragment ions, neutral losses of H2O and NH3, concerning the
undeconvoluted MS/MS spectra obtained experimentally for each
peptide of interest. Furthermore, the Peptide Fragments Anno-
tation tool available in FreeStyle was also used to annotate the same
experimental undeconvoluted MS/MS spectra.
VGF Antibody Production and Validation
The NAPP-9 peptide (human VGF 485–493) (CPC Scientific, San
Jose, CA, USA) was conjugated at its N-terminus with keyhole
limpet haemocyanin while the TLQP-10 peptide (human VGF
554–563) was synthesised (CPC Scientific) and conjugated at its
C-terminus for immunisations in guinea pigs and rabbit, re-
spectively. Antibodies were raised in rabbits against the AQEE-10
peptide at the proVGF 586–595 position (Affiniti Research
Products, Enzo Life Sciences, UK), conjugated to bovine thyro-
globulin via an additional cysteine at its C-terminus. All antibodies
Association of Lower Plasma Levels of
NAPP and AQEE with BD
Neuropsychobiology
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were affinity purified by incubation with the corresponding im-
munogen antigen covalently immobilised on a sulfolink coupling
resin (Thermo Fisher Scientific) and eluted in buffer glycine-HCl
1 M pH 2.5, after several rinses with phosphate buffer saline (PBS)
0.5 M (online supplementary material (for all online suppl. material,
see https://doi.org/10.1159/000540673; Fig. 1). Antibody validation
was carried out using either ELISA orWestern blot (WB). In ELISA,
each VGF antibody was matched to either the appropriate antigen-
peptide or other extended peptides (varying for the amino acid
counts) in order to assess antibody reactivity. Among the extended
forms, we employedNAPP-19 (CPC Scientific; [30]) as well as tVGF
peptides with demonstrated biological activity, as the TLQP-21 [36]
(CPC Scientific), TLQP-62 (Pepmic Co.) [37], AQEE-30 (Pepmic
Co.) [21], and TLQP-62 that were also included in the AQEE assay
to examine antibody cross-reactivity. Intra- and inter-assay (CV1
and CV2) and detection limit (DL) were obtained for each assay.
Every VGF antibody was examined inWB using PC12 cells (thanks
to Prof. R. Possenti), either expressing proVGF or not (PC12+ or
PC12−, respectively), as well as using TLQP-62 and AQEE-30
synthetic peptides (since short MWpeptides are scarcely retained in
WB). For WB analysis, samples were diluted in 2xSDS buffer
(Origene) to load 20 μg of each sample and then were heated to 95°C
for 5 min and subjected to SDS-PAGE using precast polyacrylamide
gradient gel (NuPAGE 4–12% Bis-Tris, Thermo Fisher Scientific)
for 20 min at 200 Volt. Internal MW standards (PageRuler Plus
prestained protein ladder 10 to 250 KDa, Thermo Scientific) were
run in parallel. Samples were transferred onto a polyvinylidene
difluoride (PVDF) membrane (Amersham Hybond-P, GE
Healthcare) for 1 h at 20 Volt. Blots were blocked by immersion in
50 mM Tris base and 150 mM Sodium Chloride (TBS) containing
0.1% Tween-20 (TBS-T) and 5% bovine serum albumin (BSA) for
1 h at room temperature. Incubation with rabbit anti-AQEE, sheep
anti-TLQP and rabbit anti-NAPP primary antibodies (1:500
common dilution in the same blocking buffer) was carried out for 1
night at 4°C. The next day, blots were rinsed 4 times with TBS-T and
incubated at room temperature for 1 h with horseradish peroxidase-
conjugated goat anti-rabbit or donkey anti-sheep secondary anti-
bodies (Jackson ImmunoResearch) diluted 1:10,000 in TBS-T. After
several washing with TBS-T, protein bands were developed using
Thermo Scientific Pierce Enhanced chemiluminescence (ECL) WB
substrate. The ImageQuant LAS-4000 was used to detect the
chemiluminescence. To reveal which peptides, among the others,
were identified by the appropriate antibody, we used dot-blot (DB)
with the same VGF synthetic peptides used in WB or ELISA. For
Table 1. Diagnostic groups and control subjects
Variables BD (n = 69) MDD (n = 37) HC (n = 38) Statistics
Gender (M/F) 29/40 12/25 21/15 χ2 = 5.12; p = 0.77
FH (Y/N/U) 35/33/1 20/13/4 0 χ2 = 1.4; p = 0.50
Suicide attempt history (Y/N/U) 17/52 6/30/1 0 χ2 = 0.87; p = 0.46
Smoke (Y/N/U) 33/32/3 18/19 13/23 χ2 = 2.29; p = 0.32
Cardio-metabolic disorders (Y/N) 17/52 10/27 8/28 χ2 = 0.27; p = 0.87
Physical activity (Y/N) 26/40 13/24 25/11 χ2 = 10.88; p = 0.004a
BMI (means ± SD) 27.8 ± 6.4 25.3 ± 5.1 23 ± 3.5 F = 9.23; p = 0.0002b
Age (means ± SD) 52.1 ± 9.9 51.2 ± 12.9 43.3 ± 10.7 F = 8.10; p = 0.0004c
Age at disease onset (means ± SD) 27.6 ± 9.9 35.0 ± 13.4 / t = −2.96; p = 0.004
Years of illness (means ± SD) 24.5 ± 11.6 16.3 ± 12.7 / t = 3.38; p = 0.001
Mood episodes (means ± SD), N 16.5 ± 12.7 3.2 ± 2.3 / t = 6.99; p = 2.86e−9
Patients under Li 40 1 / /
Duration of Li treatment (means ± SD), years 16.5 ± 10.3 / / /
Duration of VPA treatment (means ± SD), years 18.6 ± 16.9 / / /
Duration of AD treatment (means ± SD), years / 3.1 ± 2.9 / /
VPA responders/non-responders 3/21 / / /
Li responders/non-responders 12/26 / / /
Treatment-resistant depression/responders / 10/27 / /
BD, bipolar disorder; MDD, major depressive disorder; HC, healthy controls; Li, lithium; VPA, valproic acid; AD, antidepressants; M,
males; F, females; FH, family history of psychiatric disorders; Y, yes; N, no; BMI, body mass index; SD, standard deviation; N, number;
AD, antidepressants. Bold statistics are significant at a p value threshold of 0.05. Significance between aBD versus HC and MDD
versus HC; bBD versus HC; cBD versus HC and MDD versus HC.
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DB, 4 μL of each synthetic peptide was applied on PVDF mem-
branes, where it was let to spread and dry for 30 min at room
temperature. Following their blocking, the following steps were
carried out in accordance with the WB protocol.
Competitive ELISA
To measure the VGF levels in plasma samples, each VGF
peptide was coated on a separate plate (Nunc Thermo Scientific,
Milan, Italy) for 3 h at room temperature; then the plates were
treated with blocking buffer (PBS containing 90 mL/L normal
serum from donkey, 20 nmol/L aprotinin, and 1 g/L EDTA) for 2
h. Primary incubations using the corresponding VGF antibodies
diluted in blocking buffer, were carried out in triplicate, including
both serial standard synthetic peptide dilutions or plasma samples
(1:10). Biotinylated secondary antibodies (1 h, 1:10,000 dilution in
blocking buffer; Jackson, West Grove, PA, USA), streptavidin-
peroxidase conjugate (30 min, 1:10,000 dilution in PBS; Biospa,
Milan, Italy), and tetramethyl benzidine (X-tra Kem-En-Tec,
Taastrup, Denmark) were used to reveal the positive labelling. The
reaction was stopped with hydrogen chloride (1 mol/L), and the
optical density was measured at 450 nm using a multilabel plate
reader (Chameleon: Hidex, Turku, Finland).
Fig. 1. a Each antibody was tested for reactivity against its
corresponding antigen-peptide (in bold, used for immunisa-
tion) or longer peptides. The TLQP-62 was also used to test the
AQEE antibody, although the antibody’s reactivity was rather
low. R stands for reactivity, DL for detection limit, and CV1 and
CV2 for intra- and inter-assay. bUsingWB, the 70 kDa proVGF
band (inside the black rectangle) was labelled by all VGF an-
tibodies using PC12+ but not with PC12− cells. The AQEE-30
and TLQP-62 synthetic peptides were labelled by their paired
antibodies, revealing bands corresponding to the appropriate
peptide MW bands. c Using DB analysis, each VGF antibody
recognised the peptide that matched it but not the others.
d Graphs showing NAPP, AQEE, and TLQP peptide levels in
the studied groups. BD, bipolar disorder, MDD, major de-
pressive disorder; HC, healthy controls; ns, not significant; Abs,
antibodies. *p < 0.05.
Association of Lower Plasma Levels of
NAPP and AQEE with BD
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Statistics
The normality of distribution was tested with Shapiro-Wilk
and the presence of outliers with the Grubbs test. Plasma levels
of the AQEE, TLQP, and NAPP peptides were not normally
distributed (Shapiro-Wilk p < 0.001), and as such, nonpara-
metric tests were used for all the analyses. Differences among
diagnostic and response groups were tested with the χ2 test for
discrete variables or with a t test for continuous variables
(Table 1). The predictive value of the peptides in discriminating
diagnostic groups was tested using the Receiver Operating
Characteristic (ROC) curve.
The effect of discrete variables on plasma levels of VGF peptides
was tested with the Mann-Whitney U test, while the effect of
continuous variables was tested with Spearman correlation.
Considering none of the discrete variables influenced the peptide
levels, we used the Quade nonparametric ANCOVA to test dif-
ferences in VGF peptide levels among groups accounting for the
effect of covariates. Only variables showing different distributions
among groups or affecting plasma levels of VGF peptides were
included as covariates in the analytical models. The analyses were
conducted using IBM SPSS Statistics v. 28 (IBM Corporation,
Armonk, NY, USA) and Graphpad Prism V. 9 (GraphPad Soft-
ware, San Diego).
Results
Qualitative Analysis of the VGF Peptides
The qualitative analysis of the VGF peptides allowed
characterisation of three peptides: (i) the AQEE peptide
of 14 amino acids in position 585–598, corresponding to
the sequence RAQEEAEAEERRLQ (theoretical mono-
isotopic [M + 1H]+1 mass 1,714.85, experimental
monoisotopic [M + 1H]+1 mass 1,714.89); (ii) the
NAPPEPVPPPRAAPAPTHV peptide of 19 amino acids
in position 485–503 (theoretical monoisotopic [M +
1H]+1 mass 1,915.02, experimental monoisotopic [M +
1H]+1 mass 1,914.96); and (iii) the RTLQPPSALRR
peptide of 11 amino acids in position 553–563 (theo-
retical monoisotopic [M + 1H]+1 mass 1,294.77, ex-
perimental monoisotopic [M + 1H]+1 mass 1,294.77).
The structural characterisation of these three peptides is
reported in the online supplementary materials (online
suppl. Fig. 1–4; Tables 1–3).
The synthetic AQEE-30 (theoretical monoisotopic [M
+ 1H]+1 mass 3,706.84 from human) and TLQP-62
(theoretical monoisotopic [M + 1H]+1 mass 7,397.75
from rat) peptides were analysed by nano-RP-HPLC-
High Resolution ESI-MS/MS, which allows to detect
AQEE-30 at the retention time 28.6–29.3 min and TLQP-
62 at the retention time 26.0–28.0 min. Based on this
reference, we searched for the same two peptides in
human plasma by both untargeted and targeted analysis,
but were unable to detect them.
VGF Antibody Reactivity
When antibody reactivity was investigated using
ELISA (Fig. 1a), the NAPP antibody almost identically
recognised the NAPP-9 and -19; the TLQP antibody
labelled the TLQP-10, -21, and -62 (with a greater
reactivity degree for TLQP-21 and less for TLQP-62);
and the anti-AQEE antibody similarly recognised both
the short and extended AQEE forms, but not the
TLQP-62. For each assay, the values of DL, CV1, and
CV2 displayed accurate data. In WB (Fig. 1b), all VGF
antibodies, as predicted, labelled the proVGF (70 kDa
band) and other smaller bands corresponding to
cleaved peptides. The anti-TLQP and AQEE antibodies
labelled the TLQP-62 and AQEE-30 synthetic pep-
tides, respectively, with higher affinity when compared
to their reactivities against the proVGF. In DB
(Fig. 1c), the NAPP antibody labelled the NAPP-19
only, the TLQP antibody selectively identified TLQP-
62 while the AQEE antibody exclusively recognised the
AQEE-30.
VGF Peptide Levels
As mentioned in the methods section, the levels of
the three studied peptides were not normally distrib-
uted across the entire sample, as indicated by the
Shapiro-Wilk test (p < 0.05 for all peptides; online
suppl. material Fig. 5). A lack of normal distribution
could be expected in this context since the cohort in-
cluded patients with different diagnoses as well as HCs,
thus contributing to heterogeneity and skewed distri-
bution of the molecular variables, especially if these
variables are correlated with some of the studied traits
but not others (i.e., diagnosis).
The Grubbs test identified 8 outliers: one outlier for
AQEE, two outliers for NAPPE, one outlier for LTQP in
the MDD group, two outliers for NAPPE, and one
outlier for TLQP in the BD group, and one outlier for
TLQP in the HC group. These samples were removed
from the analyses.
The diagnostic groups differed in several features, as
reported in Table 1. TLQP, AQEE, and NAPP peptides
were significantly correlated with each other. Age was
significantly correlated with AQEE (rho = −0.215, p =
0.012), TLQP (rho = −0.231, p = 0.007), and NAPP
(rho = −0.225, p = 0.009), while body mass index (BMI)
was correlated with TLQP (rho = −0.273, p = 0.002) and
NAPP (rho = −0.216, p = 0.013). Years of illness were only
correlated with AQEE (rho = −0.316, p = 0.001) and the
number of episodes with AQEE (rho = 0.381, p < 0.001)
and NAPP (rho = −0.430, p < 0.001). The effect of these
variables was tested in each model. None of the discrete
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variables influenced plasma levels of any of the peptides.
The 2 samples of BD patients (treated with lithium or
other mood stabilizers) did not differ for any of the tested
variables or levels of AQEE, TLQP, and NAPP. As such,
the two samples were combined in the following analyses.
As shown in Table 1, the BD and MDD patients differed
from the HC group in terms of physical activity and age,
while BD patients had significantly higher BMI than both
MDD and HC groups. The Quade test was statistically
significant for NAPP (covariates: age, BMI and physical
activity; model F = 12.25, p = 0.00001) and for AQEE
(model F = 17.78, p = 1.49e−7), with lower levels in
patients with BD compared to those with MDD and
controls, while patients with MDD did not differ from
controls (Table 2; Fig. 1d). TLQP showed no difference
among diagnostic groups (model F = 0.201, p = 0.818).
The ROC curve had a specificity of 0.77 and sensitivity of
0.97 for NAPPE (area under the curve = 0.74, CI:
0.65–0.84), and a specificity of 0.85 and sensitivity of 0.97
for AQEE (area under the curve = 0.80, CI: 0.71–0.89),
suggesting a better classification performance of AQEE
(ROC curves reported in online suppl. material Fig. 6).
We then compared patients with treatment-resistant
depression with responders to antidepressants as well as
patients responders to lithium with non-responders
showing no significant differences among the studied
groups. Response to valproate was not included in the
analyses since only 3 patients resulted responders to the
treatment. Considering that a small proportion of MDD
patients was not under pharmacological treatment at
recruitment (5 patients), we also tested whether AQEE
was associated with being exposed or not to antide-
pressants, reporting no significant differences.
Discussion
Through the use of a top-down mass spectrometry
analysis, our study was able to show, for the first time, that
AQEE-14 and TLQP-11 are present in human blood and
also confirmed the presence of NAPP-19 [19]. Interestingly,
when compared to HC levels of AQEE and NAPP peptides
were significantly lower in patients with BD but not in those
with MDD while there was no difference for TLQP. The
latter finding does not confirm previous evidence suggesting
that TLQP-62 could be involved in depression [21].
However, it is noteworthy that the anti-TLQP antibody can
identify a range of TLQP peptides with varying lengths,
including TLQP-62. Using HPLC-MS/MS analysis, we were
only able to prove the presence of TLQP-11 but not of
TLQP-62. Nevertheless, none of the previous proteomic
studies identified TLQP-62 or any other TLQP peptide in
human plasma. Therefore, based on our findings and the
currently available evidence, we may speculate that the
TLQP-62 is either not present in human plasma or its levels
could be below the sensitivity thresholds of our techniques.
In our study, NAPP and AQEE peptides were both lower
in BD patients compared to MDD and HC. Previous works
showed that NAPP peptides are present in CSF [30, 31]
other than in plasma, and while a previous study reported
lower levels of the two peptides in obese people [19], to date,
there is no evidence of their involvement in psychiatric
diseases. On the other hand, the association of AQEE
peptides with mood disorders has been widely reported.
Neuronal biological activity has been demonstrated for
AQEE-30, affecting the synaptic activity and depressed
behaviour in animals [21], while an acute antibody-
mediated AQEE peptide sequestration has been shown to
have a pro-depressant effect in dorsal hippocampus [21].
Considering that both vgf mRNA and AQEE-11 peptide
have been reported to be lower in brain and CFS from BD
patients [23, 38] it could be speculated that the differences in
AQEE plasma lavels observed in plasma from BD indi-
viduals might be correlated with AQEE changes in the
brain. Our findings are in contrast with previous studies
showing that, compared to controls, serumVGF levels were
higher in BD patients and lower MDD patients [24–26].
However, these investigations used different VGF anti-
bodies compared to our study, such as those directed against
the C-terminus amino acid sequence, which spans from 591
Table 2. Results of the pairwise
comparisons with Quade’s analysis
of covariance controlling for age,
BMI, and physical activity
Subject groups NAPPE AQEE TLQP
t p value t p value t p value
BD versus MDD −4.592 0.00001 −5.058 1.0e−5 −0.455 0.650
BD versus NPC −3.380 0.001 −4.784 5.0e−5 −0.578 0.564
MDD versus HC 1.072 0.286 0.203 0.839 −0.114 0.910
BD, bipolar disorder; MDD, major depressive disorder; HC, healthy controls.
Association of Lower Plasma Levels of
NAPP and AQEE with BD
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to 610 [24] or the N-terminus amino acid sequence, which
spans from 23 to 174 [25, 26]. Specifically, while it is possible
that TLQP-62, NAPP-129, and AQEE-30 were recognised
by the antibody employed by Chen et al. [24], the anti-
bodies’ specificity for these peptides – as well as for other
VGF peptides – was not examined specifically. Instead, we
used antibodies specifically designed to be directed against
three different VGF peptides, including those with anti-
depressant activity, and demonstrated their presence in
plasma using the spectrometry technique.
Our findings should be viewed in the context of some
limitations. While we applied a stringent pipeline to purify
and test the antibodies’ specificity (for instance, we showed
that the anti-AQEE antibody does not react with TLQP-62),
the NAPP and AQEE antibodies we used could not dis-
tinguish between VGF peptides with similar sequences (for
instance, the anti-NAPP antibody could not distinguish
between NAPP-19 and NAPP-9, which have the same
NAPP sequence but different amino acid counts). This
limitation arises from the fact that antibodies, by nature, are
unable to differentiate between peptides or proteins that are
very similar. Furthermore, because of our incomplete
knowledge of proVGF processing mechanisms, we were
unable to measure the amount of similar VGF-cleaved
peptides with overlapping sequences that would be pres-
ent in blood. Nevertheless, even considering the possible
limitations of our methods, only AQEE-14 and NAPP-19
(and not other smaller or longer AQEE or NAPP peptides)
were identified by our analysis of the plasmatic proteome.
Hence, we can assume that in ELISA, our antibodies
specifically labelled the plasmatic AQEE-14 and NAPP-19.
A second limitation of our study was its retrospective design
with a single-time point observation, which made not
possible to explore temporal changes in VGF peptides levels
at different time points, which would have allowed us to test
their correlation with longitudinally assessed features, such
as pharmacological interventions. The strengths of our
study consist in the accurate clinical characterisation of the
cohorts of patients for whom information on response to
treatment was available, thus allowing us to explore the
effect of exposure to treatments at the time of recruitment as
well as the correlation between VGF peptides and response
or resistance to pharmacological interventions, something
that has been scarcely investigated before.
In conclusion, our research shows that the plasma levels
of the VGF peptides AQEE-14 and NAPP-19 differ sig-
nificantly among patients with a different diagnoses of
mood disorders, suggesting their potential utility as diag-
nostic biomarkers. Moreover, we suggest that VGFmay not
serve as a biomarker of response to lithium. On the other
hand, considering the limitations, more research is needed
to fully understand the pathophysiological processes that
underlie the role of VGF in mental illness and in phar-
macological interventions with psychotropic medications.
Future studies should focus on stringent identification
methods of the different VGF peptides, possibly over-
coming the limitations of currentmethodologies.Moreover,
an effort should be made to design prospective longitudinal
studies, possibly in risk studies, to clearly explore the clinical
implications of VGF in mood disorders research.
Acknowledgments
We acknowledge the CeSAR (Centro Servizi d’Ateneo per la
Ricerca) of the University of Cagliari, Italy for the Nano-RP-
HPLC-High-Resolution ESI-MS/MS experiments performed with
the LTQ Orbitrap Elite (Thermo Fisher Scientific) intrument.
Statement of Ethics
All participants signed informed written consent after a de-
tailed description of the study procedures. The research protocol
followed the principles of the Declaration of Helsinki and was
approved by the Ethics Committee of the University of Cagliari,
Italy (Approval No. 348/FC/2013 and PG/2018/11693).
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
Funding Sources
This work was partly supported with a grant funded by
“Fondazione di Sardegna and Regione Sardegna,” Call 2016,
Project ID: F72F16003090002.
Author Contributions
Barbara Noli and Elias Manca provided ELISA experiments.
Barbara Manconi and Cristina Contini provided proteomic
analysis, Cristina Cocco and Alessio Squassina designed the study;
Claudia Pisanu, Anna Meloni, Mirko Manchia, Pasquale Paribello,
Caterina Chillotti, Raffaella Ardau, and Giovanni Severino pro-
vided patients and their clinical and relevant information. All
authors reviewed the manuscript.
Data Availability Statement
All data generated or analysed during this study are included in
this article and its online supplementary material files. Further
enquiries can be directed to the corresponding author.
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