Spinal injection of newly identified cerebellin-1 and cerebellin-2 peptides induce mechanical hypersensitivity in mice

Accepted Manuscript Spinal injection of newly identified cerebellin-1 and cerebellin-2 peptides induce mechanical hypersensitivity in mice

Katalin Sandor, Shibu Krishnan, Nilesh Mohan Agalave, Emerson Krock, Jaira Villarreal Salcido, Teresa Fernandez-Zafra, Payam Emami Khoonsari, Camilla I. Svensson, Kim Kultima PII: DOI: Reference:

S0143-4179(17)30268-8 doi:10.1016/j.npep.2018.04.004 YNPEP 1859

To appear in:

Neuropeptides

Received date: Revised date: Accepted date:

27 October 2017 9 February 2018 9 April 2018

Please cite this article as: Katalin Sandor, Shibu Krishnan, Nilesh Mohan Agalave, Emerson Krock, Jaira Villarreal Salcido, Teresa Fernandez-Zafra, Payam Emami Khoonsari, Camilla I. Svensson, Kim Kultima , Spinal injection of newly identified cerebellin-1 and cerebellin-2 peptides induce mechanical hypersensitivity in mice. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ynpep(2018), doi:10.1016/j.npep.2018.04.004

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ACCEPTED MANUSCRIPT Spinal injection of newly identified cerebellin-1 and cerebellin-2 peptides induce mechanical hypersensitivity in mice

Katalin Sandora, Shibu Krishnana,b, Nilesh Mohan Agalavea, Emerson Krocka, Jaira Villarreal Salcidoa, Teresa Fernandez-Zafraa, Payam Emami Khoonsarib, Camilla I Svenssona, Kim

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

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Kultimaa,b*

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Department of Medical Sciences, Uppsala University, Uppsala, Sweden

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*Corresponding author: Dr Kim Kultima

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Department of Medical Sciences, Uppsala University Akademiska sjukhuset, SE75185 Uppsala, Sweden

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E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract By screening for neuropeptides in the mouse spinal cord using mass spectrometry (MS), we have previously demonstrated that one of the 78 peptides that is expressed predominantly (> 6-fold) in the dorsal horn compared to the ventral spinal cord is the atypical peptide desCER [des-Ser1]-cerebellin, which originates from the precursor protein cerebellin 1 (CBLN1).

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Furthermore, we found that intrathecal injection of desCER induces mechanical

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hypersensitivity in a dose dependent manner.

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The current study was designed to further investigate the relative expression of other CBLN derived peptides in the spinal cord and to examine whether they share similar nociceptive

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properties.

In addition to the peptides cerebellin (CER) and desCER we identified and relatively

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quantified nine novel peptides originating from cerebellin precursor proteins CBLN1 (two peptides), CBLN2 (three peptides) and CBLN4 (four peptides). Ten out of eleven peptides

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displayed statistically significantly (p<0.05) higher expression levels (200-350%) in the dorsal horn compared to the ventral horn. Intrathecal injection of three of the four CBLN1 and

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two of the three CBLN2 derived peptides induced mechanical hypersensitivity in response to von Frey filament testing in mice during the first 6 hours post-injection compared to saline

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injected mice, while none of the four CBLN4 derived peptides altered withdrawal thresholds. This study demonstrates that high performance MS is an effective tool for detecting novel neuropeptides in CNS tissues. We show the presence of nine novel atypical peptides originating from CBLN1, CBLN2 and CBLN4 precursor proteins in the mouse dorsal horn, whereof five peptides induce pain-like behavior upon intrathecal injection. Further studies are required to investigate the mechanisms by which CBLN1 and CBLN2 derived peptides facilitate nociceptive signal transmission. Keywords: mass spectrometry; peptidomics; neuropeptides; cerebellin; pain; nociception

ACCEPTED MANUSCRIPT Introduction The mechanism of nociception is complex and depend on activation of nociceptors followed by signal processing in the peripheral and central nervous system (CNS). Chronic pain resulting from disease or injury is a major public health problem resulting in an enormous impact and burden on society and individuals. Neuropeptides present in primary sensory

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neurons and the dorsal horn of the spinal cord have an important role in nociceptive signal

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transmission under normal conditions. Subsequent to development of pathological painful

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conditions, such as joint inflammation, peripheral nerve injury and bone cancer, the production of peptides and peptide receptors is dramatically altered, leading to a number of

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functional consequences. There is an increasing interest in developing peptides or targeting their receptors as potential avenues for new pain treatments as peptides present high

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biological activity and specificity. Thus it is critical to advance our understanding of how previously known, as well as novel peptides contribute to nociception in order to identify

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peptides that can form the basis for new strategies for pain relief.

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Technical limitations with classical techniques such as radioimmunoassay, immunohistochemistry and western blotting that are dependent on epitope recognition by

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antibodies put restrains to both detection and quantification of neuropeptides. Mass spectrometry (MS) circumvents some of the limitations as it is based on amino acid-sequence rather than antibody specificity, and peptidomics approaches have been applied to samples from various animal models and human conditions to characterize and quantify novel neuropeptides, as well as known neuropeptides, which has advanced our understanding of nervous system functions over the past decade. By using label-free (LF)-LC-high-resolution (HR) tandem mass spectrometry (MS/MS), quantitation and identification of the precise molecular forms of each peptide without a priori knowledge of the peptide identity have been

ACCEPTED MANUSCRIPT enabled (Adori et al., 2016; Kultima et al., 2009; Romanova and Sweedler, 2015; Su et al., 2014).

We previously characterized neuropeptides in naïve mouse spinal cord in an unconditional fashion. We found that 35 previously characterized neuropeptides and 53 uncharacterized

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peptides are only or predominantly (> 6-fold) expressed in the dorsal compared to the ventral

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horn of the spinal cord (Su et al., 2014). Based on location, the peptides exclusively or

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predominantly present in the dorsal horn represent interesting and potentially novel molecules

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in the transmission of nociceptive signals.

The peptide [des-Ser1]-cerebellin (desCER) that we found expressed at higher levels in the

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dorsal compared to the ventral horn had prior to our work not been associated with modulation of nociception. When administered intrathecally, desCER induces a dose

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dependent mechanical hypersensitivity in mice within the first 6 hours of injection (Su et al., 2014). This peptide originates from the precursor cerebellin 1 (CBLN1) protein, which

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belongs to the CBLN1-4 protein family. The cerebellins were first identified in the cerebellum (Slemmon et al., 1984) and characterized as glycoproteins which are organized into homo- or

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heterotrimer forms (Bao et al., 2005). It has later been shown that CBLN1, CBLN2 and CBLN4, but not CBLN3 are widely expressed in other parts of the nervous system (Miura et al., 2006; Wei et al., 2007). They are described as important molecules in the formation and maintenance of synapses between the granule and Purkinje cells by binding to the presynaptic  or  neurexins and the postsynaptic glutamate receptor (GluR) delta2 or delta1 subunit, respectively (Cheng et al., 2016; Matsuda et al., 2010; Uemura et al., 2010). It has also been shown that CBLN1 accumulates in the Bergman glia of the cerebellum indicating a possible role in neuron-glia interactions formed with the help of other membrane proteins (Wei et al.,

ACCEPTED MANUSCRIPT 2009). The current study was designed to identify and further investigate the relative expression of other CBLN1-4 protein derived peptides in naïve mouse spinal cord and to

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examine if they exert nociceptive properties after intrathecal administration.

ACCEPTED MANUSCRIPT Materials and methods

Animals All experiments were carried out in accordance with protocols approved by the Stockholm Ethical Committee for Animal Experiments (Stockholms Norra Djurförsöksetiska Nämnd,

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Sweden). Adult male C57BL/6JRj mice (Janvier, France) were used for generating spinal

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libitum and were maintained on a 12-hour light/dark cycle.

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cord tissues and for behavior experiments. All animals were provided food and water ad

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Spinal cord extraction and preparation for peptide identification and quantification The complete workflow is illustrated in Figure 1. Mice were deeply anesthetized with

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isoflurane, their spinal cord were collected with hydroextrusion and immediately heat stabilized for 30-40 s at 95ºC with the Stabilizor System (Denator AB, Gothenburg, Sweden)

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as described previously (Su et al., 2014). The lumbar spinal cords then were dissected into dorsal and ventral parts under a dissection microscope. The samples were weighed and

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transferred into low-retention Eppendorf tubes containing the extraction solution (ice cold 7.5 l 0.25% acetic acid in water / 1mg tissue). The tissues were homogenized by sonication for

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30 s and centrifuged for 40 min at 14000 g at 4ºC. The supernatants were transferred into Microcon 10 kDa cut-off spin columns (YM-10, Millipore, Bredford, MA; USA) and spinned for 90 min at 14000 g at 4ºC. The filtrate was collected and stored at -80ºC until further usage. The LC separation was performed on an EASY-nLC1000 system (Thermo Scientific™ , Bremen, Germany), through a nanospray Flex™ ion source (Thermo Scientific™ ) in brief, four microliter peptide extracts were injected and trapped on a Acclaim PepMap 100 trapping column (Dionex™ ; Sunnyvale CA, USA), 3 μm particles, 2 cm, 75 μm, C18 column and separated on a NTCC-360/100-5-153 column (Nikkyo Technos Co.,Ltd, Japan) using a

ACCEPTED MANUSCRIPT gradient of A (2% ACN, 0.1% FA) and B (96% ACN, 0.1% FA), ranging from 3 to 45% B in 40 min, going up to 98% in 10 min and back to 3% in 10 min at a flow rate of 300 nl/min. The peptide extracts were analyzed in duplicates in such a way that the 10 spinal cord pieces (5 dorsal and 5 ventral horns obtained from 5 animals) were acquired in alternates of dorsal and ventral and in repeats where the acquisition sequence was reversed. This means, 10 samples

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were analyzed twice giving a total of 20 analyses. Between two consecutive sample injection,

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20 fmol BSA peptide extract was injected and run with a 36 min short gradient to examine

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and avoid potential carry-over.

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The peptide extracts were analysed using a high performance hybrid Q Exactive quadrupole orbitrap mass spectrometer (Thermo Scientific™ , Bremen, Germany). The MS instrument was

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operated in two different settings, (a) quant setting, and (b) identification setting with two different acquisition methods. Both settings were acquired in data dependent mode where it

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switched from full MS to fragment MS/MS. In the quant mode, the full MS was acquired with the orbitrap set at 70,000-resolution power and the MS/MS was performed by collision-

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induced dissociation and acquired at 17,500 resolution. From the full MS, top 2 precursor ions were selected for MS2 analysis and the dynamic exclusion time was set to 5 sec. In the

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identification settings the top 10 intense precursor ions were selected, fragmented and MS2 acquired at 17,500 resolution.

The raw data was converted to “mzML” files using “msconvert” from ProteoWizard (Chambers et al., 2012). The following workflow was used on OpenMS platform v1.11.1 (Sturm et al., 2008) to perform the identification and quantification (unmentioned parameters were set to default value). The identification was performed using X!Tandem (Craig and Beavis, 2004) and OMSSA (Geer et al., 2004) using the following parameters:

ACCEPTED MANUSCRIPT precursor_mass_tolerance: 5 ppm, fragment_mass_tolerance: 0.03 dalton, min_precursor_charge:1, max_precursor_charge: 8, variable_modifications: Acetyl (N-term), Oxidation (M), Phospho (S), Phospho (T), Phospho (Y), Gln->pyro-Glu (N-term Q), Glu>pyro-Glu (N-term E), missed_cleavages: 62 (75 in OMSSA), cleavage_site: unspecific on an in-house database of mouse precursor containing known neuropeptides and peptide hormones

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(the database was combined with reverse sequences). The identification scores from both

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search engines were then transformed to Error posterior error probability using

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IDPosteriorErrorProbability and a consensus identification was built using “ConsensusID” tool. False discovery rate (FDR) was calculated for the peptides and the peptides with FDR

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lower than 0.05 was selected for the mapping into the quantification results (the identified CBLN derived peptides were manually curated using result from X!Tandem standalone

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software). For performing the quantification, the data was first centroided using “PeakPickerHiRes” and the features were quantified using FeatureFinderMetabo using the

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following parameters: noise_threshold_int: 25000, chrom_peak_snr:1, chrom_fwhm: 4, mass_error_ppm: 10 ppm, trace_termination_outliers: 3, width_filtering: auto, local_rt_range:

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30, local_mz_range: 30, charge_lower_bound: 1, charge_upper_bound: 12, isotope_model: peptide. The identification result was mapped to quantification using “IDMapper” allowing

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40 seconds retention time tolerance and 4 ppm mass deviation. Finally, the corresponding features across the samples were linked using “FeatureLinkerUnlabeledQT” allowing 30 seconds retention time and 4 ppm mass deviation.

Intrathecal injection of cerebellin peptides Based on the LC-MS/MS data two CBLN1, three CBLN2 and four CBLN4 derived peptides as well as previously known CER and desCER were synthesized (ProteoGenix, Schiltigheim, France). Intrathecal injections were performed as described previously (Hylden and Wilcox,

ACCEPTED MANUSCRIPT 1980). Briefly, 5 l 30 nmol CBLN derived peptide was administered to mice under light isoflurane anesthesia into the intervertebral space between the lumbar 5 and 6 segments by a 30 1/2 –gauge needle connected to a 25 ml Hamilton syringe. The dose was based on our previous experiments with the CBLN1 derived peptide desCER (Su et al., 2014). As control 5 l saline was injected intrathecally (the experimenter was blinded to the study). In a separate

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preliminary experiment, the effect of each novel peptide on the mice’s physiological state was

Assessment of locomotor and cognitive functions

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monitored for 6 hours after the injection with the help of the Irwin’s test (Irwin, 1968).

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In a separate preliminary experiment, the effect of each novel peptide on the mice’s physiological state was monitored for 6 hours after the injection with the help of the Irwin’s

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test (Irwin, 1968). Briefly, a battery of tests were selected and performed on each mouse every 15 minutes during the first hour and once an hour until the 6-hour time point

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postinjection. The mice were first put in a viewing jar and their body position, locomotion and

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bizarre behavior were observed. They were then transferred to an open arena during which their transfer arousal was also observed. In the open arena gait analysis, reflex reactions and body position were observed while the mice were unrestrained, then their reflex and cognitive

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reactions were observed while they were suspended by the tail or restrained in hand. In every single test, observation parameters were listed (based on the above mentioned paper) and each was selected which applied or each was given score if indicated. The instructions of the paper indicate which parameters and what scores indicate alterations of physiological behavior.

Assessment of mechanical hypersensitivity The mice were placed in individual enclosures on top of a wire mesh platform and habituated for the testing environment for 30-40 minutes prior to testing. Touch stimulus was applied

ACCEPTED MANUSCRIPT perpendicularly on the plantar side of both hind paws with calibrated von Frey OptiHair filaments (Marstock, Schriesheim, Germany) and the response registered after 3 s. Positive response was defined as a sharp withdrawal of the hind paw. The threshold to the touch stimulus was defined with the up-down method (Dixon, 1980) and the 50% probability of paw withdrawal was calculated (in grams) as previously described (Chaplan et al., 1994). Baseline

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threshold values were registered three separate occasions before and 2, 4, 6 and 24 hours after

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the peptide injection. The average of two paws were calculated for each mouse at each time

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point and the percent change from the baseline value was determined. Hypersensitivity index was calculated for the first 6 hours after peptide injection, which represents the area under the

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time-course curve (percent change from the baseline values over time).

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Immunohistochemistry

In a separate experiment, mice were perfused intracardially under deep isoflurane anesthesia

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with saline followed by 4% paraformaldehyde 2 and 4 hours after spinal injection of CBLN-1 (15AA), CBLN-2 (15AA) or saline as control (these two peptides were selected based on the

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hypersensitivity indeces). Lumbar spinal cords were dissected, postfixed in 4% PFA for 24 hours and cryoprotected in 20% sucrose for 48 hours. The samples were arranged into blocks

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and frozen sections (14 m) were cut with a cryostat, then mounted on glass slides. The slides were pre-incubated in 5% normal goat serum in 0.2% Triton X-100 (in PBS) to block specific binding, then the sections were incubated with primary anti-GFAP (1:1000, Millipore) or antiIba1 (1:500, Wako) antibodies to label astrocytes and microglia, respectively. Immunoreactivity was detected using Alexa-488 conjugated secondary antibodies (1:250, Invitrogen). Images were taken with a 710LSM system operated by LSM ZEN2012 software (Zeiss).

ACCEPTED MANUSCRIPT Statistical analysis For the MS data, paired t-tests were used to test for relative expression difference of CBLN14 derived peptides between dorsal and ventral horn. All behavior data were expressed as mean ± S.E.M, timeline analyses were performed with two-way ANOVA followed by Bonferroni’s posy hoc test, while hypersensitivity index

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analyses were performed with one-way ANOVA followed by Bonferroni’s post hoc test using

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Graph Pad Prism 6 (San Diego, CA, USA).

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Results

Characterization and relative quantification of atypical neuropeptides

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Using LC-MS/MS we identified and relatively quantified eleven peptides originating from CBLN1, CBLN2 and CBLN4 precursor proteins. Four CBLN1 derived peptides: Cerebellin-

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(CER) (16AA): SGSAKVAFSAIRSTNH, des-Cerebellin-(desCER) (15AA): GSAKVAFSAIRSTNH, Cerebellin-1 (15AA): SGSAKVAFSAIRSTN and Cerebellin-1

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(14AA): GSAKVAFSAIRSTN. Three CBLN2 derived peptides: Cerebellin-2 (16AA): SGSAKVAFSATRSTNH, Cerebellin-2 (15AA): SGSAKVAFSATRSTN and Cerebellin-2

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(14AA): GSAKVAFSATRSTN. Four CBLN4 derived peptides: Cerebellin-4 (16AA): AANSKVAFSAVRSTNH, Cerebellin-4 (15AA): AANSKVAFSAVRSTN, Cerebellin-4 (14AA): ANSKVAFSAVRSTN and Cerebellin-4 (12AA): SKVAFSAVRSTN. All these peptides have completely conserved sequences between man and mouse and nine of these peptides are novel peptides not previously described (Figure 2). Tandem mass spectrum for these peptides are found in supplementary file 1. No CBLN3 derived peptides were detected. Ten out of eleven peptides displayed statistically significantly (p<0.05) higher expression levels (200-350%) in the dorsal horn compared to the ventral horn of the lumbar spinal cord

ACCEPTED MANUSCRIPT (Table 1). Highest relative expression levels on both dorsal and ventral horns were found for Cerebellin, Cerebellin-1 (15AA) and the two CBLN4 derived peptides Cerebellin-4 (15AA) and Cerebellin-4 (12AA) (Figure 3).

The effect of intrathecally injected cerebellin peptides on mechanical hypersensitivity

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Intrathecal injection of five out of eleven CBLN derived peptides induced mechanical

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hypersensitivity within the first 6 hours, which was resolved by 24 hours post-injection. In the

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CBLN1 group Cerebellin-1 (15AA) induced mechanical hypersensitivity at 4 and 6 hours (Figure 4A), while in the CBLN2 group Cerebellin-2 (15AA) and Cerebellin-2 (16AA)

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evoked significant drop in the withdrawal thresholds at the 4 and 6-hour post-injection time points, respectively (Figure 4B). None of the CBLN4 derived peptides were able to induce

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statistical significant changes in the withdrawal thresholds at any observed time point (Figure 4C). Hypersensitivity index was calculated for the 0-6 hour time frame for all peptides, which

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resulted in significantly higher values in the Cerebellin, desCER, Cerebellin-1 (15AA), Cerebellin-2 (16AA) and Cerebellin-2 (15AA) injected mice, when compared to the control

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group (Figure 4A,B). None of the peptides altered the normal physiological status (locomotor and cognitive functions) of the mice during the first 6 hours after intrathecal administration in

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a separate study (data not shown).

The effect of intrathecally injected cerebellin peptides on spinal glial activation Lumbar spinal cords were collected 2 and 4 hours after intrathecal CBLN-1 (15AA) and CBLN-2 (15AA) peptide injection in order to investigate whether these two peptides induce glial activation in the spinal cord. Neither astrocytes, nor microglia activation was detected in the peptide injected groups at either time point (Figure 5).

ACCEPTED MANUSCRIPT Discussion Using high sensitive LC-MS/MS we have previously identified that the two CBLN1 derived peptides CER and desCER are present in the dorsal horn of the spinal cord of naïve mice. The current study shows for the first time that an additional nine novel CBLN derived peptides are expressed in naïve spinal cord. Two of these were derived from CBLN1, three from CBLN2

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and four from CBLN4. Furthermore, eight of the nine peptides were found to be present at

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significantly higher levels in the dorsal horn compared to the ventral horn. By administration

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of desCER to the cerebrospinal fluid by intrathecal injection we previously found that desCER is associated with modulation of nociceptive signal transmission. Here we extend this

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finding by showing that in addition to desCER, two additional CBLN1 and two CBLN2, but

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none of the CBLN4 derived peptides have pronociceptive properties.

The cerebellins, CBLN1-CBLN4, belong to the C1q/tumor necrosis factor superfamily of

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synaptic organizers which after release from neurons regulate formation, differentiation, and maintenance of synapses (Eiberger and Schilling, 2012; Hirai et al., 2005). The most studied

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member of this family is CBLN1, which was identified for the first time in 1984 in synaptosomal fractions of rat cerebellum, the reason why it was named cerebellin (Slemmon

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et al., 1984). In situ hybridization, RIA and immunohistochemistry has been used to examine the anatomical and cellular location of CBLN in the mouse spinal cord (Cagle and Honig, 2014; Su et al., 2014). Interestingly, the mRNA for Cbln1, Cbln2 and Cbln4 are expressed by distinct excitatory neuronal subpopulations, though there is some overlap, and tend to occupy different laminar positions within the dorsal spinal cord, with Cbln1 mRNA positive cells situated in lamina II and the dorsal part of lamina III, Cbln2 mRNA positive neurons the ventral part of lamina III and in lamina IV, and Cbln4 mRNA positive neurons in laminae V and VI (Cagle and Honig, 2014). In correspondence with the work by Cagle and Honing we

ACCEPTED MANUSCRIPT found the highest density of CBLN1 protein positive cells in lamina II and III in calbindin expressing cells in our previous study (Su et al., 2014). At that time, we could only detect two CBLN1 derived peptides in the spinal cord of naïve mice, but with increased instrumental sensitivity using an Q-Exactive orbitrap we were in the current study able to detect in total eleven peptides. This resulted in the identification of an additional nine CBLN derived

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peptides, of which two originates from CBLN1 (cerebellin-1 (15AA) and cerebellin-1

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(14AA)), three from CBLN2 (cerebellin-2 (16AA, 15AA and 14 AA)) and four from CBLN4

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(cerebellin-4 (16AA, 15AA, 14AA and 12AA)). Noteworthy, the amino acid sequences of these peptides are completely conserved between the human and rodent CBLN family

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proteins indicating an important role and functional similarity between humans and mice, strengthening the translational aspects of our findings. Of importance, all but one CBLN

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derived peptide, cerebellin-4 (16AA), were present at statistical significant two-fold higher levels, or more, in the dorsal horn compared to the ventral horn of the spinal cord which

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suggests an important novel role of these peptides in pain-modulation. Of note, we have not been able to identify any peptides derived from CBLN3, which is in agreement with the

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absence of detectable Cbln3 mRNA (Cagle and Honig, 2014). Furthermore, CBLN3 appears to separate from the other CBLN proteins, as CBLN1, CBLN2 and CBLN4 are released as

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glycoproteins, whereas cbln3 remains within the endoplasmic reticulum and can only be released when bound to CBLN1 (Bao et al., 2006).

In the cerebellum CBLN1 and CBLN2 are thought to have similar functions promotesing proper formation and stability of excitatory synapses between cerebellar granule neurons and Purkinje cells. The role of CBLN2 has not been established yet however there are studies showing potential functional redundancy with CBLN1 in the cerebellum (Rong et al., 2012). After release, they act as ligands that bridges the presynaptic and postsynaptic membranes by

ACCEPTED MANUSCRIPT binding to β-neurexins in granule cells and to the glutamate receptor (GluR) delta2 subunit, which is specifically expressed in Purkinje cells (Elegheert et al., 2016; Uemura et al., 2010). While GluR delta2 mRNA is present also in the dorsal spinal cord (Allen Spinal cord Atlas, http://mousespinal.brain- map.org), the role of CBLN1 and CBLN2 in pain signal transmission still remains to be investigated. In addition to its actions in the cerebellum, CBLN1 has been

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found in the spinal cord, adrenal gland, pituitary and some tumors tissues (Satoh et al., 1997;

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Seigneur and Sudhof, 2017; Su et al., 2014). Thus, it will be important to examine if CBLN1

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derived peptides are functional also in other tissues. CBLN4 is widely expressed in the brain but does not bind GluR delta 2, but instead to presynaptic deleted in colorectal cancer (DCC)

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cell-contact molecules. CBLN4 is important for the formation and maintenance of synaptic connections of GABAergic interneurons to pyramidal neurons in the mouse hippocampus

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(Chacon et al., 2015; Wei et al., 2012). The coupling to GABAergic interneurons is interesting as GABAergic interneurons in the spinal cord are associated with pain inhibition.

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Perhaps the CBLN4 derived peptides are inhibitory rather than facilitatory when it comes to nociceptive signal transmission in the spinal cord. Such actions can be difficult to detect with

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von Frey filaments in the naïve condition and in our study we could only show no or low nociceptive effects of the CBLN4 peptides. Intriguing studies to undertake would be to

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examine if CBLN4-derived peptides attenuate inflammation or tissue/nerve-damage- induced pain hypersensitivity.

In addition to mouse spinal cord, the CBLN1-derived peptides, CER and desCER have also been isolated in rat, guinea pig, porcine, and human cerebellum (Burnet et al., 1988; Slemmon et al., 1984; Yiangou et al., 1989). It has been suggested that both CER and desCER are functional neuropeptides modulating actions in the human and rat adrenal gland (Albertin et al., 2000; Hochol et al., 2001; Mazzocchi et al., 1999; Strowski et al., 2009) and we have

ACCEPTED MANUSCRIPT identified a potential role for these peptides as modulators of nociceptive information. Downregulation of CBLN1 in glutamatergic neurons in the ventral tegmental area of the brain has been indicated as a critical convergence point in the pathogenesis of sociability deficits in a genetic form of autism with epilepsy comorbidity (Krishnan et al., 2017). Though there has been major advancement on the delineation of how the full- length CBLN proteins regulate

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neurotransmission nothing is known about the mechanisms by which CBLN-derived peptides

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alter neuronal activity. Our work suggests, however, that there is an important relationship

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between structure and function, as CBLN1 and CBLN2 derived peptides containing 16 and 15 amino acids all reduced withdrawal thresholds within 6 hours of intrathecal injection, while

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the 14-amino acid long peptides did not. Serine in N-terminal position seems to be important for inducing mechanical hypersensitivity. The most potent aminoacid sequences have serine

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N-terminal and aspargine C-terminal. Loss of N-terminal serine, but histidine in C-terminal position also results in hypersensitivity, however loss of both serine and histidine in

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cerebellin-1(14AA) does not alter the baseline threshold. Furthermore, none of the four 16-14 amino-acid long CBLN4 derived peptides induced mechanical hypersensitivity. Further

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studies are warranted in order to decipher if CBLN1 and CBLN2 derived peptides regulate synaptic organization like the parent proteins, or if they function through receptor binding or

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other mechanisms.

The spinal cord and dorsal root ganglion (DRG) are important components of the signal processing system in nociceptive signaling. Peptidomics using MS has proven to be an effective tool for the discovery and characterization of novel (neuro)peptides and peptide hormones (Romanova and Sweedler, 2015). With increased instrumental sensitivity and improved reproducibility a very large number of well-characterized and novel neuropeptides can now be quantified in a single sample. This aids in understanding action mechanisms and

ACCEPTED MANUSCRIPT is hypothesis generating. Using MS helps to decrease the need for developing novel antibodies for targeting peptides. In line with this, a large number of peptides have recently been characterized in DRG using peptidomics, identifying novel peptides that may be important in nociceptive signaling (Tillmaand et al., 2015). However, in order to investigate the potential effect of novel peptides there is a need for cross laboratory efforts, using state of

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the art technologies, traditional biochemical methods and behavioral testing, that eventually

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may lead to the development of novel drugs for pain relief.

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In conclusion, this study demsonstrates the presence of nine novel atypical peptides

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originating from CBLN1, CBLN2 and CBLN4 precursor proteins in the mouse spinal cord dorsal horn, displaying a structure related coupling to their ability to induce mechanical

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hypersensitivity. Further studies aiming to provide a greater insight into the biology of the Cbln proteins and CBLN-derived peptides are important in order to decode their role in pain

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signal transmission.

ACCEPTED MANUSCRIPT Acknowledgement The authors would like to thank Jungo Kato for providing pictures for figure 1. This study was supported by Ragnar Söderberg Foundation (CIS), Knut and Alice Wallenberg Foundation (CIS), the Swedish Research Council 2013-8373 (CIS), the Karolinska Institutet Foundations (KK), Magnus Bergvalls Foundation (KK), Lars Hiertas Minne (KK), Sigurd and

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Elsa Goljes Minne Foundation (KK), O.E och Edla Johannsson Foundation (KK), the Konung

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Gustaf V:s 80-årsfond SGI2013-0013 (KS) and the IASP John J. Bonica Trainee Fellowship

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(EK).

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ACCEPTED MANUSCRIPT Legends to figures and tables

Figure 1 Experimental procedure. Spinal cords were collected from naïve C57BL/6 mice and the lumbar segments were heat stabilized and dissected into dorsal and ventral parts. The tissues

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were then homogenized, centrifuged and filtered according to protocol. The peptide extracts

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were analyzed using high performance liquid chromatography tandem mass spectroscopy

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(LC-MS/MS). The identification and quantification of the peptides were performed by X!Tandem analysis software. Based on the analysis data two CBLN1, three CBLN2 and four

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CBLN4 derived peptides, as well as previously known CER and desCER were synthesized. Each peptide was administered intrathecally to naïve C57BL/6 mice and their behavioral

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responses (mechanical hypersensitivity, locomotor and cognitive functions) were assessed.

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Figure 2

The CBLN1, CBLN2 and CBLN4 derived peptides have completely conserved sequence

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between human and mouse CBLN family proteins. The stars represent conserved regions

study.

Figure 3

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between CBLN1, CBLN2 and CBLN4 derived peptides. (#)-Novel peptides identified in this

Bar graph showing the relative expression levels of cerebellin, desCER and 9 novel peptides originating from CBLN1, CBLN2 and CBLN4. Ten out of total eleven peptides show significantly (p<0.05) higher expression levels in the dorsal compared to the ventral horn of the spinal cord, corresponding to 200-350%. (#)-Novel peptides identified in this study.

ACCEPTED MANUSCRIPT Figure 4 Timeline graphs showing changes in 50% withdrawal threshold at several time points over 24 hours after intrathecal injection and corresponding bar graphs showing the calculated hyperalgesic indeces for the first 6-hour long period (three testing time points) afther the intrathecal injection of (A) CBLN1, (B) CBLN2, (C) CBLN4 derived peptides (30 nmol i.t.)

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or saline as control. Data are presented as mean±S.E.M., *p<0.05, **p<0.01, ***p<0.001 and

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****p<0.0001 were considered significant compared to the saline group (n=8-20/group). (#)-

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Novel peptides identified in this study.

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Figure 5

Representative images showing spinal immunohistochemistry of (A) astrocytes (GFAP) and

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(B) microglia (Iba-1) reactivity in the dorsal horn 2 and 4 hours following intrathecal CBLN1

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(15AA), CBLN-2 (15AA) or saline injection. Scale bars are 100 m.

Table 1

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Detailed information on the relative expression of the eleven CBLN1, CBLN2 and CBLN4 derived peptides in the mouse spinal cord. Ten out of eleven peptides display statistically

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significantly (p<0.05) higher expression levels in the dorsal horn compared to the ventral horn of the lumbar spinal cord.

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Figure 1

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Figure 2

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Figure 3

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∆Mass (Da)

2.73

0.0062

3.33

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Dorsal/Ve pntral (fold value change)

0.0059

2.64

0.0047

3.28

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2.18

0.0060

2.37

0.0030

3.09

0.0025

3.45

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Pepti de Precur Peptide Mass (+1H) Peptide name score sor sequence (Da) (log(e )) Cerebellin-1 SGSAKVAFSAIRS CBLN1 (16AA) TNH 11.90 1632.8452 GSAKVAFSAIRS CBLN1 desCER TNH -8.00 1545.8132 Cerebellin-1 SGSAKVAFSAIRS CBLN1 (15AA) TN -6.60 1495.7863 Cerebellin-1 GSAKVAFSAIRS CBLN1 (14AA) TN -2.70 1408.7543 Cerebellin-2 SGSAKVAFSATR CBLN2 (16AA) STNH -9.70 1620.8089 Cerebellin-2 SGSAKVAFSATR CBLN2 (15AA) STN -4.70 1483.7499 Cerebellin-2 GSAKVAFSATRS CBLN2 (14AA) TN -2.40 1396.7179 Cerebellin-4 AANSKVAFSAVR CBLN4 (16AA) STNH -5.40 1659.8561 Cerebellin-4 AANSKVAFSAVR CBLN4 (15AA) STN -5.90 1522.7972 Cerebellin-4 ANSKVAFSAVRS CBLN4 (14AA) TN -6.00 1451.7601 Cerebellin-4 CBLN4 (12AA) SKVAFSAVRSTN -4.90 1266.6801

2.85E03 4.46E03 4.50E03 2.50E03 6.60E04 5.23E04 3.72E02 8.52E02 3.58E03 2.43E03 5.04E03

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Using mass spectrometry nine novel cerebellin peptides were found in mouse spinal cord The peptides were expressed at higher levels in the dorsal compared to the ventral horn of naive mice Spinal injection of five cerebellin peptides induced mechanical hypersensitivity in mice

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