Naturally occurring Antibodies against Synthetic Fragment of Neurotrophin Receptor P 75 in Sera of Cognitively Impaired Patients

Naturally occurring Antibodies against Synthetic Fragment of Neurotrophin Receptor P75 in Sera of Cognitively Impaired Patients Michala Kolarova1,3*#, Jan Ricny1, Ales Bartos1, Olga M. Volpina2, Dmitry O. Koroev2, Daniela Ripova1, Anna V. Kamynina2*# 1Department of Experimental Neurobiology, AD Center, National Institute of Mental Health, Klecany, Czech Republic 2Department of Synthetic Vaccines, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Russian Federation, Moscow 3Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic #Authors contributed equally to this work SOJ Immunology Open Access Short Communication


Introduction
To date, a lot of effort was made to clarify the pathology of Alzheimer's disease (AD), because of its prevalence in other forms of dementia.The most studied agent in the pathology of this disease has been a beta-amyloid peptide.During AD pathology, several receptors are involved in interaction with toxic betaamyloid oligomers on the neuronal cell surface which leads to irreversible changes in cells and at the end results in their death [1][2][3].We have focused on two neuronal proteins that have been shown to participate in the pathogenesis of AD -neurotrophin receptor P75 and neuronal acetylcholine receptor (nAChR) α7type [1,4].Both degraded receptors are found in neuritic plaques Belgium) (Table 1).The normal elderly controls were recruited as in-patients from the Department of Neurology.They presented with non-inflammatory conditions, mostly with polyneuropathy and peripheral Bell´s facial palsy, the rest presented with a variety of diseases (e.g., a headache, trigeminal neuralgia and transient unconsciousness).A group of 45 AD patients was categorized to a subgroup of 18 patients with mild cognitive impairment (MCI due to AD; MCI) and a subgroup of 27 patients with mild dementia (Dementia due to AD; AD).They met a diagnosis of AD according to the NIA-AA criteria [15].
Serum samples were collected, centrifuged, and aliquoted in 1 mL polypropylene tubes and stored (on average within 1.5 hours of sampling) at −80°C until analysis.The specimens were thawed just before antibody measurements

Solid-phase peptide synthesis
Peptide fragments with sequence 155-164 aa from P75 receptor and 173-193 aa from the α7-subunit of nAChR were chosen and synthesized as described previously [16,17].Peptide from P75 receptor was synthesized in a dimeric form bound by ε-aminocaproic acid for better coating on the plates without additional conjugation with a carrier protein.Synthetic peptides were purified by preparative reverse-phase HPLC.The purity of the obtained peptides was estimated as >95%.The data of peptides are summarized in Table 2.

ELISA assay
The levels of specific naturally occurring antibodies against two neuronal peptides in sera of 3 different groups were measured by ELISA.All samples were measured in duplicate.The assay was optimized to exclude non-specific signal from sample by measuring samples in non-coated, blocked wells with detection by secondary antibody and using negative and positive controls.The assay was carried out at room temperature if not stated otherwise.Blocking buffer (1% BSA in PBS-0.1% Tween 20 (PBS-T) pH 7.2) was used for dilution of all antibodies.Briefly, 0.1 ml/well 20 µg/ml peptide (155-164) from P75 and peptide (173-193) from α7 nAChR in 0.1M sodium bicarbonate buffer pH 9.5 was coated onto the wells of microplate (Gama Group, Ceske Budejovice, Czech Republic) overnight at 4°C.Plates were blocked for 2 hours and washed three times with 0.3 ml/well 0.1% BSA in PBS-T.Subsequently, 0.1 ml/well diluted serum samples (1:20 and 1:40) or rabbit polyclonal antibody against peptide AChR (173-193) [18] as a positive control (1:500) was applied to the wells and incubated for 1 hour.After washing step, 0.1 ml/ well F(ab')2-goat anti-human IgG (Fc specific, highly cross-adsorbed/HRP conjugate, Novex, Life Technologies Carlsbad, CA, USA) at dilution 1:20,000 or Goat anti-rabbit IgG HRP conjugate (Sigma-Aldrich, St. Louis, MO, USA) at dilution 1:10,000, was applied for 30 minutes.It followed by four times washing and the final incubation with TMB substrate for 25 minutes in the dark.The developing color signal was stopped by 0.1 ml/well 1M H 2 SO 4 and the absorbance was measured within 30 min after stopping by a Multiskan EX ELISA reader (Thermo Scientific) at wavelength 450 nm and 620 nm as a reference wavelength.

Statistics
Data were analyzed with GraphPad Prism software Version 5.0 from GraphPad (San Diego, CA, USA) and with software STATISTICA version 9.0 from StatSoft (Tulsa, OK, USA).The global statistical analysis was performed by the Kruskal-Wallis test and for pairwise comparisons we used Mann-Whitney-Wilcoxon test.A p-value <0.05 was considered statistically significant.

Results
Table 1 shows basic characteristics of the 74 study participants.The participants in the MCI and AD groups were older than healthy subjects.However, the analysis of age covariance did not show any effect on the analysis.Statistical analysis revealed significant drops in MMSE score in all groups with cognitive impairment when compared to controls.The CSF biomarkers showed significant differences between groups of MCI and AD patients when compared to healthy subjects.
We measured levels of naturally occurring antibodies against an extracellular non-structural loop of α7 nAChR and P75 receptors.Characteristics of peptides used in this study are indicated in Table 2.We were able to detect naturally occurring antibodies against one fragment only, particularly against P75 peptide.The AChR peptide showed very weak signal if any in all sera of subjects (Figure 1A).For P75 peptide, the presence of antibodies was shown in all the groups.We observed only a tendency for an elevation of antibodies levels against P75 fragment in the MCI group in comparison to control group (Figure 1B), but without statistical significance (Mann-Whitney test, p = 0.07).We did not observe any correlation between antibody levels and concentrations of CSF biomarkers.

Discussion
Previously, we have shown that immunization of mice either with synthetic fragment (155-164) from neurotrophin receptor P75 or with peptide (173-193) from α7-subunit of the nAChR prevented the loss of the memory and led to a significant decrease in the level of brain beta-amyloid observed in the animals with experimentally induced form of AD [16,17].Moreover, it was shown that antibodies specific to synthetic fragment (173-193) from α7 nAChR protected neuronal cells against beta-amyloid toxicity in tests in vitro [18].Thus, our previous results have demonstrated that antibodies specific to these particular sequences of each protein can play a functionally important role in the AD pathology.Therefore, the presence of naturally occurring antibodies against these significant parts of these receptors could be a hallmark of irreversible processes leading to the development of the disorder.
According to our current data from ELISA measurements of sera samples, there were naturally occurring antibodies only against the fragment (155-164) from neurotrophin receptor P75 (Figure 1).The levels of antibodies against the peptide (173-193) from nAChR turned out to be under detection limit in all investigated groups.The density of nAChR α7-type is relatively low and moreover is decreasing during AD [19] and that could explain an absence of antibodies against it in all groups of individuals.On the contrary, the P75 receptors are more abundant in the brain but also found in other tissues such as perivascular cells, dental pulp cells, lymphoidal follicular dendritic cells, basal epithelium of oral mucosa and hair follicles, prostate basal cells, myoepithelial cells and hepatocyte cells [20][21][22][23][24].However, the expression level of P75 remains stable or down-regulated in adult age and is increased only during some forms of cancer, injury or neurodegeneration that is characteristic of AD pathology [25][26][27].This is in agreement with detected basal levels of natural antibodies against P75 in cognitively normal individuals.Moreover, we observed elevated levels of antibodies against the P75 peptide in sera samples of MCI patients, which can reflect an initial pathological increase of P75 receptor levels in these patients.However, this condition could not be sustainable possibly leading to the drop of antibodies levels with the progression of neurodegeneration.The MCI group is, however, small.Therefore, these preliminary results should be confirmed by analysis of a larger cohort of MCI patients.Nevertheless, considering a difference between the two investigated fragments, the peptide (155-164) from neurotrophin receptor P75 appears promising for detection of naturally occurring antibodies in human sera and observing early changes in the course of memory impairment due to neurodegeneration.

Figure 1 :
Figure 1: Serum antibodies levels against the fragment of the α7-subunit of nAChR (173-193 aa) (A) and fragment with sequence (155-164 aa) derived from neurotrophin receptor p75.(B) in control group of cognitively normal subjects, patients with mild cognitive impairment and patients with dementia due to Alzheimer's disease.Lines represent median values with error bars showing 25 th -75 th percentiles.