BR102022009685A2 - METHOD FOR SCREENING DRUGS OR SUBSTANCES WITH NEUROPROTECTIVE POTENTIAL, KIT FOR SCREENING DRUGS OR SUBSTANCES, AND USE OF A GENE PANEL - Google Patents

Abstract

The present invention relates to a method for screening drugs or substances with neuroprotective potential. The invention also relates to a kit for screening drugs or substances with neuroprotective potential.

Description

FIELD OF INVENTION

[001] The present invention relates to the field of screening drugs or substances. More specifically, the present invention consists of a method for screening drugs or substances with neuroprotective potential. The present invention also refers to the kit for screening drugs or substances with neuroprotective potential and the use of the gene panel developed for said testing.

BASICS OF THE INVENTION

[002] The Zika virus (ZIKV) is an arbovirus from the Flaviviridae family, transmitted to humans mainly through the bite of the mosquito vector Aedes aegypti (1). Typically, the infection results in an asymptomatic form or non-specific symptoms, the most common being low-grade fever (less than 38.5°C), headaches, muscle and joint pain, maculopapular rash, non-purulent conjunctivitis and malaise. , which can be confused with those presented in other flavivirus infections, such as Dengue and Chikungunya, which can make diagnosis difficult (1).

[003] After the occurrence of some outbreaks around the world (2, 3), Brazil faced an epidemic of ZIKV infections in 2015, having first been identified in the semi-arid region of the Northeast region of the country (4, 5). Coincidentally, in the same period, a surprising increase in the number of cases of congenital microcephaly was observed, a clinical sign characterized by a reduction in the child's head circumference and which can cause harm to their cognitive and physical development. Subsequently, an association between the occurrence of infection and abnormalities in fetal and neonatal development was found, which came to be jointly described as Congenital ZIKV Syndrome (CZS) (6-8).

[004] According to the Bulletin of the Epidemiological Situation of Congenital Syndrome Associated with ZIKV Infection (6, 9), published by the Ministry of Health, between 2015 and 2020, 19,622 suspected cases of CZS were reported, among which 18.2% were confirmed. In 2020, a total of 1,007 cases were reported. Of these, 3.5% were confirmed and 59.3% still remain under investigation. In the period from 2015 to 2018, the majority of notifications were concentrated in the Northeast region of Brazil (58.3%), followed by the Southeast (25.2%) and Central-West (7.6%). However, in 2019, case notification began to be concentrated in the Southeast region (39.3%), followed by the Northeast region (36%) and Central-West region (9.2%).

[005] The ZIKV genome is composed of positive-sense single-stranded RNA. The virus has a diameter of approximately 50 nm and a nucleocapsid surrounded by a lipid bilayer, in which its membrane precursor (PrM/M) and envelope (E) structural proteins are located, organized in icosahedral symmetry (10). Its genome of approximately 10.8 kb is composed of two non-coding regions (UTR) 5' and 3' and a long open reading frame. The latter transcribes a polyprotein that cleaves into 10 proteins, three of which are structural: capsid (Cap), E and PrM; and seven nonstructural: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (11, 12). ZIKV was initially isolated from the serum of rhesus monkeys from the Zika forest in Uganda in 1947 (13).

[006] ZIKV probably originated in Africa between the end of the 19th century and the beginning of the 20th century and has two distinct lineages, African and Asian (14, 15). The isolates circulating in the Americas originate from the Asian lineage and share a common ancestor with the strain circulating in French Polynesia during the Zika outbreak that occurred in 2013 (16). In the present study, two isolates of the Asian lineage were compared with 99.9% similarity at the nucleotide level and 99.97% similarity at the amino acid level (17). The first is PE243 (ZIKV/H. sapiens/Brazil/PE243/2015; GenBank: KX197192), isolated from a patient with classic symptoms associated with Zika, in 2015, in the state of Pernambuco, Northeast region of Brazil (17). The second is SPH2015 (ZikaSPH2015; GenBank: KU321639), isolated in the state of São Paulo, southeastern Brazil (16). The main difference at the amino acid level between ZIKV PE243 and ZIKV SPH2015 is found in the nonstructural protein 1 (NS1) (17). Any change in the NS1 protein can have a relevant impact on the pathogenesis of ZIKV, since this protein has an important interaction with the host. NS1 from Zika, dengue, West Nile, Japanese encephalitis and yellow fever viruses has been shown to selectively bind to and alter the permeability of human endothelial cells of the lung, dermis, umbilical cord, brain and liver in vitro and cause tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus (18). ZIKV anti-NS1 antibodies protect against congenital infection (19). NS1 plays a crucial role in the remodeling of the endoplasmic reticulum during ZIKV replication. This process is dependent on the insertion of the protein into the hydrophobic membrane and, therefore, depends on its structure (20). Furthermore, NS1 is important in ZIKV immune evasion, suppressing the host response by manipulating the interaction between the inflammasome and type 1 IFN signaling. In this case, through specific interactions between NS1 and caspase-1, which promote the cleavage of cGAS (21).

[007] ZIKV is capable of infecting neural precursor cells, immature neurons and mature neurons. Infection of these cells induces cell cycle dysregulation and apoptosis (22-25). Fetal brain lesions caused by ZIKV infection are associated with disorganization of neural stem cells, significant loss of intermediate progenitor cells, and dysmorphia of neuronal circuitry in the dentate gyrus of the hippocampus, as demonstrated in the experimental infection of Macaca nemestrina (26). The rich vasculature and proximity to cerebrospinal fluid-filled ventricles increase hippocampal exposure to circulating ZIKV compared to other brain structures (27, 28).

[008] The hippocampus is a brain structure related to the formation of spatial memory and new episodic memories (29). A component of the limbic system, it houses neural progenitor cells in its subgranular layer of the dentate gyrus, constituting one of the main regions of brain neurogenesis (30, 31). These are capable of self-renewal or differentiation into new neurons, astrocytes or oligodendrocytes (29, 30). In the present study, a model of organotypic hippocampal cultures (COHs) of infant rats grown in a liquid-gas interface is used. This model allowed the investigation of initial processes of ZIKV infection and the assessment of the impacts of infection by ZIKV isolates PE243 and SPH2015 on phenotypic aspects and the transcriptional signature of the hippocampus. Using this model, it was possible to identify a panel of biomarker genes for the balance between neuronal death and neurogenesis and the inflammatory response characteristic of the initial stage of infection and which are determinants of the outcome observed later. The present invention uses this panel of genes in a method for screening drugs, substances or molecules with neuroprotective potential, and in the development of a kit for screening drugs or substances with neuroprotective potential, enabling the elucidation of their respective mechanisms of action.

[009] To date, there is no specific treatment for ZIKV infection, nor for SCZ. The discovery of new drugs is a generally slow process and subject to great risks of failure (“attrition”). Added to these challenges is the need to elucidate, even if only partially, the mode of action of drugs as a prerequisite for regulatory agencies to authorize their use or repositioning in humans. The use of COHs allows the evaluation of the neuroprotective potential of drugs or substances, as well as the elucidation of their mechanism of action, in a tissue context very close to the reality of the central nervous system. The COHs model significantly reduces the number of animals needed for the study and their suffering, compared to in vivo models. This model also overcomes important limitations of in vitro models, such as cell cultures, neurospheres and organoids, preserving the architecture and three-dimensional organization of the tissue, in addition to cellular diversity and interconnection (32, 33). The COHs model contains microglia, immune cells of hematopoietic origin (Reviewed in: 34), which are not normally present in in vitro models. Even in vitro models that incorporate this and other cell types, such as some brains-on-a-chip, incorporation is done artificially and the integration of different cell types is a challenge. (Ben M. Maoz. Brain-on-a-Chip: Characterizing the next generation of advanced in vitro platforms for modeling the central nervous system. APL Bioengineering 5, 030902 (2021)). Furthermore, laboratory induction of the differentiation of pluripotent stem cells to form neurospheres and organoids in certain models alters their natural epigenetic programming, potentially masking profound differences in relation to central nervous system (CNS) cells in their natural state (25).

BACKGROUND OF THE PRESENT INVENTION

[0010] There are prior art reports on the use of organotypic hippocampal cultures (COHs) as a model of ZIKV infection. Büttner and Caroline et al. (Zika Virus-Mediated Death of Hippocampal Neurons Is Independent From Maturation State. Frontiers in cellular neuroscience vol. 13 389), for example, use an organotypic culture obtained from mouse hippocampal sections to analyze the effects of ZIKV infection on hippocampal development.

[0011] There are also methods for screening drugs against ZIKV that use different types of brain organoid cultures. These models are based on different gene signatures from those disclosed in the present invention and do not always aim to screen drugs with neuroprotective potential.

[0012] Xiao Xu and collaborators, for example, reveal a method that uses three-dimensional organoid cultures to screen drugs against ZIKV, monitoring the expression of the NS1 protein, an indicator of ZIKV replication, and the activity of caspase-3 (an indicator of apoptosis of host cells), in addition to cell viability. (Miao Xu et al. Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat Med. 2016 Oct; 22(10): 1101-1107). The model of the present invention evaluates genes associated with the mechanisms of inflammation, neurogenesis and neuronal death in COHs, enabling inferences about the mechanism of action of the tested substances.

[0013] Document WO2019079360 proposes the identification of gene signatures associated with diseases (including ZIKV infection) in various types of cell cultures. One of the possible uses mentioned is to use these cultures to screen chemical compounds and drugs by monitoring changes in established markers. In the model of the said document there is no reduction to the practice with ZIKV nor are organotypic cultures used.

[0014] WO2017083705 discloses a rotating bioreactor for large-scale 3D cell cultivation. More specifically, the application describes organoids produced in this bioreactor from human pluripotent stem cells that mimic brain regions and are used to model exposure to ZIKV. The authors suggest that these organoids can be used in drug screening, including antivirals with activity against ZIKV.

SUMMARY OF THE INVENTION

[0015] The present invention describes a method for screening drugs or substances with neuroprotective potential based on a panel of biomarkers associated with neurodegeneration and the inflammatory response induced by ZIKV infection in a COHs model. This panel was identified by evaluating the impacts of infection by two ZIKV isolates on the phenotypic aspects and transcriptional signature of COHs, PE243 and SPH2015. These are genetically similar isolates that circulated in Brazil during the 2015-16 epidemic peak. Despite being genetically similar, these isolates differ at the amino acid level mainly in the non-structural protein NS1, implicated in immune escape. This panel of biomarker genes can be used to identify compounds with neuroprotective potential by comparing their expression in ZIKV COH infection models, enabling the elucidation of the mechanism of action of the selected compounds. The present invention also relates to a kit for screening drugs or substances with neuroprotective potential.

[0016] For the selection of the panel of biomarker genes of the present invention, it was first demonstrated, using immunofluorescence, confocal microscopy, RNA-Seq and Bioinformatics techniques, that ZIKV isolates PE243 and SPH2015 can infect and replicate in COHs. The hippocampal response to infection by the isolates was evaluated and the mechanisms conserved in infection by both isolates were identified, generating a panel of biomarker genes for the processes of neuronal death/neurogenesis and inflammatory response at the beginning of infection. Monitoring these biomarkers, as well as the associated phenotypic characteristics, constitutes an unprecedented process for screening drugs or substances with neuroprotective properties and makes it possible to elucidate their mechanisms of action using the ZIKV COH infection model.

[0017] In a first embodiment, the present invention provides a method for screening drugs or substances with neuroprotective potential, comprising: a) providing organotypic cultures from the hippocampus, being: i) at least one control culture, not infected and not treated with the drug or substance to be screened; ii) at least one culture that will be infected with the agent from step b) and will not be treated with the drug or substance to be screened; iii) at least one control culture that will be treated with the drug or substance to be screened; and iv) at least one culture that will be infected with the agent from step b) and will be treated with the drug or substance to be screened; b) infecting the cultures established in ii and iv with a neuroinflammatory infectious agent; c) incubate the cultures established in iii and iv with a drug or substance with neuroprotective potential to be screened; d) extract at least one messenger RNA or protein from each of at least four established subcultures; and e) determine the expression pattern based on a panel of biomarkers from each of at least four established subcultures and, thus, predict the neuroprotective potential of the screened drug or substance.

[0018] In another embodiment of the present invention, organotypic hippocampal cultures are cultivated for at least 14 days for stabilization.

[0019] In another embodiment of the present invention, the neuroinflammatory infectious agent used to infect cultures is ZIKV.

[0020] In another embodiment of the present invention, the infectious agents isolated from ZIKV are PE243 and SPH2015.

[0021] In another embodiment of the present invention, the drug or substance to be screened for neuroprotective potential is at a concentration of about 0.5µM.

[0022] In another embodiment of the present invention, the extraction of the nucleic acid from step d) occurs 16h after carrying out steps b) and c).

[0023] In another embodiment of the present invention, the determination of the expression pattern in a panel of biomarkers from step e) is carried out based on the expression of the genes Adgre1, Ccl3, Dbx2, Disp3, Gmr3, Igf1, Il1b, Rac2, Rasgrp3, Siglec5 in relation to the expression pattern of the same genes in control cultures.

[0024] In another embodiment of the present invention, the extracted nucleic acid is RNA.

[0025] In another embodiment of the present invention, the evaluation of the expression of the biomarker panel is carried out by RT-qPCR after reverse transcription, Northern Blot, digital PCR or equivalent techniques.

[0026] In a preferred form of the second embodiment, the neuroinflammatory disease is caused by infection with the ZIKV virus.

[0027] In a third embodiment, the present invention discloses a kit for screening drugs or substances with neuroprotective potential.

[0028] In a fourth embodiment, the present invention provides a method for screening drugs or substances for controlling neuroinflammation and preventing neurodegeneration associated with infection of the central nervous system by ZIKV.

BRIEF DESCRIPTION OF FIGURES

[0029] Figure 1 - Methodology for obtaining the biomarker panel of the present invention: After euthanasia, the animals' hippocampi were dissected and sliced at 400µm and the slices distributed in inserts for cultivation. According to the experimental design, COHs were infected with isolate SPH2015 or PE243 or subjected to simulated infection without the virus. Subsequently, molecular and immunofluorescence analyzes and confocal microscopy were performed to evaluate the dynamics of infection, phenotypic and transcriptional analyzes of the cultures. COHs = organotypic hippocampal cultures; p.i. = post-infection.

[0030] Figure 2 - ZIKV infection and proliferation in COHs: The Fluorescence Intensity (IF) of NS1 normalized by tissue area (DAPI+) (IF NS1/mm2) was quantified in the two isolates (PE243 and SPH2015) at 8:00 am 24 and 48h post infection (p.i.) (n > 12). Samples outside the range between the lower and upper quartiles of the median of their respective groups (interquartile range) were excluded from the analysis. Two-way ANOVA test was used followed by Tukey's multiple comparisons test. Data were expressed as mean ± standard deviation. *P< 0.05; ***P< 0.001; ****P< 0.0001..

[0031] Figure 3 - Neuronal infection by ZIKV: A - The ratio of mature neurons (NeuN+) infected by ZIKV (NeuN+ NS1+) 8, 24 or 48h p.i. was estimated (n > 12). Samples outside the range between the lower and upper quartiles of the median of their respective groups (interquartile range) were excluded from the analysis. Two-way ANOVA test was used followed by Tukey's multiple comparisons test. Data were expressed as mean ± standard deviation. ****P< 0.0001. B - Micrographs (60x) obtained from COHs 8h p.i.. Markings in blue represent cell nuclei (DAPI), in red mature neurons (NeuN) and in green ZIKV (NS1). Merge represents the overlap of the three channels, with arrows representing the colocalization of ZIKV and NeuN. Scale bar = 10µm.

[0032] Figure 4 - Neuronal density of COHs in the initial period of ZIKV infection: The number of mature neurons (NeuN+) per tissue area (DAPI+) was quantified (NeuN+/mm2) in COHs of the control (CTRL) and infected groups with ZIKV PE243 or SPH2015 at 8, 24 and 48h p.i. (n > 9). Samples outside the range between the lower and upper quartiles of the median of their respective groups (interquartile range) were excluded from the analysis. Two-way ANOVA test was used followed by Tukey's multiple comparisons test. Data were expressed as mean ± standard deviation. **P< 0.01; ****P< 0.001.

[0033] Figure 5 - Neuronal density of COHs at advanced times of ZIKV infection: The number of mature neurons (NeuN+) per tissue area (DAPI+) was quantified (NeuN+/mm2) in COHs of the control (CTRL) and infected groups with ZIKV PE243 or SPH2015 5, 7 and 10 days p.i. (n > 18). Samples outside the range between the lower and upper quartiles of the median of their respective groups (interquartile range) were excluded from the analysis. Two-way ANOVA test was used followed by Tukey's multiple comparisons test. Data were expressed as mean ± standard deviation. *P< 0.05; **P< 0.01; ****P< 0.0001.

[0034] Figure 6 - ZIKV-induced neuronal death in COHs involves apoptosis: A - Apoptotic neurons present in COHs were evaluated in the control (CTRL) and PE243 and SPH2015-infected groups 4 days p.i. through labeling with NeuN and TUNEL (NeuN+ TUNEL+) and normalized by the total number of neurons (NeuN+) (n > 3). Group variances were compared using the one-way ANOVA test followed by Tukey's multiple comparisons test. Outliers were identified with the Grubbs test (alpha = 0.1) and excluded. **P< 0.01; ***P< 0.001. B - Micrographs by confocal microscopy (100x) obtained from COHs 4 days p.i.. Markings in blue represent cell nuclei (DAPI), in red, mature neurons (NeuN) and in green, apoptotic signals (TUNEL). Merge represents the overlap of the three channels, with the colocalization of TUNEL, NeuN and DAPI markings. White arrows indicate colocalization points between TUNEL and NeuN. Scale bar = 10µm.

[0035] Figure 7 - Isolates PE243 and SPH2015 cause distinct dynamics of microglia activation in COHs: A - The area corresponding to activated microglia (Iba1+) normalized by the tissue area (DAPI+) was evaluated in isolates SPH2015 and PE243 at 8h and 24h p.i. (n > 2) and compared to the control group (CTRL). Two-way ANOVA test was used followed by Tukey's multiple comparisons test. Data were expressed as mean ± standard deviation. *P< 0.05. B - Micrographs by confocal microscopy (60x) obtained from COHs 8h p.i.. Markings in blue represent cell nuclei (DAPI), in red, mature neurons (NeuN) and in green, activated microglia (Iba1). Merge represents the overlap of the three channels, with the colocalization of Iba1, NeuN and DAPI markings. Scale bar = 10µm.

[0036] Figure 8 - Epigenetic changes induced differently by ZIKV isolates: The H3K4me3 epigenetic mark was evaluated in COHs from the control group (CTRL) and infected with isolates SPH2015 and PE243 5, 7 and 10 days p.i., estimating the fluorescence intensity (IF) of H3K4me3 in mature neurons. These values were normalized by the number of neurons positive for this mark (NeuN+ H3K4me3+) (n > 8). Samples outside the range between the lower and upper quartiles of the median of their respective groups (interquartile range) were excluded from the analysis. Two-way ANOVA test was used followed by Tukey's multiple comparison test and data were expressed according to mean ± standard deviation. *P < 0.05; ***P < 0.001; ****P < 0.0001.

[0037] Figure 9 - Venn diagram representing the genes differentially expressed by COHs infected with isolates PE243 or SPH2015 at 16h p.i.: Genes with Fold Change greater than 1.2 and P value less than 0.05 were considered differentially expressed.

[0038] Figure 10 - Changes in the processes of survival, proliferation and differentiation of neural cells induced by infection with isolates PE243 or SPH2015 (SPH). The observed pattern of neurodegeneration may imply impairments to neurodevelopment.

[0039] Figure 11 - Infection with PE243 or SPH2015 (SPH) induces distinct patterns of inflammatory response at the beginning of the infection.

[0040] Figure 12 - Validation by RT-qPCR of biomarker genes for the processes of balance between neuronal death and neurogenesis and inflammatory response regulated by ZIKV isolates PE243 and SPH2015 at the beginning of hippocampal infection. Data were expressed as mean ± standard deviation. * = P < 0.05; ** = P < 0.01; *** P < 0.001; **** = P < 0.0001. N sample > 4.

[0041] Figure 13 - Flowchart of the model established for screening drugs or molecules with neuroprotective potential in COHs. Organotypic hippocampal cultures (COHs) from approximately 7-day-old infant rats are produced from approximately 400-micrometer-thick slices of the organ grown for stabilization for at least 14 days. COHs are then challenged by ZIKV infection through exposure to culture medium suspensions containing 6x105 plaque forming units (PFU) per milliliter for two hours. At the end of these two hours, viruses not adsorbed to COH cells are removed by washing with fresh culture medium and the cultures are incubated for 16 hours in fresh medium containing the drug or substance whose neuroprotective potential will be tested. Included in the tests are falsely infected COHs, exposed to the infection procedure, but with a virus-free medium, which serve as controls for evaluating the toxicity of the substances tested in the absence of infection, and infected COHs treated with a medium containing the excipient, but not the drug or tested substance, which serve as controls on the efficiency of the infection. After incubation for 16h, RNAs (or proteins) are extracted and the expression levels of biomarker genes are quantified using techniques such as RT-qPCR, for example. Radar graphs A and B exemplify evaluations of the neuroprotective effect and possible toxicity, respectively, of a hypothetical substance tested with the model proposed here.

[0042] Figure 14 - Evaluation of the neuroprotective potential of cinnamaldehyde using the methodology of the present invention. Pharmacological treatment of organotypic hippocampal cultures infected or not with Zika virus. Assessment of the effect of the drug (A) and its toxicity (B). Drug concentration: 0.5µM. Groups were compared using the unpaired T test. Data are represented as the average of 2A(- DCt) values on a logarithmic scale. * Untreated simulated vs untreated infected; * Simulated untreated vs infected treated with the drug;* P < 0.05; ** P < 0.01.

[0043] Figure 15 - Assessment of the neuroprotective potential of diphenhydramine using the methodology of the present invention. Pharmacological treatment of organotypic hippocampal cultures infected or not with Zika virus. Assessment of the effect of the drug (A) and its toxicity (B). Drug concentration: 0.5µM. The groups were compared using the unpaired T test or Mann-Whitney test. Data is represented as the average of 2A(-DCt) values on a logarithmic scale. * Untreated simulated vs untreated infected; * Untreated sham vs drug-treated sham.* P < 0.05; ** P < 0.01.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Although the present invention may be susceptible to different embodiments, there is shown in the drawings and in the following detailed discussion a preferred embodiment with the understanding that the present description should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been illustrated and described here.

DEFINITIONS

[0045] In the present application, the expression “organotypic hippocampal cultures (COHs)” refers to hippocampi sliced at 400µm, dissected in culture medium and cooled to 4oC.

[0046] In the present application, the term “RT-qPCR” is used when the starting material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA). The cDNA is then used as a template for the qPCR reaction. RT-qPCR is used in a variety of applications, including biomarker panel expression analysis. Said expression analysis can be carried out not only by RT-qPCR, but also by Northern blot, digital PCR and equivalent techniques.

[0047] In the present application, the term “primers” refers to primers designed to be used in the qPCR reaction, to detect the expression of candidate biomarker genes Adgre1, Ccl3, Dbx2, Disp3, Gmr3, Igf1, Il1b, Rac2 , Rasgrp3, Siglec5

Method for screening drugs or substances with neuroprotective potential

[0048] In a first embodiment, the present invention provides a method for screening drugs or substances with neuroprotective potential, comprising: a) providing organotypic cultures from the hippocampus, being: i) at least one control culture, not infected and not treated with the drug or substance to be screened; ii) at least one culture that will be infected with the agent from step b) and will not be treated with the drug or substance to be screened; iii) at least one control culture that will be treated with the drug or substance to be screened; and iv) at least one culture that will be infected with the agent from step b) and will be treated with the drug or substance to be screened; b) infecting the cultures established in ii and iv with a neuroinflammatory infectious agent; c) incubate the cultures established in iii and iv with a drug or substance with neuroprotective potential to be screened; d) extract at least one messenger RNA or protein from each of at least four established subcultures; and e) determine the expression pattern based on a panel of biomarkers from each of at least four established subcultures and, thus, predict the neuroprotective potential of the screened drug or substance. In another embodiment, organotypic hippocampal cultures are grown for at least 14 days for stabilization. In another embodiment, the neuroinflammatory infectious agent used to infect the cultures is ZIKV. In another embodiment, the infectious agents are ZIKV isolates PE243 and SPH2015. In another embodiment, the drug or substance to be screened for neuroprotective potential is at a concentration of about 0.5 µM. In another embodiment, the extraction of the nucleic acid from step d) occurs 16h after performing steps b) and c). In another embodiment, the determination of the expression pattern in a panel of biomarkers from step e) is carried out based on the expression of one or more of the genes Adgre1, Ccl3, Dbx2, Disp3, Gmr3, Igf1, Il1b, Rac2, Rasgrp3, Siglec5 in relation to the expression pattern of the same genes in control cultures. In another embodiment the extracted nucleic acid is RNA. In another embodiment, evaluation of the expression of the biomarker panel is performed by RT-qPCR after reverse transcription, Northern Blot, digital PCR, and equivalent techniques. In another embodiment, the methodology is used to screen drugs or substances to control neuroinflammation and prevent neurodegeneration associated with infection of the central nervous system by ZIKV.

Kit

[0049] In a second embodiment, the present invention discloses a kit for screening drugs or substances with neuroprotective potential.

EXAMPLES

[0050] Preparation of organotypic hippocampal cultures and evaluation of infection dynamics, phenotypic and transcriptional analyzes of the cultures, as represented (Figure 1)

ZIKV

[0051] Samples of isolates PE243 and SPH2015, cultivated in C6/36 cells, were provided by the Viral Disease Immunology (IDV) laboratory of the René Rachou Institute (IRR)/FIOCRUZ to carry out the experiments and stored at -80°C .

Animals

[0052] The neonatal Wistar rats (7-10 days) used in the production of organotypic hippocampal cultures (COHs) were obtained from the Institute of Science and Technology in Biomodels (ICTB) (FIOCRUZ) and brought to the IRR/ FIOCRUZ with a lactating female the day before the experiment. They remained with water and food ad libitum, under light/dark cycles of 12-12 hours and at controlled temperature and humidity (20-26oC, 40-60%, respectively). The present method was approved by the Ethics Committee on the Use of Animals of FIOCRUZ (license LW-10/18).

Preparation of organotypic hippocampal cultures

[0053] COHs were prepared according to the method described by Stoppini et al. (35) with modifications. After euthanasia of the animals by decapitation, the brains were removed and the hippocampi dissected in culture medium (consisting of MEM Eagle medium + HEPES (Vitrocell Embriolife, Campinas, Brazil) plus 25% of 1x Hanks' balanced solution (Sigma, Basel , Switzerland)), cooled to 4oC. Then, the hippocampi were sliced at 400µm using a McIlwain tissue chopper (Mickle Laboratory Engineering Co Ltd, Gomshal, United Kingdom).

[0054] Six slices from different animals were arranged in each PICM0RG50 organotypic culture insert (Merck Millicell, Darmstadt, Germany), in a six-well culture plate. During the first week, COHs were cultivated in 1 mL of culture medium plus 25% heat-inactivated equine serum (Bio Nutrientes do Brasil Ltda., Barueri, Brazil). From the beginning of the second week, culture medium without equine serum was used. Throughout the experiment, the COHs were maintained in an oven with 5% CO2, at 37oC, changing half the volume of medium every 2-3 days. After the stabilization phase, the COHs had an average thickness close to 40µm.

Infection of COHs with ZIKV

[0055] The infection of COHs with ZIKV was carried out after at least 14 days of cultivation (stabilization phase). The cultures were divided into three groups: control (uninfected), infected with isolate PE243 and infected with isolate SPH2015.

[0056] For infection, the culture medium was replaced with fresh medium containing 6x105 pfu/mL of ZIKV, 1 mL below the membranes and 0.2 mL above in order to cover the COHs. To obtain the correct viral concentration in the medium, stock aliquots of SPH2015 and PE243 were diluted 48 times. The plates were incubated for two hours in an oven at 37°C and 5% CO2, with shaking every 20 minutes. At the end of this time, the medium with virus was removed and the membranes were washed three times with medium without ZIKV. Washing the COHs ensures that only the viruses that adsorbed the cells remain, preventing a new cycle of infection from viruses remaining in the medium. Finally, 1 mL of medium was added under the membrane and the plate was returned to the oven. Controls were also subjected to the same protocol, but using virus-free culture medium.

Immunofluorescence

[0057] The COHs were immunostained following the method described by Gogolla and collaborators (36) with adaptations. At the end of each experiment, COHs were fixed for immunostaining in the following solutions: ice-cold 4% paraformaldehyde (PFA) (pH 7.2) for 5 minutes, ice-cold 20% methanol in phosphate-buffered saline (PBS) 1x for 5 minutes , 0.05% Tween in PBS 1x overnight and bovine serum albumin (BSA) 20% in PBS 1x overnight. Washes were performed with 1x PBS between incubations. The COHs were then separated from the inserts and continued for incubation with antibodies. To determine the density of mature neurons in COHs 8h, 24h, 48h, 5, 7 and 10 days post infection (p.i.), NeuN, a protein expressed in the nucleus of mature neurons, was labeled with mouse monoclonal anti-NeuN antibody ( 1:100, MAB377C3, Merck) or rabbit polyclonal (1:100, ABN78C3, Merck) conjugated to the Cy3 fluorophore. To estimate the density of viral particles present in COHs 8, 24 and 48h p.i., the unconjugated anti-ZIKV rabbit polyclonal anti-NS1 antibody (1:1,000) was used. The evaluation of activated microglia at 8h, 24h and 48h p.i. was carried out by labeling Iba1 with the unconjugated anti-Iba1 rabbit monoclonal antibody (1:100, AB178847, ABCAM, Cambridge, United Kingdom) and detecting trimethylated histone H3 on lysine 4 (H3K4me3) 5, 7 and 10 days p.i. was performed with unconjugated mouse anti-H3K4me3 antibody (1:500, 05-1339, Merck). For labeling of unconjugated antibodies, rabbit anti-IgG (1:400, A11034, ThermoFisher, Waltham, MA) or mouse anti-IgG (1:20,000, A10684, ThermoFisher) conjugated to the Alexa Fluor 488 fluorophore was used. identification of cells undergoing apoptosis 4 days p.i., the TUNEL assay was performed with the Click-iT TUNEL AlexaFluor Imaging Assay Protocol kit (C10245, Thermo Fisher) following the manufacturer's instructions. Finally, all COHs were labeled with DAPI (4',6'-diamino-2-phenyl-indole) (1µg/mL, D1306, ThermoFisher) and slides mounted with ProLong Diamond Antifade Mountant (P36961, ThermoFisher) .

[0058] Negative control slides were produced, stained with DAPI alone or with DAPI and secondary antibody, with the aim of adjusting the parameters for obtaining images, thus excluding possible tissue autofluorescence points and background noise.

Confocal Microscopy

[0059] Images of the slides were captured using a Nikon Eclipse Ti confocal microscope (Nikon, Tokyo, Japan) with a 488/561 wavelength filter. The images were obtained at 10x magnification, looking for representative areas with greater coverage of cellular tissue. For representative photos of details of the COHs, 60x or 100x magnification was used. The photography parameters were defined according to the negative control.

[0060] The images were analyzed with the aid of NIS-Elements Analysis software (Nikon). The channels corresponding to the DAPI+, NeuN+ and Iba1+ markings were treated with the Noise Reduction, Gauss-Laplace Sharpen and Local Contrast tools. The total tissue area was determined by creating an automatic binary mask from the channel with DAPI+ fluorescence, followed by using the Interest Region Manager (ROI) tool. To count mature neurons, a binary layer was created based on the NeuN+ labeling (Cy3 channel), selecting and quantifying elements with a fluorescence threshold > 10 AU. This quantification was later normalized by the tissue area (NeuN+/mm2).

[0061] To detect the labeling area (Iba1+) and intersection regions in the labeling of Cy3 and Alexa Fluor 488 channels (NeuN+ and NS1+, NeuN+ and TUNEL+ and NeuN+ and H3K4me3+) a binary mask was also created followed by an ROI . To quantify the density of activated microglia through Iba1, the area values identified with the ROI were obtained and subsequently divided by the total area of the slice (Iba1+ area/mm2). To identify viral infection, the colocalization of NS1 labeling (Alexa Fluor 488) with NeuN+ (Cy3) was estimated (number of NeuN+ and NS1+ cells / number of NeuN+ cells) and the fluorescence intensity (IF) of NS1 per tissue area (IF NS1/mm2). TUNEL+ labeling (Alexa Fluor 488) was colocalized with NeuN+ (Cy3) to estimate the ratio of mature apoptotic neurons (number of NeuN+ and TUNEL+ cells / number of NeuN+ cells). Finally, the IF of H3K4me3 labeling (Alexa Fluor 488) was normalized by the number of NeuN+ (Cy3) and H3K4me3+ cells (IF H3K4me3+ / number of NeuN+ and H3K4me3+ cells), to estimate the intensity of this epigenetic mark in mature neurons.

Obtaining RNA samples

[0062] Total RNA from COHs was extracted using a combination of Trizol (ThermoFisher) and chloroform (Merck), following the manufacturer's protocol. Subsequently, total RNA was purified on a column of the miRNeasy Mini kit (217004, Qiagen, Hilden, Germany) according to the manufacturer's instructions, quantified by fluorometry using the Qubit RNA HS Assay kit (Q32852, Invitrogen, Carlsbad, CA) and the Qubit 2.0 Fluorometer (Invitrogen) and evaluated for purity with the NanoDrop (ThermoFisher).

RNA-Seq and Bioinformatics analyzes

[0063] Libraries for sequencing were produced using the TruSeq Stranded mRNA Kit (Illumina, San Diego, CA) and the indexed fragments were sequenced using the NextSeq 500/550 v2 high throughput Kit (Illumina, San Diego, CA ).

[0064] To process the raw reads and remove low-quality bases or very short reads (less than 36 nucleotides), the Trimmomatic software (37) was used. The clean reads were then aligned to the reference genome of Rattus norvegicus (release 94) using the STAR (Spliced Transcripts Alignment to a Reference) software (38), with the reads located in a single genomic site being used to calculate the number of reads and the number of reads per kilobase of transcript per million mapped reads of each gene (39). Contrast analysis between infected and control groups was performed using DESeq2 software (40). Genes with Fold Change greater than 1.2 and P value less than 0.05 were considered differentially expressed. After data processing, functional enrichment analysis was performed with the Ingenuity Pathways Analysis (IPA) software, with default parameterization (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis), based on ENSEMBLE GENE Gene IDs and their respective Fold Change and P values (Tables 2 and 3).

Validation of biomarker genes by RT-qPCR

[0065] RNA samples were obtained as described above, from pools of 6 COHs (two per animal) from the control groups, infected with isolate PE243 or infected with isolate SPH2015 at 16h pi. cDNA was synthesized from 0.4 to 1 µg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher), according to the manufacturer's protocol. qPCR reactions were performed with Fast SYBR™ Green Master Mix (ThermoFisher) plus 2 ng/µL of cDNA in a final volume of 10 µL. Mouse-specific primers were designed with the aid of Primer-BLAST software (NCBI, Bethesda, MD, https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to detect candidate genes for Adgre1 biomarkers. , Ccl3, Dbx2, Disp3, Gmr3, Igf1, Il1b, Rac2, Rasgrp3, Siglec5 (Table 1). The expression levels of these genes were normalized to the levels of the endogenous Ppia gene. Thermal cycling and bloom detection was performed using the ViiA 7 real-time PCR system (ThermoFisher) according to the manufacturer's recommendations. The relative expression of target genes was calculated using the 2A(-DCt) method. Table 1 - Oligonucleotides used for quantification, by RT-qPCR, of the expression of biomarker genes of neurodegeneration and inflammatory response processes regulated by ZIKV isolates PE243 and SPH2015 at the beginning of hippocampal infection

Statistical analyzes

[0066] Statistical analyzes were performed with GraphPad Prism software (version 8.0.2) (GraphPad Software Inc., Irvine, CA). The data were evaluated for normality using the Anderson-Darling, D’Agostino & Pearson, Shapiro-Wilk and Kolmogorov-Smirnov tests. For comparisons between two groups of parametric data, unpaired Student's t-test was used, with data expressed as mean ± standard deviation. For comparisons between non-parametric data, the Mann-Whitney test was used and data were expressed as median ± interquartile range. For comparison between three or more groups, with parametric data, one-way analysis of variance (ANOVA) with Tukey's multiple comparisons post-test was used. For non-parametric data, the Kruskal-Wallis test with Dunn's multiple comparisons post-test was used. Furthermore, when comparisons involved the effect of two factors (e.g., time and infection), two-way ANOVA with Tukey's multiple comparisons post-test was used. Differences were considered statistically significant when P values were less than 0.05.

Screening of drugs or substances with neuroprotective potential.

[0067] Organotypic hippocampal cultures (COHs) from infant rats around 7 days old were produced from slices of the organ approximately 400 micrometers thick cultured for stabilization for at least 14 days. COHs were then challenged by ZIKV infection by exposing them to culture medium suspensions containing 6x105 plaque forming units (PFU) per milliliter for two hours. At the end of these two hours, viruses not adsorbed to COH cells were removed by washing with fresh culture medium and the cultures were incubated for 16 hours in fresh medium containing the drug or substance whose neuroprotective potential will be tested. The tests included falsely infected COHs, exposed to the infection procedure, but with virus-free medium, which serve as controls to evaluate the toxicity of the molecules tested in the absence of infection, and infected COHs treated with medium containing the excipient, but not the drug or tested substances, which serve as controls on the efficiency of the infection. After incubation for 16h, RNAs (or proteins) were extracted and the expression levels of biomarker genes were quantified by RT-qPCR. The results were analyzed in the form of radar graphs, highlighting the neuroprotective effect and possible toxicity, respectively, of the drug, substance or molecule tested.

Example 1 Isolates PE243 and SPH2015 infect COHs with distinct proliferation dynamics

[0068] To verify the suitability of the model for the proposed study, viral infection and proliferation in COHs was evaluated by immunofluorescence and confocal microscopy, at the initial times of infection, through NS1 labeling. NS1 is an important non-structural protein of ZIKV that acts in viral replication and regulation of the antiviral immune response. It can also induce remodeling of the endoplasmic reticulum membrane in order to establish a replication compartment for the virus (Reviewed in: 41). Detection of the virus through a non-structural protein makes it possible to confirm its replication, excluding noise that could be caused by viruses remaining from the suspension used in the experimental infection process.

[0069] Quantification, by immunofluorescence and confocal microscopy, of the viral protein NS1 at early times of infection demonstrated that both ZIKV isolates infected and replicated satisfactorily in this model. The evaluation of IF by tissue area indicated that the expression of NS1 by the isolate SPH2015 increased significantly between 8h and 48h p.i.. The expression of NS1 by the isolate PE243 increased between 8 and 24h p.i. and then decreased at 48h (Figure 2). Therefore, the two ZIKV isolates present distinct replication dynamics in this model, with isolate PE243 proliferating more quickly in COHs than PE243.

Example 2 Isolates PE243 and SPH2015 infect mature neurons and have different tropisms for these cells

[0070] ZIKV is capable of infecting several CNS cells, such as neural progenitor cells (42), oligodendrocytes (43), microglia (44), astrocytes, which appear to propagate the infection in the brain of mouse embryos (45) and in mature neurons (23). ZIKV infection in neural precursor cells has already been the subject of several studies, however, less is known about infection in other cell types and susceptibility to infection in the mature CNS (22, 42, 46).

[0071] Analysis of the colocalization of NS1 and NeuN proteins proved that the two ZIKV isolates infect mature neurons in the COHs model (Figure 3). At 8 h p.i., isolate SPH2015 was able to infect approximately 10% of mature neurons, while isolate PE243 infected less than 3% of these cells. Therefore, SPH2015 has greater tropism for mature neurons in the initial periods of infection, when compared to PE243. The percentages of infected neurons decreased significantly at 24 and 48h. As the IF analysis for NS1/mm2 of COHs demonstrated high levels of infection by SPH2015 and PE243 also after 8h p.i., it is concluded that, over time, the virus infects other cell types other than neurons with greater tropism. mature.

Example 3 PE243 and SPH2015 cause different phenotypic changes in COHs

[0072] We then sought to determine the impacts of infection with these different ZIKV isolates on two critical aspects of the COHs phenotype, namely, neuronal density and microglia activation.

[0073] Initially, the effect of infection on the neuronal density (NeuN+ / mm2) of COHs was evaluated over time, from 8h to 10 days p.i. (Figures 4 and 5). The area of COHs (DAPI+) did not show significant variation throughout this period. At 8h p.i., a significant increase in neuron density was observed in infected COHs compared to control, indicating that SPH2015 or PE243 infection induces hippocampal neurogenesis. However, at subsequent times, while PE243 induced the progressive loss of neurons until 48h p.i., SPH2015 caused a pronounced loss of these cells between 8 and 24h p.i., followed by an increase between 24 and 48h p.i. (Figure 4). Thus, two clearly distinct dynamics of neurodegeneration/neurogenesis caused by ZIKV isolates PE243 and SPH2015 in the initial periods of infection are evident.

[0074] The effect of infection on neuronal density was also evaluated at more advanced times (Figure 5). Between five and ten days p.i., COHs infected with both isolates had the same density of neurons, approximately 75% of that observed in the control group (Figure 5). These findings corroborate the previous report that ZIKV also causes neuronal loss late in infection in a neonatal mouse COHs model (47). Thus, the suitability of the model proposed here of COHs from infant rats for the study of neurodegeneration associated with ZIKV infection was proven.

[0075] Next, the contribution of the process of programmed cell death (apoptosis) to the neuronal loss caused by ZIKV in COHs was investigated. For this, TUNEL and NeuN labeling were used followed by confocal microscopy in COHs on the fourth day p.i. It was observed that, at least in part, it is possible to attribute the decline in the neuronal population to death by apoptosis, which is significantly greater in infected COHs (Figure 6).

[0076] The results of immunofluorescence analysis and confocal microscopy of the expression of the Iba1 protein, a marker of activated microglia, demonstrated that microglia also behaved differently depending on the isolate evaluated (Figure 7). Microglia is a population of phagocytic and mononuclear cells residing in the CNS, which respond to potentially harmful stimuli such as infections, tissue damage and neurodegeneration by altering their proliferation patterns, transcription and morphology (Reviewed in: 48). If, on the one hand, infection of COHs with SPH2015 or PE243 did not induce microglia activation in comparison with COHs from the control group, as evidenced by the evaluation of Iba1 expression at 8h and 24h p.i., the second isolate inhibited this response in time 8h. Taken together, these data indicate that these two ZIKV isolates, collected during the same outbreak and with 99.9% similarity at the nucleotide level and 99.97% at the amino acid level (17), have different infection dynamics and induce phenotypic changes. distinct in COHs.

Example 4 Isolates SPH2015 and PE243 induce distinct patterns of epigenetic changes in mature neurons surviving infection in COHs

[0077] Epigenetic changes are modifications in the structure of chromatin or its constituent proteins that lead to an increase or decrease in gene expression. These modifications can be acetylations, methylations, among others (49). Other members of the Flaviviridae family, such as dengue virus, can induce epigenetic changes in host cells as an immune evasion strategy (50). To investigate the possible effect of ZIKV infection on the functionality of COH neurons, the epigenetic mark H3K4me3, associated with euchromatin and active state of gene expression, was evaluated late in the infection. Interestingly, isolate SPH2015 caused a pronounced and progressive increase in the H3K4me3 mark on days 7 and 10 p.i., while isolate PE243, in turn, caused a less expressive increase of this mark on days 5 and 10 p.i. (Figure 8) Therefore, isolates PE243 and SPH2015 may differentially affect the functionality of surviving mature neurons late in infection.

Example 5 Isolates PE243 and SPH2015 cause distinct patterns of changes in the COH transcriptome

[0078] In order to investigate the effect of isolates PE243 and SPH2015 and on global gene expression patterns in the hippocampus, the transcriptome of COHs was analyzed at 16h pi, an intermediate time between the periods of neurogenesis and neurodegeneration observed in infected COHs. We observed 32 differentially expressed genes in PE243-infected COHs and 113 differentially expressed genes in SPH2015-infected COHs (Figure 9 and Tables 2 and 3). Table 2 - Genes differentially expressed in COHs in response to infection with isolate PE243 16h pi Note: Genes with differential expression in response to infection with both PE243 and SPH2015 are highlighted in bold. Table 3 - Genes differentially expressed in COHs in response to infection with the SPH2015 isolate 16h pi Note: Genes with differential expression in response to infection by both PE243 and SPH2015 are highlighted in bold.

[0079] The functional enrichment analysis of the set of differentially expressed genes, carried out with the IPA software, indicated important differences in the biological processes affected in response to infection by ZIKV isolates PE243 and SPH2015 at 16h p.i.. These findings largely explain , the phenotypic differences observed.

[0080] Corroborating the drop in the neuronal population induced by the infection in the period between 8h and 24h p.i., the two isolates caused changes in the expression of genes regulating CNS cell development processes. SPH2015 infection induced downregulation of processes such as neuron development, nervous tissue differentiation and oligodendrocyte differentiation, while PE243 induced downregulation of the more generic process of brain cell proliferation. Furthermore, isolate PE243 positively regulated the processes of apoptosis and death of neurons. The results also indicate that these ZIKV isolates can induce damage to the cognitive-behavioral development of those infected, since the biological processes of spatial memory, neurotransmission and myelination of the nervous system had predicted negative regulation (Figure 10). Taken together, these results strongly corroborate the differences observed in neuronal density variations in COHs in response to infection by isolates PE243 and SPH2015.

[0081] Furthermore, isolates PE243 and SPH2015 induced distinct patterns of differential expression of inflammatory genes in COHs at 16h p.i., corroborating the result of microglia activation demonstrated by immunofluorescence for Iba1 at the beginning of experimental infection (Figure 11). The inflammatory response was predicted to be strongly activated by SPH2015 with increased expression of pro-inflammatory mediators such as Ccl2 (C-C motif chemokine ligand 2), Ccl3l3 (C-C motif chemokine ligand 3 like 3), Ccl4 (C-C motif chemokine ligand 4), Cxcl6 (C-X-C motif chemokine ligand 6), Ccl7 (C-C motif chemokine ligand 7) and Il1b. COHs infected with this isolate also had a positive prediction for the processes of neuroglial activation, astrocytosis and gliosis. For isolate PE243, a timid activation of the inflammatory response was predicted at 16h p.i. It is noted that the two isolates equally regulated the expression of the inflammatory genes Ccl3, Rac2, Il1b and Igf1. Furthermore, in COHs infected by PE243, the production of reactive oxygen species and the generation of superoxide are predicted to be inhibited and the synthesis of nitric oxide is predicted to be increased. The amount of calcium was predicted to be increased in COHs infected by SPH2015 and reduced by PE243 (Figure 11).

[0082] The two ZIKV isolates induced the differential expression of 10 genes in common: Adgre1 (adhesion G protein-coupled receptor E1), Ccl3l3 (C-C motif chemokine ligand 3 like 3), Dbx2 (developing brain homeobox 2), Disp3 ( dispatched RND transporter family member 3), Gmr3 (glutamate metabotropic receptor 3), Igf1 (insulin like growth factor 1), Il1b (interleukin 1 beta), Rac2 (Rac family small GTPase 2), Rasgrp3 (RAS guanyl releasing protein 3) and Siglec8 (sialic acid binding Ig like lectin 8) (Figure 9 and Tables 2 and 3). Differential expression induced by PE243 or SPH2015 infection was tested by RT-qPCR for eight of these genes representative of the processes of neuronal death and neurogenesis and inflammatory response in an independent set of samples and the results confirmed the RNA-Seq findings (Figure 12). .

[0083] A decrease in the expression of the Disp3 and Dbx2 genes was observed in infected COHs. Increased expression of Disp3 stimulates proliferation and inhibits differentiation of neural progenitor cells (51). In the same vein, increased Dbx2 expression inhibits primary neurogenesis in Xenopus embryos in vitro (52). Therefore, an inhibition of the expression of these genes can stimulate neurogenesis. This expression pattern may be a remnant of the host's genetic programming activated early in the infection, which leads to the phenotype observed at 8 h p.i., when infected COHs show greater neuronal density compared to controls.

[0084] Concomitantly, overexpression of Il1b and Ccl3, important inflammatory mediators, was observed. The cytokine IL-1ß, as previously highlighted, may be related to mechanisms of excitotoxicity and cell death (53). CCL3, in turn, is important for the recruitment of inflammatory cells to the site of insult and has been shown in the hippocampus to have negative effects on synaptic transmission, plasticity, and memory (54). Another important gene associated with inflammation is Adgre1, which showed increased expression at 16h p.i. This gene encodes the F4/80 antigen, expressed by hippocampal microglia (55). Its increased expression indicates the proliferation of microglia in COHs at 16h p.i.. Also with increased expression, the Rac2 gene encodes a member of the Ras superfamily of small guanosine triphosphate (GTP) metabolizing proteins. This protein regulates NADPH oxidase activity, which in turn mediates the generation of ROS in cells such as phagocytic leukocytes and microglia (56, 57). Furthermore, Siglec5 had its expression increased by infection. Siglecs are surface proteins that are members of the immunoglobulin superfamily that bind sialic acid and are expressed primarily on hematopoietic cells ( 58 ). Signaling via Siglec inhibits the pro-inflammatory immune response and phagocytosis in microglia (Reviewed in: (59)). Taken together, the increased expression of Adgre1 and Rac2 and decreased Siglec5 point to apparently antagonistic mechanisms of stimulating microglia proliferation and concomitant inhibition of their pro-inflammatory activity.

[0085] The Igf1 gene had its expression decreased by infection with ZIKV. The binding of the IGF1 protein to its main receptor IGF1R activates the PI3K/AKT (Phosphatidylinositol 3-kinase/Protein kinase B) and MAPK (Mitogen activated protein kinase) pathways, mainly the ERK (Extracellular signal-regulated kinase) pathway. The PI3K/AKT pathway is related to the regulation of processes such as cell proliferation, autophagy and apoptosis and is also important in the antiviral response. Virus-induced activation of PI3K/AKT is related to the inhibition of apoptosis, which allows for a greater potential for viral replication. However, this blockade caused by the virus generally triggers the expression of ISGs. The ERK/MAPK pathway is related to increased cell differentiation and proliferation (Reviewed in:60). Rasgrp3, whose expression was inhibited by ZIKV infection, is involved in the activation of the ERK/MAPK pathway through the Ras protein (61).

[0086] With decreased expression, the Grm3 gene encodes the G protein-coupled glutamate receptor 3 (mGluR3), expressed in neurons and glial cells in regions such as the dentate gyrus of the hippocampus (62). mGLUR3 is present in pre- and postsynaptic terminals and acts on voltage-gated calcium channels, thus participating in the modulation of neurotransmission (63). The decrease in expression of this gene at 16h p.i. may be a consequence of the loss of neurons observed between 8h and 24h p.i..

[0087] This panel of biomarkers represents a remnant of the genetic programming responsible for the neurogenesis observed up to 8h p.i., at the same time that it corroborates the phenotype of neuronal loss in the period between 8h and 24h and the important role of the inflammatory response in the initiation of infection by two ZIKV isolates tested.

Example 6 Demonstration of the neuroprotective potential of Cinnamaldehyde and Diphenhydramine in the COHs model of the present invention

[0088] Two neuroprotective drugs were selected from an analysis of the scientific literature to evaluate their effect on modulating the panel of biomarkers of the present invention and validating the present methodology: cinnamaldehyde (Drugbank ID: DB14184) and diphenhydramine (Drugbank ID : DB01075). Cinnamaldehyde (Drugbank ID: DB14184), present in the bark of Cinnamomum species, has multiple functional properties, including anti-cancer, anti-inflammatory and antioxidant properties. Suppression of NMDA receptors has been implicated as part of the mechanism of neuroprotective action of cinnamaldehyde against amyloid beta neurotoxicity (64). Diphenhydramine hydrochloride is a first-generation antihistamine, H1 receptor blocker, with anticholinergic activity, indicated for the prevention and treatment of allergic reactions related to blood or plasma transfusion and as an adjuvant to epinephrine in anaphylaxis. Recently, the neuroprotective effect of diphenhydramine was demonstrated in an experimental model of brain trauma injury in rats. The mechanism of action involves the attenuation of oxidative stress, inflammation and mitochondrial apoptosis pathways (65).

[0089] Following the flowchart in Figure 13, the neuroprotective potential of these two drugs was evaluated. As demonstrated in Figures 14 and 15, respectively, both Cinnamaldehyde and Diphenhydramine modulated the expression of the biomarker genes of this invention, partially reversing the effect of ZIKV infection on these biomarkers.

[0090] Treatment with these drugs increased the expression of the Dbx2 gene, which inhibits primary neurogenesis. In this way, the two drugs attenuate the stimulating effect of the exacerbated and early differentiation of neural progenitor cells due to infection with ZIKV, contributing to the preservation of the squad of these cells.

[0091] Furthermore, treatment with cinnamaldehyde or diphenhydramine reduced the expression of the Siglec 5 gene and increased the expression of Adgre1. Signaling via Siglec inhibits the pro-inflammatory immune response and phagocytosis in microglia and increases the expression of Adgre1, which encodes the F4/80 antigen, indicating microglia proliferation. Therefore, both drugs contribute to the proliferation of microglia and the activation of their phagocytic activity, thus favoring the elimination of infected cells and residues of dead or non-viable cells in the hippocampal tissue.

[0092] Treatment with diphenhydramine inhibited the expression of the Rac2 gene, which encodes a member of the Ras superfamily of small guanosine triphosphate (GTP) metabolizing proteins. This protein regulates NADPH oxidase activity, which in turn mediates the generation of reactive oxygen species in microglial cells. Therefore, diphenhydramine contributes to the reduction of oxidative stress in neuroinflammation caused by ZIKV infection.

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Claims (13)

1. Method for screening drugs or substances with neuroprotective potential, characterized by the fact that it comprises: a) providing organotypic hippocampal cultures (COHs), being: i) at least one control culture, not infected and not treated with the drug or substance to be screened; ii) at least one culture that will be infected with the agent from step b) and will not be treated with the drug or substance to be screened; iii) at least one control culture that will be treated with the drug or substance to be screened; and iv) at least one culture that will be infected with the agent from step b) and will be treated with the drug or substance to be screened; b) infecting the cultures established in ii and iv with a neuroinflammatory infectious agent; c) incubate the cultures established in iii and iv with a drug or substance with neuroprotective potential to be screened; d) extract at least one messenger RNA or protein from each of at least four established subcultures; and e) determine the expression pattern based on a panel of biomarkers from each of at least four established subcultures and, thus, predict the neuroprotective potential of the screened drug or substance. 2. Method according to claim 1, characterized by the fact that organotypic hippocampal cultures are cultivated for at least 14 days. 3. Method according to claim 1 or 2, characterized by the fact that the neuroinflammatory infectious agent used to infect the cultures is the Zika virus (ZIKV). 4. Method according to claim 3, characterized by the fact that the infectious agents isolated from ZIKV are PE243 and SPH2015. 5. Method according to any one of claims 1 to 4, characterized by the fact that the drug or substance to be screened for neuroprotective potential is in a concentration of about 0.1 to 1 μM. 6. Method according to any one of claims 1 to 5, characterized by the fact that the extraction of the nucleic acid from step d) occurs approximately 16h after carrying out steps b) and c). 7. Method according to any one of claims 1 to 6, characterized by the fact that the determination of the expression pattern in a panel of biomarkers from step e) is carried out based on the differential expression of one or more of the genes Adgre1, Ccl3 , Dbx2, Disp3, Gmr3, Igf1, Il1b, Rac2, Rasgrp3, Siglec5 in relation to the expression pattern of the same genes in control cultures. 8. Method according to any one of claims 1 to 7, characterized in that the extracted nucleic acid is RNA. 9. Method according to claim 7, characterized by the fact that the evaluation of the expression of the biomarker panel is carried out by RT-qPCR after reverse transcription, Northern Blot, digital PCR or equivalent techniques. 10. Method according to any of the previous claims, characterized by the fact that the drug or substance to be tested is for controlling neuroinflammation. 11. Method according to claim 10, characterized by the fact that neuroinflammation is caused by ZIKV infection. 12. Kit for screening drugs or substances characterized by the fact that it comprises organotypic hippocampal cultures and an inflammatory agent as defined in any of the previous claims, a drug or substance to be screened, reagents necessary to resolve the method as described in any one of the preceding claims and instructions for use. 13. Use of a gene panel, characterized by the fact that it is in the screening of drugs or substances with neuroprotective potential, in which the panel comprises the genes Adgre1, Ccl3, Dbx2, Disp3, Gmr3, Igf1, Il1b, Rac2, Rasgrp3, Siglec5 .