Exploring the neuroprotective potential of DHEA(S) and BDNF in cellular and animal models and subjects with Alzheimer disease

Dubravka Svob-Strac

Laboratory of Molecular Neuropsychiatry, Division of Molecular Medicine, Ruder Boskovic Institute, Bijenicka cesta 54, Zagreb, Croatia

The neurosteroids dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), as well as neurotrophin brain derived neurotrophic factor (BDNF) have been studied for their neuroprotective potential in Alzheimer's disease (AD). We have investigated potential neuroprotective effects of DHEA(S) and BDNF on the survival and viability of primary mouse neurons, exposed to the toxic Aβ oligomers, as in vitro AD model. In genetic model of AD, 3xTg-AD mice were chronically treated with DHEAS using subcutaneously implanted osmotic pumps. The pharmacologically induced AD model was established in C57BL/6 mice by intracerebroventricular injection of Aβ oligomers followed by the chronic administration of DHEA via intraperitoneal injections. Various cognitive and behavioral tests were performed on both models and analyzed using Noldus EthoVision XT software, whereas hippocampal concentrations of BDNF, Ab and Tau proteins were determined using ELISA. In human subjects with AD and mild cognitive impairment (MCI) (comparative group), cognitive symptoms were evaluated by MMSE (Mini mental state examination). DHEA(S) and BDNF plasma concentrations were determined by ELISA, whereas genotyping for rs2637125 SULT2A1 and rs6265 BDNF gene polymorphisms was conducted using qPCR. The obtained results demonstrated potential beneficial effects of DHEA(S) and BDNF suggesting these compounds as new options for the prevention and treatment of AD. The research has been supported by CSF project IP-2019-04-6100.

Specialized heparan sulfates and the vulnerability of brain regions and cell types to develop tau pathology in Alzheimer’s disease

The identity of the molecular factors responsible for the selective vulnerability of brain regions and cell subpopulations to degenerate is a fundamental question in the field of neurodegenerative diseases. Since the early 1990s, heparan sulfates were found to accumulate in neurons that develop tau pathology in Alzheimer’s disease (AD). However, the reasons for this selective heparan sulfate accumulation and their implications in disease onset and development remained unknown. Heparan sulfates (HS) are the glycan moieties of heparan sulfate proteoglycans that are synthesized by a complex machinery involving dozens of genes, some of which are expressed in a cell and tissue-specific manner. Using histology, spatial transcriptomics, scRNAseq, biochemical, and physicochemical approaches, we identified HS3ST2 as the enzyme that produces the HS that accumulate in AD. We show that this enzyme is selectively expressed in the brain regions and cells that develop tau pathology. Gain and loss of function experiments in cells and animal models of tauopathy confirmed the central role of this enzyme and its product in the mechanisms leading to disease. This is the first reported molecular factor able to mark the selective vulnerability of brain regions and cell subtypes to degenerate in AD.

Modulating cholinergic neurons in Alzheimer's Disease: new quadruple-transgenic mice model

Szidónia Farkas^1,2,3, Viktor Jasper^1,3, Kinga Nyers-Marosi^1,3, Bence Petrovai^1,3, Adrienn Szabó^2,3, Tamás Kovács^1,3, István M Ábrahám^1,3 and Dóra Zelena^1,2,3

¹Molecular Neuroendocrinology Research Group, Institute of Physiology, University of Pécs, Hungary
²Laboratory of Behavioral and Stress Studies, Institute of Physiology University of Pécs, Pécs, Hungary
³Centre for Neuroscience, Szentágothai Research Centre, Pécs, Hungary

Introduction: Alzheimer’s disease (AD) is an increasing health and social problem ranking 7^th among the most common causes of mortality worldwide​. The cholinergic system is its most affected neurocircuit, therefore, it is a common therapeutical target. However, revealing its exact role requires further studies.

Aim: Our aim was to create a genetical mouse model, that represents the progression of AD, in form of Aβ_1-42 plaques, pTau aggregates and cognitive impairments, while at the same time expresses the Cre recombinase enzyme specifically in cholinergic neurons.

Methods: Two strains were cross-bread: B6;129-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/Mmjax) and B6;129S6-Chattm2(cre)Lowl/J. After serial genotyping, a colony, homozygote for four genes (PSEN1, APPSwe, tauP301L and Cre; 3xTg-ChAT-Cre) was established. To test the functionality of the Cre enzyme a stimulating DREADD virus (AAV8-hSyn-DIO-hM3Dq-mCherry) was injected unilaterally into the nucleus basalis magnocellularis (NBM) and clozapine-N-oxide-induced c-Fos activation was compared between the two hemispheres. For behavioural characterization different tests were performed: Y-maze, single pellet skilled reaching (SPSR), fox odor test (FOT), splash test (ST) and social discrimination test (SDT). The progressive appearance of Aβ plaques and pTau aggregates were confirmed by immunohistochemistry.

Results: Immunostainings confirmed the expression of Cre-dependent fluorophore in ChAT positive cells as well as the appearance of the pathological hallmarks (Aβ and pTau). The c-Fos activity was significantly increased at the virus injected hemisphere. In the behavioral tests 3xTg-ChAT-Cre mice showed decreased locomotor activity (Y-maze, SDT, FOT), increased anxiety (FOT, ST) and weaker fine motoric skills (SPSR) compared to control animals.

Conclusions: The newly created animals have a functional Cre recombinase enzyme in cholinergic cells. Additionally, the animals showed the pathophysiological hallmark of AD in specific brain areas and kept the typical behavioral alteration found in 3xTg-AD mice before. Thus, this strain seems to be appropriate for further studies.

Neurodegeneration in the TgF344-AD rat model of Alzheimer's disease

Anett Futácsi ^1,2, Kitti Rusznák ^1,2, Gergely Szarka ^3,4, Ove Wiborg ⁵ and Boldizsár Czéh ^{1,2 *}

¹ Structural Neurobiology Research Group, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary
² Department of Laboratory Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary
³ Histology and Light Microscopy Core Facility, Szentágothai Research Centre, University of Pécs, Pécs, Hungary
⁴ Retinal Neurobiology Research Group, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary
⁵ Department of Health Science and Technology, Aalborg University, 9220 Aalborg, Denmark

Alzheimer's disease is the most common form of dementia. The incidence of the disease increases with age, affecting 1% of 60-year-olds, and 30% of 85-year-olds, altogether approximately 55 million people worldwide (WHO data). Several studies provide evidence that abnormally folded amyloid beta and tau proteins that accumulate in amyloid plaques are responsible for the neurodegeneration, causing progressive deterioration of the nervous tissue and subsequent behavioral disturbances and memory-loss. It is well documented that the aggregation of the β-amyloid leads to neuro-inflammation and neuronal cell death, however the progression and exact mechanism still remains to be clarified.

Here, we investigated neuronal and glial changes in the brains of a transgenic Alzheimer’s rat model, the TgF344-AD rats. This model shows the overexpression of human amyloid precursor protein (APPsw) and presenilin 1 (PSEN1E9), which play an important role in the progression of the disease. TgF344-AD rats express 2.6 times more human APP and 6.2 times more human PSEN1 in the brain. With post-mortem immunohistochemistry, we labelled amyloid plaques, microglial cells and astrocytes, as well as GABAergic interneurons in the hippocampus. Systematic, unbiased cell counting was carried out to assess putative cellular changes.

Our preliminary data indicate a pronounced neuro-inflammatory response in the hippocampus of the TgF344-AD model, involving mainly microglial cells, but among the GABAergic neurons only in the number of cholecystokinin-positive cells were altered.

Contrary to expectations, we could did not detect a correlation between amyloid plaque load and the neuronal and glial changes.

Acknowledgement:

This research was founded by the Hungarian Brain Research Program 3 (NAP-3), and by the TKP2021-EGA-16 project. Project TKP2021-EGA-16 has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme.

Investigation of the TRPA1 receptors in Alzheimer's disease

Viktória Kormos

Introduction: Alzheimer's disease (AD) is the most common form of dementia, associated with a progressive loss of memory and cognitive function. The neurodegeneration of the basal forebrain cholinergic cells is known in AD, however less attention has been paid to the role of other cholinergic brain areas. The cholinergic neurons of the Edinger-Westphal nucleus (EW), part of the oculomotor complex, may be affected early in the disease, causing pupillomotor dysfunction. However, no data are available on the involvement of the peptidergic neurons of the centrally projecting EW (EWcp) in AD. Both our own data and those in the literature suggest a role for the transient receptor potential ankyrin1 (TRPA1) ion channel in neurodegenerative diseases. We recently demonstrated that the EWcp area is the seat of the strongest Trpa1 mRNA expression in the mouse central nervous system that is localized to the peptidergic, UCN1-containing neurons. Considering that the EW is affected by AD, and the TRPA1 is highly expressed here, we anticipated that TRPA1 plays a role in the AD-associated neurodegeneration of the peptidergic neurons of EW. To examine the possible role of EW/TRPA1 in AD, our aimes were to describe the timecourse of the Trpa1 mRNA expression, moreover, to investigate the site-specific deletion of TRPA1 in the urocortinergic EW neurons in a mouse model of AD.

Methods: 3xTg (amyloid precursor protein, presenilin-1 and tau protein overexpressing) mice were used as a model of AD. Two, 12 and 18 months old 3xTg mice and C57BL6 mice of the same age as controls were studied. Trpa1 RNAscope in situ hybridization was combined with UCN1 immunofluorescence labeling in the EWcp to measure Trpa1 mRNA expression. An adeno-associated virus vector (AAV) was constructed that carries an UCN1 promoter-driven Cre recombinase (Ucn-Cre-AAV). The function and specificity of the AAV vector was tested on stopflox-tdTomato mice.

Results: Significantly higher Trpa1 expression was observed in 2-months-old controls than in age-matched 3xTg mice. Trpa1 expression decreased by age in the C57BL6 strain. 3xTg mice showed lower Trpa1 expression that was not affected by the course of aging. The AAV vector was specific to the UCN1 positive neurons.

Conclusions: Altered age-related dynamics of Trpa1 expression in the urocortinergic neurons of AD mice suggest that the peptidergic neurons may also be affected by AD. Further investigation is needed to test the AAV vector in TRPA1 floxed mice.

Funding: Hungarian Brain Research Programme (NAP3) 2022-2025. János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00750/22/5). New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund (ÚNKP-23-5-PTE-1991). Research grant of Medical School, University of Pécs (KA-2022-29).

The protective effects of PACAP1-38 on the retinal vasculature and hypoxic molecules in rat glaucoma model

Evelin Patko¹, Edina Szabo¹, Alexandra Vaczy¹, Dorottya Molitor¹, Eniko Tari¹, Lina Li¹, Adrienne Csutak², Gabor Toth ³, Dora Reglodi¹, Tamas Atlasz^1,4,5

¹ Department of Anatomy, MTA-PTE PACAP Research Team, University of Pecs Medical School, 7624 Pecs, Hungary
² Department of Ophthalmology, Clinical Centre , University of Pecs Medical School, 7632 Pecs, Hungary
³ Department of Medical Chemistry, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary
⁴ Szentagothai Research Center, University of Pecs, 7624 Pecs, Hungary Department of Sportbiology, University of Pecs, 7624 Pecs, Hungary
⁵ Department of Sportbiology, University of Pecs, 7624 Pecs, Hungary Szentagothai Research Center, University of Pecs, 7624 Pecs, Hungary

Introduction: Despite the critical impact of glaucoma on blindness, its cause is not fully understood. Animal models are important for better understanding the mechanism behind this disease. Elevated intraocular pressure (IOP) is a risk factor for glaucoma. In our previous study, we described an inducible, microbeads model in Sprague Dawley (SD) rat in which we were able to prove the neuroprotective effects of PACAP1-38 eye drops treatment. Vascular factors have been suggested to play an important role in the development of glaucoma, based on numerous studies.

Aim: In our present study, our aim was to examine the possible protective effects of PACAP1-38 eye drops on the retinal vasculature and the molecular patterns of hypoxia in hypertensive glaucoma model.

Methods: We induced hypertension through injection of polystyrene microbeads into the anterior chamber of SD rats, PBS receiving rats served as controls. Intraocular pressure was recorded every two weeks. We assessed retinal degeneration, vascular and molecular changes through immunofluorescence. HIF1α A protein level was also measured by western-blot.

Results: Significantly increased IOP was observed in the glaucomatous vehicle-treated group (Beads+S) however, in the PACAP1-38 treated group (Beads+P) the IOP remained in a normal range. Optical coherence tomography images suggested severe retinal degeneration in the glaucomatous group, although protective effects were measured after topical administration of PACAP1-38. We also found several vascular parameters changed in the Beads+S group. The examination of molecular patterns suggested hypoxic conditions in the Beads+S group, however after PACAP1-38 administration retinoprotective effects were observed in HIF1α protein level.

Conclusions: Our results show that PACAP1-38, given in form of eye drops, is retinoprotective in glaucoma, providing the basis for potential future therapeutic administration.

Acknowledgment: ÚNKP-23-3-II-PTE-2034; FK129190, K135457; National Brain Research Program NAP2017-1.2.1-NKP-2017-00002; MTA-TKI-14016; PTE AOK-TANDEM; GINOP-2.3.2-15-2016-00050 “PEPSYS”; EFOP-3.6.2-16-2017-00008; “The role of neuroinflammation in neurodegeneration: from molecules to clinics”; and Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary: 20765/3/2018/FEKUTSTRAT, 2020-4.1.1-TKP2020—FIKP III. Project No. TKP2020-IKA-08 has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the 2020-4.1.1-TKP2020 funding scheme.

3D online acousto-optical motion correction for in-vivo measurements

Katalin Ócsai^1,2, Attila Losonczy^3,4, Balázs Rózsa^2,5

¹Department of Mathematical Algebra and Geometry, Institute of Mathematics, Budapest University of Technology and Economics,
²Brain Vision Center, Budapest, Hungary
³Department of Neuroscience, Columbia University; New York, NY, 10027, United States.
⁴Mortimer B. Zuckerman Mind Brain Behavior Institute, Department of Neuroscience, Columbia University, New York, NY, USA
⁵Laboratory of 3D Functional Network and Dendritic Imaging, Institute of Experimental Medicine, Budapest, Hungary

In vivo measurements of behaving animals are inevitably influenced by animal motion, including both intended and unintended movements (such as visceral motion and heartbeats). However, increasing the volume around the neuron is not an option as it would result in a loss of valuable temporal resolution, which is unacceptable when using voltage-sensitive dyes to acquire signals from small regions of interest for maximal temporal precision.

Here we present an FPGA-based, clearly acousto-optical 3D online motion correction method for extracting signals at high temporal resolution up to 100 kHz. The system can handle motion up to 100 Hz on a volume of 900x900x400 um, with residual motion under 1 um and proper signal-to-noise ratio. A cell can be used as a reference for motion estimation. The correction can be combined with all scanning modes, including point-scan, squared- and ribbon-based scanning types for population and dendritic scanning, as well as raster and volume scan types (i.e. Z-Stack). Motion correction can also be combined with photostimulation to ensure precise targeting of optogenetic stimulation. The closed-loop online motion compensation has been validated with recordings in the cortex of awake mice, via ~3kHz voltage imaging using the Jedi2P sensor, and acquisition of 4D datasets (3 space + time).

Quantitative electron microscopic analysis of synapses in the hippocampus of psychiatric patients

A. Sebők-Tornai ¹, D. Csabai ^1,2, C. Szekeres-Paraczky ³, Z. Maglóczky ³, M. Simon ^1,4, C.A. Stockmeier ^5,6 and B. Czéh ^1,2

¹ Szentágothai Research Centre, University of Pécs, Pécs, Hungary
² Dept. Laboratory Medicine, Medical School, University of Pécs, Pécs, Hungary
³ Human Brain Research Laboratory, Inst. Experimental Medicine, ELKH, Budapest, Hungary
⁴ Dept. Psychiatry and Psychotherapy, Medical School, University of Pécs, Pécs, Hungary
⁵ Dept. Psychiatry and Human Behavior, Translational Research Center, University of Mississippi Medical Center, Jackson, MS, USA

Major depressive disorder (MDD) is a common multifactorial disorder, but the exact pathophysiology is still unknown. In vivo and post-mortem studies document volumetric and cellular changes in the hippocampus of depressed patients. Chemical synapses are key functional units of the central nervous system and earlier studies found reduced number of synapses in the prefrontal cortex of depressed patients (Kang HJ et al. Nature Medicine 2012;18(9):1413-1417).

Here, we investigated synapses in post-mortem hippocampal samples from psychiatric patients.

The three study groups were: 1) MDD patients (n=11); 2) patients with alcohol dependence (n=8) and 3) controls (n=10). Controls were individuals who accidentally deceased and had no neuropsychiatric disorders. Three sub-regions of the hippocampus (dentate gyrus, CA3 and CA1 areas were investigated. Ultrathin sections were examined, and photomicrographs were taken for further analysis using a JEOL JEM 1400 FLASH transmission electron microscope. Systematic quantitative analysis was conducted with the Neurolucida system using unbiased counting principles.

Synapse numbers and post-mortem volumetry revealed mild differences between the subject groups.

Our preliminary data suggest that despite our expectations hippocampal synapse densities are rather constant parameters which are not easily affected by psychopathology or alcohol consumption.

FUNDING:

This research was founded by the Hungarian Brain Research Program 3 and by the TKP2021-EGA-16 project. A.S.T. was supported by the ÚNKP-23-3-I New National Excellence program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.

Keywords: major depression, neurobiology, ultrastructure, hippocampus, synapse, mitochondria

Deciphering nano-scale characteristics of microglia using STED super-resolution microscopy

Csaba Cserép¹, Balázs Pósfai¹, Eszter Szabadits¹, Anett D. Schwarcz^1,2, Nikolett Lénárt¹, Rebeka Fekete¹, Eszter Császár¹, Ana Rita Bras^1,2, Ádám Dénes¹

¹ “Momentum” Laboratory of Neuroimmunology, Institute of Experimental Medicine, Budapest, 1083, Hungary
² Szentágothai János Doctoral School of Neurosciences, Semmelweis University, Budapest, 1083, Hungary

Microglia are the main immunocompetent cells of the CNS and the sole myeloid cells that reside in the parenchyma. Recent studies have shown that microglia are indispensable for neurodevelopment and brain homeostasis beyond their inflammatory functions, but the exact sites mediating these effects have remained unclear, mainly due to the lack of appropriate tools. We utilised advanced microscopic techniques to reveal major functional traits of microglia in health and disease.

We have used sensitive immunostaining methods, high-resolution multi-channel confocal laser scanning microscopy and 3D-reconstruction combined with stimulated-emission depletion (STED) microscopy to reveal the subcellular charcteristics of the functional connections between microglia, neurons and blood vessels, with special focus on the nanoscale distribution of a key microglial purinergic receptor.

We have shown previously that specific sites on neuronal cell bodies exist in both the mouse and the human brain, allowing the dynamic monitoring and assistance of neuronal function by microglia. Using state-of-the-art methodology, we have revealed that somatic microglia-neuron junctions possess specialized nanoarchitecture optimized for bi-directional communication. Using a multi-modal approach, we have also shown that microglia represent an integral modulatory cell type of the neurovascular unit both in the mouse and human brain, with microglial processes extending under the astrocytic endfeet to establish direct contacts with endothelial cells. A core microglial purinergic receptor, P2Y12R plays very important roles in the establishment and functioning of these intercellular communication sites. Using in vivo 2P imaging we found that P2Y12R-function is crucial to maintain normal microglial morphology and motility. We utilised STED super-resolution microscopy to assess the cell surface localisation of P2Y12R, and we described the nanoscale distribution of these receptors on microglial membranes both in mouse and human brain. Interestingly, pharmacological interventions or pathology-associated states led to a dynamic redistribution of these proteins – which can have important functional consequences concerning neuronal, glial- or vascular responses.

Collectively, our efforts to optimize and combine a wide palette of advanced microscopic modalities resulted in the description of so far unknown direct interaction sites between microglia and other brain cells with far-raching functional consequences. We have also shown that microglial P2Y12Rs play instrumental roles in the formation and functioning of these special communication sites, while the expression of these receptors is dynamically regulated. These results shed light on the complex and important roles of microglial actions in health and disease.

Dependence of immune synapse formation on the actin cytoskeleton during IL-15 trans-presentation and antigen presentation

Ádám Kenesei¹, Éva Hegedüs¹, Julianna Volkó¹, Adrienn Harsányi¹, Anna Koncz¹, Tamás Fekete², Katalin Tóth¹, Lóránt Kelemen², György Vámosi¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen
² Department of Biophysics, HUN-REN Biological Research Centre, Szeged

The long-term viability of T-cells and the development of immunological memory are dependent on interleukin-15. The receptor for this cytokine is composed of three subunits. IL-15 operates primarily via trans-presentation (TP), where an antigen-presenting cell (APC) expresses IL-15 bound to IL-15Rα and presents it to the βγ_c receptor heterodimer on an adjacent T or NK cell. The initial step of T-cell activation, known as antigen presentation (AP), is also reliant on the interaction between APCs and T-cells where the antigen-binding MHC molecules interact with T cell receptors. F-actin reorganization during AP has been documented; however, the role of the cytoskeleton in IL-15 TP is yet to be investigated. In our human Raji B-cell – Jurkat T-cell model system we monitored immune synapse (IS) formation and the redistribution, intercellular interaction and signaling of receptors during IL-15 TP and AP in intact cells and in cells deprived of actin filaments by latrunculin A treatment. We also nanofabricated novel photopolymer cages to bring cells into contact with each other by optical tweezers and to follow the kinetics of IS formation.

The presence of F-actin proved to be crucial for IS formation in both IL-15 TP and AP. When either the presenting or the T-cell was deprived of F-actin, the number of IS-s and their tensile strength decreased significantly. According to optical tweezers measurements, the average time needed to form a stable IS upon IL-15 TP was 20 seconds, which increased to 60 seconds in F-actin depleted T-cells; no IS-s were formed in the absence of IL-15. Once an IS was formed, the subsequent molecular processes were not affected: the translocation of the receptor subunits to the IS or the assembly of the IL-15 TP and AP receptor complexes did not change significantly in cells lacking F-actin. Similarly, the initial signaling steps of both IL-15 TP and AP remained unchanged when F-actin was depolymerized in the T cells. In conclusion, although F-actin is important for stable IS formation, its absence does not affect the association or signaling efficiency of IL-15 TP and AP complexes.

Uncovering mechanisms underlying target cell type-dependent short term plasticity

Noémi Holderith

Laboratory of Cellular Neurophysiology, HUN-REN Institute of Experimental Medicine, Budapest, 1083, Hungary

Postsynaptic target cell-type dependent efficacy and short-term plasticity of glutamatergic synapses enhance the computational power of the cortical network. Adult mouse hippocampal CA1 pyramidal cell connections to oriens lacunosum-moleculare (O-LM) interneurons have low release probability and display short term facilitation, while their connections to fast spiking interneurons (FS-IN) have 10 fold higher release probability and display short term depression. Active zone nano-topologies and coupling distances between Cav2.1 type Ca²⁺ channels and synaptic vesicle release sites identified by Munc13-1 clusters are not responsible for the distinct release probability. Although [Ca²⁺] transients are 40% larger in FSIN innervating boutons, when [Ca²⁺] entry is matched in the two bouton populations, EPSCs in O-LM cells remain 7-fold smaller, implying that other mechanisms contribute to the marked differences. Activation of Munc13 with a phorbol ester analog (PDBU) results in a much larger augmentation of EPSC amplitudes at the low release probability connection, suggesting incomplete docking or priming of vesicles. Similar densities of docked vesicles rule out distinct release site occupancies and demonstrate that incompletely primed, but docked vesicles limit the output of PC – O-LM synapses.

PACAP contributes to the maintenance of endotoxin fever through the regulation of pyrogenic cytokines and cyclooxygenase-2

Jason Sparks¹, Nora Furedi¹, Kata Fekete², Margit Solymar², Katalin Ordog³, Laszlo Deres^3,4, Krisztina Pohoczky⁵, Agnes Kemeny^5,6, Dora Reglodi¹, Eszter Pakai², Andras Garami²

¹ELKH-PTE PACAP Research group, Department of Anatomy, Medical School, University of Pecs, Pecs, Hungary
²Department of Thermophysiology, Institute for Translational Medicine, Medical School, University of Pecs, Pecs, Hungary
³1st Department of Medicine, Medical School, and Szentagothai Research Centre, University of Pecs, Pecs, Hungary
⁴HAS-UP Nuclear-Mitochondrial Interactions Research Group, Budapest, Hungary
⁵Department of Pharmacology and Pharmacotherapy, Medical School, University of Pecs, Pecs, Hungary
⁶Department of Medical Biology, Medical School, University of Pecs, Hungary

Introduction and Aim: Pituitary adenylate cyclase-activating polipeptide (PACAP) signaling is involved in various inflammatory processes. A common manifestation of systemic inflammation is fever, which is usually induced in animal models with the administration of bacterial lipopolysaccharide (LPS). A role for PACAP signaling was suggested in LPS-induced fever, but the underlying mechanisms of how PACAP contributes to febrile response have remained unclarified.

Methods: We administered LPS (120 µg/kg, intraperitoneally) to mice with the Pacap gene, i.e., the gene encoding the PACAP protein, either present (Pacap^+/+) (n=15) or absent (Pacap^−/−) (n=14) and measured their thermoregulatory responses, serum cytokine levels, and tissue cyclooxygenase-2 (COX-2) expression.

Results: We found that the LPS-induced febrile response was attenuated in Pacap^−/− mice compared to their Pacap^+/+ littermates starting from ~120 min postinfusion. Administration of LPS resulted in amplification of COX-2 mRNA expression in the lungs, liver, and brain of the mice in both genotypes at 210 min postinfusion. In the LPS-treated groups, the upregulation of the COX-2 mRNA in Pacap^−/− mice was significantly attenuated in the liver, whereas it was augmented in the lungs and the brain compared to Pacap^+/+ mice. Serum concentration of the pyrogenic cytokines interleukin (IL)-1α and β were significantly increased in Pacap^+/+ mice in response to LPS compared with saline, whereas the change was not significant between the treatment groups in Pacap^−/− mice. In case of IL-1α and β, the intergenotype difference between the LPS-treated groups was also significant. The serum concentrations of IL-6, IL-10, and TNFα were higher in LPS-treated than in saline-treated mice of both genotypes, however, the rise in IL-10 was significantly attenuated in Pacap^−/− mice compared to Pacap^+/+ mice.

Conclusion: PACAP signaling is necessary for normal fever maintenance. Our results suggest that PACAP contributes to the later phases of LPS-induced fever by modulation of COX-2 protein expression in the periphery and the brain, as well as by augmentation of pyrogenic cytokine levels in the circulation. These findings advance the understanding of the crosstalk between PACAP signaling and the “cytokine-COX-2” axis in systemic inflammation, thereby open up the possibilities for new therapeutic approaches.

Topiramate expressed anti-allodynic effect on the adjuvant-induced inflammatory orofacial allodynia model in the rat

Violetta Mohos¹, Máté Harmat¹, Tamás Kitka², Sándor Farkas², Erika Pintér^1,3,4,5, Zsuzsanna Helyes^1,3,4,5

¹ Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Pécs, Hungary
² Vascular Research Group, Budapest, Hungary
³ Hungarian Research Network, PTE HUN-REN Chronic Research Group, Budapest, Hungary
⁴ National Laboratory for Drug Research and Development, Budapest, Hungary
⁵ PharmInVivo Ltd., Pécs, Hungary

Introduction: Orofacial pain is a frequently occurring debilitating condition, which commonly develops due to the sensitization of extra- and intracranial trigeminal primary afferents [1, 2]. Orofacial inflammation can activate the peptidergic, trigeminal sensory neurons, inducing mechanical allodynia/hyperalgesia on the face or in the periorbital region [3]. As a large portion of patients are without appropriate therapy, it is important to identify new therapeutic approaches in different, validated animal models.

Therefore, we aimed to further characterize the Complete Freund's Adjuvant (CFA)-induced chronic orofacial allodynia model with drugs (e.g. sumatriptan, topiramate), which are widely used in disorders connected with the hypersensitization of the trigeminovascular system. Topiramate inhibits mainly the voltage-gated Na⁺ and Ca²⁺ -channels, and also modulates other targets (e.g., GABA_A receptors, glutamate receptors), while sumatriptan, the acute antimigraine drug acts on the 5-HT_1B/1D receptors [4, 5]. Our results may prove the relevance of the CFA-induced chronic orofacial allodynia model and allow its further application to test new drug candidates.

Methods: Chronic orofacial allodynia was induced by subcutaneous (s.c.) injection of CFA (0.5 mg/mL, 50 µL) into the right whisker pad of adult, male Sprague-Dawley rats, after which mechanonociceptive threshold values were measured with von Frey filaments. The effects of sumatriptan (1 mg/kg s.c., 0.1 mL/100 g volume) and topiramate (30 mg/kg per os, 0.5 mL/100 g volume) were tested on the mechanonociceptive threshold values (on days 3, 5, and 7), 60, 120, and 180 minutes after the treatments. Data were analysed using the GraphPad Prism 9 software. Results are presented as the means ± standard errors of the means (SEM).

Results: Three days after CFA administration, robust orofacial inflammatory allodynia was developed in about 60% of animals, with mechanonociceptive thresholds decreasing from 18.30 g to about 5 g. Compared to the vehicle-treated group, topiramate exerted remarkable anti-allodynic effect on all days investigated. Moreover, significant differences were also observed in mechanonociceptive thresholds before and after topiramate treatment within the same group. Although, sumatriptan also reduced the CFA-induced allodynia, this was not statistically significant compared to the vehicle-treated group.

Conclusion: This model is appropriate to investigate chronic orofacial allodynia related to trigeminovascular activation and to evaluate the effect of new drug candidates in comparison with topiramate, as the reference compound.

References: [1] Burstein R, Yamamura H, Malick A, et al. Chemical stimulation of the intracranial dura induces enhanced responses to facial stimulation in brain stem trigeminal neurons. J. Neurophysiol. 1998;79(2):964–982. doi:10.1152/jn.1998.79.2.964 [2] Romero-Reyes M, Uyanik J.M. Orofacial pain management: current perspectives. J. Pain res. 2014;7:99–115. doi: 10.2147/JPR.S37593 [3] Iwata K, Takeda M, Oh S.B, Shinoda M, (2017). “Neurophysiology of orofacial pain,” in Contemporary Oral Medicine, eds C. Farah, R. Balasubramaniam, and M. McCullough (Cham: Springer), 1–23. [4] Pearl N Z, Babin Caroline P, Catalano Nicole T, et al. Narrative Review of Topiramate: Clinical Uses and Pharmacological Considerations. Adv. Ther. 2023;40:3626–3638. doi: 10.1007/s12325-023-02586-y [5] Moein A, Ghasemi M, Jafari R M, Dehpour A R, et al. Beyond its anti-migraine properties, sumatriptan is ananti-inflammatory agent: A systematic review. Drug Dev Res. 2021;82:896–90. doi: 10.1002/ddr.21819896

Acknowledgment: Supported by TKP 2021-EGA-16, NAP-3, PTE-ÁOK-KA-2023-23, ÚNKP-23-4-II.

The central effects of PACAP on the hypothalamic-pituitary-gonadal (HPG) axis in mice

Péter Faludi¹, Lengyel Ferenc¹, Klaudia Barabás¹, Ildikó Udvarácz¹, Dániel Pham², Dóra Reglődi², Zsuzsanna Nagy¹, Gergely Kovács¹

¹ Institute of Physiology, Medical School, University of Pécs, Pécs, Hungary
² Department of Anatomy, Medical School, University of Pécs, Pécs, Hungary

Introduction: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP) neuropeptide family that is involved in the regulation of several releasing hormones and trop-hormones by stimulating intracellular cAMP production. The hypothalamic-pituitary-gonadal (HPG) axis regulates the synthesis and the release of sex hormones and the gametogenesis in all mammals. Although, the effect of PACAP on fertility is well documented, the mechanism of the effect of PACAP on hypothalamic GnRH and kisspeptin neurons, which are key elements at the highest regulatory level of the HPG axis, is not known in detail. In our previous study, we demonstrated hypothalamic changes that could contribute to the irregular estrous cycle observed in PACAP knockout (KO) female mice.

Aim: In our present experiments, we examined structural changes in the hypothalamus and the testis that might underlie the fertility problems found in male PACAP KO mice.

Methods: Our experiments were performed in brain and testis samples obtained from wild-type (WT) and PACAP KO animals using immunohistochemistry and western blot techniques. Immunohistochemistry was applied to determine the number and fiber density of GnRH neurons in WT and PACAP KO mice. Using the RNAscope technique, kisspeptin mRNA-positive cells were counted in the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus (ARC). Finally, the mRNA and protein expression of estrogen receptor alpha (ERα) and the protein expression of androgen receptor (AR) were also examined.

Results: In our experiments, we found that in PACAP KO animals, both body and testis weights were increased. We observed fewer Sertoli cells in testicular tissue obtained from PACAP KO animals. Immunohistochemical staining of the hypothalamus showed that the number and fiber density of GnRH neurons decreased in the medial preoptic area. Furthermore, the number of kisspeptin neurons increased in R3PV and the mid-portion of the arcuate nucleus. The amount of ERα mRNA increased in both the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus, in regions where kisspeptin neurons regulating GnRH neurons are located. Interestingly, the number of AR+ cells decreased while the number of ERα+ cells increased in the medial preoptic area (MPOA) region, demonstrating a misbalance between effects exerted by estrogens and testosterone.

Conclusion: Our results suggest that the observed changes in the hypothalamus and testicular tissue might be involved in the development of fertility problems in PACAP-deficient males by altering the normal function of the HPG axis. Further experiments are needed to elucidate the exact pathomechanism.