DODATEK O HOSTOVÁNÍ KE SMLOUVĚ O ÚČASTI NA PROGRAMU MERIT

DODATEK O HOSTOVÁNÍ KE SMLOUVĚ O ÚČASTI NA PROGRAMU MERIT

mezi

Středočeské inovační centrum, spolek

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Národní ústav duševního zdraví

Tento DODATEK O HOSTOVÁNÍ KE SMLOUVĚ O ÚČASTI NA PROGRAMU MERIT

uzavřené dne 11. dubna 2023 (dále jen „Dodatek o hostování“) uzavřely následující smluvní strany:

1. Středočeské inovační centrum, spolek se sídlem Xxxxxxxxx 00/00,000 00 Xxxxx 5 IČO: 042 28 235

zastoupený Mgr. Xxxxxx Xxxxxxxxxxx, statutárním ředitelem

(dále jen „Koordinátor“)

2. Národní ústav duševního zdraví

se sídlem Topolová 748, 250 67 Klecany

IČO: 00023753

Zastoupená PhDr. Xxxxxx Xxxxxxxxx, Ph.X., ředitelem č.ú. 25234081/0710 vedený u Česká národní banka

(dále jen „Partner“)

(Koordinátor a Partner společně jako „Strany“)

PREAMBULE

1. Strany uzavřely Smlouvu o účasti na programu MERIT (dále jen „Smlouva“), na jejímž základě je realizována jejich spolupráce při realizaci Programu a upraveny podmínky vzniku práva na poskytnutí Podpory.

2. Realizace Projektu postoupila do další fáze, pročež Koordinátor vyzval Partnera k uzavření tohoto Dodatku o hostování.

3. Partner v postavení Implementující organizace má zájem nabýt účinností tohoto Dodatku o hostování postavení Hostující organizace, se všemi právy a povinnostmi příslušejícími k tomuto postavení.

Strany v souladu s článkem 9 Smlouvy přistupují k uzavření Dodatku o hostování o následujícím znění:

1. Definice a výkladová pravidla

   1. Není-li v tomto Dodatku o hostování uvedeno jinak, uplatní se v plném rozsahu definice a výkladová pravidla převzatá Stranami v článku 1 Smlouvy.

2. Předmět a prohlášení stran

   1. Strany prohlašují, že

      1. Výzkumník předložil Projekt, jehož specifikace tvoří přílohu č. 1 Dodatku o hostování;

      2. Projekt předložený Výzkumníkem splňuje požadavky Programu;

      3. Výzkumník má zájem realizovat Projekt u Partnera;

      4. Výzkumník byl vybrán v rámci výběrového procesu Programu a jeho výběr, včetně přiřazení k Partnerovi, byl oficiálně schválen Řídicím výborem (Steering Committee) – Protokol o výběru Výzkumníka a jeho přiřazení k Partnerovi tvoří

přílohu č. 2 Dodatku o hostování.

2. Partner se zavazuje nejpozději do 1. října 2024, od uzavření Dodatku o hostování uzavřít s vybraným výzkumníkem, VYMAZÁNO, Pracovní smlouvu svým obsahem odpovídající Pokynům k Pracovní smlouvě (viz Příloha č. 3 Smlouvy). Nebude-li Pracovní smlouva dle předchozí věty v daném termínu uzavřena, pozbývá tento Dodatek o hostování účinnosti a Partner ztrácí postavení Hostující organizace.

3. Partner se dále zavazuje poskytnout Vybranému výzkumníku Spolufinancování za podmínek a v rozsahu stanoveném Smlouvou.

4. Partner bere na vědomí, že účinností tohoto Dodatku o hostování nabývá postavení Hostující organizace s právy a povinnostmi příslušejícími tomuto postavení. Tím nejsou dotčena další práva a povinnosti plynoucí Partnerovi ze Smlouvy.

3. Ostatní a závěrečná ustanovení

   1. Tento Dodatek o hostování nabývá účinnosti dnem jeho zveřejnění v registru smluv.

   2. Ve zbytku zůstávají ustanovení Smlouvy nedotčena.

VYMAZÁNO VYMAZÁNO

Koordinátor Xxx. Xxxxx Xxxxxxxxx, statutární ředitel Partner PhDr. Xxxx Xxxxxxx, Ph.X., ředitel

Příloha č. 1 – Specifikace Projektu

Příloha č. 2 - Protokol o výběru Výzkumníka a jeho přiřazení k Partnerovi (Zápis z jednání Steering Committee)

Obě přílohy jsou součástí Dodatku.

PROJECT PROPOSAL TECHNICAL DESCRIPTION

Decoding the Gut-Microbiome-Microglia Axis in Alzheimer's Disease Progression

1. EXCELLENCE

1. Quality and pertinence of the project’s research and innovation objectives (and the extent to which they are ambitious, and go beyond the state-of-the-art).

Introduction, State-of-the-art, specific objectives and overview of the action

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that has significant impacts on individuals and society. Despite extensive research, the underlying mechanisms of AD are still not well understood, and effective treatments are lacking. However, emerging evidence suggests that the gut microbiome, which refers to the diverse community of microorganisms in our digestive system, may play a crucial role in the development and progression of AD¹. Imbalances in the gut microbiota have been shown to cause brain inflammation, a process closely linked to the development of AD². Dysfunction in the gut microbiota can also contribute to enhanced immunosenescence, oxidative stress, cytokine secretion, and neuroinflammation, all of which are involved in the early stages of AD³.

The gut microbiota can influence various types of histone modifications and regulate the activity of enzymes involved in dynamic chromatin modifications, which are dependent on metabolites generated by the host's gut microbiome^2,3. This suggests the importance of epigenetics in the development of AD⁴. This interaction between the gut microbiota and epigenetic modification may drive disease development in response to changes in the gut microbiota⁵. Furthermore, the gut microbiome's involvement in the gut-brain axis, the communication pathway between the gut and the brain, further emphasizes its potential relevance to AD⁶.

Research on the gut microbiota of murine models of AD has revealed inconsistent findings, indicating potential associations between specific bacterial populations and AD pathology. Various AD animal models, such as Tg2576, APP/PS1-ΔE9, 5xFAD, and 3xTg-AD, have been studied to understand the changes in the gut microbiota during the progression of AD.

For instance, the analysis of fecal pellets in Tg2576 model has shown significant changes in the gut microbiota in individually housed aging female mice undergoing caloric restriction, with a decrease in Faecalibaculum rodentium and an increase in Bacteroides, correlating with reduced amyloid β plaque levels⁷. However, another study reported lower levels of Bacteroidetes and higher levels of Firmicutes in Tg2576 mice compared to xxxxxxxx⁰. Unfortunately, the study design only looked at the intestinal luminal content and had limited sampling, i.e. ≤5 mice for each condition. In the APP/PS1-ΔE9 model, aging mice showed an increasing fraction of Bacteroidetes and decreasing levels of Firmicutes in fecal pellet analysis⁹. Regarding the 5xFAD model, different studies reported inconsistent findings regarding Bacteroidetes levels, with some reporting higher levels and others reporting lower levels^10,11. Furthermore, a longitudinal analysis of the caecal and fecal microbiome and metabolome in the 5xFAD mouse model revealed disturbances in microbiota composition, diversity, and short-chain fatty acid levels¹². In the 3xTg-AD model, fecal pellet analysis demonstrated increased levels of minor gut microbial populations such as Fusobacteria and Cyanobacteria¹³. Notably, no specific bacterial species or group consistently exhibited higher or lower abundance in the gut microbiota of AD mouse models. The variations in findings may be attributed to factors like the timing of microbiome collection, environmental influences, and methodological differences for sample collection. Therefore, a comprehensive investigation and cross-study analysis are necessary to definitively determine the gut microbiota associated with AD development and progression in transgenic animal models of AD.

¹ Xxxx, N. M. et al., Sci. Rep. 7, 13537 (2017). ² Xxxx, X. X. xx al., Nutrients 10, 1765 (2018). ³ Leblhuber, F. et al., Curr. Top. Med. Chem. 20,

1415–1421 (2020). ⁴ Xxxx, X. et xx., X. Mol. Neurosci. MN 71, 1436–1455 (2021). ⁵ Xxxx, X. et al., Metabolites 13, 375 (2023). ⁶ Xxxx, X. et al.,

Clin. Epigenetics 7, 112 (2015). ⁷ Xxx, X. X. xx al., Sci. Rep. 9, 17904 (2019). ⁸ Honarpisheh, P. et al., Int. J. Mol. Sci. 21, 1711 (2020).

Fecal microbiota transplantation (FMT) studies conducted with AD mouse models have provided valuable insights into the role of the gut microbiota in AD pathogenesis. For instance, FMT from healthy wild type (WT) mice into ADLPAPT AD mouse models has shown promising outcomes, such as a reduction in amyloid β plaques, neurofibrillary tangles, glial reactivity, and cognitive impairment^14,15. Conversely, transferring the gut microbiota from 9-months-old AD 5xFAD male mice into healthy age-matched antibiotic pre-treated specific pathogen free (SPF) C57Bl/6J WT mice impaired memory function and neurogenesis and led to increased neuroinflammation in the recipient animals¹⁶, suggesting that the AD-specific gut microbiota perturbations might be a leading cause of the AD development. However, due to the short-term duration of the experiments, it is not possible to draw conclusions about the potential role of AD-derived microbiota in the formation of amyloid β plaques in the recipient WT animals. Overall, these findings strongly indicate that the composition of the gut microbiota can significantly influence AD-related pathologies.

In the context of AD, microglia, the resident immune cells of the brain, have garnered significant attention. Microglia are influenced by the gut microbiota, and dysregulation of the microbiota has been associated with microglia activation and neuroinflammation in 5xFAD mice¹⁷. FMT studies using AD mouse models have the potential to offer intriguing insights into the influence of the gut microbiota on microglia and the pathology of AD. These studies could help unravel the role of the microbiome in modulating microglial behavior during the development and progression of AD.

In the proposed project, I aim to investigate the role of the microbiome in AD development by analyzing the longitudinal microbiome composition changes in Tg2576 mice. The Tg2576 model is one of the most well characterized, and widely used, mouse models of AD. Having the Swedish mutation in the APP gene, Tg2576 mice offer a model to study amyloid formation and general AD-associated phenotypes in an accelerated manner. Further, I will assess the effects of FMT on brain development by transplanting microbiota from different AD stages into germ-free (GF) C57Bl/6J mothers and studying the resulting long-term impact of AD microbiome in their offspring.

By manipulating the gut microbiota composition, I aim to investigate how changes in the microbiota affect mouse behavior, microglia activation, neuroinflammation, and AD-related pathology and decipher the extent to which AD-related pathology is transferable in mice that are not genetically predisposed to AD development. By using mouse models, my goal is to advance our understanding of the microbiome's involvement in AD, explore the interplay between a dysbiotic microbiome and genetic factors, and potentially identify biomarkers and therapeutic interventions for AD.

The work plan contains two objectives designated below as Work Packages (WP):

WP1: Investigate the role of the microbiome in AD development by analyzing the composition changes in Tg2576 mice throughout disease progression and comparing it to age- and sex-matched C57Bl/6J control mice. Identify bacterial species associated with AD development.

WP2: Assess the effects of fecal microbiota transplantation on brain development by transplanting microbiota from different AD stages (AD-FMT) into GF C57Bl/6J mothers and studying the resulting impact on behavior, brain size, microglia phenotype and density in their offspring. Explore the relationship between dysbiotic microbiome and genetic factors in AD by differentiating the contribution of microbiome and genetic burden to the onset and progression of AD in Tg2576 and AD-FMT transplanted C57Bl/6J mice.

2. Soundness of the proposed methodology (including interdisciplinary approaches, consideration of the gender dimension and other diversity aspects if relevant for the research project).

^{2 9} Xxxxxx, X. et al., Sci. Rep. 7, 41802 (2017).¹⁰ Xxxxxxxxxxx, X. et xx., X. Alzheimers Dis. JAD 56, 775–788 (2017). ¹¹ Xxxxxx, X. X. xx al., Cells

10, 779 (2021). ¹² Xxxxxx, X. X. X. xx al., mBio 13, e0179422 (2022).¹³ Xxxxx-Xxxxxx, P. C. et al., Front. Neurosci. 15, 595583 (2021).¹⁴ Xxx, X. et

al., Transl. Psychiatry 9, 1–13 (2019).¹⁵ Xxx, X.-S. et al., Gut 69, 283–294 (2020).¹⁶ Xxx, X. et al., Brain. Behav. Immun. 98, 357–365 (2021).

Research methodology and approach

The proposed study aims to investigate the role of the microbiome in AD mouse model Tg2576 by employing an interdisciplinary approach. By integrating multiple methodologies, the project aims to provide a comprehensive understanding of the relationship between the dysbiotic microbiome, AD progression, and the onset of behavioral symptoms.

WP1: The microbiome analysis in the AD disease mouse model

The study will primarily focus on Tg2576 mice, a well-established AD mouse model, and C57Bl/6J control mice. The animals will undergo behavioral testing to evaluate the severity of symptoms and three stages of AD disease progression (early, onset, late) will be compared to analyze changes in the microbiome composition together with age-matched C57Bl/6J controls¹⁸. To control for potential confounding factors, such as sex and cage effects, both male and female mice will be included, and three cages per group with three mice per cage will be used per time-point (n=18 mice per time-point). This experimental design will provide sufficient sample size to account for the expected microbiome variability¹⁹.

The microbiome composition from caecum, ileum and fecal pellets will be analyzed using 16S rRNA sequencing, providing insights into the bacterial species present in the gut microbiota. The results will be compared to published 16S rRNA sequencing data of AD mouse models to identify common bacterial species that contribute to the AD development^12,20. The microbiome analysis will be done in collaboration with Xx. Xxxxx Xxxxxxxxxx from the Department of Zoology, Faculty of Science, Xxxxxxx University, Prague.

WP2: AD-FMT model and impact of dysbiotic microbiome on AD development and progression

To further investigate the impact of the dysbiotic microbiome on AD development and progression, FMT into GF will be conducted via collaboration with the Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences. GF C57Bl/6J females will be time-mated and at E0.5 and E1.5 will undergo FMT using Tg2576 microbiota derived from specific stages of AD, and the offspring's brain development will be assessed.

Although GF mice represent a clean-slate model for the use of FMT and thus are superior to the antibiotically treated SPF mice when it comes to studying the impact of FMT, in mice born and raised under GF conditions, microglia display an immature homeostatic phenotype defined by altered gene expression, increased numbers, and hyper-ramified morphology²¹. Recently, Xxxxxxxx-Xxxx et al. has uncovered that the microglia are increased in numbers even in neonates that have acquired microbiota at birth, underscoring the importance of the exposure to microbial metabolites during gestation²². Additionally, GF mice exhibit abnormal behavioral patterns when compared to SPF mice, such as increased motor activity and reduced anxiety-like behavior²³.

To address these behavioral differences and preserve microglial architecture, I have strategically chosen to perform FMT on GF mothers at E0.5 and E1.5. By doing so, I aim to ensure that embryonic microglia, which seed the developing mouse brain from E9.5²⁴, are not affected by the absence of microbial metabolites. The pregnant mothers with transplanted microbiomes will be transferred to the NIMH at E07 and the resulting offspring will be analyzed further (ADM mice). To control for the possible microbial changes due to the transport related stress, I will collect and sequence fecal pellets before and after the transport.

^{3 17}Mezö, C. et al., Acta Neuropathol. Commun. 8, 119 (2020). ¹⁸You, X. et al., mSystems 7, e0124821 (2022). ¹⁹Kim, D. et al., Microbiome 5, 52 (2017). ²⁰Oblak, A. L. et al., Front. Aging Neurosci. 13, (2021).²¹Erny, D. et al., Cell Metab. 33, 2260-2276.e7 (2021).²²Castillo-Xxxx, X. et al., Front. Neurosci. 17, (2023).²³Lu, J. et al., PLoS ONE 13, e0201829 (2018).²⁴Barry-Xxxxxxx, X. et al., Cell Rep. 42, 112425 (2023).

Additionally, two ADM pups per litter will be sacrificed at D07 and the paraventricular nucleus of the hypothalamus, the CA1 oriens layer of the hippocampus, the arcuate nucleus, the locus coeruleus, and the primary somatosensory cortex of the brain will be analyzed via histology to assess the microglia density and morphology, as these regions differ between neonatal GF and SPF mice²². Age and sex-matched SPF C57Bl/6J mice will be used as a control. As no phenotype is observed in the Tg2576 mice up until 6 months of age, I am expecting to see a normal brain development in the ADM pups at D07.

The adult ADM mice will be subjected to behavioral tests alongside the Tg2576 and C57Bl/6J controls from D60 onwards until 14 months of age (social interaction test - social games, open field, Morris water maze, Elevated Plus Maze). At the onset of behavioral disturbances, cerebrospinal fluid samples will be collected from living ADM mice to perform the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, which allows for the detection and quantification seeding potent of amyloid species²⁵. In cases where amyloid species are not detected, ADM mice will be kept alive until 14 months of age and sacrificed to perform the end-point testing. Based on the expected variability between mice²⁶, I plan to use 12 ADM mice for the behavioral testing per AD stage (n=36 in total for 3 AD stages).

In the case of detection of amyloid species in ADM mice and at the endpoint, histology, and single-nucleus RNA sequencing (snRNA-seq) will be performed on collected brains to analyze brain structure, amyloid β plaques, tau pathology, and microglia density. For each affected ADM animal, half of the brain will be utilized for histological analysis and the other half will be used for the snRNA-seq. The resulting transcriptional profiles of the microglia and other affected cell types (e.g hippocampal neurons) will be compared with the published datasets in the scREAD²⁷ database that is a useful resource for single-cell RNA-seq data on AD. Network-based methodologies will be used to identify molecular networks in disease-relevant immune cells of the nervous system in AD.

By employing this comprehensive approach, the project aims to elucidate the contribution of the dysbiotic microbiome to AD development, determine the timing of AD onset caused by the dysbiotic microbiome, and investigate the interaction between the microbiome and genetic factors in AD.

Originality and innovative aspects of the planned research

The proposed research project takes on a critical issue in AD research, with its timeliness and scope making it an essential and compelling endeavor. This research is timely due to the increasing recognition of the gut-brain axis and its impact on neurodegenerative disorders like AD. My project aims to gain new insights into the complex mechanisms of AD by focusing on AD-specific microbiota and its effect on microglia activation. I will incorporate behavioral analysis and microglia response assessment to comprehensively understand the impact of AD-specific microbiota on cognitive function and neuroinflammation. My innovative research involves transferring AD-FMT from Tg2576 mice to C57Bl/6J GF mothers and studying the impact on their offspring's microbiota. This enables a direct investigation of the effects of AD-specific microbiota on disease progression. By capitalizing on the growing understanding of the gut-brain axis and its relevance to AD, this project holds the potential to make significant contributions to the field and offer novel avenues for therapeutic interventions.

Integration of methods and disciplines to pursue the objectives

This project is by nature interdisciplinary, connecting immunology, neurobiology and microbiome analysis, because it is based on the idea that the dysbiotic microbiome impacts the development and progression of AD via the gut-brain axis. Through this project I will also gain new technical skills, through the behavioral analysis of FMT mouse models and state-of-the-art molecular biology, such as snRNAseq. I will strongly benefit from the culture of highly collaborative work of the host lab combined with the multidisciplinary skills offered by the qualifications of the collaborating labs. The team includes experienced neurobiologists, a gnotobiology expert and a microbiome expert. This will not only contribute to the quality and the reliability of the work but will also broaden my scientific vision and knowledge beyond my immunology background.

Gender dimension and other diversity aspects

The recent studies reveal intriguing insights into the relationship between gender differences, AD, and the gut microbiome. Notably, AD is more prevalent in women than men, and patients with the condition exhibit reduced microbial richness and diversity in their gut microbiome compared to asymptomatic individuals²⁸ . Moreover, alterations in the gut microbiota and its byproducts have been observed in AD subjects and transgenic mice in a sex-dependent manner²⁹. Surprisingly, microbiome perturbations have been found to affect brain amyloidosis and microglia gene expression differently in male and female mice. Changes in the gut microbiome have also been described in AD animal models and in AD patients. Specific gut bacterial taxa alterations have been linked to preclinical AD, indicating a potential early biomarker for the cognitive decline²⁹.

These findings suggest that sex-specific differences exist in the microbiome composition and response in AD. However, further research is needed to fully understand the gender dimension difference in the microbiome and its precise role in the development and progression of AD³⁰. The implications of these sex-specific alterations in the gut microbiome could have significant ramifications for personalized treatments in the future, but more research is required to explore this promising avenue of investigation.

In the proposed project, I will use the appropriate statistical tests and age- and sex-matched animals in all experiments to assure that the gender dimension is taken under consideration to evaluate the impact of gender on the development and progression of AD.

Research data management and management of other research outputs:

The study will comply with the EU Data Protection Regulation in line with the FAIR. Data management will be coordinated throughout the research from data collection to publication of results (to protect research data from loss, enable sharing with others, and ensure that it is discoverable, accessible, and re-usable). This will include processes for organizing research data, storage, preservation, security, data quality assurance, assignment of persistent identifiers, and data sharing rules and procedures, including licensing and access settings. The handling and protection of biological samples (collection, processing, transport, and storage of samples for further analysis for a maximum of 2.5 years) will be carried out following the legislation.

The scientific results will be disseminated according to the Open Science practices and its core commitments, including Open Access and Open Data. Data will be published in open access peer-reviewed journals with IF.

The project team is committed to promoting transparency and scientific collaboration by making the sequencing data resulting from the research publicly available in repositories. Furthermore, all relevant datasets generated throughout the study will be published as open source, ensuring that the findings are accessible to the scientific community and fostering further research and discoveries in the field. Our results will also be communicated to the professional public at scientific international conferences, and to the lay public at educational public lectures.

3. Quality of the two-way transfer of knowledge between the researcher and the host. Two-way transfer knowledge between the researcher and the host institution

⁴²⁵Manca, M. & Xxxxx, X., Biomolecules 10, 1233 (2020). ²⁶Kim, N. et al., Brain. Behav. Immun. 98, 357–365 (2021). ²⁷Jiang, J. et al., iScience 23, 101769 (2020). ²⁸Wu, S. et al., Front. Aging Neurosci. 13, 650047 (2021). ²⁹Jong, J. H. et al., PLoS ONE 17, e0278276 (2022).

³⁰Rosende-Xxxx, X. et al., Alzheimer's Research & Therapy 13, 95 (2021). ³¹Kreisinger, J. et al., bioRxiv 2023.07.10.547212; doi: xxxxx://xxx.xxx/00.0000/0000.00.00.000000

During my PhD studies in the laboratory of Xx. Xxxxxx Xxxxxxxx and Dr. Xxxxxx Xxxxx (Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic), I acquired a strong background in molecular biology, hematology and immunology. One of the major achievements was the elucidation of the mechanism of antigen-experienced memory T cell (AIMT) formation and characterization. Furthermore, my research focused on the influence of the microbiome on the development of AIMT cells. During my postdoctoral fellowship in Prof. Xxxxxx'x laboratory (Babraham Institute, Cambridge), I focused mainly on the role of regulatory T cells and microglia, which will be highly valuable for the proposed project. I also successfully carried out challenging tasks, in fields for which my host laboratories had no previous expertise and initiated and brought to a successful completion several interdisciplinary international collaborations. To achieve these objectives, I developed a strong ability to adapt, perseverance and problem-solving skills. I will bring with me to the NIMH a network of collaborators, thus creating new opportunities of collaborations for the host laboratory, i.e. with a gnotobiology expert Xx. Xxxxxx Xxxxxxxxx (The Laboratory of Gnotobiology and the Institute of Microbiology of the Czech Academy of Sciences, Czech Republic) and microbiome expert Xx. Xxxxx Xxxxxxxxxx (The Department of Zoology, Faculty of Science, Xxxxxxx University, Prague, Czech Republic), who are going to collaborate with me on the proposed project.

My experience with interdisciplinary research, and my ability to work collaboratively will allow me to bring this project, at the intersection between immunology, microbiome and neurobiology to success. Furthermore, given the wide variety of techniques I worked with (microbiota sample preparation, immunohistochemistry, 10x sequencing), I will bring skills to the laboratory, which will prove useful in this study.

After 4 years abroad, I sought an opportunity to bring my expertise back to the Czech Republic via a fellowship in a renowned institution, with an inspiring mentor, to face new scientific challenges and open my research perspectives. Through this project, I will explore a new exciting area of neurobiology and gain future understanding in the behavioral studies and neuropsychiatry. I will strongly benefit from the unique expertise of the host lab and of the world class core facilities at the NIMH. These provide technical support and high-quality training which will be instrumental to support my research and broaden my skill set. I will acquire new knowledge in advanced neuropsychiatry and bioinformatics, two essential skills which will broaden my horizons to be successful in modern biomedical research. Thanks to the renowned excellent neuropsychiatric platform and to the multidisciplinary skills of the Xxxxxx team, I am convinced that the host laboratory is the best place possible for me to acquire these new skills. I will also have the fantastic opportunity to perform innovative in vivo research, using state-of-the-art tools not available in my current hosting institution, in the best conditions possible to guarantee both the high quality and reliability of the scientific results and the wellbeing of the animals. I will also develop a new theoretical knowledge in neuropsychiatry. I will improve my communication skills by discussing my own work and the work of others at weekly lab meetings, journal club, departmental meetings and seminars (at the NIMH and international conferences). Based on the research output of the host laboratory and the potential of this project, I expect to write and publish two first-author research articles in a high-impact peer-review journal.

4. Quality and appropriateness of the researcher’s professional experience, competences and skills. The proposed project is in nature interdisciplinary, requiring a collaborative effort between three research institutes. Since I have previously collaborated with Xx. Xxxxxxxxx and Xx. Xxxxxxxxxx while working in the Xxxxxxxx and Xxxxxx laboratories and recently I have written a last-author publication with Dr. Kreisinger³¹, proving my competency to lead an independent project. During my PhD studies and my postdoctoral stay, I have gained an expertise in immunology and neuroimmunology and contributed to two studies on the intersection of immunology and microbiome in collaboration with Xx. Xxxxxxxxxx. In the proposed project, I aim to gain further skills in neuropsychiatry and behavioral studies under the guidance of Xx. Xxxxxx.

This project represents a pivotal opportunity for me to pave the way for a thriving independent career at the cutting edge of interdisciplinary research.

2. IMPACT

1. Credibility of the measures to enhance the career perspectives and employability of the researcher and contribution to his/her skills development.

The proposed project is not just a research endeavor; it represents a transformative stepping stone in my journey towards becoming an accomplished and independent interdisciplinary scientist.

Through this project, I anticipate significant skill development, both in core and advanced research skills, as well as invaluable transferable skills. The interdisciplinary nature of the research options, intersectoral experiences through proposed secondment and networking events, and international opportunities will undoubtedly enrich my expertise and broaden my perspective.

This comprehensive training will have a profound impact on my career prospects, both within and outside academia. As a MERIT fellow, I will be equipped to meet the evolving demands of industry and research organizations. Moreover, the mentoring and coaching in key areas of development, such as life coaching for maintaining mental health and overcoming academic challenges, leadership and career mentoring for aspiring principal investigators, and business mentoring for potential entrepreneurs, will provide crucial guidance in carving out my own path.

In essence, this project offers the perfect platform to cultivate a well-rounded and dynamic skill set, setting me on the trajectory to become an influential and versatile researcher. It is not merely a project; it is a transformative opportunity that holds the promise of shaping my future as an independent and impactful interdisciplinary scientist.

2. Suitability and quality of the measures to maximise expected outcomes and impacts, as set out in the dissemination and exploitation plan, including communication activities.

Dissemination

The proposed work will provide the first extensive long-term characterization of the impact of microbiota on the development and progression of AD. This study has the potential to reveal the extent to which AD-related pathology is transferable in mice that are not genetically predisposed to AD development. The therapeutic potential of such discovery will be enormous, e.g. possible diagnostic tools and targeted interventions. Therefore, this work will be of great interest for the scientific community. I will present my work in seminar series at the host institute and within Prague. I will take part in the Czech Microbiome Network, a network of researchers from academia and from biotech companies in the Czech Republic aiming to create a sense of community between researchers and to encourage research interactions. I will also communicate my research results to a larger scientific community by attending and presenting at national and international congresses, such as Cell Symposia: The Neuro-Immune Axis, Neuroscience and Microbiome World Congress.

This project has the potential to lead to the publication of at least one research article in a high-impact peer-review journal. This publication will contain the characterization of the AD-specific microbiome over the course of the mouse lifetime and will conclusively assess the impact of microbiome on AD-related pathology. All 16S and 10x sequencing data generated through this project will be deposited on the European Genome-phenome Archive (EGA) to provide full transparency and maximum utility to the research community.

Exploitation and intellectual property rights

The project's potential for commercialization will undergo evaluation by the Commercialization Council at the NIMH. The Commercialization Council plays a vital role as an advisory body to the NIMH management, focusing on assessing the commercial potential of projects and supporting the successful commercialization of the institute's scientific and research outputs. By providing expert evaluation and recommendations, the council guides strategic decisions, fosters collaborations, and promotes real-world impact in mental health research. Their expert evaluation ensures that promising discoveries and technologies are effectively harnessed to benefit society and advance the fields of mental health and neuroscience. By actively fostering collaborations with industry partners and promoting the protection and utilization of intellectual property, the council plays a pivotal role in bridging the gap between research excellence and real-world impact. As a result, the NIMH can continue to make significant strides in advancing mental health research and positively impacting the lives of countless individuals.

3. Sustainability of the candidates’ research project in the Region. Opportunities to continue the research through regional/international collaboration with relevant industry or academic sectors.

What distinguishes this project is its distinctive collaborative approach, which brings together the expertise of three renowned institutes. The Laboratory of Gnotobiology and the Institute of Microbiology of the Czech Academy of Sciences will be responsible for conducting FMT procedures, while the Department of Zoology, Faculty of Science at Xxxxxxx University, Prague will handle the analysis of the microbiome. The NIMH will perform behavioral tests and microglia analysis. All three institutes bring extensive experience in their respective areas to this project and will be a continuation of my successful collaboration spanning several years^32,33. By furthering these collaborations and successful completion of this project, I aim to open up a niche that is at the intersection of neuroimmunology, microbiome and neuropsychiatry that will allow me to establish my own line of research with the goal of becoming an independent scientist.

I believe that this area of research is highly relevant to modern neuropsychiatry and has a potential to impact the choice of therapeutic interventions offered to the AD patients. Additionally, gaining a deeper understanding in the role of microbiome in the development and progression of AD opens up possibilities for developing new diagnostic methods with a relevance to the industry through future regional/international collaborations and creates an opportunity for diagnostic spin-off with a focus on microbiome analysis in AD patients.

3. IMPLEMENTATION

   1. Quality and effectiveness of the work plan and assessment of risks.

Work plan description

The proposed work plan contains two Work Packages (WP - see section 1.1 and Gantt Chart):

WP1 The microbiome analysis in the AD disease mouse model

WP2 AD-FMT model and impact of dysbiotic microbiome on AD development and progression

To assess the progress of the project, the following milestones have been identified (see Gantt Chart):

M1.1 Analyze the microbiome changes in the AD disease mouse model

M1.2 Conclude on the impact of dysbiotic microbiome in AD-FMT mouse model

^{5 32}Paprckova, D. et al., Front. Immunol. 13, (2022). ³³Moudra, A. et xx., X. Immunol. Baltim. Md 206, 2109–2121 (2021).

The major deliverables of this project will be the publication of at least one manuscript in a peer-reviewed, high impact journal. At the beginning of the fellowship, I will establish a Career Development Plan jointly with Xx Xxxxxx including research and training objectives, planning for publications and participation at conferences. The project also includes other deliverables: a Data Management Plan, a mid-term and final report, departmental and Prague-based seminars and Poster/Oral presentations at conferences.

The Gantt Chart also includes trainings in the following areas:

Bioinformatics:

T1.1 Microbiome 16S sequencing data analysis T1.2 10X Single Cell snRNA-Seq Analysis

T1.3 snRNA-Seq Analysis

Core skills:

T2.1 Imaging training. Done on a one-to-one basis, identify and learn the imaging solution and analysis which best addresses the needs of the project.

T2.2 Behavioral testing (social interaction test - social games, open field, Morris water maze, Elevated Plus Maze)

T2.3 Behavioral data analysis

Transferable skills:

T3.1 Project management T3.2 Scientific ethics T3.3 Scientific writing T3.4 Intellectual property

Figure 1. Gantt Chart of the proposed project timeline.

Risk analysis

The project has identified and categorized the main risks associated with its execution, considering their likelihood and potential impact on the project.

WP1 focuses on analyzing changes in the microbial composition in an AD mouse model and is considered low risk as it involves a comprehensive descriptive analysis.

WP2 carries a risk that the AD-FMT into C57Bl/6J mice may not result in the development of amyloid plaques in the ADM mice. However, such an outcome would provide valuable evidence suggesting an unidentified genetic contribution essential for the development of late-onset sporadic AD. This finding would have high significance for the scientific community.

Importantly, the two aims of the project are independent of each other and not reliant on the outcome of a single initial experiment. Consequently, the project is expected to yield novel and important data that can contribute to solid publications.

2. Relevance, feasibility and benefit for the research project objectives of the planned secondment. Quality of the secondment choice in terms of at least one of the following principles: intersectorality, interdisciplinarity or international mobility.

The planned secondment, whether in Dr. Xxxxx Xxxxxxxxxx'x laboratory at the Department of Zoology, Faculty of Science, Xxxxxxx University, Prague, Czechia, or under the guidance of Xx. Xxxxxx Xxxxxxx Xxxxxxx at the Department of Food Science, University of Copenhagen, presents a transformative opportunity for me to acquire specialized knowledge and skills in the dynamic field of microbiome studies.

While I have contributed to two studies on the microbiome topic previously, my core skills in this area are currently limited to processing experimental animals and sample collections. In those studies, Dr. Xxxxxxxxxx'x laboratory handled the microbiota sample preparation for sequencing, microbiota profiling, and statistical analysis. Through the planned secondment in either Dr. Xxxxxxxxxx'x or Dr. Xxxxxxx'x laboratory, I aim to further enhance my expertise in microbiome studies.

Hands-on experience during the secondment, particularly in microbiota sample preparation for sequencing, microbiota profiling, and statistical analysis, will provide me with a deeper understanding of the microbiome field. This newfound knowledge will empower me to make significant advancements and meaningful contributions to this rapidly evolving area of research.

The intersectoral and interdisciplinary focus of the secondment offers valuable opportunities to deepen my expertise in the microbiome field. By gaining fresh insights through the analysis of microbiome data, I can contribute meaningfully to the progress of this research domain. Furthermore, the international mobility aspect of the secondment will broaden my horizons and increase my employability, paving the way for a promising scientific career in the future.

Zápis z jednání Steering Committee MERIT ze dne 11.12.2023

Přítomni:

• VYMAZÁNO

• Omluvena:

• VYMAZÁNO

Agenda:

1. Zahájení, schválení programu schůze Program schůze:

• Rekapitulace stavu úkolů z minulého jednání

• Představení výběrového procesu

• Schvalování předložených projektů

• Termíny následujících jednání + zamyšlení nad informacemi potřebnými pro monitoring vědeckých projektů

Návrh usnesení:

Řídicí komise MERIT bere na vědomí program jednání.

Výsledky hlasování Pro: 4; Proti: 0

Přijaté usnesení:

Řídicí komise MERIT bere na vědomí program jednání.

2. Úkoly z minulého jednání

• Vytvořeno sdílené úložiště pro členy SC v prostředí Microsoft 365

• Aktualizovaný jednací řád a seznam zúčastněných partnerských institucí programu MERIT poslán členům SC

• Připravena Dohoda o mlčenlivosti (Non-disclosure agreement) kvůli zajištění důvěrnosti informací členů Steering Committee, podepsána všemi členy (přílohy tvořily ještě prohlášení v AJ: Prohlášení o důvěrnosti, neexistenci střetu zájmů a nediskriminaci, kvůli splnění podmínek projektu vůči EU)

Návrh usnesení:

Řídicí komise MERIT bere na vědomí stav úkolů z minulého jednání.

Výsledky hlasování Pro: 4; Proti: 0

Přijaté usnesení: Řídicí komise MERIT bere na vědomí stav úkolů z minulého jednání.

3. Stručné představení výběrového procesu

• PPT prezentace Programové manažerky

Návrh usnesení:

Řídicí komise MERIT bere na vědomí informaci o průběhu výběrového procesu první výzvy programu MERIT.

Výsledky hlasování Pro: 4; Proti: 0

Přijaté usnesení: Řídicí komise MERIT bere na vědomí informaci o průběhu výběrového procesu první výzvy programu MERIT.

4. Schvalování předložených projektů

• Členům komise byla předem sdílena tabulka s přehledem kandidátů, jejich umístění, včetně navrhovaných secondmentů, stručné abstrakty řešených projektů, a dále kompletní projektové návrhy a hodnotící reporty z obou kol hodnocení

• Všechny projekty prošly hodnocením v mezinárodním transparentním výběrovém řízení v souladu s pravidly programu MERIT.

Návrh usnesení:

Řídicí komise schvaluje celkem 23 projektů předložených do první výzvy programu MERIT, z nichž 15 projektů, které získaly v hodnocení nejvyšší skóre, budou podpořeny, a 8 rezervních projektů může být podpořeno v případě, že některý z podpořených projektů nebude realizován.

Výsledky hlasování Pro:4 ; Proti: 0

Přijaté usnesení – Řídicí komise schvaluje celkem 23 projektů předložených do první výzvy programu MERIT, z nichž 15 projektů, které získaly v hodnocení nejvyšší skóre, budou podpořeny, a 8 rezervních projektů může být podpořeno v případě, že některý z podpořených projektů nebude realizován.

Řídící komise zároveň navrhuje prověřit zejména ruské žadatele a žadatele z třetích zemích z důvodu bezpečnostního rizika realizace jejich projektů (BIS).

5. Plán pro druhou výzvu a termíny následujících setkání

• ČERVENEC 2024 – monitoring po prvním reportovacím období (zprávy o průběhu realizace projektů, komunikační a diseminační aktivity vědců, účast vědců na akcích SIC apod.)

• ZÁŘÍ 2024 – schvalování projektů vybraných v druhé výzvě programu

• LEDEN 2025 – monitoring po druhém reportovacím období (zprávy o průběhu realizace projektů, komunikační a diseminační aktivity vědců, účast vědců na akcích SIC apod.)

Návrh usnesení:

Řídicí komise MERIT bere na vědomí plán pro druhou výzvu programu a termíny následujících jednání.

Výsledky hlasování Pro:4; Proti: 0

Přijaté usnesení

Řídicí komise MERIT bere na vědomí plán pro druhou výzvu programu a termíny následujících jednání. Řídíce komise stanovila termín na příští setkání k prvnímu reportovacímu období dne 25.7.2024.

6. Zamyšlení nad informacemi potřebnými pro monitoring projektů

• Monitoring bude probíhat jednou za 6 měsíců, informace budou pravidelně poskytovány ze strany výzkumných organizací (supervizor + vybraný stážista)

Návrh usnesení:

Řídicí komise MERIT bere na vědomí obsah monitorovacího reportu a schvalují navržené změny.

Výsledky hlasování Pro:4.; Proti:0

Přijaté usnesení

Řídicí komise MERIT bere na vědomí obsah monitorovacího reportu a schvaluje navržené následující změny, které budou vloženy do monitorovacího reportu:

• neúspěchy a případné změny projektu,

• informace, zda je projekt v souladu s harmonogramem,

• informace o tom, v jakém stavu se projekt nachází,

• milníky projektu.

Monitorovací Report bude upraven a poslán Řídicí komisi k odsouhlasení.

Zapsala: Zápis ověřil:

• VYMAZÁNO VYMAZÁNO

Příloha: Seznam schválených projektů

Seznam 15 kandidátů s nejvyšším skóre

Name of the candidate	Host organisation	Final score
VYMAZÁNO	ELI Beamlines Facility - The Extreme Light Infrastructure XXXX	95,8
VYMAZÁNO	Institute of Animal Physiology and Genetics of the Czech Academy of Sciences	95,7
VYMAZÁNO	National Institute of Mental Health	94,7
VYMAZÁNO	Astronomical Institute of the Czech Academy of Sciences	89,9
VYMAZÁNO	Astronomical Institute of the Czech Academy of Sciences	89,7
VYMAZÁNO	Institute of Animal Physiology and Genetics of the Czech Academy of Sciences	87,8
VYMAZÁNO	Institute of Thermomechanics of the Czech Academy of Sciences	85,6
VYMAZÁNO	Astronomical Institute of the Czech Academy of Sciences	84,1
VYMAZÁNO	ELI Beamlines Facility - The Extreme Light Infrastructure XXXX	83,3
VYMAZÁNO	Czech University of Life Sciences Prague	82,3
VYMAZÁNO	Institute of Physics of the Czech Academy of Sciences / HiLASE	81,5
VYMAZÁNO	Czech Technical University in Prague, Czech Institute of Informatics, Robotics, and Cybernetics	81,4
VYMAZÁNO	Research Institute of Geodesy, Topography and Cartography	81,1
VYMAZÁNO	Institute of Physics of the Czech Academy of Sciences / HiLASE	80,1
VYMAZÁNO	UCEEB Czech Technical University in Prague, University Centre for Energy Efficient Buildings	78,9

Seznam 8 rezervních kandidátů

Name of the candidate	Host organisation	Final score
VYMAZÁNO	National Institute of Mental Health	76,8
VYMAZÁNO	Institute of Microbiology of the Czech Academy of Sciences	76,1
VYMAZÁNO	Research Institute of Geodesy, Topography and Cartography	76,1
VYMAZÁNO	Astronomical Institute of the Czech Academy of Sciences	74,7
VYMAZÁNO	UCEEB Czech Technical University in Prague, University Centre for Energy Efficient Buildings	74,6
VYMAZÁNO	National Institute of Mental Health	74,5
VYMAZÁNO	Institute of Animal Physiology and Genetics of the Czech Academy of Sciences	73,8
VYMAZÁNO	ELI Beamlines Facility - The Extreme Light Infrastructure XXXX	73,6