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The success rate of regenerative medicine largely depends on the type of stem cells applied in such procedures. Consequently, to achieve the needed level for clinical standardization, we need to investigate the viability of accessible sources with sufficient quantity of cells. Since the oral region partly originates from the neural crest, which naturally develops in niche with decreased levels of oxygen, the main goal of this work was to test if human oral mucosa stem cells (hOMSC) might be used to treat neurons damaged by anoxia. Here we show that hOMSC are more resistant to anoxia than human induced pluripotent stem cells and that they secrete BDNF, GDNF, VEGF and NGF. When hOMSC were added to human neurons damaged by anoxia, they significantly improved their survival. This regenerative capability was at least partly achieved through miR-514A-3p and SHP-2 and it decreased in hOMSC exposed to neural cells for 14 or 28 days. In addition, the beneficial effect of hOMSC were also confirmed in mice affected by stroke. Hence, in this work we have confirmed that hOMSC, in a time-limited manner, improve the survival of anoxia-damaged neurons and significantly contribute to the recovery of experimental animals following stroke.

Objectives: To describe neuronal differentiation from the disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from persons with partial and full trisomy 21, and mosaic Down syndrome (DS). Methods: NSC were generated form the isogenic iPSCs following published protocol (1) and expanded in our laboratory. NSCs, between passage 5 and 10 were seeded on the commercially available mouse astrocytes and differentiated to the mature neurons 100 days in vitro (DIV) (2). Moreover, cerebral organoids were generated following published protocol (3) with our modification (4) and fixed at 30, 50, 70 and 100 DIV and analysed by immunohistochemistry. Results: In our previous work we published neuronal differentiation from EBs D3 and 47-1 (5). Here we find that Tubb3 positive cells show a specific neuronal phenotype. Disomic cells showed normal neuronal morphology, on the other hand trisomic cells showed a very specific cellular dysmorphology: neurons were smaller, shorter with thicker processes, and abnormal branching patterns. The same pattern we have observed in neurons derived form iPSCs in monolayer as well as in cerebral organoids. Both, neurons in monolayer, as well as in cerebral organoids expressed all neuronal markers: pan neuronal markers (Dcx, MAP2. Tubb3, SMI, 3R-Tau) and cortical layer specific markers (Reelen, Brn2, Ctip2, SATB2, TBR1 and TBR2). Conclusion: Trisomic cells could differentiate to mature, synaptic active neurons but morphology of trisomic cells shows a unique and specific dysmorphology pattern.

Human, mammalian, brain development is a very complex process and starts shortly after gastrulation. Human in vitro models for brain development as well as pathological changes were published in last few years, eliminating the ethical concerns that accompany the study of aborted foetal material. One of the recent approaches is the development of induced pluripotent stem cell (iPSC) technology and different types of organoids, which can be applied to any species. In our previous work we developed iPSCs from an adult individual with constitutional mosaicism for Down Syndrome (DS) (1). From those iPSCs, cerebral organoids were grown and corticogenesis and Alzheimer-like pathology in vitro were studied (2). Mixed cerebral (2), as well as midbrain (unpublished) organoids were grown in vitro from the isogenic, trisomic and disomic iPSCs. Organoids were analysed histologically by IMARIS software. Foetal human brains were stained with the same combinations of antibodies and the data were compared. Anonymized post-mortem brain samples were obtained from the Brain Bank of the Croatian Institute for Brain Research (CIBR). Isogenic cerebral organoids expressed the markers of all six cortical layers (Reelin, FOXG1, SATB2, TBR1, Ctip2 and Brn2) after 100 days differentiation in vitro (DIV). At the early time point of DIV30 the majority of cells were Nestin, SOX2, PAX6 positive. At that time point neurogenesis had already started, so we observed TRBR2, DCX, MAP2 and TUBB3 positive cells in disomic and trisomic organoids. Following further neuronal differentiation of cerebral organoids we observed decreased number of neural stem cells and increased number of neuronal markers specific for all six layers. On the other hand, midbrain organoids expressed markers specific for midbrain. At the early time points midbrain organoids expressed neuronal stem cells markers, similar to cerebral organoids, but with organoid maturation we found midbrain specific cells such as NURR1 and OTX2 specific for the early stages, and later Dopamine β Hydroxylase, NET and TH. Finally, all those markers were compared with expression in foetal human brain from individuals with Down Syndrome as well as euploid controls age, sex and brain region matched. Our data showed that isogenic cerebral organoids grown from iPSCs are good tool for multidisciplinary studies. In our group we study corticogenesis, synaptogenesis, pathology and possible drug treatments.

Background: Specific cell types from people with Down syndrome (DS) show faster accumulation of DNA damage and epigenetic senescence marks. Causative mechanisms remain un-proven ranging from amplified chromosomal instability to actions of chromosome 21 genes. Plasma immunoglobulin G (IgG) glycosylation profiles are established as a reliable predictor of biological and chronological ageing. Methods: We performed IgG glycan profiling of n=246 individuals with DS (208 adults and 38 children) clinically characterised for co- morbidities, from three European countries, and compared these to age-, sex- and demography- matched general populations. Results: We uncovered very significantly increased IgG glycosylation ageing marks associated with DS, beginning in early childhood. Average levels of IgG glycans without galactose (G0) and those with two galactoses (G2) as a function of age in persons with DS corresponded to levels detected in 19 years older euploid individuals. Sub-cohorts of DS without any known co-morbidities from different countries showed a similar result, eliminating DS- linked autoimmune diseases, frequent infections, or Alzheimer’s dementia as indirect causes of accelerated biological ageing. Importantly, no difference in slope of the curve relating IgG- glycan-age to chronological age was observed for any of the 3 analysed populations, discouraging the amplified-chromosomal-instability explanation. Remarkably, IgG-glycan profiles of a child with DS caused by a short segmental duplication of only 31 genes on chromosome-21 (DS-critical-region) had values like age-matched whole-chromosome-trisomy- 21 children, pointing to a causative contribution of one or more of the 31 genes. Conclusion: This shifts the paradigm from an (essentially untreateable) “amplified instability” mechanism, to a specific gene-overdose-driven cause of a progeria-like syndrome, opening possibilities for therapeutic amelioration strategies. We generated an induced pluripotent stem cell (iPSC) model of this child with partial trisomy 21, and editing experiments using CRISPR-Cas9 are underway in order to dissect the causative overdosed genes and mechanisms responsible for the accelerated ageing in DS.

The goal was to explore the difference in N- glycosylation between euploid disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from person with mosaic Down syndrome (DS) trisomy of 21st chromosome.....

Synaptic glycoprotein neuroplastin (Np) is a crucial synaptic junction (SJ) proteome member. Evidenced functions of Np include regulating Ca2+ clearance and its involvement in long-term potentiation. We have previously reported differential immunohistochemical pattern of Np across brain regions in Toll-like receptor 2 (TLR2) knock-out (KO) mice and assumed that detected alterations might accompany large-scale changes in the synaptic environment in TLR2KO mice. These findings guided us on a journey to address whether the absence of TLR2 affects the SJ proteome. The study was conducted on female and male mice with 4 wild types (WT) and 4 TLR2 KO per group. Tissue samples of the cortex, cerebellum, and hippocampus were isolated by neuroanatomical dissection and subjected to membrane fractionation protocol to obtain synaptic junction enriched fractions. Tryptic in- gel digestion was applied to the collected fraction, and generated peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC- MS/MS). Data analysis revealed significant changes in protein abundance across all isolated brain regions and supported our previous results demonstrating alterations of Np and P-Type ATPases expression in TLR2KO mice. The most remarkable and sex-dependent differences in protein content were observed in the cortex of TLR2KO animals compared to WT, while a lesser extent of proteome disruption was shown in the hippocampus and cerebellum of TLR2KO animals. The protein systems that underwent significant modifications are particularly those implicated in neurotransmission, overall pre-and postsynaptic arrangements, synaptic energy metabolism, axon/dendrite cytoskeleton, and myelin sheath modifications. In conclusion, investigating a synaptic proteome in the TLR2KO mouse model enables a fresh look at the potential new roles of TLR2 in intercellular communication and neuronal connectivity in a sexspecific manner.

Toll-like receptor 2 (TLR2) deficient mice model is widely used for investigation of microglial role in regulating inflammatory response following ischemic brain lesion (1). Additional functions and broader cell-type expression of TLRs have been evidenced in brain tissue which makes TLRs knock- out mice (such as TLR2-KO) a promising model for investigating neuron-microglia interactions, and microglial regulation of plasticity and excitability (2). Plasma membrane Ca2+ ATPases (PMCAs) participate significantly in ion homeostasis maintenance and signal transduction in brain tissue, thus we aimed to examine PMCAs protein expression and catalytic activity across brain regions in TLR2-KO mice. Results acquired by Western blot analysis and spectrophotometric enzyme activity measurements exhibit strikingly different protein expression and enzyme activity in brain tissue samples derived from TLR2-KO when compared with wild-type (WT) mice. Observed abundance of PMCA expression across cortex, hippocampus and cerebellum suggests that ubiquitous isoforms, PMCA 1 and 4 are less affected by TLR2 deficiency than neuron specific isoforms, PMCA 2 and 3. Notably, higher expression of PMCA2 in hippocampus and lower in cerebellum of TLR2-KO coincide with catalytic PMCA activity variations, higher activity is detected in hippocampus and lower in cerebellum of TLR2-KO compared with WT controls. Here collected evidence suggests that TLR2 deficiency is accompanied by a disturbance of neuronal Ca2+ signaling system. Further investigation should clarify in more details a potential impact of TLR2 on establishing proper neuron-microglia interactions and regulation of neurotransmission. [1.] Gorup D, Škokić S, Kriz J, Gajović S. Tlr2 Deficiency is Associated with Enhanced Elements of Neuronal Repair and Caspase 3 Activation Following Brain Ischemia. Sci Rep. 2019 ; 9(1):1–10. [2.] Yamamoto M, Kim M, Imai H, Itakura Y, Ohtsuki G. Microglia- Triggered Plasticity of Intrinsic Excitability Modulates Psychomotor Behaviors in Acute Cerebellar Inflammation. Cell Rep [Internet]. 2019 ; 28(11):2923-2938.e8. Available from: https://doi.org/10.1016/j.celrep.2019.07.078 [3.] Hajieva P, Baeken MW, Moosmann B. The role of Plasma Membrane Calcium ATPases (PMCAs) in neurodegenerative disorders. Neurosci Lett. 2018 ; 663(September 8. Available from: https://doi.org/10.1016/j.neulet.2017.09.033 Funding: Croatian Science Foundation (IP-2016-06- 8638), European Social Fund (ESF), and Centre of Excellence for basic, clinical and translational neuroscience.

Transmembrane proteins, neuroplastin (Np), and Toll-like receptor 2 (TLR2) are implicated in tuning the neuro-architecture. Np isoforms (Np55 and Np65) are related to synaptic plasticity and neuronal ion homeostasis by interactions with plasma membrane Ca2+ ATPases (PMCA). TLR2 is mainly expressed on microglia, but much broader cellular localization of TLR2 has been established, particularly during neurodevelopment. To test the hypothesis that TLR2 deficiency affects membrane dynamics, we analysed membrane protein phenotype in the brains of TLR2 knock-out (KO) mice. Cortical, cerebellar, and hippocampal tissue derived from male TLR2-KO and age-matched control (C) mice (N=36+36) underwent systematic biochemical profiling encompassing: analysis of Np55, Np65, PMCA, Na+/K+-ATPase (NKA) expression by Western blot (WB) ; transcriptional variations of selected genes by qPCR ; synaptic proteome by mass spectrometry (MS) ; activity of NKA and PMCA by spectrophotometry. MS analysis revealed astonishing differences in the synaptic proteome. Increased Np55, Np65, and NKA expression in cortex, cerebellum, and hippocampus was determined by WB in TLR2 vs. C. These findings were confirmed at a transcriptional level using qPCR. WB analysis of 4 PMCA isoforms showed that lack of TLR2 is associated with decreased expression of PMCA2 in cortex and cerebellum and increased in hippocampus. Changes in NKA and PMCA activity were also observed in TLR2 KO vs. C. Multiple-level analysis revealed that TLR2 deficiency leads to altered expression of proteins associated with synaptic plasticity and ion homeostasis. Observed proteomic changes are related to glutamatergic transmission, neuronal cytoskeleton organization, and mitochondrion energy metabolism. Further investigation may clarify previously undescribed roles of TLR2 in neuron-microglia interactions, synaptic connections arrangement, and neurotransmission.

Populations around the world are ageing faster than ever in the past. A constant and already impressive rate in the worldwide increase of life expectancy has led to the fact that the current proportion of the population above 60 years (17%) will double in the next thirty to forty years. In the next 30 years, every third person in the world will fall into the category of a senior citizen. This demographic transition will have an impact on almost all aspects of society and requires a complete and well-defined shift in the paradigm in the medical, social, and technological fields.

Aim of the study was to determine the immunoreactivity pattern and unravel potential changes of two isoforms of synaptic transmembrane glycoprotein neuroplastin (Np55 and Np65) in brains of Toll-like receptor 2 deficient (TLR2D) mice in comparison with wild-type (WT) animals. For the purposes of immunohistochemical analysis by confocal microscopy, mouse brain sections derived from TLR2D and WT mice (n=10) were subjected to immunofluorescence labeling using antibodies against Np55, Np65, TLR2, and specific cell-type markers for neurons and different glial cells. Our preliminary Western blot analysis of cortical, hippocampal and cerebellar membrane fractions revealed changed expression levels of two Np isoforms in TLR2D vs WT mice. Here presented immunohistochemical data indicate higher Np55 and Np65 signal intensity in cortical and hippocampal area of TLR2D vs WT mice, and lower Np55 signal intensity in cerebellum of TLR2D mice, which corresponds to our previous Western blot results. In addition, Np was found to be expressed only in neuronal cells in both TLR2D and WT mice. TLR2D mice are a widely used model for studying neuroinflammatory component of different neuropathological disorders and Np is known to be involved in synaptic organization and affected in neurodegenerative disorders. It may be expected that deficiency of TLR2 has impact on cell-cell communication due to changed molecular signatures of neurons and surrounding glial cells. Having in mind evidenced role of TLR2D in activated microglia, deciphering biochemical phenotype of TLR2D mice could as well contribute to understanding potential involvement of neuroplastin in neuroinflammation.

Toll-like receptors (TLRs) are widely expressed in mammalian brain. Involvement in neurodegeneration and hypoxia has been evidenced for several microglial TLRs. In order to investigate interplay of specific microglial and neuronal transmembrane proteins and its potential role in neurodegeneration, we use TLR2 knock-out (KO ) mice (C57BL/6 TLR2-/-).Our preliminary findings indicate different expression of synaptic transmembrane protein neuroplastin (Np) in brain tissue of TLR2 KO mice in comparison with wild- type littermates (WT).Neuroplastin, a cell- adhesion molecule found in two isoforms (Np65 and Np55) is an essential auxiliary subunit of plasma membrane Ca2+ ATPase (PMCA ). In addition, Np is influenced by glycosphingolipid environment of the membrane, which is also the case for another ion pumping enzyme, Na+/K+-ATPase (NKA). Since Np, PMCA and NKA all seem to favor specific lipid milieu in order to function optimally, and at least some of them are confirmed to interact with one another, we investigated the expression of these proteins in TLR2 KO compared to WT across different brain regions.We isolated membrane fractions from cortical, hippocampal and cerebellar tissue of 5 WT and 5 KO animals, and analyzed the expression of Np, NKA and PMCA by Western blotting. Data revealed increased expression level of Np isoforms and PMCA in cortex and hippocampus in TLR2 KO, and decreased expression of Np55 and PMCA in the cerebellum. NKA exhibited the same expression pattern in TLR2 KO vs WT, and increased expression in all regions analyzed (cortex, hippocampus, cerebellum). Furthermore, we investigated the cortical NKA activity which is lower in TLR2 KO mice.This data together with ongoing immunohistochemistry and transcriptional analysis will enable more detailed neurobiochemical characterization of TLR2 KO model, and give insight into functional implications of differential regional distribution of here analyzed proteins involved in synaptic plasticity and membrane ion transport.

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