Ischemic stroke (IS) remains one of the leading causes of death and disability worldwide, representing a major clinical and economic challenge. It is caused by the temporary or permanent interruption of a blood vessel, leading to disruption of the central nervous system and consequent neuronal loss. In the acute phase, therapeutic strategies include thrombolysis and thrombectomy to restore blood flow, whereas in the chronic phase, approaches aimed at promoting neural plasticity, such as cell therapy, are employed. However, these techniques face several limitations, as their efficacy is directly influenced by multiple variables, making their response unpredictable. Recent studies have attributed the effects of mesenchymal stem cells (MSCs) mainly to their paracrine action, thus positioning the use of their secretome as a promising alternative. In this work, we evaluated the impact of secretome derived from umbilical cord MSCs (UC-MSCs), obtained under normoxic and hypoxic conditions, on neuronal viability and on the recovery of synaptic structure after oxygen and glucose deprivation (OGD) in vitro. The results suggest that the secretome can modulate cell survival, preserve the length of neuronal arborization processes, and modulate synaptic markers. Although some results are promising, further studies are required to explore alternative approaches in order to better understand the action of MSC-derived secretome in the treatment of ischemic stroke and other neurological disorders associated with hypoxicischemic injury.

O Transtorno do Déficit de Atenção e Hiperatividade (TDAH) é uma desordem neurocomportamental caracterizada por graus variados de desatenção, atividade motora excessiva e impulsividade. Sua etiologia não é completamente conhecida, porém, um grande número de evidências sugere que, além de fatores de risco genéticos, a exposição gestacional ao etanol e a altas doses de cafeína contribuem para a manifestação deste transtorno. O etanol, presente na composição de bebidas alcoólicas, e a cafeína, presente na composição de diversas bebidas (refrigerantes, chás, café, e energéticos), alimentos derivados do cacau e medicamentos estão entre as substâncias neuroativas mais consumidas no mundo. Apesar das evidências epidemiológicas do uso combinado de cafeína e etanol por gestantes, as interações entre estas substâncias têm recebido pouca atenção em estudos experimentais. Este fato é particularmente importante visto que alguns estudos apontam que cafeína é capaz de ampliar alguns aspectos importantes da ação de outras substâncias com potencial neurotóxico. Nesse estudo avaliamos os efeitos da co-exposição à cafeína e ao etanol durante o desenvolvimento na atividade locomotora em camundongos adolescentes. Para tanto, camundongos Suíços foram expostos do primeiro dia gestacional até o vigésimo primeiro dia pós-natal (PN21), a solução de cafeína 0,1g/L (Grupo CAF1, 10 ninhadas), a solução de cafeína 0,3g/L (Grupo CAF3, 10 ninhadas) ou tiveram acesso à água potável (Grupo CAF0, 10 ninhadas). Em dias alternados de PN2 a PN8, os animais de cada ninhada receberam uma injeção intraperitoneal de 0,25 µl/g de etanol (grupo ETOH25), 0,5 µl/g de etanol (grupo ETOH50) ou de solução salina (grupo ETOH0). Em PN30, a atividade locomotora foi avaliada por 15 minutos no teste de Campo Aberto. A análise dos níveis de etanol sérico, realizada em uma amostra independente de animais em PN8, indicou que, para as duas doses de etanol, a alcoolemia dos animais do grupo CAF3 foi significativamente maior do que as dos grupos CAF0 e CAF1, que não diferiram entre si. No teste de campo aberto, apenas os animais expostos ao etanol apresentaram aumento da atividade locomotora. Tanto o grupo ETOH25 quanto o grupo ETOH50 tiveram a atividade maior que o grupo ETOH0. A exposição à cafeína, por si só, não afetou a atividade locomotora dos animais nem potencializou os efeitos do etanol. No grupo CAF3, a atividade locomotora não foi afetada pela exposição ao etanol. Nossos dados confirmam o papel da exposição precoce ao etanol na manifestação da hiperatividade locomotora. Além disso, a exposição à cafeína durante o desenvolvimento pode exercer um papel protetor para a manifestação da hiperatividade locomotora induzida pela exposição precoce ao etanol.

Neuronal connectivity is a highly plastic phenomenon, and a variety of stimuli can induce alterations in strength, number and structure of synaptic connections. The precise establishment of synaptic contacts during brain development is essential for proper nervous system function, and disruptions contribute to severe neurodevelopmental disorders. Ube3A is an E3 ubiquitin ligase responsible for tagging proteins to be recognized by the ubiquitin-proteasome system (UPS). Ube3A levels in the brain are correlated to different phenotypes, for instance, loss of Ube3A in the brain leads to the development of Angelman syndrome (AS), characterized by neurodevelopmental and motor delay, speech impairment and seizures, while increased levels of Ube3A lead to autism spectrum disorders. In the last years efforts have been made to identify Ube3A interacting partners and putative targets, but Ube3A’s underlying mechanisms remain unclear and its role in synaptogenesis and maintenance of synapses is not fully understood. In this project we investigated if reduced Ube3A levels result in abnormal presynaptic differentiation and impaired synapse formation. We observed that Ube3A is locally present in presynaptic terminals. We also show that loss of Ube3A leads to a reduced clustering of presynaptic proteins, indicating a role for Ube3A in presynaptic differentiation. Additionally, we developed an in vitro compartmentalized microfluidic-based system that recapitulates CNS synapse formation by co-culturing two populations of neurons. This platform will allow to address new questions and test new ideas in the context of Angelman Syndrome. Understanding the mechanism through which Ube3A modulates synapses is crucial to address AS-related symptoms and develop therapies to restore neuronal circuitry.

Injury to the central nervous system (CNS) often results in permanent neurological impairments because axons fail to regenerate and rebuild the synaptic connections that were lost. Downregulation of neuron-intrinsic growth programs and the inhibitory environment present at the injury site constitute the main obstacles to axon regeneration. Therefore, the development of a strategy capable of stimulating the regrowth of injured axons in such restrictive conditions remains crucial. Mesenchymal stem cells (MSC) have been proposed as a promising therapeutic tool for traumatic nervous system disorders, due to their neuro-regenerative effects. These outcomes have been extensively connected to paracrine mechanisms rather than long-term engraftment and cell replacement in the injured tissue. Because synaptic reintegration of injured axons into existing circuitry is required for functional recovery, we investigated the regenerative and synaptogenic potential of MSC secretome. We combined in vitro assays with a mouse model of spinal cord injury (SCI) to fully characterize the secretome’s ability to restore functional connectivity in injured neurons. Application of MSC secretome to rat hippocampal and cortical neurons induces axonal branching and presynaptic differentiation. Additionally, we show that this synaptogenic effect is axonal intrinsic. Moreover, the number of axodendritic synapses also increases after treatment with MSC secretome. This increase in synapses correlates with an enhancement in synaptic activity, revealing that the new synapses induced by MSC secretome are functional. Together, this first set of results indicates that MSC secretome promotes the formation of functional synapses in developing CNS. Although the mechanisms that promote the reorganization of lost synaptic circuits in the adult CNS remain poorly characterized, several studies indicate that the same developmental signals that instruct synaptogenesis can be reactivated following injury. By performing axotomy in microfluidic chambers, we observed that MSC secretome promotes both axonal regeneration and presynaptic differentiation in injured CNS neurons. Moreover, systemic administration of the secretome reduces inflammation, promotes motor improvements, and alleviates neuropathic pain in mice after a T8 complete spinal cord transection. Overall, our results demonstrate that MSC secretome has regenerative and synaptogenic properties and induces functional restoration after SCI, revealing a potential role of the secretome as a future cell-free therapy to restore functional connectivity in the injured CNS.

Background Animal models enable targeting autism-associated genes, such as the shank3 gene, to assess their impact on behavioural phenotypes. However, this is often limited to simple behaviours relevant for social interaction. Social contagion is a complex phenotype forming the basis of human empathic behaviour and involves attention to the behaviour of others for recognizing and sharing their emotional or affective state. Thus, it is a form of social communication, which constitutes the most common developmental impairment across autism spectrum disorders (ASD). Methods Here we describe the development of a zebrafish model that identifies the neurocognitive mechanisms by which shank3 mutation drives deficits in social contagion. We used a CRISPR-Cas9 technique to generate mutations to the shank3a gene, a zebrafish paralogue found to present greater orthology and functional conservation relative to the human gene. Mutants were first compared to wild types during a two-phase protocol that involves the observation of two conflicting states, distress and neutral, and the later recall and discrimination of others when no longer presenting such differences. Then, the whole-brain expression of different neuroplasticity markers was compared between genotypes and their contribution to cluster-specific phenotypic variation was assessed. Results The shank3 mutation markedly reduced social contagion via deficits in attention contributing to difficulties in recognising affective states. Also, the mutation changed the expression of neuronal plasticity genes. However, only downregulated neuroligins clustered with shank3a expression under a combined synaptogenesis component that contributed specifically to variation in attention. Limitations While zebrafish are extremely useful in identifying the role of shank3 mutations to composite social behaviour, they are unlikely to represent the full complexity of socio-cognitive and communication deficits presented by human ASD pathology. Moreover, zebrafish cannot represent the scaling up of these deficits to higher-order empathic and prosocial phenotypes seen in humans. Conclusions We demonstrate a causal link between the zebrafish orthologue of an ASD-associated gene and the attentional control of affect recognition and consequent social contagion. This models autistic affect-communication pathology in zebrafish and reveals a genetic attention-deficit mechanism, addressing the ongoing debate for such mechanisms accounting for emotion recognition difficulties in autistic individuals.

Diabetes durante a gravidez está associada a aumento do risco de distúrbios do neurodesenvolvimento na descendência, nomeadamente défices psicomotores, sensoriais e cognitivos. Estudos indicam que diabetes materna induz modificações estruturais e funcionais no hipocampo da descendência, uma região cerebral associada aos processos de aprendizagem e memória. No entanto, os mecanismos exatos pelos quais o ambiente uterino de uma mãe diabética afeta o hipocampo da descendência permanecem por definir.O objetivo principal da presente tese foi avaliar o impacto da diabetes materna na memória da descendência, avaliando primeiro o impacto no desenvolvimento durante o início da infância e depois analisando alterações celulares e moleculares subjacentes a alterações de memória. Além disso, evidências sugerem que diabetes materna afeta o cérebro da descendência de uma maneira dependente do gênero. No entanto, poucos estudos foram realizados simultaneamente em machos e fêmeas, a fim de estabelecer um efeito claro. Por isso, pretendemos também esclarecer o possível efeito diferencial da diabetes materna na descendência com base no género.Para avaliar a influência do ambiente intrauterino da diabetes durante a gravidez no desenvolvimento durante as primeiras semanas de vida, foi realizada uma bateria de testes para avaliar reflexos, força e locomoção durante esse período, uma vez que alterações no desempenho nesses testes podem ser sugestivas de défices do desenvolvimento do sistema sensório-motor. Diabetes materna induziu atraso no desenvolvimento de reflexos, força e comprometeu a locomoção durante as duas primeiras semanas pós-natais dos descendentes de ambos os sexos com tendência para um efeito mais evidente nos machos. O alcance de marcos físicos de desenvolvimento dos descendentes foi avaliado, revelando um atraso no desenvolvimento. Adicionalmente, este estudo longitudinal demostrou que os descendentes de ambos os sexos de mães diabéticas apresentaram défices em termos de ganho de peso. Sendo o objetivo principal desta tese avaliar o impacto da diabetes materna na memória da descendência no final da infância e, tendo em consideração que a ansiedade pode afetar a cognição, avaliou-se o comportamento ansioso utilizando o teste de campo aberto (OPF) e o teste do labirinto em cruz elevado (EPM). A atividade locomotora também foi avaliada no OPF, uma vez que deficiências locomotoras podem influenciar o desempenho da descendência em testes de memória. Não foram observadas alterações na locomoção nos machos e fêmeas descendentes de mães diabéticas. Em relação ao comportamento ansioso, observou-se que a diabetes materna teve um impacto diferencial nos descendentes consoante o género. Fêmeas demonstraram uma tendência a explorar menos a área central da arena do OPF e entraram menos vezes nos braços abertos do EPM, sugerindo um comportamento ansioso, enquanto nos machos não foram detetadas alterações. Para avaliar o impacto de diabetes materna na memória da descendência, realizou-se o teste de reconhecimento de objetos (NOR). Apesar de não ter sido possível realizar uma análise diferencial consoante o gênero, os resultados indicam que diabetes maternal afeta negativamente a memória da descendência.Para investigar os mecanismos celulares e moleculares que podem estar subjacentes aos défices de memória dos descendentes, começou-se por avaliar a organização estrutural das sub-regiões do hipocampo através da coloração de violeta de cresilo. Não foram detetadas alterações na espessura das camadas nucleares das sub-regiões do hipocampo, sugerindo que a diabetes materna não induziu perda neuronal. Adicionalmente, avaliaram-se os níveis de proteínas envolvidas na sinaptogénese tais como a PSD-95, sinapsina-1, sinaptofisina e KIF1A. A diabetes materna não induziu alterações nos níveis de proteínas sinápticas incluindo nos níveis de VGlut-1, um marcador de sinapses glutamatérgicas. Níveis proteicos de subunidades do recetores AMPA foram também avaliados sendo que estes recetores são importantes para plasticidade sináptica. Observou-se uma tendência para a diminuição dos níveis proteicos totais de GluA1 no hipocampo de fêmeas, filhas de mães diabéticas e uma tendência para o aumento de GluA2 em sinaptossomas de hipocampo de machos. Estas alterações poderão afetar funcionalmente a neurotransmissão e a plasticidade sináptica, e consequentemente os processos de memória.Este trabalho realça a importância de compreender plenamente os efeitos deletérios da diabetes materna durante o desenvolvimento do cérebro, o seu impacto a longo prazo em circuitos neuronais e no comportamento. Além disso, este estudo também demonstra a importância de estudar as diferenças de género em resposta a insultos durante o neurodesenvolvimento.Melhor compreensão dessas questões permitirá delinear estratégias terapêuticas futuras de modo a prevenir e tratar a disfunção neuronal subjacente aos défices cognitivos e de memória da descendência das mães diabéticas

Mesenchymal stem cells (MSCs) have been the target of extensive research due to their neuroprotection and neurotrophic effects, immunomodulatory action, differentiation potential and low tumorigenicity making them an attractive source for cell-based therapies. In addition to their ability to differentiate into neural progenitors, recent studies have shown that they exert a paracrine effect in the microenvironment and surrounding cells that is responsible for its beneficial effects. The array of growth factors, cytokines, chemokines, cell adhesion molecules, hormones, exosomes, microvesicles, lipid mediators and free nucleic acids secreted by the MSCs to the culture medium is considered the secretome. Interestingly, different secretomes originated from different cell niches have shown to exhibit a neuroprotective and pro-regenerative action in several neurological disorders, including the spinal cord injury. Despite mild improvements in functional recovery obtained in MSCs clinical trials, the mechanisms responsible for its regenerative action remain elusive. Recent studies have shown that the MSCs’ secretome induces neurite outgrowth and promotes cell survival. However, its effect on synapse formation is currently unknown. To address this gap, we evaluated the effect of the secretome originated from two MSC populations, the human umbilical cord perivascular cells (HUCPVC) and bone marrow-mesenchymal stem cells (BM-MSCs) on presynaptic differentiation of central nervous system neurons. We verified that the secretome of these two populations of MSC induce synaptic vesicle clustering in cortical neurons. Also, we observed a similar effect in hippocampal neurons. Furthermore, we performed a preliminary analysis of the effect of MSC secretome on mitochondria dynamics and biogenesis, an organelle imperative in all stages of neuronal development as well as in axonal regeneration. Taken together, our results show that the secretomes originated from HUCPVCs and BM-MSCs are inducers of synapse formation in neurons of the central nervous system. These results also suggest a possible role in the regulation of mitochondria biogenesis and dynamics, creating a steppingstone for future studies addressing the mechanisms that underlie these processes. These properties render the secretome of MSCs a strong candidate for future research in regenerative medicine.

The purinergic system includes P1 and P2 receptors, which are activated by ATP and its metabolites. They are expressed in adult neuronal and glial cells and are crucial in brain function, including neuromodulation and neuronal signaling. As P1 and P2 receptors are expressed throughout embryogenesis and development, purinergic signaling also has an important role in the development of the peripheral and central nervous system. In this review, we present the expression pattern and activity of purinergic receptors and of their signaling pathways during embryonic and postnatal development of the nervous system. In particular, we review the involvement of the purinergic signaling in all the crucial steps of brain development i.e. in neural stem cell proliferation, neuronal differentiation and migration as well as in astrogliogenesis and oligodendrogenesis. Then, we review data showing a crucial role of the ATP and adenosine signaling pathways in the formation of the peripheral neuromuscular junction and of central GABAergic and glutamatergic synapses. Finally, we examine the consequences of deregulation of the purinergic system during development and discuss the therapeutic potential of targeting it at adult stage in diseases with reactivation of the ATP and adenosine pathway.

Neuroinflammation, a response of the nervous system to injury, results in the release of pro-inflammatory mediators, as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). Exposure of nerve cells to a neuroinflammatory environment was shown to change the normal neurodevelopment, which can be linked to the appearance of neurological disabilities. In this work, we aimed to assess the effects of moderate levels of IL-1β and TNF-α in the establishment of neuronal arborization, growth cone morphology and synaptogenesis. An early exposure of embryonic hippocampal neurons to cytokines delay neuronal development, with an increase in the number of non-polarized cells, stage 2 of development. When analyzing stage 3 neurons, IL-1β showed to decrease total arborization, in particular at the axonal level, while TNF-α increased dendritic arborization. In fact, IL-1β reduces dendritic and axonal length and the number of axonal branches, whereas it increases the extent of dendritic and axonal branches, probably to compensate the other effects. In contrast, TNF-α increases the number of primary dendrites and dendritic branches, as well as their length. By next analyzing microtubule dynamics as the ratio of acetylated- (old) vs. tyrosinated-tubulin (newly-formed), we found that IL-1β and TNF-α induce microtubule stabilization, which may be related to a deficient axonal outgrowth. In addition, both cytokines reduced the area of growth cones, with an increase in the immunofluorescence of F-actin, indicating alterations at the cytoskeleton which may compromise axonal elongation and branching. Regarding neuronal connectivity, we demonstrated that both cytokines not only reduced the density of dendritic spines and synapses, but also the maturity of dendritic spines, suggesting a reduction in the synaptic strength. These findings establish a relation between neuroinflammation in fetal life and the emergence of neuronal damage, similar to those observed in neurodevelopmental disorders, as schizophrenia.

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.