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Neuroimmune proteins are involved in a wide array of biological functions throughout brain development. Importantly, these molecular mechanisms regulate the activity-dependent sculpting of neural circuits during the critical period. Abnormal expression of these molecular mechanisms, especially in early development, is linked to the emergence of neurodevelopmental disorders. Despite having central roles in both normal and pathological conditions, very little is known about the lifespan expression of neuroimmune proteins in the human cortex. As studies exploring the relationship between inflammation and disease tend to rely on animal models, unpacking immune lifespan trajectories in the human brain will be essential for translational research. Furthermore, it will aid the development of timely and effective therapeutic interventions for neurodevelopmental disorders. In my thesis, I characterize the development of 72 neuroimmune proteins in 30 postmortem tissue samples of the human primary visual cortex. These samples cover the lifespan from 20 days to 79 years. I compare the developmental profiles of these immune markers to those of well-studied classic neural proteins including glutamatergic, GABAergic and other synaptic plasticity-related markers. Using a data-driven approach, I found that the 72 neuroimmune proteins share approximately eight developmental patterns, most of which undulate across the lifespan. Furthermore, I used unsupervised hierarchical clustering to show that the development of neuroimmune proteins in the human visual cortex varies from that of classic neural proteins. These findings facilitate a deeper understanding of human cortical development through two classes of proteins involved in brain development and plasticity. / Thesis / Master of Science (MSc) / The human brain develops across the lifespan. This ability of the brain to change and adapt to the environment is called plasticity and it is essential for normal brain functions, such as processing visual information. Immune proteins play important roles in the visual cortex- the brain region responsible for visual information processing. They help establish brain circuits in early development and regulate ongoing neural processes important to brain plasticity. In my thesis, I measure the expression of neuroimmune proteins to unpack their developmental patterns in the human visual cortex. I found that these proteins have fluctuating levels across development, with many displaying heightened expression levels in early childhood. Additionally, I found eight common trajectory patterns that were shared between the proteins. These findings enable a better understanding of how regulators of human brain development mature.

Dopamine (DA) transmission is a key player in the rewarding aspects of ethanol as well as ethanol dependence. The current dogma is that DA transmission is increased during ethanol via the inhibition of ventral tegmental area (VTA) GABA neurons and that excitation of VTA GABA neurons during withdrawal results in decreased DA transmission. Microglia, the major neuroimmune effector in the brain, may be a key mediator in this process by releasing cytokines following activation. We evaluated the effect of ethanol on cytokine concentrations in the VTA and NAc using a cytometric bead array, and found that low dose ethanol (1.0 g/kg) decreased interleukin (IL)-10 levels, but high dose ethanol increased IL-10 levels (4.0 g/kg). We also used standard cell-attached mode electrophysiological techniques to evaluate the effects of select cytokines on VTA neuron firing rate in vitro. We found no change in firing rate in response to IL-6, but an increase in firing rate in VTA DA neurons response to IL-10. Consistent with the changes in firing rate, optically-evoked IPSCs were also found to be decreased in response to IL-10. Ex vivo voltammetry and in vivo microdialysis were done to determine whether IL-10 can directly result in an increase in DA release. Although ex vivo voltammetry showed no change in DA release, IL-10 increased DA release in vivo. These findings suggest that the rewarding and/or addictive effects of ethanol are mediated by cytokines, specifically the anti-inflammatory cytokine IL-10.

Microglia are the primary immune cell in the central nervous system and are known as the “resident” macrophages in the central nervous system (CNS). While microglia are classically known as the immune cells of the CNS, their role has more recently been shown to extend far beyond immunity. The effects of ethanol on the brain are closely linked to neuroimmune responses mediated by microglia that are present in the healthy brain from the time of development. Though microglia have been classified as the “resident” immune cells of the CNS, new research suggests that other immune cells may be implicated in the immune response. Normally, the blood-brain barrier (BBB) prevents the infiltration of cells and foreign pathogens from crossing from the periphery into the CNS. However, peripheral monocytes are known to infiltrate the CNS in response to seizures, traumatic brain injury, infection, and multiple sclerosis. Whether or not these cells engraft and become microglia is still a topic of debate. The aim of this study was to determine the effect of acute ethanol on microglia activation and monocyte infiltration into the CNS. We hypothesized that acute EtOH would lead to an increase in neuroinflammation by activating “resident” microglia to an inflammatory polarization and induce the infiltration of macrophages across the BBB. Using the Macrophage FAS-Induced Apoptosis (MaFIA) mouse model (GFP+ on Csf1r promoter), fluorescent microscopy, and flow cytometry we assessed the presence and phenotype of microglia and infiltrating macrophages following 1, 2, and 4 g/kg ethanol at .5, 1, and 2 hours post-injection. By measuring volume/surface area of microglia in the VTA and NAc following EtOH, we found that EtOH caused microglia activation in these areas, and that the microglia are shifting toward an M1 polarization. However, some of our findings were counter to our hypothesis. We found that EtOH, decreases the number of infiltrating monocytes in the VTA and NAc. It is possible that other cells like T and B cells are recruited across the BBB. These findings suggest a neuroimmune connection for acute ethanol use and challenge the dogma that ethanol has exclusively central effects on DA neuronal activity and release. Further research is being performed to examine the implications of this effect, and what effects a conditional knockdown of monocytes has on ethanol intoxication and reward.

<p>Opioid drug abuse represents a serious public health concern with few effective therapeutic strategies. A primary goal for researchers modeling substance abuse disorders has been the delineation of the biological and environmental factors that shape an individual’s susceptibility or resistance to the reinforcing properties of abused substances. Early-life environmental conditions are frequently implicated as critical mediators for later-life health outcomes, although the cellular and molecular mechanisms that underlie these effects have historically been challenging to identify. Previous work has shown that a neonatal handling procedure in rats (which promotes enriched maternal care) attenuates morphine conditioning, reduces morphine-induced glial activation in the nucleus accumbens (NAc), and increases microglial expression of the anti-inflammatory cytokine interleukin-10 (IL-10). The experiments described in this dissertation were thus designed to address if inflammatory signaling in the NAc may underlie the effects of early-life experience on later-life opioid drug-taking. The results demonstrate that neonatal handling attenuates intravenous self-administration of the opioid remifentanil in a drug concentration-dependent manner. Transcriptional profiling of the NAc reveals a suppression of pro-inflammatory cytokine and chemokine signaling molecules and an increase in anti-inflammatory IL-10 in handled rats following repeated exposure to remifentanil. To directly test the hypothesis that anti-inflammatory signaling can alter drug-taking behavior, bilateral intracranial injections of plasmid DNA encoding IL-10 (pDNA-IL-10) or control pDNA were delivered into the NAc of naïve rats. pDNA-IL-10 treatment reduces remifentanil self-administration in a drug concentration-dependent manner, similar to the previous observations in handled rats. Additional experiments confirmed that neither handling nor pDNA-IL-10 treatment alters operant responding for food or sucrose rewards. These results help define the conditions under which ventral striatal neuroimmune signaling may influence motivated behaviors for highly reinforcing opioid drugs.</p> / Dissertation

Chronic and acute stress, with associated pathophysiology, are implicated in a variety of disease states, with neuroimmunological dysregulation and inflammation as major hazards to health and functional sufficiency. Psychosocial stress and negative affect are linked to elevations in several inflammatory biomarkers. Immunosenescence, the deterioration of immune competence observed in the aged aspect of the life span, linked to a dramatic rise in morbidity and susceptibility to diseases with fatal outcomes, alters neuroimmunological function and is particularly marked in the neurodegenerative disorders, e.g., Parkinson's disease and diabetes. Physical exercise diminishes inflammation and elevates agents and factors involved in immunomodulatory function. Both the alleviatory effects of life-long physical activity upon multiple cancer forms and the palliative effects of physical activity for individuals afflicted by cancer offer advantages in health intervention. Chronic conditions of stress and affective dysregulation are associated with neuroimmunological insufficiency and inflammation, contributing to health risk and mortality. Physical exercise regimes have induced manifest anti-inflammatory benefits, mediated possibly by brain-derived neurotrophic factor. The epidemic proportions of metabolic disorders, obesity, and diabetes demand attention; several variants of exercise regimes have been found repeatedly to induce both prevention and improvement under both laboratory and clinical conditions. Physical exercise offers a unique non-pharmacologic intervention incorporating multiple activity regimes, e.g., endurance versus resistance exercise that may be adapted to conform to the particular demands of diagnosis, intervention and prognosis inherent to the staging of autoimmune disorders and related conditions.

O Transtorno do Espectro Autista (TEA), segundo o DSM-5, se enquadra nos Transtornos do Desenvolvimento e é caracterizado por uma díade comportamental: 1) prejuízos na comunicação e interação social e 2) comportamentos repetitivos ou estereotipados. Apesar da etiologia do TEA ser desconhecida, tanto fatores genéticos quanto ambientais já foram associados à desordem, incluindo a utilização de ácido valproico (VPA) durante a gestação. Observando essa relação importante, desenvolveu-se um modelo animal de autismo induzido pela exposição pré-natal ao VPA, o qual já foi amplamente validado em aspectos comportamentais e moleculares. No primeiro capítulo desta tese, utilizamos o modelo animal de autismo obtido através de uma única injeção intraperitoneal de VPA (600mg/kg) no dia E12,5 nas ratas Wistar prenhes e testamos um tratamento pré-natal com resveratrol (RSV), através de injeções subcutâneas (3,6 mg/Kg) administradas nas ratas prenhes nos dias E6,5-E18,5. O tratamento pré-natal com RSV demonstrou ser capaz de prevenir alterações na sociabilidade recíproca, porém não teve impacto nos prejuízos na memória olfativa e comportamentos repetitivos induzidos pelo VPA. No que se refere aos dados de microRNA (miRNA), RSV foi capaz de prevenir a alteração na expressão de miR134-5p, o qual também se observou alterado em pacientes com TEA, juntamente com o miR138-5p, ambos com alvos associados à modulação do citoesqueleto na estrutura de espinhos dendríticos. Em conjunto, esses resultados demonstram que o RSV altera vias importantes no modelo, possivelmente através das suas características antioxidantes e anti-inflamatórias, as quais contrapõem os aspectos inflamatórios do VPA. Este trabalho permitiu o aprimoramento e melhor conhecimento da técnica de RT-qPCR para análise de miRNA, sendo tema do segundo capítulo da presente tese, reunindo diferentes estratégias que auxiliaram a superar potenciais problemas e interferentes no uso dessa técnica. Finalmente, no terceiro capítulo, utilizamos o modelo VPA para avaliar o efeito do tratamento pós-natal com suramina, através de uma única injeção subcutânea (20 mg/kg) administrada nos filhotes machos na idade P30. Este tratamento foi capaz de reverter alterações de sociabilidade, novidade social e comportamento do tipo ansioso, enquanto os prejuízos na sociabilidade recíproca, comportamento exploratório, estereotipia e processamento sensorial não foram revertidos por esse tratamento. Nos dados moleculares, a suramina não foi capaz de reverter o aumento de expressão nos receptores purinérgicos P2X4 (hipocampo e córtex pré-frontal medial) e P2Y2 (hipocampo), porém reverteu o aumento de IL-6 promovido pelo VPA. Assim, possivelmente a modulação comportamental associada à suramina parece não estar associada a interações específicas nos receptores purinérgicos, mas sim com uma modificação neuroimune através da interleucina pró-inflamatória IL-6, demonstrando a importância do sistema imunológico na fisiopatologia do TEA. De forma geral, a tese contribuiu para elucidar mecanismos envolvidos no desenvolvimento das características do tipo autista, demonstrando o papel relevante das alterações neuroimunes e da modulação de alvos por miRNA, as quais, em conjunto, podem contribuir para o desenvolvimento de métodos diagnósticos e adequação de estratégias farmacológicas voltados ao TEA. Palavras-chave: Comportamento animal, microRNA, neuroimune, PCR, resveratrol, sistema purinérgico, suramina, transtorno do espectro autista. / According to DSM-5, Autism Spectrum Disorder (ASD) is a developmental disorder characterized by a behavioral dyad: 1) deficits in communication and social interaction, and 2) repetitive and stereotyped behaviors. Although the etiology of ASD is still unknown, both genetic and environmental factors have been associated with the disorder, including the use of valproic acid (VPA) during gestation. Observing this important relationship, an animal model of autism induced by prenatal exposure to VPA was developed, which has already been widely validated in behavioral and molecular aspects. In the first chapter of this thesis, we used the animal model obtained through a single intraperitoneal injection of VPA (600 mg/kg) at E12.5 in pregnant Wistar rats and tested a prenatal treatment with resveratrol (RSV) by subcutaneous injections (3.6 mg/kg) administered in the pregnant rats at E6.5 to E18.5. The prenatal treatment with RSV was able to prevent changes in the reciprocal sociability, but had no impact on the deficits in olfactory memory and repetitive behavior induced by VPA. Regarding the microRNA (miRNA) data, RSV treatment was able to prevent the alteration in the expression of miR134-5p, which also was altered in ASD patients along with miR138-5p, both with targets associated with cytoskeletal modulation in the structure of dendritic spines. Taken together, these results demonstrate that RSV alters important pathways in the model, possibly through its antioxidant and anti-inflammatory properties, which counteract the inflammatory aspects of VPA. This work allowed the improvement and better knowledge of the RT-qPCR technique for miRNA analysis, which was the theme of the second chapter of this thesis, combining different strategies to overcome potential problems and interferences in this methodology Finally, in the third chapter we used the same animal model to evaluate the effect of postnatal treatment with suramin after a single subcutaneous injection (20 mg/kg) administered to male pups at P30. This treatment was able to revert VPA-induced deficits in sociability, social novelty, and anxiety-like behavior, whilst present no effect on impairments in reciprocal sociability, exploratory behavior, repetitive behavior and sensory processing. In the molecular data, suramin was not able to reverse the increase of expression in the purinergic receptors P2X4 (hippocampus and medial prefrontal cortex) and P2Y2 (hippocampus), but reversed the VPA-induced increase of proinflammatory interleukin IL-6. Thus, behavioral modulation associated with suramin appears to be related not with specific interactions in purinergic receptors, but with a neuroimmune modification through the IL-6, indicating the importance of the immune system in the ASD pathophysiology. In general, the thesis contributed to elucidate the mechanisms involved in the development of autistic-like features, demonstrating the relevant role of neuroimmune alterations and the modulation of targets by miRNA, which, together, may contribute to the development of diagnostic methods and improvement of pharmacological strategies related to ASD.

La maladie de Parkinson (MP) est caractérisée par une dégénérescence des neurones dopaminergiques de la substance noire (SN) en lien avec l’agrégation de l’α-synucléine (α-Syn) sous la forme d’inclusions intraneuronales. De nombreux travaux supportent l’idée que des interactions neuro-immunes, favorisées par des altérations du système vasculaire cérébral, sont impliquées dans les mécanismes pathologiques de la MP. Au cours de ce travail, nous nous sommes intéressés à une population myéloïde particulière qui borde les vaisseaux: les macrophages périvasculaires (MPVs). Nous mettons en évidence chez des patients parkinsoniens et des souris développant une synucléinopathie dégénérative, une augmentation proliférative des MPVs. Leur ablation entraîne une aggravation du phénotype dégénératif chez les souris synucléinopathiques, suggérant un rôle protecteur qui ne serait toutefois pas lié à une régulation de la microgliose. Nous observons néanmoins que l’absence de MPVs s’associe à une augmentation vasculaire de VCAM-1 et de l’infiltrat T CD4+ et CD8+. Par ailleurs, leur absence entraîne une accélération de la diffusion de la synucléinopathie suite à l’injection de fibrilles d’α-Syn. Nous faisons l’hypothèse que les MPVs sont essentiels dans la clairance des assemblages extracellulaires d’α-Syn en raison de leur activité de phagocytose mais aussi de leur importance dans la régulation des flux paravasculaires et lymphatiques, comme le montrent nos expériences d’infusion de traceurs fluorescents. Au total, nos travaux soulignent l’importance des MPVs dans la régulation de la toxicité et la diffusion de la synucléinopathie et en font une cible thérapeutique potentielle pour la MP. / Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) associated to the aggregation of α-synuclein (α-Syn) that forms intraneuronal inclusions. In addition, mounting evidence suggest that neuro-immune interactions, favoured by cerebrovascular alterations, are involved in the pathomechanisms of PD. In this work, we focus on a myeloid population that surrounds blood vessels: perivascular macrophages (PVMs). We show that both in PD patients and in a mouse model of degenerative synucleinopathy, there is an increased proliferative recruitment of PVMs within the SN. We also found that specific ablation of PVMs aggravates the degenerative phenotype in synucleinopathic mice. PVMs-mediated neuroprotection is not likely linked to regulatory mechanisms of microglial cell response. Interestingly, the absence of PVMs is associated with higher vascular expression of VCAM-1 and enhanced infiltration of CD4+ and CD8+ T-cells, whose pathogenic role remains to be determined. We also observe that PVMs deficiency leads to an increased spreading of α-Syn pathology following striatal injections of α-Syn fibrils. We hypothesize that PVMs might be essential for the clearance of toxic α-Syn species due to their phagocytic activity, but also to their importance in regulating interstitial and cerebrospinal fluid (CSF) circulation, as shown by analysing CSF movement via the infusion of fluorescent tracers in PVMs-deficient conditions. Overall, our data highlight the importance of PVMs in regulating the toxicity and propagation of synucleinopathy and provide a rationale for therapeutic strategies aimed at boosting PVMs functions in PD.

An estimated 13.9% of Americans currently meet criteria for an alcohol (ethanol; EtOH) use disorder (AUD). While there are 4 medications approved by the Food and Drug Administration (FDA) to treat AUD, these treatments have demonstrated poor clinical efficacy. Our ongoing research program encompasses a multi-tiered screening of a natural product library and validation process to provide novel information about the mechanisms underlying EtOH-induced changes in neurobiology and to identify novel chemical scaffolds to be exploited in the development of pharmacological treatments for AUD in a rodent organotypic hippocampal slice culture model. Initial screens of several natural product compounds identified 3 compounds which attenuate 48 h EtOH-induced cytotoxicity in vitro. As analogs of natural products can be developed to have enhanced therapeutic potential over parental structures, Study 1 sought to extend on prior findings via the screening of several natural product analogs for their ability to attenuate EtOH-induced cytoxicity. Nine natural produce analogs demonstrated potent cytoprotective effects against EtOH-induced toxicity at 48 h. Several reports suggest EtOH-induced neurotoxicity may be secondary to the induction of persistent neuroimmune activation, and isoflavonoids have been shown to have effects on neuroimmune signaling. Thus, Study 2 compared the effects of compound 9b, an isoflavonoid analog identified in Study 1, to daidzein (DZ), a prototypical isoflavonoid, in the same 48 h model, with the addition of a neuroimmune component. Specifically, culture media was collected to assess for the release of the neuroimmune mediators HMGB1, TNF-α, IL-6, and IL-10 via ELISA. Compound 9b and DZ protected against EtOH-induced cytotoxicity at 48 h. EtOH exposure significantly increased secretion of HMGB1 and IL-6 into culture media at 48h. Compound 9b and DZ attenuated these increases at all concentrations tested. These results suggest potential neuroimmune modulating properties of isoflavonoids which may contribute to their neuroprotective effects against EtOH in vitro. These findings highlight the potential applications DZ and the novel isoflavonoid analog 9b for use in the treatment of AUD.