Ana João Rodrigues

Kinetic profile of the transcriptome changes induced in the choroid plexus by peripheral inflammation

The choroid plexus, being part of the blood-brain barriers and responsible for the production of cerebrospinal fluid, is ideally positioned to transmit signals into and out of the brain. This study, using microarray analysis, shows that the mouse choroid plexus displays an acute-phase response after an inflammatory stimulus induced in the periphery by lipopolysaccharide (LPS). Remarkably, the response is specific to a restricted number of genes (out of a total of 24,000 genes analyzed, 252 are up-regulated and 173 are down-regulated) and transient, as it returns to basal conditions within 72 h. The up-regulated genes cluster into families implicated in immune-mediated cascades and in extracellular matrix remodeling, whereas those down-regulated participate in maintenance of the barrier function. Importantly, several acute-phase proteins, whose blood concentrations rise in response to inflammation, may contribute to the effects observed in vivo after LPS injection, as suggested by the differential response of primary choroid plexus epithelial cell cultures to LPS alone or to serum collected from animals exposed to LPS. By modulating the composition of the cerebrospinal fluid, which will ultimately influence the brain parenchyma, the choroid plexus response to inflammation may be of relevance in brain homeostasis in health and disease.

Lipocalin 2 is a choroid plexus acute-phase protein

Lipocalin 2 (LCN2) is able to sequester iron-loaded bacterial siderophores and, therefore, is known to participate in the mammalian innate immune response. Of notice, LCN2 was shown to display bacteriostatic effects both in in vitro and in vivo. To reach the brain, bacteria must cross the blood—brain or the choroid plexus (CP)/cerebrospinal fluid (CSF) barriers. Additionally, as the CP is responsible for the production of most of the CSF, responses of the CP mediate signaling into the brain. We show here that in conditions of peripheral inflammation, LCN2 behaves as an acute phase protein in the CP. As early as 1 h after lipopolysaccharide peripheral administration, Lcn2 mRNA levels are upregulated, returning to basal levels after 72 h. Increased LCN2 protein is observed in choroidal epithelia and in endothelial cells of blood vessels in the brain parenchyma. Higher levels of LCN2 are also present in the CSF. These observations suggest that expression of LCN2 at the CP/CSF barrier might be bacteriostatic in the brain, avoiding bacteria dissemination within the CSF into the brain parenchyma. This study shows that the LCN2 is produced by the CP as a component of the innate immune response that protects the central nervous system from infection.

Behavioral characterization of the 6-hydroxidopamine model of Parkinson’s disease and pharmacological rescuing of non-motor deficits

BackgroundParkinson’s disease (PD) is a chronic neurodegenerative condition that is characterized by motor symptoms as a result of dopaminergic degeneration, particularly in the mesostriatal pathway. However, in recent years, a greater number of clinical studies have focused on the emergence of non-motor symptoms in PD patients, as a consequence of damage on the mesolimbic and mesocortical dopaminergic networks, and on their significant impact on the quality of life of PD patients. Herein, we performed a thorough behavioral analysis including motor, emotional and cognitive dimensions, of the unilateral medial forebrain bundle (MFB) 6-hydroxidopamine (6-OHDA)-lesioned model of PD, and further addressed the impact of pharmacological interventions with levodopa and antidepressants on mood dimensions.ResultsBased on apomorphine-induced turning behaviour and degree of dopaminergic degeneration, animals submitted to MFB lesions were subdivided in complete and incomplete lesion groups. Importantly, this division also translated into a different severity of motor and exploratory impairments and depressive-like symptoms; in contrast, no deficits in anxiety-like and cognitive behaviors were found in MFB-lesioned animals. Subsequently, we found that the exploratory and the anhedonic behavioural alterations of MFB-lesioned rats can be partially improved with the administration of both levodopa or the antidepressant bupropion, but not paroxetine.ConclusionsOur results suggest that this model is a relevant tool to study the pathophysiology of motor and non-motor symptoms of PD. In addition, the present data shows that pharmacological interventions modulating dopaminergic transmission are also relevant to revert the non-motor behavioral deficits found in the disease.

Functional genomics and biochemical characterization of the C. elegans orthologue of the Machado-Joseph disease protein ataxin-3

Machado‐Joseph disease (MJD) is the most common dominant spinocerebellar ataxia. MJD is caused by a CAG trinucleotide expansion in the ATXN3 gene, which encodes a protein named ataxin‐3. Ataxin‐3 has been proposed to act as a deubiquitinating enzyme in the ubiquitin‐proteasome pathway and to be involved in transcriptional repression;nevertheless, its precise biological function(s) remains unknown. To gain further insight into the function of ataxin‐3, we have identified the Caenorhabditis elegans orthologue of the ATXN3 gene and characterized its pattern of expression, developmental regulation, and subcellular localization. We demonstrate that, analogous to its human orthologue, C. elegans ataxin‐3 has deubiquitinating activity in vitro against polyubiquitin chains with four or more ubiquitins, the minimum ubiquitin length for proteasomal targeting. To further evaluate C. elegans ataxin‐3, we characterized the first known knockout animal models both phenotypically and biochemically, and found that the two C. elegans strains were viable and displayed no gross phenotype. To identify a molecular phenotype, we performed a large‐scale microarray analysis of gene expression in both knockout strains. The data revealed a significant deregulation of core sets of genes involved in the ubiquitin‐proteasome pathway, structure/motility, and signal transduction. This gene identification provides important clues that can help elucidate the specific biological role of ataxin‐3 and unveil some of the physiological effects caused by its absence or diminished function.—Rodrigues, A‐J., Coppola, G., Santos, C., do Carmo Costa, M., Ailion, M., Sequeiros, J., Geschwind, D. H., Maciel, P. Functional genomics and biochemical characterization of the C. elegans orthologue of the Machado‐Joseph disease protein ataxin‐3. FASEB J. 21, 1126–1136 (2007)

Chronic Stress and Glucocorticoids: From Neuronal Plasticity to Neurodegeneration

Stress and stress hormones, glucocorticoids (GCs), exert widespread actions in central nervous system, ranging from the regulation of gene transcription, cellular signaling, modulation of synaptic structure, and transmission and glial function to behavior. Their actions are mediated by glucocorticoid and mineralocorticoid receptors which are nuclear receptors/transcription factors. While GCs primarily act to maintain homeostasis by inducing physiological and behavioral adaptation, prolonged exposure to stress and elevated GC levels may result in neuro- and psychopathology. There is now ample evidence for cause-effect relationships between prolonged stress, elevated GC levels, and cognitive and mood disorders while the evidence for a link between chronic stress/GC and neurodegenerative disorders such as Alzheimers (AD) and Parkinsons (PD) diseases is growing. This brief review considers some of the cellular mechanisms through which stress and GC may contribute to the pathogenesis of AD and PD.

Stress shifts the response of the bed nucleus of the stria terminalis to an anxiogenic mode

The bed nucleus of the stria terminalis (BNST) is critically implicated in anxiety behavior and control of the hypothalamus–pituitary–adrenal axis. Having previously shown that chronic stress triggers dendritic/synaptic remodeling in specific nuclei of the BNST, we characterised the pattern of activation of neurons within different regions of the BNST under basal conditions and after an anxiogenic stimulus in control and stressed rats. Under basal conditions, stressed, but not control, animals displayed increased cFOS expression in the dorsomedial nucleus and decreased activation of the principal nucleus. This pattern resembled that observed in controls that had been exposed to the anxiogenic stimulus. Subsequent analysis of various BNST subnuclei revealed differential patterns of gene expression in controls and stressed animals. We found decreased levels of corticotropin‐releasing hormone 1 receptor mRNA expression in the dorsomedial and fusiform nuclei, and a global increase in the levels of corticotropin‐releasing hormone 2 receptor in the principal nucleus. In addition, we found subnuclei‐specific increases in GABAA and NR2B receptors in stressed animals, which suggest changes in the GABAergic and glutamergic innervation of the BNST. Importantly, these findings were associated with increased anxiety‐like behavior and impaired control of the hypothalamus–pituitary–adrenal axis in stressed animals. In summary, these data reveal that chronic stress shifts the pattern of response of the BNST to an anxiogenic mode and provide new information on the underlying mechanisms of the stress‐induced hypercorticalism and hyperanxious status.

Mechanisms of initiation and reversal of drug-seeking behavior induced by prenatal exposure to glucocorticoids

Stress and exposure to glucocorticoids (GC) during early life render individuals vulnerable to brain disorders by inducing structural and chemical alterations in specific neural substrates. Here we show that adult rats that had been exposed to in utero GCs (iuGC) display increased preference for opiates and ethanol, and are more responsive to the psychostimulatory actions of morphine. These animals presented prominent changes in the nucleus accumbens (NAcc), a key component of the mesolimbic reward circuitry; specifically, cell numbers and dopamine (DA) levels were significantly reduced, whereas DA receptor 2 (Drd2) mRNA expression levels were markedly upregulated in the NAcc. Interestingly, repeated morphine exposure significantly downregulated Drd2 expression in iuGC-exposed animals, in parallel with increased DNA methylation of the Drd2 gene. Administration of a therapeutic dose of L-dopa reverted the hypodopaminergic state in the NAcc of iuGC animals, normalized Drd2 expression and prevented morphine-induced hypermethylation of the Drd2 promoter. In addition, L-dopa treatment promoted dendritic and synaptic plasticity in the NAcc and, importantly, reversed drug-seeking behavior. These results reveal a new mechanism through which drug-seeking behaviors may emerge and suggest that a brief and simple pharmacological intervention can restrain these behaviors in vulnerable individuals.

Reappraising striatal D1- and D2-neurons in reward and aversion.

The striatum has been involved in complex behaviors such as motor control, learning, decision-making, reward and aversion. The striatum is mainly composed of medium spiny neurons (MSNs), typically divided into those expressing dopamine receptor D1, forming the so-called direct pathway, and those expressing D2 receptor (indirect pathway). For decades it has been proposed that these two populations exhibit opposing control over motor output, and recently, the same dichotomy has been proposed for valenced behaviors. Whereas D1-MSNs mediate reinforcement and reward, D2-MSNs have been associated with punishment and aversion. In this review we will discuss pharmacological, genetic and optogenetic studies that indicate that there is still controversy to what concerns the role of striatal D1- and D2-MSNs in this type of behaviors, highlighting the need to reconsider the early view that they mediate solely opposing aspects of valenced behaviour.

Absence of ataxin-3 leads to cytoskeletal disorganization and increased cell death.

Ataxin-3 (ATXN3) is a widely expressed protein that binds to ubiquitylated proteins, has deubiquitylating activity in vitro and is thought to modulate substrate degradation through the ubiquitin-proteasome pathway. Expansion of a polyglutamine tract in ATXN3 causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation and specific neuronal death. Although ATXN3 has been involved in transcriptional repression and in the ubiquitin-proteasome pathway, its biological function is still unknown. In this work, we show that depletion of ATXN3 using small-interference RNA (siRNA) causes a prominent phenotype in both human and mouse cell lines. A mild increase in ubiquitylation occurs and cells exhibit ubiquitin-positive foci, which is consistent with ATXN3 putative function as a deubiquitylating enzyme. In addition, siATXN3-silenced cells exhibit marked morphological changes such as rounder shape and loss of adhesion protrusions. At a structural level, the microtubule, microfilament and intermediate filament networks are severely compromised and disorganized. This cytoskeletal phenotype is reversible and dependent on ATXN3 levels. Cell-extracellular matrix connection is also affected in ATXN3-depleted cells as talin expression is reduced in the focal adhesions and lower levels of alpha-1 integrin subunit are expressed at their surface. Although the cytoskeletal and adhesion problems do not originate any major change in the cell cycle of siATXN3-depleted cells, cell death is increased in siATXN3 cultures compared to controls. In summary, in this work we show that the absence of ATXN3 leads to an overt cytoskeletal/adhesion defect raising the possibility that this protein may play a role in the cytoskeleton.

Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson’s Disease

Research in the last decade strongly suggests that mesenchymal stem cell (MSC)‐mediated therapeutic benefits are mainly due to their secretome, which has been proposed as a possible therapeutic tool for the treatment of Parkinsons disease (PD). Indeed, it has been shown that the MSC secretome increases neurogenesis and cell survival, and has numerous neuroprotective actions under different conditions. Additionally, using dynamic culturing conditions (through computer‐controlled bioreactors) can further modulate the MSC secretome, thereby generating a more potent neurotrophic factor cocktail (i.e., conditioned medium). In this study, we have characterized the MSC secretome by proteomic‐based analysis, investigating its therapeutic effects on the physiological recovery of a 6‐hydroxidopamine (6‐OHDA) PD rat model. For this purpose, we injected MSC secretome into the substantia nigra (SNc) and striatum (STR), characterizing the behavioral performance and determining histological parameters for injected animals versus untreated groups. We observed that the secretome potentiated the increase of dopaminergic neurons (i.e., tyrosine hydroxylase‐positive cells) and neuronal terminals in the SNc and STR, respectively, thereby supporting the recovery observed in the Parkinsonian rats’ motor performance outcomes (assessed by rotarod and staircase tests). Finally, proteomic characterization of the MSC secretome (through combined mass spectrometry analysis and Bioplex assays) revealed the presence of important neuroregulatory molecules, namely cystatin C, glia‐derived nexin, galectin‐1, pigment epithelium‐derived factor, vascular endothelial growth factor, brain‐derived neurotrophic factor, interleukin‐6, and glial cell line‐derived neurotrophic factor. Overall, we concluded that the use of human MSC secretome alone was able to partially revert the motor phenotype and the neuronal structure of 6‐OHDA PD animals. This indicates that the human MSC secretome could represent a novel therapeutic for the treatment of PD. Stem Cells Translational Medicine 2017;6:634–646