AGING AND THE DYNORPHINERGIC SYSTEM: EVALUATION OF MEMORY AND MOTOR SYSTEMS IN PRODYNORPHIN KNOCKOUT MICE

Dynorphins, endogenous peptide neurotransmitters expressed in the central nervous system, have been implicated in diverse pathophysiological processes, including excitotoxicity, chronic inflammation, traumatic injury, cognitive impairment, and motor dysfunction, with significant changes with aging or age-related disease processes. This has led to the hypothesis that the suppression of dynorphin expression would produce beneficial effects on learning and memory and motor function. To assess the phenotypic manifestations of chronic suppression of endogenous dynorphin, knockout (KO) mice lacking the coding exons of the gene encoding the prodynorphin (Pdyn) precursor protein, were tested in a series of behavioral, biochemical, and molecular biological studies. Moderately aged Pdyn KO perform comparatively better than similarly aged wild-type (WT) mice in the water maze task, although no Pdyn effect was seen among young adult mice. In addition, young adult Pdyn KO mice show mildly improved performance on a passive avoidance task. Minimal baseline differences were noted in spontaneous locomotor activity in an open-field assay, but Pdyn deletion produced a relative sparing of motor dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus, striatum, and frontal cortex of WT mice by quantitative radioimmunoassay. While aging produces progressive decline in Dyn B in striatum and frontal cortex, Dyn A shows an upward trend in frontal cortex without significant change in striatum. Systemic MPTP produces significant short-term elevations in dynorphin peptides that regress to below baseline by 7 days. HPLC analysis of striatal dopamine shows an age-dependent increase in basal dopamine levels in Pdyn KO mice, an effect that is abolished after MPTP. Western blotting experiments demonstrate that Pdyn deletion is associated with greater phosphorylation at the serine-40 site of tyrosine hydroxylase (TH) despite relatively less total TH immunoreactivity, suggesting a suppressive effect of dynorphins on dopamine synthesis. Microarray analysis of hippocampal tissue from young and aged WT and Pdyn KO mice reveals a number of functional groups of genes demonstrating altered expression. The results of this dissertation support a role of endogenous dynorphins in age-associated cognitive and motor dysfunction

Gut-brain axis: Role of lipids in the regulation of inflammation, pain and CNS diseases

The human gut is a composite anaerobic environment with a large, diverse and dynamic enteric microbiota, represented by more than 100 trillion microorganisms, including at least 1000 distinct species. The discovery that a different microbial composition can influence behavior and cognition, and in turn the nervous system can indirectly influence enteric microbiota composition, has significantly contributed to establish the well-accepted concept of gut-brain axis. This hypothesis is supported by several evidence showing mutual mechanisms, which involve the vague nerve, the immune system, the hypothalamic-pituitary-adrenal (HPA) axis modulation and the bacteria-derived metabolites. Many studies have focused on delineating a role for this axis in health and disease, ranging from stress-related disorders such as depression, anxiety and irritable bowel syndrome (IBS) to neurodevelopmental disorders, such as autism, and to neurodegenerative diseases, such as Parkinson Disease, Alzheimer Disease etc. Based on this background, and considering the relevance of alteration of the symbiotic state between host and microbiota, this review focuses on the role and the involvement of bioactive lipids, such as the N-acylethanolamine (NAE) family whose main members are N-arachidonoylethanolamine (AEA), palmitoylethanolamide (PEA) and oleoilethanolamide (OEA), and short chain fatty acids (SCFAs), such as butyrate, belonging to a large group of bioactive lipids able to modulate peripheral and central pathologic processes. It is well established their effective role in inflammation, acute and chronic pain, obesity and central nervous system diseases. It has been shown a possible correlation between these lipids and gut microbiota through different mechanisms. Indeed, systemic administration of specific bacteria can reduce abdominal pain through the involvement of cannabinoid receptor 1 in rat; on the other hand, PEA reduces inflammation markers in a murine model of inflammatory bowel disease (IBD), and butyrate, producted by gut microbiota, is effective in reducing inflammation and pain in irritable bowel syndrome and IBD animal models. In this review, we underline the relationship among inflammation, pain, microbiota and the different lipids, focusing on a possible involvement of NAEs and SCFAs in the gut-brain axis and their role in central nervous system diseases.    

Opioid dependence: brain structure and function : a magnetic resonance imaging, neuropsychological, and electromagnetic study

Background: Opiod dependence is a chronic severe brain disorder associated with enormous health and social problems. The relapse back to opioid abuse is very high especially in early abstinence, but neuropsychological and neurophysiological deficits during opioid abuse or soon after cessation of opioids are scarcely investigated. Also the structural brain changes and their correlations with the length of opioid abuse or abuse onset age are not known. In this study the cognitive functions, neural basis of cognitive dysfunction, and brain structural changes was studied in opioid-dependent patients and in age and sex matched healthy controls. Materials and methods: All subjects participating in the study, 23 opioid dependents of whom, 15 were also benzodiazepine and five cannabis co-dependent and 18 healthy age and sex matched controls went through Structured Clinical Interviews (SCID) to obtain DSM-IV axis I and II diagnosis and to exclude psychiatric illness not related to opioid dependence or personality disorders. Simultaneous magnetoencephalography (MEG) and electroencephalography (EEG) measurements were done on 21 opioid-dependent individuals on the day of hospitalization for withdrawal therapy. The neural basis of auditory processing was studied and pre-attentive attention and sensory memory were investigated. During the withdrawal 15 opioid-dependent patients participated in neuropsychological tests, measuring fluid intelligence, attention and working memory, verbal and visual memory, and executive functions. Fifteen healthy subjects served as controls for the MEG-EEG measurements and neuropsychological assessment. The brain magnetic resonance imaging (MRI) was obtained from 17 patients after approximately two weeks abstinence, and from 17 controls. The areas of different brain structures and the absolute and relative volumes of cerebrum, cerebral white and gray matter, and cerebrospinal fluid (CSF) spaces were measured and the Sylvian fissure ratio (SFR) and bifrontal ratio were calculated. Also correlation between the cerebral measures and neuropsychological performance was done. Results: MEG-EEG measurements showed that compared to controls the opioid-dependent patients had delayed mismatch negativity (MMN) response to novel sounds in the EEG and P3am on the contralateral hemisphere to the stimulated ear in MEG. The equivalent current dipole (ECD) of N1m response was stronger in patients with benzodiazepine co-dependence than those without benzodiazepine co-dependence or controls. In early abstinence the opioid dependents performed poorer than the controls in tests measuring attention and working memory, executive function and fluid intelligence. Test results of the Culture Fair Intelligence Test (CFIT), testing fluid intelligence, and Paced Auditory Serial Addition Test (PASAT), measuring attention and working memory correlated positively with the days of abstinence. MRI measurements showed that the relative volume of CSF was significantly larger in opioid dependents, which could also be seen in visual analysis. Also Sylvian fissures, expressed by SFR were wider in patients, which correlated negatively with the age of opioid abuse onset. In controls the relative gray matter volume had a positive correlation with composite cognitive performance, but this correlation was not found in opioid dependents in early abstinence. Conclusions: Opioid dependents had wide Sylvian fissures and CSF spaces indicating frontotemporal atrophy. Dilatation of Sylvian fissures correlated with the abuse onset age. During early withdrawal cognitive performance of opioid dependents was impaired. While intoxicated the pre-attentive attention to novel stimulus was delayed and benzodiazepine co-dependence impaired sound detection. All these changes point to disturbances on frontotemporal areas.Huumeriippuvuus on krooninen aivosairaus, joka johtaa aineen jatkuvaan käyttöön huolimatta käytön aiheuttamista vakavista haitoista kaikilla elämän alueilla. Huumeriippuvuuteen liittyy myös huomattava huumeidenkäytön uusiutumisriski erityisesti varhaisen vieroituksen aikana, mutta vielä pitkänkin päihteettömän ajanjakson jälkeen. Tutkimuksen tavoitteena oli selvittää pitkäaikaisen opioidien (heroiini ja buprenorfiini) väärinkäytön vaikutusta aivojen rakenteeseen magneettikuvauksella ja toimintaan aivojen sähköisiä vasteita mittaavilla elektroenkefalografia (EEG) - ja magnetoenkefalografiatutkimuksilla (MEG). Lisäksi kognitiivista suoriutumista selvitettiin varhaisen vieroitushoidon aikana neuropsykologisilla testeillä. Tutkimukseen osallistui 23 opioidiriippuvaista henkilöä, joista 15 oli myös bentsodiatsepiiniriippuvaisia. Tutkittavilla oli vakava pitkäaikainen opioidiriippuvuus. Tutkittavien tuloksia verrattiin iältään ja sukupuoleltaan vastaavien terveiden henkilöiden tutkimustuloksiin. Kaikille tutkittaville tehtiin strukturoidut psykiatriset haastattelut, joilla pois suljettiin sellaiset psykiatriset sairaudet, jotka eivät liity päihteiden käyttöön tai persoonallisuushäiriöihin. Kaikilla opioidiriippuvaisilla oli persoonallisuushäiriöitä, mutta verro-keilla ei niitä todettu. Vieroitushoitoon tulopäivänä opioidiriippuvaisille tehtiin samanaikainen EEG-MEG mittaus, jossa tutkittiin kuulovasteita. Tahatonta tarkkavaisuutta tutkittiin ns. poikkeavuusnegatiivisuus vasteella (mismatch negativity, MMN) ja P3a vasteella. MEG-EEG mittauksessa potilailla olivat MMN vaste EEG mittauksessa ja P3a vaste MEG mittauksessa viivästyneet viitaten tahattoman tarkkaavaisuuden muutokseen. Näitä vasteita pidetään kognitiivisina, alkeellista työmuistia edustavina vasteina. Potilailla, joilla oli myös bentsodiatsepiiniriippuvuus, oli kuuloärsykkeen aiheuttama vaste voimistunut. Aineiden sekakäyttö siis lisäsi kuulovasteen muutoksia. Vieroitushoidon aikana 15 opioidiriippuvaiselle tehtiin laaja neuropsykologinen tutkimus, jolla selvitettiin joustavaa älykkyyttä, muistin eri osa-alueita ja toiminnan ohjausta. Neuro-psykologisessa testissä potilaat menestyivät verrokeita huonommin tarkkavaisuutta ja työmuistia (PASAT) sekä toiminnan ohjausta (RFFT) ja joustavaa älykkyyttä mittaavissa tes-teissä (CFIT). PASAT ja CFIT testien tulokset korreloivat positiivisesti vieroitushoidon kestoon. Opioidiriipuvaisten kokonaissuoriutuminen neuropsykologisissa testeissä vieroitusvaiheessa oli verrokeita vaatimattomampi. Kahden viikon vieroitushoitojakson lopussa 17 opioidiriippuvaista kävi aivojen 1,5 T magneettiikuvauksessa. Kuvista analysoitiin valkean ja harmaan aineen sekä aivoselkäydin-nestetilojen tilavuudet sekä tehtiin aivorakenteiden pinta-ala mittauksia ja laskettiin rakenteiden suhteita. Myös korrelaatiot eri tutkimustulosten ja päihdehistorian välille laskettiin. Aivojen magneettiikuvauksessa todettiin, että aivojen sivukammiot olivat laajemmat kuin verrokeilla. Opioidiriipuvaisilla Sylvian uurteet oli leveämmät suhteessa aivojen poikkimittaan kuin verrokeilla ja tämä suhde korreloi opioidien käytön aloitusikään. Löydös viittaa aivojen otsa- ja ohimolohkojen surkastumiseen. Aivojen tilavuusmittauksissa todettiin aivojen kokonaisvolyymin olevan verrokeita pienempi ja suhteellisen aivoselkäydinnestemäärän olevan tutkittavilla merkittävästi verrokeita suurempi. Verrokeilla neuropsykologinen kokonaissuoriutuminen korreloi positiivisesti harmaan aineen määrään, mutta opioidirippuvaisilla henkilöillä tällaista korrelaatiota ei havaittu, voidaan siis ajatella vieroitusvaiheen muutosten heikentävän opioidiriippuvaisten suoriutumista ehkä pysyvämpiä muutoksia enemmän. Kaikissa eri tutkimuksissa saadut tulokset viittaavat pääosin otsa- ja ohimolohkojen toiminnan ja rakenteen muutoksiin opioidiriippuvaisilla

Kyotorphin derived peptides in the relationship between analgesia and alzheimer’s disease

Tese de doutoramento, Ciências Biomédicas (Bioquímica Médica), Universidade de Lisboa, Faculdade de Medicina, 2014The increased lifespan has brought about an increase in the incidence of diseases related to the aging process. Among these, there are the neurodegenerative diseases, including Alzheimer's disease (AD) and other entities associated with the phenomena of chronic pain. The possible correlation between two entities - pain and Alzheimer's disease - with such a large epidemiological burden is of enormous importance. Nowadays pain is considered the fifth vital sign and is highly prevalent, still largely under-treated. New drugs acting on different targets with a smaller range of adverse effects are needed. Moreover, Alzheimer's disease is widely spread in our population, primarily affecting the elderly but also a younger population. The ignorance of the complexity of the biochemical and physiological processes involved and progressive cognitive impairment of patients still result in ineffective treatment. The increase in scientific knowledge about the disease at various levels is mandatory in order to ensure an improvement in the provision of appropriate health care and the chance of intervening in the early stages of the disease, including at the level of prevention and diagnosis. The consequences of diagnosis and evolution of this pathology are well known: change in family / emotional environment of the patient and burden on caregivers. Therefore, a strategy for the correlation between these two entities and possible common therapeutic targets involving a single pharmacological agent is promising. As already mentioned, this project focuses on two issues of utmost clinical importance and strong social impact: Pain and Alzheimer's disease. The most innovative aspect relates to the hypothesis that both problems can be related and may, therefore, be addressed in a single therapeutic approach. This hypothesis is supported by previous experimental work in vitro, evolving now to another phase, with animal experimentation in disease models and clinical research. This thesis intended to evaluate a molecule, kyotorphin, as a molecular link in the mechanisms involved in both cases. Kyotorphin (KTP), discovered in 1979, was described as an endogenous analgesic dipeptide actuating in the brain. With na analgesic activity about 4 times higher than other endogenous peptides such as met-enkephalin, this dipeptide has very interesting features and is thought to act via opioid mechanisms. Analgesic derived molecules of kyotorphin had previously been tested for their analgesic effect in vivo in animal models and for in vitro neuroprotective effects after peripheral administration. This project brings these results to clinical research and its main objectives were: 1) to understand if there is a correlation between Alzheimer's disease and pain, 2) if analgesic peptides derived from kyotorphin are capable of neuromodulation and 3) evaluate kyotorphin as a potential molecular marker in Alzheimer's disease. To achieve these objectives, clinical and basic research were combined using diverse techniques such as questionnaires and pain scales, sophisticated analytical techniques of mass spectrometry and animal experimentation. In a first stage we assessed pain perception in Alzheimer patients and their caregivers, using validated pain scales. This study concluded that, in line with the findings in the literature, pain in Alzheimer patients is often under-evaluated and therefore undertreated. This phenomenon probably occurs because these patients are unable to value and/or express their suffering, even in moderate stages of the disease. Clinical research involved another component: the collection of cerebrospinal fluid samples of patients with AD for determination of Kyotorphin levels and its comparison with individuals without known neurodegenerative diseases; it was found that the levels of kyotorphin , a neuropeptide endogenous per se, decreases with the progression of Alzheimer's disease. This finding opens new possibilities, including the use of kyotorphin as a possible marker of neurodegenerescence and that this neuropeptide has neuromodulatory actions. Subsequently, we studied the effect of neuropeptide kyotorphin derivatives capable of crossing the blood brain barrier - IbKTP-NH2 e KTP-NH2 – in animal models with two objectives: realize if these drugs showed significant side effects compared to reference opioids used in clinical practice, and what were their effects in an animal model of neurodegenerescence. These amidated derivatives IbKTP-NH2 e KTP-NH2, unlike morphine and tramadol (two largely used drugs in clinical practice), caused no major side effects associated with opioids, which is a further indication that the mechanism of action of these peptides and opioids not fully coincide. On the other hand, the neurodegeneration animal model to which these compounds were chronically administered revealed an improvement in the behavioral pattern in comparison with animals with neurological lesions to which none of the compounds had been administered. Overall, our results indicate kyotorphin as a possible biomarker for Alzheimer's disease, and its derivatives IbKTP-NH2 and KTP-NH2 capable of neuromodulation/neuroprotection, in addition to effective analgesic molecules with reduced side effects.Fundação para a Ciência e a Tecnologia (FCT

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Cortical mapping of the neuronal circuits modulating the muscle tone. Introduction to the electrophysiological treatment of the spastic hand

L'objectiu d'aquest estudi es investigar l'organització cortical junt amb la connectivitat còrtico-subcortical en subjectes sans, com a estudi preliminar. Els mapes corticals s'han fet per TMS navegada, i els punts motors obtinguts s'han exportant per estudi tractogràfic i anàlisi de las seves connexions. El coneixement precís de la localització de l'àrea cortical motora primària i les seves connexions es la base per ser utilitzada en estudis posteriors de la reorganització cortical i sub-cortical en pacients amb infart cerebral. Aquesta reorganització es deguda a la neuroplasticitat i pot ser influenciada per els efectes neuromoduladors de la estimulació cerebral no invasiva.The purpose of this study is to investigate the motor cortex organisation together with the cortico-subcortical connectivity in healthy subjects, as a preliminary study. Cortical maps have been performed by navigated TMS and the motor points have been exported to DTI to study their subcortical connectivity. The precise knowledge of localization of the primary motor cortex area and its connectivity is the base to be used in later studies of cortical and subcortical re-organisation in stroke patients. This re-organisation is due to the neuroplascity and can be influenced by the neuromodulation effects of the non-invasive cerebral stimulation therapy by TMS

In utero and Postnatal Oxycodone Exposure: Implications for Intergenerational Effects

Prescription opioid abuse during and after pregnancy is a rising public health concern. Adding a layer of complexity is the role of heredity in the overall development of these exposed offspring. The present work uses a preclinical rat model mimicking oxycodone (oxy) exposure in utero (IUO) and postnatally (PNO) to investigate comparative and intergenerational effects in the two different exposure groups. To understand the direct effects of IUO and PNO exposure on the F1 generation, we employed a systems biology approach encompassing proton magnetic resonance spectroscopy (1H-MRS), electrophysiology RNA-sequencing, and pain assessment to elucidate molecular and behavioral changes in these offspring. 1H-MRS studies revealed significant changes in brain metabolites that were corroborated with changes in synaptic currents. RNA-sequencing of the prefrontal cortex further revealed alterations in the expression of key genes associated with synaptic transmission, neurodevelopment, mood disorders, and addiction. Von Frey testing showed lower pain thresholds in both oxy-exposed groups. Further, because addictive drugs produce significant and persistent changes in the synapse, we investigated the synaptic vesicle (SV) contents of the PNO and IUO groups. To that end, we found that the expression levels of key SV proteins associated with functional pathways and neurological disease were altered in oxy-exposed groups. While our earlier studies characterized the effects PNO and IUO exposure have on the F1 generation, we next sought to compare the overall development between F1 offspring and their progeny, the F2 generation. We observed significant differences in phenotypic attributes of both generations in each treatment group, and RNA-sequencing of the nucleus accumbens revealed alterations in the expression of key synaptic genes in both generations. Post-validation of these genes using RT-PCR highlighted the differential expression of several neuropeptides associated with the hypocretin system, a system recently implicated in addiction. Further, behavior studies revealed anxiety-like behaviors and social deficits in both treatment groups that persisted into the F2 generation. Collectively, our studies reveal a new line of investigation on the potential risks associated with oxy use during and after pregnancy, specifically the disruption of neurodevelopment and the intergenerational impact on behavior

Cardiovascular Dementia - A Different Perspective

The number of dementia patients has been growing in recent years and dementia represents a significant threat to aging people all over the world. Recent research has shown that the number of people affected by Alzheimer’s disease (AD) and dementia is growing at an epidemic pace. The rapidly increasing financial and personal costs will affect the world's economies, health care systems, and many families. Researchers are now exploring a possible connection among AD, vascular dementia (VD), diabetes mellitus (type 2, T2DM) and cardiovascular diseases (CD). This correlation may be due to a strong association of cardiovascular risk factors with AD and VD, suggesting that these diseases share some biologic pathways. Since heart failure is associated with an increased risk of AD and VD, keeping the heart healthy may prove to keep the brain healthy as well. The risk for dementia is especially high when diabetes mellitus is comorbid with severe systolic hypertension or heart disease. In addition, the degree of coronary artery disease (CAD) is independently associated with cardinal neuropathological lesions of AD. Thus, the contribution of T2DM and CD to AD and VD implies that cardiovascular therapies may prove useful in preventing AD and dementia

Plasticity related gene expression in the hippocampus

Learning and the retention of the information as a memory produces changes in synaptic efficiency, the formation of new synapses and remodelling of existing synapses in the central nervous system. These plasticity-related changes are brought about by the modification of existing proteins and the synthesis of new proteins. One structure in the brain which is particularly associated with learning and memory is the hippocampus. Manipulation of synaptic transmission in this structure (in particular the phenomenon of long-term potentiation or LTP) is widely believed to be the mechanism which underlies mammalian synaptic plasticity This work was undertaken to further investigate the proteins which are affected by plasticity-related changes within the hippocampus, to improve understanding of why these changes occur and uncover more details about the mechanisms involved