Miranda-Lourenço C, Ribeiro-Rodrigues L, Fonseca-Gomes J, Tanqueiro SR, Belo RF, Ferreira CB, Rei N, Ferreira-Manso M, de Almeida-Borlido C, Costa-Coelho T, Freitas CF, Zavalko S, Mouro FM, Sebastião AM, Xapelli S, Rodrigues TM, Diógenes MJ. Challenges of BDNF-based therapies: From common to rare diseases. Pharmacol Res. 2020 Dec;162:105281. doi: 10.1016/j.phrs.2020.105281.

Neurotrophins are a well-known family of neurotrophic factors that play an important role both in the central and peripheral nervous systems, where they modulate neuronal survival, development, function and plasticity. Brain-derived neurotrophic factor (BDNF) possesses diverse biological functions which are mediated by the activation of two main classes of receptors, the tropomyosin-related kinase (Trk) B and the p75 neurotrophin receptor (p75NTR). The therapeutic potential of BDNF has drawn attention since dysregulation of its signalling cascades has been suggested to underlie the pathogenesis of both common and rare diseases. Multiple strategies targeting this neurotrophin have been tested; most have found obstacles that ultimately hampered their effectiveness.

This review focuses on the involvement of BDNF and its receptors in the pathophysiology of Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Rett Syndrome (RTT). We describe the known mechanisms leading to the impairment of BDNF/TrkB signalling in these disorders. Such mechanistic insight highlights how BDNF signalling compromise can take various shapes, nearly disease-specific. Therefore, BDNF-based therapeutic strategies must be specifically tailored and are more likely to succeed if a combination of resources is employed.

Fonseca-Gomes J, Loureiro JA, Tanqueiro SR, Mouro FM, Ruivo P, Carvalho T, Sebastião AM, Diógenes MJ, Pereira MC. In vivo Bio-Distribution and Toxicity Evaluation of Polymeric and Lipid-Based Nanoparticles: A Potential Approach for Chronic Diseases Treatment. International Journal of Nanomedicine. 2020 Nov 5;15:8609-8621.


Nanoparticles (NPs), as drug delivery systems, appear to be a promising tool for prolonged therapeutic strategies as they allow a controlled drug release over time. However, most of the studies found in the literature simply contemplate the use of a single or low number of dosages with low NPs concentrations. In the context of chronic diseases, like Alzheimer’s disease, cancer or human immunodeficiency virus (HIV), where the therapeutic scheme is also chronic, studies with numerous repeated dosages are often neglected. We screened different NPs, polymeric and lipid-based, in a repeated-dose toxicity study, to evaluate the safety and tissue distribution of promising nanocarriers to be used in the treatment of long-lasting diseases. After administrating 24 high concentrated doses of the selected NPs intraperitoneally (i.p.) (3 times a week for 2 months), animals have presented NPs accumulation in different tissues. However, neither toxicity, bodyweight changes nor clinical signs of disease were observed. This work demonstrates no general adverse effects upon the studied NPs repeated-dose exposure, indicating the most promising NPs to be used in the different therapeutic circumstances, which may be useful in chronic diseases treatment.

Miranda-Lourenço C*, Duarte ST*, Palminha C, Gaspar C, Rodrigues TM, Magalhães-Cardoso T, Rei N, Colino-Oliveira M, Gomes R, Ferreira S, Rosa J, Xapelli S, Armstrong J, García-Cazorla À, Correia-de-Sá P, Sebastião AM*, Diógenes MJ*. Impairment of adenosinergic system in Rett syndrome: Novel therapeutic target to boost BDNF signalling. Neurobiology of Disease. 2020 Nov;145:105043.

*Equal contribution


Rett syndrome (RTT; OMIM#312750) is mainly caused by mutations in the X-linked MECP2 gene (methyl-CpG-binding protein 2 gene; OMIM*300005), which leads to impairments in the brain-derived neurotrophic factor (BDNF) signalling. The boost of BDNF mediated effects would be a significant breakthrough but it has been hampered by the difficulty to administer BDNF to the central nervous system. Adenosine, an endogenous neuromodulator, may accomplish that role since through A2AR it potentiates BDNF synaptic actions in healthy animals. We thus characterized several hallmarks of the adenosinergic and BDNF signalling in RTT and explored whether A2AR activation could boost BDNF actions. For this study, the RTT animal model, the Mecp2 knockout (Mecp2−/y) (B6.129P2 (C)-Mecp2tm1.1Bird/J) mouse was used. Whenever possible, parallel data was also obtained from post-mortem brain samples from one RTT patient. Ex vivo extracellular recordings of field excitatory post-synaptic potentials in CA1 hippocampal area were performed to evaluate synaptic transmission and long-term potentiation (LTP). RT-PCR was used to assess mRNA levels and Western Blot or radioligand binding assays were performed to evaluate protein levels. Changes in cortical and hippocampal adenosine content were assessed by liquid chromatography with diode array detection (LC/DAD). Hippocampal ex vivo experiments revealed that the facilitatory actions of BDNF upon LTP is absent in Mecp2−/y mice and that TrkB full-length (TrkB-FL) receptor levels are significantly decreased. Extracts of the hippocampus and cortex of Mecp2−/y mice revealed less adenosine amount as well as less A2AR protein levels when compared to WT littermates, which may partially explain the deficits in adenosinergic tonus in these animals. Remarkably, the lack of BDNF effect on hippocampal LTP in Mecp2−/y mice was overcome by selective activation of A2AR with CGS21680. Overall, in Mecp2−/y mice there is an impairment on adenosinergic system and BDNF signalling. These findings set the stage for adenosine-based pharmacological therapeutic strategies for RTT, highlighting A2AR as a therapeutic target in this devastating pathology.

Lourenço DM, Ribeiro-Rodrigues L, Sebastião AM, Diógenes MJ, Xapelli S. Neural Stem Cells and Cannabinoids in the Spotlight as Potential Therapy for Epilepsy. International Journal of Molecular Sciences. 2020 Oct 3;21(19):7309.


Epilepsy is one of the most common brain diseases worldwide, having a huge burden in society. The main hallmark of epilepsy is the occurrence of spontaneous recurrent seizures, having a tremendous impact on the lives of the patients and of their relatives. Currently, the therapeutic strategies are mostly based on the use of antiepileptic drugs, and because several types of epilepsies are of unknown origin, a high percentage of patients are resistant to the available pharmacotherapy, continuing to experience seizures overtime. Therefore, the search for new drugs and therapeutic targets is highly important. One key aspect to be targeted is the aberrant adult hippocampal neurogenesis (AHN) derived from Neural Stem Cells (NSCs). Indeed, targeting seizure-induced AHN may reduce recurrent seizures and shed some light on the mechanisms of disease. The endocannabinoid system is a known modulator of AHN, and due to the known endogenous antiepileptic properties, it is an interesting candidate for the generation of new antiepileptic drugs. However, further studies and clinical trials are required to investigate the putative mechanisms by which cannabinoids can be used to treat epilepsy. In this manuscript, we will review how cannabinoid-induced modulation of NSCs may promote neural plasticity and whether these drugs can be used as putative antiepileptic treatment.

Rodrigues RS, Paulo SL, Moreira JB, Tanqueiro SR, Sebastião AM, Diógenes MJ, Xapelli S. Adult Neural Stem Cells as Promising Targets in Psychiatric Disorders. Stem Cells and Development. 2020 Sep 1;29(17):1099-1117


The development of new therapies for psychiatric disorders is of utmost importance, given the enormous toll these disorders pose to society nowadays. This should be based on the identification of neural substrates and mechanisms that underlie disease etiopathophysiology. Adult neural stem cells (NSCs) have been emerging as a promising platform to counteract brain damage. In this perspective article, we put forth a detailed view of how NSCs operate in the adult brain and influence brain homeostasis, having profound implications at both behavioral and functional levels. We appraise evidence suggesting that adult NSCs play important roles in regulating several forms of brain plasticity, particularly emotional and cognitive flexibility, and that NSC dynamics are altered upon brain pathology. Furthermore, we discuss the potential therapeutic value of utilizing adult endogenous NSCs as vessels for regeneration, highlighting their importance as targets for the treatment of multiple mental illnesses, such as affective disorders, schizophrenia, and addiction. Finally, we speculate on strategies to surpass current challenges in neuropsychiatric disease modeling and brain repair.

Belo RF*, Martins MLF*, Shvachiy L, Costa-Coelho T, de Almeida-Borlido C, Fonseca-Gomes J, Neves V, Vicente Miranda H, Outeiro TF, Coelho JE, Xapelli S, Valente CA, Heras M, Bardaji E, Castanho MARB*, Diógenes MJ*, Sebastião AM*. The Neuroprotective Action of Amidated-Kyotorphin on Amyloid β Peptide-Induced Alzheimer's Disease Pathophysiology. Frontiers in Pharmacology. 2020 Jul 9;11:985

*Equal contribution


Kyotorphin (KTP, L-tyrosyl-L-arginine) is an endogenous dipeptide initially described to have analgesic properties. Recently, KTP was suggested to be an endogenous neuroprotective agent, namely for Alzheimer’s disease (AD). In fact, KTP levels were shown to be decreased in the cerebrospinal fluid of patients with AD, and recent data showed that intracerebroventricular (i.c.v.) injection of KTP ameliorates memory impairments in a sporadic rat model of AD. However, this administration route is far from being a suitable therapeutic strategy. Here, we evaluated if the blood-brain permeant KTP-derivative, KTP-NH2, when systemically administered, would be effective in preventing memory deficits in a sporadic AD animal model and if so, which would be the synaptic correlates of that action. The sporadic AD model was induced in male Wistar rats through i.c.v. injection of amyloid β peptide (Aβ). Animals were treated for 20 days with KTP-NH2 (32.3 mg/kg, intraperitoneally (i.p.), starting at day 3 after Aβ administration) before memory testing (Novel object recognition (NOR) and Y-maze (YM) tests). Animals were then sacrificed, and markers for gliosis were assessed by immunohistochemistry and Western blot analysis. Synaptic correlates were assessed by evaluating theta-burst induced long term potentiation (LTP) of field excitatory synaptic potentials (fEPSPs) recorded from hippocampal slices and cortical spine density analysis. In the absence of KTP-NH2 treatment, Aβ-injected rats had clear memory deficits, as assessed through NOR or YM tests. Importantly, these memory deficits were absent in Aβ-injected rats that had been treated with KTP-NH2, which scored in memory tests as control (sham i.c.v. injected) rats. No signs of gliosis could be detected at the end of the treatment in any group of animals. LTP magnitude was significantly impaired in hippocampal slices that had been incubated with Aβ oligomers (200 nM) in the absence of KTP-NH2. Co-incubation with KTP-NH2 (50 nM) rescued LTP toward control values. Similarly, Aβ caused a significant decrease in spine density in cortical neuronal cultures, and this was prevented by co-incubation with KTP-NH2 (50 nM). In conclusion, the present data demonstrate that i.p. KTP-NH2 treatment counteracts Aβ-induced memory impairments in an AD sporadic model, possibly through the rescuing of synaptic plasticity mechanisms.

Ruffolo G, Cifelli P, Miranda-Lourenço C, De Felice E, Limatola C, Sebastião AM, Diógenes MJ, Aronica E, Palma E. Rare Diseases of Neurodevelopment: Maintain the Mystery or Use a Dazzling Tool for Investigation? The Case of Rett Syndrome. Neuroscience. 2020 Jul 15;439:146-152


The investigation on neurotransmission function during normal and pathologic development is a pivotal component needed to understand the basic mechanisms underlying neurodevelopmental pathologies. To study these diseases, many animal models have been generated which allowed to face the limited availability of human tissues and, as a consequence, most of the electrophysiology has been performed on these models of diseases. On the other hand, the technique of membrane microtransplantation in Xenopus oocytes allows the study of human functional neurotransmitter receptors thanks to the use of tissues from autopsies or surgeries, even in quantities that would not permit other kinds of functional studies. In this short article, we intend to underline how this technique is well-fit for the study of rare diseases by characterizing the electrophysiological properties of GABAA and AMPA receptors in Rett syndrome. For our purposes, we used both tissues from Rett syndrome patients and Mecp2-null mice, a well validated murine model of the same disease, in order to strengthen the solidity of our results through the comparison of the two. Our findings retrace previous results and, in the light of this, further argue in favor of Prof. Miledi's technique of membrane microtransplantation that proves itself a very useful tool of investigation in the field of neurophysiology. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Sa de Almeida J, Vargas M, Fonseca-Gomes J, Tanqueiro SR, Belo RF, Miranda-Lourenço C, Sebastião AM, Diógenes MJ, Pais TF. Microglial Sirtuin 2 Shapes Long-Term Potentiation in Hippocampal Slices. Frontiers in Neuroscience. 2020 Jun 18;14:614.


Microglial cells have emerged as crucial players in synaptic plasticity during development and adulthood, and also in neurodegenerative and neuroinflammatory conditions. Here we found that decreased levels of Sirtuin 2 (Sirt2) deacetylase in microglia affects hippocampal synaptic plasticity under inflammatory conditions. The results show that long-term potentiation (LTP) magnitude recorded from hippocampal slices of wild type mice does not differ between those exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus, or BSA. However, LTP recorded from hippocampal slices of microglial-specific Sirt2 deficient (Sirt2–) mice was significantly impaired by LPS. Importantly, LTP values were restored by memantine, an antagonist of N-methyl-D-aspartate (NMDA) receptors. These results indicate that microglial Sirt2 prevents NMDA-mediated excitotoxicity in hippocampal slices in response to an inflammatory signal such as LPS. Overall, our data suggest a key-protective role for microglial Sirt2 in mnesic deficits associated with neuroinflammation.

Marcelino H, Nogueira VC, Santos CRA, Quelhas P, Carvalho TMA, Fonseca-Gomes J, Tomás J, Diógenes MJ, Sebastião AM, Cascalheira JF. Adenosine inhibits human astrocyte proliferation independently of adenosine receptor activation. Journal of Neurochemistry. 2020 May;153(4):455-467


Brain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3 days with test drugs. Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase release and by western blot analysis of αII-Spectrin cleavage. 30 µM-Adenosine caused a 40% ± 3% (p < .05, n = 5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100 µM-Homocysteine alone caused 16% ± 3% (p < .05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76% ± 4%, an effect higher (p < .05) than the sum of the effects of adenosine and homocysteine alone (56% ± 5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100 µM) or SAM (50-100 µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.

Xapelli S, Diógenes MJ, Crunelli V, Fitzsimons CP, Vaz SH. Editorial: Glial and Neural Stem Cells as New Therapeutic Targets for Neurodegenerative Disorders. Frontiers in Cellular Neuroscience. 2020 Apr 3;14:71.


The gradual and progressive degeneration of neural cells is a common feature of different neurological disorders culminating in a disturbed neuronal communication. The remarkable discovery that adult brain has the capacity to develop new neurons, as well as new glial cells, raised the hope to restore neuronal communication. It is now accepted that adult neurogenesis occurs in neurogenic niches which are under the control of several factors including neurotrophins. These neurogenic niches contain multipotent neural stem/progenitor cells (NSPCs) which can differentiate into neurons, astrocytes and oligodendrocytes. Interestingly, different brain injuries or pathologies, such as epilepsy, head traumas and stroke, induce neurogenesis and/or astrogliogenesis. This increase in neurogenesis has been proposed as an endogenous attempt to repair and reduce brain damage. On the contrary, aging and several neurodegenerative disorders promote a decrease in neurogenesis. Nevertheless, NSPC-based therapy is, to date, not yet a satisfactory strategy due to poor survival and differentiation levels, as well as reduced synaptic plasticity either after transplantation or neural injury. Therefore, it is important to study the molecular and cellular mechanisms of NSPC's function in detail, and their potential therapeutic applicability to improve long-term survival, differentiation, and synaptic integration of derived newborn neural cells.