Mechanisms underlying the physiological role of amyloid precursor protein glutamatergic synapses

N-methyl-D-aspartate receptors (NMDARs) are glutamatergic ionotropic receptors essential for synaptic maturation during development and synaptic plasticity in adult stages, whose properties are known to change depending on the life stage. In particular, the subunit composition of synaptic NMDARs has important implications for NMDAR function. In the hippocampus, NMDARs are mainly composed by GluN2A or GluN2B subunits, with GluN2B-NMDARs being associated with slower kinetics, more calcium charge transfer and higher mobility. GluN2B-NMDARs are predominant in immature synapses during development, contributing to the synaptic maturation process, which involves a GluN2B to GluN2A shift. Thus, GluN2A-NMDARs are the most abundant subtype in adult stages, when most synapses are in the mature state. Much less is known about the subunit contribution in aged synapses. However, previous reports showed age-related alterations in NMDAR properties, such as slower responses, lower current amplitudes and a negative correlation between GluN2B levels and memory performance. These alterations in NMDAR properties, from development to aging, might be caused by different regulation mechanisms. The amyloid precursor protein (APP), which is mainly known to be involved in Alzheimer’s Disease, has emerged as a putative regulator of NMDARs. Although the physiological role of APP is not fully understood, it is known to regulate synaptogenesis and synaptic plasticity and might have different effects when acting through the full-length protein or its derived fragments. Additionally, APP has shown to interact and regulate NMDAR surface levels and currents but the functional relevance of this interaction at different life stages, as well as the underlying mechanisms of regulation have not been explored so far. Thus, we hypothesized that APP regulates NMDARs in an age-dependent manner and defined as the main aims of this work to study APP-NMDAR regulation mechanisms in immature, mature and aged synapses. To address these questions in physiological conditions, we used as our experimental models the hippocampus of wild-type C57Bl/6 mice at different life stages (infant (7-10 days), adults (10-16 weeks) and aged (18 – 20 months), as well as postmortem brain tissue from human subjects with different ages (18-89 years old) and rodent hippocampal primary neuronal cultures. By combining patch-clamp electrophysiology and molecular approaches, we have unraveled a dual mechanism by which APP controls GluN2B-NMDARs, depending on the life stage. In the present study, we show that APP is highly abundant at the post synapse in infant mice, where it interacts with GluN2B-NMDARs, controlling its mediated currents. Moreover, APP knockdown in primary neuronal cultures caused a reduction in GluN2B-NMDAR synaptic content, suggesting that APP might be important to stabilize the receptors at the synapse. Considering the crucial role of GluN2B-NMDAR in synapse maturation, this mechanism might potentially be important to achieve functional, mature synapses during development. Although this interaction is maintained in adult/aged synapses, NMDAR-mediated currents showed to be unaltered when interfering with the APP C-terminal during a short period at these ages, contrary to the results obtained in infant mice. Thus, we concluded that the APP-NMDAR regulatory mechanisms are different in adult/aged mice when compared to infants. We hypothesize that alterations in the APP-NMDAR regulation could be the underlying mechanism for age-related alterations in NMDAR properties. Accordingly, we found that aged mice exhibit an increase in GluN2B-NMDAR relative currents, which does not correlate with alterations in subunit levels. Moreover, we found an increase in APP processing into intracellular fragments upon aging. Importantly, when we inhibited APP processing or interfered with APP intracellular signaling in aged mice, we were able to normalize GluN2B-NMDAR synaptic contribution to adult-like levels. Thus, we propose that signaling pathways mediated by APP intracellular fragments induce an increase in GluN2B-NMDAR relative currents upon aging. Additionally, we show that APP processing into intracellular fragments also tends to increase in aged humans, suggesting that a similar mechanism might occur in mice and humans. Considering the impact of NMDAR on synaptic plasticity, this increase in GluN2B-NMDAR relative currents can potentially contribute to age-related synaptic and memory impairments.Santa Casa da Misericórdia (MB-7-2018

Non-invasive viral-mediated gene therapy for Machado-Joseph disease

SARAIVA, Joana Isabel Rajão - Non-invasive viral-mediated gene therapy for Machado-Joseph disease. Coimbra : [s.n.], 2017. Dissertação de MestradoMachado-Joseph disease (MJD) is a dominant autosomal neurodegenerative disorder characterized by cerebellar dysfunction and loss of motor coordination. This disorder, which corresponds to the most common type of spinocerebellar ataxia worldwide, is caused by a CAG expansion in the coding region of the MJD1/ATXN3 gene. This mutation is translated into a toxic polyglutamine tract within ataxin-3, which triggers multiple pathogenic mechanisms, ultimately leading to neurodegeneration in several brain regions. The lack of available treatment for MJD encourages further investigation towards possible therapeutic approaches. One of the most direct, specific and effective solutions to correct MJD would be to inhibit mutant ataxin-3 expression using RNA interference (RNAi), thus targeting the initial cause of the disorder. Several studies have already investigated the impact of mutant ataxin-3 silencing in MJD rodent models, through direct administration of viral vectors into the brain parenchyma. This invasive procedure is associated with potential adverse effects and a limited vector distribution in the brain. As a result, a possible translation to human patients would benefit from a non-invasive delivery system, capable of inducing a widespread therapeutic effect throughout the CNS. Therefore, the main goal of this project was to develop a noninvasive viral-based gene therapy for MJD. For that purpose, we selected rAAV9 (recombinant adeno-associated virus serotype 9) as the RNAi delivery vector, since this serotype is able to cross the blood-brain barrier (BBB) and efficiently transduce neurons. Moreover, this viral vector mediates a long-term transgene expression and exhibits a good safety profile, being particularly suitable for CNS gene therapy. Taking all of this into account, we developed an AAV9mediated system encoding an artificial microRNA against mutant ataxin-3 (rAAV9-miR-ATXN3) and test its therapeutic impact in a transgenic mouse model of MJD. Our first task consisted on the validation of this artificial miR sequence in a neuronal cell line. We concluded that miR-ATXN3 mediates an efficient and allele-specific silencing of mutant ataxin-3, providing confidence to studies in vivo. Next, we analyzed the therapeutic potential of rAAV9-miR-ATXN3 vectors in a severely-impaired MJD transgenic mouse model following intravenous (IV) injection at postnatal day 1. Importantly, viral vectors were found to efficiently cross the BBB and transduce neurons throughout the brain of transgenic mice. Moreover, rAAV9 transduction was detected in Purkinje cells, the neuronal population that expresses mutant ataxin-3 in this particular model. Subsequently, we assessed the effects of rAAV9-miR-ATXN3 administration in the behavioral performance of MJD transgenic mice. Noteworthy, this treatment successfully alleviated gait, balance and motor coordination impairments. Finally, we also observed a significant amelioration of neuropathological changes in the cerebellum. Accordingly, rAAV9-miR-ATXN3 treated animals exhibited a reduction in the number of mutant ataxin-3 aggregates, as well as a preservation of molecular layer thickness. Altogether, our results indicate that mutant ataxin-3 silencing through a single AAV9 intravenous injection is an efficient therapeutic approach, alleviating both behavioral and neuropathological impairments. Importantly, our work constitutes the first report of long-term ataxin-3 silencing through a non-invasive viral system, supporting the use of this strategy for MJD therapy.A doença de Machado-Joseph (DMJ) é uma doença neurodegenerativa autossómica dominante, caracterizada por disfunção cerebelar e perda de coordenação motora. Esta doença corresponde ao tipo mais comum de ataxia espinocerebelosa a nível mundial e deve-se a uma expansão do número de repetições CAG na região codificante do gene MJD1/ATXN3. Esta mutação traduz-se numa longa cadeia de poliglutaminas na proteína ataxina-3, o que induz diversos mecanismos patogénicos, causando morte neuronal em várias regiões cerebrais. Neste momento não existe nenhuma terapia disponível para a DMJ. A inibição da expressão da ataxina-3 mutante usando RNA de interferência é uma potencial estratégia terapêutica capaz de corrigir a causa inicial da doença, de uma forma directa, específica e eficaz. Vários estudos investigaram anteriormente o impacto desta estratégia de silenciamento em modelos animais da DMJ, com resultados promissores. No entanto, estas experiências envolveram a administração de vectores virais através de uma injecção intracraniana, ou seja, um procedimento invasivo e associado a efeitos secundários. Além disso, este tipo de administração leva a uma dispersão reduzida dos vectores virais no cérebro. Consequentemente, a opção ideal para facilitar futuras aplicações clínicas seria uma via de administração não invasiva, capaz de induzir um efeito terapêutico em todo o Sistema Nervoso Central. Assim sendo, este projecto teve como principal objectivo desenvolver uma estratégia viral não invasiva capaz de induzir o silenciamento da ataxina-3 mutante, como possível terapia para a DMJ. Para atingir este objectivo, seleccionámos o AAV9 (virus adeno-associado do tipo sérico 9) como vector de entrega da sequência de silenciamento, visto que tem a capacidade de atravessar a barreira hematoencefálica e transduzir neurónios de forma eficaz. Além disso, este vírus induz a expressão dos transgenes durante largos períodos de tempo, sem induzir toxicidade. Neste sentido, desenvolvemos um sistema baseado no AAV9 capaz de codificar um microRNA artificial específico para a ataxina-3 mutante (rAAV9miR-ATXN3) e testámos o seu efeito terapêutico num modelo transgénico da DMJ. Em primeiro lugar, validámos a sequência miR-ATXN3 numa linha celular neuronal, sendo que verificámos um silenciamento eficaz e específico para a forma mutante da ataxina-3. Estes resultados permitiram prosseguir para estudos num modelo animal da doença. Neste contexto, testámos o efeito terapêutico dos vectores rAAV9-miR-ATXN3 em murganhos transgénicos, que receberam uma injeção intravenosa 1 dia após o nascimento. Ao analisar a distribuição do vector, concluímos que ocorreu uma transdução neuronal eficaz em várias regiões do cérebro. De seguida, focámo-nos no cerebelo e em particular nas células de Purkinje, visto que apenas esta população neuronal expressa a ataxina-3 mutante neste modelo. As células de Purkinje mostraram níveis significativos de transdução, indicando que o vector consegue aceder ao nosso principal alvo terapêutico. Consequentemente, avaliámos o efeito desta estratégia no comportamento dos murganhos tratados, sendo que observámos melhorias na coordenação motora e equilíbrio. Além disso, o tratamento permitiu atenuar a neuropatologia no cerebelo, nomeadamente ao reduzir o número de agregados e levando a uma preservação da espessura da camada molecular. Em conclusão, os resultados obtidos após uma única injeção intravenosa de vectores AAV9 indicam que esta é uma estratégia terapêutica eficaz, capaz de atenuar os problemas motores, bem como a neuropatologia. É importante salientar que este foi o primeiro estudo em que se obteve um silenciamento da ataxina-3 mutante a longo prazo e através de uma administração não invasiva, constuindo por isso uma estratégia muito promissora para a terapia da DMJ

The Amyloid Precursor Protein C-Terminal Domain Alters CA1 Neuron Firing, Modifying Hippocampus Oscillations and Impairing Spatial Memory Encoding

International audienc

The consequences of the new European reclassification of non-invasive brain stimulation devices and the medical device regulations pose an existential threat to research and treatment: An invited opinion paper

A significant amount of European basic and clinical neuroscience research includes the use of transcranial magnetic stimulation (TMS) and low intensity transcranial electrical stimulation (tES), mainly transcranial direct current stimulation (tDCS). Two recent changes in the EU regulations, the introduction of the Medical Device Regulation (MDR) (2017/745) and the Annex XVI have caused significant problems and confusions in the brain stimulation field. The negative consequences of the MDR for non-invasive brain stimulation (NIBS) have been largely overlooked and until today, have not been consequently addressed by National Competent Authorities, local ethical committees, politicians and by the scientific communities. In addition, a rushed bureaucratic decision led to seemingly wrong classification of NIBS products without an intended medical purpose into the same risk group III as invasive stimulators. Overregulation is detrimental for any research and for future developments, therefore researchers, clinicians, industry, patient representatives and an ethicist were invited to contribute to this document with the aim of starting a constructive dialogue and enacting positive changes in the regulatory environment

The consequences of the new European reclassification of non-invasive brain stimulation devices and the medical device regulations pose an existential threat to research and treatment: An invited opinion paper

A significant amount of European basic and clinical neuroscience research includes the use of transcranial magnetic stimulation (TMS) and low intensity transcranial electrical stimulation (tES), mainly transcranial direct current stimulation (tDCS). Two recent changes in the EU regulations, the introduction of the Medical Device Regulation (MDR) (2017/745) and the Annex XVI have caused significant problems and confusions in the brain stimulation field. The negative consequences of the MDR for non-invasive brain stimulation (NIBS) have been largely overlooked and until today, have not been consequently addressed by National Competent Authorities, local ethical committees, politicians and by the scientific communities. In addition, a rushed bureaucratic decision led to seemingly wrong classification of NIBS products without an intended medical purpose into the same risk group III as invasive stimulators. Overregulation is detrimental for any research and for future developments, therefore researchers, clinicians, industry, patient representatives and an ethicist were invited to contribute to this document with the aim of starting a constructive dialogue and enacting positive changes in the regulatory environment. [Abstract copyright: Copyright © 2024 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.