BDNF receptor cleavage:relevance for Alzheimer´s Disease pathophysiology

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Microglial Sirtuin 2 shapes long-term potentiation in hippocampal slices

Publication . Sa de Almeida, Joana; Vargas, Mariana; Fonseca-Gomes, João; Tanqueiro, Sara; Belo, Rita F.; Miranda-Lourenço, Catarina; Sebastião, Ana M; Diógenes, Maria José; Pais, Teresa F.

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.

2020Journal article

Open access

BDNF receptor cleavage : relevance for Alzheimer’s disease pathophysiology

Publication . Fonseca-Gomes, João Filipe; Nogueira, Maria José de Oliveira Diógenes

Alzheimer’s Disease (AD) is a chronic and progressive neurodegenerative disease, being the most common form of dementia worldwide (60-70% of all cases). AD is considered a protein misfolding disease and the accumulation of amyloid-beta (Aβ) peptide in the brain is considered one of the main hallmarks of this disease. Accumulatoin of this peptide induces a multiplicity of neurotoxic consequences, which ultimately supports the neuronal death, tightly associated with AD pathophysiology. In line with these alterations promoted by Aβ peptide also alterations in the signaling mediated by the brain-derived neurotrophic factor (BDNF) have been described. BDNF actions on neuronal survival, differentiation and plasticity are mediated by the activation of its full-length receptor (TrkB-FL). The impairment of BDNF signalling in AD might be explained by the decreased protein levels of BDNF and TrkB-FL, together with an increase of truncated TrkB isoforms (TrkB-Tc), which act as negative modulators of BDNF effects. In the last years, we have been establishing a direct relationship between Aβ peptide accumulation and TrkB-FL cleavage. Indeed, we observed, using different experimental approaches and several biological resources, that Aβ peptide, due to a dysfunctional activation of extrasynaptic N-methyl-D-aspartate receptor (eNMDAr), leads to an increase of intracellular calcium. As a consequence, calpains became overactivated and cleave TrkB-FL receptor, forming a new truncated receptor (TrkB-T’) and an intracellular fragment (TrkB-ICD), promoting the loss of BDNF signalling previously reported in the literature. Therefore, the Aβ-triggered cleavage of TrkB-FL receptor might be a strong contributor to neurodegeneration and to an overall loss of neuroprotection in AD patients. This project aimed i) to deeply assess TrkB-ICD fragmentimpact upon normal physiology, ii) to evaluate TrkB-FL cleavage in human post-mortem brain samples from AD patients, and iii) to develop and test a newly developed drug to prevent TrkB-FL cleavage. By the transfection of primary neurons, its was possible to investigate the role of TrkB-ICD. We found that TrkB-ICD i) is a stable fragment, ii) has tyrosine kinase activity and iii) accumulates in the nucleus, indicating a possible propagation of the initial Aβ toxicity. Furthermore, TrkB-ICD overexpression, by viral transduction of primary neuronal cultures with pFCK-CamKII-TrkB-ICD-IRESZsGreen induced an up-regulation of genes involved in neurotransmitter activity and in neural development, promoting in paralel a down-regulation of genes involved in chromosome structure stability and telomere organization, as analysed by whole trancriptome analysis (next generation sequencing). Remarkably, through in vivo intrahippocampal injection of the same viral particles in wild-type (WT) animals (C57BL/6, 12 weeks), we observed a disruption in recognition memory (Novel Object Recognition test) in TrkB-ICD-expressing animals. TrkB-ICD overexpression did not affect locomotion and anxiety-like behaviour. Intriguingly, we observed an increase in long-term potentiation magnitude, the molecular basis for learning and memory, recorded from hippocampal slices of TrkB-ICD-expressing-animals. This evidence is apparently not in line with the cognitive impairments detected in vivo. This discrepancy may indicate that TrkB-ICD promotes a dysfunctional increase in synaptic plasticity and could underlie, at least in part, the memory impairment well-described in AD patients. Regarding TrkB-FL cleavage validation in human brain samples, we used post-mortem inferior temporal cortical samples from human AD patients at different stages of disease severity. Results indicate that, over AD progression, TrkB-FL protein levels decreased, while TrkB-ICD protein levels increased. We must reinforce that this set of data is of an extreme importance, since it validated the enhancement of TrkB-FL cleavage in human patients diagnosed with AD. Finally, following 2D and 3D structural predictions, we designed a new compound to prevent TrkB-FL cleavage. Importantly, we validated the action of the new compound as a strategy that prevents TrkB-FL cleavage, through in vitro and ex vivo studies. Importantly, in addition to molecular studies, functional assays reinforced the potential therapeutic value of the new compound, since the prevention of TrkB-FL cleavage avoided the loss of BDNF function upon LTP and neurotransmitter release. Due to the promising collected data, this compound has a provisional patent application filled in UK. In summary, the data here presented i) indicates that TrkBICD might contribute or aggravate the Aβ toxicity, ii) validates the exacerbation of TrkB-FL cleavage in AD human brain samples, and iii) demonstrates a possible therapeutic value for our new created compound designed to prevent TrkB-FL cleavage. Interestingly, TrkB-FL cleavage was already described in other pathologies, including epilepsy, stroke and ischemia. Therefore, it should be indicated that the evidences described in this work might be transposed to other diseases where excitotoxicy plays a central role.

2020-09Doctoral thesis

Open access

Sustained hippocampal neural plasticity questions the reproducibility of an amyloid-β-induced Alzheimer’s disease model

Publication . Paulo, Sara L; Ribeiro Rodrigues, Leonor; Rodrigues, Rui S.; Mateus, Joana; Fonseca-Gomes, João; Soares, Rita; Diógenes, Maria José; Solá, Susana; Sebastião, Ana M; Ribeiro, Filipa; Xapelli, Sara

Background: The use of Alzheimer's disease (AD) models obtained by intracerebral infusion of amyloid-β (Aβ) has been increasingly reported in recent years. Nonetheless, these models may present important challenges. Objective: We have focused on canonical mechanisms of hippocampal-related neural plasticity to characterize a rat model obtained by an intracerebroventricular (icv) injection of soluble amyloid-β42 (Aβ42). Methods: Animal behavior was evaluated in the elevated plus maze, Y-Maze spontaneous or forced alternation, Morris water maze, and open field, starting 2 weeks post-Aβ42 infusion. Hippocampal neurogenesis was assessed 3 weeks after Aβ42 injection. Aβ deposition, tropomyosin receptor kinase B levels, and neuroinflammation were appraised at 3 and 14 days post-Aβ42 administration. Results: We found that immature neuronal dendritic morphology was abnormally enhanced, but proliferation and neuronal differentiation in the dentate gyrus was conserved one month after Aβ42 injection. Surprisingly, animal behavior did not reveal changes in cognitive performance nor in locomotor and anxious-related activity. Brain-derived neurotrophic factor related-signaling was also unchanged at 3 and 14 days post-Aβ icv injection. Likewise, astrocytic and microglial markers of neuroinflammation in the hippocampus were unaltered in these time points. Conclusion: Taken together, our data emphasize a high variability and lack of behavioral reproducibility associated with these Aβ injection-based models, as well as the need for its further optimization, aiming at addressing the gap between preclinical AD models and the human disorder.

2021Journal article

Restricted access

The neuroprotective action of amidated-kyotorphin on amyloid β peptide-induced Alzheimer’s disease pathophysiology

Publication . Belo, Rita F.; Martins, Margarida L. F.; Shvachiy, Liana; Costa-Coelho, Tiago; de Almeida-Borlido, Carolina; Fonseca-Gomes, João; Neves, Vera; Vicente Miranda, Hugo; Outeiro, Tiago F.; Coelho, Joana E; Xapelli, Sara; Valente, Cláudia A.; Heras, Montserrat; Bardaji, Eduard; Castanho, Miguel A. R. B.; Diógenes, Maria José; Sebastião, Ana M

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.

2020Journal article

Open access

S327 phosphorylation of the presynaptic protein SEPTIN5 increases in the early stages of neurofibrillary pathology and alters the functionality of SEPTIN5

Publication . Ferreira, Catarina B.; Marttinen, Mikael; Coelho, Joana E; Paldanius, Kaisa M.A.; Takalo, Mari; Mäkinen, Petra; Leppänen, Luukas; Miranda-Lourenço, Catarina; Fonseca-Gomes, João; Tanqueiro, Sara; Vaz, Sandra H.; Belo, Rita F.; Sebastião, Ana M; Leinonen, Ville; Soininen, Hilkka; Pike, Ian; Haapasalo, Annakaisa; Lopes, Luisa V.; De Mendonça, Alexandre; Diógenes, Maria José; Hiltunen, Mikko

Alzheimer's disease (AD) is the most common form of dementia, which is neuropathologically characterized by extracellular senile plaques containing amyloid-β and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. Previous studies have suggested a role for septin (SEPTIN) protein family members in AD-associated cellular processes. Here, we elucidated the potential role of presynaptic SEPTIN5 protein and its post-translational modifications in the molecular pathogenesis of AD. RNA and protein levels of SEPTIN5 showed a significant decrease in human temporal cortex in relation to the increasing degree of AD-related neurofibrillary pathology. Conversely, an increase in the phosphorylation of the functionally relevant SEPTIN5 phosphorylation site S327 was observed already in the early phases of AD-related neurofibrillary pathology, but not in the cerebrospinal fluid of individuals fulfilling the criteria for mild cognitive impairment due to AD. According to the mechanistic assessments, a link between SEPTIN5 S327 phosphorylation status and the effects of SEPTIN5 on amyloid precursor protein processing and markers of autophagy was discovered in mouse primary cortical neurons transduced with lentiviral constructs encoding wild type SEPTIN5 or SEPTIN5 phosphomutants (S327A and S327D). C57BL/6 J mice intrahippocampally injected with lentiviral wild type SEPTIN5 or phosphomutant constructs did not show changes in cognitive performance after five to six weeks from the start of injections. However, SEPTIN5 S327 phosphorylation status was linked to changes in short-term synaptic plasticity ex vivo at the CA3-CA1 synapse. Collectively, these data suggest that SEPTIN5 and its S327 phosphorylation status play a pivotal role in several cellular processes relevant for AD.

2022Journal article

Open access