Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia-reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO-donor, Angeli's salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 mumol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro, neuronal damage induced by oxygen-glucose-deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity - that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia-reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo.

The Gamma-Secretase-Mediated Proteolytic Processing of APP C-Terminal Fragments as a Therapeutic Target for Alzheimer's Disease

Jemila Houacine

Alzheimer's disease (AD) is a devastating neurodegenerative disorder which severely impairs cognitive functions by triggering neuronal cell death and synaptic loss, and finally leads the patients to death. Two main histopathological hallmarks can be found in the brain tissue of AD patients: the amyloid plaques composed of aggregated Amyloid-β peptides (Aβ) and the neurofibrillary tangles (NFTs) constituted of hyperphosphorylated tau protein. To explain the underlying molecular mechanisms of AD pathogenesis, the amyloid cascade hypothesis states that early accumulation of Aβ peptides (especially the Aβ42 specie identified as the most toxic one) triggers their assembly into oligomers and their subsequent extracellular aggregation into dense fibrillar deposits. The oligomeric Aβ assemblies further initiate a succession of neurotoxic events including disruptions at the synaptic level, inflammation, neuritic injury, altered calcium homeostasis, oxidative stress, and altered phosphorylation activity leading to the hyperphosphorylation of tau and its aggregation into NFTs. Finally, this cascade of neuronal deleterious effects induces cognitive dysfuntions leading to dementia. The Aβ peptides originate from the amyloidogenic processing of the amyloid precursor protein (APP), a type I membrane protein that undergoes a first cleavage by β-secretase to liberate the soluble APP domain (sAPPβ) in the extracellular space, and the membrane bound APP-C99 fragment. Then, APP-C99 is processed by the intramembrane aspartyl-protease gamma-secretase (γ-secretase) composed of four subunits (presenilin (PS), nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2)) to release the toxic Aβ peptides in the lumen, and the APP intracellular domain (AICD) in the cytosol. Alternatively, APP can undergo the non-amyloidogenic processing, in which it is first shedded by α-secretase to generate the sAPPα domain and the APP-C83 fragment. The latter is further cleaved by γ-secretase into the non toxic p3 peptide and the AICD. AD is the most frequent form of dementia in elderly humans. Despite this evidence, no treatment is currently available to prevent or cure this disease. Thus, multiple potential drugs are tested in clinical trials or are still being developed in preclinical studies. In this work, a new therapeutic approach to lower Aβ production by specifically targeting the γ-secretase-mediated processing of APP-C99 is presented. Monoclonal antibodies against APP-C99 were generated and characterized following mice immunization with the active recombinant substrate APP-C99. When tested in cells, these antibodies decreased the γ-secretase-dependent processing of APP-C99, thus lowering Aβ production. Furthermore, the intracerebroventricular injection of anti-APP-C99 antibodies in a mouse model of AD decreased total soluble Aβ levels. These results validated this new approach as a potential immunotherapeutic strategy to prevent and/or delay the neurotoxic effects caused by Aβ in AD pathogenesis. Next, the γ-secretase-mediated processing of the APP-carboxy-terminal fragments (APP-CTFs) regulating the toxic versus non-toxic pathways was further investigated with the help of in vitro activity assays. This comparative study of APP-C99 and APP-C83 processing by γ-secretase revealed that both substrates were identically processed by the enzyme, with the same AICDs being produced. In addition, the effects of familial AD (FAD) mutations in the PS subunit of highly pure and homogeneous γ-secretase complexes were also investigated, and showed a drastic loss-of-function phenotype linked to these mutants. Overall, in vitro studies of the APP-CTFs processing by γ-secretase underlined particular functional aspects of this processing, which could be related to the cellular pathways involved in AD pathogenesis such as the competition between the amyloidogenic and non-amyloidogenic pathways and the FAD-linked impaired metabolism of APP.

EPFL2011

Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Julie Laetitia Scotton

Malignant gliomas represent 80% of tumors developing in the central nervous system, with 50% being glioblastomas (GBM), the most aggressive form of the disease. The benefit from current therapies is very limited, the overall 5-year survival rate of patients with GBM being less than 5%. Thus, there is an urgent need for better therapies. It has been reported that the use of tricyclic antidepressants (TCAs) is linked with reduced incidence of gliomas. In line with these observations, our lab previously demonstrated that combining imipramine (IM), a TCA that activates adenylate cyclase and elevates production of cAMP, with ticlopidine (TIC), an inhibitor of the purinergic receptor P2Y12 that otherwise suppresses cAMP, results in a concerted increase in the levels of intracellular cAMP, which consequently stimulates increased autophagy in glioma cells, leading to autophagy-associated cell death (AACD) in culture and during orthotopic tumor growth. In several mouse models of glioma, the combination improved survival and reduced malignant grade. Seeking to assess the therapeutic translational potential of these repurposed drugs, particularly IM, we performed pre-clinical trials combining IM+/-TIC with anti-VEGF therapy, which is commonly used in patients with GBM; such anti-angiogenic therapy is associated with a transitory improvement in progression-free survival and quality of life, but does not translate into an overall survival benefit. Using genetic mouse models with enabling bioluminescent monitoring of the cancer cells, we treated established GBMs with imipramine, ticlopidine, and B20S, an anti-mouse VEGF monoclonal antibody. We observed transient stabilization of tumor growth, and the overall survival was significantly extended in the regimens combining B20S with IM +/- TIC compared to control and single treatments. Similar to clinical observations in GBM patients treated with anti-angiogenic therapy, mice treated with B20S alone had no survival benefit, implicating a synergetic effect for the combination of anti-VEGF treatment with imipramine and ticlopidine. We found that the combinatorial therapy elicited further increases in autophagy and displayed some indication of blood vessel normalization, concomitant with an elevation of CD8+T lymphocyte infiltration into the orthotopic tumors. Notably, we could partially reverse the beneficial effect of the combinations using a CD8+T cell-depleting antibody, revealing the involvement of the recruited CD8+T cells in the efficacy of the combinatorial treatment, in addition to the cell-intrinsic autophagy-associated death that is demonstrable in culture and in tumors. Together, these results suggest a strategy to improve anti-angiogenic therapy in patients with glioblastoma, by using repurposed FDA-approved drugs that markedly elevate autophagy in concert with anti-VEGF therapy. The recruitment of CD8+ T cells into the treated tumors and their evident contribution to therapeutic efficacy motivates ongoing experiments aimed to heighten and sustain the induced anti-tumor immune response.

EPFL2018

Lentiviral Transgenesis as a Tool to Study the Role of Uncoupling Protein Isoforms in the Nervous System

Hélène Danielle Perreten Lambert

A tight coupling exists between synaptic activity and glucose utilization by astrocytes. Metabolic cooperation between neurons and astrocytes mediates this coupling. During synaptic activation, glutamate that is released in the synaptic cleft as a neurotransmitter by neurons is rapidly cleared by an active uptake into astrocytes. One glutamate is co-transported with three Na+. Intracellular astrocytic Na+ homeostasis is re-established by the Na+/K+-ATPase which requires ATP provided mainly by glycolysis. The resulting lactate is released in extracellular space and contributes to the energetic balance of activated neurons. This coupling between astrocytes and neurons is known as the astrocyte-neuron lactate shuttle hypothesis (ANLSH). The role of mitochondria in this coupling remains to be determined and more specifically the role of uncoupling proteins (UCP) which have been recently identified in neural tissues. UCPs belong to a family of mitochondrial carriers which are present in the inner mitochondrial membrane. There are five isoforms named UCP1 to UCP5. The well-known characterized is UCP1, also named thermogenin, which is present in the brown adipose tissue (BAT) of the small rodents and is responsible of non-shivering thermogenesis. It dissipates the proton gradient across the inner mitochondrial membrane, thereby producing heat instead of ATP. UCP2 is ubiquitous and is implicated in protection against excessive reactive oxygen (ROS) production. The expression of UCP3 is restricted to skeletal muscle where it is linked to fatty acid metabolism. The role of the brain isoforms UCP4 and UCP5 is still poorly understood. Initially, we set out to evaluate their function in energy metabolism of astrocytes and neurons. We used the lentiviral strategy to overexpress or silence the UCPs isoforms in primary cultures of astrocytes and neurons. We found that only UCP4 and UCP5 had an uncoupling activity in brain cells. Indeed, we observed a reduced mitochondrial potential and a reduced ATP/ADP ratio in astrocytes as well as in neurons overexpressing UCP4 or UCP5. UCP4 reduced the oxidative pathway of astrocytes without modifying the basal glucose metabolism and without having a lethal effect on the cell. Oxygen consumption rate (OCR) of brain cells ovexpressing UCPs was reduced. Moreover, UCP4 increased lactate release by astrocytes, suggesting an implication in the astrocyte-neuron coupling. We also brought evidence that both UCP4 and UCP5 partially protect brain cells from oxidative damage. All these results were confirmed by experiments on UCPs silencing. In a second step, we used a co-culture model to study the impact of astrocytic uncoupling protein on neuronal survival and we demonstrated that the presence of uncoupling protein in astrocytes prevents neuronal death after glutamate excitotoxicity. Taken together, all these results support an important role of uncoupling proteins in the regulation of astrocytic energy production, thus promoting local export of lactate which can be used by neurons.

EPFL2011

Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Julie Laetitia Scotton

EPFL2018

Lentiviral Transgenesis as a Tool to Study the Role of Uncoupling Protein Isoforms in the Nervous System

Hélène Danielle Perreten Lambert

EPFL2011

The Gamma-Secretase-Mediated Proteolytic Processing of APP C-Terminal Fragments as a Therapeutic Target for Alzheimer's Disease

Jemila Houacine

EPFL2011