Miguel Díaz-Hernández

Altered P2X7-receptor level and function in mouse models of Huntington’s disease and therapeutic efficacy of antagonist administration

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntingtons disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP‐gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7‐mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7‐receptor level and altered P2X7‐mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7‐receptor stimulation. Finally, in vivo administration of the P2X7‐antagonist Brilliant Blue‐G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor‐coordination deficits. These in vivo data strongly suggest that altered P2X7‐receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.—Diaz‐Hernandez, M., Diez‐Zaera, M., Sanchez‐Nogueiro, J., Gomez‐Villafuertes, R., Canals, J.M., Alberch, J., Miras‐Portugal, M.T., Lucas, J.J. Altered P2X7‐receptor level and function in mouse models of Huntingtons disease and therapeutic efficacy of antagonist administration. FASEB J. 23, 1893–1906 (2009)

Extracellular tau is toxic to neuronal cells

The degeneration of neurons in disorders such as Alzheimers disease has an immediate consequence, the release of intracellular proteins into the extracellular space. One of these proteins, tau, has proven to be toxic when added to cultured neuronal cells. This toxicity varies according to the degree of protein aggregation. The addition of tau to cultured neuroblastoma cells provoked an increase in the levels of intracellular calcium, which is followed by cell death. We suggest that this phenomenon may be mediated by the interaction of tau with muscarinic receptors, which promotes the liberation of calcium from intracellular stores.

Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntingtons disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntingtons disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntingtons disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntingtons disease, and suggest that activation of these receptors in patients with Huntingtons disease may attenuate disease progression.

Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells.

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice

Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ‐amino butyric acid‐mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligandgated P2X receptors. Here we report that SE induced by intra‐amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure‐induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure‐suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure‐responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.—Engel, T., Gomez‐Villafuertes, R., Tanaka, K., Mesuret, G., Sanz‐Rodriguez, A., Garcia‐Huerta, P., Miras‐Portugal, M. T., Henshall, D. C., Diaz‐Hernandez, M. Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice. FASEB J. 26, 1616‐1628 (2012). www.fasebj.org

Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain

Deficits of neurotrophic support caused by reduced levels of brain‐derived neurotrophic factor (BDNF) have been implicated in the selective vulnerability of striatal neurones in Huntingtons disease (HD). Therapeutic strategies based on BDNF administration have been proposed to slow or prevent the disease progression. However, the effectiveness of BDNF may depend on the proper expression of its receptor TrkB. In this study, we analysed the expression of TrkB in several HD models and in postmortem HD brains. We found a specific reduction of TrkB receptors in transgenic exon‐1 and full‐length knock‐in HD mouse models and also in the motor cortex and caudate nucleus of HD brains. Our findings also demonstrated that continuous expression of mutant huntingtin is required to down‐regulate TrkB levels. This was shown by findings in an inducible HD mouse model showing rescue of TrkB by turning off mutant huntingtin expression. Interestingly, the length of the polyglutamine tract in huntingtin appears to modulate the reduction of TrkB. Finally, to analyse the effect of BDNF in TrkB we compared TrkB expression in mutant huntingtin R6/1 and double mutant (R6/1 : BDNF+/–) mice. Similar TrkB expression was found in both transgenic mice suggesting that reduced TrkB is not a direct consequence of decreased BDNF. Therefore, taken together our findings identify TrkB as an additional component that potentially might contribute to the altered neurotrophic support in HD.

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca2+]i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X3 subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg2+-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X7 receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca2+]i measurements in single synaptic terminals. Additionally, the P2X7 receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.

Tissue-nonspecific Alkaline Phosphatase Promotes the Neurotoxicity Effect of Extracellular Tau

There is solid evidence indicating that hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients, plays a key role in progression of this disease. However, it has been recently reported that extracellular unmodified tau protein may also induce a neurotoxic effect on hippocampal neurons by activation of M1 and M3 muscarinic receptors. In the present work we show an essential component that links both effects, which is tissue-nonspecific alkaline phosphatase (TNAP). This enzyme is abundant in the central nervous system and is mainly required to keep control of extracellular levels of phosphorylated compounds. TNAP dephosphorylates the hyperphosphorylated tau protein once it is released upon neuronal death. Only the dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase finally triggering neuronal death. Interestingly, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP in SH-SY5Y neuroblastoma cells. An increase in TNAP activity together with increases in protein and transcript levels were detected in Alzheimer disease patients when they were compared with healthy controls.

Acute Polyglutamine Expression in Inducible Mouse Model Unravels Ubiquitin/Proteasome System Impairment and Permanent Recovery Attributable to Aggregate Formation

The presence of intracellular ubiquitylated inclusions in neurodegenerative disorders and the role of the ubiquitin/proteasome system (UPS) in degrading abnormal hazardous proteins have given rise to the hypothesis that UPS-impairment underlies neurodegenerative processes. However, this remains controversial for polyglutamine disorders such as Huntington disease (HD). Whereas studies in cellular models have provided evidence in favor of UPS-impairment attributable to expression of the N-terminal fragment of mutant huntingtin (N-mutHtt), similar studies on mouse models failed to do so. Furthermore, we have recently shown that the increase in polyubiquitin conjugates reported in the brain of N-mutHtt mice occurs in the absence of a general UPS-impairment. In the present study we aim to clarify the potential of N-mutHtt to impair UPS function in vivo as well as the mechanisms by which neurons may adapt after prolonged exposure to N-mutHtt in genetic models. By combining UPS reporter mice with an inducible mouse model of HD, we demonstrate for the first time polyglutamine-induced global UPS-impairment in vivo. UPS-impairment occurred transiently after acute N-mutHtt expression and restoration correlated with appearance of inclusion bodies (IBs). Consistently, UPS recovery did not take place when IB formation was prevented through administration of N-mutHtt aggregation-inhibitors in both cellular and animal models. Finally, no UPS-impairment was detected in old mice constitutively expressing N-mutHtt despite the age-associated decrease in brain proteasome activity. Therefore, our data reconcile previous contradictory reports by showing that N-mutHtt can indeed impair UPS function in vivo and that N-mutHtt aggregation leads to long lasting restoration of UPS function.

In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3β and secretases

β-Amyloid (Aβ) peptide production from amyloid precursor protein (APP) is essential in the formation of the β-amyloid plaques characteristic of Alzheimers disease. However, the extracellular signals that maintain the balance between nonpathogenic and pathologic forms of APP processing, mediated by α-secretase and β-secretase respectively, remain poorly understood. In the present work, we describe regulation of the processing of APP via the adenosine triphosphate (ATP) receptor P2X7R. In 2 different cellular lines, the inhibition of either native or overexpressed P2X7R increased α-secretase activity through inhibition of glycogen synthase kinase 3 (GSK-3). In vivo inhibition of the P2X7R in J20 mice, transgenic for mutant human APP, induced a significant decrease in the number of hippocampal amyloid plaques. This reduction correlated with a decrease in glycogen synthase kinase 3 activity in J20 mice, increasing the proteolytic processing of APP through an increase in α-secretase activity. The in vivo findings presented here demonstrate for the first time the therapeutic potential of P2X7R antagonism in the treatment of familiar Alzheimers disease (FAD).