Juan-Alberto Arranz-Tagarro

Stabilizers of neuronal and mitochondrial calcium cycling as a strategy for developing a medicine for Alzheimer's disease.

For the last two decades, most efforts on new drug development to treat Alzheimers disease have been focused to inhibit the synthesis of amyloid beta (Aβ), to prevent Aβ deposition, or to clear up Aβ plaques from the brain of Alzheimers disease (AD) patients. Other pathogenic mechanisms such as the hyperphosphorylation of the microtubular tau protein (that forms neurofibrillary tangles) have also been addressed as, for instance, with inhibitors of the enzyme glycogen synthase-3 kinase beta (GSK3β). However, in spite of their proven efficacy in animal models of AD, all these compounds have so far failed in clinical trials done in AD patients. It seems therefore desirable to explore new concepts and strategies in the field of drug development for AD. We analyze here our hypothesis that a trifunctional chemical entity acting on the L subtype of voltage-dependent Ca(2+) channels (VDCCs) and on the mitochondrial Na(+)/Ca(2+) exchanger (MNCX), and having additional antioxidant properties, may efficiently delay or stop the death of vulnerable neurons in the brain of AD patients. In recent years, evidence has accumulated indicating that enhanced neuronal Ca(2+) cycling (NCC) and futile mitochondrial Ca(2+) cycling (MCC) are central stage in activating calpain and calcineurin, as well as the intrinsic mitochondrial pathway for apoptosis, leading to death of vulnerable neurons. An additional contributing factor to neuronal death is the excess free radical production linked to distortion of Ca(2+) homeostasis. We propose that an hybrid compound containing a dihydropyridine moiety (to block L channels and mitigate Ca(2+) entry) and a benzothiazepine moiety (to block the MNCX and slow down the rate of Ca(2+) efflux from the mitochondrial matrix into the cytosol), as well as a polyphenol moiety (to sequester excess free radicals) could break down the pathological enhanced NCC and MCC, thus delaying the initiation of apoptosis and the death of vulnerable neurons. In so doing, such a trifunctional compound could eventually become a neuroprotective medicine capable of delaying disease progression in AD patients.

The Neuroprotection Exerted by Memantine, Minocycline and Lithium, against Neurotoxicity of CSF from Patients with Amyotrophic Lateral Sclerosis, Is Antagonized by Riluzole

In a recent study we found that cerebrospinal fluids (CSFs) from amyotrophic lateral sclerosis (ALS) patients caused 20-30% loss of cell viability in primary cultures of rat embryo motor cortex neurons. We also found that the antioxidant resveratrol protected against such damaging effects and that, surprisingly, riluzole antagonized its protecting effects. Here we have extended this study to the interactions of riluzole with 3 other recognized neuroprotective agents, namely memantine, minocycline and lithium. We found: (1) by itself riluzole exerted neurotoxic effects at concentrations of 3-30 µM; this cell damage was similar to that elicited by 30 µM glutamate and a 10% dilution of ALS/CSF; (2) memantine (0.1-30 µM), minocycline (0.03-1 µM) and lithium (1-80 µg/ml) afforded 10-30% protection against ALS/CSF-elicited neurotoxicity, and (3) at 1-10 µM, riluzole antagonized the protection afforded by the 3 agents. These results strongly support the view that at the riluzole concentrations reached in the brain of patients, the neurotoxic effects of this drug could be masking the potential neuroprotective actions of new compounds being tested in clinical trials. Therefore, in the light of the present results, the inclusion of a group of patients free of riluzole treatment may be mandatory in future clinical trials performed in ALS patients with novel neuroprotective compounds.

Hypoxia-elicited catecholamine release is controlled by L-type as well as N/PQ types of calcium channels in rat embryo chromaffin cells

At early life, the adrenal chromaffin cells respond with a catecholamine surge under hypoxic conditions. This response depends on Ca(2+) entry through voltage-activated calcium channels (VACCs). We have investigated here three unresolved questions that concern this response in rat embryo chromaffin cells (ECCs): 1) the relative contribution of L (α1D, Cav1.3), N (α1B, Cav2.2), and PQ (α1A, Cav2.1) to the whole cell Ca(2+) current (ICa); 2) the relative contribution of L and N/PQ channels to the cytosolic Ca(2+) elevations triggered by hypoxia (Δ[Ca(2+)]c); and 3) the role of L and non-L high-VACCs in the regulation of the catecholamine surge occurring during prolonged (1 min) hypoxia exposure of ECCs. Nimodipine halved peak ICa and blocked 60% the total Ca(2+) entry during a 50-ms depolarizing pulse to 0 mV (QCa). Combined ω-agatoxin IVA plus ω-conotoxin GVIA (Aga/GVIA) blocked 30% of both ICa peak and QCa. This relative proportion of L- and non-L VACCs was corroborated by Western blot that indicated 55, 23, and 25% relative expression of L, N, and PQ VACCs. Exposure of ECCs to hypoxia elicited a mild but sustained Δ[Ca(2+)]c; the area of Δ[Ca(2+)]c was blocked 50% by nifedipine and 10% by Aga/GVIA. Exposure of ECCs to 1-min hypoxia elicited an initial transient burst of amperometric secretory spikes followed by scattered spikes along the time of cell exposure to hypoxia. This bulk response was blocked 85% by nimodipine and 35% by Aga/GVIA. Histograms on secretory spike frequency vs. time indicated a faster initial inactivation when Ca(2+) entry took place through N/PQ channels; more sustained secretion but at a lower rate was associated to Ca(2+) entry through L channels. The results suggest that the HIS response may initially be controlled by L and P/Q channels, but later on, N/PQ channels inactivate and the delayed HIS response is maintained at lower rate by slow-inactivating L channels.

Recent patents on calcium channel blockers: emphasis on CNS diseases

Introduction: Altered homeostasis of cell calcium movement is a central stage in multiple diseases of CNS. This explains the great therapeutic interest in blockers for the various subtypes of voltage-activated calcium channels (VACCs) expressed in neurons. Mitigation of Ca2+ entry excess elicited by those blockers may restore the altered synaptic transmission, synaptic plasticity and gene expression to normal parameters, ending the enhanced neuronal vulnerability. Areas covered: This review summarize 23 patents on ligands for L-, N- or T-type channels, claimed to have potential therapeutic interest in epilepsy, pain, migraine and neurodegenerative diseases. Expert opinion: Collections of compounds are generally screened in cell lines expressing a given subtype of VACCs. IC50 to block such channels are often, but not always, provided. In few instances, compounds exhibiting the highest potency in in vitro experiments are also tested in animal models of pain, behavior, epilepsy or Alzheimer’s disease. Attempts to develop selectivity for a given VACC subtype with non-peptidic organic ligands have so far failed. Due to their wide tissue expression, such selectivity is crucial for minimizing possible side effects. However, the few data reported by patents does not allow prediction of selectivity of the new compounds in many cases.

Plasmalemmal sodium-calcium exchanger shapes the calcium and exocytotic signals of chromaffin cells at physiological temperature.

The activity of the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) is highly sensitive to temperature. We took advantage of this fact to explore here the effects of the NCX blocker KB-R7943 (KBR) at 22 and 37°C on the kinetics of Ca(2+) currents (ICa), cytosolic Ca(2+) ([Ca(2+)]c) transients, and catecholamine release from bovine chromaffin cells (BCCs) stimulated with high K(+), caffeine, or histamine. At 22°C, the effects of KBR on those parameters were meager or nil. However, at 37°C whereby the NCX is moving Ca(2+) at a rate fivefold higher than at 22°C, various of the effects of KBR were pronounced, namely: 1) no effects on ICa; 2) reduction of the [Ca(2+)]c transient amplitude and slowing down of its rate of clearance; 3) blockade of the K(+)-elicited quantal release of catecholamine; 4) blockade of burst catecholamine release elicited by K(+); 5) no effect on catecholamine release elicited by short K(+) pulses (1-2 s) and blockade of the responses produced by longer K(+) pulses (3-5 s); and 6) potentiation of secretion elicited by histamine or caffeine. Furthermore, the more selective NCX blocker SEA0400 also potentiated the secretory responses to caffeine. The results suggest that at physiological temperature the NCX substantially contributes to shaping the kinetics of [Ca(2+)]c transients and the exocytotic responses elicited by Ca(2+) entry through Ca(2+) channels as well as by Ca(2+) release from the endoplasmic reticulum.

Cambios celulares producidos por la citotoxicidad del líquido cefalorraquídeo de pacientes con esclerosis lateral amiotrófica sobre cultivos de neuronas motoras

INTRODUCTION The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. MATERIAL AND METHODS Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. RESULTS In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. CONCLUSIONS CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed.

Cerebrospinal fluid cytotoxicity does not affect survival in amyotrophic lateral sclerosis

Cerebrospinal fluid (CSF) from some patients with amyotrophic lateral sclerosis (ALS) has been demonstrated to significantly reduce the neuronal viability of primary cell cultures of motor neurons. We aimed to study the potential clinical consequences associated with the cytotoxicity of CSF in a cohort of patients with ALS.

La infusión intracerebroventricular prolongada de líquido cefalorraquídeo procedente de pacientes con esclerosis lateral amiotrófica provoca cambios histológicos en el cerebro y la médula espinal de la rata similares a los hallados en la enfermedad

INTRODUCTION Cerebrospinal fluid (CSF) from amyotrophic lateral sclerosis (ALS) patients induces cytotoxic effects in in vitro cultured motor neurons. MATERIAL AND METHODS We selected CSF with previously reported cytotoxic effects from 32 ALS patients. Twenty-eight adult male rats were intracerebroventricularly implanted with osmotic mini-pumps and divided into 3 groups: 9 rats injected with CSF from non-ALS patients, 15 rats injected with cytotoxic ALS-CSF, and 4 rats injected with a physiological saline solution. CSF was intracerebroventricularly and continuously infused for periods of 20 or 43days after implantation. We conducted clinical assessments and electromyographic examinations, and histological analyses were conducted in rats euthanised 20, 45, and 82days after surgery. RESULTS Immunohistochemical studies revealed tissue damage with similar characteristics to those found in the sporadic forms of ALS, such as overexpression of cystatinC, transferrin, and TDP-43 protein in the cytoplasm. The earliest changes observed seemed to play a protective role due to the overexpression of peripherin, AKTpan, AKTphospho, and metallothioneins; this expression had diminished by the time we analysed rats euthanised on day 82, when an increase in apoptosis was observed. The first cellular changes identified were activated microglia followed by astrogliosis and overexpression of GFAP and S100B proteins. CONCLUSION Our data suggest that ALS could spread through CSF and that intracerebroventricular administration of cytotoxic ALS-CSF provokes changes similar to those found in sporadic forms of the disease.