Ramon Trullas

Low cerebrospinal fluid concentration of mitochondrial DNA in preclinical Alzheimer disease

To identify a novel biochemical marker that precedes clinical symptoms in Alzheimer disease (AD).

Time course of changes in serotonin and noradrenaline in rat brain after predictable or unpredictable shock

The effects of predictable and unpredictable shock on concentrations of serotonin (5-hydroxytryptamine, 5-HT), 5-hydroxyindoleacetic acid (5-HIAA), tryptophan (TP) and noradrenaline (NA) have been studied in 7 regions of rat brain. Two separate experiments have been carried out determining these substances both at 30 min and 2 h after the stress session. Unpredictable shock depleted NA levels in all brain regions except the striatum. However, at 2 h poststress NA in these regions increased significantly in comparison with both controls and predictably shocked rats. Predictable shock also decreased NA in locus coeruleus, brainstem and hypothalamus, which was not observed 2 h later. Both predictable and unpredictable shock decreased 5-HT in brainstem and hypothalamus. At 2 h poststress, 5-HT levels in these regions were still decreased in predictably shocked rats, but had attained control values in unpredictably shocked rats. 5-HT metabolism expressed as the 5-HIAA/5-HT ratio, was significantly increased 30 min after predictable shock in all regions except the locus coeruleus and hippocampus. Unpredictable shock produced a much more marked increase in 5-HIAA/5-HT ratio. At 2 h poststress 5-HT metabolism returned to control values in most of the brain regions of predictably shocked animals, but it remained high after unpredictable shock. The activation of serotonergic metabolism following each type of shock is different according to the nucleus in which the 5-HT nerve endings originate. Only slight increases in tryptophan were observed after both types of shock. Our results suggest that unpredictable shock is perceived as a more anxiogenic situation and that under this condition both 5-HT and NA levels are more effectively normalized with time.

Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons

The biochemical pathways involved in neuronal cell death in Parkinsons disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 microM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinsons disease.

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.

Lindane-induced convulsions in NMRI and OF1 mice : antagonism with (+)MK-801 and voltage-dependent calcium channel blockers

The convulsant profile of lindane was investigated in OF1 and NMRI mice lines in relation to other convulsants acting at the GABAA and NMDA receptor complexes. Thus, a specific GABA-gated chloride channel blocker, PTX, a GABAA receptor antagonist, PTZ, and an excitatory amino acid receptor agonist, NMDA, were used. Antagonism of the convulsant effects of each of these drugs was investigated with (+)MK-801, a blocker of the NMDA-operated cation channel, and with nifedipine, a voltage-dependent calcium channel antagonist. While no differences in potency for PTX or PTZ to induce seizures were observed between OF1 and NMRI mice, lindane was approximately 80 and 90% more potent in its ability to induce seizures and lethality, respectively, in OF1 than in NMRI mice. Brain lindane concentrations at the moment of convulsion, measured after ED100 doses of lindane (400 and 200 mg/kg for NMRI and OF1 mice, respectively), did not differ between OF1 and NMRI mice, suggesting that the different potency of lindane between these mouse lines is a consequence of pharmacokinetic factors. Furthermore, (+)MK-801 antagonized seizures induced by either lindane, PTX or PTZ with similar potencies in both mouse lines. These results, coupled with the different pharmacokinetics of lindane in OF1 and NMRI mice, suggest that the distinct effects of lindane in these mice are not mediated by different activities at either NMDA or GABAA receptor complexes. Nonetheless, nifedipine antagonized lindane-induced seizures with a three-fold higher potency in NMRI than in OF1 mice. In contrast, nifedipine failed to antagonize PTX and PTZ convulsions in both OF1 and NMRI mice. These results suggest that besides the GABAA receptor complex other mechanisms related to calcium mobilization may be involved in the convulsant action of lindane.

HIF-1α is neuroprotective during the early phases of mild hypoxia in rat cortical neurons

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a key role in regulating the adaptive response to hypoxia. HIF-1α is stabilised during hypoxia and, after dimerisation with hypoxia-inducible factor 1β (HIF-1β), triggers the expression of various genes involved in cell cycle control and energy metabolism associated with cell survival. However, HIF-1α also regulates the expression of proapoptotic genes. The aim of this study was to ascertain the influence of HIF-1α on neurotoxicity evoked by hypoxia in rat cortical neurons. We found that mild hypoxia induces time-dependent neuronal death involving free radical production, mitochondrial depolarisation, cytochrome c release and caspase-3 activation. Lentivirus-mediated HIF-1α knockdown markedly strengthened all of these effects during the initial 24h of hypoxia, which suggests that HIF-1α plays a neuroprotective role in hypoxia-mediated neuronal death. After this initial period, the protective actions of HIF-1α disappeared over the course of the hypoxia-mediated HIF-1α stabilisation. Moreover, lentiviral-mediated overexpression of HIF-1α increased lactate dehydrogenase (LDH) A, one of the target genes for HIF-1α, but did not show protective actions on hypoxia-mediated neuronal death, indicating that the level of endogenous HIF-1α stabilisation achieved during hypoxia was already the maximum required for HIF-1α transcription activities. These results indicate that HIF-1α is neuroprotective in the early phases of hypoxia.

Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells.

Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3beta (GSK-3beta) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3beta is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3beta activity after 2h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3beta and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3beta inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3beta inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3beta with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3beta inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li(+) and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3beta. Therefore, there is a connection between cell cycle and GSK-3beta activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal.

Role of high-affinity choline uptake on extracellular choline and acetylcholine evoked by NMDA.

Previous studies have shown that NMDA evokes a calcium‐dependent and region‐specific increase in extracellular choline that is associated with a reduction of membrane phosphatidylcholine and precedes neuronal cell death. We investigated, using in vivo microdialysis, the contribution of high‐affinity choline uptake on the increase in extracellular choline evoked by NMDA. Dialysis was performed in the presence of Neostigmine (0.5 μM), an acetylcholinesterase inhibitor, in prefrontal cortex or hippocampus of freely moving rats. Drugs were administered through the dialysis probe. In cholinergic denervation experiments, rats were subjected to sham or AMPA‐induced lesion of cholinergic nuclei at least 2 weeks before microdialysis. Excitotoxic lesion of the medial septum / ventral diagonal band nuclei reduced hippocampal choline acetyltransferase activity by 74%, [3H]hemicholinium‐3 binding by 32%, and completely abolished potassium‐evoked acetylcholine release. Despite this reduction of presynaptic cholinergic function, perfusion of NMDA (300 μM) by retrodialysis produced an increase in hippocampal extracellular choline (249 ± 22% of basal levels) that was similar to that observed in sham controls (301 ± 35%). Inhibition of choline uptake with hemicholinium‐3 in nonlesioned rats produced a sustained increase in dialysate choline (163 ± 8%) and reduced acetylcholine to 33 ± 2% of basal levels, consistent with a depletion of the acetylcholine pool due to precursor deficit. Simultaneous perfusion of hemicholinium‐3 and NMDA produced a synergistic increase in dialysate choline (664 ± 95% of basal levels), indicating that part of the choline released by NMDA is taken up. In contrast, NMDA antagonized the decrease of acetylcholine produced by hemicholinium‐3. These results show that NMDA‐evoked choline release is not mediated by inhibition of high‐affinity choline uptake and indicate that choline released by NMDA can be used to sustain acetylcholine synthesis when there is a precursor deficit secondary to uptake inhibition. Synapse 35:272–280, 2000.

Mitochondrial DNA differentiates Alzheimer's disease from Creutzfeldt-Jakob disease

Low content of cell‐free mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) is a biomarker of early stage Alzheimers disease (AD), but whether mtDNA is altered in a rapid neurodegenerative dementia such as Creutzfeldt‐Jakob disease is unknown.

Mitochondrial DNA in CSF distinguishes LRRK2 from idiopathic Parkinson's disease

Mitochondrial DNA regulates mitochondrial function which is altered in both idiopathic and familial forms of Parkinsons disease. To investigate whether these two disease forms exhibit an altered regulation of mitochondrial DNA we measured cell free mitochondrial DNA content in cerebrospinal fluid (CSF) from idiopathic and LRRK2-related Parkinsons disease patients. The concentration of mitochondrial DNA was measured using a digital droplet polymerase chain reaction technique in a total of 98 CSF samples from a cohort of subjects including: 20 LRRK2(G2019S) mutation carriers with Parkinsons disease, 26 asymptomatic LRRK2(G2019S) mutation carriers, 31 patients with idiopathic Parkinsons disease and 21 first-degree relatives of LRRK2 Parkinsons disease patients without the mutation. Here we report that LRRK2(G2019S) mutation carriers with Parkinsons disease exhibit a high concentration of mitochondrial DNA in CSF compared with asymptomatic LRRK2(G2019S) mutation carriers and with idiopathic Parkinsons disease patients. In addition, idiopathic, but not LRRK2 Parkinsons disease is associated with low CSF concentration of α-synuclein. These results show that high mitochondrial DNA content in CSF distinguishes idiopathic from LRRK2-related Parkinsons disease suggesting that different biochemical pathways underlie neurodegeneration in these two disorders.