Moisés García-Arencibia

Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration

Background Parkinsons disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

Evaluation of the neuroprotective effect of cannabinoids in a rat model of Parkinson's disease : Importance of antioxidant and cannabinoid receptor-independent properties

We have recently demonstrated that two plant-derived cannabinoids, Delta9-tetrahydrocannabinol and cannabidiol (CBD), are neuroprotective in an animal model of Parkinsons disease (PD), presumably because of their antioxidant properties. To further explore this issue, we examined the neuroprotective effects of a series of cannabinoid-based compounds, with more selectivity for different elements of the cannabinoid signalling system, in rats with unilateral lesions of nigrostriatal dopaminergic neurons caused by local application of 6-hydroxydopamine. We used the CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 receptor agonist HU-308, the non-selective agonist WIN55,212-2, and the inhibitors of the endocannabinoid inactivation AM404 and UCM707, all of them administered i.p. Daily administration of ACEA or WIN55,212-2 did not reverse 6-hydroxydopamine-induced dopamine (DA) depletion in the lesioned side, whereas HU-308 produced a small recovery that supports a possible involvement of CB2 but not CB1 receptors. AM404 produced a marked recovery of 6-hydroxydopamine-induced DA depletion and tyrosine hydroxylase deficit in the lesioned side. Possibly, this is caused by the antioxidant properties of AM404, which are derived from the presence of a phenolic group in its structure, rather than by the capability of AM404 to block the endocannabinoid transporter, because UCM707, another transporter inhibitor devoid of antioxidant properties, did not produce the same effect. None of these effects were observed in non-lesioned contralateral structures. We also examined the timing for the effect of CBD to provide neuroprotection in this rat model of PD. We found that CBD, as expected, was able to recover 6-hydroxydopamine-induced DA depletion when it was administered immediately after the lesion, but it failed to do that when the treatment started 1 week later. In addition, the effect of CBD implied an upregulation of mRNA levels for Cu,Zn-superoxide dismutase, a key enzyme in endogenous defenses against oxidative stress. In summary, our results indicate that those cannabinoids having antioxidant cannabinoid receptor-independent properties provide neuroprotection against the progressive degeneration of nigrostriatal dopaminergic neurons occurring in PD. In addition, the activation of CB2 (but not CB1) receptors, or other additional mechanisms, might also contribute to some extent to the potential of cannabinoids in this disease.

Role of CB2 receptors in neuroprotective effects of cannabinoids.

CB2 receptors, the so-called peripheral cannabinoid receptor type, were first described in the immune system, but they have been recently identified in the brain in healthy conditions and, in particular, after several types of cytotoxic stimuli. Specifically, CB2 receptors were identified in microglial cells, astrocytes and, to a lesser extent, in certain subpopulations of neurons. Given the lack of psychoactivity demonstrated by selective CB2 receptor agonists, this receptor becomes an interesting target for the treatment of neurological diseases, in particular, the case of certain neurodegenerative disorders in which induction/up-regulation of CB2 receptors has been already demonstrated. These disorders include Alzheimers disease, Huntingtons chorea, amyotrophic lateral sclerosis and others. Interestingly, in experimental models of these disorders, the activation of CB2 receptors has been related to a delayed progression of neurodegenerative events, in particular, those related to the toxic influence of microglial cells on neuronal homeostasis. The present article will review the evidence supporting that CB2 receptors might represent a key element in the endogenous response against different types of cytotoxic events, and that this receptor type may be a clinically promising target for the control of brain damage in neurodegenerative disorders.

Effects of rimonabant, a selective cannabinoid CB1 receptor antagonist, in a rat model of Parkinson's disease

Recent evidence suggest that the blockade of cannabinoid CB1 receptors might be beneficial to alleviate motor inhibition typical of Parkinsons disease (PD). In the present study, we have explored the motor effects of rimonabant, a selective antagonist of CB1 receptors, in a rat model of PD generated by an intracerebroventricular injection of 6-hydroxydopamine. Compared with rats subjected to unilateral injection of this toxin in the medial forebrain bundle, this model allows nigral dopaminergic neurons be symmetrically affected. Dose-response studies with 6-hydroxydopamine revealed that the application of 200 microg per animal caused hypokinetic signs (decreased ambulatory activity, increased inactivity, and reduced motor coordination), which paralleled several signs of degeneration of nigrostriatal dopaminergic neurons (dopamine depletion in the caudate-putamen, and decreased mRNA levels for tyrosine hydroxylase and superoxide dismutase-1 and -2 in the substantia nigra). In these conditions, the degree of hypokinesia and dopaminergic degeneration may be considered moderate, comparable to the disturbances occurring in early and middle stages of PD in humans, a period that might be appropriate to test the effects of rimonabant. There is also degeneration of other dopaminergic pathways out of the basal ganglia, but this does not appear to interfere significantly with the hypokinetic profile of these rats. Higher doses of 6-hydroxydopamine elevated significantly animal mortality and lower doses failed in general to reproduce motor inhibition. Like other animal models of PD, these rats exhibited an increase in the density of CB(1) receptors in the substantia nigra, which is indicative of the expected overactivity of the cannabinoid transmission in this disease and supports the potential of CB1 receptor blockade to attenuate hypokinesia associated with nigral cell death. Thus, the injection of 0.1 mg/kg of rimonabant partially attenuated the hypokinesia shown by these animals with no effects in control rats, whereas higher doses (0.5-1.0 mg/kg) were not effective. We also found that the antihypokinetic effects of low doses of rimonabant did not influence the dopamine deficits of these animals, as well as it did not modify GABA or glutamate transmission in the caudate-putamen. In summary, rimonabant may have potential antihypokinetic activity in moderate parkinsonism at low doses, but this effect is not related to changes in dopaminergic, GABAergic, or glutamatergic transmission in the striatum. Therefore, the elucidation of the neurochemical substrate involved in this effect remains a major challenge for the future.

A53T-Alpha-Synuclein Overexpression Impairs Dopamine Signaling and Striatal Synaptic Plasticity in Old Mice

Background Parkinsons disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Methodology/Principal Findings Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice. Conclusions/Significance Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches.

Symptom‐relieving and neuroprotective effects of the phytocannabinoid Δ9‐THCV in animal models of Parkinson's disease

BACKGROUND AND PURPOSE Previous findings have indicated that a cannabinoid, such as Δ9‐THCV, which has antioxidant properties and the ability to activate CB2 receptors but to block CB1, might be a promising therapy for alleviating symptoms and delaying neurodegeneration in Parkinsons disease (PD).

Cannabinoids and Neuroprotection in Basal Ganglia Disorders

Cannabinoids have been proposed as clinically promising neuroprotective molecules, as they are capable to reduce excitotoxicity, calcium influx, and oxidative injury. They are also able to decrease inflammation by acting on glial processes that regulate neuronal survival and to restore blood supply to injured area by reducing the vasoconstriction produced by several endothelium-derived factors. Through one or more of these processes, cannabinoids may provide neuroprotection in different neurodegenerative disorders including Parkinson’s disease and Huntington’s chorea, two chronic diseases that are originated as a consequence of the degeneration of specific nuclei of basal ganglia, resulting in a deterioration of the control of movement. Both diseases have been still scarcely explored at the clinical level for a possible application of cannabinoids to delay the progressive degeneration of the basal ganglia. However, the preclinical evidence seems to be solid and promising. There are two key mechanisms involved in the neuroprotection by cannabinoids in experimental models of these two disorders: first, a cannabinoid receptor-independent mechanism aimed at producing a decrease in the oxidative injury and second, an induction/upregulation of cannabinoid CB2 receptors, mainly in reactive microglia, that is capable to regulate the influence of these glial cells on neuronal homeostasis. Considering the relevance of these preclinical data and the lack of efficient neuroprotective strategies in both disorders, we urge the development of further studies that allow that the promising expectatives generated for these molecules progress from the present preclinical evidence till a real clinical application.

Genomic actions of 1,25-dihydroxyvitamin D3 on insulin receptor gene expression, insulin receptor number and insulin activity in the kidney, liver and adipose tissue of streptozotocin-induced diabetic rats.

Backgroundthis study set out to examine the effects of the treatment with 1,25-dihydroxyvitamin D3 (1,25D3) [150 IU/Kg (3.75 μg/Kg) one a day, for 15 days] to non-diabetic rats and in rats rendered diabetic by a single injection of streptozotocin [65 mg/kg].Resultstreatment with 1,25D3 to non-diabetic rats did not affect the biochemical parameters measured in the plasma and urine of these animals. Likewise, insulin receptor expression in the kidney, liver, or adipose tissue and insulin-stimulated glucose transport in adipocytes from these animals were not affected either. Treatment with 1,25D3 to streptozotocin-induced diabetic rats did not correct the hyperglycemia, hypoinsulinemia, glycosuria or ketonemia induced by the diabetes, although it partially reversed the over-expression of the insulin receptor gene in the liver and adipose tissue, without altering the normal expression of this gene in the kidney. These effects were accompanied by a normalization of the number of insulin receptors without altering receptor affinity but improving the insulin response to glucose transport in adipocytes from these diabetic animals. Moreover, a computer search in the rat insulin receptor promoter revealed the existence of two candidate vitamin D response element (VDRE) sequences located at -256/-219 bp and -653/-620 bp, the first overlapped by three and the second by four AP-2-like sites.Conclusionthese genomic actions of 1,25D3 could represent beneficial effects associated with the amelioration of diabetes via mechanisms that possibly involve direct transcriptional activation of the rat insulin receptor gene. The candidate VDREs identified may respond to 1,25D3 via activation of the vitamin D receptor, although this remains to be investigated.

Cannabinoids and Parkinson's Disease

Cannabinoid-based medicines have been proposed as clinically promising therapies in Parkinsons disease (PD), given the prominent modulatory function played by the cannabinoid signaling system in the basal ganglia. Supporting this pharmacological potential, the cannabinoid signaling system experiences a biphasic pattern of changes during the progression of PD. Thus, early and presymptomatic stages, characterized by neuronal malfunctioning but little evidence of neuronal death, are associated with desensitization/downregulation of CB(1) receptors. It was proposed that these losses may be part of the pathogenesis itself, since they can aggravate different cytotoxic insults which are controlled in part by cannabinoid signals, mainly excitotoxicity but also oxidative stress and glial activation. By contrast, intermediate and, in particular, advanced stages of parkinsonism characterized by a profound nigral degeneration and occurrence of major parkinsonian symptoms (e.g. bradykinesia), are associated with upregulatory responses of CB(1) receptors, possibly CB(2) receptors too, and the endocannabinoid ligands for both receptor types. This would explain the motor inhibition typical of this disease and the potential proposed for CB(1) receptor antagonists in attenuating the bradykinesia typical of PD. In addition, certain cannabinoid agonists have been proposed to serve as neuroprotective molecules in PD, given their well-demonstrated capability to reduce excitotoxicity, calcium influx, glial activation and, in particular, oxidative injury that cooperatively contribute to the degeneration of nigral neurons. However, the potential of cannabinoid-based medicines in PD have been still scarcely studied at the clinical level despite the existence of solid and promising preclinical evidence. Considering the relevance of these preclinical data, the need for finding treatments for motor symptoms that may be alternative to classic dopaminergic replacement therapy, and the lack of efficient neuroprotective strategies in PD, we believe it is of major interest to develop further studies that allow the promising expectations generated for these molecules to progress from the present preclinical evidence towards a real clinical application.

Transcriptional inhibition of the human insulin receptor gene by aldosterone

In earlier studies, we reported reduced human insulin receptor (hIR) mRNA levels, insulin binding and insulin responsiveness in U-937 human promonocytic cells treated with aldosterone. The mechanism for this inhibition could be diminished IR gene transcription, since aldosterone did not affect hIR mRNA stability. All the effects were mediated by a downregulation of the mineralocorticoid receptor (MR, NR3C2) expressed at both the RNA and protein levels, suggesting that MR could act as a transcription factor that binds to hormone response elements in the hIR gene promoter. Indeed, MR has been shown to bind glucocorticoid response elements (GREs) in target genes. Given that five GREs have been characterized in the hIR promoter, we decided to test whether these elements could mediate the aldosterone-elicited inhibition of hIR expression detected by us in U-937 cells. In the present report, we demonstrate that aldosterone inhibits the activity of the hIR wild-type promoter by 23%, and causes 23 and 31% reductions in the activity of progressive deletions of this promoter comprised of fragments up to -1473 and -876bp, respectively. This indicates that the -876 to -271bp region of the hIR promoter may be sufficient for this transcriptional inhibition by aldosterone. We also provide evidence for direct MR interaction with some of the GREs of this promoter region, specifically with the cGRE1 and cGRE3, presumably as MR-MR homodimers, and with pGRE as a MR-GR heterodimer. This heterodimer may play the most relevant role and participate in the cross-talk between mineralocorticoids, glucocorticoids and insulin signalling in U-937 cells.