André Schwambach Vieira

THE ANTIDEPRESSIVE EFFECT OF THE PHYSICAL EXERCISE CORRELATES WITH INCREASED LEVELS OF MATURE BDNF, AND proBDNF PROTEOLYTIC CLEAVAGE-RELATED GENES, p11 AND tPA

Clinical studies show an evident antidepressive effect of physical exercise and animal research corroborate such evidence. However, the neurobiological mechanisms underlying the antidepressive effect of exercise are not completely understood. Notwithstanding, it is known that exercise increases brain-derived neurotrophic factor (BDNF) expression in the hippocampus similarly to antidepressant drugs. BDNF is synthesized as a precursor molecule that undergoes a proteolytic cleavage to generate either a mature or a truncated isoform. Precursor and mature BDNF are assumed to elicit opposing biological effects in neuroplasticity. In the present study we investigated the effect of voluntary physical activity on precursor and mature brain-derived neurotrophic factor levels and on proBDNF cleavage related genes, p11 and tissue plasminogen activator (tPA), as well as the antidepressive and cognitive effects of voluntary physical activity. Mice had access to mobile or locked running wheels for 28 days and were submitted to forced-swim, tail suspension and water maze tests. Their hippocampi were dissected and analyzed by Western blot and real time RT-PCR. Voluntary physical activity, but not locked wheel exposure, induced a robust increase in hippocampal mature BDNF protein levels, as well as in p11 and tPA mRNA expression; and also promoted antidepressive effects and improved learning, when compared with sedentary mice. On the other hand, there were no significant differences between any groups in the expression of precursor or truncated isoforms of BDNF. Our data suggest that the antidepressive effect of the physical exercise may depend, at least in part, on changes in BDNF post-translational processing.

The new world of RNAs

One of the major developments that resulted from the human genome sequencing projects was a better understanding of the role of non-coding RNAs (ncRNAs). NcRNAs are divided into several different categories according to size and function; however, one shared feature is that they are not translated into proteins. In this review, we will discuss relevant aspects of ncRNAs, focusing on two main types: i) microRNAs, which negatively regulate gene expression either by translational repression or target mRNA degradation, and ii) small interfering RNAs (siRNAs), which are involved in the biological process of RNA interference (RNAi). Our knowledge regarding these two types of ncRNAs has increased dramatically over the past decade, and they have a great potential to become therapeutic alternatives for a variety of human conditions.

Ciliary neurotrophic factor (CNTF) signals through STAT3–SOCS3 pathway and protects rat pancreatic islets from cytokine-induced apoptosis

CNTF is a cytokine that promotes survival and/or differentiation in many cell types, including rat pancreatic islets. In this work, we studied the mechanism of CNTF signal in neonatal rats pancreatic islets isolated by the collagenase method and cultured for 3 days in RPMI medium without (CTL) or with 1 nM of CNTF. The medium contained, when necessary, specific inhibitors of the PI3K, MAPK and JAK/STAT3 pathways. mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of Akt, ERK1/2 and STAT3, and SOCS-3 (RT-PCR and Western blot), as well as glucose-stimulated insulin secretion (GSIS) (Radioimmunoassay), were analyzed. Our results showed that Akt, ERK1 and STAT3 mRNA expression, as well as phosphorylated Akt and ERK1/2, was not affected by CNTF treatment. CNTF increased cytoplasmatic and nuclear phosphorylated STAT3, and the SOCS3 mRNA and protein expression. In addition, CNTF lowered apoptosis and impaired GSIS. These effects were blocked by the JAK inhibitor, AG490 and by the STAT3 inhibitor Curcumin, but not by the MAPK inhibitor, PD98059, nor by the PI3K inhibitor, Wortmannin. In conclusion, CNTF signals through the JAK2/STAT3 cascade, increases SOCS3 expression, impairs GSIS and protects neonatal pancreatic rat islets from cytokine-induced apoptosis. These findings indicate that CNTF may be a potential therapeutic tool against Type 1 and/or Type 2 diabetes.

MicroRNA regulation and dysregulation in epilepsy

Epilepsy, one of the most frequent neurological disorders, represents a group of diseases that have in common the clinical occurrence of seizures. The pathogenesis of different types of epilepsy involves many important biological pathways; some of which have been shown to be regulated by microRNAs (miRNAs). In this paper, we will critically review relevant studies regarding the role of miRNAs in epilepsy. Overall, the most common type of epilepsy in the adult population is temporal lobe epilepsy (TLE), and the form associated with mesial temporal sclerosis (MTS), called mesial TLE, is particularly relevant due to the high frequency of resistance to clinical treatment. There are several target studies, as well few genome-wide miRNA expression profiling studies reporting abnormal miRNA expression in tissue with MTS, both in patients and in animal models. Overall, these studies show a fine correlation between miRNA regulation/dysregulation and inflammation, seizure-induced neuronal death and other relevant biological pathways. Furthermore, expression of many miRNAs is dynamically regulated during neurogenesis and its dysregulation may play a role in the process of cerebral corticogenesis leading to malformations of cortical development (MCD), which represent one of the major causes of drug-resistant epilepsy. In addition, there are reports of miRNAs involved in cell proliferation, fate specification, and neuronal maturation and these processes are tightly linked to the pathogenesis of MCD. Large-scale analyzes of miRNA expression in animal models with induced status epilepticus have demonstrated changes in a selected group of miRNAs thought to be involved in the regulation of cell death, synaptic reorganization, neuroinflammation, and neural excitability. In addition, knocking-down specific miRNAs in these animals have demonstrated that this may consist in a promising therapeutic intervention.

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 μL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1β (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1β. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1β in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1+ cells of the DRG, significantly increased after carrageenan or IL-1β administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCε expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1β in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.

Ciliary neurotrophic factor infused intracerebroventricularly shows reduced catabolic effects when linked to the TAT protein transduction domain

Ciliary neurotrophic factor (CNTF) regulates the differentiation and survival of a wide spectrum of developing and adult neurons, including motor neuron loss after injury. We recently described a cell‐penetrant recombinant human CNTF (rhCNTF) molecule, formed by fusion with the human immunodeficiency virus‐1 transactivator of transcription (TAT) protein transduction domain (TAT‐CNTF) that, upon subcutaneous administration, retains full neurotrophic activity without cytokine‐like side‐effects. Although the CNTF receptor is present in hypothalamic nuclei, which are involved in the control of energy, rhCNTF but not TAT‐CNTF stimulates signal transducers and activators of transcription 3 phosphorylation in the rat hypothalamus after subcutaneous administration. This could be due limited TAT‐CNTF distribution in the hypothalamus and/or altered intracellular signaling by the fusion protein. To explore these possibilities, we examined the effect of intracerebroventricular administration of TAT‐CNTF in male adult rats. TAT‐CNTF‐induced weight loss, although the effect was smaller than that seen with either rhCNTF or leptin (which exerts CNTF‐like effects via its receptor). In contrast to rhCNTF and leptin, TAT‐CNTF neither induced morphological changes in adipose tissues nor increased uncoupling protein 1 expression in brown adipose tissue, a characteristic feature of rhCNTF and leptin. Acute intracerebroventricular administration of TAT‐CNTF induced a less robust phosphorylation of signal transducers and activators of transcription 3 in the hypothalamus, compared with rhCNTF. The data show that fusion of a protein transduction domain may change rhCNTF CNS distribution, while further strengthening the utility of cell‐penetrating peptide technology to neurotrophic factor biology beyond the neuroscience field.

JAK2 inhibition is neuroprotective and reduces astrogliosis after quinolinic acid striatal lesion in adult mice

Quinolinic acid (QA) striatal lesion in rodents induces neuronal death, astrogliosis and migration of neuroblasts from subventricular zone to damaged striatum. These phenomena occur in some human neurodegenerative illnesses, but the underlying mechanisms are unknown. We investigated the effect of AG490, a Janus-kinase 2 (JAK2) inhibitor, on astrogliosis, neuronal loss and neurogenesis in the striatum of adult mice after unilateral infusion of QA (30 nmol). Animals were given subcutaneous injections of AG490 (10 mg/kg) or vehicle immediately after lesion and then once daily for six days. Brain sections were used for neuronal stereological quantification, immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of astrocytes and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI=ADPC(L)/ADPC(CL)) and neuronal ratio (NR=NN(L)/NN(CL)) were calculated. After QA administration, blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals. NR was 25% higher in mice given AG490 vs controls given vehicle. NI showed a decrease of 21% in AG490- vs vehicle-treated mice. Our results indicate that JAK2 inhibition reduces QA lesion and suggest that astrogliosis may impair neuronal survival in this model.

Bax and Bcl-2 expression and TUNEL labeling in lumbar enlargement of neonatal rats after sciatic axotomy and melatonin treatment

Peripheral axotomy in neonatal rats induces neuronal death. We studied the anti-apoptotic protein Bcl-2 and cell death promoter Bax in spinal cord of neonatal rats after sciatic transection and treatment with melatonin, a neuroprotective substance. Pups were unilaterally axotomized at P2 and received melatonin (1 mg/kg; sc) or vehicle 1 h prior to lesion, immediately after, at 1 h, 2 h and then once daily. Rats were sacrificed at 3 h, 6 h, 24 h, 72 h and 5 days postaxotomy. Intact animals were used as controls. Lumbar enlargement was processed for Nissl staining, immunohistochemistry and RT-PCR for Bax or Bcl-2 and TUNEL reaction. Motoneurons (MN) of lesioned (L) and normal (N) sides were counted, and MN survival ratio (MSR=L/N) was calculated. Bax and Bcl-2 showed cytoplasmic immunoreactivity (IR). Bax IR was noticeable in small cells but less evident in MN. In unlesioned pups, some Bax-positive small cells (B+) and TUNEL-positive nuclei (T+) were mainly seen in the dorsal horn. In lesioned animals given vehicle, Bax mRNA levels and numbers of B+ and T+ were increased in comparison with intact controls at 24 h postaxotomy. The basal IR for Bax in MN was not changed by axotomy. Bcl-2 IR was noted in all cells and, like Bcl-2 mRNA, was unaltered after lesion. Melatonin reduced MN loss at 24 h, 72 h and 5 days and T+ at 24 h after lesion but did not interfere with Bax or Bcl-2 expression. These results suggest that (1) sciatic transection at P2 increases Bax mRNA and the amount of B+ and T+ in the lumbar enlargement, (2) Bax IR in immature MN is not altered by axotomy and (3) melatonin protects MN and dorsal horn cells through a mechanism independent of Bax and Bcl-2.

mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration

Sciatic axotomy in 2-day-old rats (P2) causes lumbar motoneuron loss, which could be associated with nitric oxide (NO) production. NO may be produced by three isoforms of synthase (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We investigated NOS expression and NO synthesis in the lumbar enlargement of rats after sciatic nerve transection at P2 and treatment with the antioxidant melatonin (sc; 1 mg/kg). At time points ranging from P2 to P7, expression of each isoform was assessed by RT-PCR and immunohistochemistry; catalytic rates of calcium-dependent (nNOS, eNOS) and independent (iNOS) NOS were measured by the conversion of [3H]L-arginine to [3H]L-citrulline. All NOS isoforms were expressed and active in unlesioned animals. nNOS and iNOS were detected in some small cells in the parenchyma. Only endothelial cells were positive for eNOS. No NOS isoform was detected in motoneurons. Axotomy did not change these immunohistochemical findings, nNOS and iNOS mRNA expression and calcium-independent activity at all survival times. However, sciatic nerve transection reduced eNOS mRNA levels at P7 and increased calcium-dependent activity at 1 and 6 h. Melatonin did not alter NOS expression. Despite having no action on NOS activity in unlesioned controls the neurohormone enhanced calcium-dependent activity at 1 and 72 h and reduced calcium-independent catalysis at 72 h in lesioned rats. These results suggest that NOS isoforms are constitutive in the neonatal lumbar enlargement and are not overexpressed after sciatic axotomy. Changes in NO synthesis induced by axotomy and melatonin administration in the current model are discussed considering some beneficial and deleterious effects that NO may have.

Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus‐1 TAT (transactivator) protein. The resulting fusion protein (TAT‐CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT‐CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT‐CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20–30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT‐CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine‐like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.