SOD1G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motor neuron (MN) degeneration. Astrocytes surrounding MNs are known to modulate ALS progression. When cocultured with astrocytes overexpressing the ALS-linked mutant Cu/Zn superoxide dismutase (SOD1G93A) or when cultured with conditioned medium from SOD1G93A astrocytes, MN survival is reduced. The exact mechanism of this neurotoxic effect is unknown. Astrocytes secrete extracellular vesicles (EVs) that transport protein, mRNA, and microRNA species from one cell to another. The size and protein markers characteristic of exosomes were observed in the EVs obtained from cultured astrocytes, indicating their abundance in exosomes. Here, we analyzed the microRNA content of the exosomes derived from SOD1G93A astrocytes and evaluated their role in MN survival. Purified MNs exposed to SOD1G93A astrocyte-derived exosomes showed reduced survival and neurite length compared to those exposed to exosomes derived from non-transgenic (non-Tg) astrocytes. Analysis of the miRNA content of the exosomes revealed that miR-155-5p and miR-582-3p are differentially expressed in SOD1G93A exosomes compared with exosomes from non-Tg astrocytes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicates that miR-155-5p and miR-582-3p predicted targets are enriched in the neurotrophin signaling pathway. Importantly, when levels of miR-155-5p were reduced by incubation with a specific antagomir, SOD1G93A exosomes did not affect MN survival or neurite length. These results demonstrate that SOD1G93A-derived exosomes are sufficient to induce MN death, and miRNA-155-5p contributes to this effect. miRNA-155-5p may offer a new therapeutic target to modulate disease progression in ALS.

Mitochondrial bioenergetics, glial reactivity, and pain-related behavior can be restored by dichloroacetate treatment in rodent pain models.

Glial reactivity in the dorsal horn of the spinal cord is a hallmark in most chronic pain conditions. Neuroinflammation-associated reactive glia, in particular astrocytes, have been shown to exhibit reduced mitochondrial respiratory function. Here, we studied the mitochondrial function at the lumbar spinal cord tissue from complete Freund's adjuvant-induced inflammatory pain rat and chronic constriction injury mouse models by high-resolution respirometry. A significant decrease in mitochondrial bioenergetic parameters at the injury-related spinal cord level coincided with highest astrocytosis. Oral administration of dichloroacetate (DCA) significantly increased mitochondrial respiratory function by inhibiting pyruvate dehydrogenase kinase and decreased glial fibrillary acidic protein and Iba-1 immunoreactivity in spinal cord. Importantly, DCA treatment significantly reduced the ipsilateral pain-related behavior without affecting contralateral sensitivity in both pain models. Our results indicate that mitochondrial metabolic modulation with DCA may offer an alternative therapeutic strategy to alleviate chronic and persistent inflammatory pain.

Ibogaine Administration Modifies GDNF and BDNF Expression in Brain Regions Involved in Mesocorticolimbic and Nigral Dopaminergic Circuits.

Ibogaine is an atypical psychedelic alkaloid, which has been subject of research due to its reported ability to attenuate drug-seeking behavior. Recent work has suggested that ibogaine effects on alcohol self-administration in rats are related to the release of Glial cell Derived Neurotrophic Factor (GDNF) in the Ventral Tegmental Area (VTA), a mesencephalic region which hosts the soma of dopaminergic neurons. Although previous reports have shown ibogaine's ability to induce GDNF expression in rat midbrain, there are no studies addressing its effect on the expression of GDNF and other neurotrophic factors (NFs) such as Brain Derived Neurotrophic Factor (BDNF) or Nerve Growth Factor (NGF) in distinct brain regions containing dopaminergic neurons. In this work, we examined the effect of ibogaine acute administration on the expression of these NFs in the VTA, Prefrontal Cortex (PFC), Nucleus Accumbens (NAcc) and the Substantia Nigra (SN). Rats were i.p. treated with ibogaine 20 mg/kg (I20), 40 mg/kg (I40) or vehicle, and NFs expression was analyzed after 3 and 24 h. At 24 h an increase of the expression of the NFs transcripts was observed in a site and dose dependent manner. Only for I40, GDNF was selectively upregulated in the VTA and SN. Both doses elicited a large increase in the expression of BDNF transcripts in the NAcc, SN and PFC, while in the VTA a significant effect was found only for I40. Finally, NGF mRNA was upregulated in all regions after I40, while I20 showed a selective upregulation in PFC and VTA. Regarding protein levels, an increase of GDNF was observed in the VTA only for I40 but no significant increase for BDNF was found in all the studied areas. Interestingly, an increase of proBDNF was detected in the NAcc for both doses. These results show for the first time a selective increase of GDNF specifically in the VTA for I40 but not for I20 after 24 h of administration, which agrees with the effective dose found in previous self-administration studies in rodents. Further research is needed to understand the contribution of these changes to ibogaine's ability to attenuate drug-seeking behavior.

High Efficiency Binding Aptamers for a Wide Range of Bacterial Sepsis Agents.

Sepsis is a major health problem worldwide, with an extremely high rate of morbidity and mortality, partly due to delayed diagnosis during early disease. Currently, sepsis diagnosis requires bacterial culturing of blood samples over several days, whereas PCR-based molecular diagnosis methods are faster but lack sensitivity. The use of biosensors containing nucleic acid aptamers that bind targets with high affinity and specificity could accelerate sepsis diagnosis. Previously, we used the systematic evolution of ligands by exponential enrichment technique to develop the aptamers Antibac1 and Antibac2, targeting the ubiquitous bacterial peptidoglycan. Here, we show that these aptamers bind to four gram-positive and seven gram-negative bacterial sepsis agents with high binding efficiency. Thus, these aptamers could be used in combination as biological recognition elements in the development of biosensors that are an alternative to rapid bacteria detection, since they could provide culture and amplification-free tests for rapid clinical sepsis diagnosis.

Isolation of an Aptamer that Binds Specifically to E. coli.

Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers-called P12-31-is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies.

Anti-HCV RNA Aptamers Targeting the Genomic cis-Acting Replication Element.

Hepatitis C virus (HCV) replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE), located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80%) of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.

In vitro and ex vivo selection procedures for identifying potentially therapeutic DNA and RNA molecules.

It was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. Quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. In vitro selection and evolution strategies have been extremely useful in the analysis of functional RNA and DNA molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize DNA and RNA molecules with potential therapeutic and diagnostic applications. The great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. This review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids.