MicroRNAs (miRNAs) are post-transcriptional gene expression regulators, playing key roles in neuronal development, plasticity and disease. Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the presence of protein inclusions or Lewy bodies and a progressive loss of dopaminergic neurons in the midbrain. Here, we have evaluated miRNA expression deregulation in PD brain samples. MiRNA expression profiling revealed decreased expression of miR-34b and miR-34c in brain areas with variable neuropathological affectation at clinical (motor) stages (Braak stages 4 and 5) of the disease, including the amygdala, frontal cortex, substantia nigra and cerebellum. Furthermore, misregulation of miR-34b/c was detected in pre-motor stages (stages 1-3) of the disease, and thus in cases that did not receive any PD-related treatment during life. Depletion of miR-34b or miR-34c in differentiated SH-SY5Y dopaminergic neuronal cells resulted in a moderate reduction in cell viability that was accompanied by altered mitochondrial function and dynamics, oxidative stress and reduction in total cellular adenosin triphosphate content. MiR-34b/c downregulation was coupled to a decrease in the expression of DJ1 and Parkin, two proteins associated to familial forms of PD that also have a role in idiopathic cases. Accordingly, DJ1 and Parkin expression was reduced in PD brain samples displaying strong miR-34b/c downregulation. We propose that early deregulation of miR-34b/c in PD triggers downstream transcriptome alterations underlying mitochondrial dysfunction and oxidative stress, which ultimately compromise cell viability. A better understanding of the cellular pathways controlling and/or controlled by miR-34b/c should allow identification of targets for development of therapeutic approaches.

Lingo-1 (also known as Lern1) is a component of the Nogo receptor complex that mediates intracellular signaling in response to myelin associated inhibitors (MAIs): NogoA, MAG, and Omgp. Signaling through Nogo receptor extends to more than its well known role in preventing axon regeneration after lesion in the CNS, being implicated in neuronal functional maturation. Using Lingo-1-deficient mice, it has been demonstrated that Lingo-1 plays relevant roles in oligodendrocyte differentiation during brain development, and that treatment with Lingo-1 antagonists can improve axon regeneration after lesion in adult mice by decreasing MAI mediated signaling. However, a detailed description of the pattern of expression of Lingo-1 protein in correlation with the other partners of Nogo receptor is missing. Here, we show that components of the Nogo receptor complex, Lingo-1, NgR1, p75, and TROY coexist in mouse brain in a defined time window only at later postnatal stages. We have also determined the Lingo-1 distribution showing expression in particular subsets of neurons, but not in myelinating mature oligodendrocytes. Surprisingly, Lingo-1 is expressed at early developmental stages without NgR1, which supports the notion that Lingo-1 may participate in other activities in developing neurons different from oligodendrocyte maturation or axon extension inhibition in the adult. Finally, we propose that the intracellular domain of Lingo-1 contributes to signaling and show that it interacts with the postmitotic neuronal specific zinc finger protein Myt1l, suggesting that Lingo-1 may regulate Myt1l transcription factor activity by affecting its subcellular localization.

Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity.