Diesel Exhaust Extract Exposure Induces Neuronal Toxicity by Disrupting Autophagy.

The vast majority of neurodegenerative disease cannot be attributed to genetic causes alone and as a result, there is significant interest in identifying environmental modifiers of disease risk. Epidemiological studies have supported an association between long-term exposure to air pollutants and disease risk. Here, we investigate the mechanisms by which diesel exhaust, a major component of air pollution, induces neurotoxicity. Using a zebrafish model, we found that exposure to diesel exhaust particulate extract caused behavioral deficits and a significant decrease in neuron number. The neurotoxicity was due, at least in part, to reduced autophagic flux, which is a major pathway implicated in neurodegeneration. This neuron loss occurred alongside an increase in aggregation-prone neuronal protein. Additionally, the neurotoxicity induced by diesel exhaust particulate extract in zebrafish was mitigated by co-treatment with the autophagy-inducing drug nilotinib. This study links environmental exposure to altered proteostasis in an in vivo model system. These results shed light on why long-term exposure to traffic-related air pollution increases neurodegenerative disease risk and open up new avenues for exploring therapies to mitigate environmental exposures and promote neuroprotection.

A novel transgenic zebrafish line allows for in vivo quantification of autophagic activity in neurons.

The pathophysiology of most neurodegenerative diseases includes aberrant accumulation of protein aggregates. Recent evidence highlights the role of protein degradation pathways in neurodegeneration. Concurrently, genetic tools have been generated to enable zebrafish, Danio rerio, to be used as an animal model to study neurodegenerative processes. In addition to optical clarity and fast ex utero development, the zebrafish brain is relatively small and has conserved structures with its mammalian counterparts. To take advantage of this model organism and to aid further studies on autophagy and neurodegeneration, we created a stable transgenic zebrafish line that expresses eGFP-Map1lc3b specifically in post-mitotic neurons under the elavl3 promoter. This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagy/autophagy-modulating compounds. We determined the applicability of this transgenic line by modulating and quantifying the number of autophagosomes via treatment with a known autophagy inducer (rapamycin) and inhibitors (3-methyladenine, protease inhibitors). Additionally, we proposed an in vivo method for quantifying rates of autophagosome accumulation, which can be used to infer occurrence of autophagic flux. Last, we tested two FDA-approved drugs currently undergoing clinical studies for Parkinson disease, isradipine and nilotinib, and found that isradipine did not modulate autophagy, whereas nilotinib induced both autophagosome number and autophagic flux. It is hoped that others will find this line useful as an in vivo vertebrate model to find or validate autophagy modulators that might be used to halt the progression of neurodegenerative diseases. Abbreviations: 3MA: 3-methyladenine; BafA: bafilomycin A1; dd: dorsal diencephalon; dpf: days post fertilization; e: eye; eGFP: enhanced green fluorescent protein; Elavl3: ELAV like neuron-specific RNA binding protein 3; FDA: Food and Drug Administration; hb: habenula; hpt, hours post treatment; Map1lc3b: microtubule-associated protein 1 light chain 3 beta; nt: neural tube; ot, optic tectum; P/E: pepstatin A and E64d; PD: Parkinson disease; PMTs: photomultiplier tubes; PTU: 1-phenyl-2-thiourea; Ta: annealing temperature; Tel, telencephalon.

A transcription factor network controls cell migration and fate decisions in the developing zebrafish pineal complex.

The zebrafish pineal complex consists of four cell types (rod and cone photoreceptors, projection neurons and parapineal neurons) that are derived from a single pineal complex anlage. After specification, parapineal neurons migrate unilaterally away from the rest of the pineal complex whereas rods, cones and projection neurons are non-migratory. The transcription factor Tbx2b is important for both the correct number and migration of parapineal neurons. We find that two additional transcription factors, Flh and Nr2e3, negatively regulate parapineal formation. Flh induces non-migratory neuron fates and limits the extent of parapineal specification, in part by activation of Nr2e3 expression. Tbx2b is positively regulated by Flh, but opposes Flh action during specification of parapineal neurons. Loss of parapineal neuron specification in Tbx2b-deficient embryos can be partially rescued by loss of Nr2e3 or Flh function; however, parapineal migration absolutely requires Tbx2b activity. We conclude that cell specification and migration in the pineal complex are regulated by a network of at least three transcription factors.

Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing.

We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post-fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish.

Failure in closure of the anterior neural tube causes left isomerization of the zebrafish epithalamus.

Differences between the left and right sides of the brain are present in many animal species. For instance, in humans the left cerebral hemisphere is largely responsible for language and tool use and the right for processing spatial information. Zebrafish have prominent left-right asymmetries in their epithalamus that have been associated with differential left and right eye use and navigational behavior. In wild-type (WT) zebrafish embryos, Nodal pathway genes are expressed in the left side of the pineal anlage. Shortly thereafter, a parapineal organ forms to the left of the pineal. The parapineal organ causes differences in gene expression, neuropil density, and connectivity of the left and right habenula nuclei. In embryos that have an open neural tube, such as embryos that are deficient in Nodal signaling or the cell adhesion protein N-cadherin, the left and right sides of the developing epithalamus remain separated from one another. We find that the brains of these embryos often become left isomerized: both sides of the brain develop morphology and gene expression patterns that are characteristic of the left side. However, other aspects of epithalamic development, such as differentiation of specific neuronal cell types, are intact. We propose that there is a mechanism in embryos with closed neural tubes that prevents both sides from developing like the left side. This mechanism fails when the two sides of the epithalamus are widely separated from one another, suggesting that it is dependent upon a signaling protein with limited range.