Blaise Bossy

Nitric oxide‐induced mitochondrial fission is regulated by dynamin‐related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant‐negative Dynamin related protein 1 (Drp1K38A) inhibits mitochondrial fission induced by NO, rotenone and Amyloid‐β peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO‐induced neuronal cell death was mitigated by Mfn1 and Drp1K38A. Thus, persistent mitochondrial fission may play a causal role in NO‐mediated neurotoxicity.

Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity.

Huntingtons disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntingtons disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntingtons disease in vitro and in a mouse model of Huntingtons disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntingtons disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin–mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntingtons disease.

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.

Complex II inhibition by 3-NP causes mitochondrial fragmentation and neuronal cell death via an NMDA- and ROS-dependent pathway.

Mitochondrial respiratory complex II inhibition plays a central role in Huntingtons disease (HD). Remarkably, 3-NP, a complex II inhibitor, recapitulates HD-like symptoms. Furthermore, decreases in mitochondrial fusion or increases in mitochondrial fission have been implicated in neurodegenerative diseases. However, the relationship between mitochondrial energy defects and mitochondrial dynamics has never been explored in detail. In addition, the mechanism of neuronal cell death by complex II inhibition remains unclear. Here, we tested the temporal and spatial relationship between energy decline, impairment of mitochondrial dynamics, and neuronal cell death in response to 3-NP using quantitative fluorescence time-lapse microscopy and cortical neurons. 3-NP caused an immediate drop in ATP. This event corresponded with a mild rise in reactive oxygen species (ROS), but mitochondrial morphology remained unaltered. Unexpectedly, several hours after this initial phase, a second dramatic rise in ROS occurred, associated with profound mitochondrial fission characterized by the conversion of filamentous to punctate mitochondria and neuronal cell death. Glutamate receptor antagonist AP5 abolishes the second peak in ROS, mitochondrial fission, and cell death. Thus, secondary excitotoxicity, mediated by glutamate receptor activation of the NMDA subtype, and consequent oxidative and nitrosative stress cause mitochondrial fission, rather than energy deficits per se. These results improve our understanding of the cellular mechanisms underlying HD pathogenesis.

Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha‐like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha‐subunits is also indicated by the failure of an ALS‐beta‐galactosidase fusion protein to bind alpha‐bungarotoxin on blots in contrast to vertebrate endplate alpha‐subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5′ leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system.

Mutant SOD1G93A triggers mitochondrial fragmentation in spinal cord motor neurons: neuroprotection by SIRT3 and PGC-1α.

Mutations in the Cu/Zn Superoxide Dismutase (SOD1) gene cause an inherited form of ALS with upper and lower motor neuron loss. The mechanism underlying mutant SOD1-mediated motor neuron degeneration remains unclear. While defects in mitochondrial dynamics contribute to neurodegeneration, including ALS, previous reports remain conflicted. Here, we report an improved technique to isolate, transfect, and culture rat spinal cord motor neurons. Using this improved system, we demonstrate that mutant SOD1(G93A) triggers a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria. The increase of fragmented mitochondria coincides with an arrest in both anterograde and retrograde axonal transport and increased cell death. In addition, mutant SOD1(G93A) induces a reduction in neurite length and branching that is accompanied with an abnormal accumulation of round mitochondria in growth cones. Furthermore, restoration of the mitochondrial fission and fusion balance by dominant-negative dynamin-related protein 1 (DRP1) expression rescues the mutant SOD1(G93A)-induced defects in mitochondrial morphology, dynamics, and cell viability. Interestingly, both SIRT3 and PGC-1α protect against mitochondrial fragmentation and neuronal cell death by mutant SOD1(G93A). This data suggests that impairment in mitochondrial dynamics participates in ALS and restoring this defect might provide protection against mutant SOD1(G93A)-induced neuronal injury.

Genetic activity along 315 kb of the Drosophila chromosome

Transcripts from different tissues were mapped along a 315 kb segment of the Drosophila chromosome, a region which includes the rosy and Ace loci. Forty‐three distinct RNA species were detected, though only 12 recessive lethal complementation groups had been mapped in the interval. The sum of the sizes of the transcripts covers 33% of the genomic DNA. The distribution of transcription units along the walk is very uneven. Sixty‐three kb of genomic DNA at the proximal end of the walk encode 18 transcripts, while only seven are found in the next 153 kb. Each tissue exhibits a specific spectrum of transcripts. No clustering was seen among genes expressed coordinately. In salivary glands, the number of transcripts detected corresponds to the number of chromomeric units in the polytene chromosomes of this tissue. Moreover, the density distribution of transcripts along the DNA walk is parallel to the density distribution of chromomeric units.

Loss of OPA1 disturbs cellular calcium homeostasis and sensitizes for excitotoxicity

Optic atrophy 1 (OPA1) mutations cause dominant optic atrophy (DOA) with retinal ganglion cell (RGC) and optic nerve degeneration. The mechanism for the selective degeneration of RGCs in DOA remains elusive. To address the mechanism, we reduced OPA1 protein expression in cell lines and RGCs by RNA interference. OPA1 loss results in mitochondrial fragmentation, deficiency in oxidative phosphorylation, decreased ATP levels, decreased mitochondrial Ca2+ retention capacity, reduced mtDNA copy numbers, and sensitization to apoptotic insults. We demonstrate profound cristae depletion and loss of crista junctions in OPA1 knockdown cells, whereas the remaining crista junctions preserve their normal size. OPA1-depleted cells exhibit decreased agonist-evoked mitochondrial Ca2+ transients and corresponding reduction of NAD+ to NADH, but the impairment in NADH oxidation leads to an overall more reduced mitochondrial NADH pool. Although in our model OPA1 loss in RGCs has no apparent impact on mitochondrial morphology, it decreases buffering of cytosolic Ca2+ and sensitizes RGCs to excitotoxic injury. Exposure to glutamate triggers delayed calcium deregulation (DCD), often in a reversible manner, indicating partial resistance of RGCs to this injury. However, when OPA1 is depleted, DCD becomes irreversible. Thus, our data show that whereas OPA1 is required for mitochondrial fusion, maintenance of crista morphology and oxidative phosphorylation, loss of OPA1 also results in defective Ca2+ homeostasis.

Assessing mitochondrial morphology and dynamics using fluorescence wide-field microscopy and 3D image processing.

Mitochondrial morphology and length change during fission and fusion and mitochondrial movement varies dependent upon the cell type and the physiological conditions. Here, we describe fundamental wide-field fluorescence microscopy and 3D imaging techniques to assess mitochondrial shape, number and length in various cell types including cancer cell lines, motor neurons and astrocytes. Furthermore, we illustrate how to assess mitochondrial fission and fusion events by 3D time-lapse imaging and to calculate mitochondrial length and numbers as a function of time. These imaging methods provide useful tools to investigate mitochondrial dynamics in health, aging and disease.

Clearing the brain's cobwebs: the role of autophagy in neuroprotection.

Protein aggregates or inclusion bodies are common hallmarks of age-related neurodegenerative disorders. Why these aggregates form remains unclear. Equally debated is whether they are toxic, protective, or simple by-products. Increasing evidence, however, supports the notion that in general aggregates confer toxicity and disturb neuronal function by hampering axonal transport, synaptic integrity, transcriptional regulation, and mitochondrial function. Thus, neuroscientists in search of effective treatments to slow neural loss during neurodegeneration have long been interested in finding new ways to clear inclusion bodies. Intriguingly, two studies using conditional neuron-specific gene ablations of autophagy regulators in mice revealed that autophagy loss elicits inclusion body formation and a neurodegenerative cascade.Such studies indicate autophagy may be a built-in defense mechanism to clear the nervous system of inclusion bodies.This new finding has implications for our understanding of aging and neurodegeneration and the development of new therapies. First, we discuss the pathways underlying autophagy and its controversial role in cell death and survival regulation.We then discuss the physiological role of autophagy in the aging process of the nervous system. In the final portion of this review, we discuss the therapeutic promise of inducing autophagy and the potential side effects of such treatments.