Intermediate filament aggregates cause mitochondrial dysmotility and increase energy demands in giant axonal neuropathy.

Intermediate filaments (IFs) are cytoskeletal polymers that extend from the nucleus to the cell membrane, giving cells their shape and form. Abnormal accumulation of IFs is involved in the pathogenesis of number neurodegenerative diseases, but none as clearly as giant axonal neuropathy (GAN), a ravaging disease caused by mutations in GAN, encoding gigaxonin. Patients display early and severe degeneration of the peripheral nervous system along with IF accumulation, but it has been difficult to link GAN mutations to any particular dysfunction, in part because GAN null mice have a very mild phenotype. We therefore established a robust dorsal root ganglion neuronal model that mirrors key cellular events underlying GAN. We demonstrate that gigaxonin is crucial for ubiquitin-proteasomal degradation of neuronal IF. Moreover, IF accumulation impairs mitochondrial motility and is associated with metabolic and oxidative stress. These results have implications for other neurological disorders whose pathology includes IF accumulation.

Calcium entry and α-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons.

The core motor symptoms of Parkinson's disease (PD) are attributable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed a major factor in PD pathogenesis. Previous work has shown that activity-dependent calcium entry through L-type channels elevates perinuclear mitochondrial oxidant stress in SNc dopaminergic neurons, providing a potential basis for their selective vulnerability. What is less clear is whether this physiological stress is present in dendrites and if Lewy bodies, the major neuropathological lesion found in PD brains, exacerbate it. To pursue these questions, mesencephalic dopaminergic neurons derived from C57BL/6 transgenic mice were studied in primary cultures, allowing for visualization of soma and dendrites simultaneously. Many of the key features of in vivo adult dopaminergic neurons were recapitulated in vitro. Activity-dependent calcium entry through L-type channels increased mitochondrial oxidant stress in dendrites. This stress progressively increased with distance from the soma. Examination of SNc dopaminergic neurons ex vivo in brain slices verified this pattern. Moreover, the formation of intracellular α-synuclein Lewy-body-like aggregates increased mitochondrial oxidant stress in perinuclear and dendritic compartments. This stress appeared to be extramitochondrial in origin, because scavengers of cytosolic reactive oxygen species or inhibition of NADPH oxidase attenuated it. These results show that physiological and proteostatic stress can be additive in the soma and dendrites of vulnerable dopaminergic neurons, providing new insight into the factors underlying PD pathogenesis.

Mechanisms of Medial Wall Thinning in Chronic Total Occlusion.

BACKGROUND: The success rate of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) is lower and the risk for complications higher compared with other non-CTO PCI. Although interventionalists focus on intimal plaque characteristics, the coronary media is an important (especially for techniques involving antegrade dissection and re-entry) but poorly understood structure in CTO PCI. OBJECTIVES: The aim of the present study was to investigate coronary medial wall thinning in CTO lesions and determine how this thinning might affect CTO PCI. METHODS: A total of 2,586 sections were investigated, from arteries with evidence of CTO from 54 subjects (1,383 sections) and arteries without evidence of CTO from 54 subjects with non-coronary-related deaths (1,203 sections) after matching for age, gender, body weight, and body height. RESULTS: The medial thickness in subjects with CTO was lower than that in those with non-coronary-related death (P < 0.001). In subjects with CTO, CTO lesions had thinner medial walls compared with those with lower luminal narrowing (P < 0.001). At the CTO distal segments, the 6- to 12-mm distal segment from the distal end of the CTO had significantly less luminal narrowing (P < 0.001), and similar medial thickness, compared with the distal end of the CTO. Immunohistochemical analysis revealed that short-duration CTO had more cleaved caspase-3-positive cells in media and had significantly more CD3+, CD4+, CD8+, and CD4+CD28null T cells compared with long-duration CTO. CONCLUSIONS: CTO lesions demonstrated coronary medial thinning compared with non-CTO lesions. Further investigation of the cause-and-effect relationship among inflammation, apoptosis, and coronary medial wall thinning is warranted in future mechanistic studies.

Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor.

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms.

Cocaine-induced endocannabinoid signaling mediated by sigma-1 receptors and extracellular vesicle secretion.

Cocaine is an addictive drug that acts in brain reward areas. Recent evidence suggests that cocaine stimulates synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in midbrain, increasing dopamine neuron activity via disinhibition. Although a mechanism for cocaine-stimulated 2-AG synthesis is known, our understanding of 2-AG release is limited. In NG108 cells and mouse midbrain tissue, we find that 2-AG is localized in non-synaptic extracellular vesicles (EVs) that are secreted in the presence of cocaine via interaction with the chaperone protein sigma-1 receptor (Sig-1R). The release of EVs occurs when cocaine causes dissociation of the Sig-1R from ADP-ribosylation factor (ARF6), a G-protein regulating EV trafficking, leading to activation of myosin light chain kinase (MLCK). Blockade of Sig-1R function, or inhibition of ARF6 or MLCK also prevented cocaine-induced EV release and cocaine-stimulated 2-AG-modulation of inhibitory synapses in DA neurons. Our results implicate the Sig-1R-ARF6 complex in control of EV release and demonstrate that cocaine-mediated 2-AG release can occur via EVs.

Phasic Dopamine Signals in the Nucleus Accumbens that Cause Active Avoidance Require Endocannabinoid Mobilization in the Midbrain.

Phasic dopamine (DA) release accompanies approach toward appetitive cues. However, a role for DA in the active avoidance of negative events remains undetermined. Warning signals informing footshock avoidance are associated with accumbal DA release, whereas depression of DA is observed with unavoidable footshock. Here, we reveal a causal role of phasic DA in active avoidance learning; specifically, optogenetic activation of DA neurons facilitates avoidance, whereas optical inhibition of these cells attenuates it. Furthermore, stimulation of DA neurons during presentation of a fear-conditioned cue accelerates the extinction of a passive defensive behavior (i.e., freezing). Dopaminergic control of avoidance requires endocannabinoids (eCBs), as perturbing eCB signaling in the midbrain disrupts avoidance, which is rescued by optical stimulation of DA neurons. Interestingly, once the avoidance task is learned, neither DA nor eCB manipulations affect performance, suggesting that once acquisition occurs, expression of this behavior is subserved by other anatomical frameworks. Our findings establish an instrumental role for DA release in learning active responses to aversive stimuli and its control by eCB signaling.

Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.

Neuroblasts migrate long distances in the postnatal subventricular zone (SVZ) and rostral migratory stream (RMS) to the olfactory bulbs. Many fundamental features of SVZ migration are still poorly understood, and we addressed several important questions using two-photon time-lapse microscopy of brain slices from postnatal and adult eGFP(+) transgenic mice. 1) Longitudinal arrays of neuroblasts, so-called chain migration, have never been dynamically visualized in situ. We found that neuroblasts expressing doublecortin-eGFP (Dcx-eGFP) and glutamic acid decarboxylase-eGFP (Gad-eGFP) remained within arrays, which maintained their shape for many hours, despite the fact that there was a wide variety of movement within arrays. 2) In the dorsal SVZ, neuroblasts migrated rostrocaudally as expected, but migration shifted to dorsoventral orientations throughout ventral regions of the lateral ventricle. 3) Whereas polarized bipolar morphology has been a gold standard for inferring migration in histologic sections, our data indicated that migratory morphology was not predictive of motility. 4) Is there local motility in addition to long distance migration? 5) How fast is SVZ migration? Unexpectedly, one-third of motile neuroblasts moved locally in complex exploratory patterns and at average speeds slower than long distance movement. 6) Finally, we tested, and disproved, the hypothesis that all motile cells in the SVZ express doublecortin, indicating that Dcx is not required for migration of all SVZ cell types. These data show that cell motility in the SVZ and RMS is far more complex then previously thought and involves multiple cell types, behaviors, speeds, and directions.

RNA and protein synthesis is required for Ancylostoma caninum larval activation.

The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 µM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 µM), cycloheximide (IC(50)=50 µM), and anisomycin (IC(50)=200 µM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h. None of the RNA or protein synthesis inhibitors prevented secretion of the activation-associated protein ASP-1, despite nearly complete inhibition of feeding. The results indicate that unlike dauer recovery in C. elegans, de novo gene expression is required for hookworm larval activation, and the critical genes are expressed within 12h of exposure to activating stimuli. However, secretion of infection-associated proteins is independent of gene expression, indicating that the proteins are pre-synthesized and stored for rapid release during the initial stages of infection. The genes that are inhibited represent a subset of those required for the transition to parasitism, and therefore represent interesting targets for further investigation. Furthermore, while dauer recovery provides a useful model for hookworm infection, the differences identified here highlight the importance of exercising caution before making generalizations about parasitic nematodes based on C. elegans biology.