BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease.

Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

Mitochondrial Reactive Oxygen Species Mediate Activation of TRPV1 and Calcium Entry Following Peripheral Sensory Axotomy.

Axons that are physically separated from their soma activate a series of signaling events that results in axonal self-destruction. A critical element of this signaling pathway is an intra-axonal calcium rise that occurs just prior to axonal fragmentation. Previous studies have shown that preventing this calcium rise delays the onset of axon fragmentation, yet the ion channels responsible for the influx, and the mechanisms by which they are activated, are largely unknown. Axonal injury can be modeled in vitro by transecting murine dorsal root ganglia (DRG) sensory axons. We coupled transections with intra-axonal calcium imaging and found that Ca2+ influx is sharply reduced in axons lacking trpv1 (for transient receptor potential cation channel vanilloid 1) and in axons treated with capsazepine (CPZ), a TRPV1 antagonist. Sensory neurons from trpv1 -/- mice were partially rescued from degeneration after transection, indicating that TRPV1 normally plays a pro-degenerative role after axonal injury. TRPV1 activity can be regulated by direct post-translational modification induced by reactive oxygen species (ROS). Here, we tested the hypothesis that mitochondrial ROS production induced by axotomy is required for TRPV1 activity and subsequent axonal degeneration. We found that reducing mitochondrial depolarization with NAD+ supplementation or scavenging ROS using NAC or MitoQ sharply attenuates TRPV1-dependent calcium influx induced by axotomy. This study shows that ROS-dependent TRPV1 activation is required for Ca2+ entry after axotomy.

Local TrkB signaling: themes in development and neural plasticity.

The sensitivity of the nervous system to receive and respond to events, both internal and in the environment, depends on the ability of neural structures to remodel in response to experience (Kandel 2001; Mayford et al. 2012)⁠. Neural plasticity depends on rapid, tightly controlled rearrangements of cytoskeleton, membrane morphology, and protein content. Neurons regulate plasticity across orders of structural organization, from changes in molecular machinery that calls forth the synaptic alterations that underlie learning and memory, to events that evoke mesoscale alterations in neurite architecture, and to the birth and death of neurons. We address the concept that the events responsible for such diverse modification of neurons originate from local changes in signaling and that understanding the underlying mechanisms requires an appreciation of the nature of constraints placed upon spatial and temporal activity. During development and in the adult, both the remodeling of specific subcellular structures and induction of synaptic plasticity require local control and regulation of signaling, including those initiated by activation of surface receptors (Reichardt 2006). As an example, the receptor tyrosine kinase TrkB, activated by its ligand brain-derived neurotrophic factor (BDNF), has emerged as a potent modulator of plasticity in both development and adulthood, from neurite pruning and branching events during PNS and CNS development, to learning and memory. Here, we review the mechanisms by which TrkB signaling engages in local remodeling to support neural plasticity.

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury.

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4-/- mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4-/- mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4-/- mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.

Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx.

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro. We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca2+, and here, we address the mechanism of Ca2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca2+ influx in sensory axon degeneration.

A novel method for quantifying axon degeneration.

Axons normally degenerate during development of the mammalian nervous system, but dysregulation of the same genetically-encoded destructive cellular machinery can destroy crucial structures during adult neurodegenerative diseases. Nerve growth factor (NGF) withdrawal from dorsal root ganglia (DRG) axons is a well-established in vitro experimental model for biochemical and cell biological studies of developmental degeneration. Definitive methods for measuring axon degeneration have been lacking and here we report a novel method of axon degeneration quantification from bulk cultures of DRG that enables objective and automated measurement of axonal density over the entire field of radial axon outgrowth from the ganglion. As proof of principal, this new method, written as an R script called Axoquant 2.0, was used to examine the role of extracellular Ca2+ in the execution of cytoskeletal disassembly during degeneration of NGF-deprived DRG axons. This method can be easily applied to examine degenerative or neuroprotective effects of gene manipulations and pharmacological interventions.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Sildenafil Improves Brain Injury Recovery following Term Neonatal Hypoxia-Ischemia in Male Rat Pups.

Term asphyxiated newborns remain at risk of developing brain injury despite available neuropreventive therapies such as hypothermia. Neurorestorative treatments may be an alternative. This study investigated the effect of sildenafil on brain injury induced by neonatal hypoxia-ischemia (HI) at term-equivalent age. Neonatal HI was induced in male Long-Evans rat pups at postnatal day 10 (P10) by left common carotid ligation followed by a 2-hour exposure to 8% oxygen; sham-operated rat pups served as the control. Both groups were randomized to oral sildenafil or vehicle twice daily for 7 consecutive days. Gait analysis was performed on P27. At P30, the rats were sacrificed, and their brains were extracted. The surfaces of both hemispheres were measured on hematoxylin and eosin-stained brain sections. Mature neurons and endothelial cells were quantified near the infarct boundary zone using immunohistochemistry. HI caused significant gait impairment and a reduction in the size of the left hemisphere. Treatment with sildenafil led to an improvement in the neurological deficits as measured by gait analysis, as well as an improvement in the size of the left hemisphere. Sildenafil, especially at higher doses, also caused a significant increase in the number of neurons near the infarct boundary zone. In conclusion, sildenafil administered after neonatal HI may improve brain injury recovery by promoting neuronal populations.

Sildenafil Improves Functional and Structural Outcome of Retinal Injury Following Term Neonatal Hypoxia-Ischemia.

PURPOSE: The purpose of this study was to investigate the effects of sildenafil on retinal injury following neonatal hypoxia-ischemia (HI) at term-equivalent age in rat pups. METHODS: Hypoxia-ischemia was induced in male Long-Evans rat pups at postnatal day 10 (P10) by a left common carotid ligation followed by a 2-hour exposure to 8% oxygen. Sham-operated rats served as the control group. Both groups were administered vehicle or 2, 10, or 50 mg/kg sildenafil, twice daily for 7 consecutive days. Retinal function was assessed by flash electroretinograms (ERGs) at P29, and retinal structure was assessed by retinal histology at P30. RESULTS: Hypoxia-ischemia caused significant functional (i.e., attenuation of the ERG a-wave and b-wave amplitudes and photopic negative response) and structural (i.e., thinning of the total retina, especially the inner retinal layers) retinal damage in the left eyes (i.e., ipsilateral to the carotid ligation). Treatment with the different doses of sildenafil led to a dose-dependent increase in the amplitudes of the ERG a- and b-waves and of the photopic negative response in HI animals, with higher doses associated with greater effect sizes. Similarly, a dose response was observed in terms of improvements in the retinal layer thicknesses. CONCLUSIONS: Hypoxia-ischemia at term-equivalent age induced functional and structural damage mainly to the inner retina. Treatment with sildenafil provided a dose-dependent recovery of retinal function and structure.

The X-linked inhibitor of apoptosis regulates long-term depression and learning rate.

Hippocampal long-term depression (LTD) is an active form of synaptic plasticity that is necessary for consolidation of spatial memory, contextual fear memory, and novelty acquisition. Recent studies have shown that caspases (CASPs) play an important role in NMDA receptor-dependent LTD and are involved in postsynaptic remodeling and synaptic maturation. In the present study, we examined the role of X-linked inhibitor of apoptosis (XIAP), a putative endogenous CASP inhibitor, in synaptic plasticity in the hippocampus. Analysis in acute brain slices and in cultured hippocampal neurons revealed that XIAP deletion increases CASP-3 activity, enhances α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization, sharply increases LTD, and significantly reduces synapse density. In vivo behaviors related to memory were also altered in XIAP(-/-) mice, with faster acquisition of spatial object location and increased fear memory observed. Together, these results indicate that XIAP plays an important physiologic role in regulating sublethal CASP-3 activity within central neurons and thereby facilitates synaptic plasticity and memory acquisition.-Gibon, J., Unsain, N., Gamache, K., Thomas, R. A., De Leon, A., Johnstone, A., Nader, K., Séguéla, P., Barker, P. A. The X-linked inhibitor of apoptosis regulates long-term depression and learning rate.

XIAP regulates caspase activity in degenerating axons.

Our knowledge of the destructive events that regulate axonal degeneration is rudimentary. Here, we examine the role of caspases and their endogenous inhibitor, the X-linked inhibitor of apoptosis protein (XIAP), in axonal degeneration of dorsal root ganglion (DRG) axons. We show that caspase-3, caspase-6, and caspase-9 are present in axons and are cleaved upon nerve growth factor (NGF) withdrawal. We observed that caspase-3 activity is high in NGF-withdrawn axons and that CASP3(-/-) axons are protected from degeneration. XIAP(-/-) DRG sensory neurons degenerate more rapidly and contain more active caspase-3 than their wild-type counterparts, indicating that axonal caspases are normally regulated by XIAP. Importantly, axonal XIAP levels drop sharply after NGF withdrawal; if XIAP levels are maintained by overexpression, axonal caspase-3 activation and axonal degeneration are suppressed. Finally, we show that XIAP(-/-) embryos have stunted dermal innervation. We propose that XIAP-mediated caspase inhibition plays an important role in regulating morphogenic events that shape the nervous system during development.

Plants, like mammals, but unlike Saccharomyces, do not regulate nuclear-cytoplasmic tRNA trafficking in response to nutrient stress.

Cells respond to nutrient stress by regulating gene transcription and various key metabolic processes, including ribosome biogenesis and protein synthesis. Several studies have shown that yeasts and mammalian cells also regulate export of tRNAs from the nucleus to the cytosol in response to nutrient stress. However, nuclear export of tRNA in mammalian cells during nutrient stress is controversial, as it has been recently shown that nuclear-cytoplasmic transport of tRNAs in several mammalian cell lines is not affected by nutrient deprivation. Furthermore, contrary to previous studies, data reported recently indicate that nuclear export of mature tRNAs derived from intron-containing precursor tRNAs, but not tRNAs made from intronless precursors, is affected by nutrient availability in several Saccharomyces species, although not in Kluyveromyces lactis and Schizosaccharomyces pombe. Here, we report that plants, like mammals and some yeasts, but unlike Saccharomyces, do not directly regulate nuclear export of tRNA in response to nutrient stress, indicating that this process is not entirely conserved among evolutionarily diverse organisms.