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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.

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 Ca 2+ 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 Ca 2+ entry after axotomy.

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.

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.

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.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.

INTRODUCTION Down syndrome (DS) markedly raises the risk of Alzheimer's disease (DS‐AD). Our findings identified widespread dysregulation of the endolysosomal network (ELN) in DS and DS‐AD brains, driven by increased APP gene dose, hyperactivation of RAB5, and elevated levels of guanine nucleotide exchange factors (GEFs) for RABs 7 and 11. METHODS We investigated whether increasing APP gene dose and RAB5 hyperactivation contributed to neuropathogenesis and whether a clinically feasible intervention could reverse ELN changes. The Dp16 DS‐AD mouse model was treated with a mouse App‐specific antisense oligonucleotide (App‐ASO) and Rab5‐specific ASOs targeting Rab5a and Rab5b. RESULTS App‐ASO treatment normalized full‐length APP (fl‐APP) and its products, RAB5 activity, and downstream RABs 7 and 11 pathways. Rab5‐ASOs reduced RAB5 levels and restored endosomal Rab activity. Both ASO treatments mitigated DS‐AD‐linked pathologies. DISCUSSION These findings highlight ELN dysregulation in DS and the therapeutic potential of ASO‐based strategies targeting APP or Rab5 to counteract DS‐AD features. Highlights App‐ASO treatment reduced the levels of APP and its products and normalized endosomal Rab activity and GEF levels in Dp16 mice. Administration of Rab5‐ASOs reduced RAB5 levels and normalized endosomal Rab activity and GEF levels in Dp16 mice. Both ASO treatments were well tolerated and mitigated APP‐linked pathologies including tau hyperphosphorylation, neurotrophin signaling deficits, and synaptic protein loss. App‐ASO or Rab5‐ASOs reversed established pathological phenotypes in Dp16 mice.

Plasticity of neural structures, from synapses to neurites to entire neurons and networks, depends on tightly regulated equilibrium between cytoskeleton polymerization and depolymerisation mediated by protease activity. The conservation of signaling pathways governing cytoskeletal plasticity raises the prospect that understanding apoptotic-like degeneration can be leveraged to guide hypotheses exploring non-lethal sub-neuronal plasticity events in both the normal physiological functioning of the nervous system, and also in the earliest changes during neurodegenerative disease to halt their progression. Neurite pruning and neuronal cell death that occur normally during embryonic development establish and refine nervous systems into their mature patterning. Components of the same destructive signaling pathways appear to underlie neurodegenerative diseases such as Alzheimer’s, Parkinson’s and ALS when aberrantly reactivated in adulthood. The emerging overlap between developmental and pathological mechanisms of neurodegeneration suggests that therapeutic opportunities will be revealed by understanding the sequence of molecular events culminating in degeneration of neurites and entire neurons. Developmental axon degeneration can be modeled in vitro by nerve growth factor (NGF) withdrawal from dorsal root ganglion (DRG) neurons. We have used this model system together with a novel method of quantification to examine the role of Ca2+ influx in developmental axonal degeneration. We find that NGF deprivation from DRG sensory neurons induces a robust increase in axonal Ca2+ prior to membrane blebbing and degeneration. Chelation of divalent cations using EDTA rescues axons from degeneration when present for the final phase of NGF deprivation, indicating that cation influx plays a key role late in the degeneration cascade. Axon degeneration is significantly rescued by pharmacological inhibitors of Ca2+ channels, including capsazepine, an antagonist of transient receptor potential family member vanilloid 1 (TrpV1)-mediated Ca2+ flux, whereas inhibition of Na+ action potentials does not rescue axons. Cultured sensory neurons derived from TrpV1-null mouse embryos are partially rescued from degeneration, indicating a prodegenerative role for TrpV1 in vitro. We hypothesized that reactive oxygen species (ROS) mediate the activation of TrpV1 in this setting and consistent with this, ROS-scavengers and Nox complex inhibitors rescue axons from degeneration. Nox complexes are typically activated by PKC and we show that PKC inhibitors block degeneration whereas PMA, a PKC activator, induces potent NOXand ROS- dependent activation of TrpV1-dependent Ca2+ currents in DRG axons. We conclude that a PKC>Nox>ROS>TrpV1 axis induces toxic Ca2+ overload to drive developmental axon degeneration.

Huntingtons disease (HD) results from a CAG repeat expansion in the gene for Huntington (HTT) resulting in expansion of the polyglutamine (Q) tract in the mutant protein (mHTT). Synaptic changes are early manifestations of neuronal dysfunction in HD. However, the mechanism(s) by which mHTT impacts synapse formation and function is not well defined. Herein we explored HD pathogenesis in the BACHD and the deltaN17-BACHD mouse models of HD by examining cortical synapse formation and function in primary cultures maintained for up to 35 days (DIV35). We identified synapses by immunostaining with antibodies against pre-synaptic (Synapsin 1) and a post-synaptic (PSD95) marker. Consistent with earlier studies, cortical neurons from both WT and the HD models began to form synapses at DIV14; at this age there were no genotypic differences in synapse numbers. However, from DIV21 through DIV35 BACHD neurons showed progressively smaller numbers of synapses relative to WT neurons. Remarkably, BACHD synaptic deficits were completely rescued by treating cultures with BDNF. Building on earlier studies using reagents inspired by the chaperonin TRiC, we found that addition of the recombinant apical domain of CCT1 partially rescued synapse number. Unexpectedly, unlike BACHD cultures, synapses in deltaN17-BACHD cultures showed a progressive increase in number as compared to WT neurons, thus distinguishing synaptic changes in these HD models. Using multielectrode arrays, we discovered age-related functional deficits in BACHD cortical cultures with significant differences present by DIV28. As for synapse number, BDNF treatment prevented most synaptic deficits, including mean firing rate, spikes per burst, inter-burst interval, and synchrony. The apical domain of CCT1 showed similar, albeit less potent effects. These data are evidence that deficits in HD synapse number and function can be replicated in vitro and that treatment with either BDNF or a TRiC-inspired reagent can prevent them. Our findings support the use of cellular models to further explicate HD pathogenesis and its treatments.Competing Interest StatementThe authors have declared no competing interest.

Rivers limit the maximum elevation of active mountain belts, control the coupling between climate and tectonic processes, and archive the pace and tempo of fault-related rock uplift rates. Topographic profiles along rivers in steep, non-glaciated landscapes have led many to posit that river incision rates vary as a power function of channel discharge and slope. We used 10Be abundance in river sands and topographic analysis to test this relationship in watersheds varying by four orders of magnitude in erosion rate (4.7 × 10–3–7.1 mm yr−1), and supplemented this with a global analysis of erosion rates and topography. Our data and analyses reveal that in steep, rapidly eroding landscapes, channel morphology does not scale with erosion rate as expected. Instead, river profiles reach a threshold steepness, which may provide a bound on the topographic relief of Earth. In this case, increases in channel length may limit topographic relief, as erosion rate becomes increasingly sensitive to small changes in channel slopes in steep landscapes.