Action in the axon: generation and transport of signaling endosomes

Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of 'signaling endosomes'. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.     

Axonal transport and neuronal transcytosis of trophic factors, tracers, and pathogens

Neurons can specifically internalize macromolecules, such as trophic factors, lectins, toxins, and other pathogens. Upon internalization in terminals, proteins can move retrogradely along axons, or, upon internalization at somatodendritic domains, they can move into an anterograde axonal transport pathway. Release of internalized proteins from neurons after either retrograde or anterograde axonal transport results in transcytosis and trafficking of proteins across multiple synapses. Recent studies of binding properties of several such proteins suggest that pathogens and lectins may utilize existing transport machineries designed for trafficking of trophic factors. Specific pathways may protect trophic factors, pathogens, and toxins from degradation after internalization and may target the trophic or pathogenic cargo for transcytosis after either retrograde or anterograde transport along axons. Elucidating the molecular mechanisms of sorting steps and transport pathways will further our understanding of trophic signaling and could be relevant for an understanding and possible treatment of neurological diseases such as rabies, Alzheimer's disease, and prion encephalopathies. At present, our knowledge is remarkably sparse about the types of receptors used by pathogens for trafficking, the signals that sort trophins or pathogens into recycling or degradation pathways, and the mechanisms that regulate their release from somatodendritic domains or axon terminals. This review intends to draw attention to potential convergences and parallels in trafficking of trophic and pathogenic proteins. It discusses axonal transport/trafficking mechanisms that may help to understand and eventually treat neurological diseases by targeted drug delivery.     

A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling

A fundamental question in developmental biology is how a limited number of growth factors and their cognate receptors coordinate the formation of tissues and organs endowed with enormous morphological complexity. We report that the related neurotrophins NGF and NT-3, acting through a common receptor, TrkA, are required for sequential stages of sympathetic axon growth and, thus, innervation of target fields. Yet, while NGF supports TrkA internalization and retrograde signaling from distal axons to cell bodies to promote neuronal survival, NT-3 cannot. Interestingly, final target-derived NGF promotes expression of the p75 neurotrophin receptor, in turn causing a reduction in the sensitivity of axons to intermediate target-derived NT-3. We propose that a hierarchical neurotrophin signaling cascade coordinates sequential stages of sympathetic axon growth, innervation of targets, and survival in a manner dependent on the differential control of TrkA internalization, trafficking, and retrograde axonal signaling.     

HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology

Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission^™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease.     

Retrograde neurotrophin signaling: Trk-ing along the axon

Target-derived neurotrophins are required for the growth and survival of innervating neurons. When released by postsynaptic targets, neurotrophins bind receptors (Trks) on nerve terminals. Activated Trks signal locally within distal axons and retrogradely through long axons to distant cell bodies in order to promote gene expression and survival. Although the mechanism of retrograde neurotrophin signaling is not fully elucidated, considerable evidence supports a model in which the vesicular transport of neurotrophin-Trk complexes transmits a survival signal that involves PI3K and Erk5. Other, non-vesicular modes of retrograde signaling are likely to function in parallel. Recent studies highlight the importance of the location of stimulation as a determinant of Trk signaling. Defects in signaling from distal axons to cell bodies may be causally related to neurodegenerative disorders.     

Neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport

Recent studies suggest that excitotoxicity may contribute to neuronal damage in neurodegenerative diseases including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. Activated microglia have been observed around degenerative neurons in these diseases, and they are thought to act as effector cells in the degeneration of neural cells in the central nervous system. Neuritic beading, focal bead-like swellings in the dendrites and axons, is a neuropathological sign in epilepsy, trauma, ischemia, aging, and neurodegenerative diseases. Previous reports showed that neuritic beading is induced by various stimuli including glutamate or nitric oxide and is a neuronal response to harmful stimuli. However, the precise physiologic significance of neuritic beading is unclear. We provide evidence that neuritic beading induced by activated microglia is a feature of neuronal cell dysfunction toward neuronal death, and the neurotoxicity of activated microglia is mediated through N-methyl-d-aspartate (NMDA) receptor signaling. Neuritic beading occurred concordant with a rapid drop in intracellular ATP levels and preceded neuronal death. The actual neurite beads consisted of collapsed cytoskeletal proteins and motor proteins arising from impaired neuronal transport secondary to cellular energy loss. The drop in intracellular ATP levels was because of the inhibition of mitochondrial respiratory chain complex IV activity downstream of NMDA receptor signaling. Blockage of NMDA receptors nearly completely abrogated mitochondrial dysfunction and neurotoxicity. Thus, neuritic beading induced by activated microglia occurs through NMDA receptor signaling and represents neuronal cell dysfunction preceding neuronal death. Blockage of NMDA receptors may be an effective therapeutic approach for neurodegenerative diseases.     

On Trk for retrograde signaling.

Target-derived neurotrophins like nerve growth factor (NGF) mediate biological effects by binding to and activating Trk neurotrophin receptors at nerve terminals. The activated Trk receptors then stimulate local effects at nerve terminals, and retrograde effects at neuronal cell bodies that often reside at considerable distances from the terminals. However, the nature of the retrograde signal has been mysterious. Recent experiments suggest that the major retrograde signal required for survival and gene expression consists of activated Trk itself. Remarkably, signaling by Trk may differ at the terminal versus the neuronal cell body as a consequence of the retrograde transport mechanism, thereby allowing NGF to not only promote growth locally, but to specifically support survival and gene expression retrogradely.     

A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer's disease

Among the earliest invariant neuropathological changes in Alzheimer's disease (AD) is the degeneration of vulnerable hippocampal CA1 and subicular pyramidal neurons. Semaphorin 3A (Sema3A) is a secreted protein that functions in signaling growth cone collapse, chemorepulsion and neuronal apoptosis during early development of the central nervous system. In this report we show that accumulation of an internalized form of Sema3A is associated with degeneration of neurons in vulnerable fields of the hippocampus during AD. Accumulation of Sema3A overlaps the appearance of phosphorylated MAP1B and tau in many neurons, suggesting that Sema3A signaling at some level may be coupled to these previously identified cytoskeletal markers of neurodegeneration. Consistent with this, we isolated and partially characterized a multiprotein complex from the hippocampus of patients with AD that contains phosphorylated MAP1B, collapsin-response mediator protein 2 (CRMP-2), Plexins A1 and A2, and a processed form of Sema3A. A model is presented in which aberrant release of Sema3A from expressing neurons in the subiculum during AD results in the internalization and transport of Sema3A from this field to CA1. Within the context of the myriad of potential insults that contribute to Alzheimer's and other neurodegenerative diseases, the bioactivity of Sema3A may contribute either directly to neurodegeneration by inducing neuronal collapse, or indirectly by abrogating the recovery capabilities of adult neurons faced with these insults.     

Neuron-targeted caveolin-1 protein enhances signaling and promotes arborization of primary neurons

Decreased expression of prosurvival and progrowth-stimulatory pathways, in addition to an environment that inhibits neuronal growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration. Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav) are essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors and prosurvival kinases and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1 vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated prosurvival kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth occurred even in the presence of inhibitory cytokines (TNFα, IL-1β) and myelin-associated glycoproteins (MAG, Nogo). Targeting of Cav-1 to neurons thus enhances prosurvival and progrowth signaling and may be a novel means to repair the injured and neurodegenerative brain.     

Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.     

Prion neurodegeneration: Starts and stops at the synapse

Synaptic dysfunction is a key process in the evolution of many neurodegenerative diseases, with synaptic loss preceding that of neuronal cell bodies. In Alzheimer, Huntington, and prion diseases early synaptic changes correlate with cognitive and motor decline, and altered synaptic function may also underlie deficits in a number of psychiatric and neurodevelopmental conditions. The formation, remodelling and elimination of spines and synapses are continual physiological processes, moulding cortical architecture, underpinning the abilities to learn and remember. In disease, however, particularly in protein misfolding neurodegenerative disorders, lost synapses are not replaced and this loss is followed by neuronal death. These two processes are separately regulated, with mechanistic, spatial and temporal segregation of the death ‘routines’ of synapses and cell bodies. Recent insights into the reversibility of synaptic dysfunction in a mouse model of prion disease at neurophysiological, behavioral and morphological levels call for a deeper analysis of the mechanisms underlying neurotoxicity at the synapse, and have important implications for therapy of prion and other neurodegenerative disorders.Key words: neurodegeneration, prion, synaptic dysfunction, behavior, neurophysiology     

Retrograde transport of neurotrophins: fact and function

Retrograde signals generated by nerve growth factor (NGF) and other neurotrophins promote the survival of appropriately connected neurons during development, and failure to obtain sufficient retrograde signals may contribute to neuronal death occurring in many neurodegenerative diseases. The discovery over 25 years ago that NGF supplied to the axon terminals is retrogradely transported to the cell bodies suggested that NGF must reach the cell body to promote neuronal survival. Research during the intervening decades has produced a refinement of this hypothesis. The current hypothesis is that NGF bound to TrkA at the axon terminal is internalized into signaling endosomes, with NGF in their lumens bound to phosphorylated TrkA in their membranes, which are retrogradely transported to the cell bodies, where TrkA activates downstream signaling molecules that promote neuronal survival and regulate many aspects of neuronal gene expression. This model has been extrapolated to retrograde signaling by all neurotrophins. We consider the evidence for this model, focusing on results of experiments with neurons in compartmented cultures. Results to date indicate that while the transport of signaling endosomes containing NGF bound to TrkA may carry retrograde signals, retrograde survival signals can be carried by another mechanism that is activated by NGF at the axon terminal surface and travels to the cell body unaccompanied by the NGF that initiated it. It is hypothesized that multiple mechanisms of retrograde signaling exist and function under different circumstances. The newly discovered potential for redundancy in retrograde signaling mechanisms can complicate the interpretation of experimental results.     

The GSK3 beta signaling cascade and neurodegenerative disease

Biochemical signaling pathways are known to have a critical role in neuronal development and function. A growing body of evidence is accumulating to suggest that signaling pathways also underlie neurodegeneration and neurodegenerative disease. One pathway with a prominent role in neurodegenerative disease is the signaling pathway in which the enzyme glycogen synthase kinase 3 (GSK3) is a key component. In vitro and in vivo evidence point to a key role for GSK3 in promoting neurodegeneration and in Alzheimer's disease plaque and neurofibrillary tangle formation. How GSK3 acts in this regard is still open to debate, but it may involve both extracellular and nuclear apoptotic activities.     

Say NO to neurodegeneration: role of S-nitrosylation in neurodegenerative disorders

Nitric oxide (NO) is an important signaling molecule that controls a wide range of biological processes. One of the signaling mechanisms of NO is through the S-nitrosylation of cysteine residues on proteins. S-nitrosylation is now regarded as an important redox signaling mechanism in the regulation of different cellular and physiological functions. However, deregulation of S-nitrosylation has also been linked to various human diseases such as neurodegenerative disorders. Nitrosative stress has long been considered as a major mediator in the development of neurodegeneration, but the molecular mechanism of how NO can contribute to neurodegeneration is not completely clear. Early studies suggested that nitration of proteins, which can induce protein aggregation might contribute to the neurodegenerative process. However, several recent studies suggest that S-nitrosylation of proteins that are important for neuronal survival contributes substantially in the development of various neurodegenerative disorders. Thus, in-depth understanding of the mechanism of neurodegeneration in relation to S-nitrosylation will be critical for the development of therapeutic treatment against these neurodegenerative diseases.     

Colloquium 10: 3

Previous work has shown that neurotrophins bind to and activate Trk receptors on distal axons, and that neurotrophin‐Trk complexes are internalized and retrogradely transported to cell bodies. Whether retrograde transport of neurotrophins and retrograde neurotrophin‐Trk signalling are necessary for survival remains unclear, and recently published findings are controversial. We are using compartmentalized cultures of sympathetic neurons to address the mechanism of retrograde NGF signalling and survival. We performed survival experiments using either the Trk kinase inhibitor K252a to inhibit TrkA activity in different cellular compartments, or a dominant‐negative form of dynamin, K44A dynamin, to block internalization of NGF‐TrkA complexes. We found that sympathetic neurons supported by NGF acting on distal axons undergo apoptosis when TrkA activity in either cell bodies or distal axons is inhibited by K252a, or when internalization is blocked by K44A dynamin. Results of experiments employing three‐compartment chambers indicate that TrkA signalling is required within cell bodies and distal axons, but not in proximal axons, for retrograde support of survival. Likewise, TrkA activity within distal axons, but not in proximal axons, is required for retrograde transport of [125I] NGF. Finally, peptide‐mediated delivery of affinity‐purified anti‐NGF into cell bodies results in apoptosis of neurons. Taken together, our results support a model in which NGF internalization and retrograde transport and retrograde TrkA signalling are necessary for survival of sympathetic neurons. This work is supported by the NIH and HHMI.     

Tyrosine phosphatase SHP-2 is a mediator of activity-dependent neuronal excitotoxicity

Calcium influx can promote neuronal differentiation and survival, at least in part by activating Ras and its downstream targets, including the Erk pathway. However, excessive calcium influx can initiate molecular signals leading to neuronal death during excitotoxicity or in neurodegenerative diseases. Here we describe a new signaling pathway associated with calcium influx that contributes to neuronal cell death in cerebellar neurons. Influx of calcium, mediated either by L-type voltage-sensitive calcium channels or glutamate receptors, is associated with the suppression of brain-derived neurotrophic factor (BDNF) activation of Ras and its effectors Erk and Akt. This is the result of enhanced association of the tyrosine phosphatase Shp-2 with TrkB receptors, which inhibits BDNF-induced TrkB autophosphorylation and activation. Deletion of the Shp2 gene in neuronal cultures reverses inhibition of TrkB function and increases neuronal survival after extended depolarization or glutamate treatment. These findings implicate Shp-2 in a feedback system initiated by calcium that negatively regulates neurotrophin signaling and sensitizes neurons to excitotoxicity.     

A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.     

Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner

Glutamate released by activated microglia induces excitoneurotoxicity and may contribute to neuronal damage in neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. In addition, tumor necrosis factor-alpha (TNF-alpha) secreted from activated microglia may elicit neurodegeneration through caspase-dependent cascades and silencing cell survival signals. However, direct neurotoxicity of TNF-alpha is relatively weak, because TNF-alpha also increases production of neuroprotective factors. Accordingly, it is still controversial how TNF-alpha exerts neurotoxicity in neurodegenerative diseases. Here we have shown that TNF-alpha is the key cytokine that stimulates extensive microglial glutamate release in an autocrine manner by up-regulating glutaminase to cause excitoneurotoxicity. Further, we have demonstrated that the connexin 32 hemichannel of the gap junction is another main source of glutamate release from microglia besides glutamate transporters. Although pharmacological blockade of glutamate receptors is a promising therapeutic candidate for neurodegenerative diseases, the associated perturbation of physiological glutamate signals has severe adverse side effects. The unique mechanism of microglial glutamate release that we describe here is another potential therapeutic target. We rescued neuronal cell death in vitro by using a glutaminase inhibitor or hemichannel blockers to diminish microglial glutamate release without perturbing the physiological glutamate level. These drugs may give us a new therapeutic strategy against neurodegenerative diseases with minimum adverse side effects.