Excess phosphoserine-129 α-synuclein induces synaptic vesicle trafficking and declustering defects at a vertebrate synapse.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.

Lampreys and spinal cord regeneration: "a very special claim on the interest of zoologists," 1830s-present.

Employing history of science methods, including analyses of the scientific literature, archival documents, and interviews with scientists, this paper presents a history of lampreys in neurobiology from the 1830s to the present. We emphasize the lamprey's roles in helping to elucidate spinal cord regeneration mechanisms. Two attributes have long perpetuated studies of lampreys in neurobiology. First, they possess large neurons, including multiple classes of stereotypically located, 'identified' giant neurons in the brain, which project their large axons into the spinal cord. These giant neurons and their axonal fibers have facilitated electrophysiological recordings and imaging across biological scales, ranging from molecular to circuit-level analyses of nervous system structures and functions and including their roles in behavioral output. Second, lampreys have long been considered amongst the most basal extant vertebrates on the planet, so they have facilitated comparative studies pointing to conserved and derived characteristics of vertebrate nervous systems. These features attracted neurologists and zoologists to studies of lampreys between the 1830s and 1930s. But, the same two attributes also facilitated the rise of the lamprey in neural regeneration research after 1959, when biologists first wrote about the spontaneous, robust regeneration of some identified CNS axons in larvae after spinal cord injuries, coupled with recovery of normal swimming. Not only did large neurons promote fresh insights in the field, enabling studies incorporating multiple scales with existing and new technologies. But investigators also were able to attach a broad scope of relevance to their studies, interpreting them as suggesting conserved features of successful, and sometimes even unsuccessful, CNS regeneration. Lamprey research demonstrated that functional recovery takes place without the reformation of the original neuronal connections, for instance, by way of imperfect axonal regrowth and compensatory plasticity. Moreover, research performed in the lamprey model revealed that factors intrinsic to neurons are integral in promoting or hindering regeneration. As this work has helped illuminate why basal vertebrates accomplish CNS regeneration so well, whereas mammals do it so poorly, this history presents a case study in how biological and medical value have been, and could continue to be, gleaned from a non-traditional model organism for which molecular tools have been developed only relatively recently.

Hsc70 rescues the synaptic vesicle trafficking defects caused by α-synuclein dimers.

Aberrant buildup of α-synuclein is associated with Parkinson's disease (PD) and other neurodegenerative disorders. At synapses, α-synuclein accumulation leads to severe synaptic vesicle trafficking defects. We previously demonstrated that different molecular species of α-synuclein produce distinct effects on synaptic vesicle recycling, and that the synaptic phenotypes caused by monomeric α-synuclein were ameliorated by Hsc70. Here, we tested whether Hsc70 could also correct synaptic deficits induced by α-synuclein dimers. Indeed, co-injection of Hsc70 with α-synuclein dimers completely reversed the synaptic deficits, resulting in synapses with normal appearance. This work lends additional support for pursuing chaperone-based strategies to treat PD and other synucleinopathies.

Proprioceptive feedback amplification restores effective locomotion in a neuromechanical model of lampreys with spinal injuries.

Spinal injuries in many vertebrates can result in partial or complete loss of locomotor ability. While mammals often experience permanent loss, some nonmammals, such as lampreys, can regain swimming function, though the exact mechanism is not well understood. One hypothesis is that amplified proprioceptive (body-sensing) feedback can allow an injured lamprey to regain functional swimming even if the descending signal is lost. This study employs a multiscale, integrative, computational model of an anguilliform swimmer fully coupled to a viscous, incompressible fluid and examines the effects of amplified feedback on swimming behavior. This represents a model that analyzes spinal injury recovery by combining a closed-loop neuromechanical model with sensory feedback coupled to a full Navier-Stokes model. Our results show that in some cases, feedback amplification below a spinal lesion is sufficient to partially or entirely restore effective swimming behavior.

Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse.

We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870-61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.

Activating Transcription Factor 3 (ATF3) is a Highly Conserved Pro-regenerative Transcription Factor in the Vertebrate Nervous System.

The vertebrate nervous system exhibits dramatic variability in regenerative capacity across species and neuronal populations. For example, while the mammalian central nervous system (CNS) is limited in its regenerative capacity, the CNS of many other vertebrates readily regenerates after injury, as does the peripheral nervous system (PNS) of mammals. Comparing molecular responses across species and tissues can therefore provide valuable insights into both conserved and distinct mechanisms of successful regeneration. One gene that is emerging as a conserved pro-regenerative factor across vertebrates is activating transcription factor 3 (ATF3), which has long been associated with tissue trauma. A growing number of studies indicate that ATF3 may actively promote neuronal axon regrowth and regeneration in species ranging from lampreys to mammals. Here, we review data on the structural and functional conservation of ATF3 protein across species. Comparing RNA expression data across species that exhibit different abilities to regenerate their nervous system following traumatic nerve injury reveals that ATF3 is consistently induced in neurons within the first few days after injury. Genetic deletion or knockdown of ATF3 expression has been shown in mouse and zebrafish, respectively, to reduce axon regeneration, while inducing ATF3 promotes axon sprouting, regrowth, or regeneration. Thus, we propose that ATF3 may be an evolutionarily conserved regulator of neuronal regeneration. Identifying downstream effectors of ATF3 will be a critical next step in understanding the molecular basis of vertebrate CNS regeneration.

Synuclein Regulates Synaptic Vesicle Clustering and Docking at a Vertebrate Synapse.

Neurotransmission relies critically on the exocytotic release of neurotransmitters from small synaptic vesicles (SVs) at the active zone. Therefore, it is essential for neurons to maintain an adequate pool of SVs clustered at synapses in order to sustain efficient neurotransmission. It is well established that the phosphoprotein synapsin 1 regulates SV clustering at synapses. Here, we demonstrate that synuclein, another SV-associated protein and synapsin binding partner, also modulates SV clustering at a vertebrate synapse. When acutely introduced to unstimulated lamprey reticulospinal synapses, a pan-synuclein antibody raised against the N-terminal domain of α-synuclein induced a significant loss of SVs at the synapse. Both docked SVs and the distal reserve pool of SVs were depleted, resulting in a loss of total membrane at synapses. In contrast, antibodies against two other abundant SV-associated proteins, synaptic vesicle glycoprotein 2 (SV2) and vesicle-associated membrane protein (VAMP/synaptobrevin), had no effect on the size or distribution of SV clusters. Synuclein perturbation caused a dose-dependent reduction in the number of SVs at synapses. Interestingly, the large SV clusters appeared to disperse into smaller SV clusters, as well as individual SVs. Thus, synuclein regulates clustering of SVs at resting synapses, as well as docking of SVs at the active zone. These findings reveal new roles for synuclein at the synapse and provide critical insights into diseases associated with α-synuclein dysfunction, such as Parkinson's disease.

Swimming kinematics and performance of spinal transected lampreys with different levels of axon regeneration.

Axon regeneration is critical for restoring neural function after spinal cord injury. This has prompted a series of studies on the neural and functional recovery of lampreys after spinal cord transection. Despite this, there are still many basic questions remaining about how much functional recovery depends on axon regeneration. Our goal was to examine how swimming performance is related to degree of axon regeneration in lampreys recovering from spinal cord transection by quantifying the relationship between swimming performance and percent axon regeneration of transected lampreys after 11 weeks of recovery. We found that while swimming speeds varied, they did not relate to percent axon regeneration. In fact, swimming speeds were highly variable within individuals, meaning that most individuals could swim at both moderate and slow speeds, regardless of percent axon regeneration. However, none of the transected individuals were able to swim as fast as the control lampreys. To swim fast, control lampreys generated high amplitude body waves with long wavelengths. Transected lampreys generated body waves with lower amplitude and shorter wavelengths than controls, and to compensate, transected lampreys increased their wave frequencies to swim faster. As a result, transected lampreys had significantly higher frequencies than control lampreys at comparable swimming velocities. These data suggest that the control lampreys swam more efficiently than transected lampreys. In conclusion, there appears to be a minimal recovery threshold in terms of percent axon regeneration required for lampreys to be capable of swimming; however, there also seems to be a limit to how much they can behaviorally recover.

Resilience of neural networks for locomotion.

Locomotion is an essential behaviour for the survival of all animals. The neural circuitry underlying locomotion is therefore highly robust to a wide variety of perturbations, including injury and abrupt changes in the environment. In the short term, fault tolerance in neural networks allows locomotion to persist immediately after mild to moderate injury. In the longer term, in many invertebrates and vertebrates, neural reorganization including anatomical regeneration can restore locomotion after severe perturbations that initially caused paralysis. Despite decades of research, very little is known about the mechanisms underlying locomotor resilience at the level of the underlying neural circuits and coordination of central pattern generators (CPGs). Undulatory locomotion is an ideal behaviour for exploring principles of circuit organization, neural control and resilience of locomotion, offering a number of unique advantages including experimental accessibility and modelling tractability. In comparing three well-characterized undulatory swimmers, lampreys, larval zebrafish and Caenorhabditis elegans, we find similarities in the manifestation of locomotor resilience. To advance our understanding, we propose a comparative approach, integrating experimental and modelling studies, that will allow the field to begin identifying shared and distinct solutions for overcoming perturbations to persist in orchestrating this essential behaviour.

Effects of Excess Brain-Derived Human α-Synuclein on Synaptic Vesicle Trafficking.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle trafficking under physiological conditions. However, in several neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, α-synuclein accumulates throughout the neuron, including at synapses, leading to altered synaptic function, neurotoxicity, and motor, cognitive, and autonomic dysfunction. Neurons typically contain both monomeric and multimeric forms of α-synuclein, and it is generally accepted that disrupting the balance between them promotes aggregation and neurotoxicity. However, it remains unclear how distinct molecular species of α-synuclein affect synapses where α-synuclein is normally expressed. Using the lamprey reticulospinal synapse model, we previously showed that acute introduction of excess recombinant monomeric or dimeric α-synuclein impaired distinct stages of clathrin-mediated synaptic vesicle endocytosis, leading to a loss of synaptic vesicles. Here, we expand this knowledge by investigating the effects of native, physiological α-synuclein isolated from the brain of a neuropathologically normal human subject, which comprised predominantly helically folded multimeric α-synuclein with a minor component of monomeric α-synuclein. After acute introduction of excess brain-derived human α-synuclein, there was a moderate reduction in the synaptic vesicle cluster and an increase in the number of large, atypical vesicles called "cisternae." In addition, brain-derived α-synuclein increased synaptic vesicle and cisternae sizes and induced atypical fusion/fission events at the active zone. In contrast to monomeric or dimeric α-synuclein, the brain-derived multimeric α-synuclein did not appear to alter clathrin-mediated synaptic vesicle endocytosis. Taken together, these data suggest that excess brain-derived human α-synuclein impairs intracellular vesicle trafficking and further corroborate the idea that different molecular species of α-synuclein produce distinct trafficking defects at synapses. These findings provide insights into the mechanisms by which excess α-synuclein contributes to synaptic deficits and disease phenotypes.

Recovery of Burrowing Behavior After Spinal Cord Injury in the Larval Sea Lamprey.

AbstractFollowing traumatic spinal cord injury, most mammalian species are unable to achieve substantial neuronal regeneration and often experience loss of locomotor function. In contrast, larval sea lampreys (Petromyzon marinus) spontaneously recover normal swimming behaviors by 10-12 weeks post-injury, which is supported by robust regeneration of spinal axons. While recovery of swimming behavior is well established, the lamprey's ability to recover more complex behaviors, such as burrowing, is unknown. Here we evaluated the lamprey's ability to burrow into a sand substrate over the typical time course of functional recovery (1-11 weeks post-injury). Compared to uninjured control lampreys, which burrow rapidly and completely, spinal-transected animals did not attempt burrowing until 2 weeks post-injury; and they often did not succeed in fully covering their entire body in the sand. Burrowing behavior gradually improved over post-injury time, with most animals burrowing partially or completely by 9-11 weeks post-injury. Burrowing behavior has two components: the initial component that resembles swimming with propagated body undulations and the final component that pulls the tail under the sand. While the duration of the initial component did not differ between control and spinal-transected animals across the entire recovery period, the duration of the final component in spinal-transected animals was significantly longer at all time points measured. These data indicate that, after spinal cord injury, lampreys are able to recover burrowing behaviors, though some deficits persist.

α-Synuclein-112 Impairs Synaptic Vesicle Recycling Consistent With Its Enhanced Membrane Binding Properties.

Synucleinopathies are neurological disorders associated with α-synuclein overexpression and aggregation. While it is well-established that overexpression of wild type α-synuclein (α-syn-140) leads to cellular toxicity and neurodegeneration, much less is known about other naturally occurring α-synuclein splice isoforms. In this study we provide the first detailed examination of the synaptic effects caused by one of these splice isoforms, α-synuclein-112 (α-syn-112). α-Syn-112 is produced by an in-frame excision of exon 5, resulting in deletion of amino acids 103-130 in the C-terminal region. α-Syn-112 is upregulated in the substantia nigra, frontal cortex, and cerebellum of parkinsonian brains and higher expression levels are correlated with susceptibility to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). We report here that α-syn-112 binds strongly to anionic phospholipids when presented in highly curved liposomes, similar to α-syn-140. However, α-syn-112 bound significantly stronger to all phospholipids tested, including the phosphoinositides. α-Syn-112 also dimerized and trimerized on isolated synaptic membranes, while α-syn-140 remained largely monomeric. When introduced acutely to lamprey synapses, α-syn-112 robustly inhibited synaptic vesicle recycling. Interestingly, α-syn-112 produced effects on the plasma membrane and clathrin-mediated synaptic vesicle endocytosis that were phenotypically intermediate between those caused by monomeric and dimeric α-syn-140. These findings indicate that α-syn-112 exhibits enhanced phospholipid binding and oligomerization in vitro and consequently interferes with synaptic vesicle recycling in vivo in ways that are consistent with its biochemical properties. This study provides additional evidence suggesting that impaired vesicle endocytosis is a cellular target of excess α-synuclein and advances our understanding of potential mechanisms underlying disease pathogenesis in the synucleinopathies.

Hsc70 Ameliorates the Vesicle Recycling Defects Caused by Excess α-Synuclein at Synapses.

α-Synuclein overexpression and aggregation are linked to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and several other neurodegenerative disorders. In addition to effects in the cell body, α-synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess α-synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. We show here that acute introduction of excess human α-synuclein at a classic vertebrate synapse, the lamprey reticulospinal (RS) synapse, selectively impaired the uncoating of clathrin-coated vesicles (CCVs) during synaptic vesicle recycling, leading to an increase in endocytic intermediates and a severe depletion of synaptic vesicles. Furthermore, human α-synuclein and lamprey γ-synuclein both interact in vitro with Hsc70, the chaperone protein that uncoats CCVs at synapses. After introducing excess α-synuclein, Hsc70 availability was reduced at stimulated synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic vesicle trafficking defects. In support of this hypothesis, increasing the levels of exogenous Hsc70 along with α-synuclein ameliorated the CCV uncoating and vesicle recycling defects. These experiments identify a reduction in Hsc70 availability at synapses, and consequently its function, as the mechanism by which α-synuclein induces synaptic vesicle recycling defects. To our knowledge, this is the first report of a viable chaperone-based strategy for reversing the synaptic vesicle trafficking defects associated with excess α-synuclein, which may be of value for improving synaptic function in PD and other synuclein-linked diseases.

Thrust generation during steady swimming and acceleration from rest in anguilliform swimmers.

Escape swimming is a crucial behavior by which undulatory swimmers evade potential threats. The hydrodynamics of escape swimming have not been well studied, particularly for anguilliform swimmers, such as the sea lamprey Petromyzon marinus For this study, we compared the kinematics and hydrodynamics of larval sea lampreys with those of lampreys accelerating from rest during escape swimming. We used experimentally derived velocity fields to calculate pressure fields and distributions of thrust and drag along the body. Lampreys initiated acceleration from rest with the formation of a high-amplitude body bend at approximately one-quarter body length posterior to the head. This deep body bend produced two high-pressure regions from which the majority of thrust for acceleration was derived. In contrast, steady swimming was characterized by shallower body bends and negative-pressure-derived thrust, which was strongest near the tail. The distinct mechanisms used for steady swimming and acceleration from rest may reflect the differing demands of the two behaviors. High-pressure-based mechanisms, such as the one used for acceleration from rest, could also be important for low-speed maneuvering during which drag-based turning mechanisms are less effective. The design of swimming robots may benefit from the incorporation of such insights from unsteady swimming.

The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms.

Neurotransmission is sustained by endocytosis and refilling of synaptic vesicles (SVs) locally within the presynapse. Until recently, a consensus formed that after exocytosis, SVs are recovered by either fusion pore closure (kiss-and-run) or clathrin-mediated endocytosis directly from the plasma membrane. However, recent data have revealed that SV formation is more complex than previously envisaged. For example, two additional recycling pathways have been discovered, ultrafast endocytosis and activity-dependent bulk endocytosis, in which SVs are regenerated from the internalized membrane and synaptic endosomes. Furthermore, these diverse modes of endocytosis appear to influence both the molecular composition and subsequent physiological role of individual SVs. In addition, previously unknown complexity in SV refilling and reclustering has been revealed. This review presents a modern view of the SV life cycle and discusses how neuronal subtype, physiological temperature, and individual activity patterns can recruit different endocytic modes to generate new SVs and sculpt subsequent presynaptic performance.

Regenerative capacity in the lamprey spinal cord is not altered after a repeated transection.

The resilience of regeneration in vertebrates is not very well understood. Yet understanding if tissues can regenerate after repeated insults, and identifying limitations, is important for elucidating the underlying mechanisms of tissue plasticity. This is particularly challenging in tissues, such as the nervous system, which possess a large number of terminally differentiated cells and often exhibit limited regeneration in the first place. However, unlike mammals, which exhibit very limited regeneration of spinal cord tissues, many non-mammalian vertebrates, including lampreys, bony fishes, amphibians, and reptiles, regenerate their spinal cords and functionally recover even after a complete spinal cord transection. It is well established that lampreys undergo full functional recovery of swimming behaviors after a single spinal cord transection, which is accompanied by tissue repair at the lesion site, as well as axon and synapse regeneration. Here we begin to explore the resilience of spinal cord regeneration in lampreys after a second spinal transection (re-transection). We report that by all functional and anatomical measures tested, lampreys regenerate after spinal re-transection just as robustly as after single transections. Recovery of swimming, synapse and cytoskeletal distributions, axon regeneration, and neuronal survival were nearly identical after spinal transection or re-transection. Only minor differences in tissue repair at the lesion site were observed in re-transected spinal cords. Thus, regenerative potential in the lamprey spinal cord is largely unaffected by spinal re-transection, indicating a greater persistent regenerative potential than exists in some other highly regenerative models. These findings establish a new path for uncovering pro-regenerative targets that could be deployed in non-regenerative conditions.

Acute Manipulations of Clathrin-Mediated Endocytosis at Presynaptic Nerve Terminals.

Acute perturbations of clathrin and associated proteins at synapses have provided a wealth of knowledge on the molecular mechanisms underlying clathrin-mediated endocytosis (CME). The basic approach entails presynaptic microinjection of an inhibitory reagent targeted to the CME pathway, followed by a detailed ultrastructural analysis to identify how the perturbation affects the number and distribution of synaptic vesicles, plasma membrane, clathrin-coated pits, and clathrin-coated vesicles. This chapter describes the methodology for acutely perturbing CME at the lamprey giant reticulospinal synapse, a model vertebrate synapse that has been instrumental for identifying key protein-protein interactions that regulate CME in presynaptic nerve terminals with broader extension to nonneuronal cell types.

GABA promotes survival and axonal regeneration in identifiable descending neurons after spinal cord injury in larval lampreys.

The poor regenerative capacity of descending neurons is one of the main causes of the lack of recovery after spinal cord injury (SCI). Thus, it is of crucial importance to find ways to promote axonal regeneration. In addition, the prevention of retrograde degeneration leading to the atrophy/death of descending neurons is an obvious prerequisite to activate axonal regeneration. Lampreys show an amazing regenerative capacity after SCI. Recent histological work in lampreys suggested that GABA, which is massively released after a SCI, could promote the survival of descending neurons. Here, we aimed to study if GABA, acting through GABAB receptors, promotes the survival and axonal regeneration of descending neurons of larval sea lampreys after a complete SCI. First, we used in situ hybridization to confirm that identifiable descending neurons of late-stage larvae express the gabab1 subunit of the GABAB receptor. We also observed an acute increase in the expression of this subunit in descending neurons after SCI, which further supported the possible role of GABA and GABAB receptors in promoting the survival and regeneration of these neurons. So, we performed gain and loss of function experiments to confirm this hypothesis. Treatments with GABA and baclofen (GABAB agonist) significantly reduced caspase activation in descending neurons 2 weeks after a complete SCI. Long-term treatments with GABOB (a GABA analogue) and baclofen significantly promoted axonal regeneration of descending neurons after SCI. These data indicate that GABAergic signalling through GABAB receptors promotes the survival and regeneration of descending neurons after SCI. Finally, we used morpholinos against the gabab1 subunit to knockdown the expression of the GABAB receptor in descending neurons. Long-term morpholino treatments caused a significant inhibition of axonal regeneration. This shows that endogenous GABA promotes axonal regeneration after a complete SCI in lampreys by activating GABAB receptors.