Brian D. Ackley

Standardized assessment of concussion in football players

Article abstract-The recent formulation of guidelines for the management of concussion in sports adopted by the American Academy of Neurology specifically calls for the development of a standardized, systematic sideline evaluation for the immediate assessment of concussion in athletes. The present study involved the preliminary investigation of the feasibility and clinical validity of a standardized version of a brief sideline examination complied in accordance with these guidelines. This examination, intended for use by athletic trainers, was administered by three trainers to 141 nonconcussed high school football players at three separate schools. All players suspected of suffering a concussion (N = 6) during the fall 1995 season were also tested immediately following their injury. The examination was easily administered and scored. The concussed players as a group scored significantly below the nonconcussed controls and below their own baseline (pre-injury) performance, despite their all having been considered by the trainers to have suffered mild, grade 1 concussions. Although preliminary, these data suggest that a standardized sideline examination of this type can be useful in detecting concussion and determining fitness to return to play. NEUROLOGY 1997;48: 586-588

SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS.

A central event in synapse development is formation of the presynaptic active zone in response to positional cues. Three active zone proteins, RIM, ELKS (also known as ERC or CAST) and Liprin-α, bind each other and are implicated in linking active zone formation to synaptic vesicle release. Loss of function in Caenorhabditis eleganssyd-2 Liprin-α alters the size of presynaptic specializations and disrupts synaptic vesicle accumulation. Here we report that a missense mutation in the coiled-coil domain of SYD-2 causes a gain of function. In HSN synapses, the syd-2(gf) mutation promotes synapse formation in the absence of syd-1, which is essential for HSN synapse formation. syd-2(gf) also partially suppresses the synaptogenesis defects in syg-1 and syg-2 mutants. The activity of syd-2(gf) requires elks-1, an ELKS homolog; but not unc-10, a RIM homolog. The mutant SYD-2 shows increased association with ELKS. These results establish a functional dependency for assembly of the presynaptic active zone in which SYD-2 plays a key role.

Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans.

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

C. elegans RPM-1 Regulates Axon Termination and Synaptogenesis through the Rab GEF GLO-4 and the Rab GTPase GLO-1

C. elegans RPM-1 (for Regulator of Presynaptic Morphology) is a member of a conserved protein family that includes Drosophila Highwire and mammalian Pam and Phr1. These are large proteins recently shown to regulate synaptogenesis through E3 ubiquitin ligase activities. Here, we report the identification of an RCC1-like guanine nucleotide exchange factor, GLO-4, from mass spectrometry analysis of RPM-1-associated proteins. GLO-4 colocalizes with RPM-1 at presynaptic terminals. Loss of function in glo-4 or in its target Rab GTPase, glo-1, causes neuronal defects resembling those in rpm-1 mutants. We show that the glo pathway functions downstream of rpm-1 and acts in parallel to fsn-1, a partner of RPM-1 E3 ligase function. We find that late endosomes are specifically disorganized at the presynaptic terminals of glo-4 mutants. Our data suggest that RPM-1 positively regulates a Rab GTPase pathway to promote vesicular trafficking via late endosomes.

The two isoforms of the Caenorhabditis elegans leukocyte- common antigen related receptor tyrosine phosphatase PTP-3 function independently in axon guidance and synapse formation

Leukocyte-common antigen related (LAR)-like phosphatase receptors are conserved cell adhesion molecules that function in multiple developmental processes. The Caenorhabditis elegans ptp-3 gene encodes two LAR family isoforms that differ in the extracellular domain. We show here that the long isoform, PTP-3A, localizes specifically at synapses and that the short isoform, PTP-3B, is extrasynaptic. Mutations in ptp-3 cause defects in axon guidance that can be rescued by PTP-3B but not by PTP-3A. Mutations that specifically affect ptp-3A do not affect axon guidance but instead cause alterations in synapse morphology. Genetic double-mutant analysis is consistent with ptp-3A acting with the extracellular matrix component nidogen, nid-1, and the intracellular adaptor α-liprin, syd-2. nid-1 and syd-2 are required for the recruitment and stability of PTP-3A at synapses, and mutations in ptp-3 or nid-1 result in aberrant localization of SYD-2. Overexpression of PTP-3A is able to bypass the requirement for nid-1 for the localization of SYD-2 and RIM. We propose that PTP-3A acts as a molecular link between the extracellular matrix and α-liprin during synaptogenesis.

The Flamingo ortholog FMI-1 controls pioneer-dependent navigation of follower axons in C. elegans.

Development of a functional neuronal network during embryogenesis begins with pioneer axons creating a scaffold along which later-outgrowing axons extend. The molecular mechanism used by these follower axons to navigate along pre-existing axons remains poorly understood. We isolated loss-of-function alleles of fmi-1, which caused strong axon navigation defects of pioneer and follower axons in the ventral nerve cord (VNC) of C. elegans. Notably follower axons, which exclusively depend on pioneer axons for correct navigation, frequently separated from the pioneer. fmi-1 is the sole C. elegans ortholog of Drosophila flamingo and vertebrate Celsr genes, and this phenotype defines a new role for this important molecule in follower axon navigation. FMI-1 has a unique and strikingly conserved structure with cadherin and C-terminal G-protein coupled receptor domains and could mediate cell-cell adhesion and signaling functions. We found that follower axon navigation depended on the extracellular but not on the intracellular domain, suggesting that FMI-1 mediates primarily adhesion between pioneer and follower axons. By contrast, pioneer axon navigation required the intracellular domain, suggesting that FMI-1 acts as receptor transducing a signal in this case. Our findings indicate that FMI-1 is a cell-type dependent axon guidance factor with different domain requirements for its different functions in pioneers and followers.

Genetic analysis of synaptic target recognition and assembly

Formation of synapses by neurons onto specific targets is essential to the function of a nervous system. The isolation and analysis of Caenorhabditis elegans and Drosophila mutants with synaptogenesis defects has provided insight into the functions of evolutionarily conserved molecules at single-synapse resolution. Importantly, such studies have uncovered novel molecules and signaling mechanisms. Here, recent progress on synaptic target recognition and synaptic assembly are reviewed.

Caenorhabditis elegans Flamingo Cadherin fmi-1 Regulates GABAergic Neuronal Development

In a genetic screen for regulators of synaptic morphology, we identified the single Caenorhabditis elegans flamingo-like cadherin fmi-1. The fmi-1 mutants exhibit defective axon pathfinding, reduced synapse number, aberrant synapse size and morphology, as well as an abnormal accumulation of synaptic vesicles at nonsynaptic regions. Although FMI-1 is primarily expressed in the nervous system, it is not expressed in the ventral D-type (VD) GABAergic motorneurons, which are defective in fmi-1 mutants. The axon and synaptic defects of VD neurons could be rescued when fmi-1 was expressed exclusively in non-VD neighboring neurons, suggesting a cell nonautonomous action of FMI-1. FMI-1 protein that lacked its intracellular domain still retained its ability to rescue the vesicle accumulation defects of GABAergic motorneurons, indicating that the extracellular domain was sufficient for this function of FMI-1 in GABAergic neuromuscular junction development. Mutations in cdh-4, a Fat-like cadherin, cause similar defects in GABAergic motorneurons. The cdh-4 is expressed by the VD neurons and seems to function in the same genetic pathway as fmi-1 to regulate GABAergic neuron development. Thus, fmi-1 and cdh-4 cadherins might act together to regulate synapse development and axon pathfinding.