In order for the capital letter "I" (that starts this article) to appear onthe computer screen, two fingers had to go into action. The left-handdigit depressed "shift;" the right-hand one, the "I" key.

Before the muscles of either finger could move, neurons in thiswriter's brain had to call up the requisite action. They switched onelectrical signals, which traveled down nerve-cell axons at sub-millisecond speed, across a series of synapses along the way.

The last such synaptic cleft brought the terminal nerve axon face-to-face with the finger muscle waiting on the opposite shore of theneuromuscular junction for the command to strike the keyboard. Atthe so-called "active zone" of the synapse, facing its 50-nanometergap, some 20 to 30 vesicles or packets of neurotransmitter _ in thiscase, acetylcholine _ discharged their contents in a chemicalreaction, which galvanized the muscle receptors on the motorendplate to act.

Armies of neuroscientists around the world are busy solving thesecrets of synapses. Of some 10,000 presentations at the 24th annualmeeting of the Society for Neuroscience, last November in MiamiBeach, fully 422 dealt with synaptic subjects.

One stubborn hold-out in this nano-level exploration has been aprotein called s-laminin on the muscle side of the neuromuscularjunction. Researchers at Washington University, in St. Louis,discovered this synaptic-laminin in 1987, but could never get beyondstudying it in in vitro tissue culture. How it worked in vivo escapedthem, and others in the field, until now.

First In Vivo Synapse Organizer

Yesterday's Nature, dated March 16, carries a report by those initialdiscoverers titled "Aberrant differentiation of neuromuscularjunctions in mice lacking s-laminin." One of its two principal authors,molecular biologist John Merlie, told BioWorld Today: "This is thefirst example of a protein that organizes synapses in living animals."He observed, "I'm impressed with the fact that we can now use theknock-out-mouse approach to study the molecular genetics of a singlesynapse in vivo. It will be very useful for understanding everythingabout synapse formation."

To construct their stable of rodents totally lacking s-laminin, Merlieand his co-workers began by mutating a 10-kilobase fragment of themurine s-laminin gene. They transferred this aberrant genomicsequence into embryonic stem cells and inserted these into early-stage fertilized mouse eggs, which they then implanted in the uteri offoster mothers.

After crossing the heterozygous offspring, 68 of 325 pregnancies _21 percent as predicted _ produced s-laminin-minus knock-outmice. Though physically normal at birth, these all died between 15and 30 days later.

Under the electron microscope, neuromuscular junctions of variousmuscles in the knock-out mice revealed grossly abnormal synapses.For example: Instead of congregating at the active zones of the axonterminals, the neurotransmitter vesicles scattered randomly aroundthe synaptic site. On the muscle side, junctional folds that enrich thearchitecture of the motor endplates were few and far between. AndSchwann cells, which enwrap axons in insulating myelin, pokedintrusively into the junction space. Apropos, Merlie remarked, "Wewere very surprised to see in the mutant mice that the Schwann cell,which normally sits there and minds its own business, is now growingin between the nerve and the muscle."

In short, it takes s-laminin to generate healthy, functionallydifferentiated neuromuscular synapses in neonatal mice _ andpresumably in all mammals. "It gives us new ideas," Merlie said, "asto how two cells, synaptic partners in forming a synapse, interact toform mature synapse structure. We've been looking for several yearsfor signals the muscle might be producing that induce synapticmaturation. We think s-laminin is a great candidate for being such asignal."

Before they died, the s-laminin-missing mice developed profoundneuromuscular disease, "similar though not identical," Merlie said,"to some congenital forms of myasthenia gravis." This autoimmunedisease is a progressive muscular weakness best known for droopingeyelids. Its cause is a defect in synaptic transmission.

"In humans," Merlie said, "we can easily screen for the absence of s-laminin, by using monoclonal antibodies that detect the protein. Whatwe can't do yet is look for point mutations."

Surprise: The Kidney Connection

"No one has identified a human patient with s-laminin defect yet,"Merlie observed. But one may turn up in an unsuspected quarter.

"Of great surprise to us," he said, "these knock-out mice also hadsevere kidney defects. It looks as though their lack of s-lamininresults in some disability of the kidney glomerulus to retain proteinsthat are normally in the serum, and keep them from spilling over intothe urine. This unexpected finding," he added, "will certainly beuseful to people interested in kidney disease."

Although the neuromuscular junctions _ synapses between nervesand muscles _ are not morphologically the same as nerve-to-nervesynapses within the central nervous system (CNS), Merlie said, "wethink the two classes have things in common, and that ourneuromuscular junction makes a good model for CNS synapses."

One salient distinction between the two, he pointed out, is that "if youdamage a muscle, or nerves that innervate muscle, as by trauma orsurgery, the muscle re-forms its synapses relatively quickly.However, the CNS, where patients have head injury or neurosurgery,is another story. For some reason, central nervous synapses do notregenerate with great fidelity or efficiency.

"We are hoping," he observed " that what we learn about why theneuromuscular junction does re-form synapses relatively faithfullymay help us to figure out how to improve that situation in the CNS."He cautioned that such a project "might come to fruition only in therelatively long term."

Enhancing synapse regeneration in the brain could have somedramatic effects, Merlie pointed out, "as it's known that there aregood correlations between changes in CNS synapses, bothbiologically and morphologically, and the acquisition and retention ofmemory and learning." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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