Neuromuscular Active Zone Structure and Function in Healthy and Lambert-Eaton Myasthenic Syndrome States

Abstract

The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters.

Keywords: Lambert-Eaton myasthenic syndrome; active zone; computational modeling; neuromuscular junction.

Figure 1

The presynaptic AP waveform at the mouse NMJ is very brief. (A) BeRST 1 dye-stained image of a mammalian presynaptic motor nerve terminal. (B) Alexa Fluor 488 α-BTX stained image of the same terminal as in A. (C) normalized spline of the average presynaptic AP waveform recorded from 11 mouse motor nerve terminals. Adapted from Ojala et al. [26].

Figure 2

The structure and protein distribution of mouse NMJ AZs. (A) A mouse NMJ stained with Alexa-594 α-bungarotoxin (BTX; red) to demonstrate the shape of the NMJ and an Alexa-488 conjugated antibody to identify the bassoon protein the AZs (BSN; green). Inset shows an enlargement of one part of the NMJ to make it easier to visualize the distribution of AZs (green spots). Image adapted from [5]. (B) A freeze-fracture replica of an AZ from a mouse NMJ. The hypothesized locations of synaptic vesicles are superimposed as white circles. Scale bar = 50 nm. Adapted from [10,36]. (C) Diagram of a single AZ from a mouse NMJ based on electron microscope tomography data [36]. Diagram shows docked synaptic vesicles (gray spheres), along with AZ structures termed “pegs” (orange), “beams” (purple), and “ribs” (green). (D) The distribution of the AZ proteins bassoon (green) and P/Q-type VGCCs (magenta) at the mouse NMJ as revealed by STED super-resolution microscopy. (E) The distribution of the AZ proteins bassoon (green) and piccolo (magenta) at the mouse NMJ as revealed by STED super resolution microscopy. (F) A combined proposed overlay of all three AZ proteins (Bassoon, Piccolo, and P/Q-type VGCCs) onto the AZ fine structure based on STED super-resolution imaging. These data lead to the hypothesis that the “pegs” identified in electron microscopy tomography models from panel C represent P/Q-type VGCCs (orange), the “ribs” represent Bassoon (green), and the “beams” represent Piccolo (purple). C-F are adapted from [39] (G) The distribution of quantal content values determined from a population of mouse NMJs. Inset shows a sample miniature endplate current (mEPC; left) and a sample AP-triggered endplate current (EPC; right). (H) The distribution of AZ numbers counted from a population of mouse NMJs. Adapted from [5].

Figure 3

Freeze fracture of control and LEMS NMJs AZs showing AZ disruption after LEMS passive transfer to mice. (A) Representative control mouse AZ organization (left: diagram, right: freeze fracture replica, scale = 50 nm). (B) Representative LEMS-modified AZ organization (left: diagram of protein organization, right: two example AZs from nerve terminals of mice passively transferred with LEMS serum) Adapted from Nagel et al. [75]; Fukuoka et al. [10]; Fukunaga et al. [9].

Figure 4

Diagrams of MCell models that can be constructed of mouse AZs in healthy and LEMS conditions. (A) Diagram of a mouse NMJ MCell model environment that contains 6 AZs. The 3-dimensional enclosure depicts a portion of the motor nerve terminal with black spheres representing docked synaptic vesicles within each AZ. Dots adjacent to the docked synaptic vesicles represent AZ proteins (gray dots) and P/Q-type VGCCs (black dots). (B) Diagram of a hypothesized healthy AZ based on predictions for the number and position of docked synaptic vesicles (blue spheres), P/Q VGCCs (red circles), calcium-activated potassium channels (yellow circles), and other unknown AZ proteins (white circles). (C) Diagram of a hypothesized LEMS AZ based on predictions for the number and position of docked synaptic vesicles (blue spheres), P/Q VGCCs (red circles), calcium-activated potassium channels (yellow circles), L-type VGCCs (green circles), and other unknown AZ proteins (white circles). Panel A is modified from Laghaei et al. [5].