Cytoskeletal proteins play maintenance roles in neurotransmission

Researchers in the Cellular and Molecular Synaptic Function Unit at the Okinawa Institute of Science and Technology Graduate University (OIST) have elucidated the roles of cytoskeletal proteins at the giant presynaptic terminal, called the calyx of Held, visualized in rat brainstem slices. Initial findings were made by a graduate school student Lashmi Pyriya for her PhD thesis work under technical instructions by Dr Kohgaku Eguchi (now in IST Austria) and Dr Laurent Guillaud. This work was then substantiated by Dr Han-Ying Wang (Fig 3) and finally accepted for publication in the Journal of Neuroscience. 

The scientists’ findings provide crucial insight into the function and location of microtubules in neurotransmission. Their research may also shed light on mechanisms of synaptic dysfunctions occurring in neuronal diseases like Alzheimer’s Disease and Parkinson’s Disease.    

Fig 1 (A) Axon and presynaptic terminal. (B) Synaptic transmission. The thick filament “microtubules” (MTs) made of tubulin polymers and the thin filament “F-actin” made of actin polymers are important cytoskeletal elements supporting cell structures and functions. However, it remains unknown what role cytoskeletal proteins play in the neurotransmission process. In neurons, MTs are expressed in axons (Fig 1A) and serve as rails for transporting molecules and organelles between cell bodies and presynaptic terminals. In contrast, F-actin is expressed exclusively in presynaptic terminals. Presynaptic terminals contain “synaptic” vesicles (SVs) filled with neurotransmitter.  When the electrical signal “action potentials” (APs) reach presynaptic terminals, SVs docked on the release sites fuse into terminal membrane, thereby releasing transmitters via exocytosis (Fig 1B).  At the excitatory synapse, transmitter glutamate is released from SVs.  Glutamate diffuses and binds to postsynaptic glutamate receptors and produces the postsynaptic response “EPSP”.  When the EPSP size exceeds a threshold, APs are generated and propagate toward the axon terminal of postsynaptic neuron.

The main findings are 3-fold.

(1) Co-existence of MTs with SVs in presynaptic terminals

It has been controversial whether MTs exist in presynaptic terminals, and none of past reports support co-existence of MTs with SVs.Using a high-resolution STED microscope, at the calyx of Held, OIST researchers successfully demonstrated that ~50% of SVs co-exist with MTs with an average distance of 44 nm (Fig 2).

Fig 2 Co-localization of MTs (green) and SVs (red and yellow) in a calyx of Held presynaptic terminal. 3D reconstruction of STED image. About half SVs (red) are located within 100 nm from MTs with average distance of 44 nm (bar graphs), whereas other half SVs (yellow) are remotely located.  

(2) F-actin and MTs support fast and slow SV replenishments, respectively

Soon after SVs are docked, they release transmitter at release sites, which is then immediately (tf 0.1s) replenished by docked SVs and docking site is then replenished more slowly (ts 2s) by reserve SVs (Fig 1B). When MTs were depolymerized, the slow component of SV replenishment prolonged (Fig 3A). The magnitude of prolongation was proportional to that of MT depolymerization assayed by live imaging (Fig 3B, correlation factor, 0.98). In contrast, F-actin depolymerization specifically prolonged the fast replenishing rate tf (Fig 3C).

Fig 3 Effects of MT or F-actin depolymerization on the rate of release site replenishment.  A, Time course of SV replenishment assayed from recovery of EPSCs from synaptic depression.  MT depolymerization (red and orange) prolonged the SV replenishment (ts). B, Correlation between the SV replenishing speed and extent of MT depolymerization live-monitored using a fluorescent MT binding probe. C, F-actin depolymerization specifically slowed the fast replenishing component (tf green).  

(3) F-actin and MT support sustained high-frequency neurotransmission

Delay of SV replenishment will impair maintenance of neurotransmission, particularly at high frequency. At the calyx of Held synapse, presynaptic stimulations at 100 Hz unfailingly generate APs in postsynaptic neurons for at least 50 s. When MTs are depolymerized, postsynaptic APs start to fail at around 20s and AP failures reach 40% after 50s stimulation. The effect of F-actin depolymerization is relatively weak, with 20% failures after 50s stimulation (Fig 4). These results indicate that cytoskeletal proteins in presynaptic terminals are essential for the maintenance of high-frequency neurotransmission.

Fig 4 Left, Experimental protocol of simultaneous recording of APs from presynaptic (right crescent) and postsynaptic (left round) structures. Middle, Postsynaptic APs evoked by presynaptic APs (not shown) at 100 Hz, sampled at 1-2s (top), 25-26s (middle) and 49-50s (bottom). In controls (black) and after MT depolymerization (red) or F-actin depolymerization (green). Right, percentage of postsynaptic APs remaining at 40-50 after stimulation (number of postsynaptic APs / number of presynaptic APs).

These findings provide a new insight into the mechanisms of synaptic dysfunctions occurring in neuronal diseases. Since both a-synuclein in Parkinson’s disease and tau protein in Alzheimer disease exert toxic functions in presynaptic terminals, it is of crucial importance to elucidate presynaptic molecular mechanisms involved in the etiology of neuronal diseases.

Fig 5: The authors of the new study, published in Journal of Neuroscience. From left to right: Dr Lashmi Pyriya, Dr. Kohgaku Eguchi, Dr Laurent Guillaud, Professor Tomoyuki Takahashi, Dr. Han-Ying Wang