A Detailed Study of Axon Initial Segment Maturation and Structural Organization by Fluorescence Microscopy
by Melanie Dannemeyer
Date of Examination:2016-01-25
Date of issue:2016-02-03
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Jörg Enderlein
Referee:Dr. Andreas Neef
Persistent Address:
http://dx.doi.org/10.53846/goediss-5493
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Abstract
English
The distribution and density of neuronal voltage-gated sodium channels (VGSCs) is important for neuronal excitability, maturation and plasticity. High density of VGSCs in the axon initial segment (AIS) depends on the cytoskeletal proteins βIV-spectrin and ankyrin-G (ankG). Here, the density and structure of βIV-spectrin, βII-spectrin, ankG and VGSCs in the AIS is studied with maturation. Neuronal cultures were prepared from hippocampus and immunofluorescence was imaged between 7 and 21 days in vitro with conventional wide-field and stochastic optical reconstruction microscopy (STORM). At the earliest time point studied, DIV 7, βII-spectrin is arranged in the AIS on a very regular structure. With maturation βII-spectrin density decreases slightly, while the density of βIV-spectrin increases in the AIS. Neuronal βIV-spectrin appears in two isoforms βIV∑1 and βIV∑6. Within this detailed study the density and structural organization of βIV-spectrin is examined in detail. The mean fluorescence intensity of βIV-spectrin labeled at its N-terminus is found on a constant level starting at DIV 14. Based on the fact that N-terminal labeling targets βIV∑1, but not βIV∑6, the mean fluorescence intensity stagnates within the second week of studied maturation and a prominent periodic organization is revealed, it is suggested that βIV∑1 is highly enriched early on, arranged in the AIS on a very regular basis and functions as the main stabilizing βIV-spectrin isoform. Contrariwise, C-terminal labeling of βIV-spectrin reports an increase of the mean fluorescence intensity over the whole three weeks of studied maturation. Moreover, at DIV 6 βIV-spectrin is found on a very regular basis, whereas at DIV 14 a diffuse distribution is observed. Based on the fact that C-terminal labeling targets βIV∑1 and βIV∑6, the careful study allows to conclude that the detected increase of the mean fluorescence intensity and thus likely an increase of the density of βIV-spectrin density is due to an increase of βIV∑6. Further, a diffuse βIV-spectrin structure in mature neurons suggests an irregular placement of βIV∑6 in the existing regular structured cytoskeleton of actin-adducin rings. The adapter protein ankG mediates the contact between VGSCs and the underlying cytoskeleton. Its density is found to increase over the studied maturation period. AnkG is found organized prominently periodically in immature neurons. With maturation the periodic organization of ankG becomes less pronounced. Whereas previous studies simplify the observation by reporting the distance of ankG to the underlying network and the distribution of binding positions on a spectrin tetramer are the cause, not considering that these conditions are not only found in mature neurons, but also immature neurons, here a high quality study suggests non-periodic βIV∑6 as a mediator for the increase of irregularity of ankG. In agreement with ankG, the density of VGSCs and specifically the densities of the subtypes Nav1.2 and Nav1.6 increase with maturation. Additionally, the data suggest an early expression of Nav1.2 (measured DIV 6) and a late expression of Nav1.6 (not detected at DIV 7, but at DIV 10). Whereas, Nav1.2 is found proximal in immature and evenly distributed in mature neurons, is Nav1.6 found distal in mature neurons and evenly distributed in immature neurons. A developmental switch from one subtype to another is not found. VGSCs and specifically Nav1.6 are found (semi-)periodic. The data obtained for Nav1.2 organization do not allow to draw a final conclusion, yet. The mouse strain qv3J is a loss of function mutation in the βIV-spectrin gene (Spnb4). A single inserted base (InsT6786) causes a frameshift at amino acid G2209 and a completely changed new 49-amino-acid C-terminal extension. Here, the effect of this mutation on the expression and structural organization of βII-spectrin, βIV-spectrin, ankG and VGSCs in the AIS is studied. At DIV 7 wildtype and mutant neurons are very similar with respect to density and periodicity of ankG, VGSCs and βII-spectrin. However, only very little βIV-spectrin is observed in mutant qv3J neurons at DIV 7 and is not detectable when labeling DIV 14 neurons. The density and regularity of βII-spectrin is not altered, suggesting that it has no role within a rescue mechanism for βIV-spectrin absence. In contrast to the maturation in wildtype no increase of the ankG and VGSCs density is observed in qv3J mutant neurons. AnkG is found at a constant density and arranged strongly periodic between DIV 11 - 24. This supports its binding to βII-spectrin and suggests an absence of βIV∑6 as an irregular cytoskeletal binding partner. The VGSC density in mutant qv3J neurons decreases progressively in the three weeks of studied maturation. A detailed analysis suggests that Nav1.2 channels are not stabilized and constantly decrease in density with maturation, whereas Nav1.6 channels are rare at all ages, due to a not fully functional recruitment mechanism of VGSCs by the ankG-βIV-spectrin complex. Furthermore, the constant ankG density, but a consecutive decrease of VGSC density, suggests a direct relation between VGSCs and βIV-spectrin. The disruption of the complete mechanism is suggested to be mainly due to a disrupted βIV∑1, an isoform suggested to function as a stabilizer, and the complete absence of βIV∑6, an isoform suggested to function as a recruiter for VGSCs in neuronal maturation. To conclude, βIV-spectrin is incorporated in the existing βII-spectrin grid, βIV∑1 periodic and βIV∑6 non-periodic. The ring-like organization of proteins along the AIS does not depend on βIV-spectrin. The C-terminal mutation qv3J causes a complete loss of βIV-spectrin. The βIV-spectrin C-terminal tail is essential for its incorporation and stabilization. βIV-spectrin is a key component in ankG and VGSC recruitment. βIV-spectrin is crucial for VGSC stabilization. In addition, ankG stabilization depends strongly on βII-spectrin.
Keywords: Axon initial segment, maturation, structure, spectrin, ankyrin, sodium channels, STORM