Distinct roles of different neural cell adhesion molecule (NCAM) isoforms in synaptic maturation revealed by analysis of NCAM 180 kDa isoform-deficient mice

Abstract

Mice that lack all three major isoforms of neural cell adhesion molecule (NCAM) (180 and 140 kDa transmembrane, and 120 kDa glycosylphosphatidylinositol linked) were previously shown to exhibit major alterations in the maturation of their neuromuscular junctions (NMJs). Specifically, even by postnatal day 30, they failed to downregulate from along their axons and terminals an immature, brefeldin A-sensitive, synaptic vesicle-cycling mechanism that used L-type Ca2+ channels. In addition, these NCAM null NMJs were unable to maintain effective transmitter output with high-frequency repetitive stimulation, exhibiting both severe initial depression and subsequent cyclical periods of total transmission failures that were of presynaptic origin. As reported here, mice that lack only the 180 kDa isoform of NCAM downregulated the immature vesicle-cycling mechanism on schedule, implicating either the 140 or 120 kDa NCAM isoforms in this important maturational event. However, 180 NCAM-deficient mice still exhibited many functional transmission defects. Although 180 NCAM null NMJs did not show the severe initial depression of NCAM null NMJs, they still had cyclical periods of complete transmission failure. In addition, several presynaptic molecules were expressed at lower levels or were more diffusely localized. Thus, the 180 kDa isoform of NCAM appears to play an important role in the molecular organization of the presynaptic terminal and in ensuring effective transmitter output with repetitive stimulation. Our results also suggest that PKC and MLCK (myosin light chain kinase) may be downstream effectors of NCAM in these processes. Together, these results indicate that different isoforms of NCAM mediate distinct and important events in presynaptic maturation.

Figure 1.

The distribution and expression levels of NCAM isoforms in a mouse that lacks only the 180 isoform of NCAM. A, Western blot of hippocampal lysates from wild-type mouse (left lane) lacking all isoforms of NCAM (middle lane) and lacking only the 180 isoforms of NCAM (right lane) stained with the NCAM 13 antibody that recognizes all isoforms of NCAM. All NCAM bands are lacking from the total KO mouse, whereas only the 180 band is lacking from the 180 KO mouse. As shown in this example, however, the 120 and 140 isoforms were consistently upregulated in the 180 isoform-deficient mouse. Equal amounts of protein were loaded onto each lane and several other molecules; for example, β-tubulin did not differ in level (data not shown). B, Immunostaining of P30 NMJs with the pan-NCAM antibody R025b (bottom) and rhodamine α-BTX to visualize endplate (top) also reveals the absence of NCAM in the total KO but a similar distribution to wild type in the NMJs of the 180 isoform-specific KO. However, as observed in the hippocampal lysates, the level of NCAM in the 180-deficient NMJs was elevated compared with wild type, and this was statistically significant (p < 0.005) when quantified, as shown in the bar graphs in C. Scale bar, (in B) 40 μm.

Figure 2.

Characteristics of synaptic vesicle cycling and distribution at 180-deficient NMJs as revealed by FM1-43 and SV2 staining. A, FM1-43 uptake at 180-deficient P30 NMJs (bottom right) is primarily confined to the endplate as in wild-type NMJs (bottom left) and is absent from the entire length of the preterminal axon. Such staining along the length of the preterminal axon was previously observed in NMJs lacking all NCAM and is shown here in the bottom middle panel (arrows). However, FM1-43 uptake at 180-deficient NMJs was consistently observed from the axon immediately adjacent to the endplate (bottom right, box) where no corresponding ACh receptors were revealed with rhodamine α-BTX (top left). Because staining in this region was always less intense than at the endplate proper, the contrast within this region has been digitally increased in the bottom inset (arrow). B, Overall level of staining after complete loading with FM1-43 did not differ significantly between wild-type and 180-deficient NMJs, indicating similarly sized vesicle pools. C, FM1-43 uptake in response to electrical stimulation was not affected by the L-type Ca²⁺ channel blocker nifedipine (50 μm) or brefeldin A (10 μg/ml) but was completely blocked by ω-agatoxin TK (1 μm) and cadmium chloride (30 μm), indicating that vesicle cycling at these NMJs like that at wild-type mouse NMJs is mediated by calcium entering through P/Q-type channels. D, Immunostaining for the synaptic vesicle antigen SV2 bottom also shows staining of the axon immediately adjacent to the endplate proper in the 180-deficient NMJ (right, arrow), which was never observed in wild-type NMJs (left). E, Compared with wild type, the time it took to obtain both maximal staining and destaining was significantly faster at 180-deficient NMJs (^*p < 0.005). F, In 2 mm extracellular Ca²⁺ and in the absence of stimulation, uptake at wild-type NMJs is minimal, whereas the 180-deficient NMJs exhibit appreciable uptake.

Figure 3.

Transmitter release properties at P30 180-deficient NMJs compared with wild type and those that lack all NCAM. A, MEPP amplitude did not differ between junctions from wild-type (+/+), NCAM-deficient (-/-), or NCAM 180 isoform-deficient mice. B, MEPP frequency did not differ statistically between 180-deficient and wild-type junctions. As shown previously, it was moderately decreased in NCAM-deficient junctions. C, Quantal content in response to single stimuli also did not differ between 180-deficient and wild-type NMJs. These values both differed significantly (^*p < 0.005) from NMJs lacking all NCAM whereas, as shown previously, quantal content was slightly elevated. D, Intracellular recordings illustrating that wild-type NMJs (top trace) exhibit paired-pulse facilitation at physiological levels of Ca²⁺ and Mg²⁺ at intervals of 10, 8, 6, and 4 msec, whereas NCAM 180-deficient NMJs (bottom trace) do not. F, The relationship between extracellular Ca²⁺ levels and the amplitude of evoked EPPs for a single wild-type (left) and 180-deficient (right) NMJ.

Figure 4.

The extent that wild-type, NCAM-deficient, and 180 NCAM-deficient P30 NMJs exhibit depression or total transmission failures in response to repetitive stimulation. A, EPPs in response to a 200 Hz, 0.5 sec train from a wild-type (top trace) and NCAM-deficient NMJ (bottom trace). The NCAM-deficient NMJ exhibits both strong initial depression and periods of total transmission failures. B, EPPs from 180-deficient NMJ at different stimulus frequencies. 180 isoform-deficient NMJs do not exhibit initial depression but do exhibit transmission failures that increase in frequency with increased stimulus repetition rates. C, The number of total failures per 1 sec train at different stimulus frequencies for wild-type, NCAM-deficient, and 180 NCAM-deficient. D, Train of EPPs at 100 Hz stimulation from an NMJ from a heterozygote of the mouse that lacks all isoforms of NCAM. In contrast to the NMJ that specifically lacked only the 180 isoform of NCAM, there are no failures at this frequency of stimulation.

Figure 5.

A, Epineural recordings of passively conducted Na⁺, K⁺, and Ca²⁺ currents from the nerve terminal during high-frequency stimulus trains). Appropriate placement of the electrode in the preterminal nerve reveals an extracellular current recording consisting of two negative peaks, the first corresponding to Na⁺ influx and the second to K⁺ flux (see Results for more details). These recordings from a 180 kDa isoform null NMJ were indistinguishable from those recorded from wild-type junctions (Brigant and Mallart, 1982). B, After blockade of the Ca²⁺-activated K⁺ current with 100 μm 3,4,-diaminopyridine, an outward current that could be blocked by Cd²⁺ (data not shown) and that represents the Ca²⁺ influx at the nerve terminal is revealed. C, During a 200 Hz train from a 180 kDa isoform null NMJ, there are no large reductions or block of either Na⁺ or K⁺ currents (see Results for more details). D, Ca²⁺ currents, after blockade of the Ca²⁺-activated K⁺ currents as above, recorded from a 180 kDa isoform null NMJ during a similar train also do not reveal any large reductions or block in Ca²⁺ currents that could explain the total transmission failures (Fig. 4) seen at this stimulation frequency in these junctions. E, The amplitude of the peak Ca²⁺ currents for successive stimuli in 200 Hz trains relative to the first response in the train for wild-type (filled squares) and 180 (-/-) NMJs reveals a moderate reduction in amplitude and lengthening of the time course, but no significant differences between (+/+) and (-/-) NMJs was detected. The slight slowing of the Ca²⁺ current was also observed in both types of synapses. Importantly, there were no total blockages or large reductions in Ca²⁺ currents that could account for the total transmission failures at 180 kDa isoform null NMJs.

Figure 6.

PKC and MLCK involvement in total transmission failures at 180 NCAM-deficient synapses. A, Application of 100 μm PMA to a P30 180 null NMJ rapidly reverses the total transmission failures. The top trace is the untreated control, and the middle and bottom traces are 10 and 30 sec, respectively, after PMA application to the bath. The bar graph at the right indicates the number of failures per 1 sec 100 Hz train in untreated 180-deficient NMJs and after 30 sec of PMA application (p < 0.005). B, Application of the specific PKC inhibitor BIS-1 at 500 nm to another 180 null muscle did not increase the number of failures per 1 sec train significantly (bar graph on right). However, it completely blocked the effect of PMA in reversing the transmission failures. C, Application to the bath of the MLCK-specific inhibitor ML-9 (30 μm) increased to a moderate extent the number of failures per 1 sec train (middle trace and bar graph on right). However, it also completely blocked the effect of PMA in reversing the transmission failures (bottom trace and bar graph on right; p < 0.005).

Figure 7.

Distribution of synaptic molecules in 180-deficient and wild-type P30 NMJs. A, Immunostaining of 14 μm transverse frozen sections from wild-type (left) and 180-deficient (right) NMJs with an antibody that recognizes both smooth muscle and nonmuscle isoforms of MLCK. Although weak diffuse staining (middle panel) was detected throughout the myotubes, much more intense staining was located at the junctions as defined by rhodamine α-BTX staining shown in the top panel. The bottom panel shows a merged image with MLCK in green and α-BTX in red. When the fluorescence intensity of the MLCK was quantified, the mean value from 50 junctions was significantly increased in the 180 deficient as compared with the control (p < 0.005). B, Several presynaptic molecules involved in transmission were also altered in level or distribution in the 180-deficient NMJ. Top row, Wild type; bottom row, 180-deficient NMJs. Each junction is presented as a pair of images with α-BTX staining to define the endplate followed by staining for the P/Q Ca²⁺channel subunit Ca_v 2.1, Rim, or syntaxin. All three exhibited weaker and more diffuse staining in the 180-deficient NMJ. Scale bar, 40 μm.

Figure 8.

Morphological differences between 180-deficient and wild-type P30 NMJs. A, P30 endplates from wild-type (top) and 180 isoform-deficient (bottom) NMJs as visualized by staining with rhodamine α-bungarotoxin. The width of the junctional gutter was consistently narrower in the 180-deficient endplates. In addition, although many exhibited pretzel-like shapes similar to wild-type endplates, in many cases, portions of the endplate, as visualized by postsynaptic rhodamine α-bungarotoxin staining, were either disconnected or connected by very thin regions to the endplate proper (see middle panel for an example). Scale bar, 25 μm. B, Bar graph of the widths (mean ± SEM) of the junctional gutters from 40 wild-type and 40 180 NCAM null NMJs showing that the 180 null junctions were statistically less wide (^*p < 0.005). This apparent reduction in width, when endplates were viewed from above, may have resulted from the fact that the gutters were actually deeper when observed in transverse sections (far right). Scale bar, 10 μm.

Figure 9.

The distribution of overall NCAM in comparison with the 180 isoform in P30 NMJs. A, Left, The distribution of total NCAM expression at wild-type and 180 NCAM-deficient synapses as revealed by a polyclonal antibody (R025b) that recognizes all isoforms of NCAM. NCAM is expressed in both the endplate and at a somewhat lower level in the preterminal axon but is essentially absent from the nonsynaptic portions of the muscle fibers (staining here is not above background). Right, Higher magnification examples of pan NCAM staining showing NCAM (bottom) and rhodamine α-bungarotoxin staining (bottom). There is relatively uniform staining throughout the endplate in both wild-type and 180 NCAM-deficient NMJs. The arrows point to staining of the preterminal axons in 180 null NMJs as they enter the endplate in a region where there are no underlying ACh receptors. B, Western blots stained with a pan-NCAM antibody from P30 wild-type hippocampal lysates. When compared with the total lysate (right band), the lysate from the synaptosomal fraction (left band) shows that the 180 isoform of NCAM and, to a lesser extent, the 140 isoform, are enriched at synapses. C, Staining with a monoclonal antibody that recognizes only the 180 isoform in P30 wild-type NMJs. Left panel, Endplates viewed from above (top and middle row) show that the 180 isoform has a more punctate staining pattern at the endplate, compared with overall NCAM. Although some much less intense staining was observed in the preterminal axon close to the endplate (arrow), overall axonal staining was weak to absent. 180 NCAM staining was absent from the 180-deficient NMJ shown in the bottom panel. Right panel shows staining for 180 NCAM (middle row) in comparison with rhodamine α-bungarotoxin staining (top row) in transverse sections of P30 wild-type muscles. The bottom row shows the merged image with rhodamine α-bungarotoxin in red and 180 NCAM in green. Scale bars, 40 μm.