1 Title:
2 Sulfatide and neuronal complex gangliosides are functionally interdependent in maintaining
3 myelinating axon integrity
4 Abbreviated title (50’s): sulfatide & ganglioside interactions in the NS / membrane lipids
5 determine CNS integrity / lipid interactions determine CNS integrity
6
7 Author names: McGonigal R1, Barrie JA1, Yao D1, McLaughlin M2, Cunningham ME1, Rowan
8 EG3, Willison HJ1
9 Author affiliations: 1University of Glasgow, Institute of Infection, Immunity & Inflammation G12
10 8TA; 2University of Glasgow, School of Veterinary Biosciences G61 1QH ; 3
11 University of Strathclyde, Strathclyde Institute of Pharmacy & Biochemical
12 Sciences G4 0RE
13 Corresponding author: Hugh J. Willison
14 Institute of Infection, Immunity & Inflammation
15 SGDB, B330
16 120 University Place, University of Glasgow
17 Glasgow G12 8TA
18 +44 0141 330 8388
20 Number of pages: 36
21 Number of figures, tables: 1 Table, 8 figures
22 Number of words: 1. Abstract: 254
1
23 2. Introduction: 676
24 3. Discussion: 1617
25 Conflict of Interest: The authors declare no competing financial interests
26 Acknowledgements: **Wellcome, John Dempster
27
2
28 Abstract (250 words): reflects objectives and novelty. Include
29 Objectives
30 Methodology (include species studied and whether one or both sexes were included)
31 Key Results
32 Major Conclusion
33 Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve
34 integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knockout, CST-/-) or complex
35 gangliosides (β-1,4-N-acetylegalactosaminyltransferase 1 knockout, GalNAc-T-/-) display prominent
36 disorganization of protein localisation at the node of Ranvier (NoR) in early life, and age-dependent
37 neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T-
38 /- phenotype, as shown by neuron or glial-specific rescue whereas sulfatide is only expressed and
39 functional in glial membranes. The similarities in NoR phenotype of CST-/-, GalNAc-T-/- and axo-glial
40 protein deficient mice suggests these glycolipids may stabilise membrane proteins including
41 neurofascin 155 (NF155) and myelin-associated glycoprotein (MAG) at axo-glial junctions. To assess
42 the functional interactions between sulfatide and gangliosides, CST-/- and GalNAc-T-/- genotypes were
43 interbred. CST-/-x GalNAc-T-/- mice develop normally to P10, but all die between P20-P25, coinciding
44 with peak myelination. Ultrastructural, immunohistological and biochemical analysis reveals
45 widespread axonal degeneration and disruption to the axo-glial junction at the NoR. In the CNS, in
46 addition to sulfatide-dependent loss of NF155, CST-/-x GalNAc-T-/- mice exhibited a major reduction in
47 MAG protein levels in myelin, when compared to wild type and single lipid deficient mice. The CST-/-x
48 GalNAc-T-/- phenotype was fully restored to that of CST-/- mice by neuron-specific expression of
49 complex gangliosides, but not by glial-specific expression, and not by the global expression of a-series
50 gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and
51 neuronal membranes respectively, act in concert to promote NF155 and MAG in maintaining the
52 stable axo-glial interactions essential for normal nerve function.
3
53 Significance Statement (120 words max)
54 provide a clear explanation of the importance and relevance of the research in a manner accessible
55 to researchers without specialized knowledge in the field and informed lay readers.
56 Sulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining
57 nervous system integrity. Node of Ranvier maintenance in particular requires stable
58 compartmentalisation of multiple membrane proteins. The axo-glial adhesion molecules neurofascin
59 155 and myelin-associated glycoprotein require membrane microdomains containing either sulfatides
60 or complex gangliosides to localise and function effectively. The co-operative roles of these
61 microdomains and associated proteins are unknown. Here we show vital interdependent roles for
62 sulfatides and complex gangliosides as double (but not single) deficiency causes a rapidly lethal
63 phenotype in early age. These findings suggests that sulfatides and complex gangliosides on opposing
64 axo-glial membranes are responsible for essential tethering of the axo-glial junction proteins,
65 neurofascin155 and myelin-associated glycoprotein that interact to maintain the nodal complex. (120
66 words)
4
67 Introduction (650 words)
68 “briefly indicate the objectives of the study and provide enough background information to clarify
69 why the study was undertaken and what hypotheses were tested”
70
71 Glycosphingolipids are highly enriched in lipid rafts (Simons and Toomre, 2000), involved in the
72 topographical organisation of membrane proteins (Jackman et al., 2009; Sonnino et al., 2014;
73 Sonnino et al., 2015). Sulfatide, 3-O-sulfogalactosylceramide, synthesised from galactocerebroside
74 (GalC) by cerebroside sulfotransferase enzyme (Figure 1A) is enriched in the outer leaflet of the
75 myelin membrane (Ishizuka, 1997). Complex ganglioside biosynthesis requires β-1,4-N-
76 acetylegalactosaminyltransferase 1, GalNAc-T enzyme activity (Figure 1A) with ganglioside
77 expression being widespread in both neuronal and glial membranes. Evidence indicates that both
78 sulfatide and complex gangliosides are crucial for the maintenance and stability, rather than the
79 developmental formation of nervous system domains (Takamiya et al., 1996; Honke et al., 2002).
80 Mice deficient in these lipids thus develop relatively normally, before showing age-dependent
81 degeneration[c1] (Takamiya et al., 1996; Honke et al., 2002). Evidence in particular indicates that these
82 glycolipids are essential for stabilising the normal arrangement of axo-glial interactions, specifically
83 at the paranodal region of the node of Ranvier (NoR). Mice deficient for GalC and sulfatide (Dupree
84 et al., 1999), sulfatide alone (Ishibashi et al., 2002; Marcus et al., 2006; Hoshi et al., 2007) or complex
85 gangliosides (Susuki et al., 2007) have prominently disrupted paranodes. The paranode in both the
86 PNS and CNS tethers and seals the abutted axon and glial membranes between the node and
87 juxtaparanode (Garcia-Fresco et al., 2006; Susuki et al., 2013), thereby maintaining nodal voltage-
88 gated sodium (Nav) and juxtaparanodal potassium (Kv) channel clustering and segregation, essential
89 for normal conduction (Salzer, 1997; Poliak and Peles, 2003). The importance of appropriate
90 expression and localisation of the paranodal proteins in stabilising Nav channel clusters is also
91 revealed in paranodal protein knockout mice. Thus deficiency in contactin, contactin-associated
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92 protein (Caspr) or neurofascin (NF) 155 leads to progressive paranodal defects from P10 and
93 subsequent axon degeneration, with most mice dying by P21 (Bhat et al., 2001; Boyle et al., 2001;
94 Pillai et al., 2009). Significantly, while these adhesion complex proteins comprise the physical
95 adhesion barrier at the paranode, associated glycolipid molecules appear to be required for their
96 localisation and stabilisation, likely by the targeting and transportation of adhesion molecules in lipid
97 rafts. In support of this Schafer et al. (2004), showed that sulfatide deficiency disrupted lipid rafts,
98 leading to the loss of NF155 targeting to the paranode, thereby limiting its co-clustering function
99 with axo-glial partners. Additionally, extraction studies identified sulfatide-containing lipid rafts as
100 anchors for the axo-glial adhesion molecule myelin-associated glycoprotein (MAG), as well as NF155
101 (Pomicter et al., 2013).
102 Complex gangliosides are also an important lipid raft component required for paranode stabilisation,
103 as shown by an absence of transverse bands and dissociation of paranodal proteins in GalNAc-T-/-
104 mice (Susuki et al., 2007). As gangliosides are present in both neuronal and glial membranes, their
105 principal site of action in paranodal stabilisation cannot be presumed. To address this, we
106 demonstrated that loss of neuronal complex gangliosides underpins the GalNAc-T-/- phenotype as
107 selective reintroduction of complex ganglioside expression in neuronal but not glial membranes
108 rescues the paranodal phenotype (Yao et al., 2014). Complex gangliosides GT1b and GD1a, both
109 prominently expressed on the axonal membrane, are trans receptors for glial membrane-associated
110 MAG (indicated in Figure 1A)(Collins et al., 1997; Vinson et al., 2001). Analysis of protein extracts
111 demonstrated a reduction in MAG levels in GalNAc-T-/- mice (Sheikh et al., 1999; Kawai et al., 2001),
112 which suggests that complex neuronal gangliosides and glial MAG cooperatively contribute to the
113 stability of the axo-glial junction.
114 Importantly, loss of sulfatide does not alter ganglioside content (Honke et al., 2002) and complex
115 ganglioside deficiency does not alter sulfatide content (Yamashita et al., 2005) which suggests their
116 expression is not directly inter-linked in a compensatory way, but nevertheless might be additive
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117 functionally. Herein we considered that as these glycolipids are differentially expressed in glial and
118 axonal membranes, they may act in partnership to retain clustered proteins in their respective
119 domains. We sought to test this by interbreeding strains of single null mice and neuronal and glial
120 specific rescue mice, thereby allowing us to assess interdependency and cooperativity in the role of
121 axonal and glial glycolipids in paranodal organisation.
122
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123 Methods
124 Give sufficient detail to permit repetition of the work by others:
125 Reference should be made to published procedures wherever possible; this applies to the
126 original description and pertinent published modifications.
127 The sex of subjects should be stated
128 All companies from which materials were obtained should be listed. RRIDS
129 If materials were obtained from an individual, an affiliation for that individual should be listed.
130 Neurosci requires every research manuscript to include an Experimental Design and
131 Statistical Analysis section as a subsection of the Materials and Methods that describes the
132 experimental design and the statistical tests used in the study
133
134 Mice. Seven mouse lines on the C57Bl/6 background were used and generated: 1. Wild type (WT); 2.
135 GalNAc-T-/-; 3. CST-/-; 4. CST-/- x GalNAc-T-/--Tg(neuronal); 5. CST-/- x GD3s-/-; 6. CST-/- x GalNAc-T-/-; 7.
136 CST-/- x GalNAc-T-/--Tg(glial); (group number and age are described per experiment below).
137 Generation of GalNAc-T-/- and CST-/- transgenic mice has previously been described (Takamiya et al.,
138 1996; Honke et al., 2002) and were interbred to produce the CST-/- x GalNAc-T-/- genotype. Double
139 null mice with reconstituted site-specific expression of complex gangliosides were produced by
140 crossing the CST-/- genotype with previously described GalNAc-T-/--Tg(neuronal) or GalNAc-T-/--
141 Tg(glial) strains (Yao et al., 2014) or GD3s-/- (Okada et al., 2002) resulting in CST-/- x GalNAc-T-/--
142 Tg(neuronal), CST-/- x GalNAc-T-/--Tg(glial) and CST-/- x GD3s-/-, respectively. CST-/- x GalNAc-T-/--
143 Tg(glial) bred poorly and there were insufficient numbers to fully phenotype; therefore their
144 inclusion was restricted to survival data and phenotypic analysis. Mice were maintained under a 12
145 hour light/dark cycle in controlled temperature and humidity with unlimited access to food and
146 water. For each study, mMice of either sex [m2]were killed by rising CO2 inhalation; all experiments using
147 mice were performed in accordance with a licence approved and granted by the United Kingdom
148 Home Office and conformed to University of Glasgow institutional guidelines. Experiments complied
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149 with relevant guidelines on the care and use of animals outlined in the revised Animals (Scientific
150 Procedures) Act 1986.
151
152 Phenotypic analysis of mice. Weights were obtained at P22 from wild type (n=5), GalNAc-T-/- (n=5),
153 CST-/- (n=4), CST-/- x GalNAc-T-/--Tg(neuronal) (n=4), CST-/- x GD3s-/- (n=6) and CST-/- x GalNAc-T-/-
154 (n=10) mice. Data points from every animal per genotype were plotted, and the mean and S.E.M
155 displayed. Survival plots for all genotypes were plotted over 200 days for each genotype: wild type
156 (n=7); GalNAc-T-/- (n=10); CST-/- (n=18); CST-/- x GalNAc-T-/--Tg(neuronal) (n=6); CST-/- x GD3s-/- (n=38),
157 CST-/- x GalNAc-T-/--Tg(glial) mice (n=12); and CST-/- x GalNAc-T-/- (n=29). Mice were photographed
158 when suspended by the tail to record hind-limb leg splaying (a feature of many neurodegenerative
159 mutants), and gross brain anatomy was also recorded upon brain removal at P22.
160
161 Materials. Monoclonal antibodies used to detect complex a- or b-series gangliosides by
162 immunofluorescent staining were generated and described previously (Bowes et al., 2002; Boffey et
163 al., 2005). Anti-GM1 ganglioside antibody, DG2, and anti-GD1b antibody, MOG1, were used at 20
164 µg/ml. A new monoclonal anti-sulfatide antibody, GAMEG3, derived from mice inoculated with
165 sulfatide-bearing liposomes was used to detect sulfatide (Meehan et al., in press). Primary
166 antibodies were used as follows: mouse anti-phosphorylated neurofilament-H antibody (NF-H,
167 BioLegend; RRID:AB_2715851; 1:2000); rat anti-MBP (BioRad; RRID:AB_325004; 1:500); mouse anti-
168 pan Nav (Sigma; RRID:AB_477552; 1:100); mouse anti-ankyrin G (ThermoFisher scientific;
169 RRID:AB_2533145; 1:100); rabbit anti-Caspr (gifted from Professor Peles, Weizmann Institute, Israel;
170 1:1500); rabbit anti-Kv1.1 (Alomone labs; RRID:AB_2040144; 1:200); rabbit anti-Nav1.6 (Sigma;
171 RRID:AB_477480; 1:100); rabbit anti-pan neurofascin (gifted from Professor Brophy, University of
172 Edinburgh, UK; pNFasc 1:1000); mouse anti-MAG antibody (gifted from Professor Brophy, University
173 of Edinburgh, UK; 1:100). Primary antibodies used in Western blots were rabbit anti-MAG 248 (gifted
174 from Prof. N Groome, 1:10000[m3])(Barrie et al., 2010) and rabbit anti-NF155 (gifted from Professor
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175 Brophy, University of Edinburgh, UK; 1:5000) were prepared in 5% semi-skimmed milk in TBS
176 containing 0.1% Tween-20 (T-TBS). Secondary antibodies were made in PBS plus 1% NGS: isotype-
177 specific (IgG1, IgG3) Alexa Fluor 488- and 555-conjugated goat anti-mouse IgG antibodies (Molecular
178 Probes; RRID:AB_141780); Alexa Fluor 488- and 555-conjugated goat anti-rabbit (Molecular Probes;
179 RRID:AB_141761) and anti-rat IgG antibodies (Molecular Probes; RRID:AB_141733). Secondary
180 antibody for western blots was HRP-linked goat anti-rabbit (Dako, 1:10 000) prepared in 5%
181 skimmed milk/T-TBS.
182
183 Immunostaining , Image acquisition and analysis.
184 Lipid localisation: Snap-frozen peripheral nerves were transversely sectioned at 10µm and collected
185 onto APES-coated slides (n=3/genotype). Sections were treated with 100% EtOH for 10 min at -20C
186 then thoroughly washed in PBS. Antibodies were applied to the sections overnight at 4C in the
187 following combinations: 1) anti-ganglioside antibody DG2 or MOG1 with anti-phosphorylated NF-H
188 antibody; 2) anti-sulfatide antibody with anti-MBP antibody. Sections were PBS washed secondary
189 antibodies applied for 1h at R.T. Slides PBS washed and mounted in citifluor. Representative images
190 were captured at 40x magnification using a Zeiss AxioImager Z1 with ApoTome attachment and
191 processed using Zeiss Zen 2 blue edition software.
192 Nodal protein immunostaining: Sciatic nerves (SN) and optic nerves (OpN) were fixed for 30 min in
193 4% PFA upon removal from P22 mice (n=3/4 per genotype). Subsequently nerves were
194 cryoprotected in 30% sucrose, and either gently teased into single fibres (SN) collected on slides, or
195 frozen in OCT mounting medium and longitudinally sectioned at 10 µm (OpN). To study nodal
196 protein localization, teased SN were immunostained with primary antibodies for either anti- Nav1.6,
197 anti-Kv1.1 or pNFasc, and OpN sections were immunostained with primary antibodies for
198 combinations of anti- pNav and anti-pNFasc, or anti-Caspr and anti-AnkG. OpN sections and unfixed
199 peripheral nerve sections were stained with anti- MAG antibody. Nerves were pre-treated with
200 blocking solution (10% normal goat serum + 0.3 % Triton X-100) for 1 h at 4C before incubation
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201 overnight in the same solution plus primary antibody combinations at the same temperature. Triton
202 was omitted from blocking and incubation solutions when using unfixed peripheral nerve sections.
203 Samples were washed 3x 5 min then incubated for 2 h at room temperature in secondary antibody
204 solution. Slides were coverslipped [c4]in citifluor mount medium after three further 5 min PBS washes.
205 All nodal protein images were captured at 63x magnification, and MAG immunostained tissue at 40x
206 magnification using a Zeiss AxioImager Z1 with ApoTome attachment and processed with Zeiss Zen 2
207 blue edition software. For teased sciatic nerves, 20 NoR on average were imaged and quantified per
208 mouse for each stain. For optic nerve double-staining combinations, three 10 µm thick z-stacks (30
209 slices, step value 0.34 µm) were captured per mouse. Four 50 x 50 µm fields of view (FOV) per stack
210 were analysed for either pNav channel cluster number, number of Nav channel clusters flanked by
211 intact NFasc dimers, or intact Caspr dimers, using NIH ImageJ software (RRID:SCR_003070).
212 Clusters/dimers were included in the count if they overlaid the top and right-side borders of the
213 FOV, and excluded when they overlaid the left-side or lower borders. For MAG staining intensity
214 analysis, single slices were captured at set exposures from 3 slides per genotype. Graphs + S.E.M or
215 box-and-whisker plots were used to display the spread of all data points collected from each animal
216 then means were calculated per genotype for statistical analysis.
217
218 Ultrastructure. Mice (WT n=5, GalNAc-T-/- n=6, CST-/- n=6, CST-/- x GalNAc-T-/--Tg(neuronal) n=6, CST-
219 /- x GD3s-/-n=6, CST-/- x GalNAc-T-/- n=5) were transcardially perfused at P25 with 5%
220 glutaraldehyde/4% paraformaldehyde mixture before the optic nerve was removed and processed
221 for resin embedding, as previously described (Griffiths et al., 1981). Sections were cut for both light
222 and ultrastructural analysis. Electron micrographs from transverse sections of the optic nerve at
223 6700x magnification, were captured on a Jeol CX-100 Electron microscope. For quantification, a
224 minimum of 10 electron micrographs per animal were taken of randomly selected fields. All
225 measurements were made on scanned images using NIH ImageJ software. For axon morphometry
226 and quantification of axonal changes, all axons within or touching the top and left borders of an area
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227 of interest (AOI) were counted. The axon density and number of degenerating axons within the AOI
228 were counted. Averaged values from every animal per genotype was plotted, and the mean and
229 S.E.M displayed.
230
231 Extracellular recordings.
232 Perineural recordings were made from triangularis sterni nerve-muscle preparations set up as
233 previously described (Braga et al., 1991; McGonigal et al., 2010). Recordings were made from small
234 nerve bundles from each genotype (WT n=5, GalNAc-T-/- n=2, CST-/- n=3, CST-/- x GalNAc-T-/- n=2).
235 Perineural Nav and Kv channel waveforms were collected during a paired pulse stimulation protocol.
236 A representative graph plotting the peak Nav and Kv values as a percentage of the baseline
237 waveforms at each interstimulus interval (ISI) was used to convey recovery of the nodal ion channel
238 currents. A two-way ANOVA was used to compare the difference between genotypes, and post-hoc
239 multiple comparisons to measure differences at each ISI.
240 Conduction velocity was measured in sciatic nerve (WT n=3, GalNAc-T-/- n=4, CST-/- n=4, CST-/- x
241 GalNAc-T-/- n=5), as previously described (McGonigal et al., 2010). Optic nerves (between the eyeball
242 and the optic chiasm) were quickly removed into oxygenated (95% O2 and 5% CO2) physiological
243 Ringer’s solution containing (mM): NaCl, 129; KCl, 3; NaH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; HEPES,
244 3; NaHCO3, 20; and glucose, 10. The nerves were mounted in a recording chamber and each end
245 drawn into suction electrodes for electrophysiological studies (WT n=4, GalNAc-T-/- n=4, CST-/- n=4,
246 CST-/- x GalNAc-T-/--Tg(neuronal) n=3, CST-/- x GD3s-/-n=6, CST-/- x GalNAc-T-/- n=3). Compound action
247 potentials were evoked by a supramaximal stimulus (Grass S88 stimulator), applied via a suction
248 electrode at the proximal cut end, and recorded from a second suction electrode at the distal cut
249 end. Signals were amplified (CED1902), digitized (NIDAQ-MX A/D converter, National Instruments,
250 Austin, TX, USA) and analysed using WinWCP version 4.1.0 (J. Dempster, Strathclyde University,
251 RRID:SCR_014713). Nerves were crushed to confirm the waveforms were real at the end of the
252 experiment in order to identify and exclude electrical artefacts from the analysis. Nerve conduction
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253 velocity was calculated by dividing the length of the nerve by latency (the time between the
254 stimulation artefact and the highest peak of compound action potential). Average values from every
255 animal per genotype were plotted, and the mean and S.E.M displayed.
256
257 Western blot. Mouse brains were removed (n=4/genotype), snap frozen in liquid nitrogen, and
258 stored at -80°C until required. Myelin preparation was conducted using a modification of the
259 method by Norton and Poduslo (1973). Briefly, brains were homogenised in a buffer composed of
260 0.85 M sucrose, 10 mM Hepes, pH 7.4, 2 mM DTT, and 1 mM TLCK for 20 sec using an Ultra-Turrax
261 T8 blender ( IKA-Works Inc., Wilmington, NC) set at maximum speed and 0.25 M sucrose gently
262 layered on top of the homogenate and then centrifuged at 70,000g for 90 min at 4°C. The myelin
263 interface was collected, hypotonically lysed in chilled dH2O and pelleted at 23,000g for 30 min at
264 4°C. Following an additional two rounds of hypotonic lysis, the myelin pellet was resuspended in 10
265 mM Hepes, pH 7.4, containing 1x protease inhibitor cocktail (Sigma). The myelin fraction was stored
266 at -80°C until required. The protein concentration was determined using a bicinchoninic acid method
267 employing BSA as a standard (Pierce, Thermo Scientific). SDS-PAGE/ Western blot analysis was
268 conducted as previously described (Yool et al., 2001). In brief, 1 and 5 ug of myelin was denatured in
269 Laemmli buffer, separated on a 4-12% gel (Biorad), and transferred to nitrocellulose membrane
270 (Invitrogen). The membrane was blocked with 5% skimmed milk in TBS containing 0.1% Tween-20 (T-
271 TBS) for 1hr at room temperature, then incubated in primary antibodies overnight at 4°C on an
272 orbital shaker . Following three 10 min washes in T-TBS, the immunocomplex was detected with the
273 HRP-linked secondary antibody (Dako) and visualised using the ECL reaction as per manufacturer’s
274 instructions (Pierce, Thermo Scientific). Densitometric analysis of the Western blots was performed
275 using Image J software (NIH). Average values from every animal per genotype were plotted, and the
276 mean and S.E.M displayed.
277
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278 Experimental design and statistical analysis. The number of independent animals are described in
279 the Methods and Results sections and indicated in figure legends. Statistical differences among
280 genotypes were determined by one-way or two-way ANOVA followed by a Fisher’s or Tukey’s post-
281 hoc tests using GraphPad Prism 6 software (RRID:SCR_002798). Differences were considered
282 significant whenat p < 0.05.
283
284
285
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286 Results
287
288 present the experimental findings, clearly and succinctly. Only results essential to establish
289 the main points of the work should be included. Experimental conclusions should normally
290 be based on an adequate number of observations with statistical analysis of variance and
291 the significance of differences.
292 Authors must provide detailed information for each statistical test applied including: the type
293 of test; specific p values (not > or < ); degrees of freedom; population size; definition of
294 population (e.g., number of individual measurements, number of animals of each sex,
295 number of slices, number of times treatment was applied, etc.); and if performed, what
296 correction was used to adjust for multiple pairwise comparisons
297 JNeurosci requires every research manuscript to report in full the complete results of the
298 statistical analyses, including degrees of freedom and any estimates of effects size, should
299 be reported in the Results section. Report exact p values rather than ranges (e.g. p = 0.026
300 rather than p < 0.05). There are many types of analyses that can be reported, but examples
301 include F values (F(1, 72) = 14.5, p = 0.003, ANOVA), t values (t(10) = 2.98, p = 0.043,
302 paired t-test)
303
304
305 Ganglioside and sulfatide expression is successfully eliminated in CST-/- x GalNAc-T-/- mice
306 To investigate the interaction between ganglioside and sulfatide lipids on nervous system integrity,
307 we studied six transgenic mouse lines: wild type; GalNAc-T-/-; CST-/-; CST-/- x GalNAc-T-/--Tg(neuronal);
308 CST-/- x GD3s-/-; and CST-/- x GalNAc-T-/-. Table 1 provides an overview of the specific glycolipid
309 expression and deficiency profiles among the mice generated for this study. We confirmed the
310 presence of targeted gene disruption and reintroduction in these transgenic lines by PCR and
311 screening for complex ganglioside or sulfatide expression in neural tissue by immunostaining
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312 (FIGURE 1). An additional line, CST-/- x GalNAc-T-/--Tg(glial) was subsequently generated for
313 comparative survival plots.
314 The CST, GalNAc-T and GD3s genes are disrupted by an insert, which was assessed by PCR. From the
315 gels in Figure 1B, the larger band represents successful GalNAc-T gene disruption in GalNAc-T-/-, CST-
316 /- x GalNAc-T-/--Tg(neuronal) mice and CST-/- x GalNAc-T-/- mice, and CST disruption in CST-/-, CST-/- x
317 GalNAc-T-/--Tg(neuronal) mice, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- mice. The smaller band
318 confirms GD3 synthase (GD3s) gene disruption in CST-/- x GD3s-/- mice. Additionally, a band
319 identifying the GalNAc-T-flag [m5]in the CST-/- x GalNAc-T-/--Tg(neuronal) mouse strain confirms selective
320 GalNAc-T gene re-expression neuronally.
321 Confirmation of Aabsence or selective expression of lipids was assessed in all genotypes by staining
322 peripheral[m6] nerves with two anti-ganglioside antibodies that detected either the a- or b-series
323 complex gangliosides, and an anti-sulfatide antibody. Qualitative results for immunostaining with
324 these antibodies in the peripheral nerve are shown in (FIGURE 1C). As expected, neural tissue from
325 all genotypes except GalNAc-T-/- mice and CST-/- x GalNAc-T-/- mice was positive for anti-GM1 (a-
326 series) antibody immunolabeling. Additionally, anti-GD1b antibody (b-series) binding was
327 undetectable in the latter two genotypes confirming absence of all a- and b-series gangliosides.
328 Similarly, nerves from CST-/- x GD3s-/- mice, null for b-series ganglioside expression, had no
329 observable immunolabelling with the anti-GD1b antibody. Both a- and b-series gangliosides were
330 successfully reintroduced in the axons of the CST-/- x GalNAc-T-/--Tg(neuronal) mouse line as shown
331 by positive anti-GM1 and anti-GD1b immunoreactivity. When neural tissue was immunostained with
332 anti-sulfatide antibody, positively labelled myelin was observed in wild type and GalNAc-T-/- mice
333 nerves, while no labelling was detected in the other four genotypes, confirming the absence of
334 sulfatide expression with CST gene knock out.
335
336 Ganglioside and sulfatide double null mice have a significantly reduced lifespan
16
337 The complex ganglioside null and sulfatide null mouse strains have an age-dependent degenerative
338 phenotype that manifest clinically from 4 months and 6 weeks respectively. In contrast to each of
339 the single glycolipid deficient mice, the CST-/- x GalNAc-T-/- mice exhibited a very severe phenotype
340 from 2 weeks, lethal by 4 weeks. The mouse appears to develop normally and is similar in size to
341 wild type and single knock out mice, then subsequently fails to thrive, declining in body weight
342 beyond P15 and showing a significant reduction in weight at P22 (one-way ANOVA F(5,28) = 18.15,
343 p<0.0001[c7])(FIGURE 2A[m8]). Single pup rearing through removal of litter-mates on three occasions did
344 not prolong survival. CST-/- x GalNAc-T-/- mice exhibit a hunched, emaciated appearance, develop a
345 tremor and display hind-limb leg splaying (FIGURE 2B), a typical characteristic of the single knock out
346 mice at later stages of life. Despite reduction in weight, macroscopic brain (FIGURE 2B) and nerve
347 trunk anatomy[m9][RM10] appear normal.
348 Survival plots demonstrate that incrementally diminishing ganglioside and sulfatide expression
349 corresponds with a reduction in life expectancy, measured up to 200 days (FIGURE 2C).Wild type and
350 GalNAc-T-/- mice have a normal life expectancy up to 200 days. CST-/- x GalNAc-T-/- mice have the
351 shortest life expectancy of the lines studied, never surviving beyond 4 weeks, with the majority only
352 surviving to P21-P25. In the absence of sulfatide expression, but with normal ganglioside expression,
353 CST-/- life expectancy at 200 days more than halves. In mice lacking sulfatide and complex
354 gangliosides globally but with all complex gangliosides reintroduced specifically into neurons (i.e. the
355 CST-/- x GalNAc-T-/--Tg(neuronal) mouse), life expectancy is restored to that of sulfatide null mice,
356 with 60% of mice surviving to at least 200 days. Additionally, weight at P22 is comparable in this
357 genotype to WT, CST-/- and GalNAc-T-/- mice (FIGURE 2A). In order to determine whether a- or b-
358 series complex ganglioside deficiency was responsible for the severity of the double sulfatide and
359 complex ganglioside null mouse, we examined mice that expressed a-series gangliosides only and
360 lacked b-series gangliosides (achieved through crossing sulfatide and b-series null mice). These were
361 only modestly improved relative to the CST-/- x GalNAc-T-/- phenotype, as shown by the failure to
362 thrive and significant weight loss at P22. Life expectancy is severely reduced with only 50% survival
17
363 at 4 weeks, and less than 10% mice reaching a maximum of 21 weeks. To determine the importance
364 of neuronal relative to glial ganglioside expression, CST-/- x GalNAc-T-/- mice were generated with glial
365 expression of complex gangliosides, i.e. CST-/- x GalNAc-T-/--Tg(glial). Unlike the successful rescue of
366 the lethal phenotype with neuronal complex gangliosides, expression of compelx gangliosides in glial
367 membranes did not improve CST-/- x GalNAc-T-/- survival. Therefore based on survival data, the
368 genotypes can be categorised into three subsets: mild (WT, GalNAc-T-/-); moderate (CST-/-, CST-/- x
369 GalNAc-T-/--Tg(neuronal); and severe (CST-/- x GD3s-/-, CST-/- x GalNAc-T-/- ).
370
371 Loss of both sulfatide and complex gangliosides results in a disruption to CNS nodes of Ranvier
372 Age-dependent loss of nodal integrity is evident in both single ganglioside and sulfatide null mice
373 and could potentially contribute to loss of normal ion channel clustering, functional deficits and axon
374 degeneration. Of the single lipid deficient mice, disrupted nodal integrity is more rapid and
375 pronounced in the sulfatide null mice, beginning post-myelin development (>1 month). To assess
376 whether this process was enhanced or accelerated in CST-/- x GalNAc-T-/- mice, we initially assessed
377 changes in both the PNS [m11](sciatic nerve, internal intercostal nerve) and the CNS (optic nerve, brain) at
378 P22. When the PNS integrity was examined, We we found that combined sulfatide and complex
379 ganglioside deficiency did not augment the disorganisation of proteins at the node of Ranvier in the
380 PNS of the single sulfatide null mice (Figure 3A). Peripheral nerve Nav channel clusters were
381 lengthened both in CST-/- and CST-/- x GalNAc-T-/- sciatic nerves. Despite ANOVA reaching significance,
382 post-hoc multiple comparisons showed the Nav channel cluster lengths in the latter genotypes did
383 not reach significance compared to WT, GalNAc-T-/- or each other (one-way ANOVA, F(3,9)=.5794,
384 p=0.0491). In both CST-/- and CST-/- x GalNAc-T-/- sciatic nerve nodes of Ranvier immunostaining for
385 Kv1.1 channel domains, normally located at the juxtaparanodes, invaded the paranodal region,
386 shown by a shortening in the distance between the staining domains, compared to WT and GalNAc-
387 T-/- nerves (one-way ANOVA, F(3,9), p<0.0001). pNFasc staining did not significantly change between
18
388 genotypes (one-way ANOVA, F(3,9) = 1.157, p= 0.38) suggesting no impairment of NF155 paranodal
389 clustering in peripheral nerve at P22. Additionally, electrophysiological function did not significantly
390 worsen in CST-/- x GalNAc-T-/- peripheral nerve compared to single sulfatide deficient nerve based on
391 paired pulse stimulations and conduction velocity measurements (Figure [c12]3B). We performed
392 perineural recordings to assess the Nav and Kv channel currents of the internal intercostal nerve that
393 innervates the TS muscle. The recovery of both peaks after paired pulse stimulation was significantly
394 different among genotypes (Nav peak: two-way ANOVA, F(3,117) = 10.79, p<0.0001. Kv peak: two-
395 way ANOVA, F(3,117) = 32.24, p<0.0001). Both CST-/- and CST-/- x GalNAc-T-/- peaks were significantly
396 reduced in the initial interstimulus intervals compared to WT and GalNAc-T-/- preparations, as
397 indicated on the graphs in Figure 3B. Conduction velocity was slower in GalNAc-T-/-, CST-/- and CST-/- x
398 GalNAc-T-/- sciatic nerve compared to WT, but only reached significance in CST-/- nerve (one-way
399 ANOVA, F(3,12) = 3.939, p = 0.036). MAG immunostaining was comparable in peripheral nerves of all
400 four genotypes (Figure 3C). It is thus unlikely that PNS dysfunction and degeneration would account
401 for the lethal phenotype in double deficient mice and there was no evidence of further disruption to
402 the PNS in the double deficient mice compared to the sufatide null mice. As such, we then focussed
403 our attention on the CNS with additional Tg lines.
404 When comparing Nav channel clustering in the optic nerve from all genotypes there is a significant
405 reduction in Nav channel cluster number (one-way ANOVA (F(5,13) = 4.76, p=0.0108). CST-/- x
406 GalNAc-T-/- mice have fewer Nav channel clusters than all other genotypes, but this reduction only
407 reached significance when compared to wild type and complex ganglioside deficient mice, which
408 were not significantly different to each other (FIGURE 4A). Although reduced, the number of Nav
409 channel clusters is within the normal wild type range when sulfatide is expressed or complex
410 gangliosides are reintroduced in the neuronal membrane in CST-/- x GalNAc-T-/--Tg(neuronal) mice.
411 This is not the case when a-series gangliosides are expressed globally on the sulfatide null
412 background in CST-/- x GD3s-/- mice, which have similar cluster numbers to CST-/- x GalNAc-T-/- mice.
413 Since cluster number does not explicitly define mature nodes of Ranvier, the number of Nav channel
19
414 clusters flanked laterally by intact paranodal neurofascin dimers were assessed. Earlier reports have
415 shown that lack of sulfatide attenuates neurofascin 155 localisation to the paranodal region
416 (Ishibashi et al., 2002; Schafer et al., 2004; Marcus et al., 2006), and indeed we found all four lines
417 with sulfatide deficiency had significantly fewer neurofascin positive paranodal dimers flanking Nav
418 channel clusters as early as P22 compared to wild type and GalNAc-T-/- mice (one-way ANOVA
419 (F5,13) = 21.31, p<0.0001)(FIGURE 4B,C). A pan-neurofascin antibody was used that detects both the
420 neuronal NF186 and glial NF155 isoforms critical for Nav channel clustering and paranodal axo-glial
421 junction formation, respectively. We detected no change to NF186 [m13][RM14]presence and observed normal
422 co-localisation with Nav channel clusters suggesting this isoform is not impacted by ganglioside or
423 sulfatide lipid expression.
424 Further analysis showed paranodal Caspr dimers were significantly reduced at P22 in all genotypes,
425 with the exception of GalNAc-T-/- mice, compared to wild type mice (one-way ANOVA (F(5,14) = 8.8,
426 p=0.0006) (FIGURE 5). However, significance was greatest when wild type was compared to GalNAc-
427 T-/- x CST-/- and CST-/- x GD3s-/- mice. GalNAc-T-/- x CST-/- mice also showed a significant reduction
428 compared to CST-/- and CST-/- x GalNAc-T-/--Tg(neuronal) nerves. Expression of neuronal a- and b-
429 series gangliosides improved the feature under examination, but global a-series ganglioside
430 expression did not.
431
432 Glycolipid deficiency reduces CNS axon integrity and function
433 Age-dependent degeneration beyond 2 months of age is a feature of the CST-/- and the GalNAc-T-/-
434 mouse strains. The principal ultrastructural abnormality observed in the current study was early
435 onset CNS axon degeneration in the CST-/- x GalNAc-T-/- mice. There was a significant increase in
436 degenerating axons in the optic nerve at P25 with diminishing glycolipid expression (one-way
437 ANOVA (F(5,27) = 11.51, p<0.0001). CST-/- x GalNAc-T-/- mice had significantly more degenerate
438 axons compared to wild type and GalNAc-T-/- nerves, which are comparable (FIGURE 6A). The
20
439 number of degenerate axons in CST-/- mice was very variable and reached significance, albeit a lower
440 level compared to wild type. Additionally, the number of degenerate axons were fewer than
441 observed for CST-/- x GalNAc-T-/- optic nerve but this difference did not reach significance. A
442 protection of axons from degeneration was achieved by reinstatement of both a- and b-series
443 gangliosides neuronally, leading to a reduction in the number of degenerate axons to within wild
444 type levels. However, the number of degenerate axons was not improved by expression of global a-
445 series ganglioside expression in the CST-/- x GD3s-/- strain, which were equal to CST-/- x GalNAc-T-/-
446 levels. Electron micrographs show the normal formation of myelin in large diameter myelinated
447 fibres fibres, alongside abundant unmyelinated and thinly myelinated fibres undergoing myelin
448 maturation in all genotypes (FIGURE 6B). As peak myelination is in progress at the time-point
449 studied, abundant unmyelinated fibres and thin myelin was observed in all genotypes in the electron
450 micrographs, making it uninformative to undertake a meaningful analysis of myelin formation or
451 maintenance[m15][RM16].
452 To investigate the functional consequence of degenerating axons and nodal structure disturbance,
453 conduction velocity was recorded from isolated optic nerve at P22 (FIGURE 6C). Conduction velocity
454 was significantly reduced with diminishing glycolipid expression (one-way ANOVA (F(5,18) = 4.25,
455 p<0.01), which correlated with the survival plots, nodal and ultrastructural data. There was a
456 significant reduction in CV did not reach significance between any of the genotypes in post-hoc
457 analysis. The waveforms becamewere abnormal [c17]in CST-/-, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- optic
458 nerves as compared to wild type, and GalNAc-T-/- mouse nerves. CST-/- x GalNAc-T-/- conduction
459 velocity was reduced compared to CST-/- but this did not reach significancewith broader waveforms
460 that followed a different peak formation. Once more, tThere was an modest improvement in CST-/- x
461 GalNAc-T-/--Tg(neuronal) with optic nerve conduction velocity rates reachingwaveform within theto
462 normal wild type and GalNAc-T-/- range and significantly greater than those rates recorded from CST-
463 /- x GD3s-/- and CST-/- x GalNAc-T-/- optic nerve. Although increased, the difference in CST-/- x GalNAc-
464 T-/--Tg(neuronal) conduction velocity compared to CST-/- mice did not reach significance appearance.[c18]
21
465
466 MAG protein expression in myelin is disrupted in sulfatide and ganglioside double null mice
467 Sulfatide deficiency has previously been reported to alter expression of key myelin proteins,
468 prompting us to interrogate the differences in the expression of these proteins in the 6 genotypes
469 studied. Expression of the major myelin proteins PLP and MBP in the myelin fraction from P22 brain
470 homogenates did not significantly differ among the six genotypes, with the exception of a mildly
471 reduced expression in WT mice compared to GalNAc-T-/- genotypes (data not shown). A significant
472 reduction in MAG protein level was observed in the myelin fraction from CST-/- x GalNAc-T-/- mice
473 compared to all genotypes, which did not significantly differ from one another (one-way ANOVA
474 F(5,16) = 7.735, p=0.0007) (Figure 7A). Conversely, MAG expression in whole brain homogenates
475 and in optic nerve sections was comparably reduced in all genotypes compared to wild type,
476 suggesting an enhanced loss of appropriate trafficking or site-specific anchoring in [m19]myelin in the CST-
477 /- x GalNAc-T-/- mice (WB: one-way ANOVA F(5,18) = 13.87, p<0.0001; immunostaining [m20][RM21][m22][RM23]one-way
478 ANOVA F(5,12) = 13.36, p = 0.0001) (Figure 7A & B). This notion is strengthened by the comparable
479 expression of oligodendrocyte transcription factor 2 (Olig2) among genotypes which indicates
480 oligodendrocyte number is normal and oligodendrocytes are not lost due to glycolipid deficiency
481 (one-way ANOVA F(5,12) = 0.41, p = 0.835)(Figure 7A). Myelin NF155 protein levels were
482 significantly reduced to a comparable level in all genotypes deficient in sulfatide, which corresponds
483 with our immunostaining data and previous reports (one-way ANOVA F(5,18) = 23.41, p<0.0001)
484 (Figure 7A). This is replicated in the whole brain extract (data not shown).
485
22
486 Discussion (1500 words max)
487
488 The discussion section should be as concise as possible
489 brief statement of the principal findings
490 discussion of the validity of the observations
491 discussion of the findings in light of other published work dealing with the same or closely
492 related subjects
493 statement of the possible significance of the work
494 Extensive discussion of the literature is discouraged
495
496
497 Glycosphingolipids including sulfatide and complex gangliosides have a significant role in myelinated
498 nerve maintenance, but their distinct or interdependent roles and interactions are unclear. The
499 neurodegenerative consequence of deficiency in either sulfatides or complex ganglioside synthesis
500 through transgenic manipulation indicates a role for these lipids in maintaining myelin and axonal
501 integrity, particularly at the node of Ranvier (Takamiya et al., 1996; Sheikh et al., 1999; Honke et al.,
502 2002; Ishibashi et al., 2002; Marcus et al., 2002; Hoshi et al., 2007; Susuki et al., 2007). It was the aim
503 of this study to investigate their interdependency on interacting membranes in order to add insight
504 to the role each family of lipids plays in myelinated nerve and nodal stability.
505 We found that interbreeding sulfatide deficient mice with complex ganglioside deficient mice
506 resulted in an exaggerated neurodegenerative phenotype and early death compared to single null
507 mice. Through a combination of ultrastructural, immunohistological, functional, and biochemical
508 studies, we showed that the combined loss of both groups of lipids resulted in a more severe
509 outcome than single lipid deficiency (Takamiya et al., 1996; Honke et al., 2002). Diminishing
510 expression of lipids divided the survival of the seven genotypes produced for this study into three
511 broad groups: Mild (WT and GalNAc-T-/-); Intermediate (CST-/-, CST-/- x GalNAc-T-/--Tg(neuronal));
23
512 Severe (CST-/- x GD3s-/-, CST-/- x GalNAc-T-/--Tg(glial), CST-/- x GalNAc-T-/-). We reported a more
513 pronounced phenotype in the CNS than the PNS and thereby focused much of our analysis on the
514 CNS. Degenerate axon number increased with diminishing lipid content and conduction became
515 increasingly impaired; MAG expression in the myelin fraction from brain homogenates was
516 significantly lower in the CST-/- x GalNAc-T-/- genotype compared to all other genotypes; and Nav
517 channel cluster number, Caspr dimer number and Nav channels flanked by NF155 dimers decreased
518 with decreasing lipid expression. Rescuing the neuronal, but not the glial complex ganglioside
519 expression reversed the lethality observed in the double null mice. Examining the relative
520 importance of a- and b-series gangliosides we observed only modest improvement occurred with
521 global a-series ganglioside expression on a sulfatide null background. Collectively, these data
522 indicate the importance of neuronal b-series gangliosides (e.g. GD1b, GT1b) in maintaining survival in
523 the co-presence of sulfatide deficiency and indicate interdependency between the functions of these
524 two groups of lipids. We thus propose that sulfatide and b-series ganglioside lipid domains on
525 opposing membranes majorly contribute to a co-ordinated axo-glial adhesion and paranodal
526 organisation, a combined loss of which leads to severe impairment of nerve integrity with a fatal
527 outcome at an early age.
528 We have previously highlighted the significance of neuronal complex ganglioside expression to nerve
529 integrity in the GalNAc-T-/- age-dependent neurodegenerative genotype (Yao et al., 2014).
530 Considering the impact of neuronal ganglioside expression on survival, combined with the reduction
531 of MAG in the myelin fraction in double deficient mice, these findings suggest an underlying
532 mechanism for the lethal phenotype in CST-/- x GalNAc-T-/- mice. The complex gangliosides GD1a and
533 GT1b are prominently expressed on the axonal membrane and are receptors for MAG (Collins et al.,
534 1997; Vinson et al., 2001). MAG is expressed on the periaxonal membrane of the myelin sheath
535 (Bartsch et al., 1989; Trapp et al., 1989) and is involved in the continuous axo-glial contact and
536 bidirectional signalling along the myelinated nerve, particularly at the paranode. MAG null mice form
537 cis bonds in the myelin membrane that are stabilised by trans interactions with gangliosides in the
24
538 neuronal membrane (Pronker et al., 2016). MAG null mice exhibit delayed maturation of nodes and,
539 similar to GalNAc-T-/- mice, modest nervous system abnormalities, suggesting a role in myelinated
540 nerve maintenance rather than development (Montag et al., 1994; Li et al., 1998; Marcus et al.,
541 2002; Pan et al., 2005). Previously it has been shown that MAG levels are reduced in GalNAc-T-/- mice
542 (Sheikh et al., 1999; Kawai et al., 2001), and when considered with the comparable phenotype
543 between GalNAc-T-/- and MAG-/- mice suggests complex gangliosides and MAG cooperatively
544 contribute to the stability of the axo-glial junction[m24][RM25]. Indeed[m26], interbreeding GalNAc-T-/- and MAG-/-
545 strains did not exacerbate the phenotype of the single null mice, suggesting a complementary and
546 functional interaction between these molecules (Pan et al., 2005). Conversely, in our study,
547 combining GalNAc-T-/- and CST-/- strains exacerbates the phenotype suggesting two independent
548 roles. Notably, MAG acts as a myelin receptor for axonal gangliosides, and is localised to the myelin
549 membrane by sulfatide rich lipid rafts. Recent analysis of protein extracts from 4-week old sulfatide
550 null mouse brains confirmed a progressive reduction in MAG and a significant reduction in NF155
551 (25% compared to wild type)(Palavicini et al., 2016). Taken together with extraction studies that
552 identified sulfatide-containing lipid rafts as anchors for MAG and NF155 (Pomicter et al., 2013), it
553 seems that sulfatide acts as a wide-ranging anchor for multiple myelin and glial membrane proteins.
554 With the knowledge that MAG and sulfatide modulate axo-glial stability, Marcus et al. (2002)
555 investigated the significance of both molecules to axo-glial integrity by crossing MAG deficient mice
556 with GalC/sulfatide double deficient mice, which individually have similar phenotypes. Similar to our
557 lethal sulfatide and ganglioside double deficiency phenotype, this genetic combination resulted in a
558 lethal phenotype, with survival up to P22 (Marcus et al., 2002). Again, the NoR appear to develop
559 normally followed by subsequent generalised impairment of the paranodal axo-glial junction. Unlike
560 the minimal effect of combined MAG and complex ganglioside disruption, MAG and ceramide
561 galactosyltransferase (CGT) double deficiency aggravates the phenotype suggesting two non-
562 redundant functions are impaired. This phenotype corresponds to our lethal phenotype, indicating
563 that a loss of interaction between MAG and gangliosides in a membrane environment devoid of GalC
25
564 and sulfatide is fatal. It is likely that GalC/sulfatide has a role in promoting localisation/anchoring of
565 MAG and subsequently MAG/ganglioside promote axo-glial integrity. Indeed, we reported a striking
566 deficiency in MAG in the myelin fraction of our double null mice compared to all other genotypes
567 owing to the loss of its localising agent, sulfatide, and binding partner, complex gangliosides.
568 The severe neurodegenerative phenotype of our double lipid deficient mice reflected that of
569 paranodal protein deficient mice (Tait et al., 2000; Bhat et al., 2001; Boyle et al., 2001; Charles et al.,
570 2002), suggesting an essential role for lipids in paranodal protein maintenance. In the current study
571 we restricted our investigation of nodal disruption to those proteins related to function (Nav
572 channels) or previously identified as lipid raft associated (NF155, Caspr)(Sheikh et al., 1999; Schafer
573 et al., 2004; Susuki et al., 2007; Pomicter et al., 2013) and acknowledge that this was not an
574 exhaustive study. Adhesion molecules freely diffuse in the axon membrane and accumulate at NoR,
575 whereas ion channels and components of the cytoskeleton require transport (Zhang et al., 2012).
576 NF186 is essential for maintenance of the Nav channel clusters at the NoR (Zhang et al., 2012;
577 Desmazieres et al., 2014; Taylor et al., 2017) and appeared unperturbed in our double null mice. This
578 aligns with the fact that NF186 is likely not raft-associated (Schafer et al., 2004; Susuki et al., 2007).
579 Intact The presence of NF186 accounts for the relatively minimal reduction in Na channel clusters
580 and impairment in conduction observed in CST-/- x GalNAc-T-/- mice. Schafer et al. (2004) proposed
581 an indirect mechanism of axo-glial junction stabilisation at the paranode whereby glycosphingolipid-
582 rich lipid rafts cluster NF155 that then co-cluster with axonal binding partners Caspr and contactin.
583 Indeed, we confirmed by immunostaining and western blot that NF155 was disrupted in all
584 genotypes with sulfatide deficiency. The axonal partner of NF155, Caspr, was also disrupted as had
585 previously been shown in single ganglioside and sulfatide deficiency (Ishibashi et al., 2002; Susuki et
586 al., 2007), but to a greater degree when both sets of lipids were absent. Caspr has been associated
587 with ganglioside-rich lipid rafts on the axonal membrane and in their absence, Caspr is not anchored
588 (Susuki et al., 2007). Our results that the PNS was less perturbed than CNS likely reflects the greater
589 stability of the peripheral node of Ranvier, and greater influence that the paranode has on stability
26
590 in the CNS (Rasband and Peles, 2015). However, the combined absence of both NF155 and Caspr
591 through loss of both sulfatide and gangliosides, respectively, likely intensifies the reduction in Nav
592 channel clusters and ultimately leads to enhanced nodal instability compared to single lipid null
593 mice.
594
595 Ganglioside, sulfatide and MAG single deficient mice do not exhibit a severe phenotype, strongly
596 suggesting redundant functions: overexpression/loss of other lipids (e.g. simple gangliosides,
597 seminolipid) could, in theory, contribute to the overall phenotype in transgenic mice. Mice that lack
598 the GD3 synthase (GD3s) enzyme that underlies production of all b-series gangliosides (Figure 1A)
599 have grossly normal nodal architecture (Okada et al., 2002), demonstrating that there is
600 compensation in the ganglioside family. It is possible that ganglioside deficient mice have a less
601 severe phenotype than those lacking sulfatide due to compensation by over-expression of simple
602 gangliosides, notably GM3 & GD3 whose levels are markedly elevated in the GalNAc-T-/- phenotype
603 (Susuki et al., 2007). The increased severity of double ganglioside null (GM3 only) mice (Kawai et al.,
604 2001; Inoue et al., 2002; Yamashita et al., 2005) lends support for this theory, however, the nodal
605 protein organisation has not been characterised. It was therefore an unexpected finding that the
606 CST-/- x GD3s-/- strain had such a severe phenotype. This could be thus explained by the lack of the b-
607 series ganglioside GT1b interacting with MAG (Vinson et al., 2001).
608 It is clear from these results that sulfatide and complex gangliosides have crucial and independent
609 roles in clustering proteins on opposing membranes which is essential to normal axo-glial integrity
610 and nervous system function. Redundant features can compensate for the loss of one family, but
611 their simultaneous absence leads to a catastrophic accumulation of axon degeneration, axo-glial
612 disruption and nodal pathology, culminating in early fatalitydeath. (1617 words, 117 over)
27
613 Figure legends
614
615 Figure 1. Generation of lipid deficient transgenic mouse lines and confirmation by PCR and
616 immunostaining. Six mouse lines were used and generated: Wild type; GalNAc-T-/-; CST-/-, CST-/- x
617 GalNAc-T-/--Tg(neuronal); CST-/- x GD3s-/-; CST-/- x GalNAc-T-/-. A) Sulfatide and ganglioside
618 biosynthesis pathways (associated gene knock outs are indicated in boxes). Ceramide is the
619 precursor to sulfatide and gangliosides. The cerebroside sulfotransferase (CST) enzyme is necessary
620 for the synthesis of sulfatide from galactocerebroside (GalC). The GalNAc-transferase (GalNAc-T)
621 enzyme is necessary for generation of complex gangliosides and the GD3 synthase (GD3s) enzyme
622 for specific production of a-series complex ganglioside. Constructs were generated to drive GalNAc-T
623 expression in neurons of GalNAc-T-/- x CST-/- mice to produce the GalNAc-T-/--Tg(neuronal) mouse
624 line. Location of MAG binding site to terminal 2,3 sialic acid is indicated by a red circle. B) PCR
625 results confirm presence or absence of GalNAc-T, GD3s and CST expression in mouse lines. Large
626 bands represent the disrupted GalNAc-T and CST gene with insert, while the smaller band represents
627 the disruption of the GD3s gene. The flag identifies the re-introduction of the GalNAc-T gene into the
628 neurons. C) Expression of a- and b- series gangliosides and sulfatide was confirmed by staining
629 peripheral nerves with anti-GM1, anti-GD1b and anti-sulfatide monoclonal antibodies (green),
630 respectively. Axons and myelin were identified with neurofilament or MBP (red), respectively. Scale
631 bar = 10 µm.
632
633 Figure 2. Altering the expression of gangliosides and sulfatide in novel transgenic mouse lines effects
634 survival and phenotype. A) The weight of double null (n=10) and CST-/- x GD3s-/- (n=6) mice is normal
635 during development, but declines from P15-25 and is significantly reduced compared to other
636 genotypes at P22. Statistical differences among genotypes were determined by one-way ANOVA
637 followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as
28
638 follows: *p<0.05; **p<0.01; ***p<0.001. B) CST-/- x GalNAc-T-/- and CST-/- x GD3s-/- mice display a
639 reduction in stature, hindlimb leg splaying, a hunched appearance and tremor at P22. Gross brain
640 anatomy does not differ between genotypes. C) Survival plots demonstrate that incrementally
641 diminishing ganglioside and sulfatide expression corresponds with a reduction in life expectancy up
642 to 25 weeks. Wild type (n=7) and GalNAc-T-/- (n=10) mice have a normal life expectancy. In the
643 absence of sulfatide with normal ganglioside expression life expectancy to 25 weeks is more than
644 halved in CST-/- mice (n=18). Interestingly, in the absence of sulfatide and complex gangliosides with
645 the reintroduction of complex gangliosides into neurons alone (n=6), life expectancy is improved
646 with 60% of mice surviving to 25 weeks and beyond. Mice with no sulfatide and a-series gangliosides
647 expressed globally (n=38) can survive up to 20 weeks. Double ganglioside and sulfatide knock out
648 mice (n=29) have the worst phenotype, never surviving past 4 weeks and with death occurring at
649 P21-25. Reintroduction of gangliosides into glial (n=12) does not improve survival.
650
651 Figure 3. The additional loss of complex gangliosides on a sulfatide null background does not
652 augment disorganisation at the node of Ranvier or electrophysiological function in the peripheral
653 nervous system. A) There is modest Nav1.6 channel cluster lengthening in both CST-/- and CST-/- x
654 GalNAc-T-/- teased sciatic nerve that does not reach significance. Invasion of the sciatic nerve
655 paranode by Kv1.1 channels, shown by a decrease in the gap between positive domains, is
656 significantly and comparably enhanced in both CST-/- and CST-/- x GalNAc-T-/- mice compared to WT
657 and GalNAc-T-/- nodes of Ranvier. The length of pan-neurofascin immunostained domains does not
658 significantly differ among genotypes. Representative images are shown of sciatic nerve sections from
659 each genotype immunostained for Nav1.6 or anti-Kv1.1 antibody. Nav channel clusters were
660 observed and appeared similar in every genotype. Kv1.1 formed two distinct domains of
661 immunostaining at the juxtaparanodes in WT and GalNAcT-/- mice. Conversely, mice lacking sulfatide
662 expression had Kv1.1 staining at the paranodes, suggesting disruption to the axo-glial junction. (WT
29
663 n=3, GalNAc-T-/- n=3, CST-/- n=3, CST-/- x GalNAc-T-/- n=4 Scale bar = 5 µm. B) Perineural recordings of
664 Nav and Kv channel currents from intercostal nerves showed an increase in recovery time for both
665 peaks following paired pulse stimulation in both CST-/- and CST-/- x GalNAc-T-/- mice compared to WT
666 and GalNAc-T-/- mice. The CST-/- and CST-/- x GalNAc-T-/- mice were not significantly different from
667 each other at any interstimulus interval. Graphs display the means and +SEM for each genotype (WT
668 n=5, GalNAc-T-/- n=2, CST-/- n=3, CST-/- x GalNAc-T-/- n=2), and statistical analysis performed (two-
669 way ANOVA; # signifies WT vs. CST-/- x GalNAc-T-/- mice; * signifies WT vs CST-/-; + signifies GalNAc-T-/-
670 vs CST-/- x GalNAc-T-/-; ^ signifies GalNAc-T-/- vs CST-/- x GalNAc-T-/-). Sciatic nerve conduction velocity
671 decreased in CST-/-(n=4), GalNAc-T-/- (n=4) and CST-/- x GalNAc-T-/- (n=5) mice compared to wild type
672 (n=3), but only reached significance in CST-/- nerve. C) Peripheral nerve MAG immunostaining was
673 comparable among genotypes. Scale bar = 20 µm. Graphs display the means and +SEM for each
674 genotype, ), and statistical analysis performed (one-way ANOVA followed by Tukey’s post-hoc tests
675 to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01;
676 ***p<0.001).
677
678 Figure 4. Loss of both sulfatide and complex gangliosides results in a modest reduction in CNS Nav
679 channel cluster number and sulfatide deficiency reduces NF155 presence at parnodal loops. A) The
680 number of Nav channel clusters significantly decreases in glycolipid deficient mouse optic nerves
681 (n=3-4/genotype). Reintroducing a- and b-series gangliosides into neurons rescues this feature. B)
682 Nav channel clusters flanked by normal paranodal neurofascin are significantly reduced in number
683 when sulfatide is not expressed (n=2-3/genotype). Box-and whisker plots are used to display the
684 spread of all data points collected from each animal then means were calculated per genotype for
685 statistical analysis (one-way ANOVA followed by Tukey’s post-hoc tests to compare multiple
686 comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001). C) Representative
687 images of optic nerve sections from each genotype double-immunostained for pan-voltage gated
30
688 sodium channel (pNav) antibody (green) and pan-neurofascin (pNFasc) antibody (red). Na channel
689 clusters were observed in every genotype. pNFasc immunostaining formed a long band crossing the
690 node and paranodes in WT and GalNAc-T-/- mice suggesting labelling of the NF186 and NF155
691 isoforms, respectively. All of the genotypes lacking sulfatide expression had pNFasc staining
692 restricted to the node of Ranvier and co-localising only with pNav staining, suggesting presence of
693 only the NF186 isoform of neurofascin. Scale bar = 5 µm.
694
695 Figure 5. Paranodal Caspr dimer immunostaining in CNS tissue is progressively reduced with
696 increasing glycolipid deficiency. A) CST-/- x GalNAc-T-/- and CST-/- x GD3s-/- mice have significantly
697 fewer Caspr dimers per field of view (FOV) compared to WT and GalNAc-T-/- mice, and are not
698 significantly different to each other (n=2-5/genotype). Caspr dimer number is improved to levels
699 within GalNAc-T-/- mice range, but not WT, in CST-/- and CST-/- x GalNAc-T-/--Tg(neuronal) mice, which
700 both display significantly more Caspr dimers than CST-/- x GalNAc-T-/- mice. Box-and whisker plots
701 are used to display the spread of all data points collected from each animal then means were
702 calculated per genotype for statistical analysis (one-way ANOVA followed by Tukey’s post-hoc tests
703 to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01;
704 ***p<0.001). B) Representative images of optic nerve sections from each genotype double-
705 immunostained for Caspr (red) and nodal marker AnkyrinG (green) show the reduction in Caspr
706 dimer number with diminishing glycolipid expression. Scale bar = 10 µm.
707
708 Figure 6. Glycolipid deficiency reduces CNS axon survival and function. A) Degenerate axon number
709 increases to a significant level compared to wild type in optic nerves from CST-/-, CST-/- x GD3s-/- and
710 CST-/- x GalNAc-T-/- mice. Reintroducing a- and b-series gangliosides into neurons rescues this
711 pathology. B) Electron micrographs depict the differences among the genotypes: white arrowheads
712 indicate degenerate axons. Myelin is similar among mouse lines. One-way ANOVA followed by
31
713 Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows:
714 *p<0.05; **p<0.01; ***p<0.001. Scale bar = 2 µm C) A reduction in conduction velocity was observed
715 in optic nerves with diminishing glycolipid content. Representative CAP traces from each genotype
716 show the normal waveform in WT, GalNAc-T-/- and CST-/- x GalNAc-T-/--Tg(neuronal) optic nerves,
717 however the waveform becomes abnormal in the CST-/-, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- mice.
718 Horizontal scale bar = 2 ms, vertical scale bar = 1 mV.
719
720 Figure 7. N.B. New order of genotypes. MAG and NF155 expression in the myelin fraction is altered
721 by glycolipid deficiency. A) In the myelin fraction from P22 brain homogenates, MAG is significantly
722 reduced in CST-/- x GalNAc-T-/- mice compared to all genotypes, which do not significantly differ from
723 one another (upper graph). All genotypes have a significant reduction in MAG expression in whole
724 brain homogenate compared to WT (middle graph). Myelin NF155 was significantly reduced in all
725 genotypes compared to WT (indicated by *) and GalNAc-T-/- (indicated by #), which had similar levels
726 (lower graph). ^indicates significant difference between CST-/- x GalNAc-T-/- and CST-/- x GalNAc-T-/--
727 Tg(neuronal) mice. Olig2 is unchanged by altered glycolipid expression. Representative blots show
728 protein intensity per genotype, indicated by the corresponding number indicated in the key. B) P22
729 optic nerve MAG immunostaining is significantly reduced in all genotypes compared to wild type
730 nerve, which do not significantly differ from one another. Representative images show MAG
731 immunostaining per genotype. Means were calculated per genotype for statistical analysis (one-way
732 ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the
733 graphs as follows: *p<0.05; **p<0.01; ***p<0.001). Scale bar = 50 µm.
734
735 Figure 8. Schematic depicting the glycolipid rafts and their associated paranodal proteins Caspr,
736 NF155 and MAG under wild type conditions and in our transgenic mouse lines. GD1a and GT1b are
737 represented in the ganglioside rafts as they are the major ligands of MAG, however, in reality the
32
738 rafts would contain all complex gangliosides. Normally GD1a and GT1b in rafts will tether MAG but in
739 their absence MAG does not make the axo-glial connection. If NF155 is present, this protein can
740 partner with Caspr/Contactin to make an axo-glial junction. However, when sulfatide is absent,
741 NF155 is also lost from the paranode. In the absence of NF155, Caspr presence is also diminished,
742 especially with loss of complex ganglioside rafts. Under conditions of both ganglioside and sulfatide
743 raft deficiency, we propose an absence of the structurally supporting proteins MAG and NF155 that
744 results in the loss of a functionally competent axo-glial junction.
745 Acknowledgements
746 Include funding sources, John Dempster
747
748
749
33
750 References
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907
37
GalNAcT CST Axonal complex Global a- Non- Non- enzyme enzyme ganglioside expression series neuronal neuronal expressed expressed over GM3 over- GD3 over- globally globally a -series b - series expression expression expression
Wild type
GalNAcT-/-
-/- CST
CST-/- x GalNAcT-/- Tg
Life Life expectancy (neuronal)
-/- CST x GD3s-/-
-/- CST x GalNAcT-/- Tg(glial)
-/- CST x GalNAcT-/-
Table 1. Lipid expression profiles of genotypes in this study. A B Sulfatide & Ganglioside biosynthesis pathways 1 2 3 4 5 6
Ceramide GlcCer LacCer o-series GalNAcT Cer Cer Cer Cer Cer Cer Cer CGT-/- GA2 GA1 GM1b GD1c GalNAcT GalC a-series Cer GM3 Cer Cer Cer Cer Cer -flag
CST-/- GM2 GM1 GD1a GT1a GD3s-/- b-series GD3s Cer Cer Cer Cer Cer Cer GD3 Sulfatide GD2 GD1b GT1b GQ1b CST GalNAcT-/- GalNAc Galactose Glucose NeuNAc Sulfatide MAG binding site (enhanced if other Most abundant complex gangliosides 1. Wild type; 2. GalNAcT-/-; 3. CST-/-; 4. CST-/- x GalNAcT-/-- sialic acids on inner galactose) in nervous system Tg(neuronal); 5. CST-/- x GD3s-/- ; 6. CST-/- x GalNAcT-/-
CST-/- x GalNAcT-/-- CST-/- x CST-/- x C Wild type GalNAcT-/- CST-/- Tg(neuronal) GD3s-/- GalNAcT-/- αGM1 Nfil
αGM1
αGD1b Nfil
αGD1b
αSulfatide MBP
αSulfatide
Figure 1. Generation of lipid deficient transgenic mouse lines and confirmation by PCR and immunostaining. Six mouse lines were used and generated: Wild type; GalNAc-T-/-; CST-/-, CST-/- x GalNAc-T-/--Tg(neuronal); CST-/- x GD3s-/-; CST-/- x GalNAc-T-/-. A) Sulfatide and ganglioside biosynthesis pathways (associated gene knock outs are indicated in boxes). Ceramide is the precursor to sulfatide and gangliosides. The cerebroside sulfotransferase (CST) enzyme is necessary for the synthesis of sulfatide from galactocerebroside (GalC). The GalNAc-transferase (GalNAc-T) enzyme is necessary for generation of complex gangliosides and the GD3 synthase (GD3s) enzyme for specific production of a-series complex ganglioside. Constructs were generated to drive GalNAc-T expression in neurons of GalNAc-T-/- x CST-/- mice to produce the GalNAc-T-/--Tg(neuronal) mouse line. B) PCR results confirm presence or absence of GalNAc-T, GD3s and CST expression in mouse lines. Large bands represent the disrupted GalNAc-T and CST gene with insert, while the smaller band represents the disruption of the GD3s gene. The flag identifies the re- introduction of the GalNAc-T gene into the neurons. C) Expression of a- and b- series gangliosides and sulfatide was confirmed by staining peripheral nerves with anti-GM1, anti-GD1b and anti-sulfatide monoclonal antibodies (green), respectively. Axons and myelin were identified with neurofilament or MBP (red), respectively. Scale bar = 10 µm. A Weight at P22 B 1 5 1 2 3 4 5 6 *** *** *** ***
1 0
5 Weight Weight (g) ** * ** ***
0
1 2 3 4 5 6
1. Wild type; 2. GalNAcT-/-; 3. CST-/-; 4. CST-/- x GalNAcT-/--Tg(neuronal); 5. CST-/- x GD3s-/- ; 6. CST-/- x GalNAcT-/- S u rv iv a l p ro p o rtio n s : S u rv iv a l o f D a ta 1 C S u rv iv a l p ro p o rtSurvivalio n s : Splotsu rv iv a l o f D a ta 1 S u rv iv a l p ro p o rtio n s : S u rv iv a l o f D a ta 1 1 0 0 W T 1 0 0 WildW T type l G M 2 K O
a -/- l GalNAcTWG MT 2 K O
v 1 0 0 Mild a
i C S T K O
v
v l
i -/-
r CSTGC MS T2K O a
v Intermediate
u G M 2 -C S T -1 1 2
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i u CSTC S T Kx OGalNAcT -
% G M 2 -C S T -1 1 2
v t
s C S T -G D 3 r
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n Tg(neuronal) t u GC SMT2--GC DS T3 -1 1 2
e 5 0
n s
c G M 2 -C S T
e
r -/- -/- t CSTC S T -xG GD3sD 3
c 5 0 G M 2 -C S T
e n
r G M 2 -C S T -P L P
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c G M 2 --CCSSTT -P L P
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e Severe CSTG M-/2- x- CGalNAcTS T -P L-/P-- P 0 Tg(glial) 0 0 5 0 1 0 0 1 5 0 2 0 0 0 5 0 1 0 0 1 5 0 2 0 0 0 d a y s 0 5 0 Postnatald1a0y0s days 1 5 0 2 0 0 d a y s
Figure 2. Altering the expression of gangliosides and sulfatide in novel transgenic mouse lines effects survival and phenotype. A) The weight of double null (n=10) and CST-/- x GD3s-/- (n=6) mice is normal during development, but declines from P15-25 and is significantly reduced compared to other genotypes at P22. Statistical differences among genotypes were determined by one- way ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001. B) CST-/- x GalNAc-T-/- and CST-/- x GD3s-/- mice display a reduction in stature, hindlimb leg splaying, a hunched appearance and tremor at P22. Gross brain anatomy does not differ between genotypes. C) Survival plots demonstrate that incrementally diminishing ganglioside and sulfatide expression corresponds with a reduction in life expectancy up to 25 weeks. Wild type (n=7) and GalNAc-T-/- (n=10) mice have a normal life expectancy. In the absence of sulfatide with normal ganglioside expression life expectancy to 25 weeks is more than halved in CST-/- mice (n=18). Interestingly, in the absence of sulfatide and complex gangliosides with the reintroduction of complex gangliosides into neurons alone (n=6), life expectancy is improved with 60% of mice surviving to 25 weeks and beyond. Mice with no sulfatide and a-series gangliosides expressed globally (n=38) can survive up to 20 weeks. Double ganglioside and sulfatide knock out mice (n=29) have the worst phenotype, never surviving past 4 weeks and with death occurring at P21-25. Reintroduction of gangliosides into glial (n=12) does not improve survival. A DistanceP 1 6 betweenS c ia tic n e rv eKv1.1 Neurofascin staining Nav1.6P 1 6channel S c ia tic N e lengthr v e D is ta n c e b e tw e e n K v 1 .1 P 1 6 s c ia tic n e rv e N a v 1 .6 s ta in in g le n g th positivep o s itiv e domainsd o m a in s p N F s tlengtha in in g le n g th
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0 .0 0 0 - /- -/- - /- - /- -/- - /- - /- -/- - /- W ild typ e G a lN Ac T C S T G a lN Ac T W ild typ e G a lN Ac T C S T G a lN Ac T W ild typ e G a lN Ac T C S T G a lN Ac T - /- - /- - /- x C S T x C S T x C S T
CST-/- CST-/- Wild type GalNAcT-/- CST-/- x GalNAcT-/- Wild type GalNAcT-/- CST-/- x GalNAcT-/-
Nav1.6 Kv1.1
B N a v p e a k K v IS I
Nav channel peak recovery Kv channel peak recovery P 1 6 S SciaticN c o n d unervec tio n vCVe lo c ity e e * * **N a**v p* e a*k 1 5
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s ### ### ### # ## ## l l 1 0 0 ### # #
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i -/- -/-
l l
e G alN A c T x C S T CST-/- -/ - 5
p p d 5 0 C S T
u -/-
m m t CST x
i -/- -/-
a
a
l
G alN A-c/- T x C S T
p GalNAcT
% amplitude of 1 of amplitude %
% amplitude of 1 of amplitude %
% %
0 m 0 0
a
3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 ) ) ) ) 3 % 4 4 3 = = = = 0 n IS I (m s ) IS I (m s ) ( (n n n Interstimulus interval (ms) Interstimulus interval (ms) ( ( 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 T O O O W K K K 2 T 2 S IS I (m s ) M M G C /G T S C C Wild type GalNAcT-/- CST-/- CST-/- x GalNAcT-/-
MAG
Figure 3. The additional loss of complex gangliosides on a sulfatide null background does not augment disorganisation at the node of Ranvier or electrophysiological function in the peripheral nervous system. A) There is modest Nav1.6 channel cluster lengthening in both CST-/- and CST-/- x GalNAc-T-/- teased sciatic nerve that does not reach significance. Invasion of the sciatic nerve paranode by Kv1.1 channels, shown by a decrease in the gap between positive domains, is significantly and comparably enhanced in both CST-/- and CST-/- x GalNAc-T-/- mice compared to WT and GalNAc-T-/- nodes of Ranvier. The length of pan- neurofascin immunostained domains does not significantly differ among genotypes. Representative images are shown of sciatic nerve sections from each genotype immunostained for Nav1.6 or anti-Kv1.1 antibody. Nav channel clusters were observed and appeared similar in every genotype. Kv1.1 formed two distinct domains of immunostaining at the juxtaparanodes in WT and GalNAcT-/- mice. Conversely, mice lacking sulfatide expression had Kv1.1 staining at the paranodes, suggesting disruption to the axo-glial junction. (WT n=3, GalNAc-T-/- n=3, CST-/- n=3, CST-/- x GalNAc-T-/- n=4 Scale bar = 5 µm. B) Perineural recordings of Nav and Kv channel currents from intercostal nerves showed an increase in recovery time for both peaks following paired pulse stimulation in both CST-/- and CST-/- x GalNAc-T-/- mice compared to WT and GalNAc-T-/- mice. The CST-/- and CST-/- x GalNAc-T-/- mice were not significantly different from each other at any interstimulus interval. Graphs display the means and +SEM for each genotype (WT n=5, GalNAc-T-/- n=2, CST-/- n=3, CST-/- x GalNAc-T-/- n=2), and statistical analysis performed (two-way ANOVA; # signifies WT vs. CST-/- x GalNAc-T-/- mice; * signifies WT vs CST-/-; + signifies GalNAc-T-/- vs CST-/- x GalNAc-T-/-; ^ signifies GalNAc-T- /- vs CST-/- x GalNAc-T-/-). Sciatic nerve conduction velocity decreased in CST-/-(n=4), GalNAc-T-/- (n=4) and CST-/- x GalNAc-T-/- (n=5) mice compared to wild type (n=3), but only reached significance in CST-/- nerve. C) Peripheral nerve MAG immunostaining was comparable among genotypes. Scale bar = 20 µm. Graphs display the means and +SEM for each genotype, ), and statistical analysis performed (one-way ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001). A B Nav channel clusters Nav channel clusters flanked with with Nfasc dimers
8 0 *** 1 2 5
1 0 0 * ** 6 0
7 5
4 0 ** # / # FOV # / # FOV 5 0 * * ** 2 0 2 5 ***
0 0
CST-/- x C -/- CST-/- x CST-/- x -/- -/- GalNAcT - Wild type GalNAcT CST -/- -/- Tg(neuronal) GD3s GalNAcT pNav pNFasc
pNav
pNFasc
Figure 4. Loss of both sulfatide and complex gangliosides results in a modest reduction in CNS Nav channel cluster number and sulfatide deficiency reduces NF155 presence at parnodal loops. A) The number of Nav channel clusters significantly decreases in glycolipid deficient mouse optic nerves (n=3-4/genotype). Reintroducing a- and b-series gangliosides into neurons rescues this feature. B) Nav channel clusters flanked by normal paranodal neurofascin are significantly reduced in number when sulfatide is not expressed (n=2-3/genotype). Box-and whisker plots are used to display the spread of all data points collected from each animal then means were calculated per genotype for statistical analysis (one-way ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001). C) Representative images of optic nerve sections from each genotype double-immunostained for pan-voltage gated sodium channel (pNav) antibody (green) and pan-neurofascin (pNFasc) antibody (red). Na channel clusters were observed in every genotype. pNFasc immunostaining formed a long band crossing the node and paranodes in WT and GalNAc-T-/- mice suggesting labelling of the NF186 and NF155 isoforms, respectively. All of the genotypes lacking sulfatide expression had pNFasc staining restricted to the node of Ranvier and co-localising only with pNav staining, suggesting presence of only the NF186 isoform of neurofascin. Scale bar = 5 µm. A Caspr dimers
** * *** ***
* **
*
* # / # FOV
B Wild type GalNAcT-/- CST-/-
Caspr AnkG
CST-/- x GalNAcT-/-- CST-/- x GD3s-/- CST-/- x GalNAcT-/- Tg(neuronal)
Figure 5. Paranodal Caspr dimer immunostaining in CNS tissue is progressively reduced with increasing glycolipid deficiency. A) CST-/- x GalNAc-T-/- and CST-/- x GD3s-/- mice have significantly fewer Caspr dimers per field of view (FOV) compared to WT and GalNAc-T-/- mice, and are not significantly different to each other (n=2-5/genotype). Caspr dimer number is improved to levels within GalNAc-T-/- mice range, but not WT, in CST-/- and CST-/- x GalNAc-T-/--Tg(neuronal) mice, which both display significantly more Caspr dimers than CST-/- x GalNAc-T-/- mice. Box-and whisker plots are used to display the spread of all data points collected from each animal then means were calculated per genotype for statistical analysis (one-way ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001). B) Representative images of optic nerve sections from each genotype double-immunostained for Caspr (red) and nodal marker AnkyrinG (green) show the reduction in Caspr dimer number with diminishing glycolipid expression. Scale bar = 10 µm. A Degenerate axon density C Conduction velocity (Optic nerve) (Optic nerve) O p tic n e r v e C V P 2 2
* *** *** 4 *** *** *** ***
** 3 2
) ** *** **
s
/ m
( 2
*** *
V
CV (m/s) CV Axon/mm * C 1
0
T o o 1 3 2 k k y D W 2 T h M S T G G M T T G C T S S S C C C
-/- B Wild type GalNAcT CST-/-
CST-/- x GalNAcT-/--Tg(neuronal) CST-/- x GD3s-/- CST-/- x GalNAcT-/-
Figure 6. Glycolipid deficiency reduces CNS axon survival and function. A) Degenerate axon number increases to a significant level compared to wild type in optic nerves from CST-/-, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- mice. Reintroducing a- and b- series gangliosides into neurons rescues this pathology. B) Electron micrographs depict the differences among the genotypes: white arrowheads indicate degenerate axons. Myelin is similar among mouse lines. C) A reduction in conduction velocity was observed in optic nerves from CST-/-, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- mice. There was a reduction in conduction velocity in CST-/- x GalNAc-T-/--Tg(neuronal) mice, but this did not reach significance and was improved to within wild type and GalNAc-T-/- rates. One-way ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001. Scale bar = 2 µm. A Degenerate axon density B (Optic nerve)
-/- * *** *** Wild type GalNAcT CST-/- ***
** 2
Axon/mm *
CST-/- x GalNAcT-/-- CST-/- x GD3s-/- CST-/- x GalNAcT-/- Tg(neuronal)
C -/- CV to highest peak Wild type GalNAcT CST-/- C V(Optic to h i gnerve)h e s t p e a k 2 .5
Ch.0 Ch.0 Ch.0
2 .0 )
s 1 .5
(
-1 mV -1 mV -1 mV e
m/s 1.638 ms 1.638 ms 1.638 ms m i 1 .0 CST-/- x GalNAcT-/-- CST-/- x GD3s-/- CST-/- x GalNAcT-/- T Tg(neuronal)
0 .5
0 .0 Ch.0 Ch.0 Ch.0
T o o 1 3 2 k k y D W 2 T h M S T G G M T T G C T S S S C C C -1 mV -1 mV -1 mV
1.638 ms 1.638 ms 1.638 ms
Figure 6. Glycolipid deficiency reduces CNS axon survival and function. A) Degenerate axon number increases to a significant level compared to wild type in optic nerves from CST-/-, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- mice. Reintroducing a- and b- series gangliosides into neurons rescues this pathology. B) Electron micrographs depict the differences among the genotypes: white arrowheads indicate degenerate axons. Myelin is similar among mouse lines. Scale bar = 2 µm C) A reduction in conduction velocity was observed in optic nerves with diminishing glycolipid content. Representative CAP traces from each genotype show the normal waveform in WT, GalNAc-T-/- and CST-/- x GalNAc-T-/--Tg(neuronal) optic nerves, however the waveform becomes abnormal in the CST-/-, CST-/- x GD3s-/- and CST-/- x GalNAc-T-/- mice. One-way ANOVA followed by Tukey’s post-hoc tests to compare multiple comparisons, indicated on the graphs as follows: *p<0.05; **p<0.01; ***p<0.001. Horizontal scale bar = 2 ms, vertical scale bar = 1 mV. A 1Myelin:u g m y e lin MAG M A G 2 Myelin: MAG 2 5 0 0 0
d 2 0 0 0 0 * *** *
n ** 98kDa
a b
Figure 7. N.B. New order of
f 1 5 0 0 0 **
o
y genotypes. MAG and NF155
t i
s 1 0 0 0 0
n expression in the myelin e t 62kDa 1 2 3 4 5 6 n fraction is altered by I 5 0 0 0
Arbitrary intensity Arbitrary glycolipid deficiency. A) In 0 the myelin fraction from P22 T 2 T T T 1 S S S y W M h G C /C /C T 2 3 / brain homogenates T M D S G G Total brain: MAG/C Total brain: MAG 2 (n=4/genotype), MAG is 2 5 0 0 0 M G significantly reduced in CST-/- P 2 2 2 0 0 0 0 x GalNAc-T-/- mice compared ** 98 kDa 1 5 0 0 0 ** ** to all genotypes, which do *** *** not significantly differ from 1 0 0 0 0 62 kDa 1 2 3 4 5 6 one another (upper graph).
5 0 0 0 ^ All genotypes have a Arbitrary intensity Arbitrary 0 significant reduction in MAG
T 2 T T 1 T S S y S expression in whole brain W M h G C /C T /C 2 / 3 T M S D homogenate compared to G G /C Myelin: NF1552 M Myelin: NF155 WT (middle graph). Myelin 2 5 0 0 0 G NF155 was significantly 2 0 0 0 0 ^ 198 kDa reduced in all genotypes
1 5 0 0 0 * compared to WT (indicated ## by *) and GalNAc-T-/- *** *** 98 kDa 1 0 0 0 0 ### *** ### ### 1 2 3 4 5 6 (indicated by #), which had 5 0 0 0 similar levels (lower graph).
Arbitrary intensity Arbitrary ^indicates significant 0 difference between CST-/- x GalNAc-T-/- and CST-/- x O liTotalg 2 1 0 u gbrain: to ta l b rOlig2a in h o m o g e n a te Total: Olig2 GalNAc-T-/--Tg(neuronal) 2 5 0 0 0 mice. Olig2 is unchanged by
d 2 0 0 0 0 altered glycolipid expression. n
a 38 kDa
b Representative blots show
f 1 5 0 0 0 o
protein intensity per
y t
i 26 kDa 1 2 3 4 5 6
s 1 0 0 0 0 genotype, indicated by the
n
e t
n corresponding number
I 5 0 0 0
Arbitrary intensity Arbitrary indicated in the key. B) P22 Key: 0 -/- -/- optic nerve MAG T 2 T T T 1 1. Wild type 2. GalNAc-T 3. CST S S S y W M h -/- -/- -/- -/- G C /C /C T 4. CST x GalNAc-T 5. CST x GD3 immunostaining 2 3 / T M D S -/- -/- G G /C 6. CST xGalNAc-T -Tg(neuronal) 2 (n=3/genotype) is M G significantly reduced in all P 2 2 genotypes compared to wild type nerve, which do not significantly differ from one B -/- 1. Wild type 2. GalNAcT 3. CST-/- another. Representative images show MAG M A GMAG P 2 2 O p N immunostaining per 7 0 genotype. Means were
6 0 calculated per genotype for statistical analysis (one-way 5 0 ** ANOVA followed by Tukey’s ** ** MAG *** *** post-hoc tests to compare 4 0 multiple comparisons,
Arbitrary intensity Arbitrary indicated on the graphs as 3 0
T o o o 1 3 follows: *p<0.05; **p<0.01; k k k y D W 2 T L h T G M S B T C D T G S S ***p<0.001). Scale bar = 50 C C µm. 4. CST-/- x GalNAcT-/- 5. CST-/- x GalNAcT-/- 6. CST-/- x GD3s-/- -Tg(neuronal) Wild type GalNAcT -/- CST-/-
Paranodal loop
Sulfatide raft Caspr
MAG Nf155 ganglioside raft GT1b Contactin GD1a
Axonal cytoplasm
CST-/- x GalNAcT-/- CST-/- x GD3s-/- CST-/- x GalNAcT-/- -Tg(neuronal)
a-series raft
Figure 8. Schematic depicting the glycolipid rafts and changes to their associated paranodal proteins Caspr, NF155 and MAG composition in our transgenic mouse lines. GD1a and GT1b are represented in the ganglioside rafts as they are the major ligands of MAG, however, in reality the rafts would contain all complex gangliosides. Normally GD1a and GT1b in rafts will tether MAG but in their absence MAG does not make the axo-glial connection. If NF155 is present, this protein can partner with Caspr/Contactin to make an axo-glial junction. However, when sulfatide is absent, NF155 is also lost from the paranode. In the absence of NF155, Caspr presence is also diminished, especially with loss of complex ganglioside rafts. Under both ganglioside and sulfatide raft deficiency, we propose that MAG and NF155 are both absent from the paranodal axo-glial domain.