Tau-mediated axonal degeneration is prevented by activation of the WldS pathway

Tauopathy is characterised by neuronal dysfunction and degeneration occurring as a result of changes to the microtubule associated protein tau. The neuronal changes evident in Tauopathy bear striking morphological resemblance to those reported in models of Wallerian degeneration. The mechanisms underpinning Wallerian degeneration are not fully understood although it can be delayed by the expression of the slow Wallerian degeneration (WldS) protein, which has also been demonstrated to delay axonal degeneration in some models of neurodegenerative disease. Given the morphological similarities between tauopathy and Wallerian degeneration, this study investigated whether tau-mediated phenotypes can be modulated by expression of WldS. In a Drosophila model of tauopathy in which expression of human Tau protein (hTau0N3R) leads to progressive age-dependent phenotypes, activation of the pathway downstream of WldS completely suppressed tau-mediated degeneration. This protective effect was evident even if the pathway downstream of WldS was activated several weeks after hTau-mediated degeneration had become established. In contrast, WldS expression without activation of the downstream protective pathway did not rescue tau-mediated degeneration in adults or improve tau-mediated neuronal dysfunction including deficits in axonal transport, synaptic alterations and locomotor behaviour in hTau0N3R –expressing larvae. This collectively implies that the pathway mediating the protective effect of WldS intersects with the mechanism(s) of degeneration initiated by hTau and can effectively halt tau-mediated degeneration at both early and late stages. Understanding the mechanisms underpinning this protection could identify much-needed disease-modifying targets for tauopathies.


Introduction 45
Tau pathology is observed in numerous neurodegenerative diseases, including Alzheimer's 46 disease, Parkinson's disease (PD), motor neuron disease (MND) and a variety of other 47 tauopathies such as fronto-temporal dementia, Pick's Disease, progressive supra-nuclear palsy 48 and others. The axon is susceptible to tau pathology in these neurodegenerative diseases, with 49 evidence of white matter changes indicative of axonal degeneration in tauopathies such as AD 50 (1-3). Studies in animal models have demonstrated that axonal dysfunction in tauopathy is 51 typified by disrupted axonal transport (4, 5) (6), due to tau hyperphosphorylation resulting in 52 reduced cytoskeletal integrity (7). Axonal swellings and loss of white matter, hallmarks of 53 axonal degeneration have been observed in P301L-tau mice, a model of familial fronto-54 temporal dementia (8) (9) (10). 55 56 Wallerian degeneration describes the sequential degeneration of axons following axonal injury 57 which begins with breakdown of the cytoskeleton and ends with the fragmentation and loss of 58 the separated distal axon (11). Wallerian degeneration and axonal degeneration in 59 neurodegenerative disease share similarities including cytoskeletal breakdown (7) (12), 60 disrupted axonal transport (13) (14), alterations to mitochondrial morphology (15) (16), and in 61 the central nervous system (CNS), axonal swellings (12) (17). These similarities suggest that 62 the mechanisms overlap and the term Wallerian-like may be used to describe degeneration that 63 is not due to an acute injury. Considering that the axon is a site of tau-mediated dysfunction and degeneration, the aim of 75 the present study was to investigate whether the axonal protection mediated by Wld S was able 76 to rescue tau-mediated axonal dysfunction and degeneration. Drosophila melanogaster has 77 been used in the study of Wallerian degeneration and Wld S (29) (30) (31) (32) (33) (34), and 78 Drosophila models of tauopathy are similarly well-established (5)  The third antennal segment was removed from flies, 1 or 3 weeks after eclosion from Or47b-149 GAL4 driven crosses, under CO2 anaesthesia using Dumont #5 forceps. This induced an axonal 150 injury in olfactory receptor neurons (ORNs), whose cell bodies are located in the third antennal 151 segment. At the relevant time points, brains were dissected as described above. Degeneration 152 was quantified by previously described methods (42). Briefly, with the assessor blind to 153 genotype and time point, the presence of the commissural axons was recorded (Y/N) and the 154 percentage of brains of each genotype at each time point with intact axons was calculated. The 155 intensity of GFP signal within glomeruli was measured using ImageJ and the background 156 intensity was subtracted. 157 158 Statistics 159 Statistical analysis was conducted using GraphPad Prism, version 6.0 (GraphPad Software, 160 Inc.), using analysis of variance and the Bonferroni correction for the comparison of groups. 161 The Mantel-Cox test was used for survival analysis, with the Bonferroni correction used for 162 the comparison of multiple groups. Values are presented as the mean ± standard error. P<0.05 163 was considered to indicate a statistically significant difference. 164

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Activation of the pathway downstream of Wld S protects against hTau 0N3R -induced degeneration 166 Though previous studies of injury models indicate that the presence of Wld S within the axon is 167 crucial for its protection (33-35), the findings from chronic models of disease do not show any 168 consistent or significant Wld S -mediated protection despite clear evidence of Wallerian-like 169 degeneration in these models (31) (32) (34). One explanation for this lack of rescue could be 170 that the pathway that Wld S is acting in is not "activated" in these models of chronic 171 degeneration, raising the possibility that the protein may require some form of injury to unmask 172 its protective effect. Indeed, in all cases where Wld S has been reported to rescue axonal 173 degeneration, the neurons are injured by default as part of the experimental paradigm (22)  To explore whether injury-induced "activation" was required for the Wld S pathway to protect 177 against tau-mediated degeneration, hTau 0N3R ;Wld S axons of the olfactory receptor neurons 178 (ORNs) expressing membrane bound GFP were injured (axotomised) by removal of the third 179 antennal segments as previously described (42). Adult brains were analysed after ecclosion at 180 hourly (h) or weekly (w) time points post-axotomy induced Wld S pathway activation (referred 181 to as "pa" from here on). This revealed that control and tau expressing axons had degenerated 182 at 2w after eclosion/1wpa. In contrast hTau 0N3R ;Wld S expressing axons were intact at this time 183 point (data not shown) confirming that the axotomy paradigm "activated" the pathway 184 downstream of Wld S . To ascertain the extent to which activation of the Wld S pathway protected 185 against tau-mediated degeneration, the prominent degenerative features of tau-expressing 186 axons were quantified. Axonal swellings, which are characteristic of tau-mediated axonal 187 degeneration, were present in naïve hTau 0N3R ;Wld S axons (arrowheads Fig 1ai). In contrast 188 these were not found in hTau-animals of the same genotype after activation of the Wld S 189 pathway (Fig. 1aii). The progressive accumulation of axonal swellings is evident in hTau 190 expressing animals within 2 weeks after eclosion and trebles by week 5. In contrast swellings 191 were not seen at any time point in hTau 0N3R ;Wld S expressing animals where the activation of 192 the Wld S pathway was elicited through axotomy (Fig. 1b). This illustrates that once activated, The results indicate that activation of the pathway downstream of Wld S potently suppresses 218 hTau-mediated degeneration. This supports our hypothesis that the lack of protection through 219 co-expression of Wld S in chronic models of degeneration (31) (32) (33) (34) is because the 220 pathway that Wld S acts in is not normally "activated" in otherwise naïve axons. Acute injury 221 to an axon "activates" it unmasking its protective effect. However, as the previous studies were 222 conducted in rodents, we sought to ascertain whether the "protection requires pathway 223 activation" phenomenon that we described in Figs 1 and 2 holds true in our invertebrate model 224 as well. 225

226
To prove that expression of Wld S is insufficient for protection against hTau 0N3R -mediated 227 degeneration and that "activation" of the pathway downstream is required, degeneration in 228 naïve hTau 0N3R ;Wld S flies was studied in the absence of "activation". ORNs expressing 229 membrane-bound GFP underwent progressive age-related axonal degeneration in all hTau 0N3R 230 expressing flies. This was characterised by the appearance of axonal swellings at 2-3 weeks 231 after eclosion, which increased in number and size as the flies aged ( Fig. 3a/b hTau 0N3R 232 column). Axonal swellings were also evident in controls and Wld S flies, but only at older, 5-7 233 week time points (Fig. 3a control and Wld s columns). Noticeably, these swellings were also 234 apparent in hTau 0N3R ;Wld S flies (where the Wld s pathway had not been activated - Fig 3a  235 hTau 0N3R ;Wld s column). Quantification confirmed that there was no significant difference in 236 onset, extent or progression of axonal swellings in the hTau 0N3R ;Wld S flies when compared 237 with hTau 0N3R alone ( Fig. 3a/b). 238 These results suggest that simply co-expressing Wld S does not protect against tau-mediated 239 degeneration. We next sought to investigate whether this is also the case in larvae, where tau-240 mediated neuronal dysfunction manifests in profound Wallerian-like axonal phenotypes 241 incuding disrupted axonal transport and destabilisation of the cytoskeleton (5) (7). Using a 242 Drosophila line expressing GFP-tagged neuropeptide Y in motor neurons, axonal transport was 243 visualised using microscopy in live intact third instar larvae. As reported previously, numerous 244 large vesicular aggregates were found in tau-expressing larvae indicative of axonal transport 245 disruption (Fig. 4a). However, these aggregates were also observed in hTau 0N3R ;Wld S larvae. 246 Quantification of the coverage areas of the aggregates indicated that the aggregates were not 247 significantly reduced in hTau 0N3R ;Wld S larvae compared with hTau 0N3R larvae (Fig. 4b).  (Fig. 4h). However, the co-expression of Wld S 263 with hTau 0N3R did not improve locomotor behaviour, with no significant difference between 264 hTau 0N3R ;Wld S and hTau 0N3R expressing larvae (Fig. 4f-h). 265 The adult and larval data collectively shows that without "activation" of the pathway 266 downstream of Wld S , the protective effect of Wld S on hTau 0N3R -mediated dysfunction (in 267 larvae) or degeneration (in adult flies) is not uncovered. 268 269 Activation of the Wld S -pathway protects against the effects of hTau 0N3R without influencing 270 total or phosphorylated tau levels 271 The most parsimonious explanation for this curious phenomenon of injured-activated 272 protection against hTau 0N3R pathology, may simply be that hTau is lost from injured 273 hTau 0N3R ;Wld S axons and therefore cannot exert it's detrimental effects to cause axonal 274 degeneration. To investigate this, hTau immunoreactivity was assessed in hTau-expressing 275 animals with and without Wld S -pathway activation and both the amount of hTau and its cellular 276 localisation was examined. No significant differences were found in hTau distribution or total 277 hTau expression between these two groups; hTau staining persisted in injured hTau 0N3R ;Wld S 278 axons even 5 weeks after Wld S activation (Fig. 5a) and there was no difference in total Tau 279 levels (Fig 5b). This is remarkable because it implies that despite expression within the axon 280 for 6 weeks, hTau has not caused degeneration in the hTau 0N3R ;Wld S axons once the Wld S 281 pathway is activated. This begs the question as to how activation, of the Wld S -pathway protects 282 against the human tau induced degeneration across this length of time. 283 284 Since hyper-phosphorylation has been shown to mediate tau toxicity in many Drosophila 285 models (5) (7) (44), it is conceivable that the activated Wld S -pathway is altering the 286 degenerative changes by reducing the levels of phosphorylated hTau. To investigate this, hTau 287 phosphorylated at the PHF-1 site was quantified in hTau 0N3R ;Wld S axons with and without 288 Wld S -pathway activation. There was a trend for a reduction in the PHF-1 signal in the 289 hTau 0N3R ;Wld S flies where Wld S was activated but this was not significant (Fig 5c). 290

Discussion 291
The axonal compartment of neurons is susceptible to tau-mediated dysfunction and 292 degeneration making it a potential therapeutic target in the treatment of neurodegenerative 293 disease. This study demonstrates that when the pathway downstream of Wld S is "activated" in 294 hTau 0N3R ;Wld S axons, tau-mediated axonal swellings were prevented from forming. 295 Significantly, in animals allowed to develop axonal swellings due to hTau 0N3R expression any 296 further progression of pathology was halted after the Wld S -pathway was activated. This neurodegeneration and their variable sensitivity to Wld S could be a different mechanism of 319 axonal degeneration occurring in acute compared with chronic neurodegenerative conditions. 320 Another explanation for the variable sensitivity to Wld S in models of chronic 321 neurodegeneration could simply be that its protective effect is a general delaying of 322 degeneration which is not always apparent in the time period assayed in the chronic models in 323 question. Our data imply that this is unlikely to be the case since no protective effect emerged 324 at even very late time points when Wld S was simply expressed with hTau 0N3R (Fig 1). Instead, 325 we propose and that another explanation may be provided by a key difference between the 326 experimental paradigms employed to study acute degeneration, which is missing in the models 327 of chronic neurodegeneration. This is that in all acute models, injury has to be simulated to 328 create the acute condition and this may set in motion a series of events that "activate" the Wld S 329 protective pathway. This is never done in models of chronic neurodegeneration so it is 330 conceivable that in those models the protective effect of Wld S is not induced due to inadequate 331 "activation" of the pathway that Wld S acts upon. This would limit the impact of Wld S on the 332 ensuing neurodegeneration. Where there is partial rescue of phenotype in chronic models, the 333 Wld S pathway may start to become "activated" as the degeneration sets in. "Activation" of the 334 Wld S pathway by simulating injury or established neurodegeneration is a novel concept. There 335 is no precedence for this idea from studies published to date because no one has reported 336 overlaying an acute injury in a chronic model. Our data indicates that Wld S behaves differently 337 in uninjured axons compared to injured ones -the mechanisms responsible for this need to be 338 What is the mechanism by which activated Wld S pathway protects against hTau? 358 Tau-mediated degeneration is dependent upon factors including total tau levels (55), 359 phosphorylation at pathological sites (56) (35) and tau aggregation (57). The presence of 360 human tau within hTau 0N3R ;Wld S axons, even weeks after activation of the Wld S pathway, 361 indicates that the protection seen was not due to a reduction in total tau level as a result of loss 362 of human tau from the axon. Nor is it likely to be due to any significant reduction in its 363 phosphorylation status at the one pathological site, PHF1 (ser/thr 396 and 404) that we 364 examined. This surprising observation implies that upon activation, the Wld S -pathway acts to 365 negate the degenerative effects of tau, despite the persistence of pathologically phosphorylated 366 human tau within the axon. Nonetheless it is possible that this protective effect was conferred 367 by reduced misfolding or phosphorylation at other pathological sites that have previously been 368 which the activated Wld S pathway intersects with and therefore protects against tau-mediated 404 degeneration. In particular it will be vital to explore whether expression of downstream 405 meditors of the Wld S pathway that potentially block injury-induced axon degeneration (such 406 as NMNAT, dSarm, axed, highwire, or even NAD + ) also modulate tau-mediated degeneration 407 to emulate Wld S pathway activation. 408

Conclusion 409
We show that activation of the Wld S -pathway reliably protects against tau-mediated axonal 410