Microtubule stabilising peptides rescue tau phenotypes in-vivo

The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer’s disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP’s protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative ‘SIP’ (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer’s disease.

Scientific RepoRts | 6:38224 | DOI: 10.1038/srep38224 within its amino acid sequence (NAPVSIPQ). It interacts in-vitro 18 with both EB1, a key regulator of MT dynamics and polymerisation 23,24 and EB3 a central component in dendritic spine formation 25 . Silencing of either EB1 or EB3 abolishes NAP's protective activity in PC12 cells. Furthermore, silencing of EB3 in primary cortical neurons inhibits NAP-mediated dendritic spine formation 18 . A novel NAP analogue, SKIP, is reported to bind NAP and enhance axonal transport in ADNP-deficient mice 26 . It is therefore conceivable that NAP restores MT integrity and function in our Drosophila model of tauopathy by interacting with EB's via its SIP domain. The data presented here tests this hypothesis by exploring the MT stabilising potential of another analogous peptide called SAL (SALLRSIPA also termed ADNF-9), which also contains a SIP domain. SAL is derived from the glial precursor protein, activity dependent neurotrophic factor (ADNF). It exhibits similar neuroprotective capabilities to NAP in numerous animal and cell models of injury and disease [27][28][29][30][31] .
In this study we investigated whether SAL, like NAP could also protect against htau 0N3R -mediated neuronal dysfunction in our Drosophila model of tauopathy. The phenotypes that arise in this model occur as a direct or indirect consequence of MT breakdown. This model was therefore ideally suited to test SAL's ability to modulate MT integrity and thus determine the importance of the SIP domain in MT stabilising therapeutic approaches.

Discussion
In this study, we demonstrate that the neuroprotective peptide SAL (SALLRSIPA) provides protection against htau 0N3R -mediated phenotypes as also demonstrated for an analogous neuroprotective peptide NAP (NAPVSIPQ), albeit at higher doses (5 and 10 μg/ml for SAL compared to 2.5 μg/ml for NAP). SAL significantly improved htau 0N3R -mediated phenotypes in-vivo, including axonal transport disruption and behavioural defects. SAL was able to rescue htau 0N3R phenotypes without altering phosphorylation at key disease-associated epitopes. These results are reminiscent of those observed with NAP-treatment in this model of tauopathy 14 . SAL is reported to exhibit a similar neuroprotective profile compared to NAP 36,37 . However, several studies have also shown that NAP is more efficacious than SAL 15,29 . This is consistent with our observations in the present study.
SAL and NAP -two neuroprotective peptides. SAL and NAP are short peptides derived from two secreted astroglial parent proteins, ADNF and ADNP (respectively) 15,38,39 . NAP and SAL were identified as the essential regions of their respective parent proteins for conferring neuroprotection 39 . In subsequent studies, SAL and NAP were found to protect against a variety of cellular insults including neurotoxic drugs such as NMDA 40 and ethanol 28,41 . They were also found to be protective in models of Alzheimer's disease 7,27,42,43 , diabetic neuropathy 44 , amylolateral sclerosis and ADNP induced tauopathy 10,17,45 . The molecular mechanism underpinning NAP's neuroprotective ability is thought to occur by MT stabilisation 46 , which counters axonal transport defects 14,17 . It is conceivable that like NAP, SAL also has MT stabilising effects as it has been shown to displace NAP in an in-vitro MT binding assay 47 . SAL also promotes neurite outgrowth in rat hippocampal cultures, a function reliant on MT stabilisation and plasticity 48 . The data presented herein supports this further by showing that like NAP, SAL also protects against htau 0N3R -mediated behavioral and axonal transport defects, which arise due to cytoskeletal destabilisation 6,14 . These htau 0N3R -phenotypes could be attributed to neurodevelopmental effects given the drivers used in this study (D42-and Elav-Gal4) are not exclusively post-mitotic and motor-neuron specific 49,50 . D42-Gal4 is expressed in all post-mitotic motor neurons and in some sensory neurons in the peripheral nervous system 49 . Elav-Gal4 is also expressed in post-mitotic neurons, but is transiently expressed in embryonic glial cells and neuroblasts 50 . Importantly, expression of htau 0N3R with these drivers results in robust phenotypes, including MT destabilisation and disrupted axonal transport within the motor neurons of Drosophila. We have previously shown rescue of htau 0N3R -mediated axonal transport deficits after just 24 hours NAP treatment 14 . This implies that the protective effects of NAP and SAL target htau 0N3R -phenotypes that arise in this model due to MT-associated neuronal dysfunction 11 . We previously demonstrated by EM that cytoskeletal integrity in wt (OreR) controls was unaffected by NAP treatment 14 . Unsurprisingly, no effect of NAP was evident in axonal transport or behavioural assays in these wt controls. In contrast, the same ultrastructural analysis showed a disrupted cytoskeleton, which was restored by NAP-treatment in htau 0N3R -expressing animals 14 . Likewise, in the current study, wt controls treated with SAL did not show any statistically significant differences in locomotor performance compared to untreated controls, as illustrated in Supplementary Figure 1b-d. An important point to note is that wt strains such as OreR are more robust when compared to isogenised, Gal4/UAS strains which may be susceptible to Gal4 titrations. It would have been ideal to confirm the tau-specific, neuroprotective effect of these peptides, by assessing Wild-type, control untreated larvae exhibited a homogeneous distribution of vGFP fluorescence in peripheral nerves indicative of an efficient axonal transport system (a). Htau 0N3R larvae exhibited accumulation of vGFP, indicative of disrupted axonal transport (b). 2.5 μg/ml NAP prevented accumulation of vGFP in htau 0N3R larval motor neurons (c). 10 μg/ml SAL also prevented accumulation of vGFP in htau 0N3R larval motor neurons (d). The total area of axons (within a defined region) encompassed by vGFP accumulates was greater in htau 0N3R larvae compared to controls. 2.5 μg/ml NAP and 10 μg/ml SAL reduced the area covered by vesicular accumulates back to control levels (e). Data were analysed by one-way ANOVA with Bonferroni's correction. Error bars represent mean ± S.E.M., P**** < 0.0001; n = wt (10), htau 0N3R (9), 2.5 μg/ml NAP (5), 10 μg/ml SAL (9). Scale bar: 10 μm.

Figure 3. SAL does not alter total htau 0N3R levels or phosphorylation at a number of sites relevant to AD.
For each phospho-tau antigen, intensity of signal (pixels/mm 2 ) was normalised to total tau levels. There was no significant change in the levels of the phospho-tau epitopes detected by AT180 (a), AT8 (b), and PHF-1 (c) after treatment with 5 μg/ml SAL (green bars) and 10 μg/ml SAL (dark green bars) SAL. Total dtau levels were not altered by SAL treatment (d). Representative blots are shown (a-d). All lanes were run on the same gel. Data were analysed by one-way ANOVA with Bonferroni's correction. Error bars represent mean ± S.E.M; n = htau 0N3R (5), 5 μg/ml SAL (5), 10 μg/ml SAL (5).
Scientific RepoRts | 6:38224 | DOI: 10.1038/srep38224 their impact on transgenic control lines with a UAS background (e.g. UAS-LacZ). However, our primary aim in this study was to determine if SAL-treatment significantly affected htau 0N3R -phenotypes arising because of MT destabilisation. As such, treated and untreated Drosophila expressing htau 0N3R were reared and tested alongside each other to minimise any titration and genetic artefacts. Interestingly, we found that axonal transport deficits could be prevented and efficient axonal transport maintained in htau 0N3R -expressing animals at a comparable level to robust wt OreR control larvae.

Mode of action of SAL and NAP. Hyper-phosphorylated tau is considered to be the toxic species in
tauopathies. It is believed to cause degeneration both due to loss of MT-binding function and accumulation of toxic tau aggregates 51,52 . Tau-centric disease-modifying strategies rescue tau phenotypes by reducing tau phosphorylation 11,53 , increasing MT stabilisation 9,14,46,54 or reducing tau aggregation 55 . Our data imply neuroprotective effects independent of reductions in tau phosphorylation, but whether these peptides impact on tau aggregation remains to be determined. Interestingly, we have previously shown that inhibition of GSK-3β rescues tau phenotypes and restores MT integrity, by reducing tau phosphorylation with a consequent increase in tau protein levels and insoluble granular tau oligomers 56 . In the present study, we did not observe any significant changes in total htau 0N3R protein and phosphorylation levels after treatment with SAL and as demonstrated for NAP 14 . Previous findings from our lab, as well as other studies conducted in rodent and cell culture models of tauopathy, strongly suggest that NAP neuroprotects by fortification of MTs 14,[16][17][18]57 .
In this study, we assessed the neuroprotective capabilities of both NAP and SAL peptides individually. SAL showed dose-dependent neuroprotective effects, consistent with our previous observations for NAP 14 . Other studies have also reported dose-dependent neuroprotective effects for both peptides 29 . In a rat model of cholinotoxicity, NAP was more efficacious compared to SAL in cholinergic protection 29 . NAP has also been shown to be more effective then SAL in providing long term protection against loss of spatial memory in apolipoprotein E-deficient mice 15 and AF64A-treated animals 29 . A combinatorial peptide approach would also have been interesting but is beyond the scope of the current study. However, previous studies have investigated the protective effects of combining both NAP and SAL. A few studies have shown that both peptides are more efficacious together, than either alone [58][59][60][61] . These peptides do not exhibit stereo-selectivity 59 . The more stable, all D-amino acid SAL (D-SAL) showed efficacy in-vivo and in-vitro models of disease 30,31 . In a model of fetal alcohol syndrome (FAS), administration of both D-NAP and D-SAL reduced fetal demise, however, no significant differences between combination and individual drug treatments were seen. In the same study, apolipoprotein E knockout mice treated with both D-NAP and D-SAL showed improved performance in the Morris water maze 59 . In another study, NAP alone was effective in preventing alcohol-induced fetal death, whereas SAL at the same dose was not protective. However, a combinatorial treatment with NAP and SAL was more effective in preventing growth restriction due to prenatal alcohol treatment 60 . These studies suggest a dose-dependent, synergistic effect rather than an additive effect. The differences reported in the literature and the differences in efficacious dose that we too observe may be attributable to the non-homologous amino acids either side of the SIP motif in the two peptides.
Interestingly, both peptides are derived from parent proteins that are secreted by glial cells in response to vasoactive intestinal peptide (VIP) 15,38 . VIP is expressed under conditions of stress and one of the early events that occurs during stress or insult mediated injury is a dynamic reorganisation of the cytoskeleton 62 . Both NAP and SAL (NAPVSIPQ and SALLRSIPA) contain a SIP motif 63 . The 'SIP' motif within NAP has been implicated in protection against ethanol and tetrodotoxin toxicity in cortical neurons 40 . Substitution of proline (P) with alanine (A) abolishes neuroprotection against oxidative stress (H 2 O 2 ) in pheochromocytoma (PC12) cells 64 . Indeed, the SIP motif of NAP and SAL is essential for neuroprotection and interaction with key MT end-binding proteins EB1 and EB3, promoting MT assembly and neuronal plasticity 18,26 . Collectively, our data and the studies discussed imply that SAL acts in a similar manner as NAP to confer neuroprotection. However, since the molecular mode of action of SAL has not been explored as comprehensively as that of NAP, this cannot be concluded unequivocally without further investigations.
The data presented here supports the use of SIP containing neuropeptides like NAP and SAL for protection against MT destabilisation such as that seen in tauopathies. Importantly, this work highlights MT stabilisation as a disease-modifying therapeutic strategy that holds great promise for tauopathies like AD where abnormal tau-mediated MT dysfunction is evident.

Methods
Drosophila genotypes and drug treatments. Transgenic expression of htau 0N3R (y 1 w 1118 ; P{UAS-MAPT.A}59 A: Bloomington Stock Centre, stock no. 181) was directed to Drosophila melanogaster motor neurons using either D42-Gal4, or the D42-Gal4 driver fused to vesicular GFP-tagged neuropeptide-Y (D42-GAL4.UAS-NPY:GFP) as previously described 11 . Pan-neural expression was established with the Elav-Gal4 driver. Female virgin flies homozygous for the D42 or Elav driver were crossed to male flies homozygous for htau 0N3R under the UAS promoter (+;+; UAS-htau 0N3R ), or with Oregon-R (OreR) wt, control males. Stocks and transgenic crosses were maintained at 23 °C on a 12 h light/dark cycle. Flies were raised on basic food consisting of malt extract, maize meal, soya flour, agar, granulated sugar, yeast, and propionic acid. NAP (NAPVSIPQ) and SAL (SALLRSIPA) (L-isoforms synthesised by Peptide Protein Research Ltd, UK) were delivered to basic fly food at a final concentration of 2.5 μg/ml (NAP) and 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml or 10 μg/ml (SAL). Late L3-stage larvae were selected for by size and wandering behaviour.
Larval locomotion assay. Larval locomotion analysis was conducted using a semi-quantitative assay as previously described 32 . Briefly, crawling behaviour was analysed on 1% agarose plates dyed with 0.1% w/v Alcian blue (Hopkin and Williams, UK). L3 larvae were positioned in the centre of each plate and allowed to acclimatise for 2 min prior to testing. Open field activity was recorded for 2 min (trial 1). This was further repeated for 2 more trials. Wherever possible, genotypes and treatments were randomised between adjacent plates. Videos of larval locomotion were analysed in Ethovision 3.0 software (Noldus) to determine velocity, angular velocity and meander.
In-vivo axonal transport analysis. All treatment groups were subjected to a 3-4 hour timed lay on apple juice agar plates. F1 eggs were transferred to either basic, NAP or SAL treated food. Larvae were left to develop to L3 wandering stage (day 5). Axonal transport analysis was conducted as previously described 11,65 . Briefly, L3 larvae were anaesthetised in diethylether vapour for 15 min, immobilised on glass slides in 1% agarose ventral face up and mounted under coverslips. Peripheral nerves were analysed between the 2 nd and 4 th denticle bands. For total area acquisition, vGFP accumulates were imaged at x63 on an Axioplan2 Epifluorescence Microscope (Zeiss), and thresholded in Metamorph software (Molecular Devices, CA, USA).