nap (davunetide) preferential interaction with dynamic 3 ... · adnp), a protein vital for brain...

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Full Title: NAP (davunetide) preferential interaction with dynamic 4

3-repeat Tau explains differential protection in selected tauopathies 5

Short Title: NAP preferential interactions explain neuroprotective 6

mechanisms. 7

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Yanina Ivashko-Pachima1, Maya Maor1 and Illana Gozes1,2* 10

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1Elton Laboratory for Molecular Neuroendocrinology, 2Lily and Avraham Gildor Chair 14

for the Investigation of Growth Factors, Department of Human Molecular Genetics and 15

Biochemistry, Sackler Faculty of Medicine, Sagol School of Neuroscience and Adams 16

Super Center for Brain Studies, Tel Aviv University, Tel Aviv, Israel. 17

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*Corresponding author 20

E-mail: [email protected] 21

certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted October 11, 2018. . https://doi.org/10.1101/440941doi: bioRxiv preprint

https://doi.org/10.1101/440941

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Abstract 22

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT 23

assembly and dynamic instability, orchestrating MT-dependent cellular processes. 24

Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 25

3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. 26

Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and 27

cognitive function. ADNP deficiency in mice causes pathological Tau 28

hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has 29

been previously shown that the ADNP-derived peptide NAP protects against ADNP 30

deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP 31

prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and 32

expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, 33

CP201) exhibited efficacy in prodromal Alzheimer’s disease patients (Tau3R/4R 34

tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, 35

we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward 36

personalized treatment of tauopathies. Affinity-chromatography showed that NAP 37

preferentially interacted with Tau3R protein from rat brain extracts and fluorescence 38

recovery after photobleaching assay indicated that NAP induced increased recruitment of 39

human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we 40

showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-41

binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. 42

The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. 43

Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders. 44


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Introduction 45

Microtubules (MTs) are the major component of the neuronal cytoskeleton, and MT 46

stability and organization play a critical regulatory role during axonal transport and 47

synaptic transmission (1). The MT-associated protein Tau is widely expressed in neurons 48

and serves as a primary protein marker for axons (2, 3). Tau promotes MT assembly and 49

regulates MT dynamic instability, which is essential for establishing neuronal polarity, 50

axonal elongation, and neural outgrowth (4). Neurodegenerative disorders with Tau 51

involvement are referred to as tauopathies (5). The Tau protein consists of an N-terminus 52

region projecting outward from the MTs and a C-terminus part directly interacting with the 53

MTs through MT-binding domains (6). Tau3R and 4R (containing either three or four MT-54

tubulin - binding repeats, respectively) are produced by alternative splicing around exon 55

10 of the Tau transcript (7). The healthy human brain exhibits a 1/1 ratio of Tau3R/4R and 56

deviation from this ratio are the pathological feature of several tauopathies (8). 57

Phosphorylation of Tau protein controls its binding to MT and is associated with Tau 58

aggregation in neurodegenerative diseases (5, 9). In general, Tau3R has been linked to 59

neurodevelopment (7), while Tau4R with aging (10). 60

We have previously shown that the expression of activity-dependent neuroprotective 61

protein (ADNP), a protein vital for brain formation (11, 12)), is correlated with Tau3R 62

expression (13) and Adnp+/- mice exhibit tauopathy features - significant increase in 63

phosphorylated Tau, prevented by treatment of ADNP-derived peptide NAP (NAPVSIPQ) 64

(14) as well as and tangle-like structures. Our cell culture results have indicated that NAP 65

enhances Tau-MT interaction in the face of zinc intoxication (15) and NAP protective 66


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activity requires Tau expression (16). We have further revealed that NAP-Tau association 67

is mediated by direct interaction of NAP and Tau with MT end-binding proteins (EBs) (15, 68

17). 69

Clinical trials identified the potential efficacy of NAP (davunetide, CP201) in enhancing 70

short term memory in amnestic mild cognitive impairment patients (18). However, it was 71

not found to be an effective (though, well tolerated) treatment for progressive supranuclear 72

palsy (PSP) patients (19). Because abnormal aggregation of Tau4R is a hallmark of PSP 73

pathophysiology (20), the current study aimed to determine whether NAP had a different 74

activity on either Tau3R or 4R. Our results now showed that NAP preferentially interacted 75

with Tau3R protein from rat brains and induced increased recruitment of human Tau3R to 76

MTs under zinc toxic condition in comparison to Tau4R. Furthermore, we showed that 77

NAP interaction with tubulin was inhibited by paclitaxel obstruction of Tau-binding sites 78

on MTs, confirming the requirement of Tau-MT interaction for NAP activity. 79


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Results 80

Tau from 60-day-old rat brain does not associate with NAP under conditions that 81

Tau from newborn rat brain does 82

Different tubulin and Tau isotypes are expressed in the course of a rat brain development 83

(10, 21). Newborn-rats predominantly express the Tau3R isoform while adults 84

predominantly express the Tau4R isoform (22). Here, newborn and 60-day-old rat cerebral 85

cortex extracts were analyzed by immunoblotting, and prevailing expression of Tau3R or 86

4R was observed in the newborn or 60-day-old cortex protein lysate, respectively, as 87

expected (Fig 1A). Then, newborn and 60-day-old rat cortex protein lysates were exposed 88

to NAP affinity chromatography, and eluted proteins were analyzed by immunoblotting 89

with Tau3R and 4R, total-Tau (identifying all Tau isoforms), tubulin Tub2.1 (identifying 90

neuronal-enriched tubulin (23)) and TubβIII (identifying neuronal-specific tubulin (24)) 91

antibodies. Immunoreactivity for all tested antibodies was detected in the acid eluted 92

fraction from newborn rat brain extract (Fig 1B). However, no significant Tau or tubulin-93

like bands were observed in the eluted fractions from the mature rat extract under the 94

current experimental conditions (Fig 1B). 95

Fig 1. NAP preferentially associates with 3R-tau. (A) Western blot analysis of equal 96

amounts of protein extracts from newborn (NB) rat brain cortex and 60-day-old (60d) rat 97

brain cortex. Significantly larger amount of Tau3R was detected in the one-day-old cortical 98

extract compared to the 60-day cortical extract, and Tau4R was recognized in the 60-day 99

cortical extract only. (B) Western blot analysis of elution fractions obtained by NAP-100

affinity chromatography with the same protein extracts of rat brain cortex. Tau3R, total-101


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Tau, and tubulin were identified in the NAP-binding fraction of the newborn rat cortical 102

brain extract, but essentially neither Tau nor tubulin was identified in the elution fraction 103

of the 60-day-old cortical brain extract (three independent experiments). 104

NAP induces increased recruitment of human Tau3R to MTs under zinc toxic 105

condition in comparison to Tau4R 106

In order to test the effect of NAP on the interactions of different Tau isoforms with MTs, 107

fluorescent recovery after photobleaching (FRAP) assay was performed (Fig 2). mCherry-108

tagged human Tau3R and 4R proteins (S1 Fig.) were over-expressed in differentiated 109

neuroblastoma N1E-115 cells and extracellular zinc (400µM, 1 hour) was used as a MT 110

disruptor, inducer of Tau release from MTs (15). In general, after photobleaching of the 111

region of interest (ROI), the unrecovered portion of initial fluorescence intensity within a 112

bleached area is referred to the immobile fraction of bleached mCherry-Tau proteins 113

because it does not release binding sites on MTs for the entry of un-bleached mCherry-Tau 114

molecules and thus does not allow fluorescence recovery. Therefore, the immobile 115

mCherry-Tau fraction represents MT-bound Tau and reflects the MT-Tau interaction. 116

Here, we observed that treatment with extracellular zinc increased fluorescence recovery 117

87sec after photobleaching of both mCherry-Tau3R and 4R molecules (Fig 2A). Analysis 118

with one-phase exponential association showed a significant decrease of Tau3R and 4R 119

immobile fractions (Fig 2B and C). NAP added together with zinc decreased fluorescence 120

recovery (Fig 2A) and thus significantly enhanced the immobile fraction of Tau3R and 4R 121

compared to treatment with zinc alone (Fig 2B and C). However, while the Tau4R 122

immobile fraction was restored to untreated control level, while the immobile fraction of 123

Tau3R was further increased in comparison to control values and the difference between 124


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Tau3R and 4R immobile fractions was found statistically significant (Fig 2C). Thus, NAP 125

treatment produces a more potent impact on 3R-, rather than on 4R-Tau association with 126

MTs. 127

Fig 2. NAP induces increased recruitment of human Tau3R to MTs under zinc toxic 128

condition in comparison to Tau4R. (A) Representative images of photo-bleaching and 129

fluorescence recovery of mCherry-tagged Tau3R and 4R in differentiated N1E-115 cells 130

treated with extracellular zinc (400μM, 2hrs) with or without NAP treatment (10-12M, 131

2hrs). N1E-115 cells expressing m-Cherry-Tau3R/4R without any treatment represented 132

the control. (B) FRAP recovery curves of normalized data (see “Materials and Methods”). 133

(C) The graph represents percentages (±SEM) of the fitted data (from three independent 134

experiments) of immobile fractions relative to control – 100%. Normalized FRAP data 135

were fitted with one-exponential functions (GraphPad Prism 6), and statistical analysis was 136

performed by Two Way ANOVA (SigmaPlot 11). Statistical significance is presented by 137

*P<0.05, **P<0.01, *** P<0.001. Tau3R: Control n=58, zinc n=85, zinc + NAP n=58; 138

Tau4R: Control n=56, zinc n=47, zinc + NAP n=60. 139

Tau interaction with MTs is required for NAP activity 140

Further, we aimed to test requirement of Tau-MT association for NAP interaction with 141

tubulin/MTs. Because paclitaxel obstructs Tau-binding sites on tubulin (25) we incubated 142

NAP-affinity columns with newborn cerebral cortical extracts in the presence of paclitaxel 143

dissolved in DMSO or in the presence of DMSO alone (used as a control). Paclitaxel 144

markedly decreased tubulin immunoreactivity in the acid-eluted fractions compared to the 145

control (Fig 3A and B). Immunoreactivity of Tau3R appeared in both fractions – with and 146


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without paclitaxel exposure (Fig 3C). As tubulin was washed away in the presence of 147

paclitaxel, whereas Tau3R remained bound to the NAP column, we suggest that NAP 148

interaction with tubulin required mediation of Tau (Fig 3D). To ascertain the specificity of 149

NAP binding, an affinity control column with eight-amino-acids inactive peptide 150

(VLGGGSALL) was prepared, as well. The peptide VLGGGSALL has previously shown 151

no MT-related neuroprotective activity (26). Affinity chromatography with the control 152

peptide showed Tau3R presence in the loaded material, column flow-through, and column 153

wash, but did not detect Tau3R in the acid elution fractions of both columns, neither in the 154

absence no in the presence of paclitaxel (S2 Fig). Also, tubulin was associated with 155

VLGGGSALL regardless of paclitaxel presence, demonstrating nonspecific interaction 156

(S2 Fig). 157

Then, we assessed the protective activity of NAP against increased concentrations of 158

paclitaxel. For this purpose, differentiated N1E-115 cells were exposed to paclitaxel (5, 6 159

and 7 µM) with or without NAP (10-15, 10-12, 10-9M) for 4hrs. Cell viability, measured by 160

mitochondrial activity, was significantly reduced following the 4hr-incubation period with 161

paclitaxel. However, co-treatment with NAP (10-12 and 10-9M, but not 10-15M) protected 162

against the lowest tested concentration of paclitaxel - 5µM (Fig 4), but not against increased 163

concentrations of paclitaxel – 6 and 7µM. These results suggest a requirement of direct 164

Tau-MT interaction for NAP activity, confirming our previously published data that 165

showed requirement of Tau expression for NAP protective capabilities (16). 166

Fig 3. NAP interaction with tubulin, but not with Tau, is inhibited by paclitaxel. (A) 167

Bio-SafeTM Coomassie protein staining of the different fractions (F – flow-through, W - 168

first wash, Wl - last wash, E1/2/3 – elution fractions by order) obtained from NAP-affinity 169


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column loaded with protein extracts of newborn rat cerebral cortex with 4 mg paclitaxel, 170

dissolved in 80µl DMSO, or equal volume of DMSO, alone. Almost no tubulin is evident 171

in the elution fractions in the paclitaxel column, in contrast to the control one. (B, C) 172

Western analysis of elution fractions obtained similarly as in panel (A). (B) Tubulin is not 173

detected in the elution fractions of the pre-incubated paclitaxel column in comparison to 174

the control column. (C) Tau3R is observed in the elution fractions of both the pre-incubated 175

paclitaxel column and the control column. (D) Graphic depiction of the hypothesis that 176

NAP, bound to an affinity column, interacts with tubulin throughout mediation of Tau. 177

Fig 4. NAP protective activity is inhibited by paclitaxel. Cell viability test performed by 178

the MTS assay (measures mitochondrial activity, see “Materials and Methods”). 179

Differentiated N1E-115 cells are exposed to different concentrations of NAP (10-15M, 10-180

12M, 10-9M) and paclitaxel (5, 6 and 7 µM) for 2 hrs. Average of mitochondrial activity 181

results (MTS reduction) are displayed in relation to the control (non-treated cell) value – 182

0.1411±0.02989. Statistical analysis of the data was performed using one-way ANOVA 183

with Tukey post hoc test, n=5. Statistical significance is presented relative to control as 184

*P<0.05, **P<0.01, *** P<0.001; to “w/o NAP” (cells treated with paclitaxel, alone) as 185

#P<0.05, ##P<0.01, ###P<0.001. 186


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Discussion 187

As dynamic tracks for motor proteins, MTs are involved in axonal transport and synaptic 188

transmission. We have previously shown that NAP provides neuroprotection (26, 27) and 189

neurotrophic activities (28) through interaction with MTs (15), rescues impaired axonal 190

transport (29-31), regulates dendritic spines (29, 32, 33) and enhances memory (33). NAP 191

also protects against the accumulation of pathologically modified, hyperphosphorylated 192

Tau (14, 34-36). We have previously suggested the mechanism of NAP protective activity 193

on MT-mediated cellular processes through the involvement of Tau and MT end-binding 194

proteins (EBs) (16, 29). Specifically, NAP contains an ADNP association site (SIP) a 195

signature motif for direct interaction with the EB1 and the EB3 proteins (29), which in turn 196

bind to MTs (37) and Tau (17). Furthermore, Tau has been identified as a regulator of EB’s 197

action, and localization on MTs in developing neuronal cells (17) and NAP increases Tau-198

EB1/3 association (16). Tau is essential for the establishment of MT dynamic instability 199

and axonal transport, while EB1 is more prevalent in neuronal axons (38) and EB3 in 200

dendritic spines (39). Formerly, it has been shown that expression of Tau and EB1/3 201

proteins are required for NAP-dependent neuronal survival (16, 29). Here, we added details 202

to the understanding of the molecular mechanism underlying MT-related activity of NAP. 203

Our current experiments showed that NAP preferentially interacted with rodent Tau3R and 204

induced enhanced recruitment of human Tau3R to MTs under zinc toxic condition in 205

comparison to Tau4R. Furthermore, we demonstrated that paclitaxel-disturbed Tau-tubulin 206

interaction prevented NAP association with tubulin/MTs and inhibited NAP protective 207

activity, suggesting the requirement of not only the expression of Tau, but also Tau-tubulin 208

direct association for sufficient action of NAP. 209


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The only difference between the two Tau isoforms (3R and 4R) is the presence of the exon 210

10 coding sequence comprising an extra MT-binding repeat in Tau4R (Fig 5A, red 211

sequence) which is excluded during alternative splicing in Tau3R (40). Elm prediction 212

analysis (41) of the whole Tau sequence identified cyclin A-docking motif within the 213

translated sequence of exon 10 (Fig 5A, S1 Table). Whereas cyclin-dependent kinase (Cdk) 214

5 is activated by non-cyclin proteins, Cdk1/2 requires direct association with cyclin A 215

imposing an active conformation on the kinase (42). Furthermore, six 216

docking/phosphorylation sites of the cyclin-dependent kinase subunit 1 (Cks1) were 217

identified by Elm prediction on Tau (Fig 5A, S1 Table). Cks1 associated with Cdk-cyclin 218

complex increases the specificity and efficiency of Cdk substrate phosphorylation (43). It 219

has been reported that Cdk2 and Cdk5 provide different Tau phosphorylation profiles (44). 220

It was further reported that region-specific Tau phosphorylation might attenuate Tau-EB 221

association (45). Because NAP interacts with Tau through EB proteins, and Tau3R and 4R 222

may present differences in the phosphorylation profiles, we speculate that observed 223

attenuation of NAP-Tau4R interaction occurs due to some phosphate incorporation on Tau 224

and ensuing decrease of EB protein association (Fig 5B). In this respect, we have 225

previously reported that NAP reduces Tau phosphorylation at Ser262 (30), Ser202/Thr205, 226

and Thr231 (46) residues, but does not exhibit a significant impact on Tau phosphorylation 227

level at Thr181 (19). Intriguingly, Thr181 is on one of the predicted Tau 228

binding/phosphorylation motifs of Cks1 modulating the activity of Cdk (Fig 5A, S1 Table). 229

As opposed to rodents, Tau3R is abundant alongside with Tau4R in the human adult brain. 230

The ratio of Tau 3R:4R proteins is important, and changes in the ratio are observed in 231

tauopathies (47). NAP does not interact with MT proteins from various cancer cell lines or 232


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fibroblasts (27), which may not express any MT-associated proteins with properties that 233

are similar to Tau3R, and NAP does not affect cell division (48). Furthermore, NAP does 234

not protect cells from fibroblast origin unless those cells are transfected with Tau3R-235

expressing plasmid (16). However, NAP protects MT organization in mature neurons and 236

glia (26, 27, 49). We have previously shown that by promoting the interactions of Tau and 237

EBs with MTs, NAP protects MTs against degradation and concurrently enhances MT 238

dynamics (16). In vivo, chronic NAP treatment reduces excess Tau accumulation and 239

pathological hyperphosphorylation (14, 35, 46). Our studies explain, in part, the efficacy 240

of NAP (davuntide, CP201) in enhancing cognitive functions in mild cognitive impairment 241

patients (18), while showing no efficacy (although high safety) in the 4R tauopathy 242

progressive supranuclear palsy (PSP) (19). Since tauopathy underlies a verity of 243

neurodegenerative conditions, our current findings may pave the path for treatment by 244

peptide drugs that have an impact on tubulin-Tau interaction and specific neurofibrillary 245

tangle populations (50). As neurodevelopment has been linked to Tau3R (7, 10), our results 246

pave the path to the development of NAP (davunetide, CP201) for ADNP deficiencies 247

associated with neurodevelopment, for example, the autism-like ADNP syndrome, 248

resulting from de novo truncating mutations in ADNP (32). 249

Fig 5. Suggested explanation for the preference of NAP binding to Tau3R over 250

Tau4R. (A) Amino-acid sequence of the Tau isoform 2 (NP_005901, 441aa) (Tau4R). The 251

translated protein sequence of exon 10, which is spliced in Tau3R, is marked by red. 252

Functional motifs, predicted by Elm analysis (41) (S1 Table) are indicated. (B) Graphic 253

depiction of speculated hypothesis suggesting the preference of NAP to interact with 254

Tau3R based on the findings of Elm prediction analysis. The second MT-binding repeat of 255


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Tau4R (spliced in Tau3R) includes cyclin A-docking motif and thus may enable Tau 256

binding to cyclin A, essential for the activation of Cdk1/2 (42). Cks1 may also associate 257

with Cdk and cyclin A to form more efficient cyclin A-Cdk-Csk1 phosphorylation 258

complex. Tau4R phosphorylation by Cdk1/2 differs from Cdk5 (a conventional Tau 259

kinase) (44) and may disturb/attenuate Tau-EB interaction, which has been previously 260

indicated as a crucial for NAP interaction with MTs (16, 29). The EB dimer structure was 261

constructed according to a published review (37). 262


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Materials and Methods 263

Brain extract preparation 264

Protein lysate was prepared from either one- or sixty-day-old rat cerebral cortex in a lysis 265

buffer containing: 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 4.5 (or 7.5 as 266

indicated), 0.1% Triton X-100, 1% Nonidet P-40, and a protease inhibitor cocktail (Roche 267

Diagnostics, Mannheim, Germany). DNA was fragmented by sonication. Cell debris was 268

discarded following 20 minutes of centrifugation at 30,000g at 4oC, as described previously 269

(26). 270

NAP affinity chromatography 271

Affinity columns contained extended NAP (CKKKGGNAPVSIPQ, the linker peptide is 272

in bold). Peptides were purchased from Genemed Synthesis, Inc., San Antonio, TX, USA 273

or synthesized as before (51). Columns and peptide binding were prepared using Sulfolink 274

coupling gel (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions as 275

before (26). 2 ml Sulfolink coupling gel was loaded onto Poly-Prep Chromatography 276

Columns (Bio-Rad, Hercules, CA, USA) and coupled with 2mg/ml peptide. Coupling was 277

ascertained by free peptide measurements. 278

In order to determine the binding specificity of Tau and tubulin to NAP, multiple 279

experiments were performed under stringent comparable experimental conditions as 280

follows: 281

1] Proteins from either newborn or sixty-day-old rat cerebral cortical extracts (2mg 282

protein/ml, total volume 2 ml) were loaded onto the NAP-affinity columns at pH 7.5 and 283


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incubated for 16 hours at 4°C, columns were washed with phosphate buffered saline (PBS, 284

20-25 ml) until all unbound protein had eluted as confirmed by the Bradford protein assay 285

(Bradford, BioRad, Hercules, CA, USA). The bound protein was then eluted with 0.1 M 286

glycine pH 2.6. 287

2] Proteins from one-day-old rat (expressing Tau3R, solely) cerebral cortical extracts were 288

loaded onto the NAP column with 4 mg paclitaxel (Haorui Pharma-Chem Inc., New-Jersey, 289

USA) dissolved in 80µl dimethyl sulfoxide (DMSO, Sigma, Rehovot, Israel). A control 290

NAP column was treated similarly with 80µl DMSO but without paclitaxel. The columns 291

were incubated with brain extract, washed as described above, and eluted with glycine 0.1 292

M pH 2.6. 293

3] The control column contained an inactive peptide CKKKGGVLGGGSALL (the linker 294

peptide is in bold) described in supplemental materials and methods (S1 File and S2 Fig). 295

SDS-PAGE and western blot analysis 296

The flow-through, wash fractions and elution fractions were separated by 10% or 12% 297

SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining using 298

Bio-SafeTM Coomassie (Bio-Rad, Hercules, CA, USA) according to manufacturer’s 299

instructions or transferred to nitrocellulose membranes (Schleicher and Schull, Dassel, 300

Germany) for western blot analysis. In comparative experiments, run in parallel, the same 301

amounts were loaded on the gels, for each parallel fraction. Non-specific sites on the 302

nitrocellulose membranes used for western analysis were blocked in a blocking solution 303

(10 mM Tris pH 8, 150 mM NaCl, and 0.05% Tween 20 [TBST]) supplemented with 5% 304

non-fat dried milk (1 hour, at room temperature). The Protein complexes were visualized 305


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by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).and 306

exposed on Fuji Film Medical X-ray film (Fuji Corporation, Tokyo, Japan). 307

Antibodies 308

Total-Tau - mouse monoclonal antibody Tau5 (antibody recognizing all Tau forms) was 309

obtained from MBL International Corporation (Woburn, MA, USA). Tau3R and Tau4R - 310

mouse monoclonal anti-Tau RD3 (3-repeat isoform) and mouse monoclonal anti-Tau RD4 311

(4-repeat isoform) were obtained from Millipore Corporation (Billerica, MA, USA). 312

Tub2.1 and Tub2.5 - mouse monoclonal tubulin antibodies maintained and kindly provided 313

by Professor Colin Barnstable, and were used as before (23). TubβIII - mouse monoclonal 314

antibody β tubulin isotype III was obtained from Sigma-Aldrich (St. Louis, MO, USA). 315

Secondary antibodies were goat anti-mouse-horseradish peroxidase - HRP (Jackson 316

ImmunoResearch, West Grove, PA, USA). All antibodies were used at the dilution of 317

1:1000, except when otherwise indicated. 318

Plasmid construction 319

DNA inserts carrying Tau3R and 4R were obtained from human Tau3R and 4R cDNA 320

containing plasmids (a kind gift of Professor M. Goedert, MRC Laboratory of Molecular 321

Biology, Cambridge, UK) and then cloned into the backbone of the newly constructed 322

pmCherry-C1 plasmid. For more details, see supplemental materials and methods (S1 File 323

and S1 Fig). 324

Cell culture and treatments 325


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Mouse neuroblastoma N1E-115 cells (ATCC, Bethesda, MD; passage numbers from 10 to 326

13) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine 327

serum (FBS), 2 mM glutamine and 100 U/ml penicillin, 100 mg/ml streptomycin 328

(Biological Industries, Beit Haemek, Israel). The cells were incubated in 95% air/5% CO2 329

in a humidified incubator at 37°C. N1E-115 cells were plated on 35mm dishes (81156, 60 330

µ-Dish, Ibidi, Martinsried, Germany) at a concentration of 25*104 cells/dish and then were 331

differentiated with reduced FBS (2%) and DMSO (1.25%) containing medium during five 332

days before transfection and seven days before the experiment. On the day of experiment 333

differentiated N1E-115 cells were treated for 1 hrs with zinc chloride (ZnCl2; final 334

concentration, 400 μM, Sigma, Rehovot, Israel) with or without NAP (10−12M). 335

Transfection of over-expression plasmids and Fluorescence recovery after 336

photobleaching (FRAP) 337

5-day differentiated N1E-115 cells were transfected with a 1µg pm Cherry-C1-Tau3R/4R 338

plasmid. 48 hrs after transfection, cultured N1E-115 cells were incubated at 37 °C with a 339

5% CO2/95% air mixture in a thermostatic chamber placed on the stage of a Leica TCS 340

SP5 confocal microscope [objective 100x (PL Apo) oil immersion, NA 1.4]. An ROI 341

(region of interest) for photo-bleaching was drawn in the proximal cell branches. mCherry-342

Tau3R/4R was bleached with a 587nm argon laser, and fluorescence recovery was at 610-343

650nm. Immediately after bleaching, 80 images were collected every 0.74s. Fluorescence 344

signals were quantified with ImageJ (NIH), obtained data were normalized with 345

easyFRAP43, and FRAP recovery curves were fitted by a one-phase exponential 346

association function using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). 347

Samples with R2<0.9 were excluded. 348


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Cell viability assay 349

7-day differentiated N1E-115 cells were treated with different concentrations of NAP (10-350

15M, 10-12M and 10-9M) and paclitaxel (5, 6 and 7µM diluted in DMSO) for 2 hours. 351

Treatments with 5, 6 and 7µM of DMSO alone were used as controls. Cell viability was 352

measured using the MTS assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation 353

Assay; Promega, Madison, WI, USA), which was performed according to the 354

manufacturer's instructions and read in an ELISA plate reader at 490nm. 355

Statistical Analysis 356

Data are presented as the mean ± SEM from 3 independent experiments. Statistical analysis 357

of the data was performed by using one-way ANOVA test (followed by the Tukey post hoc 358

test) by the IBM SPSS Statistics software version 23. * P<0.05, ** P<0.01, *** P<0.001. 359


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Acknowledgements: This work is in partial fulfillment of the PhD thesis requirements of 360

Msc. Yanina Ivashko-Pachima. Work was supported in part by the following grants, ISF 361

1424/14, ERA-NET neuron AUTISYN, BSF-NSF 2016746, AMN Foundation as well as 362

Drs. Ronith and Armand Stemmer, Mr Arthur Gerbi (French Friends of Tel Aviv 363

University), and Spanish Friends of Tel Aviv University. Professor Illana Gozes is the 364

incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors, 365

and the Director of the Dr. Diana and Zelman Elton (Elbaum) Laboratory for Molecular 366

Neuroendocrinology at Tel Aviv University. Professor Gozes also serves as the Chief 367

Scientific Officer of Coronis Neurosciences, developing CP201 for the ADNP syndrome. 368


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Supporting Information 532

S1 Fig. Plasmid maps. pmCherry-C1 vector (A), human Tau3R (B) and 4R (C) expressing 533

plasmids based on pmCherry-C1 vector. The plasmid maps were constructed with 534

Benchling platform (www.benchling.com). 535

S2 Fig. VLGGGSALL does not interact with Tau3R. The gel lanes contain the protein 536

loaded (load) flow-through (FT) PBS wash, pH7.5 (W1-30) and acid elutes (E1-E4) from 537

the columns linked to eight-amino-acid inactive peptide VLGGGCALL P (has previously 538

shown no microtubule-related neuroprotective activity (26)) that were incubated with brain 539

extracts and DMSO in the absence and presence of paclitaxel. Western blotting analysis 540

with anti Tau RD3 detected Tau3R presence in the loaded material, column flow-through 541

and column wash, but did not detect Tau3R in the acid elution fractions of both the 542

columns. In contrast, tubulin antibodies - Tub2.5 identified tubulin-like bands also in the 543

elution fractions with no apparent influence of paclitaxel treatment. 544

S1 Table. Elm prediction analysis of Tau (NP_005901) exon 10 translation sequence. 545

Elm analysis (41) predicted functional motifs of the translation sequence of spliced exon 546

10 (VQIINKKLDLSNVQSKCGSKDNIKHVPGGGS) of Tau isoform 2 (NP_005901). 547

DOC_CYCLIN_RxL_1 motif appeared only once in full Tau sequence. 548


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Figure 1 549


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Figure 2 550


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Figure 3 551


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Figure 4 552


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Figure 5 553


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nap (davunetide) preferential interaction with dynamic 3 ... · adnp), a protein vital for brain...

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