nap (davunetide) preferential interaction with dynamic 3 ... · adnp), a protein vital for brain...
Post on 15-Jun-2020
0 Views
Preview:
TRANSCRIPT
1
1
2
3
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
8
9
Yanina Ivashko-Pachima1, Maya Maor1 and Illana Gozes1,2* 10
11
12
13
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
18
19
*Corresponding author 20
E-mail: igozes@tauex.tau.ac.il 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
2
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
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
3
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
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
4
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
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
5
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
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
6
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
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
7
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
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
8
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
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
9
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
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
10
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
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
11
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
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
12
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
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
13
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
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
14
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
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
15
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
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
16
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
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
17
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
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
18
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
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
19
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
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
20
References 369
1. Witte H, Neukirchen D, Bradke F. Microtubule stabilization specifies initial neuronal 370 polarization. The Journal of cell biology. 2008 Feb 11;180(3):619-32. PubMed PMID: 18268107. 371 Pubmed Central PMCID: 2234250. 372 2. Lee VM, Balin BJ, Otvos L, Jr., Trojanowski JQ. A68: a major subunit of paired helical 373 filaments and derivatized forms of normal Tau. Science. 1991 Feb 8;251(4994):675-8. PubMed 374 PMID: 1899488. 375 3. Trojanowski JQ, Schuck T, Schmidt ML, Lee VM. Distribution of tau proteins in the normal 376 human central and peripheral nervous system. The journal of histochemistry and cytochemistry : 377 official journal of the Histochemistry Society. 1989 Feb;37(2):209-15. PubMed PMID: 2492045. 378 4. Morris M, Maeda S, Vossel K, Mucke L. The many faces of tau. Neuron. 2011 May 379 12;70(3):410-26. PubMed PMID: 21555069. Pubmed Central PMCID: 3319390. 380 5. Lee VM, Goedert M, Trojanowski JQ. Neurodegenerative tauopathies. Annual review of 381 neuroscience. 2001;24:1121-59. PubMed PMID: 11520930. 382 6. Mandelkow EM, Schweers O, Drewes G, Biernat J, Gustke N, Trinczek B, et al. Structure, 383 microtubule interactions, and phosphorylation of tau protein. Annals of the New York Academy 384 of Sciences. 1996 Jan 17;777:96-106. PubMed PMID: 8624133. 385 7. Goedert M, Jakes R. Expression of separate isoforms of human tau protein: correlation 386 with the tau pattern in brain and effects on tubulin polymerization. The EMBO journal. 1990 387 Dec;9(13):4225-30. PubMed PMID: 2124967. Pubmed Central PMCID: 552204. 388 8. Kalbfuss B, Mabon SA, Misteli T. Correction of alternative splicing of tau in frontotemporal 389 dementia and parkinsonism linked to chromosome 17. The Journal of biological chemistry. 2001 390 Nov 16;276(46):42986-93. PubMed PMID: 11560926. 391 9. von Bergen M, Barghorn S, Biernat J, Mandelkow EM, Mandelkow E. Tau aggregation is 392 driven by a transition from random coil to beta sheet structure. Biochimica et biophysica acta. 393 2005 Jan 3;1739(2-3):158-66. PubMed PMID: 15615635. 394 10. Francon J, Lennon AM, Fellous A, Mareck A, Pierre M, Nunez J. Heterogeneity of 395 microtubule-associated proteins and brain development. European journal of biochemistry. 1982 396 Dec 15;129(2):465-71. PubMed PMID: 7151809. 397 11. Mandel S, Rechavi G, Gozes I. Activity-dependent neuroprotective protein (ADNP) 398 differentially interacts with chromatin to regulate genes essential for embryogenesis. 399 Developmental biology. 2007 Mar 15;303(2):814-24. PubMed PMID: 17222401. 400 12. Pinhasov A, Mandel S, Torchinsky A, Giladi E, Pittel Z, Goldsweig AM, et al. Activity-401 dependent neuroprotective protein: a novel gene essential for brain formation. Brain research 402 Developmental brain research. 2003 Aug 12;144(1):83-90. PubMed PMID: 12888219. 403 13. Schirer Y, Malishkevich A, Ophir Y, Lewis J, Giladi E, Gozes I. Novel marker for the onset of 404 frontotemporal dementia: early increase in activity-dependent neuroprotective protein (ADNP) 405 in the face of Tau mutation. PloS one. 2014;9(1):e87383. PubMed PMID: 24489906. Pubmed 406 Central PMCID: 3906161. 407 14. Vulih-Shultzman I, Pinhasov A, Mandel S, Grigoriadis N, Touloumi O, Pittel Z, et al. Activity-408 dependent neuroprotective protein snippet NAP reduces tau hyperphosphorylation and 409 enhances learning in a novel transgenic mouse model. The Journal of pharmacology and 410 experimental therapeutics. 2007 Nov;323(2):438-49. PubMed PMID: 17720885. 411
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
21
15. Oz S, Ivashko-Pachima Y, Gozes I. The ADNP derived peptide, NAP modulates the tubulin 412 pool: implication for neurotrophic and neuroprotective activities. PloS one. 2012;7(12):e51458. 413 PubMed PMID: 23272107. Pubmed Central PMCID: 3522725. 414 16. Ivashko-Pachima Y, Sayas CL, Malishkevich A, Gozes I. ADNP/NAP dramatically increase 415 microtubule end-binding protein-Tau interaction: a novel avenue for protection against 416 tauopathy. Molecular psychiatry. 2017 Sep;22(9):1335-44. PubMed PMID: 28115743. 417 17. Sayas CL, Tortosa E, Bollati F, Ramirez-Rios S, Arnal I, Avila J. Tau regulates the localization 418 and function of End-binding proteins 1 and 3 in developing neuronal cells. Journal of 419 neurochemistry. 2015 Jun;133(5):653-67. PubMed PMID: 25761518. 420 18. Morimoto BH, Schmechel D, Hirman J, Blackwell A, Keith J, Gold M, et al. A double-blind, 421 placebo-controlled, ascending-dose, randomized study to evaluate the safety, tolerability and 422 effects on cognition of AL-108 after 12 weeks of intranasal administration in subjects with mild 423 cognitive impairment. Dementia and geriatric cognitive disorders. 2013;35(5-6):325-36. PubMed 424 PMID: 23594991. 425 19. Boxer AL, Lang AE, Grossman M, Knopman DS, Miller BL, Schneider LS, et al. Davunetide 426 in patients with progressive supranuclear palsy: a randomised, double-blind, placebo-controlled 427 phase 2/3 trial. The Lancet Neurology. 2014 Jul;13(7):676-85. PubMed PMID: 24873720. Pubmed 428 Central PMCID: 4129545. 429 20. Dickson DW. Neuropathologic differentiation of progressive supranuclear palsy and 430 corticobasal degeneration. Journal of neurology. 1999 Sep;246 Suppl 2:II6-15. PubMed PMID: 431 10525997. 432 21. Gozes I, Littauer UZ. Tubulin microheterogeneity increases with rat brain maturation. 433 Nature. 1978 Nov 23;276(5686):411-3. PubMed PMID: 714168. 434 22. Takuma H, Arawaka S, Mori H. Isoforms changes of tau protein during development in 435 various species. Brain research Developmental brain research. 2003 May 14;142(2):121-7. 436 PubMed PMID: 12711363. 437 23. Gozes I, Barnstable CJ. Monoclonal antibodies that recognize discrete forms of tubulin. 438 Proceedings of the National Academy of Sciences of the United States of America. 1982 439 Apr;79(8):2579-83. PubMed PMID: 6178107. Pubmed Central PMCID: 346243. 440 24. Joshi HC, Cleveland DW. Differential utilization of beta-tubulin isotypes in differentiating 441 neurites. The Journal of cell biology. 1989 Aug;109(2):663-73. PubMed PMID: 2503525. Pubmed 442 Central PMCID: 2115728. 443 25. Kar S, Fan J, Smith MJ, Goedert M, Amos LA. Repeat motifs of tau bind to the insides of 444 microtubules in the absence of taxol. The EMBO journal. 2003 Jan 2;22(1):70-7. PubMed PMID: 445 12505985. Pubmed Central PMCID: 140040. 446 26. Divinski I, Mittelman L, Gozes I. A femtomolar acting octapeptide interacts with tubulin 447 and protects astrocytes against zinc intoxication. The Journal of biological chemistry. 2004 Jul 448 2;279(27):28531-8. PubMed PMID: 15123709. 449 27. Divinski I, Holtser-Cochav M, Vulih-Schultzman I, Steingart RA, Gozes I. Peptide 450 neuroprotection through specific interaction with brain tubulin. Journal of neurochemistry. 2006 451 Aug;98(3):973-84. PubMed PMID: 16893427. 452 28. Gozes I, Spivak-Pohis I. Neurotrophic effects of the peptide NAP: a novel neuroprotective 453 drug candidate. Current Alzheimer research. 2006 Jul;3(3):197-9. PubMed PMID: 16842095. 454 29. Esteves AR, Gozes I, Cardoso SM. The rescue of microtubule-dependent traffic recovers 455 mitochondrial function in Parkinson's disease. Biochimica et biophysica acta. 2014 Jan;1842(1):7-456 21. PubMed PMID: 24120997. 457 30. Jouroukhin Y, Ostritsky R, Assaf Y, Pelled G, Giladi E, Gozes I. NAP (davunetide) modifies 458 disease progression in a mouse model of severe neurodegeneration: protection against 459
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
22
impairments in axonal transport. Neurobiology of disease. 2013 Aug;56:79-94. PubMed PMID: 460 23631872. 461 31. Amram N, Hacohen-Kleiman G, Sragovich S, Malishkevich A, Katz J, Touloumi O, et al. 462 Sexual divergence in microtubule function: the novel intranasal microtubule targeting SKIP 463 normalizes axonal transport and enhances memory. Molecular psychiatry. 2016 Oct;21(10):1467-464 76. PubMed PMID: 26782054. 465 32. Oz S, Kapitansky O, Ivashco-Pachima Y, Malishkevich A, Giladi E, Skalka N, et al. The NAP 466 motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through 467 microtubule end binding proteins. Molecular psychiatry. 2014 Oct;19(10):1115-24. PubMed 468 PMID: 25178163. 469 33. Hacohen-Kleiman G, Sragovich S, Karmon G, Gao AYL, Grigg I, Pasmanik-Chor M, et al. 470 Activity-dependent neuroprotective protein deficiency models synaptic and developmental 471 phenotypes of autism-like syndrome. The Journal of clinical investigation. 2018 Aug 14. PubMed 472 PMID: 30106381. 473 34. Matsuoka Y, Gray AJ, Hirata-Fukae C, Minami SS, Waterhouse EG, Mattson MP, et al. 474 Intranasal NAP administration reduces accumulation of amyloid peptide and tau 475 hyperphosphorylation in a transgenic mouse model of Alzheimer's disease at early pathological 476 stage. Journal of molecular neuroscience : MN. 2007;31(2):165-70. PubMed PMID: 17478890. 477 35. Shiryaev N, Jouroukhin Y, Giladi E, Polyzoidou E, Grigoriadis NC, Rosenmann H, et al. NAP 478 protects memory, increases soluble tau and reduces tau hyperphosphorylation in a tauopathy 479 model. Neurobiology of disease. 2009 May;34(2):381-8. PubMed PMID: 19264130. 480 36. Magen I, Ostritsky R, Richter F, Zhu C, Fleming SM, Lemesre V, et al. Intranasal NAP 481 (davunetide) decreases tau hyperphosphorylation and moderately improves behavioral deficits in 482 mice overexpressing alpha-synuclein. Pharmacology research & perspectives. 2014 483 Oct;2(5):e00065. PubMed PMID: 25505609. Pubmed Central PMCID: 4186425. 484 37. Akhmanova A, Steinmetz MO. Control of microtubule organization and dynamics: two 485 ends in the limelight. Nature reviews Molecular cell biology. 2015 Dec;16(12):711-26. PubMed 486 PMID: 26562752. 487 38. Alves-Silva J, Sanchez-Soriano N, Beaven R, Klein M, Parkin J, Millard TH, et al. 488 Spectraplakins promote microtubule-mediated axonal growth by functioning as structural 489 microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins). The Journal 490 of neuroscience : the official journal of the Society for Neuroscience. 2012 Jul 4;32(27):9143-58. 491 PubMed PMID: 22764224. Pubmed Central PMCID: 3666083. 492 39. Jaworski J, Kapitein LC, Gouveia SM, Dortland BR, Wulf PS, Grigoriev I, et al. Dynamic 493 microtubules regulate dendritic spine morphology and synaptic plasticity. Neuron. 2009 Jan 494 15;61(1):85-100. PubMed PMID: 19146815. 495 40. Dehmelt L, Halpain S. The MAP2/Tau family of microtubule-associated proteins. Genome 496 biology. 2005;6(1):204. PubMed PMID: 15642108. Pubmed Central PMCID: 549057. 497 41. Dinkel H, Van Roey K, Michael S, Kumar M, Uyar B, Altenberg B, et al. ELM 2016--data 498 update and new functionality of the eukaryotic linear motif resource. Nucleic acids research. 2016 499 Jan 4;44(D1):D294-300. PubMed PMID: 26615199. Pubmed Central PMCID: 4702912. 500 42. Malumbres M. Cyclin-dependent kinases. Genome biology. 2014;15(6):122. PubMed 501 PMID: 25180339. Pubmed Central PMCID: 4097832. 502 43. Koivomagi M, Ord M, Iofik A, Valk E, Venta R, Faustova I, et al. Multisite phosphorylation 503 networks as signal processors for Cdk1. Nature structural & molecular biology. 2013 504 Dec;20(12):1415-24. PubMed PMID: 24186061. Pubmed Central PMCID: 3855452. 505
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
23
44. Landrieu I, Leroy A, Smet-Nocca C, Huvent I, Amniai L, Hamdane M, et al. NMR 506 spectroscopy of the neuronal tau protein: normal function and implication in Alzheimer's disease. 507 Biochemical Society transactions. 2010 Aug;38(4):1006-11. PubMed PMID: 20658994. 508 45. Ramirez-Rios S, Denarier E, Prezel E, Vinit A, Stoppin-Mellet V, Devred F, et al. Tau 509 antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-510 dependent mechanism. Molecular biology of the cell. 2016 Oct 1;27(19):2924-34. PubMed PMID: 511 27466319. Pubmed Central PMCID: 5042579. 512 46. Matsuoka Y, Jouroukhin Y, Gray AJ, Ma L, Hirata-Fukae C, Li HF, et al. A neuronal 513 microtubule-interacting agent, NAPVSIPQ, reduces tau pathology and enhances cognitive 514 function in a mouse model of Alzheimer's disease. The Journal of pharmacology and experimental 515 therapeutics. 2008 Apr;325(1):146-53. PubMed PMID: 18199809. 516 47. Goedert M, Jakes R. Mutations causing neurodegenerative tauopathies. Biochimica et 517 biophysica acta. 2005 Jan 3;1739(2-3):240-50. PubMed PMID: 15615642. 518 48. Gozes I, Divinsky I, Pilzer I, Fridkin M, Brenneman DE, Spier AD. From vasoactive intestinal 519 peptide (VIP) through activity-dependent neuroprotective protein (ADNP) to NAP: a view of 520 neuroprotection and cell division. Journal of molecular neuroscience : MN. 2003;20(3):315-22. 521 PubMed PMID: 14501014. 522 49. Gozes I, Divinski I. NAP, a neuroprotective drug candidate in clinical trials, stimulates 523 microtubule assembly in the living cell. Current Alzheimer research. 2007 Dec;4(5):507-9. PubMed 524 PMID: 18220512. 525 50. Gozes I, Hoglinger G, Quinn JP, Hooper NM, Hoglund K. Tau Diagnostics and Clinical 526 Studies. Journal of molecular neuroscience : MN. 2017 Oct;63(2):123-30. PubMed PMID: 527 29082468. 528 51. Bassan M, Zamostiano R, Davidson A, Pinhasov A, Giladi E, Perl O, et al. Complete 529 sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective 530 peptide. Journal of neurochemistry. 1999 Mar;72(3):1283-93. PubMed PMID: 10037502. 531
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
24
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
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
25
Figure 1 549
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
26
Figure 2 550
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
27
Figure 3 551
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
28
Figure 4 552
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
29
Figure 5 553
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
top related