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Neuroprotective effects of Cerebrolysin in triple repeat Tau transgenic model of Pick’s disease and fronto-temporal tauopathies

Neuroprotective effects of Cerebrolysin in triple repeat Tau transgenic model of Pick’s disease... Background: Tauopathies are a group of neurodegenerative disorders with accumulation of three-repeat (3R) or four-repeat (4R) Tau. While 3R tau is found in Pick’s disease and Alzheimer’s disease (AD), 4R tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and AD. We have previously shown that Cerebrolysin (CBL), a neuropeptide mixture with neurotrophic effects, ameliorates the pathology in amyloid precursor protein transgenic (tg) mouse model of AD and 4R tau, however it is unclear if CBL ameliorates the deficits and neuropathol- ogy in the mouse model of Pick’s disease over expressing 3R tau. Results: Mice expressing 3R tau (L266V and G272V mutations) under the mThy-1 promoter were treated with CBL in two separate groups, the first was 3 months old (treated for 3 months, IP) and the second was 6 months old (treated for 3 months, IP) at the start of the treatment. We found that although the levels of total 3R tau were unchanged, CBL reduced the levels of hyper-phosphorylated tau in both groups of mice. This was accompanied by reduced neuro- degenerative pathology in the neocortex and hippocampus in both groups and by improvements in the behavioral deficits in the nest-building test and water maze in the 3–6 month group. Conclusion: Taken together these results support the notion that CBL may be beneficial in other taupathy models by reducing the levels of aberrantly phosphorylated tau. Keywords: 3 repeat Tau, Pick’s disease, Cerebrolysin, Neuronal loss, Transgenic degeneration (CBD) and progressive supranuclear palsy Background (PSP) in Pick’s disease (PiD) only 3R tau accumulates. A Tau is a major neuronal cytoskeletal protein encoded by mixture of 3R and 4R tau is found in Alzheimer’s Disease an alternatively spliced gene (MAPT) present on chro- (AD) and FTDP-17T [3]. mosome 17 (MAPT) [1]. Six different isoforms of tau are Pick’s disease is a rare neurodegenerative disorder asso- found in the CNS and, depending on the number of the ciated with dementia and fronto-temporal lobar degen- ~32 amino acid microtubule binding domain repeats, can be either three-repeat (3R) and four-repeat (4R) tau eration [6]. Patients with PiD display cortical atrophy, [2]. Taupathies are common neurodegenerative disorders neuronal loss, astrogliosis and formation of 3R tau-pos- of the aging population that lead to behavioral altera itive, globular, intra-neuronal inclusions in the neocortex and limbic system denominated pick bodies (PBs) [3]. tions and dementia [3–5]. Tauopathies are divided into There are sporadic and familial forms and mutations in those containing 3R, 4R or both species of tau. While MAPT account for the majority of these cases [7–10]. 4R tau alone is predominantly present in corticobasal Cerebrolysin (CBL) is a peptide mixture with neuro- trophic-like properties that amliorates behavioral defi - *Correspondence: emasliah@ucsd.edu cits in patients with mild to moderate AD [11]. Likewise, Department of Neurosciences, University of California, La Jolla, San Diego, CA 92093-0624, USA we have previously shown that CBL ameliorates the Full list of author information is available at the end of the article © 2015 Rockenstein et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rockenstein et al. BMC Neurosci (2015) 16:85 Page 2 of 12 neurodegenerative pathology in amyloid precursor pro- non-tg and 40 tg) were included in this study. Mice were tein (APP) transgenic (tg) models of AD [12–15] as well killed 24  h after the last injection of vehicle or CBL was as in models of tauopathy expressing 4R tau [16, 17]. The administered. Cerebrolysin is a mixture of peptides and protective effects of CBL in these models of AD and tau - amino acids obtained after high quality hydrolyzing and pathy might involve different mechanisms including reg - purification from porcine brain, more information is ulation of GSK3β and CDK5 signaling and anti-apoptotic available at the web site (http://www.hypermed.com.au/ effects mediated by expression of endogenous neuro - Clinical%20Research/EVER2010_Monograph_screen. trophic factors [18]. However, it is unclear if CBL might pdf ). Cerebrolysin was a gift from EverPharma. display similar neuroprotective effects in models of 3R tau accumulation that mimic PiD. Behavioral analysis We recently developed a tg mouse model expressing 3R One month prior to the end of the experiments, mice tau bearing mutations associated with familial forms of were tested in the behavioral paradigms. Mice were PiD (L266V and G272V) under the neuronal my Th -1 pro - continued with vehicle or CBL during the course of the moter [19]. These mice display extensive time-dependent testing. Spatial learning and memory was investigated accumulation of 3R tau in the neocortex and hippocam- using the water maze. For this purpose, a pool (diameter pus, with inclusion formation, behavioral deficits, and 180  cm) was filled with opaque water (24  °C) and mice neurodegeneration that mimic some aspects of PiD [19]. were first trained to locate a visible platform (days 1–3) In the present study, these 3R tau tg mice were treated and then a submerged hidden platform (days 4–7) in with CBL starting at 3 month old (for 3 month, IP) or at three daily trials 2–3  min apart. Mice that failed to find 6  months of age (for 3  month, IP) and evaluated neuro- the hidden platform within 90  s were placed on it for pathologically and behaviorally. We found that although 30 s. The same platform location was used for all sessions total levels of 3R tau were unchanged, CBL reduced the and all mice. The starting point at which each mouse was levels of hyper-phosphorylated tau in both groups of placed into the water was changed randomly between mice. This was accompanied by reductions in the neuro - two alternative entry points located at a similar distance degenerative pathology in both groups and by improve- from the platform. In addition, on the final day of test - ments in the behavioral deficits in the younger group. ing the platform was removed and the time spent by Taken together these results suggest that CBL might be mice in the correct quadrant was measured (Probe test). beneficial in orphan disease tauopathies such as PiD. The duration of the probe test was 40  s. Time to reach the platform (escape latency) was recorded with a Nol- Methods dus Instruments EthoVision video tracking system (San Generation of mThy‑1 3R Tau mutant transgenic mice Diego Instruments, San Diego, CA) set to analyze two and treatments samples per second. The nesting test and open field was All animal experiments were approved by The University performed in plastic cages with beam break photodetec- of California at San Diego’s animal subjects committee. tors as previously described [20]. Mice expressing human 3R Tau-bearing the mutations associated with familial PiD (L266V and G272V) under Tissue preparation the neuronal myTh -1 promoter cassette (provided by Following behavioral analysis, mice were killed following Dr. H. van der Putten) were generated on the C57BL/6 NIH guidelines. The right hemi-brain was post-fixed for background, as previously described [19]. The high 48  h in 4  % phosphate-buffered paraformaldehyde (pH expressing Line 13 mice were chosen for these studies. 7.4) at 4 °C and sagittal sectioned vibratome 2000 (40 µm To differentiate preventative versus therapeutic effects Leica, Deerfield, IL). The left hemi-brain was snap-frozen of CBL, the mice were divided into two groups, the first and stored at −70 °C. were 3  months old at the start of the experiment and were treated for 3 months (IP, 5 ml/kg) with CBL or vehi- Immunohistochemistry and image analysis cle (n  =  10 per group). This group is hereafter denomi - Analysis of tau expression was performed using free- nated as the “3–6  month” group. The second group was floating, blind-coded vibratome (50  µm thick) sections 6 months old at the beginning of the experiment and was [21]. Sections were incubated overnight at 4 °C with anti- treated for 3  months (IP, 5  ml/kg) with CBL or vehicle bodies against 3R tau (1:250, Millipore) and pTau (PHF-1 (n = 10 per group). This group is hereafter denominated 1:500, gift from Peter Davies) followed by biotin-tagged as the “6–9 month” group. For both groups, control, non- anti-rabbit or anti-mouse IgG1 secondary antibodies tg littermates of the same age and gender were included (1:100, Vector Laboratories, Inc., Burlingame, CA), Avi- and treated with either vehicle or CBL (IP, 5  ml/kg) for din D-HRP (1:200, ABC Elite, Vector), and visualized 3  months (n  =  10 per group). A total of 80 mice (40 with diaminobenzidine. Sections were scanned with a Rockenstein et al. BMC Neurosci (2015) 16:85 Page 3 of 12 digital Olympus bright field digital microscope (BX41). Statistical analysis For comparison of the neuropathological pathology in All analyses were performed using GraphPad Prism human PiD samples with the 3R tau mice, samples from (Version 5.0). Differences among means were assessed the frontal cortex from three PiD cases were obtained by one-way ANOVA with Dunnett’s post hoc test when from patients evaluated neurologically and psychometri- compared to non-tg and by Tukey–Kramer when com- cally at the Alzheimer Disease Research Center/Univer- paring tg groups. Two-way ANOVA with repeated meas- sity of California, San Diego [22]. ures followed by a Bonferroni multiple comparisons post Neurodegenerative pathology was analyzed using sec- hoc test was used for analyzing the interactions between tions immunolabeled overnight with antibodies against groups and time. The null hypothesis was rejected at the the neuronal marker NeuN (1:500, Millipore) and the 0.05 level. astroglial marker glial fibrillary acidic protein (GFAP, 1:1000, Millipore). Sections reacted with antibodies Results against NeuN or GFAP were incubated with secondary Cerebrolysin treatment reduced tau antibodies, Avidin D-HRP, and visualized with diamin- hyper‑phosphorylation in 3R tau transgenic mouse obenzidine. Analysis of levels of Tau and GFAP immu- without affecting total levels of 3R tau noreactivity was performed in layer 5 of the neocortex To investigate the neuroprotective versus the therapeutic and in the hippocampus dentate gyrus with Image J and effects of CBL in a 3R tau tg mouse model of an orphan expressed as optical density. Briefly, for each section disorder namely PiD, mice were divided into two treat- a total of four images were captured at 400× and con- ment groups: those with a treatment start at 3 month of verted to gray scale, opened with Image J, thresholded age (3–6  month group) and those beginning at 6  month and a dynamic scale set to determine optical density. of age (6–9  month group). The choice of these groups The numbers of NeuN-immunoreactive neurons were was based on our previous time course studies that have estimated utilizing unbiased stereological methods [23]. shown progressive tau pathology in this mouse model Hemi-sections containing the neocortex, hippocampus starting at 3 month of age [19]. As previously described, and striatum were outlined using an Olympus BX51 both groups of Line 13 mutant 3R tau tg mice displayed microscope running StereoInvestigator 8.21.1 software abundant 3R tau immunoreactivity in neuronal cells in (Micro-BrightField, Cochester, VT). Grid size for the the neocortex and hippocampus including pyramidal hippocampal dentate was: 300 × 300 µm, and the count- layers and dentate gyrus (Fig.  1) [19]. In the 3–6  month ing frame was 50  ×  50  µm. The average coefficient of group, no 3R tau was detected in the non-tg mice treated error for each region was 0.09. Sections were analyzed with either vehicle or CBL (Fig. 1a). In the tg mice treated using a 100  ×  1.4 PlanApo oil-immersion objective. A with vehicle or CBL similar numbers of 3R tau positive 5  µm high dissector, allowed for 2  µm top and bottom cells were detected in the neocortex and hippocampus guard-zones. (Fig.  1a, b). In the 6–9  month group overall there was abundant 3R tau accumulation in the tg mice while no 3R Tissue fractionation and immunoblot analysis tau was detected in non-tg littermates (Fig. 1c). In the 3R The levels of tau were analyzed using lysates that were tau tg mice, CBL treatment reduced the accumulation of extracted and fractioned into soluble and insoluble frac- 3R tau in neuronal cell bodies and in the neuropil com- tions by ultracentrifugation utilizing the posterior half pared to vehicle treatment (Fig.  1c, d). In the hippocam- of the right hemi-brain that includes the neocortex and pus there were no significant effects of CBL compared to hippocampus [20]. Protein (20 µg/lane) from the insolu- vehicle (Fig. 1c, d). ble fraction was loaded onto 4–12  % SDS/PAGE gels Next we analyzed the levels of tau phosphorylation and blotted onto PVDF membranes, incubated mouse with the PHF-1 antibody that is sensitive at detecting monoclonal antibodies against total Tau (tTau 1:1000), pathological forms of tau found in patients with PiD 3R tau (1:2000), p-tau (PHF-1 1:1500), t-GSK3β (1:500, and in the 3R tau tg model. In the non-tg mice from the Cell Signaling), p-GSK3β (GSK3βY216, 1:500, Life Tech- 3–6 month group we observed mild immunoreactivity in nologies), t-Akt (1:1000, Cell Signaling), p-Akt (Ser473, the neuropil but no neuronal immunolabeling (Fig.  2a). 1:500, Santa Cruz) followed by HRP-tagged secondary In the 3R tau tg mice from the 3–6  month group there antibodies (1:5000 Santa Cruz Biotechnology). Bands was abundant p-tau immunostaining of neuronal cell were visualized by enhanced chemiluminescence (ECL, bodies and axons in the neocortex and hippocampus PerkinElmer, Boston, MA) and analyzed with a quanti- that was more abundant in the vehicle tg mice com- tative Versadoc XL imaging apparatus (BioRad). β-Actin pared to the CBL treated animals (Fig.  2a, b). Likewise, (1:3000, Sigma) was used as the loading control. in the 6–9 month group there was greater levels of p-tau Rockenstein et al. BMC Neurosci (2015) 16:85 Page 4 of 12 Fig. 1 Immunocytochemical analysis of effects of CBL on levels of 3R Tau transgenic mice. a Immunocytochemistry with an antibody against 3R Tau in the 3–6 month group. b Image analysis of levels of 3R Tau immunoreactivity in the neocortex and hippocampus. c Immunocytochemistry with an antibody against 3R Tau in the 6–9 month group. d Image analysis of levels of 3R Tau immunoreactivity in the neocortex and hippocampus of the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test immunoreactivity in neuronal cell and axons in the neo- MW of 50–60  kDa (Fig.  3a). No bands were detected in cortex and hippocampus of the vehicle-treated 3R tau tg the non-tg groups (Fig. 3a). The levels of 3R tau were sim - with a significant reduction in both regions in animals ilar between the vehicle and CBL-treated tg mice (Fig. 3a, treated with CBL (Fig. 2c, d). b). The p-tau band was identified in the 3R tau tg mice To further verify these results by an independ- as doublet at an estimated MW of 50–60  kDa; a light ent method, immunoblot analysis was performed. As single band was detected in the non-tg mice (Fig.  3a). expected, in the 3–6  month group abundant 3R tau was The vehicle-treated tg mice displayed higher levels of detected in the tg mice as a triplet band at an estimated p-tau immunoreactivity, levels were decreased in 3R tau Rockenstein et al. BMC Neurosci (2015) 16:85 Page 5 of 12 Fig. 2 Immunocytochemical analysis of effects of CBL on levels of p-tau in 3R tau transgenic mice. a Immunocytochemistry with an antibody against p-tau (PHF1) in the 3–6 month group. b Image analysis of levels of p-tau immunoreactivity in the neocortex and hippocampus. c Immuno- cytochemistry with an antibody against p-Tau in the 6–9 month group. d Image analysis of levels of p-tau tau immunoreactivity in the neocortex and hippocampus of the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test tg mice treated with CBL (Fig.  3a, c). In the 6–9  month levels of p-tau immunoreactivity, levels were decreased in group there was more abundant 3R tau and p-tau 3R tau tg mice treated with CBL (Fig. 3d, f ). immunoreactivity (compared to the 3–6  month group), detected in the tg mice as a triplet band at an estimated Cerebrolysin treatment prevented and rescued the MW of 50–60  kDa (Fig.  3d). No bands were detected in neurodegenerative pathology in 3R tau transgenic mice the non-tg groups (Fig.  3d). The levels of 3R tau were The neurodegenerative pathology in the mice was inves - similar between the vehicle and CBL-treated tg mice tigated by immunocytochemistry and image analysis (Fig.  3d, e). The vehicle-treated tg mice displayed higher with antibodies against the neuronal marker, NeuN, and Rockenstein et al. BMC Neurosci (2015) 16:85 Page 6 of 12 Fig. 3 Western blot analysis of effects of CBL on levels of tau in 3R tau transgenic mice a Representative immunoblots of samples from the 3–6 month group analyzed with antibodies against 3R Tau tTau and p-tau (PHF1). b, c Image analysis of levels of 3R Tau and ratio of p-tau/tTau in the brains of mice from the 3–6 month group. d Representative immunoblots of samples from the 6–9 month group analyzed with antibodies against 3R Tau, tTau and p-tau (PHF1). e, f Image analysis of levels of 3R tau and p-tau/tTau in the brains of mice from the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test the astroglial marker, GFAP. In the 3–6 month group, as the 3R tau tg mice, bringing it in line with that observed previously described, we observed in the vehicle-treated in the vehicle-treated non-tg mice (Fig. 4e, f ). 3R tau tg mice a reduction in the number of cells in the In these tg mice the neuronal loss is accompanied by frontal cortex and in the dentate gyrus of the hippocam- astrogliosis. In the 3–6  month group there was some pus (Fig.  4a, b). Treatment with CBL was protective as astrogliosis in the neocortex of the vehicle treated 3R tau the neuronal cell counts in both of these regions were tg mice compared to the non-tg mice (Fig.  5a, b). Treat- similar in the non-tg groups and in the 3R tau tg treated ment with CBL rescued this effect in the neocortex of the with CBL (Fig.  4a, b). In the 6–9  month group, the loss 3R tau tg mice (Fig. 5a, b). In the 6–9 month group there of neurons in the neocortex and dentate gyrus of the was astrogliosis in the neocortex and hippocampus of the vehicle-treated 3R tau tg mice was greater (Fig.  4c, d). vehicle treated 3R tau tg mice compared to the non-tg Compared to the non-tg groups, there was an ameliora- mice (Fig.  5c, d). Treatment with CBL partially reduced tion of the neuronal deficits in the CBL-treated 3R tau astrogliosis in the 3R tau tg mice (Fig.  5c, d). There was tg mice (Fig.  4c, d). These results were confirmed with no significant difference between CBL-treated non-tg MAP2, a second neuronal-specific marker (Fig.  4e, f ). As mice and 3R tau tg mice treated with CBL. with the NeuN, MAP2 immunoreactivity in the dentate gyrus demonstrated neuronal loss in the vehicle-treated Cerebrolysin treatment ameliorated the behavioral deficits 3R tau tg mice and vehicle-treated non-tg mice, this was in 3R tau transgenic mice observed in both the 3–6 and 6–9 month groups (Fig. 4e, To investigate the functional effects of CBL in the 3R tau f ). CBL treatment increased MAP2 immunoreactivity in tg mice behavioral analysis was performed in the open (See figure on next page.) Fig. 4 Analysis of NeuN cell counts in 3R tau transgenic mice treated with CBL. a Immunocytochemistry with an antibody against NeuN in the 3–6 month group. b Stereological analysis with the dissector method to estimate the numbers of NeuN positive cells in the neocortex and hip- pocampus in the 3–6 month group. c Immunocytochemistry with an antibody against NeuN in the brains of the 6–9 month group. d Stereological analysis with the dissector method to estimate the numbers of NeuN positive cells in the neocortex and hippocampus in the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test Rockenstein et al. BMC Neurosci (2015) 16:85 Page 7 of 12 Rockenstein et al. BMC Neurosci (2015) 16:85 Page 8 of 12 Fig. 5 Analysis of astrogliosis in 3R Tau transgenic mice treated with CBL a Immunocytochemistry with an antibody against GFAP in the 3–6 month group. b Computer aided image analysis of levels of GFAP immunoreactivity in the neocortex and hippocampus in the 3–6 month group. c Immu- nocytochemistry with an antibody against GFAP in the brains of the 6–9 month group. d Computer aided image analysis of levels of GFAP immu- noreactivity in the neocortex and hippocampus in the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test field, nesting test, and water maze. In the 3–6  month (Fig. 6c). Treatment with CBL partially rescued the alter- group analysis in the open field showed a trend toward ations in this behavior in the 3R tau tg mice (Fig.  6c). In increased total activity and rearing in the 3R tg tau mice, the 6–9 month group in the open field no statistically sig - but no statistically significant differences were observed nificant differences among the four groups were detected among the four groups (Fig. 6a, b). However, in the nest- (Fig. 6d, e). In the nesting test, the vehicle treated 3R tau ing test, the vehicle treated 3R tau tg mice displayed tg mice displayed significant alterations that were not a profound reduction in the ability to build the nest fully rescued by CBL treatment (Fig. 6f ). Next analysis of Rockenstein et al. BMC Neurosci (2015) 16:85 Page 9 of 12 Fig. 6 Eec ff ts of CBL treatment in behavioral alterations in 3R Tau transgenic mice. Mice were evaluated for context-dependent learning in an open field area using a Kinder SmartFrame Cage Rack Station activity monitor system. a, b Levels of total activity and rearing in the cage in the 3–6 month group. c Nesting behavior in the cage of the 3–6 month group. d, e Levels of total activity and rearing in the cage in the 6–9 month group. f Nesting behavior in the cage of the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test memory and spatial learning was performed in the water age group some visual impairment is present that might maze. In the 3–6  month group compared to the non-tg account for the lack of effects of CBL in the hidden seg - mice the vehicle treated 3R tau tg mice displayed defi - ment of the platform test. cits in spatial learning acquisition in the hidden platform segment of the test (Fig.  7a). In contrast, CBL treatment Cerebrolysin effects on 3RTau phosphorylation might be prevented these deficits in the 3R tau tg mice (Fig.  7a). mediated via Akt/GKS3β Memory retention in the probe test showed that com- We have previously shown that CBL rescues the hyper- pared to non-tg mice the vehicle treated 3R tau tg mice activation of GSK3β in other transgenic models [15, 17], expended less time in the target quadrant (Fig. 7b), treat- in order to examine the mechanisms through which CBL ment with CBL rescued this deficit in the 3R tau tg mice might reduce the hyper-phosphorylation of 3R Tau and (Fig.  7b), which performed similar to the non-tg control ameliorate the neurodegenerative pathology we analyzed groups (Fig.  7b). Visual success was comparable among levels of GSK3β and Akt by immunoblot. We found that the four groups of mice for the 3–6 month group (Fig. 7c). there was increased activation of p-GSK3β (Y216) in the Next, analysis of memory and spatial learning was per- vehicle-treated 3R Tau tg mice compared to non-tg con- formed in the 6–9 month group. Compared to the non-tg trols Fig.  8a, b), and therefore the ratio of p-GSK3β to mice, the vehicle treated 3R tau tg mice displayed deficits t-GSK3β was higher (Fig. 8b). In contrast, treatment with in spatial learning acquisition in the hidden platform seg- CBL reduced p-GSK3β (Y216) and decreased the ratio ment of the test (Fig.  7d), CBL did not fully ameliorate of p-GSK3β to total (Fig.  8a, b). Next we analyzed Akt these deficits (Fig.  7d). Memory retention in the probe and found that there was decreased activation of p-Akt test showed that compared to non-tg mice the vehicle (Ser473) in the vehicle treated 3R Tau tg mice compared treated 3R tau tg mice expended less time in the target to non-tg controls (Fig.  8a, c), and therefore the ratio of quadrant (Fig. 7e), however, here CBL treatment amelio- p-Akt to total was lower (Fig.  8c). Treatment with CBL rated the deficits in the 3R tau tg mice (Fig.  7e), which increased p-Akt (Ser473) and increased the ratio of p-Akt performed similar to the non-tg control groups (Fig. 7e). to total (Fig. 8a, c). Therefore, the neuroprotective effects Visual success score was higher in the non-tg compared of CBL in the 3R Tau tg mice might be related to regula- to the 3R tau tg mice (Fig. 7f ), indicating that in this older tion of the Akt/GSK3β axis. Rockenstein et al. BMC Neurosci (2015) 16:85 Page 10 of 12 Fig. 7 Eec ff ts of CBL on learning memory in the water maze in 3R Tau tg mice. a Water maze testing presented as distance traveled to find the platform, respectively in the 3–6 month group. During the cued portion of the test both the vehicle and CBL treated non-tg and 3R tau tg mice performed as expected. However during the hidden portion of the test, the vehicle treated 3R tau tg mice went a farther distance to find the platform compared to the non-tg, CBL prevented these effects. b At day 8, during the probe portion of the test (with platform removed) memory retention was evaluated in the 3–6 month group. c In the 3–6 month group, probe test with the visual platform confirmed that no visual altera- tions were detected. d Water maze testing presented as distance traveled to find the platform in the 6–9 month group. During the cued portion of the test both the vehicle and CBL treated non-tg and 3R tau tg mice performed as expected. However during the hidden portion of the test, the vehicle treated 3R Tau tg mice went a farther distance to find the platform compared to the non-tg. e At day 8, during the probe portion of the test (with platform removed) memory retention was evaluated in the 6–9 month group. f In the 6–9 month group, probe test with the visual platform showed visual alterations in the 3R tau tg group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test Fig. 8 Western blot analysis of effects of CBL on levels of Akt/GSK3β in the 3R Tau transgenic mice. a Representative immunoblots of samples from the 6–9 month group analyzed with antibodies against p-GSK3β (Y216), t-GSK3β, t-Akt and p-Akt (Ser473). b, c Image analysis of the ration between p-GSK3β (Y216) and total and p-Akt (Ser473) and total respectively. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to tg control using one way ANOVA with Tukey post hoc test the neurodegenerative pathology and behavioral defi - Discussion cits, although levels of 3R tau were not affected by the In the present study, we investigated the neuropro- treatment. When CBL treatment occurred at a more tective versus therapeutic effects of CBL at rescuing advanced stage (6 month old mice) we still observed that pathology in mutant 3R Tau in tg mouse that models this neurotrophic peptide mixture was able to reduce the aspects of the pathogenesis in PiD. We found that early levels of p-tau and ameliorated the neurodegenerative adminstration of CBL (at 3  month of age, for 3  months) pathology, however only partially rescued the behavioral resulted in decreased p-tau and completely rescued Rockenstein et al. BMC Neurosci (2015) 16:85 Page 11 of 12 deficits. These results in the 3R tau tg mice are consist - oligomerization and fibril formation, mitigating the for - ent with previous studies showing that CBL rescues the mation of potentially neurotoxic tau species, and enhanc- neurodegenerative pathology associated with p-tau accu- ing degradation of pathological tau through increasing mulation. However in previous studies we investigated clearance mechanisms [37]. Finally, a new provocative the effects of CBL at rescuing pathology associated with approach is to reduce the accumulation and propagation mutant 4R tau over-expression (AAV-mediated) in APP of tau utilizing specific monoclonal antibodies against tg mice [16] or in crosses between mutant 4R tau tg and various tau species [38–41]. GSK3β tg mice [17]. In the bigenic mice, CBL treatment reduced the accumulation of p-Tau, ameliorated the neu- Conclusions rodegenerative pathology, decreased Drp-1 and p-Drp-1 In summary, in the present study we found that although expression, and returned mitochondria to characteristics the levels of total 3R tau were unchanged, CBL reduced comparable to non-tg mice [17]. the levels of hyper-phosphorylated tau in both groups of The precise mechanisms of action of CBL are unknown, mice. This was accompanied by reductions in the neuro - however the rescue effects of CBL were likely related degenerative pathology in both groups and by improve- its ability to regulate levels of p-tau rather than levels of ments in the behavioral deficits in the younger group. total tau (either 3R or 4R tau) [24]. This suggests that, These results suggest that CBL might be beneficial in at least in part, the neuroprotective and neurotrophic orphan disease taupathies such as PiD. effects of CBL might be associated with its ability to reg - ulate CDK5 and GSK3β activity. Both of these kinases Abbreviations have been previously shown to play an important role in 3R: three-repeat tau; 4R: four-repeat tau; AD: Alzheimer’s disease; APP: amyloid hyper-phosphorylating tau [25–27] and mediating the precursor protein; CBD: corticobasal degeneration; CBL: Cerebrolysin; GFAP: glial fibrillary acidic protein; PiD: Pick’s disease; PSP: progressive supranuclear neuropathology in models of AD and FTD [28, 29]. We palsy; tg: transgenic. have previously shown that the CBL neurotrophic pep- tide mixture is capable of reducing behavioral deficits Authors’ contributions ER designed the experimental plan, coordinated and conducted the animal in an APP tg mouse model of AD-like pathology [13] by studies. KU analyzed and reviewed the results and edited the manuscript. blocking CDK5 and GSK3β [16], resulting in decreased MM, JF conducted the animal studies and analyzed the data. AA performed APP maturation and Aβ biosynthesis [30], increased the immunohistochemistry. SW, HB edited manuscript and provided intel- lectual input on the experimental plan. DM provided intellecutal input on the neurogenesis [15] and synaptic formation [13]. Further experimental plan. EM provided intellecutal input on the experimental plan, supporting a role of this pathway a recent in  vitro study analyzed the immunohistochemistry and wrote manuscript. All authors read showed that CBL protects neuronal cell lines from the and approved the final manuscript. neurotoxic effects of CoCl via regulation of GSK3β acti- Author details vation [31]. Interestingly, tau hyper-phosphorylation in 1 Department of Neurosciences, University of California, La Jolla, San Diego, CA PiD [32, 33] as well as in our model [19] is likely associ- 92093-0624, USA. Clinical Research and Pharmacology, EVER Neuro Pharma GmbH, Unterach, Austria. Department of Pathology, University of California, ated with GSK3β. La Jolla, San Diego, CA, USA. Pick’s disease belongs to the FTLD-tau with Picks bodies as defined by the working group [34–36]. Pick’s Acknowledgements This work was partially funded by a grant from EVERPharma and NIH grant disease is a rare cause of dementia in the elderly, while AG18440. some anti-depressants such as selective serotonin reup- take inhibitors have been shown to ameliorate some of Competing interests Stefan Winter, Hemma Brandstaetter and Dieter Meier are employed by EVER the symptoms and obsessive–compulsive behaviors asso- Neuro Pharma GmbH, Unterach, Austria. None of the other authors have any ciated with PiD, there are currently no known disease- competing interests. modifying treatments available. 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Neuroprotective effects of Cerebrolysin in triple repeat Tau transgenic model of Pick’s disease and fronto-temporal tauopathies

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Springer Journals
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Copyright © 2015 by Rockenstein et al.
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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10.1186/s12868-015-0218-7
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26611895
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Abstract

Background: Tauopathies are a group of neurodegenerative disorders with accumulation of three-repeat (3R) or four-repeat (4R) Tau. While 3R tau is found in Pick’s disease and Alzheimer’s disease (AD), 4R tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and AD. We have previously shown that Cerebrolysin (CBL), a neuropeptide mixture with neurotrophic effects, ameliorates the pathology in amyloid precursor protein transgenic (tg) mouse model of AD and 4R tau, however it is unclear if CBL ameliorates the deficits and neuropathol- ogy in the mouse model of Pick’s disease over expressing 3R tau. Results: Mice expressing 3R tau (L266V and G272V mutations) under the mThy-1 promoter were treated with CBL in two separate groups, the first was 3 months old (treated for 3 months, IP) and the second was 6 months old (treated for 3 months, IP) at the start of the treatment. We found that although the levels of total 3R tau were unchanged, CBL reduced the levels of hyper-phosphorylated tau in both groups of mice. This was accompanied by reduced neuro- degenerative pathology in the neocortex and hippocampus in both groups and by improvements in the behavioral deficits in the nest-building test and water maze in the 3–6 month group. Conclusion: Taken together these results support the notion that CBL may be beneficial in other taupathy models by reducing the levels of aberrantly phosphorylated tau. Keywords: 3 repeat Tau, Pick’s disease, Cerebrolysin, Neuronal loss, Transgenic degeneration (CBD) and progressive supranuclear palsy Background (PSP) in Pick’s disease (PiD) only 3R tau accumulates. A Tau is a major neuronal cytoskeletal protein encoded by mixture of 3R and 4R tau is found in Alzheimer’s Disease an alternatively spliced gene (MAPT) present on chro- (AD) and FTDP-17T [3]. mosome 17 (MAPT) [1]. Six different isoforms of tau are Pick’s disease is a rare neurodegenerative disorder asso- found in the CNS and, depending on the number of the ciated with dementia and fronto-temporal lobar degen- ~32 amino acid microtubule binding domain repeats, can be either three-repeat (3R) and four-repeat (4R) tau eration [6]. Patients with PiD display cortical atrophy, [2]. Taupathies are common neurodegenerative disorders neuronal loss, astrogliosis and formation of 3R tau-pos- of the aging population that lead to behavioral altera itive, globular, intra-neuronal inclusions in the neocortex and limbic system denominated pick bodies (PBs) [3]. tions and dementia [3–5]. Tauopathies are divided into There are sporadic and familial forms and mutations in those containing 3R, 4R or both species of tau. While MAPT account for the majority of these cases [7–10]. 4R tau alone is predominantly present in corticobasal Cerebrolysin (CBL) is a peptide mixture with neuro- trophic-like properties that amliorates behavioral defi - *Correspondence: emasliah@ucsd.edu cits in patients with mild to moderate AD [11]. Likewise, Department of Neurosciences, University of California, La Jolla, San Diego, CA 92093-0624, USA we have previously shown that CBL ameliorates the Full list of author information is available at the end of the article © 2015 Rockenstein et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rockenstein et al. BMC Neurosci (2015) 16:85 Page 2 of 12 neurodegenerative pathology in amyloid precursor pro- non-tg and 40 tg) were included in this study. Mice were tein (APP) transgenic (tg) models of AD [12–15] as well killed 24  h after the last injection of vehicle or CBL was as in models of tauopathy expressing 4R tau [16, 17]. The administered. Cerebrolysin is a mixture of peptides and protective effects of CBL in these models of AD and tau - amino acids obtained after high quality hydrolyzing and pathy might involve different mechanisms including reg - purification from porcine brain, more information is ulation of GSK3β and CDK5 signaling and anti-apoptotic available at the web site (http://www.hypermed.com.au/ effects mediated by expression of endogenous neuro - Clinical%20Research/EVER2010_Monograph_screen. trophic factors [18]. However, it is unclear if CBL might pdf ). Cerebrolysin was a gift from EverPharma. display similar neuroprotective effects in models of 3R tau accumulation that mimic PiD. Behavioral analysis We recently developed a tg mouse model expressing 3R One month prior to the end of the experiments, mice tau bearing mutations associated with familial forms of were tested in the behavioral paradigms. Mice were PiD (L266V and G272V) under the neuronal my Th -1 pro - continued with vehicle or CBL during the course of the moter [19]. These mice display extensive time-dependent testing. Spatial learning and memory was investigated accumulation of 3R tau in the neocortex and hippocam- using the water maze. For this purpose, a pool (diameter pus, with inclusion formation, behavioral deficits, and 180  cm) was filled with opaque water (24  °C) and mice neurodegeneration that mimic some aspects of PiD [19]. were first trained to locate a visible platform (days 1–3) In the present study, these 3R tau tg mice were treated and then a submerged hidden platform (days 4–7) in with CBL starting at 3 month old (for 3 month, IP) or at three daily trials 2–3  min apart. Mice that failed to find 6  months of age (for 3  month, IP) and evaluated neuro- the hidden platform within 90  s were placed on it for pathologically and behaviorally. We found that although 30 s. The same platform location was used for all sessions total levels of 3R tau were unchanged, CBL reduced the and all mice. The starting point at which each mouse was levels of hyper-phosphorylated tau in both groups of placed into the water was changed randomly between mice. This was accompanied by reductions in the neuro - two alternative entry points located at a similar distance degenerative pathology in both groups and by improve- from the platform. In addition, on the final day of test - ments in the behavioral deficits in the younger group. ing the platform was removed and the time spent by Taken together these results suggest that CBL might be mice in the correct quadrant was measured (Probe test). beneficial in orphan disease tauopathies such as PiD. The duration of the probe test was 40  s. Time to reach the platform (escape latency) was recorded with a Nol- Methods dus Instruments EthoVision video tracking system (San Generation of mThy‑1 3R Tau mutant transgenic mice Diego Instruments, San Diego, CA) set to analyze two and treatments samples per second. The nesting test and open field was All animal experiments were approved by The University performed in plastic cages with beam break photodetec- of California at San Diego’s animal subjects committee. tors as previously described [20]. Mice expressing human 3R Tau-bearing the mutations associated with familial PiD (L266V and G272V) under Tissue preparation the neuronal myTh -1 promoter cassette (provided by Following behavioral analysis, mice were killed following Dr. H. van der Putten) were generated on the C57BL/6 NIH guidelines. The right hemi-brain was post-fixed for background, as previously described [19]. The high 48  h in 4  % phosphate-buffered paraformaldehyde (pH expressing Line 13 mice were chosen for these studies. 7.4) at 4 °C and sagittal sectioned vibratome 2000 (40 µm To differentiate preventative versus therapeutic effects Leica, Deerfield, IL). The left hemi-brain was snap-frozen of CBL, the mice were divided into two groups, the first and stored at −70 °C. were 3  months old at the start of the experiment and were treated for 3 months (IP, 5 ml/kg) with CBL or vehi- Immunohistochemistry and image analysis cle (n  =  10 per group). This group is hereafter denomi - Analysis of tau expression was performed using free- nated as the “3–6  month” group. The second group was floating, blind-coded vibratome (50  µm thick) sections 6 months old at the beginning of the experiment and was [21]. Sections were incubated overnight at 4 °C with anti- treated for 3  months (IP, 5  ml/kg) with CBL or vehicle bodies against 3R tau (1:250, Millipore) and pTau (PHF-1 (n = 10 per group). This group is hereafter denominated 1:500, gift from Peter Davies) followed by biotin-tagged as the “6–9 month” group. For both groups, control, non- anti-rabbit or anti-mouse IgG1 secondary antibodies tg littermates of the same age and gender were included (1:100, Vector Laboratories, Inc., Burlingame, CA), Avi- and treated with either vehicle or CBL (IP, 5  ml/kg) for din D-HRP (1:200, ABC Elite, Vector), and visualized 3  months (n  =  10 per group). A total of 80 mice (40 with diaminobenzidine. Sections were scanned with a Rockenstein et al. BMC Neurosci (2015) 16:85 Page 3 of 12 digital Olympus bright field digital microscope (BX41). Statistical analysis For comparison of the neuropathological pathology in All analyses were performed using GraphPad Prism human PiD samples with the 3R tau mice, samples from (Version 5.0). Differences among means were assessed the frontal cortex from three PiD cases were obtained by one-way ANOVA with Dunnett’s post hoc test when from patients evaluated neurologically and psychometri- compared to non-tg and by Tukey–Kramer when com- cally at the Alzheimer Disease Research Center/Univer- paring tg groups. Two-way ANOVA with repeated meas- sity of California, San Diego [22]. ures followed by a Bonferroni multiple comparisons post Neurodegenerative pathology was analyzed using sec- hoc test was used for analyzing the interactions between tions immunolabeled overnight with antibodies against groups and time. The null hypothesis was rejected at the the neuronal marker NeuN (1:500, Millipore) and the 0.05 level. astroglial marker glial fibrillary acidic protein (GFAP, 1:1000, Millipore). Sections reacted with antibodies Results against NeuN or GFAP were incubated with secondary Cerebrolysin treatment reduced tau antibodies, Avidin D-HRP, and visualized with diamin- hyper‑phosphorylation in 3R tau transgenic mouse obenzidine. Analysis of levels of Tau and GFAP immu- without affecting total levels of 3R tau noreactivity was performed in layer 5 of the neocortex To investigate the neuroprotective versus the therapeutic and in the hippocampus dentate gyrus with Image J and effects of CBL in a 3R tau tg mouse model of an orphan expressed as optical density. Briefly, for each section disorder namely PiD, mice were divided into two treat- a total of four images were captured at 400× and con- ment groups: those with a treatment start at 3 month of verted to gray scale, opened with Image J, thresholded age (3–6  month group) and those beginning at 6  month and a dynamic scale set to determine optical density. of age (6–9  month group). The choice of these groups The numbers of NeuN-immunoreactive neurons were was based on our previous time course studies that have estimated utilizing unbiased stereological methods [23]. shown progressive tau pathology in this mouse model Hemi-sections containing the neocortex, hippocampus starting at 3 month of age [19]. As previously described, and striatum were outlined using an Olympus BX51 both groups of Line 13 mutant 3R tau tg mice displayed microscope running StereoInvestigator 8.21.1 software abundant 3R tau immunoreactivity in neuronal cells in (Micro-BrightField, Cochester, VT). Grid size for the the neocortex and hippocampus including pyramidal hippocampal dentate was: 300 × 300 µm, and the count- layers and dentate gyrus (Fig.  1) [19]. In the 3–6  month ing frame was 50  ×  50  µm. The average coefficient of group, no 3R tau was detected in the non-tg mice treated error for each region was 0.09. Sections were analyzed with either vehicle or CBL (Fig. 1a). In the tg mice treated using a 100  ×  1.4 PlanApo oil-immersion objective. A with vehicle or CBL similar numbers of 3R tau positive 5  µm high dissector, allowed for 2  µm top and bottom cells were detected in the neocortex and hippocampus guard-zones. (Fig.  1a, b). In the 6–9  month group overall there was abundant 3R tau accumulation in the tg mice while no 3R Tissue fractionation and immunoblot analysis tau was detected in non-tg littermates (Fig. 1c). In the 3R The levels of tau were analyzed using lysates that were tau tg mice, CBL treatment reduced the accumulation of extracted and fractioned into soluble and insoluble frac- 3R tau in neuronal cell bodies and in the neuropil com- tions by ultracentrifugation utilizing the posterior half pared to vehicle treatment (Fig.  1c, d). In the hippocam- of the right hemi-brain that includes the neocortex and pus there were no significant effects of CBL compared to hippocampus [20]. Protein (20 µg/lane) from the insolu- vehicle (Fig. 1c, d). ble fraction was loaded onto 4–12  % SDS/PAGE gels Next we analyzed the levels of tau phosphorylation and blotted onto PVDF membranes, incubated mouse with the PHF-1 antibody that is sensitive at detecting monoclonal antibodies against total Tau (tTau 1:1000), pathological forms of tau found in patients with PiD 3R tau (1:2000), p-tau (PHF-1 1:1500), t-GSK3β (1:500, and in the 3R tau tg model. In the non-tg mice from the Cell Signaling), p-GSK3β (GSK3βY216, 1:500, Life Tech- 3–6 month group we observed mild immunoreactivity in nologies), t-Akt (1:1000, Cell Signaling), p-Akt (Ser473, the neuropil but no neuronal immunolabeling (Fig.  2a). 1:500, Santa Cruz) followed by HRP-tagged secondary In the 3R tau tg mice from the 3–6  month group there antibodies (1:5000 Santa Cruz Biotechnology). Bands was abundant p-tau immunostaining of neuronal cell were visualized by enhanced chemiluminescence (ECL, bodies and axons in the neocortex and hippocampus PerkinElmer, Boston, MA) and analyzed with a quanti- that was more abundant in the vehicle tg mice com- tative Versadoc XL imaging apparatus (BioRad). β-Actin pared to the CBL treated animals (Fig.  2a, b). Likewise, (1:3000, Sigma) was used as the loading control. in the 6–9 month group there was greater levels of p-tau Rockenstein et al. BMC Neurosci (2015) 16:85 Page 4 of 12 Fig. 1 Immunocytochemical analysis of effects of CBL on levels of 3R Tau transgenic mice. a Immunocytochemistry with an antibody against 3R Tau in the 3–6 month group. b Image analysis of levels of 3R Tau immunoreactivity in the neocortex and hippocampus. c Immunocytochemistry with an antibody against 3R Tau in the 6–9 month group. d Image analysis of levels of 3R Tau immunoreactivity in the neocortex and hippocampus of the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test immunoreactivity in neuronal cell and axons in the neo- MW of 50–60  kDa (Fig.  3a). No bands were detected in cortex and hippocampus of the vehicle-treated 3R tau tg the non-tg groups (Fig. 3a). The levels of 3R tau were sim - with a significant reduction in both regions in animals ilar between the vehicle and CBL-treated tg mice (Fig. 3a, treated with CBL (Fig. 2c, d). b). The p-tau band was identified in the 3R tau tg mice To further verify these results by an independ- as doublet at an estimated MW of 50–60  kDa; a light ent method, immunoblot analysis was performed. As single band was detected in the non-tg mice (Fig.  3a). expected, in the 3–6  month group abundant 3R tau was The vehicle-treated tg mice displayed higher levels of detected in the tg mice as a triplet band at an estimated p-tau immunoreactivity, levels were decreased in 3R tau Rockenstein et al. BMC Neurosci (2015) 16:85 Page 5 of 12 Fig. 2 Immunocytochemical analysis of effects of CBL on levels of p-tau in 3R tau transgenic mice. a Immunocytochemistry with an antibody against p-tau (PHF1) in the 3–6 month group. b Image analysis of levels of p-tau immunoreactivity in the neocortex and hippocampus. c Immuno- cytochemistry with an antibody against p-Tau in the 6–9 month group. d Image analysis of levels of p-tau tau immunoreactivity in the neocortex and hippocampus of the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test tg mice treated with CBL (Fig.  3a, c). In the 6–9  month levels of p-tau immunoreactivity, levels were decreased in group there was more abundant 3R tau and p-tau 3R tau tg mice treated with CBL (Fig. 3d, f ). immunoreactivity (compared to the 3–6  month group), detected in the tg mice as a triplet band at an estimated Cerebrolysin treatment prevented and rescued the MW of 50–60  kDa (Fig.  3d). No bands were detected in neurodegenerative pathology in 3R tau transgenic mice the non-tg groups (Fig.  3d). The levels of 3R tau were The neurodegenerative pathology in the mice was inves - similar between the vehicle and CBL-treated tg mice tigated by immunocytochemistry and image analysis (Fig.  3d, e). The vehicle-treated tg mice displayed higher with antibodies against the neuronal marker, NeuN, and Rockenstein et al. BMC Neurosci (2015) 16:85 Page 6 of 12 Fig. 3 Western blot analysis of effects of CBL on levels of tau in 3R tau transgenic mice a Representative immunoblots of samples from the 3–6 month group analyzed with antibodies against 3R Tau tTau and p-tau (PHF1). b, c Image analysis of levels of 3R Tau and ratio of p-tau/tTau in the brains of mice from the 3–6 month group. d Representative immunoblots of samples from the 6–9 month group analyzed with antibodies against 3R Tau, tTau and p-tau (PHF1). e, f Image analysis of levels of 3R tau and p-tau/tTau in the brains of mice from the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test the astroglial marker, GFAP. In the 3–6 month group, as the 3R tau tg mice, bringing it in line with that observed previously described, we observed in the vehicle-treated in the vehicle-treated non-tg mice (Fig. 4e, f ). 3R tau tg mice a reduction in the number of cells in the In these tg mice the neuronal loss is accompanied by frontal cortex and in the dentate gyrus of the hippocam- astrogliosis. In the 3–6  month group there was some pus (Fig.  4a, b). Treatment with CBL was protective as astrogliosis in the neocortex of the vehicle treated 3R tau the neuronal cell counts in both of these regions were tg mice compared to the non-tg mice (Fig.  5a, b). Treat- similar in the non-tg groups and in the 3R tau tg treated ment with CBL rescued this effect in the neocortex of the with CBL (Fig.  4a, b). In the 6–9  month group, the loss 3R tau tg mice (Fig. 5a, b). In the 6–9 month group there of neurons in the neocortex and dentate gyrus of the was astrogliosis in the neocortex and hippocampus of the vehicle-treated 3R tau tg mice was greater (Fig.  4c, d). vehicle treated 3R tau tg mice compared to the non-tg Compared to the non-tg groups, there was an ameliora- mice (Fig.  5c, d). Treatment with CBL partially reduced tion of the neuronal deficits in the CBL-treated 3R tau astrogliosis in the 3R tau tg mice (Fig.  5c, d). There was tg mice (Fig.  4c, d). These results were confirmed with no significant difference between CBL-treated non-tg MAP2, a second neuronal-specific marker (Fig.  4e, f ). As mice and 3R tau tg mice treated with CBL. with the NeuN, MAP2 immunoreactivity in the dentate gyrus demonstrated neuronal loss in the vehicle-treated Cerebrolysin treatment ameliorated the behavioral deficits 3R tau tg mice and vehicle-treated non-tg mice, this was in 3R tau transgenic mice observed in both the 3–6 and 6–9 month groups (Fig. 4e, To investigate the functional effects of CBL in the 3R tau f ). CBL treatment increased MAP2 immunoreactivity in tg mice behavioral analysis was performed in the open (See figure on next page.) Fig. 4 Analysis of NeuN cell counts in 3R tau transgenic mice treated with CBL. a Immunocytochemistry with an antibody against NeuN in the 3–6 month group. b Stereological analysis with the dissector method to estimate the numbers of NeuN positive cells in the neocortex and hip- pocampus in the 3–6 month group. c Immunocytochemistry with an antibody against NeuN in the brains of the 6–9 month group. d Stereological analysis with the dissector method to estimate the numbers of NeuN positive cells in the neocortex and hippocampus in the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test Rockenstein et al. BMC Neurosci (2015) 16:85 Page 7 of 12 Rockenstein et al. BMC Neurosci (2015) 16:85 Page 8 of 12 Fig. 5 Analysis of astrogliosis in 3R Tau transgenic mice treated with CBL a Immunocytochemistry with an antibody against GFAP in the 3–6 month group. b Computer aided image analysis of levels of GFAP immunoreactivity in the neocortex and hippocampus in the 3–6 month group. c Immu- nocytochemistry with an antibody against GFAP in the brains of the 6–9 month group. d Computer aided image analysis of levels of GFAP immu- noreactivity in the neocortex and hippocampus in the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test field, nesting test, and water maze. In the 3–6  month (Fig. 6c). Treatment with CBL partially rescued the alter- group analysis in the open field showed a trend toward ations in this behavior in the 3R tau tg mice (Fig.  6c). In increased total activity and rearing in the 3R tg tau mice, the 6–9 month group in the open field no statistically sig - but no statistically significant differences were observed nificant differences among the four groups were detected among the four groups (Fig. 6a, b). However, in the nest- (Fig. 6d, e). In the nesting test, the vehicle treated 3R tau ing test, the vehicle treated 3R tau tg mice displayed tg mice displayed significant alterations that were not a profound reduction in the ability to build the nest fully rescued by CBL treatment (Fig. 6f ). Next analysis of Rockenstein et al. BMC Neurosci (2015) 16:85 Page 9 of 12 Fig. 6 Eec ff ts of CBL treatment in behavioral alterations in 3R Tau transgenic mice. Mice were evaluated for context-dependent learning in an open field area using a Kinder SmartFrame Cage Rack Station activity monitor system. a, b Levels of total activity and rearing in the cage in the 3–6 month group. c Nesting behavior in the cage of the 3–6 month group. d, e Levels of total activity and rearing in the cage in the 6–9 month group. f Nesting behavior in the cage of the 6–9 month group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test memory and spatial learning was performed in the water age group some visual impairment is present that might maze. In the 3–6  month group compared to the non-tg account for the lack of effects of CBL in the hidden seg - mice the vehicle treated 3R tau tg mice displayed defi - ment of the platform test. cits in spatial learning acquisition in the hidden platform segment of the test (Fig.  7a). In contrast, CBL treatment Cerebrolysin effects on 3RTau phosphorylation might be prevented these deficits in the 3R tau tg mice (Fig.  7a). mediated via Akt/GKS3β Memory retention in the probe test showed that com- We have previously shown that CBL rescues the hyper- pared to non-tg mice the vehicle treated 3R tau tg mice activation of GSK3β in other transgenic models [15, 17], expended less time in the target quadrant (Fig. 7b), treat- in order to examine the mechanisms through which CBL ment with CBL rescued this deficit in the 3R tau tg mice might reduce the hyper-phosphorylation of 3R Tau and (Fig.  7b), which performed similar to the non-tg control ameliorate the neurodegenerative pathology we analyzed groups (Fig.  7b). Visual success was comparable among levels of GSK3β and Akt by immunoblot. We found that the four groups of mice for the 3–6 month group (Fig. 7c). there was increased activation of p-GSK3β (Y216) in the Next, analysis of memory and spatial learning was per- vehicle-treated 3R Tau tg mice compared to non-tg con- formed in the 6–9 month group. Compared to the non-tg trols Fig.  8a, b), and therefore the ratio of p-GSK3β to mice, the vehicle treated 3R tau tg mice displayed deficits t-GSK3β was higher (Fig. 8b). In contrast, treatment with in spatial learning acquisition in the hidden platform seg- CBL reduced p-GSK3β (Y216) and decreased the ratio ment of the test (Fig.  7d), CBL did not fully ameliorate of p-GSK3β to total (Fig.  8a, b). Next we analyzed Akt these deficits (Fig.  7d). Memory retention in the probe and found that there was decreased activation of p-Akt test showed that compared to non-tg mice the vehicle (Ser473) in the vehicle treated 3R Tau tg mice compared treated 3R tau tg mice expended less time in the target to non-tg controls (Fig.  8a, c), and therefore the ratio of quadrant (Fig. 7e), however, here CBL treatment amelio- p-Akt to total was lower (Fig.  8c). Treatment with CBL rated the deficits in the 3R tau tg mice (Fig.  7e), which increased p-Akt (Ser473) and increased the ratio of p-Akt performed similar to the non-tg control groups (Fig. 7e). to total (Fig. 8a, c). Therefore, the neuroprotective effects Visual success score was higher in the non-tg compared of CBL in the 3R Tau tg mice might be related to regula- to the 3R tau tg mice (Fig. 7f ), indicating that in this older tion of the Akt/GSK3β axis. Rockenstein et al. BMC Neurosci (2015) 16:85 Page 10 of 12 Fig. 7 Eec ff ts of CBL on learning memory in the water maze in 3R Tau tg mice. a Water maze testing presented as distance traveled to find the platform, respectively in the 3–6 month group. During the cued portion of the test both the vehicle and CBL treated non-tg and 3R tau tg mice performed as expected. However during the hidden portion of the test, the vehicle treated 3R tau tg mice went a farther distance to find the platform compared to the non-tg, CBL prevented these effects. b At day 8, during the probe portion of the test (with platform removed) memory retention was evaluated in the 3–6 month group. c In the 3–6 month group, probe test with the visual platform confirmed that no visual altera- tions were detected. d Water maze testing presented as distance traveled to find the platform in the 6–9 month group. During the cued portion of the test both the vehicle and CBL treated non-tg and 3R tau tg mice performed as expected. However during the hidden portion of the test, the vehicle treated 3R Tau tg mice went a farther distance to find the platform compared to the non-tg. e At day 8, during the probe portion of the test (with platform removed) memory retention was evaluated in the 6–9 month group. f In the 6–9 month group, probe test with the visual platform showed visual alterations in the 3R tau tg group. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to vehicle-treated tg mice control using one way ANOVA with Dunnet’s post hoc test Fig. 8 Western blot analysis of effects of CBL on levels of Akt/GSK3β in the 3R Tau transgenic mice. a Representative immunoblots of samples from the 6–9 month group analyzed with antibodies against p-GSK3β (Y216), t-GSK3β, t-Akt and p-Akt (Ser473). b, c Image analysis of the ration between p-GSK3β (Y216) and total and p-Akt (Ser473) and total respectively. For analysis, N = 10 non-tg and N = 10 3R Tau tg mice from each age and treatment group. *P < 0.05 when compared to non-tg control using one way ANOVA with Dunnet’s post hoc test. P < 0.05 when compared to tg control using one way ANOVA with Tukey post hoc test the neurodegenerative pathology and behavioral defi - Discussion cits, although levels of 3R tau were not affected by the In the present study, we investigated the neuropro- treatment. When CBL treatment occurred at a more tective versus therapeutic effects of CBL at rescuing advanced stage (6 month old mice) we still observed that pathology in mutant 3R Tau in tg mouse that models this neurotrophic peptide mixture was able to reduce the aspects of the pathogenesis in PiD. We found that early levels of p-tau and ameliorated the neurodegenerative adminstration of CBL (at 3  month of age, for 3  months) pathology, however only partially rescued the behavioral resulted in decreased p-tau and completely rescued Rockenstein et al. BMC Neurosci (2015) 16:85 Page 11 of 12 deficits. These results in the 3R tau tg mice are consist - oligomerization and fibril formation, mitigating the for - ent with previous studies showing that CBL rescues the mation of potentially neurotoxic tau species, and enhanc- neurodegenerative pathology associated with p-tau accu- ing degradation of pathological tau through increasing mulation. However in previous studies we investigated clearance mechanisms [37]. Finally, a new provocative the effects of CBL at rescuing pathology associated with approach is to reduce the accumulation and propagation mutant 4R tau over-expression (AAV-mediated) in APP of tau utilizing specific monoclonal antibodies against tg mice [16] or in crosses between mutant 4R tau tg and various tau species [38–41]. GSK3β tg mice [17]. In the bigenic mice, CBL treatment reduced the accumulation of p-Tau, ameliorated the neu- Conclusions rodegenerative pathology, decreased Drp-1 and p-Drp-1 In summary, in the present study we found that although expression, and returned mitochondria to characteristics the levels of total 3R tau were unchanged, CBL reduced comparable to non-tg mice [17]. the levels of hyper-phosphorylated tau in both groups of The precise mechanisms of action of CBL are unknown, mice. This was accompanied by reductions in the neuro - however the rescue effects of CBL were likely related degenerative pathology in both groups and by improve- its ability to regulate levels of p-tau rather than levels of ments in the behavioral deficits in the younger group. total tau (either 3R or 4R tau) [24]. This suggests that, These results suggest that CBL might be beneficial in at least in part, the neuroprotective and neurotrophic orphan disease taupathies such as PiD. effects of CBL might be associated with its ability to reg - ulate CDK5 and GSK3β activity. Both of these kinases Abbreviations have been previously shown to play an important role in 3R: three-repeat tau; 4R: four-repeat tau; AD: Alzheimer’s disease; APP: amyloid hyper-phosphorylating tau [25–27] and mediating the precursor protein; CBD: corticobasal degeneration; CBL: Cerebrolysin; GFAP: glial fibrillary acidic protein; PiD: Pick’s disease; PSP: progressive supranuclear neuropathology in models of AD and FTD [28, 29]. We palsy; tg: transgenic. have previously shown that the CBL neurotrophic pep- tide mixture is capable of reducing behavioral deficits Authors’ contributions ER designed the experimental plan, coordinated and conducted the animal in an APP tg mouse model of AD-like pathology [13] by studies. KU analyzed and reviewed the results and edited the manuscript. blocking CDK5 and GSK3β [16], resulting in decreased MM, JF conducted the animal studies and analyzed the data. AA performed APP maturation and Aβ biosynthesis [30], increased the immunohistochemistry. SW, HB edited manuscript and provided intel- lectual input on the experimental plan. DM provided intellecutal input on the neurogenesis [15] and synaptic formation [13]. Further experimental plan. EM provided intellecutal input on the experimental plan, supporting a role of this pathway a recent in  vitro study analyzed the immunohistochemistry and wrote manuscript. All authors read showed that CBL protects neuronal cell lines from the and approved the final manuscript. neurotoxic effects of CoCl via regulation of GSK3β acti- Author details vation [31]. Interestingly, tau hyper-phosphorylation in 1 Department of Neurosciences, University of California, La Jolla, San Diego, CA PiD [32, 33] as well as in our model [19] is likely associ- 92093-0624, USA. Clinical Research and Pharmacology, EVER Neuro Pharma GmbH, Unterach, Austria. Department of Pathology, University of California, ated with GSK3β. La Jolla, San Diego, CA, USA. Pick’s disease belongs to the FTLD-tau with Picks bodies as defined by the working group [34–36]. Pick’s Acknowledgements This work was partially funded by a grant from EVERPharma and NIH grant disease is a rare cause of dementia in the elderly, while AG18440. some anti-depressants such as selective serotonin reup- take inhibitors have been shown to ameliorate some of Competing interests Stefan Winter, Hemma Brandstaetter and Dieter Meier are employed by EVER the symptoms and obsessive–compulsive behaviors asso- Neuro Pharma GmbH, Unterach, Austria. None of the other authors have any ciated with PiD, there are currently no known disease- competing interests. modifying treatments available. 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Published: Nov 26, 2015

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