Methylene Blue (Tetramethylthionine Chloride) Influences the Mobility of Adult Neural Stem Cells: A Potentially Novel Therapeutic Mechanism of a Therapeutic Approach in the Treatment of Alzheimer’s Disease

Abstract

An interest in neurogenesis in the adult human brain as a relevant and targetable process has emerged as a potential treatment option for Alzheimer’s disease and other neurodegenerative conditions. The aim of this study was to investigate the effects of tetramethylthionine chloride (methylene blue, MB) on properties of adult murine neural stem cells. Based on recent clinical studies, MB has increasingly been discussed as a potential treatment for Alzheimer’s disease. While no differences in the proliferative capacity were identified, a general potential of MB in modulating the migratory capacity of adult neural stem cells was indicated in a cell mobility assay. To our knowledge, this is the first time that MB could be associated with neural mobility. The results of this study add insight to the spectrum of features of MB within the central nervous system and may be helpful for understanding the molecular mechanisms underlying a potential therapeutic effect of MB.

Keywords

Adult stem cells Alzheimer’s disease methylene blue neural plasticity

INTRODUCTION

First described by Alois Alzheimer in 1907 [1, 2], Alzheimer’s disease (AD) is nowadays considered one of the most social-economically relevant disorders in the western world. AD is the most common type of dementia (≈60%) and accounts for more than 35 million affected individuals worldwide [3]. The enormous financial demand on welfare funds and the suffering of patients and their familiesas well as the demographic change especially in western countries contribute to the urgent need for research and development of effective therapies for this progressive neurodegenerative condition.

Despite the vast number of AD patients, the causes of the disease and hence the specific targets for drugs against AD are largely unknown and remain to be elucidated. AD results in a significant atrophy especially in hippocampal and frontotemporal regions of the brain [4] and is thought to belong to the class of cerebral proteinopathies [5]. However, while extracellular plaques of amyloid-β [1, 2] and intracellular neurofibrillary tangles of hyperphosphorylated tau [6] are the most established histological hallmarks of AD, it is still controversial whether and to which extent these protein deposits contribute to disease pathogenesis [7]. Many studies indicate pathologies such as neuroinflammation [7], mitochondrial dysfunction [8] and oxidative damage [9] in addition to the above-mentioned abnormalities. Moreover, it remains unclear which of these alterations are causal and whether they result from one another.

To date treatments that specifically target amyloid-β and hyperphosphorylated tau have not achieved a striking success and established themselves in the medical care of affected individuals [5]. Currently available medical therapies address imbalances in the cholinergic or the glutamatergic neurotransmitter system within the central nervous system (CNS) [10]. However, these drugs can improve the symptoms of AD in a subset of affected patients at most but fail to impact the overall progression of AD [5].

More recently the interest in neurogenesis in the adult human brain has grown, since it constitutes a new approach to finding novel treatment options for neurodegenerative diseases. While highly controversial in the past, it is now accepted that adult neural stem cells (ANSC) located in certain regions of the adult mammalian brain are able to contribute to the de novo formation of neurons and other cells of the neural lineage [11]. These regions of the adult mammalian brain containing adult neural stem cells include the subgranular zone of the dental gyrus of the hippocampus as well as the subventricular zone along the lateral ventricles [12]. Although neurogenic processes in humans appear to be limited in comparison to other mammals [12, 13], alterations in adult neurogenesis are increasingly being discussed in the context of the cognitive decline that occurs during the regular aging process and as a possible contributor to a number of neuropsychiatric disorders including AD [14, 15].

The aim of this study was to investigate a possible influence of tetramethylthionine chloride (methylthionium, methylene blue, MB, C₁₆H₁₈ClN₃S, CAS 61-73-4) on adult neurogenesis in vitro.

Originally developed as a dye in 1876 [16], MB soon established itself as a treatment option in a number of medical conditions including methemoglobinemia, vasoplegia, and ifosfamide-induced encephalopathy [17]. MB furthermore served as the template for the development of the pharmacologic class of phenothiazines, which are still being used as antihistaminic and neuroleptic drugs today [18]. Due to its low toxicity [19] and its permeability through cellular membranes and the blood-brain barrier, MB is an attractive therapeutic agent [16]. MB has a high affinity to neural tissue [18] and shows a pronounced accumulation in certain neural cell types including intrinsic interneurons and other cells within the murine hippocampus [20].

MB first raised public interest as a potential medication for AD in 2008, when Claude Wischik presented promising results of a clinical study at the annual meeting of the Alzheimer’s Society in Chicago [21]. This initial study as well as a recently published multicentered, dose-finding trial of MB in 321 patients indicates that mild to moderate forms of AD can benefit from a daily application of MB [22, 23]. 24 weeks after a daily treatment with either placebo, 69 mg MB, 138 mg MB, or 228 mg MB the patients in the subgroup with 138 mg MB per day showed a statistically significant treatment effect, which resulted in better scores in clinical evaluation scales in patients with moderate disease (treatment effect: –5.42 units, corrected p = 0.047). Participants with mild AD and a daily treatment with 138 mg MB initially only showed significant improvements in the regional cerebral blood flow measure (treatment effect: 1.97%, corrected p < 0.001) but after continuing treatment for a total of 50 weeks statistically significant improvements in the clinical evaluation scale (ADAS-cog) were evident in the group of mildly affected patients as well [23]. Moreover, very recently a derivate of MB— leuco-methylthionium-bis (hydromethanesulfonate – LTM; TRx-0237)—was assessed in a phase III trial in mild to moderate AD. LMTM is a stabilized reduced form of the methylthionium moiety. Patients were treated for 15 months with 150 or 250 mg LMTM per day or placebo in a placebo-controlled trial [24].

In the 2015 study, the authors attribute the apparent benefits of MB mainly to an effect of the drug on aggregates of tau [23]. In fact, the observation that pharmacological relatives of MB (azur A/B, tolonium chloride) enhance the proteolytic cleavage of intracellular tangles of the tau protein in vitro [25] first initiated the hypothesis that MB could potentially be effective in the medical treatment of AD in vivo where tangles of hyperphosphorylated tau constitute a significant proportion of histological pathologies. Many groups have further demonstrated various interactions between MB, MB-related substances, and the tau protein [26, 27].

It is important to note, however, that other studies also indicate additional target structures of MB, many of which are being discussed in the context of the pathogenesis of AD. These include amyloid-β [28] and acetylcholinesterase [29] and thus they constitute additional potential mechanisms of the therapeutic effects of MB.

The approach of this study was to analyze key features of mANSC (murine adult neural stem cells) as an in vitro model for neurogenesis and to investigate whether MB has a potential impact on these properties in addition to its known effects. One of the key features of stem cells (including, but not limited to (m)ANSC) is their proliferative capacity which was evaluated using short-term (24 h) as well as long-term (28 days) proliferation assays. Another key feature of (m)ANSC is a directed migration in vivo by which newly generated cells travel from circumscribed neurogenic niches into other regions of the adult brain. This feature was evaluated in vitro using the Boyden Chamber mobility assay. Furthermore, the expression levels of genes of interest were evaluated by means of quantitative real-time PCR (polymerase chain reaction). Four enzymes and signaling molecules that are presumably involved in pathogenetic processes underlying AD were selected as genes of interest to evaluate a possible impact of MB in this context. Relative expressions of the beta-site APP cleaving enzyme 1 (Bace 1) and Synaptophysin (Syp) served as indicators for two of the most discussed pathogenic mechanisms in AD: the production of amyloid-β [30]and impairments of synaptic plasticity [31]. Furthermore, expression levels of two neurogenesis-associated genes, brain-derived neurotrophic factor (BDNF) [32] and tryptophan hydroxylase 2 (TPH2) [33] were evaluated. BDNF is of special interest in this respect, since BDNF acts on adult neurogenesis to facilitate memory consolidation, a core memory function impaired in AD [34, 35].

METHODS

Establishment of primary ANSC cultures

Animals were treated according to NIH (National Institutes of Health Guide for the Care and Use of Laboratory Animals) equivalent animal care rules. The study was carried out in accordance with the Animal Welfare Act. Adult wild-type mice (Charles River Italia, 14 weeks old) were anesthetized by intraperitoneal injection of pentobarbital (120 mg/kg) and sacrificed by cervical dislocation. Brains were removed and placed in chilled phosphate-buffered saline (PBS). The dentate gyrus of the hippocampi was carefully removed and put in digestion solution [EBSS (Earle’s balanced salt solution) containing 0.94 mg/ml papain (Worthington Biochemicals)], 0.2 mg/ml cysteine and ethylenediaminetetraacetic acid (EDTA; both from Sigma-Aldrich) for 50 min at 37°C under gentle rocking. After digestion, tissues were washed twice in Dulbecco’s modified eagle medium (DMEM, Gibco Life), mechanically dissociated using a fire-polished Pasteur pipette, and finally placed in serum-free DMEM/F12 (1:1 v/v; Gibco Life) containing 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml fibroblast growth factor (FGF-2) (both human recombinant; Peprotech), 2 mM L- glutamine, 0.6% glucose, 9.6 μg/ml putrescine, 6.3 μg/ml progesterone, 5.2 ng/ml sodium selenite, 0.025 mg/ml insulin, 0.1 mg/ml transferrin, and 0.2 μg/ ml heparin (all Sigma) (growth medium) at a density of 20,000 cells/ ml onto sterile, non-coated Petri dishes (Corning).

Cell culturing and propagation

Hippocampal stem cells from the dentate gyrus were serially subcultured by mechanical dissociation every 4–7 days. Cells were collected as neurospheres and the total number of viable cells was assessed at each passage by trypan blue (Sigma) exclusion on a cell counter (Beckman Instrument Particle count & Size Analyzer). Primary neurospheres consistently gave rise to continuously expanding cultures, which were used for long-term proliferation assessment. Following evaluation of ANSC counts, cells were always replated at the same density (200,000 cells). By taking into account the previous cell number and the amount of plated cells over a period of 28 days, we were subsequently able to establish growth curves and compare the proliferation rates of ANSC in different culture media (control and MB-containing (100 nM) culture media). For growth curves each experiment was performed at least three times per passage. Data obtained from each experiment represent triplicates. For further experiments (migration, short-term proliferation, quantitative PCR), ANSC were collected 5 days after the last subculturingpassage.

Cell culture treatments with methylene blue

ANSC were treated with methylene blue (tetramethylthionine chloride, C₁₆H₁₈ClN₃S, CAS 61-73-4). In preliminary experiments, the appropriate concentration of MB was determined by toxicity assays using Alamar blue^® (Invitrogen) in order to exclude cytotoxic effects, which could be mistaken for the results of survival or proliferation. As the absorption wave length of Alamar blue^® closely resembles MB (≈600 nm) additional controls and outcomes of long-term proliferation studies were used to determine the ideal concentration of MB for the subsequent cell culture. 100 nM MB was the highest non-toxic concentration and therefore determined appropriate for further use in the ANSC cell culture. For assessing the impact of MB at the cellular level, pulverised methylene blue trihydrate was used (Sigma, CAS-Nr. 7220-79-3) due to the ethanol contained in liquid preparations, which was considered unfavorable for cell culture. MB was soluble in culture media and was freshly prepared for each experiment.

Mobility assay

For mobility experiments Boyden chambers were used. PVP (Polyvinylpyrrolidone)-free polycarbonate filters with 8 μm pores (Micron) were coated with Matrigel. DMEM (control), DMEM plus 100 nM MB, or regular growth medium as an internal standard containing EGF and FGF2 were placed in the lower chambers. Undifferentiated ANSCs with a regular cell diameter of ∼16 μm were used for the assays 24 h after the last subculturing passage. 50,000 cells were resuspended in 200 μL DMEM and then placed in the upper chambers, where they were incubated for 6 h at 37°C in 5% CO₂. ANSCs remaining on the upper surface of the filters were mechanically removed, while those that had migrated to the lower surface were fixed with ethanol, Giemsa stained, and counted at 400-fold magnification in 5 random fields per filter.

Immunocytochemistry/short-term proliferation assay

For short-term proliferation experiments 35,000 undifferentiated ANSC in growth medium plus 100 nM methylene blue or regular growth medium (control) were plated onto Matrigel-coated glass coverslips (Menzel Deckgläser). Cells were incubated at 37°C in 5% CO₂ for 24 h. After incubation, the cell suspension was largely removed and replaced with BrdU- (bromodeoxyuridine) and 5-flouro-2’desoxyuridin-containing labeling reagent (Amersham Cell Proliferation Biotrak ELISA System). After 60 min of incubation at 37°C in 5% CO₂ the media were entirely removed and glass coverslips were washed with PBS. Remaining cells were fixed (4% paraformaldehyde in PBS, pH 7.4, for 20 min). Freeze-dried nuclease (Amersham Cell Proliferation Biotrak ELISA System) and Anti-BrdU were added. DNA-digestion and binding of anti-BrdU were allowed within the following 60 min. After washing with PBS, Cy3-labeled anti-BrdU-antibody (Cy3-goat-anti-mouse, whole IgG H + L, Dianova) was added (1:1000, 30 min at room temperature) for selective visualization of proliferating cells. All cell nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole, 1:1000 dilution of stock solution, 10 min at room temperature) in order to assess the total number of cells independently of their proliferation status. Samples were examined and photographed using a fluorescence microscope at 200-fold magnification. Immunoreactive cells were counted in at least 5 non-overlapping fields in each sample and expressed as a percentage of the total number of nuclei. Fluorescent signals from single optical sections were sequentially acquired and analyzed by Photoshop CC(Adobe).

Reverse transcriptase-PCR (RT-PCR)

Total RNA from ANSC was extracted using the RNeasy mini kit (Qiagen). cDNA was obtained using Superscript II RNA transcriptase (15 U/μl,Invitrogen) and random hexamer primers (Invitrogen). Reverse-transcriptase PCR was performed using a mastercycler thermocycler (Eppendorf). The thermal conditions were: 1 cycle, 10 min at 25°C (room temperature); 1 cycle, 50 min at 42°C; 15 min at 70°C and subsequent cool down to 4°C.

Semi-quantitative PCR (q-PCR)

Semi-quantitative real-time PCR was performed using a Rotorgene 2000 real-time PCR cycler (Qiagen) in the presence of SYBR Green (Invitrogen). Each PCR reaction (25 μl total volume) contained 10 μl of cDNA template, 12.5 μl of SYBR Green Taq DNA polymerase (Invitrogen) and 1 μl of gene-specific forward and reverse primers. Primers for the genes of interest (Bace1, BDNF, Syp, and Tph2) were designed using the Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3). Selection criteria included: product size range: 50–150 bp, primer size: 18–22 bp, primer melting temperature (T_m): 59–60°C, primer GC% : 40–60%, max. 3’ self-complementary: 3. Primer sequences were subsequently screened for gene-specificity using the basic local alignment search tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Primer sequences were as follows: Bace1-fwd5’-TCG CTT GCC CAA GAA AGT AT, Bace1-rev 3’-CTG CTC CCC TAG CCA AAA G; BDNF-fwd 5’-CCC ATG AAA GAA GTA AAC GTC C, BDNF-rev 3’-GTC GCT AGA CCT CTC GAA CC, Syp-fwd 5’-CAA GGA GAT GCC TAT GTG CC, Syp-rev 3’-AAC ACG AAC CAT AGG TTG CC, Tph2-fwd 5’-AAT TGA AGC ATG CTC TTT CC, Tph2-rev 3’-TTC AAA ACT GTC CGA AAC AAA.

Primer sequences for housekeeping-genes (hypoxanthine phosphoribosyltransferase (Hprt), acidic ribosomal phosphoprotein PO (Arbp), TATA-box binding protein (Tbp), and Peptidylprolyl Isomerase A (Ppia)) were as follows: Hprt-fwd 5’-CTC ATG GAC TGA TTA TGG ACA GGA C, Hprt-rev 3’-GCA GGT CAG CAA AGA ACT TAT AGC C; Arbp-fwd 5’-GAA AAT CTC CAG AGG CAC CAT TG, Arbp-rev 3’-TCC CAC CTT GTC TCC AGT CTT TAT C; Tbp-fwd 5’-CCC TGA ATC TTG GCT GTA AAC TTG, Tbp-rev 3’-GTT GTC CGT GGC TCT CTT ATT CTC; Ppia-fwd 5’-TAT CTG CAC TGC TAA GAC TGA ATG, Ppia-rev 3’-CTT CTT GCT GGT CTT GCC ATT CC.

The thermal conditions were: 1 cycle, 15 min at 95°C; 35 cycles of 30 s at 95°C, 30 s at 60°C. During the first cycle of the protocol the separation of double stranded DNA (melting step) was performed at 60°C for 10 s. The temperature during this step was gradually decreased by 0.5°C for every subsequent cycle of the PCR protocol in order to gain a melting curve. To confirm amplification specificity the resulting PCR products were subjected to a melting curve analysis. PCR runs without cDNA served as negative controls. Stability of housekeeping genes was assessed by “qBase+-software”. According to the results of the statistical analysis, hypoxanthine phosphoribosyltransferase (Hprt), acidic ribosomal phosphoprotein PO (Arbp) and TATA-box binding protein (Tbp) were considered sufficiently stable and subsequently used to normalize each template (“qBase+”).

Statistical analysis

Given the Gaussian distribution of the data, experimental data are reported as mean±standard deviation (SD). Differences between means of MB and control groups were determined by unpaired Student’s t-test, with p < 0.05 being considered significant. Migration assay data were analyzed statistically by ANOVA following Student’s t-test.

RESULTS

We studied the impact of MB on proliferation, migration, and expression of selected genes of adult neural stem cells derived from mouse hippocampus. In our in vitro model, only ANSCs and highly undifferentiated progenitors proliferate while the population of committed precursors and terminally differentiated cells progressively decreases, thus allowing the establishment of long-term expanding NSC lines that maintain stable proliferation and multipotency, i.e., the ability to give rise to astrocytes, oligodendrocytes, and neurons.

Proliferation outcome

To compare the proliferative capacity of adult neural stem cells in MB-containing culture media versus control, long-term proliferation was assessed for a period of one month. Both control and MB-treated (100 nM) cell populations displayed parallel growth curves indicating no statistically significant difference (p = 0.99) in long-term proliferation between groups throughout the observation period (Fig. 1A). Comparably, short-term proliferation as evidenced by the incorporation of the thymidine-analog BrdU (bromodeoxyuridine) after 24 h did not reveal a statistically significant difference (p = 0.067) between the two groups (Fig. 1B).

Migration outcome

We further investigated the potential effect of MB on another ANSC functional property, their migratory activity (Fig. 2). Migration assays using Boyden chambers revealed that exposure of ANSC to 100 nM MB for a period of 6 h resulted in an approximately 7-fold increase in the number of migrated cells (37.27±15.15) in comparison to ANSC grown in DMEM (control, 5.12±5.7). Media enriched with growth factors (EGF and FGF2) served as a positive control/internal standard. Outcomes of migration assays were analyzed using analysis of variance (ANOVA). Observed differences in migratory activity were statistically significant (p < 0.05).

Gene expression analysis

As part of our investigations we examined the expression patterns in adult neural stem cells of four genes relevant to adult neurogenesis or the pathophysiology of AD, namely beta-site APP-cleaving enzyme 1 (Bace1), brain-derived neurotrophic factor (BDNF), Synaptophysin (Syp) and tryptophan hydroxylase 2 (Tph2). By analyzing expression levels of Bace1, a pivotal enzyme in amyloid metabolism [3] we sought insights into possible effects of MB on Amyloid-β metabolism. Moreover, Bace1 regulates the balance of hippocampal neurogenesis and astrogenesis, although this has to date been investigated only in early development and not in adult hippocampal neurogenesis [36]. Expression levels of Syp and BDNF provided information about a possible influence of MB on synaptogenesis and neural growth, respectively. Levels of Tph2 served to identify a possible impact of MB on the serotonergic system, a neurotransmitter system involved in adult neurogenesis [33, 37]. Interestingly, BDNF also functions as a serotonergic growth factor [38 –40].

Analysis of gene expression patterns (qBase +) did not reveal statistically significant differences between ANSC cultivated in MB-containing media (100 nM MB) versus media without MB (p > 0.05).

DISCUSSION

The results of this study suggest that MB at a concentration of 100 nM (as determined by the Alamar blue^® cell viability assay as well as subsequent growth curves) does not influence the proliferative capacities of murine neural progenitor cells. A general potential of MB in modulating the behavior of mANSC, however, is indicated by the mobility assay. Thus, mANSC display a 7-fold increase in migratory activity toward MB in comparison to media without MB. The affinity of mANSC to MB corresponded to 47.5% of the migratory activity that could be measured toward a positive control (growth-factor containing, internal standard). The results of this study therefore indicate a significant influence of MB on neuroplastic properties of mANSC, in this case cell mobility.

In general, the term neuroplasticity summarizes synaptic and non-synaptic processes that enable a dynamic modification of neural structures and pathways in response to internal and external stimuli [41]. This includes the generation of new neurons (i.e., neurogenesis), neural migration, and transformation of existing synaptic connections.

Our understanding of plastic changes within the adult CNS has increased considerably during the past decades [11]. Migratory processes within the adult brain are nowadays considered to be necessary for the maintenance and modulation of existing neurological circuits [42]. Migratory movements of progenitor cells may thereby contribute to the life-long functional plasticity of some areas of the adult brain such as the hippocampus. This is of course of particular relevance for AD, given the well-known role of the hippocampus in the pathophysiology of AD.

There is evidence that the characteristics of newly generated neurons and the features of directed migration in the adult CNS change with aging [43, 44]. These findings have led to an ongoing discussion regarding a possible relevance of deficits in adult neurogenesis or neuroplasticity for aging and the pathogenesis of neurodegenerative diseases.

A number of studies indicate that molecules believed to play a role in the pathogenesis of familial AD have the ability to influence neurogenic processes of mice. Knock-out mouse models of the AD-predisposing genes ApoE and PS1 show an increased proliferative rate of neural precursors in the murine dentate gyrus [45] and an increase in differentiation toward neural cells [46], respectively. While the soluble component of the amyloid-βprotein precursor alpha (sAβPPα) appears to promote neurogenic activity [47], an overexpression of the AβPP intracellular domain (AICD) decreases both proliferative activity and survival of hippocampal precursor cells [48].

In the same way that impairments in adult neurogenesis may contribute to the pathogenesis of AD, there is increasing evidence that non-pharmacological treatments may also achieve their beneficial effects by influencing adult neurogenesis. Physical activity, which is generally recommended as supporting treatment in AD, has been shown to positively regulate adult neurogenesis [15]. Moreover, in genetic mouse models with AD, physical activity results in an improvement of cognitive capacities [49]. Furthermore, a complex and stimulating environment leads to reduced levels of amyloid-β reduced accumulation of amyloid deposits, and restoration of impaired neurogenesis in mice [50].

The increasing knowledge about directed migration of neural precursors has raised hopes for a potential therapeutic use of these mechanisms in degenerative or traumatic conditions of the CNS. Thus, in 2002, Magavi et al. [51] and Nakatomi et al. [52] observed that limited neural destruction and hypoxia were able to induce neurogenesis and the replacement of damaged neural cells in the adult cerebral cortex of rodents. It was not possible to determine from which brain area these newly generated cells originally arose before migrating into the damaged areas or to which extent they were able to functionally integrate into the preexisting networks [14]. However, these findings support the idea that the localized, short-distance migration of single neural progenitor cells is possible and may represent a promising therapeutic target in the future [42]. In this respect, our findings on the increased migration induced by MB are of particular interest.

Further to our findings, the question arises regarding the molecular mechanisms of the pro-migratory effect of MB. Many of the identified properties of MB appear to be closely associated with the specific physico-chemical characteristics of MB such as redox potential, charge and light-absorption and excitation [16]. MB targets many structures within the CNS as well as outside of the CNS. The peripheral structures that are targeted by MB include nitric oxide (NO)-synthase [53], methemoglobin and glutathione reductase [54]. However, due to its high permeability through the blood-brain barrier and its affinity for neural tissue, MB also influences neural tissue homeostasis in many other ways. MB targets multiple signaling processes by influencing neurotransmitter systems in the peripheral and CNS including the cholinergic [55] and the glutamatergic system [56]. Furthermore, MB affects the properties of a number of current-gated ion channels [57 –59].

We have attempted to dissect the molecular mechanisms by which MB achieves the observed increase in migratory activity of ANSC. However, the expression patterns of the four genes selected for relevance to adult neurogenesis or the pathophysiology of AD were not altered in adult neural stem cells exposed to MB. Further insights may be gained by the investigations by Werner et al. [60], who demonstrated an influence of MB on the migratory behavior of hematologic cells. Given the therapeutic use of MB in post-surgical vasoplegic syndrome, Werner et al. [60] investigated the influence of MB on the transendothelial migration of peripheral blood cells. According to their results, MB is able to affect the transmigration of certain peripheral blood cells through endothelial monolayers in a dose-dependent manner in vitro (60 μM and above) [60]. The authors suggest an influence of MB on NO-synthase as the main mechanism of the observed changes in transmigratory activity [60]. NO is a signaling molecule which has been associated with the adhesion and migration of leucocytes [61, 62]. NO has also been linked to angiogenesis and other transmigration processes [63]. It remains unclear, however, if or to which extent the increase in migratory activity of mANSCs shows any molecular similarities to the mechanisms proposed by Werner et al. [60]. Limitations regarding the comparability of both studies include the use of different cell types (neural precursors versus hematologic cells), significantly different concentrations of MB (100 nM versus 60 μM) and different assays for assessment of the cellular migration in vitro.

It is a limitation of this study that the mechanisms of action remain to be clarified. Given the link between MB and tau [23, 25] or amyloid-β [28], future studies should evaluate changes in biomarkers in the cell culture media and relate them to the degree of ANSC mobility. Still, the results of this study add to the spectrum of effects of MB which may be relevant for the therapy of AD patients with MB.

DISCLOSURE STATEMENT

Authors’ disclosures available online (http://j-alz.com/manuscript-disclosures/16-0755r2).

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