Microtubule defects in mesenchymal stromal cells distinguish patients with Progressive Supranuclear Palsy

Abstract Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP‐linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti‐inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease‐linked defect occurs in this non‐neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α‐tubulin post‐translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non‐neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.

involves the basal ganglia, the prefrontal cortex and the cerebellum, with accumulation of tau protein, hence the classification as tauopathy. 5 Tau is a MT-binding protein encoded by the MAPT gene into 6 isoforms that are commonly referred to as 3R or 4R (with 3 or 4 MT-binding domains, respectively). Tau binds to and stabilizes MTs, and promotes MT polymerization. 6 The binding to MTs is regulated by phosphorylation of many residues; indeed, when hyperphosphorylated, tau detaches from MTs and accumulates forming neurofibrillary tangles (NFTs). All tauopathies are characterized by the presence of aggregates of abnormally phosphorylated tau protein, although the isoforms that aggregate vary. 7 Both hyperphosphorylation and accumulation of 4R tau protein in neurons and glia, in basal ganglia and in the brain stem, are characteristic features of PSP. 8 In PSP, the abnormal phosphorylation of tau triggers its detachment from MTs, mislocalization from the axon to dendrites and accumulation of still-soluble "oligomers." 9 MTs are cytoskeletal polymers built up by a/b tubulin heterodimers, which participate in many cellular functions, such as maintenance of cell shape, cell migration and intracellular transport. MTs show a dynamic behaviour, switching between slow growth and rapid depolymerization 10 and are finely regulated by the incorporation of specific a/b tubulin isotypes, by a plethora of MT-binding proteins and by tubulin post-translational modifications (PTMs). 11,12 Notably, a-tubulin PTMs have been correlated with different MT subsets: tyrosinated MTs are the most dynamic ones, whereas acetylated or detyrosinated MTs are associated with more stable pools.
The wide range of PTMs might, alone or in combination, generate chemical differences that are sufficient to confer cellular functions on MTs. Tubulin PTMs have important roles in regulating not only MT dynamics, but also motor traffic. Interestingly, defects in MTbased transport in neurons, which are often linked to the accumulation of aggregated proteins, are typical of many neurodegenerative disorders, including Alzheimer's 13 and Parkinson's (PD) diseases. 14 In addition, it has been shown that MT stability and PTMs of tubulin are impaired in human fibroblasts derived from patients with PD. 15 For PSP, there are currently no effective symptomatic or disease-modifying treatments. In the last years, few clinical trials targeting mitochondria dysfunction, tau aggregation or MT stability have been performed or are ongoing. 16 Besides other promising drugs, davunetide, which promotes MT stability, was effective as neuroprotective agent in a mouse model of tauopathy 17 but it failed in a phase 2/3 clinical trial on patients with PSP, 18 while TPI-287, another MT stabilizer molecule, has recently entered a phase 1 clinical trial (Trial registration: ClinicalTrials.gov identifier NCT02133846).
Among the ongoing trials, a therapy based on transplantation of undifferentiated human bone marrow MSCs has been proposed.
MSCs are multipotent cells that can be isolated from many sources and whose therapeutic relevance is mostly due to their immunosuppressive and anti-inflammatory properties. 19,20 Interestingly, beneficial effects of intravenous delivery of MSCs have been reported in rotenone-treated mice, a PD model. 21 Starting from encouraging pre-clinical data, where MSCs show the ability to in vitro rescue 6hydroxydopamine-damaged neural cell lines and to synthesize and secrete neurotrophines, 22 we moved to a first pilot phase 1 study. In this trial, we had the dual aim to assess the safety of MSC therapy in a "first-in-man" context and the efficacy of autologous MSC treatment. Five patients have been treated in the open phase of our trial and at the end of this first step, we demonstrated the feasibility of autologous MSC administration in subjects with PSP and we recorded a clinical stabilization for at least 6 months (Trial registration ClinicalTrials.gov NCT01824121). 23 To understand the real potential of patient-derived MSCs, we performed in-depth investigation of their biology. Specifically, we characterized the MT cytoskeleton of MSCs from patients affected by PSP, highlighting their characteristics in terms of MT stability and imbalance in a-tubulin PTMs.

| Diagnostic criteria for PSP diagnosis
The criteria used for the diagnosis of PSP followed in this study are as follows: 1-diagnosis of "probable Progressive Supranuclear Palsy-Richardson's disease subtype" according to current diagnostic criteria, 2,24,25 including akinetic-rigid syndrome: gradually progressive disorder with age at onset of 40 years or later, vertical supranuclear palsy and prominent postural instability with falls within first year of disease onset; 2-positive MRI for PSP criteria 26 ; 3-lack of response to chronic levodopa (at least 12-month treatment).

| Cell culture, subculture and cumulative population doublings
MSCs were obtained as previously reported in. 22 Briefly, bone marrow was obtained after informed consent from aspiration of iliac crest and directly seeded in alpha-modified Eagle's medium (alpha-MEM; Macopharma, Mouvaux, France) supplemented with 10% high-quality gamma-irradiated foetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), at the concentration of 50 000 white blood cell (WBC)/cm 2 , at 37°C in a humidified atmosphere, 5% CO 2 . After 72 hours, non-adherent cells were removed by washing with PBS (Macopharma) with complete medium change.
On day 14, MSCs at P0 were detached using 0.04 mL/cm 2 of Try-

| Morphometric analysis
For morphometric analyses, 10 random images per well were captured using a Nikon Eclipse Ti-S microscope (Nikon, Chiyoda, Japan), and analyses were made using ImageJ software (National Institute of Health, Bethesda, MD, USA) as previously described. 15 For each cell, the maximum and minimum axes and the cell area were measured.
The ratio between the maximum and minimum axis has been calculated. Only intact cells fully present into the image were considered.   antibodies for 1 hour at room temperature was performed using the  (Table S1 and Figure S1). After that, we investigated the

| DISCUSSION
PSP is a rare neurodegenerative disorder that affects various areas of the brain, including basal ganglia, brainstem, cerebral cortex, dentate nucleus and spinal cord regions, and that can affect not only neurons, but also glial cells. 30,31 The mechanism(s) that can lead to PSP are not yet understood, as well as its causes, that include both environmental  Notably, specific properties of MSCs cells include the secretion of a set of factors/molecules to the extracellular space, the so-called secretome, whose crucial role in anti-inflammatory response makes them a promising therapeutic tool. 46 Here, our detailed analysis of MSCs of patients with PSP unravels MT defects, reduced proliferative capacity and predisposition to ageing in culture conditions.
Whether these defects can affect their secretory profile and therefore to their paracrine reparative effects is still under investigation, but the clinical use of early passage MSCs is strongly recommended when autologous MSCs are tested in phase 1/2 clinical trials. Notably, also in the clinical trial performed by our group based on the autologous use of MSCs in patients with PSP, the administered cellular products were composed by maximum passage 2 MSCs. 22 Peripheral tissues are a source of human living cells, and in the last few years, they have become reliable models for the identification of molecular alterations and possible therapeutic targets in neurodegenerative disorders. For example, skin primary fibroblasts are considered a good model system for PD 47 and, interestingly, we have previously reported that fibroblasts of patients with PD are characterized by changes in MT mass compared to controls. 15 Focusing on MSCs, they are relatively easy to obtain, to maintain and to expand in culture. The relevance of studying this cell type for unravelling defects linked to neuronal disorders is completely unexplored.
For this reason, our detailed analysis of MT system is a starting point for moving to neuronal models obtained from patients that are actually not available (eg iPSC-derived neurons). Beyond being a potential model for studying PSP-linked dysfunction, MCSc could be used for drug screening. Indeed, Polioudaki et al 48 showed that taxol and nocodazole, 2 well-known MT-interacting drugs, can induce moderate and reversible damage to MSCs of healthy donors. As we found that control and PSP-derived MSCs are characterized by differences in MT system, it will be intriguing to evaluate if they differently respond to anti-MT drugs. Thus, MSCs are also a very precious and promising tool for personalization of drug screening and therapies.
In conclusion, for the first time, our study unravels the characteristics of MT cytoskeleton in MSCs from patients affected by PSP, a rare neurodegenerative disorder, and demonstrates that these cells differ from healthy controls in terms of MT stability, a-tubulin PTMs, cell morphology and growth. This is in accordance with a very recent study by a part of our group showing that mitochondrial dysfunction occurs in MSCs from patients with PSP. 49 This suggests that the mechanisms leading to PSP might also affect undifferentiated nonneural cells. Our results pave the way to the experimental use of alternative cellular models as in vitro system for deciphering the intracellular mechanisms of PSP and identifying novel pharmacological targets, thus ultimately helping in finding new therapeutic approaches to PSP as well as other still orphan neurologic diseases.

ACKNOWLEDG EMENTS
The authors thank all the patients and healthy individuals who participated to this study. The authors are grateful to Dr. Jennifer S.