Bone marrow transplantation improves motor activity in a mouse … · 2020. 2. 11. · Bone marrow transplantation improves motor activity in a mouse model of ataxia David Díaz 1,

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/term.2626

This article is protected by copyright. All rights reserved.

Bone marrow transplantation improves motor activity in a mouse model of

ataxia

David Díaz 1, 2*, Marina Piquer-Gil 3*, Javier Sánchez Recio 1, María Magdalena Martínez-Losa3, José

Ramón Alonso 1, 2, 4, Eduardo Weruaga 1, 2#, Manuel Álvarez-Dolado 4#

1 Laboratory of Neuronal Plasticity and Neurorepair, Institute for Neuroscience of Castile and

León (INCyL), Universidad de Salamanca, Salamanca, Spain.

2 Institute of Biomedical Research of Salamanca, IBSAL. Salamanca, Spain.

3 Laboratory of Cell Therapy for Neuropathologies, Andalusian Center for Molecular Biology and

Regenerative Medicine (CABIMER), Seville, Spain.

4 Instituto de Alta Investigación, Universidad de Tarapacá, Arica, Chile.

* and # These authors contributed equally.

Corresponding authors

Dr. Eduardo Weruaga, INCyL, Universidad de Salamanca, C/ Pintor Fernando Gallego 1, E-

37007, Salamanca, Spain; phone: +34 923294500 ext. 5324; fax: + 34 923294750; E-mail:

[email protected]

Dr. Manuel Álvarez-Dolado, CABIMER, Avda. Americo Vespucio s/n. Parque Científico y

Tecnológico Cartuja, E-41092, Sevilla, Spain; phone: +34 954468004; E-mail:

[email protected]

Abbreviated Title: Bone marrow stem cells for ataxia

Keywords: ataxia, bone marrow transplantation, cerebellum, muscle, PCD mouse, stem cells.

Abstract word count: 243 Manuscript word count: 5250

Figures: 6 (+ 3 supplementary)

Conflict of interest: The authors declare no competing financial interest.

Financial support: This work was supported by the Fondo de Investigaciones Sanitarias (FIS

04/2744), the Ministerio de Ciencia e Innovación (BFU2010-18284), the Ministerio de Economía

y Competitividad (SAF2013-41175R), the Generalitat Valenciana (ACOMP06/131), the Junta de

Castilla y León, the Junta de Andalucía (PI-0736-2010), the Centre for Regenerative Medicine and

Cell Therapy of Castilla y León, the Samuel Solórzano, FOLTRA, MMA, Colectivo Ataxias en

Movimiento, the Alicia Koplowitz Foundation, and the University of Salamanca.

mailto:[email protected]

mailto:[email protected]


Abstract

Ataxias are locomotor disorders that can have an origin both neural and muscular, although

both impairments are related. Unfortunately, ataxia has no cure and the current therapies are

aimed at motor re-education or muscular reinforcement. Nevertheless, cell therapy is becoming

a promising approach to deal with incurable neural diseases, including neuro-muscular ataxias.

Here we have used a model of ataxia, the PCD mutant mouse, to study the effect of healthy

(wild-type) bone marrow transplantation on the restoration of defective mobility. Bone marrow

transplants (from both mutant and healthy donors) were performed in wild-type and PCD mice.

Then, a wide battery of behavioral tests was employed to determine possible motor

amelioration in mutants. Finally, cerebellum, spinal cord and muscle were analyzed to study

the integration of the transplant-derived cells and the origin of the behavioral changes. Our

results demonstrated that the transplant of wild-type bone marrow restores the mobility of PCD

mice, increasing their capabilities of movement (52-100% of recovery), exploration (20-71%

of recovery), speed (35% of recovery), and motor coordination (25% of recovery).

Surprisingly, our results showed that bone marrow transplant notably improves the skeletal

muscle structure, which is severely damaged in the mutants, rather than ameliorating the central

nervous system. Although a multimodal effect of the transplant is not discarded, muscular

improvements appear to be the basis of this motor recovery. Furthermore, the results from our

study indicate that bone marrow stem cell therapy can be a safe and effective alternative for

dealing with movement disorders such as ataxias.

Abbreviations

Ccp1/CCP1 cytosolic carboxypeptidase 1

GFP green fluorescent protein

IGF1 insulin-like growth factor 1

IMDM Iscove´s Modified Dulbecco´s Medium

PBS phosphate-buffered saline

PCD Purkinje Cell Degeneration


Introduction

Ataxias are locomotor disorders characterized by an abnormal gait, including trembling

movements and/or loss of directionality (Koeppen, 1998). Cerebellar and spinocerebellar

ataxias are of neural origin and usually involve the death of Purkinje cells or, at least,

dysfunctions in their physiology (Butterworth, 1993; Zoghbi, 1995; Koeppen, 1998; Vanier,

2010). Likewise, ataxic symptoms can also be due to muscle impairments, where cerebellar

ataxias cause secondary muscle atrophy due to impaired mobility, which can in turn exacerbate

the patient’s gait problems (Milne et al., 2016).

Unfortunately, most ataxias have no effective cure, and most treatments aim to palliate the

locomotor symptoms through motor re-education or muscular reinforcement (Milne et al.,

2016). Notwithstanding, cell-based therapies using different animal models of the disease have

provided interesting results regarding both cerebellar ataxias (Bae et al., 2005; Bae et al., 2007;

Chen et al., 2011; Jones et al., 2015) and muscular impairments (Ferrari et al., 1998; Gussoni

et al., 1999; Galli et al., 2014).

The use of bone marrow-derived stem cells is very suitable for cell therapy for several reasons:

they constitute a population that has been well characterized over a long period of time (Becker

et al., 1963); they integrate physiologically into several organs including both encephalon and

muscle (Mezey et al., 2000; Álvarez-Dolado et al., 2003; Mezey, 2005; Nern et al., 2009; Fujita

et al., 2015); and their mechanisms of regeneration are diverse, ranging from direct

differentiation into neurons to cell fusion, or the secretion of trophic factors (Díaz et al., 2015).

These processes usually take place simultaneously during tissue regeneration after a bone

marrow stem cell transplant. Nonetheless, few detailed studies describe the exact mechanism

by which these stem cells improve the symptoms of ataxia.

Accordingly, over the past years, our group has experimented with Purkinje Cell Degeneration

(PCD) mutant mice, a model of cerebellar ataxia. These mice lack the expression of the Ccp1


gene (cytosolic carboxypeptidase 1), also known as Agtpbp1 or Nna1 (Harris et al., 2000;

Fernández-González et al., 2002; Berezniuk et al., 2012). Ccp1 is specifically expressed in cells

that degenerate in PCD animals, and has been shown to be related to axon growth, and more

precisely, to the dynamics of microtubules (Harris et al., 2000; Berezniuk et al., 2012) which

play a key role in the functioning of the cytoskeleton of neurons (Berezniuk et al., 2012). Little

is known about other associated impairments or cellular malfunctions that PCD mutants may

suffer because of the mutation. For instance, previous reports have suggested muscular

impairment to be involved with this phenotype (Carrascosa et al., 2004), but it is unclear if it

is a concomitant secondary atrophy derived from the cerebellar ataxia.

In parallel, the PCD results ideal for deciphering the mechanisms involved in a cell-based

therapy for ataxia, as it has previously allowed us to show the features of bone marrow-derived

cells integration into the brain (Recio et al., 2011). Furthermore, and interestingly, cell therapy

using stem cells from healthy bone marrow in the PCD reduced the degeneration of mitral cells

in the olfactory bulb, leading to a restoration of the olfaction (Díaz et al., 2012a), although such

therapy did not palliate Purkinje cell death (Recio et al., 2011). However, it has been reported

that PCD mice suffer other secondary impairments in both cerebellum (Triarhou et al., 1987;

Zhang et al., 1997; Baurle et al., 1997; Wang and Morgan, 2007) and muscle (Carrascosa et

al., 2004), and possible improvement in locomotor defects after cell therapy, to date, has not

been investigated.

Therefore, our hypothesis is that a healthy bone marrow transplantation can restore the

movement of PCD mice. To verify this hypothesis, we have transplanted healthy (wild-type) or

mutant (PCD) bone marrow in PCD animals (with the adequate controls) and performed a wide

battery of motor tests. The integration and possible effects of the transplant-derived cells were

also analyzed in situ in the cerebellum, the spinal cord and the skeletal muscle.


Material and methods

Animals

Mice were housed at the Animal Facilities of the University of Salamanca or the CABIMER

at constant temperature and humidity, with a 12/12-hour photoperiod, and with water and

special rodent chow ad libitum (Rodent toxicology diet, B&K Universal G.J., S.L. Molins de

Rei, Barcelona). All animals were housed, manipulated and sacrificed in accordance with

current European (2010/63/UE, Recommendation 2007/526/CE) and Spanish Legislation (Law

32/2007, RD 53/2013); the experiments were approved by the Bioethical Committees of the

University of Salamanca and CABIMER.

Bone marrow transplants were carried out on both wild-type (+/+) and PCD (pcd/pcd) mice of

the C57BL/DBA strain (Jackson Bar Harbor, ME, USA). They were produced from mating

heterozygous PCD mice (+/pcd) within the animal facilities. Mice with the same genotypes

were also used as non-transplanted control animals.

Both wild-type or PCD green fluorescent protein (GFP) transgenic mice (Jackson) were used

as the bone marrow donors (Recio et al., 2011; Díaz et al., 2012b).

Mouse genotyping

Wild-type and heterozygous mice are phenotypically indistinguishable and in addition, PCD

animals are not suitable for breeding (Wang and Morgan, 2007). Therefore, a genotyping

method was necessary to identify the different animals. PCR was performed on the

microsatellite regions D13Mit250 and D13Mit283, associated with the locus of the mutant or

wild-type alleles, respectively, as previously described (Valero et al., 2006; Díaz et al., 2012b).

The sequences of the primers employed were


http://www.informatics.jax.org/searches/probe.cgi?38700 and

http://www.informatics.jax.org/searches/probe.cgi?41581.

Bone marrow ablation and transplantation

Recipient animals were irradiated at postnatal day 19 (P19) with a 137Cs source for mice

(Gammacell 1000 Elite; radiation rate, 243 cGy/min; energy, 0.662 MeV; MDS Nordion,

Ottawa, Canada). The dose used was 7.5 Gy, enough to ablate the bone marrow of recipients

and allow subsequent transplantation (Álvarez-Dolado et al., 2003; Recio et al., 2011; Díaz et

al., 2011).

The bone marrow extraction and transplantation was performed as previously described (Díaz

et al., 2011; Recio et al., 2011; Díaz et al., 2012a; Díaz et al., 2012b). Briefly, after sacrifice

donors and dissect their femurs and tibias, bone marrow extraction was performed by injecting

IMDM (Iscove´s Modified Dulbecco´s Medium; Invitrogen; Carlsbad, CA, USA) with a

syringe at both epiphyses of each bone. The bone marrow wash was filtered and centrifuged at

1,500 rpm for 5 min. The supernatant was removed and the pellet was re-suspended in 5 ml of

lysis buffer (140 mM NH4Cl, 17 mM Tris-base, pH 7.4) for 5 min to destroy the erythrocytes.

The reaction was stopped by adding 45 ml of 0.1 M phosphate-buffered saline, pH 7.4 (PBS)

and the number of cells of each vial was estimated using a Thoma chamber. The cell suspension

was centrifuged again at 1,500 rpm for 5 min and the pellet was re-suspended in PBS for

transplantation into the recipients. Transplants were performed twenty-four hours after the

irradiation (at P20), and each mouse received a single injection in the tail vein containing a

suspension of 7.5 million bone marrow cells (from the wild-type or PCD donor animals) in

PBS. The constitutive expression of GFP in donor mice allows their derived cells to be tracked

in the tissues of recipients (Recio et al., 2011). In every radiation/transplant round two mice

were left without transplant, as radiation controls, to confirm the effectivity of the ablation,

http://www.informatics.jax.org/searches/probe.cgi?38700

http://www.informatics.jax.org/searches/probe.cgi?41581


since it was performed with a minimal lethal radiation dose (Díaz et al., 2011). In every case,

these mice died after 7-10 days post radiation, thus verifying the quality of the procedure.

Recipients were housed separately for three weeks in an insulated rack for immunodepressed

animals. Then, animals were returned to their cages for further analysis. Hematopoietic

engraftment was quantified 1 month after transplantation by flow cytometry (Becton

Dickinson; Franklin Lakes, NJ, USA) based on the frequency of cells expressing GFP in

peripheral blood after erythrocyte lysis.

To summarize, six experimental groups of mice (n=5-6, each) were used: non-transplanted

wild-type (1) and PCD (2) mice, as the wild-type or PCD control groups, respectively; wild-

type mice transplanted with healthy (wild-type) bone marrow (3), as wild-type sham group, to

analyze putative deleterious effects of the radiation; PCD mice transplanted with PCD bone

marrow (4), as PCD sham, to discard beneficial effects derived from the technical procedures

themselves; and PCD mice transplanted with healthy bone marrow (5) used as the PCD

experimental group to determine the positive effects of the treatment (healthy bone marrow

transplantation). Additionally, a sixth group was employed: wild-type animals transplanted

with PCD bone marrow (wild-type experimental). This group was only used to analyze the

properties of migration and nesting of the PCD-derived bone marrow cells.

Behavioral tests

All mice underwent the same sequence of behavioral tests (open field, measurement of their

average locomotion speed, footprint analysis, and rotarod). The tests were conducted weekly

during 4 weeks, from P40 and onward. All tests were conducted by an operator blind to the

experimental condition, in the same testing room, under the same indirect light and daytime

intervals (always during the light phase of the circadian cycle, between 08:00 and 18:00 hours).

The mice were habituated in their home cages and transferred to the testing room at least 1


hour before the start of the experiment, and returned to their cages after the tests. The room

environment was set up to minimize olfactory, auditory and visual cues. All of the testing

apparatus were cleaned with soapy water and 70% ethanol before and after every trial, and

tasks were recorded by a video-camera for subsequent observer-blind analysis.

The open field test provides information related to the motor capacity, exploratory behavior

and emotional reactivity of animals (Takahashi et al., 2006). Its device consisted of a

rectangular plastic cage of 50 x 29 cm and a depth of 17.5 cm. At the bottom of the cage the

field was marked with a grid containing 5 x 3 squares (9 x 9 cm each; Supplementary Fig. 1).

Each mouse was placed in the central square and five parameters were quantified during 5 min:

1) complete crossings, where the mouse crossed, with all four paws, from one square to the

adjacent square; 2) half crossings, where the animal crossed a line with only two of its paws

and then returned back to the original square; 3) rearing events, where the animal stood up on

its hind paws; 4) grooming events, where the animal cleaned its face, snout and whiskers; and

5) fecal pellets. The two first parameters indicated the directionality and decision of movement,

whereas the two vertical movements (parameters 3 and 4) were related with their exploratory

behavior. Fecal pellets were considered as an indicator of stress during the execution of the

task.

To determine the speed of movement, the time it took the animals to pass through a narrow

corridor 1 m in length was measured. The width of the passage was 3 cm, preventing the

animals from turning around. In cases where the animal changed its direction, the test was

restarted.

The footprint test allowed a qualitative analysis of the locomotion of the mouse. The forepaws

were painted with a nontoxic red ink, and the hind paws with a nontoxic blue ink. Then, the

animals were allowed to walk over a filter paper, where the pattern of their tracks was obtained.


The animals were also subjected to the rotarod test (Panlab, Barcelona, Spain). This is a more

complex motor task that assesses the animals’ motor coordination and equilibrium. The test

consists of a horizontal rod that rotates at constant acceleration (from 4 to 40 rpm along 10

min). The mice were placed onto the rod and had to walk in the direction opposite to its rotation

to avoid falling off, their latency to fall being scored. Three trials were performed per daily

session during a period of 7 days. Animals were allowed to rest for a minimum of 10 min

between trials to avoid fatigue.

Sacrifice and tissue processing

Mice were deeply anaesthetized with 10 µl/g b.w. of chloral hydrate (Prolabo, Fontenay-sous-

Bois, France). Then, they were sacrificed by intracardiac perfusion of PBS, followed by

Zamboni’s fixative (4% w/v depolymerized paraformaldehyde and 15% v/v saturated picric

acid in PBS) during 15 min. After perfusion, the brain, an ischiotibial muscle and a cervical-

thoracic portion of the spinal cord were dissected out and rinsed for 2 hours with PBS.

Cerebella were cryoprotected by immersing them in 30% (w/v) sucrose in PBS, frozen, and

sectioned in 40-μm thick parasagittal slices using a freezing-sliding microtome (Jung SM 2000,

Leica Instruments, Nussloch, Germany) attached to a freezing unit (Frigomobil, Leica). Muscle

samples were similarly cryoprotected and divided to obtain both longitudinal and transversal

sections 14-μm thick with a cryostat (Microm HM 560, Thermo Scientific, MA, USA). Spinal

cord samples were also divided in two parts, one of them was cryoprotected, sectioned into 14-

μm thick coronal slices using a cryostat (Thermo Scientific) and destined for

immunohistochemistry. The other part was embedded in paraffin and sectioned in 14-μm thick

coronal slices using a rotary microtome (Leica 2065, Leica) and destined for general histology.


Tissue staining

Both paraffin slices of the spinal cord and muscle slices were stained with hematoxylin-eosin

to reveal the general distribution of cells within the tissue. Nissl staining was also performed

on the spinal cord sections for the same purpose. Masson’s trichrome staining was performed

on muscle slices to study the morphology and collagen distribution within the muscle fibers.

Immunofluorescence

Slices were washed in PBS (3 x 10 min) and incubating them overnight at room temperature

under continuous rotary shaking in a medium containing 0.2% (w/v) Triton X-100, 5% (v/v)

donkey serum, the primary anti-GFP polyclonal goat antibody (1:2,000; Abcam, Cambridge,

UK), the primary anti-calbindin D28k monoclonal mouse antibody (1:2,000; Swant,

Bellinzona, Switzerland) to label Purkinje cells, and/or an anti-Iba1 polyclonal rabbit IgG

(1:1,000; Wako, Osaka, Japan) to label microglia, in PBS. Following this, slices were washed

in PBS and incubated in a second medium with Cy2-conjugated donkey anti-goat fluorescent

antibody (1:500; Jackson Laboratories, West Grove, PA, USA), Cy3-conjugated donkey anti-

mouse fluorescent antibody (1:500; Jackson) and/or Cy5-conjugated donkey anti-rabbit

fluorescent antibody (1:500; Jackson) in PBS for 2 hours at room temperature under continuous

rotary shaking. Finally, sections were rinsed in PBS and mounted with coverslips and a freshly

prepared anti-fade medium composed by 0.42% (w/v) glycine, 0.021% (w/v) NaOH, 0.51%

(w/v) NaCl, 5% (w/v) N-propyl-gallate, 70% (v/v) glycerol and 0.002% (w/v) Thimerosal.

Data analysis

GFP+ cells were counted with the Neurolucida (V8.23, MicroBrightField, Colchester, VT,

USA) and Neuroexplorer programs (V4.70.3, MicroBrightField), and referred to a surface to

obtain the corresponding cell density (when required). The thickness of muscle fibers was

estimated by measuring the diameter of these cells in transversal sections of muscle samples


and calculating its mean for each animal. Then, using these data, the mean fiber diameter for

each experimental group was calculated. More than 2000 muscle fibers were counted for each

group.

Homoscedasticity was checked before any statistical analysis was carried out (Kolmogorov-

Smirnov test). Then, different statistical tests were performed depending on the experiments.

To make comprehension easier, each test will be described where applicable, in both the

Results section and in the figure legends. The statistic suite SPSS (v23.0 for Windows, SPSS,

Chicago, IL, USA) was used for all statistical analyses.

Results

Transplant integration

All of the transplanted mice survived the quarantine period after radiation and transplant, and

had a healthy appearance. The analysis of blood, one month after the transplant, by flow

cytometry confirmed bone marrow integration in all of the different groups, with a chimerism

level of GFP+ blood cells similar to the donors (60-85%, Supp. Fig. 2). The analysis confirmed

no significant differences among the groups that had received a transplant (both sham and

experimental; one-way ANOVA test, GL = 3, F = 0.078, p = 0.971). A second blood analysis

was performed before euthanasia with similar results. These data suggested that neither

transplant homing nor bone marrow incorporation were affected by the pcd mutation.

Motor tests

As PCD mice suffer severe ataxia at the age when behavioral tests are usually carried out

(Mullen et al., 1976; Wang and Morgan, 2007), they are incapable of properly performing very

complicated motor tasks. Thus, the mice were first subjected to an open field test, followed by


measuring of the average speed of movement, the footprint analysis, and finally, the rotarod

test.

1. Open field. A relevant improvement in the movement of PCD experimental mice was

detected. The positive effects appeared in both movement directionality (complete and

half crossings; Fig. 1 A, B; Supp. Table 1; one-way ANOVA, GL = 4, F = 5.095, p =

0.001, and F = 10.270, p < 0.001, respectively) and explorative behavior (rearing and

grooming; Fig. 1 C, D; Supp. Table 1; one-way ANOVA, GL = 4, F = 78.798, p <

0.001, and F = 6.140, p < 0.001, respectively). Thus, the PCD experimental group

presented similar values to both groups of wild-type mice (control and sham) in

complete crossings (Fig. 1 A; Supp. Table 1; PCD Bonferroni post hoc test, p ≥ 0.05)

and grooming events (Fig. 1 D; Supp. Table 1; Bonferroni post hoc test, p ≥ 0.05). In

addition, statistically significant differences were observed upon comparing the PCD

experimental group with both the PCD control and the PCD sham (ataxic) groups, for

these same parameters (Fig. 1 A, Supp. Table 1; D; Bonferroni post hoc tests, p < 0.05

and p < 0.01, respectively). Concerning the other parameters, the PCD experimental

mice presented intermediate values between those corresponding to both groups of

wild-type and both PCD mutant mice: half crossings (Fig. 1 B; Supp. Table 1;

Bonferroni post hoc tests, p < 0.05, p < 0.01) and rearing events (Fig. 1 C; Supp. Table

1; one-way ANOVA and Bonferroni post hoc tests, p < 0.05, p < 0.01). Possible

differences related to stress were discarded since no statistical differences were

observed in the number of fecal pellets of the different groups (Fig. 1 E; Supp. Table 1;

one-way ANOVA, GL = 4, F = 0.861, p = 0.489).

2. Locomotion speed and footprint analysis. PCD experimental mice, although slower

than the wild-type control and sham animals (Fig. 1 F; Supp. Table 1; one-way


ANOVA, GL = 4, F = 28.875, p < 0.001; and Bonferroni post hoc tests, p < 0.01),

presented average speed values higher than those corresponding to the other two PCD

groups (Fig. 1 F; Supp. Table 1; Bonferroni post hoc tests, p < 0.05). Thus, healthy

bone marrow transplantation also increased the speed of movement of PCD mice.

Moreover, the locomotion pattern of PCD experimental mice also presented marked

improvements, as shown by the footprint analysis (Fig. 1 G, I, K). It is also necessary

to comment that both PCD control and sham mice advanced by standing on the heels

of their back paws, whereas the PCD experimental mice advanced on their toes, as

observed in wild-type mice (Fig. 1 H, J, L, arrows).

3. Rotarod. As the previous locomotor tests demonstrated a clear improvement in the

motor activity of PCD experimental mice, a new cohort of animals was subjected to the

rotarod test to confirm these results and evaluate coordination, equilibrium, and

progressive motor skill acquisition. Also, the possible effect of the techniques employed

was discarded since both sham groups showed results similar to their corresponding

control groups. Therefore, the animals within the sham groups were not included in this

study in order to reduce the number of animals.

The aforementioned motor tests were corroborated: PCD mice were almost incapable

of remaining on the rod, presenting latency values close to 0 along the seven sessions

included within the test (Fig. 2). By contrast, both wild-type and PCD experimental

mice were able to remain on the rod (Fig. 2). A two-way repeated-measures ANOVA

analysis confirmed significant differences among the sessions (GL = 6, F = 8.178, p <

0.001) and type of treatment (GL = 2, F = 22.580; p < 0.001). In addition, after

combining the mean latency values of each group along the sessions (Fig. 2B), we

observed that PCD experimental mice presented an intermediate latency in comparison

with both wild-type and PCD animals (Fig. 2B; PCD experimental 0.694 ± 0.124 SEM,


wild-type 2.740 ± 0.178 SEM, PCD 0.017 ± 0.02 SEM; one-way ANOVA, GL = 2, F

= 115.876, p < 0.001; Bonferroni post hoc test, p < 0.01). These results confirmed a

partial rescue of the locomotor activity in PCD mice after a healthy bone marrow

transplant.

Tissue analysis

After the behavioral tests were completed, the mice were euthanized and the cerebellum, spinal

cord and skeletal muscle were analyzed. First, we analyzed the cerebellum of the transplanted

mice to detect cells derived from the transplant. The main cell type found in all of the

transplanted animals (both PCD and wild-type mice, whether transplanted with wild-type or

PCD bone marrow) was microglia (Iba1+), as has been previously reported (Recio et al., 2011),

with no differences among the different groups (wild-type sham 393.002 ± 47.841 SEM, wild-

type experimental 365.920 ± 44.894 SEM, PCD sham 375.383 ± 55.627 SEM, PCD

experimental 359.500 ± 86.559 SEM cells in each section of cerebellar vermis; one-way

ANOVA, GL = 3, F = 2.096, p = 0.169). In both PCD sham and experimental groups no

additional cell types derived from the transplant were detected in the vermis of the cerebellum.

However, in both wild-type transplanted groups (whether with healthy or PCD bone marrow),

GFP+ Purkinje cells derived from the transplant appeared (Fig. 3), with no significant

differences between them (Fig. 3D; wild-type sham 1.382 ± 0.627 SEM, wild-type

experimental 2.000 ± 0.894 SEM; Student’s t test, GL = 10, p = 0.584). Thus, PCD bone

marrow-derived cells showed features of migration and integration similar to those of wild-

type cells. On the other hand, the cerebellar degenerative pattern in the three PCD groups

(control, sham or experimental) was similar. No Purkinje cells were detected, thus confirming

a complete degeneration of these neurons as previously reported (Recio et al., 2011). Therefore,

the origin of the partial rescue of the locomotor system in PCD experimental mice appears to

be independent of the cerebellar degeneration.


Then, we analyzed the spinal cord, observing that its general histology was not different among

the groups (Supplementary Fig. 3); thus, spinal neurons do not seem to be affected by the pcd

mutation. Moreover, the immunohistochemistry revealed only GFP+ microglial cells in the

spinal cord sections of all of the transplanted groups without any further differences (one-way

ANOVA, GL = 3, F = 0.160, p = 0.855). Therefore, the spinal cord also seemed to be unrelated

to the partial motor recovery of the PCD experimental mice.

Finally, we analyzed the muscle samples. Here, we found GFP+ cells in all of the samples of

transplanted animals, corresponding to macrophages (identified by CD45 labeling, data not

shown) and muscle fibers with their characteristic morphologies (Fig. 4A). The number of these

GFP+ muscle fibers was higher in transplanted PCD groups compared to their wild-type

counterparts, suggesting that the mutant environment exerts an attraction of donor cells or favor

their differentiation into the muscle (Fig. 4B; Student’s t test, wild-type sham 0.113 ± 0.023

SEM cells/mm2, PCD experimental 0.213 ± 0.072 SEM cells/mm2; GL = 10, p = 0.045). Then,

we analyzed the histology and morphology of muscular fibers by hematoxylin-eosin and

Masson’s trichrome staining. No accumulation of collagen or fibrosis was observed in any of

the analyzed groups. The general muscle structure was otherwise normal, except in PCD mice

(both control and sham), which presented a smaller size, most likely due to ataxia-derived

atrophy. PCD muscle fibers were thinner in comparison to the wild-type groups (Fig. 5).

Interestingly, the organization of the muscle fibers in PCD experimental mice was more similar

to the wild-type. In addition, the thickness of these muscle cells was larger than those

corresponding to the other PCD groups, and similar to the thickness observed in the wild-type

animals (Fig. 5 and 6; one-way ANOVA, GL = 2, F = 43.390, p < 0.001; Bonferroni post hoc

test, p < 0.01). These results indicate that the healthy bone marrow transplant had a positive

effect on the musculature of the PCD experimental mice, which may account for their partially

improved locomotor activity.


Discussion

Ataxias are severe degenerative neuromuscular disorders with diverse etiology and a general

lack of effective treatments. Recently, cell-based therapy with bone marrow-derived stem cells

has been assayed in animal models of ataxia as a new therapeutic approach. Transplanted cells

usually originate from healthy donors, but they can derive from mutant animals (used as

negative controls; Chen et al., 2011; Baltanás et al., 2013b; Díaz et al., 2015). In the latter

situation, the viability of the mutant transplanted cells and their integration should be checked.

This was the case of the negative controls used in this study, which involved transplantation

using bone marrow cells carrying the pcd mutation in the Ccp1 gene. We employed PCD mice

as donors of bone marrow cells and demonstrated that the processes of cell homing, migration,

and transdifferentiation after bone marrow transplantation were not affected by the Ccp1

mutation, either by affecting the bone marrow physiology directly or generating a non-

permissive environment in PCD mice. First, both wild-type and mutant animals transplanted

with PCD bone marrow survived along all the experiments after a (minimal) lethal dose of

radiation, as well as the animals transplanted with wild-type bone marrow (Díaz et al., 2011).

Thus, the cell homing of the new transplanted cells and the subsequent restoration of the

hematopoietic/immune systems were not affected (Whetton and Graham, 1999). Second, we

detected mutant (PCD) bone marrow-derived both microglia and Purkinje cells (in the wild-

type recipients) of the same origin, either by transdifferentiation or cell fusion, as previously

described (Recio et al., 2011). Finally, the proportion of mutant bone marrow-derived neural

cells was similar to that derived from wild-type bone marrow, which demonstrates similar

features of integration.

To date, the locomotor activity of PCD mice after a bone marrow transplantation has not been

tested. However, the results of our behavioral tests demonstrated that the transplantation of

healthy bone marrow in PCD mice elicited a beneficial effect, with no apparent secondary


effects related to the experimental procedures. The loss of Purkinje cells is considered to be the

cause of the severe ataxia suffered by PCD mice (Mullen et al., 1976). Hence, it was thought

that the improved motor activity would be the result of the rescue of Purkinje cells in the

cerebellum by cell fusion between healthy/genetically modified bone marrow cells and

Purkinje cells, as previously shown in other cerebellar degeneration models (Weimann et al.,

2003; Bae et al., 2007; Álvarez-Dolado, 2007; Chen et al., 2011). This hypothesis, however,

was discarded as no Purkinje neurons (either fused with transplant-derived cells or not) were

detected in the cerebellum of all our PCD groups, which in fact exhibited similar cerebellar

degeneration. It is likely that the degeneration of Purkinje cells was already quite advanced

before the reconstitution of the bone marrow was completed, and therefore, the possible cell

fusion events between Purkinje and bone marrow-derived cells could not have taken place, as

previously suggested (Recio et al., 2011). Besides, a neuroprotective effect mediated by bone

marrow-derived microglia cannot be discarded (Derecki et al., 2012; Díaz et al., 2012a).

However, a putative reduction in the loss of Purkinje cells, if this actually occurs, would be

very transient since similar transplants with shorter survival times show no neuroprotection

(Recio et al., 2011).

The only reference mentioning tissue damage in structures related to motor behavior in PCD

mice, except for the cerebellum, has merely suggested some undefined muscle impairments

(Carrascosa et al., 2004). Considering that Ccp1 was discovered by inducing axotomy in the

motoneurons of the spinal cord and is involved in axon regeneration and reinnervation (Harris

et al., 2000), we decided to investigate both the spinal cord and the musculature of PCD mice.

No apparent differences in the tissue morphology of the spinal cord were found within the

experimental groups, and a similar number of bone marrow-derived microglial cells were

observed in transplanted animals, independently of their genotype. Previous findings verified

this integration of bone marrow-derived cells in the spinal cord, also showing that their number


increases after spinal cord damage (Lewis et al., 2013). Therefore, these data indicate that PCD

mice have no additional spinal cord impairments, and that the transplant had no effect on this

structure.

Additionally, the analysis of muscle fibers helped to clarify the origin of the improved motor

activity in PCD experimental mice: a partial rescue of the muscle fiber thickness within the

muscle was observed. This may be interpreted as the direct cause of the improved locomotion

observed in PCD experimental mice or as a consequence of reduced muscle atrophy caused by

improved mobility. Some studies have suggested that low doses of radiation can increase cell

proliferation (Balentova et al., 2006; Balentova et al., 2007) and stimulate local muscle stem

(satellite) cells. To address this question, we included the PCD sham group of mice. Since no

positive (or negative) effects of the treatment were detected in this group, we discarded the

possibility of any amelioration of muscular degeneration due to the experimental procedures.

Conversely, high doses of radiation could lead to muscle alterations (Russell and Connor, 2014;

Mericli et al., 2015). However, the dose applied in our experiments was low and, in addition,

no differences were found between the wild-type groups. Therefore, the observed enhanced

locomotor behavior in PCD mice should not be related to the experimental radiation and be

exclusively due to the transplantation of healthy/wild-type bone marrow.

Previous reports have hypothesized muscle deterioration in PCD animals (Carrascosa et al.,

2004), but to our knowledge, this is the first time that the muscle fibers of PCD mice have been

measured, showing a dramatic weakness. Ccp1 is expressed in the striatal muscle of wild-type

animals (Harris et al., 2000), but it is unclear whether the muscular damage observed in PCD

mice is caused directly by the lack of Ccp1 expression or by a secondary atrophy concomitant

to neural ataxia. In any case, only cells derived from wild-type bone marrow were able to

restore muscle morphology, either by compensating for the lack of Ccp1 expression in the

muscle or by palliating the ataxia-related atrophy. Moreover, the increase in thickness was


detected in all of the muscle fibers of PCD experimental mice, independently of whether these

cells came from the donor bone marrow or were already present. This suggests that healthy

bone marrow-derived cells could mediate a paracrine effect over the whole musculature, rather

than replace degenerated fibers, achieving better muscle performance. Thus, bone marrow-

derived cells express and secrete insulin-like growth factor 1 (IGF1), which mediates

therapeutic effects on different disease models (Feng et al., 2012; Cervelló et al., 2015). These

observations are also in agreement with previous findings that suggest that a substitutive

treatment of IGF1 can improve the mobility of PCD mice, and propose some muscle recovery

by means of weight gain and survival extension (Carrascosa et al., 2004). Other relevant factors

of the neuromuscular system are the neurotrophins which can modulate the development and

differentiation of muscle fibers (Chevrel et al., 2006). Interestingly, transplanted bone marrow

mesenchymal stem cells mediate a therapeutic effect on a mouse model of Friedreich ataxia

through the expression of NT3, NT4, and the brain derived neurotrophic factor (Jones et al.,

2015).

Another possible explanation for the enhanced mobility is the physiological integration of some

bone marrow stem cell populations into the striatal muscle, what ensures its normal functioning

and turnover (Fujita et al., 2015). This integration is especially important after muscular

damage, given the anti-inflammatory properties of the bone marrow-derived cells (Fujita et al.,

2015). Hence, it is plausible to consider that in PCD mice this endogenous mechanism of

muscle turnover may be impaired, since these animals may present abnormal patterns of

inflammation, as it occurs in their central nervous system (Baltanás et al., 2013a). Conversely,

when wild-type bone marrow cells substitute the bone marrow cells of the mutant animals, the

process could take place properly and mediate normal turnover in the muscle.

Finally, the transplantation of healthy bone marrow reduces mitral cell degeneration in PCD

mice and improves their olfactory capabilities (Díaz et al., 2012a). Accordingly, these


transplanted PCD mutants with restored olfaction would receive more olfactory inputs than

standard PCD mice, exploring more actively the source of such inputs. This idea is supported

by the results regarding the number of grooming and rearing events in the open field test. This

increase in exploratory behavior consequently induces greater mobility in these PCD mice, and

moderate exercise has been demonstrated to reduce the symptoms of muscular dystrophies

(Chan et al., 2014; Rowe et al., 2014; Alemdaroglu et al., 2015; Hyzewicz et al., 2015). Thus,

the improvement in olfaction should drive the PCD experimental mice to explore and move

more, indirectly reducing secondary muscular atrophy.

Most probably there is not just one explanation for the improved locomotor ability in PCD

experimental mice, and hence additional hypotheses cannot be discarded. Concerning strict

muscular damage, previous findings have reported the benefits of both systemic bone marrow

transplantation or direct muscular injections (Ferrari et al., 1998; Gussoni et al., 1999; amongst

others), but the PCD is a more complex model. Thus, the positive effect of healthy bone marrow

transplantation on the movement of PCD mice is quite likely a multi-factorial process, and the

exact mechanism of each of these factors will need to be deciphered using other specific

approaches. For instance, wild-type bone marrow transplantation in a strict inducible model of

Purkinje cell degeneration should help to clarify both whether the muscular damage is

secondary to this neuronal death, and to discard maybe the possible effect of the olfactory

loss/regeneration.

The present work was designed to describe a putative positive effect of bone marrow

transplantation on the locomotor activity of ataxic mice. Although the significance of our

results is still being contemplated, this type of cell therapy seems to be a promising palliative

tool for the treatment of ataxias. Accordingly, cells derived from a healthy bone marrow can

be administered as a complementary therapy of the motor re-education (Milne et al., 2016).

Indeed, as the bone marrow cells could exert their benefits without interfering with the


cerebellar functioning, this complementary therapy could be useful for several types of ataxia

(Vanier, 2010; Milne et al., 2016), and maybe for other motor disorders.

Reference List

Alemdaroglu I, Karaduman A, Yilmaz OT, Topaloglu H (2015) Different types of upper

extremity exercise training in Duchenne muscular dystrophy: Effects on functional

performance, strength, endurance, and ambulation. Muscle Nerve 51:697-705.

Álvarez-Dolado M (2007) Cell fusion: biological perspectives and potential for regenerative

medicine. Front Biosci 12:1-12.

Álvarez-Dolado M, Pardal R, García-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C,

Morrison SJ, Álvarez-Buylla A (2003) Fusion of bone-marrow-derived cells with Purkinje

neurons, cardiomyocytes and hepatocytes. Nature 425:968-973.

Bae JS, Furuya S, Shinoda Y, Endo S, Schuchman EH, Hirabayashi Y, Jin HK (2005)

Neurodegeneration augments the ability of bone marrow-derived mesenchymal stem cells to

fuse with Purkinje neurons in Niemann-Pick type C mice. Hum Gene Ther 16:1006-1011.

Bae JS, Han HS, Youn DH, Carter JE, Modo M, Schuchman EH, Jin HK (2007) Bone marrow-

derived mesenchymal stem cells promote neuronal networks with functional synaptic

transmission after transplantation into mice with neurodegeneration. Stem Cells 25:1307-1316.

Balentova S, Racekova E, Martoncikova M, Misurova E (2006) Cell proliferation in the adult

rat rostral migratory stream following exposure to gamma irradiation. Cell Mol Neurobiol

26:1131-1139.

Balentova S, Racekova E, Misurova E (2007) Effect of low-dose irradiation on proliferation

dynamics in the rostral migratory stream of adult rats. Folia Biol (Praha) 53:74-78.

Baltanás FC, Berciano MT, Valero J, Gómez C, Díaz D, Alonso JR, Lafarga M, Weruaga E

(2013a) Differential glial activation during the degeneration of Purkinje cells and mitral cells

in the PCD mutant mice. Glia 61:254-272.

Baltanás FC, Pérez-Andrés M, Ginel-Picardo A, Díaz D, Jimeno D, Liceras-Boillos P, Kortum

RL, Samelson LE, Orfao A, Santos E (2013b) Functional redundancy of Sos1 and Sos2 for

lymphopoiesis and organismal homeostasis and survival. Mol Cell Biol 33:4562-4578.

Baurle J, Helmchen C, Grusser-Cornehls U (1997) Diverse effects of Purkinje cell loss on deep

cerebellar and vestibular nuclei neurons in Purkinje cell degeneration mutant mice: a possible

compensatory mechanism. J Comp Neurol 384:580-596.

Becker AJ, McCulloch EA, Till JE (1963) Cytological demonstration of the clonal nature of

spleen colonies derived from transplanted mouse marrow cells. Nature 197:452-454.


Berezniuk I, Vu HT, Lyons PJ, Sironi JJ, Xiao H, Burd B, Setou M, Angeletti RH, Ikegami K,

Fricker LD (2012) Cytosolic carboxypeptidase 1 is involved in processing alpha- and beta-

tubulin. J Biol Chem 287:6503-6517.

Butterworth RF (1993) Pathophysiology of cerebellar dysfunction in the Wernicke-Korsakoff

syndrome. Can J Neurol Sci 20 Suppl 3:S123-S126.

Carrascosa C, Torres-Aleman I, Lopez-Lopez C, Carro E, Espejo L, Torrado S, Torrado JJ

(2004) Microspheres containing insulin-like growth factor I for treatment of chronic

neurodegeneration. Biomaterials 25:707-714.

Cervelló I, Gil-Sanchís C, Santamaría X, Cabanillas S, Díaz A, Faus A, Pellicer A, Simón C

(2015) Human CD133(+) bone marrow-derived stem cells promote endometrial proliferation

in a murine model of Asherman syndrome. Fertil Steril 104:1552-1560.

Chan MC, Rowe GC, Raghuram S, Patten IS, Farrell C, Arany Z (2014) Post-natal induction

of PGC-1alpha protects against severe muscle dystrophy independently of utrophin. Skelet

Muscle 4:2.

Chen KA, Cruz PE, Lanuto DJ, Flotte TR, Borchelt DR, Srivastava A, Zhang J, Steindler DA,

Zheng T (2011) Cellular fusion for gene delivery to SCA1 affected Purkinje neurons. Mol Cell

Neurosci 47:61-70.

Chevrel G, Hohlfeld R, Sendtner M (2006) The role of neurotrophins in muscle under

physiological and pathological conditions. Muscle Nerve 33:462-476.

Derecki NC, Cronk JC, Lu Z, Xu E, Abbott SB, Guyenet PG, Kipnis J (2012) Wild-type

microglia arrest pathology in a mouse model of Rett syndrome. Nature 484:105-109.

Díaz D, Lepousez G, Gheusi G, Alonso JR, Lledo PM, Weruaga E (2012a) Bone marrow cell

transplantation restores olfaction in the degenerated olfactory bulb. J Neurosci 32:9053-9058.

Díaz D, Muñoz-Castañeda R, Alonso JR, Weruaga E (2015) Bone Marrow-Derived Stem Cells

and Strategies for Treatment of Nervous System Disorders: Many Protocols, and Many Results.

Neuroscientist 21:637-652.

Díaz D, Recio JS, Baltanás FC, Gómez C, Weruaga E, Alonso JR (2011) Long-lasting changes

in the anatomy of the olfactory bulb after ionizing irradiation and bone marrow transplantation.

Neuroscience 173:190-205.

Díaz D, Recio JS, Weruaga E, Alonso JR (2012b) Mild cerebellar neurodegeneration of aged

heterozygous PCD mice increases cell fusion of Purkinje and bone marrow-derived cells. Cell

Transplant 21:1595-1602.

Feng J, Xiong J, Xiong Q, Chen XD, Wang B, Chen DF (2012) Insulin-like growth factor-1-

mediated paracrine pro-proliferative effect of bone marrow mesenchymal stem cells on injured

L02 hepatocytes. Zhonghua Gan Zang Bing Za Zhi 20:853-858.

Fernández-González A, La Spada AR, Treadaway J, Higdon JC, Harris BS, Sidman RL,

Morgan JI, Zuo J (2002) Purkinje cell degeneration (pcd) phenotypes caused by mutations in

the axotomy-induced gene, Nna1. Science 295:1904-1906.


Ferrari G, Cusella-De AG, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F (1998)

Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279:1528-1530.

Fujita R, Tamai K, Aikawa E, Nimura K, Ishino S, Kikuchi Y, Kaneda Y (2015) Endogenous

mesenchymal stromal cells in bone marrow are required to preserve muscle function in mdx

mice. Stem Cells 33:962-975.

Galli D, Vitale M, Vaccarezza M (2014) Bone marrow-derived mesenchymal cell

differentiation toward myogenic lineages: facts and perspectives. Biomed Res Int

2014:762695.

Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan

RC (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation.

Nature 401:390-394.

Harris A, Morgan JI, Pecot M, Soumare A, Osborne A, Soares HD (2000) Regenerating motor

neurons express Nna1, a novel ATP/GTP-binding protein related to zinc carboxypeptidases.

Mol Cell Neurosci 16:578-596.

Hyzewicz J, Tanihata J, Kuraoka M, Ito N, Miyagoe-Suzuki Y, Takeda S (2015) Low intensity

training of mdx mice reduces carbonylation and increases expression levels of proteins

involved in energy metabolism and muscle contraction. Free Radic Biol Med 82:122-136.

Jones J, Estirado A, Redondo C, Pacheco-Torres J, Sirerol-Piquer MS, Garcia-Verdugo JM,

Martinez S (2015) Mesenchymal stem cells improve motor functions and decrease

neurodegeneration in ataxic mice. Mol Ther 23:130-138.

Koeppen AH (1998) The hereditary ataxias. J Neuropathol Exp Neurol 57:531-543.

Lewis CA, Manning J, Barr C, Peake K, Humphries RK, Rossi F, Krieger C (2013)

Myelosuppressive conditioning using busulfan enables bone marrow cell accumulation in the

spinal cord of a mouse model of amyotrophic lateral sclerosis. PLoS One 8:e60661.

Mericli AF, Das A, Best R, Rodeheaver P, Rodeheaver G, Lin KY (2015) Deferoxamine

mitigates radiation-induced tissue injury in a rat irradiated TRAM flap model. Plast Reconstr

Surg 135:124e-134e.

Mezey E (2005) Bone marrow and brain: unexpected allies or accidental acquaintances? Stem

Cell Rev 1:15-19.

Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR (2000) Turning blood into brain:

cells bearing neuronal antigens generated in vivo from bone marrow. Science 290:1779-1782.

Milne SC, Corben LA, Yiu E, Delatycki MB, Georgiou-Karistianis N (2016) Gastrocnemius

and soleus spasticity and muscle length in Friedreich's ataxia. J Clin Neurosci.

Mullen RJ, Eicher EM, Sidman RL (1976) Purkinje cell degeneration, a new neurological

mutation in the mouse. Proc Natl Acad Sci USA 73:208-212.

Nern C, Wolff I, Macas J, von RJ, Scharenberg C, Priller J, Momma S (2009) Fusion of

hematopoietic cells with Purkinje neurons does not lead to stable heterokaryon formation under

noninvasive conditions. J Neurosci 29:3799-3807.


Recio JS, Álvarez-Dolado M, Díaz D, Baltanás FC, Piquer-Gil M, Alonso JR, Weruaga E

(2011) Bone marrow contributes simultaneously to different neural types in the central nervous

system through different mechanisms of plasticity. Cell Transplant 20:1179-1192.

Rowe GC, Raghuram S, Jang C, Nagy JA, Patten IS, Goyal A, Chan MC, Liu LX, Jiang A,

Spokes KC, Beeler D, Dvorak H, Aird WC, Arany Z (2014) PGC-1alpha induces SPP1 to

activate macrophages and orchestrate functional angiogenesis in skeletal muscle. Circ Res

115:504-517.

Russell JA, Connor NP (2014) Effects of age and radiation treatment on function of extrinsic

tongue muscles. Radiat Oncol 9:254.

Takahashi A, Kato K, Makino J, Shiroishi T, Koide T (2006) Multivariate analysis of temporal

descriptions of open-field behavior in wild-derived mouse strains. Behav Genet 36:763-774.

Triarhou LC, Norton J, Ghetti B (1987) Anterograde transsynaptic degeneration in the deep

cerebellar nuclei of Purkinje cell degeneration (pcd) mutant mice. Exp Brain Res 66:577-588.

Valero J, Berciano MT, Weruaga E, Lafarga M, Alonso JR (2006) Pre-neurodegeneration of

mitral cells in the pcd mutant mouse is associated with DNA damage, transcriptional

repression, and reorganization of nuclear speckles and Cajal bodies. Mol Cell Neurosci 33:283-

295.

Vanier MT (2010) Niemann-Pick disease type C. Orphanet J Rare Dis 5:16.

Wang T, Morgan JI (2007) The Purkinje cell degeneration (pcd) mouse: an unexpected

molecular link between neuronal degeneration and regeneration. Brain Res 1140:26-40.

Weimann JM, Johansson CB, Trejo A, Blau HM (2003) Stable reprogrammed heterokaryons

form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 5:959-

966.

Whetton AD, Graham GJ (1999) Homing and mobilization in the stem cell niche. Trends Cell

Biol 9:233-238.

Zhang W, Ghetti B, Lee WH (1997) Decreased IGF-I gene expression during the apoptosis of

Purkinje cells in pcd mice. Brain Res Dev Brain Res 98:164-176.

Zoghbi HY (1995) Spinocerebellar ataxia type 1. Clin Neurosci 3:5-11.

Acknowledgements: We thank E. Keck for English language revision. DD, MP-G JSR and

MMM-L performed the experiments and quantitative analyses; DD, EW and MA-D performed

the statistical analyses; DD and EW wrote the manuscript and made figures; JRA, EW and

MA-D revised the manuscript.


Figure 1. Motor tests I: open field, locomotion speed, and footprinting. A-E, charts showing

the results of the open field test: A (complete crossings) and B (half crossings), indicative of

movement directionality; C (rearing events) and D (grooming events), indicative of exploratory


behavior; E (defecations), estimation of mouse anxiety levels. Note that for all of the

parameters measured, except for defecations (E), the two groups of wild-type mice (control

and sham) presented significant differences with both PCD mutant control mice and PCD sham

mice. Conversely, PCD experimental mice showed scores either similar to the wild-type groups

(A, D) or intermediate between the wild-type and the other PCD groups (B, C). For grooming

events (D), PCD sham mice only showed significant differences with the two wild-type groups

(#). F, speed of gating; the two groups of wild-type mice (control and sham) were significantly

faster than the three PCD groups; however, the PCD experimental mice were also faster than

both control and sham PCD groups, thus having an intermediate speed amongst wild-type and

the other PCD mice. G, I and K, footprints representing the gait of wild-type control (G), PCD

control (similar to PCD sham animals; I) and PCD experimental mice (K); the footprints of

both wild-type control (G) and PCD experimental mice (K) followed a straight pattern, whereas

the gait of the PCD mutant control animals was completely blurred and ataxic (I). H, J, and L,

photograms of the mice during the measurement of locomotion speed; note that both wild-type

control (H) and PCD experimental mice (L) advanced on their toes, while both the PCD control

(J) and PCD sham (not shown) mice advanced by standing on the heels of their back paws.

One-way ANOVA and Bonferroni post hoc tests; *, p < 0.05; **, p < 0.01.


Figure 2. Motor tests II: rotarod. Charts showing the latency to fall from the rod throughout

the different sessions (A) and the mean global values (B). PCD experimental mice reached

values intermediate to those observed for the wild-type and PCD control animals. Note that

PCD control animals barely remained on the rod during almost all of the sessions, whereas

PCD experimental mice exhibited a consistent and significant motor improvement. Two-way

repeated-measures ANOVA analysis (A) and one-way ANOVA analysis with Bonferroni's

post hoc tests (B); **, p < 0.01.


Figure 3. Bone marrow-derived cells in the cerebellum. Neurons derived from the transplant

(GFP, green) into the cerebellum of wild-type sham mice (A-C) and wild-type experimental

mice (E-H). Purkinje cells are labelled with calbindin (CB, red). There is no significant

difference in the number of bone marrow-derived Purkinje cells between both groups (D;

Student’s t test). H, magnification of a mutant bone marrow-derived Purkinje cell; note the

characteristic morphology of a standard Purkinje neuron, even showing an axon passing

through the granular layer (arrows). Scale bar: 50 μm.


Figure 4. Bone marrow-derived cells in the muscle. A, representative GFP+ (green) muscular

fibers (arrow) and macrophages (arrowheads) derived from the transplant. B, PCD mice

showed a higher GFP muscular cell density than wild-type animals. Student’s t test; *, p < 0.05.

Scale bar: 250 μm.


Figure 5. Histological characterization of the muscle. A-C, longitudinal view of fibers labeled

with Masson’s trichrome (TriM); D-I, hematoxylin and eosin (H & E) staining of muscular

cells in longitudinal (D-F) and transversal (G-I) views. Note differences in thickness amongst

experimental groups. Scale bar: 100 μm for A-C; 30 μm for D-I.


Figure 6. Quantification of muscle fibers diameter. Wild-type sham and PCD experimental

mice presented thicker muscle fibers than PCD sham animals. One-way ANOVA and

Bonferroni post hoc tests; **, p < 0.01.

Documents

Bone marrow transplantation improves motor activity in a mouse … · 2020. 2. 11. · Bone marrow transplantation improves motor activity in a mouse model of ataxia David Díaz 1,