Rémi Mounier

Louise Lantier

Jocelyne Leclerc

Athanassia Sotiropoulos

Mario Pende

Dominique Daegelen

Kei Sakamoto

Marc Foretz

Benoit Viollet
email: benoit.viollet@inserm.fr

Important role for AMPKalpha1 in limiting

Keywords: muscle functional overload, protein synthesis, mTOR-S6K signaling

Introduction

The 5'-AMP-activated protein kinase (AMPK) is a sensor of cellular energy homeostasis well conserved in all eukaryotic cells [START_REF] Viollet | Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders[END_REF]. AMPK is activated by metabolic stresses that elevate the AMP/ATP ratio by lowering ATP production following inhibition of mitochondrial oxidative phosphorylation (e.g., metabolic poisons) or increasing ATP consumption (e.g., muscle contraction). Once activated, at the level of the individual cell, AMPK switches on catabolic pathways (such as fatty acid oxidation and glycolysis) and switches off ATP-consuming pathways (such as lipogenesis) with short-term effects on phosphorylation of regulatory proteins and long-term effects on gene expression. Mammalian AMPK is a heterotrimeric enzyme complex consisting of one catalytic subunit (α) and two regulatory (β and γ) subunits and isoforms of all three subunits have been identified (α1, α2, β1, β2, γ1, γ2 and γ3). The phosphorylation of a conserved threonine residue (Thr172) within the kinase domain of the α-catalytic subunit is absolutely required for AMPK activation. Three upstream kinases have been recently identified and correspond to the tumor suppressor LKB1/STK11 kinase, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) and TAK1 (TGFβ-activated kinase-1, a member of the mitogen-activated protein kinase kinase family) [START_REF] Jorgensen | How is AMPK activity regulated in skeletal muscles during exercise?[END_REF]. Binding of AMP to the γ-subunits leads to allosteric activation of AMPK as well as to enhanced phosphorylation of Thr172 by inhibiting its dephosphorylation by protein phosphatase(s) [START_REF] Sanders | Defining the mechanism of activation of AMP-activated protein kinase by the small molecule A-769662, a member of the thienopyridone family[END_REF].

In skeletal muscle, AMPK is activated by multiple stimuli including hypoxia, osmotic stress, endurance exercise and electrically-stimulated contraction [START_REF] Hardie | AMPK: a key sensor of fuel and energy status in skeletal muscle[END_REF]. AMPK activation causes metabolic changes that assist muscle cells in making chronic adaptations to energy deprivation, such as an increase in oxidative capacity via increased mitochondrial biogenesis, enzyme expression and uptake of nutrients such as glucose and fatty acids. Importantly, some of these metabolic adaptations seem to preferentially occur through the regulation of AMPKα2 but not via AMPKα1 isoform [START_REF] Jorgensen | Knockout of the alpha2 but not alpha1 5'-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4carboxamide-1-beta-4-ribofuranosidebut not contraction-induced glucose uptake in skeletal muscle[END_REF][START_REF] Jorgensen | The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading[END_REF][START_REF] Jorgensen | Role of AMPKalpha2 in basal, training-, and AICARinduced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle[END_REF]. These results suggest distinct physiological roles for α1and α2-containing AMPK complexes in the regulation of skeletal muscle adaptations. Activation of the various AMPK complexes may depend on the specificity of upstream kinase for α-catalytic subunits phosphorylation. Indeed, in LKB1-deficient muscle, AMPKα2 activation is severely blunted whereas AMPKα1 activation is substantially induced in response to contraction, indicating discordant regulation of α1and α2-containing AMPK complexes [START_REF] Koh | Skeletal muscleselective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3[END_REF]. Similar results have been obtained in LKB1-deficient heart in response to ischemia or anoxia [START_REF] Sakamoto | Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPKalpha2 but not AMPKalpha1[END_REF].

One consequence of endurance exercise training is a partial fiber type transition towards a slow-oxidative phenotype associated with mitochondrial biogenesis but no growth. In contrast, resistance exercise training mainly stimulates muscle protein synthesis and increases muscle fiber volume resulting in hypertrophy. Since protein synthesis can account for up to 30-50% of the cellular energy expenditure, a reduction in protein synthesis seems an efficient mechanism to save energy. Activation of skeletal muscle AMPK during conditions of energetic stress is thought to act as a negative regulator of protein synthesis and may therefore modulate skeletal muscle mass and hypertrophy [START_REF] Bolster | AMPactivated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling[END_REF][START_REF] Nader | Translational control: implications for skeletal muscle hypertrophy[END_REF]. AMPK activation has been also reported to inhibit protein synthesis associated with hypertrophy in cardiac muscle [START_REF] Chan | Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte[END_REF][START_REF] Blair | Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis[END_REF].

Several lines of evidence suggest that AMPK reduces both the initiation and the elongation of ribosomal peptide synthesis [START_REF] Kimura | A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway[END_REF]. One potential mechanism is through AMPK-mediated phosphorylation of the eukaryotic elongation factor 2 (eEF-2) kinase at Ser 398 (15), leading to increase Thr 56 phosphorylation and inactivation of eEF2, a key component in protein elongation [START_REF] Carlberg | Functional properties of phosphorylated elongation factor 2[END_REF]. Another mechanism is linked to the ability of AMPK to block the activation of the mammalian target of rapamycin (mTOR) signaling pathway [START_REF] Bolster | AMPactivated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling[END_REF][START_REF] Bodine | Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[END_REF][START_REF] Bolster | Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle[END_REF][START_REF] Reynolds | Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load[END_REF][START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF]. While AMPK is active in response to nutrient deprivation and inactive under nutrient-rich conditions, mTOR is activated in the inverse pattern. mTOR is part of two distinct multiprotein complexes, mTORC1 (containing mTOR, mLST8 and Raptor) which is responsible for cell growth and mTORC2 (containing mTOR, mLST8, protor and Rictor) which is important in glucose metabolism and cytoskeletal organization [START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF][START_REF] Kumar | Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity[END_REF]. mTORC1 is activated in skeletal muscle by a variety of anabolic signals, such as resistance exercise, insulin, growth factors and amino acids, to stimulate protein synthesis and hence muscle cell growth and hypertrophy [START_REF] Bodine | Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[END_REF][START_REF] Pallafacchina | A protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type specification[END_REF][START_REF] Rommel | Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways[END_REF]. It has been well established that insulin primarily regulates mTOR through PI3K-Akt signaling to stimulate cell growth [START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF]. It was recently shown that Rag GTPases play a primary role in amino acids signaling to mTOR [START_REF] Sancak | The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1[END_REF]. The mTORC1 signaling pathway contains multiple potential sites for regulatory integration with AMPK [START_REF] Bolster | AMPactivated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling[END_REF][START_REF] Inoki | TSC2 mediates cellular energy response to control cell growth and survival[END_REF]. AMPK phosphorylates and activates the tuberous sclerosis complex 2 (TSC2) kinase, which subsequently inactivates the G-protein Rheb thereby blocking activation of mTOR [START_REF] Inoki | TSC2 mediates cellular energy response to control cell growth and survival[END_REF][START_REF] Li | Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity[END_REF].

Recently, it has been shown that AMPK-dependent phosphorylation of Raptor leads also to the inhibition of mTORC1 [START_REF] Gwinn | AMPK phosphorylation of raptor mediates a metabolic checkpoint[END_REF]. Skeletal muscle ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor (eIF)-4E binding protein 1 (4E-BP1) are well-characterized downstream components of mTORC1 signaling pathway and phosphorylation of these proteins have been associated with increased muscle mass following hypertrophic stimuli.

Conversely, deletion of p70S6K reduces muscle cell size to the same extent as that observed with the mTORC1 inhibitor rapamycin. Interestingly, the suppression of the p70S6K kinases is sufficient to trigger an energy stress response associated with AMPK activation, leading to coordinated changes in cell growth and metabolic rates [START_REF] Aguilar | S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase[END_REF].

A strong negative correlation has been reported between the degree of AMPK phosphorylation and the degree of hypertrophy in overloaded plantaris (PLN) muscles, implicating AMPK as a potentially important negative regulator of overload-induced skeletal muscle hypertrophy [START_REF] Thomson | Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation[END_REF]. AMPK phosphorylation was negatively correlated with the amount of phosphorylated p70S6K at the mTOR-specific Thr 389 residue and it was postulated that AMPK inhibited p70S6K phosphorylation via its reported suppression of mTOR pathway [START_REF] Bolster | AMPactivated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling[END_REF][START_REF] Aguilar | S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase[END_REF][START_REF] Thomson | Impaired overload-induced muscle growth is associated with diminished translational signalling in aged rat fast-twitch skeletal muscle[END_REF]. It has recently been reported that continuous AICAR (5-aminoimidazole-4carboxamide-1-β-D-ribofuranoside) infusion inhibits overloading-induced muscle hypertrophy [START_REF] Gordon | Does AMP-activated protein kinase negatively mediate aged fast-twitch skeletal muscle mass?[END_REF]. Interestingly, McGee et al. have shown that the hypertrophy produced by overload was associated with a marked activation of AMPKα1 in both WT and LKB1deficient mice and they proposed that AMPKα1 might play an important role in the regulation of skeletal muscle growth [START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF].

Therefore, the aim of this investigation was to examine the potential role of AMPKα1 catalytic subunit in the size control of muscle cells in unstimulated and in response to hypertrophic stimuli. For this purpose, we analyzed the size of AMPKα1-deficient muscle cells in response to cell growth stimulation and determined the degree of muscle hypertrophy following muscle overload in AMPKα1-deficient mice.

Methods

Animals

The generation of AMPKα1 (-/-) mice has been described previously [START_REF] Jorgensen | Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle[END_REF]. AMPKα1 (-/-) and WT mice (20-26 weeks old) were maintained on a 12h:12h light-dark cycle and received standard rodent chow and water ad libitum. Genotyping was performed by PCR using DNA from a tail-piece (forward 5'-AGCACAGTACCTGGTATCTTATAGG-3' and reverse 5'-GGACTTATTACTAAACAGACCTCTG-3' primers for WT allele; forward 5'-ACCAGAAGCGGTGCCGGAAAGCTGG-3' and reverse 5'-TGTAGTCGGTTTATGCAGCAACGAG-3' primers for AMPKα1 deleted allele). All procedures were performed in accordance with the principles and guidelines established by the European Convention for the Protection of Laboratory Animals.

Cell culture and adenovirus infection

Primary muscle cell cultures were derived from gastrocnemius and tibialis anterior (TA) muscles of 4-week-old mice as described [START_REF] Ohanna | Atrophy of S6K1(-/-) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control[END_REF]. Myotubes at day 2 of differentiation were infected with an adenovirus expressing GFP or a constitutively active Akt, NH(2)-terminally myristoylation signal-attached Akt (MyrAkt) [START_REF] Aguilar | S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase[END_REF] at the multiplicity of infection of 75 and incubated for 2 days in DMEM/Ham's F12 medium containing 2% horse serum. Then, brightfield images were taken and analysed using Metamorph software (version 7.04, Molecular Devices). The diameter of at least 400 myotubes was measured at day 4 of differentiation in a region where myonuclei were absent and diameter was constant. Cells were harvested the same day for western blot analysis.

In vivo transfection of mouse skeletal muscle by electroporation Hyalurodinase (0.4 U) was injected along the TA muscle length and after two hours, animals anaesthetized with xylazine-ketamine were injected with 15 µg of plasmids encoding GFP or MyrAkt. Electrical pulses were then applied by two stainless steel spatula placed on each side of the muscle (130 Volts/cm, 6 pulses, 100 ms interval). Muscles were removed and analyzed 10 days later.

Bilateral synergist ablation model

The bilateral synergist ablation model was used to induce hypertrophy of the plantaris (PLN)

muscle by functional overload in AMPKα1 (-/-) and control mice, as previously described [START_REF] Tsika | M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice[END_REF]. On the day of surgery (D0), animals were anaesthetized with xylazine-ketamine and maintained in the surgical plane. A longitudinal incision on the dorsal aspect of the lower hindlimb was made exposing the gastrocnemius muscle. The tendons of the gastrocnemius and soleus muscles were isolated and used to guide in the excision of these muscles without disturbing the PLN muscle. The overlying skin was closed using sterile suture and the mice were allowed to recover in a temperature-controlled area prior to being returned to their cages.

Animals were monitored for signs of pain or post-operative infection.

Tissue collection

Animals were anaesthetized with xylazine-ketamine and overloaded TA muscles or transfected PLN muscles of both legs were removed, cleaned and precisely weighed. Muscles were then frozen in liquid nitrogen for protein extraction or in liquid nitrogen-chilled isopentane for preservation of fiber morphology and stored at -80°C until processed.

Protein extraction, western blotting and AMPK activity assays

Total protein extracts from PLN muscles and muscle cells samples were obtained as described previously [START_REF] Koh | Skeletal muscleselective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3[END_REF]. Fifty µg of protein were subjected to SDS-PAGE and transferred to nitrocellulose membrane. Blots were probed with antibodies against total AMPKα1, AMPKα2 (a kind gift from G. Hardie, University of Dundee, Scotland), phosphorylated forms of eEF2 (Thr56), S6 (Ser235/236), p70S6K (Thr389) and 4E-BP1 (Thr37/46) (Cell Signaling). Measure of AMPK α1 and α2 activities was performed as described previously [START_REF] Sakamoto | Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR[END_REF].

Immunohistochemistry

Serial transverse sections (12-16 µm thick) were cut from TA and PLN muscles in a cryostat microtome (Leica, Germany) maintained at -25°C and stained with hematoxylin and eosin to visualize tissue morphology. We analyzed fiber size by incubating muscle sections with mouse anti-dystrophin Dys2 (Novocastra) and anti-HA (Roche) antibodies and staining with Hoechst dye. The fiber cross-sectional area (CSA) was determined using Metamorph software (version 7.04, Molecular Devices).

Statistical analysis

Results are expressed as means ± SD. We used Student's t test for unpaired data. Differences were considered significant if P < 0.05.

Results

Cell size control in AMPKα α α α1-deficient muscle cells

In order to examine the role of AMPKα1 in the control of muscle cell size, we isolated and analyzed primary muscle cells from AMPKα1 (-/-) and control mice. Western blotting analysis revealed that AMPKα1 protein was undetectable in AMPKα1 (-/-) myotubes and the levels of AMPKα2 protein were similar between AMPKα1 (-/-) and WT myotubes (Figure 1A). The deletion of AMPKα1 caused a 15% increase in myotube diameter (Figure 1B).

Furthermore, myotubes size distribution for AMPKα1 (-/-) cells showed fewer smalldiameter myotubes and increased number of large-diameter myotubes compared to WT cells (Figure 1C). To evaluate the functional consequence of AMPK deletion on mTOR-pathwaymediated hypertrophy, we introduced WT and AMPKα1 (-/-) muscle cells with an adenovirus expressing MyrAkt, an established way of activating the mTOR signaling pathway [START_REF] Pallafacchina | A protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type specification[END_REF][START_REF] Rommel | Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways[END_REF].

We found that the hypertrophic action of MyrAkt was two-fold higher in AMPKα1 (-/-) myotubes compared to WT cells (Figure 1B). The diameter of myotubes increased significantly by 29% in AMPKα1 (-/-) cells vs 16% in WT cells. Furthermore, the degree of hypertrophy (determined by the shift on the right of the cumulated percentage of myotube size distribution) was greater for AMPKα1 (-/-) cells in response to MyrAkt overexpression (Figures 1C). Finally, we observed an increase in p70S6K phosphorylation (+39%) in AMPKα1 (-/-) myotubes introduced with MyrAkt, consistent with a negative action of AMPKα1 on mTOR signaling network (Figure 1D).

MyrAkt-induced cell growth in AMPKα α α α1-deficient muscle

In order to gain further insights into the role of AMPKα1 in the regulation of muscle cell growth in vivo, we transfected AMPKα1 (-/-) and control TA muscles with a plasmid expressing MyrAkt or GFP by electroporation. The hypertrophic action of MyrAkt was significantly higher in AMPKα1 (-/-) skeletal muscle fibers compared to WT fibers (Figure 2). Indeed, the cross sectional area of AMPKα1 (-/-) myofibers increased significantly by 125% compared to 68% for WT myofibers (Figure 2).

Changes in AMPK activity and expression following overload-induced hypertrophy

Since robust activation of AMPKα1 has recently been reported during muscle hypertrophy [START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF], we examined the overloading-induced muscle growth in mice lacking AMPKα1.

Following 7 days of overload, AMPKα1 activity was markedly increased in the PLN of WT mice (+446%; Figure 3A) and remained significantly higher at D21 compared to D0 (+106%;

Figure 3A), although it was significantly lower compared to D7 (Figure 3A). Coincidentally, AMPKα1 expression increased dramatically at D7 (+441%; Figure 4) and at D21 (+336%;

Figure 4) in WT mice. In the PLN muscles of the AMPKα1 (-/-) mice, AMPKα1 activity was ablated and only background counts were detected (Figure 3A). The absence of AMPKα1 expression in PLN muscle of AMPKα1 (-/-) mice was confirmed by western blot analysis (Figure 4). Loss of AMPKα1 resulted in an increase in the basal level of AMPKα2 activity (Figure 3B). In contrast to AMPKα1 activity, AMPKα2 activity was reduced in both WT (-67%; Figure 3B) and AMPKα1 (-/-) mice at D7 (-65%; Figure 3B). The activity of AMPKα2 at D21 for both WT and AMPKα1 (-/-) mice returned to a value closed to the basal level of WT mice (Figure 3B). AMPKα2 expression was unaffected in PLN from AMPKα1 (-/-) and WT mice at D7, but significantly decreased in AMPKα1 (-/-) mice at D21 (Figure 4).

Muscle mass and myofiber size in AMPKα α α α1-deficient mice

The ratio of PLN mass to tibia length was significantly lower in AMPKα1 (-/-) mice compared to WT mice (Figure 5A). Myofiber size distribution of AMPKα1 (-/-) PLN exhibits increased number of small myofibers and fewer large myofibers compared to WT PLN (Figures 5B). Morever, the number of nuclei per fiber in AMPKα1 (-/-) mice was smaller (-22%) compared to WT mice (P<0.01).

Muscle mass and myofiber size following overload-induced hypertrophy After 7 and 21 days of overload, the degree of muscle hypertrophy (determined by PLN mass to tibia length) produced by bilateral functional overload is more pronounced in AMPKα1 (-/-) mice than in WT mice (Figure 5A). PLN mass of AMPKα1 (-/-) mice was increased by 53% (P<0.01) and 76% (P<0.01) at D7 and D21 respectively, whereas PLN mass of WT mice was significantly increased by 32% (P<0.05) and 57% (P<0.01) during the same period (Figure 5A). The degree of hypertrophy, as determined by the shift of myofibers size distribution between D0 and D7 and between D0 and D21, was more pronounced in AMPKα1 (-/-) mice than in WT mice (Figures 5B and5C). Furthermore, analysis of myofiber size distribution shows a clear shift to fibers with larger size at D21 compared to D0 and D7 for both AMPKα1 (-/-) and WT mice (Figures 5C and5D).

mTOR signaling in AMPKα α α α1 (-/-) mice following overload-induced hypertrophy After 7 days of overload, we observed a significant increase of p70S6K phosphorylation in both WT and AMPKα1 (-/-) mice (Figure 6A). This increase in p70S6K phosphorylation between D0 and D7 was greater for AMPKα1 (-/-) mice compared to WT mice (Figure 6A).

In addition, the enhanced phosphorylation p70S6K (Figure 6A) was still present at D21 in AMPKα1 (-/-) mice, whereas it returned to basal level in WT mice (Figure 6A).

Consequently, phosphorylation of p70S6K was significantly higher in AMPKα1 (-/-) mice compared to WT mice at D21. Following 7 and 21 days of overload, phosphorylation of S6 was also significantly increased in AMPKα1 (-/-) and WT mice (Figure 6A). The deletion of AMPKα1 caused an enhanced increase in 4E-BP1 phosphorylation at D7 (P<0.001) and D21 (P=0.051) (Figure 6B). Finally, phosphorylation of eEF2 was significantly higher in AMPKα1 (-/-) mice compared to WT mice at D0 (Figure 6B). Furthermore, we observed a significant increase in phosphorylation of eEF2 in response to 21 days of overload in WT mice (Figure 6B), whereas phosphorylation of eEF2 significantly decreased in AMPKα1 (-/-) mice (-59%; P<0.01; Figure 6B).

Discussion

The major aim of this study was to investigate the role of AMPKα1 in the control of muscle cell size and growth. Previous studies have demonstrated that AMPK integrates fuel sensing with cell growth control and contributes to the atrophic adaptations of p70S6K-deficient muscle [START_REF] Aguilar | S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase[END_REF]. Here, we show that the size of AMPKα1-deficient myotubes is larger than AMPKα1-expressing control cells, demonstrating a unique role for AMPKα1 in the control of cell size. However unexpectedly, the mass of PLN muscle in basal state was significantly lower in AMPKα1 (-/-) mice compared to WT mice. One potential reason for this lower muscle mass in AMPKα1 (-/-) mice is a reduced ATP production, thus providing less energy for protein synthesis and growth. However, this should not be the case in cultured AMPKα1

(-/-) myotubes as their size is larger than AMPKα1-expressing control myotubes.

Interestingly, the role for AMPKα1 isoform in the control of muscle energy balance has been recently challenged and data obtained from LKB1-deficient muscles suggest that skeletal muscle metabolic adaptation is rather dependent on AMPKα2 than on AMPKα1 activation [START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF]. Furthermore, we previously reported that the lack of AMPKα2 isoform was associated with a lower ATP content in skeletal muscle [START_REF] Jorgensen | Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle[END_REF], indicating that remaining AMPKα1

isoform was not able to compensate for the loss of AMPKα2 isoform to maintain muscle energy balance. Lastly, it should be also noted that in AMPKα1 (-/-) PLN muscle, AMPKα2 activity is increased compared to WT muscle in the basal state, resulting probably in a compensatory response for ATP production. Another hypothesis for the lower basal PLN mass in AMPKα1 (-/-) mice is a role for satellite cells that are partly responsible for postnatal muscle growth [START_REF] Grand | Satellite and stem cells in muscle growth and repair[END_REF][START_REF] Grand | Skeletal muscle satellite cells and adult myogenesis[END_REF]. Although AMPK function in satellite cells has not been investigated yet, an impaired or reduced satellite cell proliferation in the absence of AMPKα1, as illustrated by the lower number of nuclei per fiber in AMPKα1 (-/-) PLN muscle, might explain this unexpected result [START_REF] Fulco | Comparing and contrasting the roles of AMPK and SIRT1 in metabolic tissues[END_REF].

Recent evidence supports the existence of distinct regulatory pathways and functions for AMPKα1 and AMPKα2 isoforms. The overloading-induced hypertrophy is associated with a significant increase in AMPKα1 expression and activity in WT muscle, whereas the activity of AMPKα2 is reduced at D7 and returns close to basal value at D21 for both WT and AMPKα1 (-/-) muscles. This marked activation of AMPKα1 following 7 and 21 days of chronic overload has been suggested to limit muscle mass growth [START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF]. AMPKα2 appears to be preferentially activated in response to acute exercise [START_REF] Fujii | Exercise induces isoform-specific increase in 5'AMP-activated protein kinase activity in human skeletal muscle[END_REF][START_REF] Wojtaszewski | Isoform-specific and exercise intensity-dependent activation of 5'-AMP-activated protein kinase in human skeletal muscle[END_REF][START_REF] Durante | Effects of endurance training on activity and expression of AMP-activated protein kinase isoforms in rat muscles[END_REF][START_REF] Stephens | Progressive increase in human skeletal muscle AMPKalpha2 activity and ACC phosphorylation during exercise[END_REF][START_REF] Nielsen | 5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle[END_REF][START_REF] Yu | Metabolic and mitogenic signal transduction in human skeletal muscle after intense cycling exercise[END_REF][START_REF] Lee-Young | Acute Exercise Does Not Cause Sustained Elevations in AMPK Signaling or Expression[END_REF] or by endurance training program (47) whereas low intensity contraction stimulation activates AMPKα1 in skeletal muscle [START_REF] Toyoda | Low-intensity contraction activates the alpha1isoform of 5'-AMP-activated protein kinase in rat skeletal muscle[END_REF]. Interestingly, it has been shown that only AMPKα1 activation is required for stimulation of glucose uptake by twitch contraction [START_REF] Jensen | AMPK alpha1 activation is required for stimulation of glucose uptake by twitch contraction, but not by H2O2, in mouse skeletal muscle[END_REF]. In this condition, AMPKα1 activation was not accompanied by an increase in AMP level and AMP/ATP ratio, suggesting that AMPKα1 activation induced by low-intensity contraction is regulated by an AMPindependent [START_REF] Toyoda | Low-intensity contraction activates the alpha1isoform of 5'-AMP-activated protein kinase in rat skeletal muscle[END_REF] and/or LKB1-independent mechanism. Accordingly, LKB1 appears to be essential for AMPKα2 activity in different muscle types whereas AMPKα1 activity is only partially affected in LKB1-deficient skeletal muscle [START_REF] Koh | Skeletal muscleselective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3[END_REF][START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF]. Recently, it has been demonstrated that skeletal muscle hypertrophy is normal in response to chronic mechanical overload in the absence of LKB1 due to significant increase in the activation of the AMPKα1 isoform [START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF]. One mechanism for AMPKα1 activation could be through the Ca 2+ /calmodulindependent protein kinase kinases (CaMKKs) and the TGFβ-activated protein kinase (TAK-1) [START_REF] Mcgee | Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice[END_REF].

In the present study, we provide genetic evidence for a negative role for AMPKα1 in the control of mTOR signaling and growth in muscle cells. The hypertrophic action of MyrAkt, an effector known to stimulate skeletal muscle protein synthesis through activation of mTOR signaling, was enhanced in primary cultured myotubes and myofibers deleted for AMPKα1.

However, the effect of rapamycin or Torin1 (potent allosteric mTORC1 inhibitors) in AMPKα1-deficient muscle would bring additional evidence for a negative role on mTOR signaling. We also show that muscle hypertrophy produced by functional overload is greater in mice deleted for AMPKα1 catalytic subunit compared to WT mice. A strong correlation between increased AMPK activity in the PLN muscle from old rats and impaired overloadinduced skeletal muscle hypertrophy has been recently reported [START_REF] Thomson | Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation[END_REF]. AMPK phosphorylation was negatively correlated with the amount of p70S6K phosphorylated at the mTOR-specific Thr 389 residue in response to 7 days of chronic overload, supporting the notion that AMPK inhibited p70S6K phosphorylation in skeletal muscle via suppression of mTOR [START_REF] Bolster | AMPactivated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling[END_REF][START_REF] Thomson | Impaired overload-induced muscle growth is associated with diminished translational signalling in aged rat fast-twitch skeletal muscle[END_REF].

Conversely, enhanced hypertrophy in AMPKα1 (-/-) muscle is associated with higher activation of mTORC1 signaling pathway, as determined by increased phosphorylation of p70S6K and 4E-BP1 at D7 and D21. These results are in accordance with studies showing the importance of mTOR-p70S6K signaling and 4E-BP1 phosphorylation in the hypertrophic response of chronically-overloaded muscle [START_REF] Bodine | Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[END_REF][START_REF] Reynolds | Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load[END_REF][START_REF] Thomson | Impaired overload-induced muscle growth is associated with diminished translational signalling in aged rat fast-twitch skeletal muscle[END_REF]. In addition, we observed a decrease in eEF2 phosphorylation in AMPKα1 (-/-) muscle following 7 and 21 days of overload, indicating an improvement of protein elongation. Thus, in the absence of AMPKα1, unphosphorylated (active) eEF2 levels are higher in response to chronic overload with a parallel increase in mTORC1 signaling leading to a greater muscle hypertrophy. Hence, AMPKα1 behaves as a negative effector required to limit mTORC1 activity and to inhibit overgrowth of skeletal muscle in response to hypertrophic stimuli.

In conclusion, we demonstrate an essential role for AMPKα1 in the adaptation of skeletal muscle growth to hypertrophy. The major new findings of this study are as follows: [START_REF] Viollet | Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders[END_REF] muscle cells hypertrophy in vitro and in vivo is more pronounced in mice deleted for AMPKα1 catalytic subunit compared to control mice; (2) phosphorylation of mTORC1 signaling downstream targets, controlling protein synthesis, is enhanced in the absence of AMPKα1 following chronic overload and is associated with a greater muscle hypertrophy.

Our study provides important knowledge on the role of AMPK in the molecular mechanisms underlying the cell size control that may open new avenues of intervention against age-related skeletal muscle atrophy. Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older humans is limited. The prevention of sarcopenia, which is the consequence of a reduction of protein synthesis and an increase in muscle protein degradation, would provide an obvious clinical benefit.

Figure 1 :

 1 Figure 1 : Cell size control in primary muscle cells lacking AMPKα α α α1. (A) Primary muscle cells isolated from AMPKα1 (-/-) and control mice were differentiated into myotubes and AMPKα1 and AMPKα2 protein content was assessed by western blot analysis. A representative immunoblot of AMPKα1 (upper panel) and AMPKα2 (lower panel) is shown. (B) Size of WT and AMPKα1 (-/-) myotubes transduced with 75 moi GFP and MyrAkt adenoviruses as indicated. Results are presented as a percentage of WT muscle cells transduced with GFP adenovirus. Representative bright-field images of myotubes transduced with GFP and MyrAkt adenoviruses are shown in the upper part of the figure. (C) Frequency distribution of size of WT and AMPKα1 (-/-) myotubes transduced with GFP and MyrAkt adenoviruses. Size of myotubes was determined with the diameter of at least 400 myotubes measured in a region where myonuclei were absent and diameter was constant. Data from five experiments on two different cultures are shown in B and C. Results are represented as means ± SD in B and C. Different from GFP cells of the same genotype in B : $ P<0.05, $$ P<0.01; Different for the same conditions of infection in B : # P<0.05, ## P<0.01. WT myotubes different from AMPKα1 (-/-) myotubes both transduced with GFP adenovirus in C : # P<0.05, ## P<0.01, ### P<0.001. WT myotubes different from AMPKα1 (-/-) myotubes both transduced with MyrAkt adenovirus in C : § P<0.05, § § § P<0.001. (D) Thr389 p70S6K phosphorylation was determined 2 days after infection. Representative immunoblot is shown in the upper part of the figure. Protein content is expressed in arbitrary units relative to GFP infected cells for each genotype. Results are represented as means ± SD. Different from GFP cells of the same genotype : $ P<0.05, $$ P<0.01.

Figure 2 :

 2 Figure 2 : Change in cross-sectional area of WT and AMPKα α α α1 (-/-) myofibers in tibialis anterior muscle transfected with MyrAkt. Changes in size of WT and AMPKα1 (-/-)

Figure 3 :

 3 Figure 3 : AMPK activity in plantaris muscle following overload-induced hypertrophy in WT and AMPKα α α α1 (-/-) mice. (A) AMPKα1 and (B) AMPKα2 activity (mU/mg) measured using immunoprecipitate kinase assays in plantaris muscle following 0 (D0), 7 (D7) and 21 (D21) days of overload. Results are represented as means ± SD, n = 6 for each genotype. Different from Day 0 : $ P<0.05; $$ P<0.01, $$$ P<0.001; Different from Day 7 : § P<0.05, § § § P<0.001; Different from WT : # P<0.05; ## P<0.01.

Figure 4 :

 4 Figure 4 : AMPK expression in plantaris muscle following overload-induced hypertrophy in WT and AMPKα α α α1 (-/-) mice. AMPKα1 and AMPKα2 expression measured by western blot analysis in plantaris muscle following 0 (D0), 7 (D7) and 21 (D21) days of overload. Quantification by densitometry of immunoblots is reported alongside the figure. Results are represented as means ± SD, n = 4 for each genotype. Different from Day 0 : $$$ P<0.001; Different from WT : ## P<0.01.

Figure 5 :

 5 Figure 5 : Muscle mass and fiber size in response to overload-induced hypertrophy in WT and AMPKα α α α1 (-/-) mice. (A) Mass (g) of plantaris muscles normalized to the tibia length (cm) following 0 (D0), 7 (D7) and 21 (D21) days of overload is shown for WT and AMPKα1 (-/-) mice. Percentage of plantaris muscles mass increase following 7 and 21 days

Figure 6 :

 6 Figure 6 : Changes in phosphorylation levels of p70S6K, S6K, 4E-BP1 and eEF2 in plantaris muscle following overload-induced hypertrophy in WT and AMPKα α α α1 (-/-) mice. (A) p70S6K phosphorylation at Thr389 (upper part) and S6 ribosomal protein phosphorylation at Ser235/236 (lower part) measured by western blot analysis in plantaris muscle before (D0) and following 7 (D7) and 21 (D21) days of overload. (B) 4E-BP1 phosphorylation at Thr37/46 (upper part) and eEF2 phophorylation at Thr56 (lower part) measured by western blot analysis in plantaris muscle before (D0) and following 7 (D7) and 21 (D21) days of overload. Quantification by densitometry of all immunoblots is reported alonside the figure. Results are represented as means ± SD, n = 4 for each genotype. Different from Day 0 : $ P<0.05, $$ P<0.01, $$$ P<0.001; Different from WT : # P<0.05, ## P<0.01, ### P<0.001.

Acknowledgments

This work was supported by the European Union FP6 program (EXGENESIS Integrated Project LSHM-CT-2004-005272), ANR (Agence Nationale de la Recherche) and AFM (Association Française contre les Myopathies). KS is supported by the UK Medical Research Council and Diabetes UK and by the companies (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck & Co. Inc, Merck KGaA and Pfizer).

The technical assistance of Charlotte Lahoute and Véronique Fauveau is gratefully acknowledged. We thank Grahame Hardie, University of Dundee, for providing AMPKα1 and α2 antibodies.


Rikesh M Rajani

Nicolas Dupré

Valérie Domenga-Denier

Guillaume Van Niel

Xavier Heiligenstein

Anne Joutel
email: anne.joutel@inserm.fr.

Affiliations

Characterization of early ultrastructural changes in the cerebral white matter of CADASIL small vessel disease using high pressure freezing/freeze-substitution Authors

Keywords: CADASIL, small vessel disease, white matter, high pressure freezing/freeze-substitution, myelin splitting, inner tongue

Aims. The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease.

Methods. We used high pressure freezing and freeze substitution (HPF/FS) in combination with high resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3 R169C ) mice aged 4 to 15 months to study white matter lesions in the corpus callosum.

Results. We first optimized the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months, then vesiculation of the inner tongue and myelin sheath thinning at 15 months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age.

Conclusion.

We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism.

Introduction

Cerebral small vessel disease (SVD) is the leading cause of vascular cognitive impairment that typically manifests with lacunar infarcts and white matter (WM) lesions [START_REF] Pantoni | Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges[END_REF]. Cerebral autosomal dominant angiopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of SVD, caused by highly stereotyped mutations in NOTCH3, a receptor which is predominantly expressed in pericytes and smooth muscle cells around small vessels. Unlike sporadic SVD, comorbidities and age-related pathologies are uncommon in CADASIL patients. Therefore, CADASIL is now widely recognized as an invaluable model for studying mechanisms of SVD [START_REF] Chabriat | CADASIL: yesterday, today, tomorrow[END_REF].

One of the earliest and most consistent features of CADASIL observed on brain magnetic resonance imaging is the presence of WM hyperintensities. These present as punctiform lesions from 30 years of age, and become more diffuse and symmetrical with age [START_REF] Chabriat | CADASIL: yesterday, today, tomorrow[END_REF]. The volume of these WM hyperintensities is mainly related to deficits in executive functioning, and especially in the speed of cognitive processing, the earliest and most prominent cognitive manifestations of CADASIL and SVD in general [START_REF] Benisty | White-matter lesions without lacunar infarcts in CADASIL[END_REF][START_REF] Duering | Strategic role of white matter tracts in vascular cognitive impairment: a voxel-based lesion-symptom mapping study in CADASIL[END_REF]. Several studies strongly suggest that histopathological correlates of WM changes in SVD are heterogeneous [START_REF] Schmidt | Heterogeneity in age-related white matter changes[END_REF]. Our recent analysis of post-mortem brain tissue from patients with CADASIL confirmed this. Staining of tissue sections with Luxol fast blue, Haematoxylin & Eosin or anti-fibrinogen antibody, used as a marker of blood brain barrier leakage, revealed that WM lesions encompass at least 3 distinct categories of damage, namely: (i) WM lesions around lacunes, characterized by WM pallor with strong anti-fibrinogen staining; (ii) WM lesions surrounding enlarged perivascular spaces characterized by a non-uniform disorganization and pallor of the WM, with marked anti-fibrinogen staining, that bear some similarity to lesions which have previously been described as microinfarcts; and (iii) a category which we termed pure WM lesions that exhibit myelin pallor, but do not contain any overt pathology and is essentially devoid of fibrinogen leakage [START_REF] Rajani | Blood brain barrier leakage is not a consistent feature of white matter lesions in CADASIL[END_REF]. Because postmortem tissue from CADASIL patients generally represents a late stage of the disease, the chronological order of these 3 categories of WM damage cannot be determined. The TgNotch3 R169C mouse, a well characterized model of CADASIL, exhibits WM changes initially in the form of increased punctate staining using a specific monoclonal antibody (SMI-94) against myelin basic protein (MBP) which is thought to mark uncompacted or degraded myelinand later in life (20 months) as a pallor and vacuolization of WM tracts using Luxol fast blue staining [START_REF] Joutel | Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease[END_REF][START_REF] Cognat | Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model[END_REF][START_REF] Capone | Reducing Timp3 or vitronectin ameliorates disease manifestations in CADASIL mice[END_REF]. Importantly, the TgNotch3 R169C mouse does not show any signs of lacunes or enlarged perivascular spaces, suggesting that pure WM lesions are the earliest WM changes in CADASIL [START_REF] Joutel | Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease[END_REF].

The mechanism that leads to these pure WM lesions is still unknown. Experimental studies have bolstered the idea that WM lesions might be caused by an increase in blood brain barrier permeability as a result of pericyte deficiency [START_REF] Montagne | Pericyte degeneration causes white matter dysfunction in the mouse central nervous system[END_REF]. However, our recent work studying pericyte labelling, endogenous blood-borne proteins, and injected exogenous small or large tracers has shown that pericyte coverage is unaltered and blood brain barrier integrity is preserved in the damaged WM of TgNotch3 R169C mice, a finding consistent with the lack of fibrinogen leakage in pure WM lesions in tissue sections from patients [START_REF] Rajani | Blood brain barrier leakage is not a consistent feature of white matter lesions in CADASIL[END_REF]. This suggests that in order to understand this important feature of CADASIL, it is necessary to study the WM directly at an early stage of the disease.

WM fibres are axons surrounded by a myelin sheath, a multi-layered stack of self-associating plasma membranes tightly wrapped around the axon. Electron microscopy (EM) provides the necessary and required resolution to study WM fibres. However, in samples fixed and dehydrated using conventional methods, at 4°C or room temperature, preservation of myelin is a notorious problem, since the multilamellar structure tends to split upon chemical fixation or dehydration. While optimization of this technique can lead to excellent preservation in younger animals, this becomes particularly deleterious in older animals or those with severe WM damage. High pressure freezing (HPF), with or without prior chemical fixation, in combination with freeze substitution (FS) has recently emerged as a valuable alternative to the standard preparation of EM samples. This technique involves freezing the sample rapidly at a high pressure (2100 bar) to transform water into an amorphous state, preventing the formation of ice crystals, and then carrying out the dehydration step at temperatures below freezing over several days to hold the tissue structure in place and reduce artefacts [START_REF] Möbius | Electron microscopy of the mouse central nervous system[END_REF][START_REF] Möbius | Electron microscopy of myelin: Structure preservation by high-pressure freezing[END_REF].

In this study, we assessed WM changes at an early stage of CADASIL, at both the ultrastructural and cellular levels. To achieve this, we used the TgNotch3 R169C mouse model, and because WM changes in this model develop with an anteroposterior and medio-lateral gradient (6), we selected the medial part of the genu of the corpus callosum. To study the WM at the ultrastructural level, we optimized a novel protocol including chemical fixation of the samples followed by HPF/FS in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. To further analyse the WM at the cellular level, we used immunohistochemistry in combination with confocal microscopy.

Results presented in this study provide insight into the earliest substrates of WM changes in CADASIL that suggest a primary myelin defect in this disease, and help to understand the mechanisms of this defect.

Methods

Animals

TgNotch3 R169C mice overexpress the rat Notch3 gene containing the mutation R169C found in CADASIL patients and were maintained on an FVB/N background [START_REF] Joutel | Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease[END_REF]. As controls, we used TgNotch3 WT mice, overexpressing the wild type rat Notch3 gene, and wild type mice interchangeably, as it has previously been shown that there are no white matter differences between TgNotch3 WT mice and wild type mice [START_REF] Joutel | Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease[END_REF][START_REF] Cognat | Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model[END_REF]. One 15-month-old wild type mouse and one 15-month-old TgNotch3 R169C mouse was male; the rest of the animals used in this study were female. Preparation of tissue samples for EM analysis Mice were deeply anaesthetized with sodium pentobarbital (80 mg/kg) and transcardially perfused with 0.1M heparinized phosphate buffer (PB) followed by Karlsson-Schultz fixative (K-S fix; 4% paraformaldehyde (PFA), 2.5% glutaraldehyde in 0.1M PB). Brains were extracted, split into hemispheres, and fixed overnight in K-S fix at 4 o C. For processing using HPF/FS, 100μm thick sagittal sections were cut using a Leica VT1200S vibratome. For processing using conventional techniques, samples were further fixed for 1 week in 1% PFA in PB, before 1 mm thick sagittal sections were cut using a brain matrix. From these sections, a 2 mm diameter disc containing the most anterior portion of the corpus callosum (genu) at the midline was punched out of the section using a biopsy punch.

Processing using HPF/FS Prefixed samples were immediately transported to the HPF machine in a solution of 1% PFA in PB on ice. Specimens were placed in 3 mm diameter aluminium sample carriers within a solution of 20% polyvinylpyrrolidone (PVP; MW=10,000) in PB, and frozen using a HPM Live µ high pressure freezing machine (CryoCapCell, France). Samples were stored for up to 1 week in liquid nitrogen before freeze substitution. Freeze substitution was carried out using a Leica EM AFS machine by modifying a previously used protocol [START_REF] Weil | Intracerebral injections and ultrastructural analysis of high-pressure frozen brain tissue[END_REF]. Briefly, samples were incubated in 0.1% tannic acid in acetone for 24h at -90 o C followed by 4 acetone washes each of 30mins at -90 o C. Samples were then incubated in 2% osmium tetroxide, 0.1% uranyl acetate in acetone for 32-56h at -90 o C, before the temperature was raised at 5 o C/h to -20 o C, then incubated for 16h at -20 o C, followed by a temperature rise of 10 o C/h to 4 o C, followed by a 1h incubation at 4 o C. Samples were then washed in acetone (4x 30 minutes at 4 o C, followed by 1 hour while temperature was raised to room temperature), before being incubated in increasing concentrations of Epon in acetone (33% for 2h, 50% for 2h, 67% for 2h, 90% overnight, 100% for 2h, 100% for 2h), and finally placed in silicone moulds in Epon at 60 o C overnight for polymerization.

Processing using conventional techniques

Prefixed samples were incubated in 1% osmium tetroxide in PB for 2h at 4 o C, before being dehydrated in increasing concentrations of ethanol at room temperature over 2.5h. Samples were then incubated in increasing concentrations of Epon in propylene oxide before being incubated in pure Epon overnight and finally being placed in moulds in Epon at 60 o C for 24h for polymerization.

Sectioning, imaging and quantification of EM samples Samples were sectioned using a Leica Ultracut UCT. Semithin sections (1μm) were stained with toluidine blue to reveal myelin, and blocks were trimmed to leave only the most anterior and ventral portion of the corpus callosum. 3-5 serial ultrathin sections (70nm) were mounted on uncoated copperpalladium grids, and stained with 2% uranyl acetate (1min) and 0.2% lead citrate (4mins). Samples were imaged using a Tecnai Spirit transmission electron microscope (Thermo Fisher Scientific).

For assessment of pathology, randomly selected, non-overlapping images were taken at 6,500x magnification and the experimenter was blinded throughout the process (imaging, image analysis and quantifications). Images were analysed using the Fiji distribution of ImageJ (open source).

Quantification was performed on 4-5 animals per group with at least 6 fields of view covering at least 250 μm 2 and at least 250 (quantification of pathologies) or 150 (g-ratios and t-ratios) myelinated axons per animal; exact numbers of features in each animal are given in Supplementary Table 1. A myelinated axon was defined as having: inner tongue vesiculation if there were multiple vesicles within the inner tongue; a split myelin sheath if there was more than one point of decompaction; myelin outfolding if the myelin sheath bulged to an extent which significantly disrupted circularity. Myelin debris was defined as multilamellar myelin sheaths lacking an axon. A myelinated axon was defined as having a degenerating axon if the axon became dark and its content of organelles very difficult to distinguish.

Segmentation of the axon, the inner tongue and the myelin was carried out manually using the Fiji distribution of ImageJ. G-ratios, a measure of myelin thickness, were corrected for inner tongue diameter and calculated as: [axon diameter]/[axon+myelin diameter]-([axon+inner tongue diameter]-[axon diameter]). T-ratios, a measure of inner tongue thickness, were calculated as: [axon diameter]/[axon+inner tongue diameter]. Diameters were calculated assuming circularity based on areas, where circularity tests were met. T-ratios are presented separately for small and large calibre axons, using a cut-off of 0.6µm as previously defined [START_REF] Poggi | Cortical network dysfunction caused by a subtle defect of myelination[END_REF].

Immunohistochemistry

Mice were deeply anaesthetized with sodium pentobarbital (80 mg/kg) and transcardially perfused with 0.1M heparinized PB followed by 4% PFA. Brains were extracted, split into hemispheres, and postfixed for either 1 hour (oligodendroglia) or overnight (microglia) before cryopreservation and freezing.

Brains were sectioned sagittally using a cryostat at either 10µm onto glass slides (oligodendroglia) or 16µm and processed as floating sections (microglia). Sections were blocked in 10% goat serum, 0.3% triton in PBS, before being incubated with primary antibodies [Olig2 (1:200, AB9610, Millipore), APC Imaging and analysis of fluorescent images Fluorescent images were acquired using a Leica SP8 confocal microscope, capturing a 10 µm (oligodendrocytes) or 12μm (microglia) thick stack with optical sections taken at 1µm intervals (objective 40x). At least 2 fields of view (290 x 290 µm) were acquired from the most anterior part of the corpus callosum, and at least 3 sections were analysed per animal. All quantifications were performed using the Fiji distribution of ImageJ and all procedures were performed with prefixed parameters. The experimenter was blinded to the genotype throughout quantifications.

The number of oligodendrocyte precursor cells and oligodendrocytes was counted manually on sections double stained for Olig2 and APC (CC-1). The number of microglial cells was counted manually on sections double stained for Iba1 and CD68. Results are expressed as the number of cells per mm 2 .

Statistics

Graphs were created and statistics calculated using GraphPad Prism 7. All data are shown as mean ± standard error of the mean (SEM). The number of biological replicates for each experiment is indicated in the appropriate figure legend. Significance was calculated using either a Student's t test or one-way or two-way ANOVA as appropriate with Tukey's or Sidak's post-hoc tests.

Results

Optimization of the HPF/FS protocol to assess WM integrity in the corpus callosum genu from aged mice

In the traditional HPF/FS protocol, fresh tissue is quickly dissected and frozen at high pressure without any prior chemical fixation. While this technique allows excellent ultrastructural preservation of myelin in the optic nerve, it has rarely been used on the corpus callosum [START_REF] Weil | Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases[END_REF] and, to the best of our knowledge, never on the mouse corpus callosum from a chronic disease model. The challenge is that the brain must be rapidly extracted, sectioned at <200µm, and the tissue of interest dissected and frozen in 5-10 minutes, something which, unlike the optic nerve, is extremely difficult to achieve with a tiny sample of the mouse corpus callosum. Moreover, fresh brain tissue is difficult to handle and stretching fresh tissue is suspected to cause detachment of myelin lamellae from the axon [START_REF] Möbius | Electron microscopy of the mouse central nervous system[END_REF]. Therefore, we optimized this technique to the study of the genu of the corpus callosum in mice aged from 6 to 15 months, by combining the advantages of a standard chemical fixation prior to freezing with those of HPF and FS (Figure 1A). While a similar approach has been previously used to study brain ultrastructure in young rats, it has not been optimized for myelin preservation in older animals [START_REF] Sosinsky | The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications[END_REF].

We found that a glutaraldehyde-paraformaldehyde fixation combining perfusion/immersion fixation preserved the ultrastructure of the WM much better than either an isolated perfusion or a sole immersion fixation (not shown). Testing different space fillers in the sample carrier revealed that PVP outperformed bovine serum albumin and yeast paste, in terms of myelin splitting. Moreover, consistent with a previous report [START_REF] Möbius | Electron microscopy of the mouse central nervous system[END_REF], extracellular spaces seemed artefactually larger using yeast paste (Supplementary Figure 1). Finally, we noticed that thinner sections (100µm) resulted in better quality freezing, although we did not attempt to quantify this effect.

It has been suggested that myelin splitting is mainly an effect of chemical fixation and tissue dehydration. Therefore, we sought to compare qualitatively the effect of freeze-substitution with a conventional dehydration performed at room temperature, using samples of corpus callosum genu from the same prefixed specimens. The most astonishing difference was the superior preservation of myelin in the freeze-substituted samples. Indeed, myelin membranes in the freeze-substituted samples were sharper and smoother, whereas membranes in the conventionally processed samples were frequently undulating and split (Figure 1B-C). Moreover, the outer and the inner tonguesthe ab and adaxonal non-myelin compartments respectivelywere more visible and displayed finer details in the freezesubstituted compared to the conventionally processed samples (Figure 2).

Ultrastructural analysis of WM changes in the corpus callosum of CADASIL mice using HPF/FS

The TgNotch3 R169C mouse model (which we will refer to as CADASIL mice), is a well characterized model of CADASIL [START_REF] Joutel | Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease[END_REF]. Mutant mice develop WM changes in the form of increased punctate staining using a specific monoclonal antibody (SMI-94) against MBP, which is thought to mark uncompacted or degraded myelin, starting at 6 months of age and progressively worsening [START_REF] Cognat | Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model[END_REF][START_REF] Capone | Reducing Timp3 or vitronectin ameliorates disease manifestations in CADASIL mice[END_REF]. These defects are more severe in the anterior and medial part of corpus callosum (6), therefore we focused our analysis on this brain region. WM samples from CADASIL and control mice were prefixed and processed in parallel using HPF/FS as described above. We assessed WM changes on electron micrographs from 4 to 5 mice per group, first at 6 months and then at 15 months of age, to examine the temporal dynamics of emerging lesions (Supplementary Table 1).

The most striking and earliest change we observed in CADASIL mice was a significant increase in the percentage of axons with myelin lamellae splitting at 6 months of age. The observation of noncompacted cytoplasmic channels within these split membranes suggests that these are true defects rather than stretching or fixation artefacts (Figure 3A-C and Figure 2 for comparison of a normal myelin sheath). This was followed at 15 months by an increase in the percentage of axons whose myelin sheath displayed vesiculation or tubulation at the inner tongue (Figure 3D-F). We also observed focal bulging of entire stacks of compacted myelin membrane, also called myelin outfolding (Figure 3G-H) and myelin debris (Figure 3J-K), pathologies which have been described in myelin mutants or brain ageing [START_REF] Erwig | Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin[END_REF][START_REF] Safaiyan | Age-related myelin degradation burdens the clearance function of microglia during aging[END_REF], but in comparable numbers in CADASIL and control samples (Figure 3I,L). To further quantify changes at the level of the inner tongue, we calculated the t-ratio, a measure of the thickness of the inner tongue relative to the axon diameter (Supplementary Figure 2) [START_REF] Poggi | Cortical network dysfunction caused by a subtle defect of myelination[END_REF]. T-ratio measurements suggested an enlargement of the inner tongue in small calibre axons of CADASIL mice at both ages. This was further confirmed by separately analysing the t-ratio in the small (< 0.6m) versus large (> 0.6µm) calibre axons (Figure 4).

Although, the percentage of myelinated axons was comparable between CADASIL and control mice at both 6 and 15 months of age (Figure 5A), the frequency distribution of myelinated axons showed that in older CADASIL animals (15 months) there were fewer large calibre myelinated axons (Figure 5B-C). To better understand any changes in myelin of CADASIL mice, we calculated the g-ratio, a measure of the thickness of the myelin sheath relative to the axon diameter, and plotted it against the axon calibre. In this analysis, we excluded the severely split fibres, as shown on Figure 3B, that might artificially increase the myelinated fibre diameter and bias the g-ratio. Moreover, due to the phenotype at the inner tongue, we used a corrected g-ratio, in which the area of the inner tongue was subtracted (Supplementary Figure 2). At both 6 and 15 months of age, the "corrected g-ratio" scatter plot showed an upshift of the cloud in CADASIL mice, suggesting myelin thinning especially in larger axons (Figure 5D-E).

Remarkably, myelin defects in CADASIL mice were not associated with overt axonal lesions at the ultrastructural level. Indeed, we found almost no degenerating axons at 6 months and a low but comparable number of degenerating axons in WT and CADASIL mice at 15 months (Supplementary Figure 3).

Analysis of cellular changes in the corpus callosum of CADASIL mice using immunohistochemistry

We then sought to determine whether myelin defects observed in CADASIL mice were caused by the loss of oligodendrocytes. To achieve this, we labelled brain sections with Olig2 and CC-1 antibodies and quantified the number of oligodendrocytes and oligodendrocyte precursor cells (OPCs) (Figure 6A). To align with our ultrastructural studies, we focused our quantifications on the anterior and medial part of the corpus callosum. We selected 3 time points: before myelin pathology (4 months), and at early (6 months) and established stages of myelin defect (12 months). We found no evidence of a reduction in the number of mature oligodendrocytes (Olig2 + and CC-1 + cells) in CADASIL mice at any age (Figure 6B). We did, however, find an increase in the number of OPCs (Olig2 + and CC-1 -) at 12 months of age (Figure 6C). The fact that OPC numbers were only significantly increased in older animals, but neither at 6 nor at 4 months, suggests that this increase in OPC numbers is as a response to myelin damage. Finally, we examined microglia cells, as these cells have been implicated in both exacerbating and repairing WM lesions. We labelled brain sections with Iba1 and CD68 antibodies and quantified the number of positive cells again in the anterior and medial part of the corpus callosum (Figure 7A).

We found an increase in the number of Iba1 positive cells in older (15-month-old) but not in younger (6-month-old) CADASIL mice. However, we did not see, at any age, a significant increase in the number of Iba1 positive cells also positive for CD68, a marker of activated microglia (Figure 7B-C).

These results suggest that WM changes in CADASIL mice are associated with a low-grade inflammation, which is secondary rather than being causative of them.

Discussion

In this study, we took advantage of recent advances in the development of HPF/FS to improve visualization of the ultrastructure of cerebral WM and to elucidate the early myelin pathology that occurs in CADASIL, an archetypal SVD. We first optimized the protocol for the analysis of murine corpus callosum by including a prefixation step, sectioning the samples at 100µm and using PVP as a space filler in the sample carrier. Our high-resolution EM analysis of CADASIL mice shows that the pathological process affects first and foremost the myelin sheath and the inner tongue. Specifically, our ultrastructural analysis reveals that mutant mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months. We further detect vesiculation of the inner tongue and myelin sheath thinning, especially in larger axons, at 15 months of age. Our cellular analysis suggests that these defects are not caused by a loss of oligodendrocytes, nor by a block in the differentiation of OPCs, and that they are associated at later stages with a low-grade microglial activation. Finally, our ultrastructural analysis supports the notion that myelinated axons are preserved, at least at this stage of the disease.

The use of HPF/FS has been instrumental in the elucidation of WM changes in CADASIL mice. Indeed, splitting of myelin lamellae is frequently considered as a fixation/dehydration artefact in EM samples that are conventionally processed. During the optimization of the HPF/FS procedure, we found that HPF/FS compared to the conventional approach strongly reduced splitting of myelin lamellae despite the use of prefixed specimens, which we deemed necessary. This suggests that in our hands myelin splitting is mainly caused by the room temperature dehydration steps of conventional EM sample preparation. Although we cannot formally exclude that myelin splitting might have been exacerbated in the CADASIL specimens, the observation of cytoplasm within the split area makes us confident that what we observed is a true splitting of the myelin lamellae. A second added value of the use of HPF/FS was the opportunity to better visualize pathological changes at the level of the inner tongue, which is much less visible and displays much less detail in conventionally processed samples.

In fact, we have been able to detect an abnormal vesiculation at the inner tongue and an enlarged inner tongue of small calibre axons in CADASIL mice. In a previous EM study using samples processed conventionally, we reported that myelinated axons in CADASIL mice aged 20 months displayed large intra-myelinic vacuoles containing aberrant myelin sheets usually located in the innermost layer of the myelin [START_REF] Cognat | Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model[END_REF]. Because it is likely that pathological changes of myelin worsen with age, we surmise that such vacuoles may correspond to the ultimate stage of a very severe vesiculation of the inner tongue.

Nevertheless, we believe that the size of these vacuoles is likely exaggerated by the conventional preparation of samples. Also, using this conventional approach, the specific splitting of myelin layers, which we interpreted as an artefact, could not be appreciated in CADASIL mice. An additional strength in current experimental design has been the use in all of our EM analyses of the same region of the corpus callosum, i.e., the medial part of the genu, that we sectioned in a way to get cross sections of transcallosal fibres. Thus, we are confident that the myelin defects we report in this study are not merely regional variations within the corpus callosum.

Our results are highly suggestive of a primary myelin injury at the early stage of CADASIL disease. In support of this interpretation is the observation that myelin defects exhibited by CADASIL mice are highly reminiscent of those recently reported in models of demyelinating diseases [START_REF] Weil | Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases[END_REF]. In those models, induced either by administration of Cuprizone, anti-aquaporin 4 or anti-myelin oligodendrocyte glycoprotein antibodies into the corpus callosum, the WM exhibits a common pattern of myelin breakdown with vesiculation and enlargement of the inner tongue, and splitting of the myelin sheath. Such defects have been interpreted as a common pathological feature of degenerating myelin in diseases [START_REF] Weil | Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases[END_REF]. Myelin splitting is also a common age-related myelin defect [START_REF] Stadelmann | Myelin in the Central Nervous System: Structure, Function, and Pathology[END_REF], however it is usually associated with focal myelin bulging and accumulation of myelin debris [START_REF] Safaiyan | Age-related myelin degradation burdens the clearance function of microglia during aging[END_REF], that we found not to be significantly increased in CADASIL mice. Several elegant studies from the Simons lab suggest that this pathology (myelin splitting and vesiculation) is caused by an aberrant switching of the polymerization state of MBP molecules to a non-physiological soluble state [START_REF] Weil | Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases[END_REF][START_REF] Bakhti | Myelin architecture: zippering membranes tightly together[END_REF][START_REF] Aggarwal | Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork[END_REF].

Previously, we reported that WM damage in CADASIL mice initially manifests as punctate staining on free-floating brain sections using a specific monoclonal antibody (SMI-94) raised against human MBP residues 70-89 [START_REF] Cognat | Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model[END_REF]. Notably, this epitope is immediately adjacent to the two phenylalanine residues in position 90 and 91 of human MBP that are required for the polymerization of MBP [START_REF] Aggarwal | Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork[END_REF].

Moreover, the Simons lab showed that the QD9 antibody raised against human MBP residues 82-88 only recognizes MBP molecules when converted to their non-physiological soluble state and thus stains only myelin fibres undergoing myelin breakdown [START_REF] Weil | Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases[END_REF]. Therefore, together these data strongly suggest that the punctate staining detected by the SMI-94 antibody in CADASIL mice corresponds to the myelin breakdown as detected in our EM analysis.

One attractive mechanism by which vessel changes associated with CADASIL could lead to a primary defect at the level of the myelin sheath is by a chronic hypoxic/ischaemic mechanism. Nawaz et al. have shown that phosphatidylinositol 4,5-bisphosphate (PIP2) is critically required for the stable membrane association of MBP, and in extension for the function of MBP. Specifically they showed in acute brain slices that hydrolysis of PIP2, by increasing Ca 2+ entry, or altering PIP2 levels by depleting the cellular ATP pool, leads to the dissociation of MBP from membranes, resulting in myelin vesiculation and breakdown [START_REF] Nawaz | Phosphatidylinositol 4,5bisphosphate-dependent interaction of myelin basic protein with the plasma membrane in oligodendroglial cells and its rapid perturbation by elevated calcium[END_REF]. Calcium elevation and ATP depletion in oligodendrocytes are relevant in the context of hypoxia/ischemia [START_REF] Hamilton | Proton-gated Ca(2+)-permeable TRP channels damage myelin in conditions mimicking ischaemia[END_REF][START_REF] Micu | NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia[END_REF]. Moreover, the inner tongue, where myelin breakdown is prominent, might be especially vulnerable because it is located far away from the oligodendrocyte cell body and because of its high energy requirement [START_REF] Simons | Oligodendrocytes: Myelination and Axonal Support[END_REF]. Yet, the reason why the enlargement of the inner tongue predominates in small calibre axons is unclear. Consistent with such a mechanism, we reported that adaptive responses of the cerebral circulation to blood pressure changes or neuronal activation were impaired early, and that resting cerebral blood flow was reduced later in CADASIL mice [START_REF] Joutel | Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease[END_REF][START_REF] Capone | Reducing Timp3 or vitronectin ameliorates disease manifestations in CADASIL mice[END_REF]. Future studies are needed to record Ca 2+ and ATP changes in the WM of CADASIL mice to further test this hypothesis.

In conclusion, we have used high resolution EM analysis in combination with HPF/FS to study WM changes for the first time in a chronic SVD model. Our results suggest that WM lesions in CADASIL are the result of a primary myelin injury originating at the level of the myelin sheath. These defects are consistent with a hypoxia/ischemia mechanism leading to myelin splitting and vesiculation. shows that the inner tongue is larger in small calibre axons in CADASIL mice at both ages. n=4-5 mice per group.

Legends to Figures

  Ethics approvalAnimal (mouse) experiments were approved by our local institutional Animal Care and Use Committees (CEEA9 and CEEA34, registration numbers 95 and 19-031) and by the French government (Ministère de l'Enseignement Supérieur et de la Recherche, authorizations #03653.02 and APAFiS #21100-2019041816189580v4).

( 1 :

 1 200, clone CC-1, OP80, Merck Millipore), Iba1 (1:500, 019-19741, Wako), CD68 (1:1000, clone FA-11, MCA1957, BioRad), overnight at 4 o C. Sections were washed, incubated with Alexa Fluor secondary antibodies (1:500; Life Technologies, Saint-Aubin, France) at room temperature, counterstained with DAPI, and mounted with Dako Mounting Medium.

Figure 1 .

 1 Figure 1. Comparison between HFP/FS and conventional processing on the ultrastructure of WM corpus callosum WM. A: Schematic of the HPF/FS protocol used in this study. B-C: Representative electron micrographs of corpus callosum samples prepared from the left and right hemispheres of the same 15-month-old WT mouse prefixed in Karlsson-Schultz fixative, and processed either by HPF/FS (B) or by conventional dehydration at room temperature (C). Here we have selected micrographs that represent some of the frequent artefacts than can occur in old mice with the conventional processing. The sample prepared by HPF/FS displays reduced artefacts of jagged myelin sheaths and myelin sheath splitting. Scale bars= 1µm.

Figure 2 .

 2 Figure 2. The optimized HFP/FS protocol provides finer details at the level of both compact and uncompact myelin than conventional processing. A-B: Representative electron micrographs of a myelinated axon from the corpus callosum of a 15month-old WT mouse prefixed in Karlsson-Schultz fixative and conventionally processed (A) or processed by HPF/FS (B). C: Schematic of a myelinated axon in the perinodal region. D: Higher magnification view of the boxed regions in B showing the outer (1) and inner (2) tongues. Fine details of the myelin sheath as well as of the inner and outer tongues are better visible on the sample processed by HPF/FS. Scale bar = 200 nm (A, B) and 46 nm (D).

Figure 3 .

 3 Figure 3. Qualitative and quantitative assessment of ultrastructural changes in the myelin sheath in the corpus callosum of CADASIL mice. Schematics (A, D, G, J) and representative electron micrographs (B, E, H, K) of the different pathological changes at the level of the myelin sheath detected in CADASIL mice: splitting (arrows in B) of the myelin sheath (A,B) with sleeves of uncompacted myelin, vesiculation at the inner tongue (D-E), myelin outfoldings (G-H), and myelin debris away from an axon (J-K). C, F, I, L: Genotypedependent quantification of these changes in control (WT, blue dots) and CADASIL mice (TgNotch3 R169C , orange squares) at 6 and 15 months of age: percentage of myelinated axons with a split myelin sheath (C), percentage of myelinated axons with vesiculation/tubulation at the inner tongue (F), percentage of myelinated axons with myelin outfoldings (I), number of myelin debris (L) showing in CADASIL mice significant increase in myelin splitting at both ages and significant increase in vesiculation at the inner tongue at 15 months of age. Mean ± SEM; *: P <0.05, **: P <0.01; two-way ANOVA with Tukey's post-hoc tests; n=4-5 mice per group. Scale bar = 200 nm

Figure 4 .

 4 Figure 4. Quantitative measurement of the inner tongue thickness in the corpus callosum ofCADASIL miceCalculation of the t-ratio against axon diameter in small calibre (< 0.6µm) axons (A-B) and large calibre axons (> 0.6µm) axons (C-D) in WT and CADASIL mice, at 6 months (A, C) and 15 months (B, D)

Figure 5 .

 5 Figure 5. Fewer large calibre myelinated axons with thinner myelin in the corpus callosum of CADASIL mice A: Percentage of axons which are myelinated in WT and CADASIL mice is comparable at both 6 and 15 months of age. B-C: Size distribution of myelinated axons with respect to the axon diameter at 6 months (B) and 15 months of age (C) shows a shift to the left in 15-month-old TgNotch3 R169C mice, indicating fewer large calibre myelinated axons. D-E: Calculation of the corrected g-ratio against axondiameter shows that myelin is thinner (higher g-ratio) in larger axons of TgNotch3 R169C mice at 6 months (D) and 15 months (E). n=4-5 mice per group.

Figure 6 .

 6 Figure 6. No loss of oligodendrocytes but an increase in the number of OPCs in the corpus callosum of aged CADASIL mice. A: Representative immunofluorescent images showing staining for Olig2 (green; a marker of all oligodendroglia) and CC-1 (red; a marker of oligodendrocytes) in the anterior corpus callosum of 12month-old WT and TgNotch3 R169C mice. White arrows mark OPCs (Olig2 + CC-1 -). B: Quantification of the number of oligodendrocytes (Olig2 + CC-1 + ) at 4, 6 and 12 months of age shows no difference between WT and TgNotch3 R169C mice. C: Quantification of the number of OPCs (Olig2 + CC-1 -) at 4, 6 and 12 months of age shows an increase in OPC number, but only at 12 months of age. Mean ± SEM; **: P <0.01; two-way ANOVA with Tukey's post-hoc tests (B, C). N= 5-8 mice per group. Scale bar = 30 µm.

Figure 7 .

 7 Figure 7. Increase in microglia number in the corpus callosum of aged CADASIL. A: Representative immunofluorescent images showing Iba1 (green; a marker of all microglia), CD68 (red; a marker of activated microglia), and DAPI (blue; nuclear marker) in the anterior corpus callosum of 15-month-old WT and TgNotch3 R169C mice. B-C: Quantification at 6 and 15 months of the total number of microglia (Iba1 + ; B) and the number of activated microglia (Iba1 + CD68 + ; C) shows a moderate increase in the total number of microglia in 15-month-old TgNotch3 R169C mice but no difference in the number of activated microglia. Mean ± SEM; *: P <0.05; two-way ANOVA with Sidak's post-hot tests. N= 5-8 mice per group. Scale bar = 50 µm.

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Acknowledgements

We are grateful to Wiebke Möbius for technical advice on the use of HPF/FS and Ilse Hurbain for technical assistance with the HPF/FS from the cell and tissue imaging facility (PICT) of Institut Curie. Confocal images were acquired at the Institute of Psychiatry and Neuroscience of Paris (NeurImag platform); the SP8 confocal microscope was funded by a grant from Fondation Leducq. EM images were acquired at the cell and tissue imaging facility of Institut Curie or at the Imagoseine imaging core facility, member of the France BioImaging infrastructure supported by grant ANR-10-INBS-04 from the French National Research Agency. This work was supported by Fondation Leducq (Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease, 16 CVD 05) and the National Research Agency, France (ANR-16-RHUS-0004) to AJ.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

List of abbreviations

Conflicts of interest

Xavier Heiligenstein is the chief strategy officer and co-founder of CryoCapCell.

Figure 2