extfieldsofstudy: Chemistry Medicine

text:
Myosin Isoforms and Contractile Properties of Single Fibers of Human Latissimus Dorsi Muscle

The aim of our study was to investigate fiber type distribution and contractile characteristics of Latissimus Dorsi muscle (LDM). Samples were collected from 18 young healthy subjects (9 males and 9 females) through percutaneous fine needle muscle biopsy. The results showed a predominance of fast myosin heavy chain isoforms (MyHC) with 42% of MyHC 2A and 25% of MyHC 2X, while MyHC 1 represented only 33%. The unbalance toward fast isoforms was even greater in males (71%) than in females (64%). Fiber type distribution partially reflected MyHC isoform distribution with 28% type 1/slow fibers and 5% hybrid 1/2A fibers, while fast fibers were divided into 30% type 2A, 31% type A/X, 4% type X, and 2% type 1/2X. Type 1/slow fibers were not only less abundant but also smaller in cross-sectional area than fast fibers. During maximal isometric contraction, type 1/slow fibers developed force and tension significantly lower than the two major groups of fast fibers. In conclusion, the predominance of fast fibers and their greater size and strength compared to slow fibers reveal that LDM is a muscle specialized mainly in phasic and powerful activity. Importantly, such specialization is more pronounced in males than in females.

Introduction
Quadriceps femoris and even more its lateral portion, vastus lateralis (VL), has been the source of most of the present knowledge on "in vitro" human muscle fibers characteristics. The choice of VL has been based on the possibility to collect biopsy samples from the bulk of the muscle without the risk of touching important nerve and blood vessels [1]. As additional reasons, the choice has been driven by the possible correlation between muscle characteristics and physical performance as the VL muscle belongs to the group of the leg extensors which can be easily tested (leg extension, jump, etc.) and play an important role in posture and many motor activity (raising from a chair as well as jumping) [2]. In upper limb muscles, biopsy sampling and single fiber characterization have been performed less often, even if upper limb muscles are specifically affected by some muscle diseases (e.g., FSHD or Facio-Scapulo-Humeral muscular Dystrophy [3]) and are specifically involved in some athletic performances and daily activity. Upper limb muscles are in a condition clearly different from lower limb muscles, with respect to postural activity, applied load, and kind of "natural" movement (i.e., walking, running, and jumping for lower limbs versus throwing, climbing, lifting objects for upper limbs). This is reflected in the distinct fiber type composition and, taking MyHC isoforms as markers of fiber type, the proportion of fast MyHC appears definitely higher in upper limbs compared to lower limbs [4].
From the physiological point of view, LDM is involved in many daily life activities like pulling an object and in sport movements (rowing, throwing objects, etc.); moreover LDM coactivation during active arm abduction has been reported to have a stabilization action on the shoulder [20]. It is thus surprising that, despite the previously mentioned important functions, studies on LDM fiber distribution and mechanical characteristics are lacking. This prompted us to study LDM at single fiber level in 18 healthy young volunteers. Each single fiber was characterized by its ability to develop isometric force and this functional parameter was related to fiber size or cross-sectional area, to fiber type, based on myosin isoform composition, and to myonuclear density, which seems to be also a determinant of muscle fiber strength [21]. We compared the results with available information on single muscle fibers from VL, taken as representative example of lower limb muscles.

Subjects.
Eighteen undergraduate students (9 males and 9 females; age 24.9 ± 5.3 years; 71.15 ± 12.15 kg body weight 176.53 ± 9.35 cm height) of the course of "Human Movement Science" of the University of Padova responded to an invitation to participate in the study. Respondents provided written informed consent to take part in the study and were screened for the presence of diseases or conditions that would place them at risk for adverse responses to exercise. All participants were healthy, nonobese, nonsmokers and were not taking any medications. The study was approved by the Ethical Commission of the Salvatore Maugeri Foundation (Pavia, Italy) where biopsy and medical tests were performed, in accordance with Helsinki's declaration of 1995 as modified in 2000.

Muscle Biopsy and Muscle Fibers
Analysis. Biopsy samples were collected from the lateral edge of the LDM at the level of the 5th rib. Tru-cut needles (PRECISA 1410 HS Hospital Service S.p.A. Latina. Italy) with a diameter of 14 G and an insertion cannula length of 100 mm were used. The utilization of a thin try cut needle has been validated in a previous publication [22]. The protocol adopted can be summarized as follows. To localize the spatial coordinates of LDM a strict anatomy palpation protocol was adopted. A careful identification of the biopsy site is extremely important, particularly in large muscles, as LDM, where different regions can play distinct functional roles and therefore possess specific fiber type complement. The tendon of the LDM was identified through palpation as it passes under teres major, forming the posterior wall of the axilla. If extension and medial rotation of the arm is resisted, the tendon can be traced along the medial side of the humerus towards its insertion into the floor of the intertubercular groove of the humerus. Thus, the LDM fascicles can be followed by palpation from under axilla toward waist and iliac crest as far as possible. After locating LDM fascicles the subjects were asked to lie down flat on a bed on the nondominant side with legs on top of each other, knees slightly bent, and the nondominant forearm under the head. The dominant arm was relaxed and bent on the anterior side of the trunk and the skin was marked with a skin pen to define the needle entry point and the needle direction.
To further check the location and the orientation of the LDM fibers ultrasonography was performed by ultrasound probe (Carsi Plus ESAOTE, 7.5-10 MHz probe) first with the muscle relaxed (muscle thickness 0.5-0.8 cm) and then during a maximum voluntary isometric contraction (muscle thickness 0.7-1.8 cm). This allowed the surgeon to confirm the localization of the muscle belly. In a few subjects ultrasonography was performed also during needle insertion.
After identifying the biopsy location and after local anaesthesia with 2 cc of xylocaine 2% and sterilization with Betadine, the needle was inserted into the muscle. In each subject, three samples were collected from the same location in subsequent insertions of the inner notched rod of the needle. The average yield from each sampling was 4 mg, which corresponds to a cylinder of approximately 0.7-0.9 mm diameter, cross-sectional area of approximately 0.5 mm 2 and length of about 8 mm (i.e., the full length of the notch on the inner rod). Approximately 500 fiber segments were present in each sample and a good fraction of them reached a length above 0.5 mm, thus allowing their use in single fiber mechanical experiments. The sample to be used for this purpose was immediately immersed in high potassium, high EGTA solution, usually indicated as skinning solution (see below), mixed in equal part with glycerol. Once immersed in this solution, the sample could be stored at −20 ∘ C for up to two weeks.
On the day of the experiment, the samples were washed with skinning solution with ATP; single fibers were manually dissected under a stereomicroscope and permeabilized with 1% Triton X-100. Fiber segments of the average length of 0.7 mm were mounted in the setup between the force transducer (model AME-801; SensorOne, Sausalito, CA) and the motor (SI, Heidelberg, Germany) equipped with a displacement transducer by means of light aluminium clips. The fiber segment was immersed in a drop of relaxing solution and, after measuring length, diameters and sarcomere length at 400X magnification was stretched by 20%. It was then transferred into the preactivating solution and finally maximally activated by immersion in the activating solution (pCa 4.6) at 12 ∘ C. Isometric force ( 0 ) was measured in four subsequent maximal activations and average value was calculated. The fiber segment was then removed from the setup and stored in Laemmli solution for electrophoretic determination of MyHC isoform composition.
Skinning, relaxing, preactivating, and activating solutions were prepared as previously described [23]. Their millimolar composition was as follows: (1) skinning solution contained 150 potassium propionate, 5 magnesium acetate, 5 ATP, 5 EGTA, and 5 KH 2 PO 4 ; (2) relaxing solution contained 100 KCl, 20 imidazole, 5 MgCl 2 , 5 ATP, and 5 EGTA. Preactivating solution was similar to relaxing solution except that EGTA concentration was reduced to 0.5 mM and 25 mM creatine phosphate and 300 U/mL creatine phosphokinase were added, whereas activating solution was similar to relaxing solution with the addition of 5 mM CaCl 2 , 25 mM creatine phosphate, and 300 U/mL creatine phosphokinase. The pH of 3 all solutions was adjusted to 7.0 at the temperature at which solutions were used (12 ∘ C). Protease inhibitors (10 M E-64 and 40 M leupeptin) were present in all solutions.
Each fiber was classified by its MyHC isoform composition and characterized by its cross-sectional area (CSA) calculated from three diameters, its isometric force ( 0 ), and isometric tension ( 0 ) obtained by normalizing 0 to CSA.
Proteins for gel electrophoresis were prepared from the remaining of the sample after single fiber dissection and from a sample, specifically collected for this purpose. The tissue samples were solubilized in Laemmli solution (62.5 mM Tris, pH 6.8, 10% glycerol, 2.3% SDS, 5% -mercaptoethanol, with 0.1% E-64 and 0.1% leupeptin as antiproteolytic factors). After heating for 5 min at 80 ∘ C, appropriate amounts of the protein suspension were loaded onto polyacrylamide gels (about 1 g of total protein/lane). For single fibers identification, the whole fiber segment was solubilized in 10 L of Laemmli solution, and 2-3 L was loaded onto gels. The separation of MyHC isoforms was achieved on 8% polyacrylamide slab gels with a protocol derived from Talmadge and Roy [24] with some modifications. Slabs 18 cm wide, 16 cm high, and 1 mm thick were used. Electrophoresis was run at 4 ∘ C for 24 h, at 70 V for 1 h and 230 V for the remaining time. Three bands were separated in the region of 200 kDa, corresponding (in order of migration from the fastest to the slowest) to MyHC-1, MyHC-2A, and MyHC-2X. Gels were silver stained (Bio-Rad Silver stain plus) for single fiber identification or stained with Coomassie Blue for determination of the relative proportions of the three MyHC isoforms. The relative proportions of MyHC isoforms were determined by the measurement of the brightness-area product B. A. P. (i.e., the product of the area of the band by the average brightness. subtracted local background after black-white inversion) with the accuracy of 600 dpi.

Immunofluorescence.
In order to determine the nuclear domain size, from the fiber bundles immersed in skinning solution (see above) single muscle fibers were manually dissected and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The fibers were permeabilized with 0.1% Triton X-100 in PBS at room temperature and then incubated in 10% normal goat serum for at least 30 min to block nonspecific antibody binding. Mouse anti--actinin (clone EA-53 Sigma), a monoclonal antibody, was applied (1:2000) at room temperature in PBS. After 3 washes (10 min each), fluorescent secondary Alexa-568 anti-mouse (Molecular Probes) antibody was incubated for 2 h at room temperature. For visualization of nuclei, single fibers were stained with Hoechst (25 g/mL; SIGMA) for 10 min. After a final wash in 0.1 M PB, the fibers were mounted in 100% glycerol (Sigma-Aldrich) and covered with a coverslip. The fibers were viewed with a confocal microscope (VICO, Nikon). Serial confocal optical sections (step size: 0.5 m) were collected by scanning the fiber in only one direction (from top to bottom). The fiber segment volume was reconstructed by adding the volume of the individual sections, each obtained as the product of thickness section (z-axis) by surface area (xy-axis). The sections were then collapsed on the z-axis and the number of nuclei was counted. From nuclei number and volume, the nuclear density (nuclei/10 6 m 3 ) and the nuclear domain size ( m 3 /nucleus) were obtained. It is worth to observe that the combination of fixation and compression between slides leads to values comparable to other data obtained with similar procedure [25], but lower than those obtained with skinned unfixed fibers [26].

Statistical
Analysis. Data are expressed as means and standard error mean unless otherwise indicated. Unpaired ttest was used to asset significant differences between male and female. Significance was settled as < 0.05.

Results
The electrophoretic separation and densitometric determination of MyHC isoform were performed in each of the 18 subjects to delineate the general distribution of fast and slow isoforms. The results showed a clear predominance of fast isoforms with 41.29 ± 2.06% of MyHC 2A and 24.89 ± 1.88 of MyHC 2X, while MyHC 1 represented only 32.84 ± 1.3% (means and SEM. = 18). The predominance of fast isoforms was more pronounced in males. Statistical analysis showed a significant greater percentage of MyHC 1 isoform ( < 0.05) and lower percentage of MyHC 2A isoform ( < 0.05) fibers in females compared to males, whilst no significant difference was detected for MyHC 2X isoform (see Table 1).
From each subject, at least 15 single muscle fibers were successfully analysed. In each fiber, MyHC isoform composition was determined, cross-sectional area was calculated, and maximal isometric force was measured. The predominance of fast fibers was confirmed as slow or type 1 fibers represented 28%, hybrid 1/2A fibers represented 5%, while fast fibers were divided into 30% type 2A, 31% type A/X, 4% type X, and 2% type 1/2X. Moreover, the proportion of slow fibers was lower in males compared to females (see Figure 1).
In the same three groups, as can be seen in Figure 2, the mean isometric force ( 0 ) was 0.66 ± 0.07 mN for 2A/X fibers, 0.8 ± 0.09 mN for 2A fibers, and 0.37 ± 0.03 mN for type 1 fibers. The isometric specific tension ( 0 ) was 134.48 ± 13.26 mN/mm 2 for type 2A/X, 161.3 ± 12.55 mN/mm 2 for type 2A, and 115.89 ± 17.26 mN/mm 2 for type 1 fibers; when all fibers were pooled together, 0 was 142.81 ± 12.55 mN/mm 2 . Thus, slow fibers developed less force and less tension than fast 2A fibers. Interestingly, isometric force was lower in muscle fibers in females compared to males (see Table 1).
In a subset of fibers from 6 randomly selected subjects myonuclear density was calculated as described in Section 2. The results showed that the average myonuclear density was 119.5 ± 3.7 nuclei/10 6 m 3 (mean and SEM. = 78) which

Discussion
In this study we determined mechanical characteristics of Latissimus Dorsi muscle (LDM) fibers obtained with fine needle biopsy. Only few papers have described LDM fiber type distribution using histochemical or immunohistochemical methods [13,14,16,17,19,27,28] and only one study has also considered the mechanical characteristics [13], although this aspect was not analysed extensively as the main focus was on the regeneration and reinnervation processes. The emerging picture of the LDM viewed at fiber level is that the muscle is mainly composed of fast fibers, which are not only more abundant but also thicker than slow fibers. Among fast fibers 2A fibers are the majority followed by the group of mixed 2A/X fibers, while pure 2X fibers, although present, are very rare. This fiber type distribution fits well the role of LDM as a muscle with limited postural function, whilst it is commonly used for short, moderate to intense effort [29,30].
To fully understand the relation between specialization at single fiber level and the functional role of the whole muscles, it is worth to compare the present results obtained in LDM with the characterization of single muscle fibers in VL, a lower limb muscle which combines a pronounced postural duties with the involvement in phasic and powerful activity from raising from the chair to jumping. Among the many studies available published by us [31][32][33][34] and other workers [35,36], we selected for the comparison between LDM and VL the recent paper by Doria et al. [31], as precisely the same methods and the same experimental conditions were applied, and we restricted the comparison to the pretrekking conditions, to avoid confounding effects of a training period. In that study, VL muscle fibers from 7 male young (age 39.4. height 1.72 m, and weight 76 kg) and moderately active subjects were studied.
The myosin isoform expression of VL and LDM appears clearly different as the slow MyHC isoform expression is greater in VL than in LDM: 40.5 ± 0.8% versus 32.84 ± 1.3% ( < 0.05). Among the fast isoforms, MyHC 2A proportion is very similar with 41.6 ± 1.6% in VL and 41.29 ± 2.06% in LDM, while MyHC 2X is more abundant in LDM with 24.89 ± 1.88 versus 18.3 ± 1.5% in VL ( < 0.05). The fiber type distribution, based on MyHC isoform composition, reflects the proportion of MyHC isoforms with the slow fibers being 35% in VL and only 28% in LDM. The proportion of hybrid fibers is greater in LDM than in VL: 37% versus 30%. A point which deserves a comment is the difference between VL and LDM in cross-sectional area of the slow and fast fibers. Table 2 shows the values of CSA, 0 , and 0 obtained in the VL fibers analysed in Doria et al. 2011 [31] in pretrekking conditions, divided in groups on the basis of their MyHC isoform composition and compared with our data of LDM. These data were not present in the paper by Doria and coworkers. As can be seen, in VL the CSA of slow fibers is similar to that of fast fibers, while in LDM slow fibers have a significantly smaller size (see also Figure 2). This is likely related to the different functional tasks of the two muscles: slow fibers which fulfil postural duties are not only more abundant but also thicker in VL than LDM. Importantly, type 1 fibers showed a greater CSA not only in VL but also in other muscles with even broader postural tasks, such as erector spinae muscle. For example, Mannion et al. showed that type 1 fibers had a diameter 62.5±10.0 m for men and 52.6±4.6 m for women compared to 59.4 ± 8.4 m and 41.6 ± 4.7 m for type 2A [37]. The comparison of the isometric performance in terms of force ( 0 ) and tension or specific force ( 0 ) shows that both in VL and LDM the slow fibers are weaker than fast fibers, a feature which can be observed also in other human muscles and in skeletal muscles of other animal species [38]. The lower force of slow fibers seems, thus, an intrinsic functional feature, at least under the experimental conditions adopted to study permeabilized fibers in vitro.
The values of nuclear density or of nuclear domain volume which is the reciprocal are worth some comments. The values obtained in LDM are very similar to those obtained by us in a previous study on VL fibers: 119 nuclei/10 6 m 3 versus 122 nuclei/10 6 m 3 [34]. This might indicate that the myoplasm volume controlled by a single myonucleus is not different in upper and lower limb muscles. Two further comments are also useful: (1) the values of nuclear domain volume obtained in this study are lower than those reported in other studies [26] where a different method was adopted. They are comparable to those reported by Mantilla and coworkers [25] who also counted the nuclei and measured the fiber size after the fiber was inserted among two cover slips with a possible effect of compression. (2) The fibers used for nuclear domain determination were not characterized as to the MyHC isoform composition. They were randomly selected and possibly with a different contribution of fast and slow fibers. There is published evidence [26] that slow fibers have a higher nuclear density, or lower nuclear domain volume, than fast fibers.
The results obtained in this study also provide evidence of a sex-related dimorphism. Some previous papers have also analysed the differences in muscle fiber composition between males and females. For example, Staron and coworkers have analysed sex-related muscle fiber differences in vastus lateralis of young subjects [39]. Fibers were classified as types I, IC, IIC, IIA, IIAB, and IIB and no significant differences between men and women for muscle fiber type distribution were found except for fiber type IC. The percentage distribution of pooled subjects was 41% I, 1% IC, 1% IIC, 31% IIA, 6% IIAB, and 20% IIB. The cross-sectional area of all major fiber types was larger for the men compared to the women, with the exception of type 1 fibers that showed an inverse trend, being larger in women. These data are consistent with those of Yasuda et al. who reported a slight predominance in women compared to men of type I (M: 45.2 ± 10.6%. W: 47.8 ± 9.5%) over type IIa (M: 37.0 ± 6.7%. W: 35.0 ± 3.4%) and type IIx (M: 17.8 ± 8.3%. W: 17.2 ± 10.2%) of vastus lateralis. The comparison of the total area for each fiber type showed that when type 2 fibers are considered together (IIa + IIx), mean fiber area percentage was superimposable between male and female (M: 72.8 ± 3.3% versus W: 68.6 ± 4.6%) whereas women showed a higher type I area compared with men (M: 27.2 ± 3.3% versus W: 31.4 ± 4.6%). Considering the high correlation between the histochemically assessed fractional fiber type area and the electrophoretically assessed MyHC content [40], the data from Yasuda et al. [41] appears to be in agreement with our results on latissimus dorsi: a higher type 1 fiber and a lower type 2 fiber proportion in females compared to males. Further support comes from the data of Martel et al. [42] who showed that in VL type 1 fibers represent 40 ± 3% in males and 47 ± 3% in females, whilst 2A fibers were 31 ± 3% and 32 ± 3%, respectively. Type 2X fibers were 29 ± 3% and 21 ± 4% for males and females. A higher percentage of type 1 fibers in women (67.8 ± 10.5 versus 62.0 ± 9.3) has been found also in Erector spinae muscles counterbalanced by a lower percentage of 2X fibers (4.6 ± 4.7 versus 10.9 ± 6.3) [37]. Thus, the greater abundance of slow fibers in women seems generally supported by published evidence.
In conclusion, the present study provides the first comprehensive analysis of single muscle fibers from human LDM. LDM shares with many upper limb muscles the predominance of fast fibers in causal relation with the low postural duties. Interestingly, the comparison between males and females show that regardless of the postural duties, female muscles are richer in slow fibers.

Conflict of Interests
The authors declare that there no conflict of interests.


extfieldsofstudy: Biology Medicine

text:
Innate immunity in the Grid2Lc/+ mouse model of cerebellar neurodegeneration: glial CD95/CD95L plays a non-apoptotic role in persistent neuron loss-associated inflammatory reactions in the cerebellum

Background There is growing evidence that the death receptor CD95 has a wider role in non-apoptotic functions. In the brain, it may contribute to neural death and to the associated inflammatory reaction via a non-apoptotic pathway. Brain injury triggers an inflammatory reaction in which the CD95/CD95L system acts principally through peripheral cells recruited to the lesion. In cases of inflammation within the brain, with no blood–brain barrier leakage, the role of the CD95/CD95L system is thus unclear. We investigated the possible role of CD95 and CD95L in such conditions, by studying the relationships between glial cell activation, neuron death and CD95/CD95L expression in the cerebellum of the Lurcher (Grid2Lc/+) mutant mouse, a model of cerebellar neurodegeneration. Methods Glial cells in slices of wild-type and Lurcher mouse cerebella were observed by light microscopy at various ages overlapping periods of neuron loss and of pre- and post-neurodegeneration. Subcellular organization was studied by electron microscopy. We assessed CD95 levels by western blotting, RT-PCR and glial cell cultures. The levels of CD95L and IL-6 were studied by ELISA and a biological assay, respectively. Results In the Grid2Lc/+cerebellum, neuron loss triggers a typical, but abnormally persistent, inflammatory reaction. We identified two phases of astrogliosis: an early burst of large glial cell activation, peaking at postnatal days 25 to 26, coinciding with peak cerebellar neuron loss, followed by a long period of slow decline indicating that the strength of the glial reaction is modulated by neuron mortality rates. Comparisons of time-courses of glial cell activation, cytokine production and neuron loss revealed that the number of surviving neurons decreased as CD95 increased. Thus, CD95 cannot be directly involved in neuron death, and its role must be limited to a contribution to the inflammatory reaction. The upregulation of CD95 likely on astrocytes coincides with increases in the levels of IL-6, a cytokine produced principally by astrocytes, and soluble CD95L. Conclusions These results suggest that CD95 and soluble CD95L contribute, via non-apoptotic signaling, to the inflammatory reaction initiated early in neuron death within the Grid2Lc/+ cerebellum.

Background
Central nervous system (CNS) inflammation may develop in response to brain injury or in association with neurodegeneration in human neurodegenerative diseases, such as Parkinson's, Huntington's and Alzheimer's diseases. Inflammation resulting from trauma or ischemia reperfusion injury includes endothelial damage, the release of proinflammatory mediators, changes in vascular permeability, infiltration with peripheral inflammatory cells and the activation of microglia, the innate immune cells of the brain, and astrocytes. It is marked by the recruitment of peripheral blood cells and the formation of a scar. CD95 (Fas, APO-1), which was initially identified as a death receptor, and its cognate ligand (CD95L, Fas-L) are both expressed in the brain. They are thought to contribute to the inflammatory reaction through non-apoptotic signaling, mediating proinflammatory responses and triggering the recruitment of peripheral macrophages and leukocytes to the site of inflammation [1,2]. Recent data obtained by Letellier et al. [3] for the spinal cord lesion model indicate that the expression of CD95L on peripheral myeloid cells plays a crucial role in the neural cell reaction at the lesion. These authors demonstrated that peripheral cell CD95L expression, which is upregulated by the lesion, is required for the recruitment of peripheral myeloid cells to the injured tissue and contributes to neural cell loss by acting on peripheral myeloid cells, rather than via direct CD95-induced apoptosis in CD95-bearing resident neural cells [3]. In neurodegenerative diseases, the glial cell activation associated with neurodegeneration is chronic. The inflammatory reaction is mild and features an absence of leukocytes within the neural parenchyma tissue; the possible occurrence of peripheral monocyte infiltration remains a matter of debate [4][5][6]. Despite these important differences between the two types of inflammatory reaction, the CD95/CD95L system has long been considered to play similar roles in the two situations [1,7]. However, in the absence of infiltrating peripheral cells, the contribution of the CD95/CD95L system to neural cell reactions (neural degeneration and the glial cell inflammatory reaction) remains unclear. Natural mutant mice, such as the ataxic Lurcher (Grid2 Lc/+ ) mouse, which displays cerebellar neurodegeneration, are valuable for investigations of the role of the CD95/CD95L system in this context making use of our extensive knowledge of cerebellar neurodegeneration in this mutant [8].
Ataxia becomes clinically detectable in the Lurcher mouse at about postnatal day 15 (P15). It results from the progressive postnatal loss of cerebellar neurons due to a mutation in the ∂2 glutamate receptor subunit (GluR∂2) gene (Grid2). The Lurcher mutation is a semi-dominant gain-of-function mutation involving a base-pair change [9]. Within the cerebellum, GluR∂2 is present specifically in Purkinje cells [10,11]. The expression of the mutated allele leads to a cell-autonomous process of Purkinje cell degeneration occurring between P7 and P30 [8]. As a result, 90% of the granule cells and 60 to 75% of the inferior olivary neurons die within the first 3 months after birth, due to the loss of their target [10,11]. The mechanisms by which Purkinje cells die have remained elusive. Activated caspase-3, DNA fragmentation and autophagosomes in dying Purkinje cells were thought to be the results of the activation of various cell-death execution pathways [12][13][14]. Recent in vitro and in vivo studies supported a necrosis mechanism caused by excitotoxicity and accompanied with features of autophagy [15]. In the Grid2 Lc/+ cerebellum, this degenerative process takes place in an intact brain with a fully functional blood-brain barrier (N Delhaye-Bouchaud and J Mariani, unpublished data).
The glial reaction to neuron loss has remained largely unexplored in the Grid2 Lc/+ cerebellum. The presence of reactive astrocytes and high IL-1ß concentrations reported in previous studies suggest that an inflammatory process occurs in the Grid2 Lc/+ cerebellum [16,17].
In this study, we investigated the possible role of the CD95/CD95L system in the mechanism of neuronal death and in the inflammatory response, as an intrinsic component of the glial reaction in the Grid2 Lc/+ cerebellum. We analyzed the relationship between the death of cerebellar neurons and glial activation. Correlations between glial cell activation and CD95 and CD95L expression on the one hand, and neurodegeneration events occurring from birth into adulthood on the other, indicate that the CD95/CD95L system is involved in the inflammatory reaction, mostly through astrocytes.

Animals
Heterozygous Grid2 Lc/+ mutant mice (Lc/+) and the corresponding wild-type strain, C57BL/6J X CBA (+/+), were obtained by crossing Lc/+ males with +/+ females. Mutant offspring were identified on the basis of ataxic behavior at about P15, or by genotyping for younger animals, as previously described [12]. Animals were cared for and killed in accordance with the guidelines of the French National Ethics Committee for Life and Health Sciences.

Glial cell cultures
High-purity cultures of astrocytes and microglia were prepared from mice from 5-and 1-day-old Lurcher litters, respectively, as previously described [18].

Tissue collection
Cerebella and cerebral cortices were collected on ice in an RPMI bath. The visible meninges and vessels were stripped away under a binocular microscope [17]. The dissected tissue was then immediately frozen in liquid nitrogen and stored at −80°C until use.

Immunohistochemistry
Mice were anesthetized by an intraperitoneal injection of 3.5% chloral hydrate (0.1 ml/g body weight) and transcardially perfused with buffer A (0.1 M cacodylate buffer pH 7.2, containing 4% paraformaldehyde, 8% saccharose and 5% dimethylsulfoxide). Brains were postfixed by overnight incubation in buffer A at 4°C and rinsed by incubation for 1 day with buffer A without dimethylsulfoxide. A vibratome (LEICA VT 1000S) was then used to cut one hemisphere into 30 μm sagittal sections, which were placed in 0.2 M Tris-maleate buffer (pH 7.2) supplemented with 8% saccharose. Microglia were detected and stained by the nucleoside diphosphatase histo-enzymological method, as previously described [19]. Briefly, sections were incubated with 2 mM 5′-uridine-diphosphate in 80 mM Trismaleate buffer (pH 7.2) supplemented with 0.12% lead citrate, 5 mM manganese chloride and 1% dimethyl sulfoxide for 30 minutes at 37°C. The reaction product was detected as a brown precipitate by incubating sections in 2% ammonium sulfide. For glial fibrillary acidic protein (GFAP) labeling, sections prepared as described above were washed three times in TBS and incubated in TBS supplemented with 1% Triton X-100 and 10% normal goat serum for 20 minutes. Rabbit polyclonal anti-GFAP antibody (Sigma; 1/200 dilution in TBS), and biotinylated goat anti-rabbit IgG antibody (affinity-purified, from Vector Laboratories; 1/500 dilution), were applied to the sections, which were then incubated overnight at 4°C and for 3 hours at room temperature, respectively. Labeling was visualized with the Vectastain ABC kit, using diaminobenzidine (Sigma) as the chromogen.

Electron microscopy
Two Grid2 Lc/+ mice were anesthetized with sodium pentobarbital (15 mg/50 g body weight, Sanofi Diagnostic Pasteur) and fixed by transcardial perfusion with 300 ml 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Sagittal sections (1 mm) of the cerebellar vermis were cut with a vibratome and postfixed by incubation for 1 hour in 1% osmium tetroxide. They were then dehydraded by passage through a series of concentrations of ethanol and embedded in Araldite. Ultrathin sections (60 nm thick) cut from blocks (1 mm 3 ) sampled from the vibratome sections were counterstained with uranyl acetate and examined with a Hitachi 7500 transmission electron microscope.

Semiquantitative RT-PCR
Total RNA was isolated from frozen brain tissues with the RNeasy Mini kit (Qiagen, Hilden, Germany) and from cultured cells with Trizol reagent. RNA concentration and purity were determined with a Nanodrop ND-1000 spectrophotometer. The reverse transcription reaction was performed with a Reverse Transcription System kit (Promega), with avian myeloblastosis virus transcriptase and oligo(dT)15 primers, according to the manufacturer's instructions. The first-strand cDNA was amplified by PCR in a Perkin Elmer Thermal Cycler, with Taq polymerase and the primers for GFAP (sense primer TTC CTG TAC AGA CTT TCT CC and antisense primer CCC TTC AGG ACT GCC TTA GT); Fas (sense primer CAT CTC CGA GAT TTT AAA GC and antisense primer GTT TCC TGC AGT TTG TAT TGC T) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) (sense primer CCT GCT GGA TTA CAT TAA AGC ACT G and antisense primer GTC AAG GGC ATA TCC AAC AAC AAA C). We used appropriate concentrations of cDNA to ensure that PCR was carried out in non-saturated conditions. Amplification products were visualized by electrophoresis in a 2% agarose gel containing ethidium bromide (0.6 mg/ml), and quantified by densitometry with Densylab software (Microvision Instruments, Evry, France). Values were normalized with respect to the amplicon for the HPRT housekeeping gene. A stimulation index (SI) was calculated for the kinetic study. SI is defined as the normalized CD95 mRNA level for stimulated cells from which we subtracted the normalized CD95 mRNA level for unstimulated cells, before dividing the result by the normalized CD95 mRNA level for unstimulated cells.
Determination of cytokine levels IL-6 activity and CD95L concentration were determined on tissue extracts prepared as previously described [17]. Briefly, frozen tissues were rapidly weighed, dissociated with a Dounce Potter homogenizer on ice with five parts by weight of RPMI containing antiproteases (10 mM amino-n-caproic acid, 0.5 mM benzamidine, 1 mM ethylenediaminetetraacetic acid (EDTA) and 20 μM AEBSF for CD95L and only EDTA and AEBSF for IL-6 ). The resulting homogenate was centrifuged for 30 minutes at 13400 × g at 4°C. Supernatants were collected, and small aliquots were immediately frozen at −20°C until use. Total protein concentrations were determined as described above for immunoblotting. Two cerebella from different animals were used to prepare a single extract. Each sample was analyzed in duplicate. IL-6 activity was determined in a biological assay with the IL-6-dependent B-cell hybridoma B9. B9 cells (5 × 10 3 cells/well, in 100 μl) were plated in 96-well microtiter plates, and 100 μl of a serial dilution of extract and murine IL-6 as a standard were added. The plates were incubated for 72 hours at 37°C in a humidified chamber containing 5% CO 2 -95% air. B9 cell growth was then assessed in an MTT assay. We incubated 0.5 mg/ml MTT with cells for 3.5 hours in a culture incubator. Insoluble purple formazan salts were solubilized with 150 μl isopropanol plus 0.1 M HCl and absorbance was determined at 560 nm. Fas-L concentrations were measured in a sandwich ELISA with the Quantikine M kit from R&D Systems, in accordance with the manufacturer's instructions. Experiments were repeated two or three times.

Statistical analysis
Group difference was established by using the nonparametric Mann Whitney test or with a one-way analysis of variance with de Scheffé multiple comparison test for multiple group comparison. Homogeneity of the variance was assessed using F-test. Statistical analyses were carried out using StatView software (Abacus Concept).

Results
Early neurodegeneration in the Grid2 Lc/+ cerebellum is associated with the persistent activation of glial cells.
Grid2 Lc/+ cerebellar neurons die between P7 and P90, with large-scale degeneration occurring between P17 and P25 [11]. We studied the time course of the glial cell reaction to neuron loss by following glial morphological changes from P5 to adulthood by GFAP immunolabeling for astrocytes and the histoenzymatic nucleoside diphosphatase method for microglia. Cerebella from P5, P13, P21 and P150 mice were studied. At P5, round or ameboid microglial cells were present in the cerebellar white matter of both wild-type and mutant mice (data not shown). These microglia were of the macrophage type and were subsequently replaced by microglia displaying signs of activation, with an enlarged cell body and short thick processes, in the Grid2 Lc/+ cerebellum, and by non-activated microglia in the wild type (see Figures 1A and 1B, respectively). At P13, activated microglia were detected either as isolated cells or in sparse clusters, indicating the occurrence of a microglial activation process that began before this time point. Clusters were first observed in the vicinity of Purkinje cell bodies at about P13. They were later found throughout the cerebellum of Grid2 Lc/+ adult mice, particularly in the vicinity of blood vessels ( Figure 1D). In the adult wild-type cerebellum ( Figure 1C) and in the other brain structures, such as the brainstem, the microglia remained non-activated (data not shown).
Paralleling the microglial reaction, astrocytes displayed changes in morphology from P13 and P21 into adulthood. In the P21 degenerating Grid2 Lc/+ cerebellum, fibers of Bergmann glia were shorter and less regularly spaced than in the cerebella of control littermates (data not shown). The granular layer was invaded by numerous strongly stained astrocytes, which persisted during adulthood ( Figure 2B). Electron microscopy showed that all surviving neuronal processes -a few parallel fibers, basket and stellate interneurons in the molecular layer and rare glomeruli in the internal granular layer -were rolled up into hyperdeveloped, superimposed astrocytic processes extending throughout the atrophied cerebellar cortex of the Grid2 Lc/+ mutants (Figures 3A,B).
Long-term astrocyte activation is preceded by acute gfap gene activation GFAP upregulation is a hallmark of astrocyte activation. We investigated the transcriptional regulation of GFAP in the Grid2 Lc/+ cerebellum by analyzing gfap mRNA levels at various time points in the Grid2 Lc/+ and wildtype cerebellum (Figure 4). In the wild-type cerebellum, gfap mRNA levels decreased from P5 to P18 and remained stable and low throughout the rest of the study period (data not shown). In the Grid2 Lc/+ cerebellum, gfap mRNA levels increased strongly between P22 and P25, peaking sharply at a level 93 times higher than that in the wild type. In parallel to the age-related neuron survival, according to the estimates reported by Caddy and Biscoe for the Lurcher cerebellar neuron population [11], a burst of gfap mRNA production was found to occur at the end of a period of extensive neuron (Purkinje and granule cell) loss. From P36 until P110 gfap mRNA levels remained high. They stabilized in adult Grid2 Lc/+ cerebellum at a level 10 times that in wild-type cerebellum.

Increases in CD95 expression reflect astroglial cell activation
CD95 is expressed principally on neurons and, to a lesser extent, on glial cells in the adult brain [2]. Its expression is upregulated in pathological conditions. We investigated the contribution of CD95 to Grid2 Lc/+ cerebellar degeneration by studying CD95 expression in the cerebellum of Grid2 Lc/+ and wild-type mice by western blot analyses of crude cerebellar extracts ( Figure 5). The anti-CD95 antibody used cross-reacts with undefined proteins, as previously described [20]. We thus included a negative control in our study: thymic extracts from lpr mice, which have only very low levels of CD95 [21]. CD95 was identified as a 52 kDa band. A 46 kDa band was consistently detected in all tested brain tissue extracts and may correspond to a non-glycosylated form of CD95 ( Figure 5A) [20]. We evaluated CD95 expression in the Grid2 Lc/+ mutant and wild-type littermate cerebella at P22 and P52. P22 corresponds to a period of massive neuron loss. One month later, at P52, the neuronal death process is coming to an end [11]. CD95 was significantly more strongly expressed in the Grid2 Lc/+  than in the wild-type cerebellum, and its expression increased steadily from P22 to P52 ( Figure 5B).
We characterized CD95 upregulation in the Grid2 Lc/+ cerebellum further, by studying CD95 transcripts from P5 to P110. In the Grid2 Lc/+ cerebellum, transcripts levels were similar to those in the wild type until P18. From P18 onwards, they increased over a period of 8 weeks, eventually reaching a plateau at a level 10 times that in the wild type ( Figure 5C). CD95 mRNA levels were still high in 16-month-old Grid2 Lc/+ mice (data not shown).
Thus, the increase in CD95 expression was due to an increase in the number of CD95 transcripts, indicating the involvement of a transcriptional mechanism.
We investigated the cellular distribution of CD95 by carrying out immunohistochemistry studies in brain slices with several anti-CD95 antibodies. Studies of CD95-deficient mice, used as a negative control, showed that none of the antibodies tested, including those used for western blotting, was entirely specific for CD95 in brain slices, as previously reported [22]. Thus, we looked for CD95 transcripts in cultures highly enriched in wildtype and Grid2 Lc/+ astrocytes and microglia ( Figure 6). No difference was found between wild-type and mutant cells. Non-stimulated resting astrocytes expressed CD95, whereas microglia did not. CD95 transcript levels were upregulated by proinflammatory treatment in astrocytes, but not in microglia, in which neither lipopolysaccharide (LPS) nor proinflammatory cytokines (data not shown) induced CD95 expression ( Figure 6A). In cultured astrocytes, high levels of CD95 were detected after 40 minutes of stimulation with proinflammatory cytokines, these levels increasing for up to 2 hours ( Figure 6B).

Upregulation of soluble CD95L and inflammatory cytokines
In normal brain, inflammatory cytokines are either absent or are produced in quantities only just exceeding the detection threshold. These molecules are induced or Representative electron microscopy views of the molecular layer (A) and of the internal granular layer (B). In the molecular layer, a few surviving parallel fibers (pf) are wrapped in abnormally abundant, superimposed astrocytic processes (asterisk); G, glial cell soma with electrolucent cytoplasm; n, nucleus. In the internal granular layer, abundant astrocytic processes are stacked around a cerebellar glomerulus made of a mossy fiber-like terminal (mf) forming asymmetric synapses (arrowheads) on a few granule cell dendrites (gc), which receive inputs from Golgi neuron terminals (go) at symmetric synapses (arrows). Bar: 500 nm. Gfap mRNA is present in large amounts from P22 to P120. The gfap mRNA is produced in a large burst coinciding with the end of the episode of massive neuron degeneration. The results are expressed as the ratio of Grid2 Lc/+ gfap mRNA to wild-type gfap mRNA, and the means ± SEM of four to six paired age-matched Grid2 Lc/+ and wild-type mice are shown. Age-related survival of neurons (shaded in gray), including Purkinje and granule cells, was determined from the estimates of Caddy and Biscoe [11]. Survival is expressed as a percentage of the number of cerebellar neurons on P4. upregulated during brain inflammation featuring glial cell activation. We previously showed that levels of the proinflammatory cytokine IL-1ß were higher in 4-month -old Grid2 Lc/+ mice than in the wild type [17]. We investigated the involvement of CD95L in the glial reaction in Grid2 Lc/+ cerebellum by studying soluble CD95L levels in wild-type and mutant mouse cerebellum. Determinations were carried out at two time points -during the period of intense degeneration and much later, at P21 and P150, respectively ( Figure 7A). CD95L was detected in the cerebellum of young wild-type mice, but it increased in abundance with age. CD95L levels in the Grid2 Lc/+ cerebellum were higher than those in the wild type at both ages. In the cerebral cortex of mutant and wild-type mice, CD95L levels were in the range of values found for the P150 wild-type cerebellum (data not shown). We also investigated changes in the amount of IL-6, an inflammatory cytokine produced mostly by astrocytes and, to a lesser extent, microglia, during brain inflammation. We determined IL-6 levels during and after neurodegeneration, from P5 to P130 ( Figure 7B). IL-6 was undetectable during the first 3 weeks after birth in both wild-type and Grid2 Lc/+ cerebella. From P50 to adulthood, IL-6 was present at stable, low levels in the cerebellum of the wild-type mice but at levels three times higher in Grid2 Lc/+ mice.

Discussion
The CD95 receptor and its ligand, CD95L, are thought to contribute to brain neural death and inflammation processes through non-apoptotic signaling. In this study, we investigated the role of the CD95/CD95L system in the cerebellum of the Grid2 Lc/+ mutant mouse, a model of naturally occurring brain parenchyma neurodegeneration without damage to the blood-brain barrier. We focused on the associated inflammatory reaction. We studied glial cell activation and CD95 expression before, during and after cerebellar neuron loss. Levels of CD95 and its ligand were high in mutant mice, and we observed a protracted inflammatory reaction triggered by the early onset of cerebellar neuron death. The correlations identified in studies of the time courses of CD95 expression, neuron survival and astrocyte activation indicated that CD95 was expressed on glial cells, but not on neurons, and might therefore contribute exclusively to the inflammatory process.
The time course and mechanisms of death of the various types of neurons in mutant mice have been widely studied and are well understood [8]. In this study, we studied the glial reaction to neuron loss. Analyses of the morphological and biochemical criteria for glial cell activation showed that astrocytes and microglia were activated in a process characterized by an early onset and a burst of activation between P22 and P25, coinciding Values are the means ± SEM of four independent determinations (one-way analysis of variance followed by de Sheffé multiple comparison test, *P <0.05, **P <0.01). (C) Time course of CD95 mRNA production in the cerebellum of Lc and wt mice from P5 to adulthood. The results are expressed as the ratio of the Lc CD95 mRNA to wt CD95 mRNA, and the means ± SEM of four paired age-matched Lc and wt mice are shown. Note that CD95 levels begin to increase at the end of the episode of massive neuron degeneration (shaded in gray; see Figure 4). with peak levels of neuron cell death. From P7 to P30, 90% of the Purkinje cells and granular neurons disappeared from the Grid2 Lc/+ cerebellum. Purkinje cells genetically committed to death display morphological and physiological alterations that can be detected as early as P6 and they die between P10 and P30 [11]. Our findings, consistent with the kinetics of Purkinje cell and granular neuron degeneration, show that microglia are activated after P5 and before P13 and that not all microglial cells are reactive at P13, probably due to the gradual loss of cerebellar neurons. Furthermore, the clusters of microglia found in a few areas of the Purkinje cell layer were consistent with the "holes" lacking Purkinje cells in this layer described in P12 Grid2 Lc/+ by Dumesnil-Bousez and Sotelo [23]. Clustered microglia are probably of the macrophage type, playing a role in the removal of dead cells [24,25]. Complete microglial activation throughout the cerebellum occurs during the period of mass neuron death between P17 and P25 [11]. Astrocyte activation parallels microglial activation, but with a small time lag, in the stereotypical glial reaction to brain injury. Microglial cells are fully activated at about P25 to P26, whereas astrocytes are only just beginning to be activated at this time point, as shown by the upregulation of GFAP. Astrocyte activation begins with a huge burst of gfap transcription (resulting in transcript levels 93 times higher than those in the wild type) coinciding with a period of mass neuron death in the Grid2 Lc/+ cerebellum. In the adult Grid2 Lc/+ cerebellum, gfap mRNA levels remain 10 times higher than those in the wild type, and microglial clusters may persist for up to 5 months. Electron microscopy revealed that, throughout the highly disturbed cytoarchitecture of the Grid2 Lc/+ cerebellum, astrocytes ensheathed the surviving neurons -basket, stellate and Golgi cells [11] -in a large number of stacked astrocytic processes forming a cocoon-like structure.
The Grid2 Lc/+ glial response resembles experimental models of acute neuronal degeneration, in which dead neurons are rapidly removed by phagocytic microglia, and neuron-depleted areas are filled with astrocytes, leading to the formation of a scar [25,26]. Nevertheless, it differs from these models in the persistence of activated microglial cells long after the neurodegenerative event. For example, in the mouse model of kainic acidinduced hippocampal neuronal death, for doses of kainic acid destroying all pyramidal cells in the injected hippocampus without affecting non-neuronal cells, the number of microglia with a quiescent cell morphology returns to normal after about 3 months [27]. Thus, the Grid2 Lc/+ microglial reaction triggered by early neuron loss seems to decline very slowly, taking a particularly long time to come to an end.
In the Grid2 Lc/+ cerebellum, the onset of the neuron loss takes place early after birth, overlapping a period known to have specific inflammatory properties. Lawson and Perry [28] have shown that, during postnatal development, there is a window of susceptibility to induce a classical inflammatory response, resembling that seen in non-CNS tissues. They showed that, in contrast to adult mice, 7-day-old mice injected with LPS in the parenchyme develop a vigorous inflammatory response with prolific neutrophil recruitment and a brisk mononuclear phagocyte response. In contrast to the model of Lawson and Perry, Grid2 Lc/+ glial cell activation occurs without leakage of the blood-brain barrier and peripheral cell intrusion (N Delhaye-Bouchaud and J Mariani, unpublished data). Thus, the glial activation intensity may be related to the age-related properties of the inflammatory response while the absence of infiltrating peripheral cells, known to contribute to the healing mechanisms [29], may be responsible for the persistence of the glial reaction. Taken together, this may give an unusual dynamic to the glial reaction in the Grid2 Lc/+ cerebellum.
The cellular origin of CD95 in the brain has been the subject of lively debate. Neurons, astrocytes and oligodendrocytes have all been shown to express CD95, and it is now thought that neurons are the strongest expressers of CD95 in the normal adult brain, whereas CD95 expression is detected on glial cells in pathological conditions. CD95L is present in the body as a membrane-bound protein mediating apoptotic signaling, or as a metalloprotease-cleaved soluble form. In the brain, CD95L has been reported to be constitutively expressed on neurons and astrocytes and upregulated by inflammation [2,30].
In the Grid2 Lc/+ cerebellum, increases in the levels of both GFAP and CD95, and decreases in neuron survival occur simultaneously, suggesting that the CD95 synthesized de novo originates from astrocytes. Unfortunately, we were unable to show that CD95 was located on astrocytes by immunohistochemical methods, due to the known lack of specificity of antibodies directed against the mouse protein [22]. However, increase in CD95 levels in response to stimulation on cultured astrocytes [31,32] but not on microglia suggests that the newly synthesized CD95 is of astrocytic origin. The soluble form of CD95L (sCD95L) is also abnormally expressed in the Grid2 Lc/+ cerebellum. Peripheral cells, such as T cells or cells of the monocyte lineage, cannot be held responsible for the production of CD95L in this context, because peripheral immune cells are not recruited to the brain during cerebellar degeneration in Grid2 Lc/+ mice (N Delhaye-Bouchaud and J Mariani, unpublished data). However, the CD95L producing cells within the Grid2 Lc/+ cerebellum remain to be identified.
In the Grid2 Lc/+ cerebellar inflammatory reaction, CD95L might be involved in several of the mechanisms of reactive gliosis. Recently, sCD95L has been implicated in cell migration via a non-apoptotic pathway. The underlying mechanism involves the formation of a specific signaling complex containing CD95 [30]. Cell migration is an important process in the cascade of events occurring during the inflammatory reaction, and is a feature of microglial cells in the CNS. Several mechanisms involving different microglial attractants have been described [33] and it is not possible to rule out the possibility that sCD95L belongs to the microglial motility machinery. However, sCD95L might play a role in the motility of the astrocytes, which grow and fill the spaces vacated by the lost neurons. Less speculative is the role of CD95 activation in the induction or upregulation of inflammatory mediators, such as IL-1, IL-6, IL-8 and cell surface integrins [34]. We found that IL-6 concentration increased with glial cell activation. In a previous study, abnormally high basal levels of IL-1ß were detected in adult Grid2 Lc/+ cerebellum [17]. These two cytokines play an essential role in the inflammatory Representative time-course study of IL-6 levels in the cerebellum of Grid2 Lc/+ (open diamonds) and wild-type (black diamonds) mice. Levels were determined in a biological activity assay. Note that IL-6 levels begin to increase at the end of the period of massive neuron degeneration (shaded in gray; see Figure 4). The mean values for four determinations are shown, with range error bars.
cascade. IL-6, which is mostly produced by astrocytes during brain inflammation, triggers astrocyte proliferation and is believed to be involved in astrogliosis and the activation of microglia [31,35]. Microglia are the principal inducible source of IL-1ß, a strong inducer of astrocyte proliferation [36]. Thus, within the adult Grid2 Lc/+ cerebellum, reactive gliosis may self-perpetuate for some time through the cytokine and chemokine network, including the CD95/CD95L system, in the absence of infiltrating peripheral immune cells. Such peripheral immune cells play a key role in the repair cascade resulting in the healing of brain wound [29].
In conclusion, our findings support the involvement of the CD95/CD95L system in the glial reaction triggered by neuron death, through non-apoptotic signaling in the neurodegenerative cerebellum of Grid2 Lc/+ mutant mice. The persistence of microglial activation in this model is consistent with the misregulation of molecular mechanisms responsible for the termination of glial activation, in which the role of the CD95/CD95L system remains to be determined.