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Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease in adults with no curative treatment. Neurofilament (NF) level in patient’ fluids have recently emerged as the prime biomarker of ALS disease progression, while NF accumulation in MNs of patients is the oldest and one of the best pathological hallmarks. However, the way NF accumulations could lead to MN degeneration remains unknown. To assess NF accumulations and study the impact on MNs, we compared MNs derived from induced pluripotent stem cells (iPSC) of patients carrying mutations in C9orf72, SOD1 and TARDBP genes, the three main ALS genetic causes. We show that in all mutant MNs, light NF (NF-L) chains rapidly accumulate in MN soma, while the phosphorylated heavy/medium NF (pNF-M/H) chains pile up in axonal proximal regions of only C9orf72 and SOD1 MNs. Excitability abnormalities were also only observed in these latter MNs. We demonstrate that the integrity of the MN axonal initial segment (AIS), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of pNF-M/H accumulations in C9orf72 and SOD1 MNs. We establish a strong correlation between these pNF-M/H accumulations, an AIS distal shift, increased axonal calibers and modified repartition of sodium channels. The results expand our understanding of how NF accumulation could dysregulate components of the axonal cytoskeleton and disrupt MN homeostasis. With recent cumulative evidence that AIS alterations are implicated in different brain diseases, preserving AIS integrity could have important therapeutic implications for ALS.

Traumatic spinal cord injury (SCI) is a neurological condition characterized by long-term motor and sensory neurological deficits as a consequence of an external physical impact damaging the spinal cord. Anatomical magnetic resonance imaging (MRI) is considered the gold-standard diagnostic tool to obtain structural information for the prognosis of acute SCI, however, it lacks functional objective information to assess SCI progression and recovery. In this study, we explored the use of synaptic vesicle glycoprotein 2A (SV2A) positron emission tomography (PET) imaging to detect spinal cord lesions non-invasively following SCI. METHODS: Mice (n=7) and rats (n=8) subjected to unilateral moderate cervical (C5) contusion were euthanized 1-week post-SCI for histological and autoradiographic (3H-UCB-J) investigation of SV2A levels. Longitudinal 11C-UCB-J PET/computed tomography (CT) imaging was performed in sham (n=7) and SCI rats (n=8) 1 week and 6 weeks post-SCI. Animals also underwent an 18F-FDG PET scan during the latter time point. Post-mortem tissue SV2A analysis to corroborate in vivo PET findings was performed 6 weeks post-SCI. RESULTS: A significant SV2A loss (ranging from -70.3% to -87.3%; P11C-UCB-J PET imaging detected SV2A loss in SCI rats (- 49.0±8.1% at 1 week and -52.0±12.9% at 6 weeks post-SCI), with no change observed in sham rats. In contrast, 18F-FDG PET imaging measured only subtle hypometabolism (-17.6±14.7%). Finally, post-mortem 3H-UCB-J autoradiography correlated with the in vivo SV2A PET findings (r=0.92, P11C-UCB-J PET/CT imaging is a non-invasive marker for SV2A loss following SCI. Collectively, these findings indicate SV2A PET may provide an objective measure of SCI, and thus a valuable tool to evaluate novel therapeutics. Clinical assessment of SCI with SV2A PET imaging is highly recommended.

Following spinal cord injury (SCI) the degree of functional (motor, autonomous, or sensory) loss correlates with the severity of nervous tissue damage. An imaging technique able to capture non-invasively and simultaneously the complex mechanisms of neuronal loss, vascular damage, and peri-lesional tissue reorganization is currently lacking in experimental SCI studies. Synchrotron X-ray phase-contrast tomography (SXPCT) has emerged as a non-destructive three-dimensional (3D) neuroimaging technique with high contrast and spatial resolution. In this framework, we developed a multi-modal approach combining SXPCT, histology and correlative methods to study neurovascular architecture in normal and spinal level C4-contused mouse spinal cords (C57BL/6J mice, age 2-3 months). The evolution of SCI lesion was imaged at the cell resolution level during the acute (30 min) and subacute (7 day) phases. Spared motor neurons (MNs) were segmented and quantified in different volumes localized at and away from the epicenter. SXPCT was able to capture neuronal loss and blood-brain barrier breakdown following SCI. Three-dimensional quantification based on SXPCT acquisitions showed no additional MN loss between 30 min and 7 days post-SCI. In addition, the analysis of hemorrhagic (at 30 min) and lesion (at 7 days) volumes revealed a high similarity in size, suggesting no extension of tissue degeneration between early and later time-points. Moreover, glial scar borders were unevenly distributed, with rostral edges being the most extended. In conclusion, SXPCT capability to image at high resolution cellular changes in 3D enables the understanding of the relationship between hemorrhagic events and nervous structure damage in SCI.

Tibialis anterior muscles of 45-week-old female obese Zucker rats with defective leptin receptor and non-insulin dependent diabetes mellitus (NIDDM) showed a significative atrophy compared to lean muscles, based on histochemical-stained section’s measurements in the sequence: oxidative slow twitch (SO, type I) < oxidative fast twitch (FOG, type IIa) < fast glycolytic (FG, type IIb). Both oxidative fiber’s outskirts resembled ‘ragged’ fibers and, in these zones, ultrastructure revealed small clusters of endoplasm-like reticulum filled with unidentified electron contrasted compounds, contiguous and continuous with adjacent mitochondria envelope. The linings appeared crenated stabbed by circular patterns resembling those found of ceramides. The same fibers contained scattered degraded mitochondria that tethered electron contrasted droplets favoring larger depots while mitoptosis were widespread in FG fibers. Based on other interdisciplinary investigations on the lipid depots of diabetes 2 muscles made us to propose these accumulated contrasted contents to be made of peculiar lipids, including acyl-ceramides, as those were only found while diabetes 2 progresses in aging obese rats. These could interfere in NIDDM with mitochondrial oxidative energetic demands and muscle functions.

Aim: To model in vitro the contact between adult dental pulp stem cells (DPSCs) and lipoteichoic acid (LTA), a cell wall component expressed at the surface of most Gram-positive bacteria. Methodology: Human DPSCs obtained from impacted third molars were cultured and exposed to various concentrations of S. aureus LTA (0.1, 1.0 and 10 µg mL −1). The effects of LTA on DPSCs proliferation and apoptosis were investigated by MTT assay and flow cytometry. Mineralization of DPSCs was evaluated by alizarin red staining assay. Migration was investigated by microphotographs of wound-healing and Transwell migration assays. Reverse transcription polymerase chain reaction was used to examine the effects of LTA on p65 NF-κB translocation and TLR1, TLR2 or TLR6 regulation. Enzyme-linked immunosorbent assay was used to investigate LTA-stimulated DPSCs cytokine production. One-way or two-way ANOVA and Tukey post hoc multiple comparison were used for statistical analysis. Results: DPSCs expressed TLR1, TLR2 and TLR6 involved in the recognition of various forms of LTA or lipoproteins. Exposure to LTA did not up- or down-regulate the mRNAs of TLR1, TLR2 or TLR6 whilst LPS acted as a potent inducer of them [TLR1 (P ≤ 0.05), TLR2 (P ≤ 0.001) and TLR6 (P ≤ 0.001)]. Translocation of p65 NF-κB to the nucleus was detected in LTA-stimulated cells, but to a lesser extent than LPS-stimulated DPSCs (P ≤ 0.001). The viability of cells exposed to LTA was greater than unstimulated cells, which was attributed to an increased proliferation and not to less cell death [LTA 1 μg mL −1 (P ≤ 0.001) and 10 μg mL −1 (P ≤ 0.01)]. For specific doses of LTA (1.0 µg mL −1), adhesion of DPSCs to collagen matrix was disturbed (P ≤ 0.05) and cells enhanced their horizontal mobility (P ≤ 0.001). LTA-stimulated DPSCs released IL-6 and IL-8 in a dose-dependent manner (P ≤ 0.0001). At all concentrations investigated, LTA did not influence osteogenic/odontoblastic differentiation. Conclusions: Human DPSCs were able to sense the wall components of Gram-positive bacteria likely through TLR2 signalling. Consequently, cells modestly proliferated, increased their migratory behaviour and contributed significantly to the local inflammatory response through cytokine release.

Foot pain is a frequent cause of lameness in horses and can involve multiple structures within the hoof. The T-ligament (an anatomical structure connecting the synovium of the distal interphalangeal joint, the digital flexor tendon sheath and the navicular bursa) is poorly described. Five pairs of equine cadaver distal forelimbs were collected from a slaughterhouse. Sagittal sections (medial, middle and lateral) were obtained and processed with Haematoxylin Eosin Safran, Unna's Orcein, and Picrosirius red stains. Histological assessment revealed that the T-ligament was covered by the surrounding synovia of the distal interphalangeal joint, the digital flexor tendon sheath and the navicular bursa. Its collagen content was lower (30.01%±10.15) than that of the collateral sesamoidean ligament (89.48%±5.8; P = .0008) and the middle phalanx (85.72%±3.67; P = .0008). Under polarized light microscopy, it showed a slight heterogeneous pattern of birefringence, with angle-related changes. Elastic fibres were more numerous (21.76%±8.72) than in the collateral sesamoidean ligament (0.28%±0.45), or deep digital flexor tendon (0.04%±0.02); and were more densely packed. Mean cell count was higher for the T-ligament than for other tissues (P = .0007). Blood vessels were identified in the T-ligament and were penetrating the deep digital flexor tendon (8/10 limbs, 5/5 horses). In conclusion, the T-ligament looked like a vinculum for the deep digital flexor tendon, with a central elastic core, surrounding loose connective tissue and blood vessels. It is not a ligament. Its clinical relevance still needs to be determined.

Although shedding of zoonotic brucellae in milk has been demonstrated in natural hosts, these data are still missing for the standard murine infection model. We therefore analysed shedding kinetics and the niche of B. melitensis in murine milk. Pregnant Balb/cByJ mice were intraperitoneally infected with 105 CFU of the 16 M reference strain, a 16 M mCherry mutant or a human isolate. Milk was collected over the course of lactation, and subjected to culture and immunofluorescence assays. Bacteria were also quantified in spleen and mammary glands of maternal mice and in spleen of the litter. The shedding of the three strains did not differ significantly (p = 0.301), ranging from log10 1.5 to 4.04 CFU/ml. A total of 73% of the mice excreted B. melitensis into the milk with peak values at mid-lactation; up to 30 bacteria/cell were found in macrophages and neutrophils. While the bacterial counts in the spleen of lactating females confirmed a well-established infection, only 50% of the pups harboured brucellae in their spleen, including the spleen of an uninfected pup fed by an infected foster mother. In conclusion, the murine model of infection may contribute to a better understanding of the zoonotic transmission of brucellosis.

Diffusion of drugs injected into the distal interphalangeal joint or the navicular (podotrochlear) bursa can influence diagnosis and treatment of foot pain. Previous anatomical and radiographic studies of the communication between these synovial structures have produced conflicting results and did not identify the location of any communication if present. This anatomic study aimed to assess the presence and site of communication between the distal interphalangeal joint and the navicular bursa in the horse by computed tomography arthrography. Sixty‐six pairs of cadaver forelimbs were injected with contrast medium into the distal interphalangeal joint and imaged by computed tomography arthrography. The presence of a communication, location of the communication and additional structural changes were assessed. Navicular bursa opacification occurred in 7 distal limbs (5.3%) following distal interphalangealjoint injection. One limb showed a communication through the T‐ligament and 6 limbs showed a communication through the distal sesamoidean impar ligament. In 3 cases, the communication through the distal sesamoidean impar ligament was associated with a distal border fragment. Our study showed that communication between the distal interphalangeal joint and navicular bursa is uncommon and inconsistent. Clinically, the presence of a communication could (1) influence the interpretation of diagnostic analgesia of the distal interphalangeal joint or the navicular bursa by facilitating the diffusion of local anaesthetic between these structures; (2) allow the drug and its potential adverse effects to spread from the treated synovial cavity to the non‐targeted synovial cavity; (3) be responsible for the failure of joint drainage in the case of sepsis.

Despite ample evidence for the therapeutic potential of inhibition of the cystine/glutamate antiporter system xc− in neurological disorders and in cancer, none of the proposed inhibitors is selective. In this context, a lot of research has been performed using the EMA- and FDA-approved drug sulfasalazine (SAS). Even though this molecule is already on the market for decades as an anti-inflammatory drug, serious side effects due to its use have been reported. Whereas for the treatment of the main indications, SAS needs to be cleaved in the intestine into the anti-inflammatory compound mesalazine, it needs to reach the systemic circulation in its intact form to allow inhibition of system xc−. The higher plasma levels of intact SAS (or its metabolites) might induce adverse effects, independent of its action on system xc−. Some of these effects have however been attributed to system xc− inhibition, calling into question the safety of targeting system xc−. In this study we chronically treated system xc− - deficient mice and their wildtype littermates with two different doses of SAS (160 mg/kg twice daily or 320 mg/kg once daily, i.p.) and studied some of the adverse effects that were previously reported. SAS had a negative impact on the survival rate, the body weight, the thermoregulation and/or stress reaction of mice of both genotypes, and thus independent of its inhibitory action on system xc−. While SAS decreased the total distance travelled in the open-field test the first time the mice encountered the test, it did not influence this parameter on the long-term and it did not induce other behavioral changes such as anxiety- or depressive-like behavior. Finally, no major histological abnormalities were observed in the spinal cord. To conclude, we were unable to identify any undesirable system xc−-dependent effect of chronic administration of SAS.

Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes – needed for invading epithelial cells – were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl. Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

A murine osmotic demyelination syndrome (ODS) model of the central nervous system included the relay thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei. Morphologic comparisons between treatments have revealed oligodendrocyte changes and, already 12 hours following the osmolality restoration, some heavily contrasted oligodendrocytes formed a unique intracellular primary cilium. This unique structure, found in vivo, in mature CNS oligodendrocytes, could account for a local awakening of some of the developmental proteome as it can be expressed in oligodendrocyte precursor cells. This resilience accompanied the emergence of arl13b protein expression along with restoration of nerve cell body axon hillocks shown in a previous issue of this journal. Additionally, the return of several thalamic oligodendrocyte fine features (nucleus, organelles) was shown 36 h later, including some mitosis. Those cell restorations and recognized translational activities comforted that local repairs could again take place, due to oligodendrocyte resilience after ODS instead or added to a postulated immigration of oligodendrocyte precursor cells distant from the sites of myelinolysis., info:eu-repo/semantics/published

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS.

To investigate the role of the interleukin IL-33/ST2 axis in systemic lupus erythematosus (SLE).Serum concentrations of IL-33 and sST2 were measured by sandwich ELISA in SLE patients (n=111) compared to sex- and age-matched healthy controls (n=36). The serum concentrations of IL-33 and sST2 were correlated with various clinical and biological parameters. The expressions of IL-33 and ST2L were investigated in kidney sections by immunohistochemistry in lupus nephritis patients (n=23) and controls (n=10).Serum levels of IL-33 were significantly higher in SLE patients (11.64±3.141 pg/mL) than in controls (1.043±0.8526 pg/mL) (p0.0001). Similarly, the serum concentrations of sST2 were significantly higher in SLE patients (34.013±2.043 pg/mL) than in controls (25.278±2.258 pg/mL) (p=0.046). sST2, but not IL-33, correlated significantly with disease activity index (SLEDAI). In addition, serum levels of sST2 were significantly higher in patients with lupus nephritis (45.438±5.661 pg/mL) that in SLE patients without renal involvement (30.691±1.941 pg/mL) (p=0.016). The immunoreactivity of IL-33 in renal biopsies of patients with lupus nephritis was not increased compared to controls, while the glomerular expression of ST2L was significantly higher in nephritis patients compared to controls.Although IL-33 and sST2 levels are both increased in SLE, sST2 represents a surrogate marker of disease activity and complications of nephritis.

Purpose Image processing plays a fundamental role in the study of central nervous system, for example in the analysis of the vascular network in neurodegenerative diseases. Synchrotron X-ray Phase-contrast micro-Tomography (SXPCT) is a very attractive method to study weakly absorbing samples and features, such as the vascular network in the spinal cord (SC). However, the identification and segmentation of vascular structures in SXPCT images is seriously hampered by the presence of image noise and strong contrast inhomogeneities, due to the sensitivity of the technique to small electronic density variations. In order to help with these tasks, we implemented a user-friendly ImageJ plugin based on a 3D Gaussian steerable filter, tuned up for the enhancement of tubular structures in SXPCT images. Methods The developed 3D Gaussian steerable filter plugin for ImageJ is based on the steerability properties of Gaussian derivatives. We applied it to SXPCT images of ex-vivo mouse SCs acquired at different experimental conditions. Results The filter response shows a strong amplification of the source image contrast-to-background ratio (CBR), independently of structures orientation. We found that after the filter application, the CBR ratio increases by a factor ranging from ~6 to ~60. In addition, we also observed an increase of 35% of the contrast to noise ratio in the case of injured mouse SC. Conclusion The developed tool can generally facilitate the detection/segmentation of capillaries, veins and arteries that were not clearly observable in non-filtered SXPCT images. Its systematic application could allow obtaining quantitative information from pre-clinical and clinical images.

The development of a murine model of osmotic demyelinating syndrome (ODS) allowed to study changes incurred in extrapontine zones of the CNS and featured neuron and glial cell changes in the relay thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei before, during and after ODS induction, and characterized without immune response. There, the neuron Wallerian-type deteriorations were verified with fine structure modifications of the neuron cell body, including some nucleus topology and its nucleolus changes. Morphologic analyses showed a transient stoppage of transcriptional activities while myelinated axons in the surrounding neuropil incurred diverse damages, previously reported. Even though the regional thalamus myelin deterioration was clearly recognized with light microscopy 248 h after osmotic recovery of ODS, ultrastructure analyses demonstrated that, at that time, the same damaged parenchyma regions contained nerve cell bodies that have already reactivated nucleus transcriptions and neuroplasm translations because peculiar accumulations of fibro-granular materials, similar to those detected in restored ODS astrocytes, were revealed in these restructuring nerve cell bodies. Their aspects suggested to be accumulations of ribonucleoproteins. The findings suggested that progressive neural function's recovery in the murine model could imitate some aspects of human ODS recovery cases., info:eu-repo/semantics/published

xCT is the specific subunit of System xc-, an antiporter importing cystine while releasing glutamate. Although xCT expression has been found in the spinal cord, its expression and role after spinal cord injury (SCI) remain unknown. The aim of this study was to characterize the role of xCT on functional and histological outcomes following SCI induced in wild-type (xCT+/+) and in xCT-deficient mice (xCT−/−). In the normal mouse spinal cord, slc7a11/xCT mRNA was detected in meningeal fibroblasts, vascular mural cells, astrocytes, motor neurons and to a lesser extent in microglia. slc7a11/xCT gene and protein were upregulated within two weeks post-SCI. xCT−/− mice recovered muscular grip strength as well as pre-SCI weight faster than xCT+/+ mice. Histology of xCT−/− spinal cords revealed significantly more spared motor neurons and a higher number of quiescent microglia. In xCT−/− mice, inflammatory polarization shifted towards higher mRNA expression of ym1 and igf1 (anti-inflammatory) while lower levels of nox2 and tnf-a (pro-inflammatory). Although astrocyte polarization did not differ, we quantified an increased expression of lcn2 mRNA. Our results show that slc7a11/xCT is overexpressed early following SCI and is detrimental to motor neuron survival. xCT deletion modulates intraspinal glial activation by shifting towards an anti-inflammatory profile.

Young male Zucker rats with a leptin receptor mutation are obese, have a non-insulin-dependent diabetes mellitus (NIDDM), and other endocrinopathies. Tibial branches of the sciatic nerve reveal a progressive demyelination that progresses out of the Schwann cells (SCs) where electron-contrast deposits are accumulated while the minor lines or intermembranous SC contacts display exaggerated spacings. Cajal bands contain diversely contrasted vesicles adjacent to the abaxonal myelin layer with blemishes; they appear dispatched centripetally out of many narrow electron densities, regularly spaced around the myelin annulus. These anomalies widen and yield into sectors across the stacked myelin layers. Throughout the worse degradations, the adaxonal membrane remains along the axonal neuroplasm. This peripheral neuropathy with irresponsive leptin cannot modulate hypothalamic-pituitary-adrenal axis and SC neurosteroids, thus exacerbates NIDDM condition. Additionally, the ultrastructure of the progressive myelin alterations may have unraveled a peculiar, centripetal mode of trafficking maintenance of the peripheral nervous system myelin, while some adhesive glycoproteins remain between myelin layers, somewhat hindering the axon mutilation. Heading title: Peripheral neuropathy and myelin.

The osmotic demyelination syndrome (ODS) is a non-primary inflammatory disorder of the central nervous system myelin that is often associated with a precipitous rise of serum sodium concentration. To investigate the physiopathology of ODS in vivo, we generated a novel murine model based on the abrupt correction of chronic hyponatremia. Accordingly, ODS mice developed impairments in brainstem auditory evoked potentials and in grip strength. At 24 hr post-correction, oligodendrocyte markers (APC and Cx47) were downregulated, prior to any detectable demyelination. Oligodendrocytopathy was temporally and spatially correlated with the loss of astrocyte markers (ALDH1L1 and Cx43), and both with the brain areas that will develop demyelination. Oligodendrocytopathy and astrocytopathy were confirmed at the ultrastructural level and culminated with necroptotic cell death, as demonstrated by pMLKL immunoreactivity. At 48 hr post-correction, ODS brains contained pathognomonic demyelinating lesions in the pons, mesencephalon, thalamus and cortical regions. These damages were accompanied by blood–brain barrier (BBB) leakages. Expression levels of IL-1β, FasL, TNFRSF6 and LIF factors were significantly upregulated in the ODS lesions. Quiescent microglial cells type A acquired an activated type B morphology within 24 hr post-correction, and reached type D at 48 hr. In conclusion, this murine model of ODS reproduces the CNS demyelination observed in human pathology and indicates ambiguous causes that is regional vulnerability of oligodendrocytes and astrocytes, while it discards BBB disruption as a primary cause of demyelination. This study also raises new queries about the glial heterogeneity in susceptible brain regions as well as about the early microglial activation associated with ODS., SCOPUS: ar.j, info:eu-repo/semantics/published

A murine model used to investigate the osmotic demyelination syndrome (ODS) demonstrated ultrastructural damages in thalamus nuclei. Following chronic hyponatremia, significant myelinolysis was merely detected 48 h after the rapid reinstatement of normonatremia (ODS 48 h). In ODS samples, oligodendrocytes and astrocytes revealed injurious changes associated with a few cell deaths while both cell types seemed to endure a sort of survival strategy: (a) ODS 12 h oligodendrocytes displayed nucleoplasm with huge heterochromatic compaction, mitochondria hypertrophy, and most reclaimed an active NN cell aspect at ODS 48 h. (b) Astrocytes responded to the osmotic stress by overall cell shrinkage with clasmatodendrosis, these changes accompanied nucleus wrinkling, compacted and segregated nucleolus, destabilization of astrocyte-oligodendrocyte junctions, loss of typical GFAP filaments, and detection of round to oblong woolly, proteinaceous aggregates. ODS 48 h astrocytes regained an active nucleus aspect, without restituting GFAP filaments and still contained cytoplasmic proteinaceous deposits. (c) Sustaining minor shrinking defects at ODS 12 h, neurons showed slight axonal injury. At ODS 48 h, neuron cell bodies emerged again with deeply indented nucleus and, owing nucleolus translational activation, huge amounts of polysomes along with secretory-like activities. (d) In ODS, activated microglial cells got stuffed with huge lysosome bodies out of captures cell damages, leaving voids in interfascicular and sub-vascular neuropil. Following chronic hyponatremia, the murine thalamus restoration showed macroglial cells acutely turned off transcriptional and translational activities during ODS and progressively recovered activities, unless severely damaged cells underwent cell death, leading to neuropil disruption and demyelination., SCOPUS: ar.j, info:eu-repo/semantics/published

Osmotic demyelination syndrome (ODS) is a disorder of the central myelin that is often associated with a precipitous rise of serum sodium. Remarkably, while the myelin and oligodendrocytes of specific brain areas degenerate during the disease, neighboring neurons and axons appear unspoiled, and neuroinflammation appears only once demyelination is well established. In addition to blood‒brain barrier breakdown and microglia activation, astrocyte death is among one of the earliest events during ODS pathology. This review will focus on various aspects of biochemical, molecular and cellular aspects of oligodendrocyte and astrocyte changes in ODS-susceptible brain regions, with an emphasis on the crosstalk between those two glial cells. Emerging evidence pointing to the initiating role of astrocytes in region-specific degeneration are discussed.

Young male Zucker rats with a leptin receptor mutation are obese, have a non-insulin-dependent diabetes mellitus (NIDDM), and other endocrinopathies. Fine structure aspects of the tibial branches of the sciatic nerve of lean (Fa/?) and obese (fa/fa) perfused rats were compared and revealed a progressive demyelination caused by Schwann cells (SCs). There, stacked myelin layers membranes and other adhering junctions were defective in many nerve fibers of the obese rats, including of the mesaxons of the smallest fibers. Additionally, the progressive myelin alterations caused by metabolomic alterations in membrane components sorted by the SCs may also have revealed a peculiar, centripetal mode of sorting and trafficking maintenance of the peripheral nerve myelin.

xCT protein is the specific subunit of System xc-, a multi-proteic complex importing cystine into cell while releasing extracellular glutamate. Although xCT protein expression is detected in brain glial cells, its expression in the spinal cord and during spinal cord disorders remain elusive. The aim of this study is to characterize the contribution of xCT to functional and histological outcomes following SCI, using wild-type mice (xCT+/+) and genetically-invalidated mice (xCT-/-). In situ hybridization allowed to detect xCT mRNA mostly in astrocyte subpopulations and in meningeal fibroblasts in the normal spinal cord. During the subacute phase of spinal cord injury, xCT mRNA could also be detected in significant amount in microglial cells. Moreover, overall xCT mRNA levels were upregulated, peaking at 4 days post-injury. While both injured xCT+/+ and xCT-/- mice recovered partly their motor functions, xCT-/- mice recovered muscular grip strength as well as pre-SCI weight substantially faster than xCT+/+ mice.Histology of injured spinal cords revealed increased number of motor neurons in xCT-/- mice at multiple distances around lesion epicenter. Intraspinal glutathione levels were unchanged in both injured groups. As xCT has been demonstrated as a regulator of microglial function (Mesci et al., Brain, 2015), we assessed markers of microglial activation. At 2 weeks post-SCI, the number of type A Iba1+ cells was unexpectedly much higher in contused xCT-/- than in xCT+/+ spinal cords. Analysis of M1/M2 polarization showed that contused xCT-/- spinal cords contained higher mRNA levels of Ym1 and IGF-1 (M2) while lower levels of NOX2 and TNF-a (M1). Additionally, the number of reactive astrocytes was slightly higher in xCT-/- mice. The number of surviving oligodendrocytes was unchanged between the two injured groups. This study suggests that, following SCI trauma, an early xCT upregulation (if confirmed at the protein level) is detrimental to motor neuron survival and influences inflammation by dysbalancing M1/M2 polarization.

Interleukin-33 (IL-33) is a member of the IL-1 family and has dual functions as a nuclear factor as well as a cytokine. The pivotal role of IL-33 as an active player contributing to aberrant local and systemic damage has been highlighted in several inflammatory and autoimmune diseases. Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by dry eyes and mouth syndrome due to local dysfunctions of exocrine glands, but also accompanied with systemic manifestations. The pathophysiology of pSS has been advocated as a conjecture of activated B and T cells as well as the production of inflammatory cytokines and autoantibodies, driving epithelial tissue damage and disease progression. In pSS, IL-33 is released in the extracellular space from damaged salivary cells upon pro-inflammatory stimuli and/or dysfunction of epithelial barrier. Counter-regulatory mechanisms are initiated to limit the pro-inflammatory actions of IL-33 as portrayed by an increase in the decoy receptor for IL-33, the soluble form of ST2 (sST2). In pSS and associated diseases, the levels of IL-33 are significantly elevated in the serum or tears of patients. Mechanistically, IL-33 acts in synergy with IL-12 and IL-23 on NK and NKT cells to boost the production of IFN-γ contributing to inflammation. TNF-α, IL-1β and IFN-γ in turn further increase the activation of IL-33/ST2 pathway, thereby constituting a vicious inflammatory loop leading to disease exacerbation. IL-33/ST2 axis is involved in Sjögren's syndrome and opens new perspectives as therapeutic target of one of the culprits in the inflammatory perpetuation.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease affecting primarily the population of motor neurons, even though a non-cell autonomous component, involving neighbouring non-neuronal cells, is more and more described. Despite 140 years of disease experience, still no efficient treatment exists against ALS. The inability to readily obtain the faulty cell types relevant to ALS has impeded progress in drug discovery for decades. However, the pioneer work of Shinya Yamanaka in 2007 in the stem cell field was a real breakthrough. Recent advances in cell reprogramming now grant access to significant quantities of CNS disease-affected cells. Induced pluripotent stem cells (iPSc) have been recently derived from patients carrying mutations linked to familial forms of ALS as well as from sporadic patients. Precise and mature protocols allow now their differentiation into ALS-relevant cell subtypes; sustainable and renewable sources of human motor neurons or glia are being available for ALS disease modelling, drug screening or for the development of cell therapies. In few years, the proof-of-concept was made that ALS disease-related phenotypes can be reproduced with iPSc and despite some remaining challenges, we are now not so far to provide platforms for the investigation of ALS therapeutics. This paper also reviews the pioneering studies regarding the applicability of iPSc technology in ALS animal models. From modest slowing down of ALS progression to no severe adverse effects, iPSc-based cell therapy resulted in promising premises in ALS preclinical paradigms, although long-term surveys are highly recommended.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

xCT protein is the specific subunit of System xc-, a multi-proteic complex importing cystine into cell while releasing extracellular glutamate. Although xCT protein expression is detected in brain glial cells, its expression in the spinal cord and during spinal cord disorders remain elusive. The aim of this study is to characterize the contribution of xCT to functional and histological outcomes following SCI, using wild-type mice (xCT+/+) and genetically-invalidated mice (xCT-/-). In situ hybridization allowed to detect xCT mRNA in astrocyte subpopulations and in meningeal fibroblasts in the normal spinal cord. During the early phase of spinal cord injury, xCT mRNA could also be detected in microglial cells and overall mRNA levels were upregulated, peaking at 4 days post-injury. While both injured xCT+/+ and xCT-/- mice recovered partly their motor functions, xCT-/- mice recovered muscular grip strength as well as pre-SCI weight substantially faster than xCT+/+ mice. Histology of injured spinal cords revealedincreased number of motor neurons in xCT-/- mice at multiple distances around lesion epicenter. As xCT has been demonstrated as a regulator of microglial function (Mesci et al., Brain, 2015), we assessed markers of microglial activation. At 2 weeks post-SCI, the number of type A Iba1+ cells was unexpectedly much higher in contused xCT-/- than in xCT+/+ spinal cords. Analysis of M1/M2 polarization showed that contused xCT-/- spinal cords contained higher mRNA levels of Ym1 and IGF-1 (M2) while lower levels of NOX2 and TNF-a (M1). Additionally, the number of astrocytes and oligodendrocytes were unchanged between the two injured groups. This study suggests that, following SCI trauma, an early xCT upregulation (if confirmed at the protein level) exacerbates microglia-driven inflammation and influences motor neuron survival.

info:eu-repo/semantics/published

Aim. Tracking human cells xenotransplanted into animal models is crucial for studying their in vivo biologic potential and essential to follow their fate in the field of cell therapy or tumor xenografting. Preclinical assessment for cellular therapy requires data on biodistribution, viability, integration into host tissue, differentiation into functional cells, tumorigenic potential and safety of delivery. Often injected in limited amount, these cells, from human origin, must be recognized with high sensitivity. Post-mortem immunohistochemistry is currently the most sensitive method for cell tracking affording the detection of specific cell populations, while preserving both their morphology and that of the surrounding environment. Here, we focused our work on the specific expression throughout the human body of three human-specific biomarkers: Ku80, human mitochondria (hMito) and Alu after transplantation of human stem cell in animals. Results. We showed that Ku80, hMito and Alu biomarkers are broadly expressed in human tissues with no or low cross-reactivity towards rat, mouse or pig tissues. Through in vitro studies, we demonstrated that the expression of Ku80 and Alu biomarkers is stable over time and does not change upon the differentiation process of human-derived induced pluripotent stem cells or glial-restricted precursors. We also showed that these human biomarkers could be reliably used to track human neural or human glial stem cells transplanted in the rodent spinal cord. Using computer-assisted image analysis, we were able to quantify the engraftment of these human stem cells in animal tissue. Human-specific antibodies helping at detecting apoptotic, proliferative or neural-committed cells were also characterized. Conclusions. Based on immunohistochemistry and in situ hybridization, this methodological study assesses the human-species specificity of Ku80, hMito and Alu biomarkers and proposes useful tools for the critical analysis of human stem cells tracking after transplantation, which is needed for both preclinical cell therapy evaluation and tumor xenografting analysis.

A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP+astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.

Fractal analysis is a powerful method for the morphological study of complex systems that is increasingly applied to biomedical images. Spatial resolution and image segmentation are crucial for the discrimination of tissue structures at the multiscale level. In this work, we have applied fractal analysis to high-resolution X-ray phase contrast micro-tomography (XrPC&mu, T) images in both uninjured and injured tissue of a mouse spinal cord. We estimated the fractal dimension (FD) using the box-counting method on tomographic slices segmented at different threshold levels. We observed an increased FD in the ipsilateral injured hemicord compared with the contralateral uninjured tissue, which was almost independent of the chosen threshold. Moreover, we found that images exhibited the highest fractality close to the global histogram threshold level. Finally, we showed that the FD estimate largely depends on the image histogram regardless of tissue appearance. Our results demonstrate that the pre-processing of XrPC&mu, T images is critical to fractal analysis and the estimation of FD.

Background/Aims: The expression and localization of several distinct glucose transporters (GLUT1, GLUT2, GLUT4, and SGLT1) was recently characterized in the parotid gland of normal rats by quantitative real-time PCR analysis, immunohistochemistry and Western blotting. The major aims of the present study was to compare the mRNA expression of these glucose transporters in both the parotid gland and submaxillary gland of control rats, streptozotocin-induced diabetic rats and hereditarily diabetic Goto-Kakizaki rats. Methods: Quantitative real-time PCR analysis was performed in the parotid and submaxillary salivary glands and, for purpose of comparison, also in the heart, kidney, liver, lung, muscle and pancreas from control animals and either streptozotocin-treated or Goto-Kakizaki rats. Results: The expression of GLUT4, but not GLUT1 or SGLT1, mRNA was decreased in the diabetic rats. The results also allow comparing both the mRNA expression level of the four glucose transporters in salivary glands and six other organs, and the diabetes-induced changes in such an expression in distinct locations. Conclusion: The mRNA expression of the insulin-dependent GLUT4 transporter was the sole to be significantly decreased in the salivary glands of diabetic animals. The possible consequence of such a decrease in terms of the control of salivary glucose concentration requires further investigation. Copyright © 2012 S. Karger AG, Basel., SCOPUS: ar.j, info:eu-repo/semantics/published

A primary cause of morbidity and mortality following cervical spinal cord injury (SCI) is respiratory compromise, regardless of the level of trauma. In particular, SCI at mid-cervical regions targets degeneration of both descending bulbospinal respiratory axons and cell bodies of phrenic motor neurons, resulting in deficits in the function of the diaphragm, the primary muscle of inspiration. Contusion-type trauma to the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. We have generated a mouse model of cervical contusion SCI that unilaterally targets both C4 and C5 levels, the location of the phrenic motor neuron pool, and have examined histological and functional outcomes for up to 6 weeks post-injury. We report that phrenic motor neuron loss in cervical spinal cord, phrenic nerve axonal degeneration, and denervation at diaphragm neuromuscular junctions (NMJ) resulted in compromised ipsilateral diaphragm function, as demonstrated by persistent reduction in diaphragm compound muscle action potential amplitudes following phrenic nerve stimulation and abnormalities in spontaneous diaphragm electromyography (EMG) recordings. This injury paradigm is reproducible, does not require ventilatory assistance, and provides proof-of-principle that generation of unilateral cervical contusion is a feasible strategy for modeling diaphragmatic/respiratory deficits in mice. This study and its accompanying analyses pave the way for using transgenic mouse technology to explore the function of specific genes in the pathophysiology of phrenic motor neuron degeneration and respiratory dysfunction following cervical SCI.

To determine the role of S100A8/A9 in the pathogenesis of primary Sjögren's syndrome (pSS).The serum levels of S100A8/A9 were determined in pSS patients and healthy controls by ELISA. The expression of S100A8/A9 in salivary glands was assessed by immunohistochemistry. The phenotype of S100A8+ and S100A9+ cells was identified using double immunofluorescence. The effects of S100A8/A9 on cytokine production by peripheral blood mononuclear cells (PBMCs) from pSS patients were determined in vitro by flow cytometry. The effects of pro-inflammatory cytokines on S100A8/A9 secretion were additionally investigated in vitro by ELISA in PBMCs from pSS patients and control subjects.Serum levels of S100A8/A9 were significantly increased in pSS patients compared to healthy controls. The tissular expression of S100A8 and S100A9, identified in professional phagocytes (neutrophils, monocytes and plasmacytoid dendritic cells), was increased in the salivary glands of pSS patients and correlated with focus score. In vitro, recombinant S100A8/A9 increased the production of IL-1β, IL-6, TNF-α, IFN-γ, IL-10, IL-17A and IL-22 by PBMCs. The S100A8/A9-induced increase in TNF-α production in pSS patients was significant relative to controls. Furthermore, IL-1β, TNF-α, IL-6, and IL-17A stimulated release of S100A8/A9 from PBMCs in pSS patients.S100A8/A9 is increased in pSS patients contributing to the in vitro increased production of pro-inflammatory cytokines. As such, S100A8/A9 in concert with other cytokines might contribute to the pathogenesis of pSS.

Respiratory dysfunction is the leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). Injuries targeting mid-cervical spinal cord regions affect the phrenic motor neuron pool that innervates the diaphragm, the primary respiratory muscle of inspiration. Contusion-type injury in the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. In an attempt to target the phrenic motor neuron pool, two unilateral contusion injury paradigms were tested, a single injury at level C4 and a double injury both at levels C3 and C4, and animals were followed for up to 6 weeks post-injury. Both unilateral cervical injury paradigms are reproducible with no mortality or need for breathing assistance, and are accompanied by phrenic motor neuron loss, phrenic nerve axon degeneration, diaphragm atrophy, denervation and subsequent partial reinnervation at the diaphragm neuromuscular junction, changes in spontaneous diaphragm EMG recordings, and reduction in phrenic nerve compound muscle action potential amplitude. These findings demonstrate significant and chronically persistent respiratory compromise following mid-cervical SCI due to phrenic motor neuron degeneration. These injury paradigms and accompanying analyses provide important tools both for understanding mechanisms of phrenic motor neuron and diaphragm pathology following SCI and for evaluating therapeutic strategies in clinically relevant cervical SCI models.

Oral Diseases (2012) 18, 568–574 Objective: To determine whether a link exists between inflammation and aquaporin-5 distribution in submandibular glands from three animal models for Sjogren’s syndrome: IQI/JIC, r1ΔT/r2n and non-obese diabetic mice. Methods: Mice of different ages were used. Inflammatory infiltrates were quantified using the focus score. Acinar aquaporin-5 subcellular distribution was determined by immunohistochemistry and quantified using labelling indices. Results: Minor inflammatory infiltrates were present in r1f/r2n mice. Massive inflammatory infiltrates and acinar destruction were observed in 24-week-old non-obese diabetic mice, 10-and 13-month-old IQI/JIC mice and some r1ΔT/r2n mice. Aquaporin-5 immunoreactivity was primarily apical in submandibular glands from 8- and 24-week-old Balb/C mice, 8-week-old non-obese diabetic mice, 2-, 4- and 7-month-old IQI/JIC mice and r1f/r2n mice. In contrast, decreased apical aquaporin-5 labelling index with concomitant increased apical-basolateral, apical-cytoplasmic and/or apical-basolateral-cytoplasmic aquaporin-5 labelling indices was observed in 24-week-old non-obese diabetic, 10- and 13-month-old IQI/JIC and r1ΔT/r2n mice with a focus score ≥ 1. Conclusions: Altered aquaporin-5 distribution in submandibular acinar cells from IQI/JIC, non-obese diabetic and r1ΔT/r2n mice with a focus score ≥ 1 appears to be concomitant to the presence of inflammatory infiltrates and acinar destruction.

To determine serum levels of high-mobility group box 1 (HMGB1) in patients with primary Sjögren's syndrome (pSS) as compared to healthy volunteers and patients with sicca symptoms, and to determine whether serum HMGB1 levels are correlated with disease activity in pSS.Serum HMGB1 levels were determined by enzyme-linked immunosorbent assay (ELISA) in 101 patients with pSS, 13 patients with sicca symptoms, and 40 healthy volunteers. Clinical and laboratory variables were also analysed and serum HMGB1 levels were correlated with the Sjögren's Syndrome Disease Activity Index (SSDAI).The serum levels of HMGB1 were significantly increased in pSS patients as compared to patients with sicca symptoms and healthy controls (p = 0.04 and p = 0.01, respectively). In the subgroups of patients with anti-SSA autoantibodies, the serum levels of HMGB1 were significantly higher than those in the subgroup of pSS patients who were anti-SSA negative and in healthy controls and patients with sicca symptoms (p0.001, p0.001, and p = 0.004, respectively). There was no significant correlation between serum HMGB1 levels (in pSS patients with anti-SSA autoantibodies) and SSDAI score (r = 0.03, p = 0.83). Patients with active disease had higher HMGB1 levels than patients with low disease activity (p = 0.04), but HMGB1 levels did not correlate with the SSDAI.Serum HMGB1 levels are increased in pSS patients and more specifically in patients with SSA autoantibodies. There was, however, no correlation of HMGB1 with the SSDAI.

Fast anterograde and retrograde axoplasmic transports in neurons rely on the activity of molecular motors and are critical for maintenance of neuronal and synaptic functions. Disturbances of axoplasmic transport have been identified in Alzheimer's disease and in animal models of this disease, but their mechanisms are not well understood. In this study we have investigated the distribution and the level of expression of kinesin light chains (KLCs) (responsible for binding of cargos during anterograde transport) and of dynein intermediate chain (DIC) (a component of the dynein complex during retrograde transport) in frontal cortex and cerebellar cortex of control subjects and Alzheimer's disease patients. By immunoblotting, we found a significant decrease in the levels of expression of KLC1 and 2 and DIC in the frontal cortex, but not in the cerebellar cortex, of Alzheimer's disease patients. A significant decrease in the levels of synaptophysin and of tubulin-β3 proteins, two neuronal markers, was also observed. KLC1 and DIC immunoreactivities did not co-localize with neurofibrillary tangles. The mean mRNA levels of KLC1, 2 and DIC were not significantly different between controls and AD patients. In SH-SY5Y neural cells, GSK-3β phosphorylated KLC1, a change associated to decreased association of KLC1 with its cargoes. Increased levels of active GSK-3β and of phosphorylated KLC1 were also observed in AD frontal cortex. We suggest that reduction of KLCs and DIC proteins in AD cortex results from both reduced expression and neuronal loss, and that these reductions and GSK-3β-mediated phosphorylation of KLC1 contribute to disturbances of axoplasmic flows and synaptic integrity in Alzheimer's disease., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Background/Aims: Salivary glucose is often considered as being from glandular origin. Little information is available, however, on the possible role of glucose transporters in the secretion of the hexose by salivary glands. The major aim of the present study was to investigate the expression and localization of several distinct glucose transporters in acinar cells of rat parotid glands. Methods: Quantitative real-time PCR analysis, immunohistochemistry and western blotting techniques were used to assess the presence of SGLT1, GLUT1, GLUT2 and GLUT4 in acinar cells of rat parotid glands. Results: Quantitative real-time PCR documented the expression of SGLT1 and GLUT1 in parotid tissues, with a much lower level of GLUT4 mRNA and no expression of GLUT2 mRNA. Western blot analysis revealed the presence of SGLT1, GLUT1 and GLUT4 proteins, but not GLUT2 proteins in the parotid extract. Immunohistochemistry confirmed these findings. SGLT1 was specifically located at the baso-lateral membrane, co-localizing with Na + /K + ATPase. GLUT1 was found both at the baso-lateral and apical level. GLUT4 appeared to be also located at the baso-lateral level. However, too little GLUT4 was present to allow co-localization labeling. Conclusion: Based on these findings, a model is proposed for the transport of glucose into the acinar cells and thereafter into the acinar lumen.