Altered glycosylation in secreting cells of the gastric glands of aquaporin-4-deficient mice.

Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin-4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4-deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4-deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5-µm thick, were analyzed by histochemical methods (Periodic acid-Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA-I, AAA, LTA). Immunohistochemical methods such as anti-Muc6 for neck cells and anti- ß- H+/K+-ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions. RESEARCH HIGHLIGHTS: Glycopatterns in gastric glands were compared between wild type (WT) and AQP4-deficient knockout (KO) mice by histochemical and lectin-binding methods. In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed. In the parietal cells lower fucosylation also resulted. AQP4-deficiency affects glycosylation and could result in altered functionality and pathological conditions.

Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

AQP4-independent TRPV4 modulation of plasma membrane water permeability.

Despite of the major role of aquaporin (AQP) water channels in controlling transmembrane water fluxes, alternative ways for modulating water permeation have been proposed. In the Central Nervous System (CNS), Aquaporin-4 (AQP4) is reported to be functionally coupled with the calcium-channel Transient-Receptor Potential Vanilloid member-4 (TRPV4), which is controversially involved in cell volume regulation mechanisms and water transport dynamics. The present work aims to investigate the selective role of TRPV4 in regulating plasma membrane water permeability in an AQP4-independent way. Fluorescence-quenching water transport experiments in Aqp4-/- astrocytes revealed that cell swelling rate is significantly increased upon TRPV4 activation and in the absence of AQP4. The biophysical properties of TRPV4-dependent water transport were therefore assessed using the HEK-293 cell model. Calcein quenching experiments showed that chemical and thermal activation of TRPV4 overexpressed in HEK-293 cells leads to faster swelling kinetics. Stopped-flow light scattering water transport assay was used to measure the osmotic permeability coefficient (Pf, cm/s) and activation energy (Ea, kcal/mol) conferred by TRPV4. Results provided evidence that although the Pf measured upon TRPV4 activation is lower than the one obtained in AQP4-overexpressing cells (Pf of AQP4 = 0.01667 ± 0.0007; Pf of TRPV4 = 0.002261 ± 0.0004; Pf of TRPV4 + 4αPDD = 0.007985 ± 0.0006; Pf of WT = 0.002249 ± 0.0002), along with activation energy values (Ea of AQP4 = 0.86 ± 0.0006; Ea of TRPV4 + 4αPDD = 2.73 ± 1.9; Ea of WT = 8.532 ± 0.4), these parameters were compatible with a facilitated pathway for water movement rather than simple diffusion. The possibility to tune plasma membrane water permeability more finely through TRPV4 might represent a protective mechanism in cells constantly facing severe osmotic challenges to avoid the potential deleterious effects of the rapid cell swelling occurring via AQP channels.

The emerging science of Glioception: Contribution of glia in sensing, transduction, circuit integration of interoception.

Interoception is the process by which the nervous system regulates internal functions to achieve homeostasis. The role of neurons in interoception has received considerable recent attention, but glial cells also contribute. Glial cells can sense and transduce signals including osmotic, chemical, and mechanical status of extracellular milieu. Their ability to dynamically communicate "listening" and "talking" to neurons is necessary to monitor and regulate homeostasis and information integration in the nervous system. This review introduces the concept of "Glioception" and focuses on the process by which glial cells sense, interpret and integrate information about the inner state of the organism. Glial cells are ideally positioned to act as sensors and integrators of diverse interoceptive signals and can trigger regulatory responses via modulation of the activity of neuronal networks, both in physiological and pathological conditions. We believe that understanding and manipulating glioceptive processes and underlying molecular mechanisms provide a key path to develop new therapies for the prevention and alleviation of devastating interoceptive dysfunctions, among which pain is emphasized here with more focused details.

Serum Neurofilament Light Chain and Glial Fibrillary Acidic Protein as Potential Diagnostic Biomarkers in Autism Spectrum Disorders: A Preliminary Study.

Autism spectrum disorder (ASD) is one of the most common neurodevelopment disorders, characterized by a multifactorial etiology based on the interaction of genetic and environmental factors. Recent evidence supports the neurobiological hypothesis based on neuroinflammation theory. To date, there are no sufficiently validated diagnostic and prognostic biomarkers for ASD. Therefore, we decided to investigate the potential diagnostic role for ASD of two biomarkers well known for other neurological inflammatory conditions: the glial fibrillary acidic protein (GFAP) and the neurofilament (Nfl). Nfl and GFAP serum levels were analyzed using SiMoA technology in a group of ASD patients and in a healthy control group (CTRS), age- and gender-matched. Then we investigated the distribution, frequency, and correlation between serum Nfl and GFAP levels and clinical data among the ASD group. The comparison of Nfl and GFAP serum levels between ASD children and the control group showed a mean value of these two markers significantly higher in the ASD group (sNfL mean value ASD pt 6.86 pg/mL median value ASD pt 5.7 pg/mL; mean value CTRS 3.55 pg/mL; median value CTRS 3.1 pg; GFAP mean value ASD pt 205.7 pg/mL median value ASD pt 155.4 pg/mL; mean value CTRS 77.12 pg/mL; median value CTRS 63.94 pg/mL). Interestingly, we also found a statistically significant positive correlation between GFAP levels and hyperactivity symptoms (p-value <0.001). Further investigations using larger groups are necessary to confirm our data and to verify in more depth the potential correlation between these biomarkers and ASD clinical features, such as the severity of the core symptoms, the presence of associated symptoms, and/or the evaluation of a therapeutic intervention. However, these data not only might shed a light on the neurobiology of ASD, supporting the neuroinflammation and neurodegeneration hypothesis, but they also might support the use of these biomarkers in the early diagnosis of ASD, to longitudinally monitor the disease activity, and even more as future prognostic biomarkers.

Real-Life Experience of the Effects of Cladribine Tablets on Lymphocyte Subsets and Serum Neurofilament Light Chain Levels in Relapsing Multiple Sclerosis Patients.

Although cladribine induces sustained reductions in peripheral T and B lymphocytes, little is known about its effect on axonal damage reduction in multiple sclerosis (MS), which could be demonstrated by assessing the serum neurofilament light chain (sNfL) levels. We investigated the reduction/reconstitution of different lymphocyte subsets (LS) by verifying the correlation with no evidence of disease activity (NEDA) and the variation in sNfL levels during cladribine treatment. We analysed 33 highly active relapsing MS patients and followed them up for 12 ± 3.3 months; blood samples were collected at treatment start (W0) and after 8, 24 and 48 weeks. Seventeen patients (60.7%) showed NEDA during the first treatment. At week 8, we observed a significant decrease in B memory cells, B regulatory 1 CD19+/CD38+ and B regulatory 2 CD19+/CD25+, a significant increase in T regulatory CD4+/CD25+, a slight increase in T cytotoxic CD3+/CD8+ and a non-significant decrease in T helper CD3+/CD4+. Starting from week 24, the B subsets recovered; however, at week 48, CD19+/CD38+ and CD19+/CD25+ reached values near the baseline, while the Bmem were significantly lower. The T cell subsets remained unchanged except for CD4+/CD25+, which increased compared to W0. The LS changes were not predictive of NEDA achievement. The sNfL levels were significantly lower at week 24 (p = 0.046) vs. baseline. These results could demonstrate how cladribine, by inflammatory activity depletion, can also reduce axonal damage, according to the sNfL levels.

AQP4-dependent glioma cell features affect the phenotype of surrounding cells via extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) are membrane-enclosed particles released systemically by all cells, including tumours. Tumour EVs have been shown to manipulate their local environments as well as distal targets to sustain the tumour in a variety of tumours, including glioblastoma (GBM). We have previously demonstrated the dual role of the glial water channel aquaporin-4 (AQP4) protein in glioma progression or suppression depending on its aggregation state. However, its possible role in communication mechanisms in the microenvironment of malignant gliomas remains to be unveiled. RESULTS: Here we show that in GBM cells AQP4 is released via EVs that are able to affect the GBM microenvironment. To explore this role, EVs derived from invasive GBM cells expressing AQP4-tetramers or apoptotic GBM cells expressing orthogonal arrays of particles (AQP4-OAPs) were isolated, using a differential ultracentrifugation method, and were added to pre-seeded GBM cells. Confocal microscopy analysis was used to visualize the interaction and uptake of AQP4-containing EVs by recipient cells. Chemoinvasion and Caspase3/7 activation assay, performed on recipient cells after EVs uptake, revealed that EVs produced by AQP4-tetramers expressing cells were able to drive surrounding tumour cells toward the migratory phenotype, whereas EVs produced by AQP4-OAPs expressing cells drive them toward the apoptosis pathway. CONCLUSION: This study demonstrates that the different GBM cell phenotypes can be transferred by AQP4-containing EVs able to influence tumour cell fate toward invasiveness or apoptosis. This study opens a new perspective on the role of AQP4 in the brain tumour microenvironment associated with the EV-dependent communication mechanism.

Strategies to Screen Anti-AQP4 Antibodies from Yeast Surface Display Libraries.

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.

The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain.

AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine331 and serine335, located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser335 is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser335 was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser335 in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis.

Alteration of the translational readthrough isoform AQP4ex induces redistribution and downregulation of AQP4 in human glioblastoma.

Glioblastoma multiforme (GBM) is characterized by a remarkable cellular and molecular heterogeneity that make the behavior of this tumor highly variable and resistant to therapy. In addition, the most serious clinical complication of GBM and other brain tumors is the development of vasogenic edema which dramatically increase the intracranial pressure. In the present study we evaluate the expression, supramolecular organization and spatial distribution of AQP4 and AQP4ex, the new readthrough isoform of AQP4, in relationship with the degree of vasogenic brain edema and tumor progression. To this purpose, tissue samples from regions of tumor core, peritumoral and non-infiltrated tissues of each GBM patient (n = 31) were analyzed. Immunofluorescence experiments revealed that the expression of AQP4ex was almost absent in tumoral regions while the canonical AQP4 isoforms appear mostly delocalized. In peritumoral tissues, AQP4 expression was found altered in those perivascular astrocyte processes where AQP4ex appeared reduced and partially delocalized. Protein expression levels measured by immunoblot showed that global AQP4 was reduced mainly in the tumor core. Notably, the relative amount of AQP4ex was more severely reduced starting from the peritumoral region. BN-PAGE experiments showed that the supramolecular organization of AQP4 is only partially affected in GBM. Edema assessment by magnetic resonance imaging revealed that the level of AQP4ex downregulation correlated with edema severity. Finally, the degree of BBB alteration, measured with sodium fluorescein content in GBM biopsies, correlated with the edema index and AQP4ex downregulation. Altogether these data suggest that the AQP4ex isoform is critical in the triggering event of progressive downregulation and mislocalization of AQP4 in GBM, which may affect the integrity of the BBB and contributes to accumulation of edema in the peritumoral tissue. Thus, AQP4ex could be considered as a potential early biomarker of GBM progression.

Serum Neurofilament Light Chain Levels and Myelin Oligodendrocyte Glycoprotein Antibodies in Pediatric Acquired Demyelinating Syndromes.

Introduction: The relationship between serum neurofilament light chain (sNfL) and myelin oligodendrocyte glycoprotein antibody (MOG-Ab) status has not been yet investigated in children with the acquired demyelinating syndrome (ADS). Objective and Methods: The sNfL levels and MOG-Abs were measured by ultrasensitive single-molecule array and cell-based assay in a cohort of 37 children with ADS and negativity for serum anti-aquaporin 4 (AQP4) antibodies. The sNfL levels were compared in MOG-Ab+/MOG-Ab- and in two subgroups MOG-Ab+ with/without encephalopathy. Results: About 40% ADS resulted in MOG-Ab+. MOG-Ab+ were younger at sampling (median = 9.8; range = 2.17-17.5 vs. 14.7/9-17; p = 0.002) with lower frequency of cerebrospinal fluid oligoclonal bands positivity (27% vs. 70%; p = 0.013) compared to MOG-Ab-. About 53% of MOG-Ab+ presented encephalopathy at onset, 1/22 of MOG-Ab- (p = 0.0006). Higher sNfL levels (p = 0.0001) were found in MOG-Ab+ (median/range = 11.11/6.8-1,129) and MOG-Ab- (median/range = 11.6/4.3-788) compared to age-matched controls (median/range = 2.98/1-4.53), without significant difference. MOG-Ab+ with encephalopathy resulted significantly younger at sampling (median/range: 4.5/2.17-11.17 vs. 14.16/9.8-17.5; p = 0.004), had higher sNfL levels (median/range:75.24/9.1-1,129 vs. 10.22/6.83-50.53; p = 0.04), and showed a trend for higher MOG-Ab titer (0.28/0.04-0.69 vs. 0.05/0.04-0.28; p = 0.1) in comparison to those without encephalopathy. Discussion: We confirmed high sNfL levels in pediatric ADS independently from the MOG-Ab status. Encephalopathy at onset is associated more frequently with MOG Ab+ children with higher sNfL levels and MOG titer. These findings suggest a role of acute demyelination in association with axonal damage in the pathogenesis of encephalopathy in pediatric ADS.

Cell Volume Regulation Mechanisms in Differentiated Astrocytes.

BACKGROUND/AIMS: The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K+ channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4(TRPV4), and Volume Regulated Anion Channel (VRAC). METHODS: The calcein quenching method was used to study water transport and cell volume regulation. Calcium imaging and electrophysiology (patch-clamp) were used for functional analyses of calcium dynamics and chloride currents. Western blot and immunofluorescence were used to analyse the expression and localization of the channel proteins of interest. RESULTS: We found that the increase in water permeability, previously observed in differentiated astrocytes, occurs simultaneously with more efficient regulatory volume increase and regulatory volume decrease. Accordingly, the magnitude of the hypotonic induced intracellular calcium response, typically mediated by TRPV4, as well as the hypotonic induced VRAC current, was almost twice as high in differentiated astrocytes. Interestingly, while we confirmed increased AQP4 expression in the membrane of differentiated astrocytes, the expression of the channels TRPV4 and Leucine-Rich Repeats-Containing 8-A (LRRC8-A) were comparable between differentiated and non-differentiated astrocytes. CONCLUSION: The reported results indicate that AQP4 up-regulation observed in differentiated astrocytes might promote higher sensitivity of the cell to osmotic changes, resulting in increased magnitude of calcium signaling and faster kinetics of the RVD and RVI processes. The implications for cell physiology and the mechanisms underlying astrocytic interaction with nanostructured interfaces are discussed.

Regulation of aquaporin-4 expression in the central nervous system investigated using M23-AQP4 null mouse.

In astrocytes, unknown mechanisms regulate the expression of M1 and M23 isoforms of water channel aquaporin-4 (M1-AQP4 and M23-AQP4). The ratio between these two isoforms controls the AQP4 assembly state in the plasma membrane known as orthogonal arrays of particles (OAPs). To give new insights into these mechanisms, here, we explore the regulation of AQP4 expression in the spinal cord of a CRISPR/Cas9 M23-null mouse model (M23-null). In the M23-null spinal cord OAP assembly, the perivascular localization of AQP4 and M1-AQP4 protein were drastically reduced. In heterozygous, M1-AQP4 was proportionally reduced with M23-AQP4, maintaining the isoform ratio unaffected. We hypothesize a role of the M23-AQP4 in the regulation of M1-AQP4 expression. M1-AQP4 transcription, splicing and M1-AQP4 protein degradation were found to be unaffected in M23-null spinal cord and in M23-null astrocyte primary culture. The translational control was investigated by mRNA-protein pull down and quantitative mass spectrometry, to isolate and quantify AQP4 mRNA binding proteins (AQP4-RBPs). Compared to WT, in M23-null spinal cord, the interaction between AQP4 mRNA and polypyrimidine tract binding protein 1, a positive regulator of AQP4 translation, was higher, while interaction with the RNA helicase DDX17 was lower. In astrocyte primary cultures, DDX17 knockdown upregulated AQP4 protein expression and increased cell swelling, leaving AQP4 mRNA levels unchanged. Here, we identify AQP4-RBPs and provide evidence that in mouse spinal cord M23-AQP4 deletion changes the interaction between AQP4 mRNA and some RBPs involved in AQP4 translation. We describe for the first time the RNA helicase DDX17 as a regulator of AQP4 expression in astrocytes.

Orchestrating aquaporin-4 and connexin-43 expression in brain: Differential roles of α1- and ß1-syntrophin.

Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether ß1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of ß1-syntrophin deletion is rather limited, double knockout of α1- and ß1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.

Longitudinal Evaluation of Serum MOG-IgG and AQP4-IgG Antibodies in NMOSD by a Semiquantitative Ratiometric Method.

Background and purpose: Immunoadsorption (IA) is an antibody-depleting therapy used to treat neuromyelitis optica spectrum disorder (NMOSD) associated to antiaquaporin 4 (anti-AQP4-IgG) and antimyelin oligodendrocyte glycoprotein (anti-MOG-IgG) serum autoantibodies. Our aim was to evaluate longitudinal changes of serum MOG-IgG and AQP4-IgG antibody titer and to correlate it with the clinical status. Methods: Autoantibody titer and clinical features of two MOG-IgG+/AQP4-IgG- and two AQP4-IgG+/MOG-IgG- patients with NMOSD were collected at baseline (T0), after 6 IA courses (T1), and then 2 weeks (T2) and 6 months after treatment (T3). A fluorescent ratiometric assay was used for a quantitative detection of MOG and AQP4 antibodies, based on HEK-293 cells transfected with the full-length hMOG fused to GFP or h-AQP4-M23 isoform fused to m-cherry, respectively. We defined the antibody titer as MOG quantitative ratio (MOGqr) and AQP4 quantitative ratio (AQP4qr). Results: In Case 1, the MOGqr dropped from 0.98 at T0 to 0.14 at T3, and in Case 2, it decreased from 0.96 at T0 to undetectable at T3. In Case3, the AQP4qr remained high: 0.90 at T0 and 0.92 at T3. In Case 4, the AQP4qr decreased from 0.50 at T0 to undetectable at T3. Complete recovery was found in Cases 1, 2, and 4. Conclusions: Semiquantitative ratiometric method accurately detects even slight variation of MOG-IgG and AQP4-IgG titer, suggesting it may be useful to monitor the antibody titer during the disease course and maintenance immunotherapy.

Orthogonal arrays of particle assembly are essential for normal aquaporin-4 expression level in the brain.

Astrocyte endfeet are endowed with aquaporin-4 (AQP4)-based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 tetramers, we have generated a novel "OAP-null" mouse model selectively lacking the OAP forming M23-AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS-PAGE and Western blot performed on OAP-null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1-AQP4-based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super-resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild-type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP-null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.

Effect of Cladribine on Neuronal Apoptosis: New Insight of In Vitro Study in Multiple Sclerosis Therapy.

BACKGROUND: Cladribine (2-CdA) can cross the blood-brain barrier, resulting in inhibition of DNA synthesis and repair and disruption of cellular proliferation in actively dividing lymphocytes. No data on effect on neurons are available. AIM: To study "in vitro" 2-CdA apoptotic effects on neurons in healthy donor and multiple sclerosis patient lymphocytes. METHODS: Neuroblastoma cells were co-cultured with lymphocytes, with and without 2-CdA. RESULTS: Apoptosis increased in lymphocytes with 2-CdA; increase was also observed when lymphocytes were cultured with neuronal cells. However, neurons were not affected by 2-CdA for apoptosis. CONCLUSIONS: 2-CdA causes peripheral and central lymphocyte death preserving neurons, with a reasonable impact on inflammation and neuroprotection.

Stimulation of water and calcium dynamics in astrocytes with pulsed infrared light.

Astrocytes are non-neuronal cells that govern the homeostatic regulation of the brain through ions and water transport, and Ca2+ -mediated signaling. As they are tightly integrated into neural networks, label-free tools that can modulate cell function are needed to evaluate the role of astrocytes in brain physiology and dysfunction. Using live-cell fluorescence imaging, pharmacology, electrophysiology, and genetic manipulation, we show that pulsed infrared light can modulate astrocyte function through changes in intracellular Ca2+ and water dynamics, providing unique mechanistic insight into the effect of pulsed infrared laser light on astroglial cells. Water transport is activated and, IP3 R, TRPA1, TRPV4, and Aquaporin-4 are all involved in shaping the dynamics of infrared pulse-evoked intracellular calcium signal. These results demonstrate that astrocyte function can be modulated with infrared light. We expect that targeted control over calcium dynamics and water transport will help to study the crucial role of astrocytes in edema, ischemia, glioma progression, stroke, and epilepsy.

Tissue Distribution of the Readthrough Isoform of AQP4 Reveals a Dual Role of AQP4ex Limited to CNS.

Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.