Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions.

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood-brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca2+ indicator GCaMP6f to study Ca2+ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two-photon microscopy. Ca2+ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca2+ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau-type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5-triphosphate receptor (IP3R2) severely suppressed the Ca2+ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca2+ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin-4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca2+ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca2+ signals only in subpial and perivascular endfeet.

Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood-brain water uptake and confers barrier function on perivascular astrocyte endfeet.

Tissue- and cell-specific deletion of the Aqp4 gene is required to differentiate between the numerous pools of aquaporin-4 (AQP4) water channels. A glial-conditional Aqp4 knockout mouse line was generated to resolve whether astroglial AQP4 controls water exchange across the blood-brain interface. The conditional knockout was driven by the glial fibrillary acidic protein promoter. Brains from conditional Aqp4 knockouts were devoid of AQP4 as assessed by Western blots, ruling out the presence of a significant endothelial pool of AQP4. In agreement, immunofluorescence analysis of cryostate sections and quantitative immunogold analysis of ultrathin sections revealed no AQP4 signals in capillary endothelia. Compared with litter controls, glial-conditional Aqp4 knockout mice showed a 31% reduction in brain water uptake after systemic hypoosmotic stress and a delayed postnatal resorption of brain water. Deletion of astroglial Aqp4 did not affect the barrier function to macromolecules. Our data suggest that the blood-brain barrier (BBB) is more complex than anticipated. Notably, under certain conditions, the astrocyte covering of brain microvessels is rate limiting to water movement.

Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse.

Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin-4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer's disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here, we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus, and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α-syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α-syntrophin--while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes--had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. In addition, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α-syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization.

Deletion of aquaporin-4 changes the perivascular glial protein scaffold without disrupting the brain endothelial barrier.

Expression of the water channel aquaporin-4 (AQP4) at the blood-brain interface is dependent upon the dystrophin associated protein complex. Here we investigated whether deletion of the Aqp4 gene affects the molecular composition of this protein scaffold and the integrity of the blood-brain barrier. High-resolution immunogold cytochemistry revealed that perivascular expression of α-syntrophin was reduced by 60% in Aqp4(-/-) mice. Additionally, perivascular AQP4 expression was reduced by 88% in α-syn(-/-) mice, in accordance with earlier reports. Immunofluorescence showed that Aqp4 deletion also caused a modest reduction in perivascular dystrophin, whereas ß-dystroglycan labeling was unaltered. Perivascular microglia were devoid of AQP4 immunoreactivity. Deletion of Aqp4 did not alter the ultrastructure of capillary endothelial cells, the expression of tight junction proteins (claudin-5, occludin, and zonula occludens 1), or the vascular permeability to horseradish peroxidase and Evans blue albumin dye. We conclude that Aqp4 deletion reduces the expression of perivascular glial scaffolding proteins without affecting the endothelial barrier. Our data also indicate that AQP4 and α-syntrophin are mutually dependent upon each other for proper perivascular expression.

Presence of a t(12;18)(q14;q21) Chromosome Translocation and Fusion of the Genes for High-mobility Group AT-Hook 2 (HMGA2) and WNT Inhibitory Factor 1 (WIF1) in Infrapatellar Fat Pad Cells from a Patient With Hoffa's Disease.

BACKGROUND/AIM: Hoffa's disease is anterior knee pain presumably stemming from inflammatory fibrous hyperplasia of the infrapatellar fat pad (Hoffa's pad). The etiology and pathogenesis are unclear, however, and no genetic information about the disease has been published. We report the genetic findings in cells from the fat pad of a patient with Hoffa's disease. MATERIALS AND METHODS: Infrapatellar fat pad cells from a patient with Hoffa's disease were examined using cytogenetic, RNA sequencing, reverse transcription-polymerase chain reaction, and Sanger sequencing techniques. RESULTS: Cytogenetic examination of short-term cultured cells from the Hoffa's pad revealed a balanced t(12;18)(q14;q21) translocation as the sole chromosomal aberration. RNA sequencing detected an out-of-frame fusion of exon 3 of the gene coding for high mobility group AT-hook 2 (HMGA2) with exon 9 of the gene coding for WNT inhibitory factor 1 (WIF1). The fusion was subsequently verified by reverse transcription-polymerase chain reaction together with Sanger sequencing. CONCLUSION: Our data indicate that Hoffa's disease is a neoplastic process with acquired genetic aberrations similar to those found in many benign tumors of connective tissues. The genetic aberrations are presumably acquired by mesenchymal stem cells of the infrapatellar fat pad inducing proliferation and differentiation into adipocytes or other mature connective tissue cells.

Rare KMT2A-ELL and Novel ZNF56-KMT2A Fusion Genes in Pediatric T-cell Acute Lymphoblastic Leukemia.

BACKGROUND/AIM: Previous reports have associated the KMT2A-ELL fusion gene, generated by t(11;19)(q23;p13.1), with acute myeloid leukemia (AML). We herein report a KMT2A-ELL and a novel ZNF56-KMT2A fusion genes in a pediatric T-lineage acute lymphoblastic leukemia (T-ALL). MATERIALS AND METHODS: Genetic investigations were performed on bone marrow of a 13-year-old boy diagnosed with T-ALL. RESULTS: A KMT2A-ELL and a novel ZNF56-KMT2A fusion genes were generated on der(11)t(11;19)(q23;p13.1) and der(19)t(11;19)(q23;p13.1), respectively. Exon 20 of KMT2A fused to exon 2 of ELL in KMT2A-ELL chimeric transcript whereas exon 1 of ZNF56 fused to exon 21 of KMT2A in ZNF56-KMT2A transcript. A literature search revealed four more T-ALL patients carrying a KMT2A-ELL fusion. All of them were males aged 11, 11, 17, and 20 years. CONCLUSION: KMT2A-ELL fusion is a rare recurrent genetic event in T-ALL with uncertain prognostic implications. The frequency and impact of ZNF56-KMT2A in T-ALL are unknown.

Therapy-induced Deletion in 11q23 Leading to Fusion of KMT2A With ARHGEF12 and Development of B Lineage Acute Lymphoplastic Leukemia in a Child Treated for Acute Myeloid Leukemia Caused by t(9;11)(p21;q23)/KMT2A-MLLT3.

BACKGROUND/AIM: Fusion of histone-lysine N-methyltransferase 2A gene (KMT2A) with the Rho guanine nucleotide exchange factor 12 gene (ARHGEF12), both located in 11q23, was reported in some leukemic patients. We report a KMT2A-ARHGEF12 fusion occurring during treatment of a pediatric acute myeloid leukemia (AML) with topoisomerase II inhibitors leading to a secondary acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: Multiple genetic analyses were performed on bone marrow cells of a girl initially diagnosed with AML. RESULTS: At the time of diagnosis with AML, the t(9;11)(p21;q23)/KMT2A-MLLT3 genetic abnormality was found. After chemotherapy resulting in AML clinical remission, a 2 Mb deletion in 11q23 was found generating a KMT2A-ARHGEF12 fusion gene. When the patient later developed B lineage ALL, a t(14;19)(q32;q13), loss of one chromosome 9, and KMT2A-ARHGEF12 were detected. CONCLUSION: The patient sequentially developed AML and ALL with three leukemia-specific genomic abnormalities in her bone marrow cells, two of which were KMT2A-rearrangements.