Biology subjects

Spray, D. C.

Publications and source records attributed to Spray, D. C..

6 recordsLinked to original sources

bioRxiv2026

Patient CACNA1A variant uncouples calcium channel conductance from synaptic output

Channelopathies are a class of neurodevelopmental disorders with often devastating consequences, and effective therapies depend on understanding how patient variants alter channel function. These effects are typically assessed by biophysical characterization in heterologous expression systems. Mutations in CACNA1A, which encodes the P/Q-type calcium channel CaV2.1, underlie a spectrum of neurological disorders in which symptoms are generally classified as loss-of-function (LoF) or gain-of-function (GoF). However, some patients present with overlapping phenotypes that defy this binary framework. Here we describe a CACNA1A variant for which heterologous assays fail to capture a key in vivo functional effect. We characterize a de novo variant of a highly conserved residue, D1634N, identified in a patient with a mixed clinical presentation that includes both LoF- and GoF-associated symptoms. Biophysical characterization in HEK293T cells supports a classic and severe LoF effect, including reduced current density and a right-shifted current-voltage relationship. In contrast, in vivo analysis of the corresponding endogenous variant in the C. elegans homolog reveals a paradoxical increase in spontaneous synaptic vesicle release, despite reduced channel expression. Molecular dynamics simulations predict that the mutation prolongs dwell time in a partially open state, potentially increasing calcium influx at rest. This model is supported by biophysical recordings of the human channel showing increased current at hyperpolarized potentials and by rescue of the C. elegans phenotype through genetic elevation of resting membrane potential. Together, these findings reconcile the patients clinical presentation by describing a complex, mixed-function variant, highlight the importance of cellular context in variant interpretation and therapeutic development, and establish C. elegans as a powerful in vivo platform for evaluating the functional consequences of pathogenic ion channel variants.

neuroscience↗

bioRxiv2023

Astrocyte sensitivity to glymphatic shear stress is amplified by albumin and mediated by the interaction of sphingosine 1 phosphate with Piezo1

Astrocyte endfeet enwrap brain vasculature, forming a boundary for perivascular glymphatic flow of fluid and solutes along and across the astrocyte endfeet into the brain parenchyma. To determine whether astrocytes may sense and respond to the shear forces generated by glymphatic flow, we examined intracellular calcium (Ca2+) changes evoked in astrocytes to brief fluid flow applied in calibrated microfluidic chambers. Shear stresses < 20 dyn/cm2 failed to evoke Ca2+ responses in the absence of albumin, but cells responded to shear stress below 1 dyn/cm2 when as little as 5 M albumin was present in flow medium. A role for extracellular matrix in mechanotransduction was indicated by reduced sensitivity after degradation of heparan sulfate proteoglycan. Sphingosine-1-phosphate (S1P) amplified shear responses in the absence of albumin, whereas mechanosensitivity was attenuated by the S1P receptor blocker fingolimod. Piezo1 participated in the transduction as revealed by blockade by the spider toxin GsMTX and amplification by the chemical modulator Yoda1, even in absence of albumin or S1P. Our findings that astrocytes are exquisitely sensitive to shear stress and that sensitivity is greatly amplified by albumin concentrations encountered in normal and pathological CSF predict that perivascular astrocytes are responsive to glymphatic shear stress and that responsiveness is augmented by elevated CSF protein. S1P receptor signaling thus establishes a setpoint for Piezo1 activation that is finely tuned to coincide with albumin level in CSF and to the low shear forces resulting from glymphatic flow. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/565884v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@17086e6org.highwire.dtl.DTLVardef@a89f32org.highwire.dtl.DTLVardef@6baed5org.highwire.dtl.DTLVardef@1a32f80_HPS_FORMAT_FIGEXP M_FIG C_FIG Astrocyte endfoot responds to glymphatic shear stress when albumin is present. Mechanism involves sphingosine-1-phosphate (S1P) binding to its receptor (S1PR), activating phospholipase C (PLC) and thereby sensitizing the response of Piezo1 to flow. Ca2+ influx triggers Ca2+ release from intracellular stores and further downstream signaling, thereby modulating parenchymal perfusion. Illustration created using BioRender.com

neuroscience↗

bioRxiv2023

Differential activation of mouse and human Panx1 channel variants.

Pannexins are ubiquitously expressed in human and mouse tissues. Pannexin 1 (Panx1), the most thoroughly characterized isoform of this family, forms plasmalemmal membrane channels permeable to relatively large molecules, such as ATP. Although human and mouse Panx1 amino acid sequences are conserved in the presently known regulatory sites involved in trafficking and modulation of the channel, differences are reported in the N- and C-termini of the protein, and the mechanisms of channel activation by different stimuli remain controversial. Here we used a neuroblastoma cell line to study the activation properties of endogenous mPanx1 and exogenously expressed hPanx1. Dye uptake and electrophysiological recordings revealed that in contrast to mouse Panx1, the human ortholog is insensitive to stimulation with high extracellular [K+] but responds similarly to activation of the purinergic P2X7 receptor. The two most frequent Panx1 polymorphisms found in the human population, Q5H (rs1138800) and E390D (rs74549886), exogenously expressed in Panx1-null N2a cells revealed that regarding P2X7 receptor mediated Panx1 activation, the Q5H mutant is a gain of function whereas the E390D mutant is a loss of function variant. Collectively, we demonstrate differences in the activation between human and mouse Panx1 orthologs and suggest that these differences may have translational implications for studies where Panx1 has been shown to have significant impact.

cell biology↗

bioRxiv2021

Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if blood-brain-barrier integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

neuroscience↗

bioRxiv2020

GENERATION AND CHARACTERIZATION OF IMMORTALIZED MOUSE CORTICAL ASTROCYTES FROM WILDTYPE AND CONNEXIN43 KNOCKOUT MICE

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1 (ALDH1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

neuroscience↗

bioRxiv2020

The dynamic Nexus: Gap junctions control protein localization and mobility in distinct and surprising ways

Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching (FRAP) of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters. Summary StatementGap junctions are clustered membrane channels in plasma membrane of astrocytes and other cells. We report new information on how gap junctions control location and mobility of other astrocyte proteins.

cell biology↗