Vishnu Anand Cuddapah

A neurocentric perspective on glioma invasion.

Malignant gliomas are devastating tumours that frequently kill patients within 1 year of diagnosis. The major obstacle to a cure is diffuse invasion, which enables tumours to escape complete surgical resection and chemo- and radiation therapy. Gliomas use the same tortuous extracellular routes of migration that are travelled by immature neurons and stem cells, frequently using blood vessels as guides. They repurpose ion channels to dynamically adjust their cell volume to accommodate to narrow spaces and breach the blood–brain barrier through disruption of astrocytic endfeet, which envelop blood vessels. The unique biology of glioma invasion provides hitherto unexplored brain-specific therapeutic targets for this devastating disease.

Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome

Background Rett syndrome (RTT), a neurodevelopmental disorder that primarily affects girls, is characterised by a period of apparently normal development until 6–18 months of age when motor and communication abilities regress. More than 95% of individuals with RTT have mutations in methyl-CpG-binding protein 2 (MECP2), whose protein product modulates gene transcription. Surprisingly, although the disorder is caused by mutations in a single gene, disease severity in affected individuals can be quite variable. To explore the source of this phenotypic variability, we propose that specific MECP2 mutations lead to different degrees of disease severity. Methods Using a database of 1052 participants assessed over 4940 unique visits, the largest cohort of both typical and atypical RTT patients studied to date, we examined the relationship between MECP2 mutation status and various phenotypic measures over time. Results In general agreement with previous studies, we found that particular mutations, such as p.Arg133Cys, p.Arg294X, p.Arg306Cys, 3° truncations and other point mutations, were relatively less severe in both typical and atypical RTT. In contrast, p.Arg106Trp, p.Arg168X, p.Arg255X, p.Arg270X, splice sites, deletions, insertions and deletions were significantly more severe. We also demonstrated that, for most mutation types, clinical severity increases with age. Furthermore, of the clinical features of RTT, ambulation, hand use and age at onset of stereotypies are strongly linked to overall disease severity. Conclusions We have confirmed that MECP2 mutation type is a strong predictor of disease severity. These data also indicate that clinical severity continues to become progressively worse regardless of initial severity. These findings will allow clinicians and families to anticipate and prepare better for the needs of individuals with RTT.

Molecular interaction and functional regulation of ClC-3 by Ca2+/calmodulin-dependent protein kinase II (CaMKII) in human malignant glioma.

Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.

Bradykinin-induced chemotaxis of human gliomas requires the activation of KCa3.1 and ClC-3.

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca2+]i), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca2+]i regulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers that spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin. Using simultaneous fura-2 Ca2+ imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca2+]i and induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca2+-dependent, TRAM-34-sensitive KCa3.1 channels, and Ca2+-dependent, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive and gluconate-sensitive Cl− channels. A significant portion of these Cl− currents can be attributed to Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation of ClC-3, a voltage-gated Cl− channel/transporter, because pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca2+-activated Cl− currents. Western blots show that KCa3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both KCa3.1 and ClC-3 colocalize to the invading processes of glioma cells. Importantly, inhibition of either channel abrogates bradykinin-induced chemotaxis and reduces tumor expansion in mouse brain slices in situ. These channels should be further explored as future targets for anti-invasive drugs. Furthermore, these data elucidate a novel mechanism placing cation and anion channels downstream of ligand-mediated [Ca2+]i increases, which likely play similar roles in other migratory cells in the nervous system.

Differential role of IK and BK potassium channels as mediators of intrinsic and extrinsic apoptotic cell death.

An important event during apoptosis is regulated cell condensation known as apoptotic volume decrease (AVD). Ion channels have emerged as essential regulators of this process mediating the release of K(+) and Cl(-), which together with osmotically obliged water, results in the condensation of cell volume. Using a Grade IV human glioblastoma cell line, we examined the contribution of calcium-activated K(+) channels (K(Ca) channels) to AVD after the addition of either staurosporine (Stsp) or TNF-α-related apoptosis-inducing ligand (TRAIL) to activate the intrinsic or extrinsic pathway of apoptosis, respectively. We show that AVD can be inhibited in both pathways by high extracellular K(+) or the removal of calcium. However, BAPTA-AM was only able to inhibit Stsp-initiated AVD, whereas TRAIL-induced AVD was unaffected. Specific K(Ca) channel inhibitors revealed that Stsp-induced AVD was dependent on K(+) efflux through intermediate-conductance calcium-activated potassium (IK) channels, while TRAIL-induced AVD was mediated by large-conductance calcium-activated potassium (BK) channels. Fura-2 imaging demonstrated that Stsp induced a rapid and modest rise in calcium that was sustained over the course of AVD, while TRAIL produced no detectable rise in global intracellular calcium. Inhibition of IK channels with clotrimazole or 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) blocked downstream caspase-3 activation after Stsp addition, while paxilline, a specific BK channel inhibitor, had no effect. Treatment with ionomycin also induced an IK-dependent cell volume decrease. Together these results show that calcium is both necessary and sufficient to achieve volume decrease and that the two major pathways of apoptosis use unique calcium signaling to efflux K(+) through different K(Ca) channels.

Ion Channels and the Control of Cancer Cell Migration

A hallmark of high-grade cancers is the ability of malignant cells to invade unaffected tissue and spread disease. This is particularly apparent in gliomas, the most common and lethal type of primary brain cancer affecting adults. Migrating cells encounter restricted spaces and appear able to adjust their shape to accommodate to narrow extracellular spaces. A growing body of work suggests that cell migration/invasion is facilitated by ion channels and transporters. The emerging concept is that K(+) and Cl(-) function as osmotically active ions, which cross the plasma membrane in concert with obligated water thereby adjusting a cells shape and volume. In glioma cells Na(+)-K(+)-Cl(-) cotransporters (NKCC1) actively accumulate K(+) and Cl(-), establishing a gradient for KCl efflux. Ca(2+)-activated K(+) channels and voltage-gated Cl(-) channels are largely responsible for effluxing KCl promoting hydrodynamic volume changes. In other cancers, different K(+) or even Na(+) channels may function in concert with a variety of Cl(-) channels to support similar volume changes. Channels involved in migration are frequently regulated by Ca(2+) signaling, most likely coupling extracellular stimuli to cell migration. Importantly, the inhibition of ion channels and transporters appears to be clinically relevant for the treatment of cancer. Recent preclinical data indicates that inhibition of NKCC1 with an FDA-approved drug decreases neoplastic migration. Additionally, ongoing clinical trials demonstrate that an inhibitor of chloride channels may be a therapy for the treatment of gliomas. Data reviewed here strongly indicate that ion channels are a promising target for the development of novel therapeutics to combat cancer.

GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor-associated epilepsy

Seizures frequently accompany gliomas and often escalate to peritumoral epilepsy. Previous work revealed the importance of tumor‐derived excitatory glutamate (Glu) release mediated by the cystine‐glutamate transporter (SXC) in epileptogenesis. We now show a novel contribution of GABAergic disinhibition to disease pathophysiology. In a validated mouse glioma model, we found that peritumoral parvalbumin‐positive GABAergic inhibitory interneurons are significantly reduced, corresponding with deficits in spontaneous and evoked inhibitory neurotransmission. Most remaining peritumoral neurons exhibit elevated intracellular Cl− concentration ([Cl−]i) and consequently depolarizing, excitatory gamma‐aminobutyric acid (GABA) responses. In these neurons, the plasmalemmal expression of KCC2, which establishes the low [Cl−]i required for GABAAR‐mediated inhibition, is significantly decreased. Interestingly, reductions in inhibition are independent of Glu release, but the presence of both decreased inhibition and decreased SXC expression is required for epileptogenesis. We suggest GABAergic disinhibition renders peritumoral neuronal networks hyper‐excitable and susceptible to seizures triggered by excitatory stimuli, and propose KCC2 as a therapeutic target. GLIA 2015;63:23–36

With-No-Lysine Kinase 3 (WNK3) stimulates glioma invasion by regulating cell volume.

Among the most prevalent and deadly primary brain tumors, high-grade gliomas evade complete surgical resection by diffuse invasion into surrounding brain parenchyma. Navigating through tight extracellular spaces requires invading glioma cells to alter their shape and volume. Cell volume changes are achieved through transmembrane transport of osmolytes along with obligated water. The sodium-potassium-chloride cotransporter isoform-1 (NKCC1) plays a pivotal role in this process, and previous work has demonstrated that NKCC1 inhibition compromises glioma invasion in vitro and in vivo by interfering with the required cell volume changes. In this study, we show that NKCC1 activity in gliomas requires the With-No-Lysine Kinase-3 (WNK3) kinase. Western blots of patient biopsies and patient-derived cell lines shows prominent expression of Ste-20-related, proline-alanine-rich kinase (SPAK), oxidative stress response kinase (OSR1), and WNK family members 1, 3, and 4. Of these, only WNK3 colocalized and coimmunoprecipitated with NKCC1 upon changes in cell volume. Stable knockdown of WNK3 using specific short hairpin RNA constructs completely abolished NKCC1 activity, as measured by the loss of bumetanide-sensitive cell volume regulation. Consequently, WNK3 knockdown cells showed a reduced ability to invade across Transwell barriers and lacked bumetanide-sensitive migration. This data indicates that WNK3 is an essential regulator of NKCC1 and that WNK3 activates NKCC1-mediated ion transport necessary for cell volume changes associated with cell invasion.

The role of glial-specific Kir4.1 in normal and pathological states of the CNS.

Kir4.1 is an inwardly rectifying K+ channel expressed exclusively in glial cells in the central nervous system. In glia, Kir4.1 is implicated in several functions including extracellular K+ homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and facilitation of glutamate uptake. Knockout of Kir4.1 in rodent models leads to severe neurological deficits, including ataxia, seizures, sensorineural deafness, and early postnatal death. Accumulating evidence indicates that Kir4.1 plays an integral role in the central nervous system, prompting many laboratories to study the potential role that Kir4.1 plays in human disease. In this article, we review the growing evidence implicating Kir4.1 in a wide array of neurological disease. Recent literature suggests Kir4.1 dysfunction facilitates neuronal hyperexcitability and may contribute to epilepsy. Genetic screens demonstrate that mutations of KCNJ10, the gene encoding Kir4.1, causes SeSAME/EAST syndrome, which is characterized by early onset seizures, compromised verbal and motor skills, profound cognitive deficits, and salt-wasting. KCNJ10 has also been linked to developmental disorders including autism. Cerebral trauma, ischemia, and inflammation are all associated with decreased astrocytic Kir4.1 current amplitude and astrocytic dysfunction. Additionally, neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis demonstrate loss of Kir4.1. This is particularly exciting in the context of Huntington disease, another neurodegenerative disorder in which restoration of Kir4.1 ameliorated motor deficits, decreased medium spiny neuron hyperexcitability, and extended survival in mouse models. Understanding the expression and regulation of Kir4.1 will be critical in determining if this channel can be exploited for therapeutic benefit.

Calcium entry via TRPC1 channels activates chloride currents in human glioma cells.

Malignant gliomas are highly invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl(-) channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl(-) channels are regulated in the context of migration. Using patch-clamp recordings we show Cl(-) currents are activated by physiological increases of [Ca(2+)]i to 65 and 180nM. Cl(-) currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl(-) channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl(-) currents in a Ca(2+)-dependent fashion, placing Cl(-) channels under the regulation of Ca(2+) entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl(-) channels. Thus endogenous glioma Cl(-) channels are regulated by TRPC1. Cl(-) channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca(2+)]i to the shape and volume changes associated with migrating cells.