Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

PURPOSE: Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. METHODS: Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. RESULTS: OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. CONCLUSIONS: OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. TRANSLATIONAL RELEVANCE: Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

G protein-activated inwardly rectifying potassium channels mediate depotentiation of long-term potentiation.

Excitatory synapses in the brain undergo activity-dependent changes in the strength of synaptic transmission. Such synaptic plasticity as exemplified by long-term potentiation (LTP) is considered a cellular correlate of learning and memory. The presence of G protein-activated inwardly rectifying K(+) (GIRK) channels near excitatory synapses on dendritic spines suggests their possible involvement in synaptic plasticity. However, whether activity-dependent regulation of GIRK channels affects excitatory synaptic plasticity is unknown. In a companion article we have reported activity-dependent regulation of GIRK channel density in cultured hippocampal neurons that requires activity of NMDA receptors (NMDAR) and protein phosphatase-1 (PP1) and takes place within 15 min. In this study, we performed whole-cell recordings of cultured hippocampal neurons and found that NMDAR activation increases basal GIRK current and GIRK channel activation mediated by adenosine A(1) receptors, but not GABA(B) receptors. Given the similar involvement of NMDARs, adenosine A(1) receptors, and PP1 in depotentiation of LTP caused by low-frequency stimulation that immediately follows LTP-inducing high-frequency stimulation, we wondered whether NMDAR-induced increase in GIRK channel surface density and current may contribute to the molecular mechanisms underlying this specific depotentiation. Remarkably, GIRK2 null mutation or GIRK channel blockade abolishes depotentiation of LTP, demonstrating that GIRK channels are critical for depotentiation, one form of excitatory synaptic plasticity.

Turning G proteins on and off using peptide ligands.

Intracellular Galpha subunits represent potential therapeutic targets for a number of diseases. Here we describe three classes of new molecules that modulate G protein signaling by direct targeting of Galpha. Using messenger RNA display, we have identified unique peptide sequences that bind Galpha i1 . Functionally, individual peptides were found that either enhance or repress basal levels of G protein-activated inwardly rectifying potassium (GIRK) channel signaling, a downstream effector of G protein activation, indicating that the peptides directly turn G proteins on or off in vivo . A third functional class acts as a signaling attenuator; basal GIRK channel activity is unaffected but responses to repeated G protein activation are reduced. These data demonstrate that G protein-directed ligands can achieve physiological effects similar to those resulting from classical receptor targeting and may serve as leads for developing new classes of therapeutics.

Modulation of basal and receptor-induced GIRK potassium channel activity and neuronal excitability by the mammalian PINS homolog LGN.

G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). Here we report that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, we further show an essential role of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability.

Oncogenic potential of TASK3 (Kcnk9) depends on K+ channel function.

TASK3 gene (Kcnk9) is amplified and overexpressed in several types of human carcinomas. In this report, we demonstrate that a point mutation (G95E) within the consensus K+ filter of TASK3 not only abolished TASK3 potassium channel activity but also abrogated its oncogenic functions, including proliferation in low serum, resistance to apoptosis, and promotion of tumor growth. Furthermore, we provide evidence that TASK3G95E is a dominant-negative mutation, because coexpression of the wild-type and the mutant TASK3 resulted in inhibition of K+ current of wild-type TASK3 and its tumorigenicity in nude mice. These results establish a direct link between the potassium channel activity of TASK3 and its oncogenic functions and imply that blockers for this potassium channel may have therapeutic potential for the treatment of cancers.

Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner.

Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.

Ionic dependence of Ca2+ channel modulation by syntaxin 1A.

Alteration of the kinetic properties of voltage-gated Ca(2+) channels, Ca(v)1.2 (Lc-type), Ca(v)2.2 (N type), and Ca(v)2.3 (R type), by syntaxin 1A (Syn1A) and synaptotagmin could modulate exocytosis. We tested how switching divalent charge carriers from Ca(2+) to Sr(2+) and Ba(2+) affected Syn1A and synaptotagmin modulation of Ca(2+)-channel activation. Syn1A accelerated Ca(v)1.2 activation if Ca(2+) was the charge carrier; and by substituting for Ba(2+), Syn1A slowed Ca(v)1.2 activation. Syn1A also significantly accelerated Ca(v)2.3 activation in Ca(2+) and marginally in Ba(2+). Synaptotagmin, on the other hand, increased the rate of activation of Ca(v)2.3 and Ca(v)2.2 in all permeating ions tested. The Syn1A-channel interaction, unlike the synaptotagmin-channel interaction, proved significantly more sensitive to the type of permeating ion. It is well established that exocytosis is affected by switching the charge carriers. Based on the present results, we suggest that the channel-Syn1A interaction could respond to the conformational changes induced within the channel during membrane depolarization and divalent ion binding. These changes could partially account for the charge specificity of synaptic transmission as well as for the fast signaling between the Ca(2+) source and the fusion apparatus of channel-associated-vesicles (CAV). Furthermore, propagation of conformational changes induced by the divalent ions appear to affect the concerted interaction of the channel with the fusion/docking machinery upstream to free Ca(2+) buildup and/or binding to a cytosolic Ca(2+) sensor. These results raise the intriguing possibility that the channel is the Ca(2+) sensor in the process of fast neurotransmitter release.