Elena Abad

Profilin induces lamellipodia by growth factor-independent mechanism

Profilin has been implicated in cell mo tility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP‐actin monomers by increasing the rate of nucleotide exchange of ADP‐actin for ATP‐actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane‐permeable version of profilin I (PTD4‐PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time‐ and concentration‐dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipo‐ dia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) ‐induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time‐lapse microscopy confirmed the effects of profilin on lamel‐ lipodia extension with a higher spreading velocity than FBS. PTD4‐Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS‐induced lamellipodia formation activates Rac1, PTD4‐Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.—Syriani, E., Gomez‐Cabrero, A., Bosch, M., Moya, A., Abad, E., Gual, A., Gasull, X., Morales, M. Profilin induces lamellipodia by growth factor independent mechanism. FASEB J. 22, 1581–1596 (2008)

Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity

BackgroundMultiple Sclerosis (MS) is considered a T-cell-mediated autoimmune disease with a prototypical oscillatory behavior, as evidenced by the presence of clinical relapses. Understanding the dynamics of immune cells governing the course of MS, therefore, has many implications for immunotherapy. Here, we used flow cytometry to analyze the time-dependent behavior of antigen-specific effector (Teff) and regulatory (Treg) T cells and microglia in mice model of MS, Experimental Autoimmune Encephalomyelitis (EAE), and compared the observations with a mathematical cross-regulation model of T-cell dynamics in autoimmune disease.ResultsWe found that Teff and Treg cells specific to myelin olygodendrocyte glycoprotein (MOG) developed coupled oscillatory dynamics with a 4- to 5-day period and decreasing amplitude that was always higher for the Teff populations, in agreement with the mathematical model. Microglia activation followed the oscillations of MOG-specific Teff cells in the secondary lymphoid organs, but they were activated before MOG-specific T-cell peaks in the CNS. Finally, we assessed the role of B-cell depletion induced by anti-CD20 therapy in the dynamics of T cells in an EAE model with more severe disease after therapy. We observed that B-cell depletion decreases Teff expansion, although its oscillatory behavior persists. However, the effect of B cell depletion was more significant in the Treg population within the CNS, which matched with activation of microglia and worsening of the disease. Mathematical modeling of T-cell cross-regulation after anti-CD20 therapy suggests that B-cell depletion may influence the dynamics of T cells by fine-tuning their activation.ConclusionsThe oscillatory dynamics of T-cells have an intrinsic origin in the physiological regulation of the adaptive immune response, which influences both disease phenotype and response to immunotherapy.

Activation of Store-Operated Ca 2 Channels in Trabecular Meshwork Cells

PURPOSE In nonexcitable cells, G(q)-coupled membrane receptor activation induces a biphasic increase in intracellular calcium ([Ca(2+)](i)) expressed as an initial IP(3)-dependent release from intracellular stores followed by a sustained Ca(2+) influx from the extracellular space that involves store-operated Ca(2+) channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca(2+) signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility. METHODS [Ca(2+)](i) was measured in cultured bovine TM cells loaded with Fura-2. Ca(2+) currents were recorded using the patch clamp technique. Cell contractility measurements were assessed by traction microscopy. RESULTS BK and ET-1 activate a store-operated Ca(2+) entry that was greatly reduced in the absence of extracellular Ca(2+) or by preincubation with SOC blocker 2-APB or SKF96365. Store-operated Ca(2+) currents were also activated by intracellular dialysis with IP(3) + EGTA or after stimulation with thapsigargin. Electrophysiological characterization supports the presence of Ca(2+) release-activated Ca(2+) channels (CRACs) and nonselective cation channels, of which TRPC1 and TRPC4 channels may be candidate TRPs detected in TM cells. Extracellular Ca(2+) entry through SOCs is not required for TM cell contraction in response to BK or ET-1, but it modulates this process. CONCLUSIONS Extracellular Ca(2+) entry in TM cells in response to agonist stimulation and store-depletion is mediated by the activation of SOCs, which do not contribute to cell contraction but which may activate regulatory mechanisms to prevent excessive contraction. CRAC and TRPC channels involved represent interesting modulators of TM function to improve aqueous humor outflow.

Effects of platelet-derived growth factor on aqueous humor dynamics.

PURPOSE It is well known that the small GTPase RhoA modulates actin cytoskeleton and cellular contractility in the trabecular meshwork (TM). Several substances known to contract the TM reduce outflow facility, whereas cellular relaxation is commonly associated with the opposite effect. Inhibitors of the RhoA pathway are under development as antiglaucoma drugs. Here the authors investigate the role of platelet-derived growth factor (PDGF), a known activator of the Rac1 pathway, in cell cytoskeleton, outflow facility, and intraocular pressure (IOP). METHODS Effects of PDGF on actin cytoskeleton, Rac1, and AKT activation were tested in preconfluent and confluent bovine TM cells in culture. Rac1 and AKT/P-AKT activation were assessed by Western blot analysis. Trabecular outflow facility was measured in bovine perfused anterior segments. Changes in IOP were measured for up to 6 hours after topical application in the cornea of rabbit eyes by means of a contact tonometer. RESULTS In TM cells, PDGF (10 ng/mL) activated Rac1 through AKT and induced actin cytoskeleton rearrangement with lamellipodia formation. In this sense, lamellipodia formation in TM cells was prevented by NSC23766, a Rac1 inhibitor, and LY294002, a PI3K inhibitor. In perfused anterior segments, PDGF (100 ng/mL) increased trabecular outflow facility by 26%. In vivo, when topically applied to rabbit corneas, PDGF induced a 20% decrease in IOP (100 ng/mL). This reduction was concentration dependent and presented an EC(50) value of 2.7 nM. CONCLUSIONS PDGF, by activating the Rac1 pathway, induces cytoskeletal changes in TM cells that enhance outflow facility. Decreased IOP after PDGF application is likely caused by the facilitation of aqueous humor outflow. Rac1 pathway activation appears to be a positive modulator of outflow facility and an interesting target for decreasing IOP after ocular hypertension.

Dynamics and heterogeneity of brain damage in multiple sclerosis

Multiple Sclerosis (MS) is an autoimmune disease driving inflammatory and degenerative processes that damage the central nervous system (CNS). However, it is not well understood how these events interact and evolve to evoke such a highly dynamic and heterogeneous disease. We established a hypothesis whereby the variability in the course of MS is driven by the very same pathogenic mechanisms responsible for the disease, the autoimmune attack on the CNS that leads to chronic inflammation, neuroaxonal degeneration and remyelination. We propose that each of these processes acts more or less severely and at different times in each of the clinical subgroups. To test this hypothesis, we developed a mathematical model that was constrained by experimental data (the expanded disability status scale [EDSS] time series) obtained from a retrospective longitudinal cohort of 66 MS patients with a long-term follow-up (up to 20 years). Moreover, we validated this model in a second prospective cohort of 120 MS patients with a three-year follow-up, for which EDSS data and brain volume time series were available. The clinical heterogeneity in the datasets was reduced by grouping the EDSS time series using an unsupervised clustering analysis. We found that by adjusting certain parameters, albeit within their biological range, the mathematical model reproduced the different disease courses, supporting the dynamic CNS damage hypothesis to explain MS heterogeneity. Our analysis suggests that the irreversible axon degeneration produced in the early stages of progressive MS is mainly due to the higher rate of myelinated axon degeneration, coupled to the lower capacity for remyelination. However, and in agreement with recent pathological studies, degeneration of chronically demyelinated axons is not a key feature that distinguishes this phenotype. Moreover, the model reveals that lower rates of axon degeneration and more rapid remyelination make relapsing MS more resilient than the progressive subtype. Therefore, our results support the hypothesis of a common pathogenesis for the different MS subtypes, even in the presence of genetic and environmental heterogeneity. Hence, MS can be considered as a single disease in which specific dynamics can provoke a variety of clinical outcomes in different patient groups. These results have important implications for the design of therapeutic interventions for MS at different stages of the disease.

Impact of Zygosity on Bimodal Phenotype Distributions

Allele number, or zygosity, is a clear determinant of gene expression in diploid cells. However, the relationship between the number of copies of a gene and its expression can be hard to anticipate, especially when the gene in question is embedded in a regulatory circuit that contains feedback. Here, we study this question making use of the natural genetic variability of human populations, which allows us to compare the expression profiles of a receptor protein in natural killer cells among donors infected with human cytomegalovirus with one or two copies of the allele. Crucially, the distribution of gene expression in many of the donors is bimodal, which indicates the presence of a positive feedback loop somewhere in the regulatory environment of the gene. Three separate gene-circuit models differing in the location of the positive feedback loop with respect to the gene can all reproduce the homozygous data. However, when the resulting fitted models are applied to the hemizygous donors, one model (the one with the positive feedback located at the level of gene transcription) is superior in describing the experimentally observed gene-expression profile. In that way, our work shows that zygosity can help us relate the structure and function of gene regulatory networks.