Inhibition of calpain prevents NMDA-induced cell death and β-amyloid-induced synaptic dysfunction in hippocampal slice cultures

Background and purpose:

Alzheimer''s disease (AD) is a multifactorial, neurodegenerative disease, which is in part caused by an impairment of synaptic function, probably mediated by oligomeric forms of amyloid-β (Aβ). While the Aβ pathology mainly affects the physiology of neurotransmission, neuronal decline is caused by excitotoxic cell death, which is mediated by the NMDA receptor. A comprehensive therapeutic approach should address both Aβ-induced synaptic deficits, as well as NMDA receptor-mediated neurodegeneration, via one molecular target. This study was designed to test whether calpain could be involved in both pathological pathways, which would offer a promising avenue for new treatments.

Experimental approach:

Application of the specific, water-soluble calpain inhibitor A-705253 was used to inhibit calpain in hippocampal slice cultures. We examined whether inhibition of calpain would prevent Aβ-induced deficits in neurotransmission in CA1, as well as NMDA-induced neuronal cell death.

Key results:

A-705253 dose-dependently prevented excitotoxicity-induced neurodegeneration at low nanomolar concentrations, determined by propidium iodide histochemistry. Inhibition of the NMDA receptor similarly protected from neuronal damage. Caspase staining indicated that calpain inhibition was protective by reducing apoptosis. Electrophysiological analysis revealed that inhibition of calpain by A-705253 also fully prevented Aβ oligomer-induced deficits in neurotransmission. The protective effect of calpain was compared to the clinically available NMDA receptor antagonist memantine, which was also effective in this model.

Conclusions and implications:

We suggest that inhibition of calpain exhibits a promising strategy to address several aspects of the pathology of AD that may go beyond the available therapeutic intervention by memantine.     

Fiber laser-microscope system for femtosecond photodisruption of biological samples

We report on the development of a ultrafast fiber laser-microscope system for femtosecond photodisruption of biological targets. A mode-locked Yb-fiber laser oscillator generates few-nJ pulses at 32.7 MHz repetition rate, amplified up to ～125 nJ at 1030 nm. Following dechirping in a grating compressor, ～240 fs-long pulses are delivered to the sample through a diffraction-limited microscope, which allows real-time imaging and control. The laser can generate arbitrary pulse patterns, formed by two acousto-optic modulators (AOM) controlled by a custom-developed field-programmable gate array (FPGA) controller. This capability opens the route to fine optimization of the ablation processes and management of thermal effects. Sample position, exposure time and imaging are all computerized. The capability of the system to perform femtosecond photodisruption is demonstrated through experiments on tissue and individual cells.     

L-selectin mediates neutrophil rolling in inflamed venules through sialyl LewisX-dependent and -independent recognition pathways

The glycoprotein (GP) L-selectin initiates adhesive interactions between leukocytes and endothelial cells (EC). It functions as a lymphocyte-lectin homing receptor recognizing carbohydrate determinants of the peripheral lymph node addressing on high endothelial venules. It also mediates neutrophil rolling, the earliest interaction of neutrophils with acutely inflamed venules. Neutrophil L-selectin presents sialyl-LewisX (sLe(X)) as a ligand to P- and E-selectin in vitro, and we have proposed that this is a major mechanism of L- selectin-mediated rolling in vivo. In contrast, the contribution of neutrophil L-selectin as a receptor protein recognizing one (or more) ligand(s) on inflamed EC is unclear. To address this question, an sLe(X)-negative murine pre-B cell line, L1-2, that can neither bind vascular selectins nor roll in inflamed rabbit venules, was transfected with human L-selectin cDNA. L-selectin expression in stable transfectants was sufficient to confer significant rolling in vivo. Rolling was unaffected by neuraminidase treatment but completely blocked by anti-L-selectin monoclonal antibody (MoAb) DREG-56. Thus, L- selectin can initiate leukocyte interactions with EC determinants potentially through recognition of endothelial carbohydrates. In contrast, when human neutrophils were tested, rolling was reduced, but not abolished, by MoAb DREG-56. Likewise, treatment with neuraminidase or anti-sLe(X) MoAbs decreased, but did not abrogate, neutrophil rolling, consistent with residual EC recognition via L-selectin. Combination of MoAb DREG-56 and neuraminidase resulted in almost complete loss of rolling, as did removal of glycosylated L-selectin by chymotrypsin. Together with the demonstrable rolling of L-selectin transfectants, our results support the concept of a bidirectional interaction between L-selectin bearing sLe(X) on neutrophils and activated EC in vivo. These findings also suggest that L-selectin may mediate rolling of lymphocytes that lack carbohydrate ligands for E- or P-selectin, although probably less efficiently than through bidirectional recognition.     

GNAS1 T393C polymorphism and disease progression in patients with malignant melanoma

Background: Once metastasized, despite a variety of therapeutic options, the prognosis of patients with malignant melanoma (MM) is still poor. Therefore, the search for reliable markers to identify patients with high risk of disease progression is of high clinical importance. We have recently shown that TT genotypes of the single-nucleotide polymorphism (SNP) T393C in the gene GNAS1 are significantly associated with better outcome in a variety of carcinomas. - Patients: In the present study we assessed whether the T393C SNP is also related to the clinical course in MM. 328 patients with MM were retrospectively genotyped and genotypes were correlated with clinical outcome. - Results: While the allele frequency in the MM group (fC 0.52) did not significantly differ from that of healthy blood donors, the T393C SNP was associated with tumor progression of MM. Carriers of the C-allele showed a significantly more severe tumor progression as estimated from the time period to develop metastasis (HR 2.2, 95% CI 1.1-3.2, p = 0.017). Proportions of 5-year metastasis-free intervals were 87.1% for TT genotypes and 66.0% for C-allele carriers. Moreover, multivariable Cox regression analysis including tumor stage and melanoma subtype proved the T393C polymorphism to be an independent factor for metastasis (p = 0.012). - Conclusions: In summary, the GNAS1 T393C SNP represents a genetic host factor for predicting tumor progression also in patients with MM; genotyping of this SNP may contribute to better define patients who could benefit from an early individualized therapy.     

Onychomycosis in a premature infant caused by Candida tropicalis

We report a case of onychomycosis caused by Candida tropicalis in a 107-day-old infant.     

Recombinant human interleukin-1 receptor antagonist protects early myeloid progenitors in a murine model of cyclophosphamide-induced myelotoxicity

Expression of hematoregulatory cytokines such as interleukin-1 (IL-1) in response to cytotoxic chemotherapy hastens hematopoietic recovery, but may also potentiate myelotoxicity if myeloid progenitors enter cell cycle before drug clearance. In the present study, the ability of recombinant human IL-1 receptor antagonist (IL-1ra) to protect hematopoietic progenitors was studied in a murine model of cyclophosphamide (CPA)-induced myelotoxicity. CF-1 female mice received 200 mg/kg CPA and either 10 mg/kg IL-1ra or an equal volume of 0.05% human serum albumin (HSA) intraperitoneally (i.p.), followed 12 hours later by IL-1ra or HSA. CPA and IL-1ra increased absolute neutrophil counts (ANCs) at days 2 (P = .001) and 14 (P = .0025) after CPA. In IL- 1ra-treated mice, colony-forming units granulocyte-macrophage (CFU- GM)/tibia were increased twofold and threefold at days 2 (P = .0047) and 7 (P = .023), respectively, whereas high proliferative potential colony-forming cells (HPP-CFC)/tibia were decreased twofold to threefold at 8 hours (P = .039) and 24 hours (P = .0033), but were approximately threefold higher than HSA-treated mice at day 7 after CPA. Coadministration of CPA and IL-1 enhanced myelotoxicity compared with mice injected with CPA and IL-1ra or HSA. In vivo, IL-1ra protected HPP-CFC, but not CFU-GM, from hydroxyurea suicide after a single dose of CPA, suggesting that IL-1ra inhibited cycling of HPP- CFC. In vitro, IL-1ra did not alter proliferation of CFU-GM, but inhibited IL-1-enhanced proliferation of HPP-CFC. These data suggest that IL-1ra acts as an indirect negative regulator of hematopoiesis and protects HPP-CFC from CPA, possibly by inhibiting IL-1-enhanced proliferation of early myeloid progenitors.     

Benzimidazolones enhance the function of epithelial Na+ transport

Background and Purpose

Pharmacological enhancement of vectorial Na⁺ transport may be useful to increase alveolar fluid clearance. Herein, we investigated the influence of the benzimidazolones 1-ethyl-1,3-dihydro-2-benzimidazolone (1-EBIO), 5,6-dichloro-1-EBIO (DC-EBIO) and chlorzoxazone on vectorial epithelial Na⁺ transport.

Experimental Approach

Effects on vectorial Na⁺ transport and amiloride-sensitive apical membrane Na⁺ permeability were determined by measuring short-circuit currents (I_SC) in rat fetal distal lung epithelial (FDLE) monolayers. Furthermore, amiloride-sensitive membrane conductance and the open probability of epithelial Na⁺ channels (ENaC) were determined by patch clamp experiments using A549 cells.

Key Results

I_SC was increased by approximately 50% after addition of 1-EBIO, DC-EBIO and chlorzoxazone. With permeabilized basolateral membranes in the presence of a 145:5 apical to basolateral Na⁺ gradient, the benzimidazolones markedly increased amiloride-sensitive I_SC. 5-(N-Ethyl-N-isopropyl)amiloride-induced inhibition of I_SC was not affected. The benzamil-sensitive I_SC was increased in benzimidazolone-stimulated monolayers. Pretreating the apical membrane with amiloride, which inhibits ENaC, completely prevented the stimulating effects of benzimidazolones on I_SC. Furthermore, 1-EBIO (1 mM) and DC-EBIO (0.1 mM) significantly increased (threefold) the open probability of ENaC without influencing current amplitude. Whole cell measurements showed that DC-EBIO (0.1 mM) induced an amiloride-sensitive increase in membrane conductance.

Conclusion and Implications

Benzimidazolones have a stimulating effect on vectorial Na⁺ transport. The antagonist sensitivity of this effect suggests the benzimidazolones elicit this action by activating the highly selective ENaC currents. Thus, the results demonstrate a possible new strategy for directly enhancing epithelial Na⁺ transport.