Good long-term visual outcomes of parapapillary choroidal melanoma patients treated with proton therapy: a comparative study.

PURPOSE: To evaluate why small- and certain medium-sized parapapillary choroidal melanoma (pcM) patients treated with hypo-fractionated proton therapy (PT) retain excellent long-term visual acuity (VA) and assess the negative predictive factors for retaining good vision (≤ 0.2 logMAR (≥ 0.6 decimal) after 5 years. METHODS: This single-center, retrospective, comparative study recruited consecutive pcM patients that were treated with PT. Between 1984 and 2005, 609 patients received a total of 60 CGE, of whom 310 met the following inclusion criteria: posterior tumor border ≤ 2.5 mm from the optic disc, largest tumor diameter ≤ 17.9 mm, tumor thickness ≤ 5.2 mm and available follow-up data for at least 5 years. RESULTS: Mean follow-up was 120.8 ± 48.8 months (54.0-295.0). Out of 310 patients, 64 (21%) maintained a VA ≤ 0.2 logMAR (≥ 0.6 decimal) for at least 5 years following PT and were allocated to the "good visual outcome" (GVO) group, while the remaining 246 (79%) constituted the "poor visual outcome" (PVO) group, subdivided into 70 (22%) with a VA of 0.3-1.0 logMAR (0.1-0.5 decimal) and 157 (57%) patients with a VA > 1.0 logMAR (< 0.1 decimal). On multivariate analysis, older age (P = 0.04), tumor localization ≤ 0.5 mm to the fovea (P < 0.03), volume of the optic disc and macula receiving 50% of dose (30 CGE) (P = 0.02 and P < 0.001, respectively) were independent negative predictors of GVO. CONCLUSIONS: Of 310 small- to medium-sized pcM patients successfully treated with PT, 21% retained a VA ≤ 0.2 logMAR (≥ 0.6 decimal) for at least 5 years. Strongest negative predictive factor for retaining good long-term vision was the volume of the macula irradiated with at least 30 Gy.

Levels of the oxidative stress biomarker malondialdehyde in tears of patients with central serous chorioretinopathy relate to disease activity.

Purpose: Central serous chorioretinopathy (CSCR) has been associated with oxidative stress-related risk factors. The objective of this study was to optimize an analytical method for evaluating the oxidative stress biomarker malondialdehyde (MDA) in human tears and determine its level in the tears of patients with CSCR. Methods: In this pilot study, tear samples were obtained from 34 healthy donors and 31 treatment-naïve CSCR male patients (eight with acute CSCR and 23 with chronic CSCR). Two analytical methods based on high-performance liquid chromatography followed by fluorescence detection were evaluated, with either 2-thiobarbituric derivative (TBA) or 2-aminoacridone (2-AA). Activity of CSCR was defined by the serous retinal detachment (SRD) height, which was measured by two independent observers on spectral-domain optical coherence tomography. Results: The 2-AA method showed higher sensitivity and precision compared to the TBA method. When the 2-AA method was applied to tears from healthy donors, the levels of MDA were statistically significantly higher in men compared to women (mean ± standard deviation, SD: 9,914 nM ± 6,126 versus 4,635 nM ± 1,173, p = 0.006). No difference was found in tear MDA levels between male patients with CSCR and age-matched control men (p = 0.17). However, MDA levels were statistically significantly higher in acute compared to chronic CSCR cases (mean ± SD: 12,295 nM ± 8,495 versus 6,790 ± 3,969 nM, p = 0.03). Additionally, there was a correlation between MDA levels and RPE leakage, quantified by the height of the serous retinal detachment (p = 0.02, r = 0.40). Conclusions: Levels of MDA in tears, measured with an optimized analytical method, correlate with RPE leakage in CSCR.

Clinical Outcomes in AYAs (Adolescents and Young Adults) Treated with Proton Therapy for Uveal Melanoma: A Comparative Matching Study with Elder Adults.

OBJECTIVE: The aim of this study was to compare the clinical outcomes of adolescents and young adults (AYAs) with those of elder adult patients treated with proton therapy (PT) for uveal melanoma (UM). MATERIAL AND METHODS: A retrospective, comparative study was conducted in UM patients who underwent PT at the Ocular Oncology Unit of the Jules-Gonin Eye Hospital (University of Lausanne, Lausanne, Switzerland) and the Paul Scherrer Institute (PSI); (Villigen, Switzerland) between January 1997 and December 2007. Propensity score matching (PSM) was used to select for each AYA (between 15-39 years old) an elder adult patient (≥40 years) with similar characteristics. We assessed ocular follow-up, local tumor control, metastasis incidence, and overall and relative survival (OS and RS). Non-terminal outcomes were then compared between the two groups using competing risk survival analysis. RESULTS: Out of a total of 2261 consecutive UM patients, after excluding 4 children (<15 years) and 6 patients who were metastatic at presentation, we identified 272 AYA patients and matched 270 of them with 270 elder adult patients. Before PSM, the AYA patients had a higher incidence of primary iris melanoma (4.0% vs. 1.4%; p = 0.005), while the elder patients were more likely to have other neoplastic diseases at presentation (9% vs. 3.7%; p = 0.004). Ocular outcomes and local tumor control were similar in both groups. Cumulative metastasis incidence for the AYA and elder adult groups was 13% and 7.9% at 5 years and 19.7% and 12.7% at 10 years, respectively, which was not significantly different between the groups (p = 0.214). The OS was similar in the two groups (p = 0.602), with estimates in the AYA and elder adult groups of 95.5% and 96.6% at 5 years and 94.6% and 91.4% at 10 years, respectively. However, the relative survival (RS) estimation was worse in the AYA group than the elder group (p = 0.036). CONCLUSION: While AYAs treated with PT for UM have similar ocular outcomes and present the same metastasis incidence and OS as elder adults, their RS is worse than that in elder adults, when compared with the population in general.

Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer's disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Processing of the synaptic cell adhesion molecule neurexin-3beta by Alzheimer disease alpha- and gamma-secretases.

Neurexins (NRXNs) are synaptic cell adhesion molecules having essential roles in the assembly and maturation of synapses into fully functional units. Immunocytochemical and electrophysiological studies have shown that specific binding across the synaptic cleft of the ectodomains of presynaptic NRXNs and postsynaptic neuroligins have the potential to bidirectionally coordinate and trigger synapse formation. Moreover, in vivo studies as well as genome-wide association studies pointed out implication of NRXNs in the pathogenesis of cognitive disorders including autism spectrum disorders and different types of addictions including opioid and alcohol dependences, suggesting an important role in synaptic function. Despite extensive investigations, the mechanisms by which NRXNs modulate the properties of synapses remain largely unknown. We report here that α- and γ-secretases can sequentially process NRXN3ß, leading to the formation of two final products, an ∼80-kDa N-terminal extracellular domain of Neurexin-3ß (sNRXN3ß) and an ∼12-kDa C-terminal intracellular NRXN3ß domain (NRXN3ß-ICD), both of them being potentially implicated in the regulation of NRXNs and neuroligins functions at the synapses or in yet unidentified signal transduction pathways. We further report that this processing is altered by several PS1 mutations in the catalytic subunit of the γ-secretase that cause early-onset familial Alzheimer disease.

Novel gamma-secretase inhibitors uncover a common nucleotide-binding site in JAK3, SIRT2, and PS1.

Gamma-secretase is an intramembrane-cleaving protease responsible for the final proteolytic event in the production of the amyloid-beta peptides (Abeta) implicated in Alzheimer's disease (AD). Inhibition of gamma-secretase activity is thus an attractive therapeutic strategy to slow down the pathogenesis of AD. Drugs often target more than one biomolecule because of conserved 3-dimensional structures in prospective protein binding sites. We have capitalized on this phenomenon of nature to identify new gamma-secretase inhibitors. Here we show that 2-hydroxy naphthyl derivatives, a previously identified subclass of NAD(+) analog inhibitors of sirtuin 2 (SIRT2), are direct gamma-secretase inhibitors. Subsequent structure-activity relationship studies further showed that 2-hydroxy-1-naphthaldehyde is the minimal pharmacophore for gamma-secretase inhibition. In evaluating target protein determinants of inhibition, we identified a common GXG signature nucleotide-binding site (NBS) shared by the gamma-secretase subunit presenilin-1 C-terminal fragment (PS1-CTF), SIRT2, and Janus kinase 3 (JAK3). Because a detailed 3-dimensional structure of gamma-secretase is beyond our knowledge, we took advantage of the known crystal structure of human JAK3 to model the NBS of the PS1-CTF, which includes the catalytic residue D385. Our results suggest that the flexible PS1-CTF (381)LGLG(384) loop comprises a substrate-docking site capable of recognizing specifically different gamma-secretase substrates.

Molecular Biomarkers of Neovascular Age-Related Macular Degeneration With Incomplete Response to Anti-Vascular Endothelial Growth Factor Treatment.

The standard treatment for neovascular age-related macular degeneration (nAMD) consists of intravitreal anti-vascular endothelial growth factors (VEGF). However, for some patients, even maximal anti-VEGF treatment does not entirely suppress exudative activity. The goal of this study was to identify molecular biomarkers in nAMD with incomplete response to anti-VEGF treatment. Aqueous humor (AH) samples were collected from three groups of patients: 17 patients with nAMD responding incompletely to anti-VEGF (18 eyes), 17 patients affected by nAMD with normal treatment response (21 eyes), and 16 control patients without any retinopathy (16 eyes). Proteomic and multiplex analyses were performed on these samples. Proteomic analyses showed that nAMD patients with incomplete anti-VEGF response displayed an increased inflammatory response, complement activation, cytolysis, protein-lipid complex, and vasculature development pathways. Multiplex analyses revealed a significant increase of soluble vascular cell adhesion molecule-1 (sVCAM-1) [ p = 0.001], interleukin-6 (IL-6) [ p = 0.009], bioactive interleukin-12 (IL-12p40) [ p = 0.03], plasminogen activator inhibitor type 1 (PAI-1) [ p = 0.004], and hepatocyte growth factor (HGF) [ p = 0.004] levels in incomplete responders in comparison to normal responders. Interestingly, the same biomarkers showed a high intercorrelation with r2 values between 0.58 and 0.94. In addition, we confirmed by AlphaLISA the increase of sVCAM-1 [ p < 0.0001] and IL-6 [ p = 0.043] in the incomplete responder group. Incomplete responders in nAMD are associated with activated angiogenic and inflammatory pathways. The residual exudative activity of nAMD despite maximal anti-VEGF treatment may be related to both angiogenic and inflammatory responses requiring specific adjuvant therapy. Data are available via ProteomeXchange with identifier PXD02247.

Gait analysis following single-shot hyaluronic acid supplementation: a pilot randomized double-blinded controlled trial.

OBJECTIVES: Viscosupplementation with new-generation, polyol-containing, cross-linked hyaluronic acid (HA) gels reduces joint inflammation in patients with knee osteoarthritis. Gait analysis is a complementary outcome measure to standard patient-reported scores and physical measures for testing the effect of HA injection. This three-arm, prospective, randomized, controlled, double-blind, feasibility pilot study investigated which gait parameters are more sensitive following a single bolus injection of polyol-containing HA for knee osteoarthritis. METHODS: Twenty-two patients with Ahlbäck grade II-III knee osteoarthritis were randomly allocated into three groups: (1) HA + mannitol (n = 9), (2) HA + sorbitol (n = 5), and (3) saline placebo (n = 8). Patients were assessed by blinded observers prior to injection and at 4 weeks post-injection (4W). Outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Knee Society score (KSS), EuroQol in five-dimensions (EQ-5D), VAS pain, and VAS stiffness. Gait was assessed over 30 m using a portable inertial-based data logger (Physilog®). RESULTS: Differences between 4W and baseline were statistically significant for the mannitol-containing viscosupplement, with a median increase of 0.076 m/s on gait speed (p = 0.039), 0.055 m on stride length (p = 0.027), and 15 points on the KSS (p = 0.047). In contrast, the HA + sorbitol and saline groups demonstrated no significant changes from baseline to 4W in any gait parameters or self-reported outcome measures (all p > 0.3). The observed increase in gait speed is approximately 13% greater than the mean difference between healthy subjects and those with knee osteoarthritis, is clinically important, and thus is a sensitive gait parameter. CONCLUSIONS: This study demonstrated gait speed and stride length are the most relevant gait parameters to investigate when assessing the effect of polyol-containing HA viscosupplementation. This study supports the need for a larger, randomized, controlled, clinical trial to assess the effect of a single-bolus HA injection versus multiple injections in people with knee osteoarthritis using both gait performance and self-reported parameters of knee function. TRIAL REGISTRATION: This study was retrospectively registered at clinicaltrials.gov on August 20, 2018, and assigned #NCT03636971. LEVEL OF EVIDENCE: I.

GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states.

Stressful experiences in early life are known risk factors for anxiety and depressive illnesses, and they inhibit hippocampal neurogenesis and the expression of GABA(A) receptors in adulthood. Conversely, deficits in GABAergic neurotransmission and reduced neurogenesis are implicated in the etiology of pathological anxiety and diverse mood disorders. Mice that are heterozygous for the gamma2 subunit of GABA(A) receptors exhibit a modest functional deficit in mainly postsynaptic GABA(A) receptors that is associated with a behavioral, cognitive, and pharmacological phenotype indicative of heightened trait anxiety. Here we used cell type-specific and developmentally controlled inactivation of the gamma2 subunit gene to further analyze the mechanism and brain substrate underlying this phenotype. Heterozygous deletion of the gamma2 subunit induced selectively in immature neurons of the embryonic and adult forebrain resulted in reduced adult hippocampal neurogenesis associated with heightened behavioral inhibition to naturally aversive situations, including stressful situations known to be sensitive to antidepressant drug treatment. Reduced adult hippocampal neurogenesis was associated with normal cell proliferation, indicating a selective vulnerability of postmitotic immature neurons to modest functional deficits in gamma2 subunit-containing GABA(A) receptors. In contrast, a comparable forebrain-specific GABA(A) receptor deficit induced selectively in mature neurons during adolescence lacked neurogenic and behavioral consequences. These results suggest that modestly reduced GABA(A) receptor function in immature neurons of the developing and adult brain can serve as a common molecular substrate for deficits in adult neurogenesis and behavior indicative of anxious and depressive-like mood states.

Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice.

Although glycine is a major inhibitory transmitter in the mammalian CNS, the role of glycinergic neurons in defined neuronal circuits remains ill defined. This is due in part to difficulties in identifying these cells in living slice preparations for electrophysiological recordings and visualizing their axonal projections. To facilitate the morphological and functional analysis of glycinergic neurons, we generated bacterial artificial chromosome (BAC) transgenic mice, which specifically express enhanced green fluorescent protein (EGFP) under the control of the promotor of the glycine transporter (GlyT) 2 gene, which is a reliable marker for glycinergic neurons. Neurons expressing GlyT2-EGFP were intensely fluorescent, and their dendrites and axons could be visualized in great detail. Numerous positive neurons were detected in the spinal cord, brainstem, and cerebellum. The hypothalamus, intralaminar nuclei of the thalamus, and basal forebrain also received a dense GlyT2-EGFP innervation, whereas in the olfactory bulb, striatum, neocortex, hippocampus, and amygdala positive fibers were much less abundant. No GlyT2-EGFP-positive cell bodies were seen in the forebrain. On the subcellular level, GlyT2-EGFP fluorescence was colocalized extensively with glycine immunoreactivity in somata and dendrites and with both glycine and GlyT2 immunoreactivity in axon terminals, as shown by triple staining at all levels of the neuraxis, confirming the selective expression of the transgene in glycinergic neurons. In slice preparations of the spinal cord, no difference between the functional properties of EGFP-positive and negative neurons could be detected, confirming the utility of visually identifying glycinergic neurons to investigate their functional role in electrophysiological studies.

Ferritin H gene deletion in the choroid plexus and forebrain results in hydrocephalus.

Ferritin H, the major iron storage protein, has essential functions in early embryonic development as well as in adult liver and intestine. To address the question whether ferritin H has similarly essential functions in the brain we used the Cre/loxP system to generate mice with a forebrain-specific inactivation of the ferritin H gene. Ferritin H deficiency in most cells of the forebrain including cells of the choroid plexus caused accumulation of cerebrospinal fluid in the lateral ventricles and the subarachnoid space. Brain tissue iron content was unchanged.

Diffusion barriers constrain receptors at synapses.

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the γ-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (γ2) or not (α5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, γ2 and α5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, γ2 was ~3-fold more confined and dwelled ~3-fold longer in inhibitory synapses than α5, indicating faster synaptic escape of α5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of γ2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit (GluA2) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.

Activity-dependent tuning of inhibitory neurotransmission based on GABAAR diffusion dynamics.

An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA(A)R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA(A)Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA(A)R gamma2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA(A)R increases in parallel with neuronal activity, depending on Ca(2+) influx and calcineurin activity. These results indicate that GABA(A)R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca(2+) concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.

Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion.

In the spinal cord, most inhibitory synapses have a mixed glycine-GABA phenotype. Using a pharmacological approach, we report an NMDAR activity-dependent regulation of the mobility of GlyRs but not GABA(A)Rs at inhibitory synapses in cultured rat spinal cord neurons. The NMDAR-induced decrease in GlyR lateral diffusion was correlated with an increase in receptor cluster number and glycinergic mIPSC amplitude. Changes in GlyR diffusion properties occurred rapidly and before the changes in the number of synaptic receptors. Regulation of synaptic GlyR content occurred without change in the amount of gephyrin. Moreover, NMDAR-dependent regulation of GlyR lateral diffusion required calcium influx and calcium release from stores. Therefore, excitation may increase GlyR levels at synapses by a calcium-mediated increase in postsynaptic GlyR trapping involving regulation of receptor-scaffold interactions. This provides a mechanism for a rapid homeostatic regulation of the inhibitory glycinergic component at mixed glycine-GABA synapses in response to increased NMDA excitatory transmission.

Imaging the lateral diffusion of membrane molecules with quantum dots.

This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor alpha3 subunit-null mice.

Multiple GABAA-receptor subtypes are assembled from alpha, beta and gamma subunit variants. GABAA receptors containing the alpha3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the alpha3 subunit gene (alpha3(0/0)) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of alpha3 subunit for maturation of GABAA receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the alpha1, alpha2, or alpha5 subunit was unaltered in alpha3(0/0) mice and the prominent alpha3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the alpha3 and gamma2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In alpha3(0/0) mice, gamma2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABAA receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABAA receptors are required for gephyrin clustering and long-term synapse maintenance. The absence of GABAA-mediated transmission in the nRT may have a significant impact on the function of the thalamo-cortical loop of alpha3(0/0) mice.

The gamma 2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses.

The gamma2 subunit of GABA(A) receptor chloride channels is required for normal channel function and for postsynaptic clustering of these receptors during synaptogenesis. In addition, GABA(A) receptor function is thought to contribute to normal postnatal maturation of neurons. Loss of postsynaptic GABA(A) receptors in gamma2-deficient neurons might therefore reflect a deficit in maturation of neurons due to the reduced channel function. Here, we have used the Cre-loxP strategy to examine the clustering function of the gamma2 subunit at mature synapses. Deletion of the gamma2 subunit in the third postnatal week resulted in loss of benzodiazepine-binding sites and parallel loss of punctate immunoreactivity for postsynaptic GABA(A) receptors and gephyrin. Thus, the gamma2 subunit contributes to postsynaptic localization of GABA(A) receptors and gephyrin by a mechanism that is operant in mature neurons and not limited to immature neurons, most likely through interaction with proteins involved in trafficking of synaptic GABA(A) receptors.