GAL-101 prevents amyloid beta-induced membrane depolarization in two different types of retinal cells

GAL-101 prevents amyloid beta-induced membrane depolarization in two different types of retinal cells | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results GAL-101 prevents amyloid beta-induced membrane depolarization in two different types of retinal cells Erika Pizzi , Simona Gornati , Stefano Stabilini , Dario Brambilla , Chiara A. Mercurio , View ORCID Profile Hermann Russ , Christopher G. Parsons , Michele Mazzanti doi: https://doi.org/10.1101/2025.01.21.634046 Erika Pizzi 1 Dept. Bioscienze, University of Milan , Via Celoria 26, 20133 Milano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Simona Gornati 1 Dept. Bioscienze, University of Milan , Via Celoria 26, 20133 Milano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stefano Stabilini 1 Dept. Bioscienze, University of Milan , Via Celoria 26, 20133 Milano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dario Brambilla 2 Dept. Fisiopatologia e dei Trapianti, University of Milan , Via Mangiagalli 32, 20133 Milano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chiara A. Mercurio 1 Dept. Bioscienze, University of Milan , Via Celoria 26, 20133 Milano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hermann Russ 3 Galimedix Therapeutics Inc. Kensington , MD 20895, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hermann Russ For correspondence: HRuss{at}galimedix.com Christopher G. Parsons 3 Galimedix Therapeutics Inc. Kensington , MD 20895, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michele Mazzanti 1 Dept. Bioscienze, University of Milan , Via Celoria 26, 20133 Milano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Glaucoma and age-related macular degeneration (AMD) are two of the major causes of progressive vision loss and ultimately blindness worldwide. Both retinopathies share several pathological features with Alzheimer’s disease (AD) such as: impairment of neuronal function, astrocytosis, and activation of immune-competent microglia and Müller cells. It also has been shown that these conditions are characterized by the presence of an elevated concentration of amyloid beta (Aβ). Under pathological conditions, Aβ 1-42 tends to aggregate, forming toxic soluble oligomers, considered to be the most harmful amyloid species. One strategy adopted to prevent cell damage caused by these oligomers is to impair their aggregation. Here we studied GAL-101, a small molecule designed to modify the aggregation of Aβ 1-42. To assess the role of GAL-101 in the aggregation of Aβ 1-42 , in vitro electrophysiological measurements on retinal ganglion cells (RGCs) and retinal pigment epithelial (RPE) cells were performed to determine the polarization of the resting membrane potential. Cells treated only with Aβ 1-42 oligomers showed a strong depolarization of the resting membrane potential, which is believed to be the main reason for retinal cells malfunctioning in neurodegenerative diseases of the eye. Pre-incubation with GAL-101 stabilized the cell resting potential to around -50mV during exposure to Aβ 1-42 , in both RGCs and RPE cells. GAL-101 was able to prevent changes in resting membrane potential and thus would be expected to prevent impairment of retinal cell function. These results are supportive of evaluating GAL-101 as a potential treatment of Aβ-associated retinopathies like glaucoma and dry AMD. 1. Introduction Glaucoma and age-related macular degeneration (AMD) are leading causes of progressive vision loss and blindness worldwide, and their incidence and prevalence increase with age ( 1 – 3 ). While recognized as distinct pathologies, similarities exist between these two retinal diseases, as well as with Alzheimer’s disease (AD). AD is a neurodegenerative disease characterized histopathologically by the presence of extracellular deposits of misfolded amyloid-beta (Aβ) in the form of senile plaques and the intracellular accumulation of hyperphosphorylated tau in the form of neurofibrillary tangles ( 4 ). The incidence of all three conditions increases with age and the chronic neurodegenerative changes seen in the eyes of glaucoma and AMD patients are similar to changes characteristic of the brains of AD patients ( 1 – 3 , 5 – 7 ). The association between glaucoma and AD has emerged from studies showing that AD is associated with glaucomatous changes, such as RGC loss, optic neuropathy and impaired visual function ( 8 – 13 ). Aβ has been reported to be implicated in the development of RGC apoptosis in glaucoma, with evidence of caspase-3-mediated abnormal APP processing ( 14 ), increased expression of Aβ 1-42 in RGCs and optic nerves ( 14 – 19 ) and decreased vitreous Aβ levels (consistent with retinal Aβ deposits) in patients with glaucoma ( 20 ). AMD is characterized by drusen, extracellular waste deposited between the basal surface of the retinal pigment epithelium (RPE) and Bruch’s membrane ( 21 ). Drusen are a clear hallmark of early, or “dry” AMD ( 22 ), and a significant risk factor for progression to “wet” AMD involving neovascularization ( 23 ). Drusen include deposits of Aβ, remnants of RPE cells ( 24 ), and a variety of immune system-related molecules, including immunoglobulins and complement system components ( 25 ). The deposition of Aβ, in particular Aβ oligomers, in drusen has been shown to increase with age ( 26 , 27 ). Small soluble oligomeric forms of Aβ are able to induce significant RGC apoptosis in vivo and in vitro ( 16 , 28 , 29 ). It has been shown that targeting Aβ 1-42 formation and aggregation reduces glaucomatous RGC apoptosis in vivo and therefore raises the possibility of using antiaggregant therapy to provide neuroprotection against glaucoma progression ( 16 ). GAL-101 (formerly MRZ-99030, EG30) is a small D-amino acid dipeptide that modulates Aβ 1-42 aggregation by triggering a non-amyloidogenic aggregation pathway and thereby reduces the amount of toxic soluble oligomeric Aβ 1-42 species ( 30 – 32 ). According to recent publications, Aβ is able to form ionic pathways of different conductance depending on the size of the oligomeric species ( 33 – 36 ). This might be related to the ability of Aβ to infiltrate biological membranes, as has been described in detail, into an artificial lipid bilayer ( 37 , 38 ). In the present study, we monitored the resting membrane potential (RMP) of RGC and RPE cells before and after acute application of 50nM Aβ 1−42 . The gating of unspecific ionic pathways mediated by Aβ 1−42 caused a net inward current, thus inducing RMP depolarization. Preincubation of Aβ 1-42 with GAL-101 was able to reduce or even prevent RMP depolarization. 2. Materials and Methods 2.1.Cell culture RGCs were isolated from P5-P6 mouse retinas (neonatal mice C57BL/6N, strain code 027, Charles River) using the standardized procedure in the Miltenyi Biotec kit. By the 4th day in vitro , three types of cells were distinguishable: RGCs, amacrine cells, and astrocytes. RGCs were recognized by the presence of one long protrusion representing the axon. RGCs were maintained for 7 days in Neurobasal medium supplemented with Sato 1X, B27 2X, T3 1X, Sodium Pyruvate 1 mM, Glutamine 1 mM, Insulin 1X, CNTF 1X, BDNF 1X and Forskolin 1X at 37°C and 5% CO2 before recording. RPE cells were obtained from the American Tissue Collection (ARPE-19 - ATCC ® CRL-2302TM) and maintained in 1:1 DMEM/F12 with 10% FBS at 37°C and 5% CO 2 . 2.2. GAL-101 molecule GAL-101 (see Figure 1 ) is designed to prevent the formation of all forms of toxic Aβ oligomers by binding with high affinity to the misfolded Aβ monomers before they can form toxic soluble oligomers. These oligomers then rapidly form amorphous, non-beta-sheet “clusters,” which are innocuous. Interestingly, once GAL-101 concentration reaches effective levels, it “triggers” formation of the “clusters”, which have shown their ability to collect additional misfolded Aβ monomers, even in the absence of additional GAL-101 molecules, through a self-propagating mechanism. This novel “trigger effect,” protected by Galimedix’s patent portfolio, results in a sustained effect. The effect lasts far longer than the time a single administration of the drug remains at therapeutic levels in the retina, potentially allowing for a convenient interval application regimen for patients. Thus, GAL-101 drops may potentially provide sustained prevention of formation of toxic Aβ oligomers in the retina, leading to a reduction in complement response and its consequent damage. Thus GAL-101 could contribute to slowing or stopping progression, and possibly restoring neural function depressed by the chronic toxic attack. Download figure Open in new tab Figure 1: Chemical structure of GAL-101 2.3. Electrophysiological recordings 2.3.1. Resting membrane potential measurements Membrane potential recordings were conducted on single cells using the perforated whole-cell configuration in current-clamp mode. Data were collected using an Axopatch 200B amplifier (Molecular Device, CA, US) and experimental traces were digitized at 5 kHz and filtered at 1 kHz with a Digidata 1322 acquisition system. Clampex-8 was used as the acquisition software. Patch pipettes (GB150F-8P with filament, Science Products) were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller (Sutter Instruments, Novato, CA, US) and fire-polished to a final electrical resistance of 4-5 MΩ. Shortly before starting the recordings, the culture medium was replaced with the external recording solution. The perforated whole-cell configuration was achieved by using the antibiotic Gramicidin (Sigma Aldrich) diluted in the internal solution at a final concentration of 5 μg/ml and 10 μg/ml for RGCs and RPEs, respectively. Electrical access to the cell was thereby achieved after about 5-10 minutes. The solutions used were (in mM): RGCs –solution ext.: 140 NaCl, 2.5 KCl, 1.8 CaCl 2 , 0.5 MgCl 2 , 10 Glucose, 10 HEPES, pH 7.4. RGCs - solution int.: 140 KCl, 10 NaCl, 2 MgCl 2 , 0.1 CaCl 2 , 10 Glucose, 10 HEPES, pH 6.9. RPE cells – external solution: 132 NaCl, 2 CaCl 2 , 1 MgCl 2 , 10 Glucose, 10 HEPES, pH 7.4. RPE cells - internal solution: 132 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 Glucose, 10 HEPES, pH 7.0. 2.3.2. HEK293 whole-cell voltage-clamp recordings Patch-clamp electrophysiology was performed in whole-cell configuration in HEK293 cells. The intracellular solution was (in mM): 20 KCl, 120 K-aspartate, 10 HEPES, and 1 MgCl 2 . The potassium channel blocker tetraethyl-ammonium (TEA 40 mM; Sigma, St Louis, MO, USA) was used to minimize the contribution of these ions. To obtain voltage-current relationships (i/V), the cell voltage was held at -50 mV and the current was measured at the end of 800 ms voltage steps from -70 to + 50 mV. A subtraction method using 50 µM indanyloxyacetic acid 94 (IAA94, Sigma, St Louis, MO, USA) a Cl-channel inhibitor, was used to isolate the inhibitor-sensitive current. The Axopatch 200 B amplifier and the pClamp-9 acquisition software and Clampfit 9 (both from Molecular Device, Novato, CA) were used to record and analyze whole-cell traces. Current recordings were digitized at 5 kHz and filtered at 1000 Hz. Patch electrodes (GB150F-8P with filament, Science Products) were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller (Sutter Instruments, Novato, USA) and fire-polished to a tip diameter of 1-1.5 µm and electrical resistance of 5-7 MΩ. 2.3.3. Tip-dip lipid bilayer current recording Single-channel recordings from lipid bilayers were obtained using the tip-dip method. In brief, patch clamp pipettes (Garner Glass 7052) were made using a P97 Sutter Instruments puller (Novato, CA) and fire-polished to a tip diameter of 1–1.5 μm and 5–7 MΩ resistance. The same solution was used both in the bath and in the pipette (140 mM KCl, 10 mM Hepes, pH 6). Purified recombinant CLIC1 protein (2 μg/ml) was added to the pipette solution. As soon as the pipette tip reached the bath solution, a phospholipid monolayer (phosphatidylcholine, Avanti Polar Lipids, Inc., Birmingham, AL) was spread on the surface. The electrode was repeatedly passed through the surface of the solution until the pipette resistance rose above 5 GΩ. An Axopatch 1D amplifier and pClamp 7 (both from Axon Instruments, Novato, CA) were used to record and analyze single-channel currents. Current recordings were digitized at 5 kHz and filtered at 800 Hz. 2.4. Amyloid beta preparation Aβ was prepared according to the protocol originally developed by the group of Klein ( 32 , 39 ). In brief: lyophilized Aβ 1-42 from Bachem (Code. H-1368 lot number 1030255) was dissolved in HFIP (Sigma Aldrich) at a concentration of 1 mg/ml under 90 min of continuous shaking. 50 ml aliquots were then frozen at –80 °C for 45 minutes. After overnight lyophilization (−20 °C) the aliquots were kept stored at –20 °C until use. Right before use, Aβ 1-42 was dissolved in DMSO at a final concentration of 100 μM and sonicated in an ultrasonic water bath for 1 hour. Aβ 1-42 was quantified by using a BCA protein assay kit (Pierce). DMSO concentration never exceeded 1% (ranging from 0.1% to 0.5%). No previous data have been reported in the literature on the use of Aβ 1-42 on single isolated RGCs or RPE cells. We therefore designed our experiment based on our own preliminary experiments. The final Aβ 1-42 concentration was different depending on the cell type. The concentration-response analysis revealed the optimal concentration for RGCs to be 50 nM. In contrast, the equivalent concentration for RPE cells was 1 μM as RPE cells were considerably less sensitive. In all experiments, Aβ 1-42 was used after a 90-minute incubation period at 36 °C and within 2 hours from its dilution in the external solution. Aβ 1-42 was applied using a gravity-driven perfusion system (RSC-200, BioLogic) through a micropipette positioned close to the recorded cell. GAL-101 was used at the final concentration of 0.5 and 1 μM for RGCs and 10 and 20 μM for RPE cells, corresponding to ten or twenty times of the Aβ 1-42 concentration. GAL-101 was added to Aβ 1-42 before incubation. 2.5. Voltage-clamp, whole-cell configuration Aβ 1-42 was incubated at 37 °C at different times at the concentration of 1 µM (see methods). HEK cells were exposed to different preparations of Aβ 1-42 for 15 minutes prior to the measurement of membrane conductance. The final Aβ 1-42 concentration in contact with cells was 50 nM. 2.6. Statistical analysis The statistical significance of the data was assessed using a two-tailed paired Student’s t test. All data are shown as mean ± SEM. Origin 9 was used for plotting data and for statistical analysis. 3. Results 3.1. Amyloid beta impact on resting membrane potential of retinal ganglion cells To evaluate the impact of Aβ 1-42 on the RMP of RGCs, we first measured the resting potential of the cells after performing whole-cell patch-clamp recordings in current-clamp mode. Under controlled conditions, RGCs had an average RMP of -68.3 ± 1.9 mV (n=26; Figure 2A ). The RMP of RGCs also was measured under control conditions up to 1 hour ( Figure 2B ) to be sure that our experimental recording conditions were not affecting cell functions. In some cells, it was not unusual to observe spontaneous firing activity ( Figure 2C ). Download figure Open in new tab Figure 2: Measuring resting potential in RGC-isolated cell. A: Distribution of cell resting potential (average -68.3 ±1.9mV;n = 26). B: Stability of membrane potential over 40 minutes of continuous recording (n = 12). C: Spontaneous action potential in RGC cell during resting membrane potential monitoring. The Aβ 1-42 concentration was able to induce a relevant membrane voltage depolarization without being toxic for the cell. This was estimated by performing a dose/response curve to calculate the EC50 (half maximal effective concentration) of Aβ 1-42 . To determine this, we used three different procedures; Figure 3 shows two of the three. The first method ( Figure 3A ) consisted in measuring the RMP in time course experiments under controlled conditions for several minutes. Aβ 1-42 was applied to the cells from 1 nM up to 10 nM Aβ 1-42 , with 1 nM increments every 4 minutes, and from 50 to 100 nM. Using the second method ( Figure 3B ), the Aβ 1-42 effect on RMP was measured using a peptide concentration from 10 nM up to 100 nM delivered every 4 minutes. Average RMP values at each concentration (n = 3) obtained by these two experimental procedures were used to generate the dose-response curve shown in Figure 3C . The plot depicts combined data from the two protocols previously described (squares) as well as from a third method in which Aβ 1-42 was applied on a current-clamped RGC as a single concentration (n = 3; circles). From the curve fitting, we obtained an EC50 of 44.06 ± 6.3 nM (membrane potential depolarization -35.6 ± 1.8 mV) and a maximal effect at 100 nM. According to this result, all the experiments on RGCs with GAL-101 were performed using 50 nM Aβ 1-42 , an effective concentration to induce a membrane potential depolarization without causing short term toxicity. Download figure Open in new tab Figure 3: Time course of whole-cell current-clamp experiments. A and B: Depolarization ofRGC cells after addition of Aβ 1-42 at increasing concentrations (A:n = 3 B:n = 3). The insert in B shows control measurement of a stable membrane potential over 80 minutes. C: Dose/response curve of depolarization rate, recorded in a dynamic condition (circles, n = 3) and from steady-state experiments (squares, n = 3). Calculated EC50 is 44.1 ±6.3 nM at a membrane voltage of-35.6 ± 1.8 m V. 3.2. GAL-101 prevents beta amyloid aggregation and reduces membrane depolarization in RGCs We have previously demonstrated that Aβ 1-42 aggregation into oligomers increases the permeability to cations in biological membranes ( 35 ). Since GAL-101 is a modulator of Aβ 1-42 oligomerization, we assessed whether it is able to prevent the depolarization of the RMP induced by oligomeric Aβ 1-42 in RGCs. To this end, the RMP of isolated RGCs was monitored under whole-cell, current-clamp conditions and perfused with a solution containing 50 nM Aβ 1-42 preincubated with different concentrations of GAL-101. As previously shown, Aβ 1-42 is already effective in causing a membrane potential depolarization at 20 nM. As shown in Figure 4 , GAL-101 inhibited Aβ 1-42 -induced depolarization in a concentration-dependent manner. Indeed, the GAL-101 effect was statistically significant at a 20-fold (1 µM) stoichiometric increase over Aβ 1-42 compared to 0.5 µM GAL-101. Download figure Open in new tab Figure 4: Chart plot of membrane potential measurements in control conditions (n=11), after addition of 50 nM Aβ (n = 8), and in the simultaneous presence of 50 nM Aβ and 0.5 μM (n = 9), or 1 μM GAL-101 (n = 9;p**** = 0.0001). Figure 5A (shows the effect of 50 nM Aβ 1-42 on the RMP of six RGC cells. After a variable time (1-15 minutes), the cells showed a membrane depolarization of several millivolts, reaching ∼ -20 mV. In figure 5B , six RGC cells were perfused with 50 nM Aβ 1-42 in addition to 1 µM GAL-101. Even then, the RMP depolarized several minutes after the application of Aβ 1-42 and GAL-101, reaching, however, less depolarized values (∼ -50 mV) than those recorded in the absence of 1 µM GAL-101. The time between Aβ 1-42 perfusion and the onset of RMP depolarization was independent from the presence of GAL-101. Download figure Open in new tab Figure 5: GAL-101 reduces Aβ 1-42 oligomer-induced depolarization. Time-course current-clamp recordings of the individual RGCs’ resting membrane potential treated under control conditions (n = 6) (A), Aβ 1-42 alone (n = 6) (B) and 50 nM Aβ 1-42 together with 1 pM GAL-101 (n = 6) (C). Figure 6 shows the RMP values under control conditions and after the application of Aβ 1-42 alone or Aβ 1-42 pre-incubated with 1 µM GAL-101. GAL-101 prevented major depolarization of RGCs. Download figure Open in new tab Figure 6: GAL-101 prevents depolarization of RGCs after 20 minutes. The chart plot shows the comparison between membrane potential values at the end of 20 minutes of membrane potential recording of single RGC cells in control conditions (n = 23), exposed to 50 nM Aβ (n = 6,p = 0.001) and 50 nMAβ+1 μM GAL-101 (n = 6, p = 0.001). Since 50 nM Aβ 1-42 significantly depolarizes the resting potential of RGCs, higher concentrations of Aβ 1-42 could cause an inhibition of neuronal functions such as the initiation/conduction of action potentials and thus synaptic transmission. By preventing membrane depolarization, GAL-101 is expected to be able to counteract these negative effects. 3.3. GAL-101 effect on non-excitable cells Long-term chronic depolarization is harmful for any kind of cell because most of the membrane transport systems are driven by the membrane potential. To test the ability of GAL-101 to interfere with Aβ 1-42 effect in non-excitable cells, we performed electrophysiological recordings on RPE cells. Figure 7A shows the RMP values recorded from 29 RPE cells in current-clamp mode. Average RPE resting potential was -40.2 ± 2.4 mV. The same experiments were repeated, perfusing the cells with the extracellular solution containing the vehicle 0.1% DMSO. No differences were detected compared with control (data not shown). The difference between RGCs and RPE cells was the Aβ 1-42 concentration required to induce a significant depolarization. The dose response/curve was obtained by incubating RPE cells at different Aβ 1-42 concentrations and measuring the resting potential of the cells after 15 minutes ( Figure 7B ). From the best fit of the experimental points, it was possible to calculate an EC50 of Aβ 1-42 for RPE cells (790 ± 80 nM) that was almost 18-fold less potent compared to the EC50 for RGCs (44.1 ± 6.3 nM). As with the RGCs, we used a concentration of Aβ 1-42 able to depolarize the RMP without killing the cells. Figure 7C shows an example of the effect caused by 1 μM Aβ 1-42 in a time course experiment in which the RMP was monitored for several minutes before and after the addition of Aβ 1-42 (arrow). The effect of 1 μM Aβ 1-42 under both control conditions and Aβ 1-42 previously incubated with GAL-101 at two different concentrations is shown in Figure 8 . GAL-101 at 10 times higher concentration than Aβ 1-42 was not able to counteract the effect caused by 1 μM Aβ 1-42 ( Figure 8B ; n = 7). However, GAL-101 at 20 times higher concentration than Aβ 1-42 was able to inhibit the RMP depolarization seen in Figure 8A ( Figure 8C ; n = 11). As for RGCs, GAL-101 was able to significantly reduce the depolarization of the RPE cells but at a concentration 20-fold higher compared to Aβ 1-42 . Download figure Open in new tab Figure 7: Aβ induces depolarization of retinal pigment epithelium (RPE) cells. A: Distribution of resting membrane values in RPE cells (n = 30). B: Distribution of resting membrane values in RPE cells perfused with the vehicle 0.1 % DMSO (n = 8). C: Time course experiment monitoring single RPE cell resting membrane potential during exposition to 1 μM Aβ (n = 5). D: Dose/response curve of RPE cells challenged with different Aβ concentrations EC50 (790 ÷ 80 nM, n = 8). Download figure Open in new tab Figure 8: RPE depolarization induced by Aβ decrease in the presence of GAL-101. A: RPE resting membrane potential measurements under controlled conditions and in the presence of 1 μMAβ (−46.44 ±7.86 mV and -15.66 ±4.9 mV, respectively). B and C: Effect of GAL-101 at 10 μM (control vs. treated: -45.45 ± 5.43 mV and -23.6 ± 4.8 mV) or 20 pM (control vs. treated: -44.94 ± 5.69 mV and -41.56 ± 5.2 mV) respectively on RPE single cell depolarization induced by 1 μM Aβ. A andB:n=10;p**** = 0.0001.C:n= 10. 3.4. GAL-101 and amyloid ionic conductance in cell and artificial membranes The ability of Aβ 1-42 to operate as a transmembrane ionic pathway was tested in HEK cells under voltage-clamp, whole-cell configuration. The results presented in Figure 9 , showed that 50 nM Aβ 1-42 at 0 incubation time demonstrated the same conductance recorded once Aβ 1-42 was incubated for 240 minutes ( Figure 9 , first and last open chart box from the left). Download figure Open in new tab Figure 9: Membrane conductance is modified by Aβ 1-42 Chart plot of whole cell HEK cell membrane conductance in the presence of 1 μM Aβ (white boxes) and with the addition of 20 μM GAL-101 (grey boxes). The gray line represents the experiments (n = 5at each time point) in the control condition (average and SE). 0 min: n= 10; 60 min: n= 10; 120 min: n =10; 240 min: n= 10. However, Aβ 1-42 maintained at 37 °C between 60 and 120 minutes and then added to the external solution caused a robust increase of HEK cells membrane conductance ( Figure 9 , second and third open chart boxes from the left). The same experiments were repeated with the Aβ 1-42 previously incubated at 37 °C in the presence of 20 µM GAL-101, in accordance with the experiment presented in Figure 8 . The final GAL-101 concentration in contact with the cells was then 200 nM in addition to 50 nM Aβ 1-42 . The membrane conductance measured under these conditions is plotted in Figure 9 at each incubation time as a grey filled chart box. In this case, the membrane conductance result was not significantly different under various conditions. Aβ 1-42 preparation in the presence or in the absence of GAL-101 was also used to challenge an artificial bilayer. Tip-dip experiments resume, at single channel level, the results obtained in HEK cells. Figure 10 depicts current recordings after delivering Aβ 1-42 after 120 and 240 minutes of incubation at 37 °C in the trans solution. The membrane conductance formed by the Aβ 1-42 at different aggregation times show a marked difference in the current level between two- and four-hours incubation periods. Download figure Open in new tab Figure 10: Tip-dip current recordings of Aβ 1-42 -induced membrane conductance in artificial lipid bilayer. A: Single channel recording at different membrane potentials under control conditions (left), in presence of 1 μM Aβ 1-42 (incubated at37°C) in the absence (middle) and in the presence (right) of 20 μM GAL-101. B: Relative single channel current/voltage relationships. Control: n = 5; Aβ 1-42 : n = 5; A 1-42 + GAL-101: n = 5. 4. Discussion Amyloid peptide aggregation and accumulation in the central nervous system (CNS) is a hallmark of several neurodegenerative diseases. In particular, Aβ 1-42 concentration in the CNS fluid is one of the tools used to identify patients with AD, and it is combined with other biomarkers such as phosphorylated tau and advanced imaging techniques to get a more accurate AD diagnosis ( 40 , 41 ). Several reports have shown that Aβ 1-42 deposits are also present in different retinal cell layers and in the optic nerve ( 15 , 16 ). Both the presence of the peptide and several degenerative processes in the eye have been proposed as biomarkers to identify neurodegenerative pathologies ( 42 ). For example, it is not unusual that AD patients also suffer from degeneration of the retina ( 26 , 43 ). Glaucoma and AMD are the most recognized causes of irreversible vision loss worldwide. Indeed, both these pathological conditions show Aβ 1-42 deposits in the retina ( 44 , 45 ). Although the molecular mechanism by which Aβ 1-42 exerts its cytotoxicity has still not been completely elucidated, it is widely accepted that the toxic form of the amyloid peptide is represented by the soluble oligomeric aggregates ( 46 – 49 ). The neurodegenerative action of Aβ 1-42 oligomers is believed to be exerted both at the membrane level ( 50 , 51 ) as well as inside the cell after cytoplasmic inclusion of Aβ 1-42 agglomerates ( 52 – 54 ). Several membrane receptors have been identified to react to Aβ 1-42 , either by increasing their activity or inhibiting their functions ( 55 – 60 ). It is still controversial if Aβ 1-42 can directly cause membrane instability and/or have the ability to form ion-conducting channels in vivo. Certainly this is true for artificial lipid bilayers where Aβ 1-42 is responsible for generating conductance directly proportional to the Aβ 1-42 aggregation state ( 35 ). Despite not evaluating the exact mechanism of action of Aβ 1-42 , in these experiments, we demonstrated that Aβ 1-42 oligomer-enriched solution at 50 nM concentration was able to cause RMP depolarization of isolated mouse RGCs. According to the cell properties, opening of non-selective ionic conductance should move the cell resting potential to more depolarized values. This is exactly what happened in our experiments, suggesting the formation of ionic conductance more than overt, acute membrane damage. Furthermore, the kinetics of the change in the membrane potential was a relatively slow process, suggesting a step-by-step addition of small ionic pathways, more than the rapid acute response of e.g., ionic membrane receptors. However, we cannot exclude the partial or total contribution of a slowly activating receptor such as a G protein-coupled receptors (GPCR) in causing membrane depolarization. In light of these results, a strategy to reduce RGC depolarization could be to block Aβ 1-42 aggregation to prevent the formation of the toxic Aβ 1-42 oligomers. For GAL-101, it was demonstrated that this compound prevents and even reverses the formation of Aβ 1-42 oligomers ( 61 ). The results on RGCs demonstrated that GAL-101 is able to prevent amyloid-induced GC depolarization, possibly acting on amyloid aggregation formation. Since the effect of Aβ 1-42 was the depolarization of membrane potential, the final outcome would be the inhibition of neuronal functions such as the generation and conduction of action potentials and synaptic transmission. The antiaggregant action of GAL-101 fully prevents the depolarization of the RMP, thus preserving the neuronal activity of RGCs. Aβ 1-42 -induced membrane potential depolarization was also effective on non-excitable cells as shown by RPE recordings. However, the concentration of Aβ 1-42 able to cause a change in the RMP was 20-fold higher for RPE cells compared to RGCs. A 20-fold difference in the EC50 of Aβ 1-42 as determined in RPE cells and in RGCs indicates that the mechanism of toxic action is different in these two cell types: the first has epithelial origin and the second has neuronal origin. The toxicity to the neuronal RGCs might be caused by an interaction of Aβ 1-42 with one of the described binding sites located on neuronal receptors, e.g., glutamatergic, GABAergic, or nicotinic receptors ( 49 ), while the toxicity to the epithelial RPE cells might be caused by a lower affinity interaction with the membrane. The toxic effect of Aβ 1-42 on the neuronal RGCs was very strong, with an EC50 of 44 nM calculated from the dose/response curve ( Figure 3C ). RPE cells required a much higher Aβ 1-42 concentration able to induce a significant depolarization ( Figure 6C ). It is possible that the lipid membrane composition is different between these two cell types, making the insertion of Aβ 1-42 aggregates into the cells’ plasma membrane more difficult. A second possibility is that RGCs express a higher number of membrane receptors than RPE cells able to react with Aβ 1-42 , thus contributing to the cell depolarization. Both hypotheses are supported by the similar action of GAL-101 on the two cell types. Our experiments show that this anti-aggregation compound must be 20 times more concentrated than Aβ 1-42 to exert its activity. In conclusion, we have shown that Aβ 1-42 oligomers are able to cause membrane potential depolarization both in neuronal and epithelial cells from retinal tissue. We suggest that the mechanism of action is the insertion of oligomeric Aβ 1-42 in the cell membrane and the opening of non-selective ionic permeability pathways. In addition to this non-specific effect, it is possible that RGCs expressing membrane receptor are able to react with Aβ 1-42 , contributing to membrane depolarization. Evidence that the RMP in the presence of Aβ 1-42 does not achieve 0 mV strongly suggests that the change in the membrane permeability is not due to membrane disruption. With membrane breaks, in a very short time, the cytoplasm would wash out by the external solution. On the contrary, the activation of membrane receptors or, more likely, the formation of non-selective pores, for the physiological characteristics of the cell, stabilizes the membrane resting potential to -10/-15 mV. Pre-incubation of Aβ 1-42 during the aggregation process with GAL-101, at the concentration tested in our experiments, inhibits amyloid peptide aggregation. This should prevent strong changes in resting membrane potential and thus would prevent impairment of cell function. A possible future therapeutic option could be the chronic delivery of GAL-101. After treatment, Aβ 1-42 aggregation process should slow down and, consequently, this would eventually slow down membrane depolarization during various retinal pathological stages. A potential treatment approach for glaucoma and dry AMD, which are caused by the neurodegeneration of RGCs and RPE cells, could be based on the elimination of Aβ 1-42 oligomers from the retina that have detrimental effects on the RMP of these cells, leading to long-term toxic effects. A potential drug candidate for this approach could be the Aβ 1-42 aggregation modulator GAL-101, which has already shown beneficial effects in an animal glaucoma model ( 62 ). The prevention of Aβ 1-42 -induced depolarization in both RGCs and RPE cells, together with the known effects of GAL-101 on Aβ-suppressed synaptic plasticity ( 61 , 63 ), qualifies GAL-101 as a new candidate for further clinical investigation in Aβ-associated retinopathies. 5. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . 6. Author Contributions Individual contributions. “Conceptualization, C. P. and M.M.; methodology, E.P., S.G and S.S.; validation, D.B., C.P. and M. M.; formal analysis, E.P, S.G., C.A.M; investigation, C.P. and M.M.; resources, M.M.; data curation, S.S and D.B.; writing—original draft preparation, C.P., E.P., S.G., S.S., and D.B.; writing—review and editing, C.A.M., H.R., M.M.. All authors have read and agreed to the published version of the manuscript.” 7. Funding This research received no external funding 8. Acknowledgments This manuscript is dedicated to the late Christopher G. Parsons, an exceptional pharmacologist who made a significant contribution to this scientific work with his passion for the fascinating mechanism of action of GAL-101. 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