Bruno P. Meloni

Background/Objectives: Effective disease-modifying therapies targeting pathogenic proteins associated with Alzheimer’s disease (AD) remain limited. This study investigated the therapeutic potential of the neuroprotective, cationic arginine-rich peptide R18D to mitigate the pathogenic effects of amyloid-beta (Aβ) and tau associated with AD. Methods: R18D was examined for its ability to inhibit Aβ aggregation in a cell-free assay, attenuate Aβ-induced cytotoxicity in MC65 cells, and suppress intracellular tau aggregation in two neural cell models. Intracellular tau aggregation was quantified using a homogeneous time-resolved fluorescence assay. Additionally, a pilot pharmacokinetic study of R18D was conducted in mice following oral gavage administration. Results: In the cell-free assay, R18D inhibited Aβ aggregation by up to 65%. In human MC65 cells induced to overexpress APP-C99 and accumulate Aβ, treatment with R18D inhibited cellular toxicity by as much as 100%. Preformed tau seeds were applied to human SH-SY5Y cells and rat primary cortical neurons to induce intracellular tau aggregation, and tau levels were quantified after 48 h. Exposure to tau seeds induced robust tau aggregation in both cellular models, which was significantly attenuated by R18D treatment, reducing aggregation by 34.8% in SH-SY5Y cells and 49.9% in cortical neurons. Pharmacokinetic studies demonstrated that R18D was detectable in plasma at 30 and 60 min following oral administration in mice. Conclusions: Together, these results demonstrate that R18D can modulate both Aβ and tau pathologies in vitro and is orally bioavailable, supporting its further evaluation as a therapeutic candidate for AD and other tau-associated neurodegenerative disorders.

Background: This study examined the dose‐dependent efficacy and pharmacokinetics of R18D in a rat transient middle cerebral artery occlusion (MCAO) stroke model. Methods: R18D was intravenously administered 60 min after the onset of a 90‐min MCAO induced by intraluminal filament insertion. For pharmacokinetic profiling, blood samples were collected 10, 15, 30 and 60 min, and at 2, 4 and 8 h after the start of a 10‐min intravenous infusion of R18D. Infarct volume and body weight were assessed at 24 h after MCAO. Results: R18D reduced mean total infarct volume by 47.2% ( p = 0.0003), 28.4% ( p = 0.008), and 27.9% ( p = 0.003) at doses of 325, 650 and 1100 nmol/kg, respectively. The 650 nmol/kg dose significantly reduced cerebral hemisphere swelling by 8.3% ( p = 0.01), whereas the 325 nmol/kg (5.6%; p = 0.15) and 1100 nmol/kg (4.5%; p = 0.14) doses produced non‐significant reductions in swelling. R18D did not significantly affect post‐stroke weight loss, however rats treated with the lowest dose (325 nmol/kg) exhibited the smallest reduction in body weight compared with vehicle‐treated animals (7.35% vs. 9.35). Pharmacokinetic analysis of the three R18D doses after stroke demonstrated a half‐life of 72 to 78 min, with dose‐dependent plasma Cmax values of 5.3 μg/ mL , 11.9 μg/mL and 17.3 μg/mL for the low‐, medium‐ and high‐dose groups, respectively. Conclusions: These results further support the neuroprotective properties of R18D and provide strong justification for its continued development as a neuroprotective treatment for ischemic stroke.

Background/Objectives: The role of α-synuclein (α-syn) pathology in Parkinson’s disease (PD) is well established; however, effective therapies remain elusive. Two mechanisms central to PD neurodegeneration are the intracellular aggregation of misfolded α-syn and the uptake of α-syn aggregates into neurons. Cationic arginine-rich peptides (CARPs) are an emerging class of molecule with multiple neuroprotective mechanisms of action, including protein stabilisation. This study characterised both intracellular α-syn aggregation and α-syn uptake in cortical neurons in vitro. Thereafter, this study examined the therapeutic potential of the neuroprotective CARP, R18D (18-mer of D-arginine), to prevent the aforementioned PD pathogenic processes through a cell-free thioflavin-T (ThT) assay and in cortical neurons. Methods: To induce intracellular α-syn aggregation, rat primary cortical neurons were exposed to α-syn seed (0.14 μM) for 2 h to allow uptake of the protein, followed by R18D treatment (0.0625, 0.125, 0.25, 0.5 μM), and a subsequent measurement of α-syn aggregates 48 h later using a homogenous time-resolved fluorescence (HTRF) assay. To assess neuronal uptake, α-syn seeds were covalently labelled with an Alexa-Fluor 488 fluorescent tag, pre-incubated with R18D (0.125, 0.25, 0.5 μM), and then exposed to cortical neurons for 24 h and assessed via confocal microscopy. Results: It was demonstrated that R18D significantly reduced both intracellular α-syn aggregation and α-syn seed uptake in neurons by 37.8% and 77.7%, respectively. Also, R18D reduced the aggregation of α-syn monomers in the cell-free assay. Conclusions: These findings highlight the therapeutic potential of R18D to inhibit key α-syn pathological processes and PD progression.

In Parkinson’s disease (PD), gut inflammation is hypothesised to contribute to α-synuclein aggregation, but gastrointestinal α-synuclein expression is poorly characterised. Cationic arginine-rich peptides (CARPs) are an emerging therapeutic option that exerts various neuroprotective effects and may target the transmission of protein aggregates. This study aimed to investigate endogenous α-synuclein expression in enteroendocrine STC-1 cells and the potential of the CARP, R18D (18-mer of D-arginine), to prevent internalisation of pre-formed α-synuclein fibrils (PFFs) in enteroendocrine cells in vitro. Through confocal microscopy, the immunoreactivity of full-length α-synuclein and the serine-129 phosphorylated form (pS129) was investigated in STC-1 (mouse enteroendocrine) cells. Thereafter, STC-1 cells were exposed to PFFs tagged with Alexa-Fluor 488 (PFF-488) for 2 and 24 h and R18D-FITC for 10 min. After confirming the uptake of both PFFs and R18D-FITC through fluorescent microscopy, STC-1 cells were pre-treated with R18D (5 or 10 μM) for 10 min prior to 2 h of PFF-488 exposure. Immunoreactivity for endogenous α-synuclein and pS129 was evident in STC-1 cells, with prominent pS129 staining along cytoplasmic processes and in perinuclear areas. STC-1 cells internalised PFFs, confirmed through co-localisation of PFF-488 and human-specific α-synuclein immunoreactivity. R18D-FITC entered STC-1 cells within 10 min and pre-treatment of STC-1 cells with R18D interfered with PFF uptake. The endogenous presence of α-synuclein in enteroendocrine cells, coupled with their rapid uptake of PFFs, demonstrates a potential for pathogenic spread of α-synuclein aggregates in the gut. R18D is a novel therapeutic approach to reduce the intercellular transmission of α-synuclein pathology.

Topographic mapping of neural circuits is fundamental in shaping the structural and functional organization of brain regions. This developmentally important process is crucial not only for the representation of different sensory inputs but also for their integration. Disruption of topographic organization has been associated with several neurodevelopmental disorders. The aim of this review is to highlight the mechanisms involved in creating and refining such well-defined maps in the brain with a focus on the Eph and ephrin families of axon guidance cues. We first describe the transgenic models where ephrin-A expression has been manipulated to understand the role of these guidance cues in defining topography in various sensory systems. We further describe the behavioral consequences of lacking ephrin-A guidance cues in these animal models. These studies have given us unexpected insight into how neuronal activity is equally important in refining neural circuits in different brain regions. We conclude the review by discussing studies that have used treatments such as repetitive transcranial magnetic stimulation (rTMS) to manipulate activity in the brain to compensate for the lack of guidance cues in ephrin-knockout animal models. We describe how rTMS could have therapeutic relevance in neurodevelopmental disorders with disrupted brain organization.

The poly-arginine peptides R18D and R18 represent novel potential neuroprotective treatments for acute ischaemic stroke. Here we examined whether R18D and R18 had any significant effects on the thrombolytic activity of alteplase (tPA) and tenecteplase (TNK) on clots formed from whole blood in an in vitro thrombolysis plate assay. R18D and R18 were examined at concentrations of 0.25, 0.5, 1, 2, 4, 8 and 16 µM during the 1-h thrombolytic assay. We also included the well-characterised neuroprotective NA-1 peptide as a control. R18D, R18 and NA-1 all reduced tPA or TNK percentage clot lysis by 0–9.35%, 0–3.44% and 0–4.8%, respectively. R18D, R18 and NA-1 had a modest and variable effect on the lag time, increasing the time to the commencement of thrombolysis by 0–9.9 min, 0–5.53 min and 0–7.16 min, respectively. Lastly, R18 and NA-1 appeared to increase the maximal activity of the thrombolysis reaction. In addition, the in vitro anti-excitotoxic neuroprotective efficacy of R18D and R18 was not affected by pre-incubation for 1–2 h or overnight with tPA or TNK, whereas only R18D retained high anti-excitotoxic neuroprotective efficacy when pre-incubated in a synthetic trypsin (TrypLE Express). The present in vitro findings suggest that neither R18D or R18 when co-administered with the thrombolytic inducing agents tPA or TNK are likely to have a significant impact when used clinically during clot thrombolysis and confirm the superior proteolytic stability of the R18D peptide.

Poly-arginine peptides R18 and R18D have previously been demonstrated to be neuroprotective in ischaemic stroke models. Here we examined the proteolytic stability and efficacy of R18 and R18D in reducing infarct core growth and preserving the ischaemic penumbra following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. R18 (300 or 1000 nmol/kg), R18D (300 nmol/kg) or saline were administered intravenously 10 min after MCAO induced using a filament. Serial perfusion and diffusion-weighted MRI imaging was performed to measure changes in the infarct core and penumbra from time points between 45- and 225-min post-occlusion. Repeated measures analyses of infarct growth and penumbral tissue size were evaluated using generalised linear mixed models (GLMMs). R18D (300 nmol/kg) was most effective in slowing infarct core growth (46.8 mm³ reduction; p < 0.001) and preserving penumbral tissue (21.6% increase; p < 0.001), followed by R18 at the 300 nmol/kg dose (core: 29.5 mm³ reduction; p < 0.001, penumbra: 12.5% increase; p < 0.001). R18 at the 1000 nmol/kg dose had a significant impact in slowing core growth (19.5 mm³ reduction; p = 0.026), but only a modest impact on penumbral preservation (6.9% increase; p = 0.062). The in vitro anti-excitotoxic neuroprotective efficacy of R18D was also demonstrated to be unaffected when preincubated for 1–3 h or overnight, in a cell lysate prepared from dying neurons or with the proteolytic enzyme, plasmin, whereas the neuroprotective efficacy of R18 was significantly reduced after a 2-h incubation. These findings highlight the capacity of poly-arginine peptides to reduce infarct growth and preserve the ischaemic penumbra, and confirm the superior efficacy and proteolytic stability of R18D, which indicates that this peptide is likely to retain its neuroprotective properties when co-administered with alteplase during thrombolysis for acute ischaemic stroke.

Emerging cytoprotective peptide therapies for stroke. Expert Review of Neurotherapeutics. Accepted 24 June 2020.

Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.