Whole and fractionated human platelet lysate biomaterials-based biotherapy induces strong neuroprotection in experimental models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons leading to death within 3 years and without a curative treatment. Neurotrophic growth factors (NTFs) are pivotal for cell survival. A reason for the lack of patient efficacy with single recombinant NTF brain infusion is likely to be due to the synergistic neuroprotective action of multiple NTFs on a diverse set of signaling pathways. Fractionated (protein size <50, <30, <10, <3 kDa) heat-treated human platelet lysate (HHPL) preparations were adapted for use in brain tissue with the aim of demonstrating therapeutic value in ALS models and further elucidation of the mechanisms of action. In neuronal culture all fractions induced Akt-dependent neuroprotection as well as a strong anti-apoptotic and anti-ferroptotic action. In the <3 kDa fraction anti-ferroptotic properties were shown to be GPX4 dependent highlighting a role for other platelet elements associated with NTFs. In the SOD1G86R mouse model, lifespan was strongly increased by intracerebroventricular delivery of HHPL or by intranasal administration of <3 kDa fraction. Our results suggest that the platelet lysate biomaterials are neuroprotective in ALS. Further studies would now validate theragnostic biomarker on its antiferroptotic action, for further clinical development.

Conservative iron chelation for neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis.

Focal iron accumulation associated with brain iron dyshomeostasis is a pathological hallmark of various neurodegenerative diseases (NDD). The application of iron-sensitive sequences in magnetic resonance imaging has provided a useful tool to identify the underlying NDD pathology. In the three major NDD, degeneration occurs in central nervous system (CNS) regions associated with memory (Alzheimer's disease, AD), automaticity (Parkinson's disease, PD) and motor function (amyotrophic lateral sclerosis, ALS), all of which require a high oxygen demand for harnessing neuronal energy. In PD, a progressive degeneration of the substantia nigra pars compacta (SNc) is associated with the appearance of siderotic foci, largely caused by increased labile iron levels resulting from an imbalance between cell iron import, storage and export. At a molecular level, α-synuclein regulates dopamine and iron transport with PD-associated mutations in this protein causing functional disruption to these processes. Equally, in ALS, an early iron accumulation is present in neurons of the cortico-spinal motor pathway before neuropathology and secondary iron accumulation in microglia. High serum ferritin is an indicator of poor prognosis in ALS and the application of iron-sensitive sequences in magnetic resonance imaging has become a useful tool in identifying pathology. The molecular pathways that cascade down from such dyshomeostasis still remain to be fully elucidated but strong inroads have been made in recent years. Far from being a simple cause or consequence, it has recently been discovered that these alterations can trigger susceptibility to an iron-dependent cell-death pathway with unique lipoperoxidation signatures called ferroptosis. In turn, this has now provided insight into some key modulators of this cell-death pathway that could be therapeutic targets for the NDD. Interestingly, iron accumulation and ferroptosis are highly sensitive to iron chelation. However, whilst chelators that strongly scavenge intracellular iron protect against oxidative neuronal damage in mammalian models and are proven to be effective in treating systemic siderosis, these compounds are not clinically suitable due to the high risk of developing iatrogenic iron depletion and ensuing anaemia. Instead, a moderate iron chelation modality that conserves systemic iron offers a novel therapeutic strategy for neuroprotection. As demonstrated with the prototype chelator deferiprone, iron can be scavenged from labile iron complexes in the brain and transferred (conservatively) either to higher affinity acceptors in cells or extracellular transferrin. Promising preclinical and clinical proof of concept trials has led to several current large randomized clinical trials that aim to demonstrate the efficacy and safety of conservative iron chelation for NDD, notably in a long-term treatment regimen.

Past and Future of Neurotrophic Growth Factors Therapies in ALS: From Single Neurotrophic Growth Factor to Stem Cells and Human Platelet Lysates.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that typically results in death within 3-5 years after diagnosis. To date, there is no curative treatment and therefore an urgent unmet need of neuroprotective and/or neurorestorative treatments. Due to their spectrum of capacities in the central nervous system-e.g., development, plasticity, maintenance, neurogenesis-neurotrophic growth factors (NTF) have been exploited for therapeutic strategies in ALS for decades. In this review we present the initial strategy of using single NTF by different routes of administration to the use of stem cells transplantation to express a multiple NTFs-rich secretome to finally focus on a new biotherapy based on the human platelet lysates, the natural healing system containing a mix of pleitropic NTF and having immunomodulatory function. This review highlights that this latter treatment may be crucial to power the neuroprotection and/or neurorestoration therapy requested in this devastating disease.

Could Conservative Iron Chelation Lead to Neuroprotection in Amyotrophic Lateral Sclerosis?

Iron accumulation has been observed in mouse models and in both sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Iron chelation could reduce iron accumulation and the related excess of oxidative stress in the motor pathways. However, classical iron chelation would induce systemic iron depletion. We assess the safety and efficacy of conservative iron chelation (i.e., chelation with low risk of iron depletion) in a murine preclinical model and pilot clinical trial. In Sod1G86R mice, deferiprone increased the mean life span compared with placebo. The safety was good, without anemia after 12 months of deferiprone in the 23 ALS patients enrolled in the clinical trial. The decreases in the ALS Functional Rating Scale and the body mass index were significantly smaller for the first 3 months of deferiprone treatment (30 mg/kg/day) than for the first treatment-free period. Iron levels in the cervical spinal cord, medulla oblongata, and motor cortex (according to magnetic resonance imaging), as well as cerebrospinal fluid levels of oxidative stress and neurofilament light chains were lower after deferiprone treatment. Our observation leads to the hypothesis that moderate iron chelation regimen that avoids changes in systemic iron levels may constitute a novel therapeutic modality of neuroprotection for ALS. Antioxid. Redox Signal. 29, 742-748.

Tailor-made purified human platelet lysate concentrated in neurotrophins for treatment of Parkinson's disease.

Human platelet lysates (PLs), which contain multiple neurotrophins, have been proposed for treating neurodegenerative disorders, including Parkinson's disease (PD). However, current PLs suspended in plasma have high protein content and contain fibrinogen/fibrin and, following activation, also proteolytic and thrombogenic enzymes. Upon brain administration, such PLs may saturate the cerebrospinal fluid and exert neurotoxicity. We assessed whether purified PLs, concentrated in neurotrophins, protected dopaminergic neurons in PD models. Platelet concentrates were collected by apheresis and centrifuged to eliminate plasma and recover the platelets. Platelets were lysed by freeze-thaw cycles, and the 10-fold concentrated platelet pellet lysates (PPLs) were heat-treated (at 56 °C for 30 min). The heat-treated PPLs were low in total proteins, depleted in both plasma and platelet fibrinogen, and devoid of thrombogenic and proteolytic activities. They exerted very high neuroprotective activity when non-oncogenic, Lund human mesencephalic (LUHMES) cells that had differentiated into dopaminergic neurons were exposed to the MPP+ neurotoxin. Heat treatment improved the neuroprotection and inactivated the neurotoxic blood-borne hepatitis C virus. PPL did not induce inflammation in BV2 microglial cells and inhibited COX-2 expression upon lipopolysaccharide exposure. Intranasal administration in mice revealed (a) diffusion of neurotrophins in the striatum and cortex, and (b) MPTP intoxication neuroprotection in the substantia nigra and striatum and the absence of neuroinflammation. These dedicated heat-treated PPLs can be a safe and valuable candidate for a therapeutic strategy for PD.

The protective effect of human platelet lysate in models of neurodegenerative disease: involvement of the Akt and MEK pathways.

Neurodegenerative diseases have huge economic and societal impacts, and place an immense emotional burden on patients and caregivers. Given that platelets have an essential physiological role in wound healing and tissue repair, human platelet lysates (HPLs) are being developed as a novel, effective biotherapy for neurodegenerative diseases. HPLs constitute abundant, readily accessible sources of physiological mixtures of many growth factors (GFs), with demonstrable effects on neuron survival and thus the development, maintenance, function and plasticity of the vertebrate nervous system. Here, we found that HPLs had marked neuroprotective abilities in cell-based models of Parkinson's disease and amyotrophic lateral sclerosis (the LUHMES and NSC-34 cell lines, respectively). The HPLs protected against specific cell death pathways (apoptosis and ferroptosis) and specific oxidative stress inducers [1-methyl-4-phenylpyridinium (MPP+) and menadione], and always afforded more protection than commonly used recombinant GFs (rGFs). The mechanism of protection of HPLs involved specific signalling pathways: whereas the Akt pathway was activated by HPLs under all conditions, the MEK pathway appeared to be more specifically involved in protection against MPP+ toxicity in LUHMES and, in a lesser extent, in staurosporine toxicity in NSC-34. Our present results suggest that HPLs-based therapies could be used to prevent neuronal loss in neurodegenerative diseases while overcoming the limitations currently associated with use of rGFs. Copyright © 2016 John Wiley & Sons, Ltd.

Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC.

Parkinson's disease (PD) is a complex illness characterized by progressive dopaminergic neuronal loss. Several mechanisms associated with the iron-induced death of dopaminergic cells have been described. Ferroptosis is an iron-dependent, regulated cell death process that was recently described in cancer. Our present work show that ferroptosis is an important cell death pathway for dopaminergic neurons. Ferroptosis was characterized in Lund human mesencephalic cells and then confirmed ex vivo (in organotypic slice cultures) and in vivo (in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model). Some of the observed characteristics of ferroptosis differed from those reported previously. For example, ferroptosis may be initiated by PKCα activation, which then activates MEK in a RAS-independent manner. The present study is the first to emphasize the importance of ferroptosis dysregulation in PD. In neurodegenerative diseases like PD, iron chelators, Fer-1 derivatives and PKC inhibitors may be strong drug candidates to pharmacologically modulate the ferroptotic signaling cascade.

Disruption of TCA Cycle and Glutamate Metabolism Identified by Metabolomics in an In Vitro Model of Amyotrophic Lateral Sclerosis.

This study aims to develop a cellular metabolomics model that reproduces the pathophysiological conditions found in amyotrophic lateral sclerosis in order to improve knowledge of disease physiology. We used a co-culture model combining the motor neuron-like cell line NSC-34 and the astrocyte clone C8-D1A, with each over-expressing wild-type or G93C mutant human SOD1, to examine amyotrophic lateral sclerosis (ALS) physiology. We focused on the effects of mutant human SOD1 as well as oxidative stress induced by menadione on intracellular metabolism using a metabolomics approach through gas chromatography coupled with mass spectrometry (GC-MS) analysis. Preliminary non-supervised analysis by Principal Component Analysis (PCA) revealed that cell type, genetic environment, and time of culture influenced the metabolomics profiles. Supervised analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) on data from intracellular metabolomics profiles of SOD1G93C co-cultures produced metabolites involved in glutamate metabolism and the tricarboxylic acid cycle (TCA) cycle. This study revealed the feasibility of using a metabolomics approach in a cellular model of ALS. We identified potential disruption of the TCA cycle and glutamate metabolism under oxidative stress, which is consistent with prior research in the disease. Analysis of metabolic alterations in an in vitro model is a novel approach to investigation of disease physiology.

Importance of local hypoxia on endothelial phenotype for an in vitro approach to bone marrow angiogenesis.

The vasculature of bone marrow differs from that in other organs, and its characteristics should be considered when exploring the medullar angiogenesis associated with hematological malignancies. We show here that the human bone marrow sinusoidal cell line HBME-1 has a specific expression pattern of angiogenic factors and receptors, characterized by a unique VEGFR3(+), Tie2(-) signature, that resembles the in vivo pattern. Moreover, the HBME-1 cultured for up to 3 days in hypoxic conditions, similar to those found in the bone marrow, specifically downregulated expression of VEGFR1, VEGFR2 and ETAR. Thus, a model using bone marrow sinusoidal cells cultured under reduced oxygen tension may be more relevant than classical in vitro endothelial cultures for understanding the interactions between endothelial and malignant cells in the medullar microenvironment.

Rac-1 GTPase controls the capacity of human leukaemic lymphoblasts to migrate on fibronectin in response to SDF-1α (CXCL12).

Acute lymphoblastic leukaemia (ALL) is characterized by malignant cell infiltration of bone marrow, requiring chemotactic response to SDF-1α. Using time-lapse video, we measured the velocity of ALL cells on fibronectin, and found that SDF-1α increased their migration activity for 2 h, but had no effect after 4h, following internalization of its receptor CXCR4. Transfection of ALL cells with dominant-negative Rac1 mutant significantly prolonged their chemotactic response to SDF-1α, and this effect was associated with an alteration of CXCR4 internalization. These data suggest a regulatory role for Rac1 in the chemotactic response of ALL cells to SDF-1α via receptor processing.