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A breadth of preclinical studies now support the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, most cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such a format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with cryopreserved single-cell DA progenitors. Here, we used a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabling the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods, including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner using a Parkinsonian rat model. The survival of the cells was confirmed by post-mortem histological analysis, characterised by the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson's disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks after transplantation using cryopreserved 3D format., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nicolas Prudon reports financial support was provided by TreeFrog Therapeutics. Erwan Bezard reports a relationship with TreeFrog Therapeutics that includes: consulting or advisory. Maxime Feyeux has patent issued to University of Bordeaux. Kevin Alessandri has patent issued to University of Bordeaux. Erwan Bezard has patent issued to University of Bordeaux. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Triggering receptor expressed on myeloid cells 2 (Trem2) is a myeloid cell-specific gene expressed in brain microglia, with variants that are associated with neurodegenerative diseases, including Alzheimer's disease. Trem2 is essential for microglia-mediated synaptic refinement, but whether Trem2 contributes to shaping neuronal development remains unclear. Here, we demonstrate that Trem2 plays a key role in controlling the bioenergetic profile of pyramidal neurons during development. In the absence of Trem2, developing neurons in the hippocampal cornus ammonis (CA)1 but not in CA3 subfield displayed compromised energetic metabolism, accompanied by reduced mitochondrial mass and abnormal organelle ultrastructure. This was paralleled by the transcriptional rearrangement of hippocampal pyramidal neurons at birth, with a pervasive alteration of metabolic, oxidative phosphorylation, and mitochondrial gene signatures, accompanied by a delay in the maturation of CA1 neurons. Our results unveil a role of Trem2 in controlling neuronal development by regulating the metabolic fitness of neurons in a region-specific manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intracerebral hemorrhages are recognized risk factors for neurodevelopmental disorders and represent early biomarkers for cognitive dysfunction and mental disability, but the pathways leading to their occurrence are not well defined. We report that a single intrauterine exposure of the immunostimulant Poly I:C to pregnant mice at gestational day 9, which models a prenatal viral infection and the consequent maternal immune activation, induces the defective formation of brain vessels and causes intracerebral hemorrhagic events, specifically in male offspring. We demonstrate that maternal immune activation promotes the production of the TGF-β1 active form and the consequent enhancement of pSMAD1-5 in males' brain endothelial cells. TGF-β1, in combination with IL-1β, reduces the endothelial expression of CD146 and claudin-5, alters the endothelium-pericyte interplay resulting in low pericyte coverage, and increases hemorrhagic events in the adult offspring. By showing that exposure to Poly I:C at the beginning of fetal cerebral angiogenesis results in sex-specific alterations of brain vessels, we provide a mechanistic framework for the association between intragravidic infections and anomalies of the neural vasculature, which may contribute to neuropsychiatric disorders., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Citrus sour rot caused by Geotrichum citri-aurantii is one of the most important postharvest diseases in citrus fruit, causing huge economic losses. Traditionally, it has been controlled by the postharvest application of guazatine and propiconazole fungicides, but restrictions in their use make it urgent to find an alternative for sour rot management. Natamycin, a common food preservative, and the organosulfuric compounds extracted from Allium species are safe food additives that control different foodborne pathogens. In the present study, the curative activities of commercial formulations of natamycin (Fruitgard Nat 20) and an Allium extract (PTSO: propyl thiosulfinate oxide; Proallium FRD®), were evaluated for the control of G. citri-aurantii in artificially inoculated lemon fruit. Trials in laboratory and in commercial conditions were carried out to explore the feasibility of including both compounds as part of a safe postharvest sour rot disease control strategy. Under controlled laboratory conditions, sour rot was significatively reduced by 500 mg L -1 of natamycin, 580 mL L -1 of PTSO and 290 mL L -1 of PTSO + 4% of a food coat, applied by immersion. Nevertheless, the maximum dose of PTSO (580 mL L -1 ) caused phytotoxicity on the fruit rind. In commercial drenching conditions, 290 mL L -1 of PTSO + 4% of a food coat reduced sour rot incidence similar to conventional treatment. In a packing line treatment, spray application of 500 mg L -1 of natamycin with a previous dip in sodium bicarbonate, resulted in nearly 70% reduction of disease incidence compared to conventional salt application. A second commercial experiment revealed that fruit drenching with 290 mL L -1 of PTSO + 4% food coat followed by an in-line cascade application of 500 mg L -1 of natamycin is completely effective for sour rot control after 20 days at 5 °C. Further exposure at room temperature for 7 d showed a 61% reduction in sour rot incidence compared to the control. Results revealed that natamycin and PTSO are promising tools for sour rot control used alone or combined as part of an integrated postharvest strategy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar α-synuclein (α-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson's disease (PD) where the pathological α-syn aggregates are almost exclusively neuronal, the α-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of α-syn, yet pathogenic amyloid α-syn seeds require significant amounts of α-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human α-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated α-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated α-syn monomers/oligomers and not to the appearance of the distinctive fibrillar α-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-αSyn mice we observed a variable association of S129-phosphorylated α-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-α-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with α-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of α-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between α-syn aggregation and the development of a clinical phenotype in these transgenic animals.

The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ - ). The generation of τ - fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Parkinson's disease (PD) affects millions of patients worldwide and is characterized by alpha-synuclein aggregation in dopamine neurons. Molecular tweezers have shown high potential as anti-aggregation agents targeting positively charged residues of proteins undergoing amyloidogenic processes. Here we report that the molecular tweezer CLR01 decreased aggregation and toxicity in induced pluripotent stem cell-derived dopaminergic cultures treated with PD brain protein extracts. In microfluidic devices CLR01 reduced alpha-synuclein aggregation in cell somas when axonal terminals were exposed to alpha-synuclein oligomers. We then tested CLR01 in vivo in a humanized alpha-synuclein overexpressing mouse model; mice treated at 12 months of age when motor defects are mild exhibited an improvement in motor defects and a decreased oligomeric alpha-synuclein burden. Finally, CLR01 reduced alpha-synuclein-associated pathology in mice injected with alpha-synuclein aggregates into the striatum or substantia nigra. Taken together, these results highlight CLR01 as a disease-modifying therapy for PD and support further clinical investigation.

Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine (PS) represents a neuronal "eat-me" signal involved in microglial-mediated pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS-labeled material by microglia occurs during established developmental periods of microglial-mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Parkinson's Disease (PD) is characterized both by the loss of dopaminergic neurons in the substantia nigra and the presence of cytoplasmic inclusions called Lewy Bodies. These Lewy Bodies contain the aggregated α-synuclein (α-syn) protein, which has been shown to be able to propagate from cell to cell and throughout different regions in the brain. Due to its central role in the pathology and the lack of a curative treatment for PD, an increasing number of studies have aimed at targeting this protein for therapeutics. Here, we reviewed and discussed the many different approaches that have been studied to inhibit α-syn accumulation via direct and indirect targeting. These analyses have led to the generation of multiple clinical trials that are either completed or currently active. These clinical trials and the current preclinical studies must still face obstacles ahead, but give hope of finding a therapy for PD with time., Competing Interests: The authors declare no conflict of interest.

Background: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is considered as a gold standard therapy for the alleviation of motor symptoms in Parkinson's disease (PD). This success paved the way to its application for other neurological and psychiatric disorders. In this context, we aimed to develop a rodent-specific stimulator with characteristics similar to those used in patients., New Method: We designed a stimulator that can be connected to an electrode container with options for bilateral or unilateral stimulation selection and offers a wide range of frequencies, pulse widths and intensities, constant current, biphasic current-control and charge balancing. Dedicated software was developed to program these parameters and the device was tested on a bilateral 6-hydroxydopamine (6-OHDA) rat model of PD., Results: The equipment was well tolerated by the animals with a good general welfare. STN stimulation (130 Hz frequency, 0.06 ms pulse width, 150 μA average intensity) improved the motor deficits induced by 6-OHDA as it significantly increased the number of movements compared to the values obtained in the same animals without STN stimulation. Furthermore, it restored motor coordination by significantly increasing the time spent on the rotarod bar., Conclusion: We successfully developed and validated a new portable and programmable stimulator for freely moving rats that delivers a large range of stimulation parameters using bilateral biphasic current-control and charge balancing to maximize tissue safety. This device can be used to test deep brain stimulation in different animal models of human brain diseases., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Anxiety in Parkinson's disease is a comorbid non-motor symptom that alters the quality of life of patients. Its neuronal substrates and those of l-Dopa treatment are still poorly known. Using different combinations of monoaminergic system lesions in the rat, we addressed the contribution of these systems in the efficacy of l-DOPA on anxiety and on the neuronal activity of basolateral amygdala (BLA), a brain structure involved in anxiety. Anxiety, locomotor activity and motor performance were assessed using the elevated plus maze, the open field and the skinner box, respectively. The neuronal activity of BLA was electrophysiologically recorded and the loss of dopamine, noradrenaline and serotonin neurons was quantified by immunohistochemistry and stereology. Selective bilateral lesion of dopamine neurons, with or without the additional lesions of noradrenaline and/or serotonin neurons, induced anxiety disorder. l-Dopa significantly decreased anxiety in animals with bilateral lesion of dopamine neurons alone or combined with that of noradrenaline neurons. In these two groups, l-DOPA enhanced the firing rate of BLA neurons. However, in animals with combined lesions of dopamine and serotonin neurons or in animals with lesions of the three monoaminergic systems, l-Dopa was no longer able to decrease anxiety behavior or to change the electrophysiological parameters of BLA neurons. Our data provide the first evidence of the key and positive role of the serotonergic system in the combined efficacy of l-Dopa on anxiety and the paralleled BLA neuronal activity, suggesting that the enhancement of the activity of serotonin neurons may boost the anxiolytic action of l-DOPA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Multiple Sclerosis (MS) is an inflammatory disease with neurodegenerative alterations, ultimately progressing to neurological handicap. Therapies are effective in counteracting inflammation but not neurodegeneration. Biomarkers predicting disease course or treatment response are lacking. We investigated whether altered gene and protein expression profiles were detectable in the peripheral blood of 78 relapsing remitting MS (RR-MS) patients treated by disease-modifying therapies. A discovery/validation study on RR-MS responsive to glatiramer acetate identified 8 differentially expressed genes: ITGA2B, ITGB3, CD177, IGJ, IL5RA, MMP8, P2RY12, and S100β. A longitudinal study on glatiramer acetate, Interferon-β, or Fingolimod treated RR-MS patients confirmed that 7 out of 8 genes were downregulated with reference to the different therapies, whereas S100β was always upregulated. Thus, we identified a peripheral gene signature associated with positive response in RR-MS which may also explain drug immunomodulatory effects. The usefulness of this signature as a biomarker needs confirmation on larger series of patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Myasthenia gravis (MG) is a T-cell dependent autoimmune disorder of the neuromuscular junction, characterised by muscle weakness and fatigability. Autoimmunity is thought to initiate in the thymus of acetylcholine receptor (AChR)-positive MG patients; however, the molecular mechanisms linking intra-thymic MG pathogenesis with autoreactivity via the circulation to the muscle target organ are poorly understood. Using whole-transcriptome sequencing, we compared the transcriptional profile of peripheral blood mononuclear cells from AChR-early onset MG (AChR-EOMG) patients with healthy controls: 178 coding transcripts and 229 long non-coding RNAs, including 11 pre-miRNAs, were differentially expressed. Among the 178 coding transcripts, 128 were annotated of which 17% were associated with the 'infectious disease' functional category and 46% with 'inflammatory disease' and 'inflammatory response-associated' categories. Validation of selected transcripts by qPCR indicated that of the infectious disease-related transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were upregulated, whereas CLC and IL4 were downregulated in AChR-EOMG patients; in the 'inflammatory' categories, ABCA1, FUS, and RELB were upregulated, suggesting a contribution of these molecules to immunological dysfunctions in MG. Data selection and validation were also based on predicted microRNA-mRNA interactions. We found that miR-612, miR-3654, and miR-3651 were increased, whereas miR-612-putative AKAp12 and HRH4 targets and the miR-3651-putative CRISP3 target were downregulated in AChR-EOMG, also suggesting altered immunoregulation. Our findings reveal a novel peripheral molecular signature in AChR-EOMG, reflecting a critical involvement of inflammatory- and infectious disease-related immune responses in disease pathogenesis., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

This paper describes a Deep Brain Stimulation device, portable, for chronic experiments on rodents in the context of Parkinson's disease. Our goal is to equip the animal with a device that mimics the human therapeutic conditions. It implies to respect a set of properties such as bilateral current-mode and charge-balanced stimulation, as well as programmability, low power consumption and re-usability to finally reach a suitable weight for long-term experiments. After the analysis of the solutions found in the literature, the full design of the device is explained. First, the stimulation front-end circuit driven by a processor unit, then the choice of supply sources which is a critical point for the weight and life-time of our system. Our low cost system has been realized using commercial discrete components and the overall power consumption was minimized. We achieved 6 days of maximal current stimulation with the chosen battery for a weight of 13.8 g . Finally, the device was carried out in vivo on rats during a 3 weeks experiment as the used implantation technique allows battery changing. This experiment also permits to emphasize the mechanical aspects including the packaging and electrodes holding.

Non-motor symptoms of Parkinson's disease are under-studied and therefore not well treated. Here, we investigated the role of combined depletions of dopamine, norepinephrine and/or serotonin in the manifestation of motor and non-motor deficits in the rat. Then, we studied the impact of these depletions on the efficacy of deep brain stimulation of the subthalamic nucleus (STN-DBS). We performed selective depletions of dopamine, norepinephrine and serotonin, and the behavioral effects of different combined depletions were investigated using the open field, the elevated plus maze and the forced swim test. Bilateral dopamine depletion alone induced locomotor deficits associated with anxiety and mild "depressive-like" behaviors. Although additional depletions of norepinephrine and/or serotonin did not potentiate locomotor and anxiety disorders, combined depletions of the three monoamines dramatically exacerbated "depressive-like" behavior. STN-DBS markedly reversed locomotor deficits and anxiety behavior in animals with bilateral dopamine depletion alone. However, these improvements were reduced or lost by the additional depletion of norepinephrine and/or serotonin, indicating that the depletion of these monoamines may interfere with the antiparkinsonian efficacy of STN-DBS. Furthermore, our results showed that acute STN-DBS improved "depressive-like" disorder in animals with bilateral depletion of dopamine and also in animals with combined depletions of the three monoamines, which induced severe immobility in the forced swim test. Our data highlight the key role of monoamine depletions in the pathophysiology of anxiety and depressive-like disorders and provide the first evidence of their negative consequences on the efficacy of STN-DBS upon the motor and anxiety disorders in the context of Parkinson's disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)