C9-Functionalized Doxycycline Analogs as Drug Candidates to Prevent Pathological α-Synuclein Aggregation and Neuroinflammation in Parkinson's Disease Degeneration.

Doxycycline, a semi-synthetic tetracycline, is a widely used antibiotic for treating mild-to-moderate infections, including skin problems. However, its anti-inflammatory and antioxidant properties, combined with its ability to interfere with α-synuclein aggregation, make it an attractive candidate for repositioning in Parkinson's disease. Nevertheless, the antibiotic activity of doxycycline restricts its potential use for long-term treatment of Parkinsonian patients. In the search for non-antibiotic tetracyclines that could operate against Parkinson's disease pathomechanisms, eighteen novel doxycycline derivatives were designed. Specifically, the dimethyl-amino group at C4 was reduced, resulting in limited antimicrobial activity, and several coupling reactions were performed at position C9 of the aromatic D ring, this position being one of the most reactive for introducing substituents. Using the Thioflavin-T assay, we found seven compounds were more effective than doxycycline in inhibiting α-synuclein aggregation. Furthermore, two of these derivatives exhibited better anti-inflammatory effects than doxycycline in a culture system of microglial cells used to model Parkinson's disease neuroinflammatory processes. Overall, through structure-activity relationship studies, we identified two newly designed tetracyclines as promising drug candidates for Parkinson's disease treatment.

4'-fluorocannabidiol associated with capsazepine restrains L-DOPA-induced dyskinesia in hemiparkinsonian mice: Contribution of anti-inflammatory and anti-glutamatergic mechanisms.

We tested the efficacy of 4'-fluorocannabidiol (4'-F-CBD), a semisynthetic cannabidiol derivative, and HU-910, a cannabinoid receptor 2 (CB2) agonist in resolving l-DOPA-induced dyskinesia (LID). Specifically, we were interested in studying whether these compounds could restrain striatal inflammatory responses and rescue glutamatergic disturbances characteristic of the dyskinetic state. C57BL/6 mice were rendered hemiparkinsonian by unilateral striatal lesioning with 6-OHDA. Abnormal involuntary movements were then induced by repeated i.p. injections of l-DOPA + benserazide. After LID was installed, the effects of a 3-day treatment with 4'-F-CBD or HU-910 in combination or not with the TRPV1 antagonist capsazepine (CPZ) or CB2 agonists HU-308 and JWH015 were assessed. Immunostaining was conducted to investigate the impacts of 4'-F-CBD and HU-910 (with CPZ) on inflammation and glutamatergic synapses. Our results showed that the combination of 4'-F-CBD + CPZ, but not when administered alone, decreased LID. Neither HU-910 alone nor HU-910+CPZ were effective. The CB2 agonists HU-308 and JWH015 were also ineffective in decreasing LID. Both combination treatments efficiently reduced microglial and astrocyte activation in the dorsal striatum of dyskinetic mice. However, only 4'-F-CBD + CPZ normalized the density of glutamate vesicular transporter-1 (vGluT1) puncta colocalized with the postsynaptic density marker PSD95. These findings suggest that 4'-F-CBD + CPZ normalizes dysregulated cortico-striatal glutamatergic inputs, which could be involved in their anti-dyskinetic effects. Although it is not possible to rule out the involvement of anti-inflammatory mechanisms, the decrease in striatal neuroinflammation markers by 4'-F-CBD and HU-910 without an associated reduction in LID indicates that they are insufficient per se to prevent LID manifestations.

The two synthetic cannabinoid compounds 4'-F-CBD and HU-910 efficiently restrain inflammatory responses of brain microglia and astrocytes.

To study the anti-inflammatory potential of the two synthetic cannabinoids 4'-F-CBD and HU-910, we used post-natal brain cultures of mouse microglial cells and astrocytes activated by reference inflammogens. We found that 4'-F-CBD and HU-910 efficiently curtailed the release of TNF-α, IL-6, and IL-1ß in microglia and astrocytes activated by the bacterial Toll-Like Receptor (TLR)4 ligand LPS. Upon LPS challenge, 4'-F-CBD and HU-910 also prevented the activation of phenotypic activation markers specific to microglia and astrocytes, that is, Iba-1 and GFAP, respectively. In microglial cells, the two test compounds also efficiently restrained LPS-stimulated release of glutamate, a non-cytokine inflammation marker for these cells. The immunosuppressive effects of the two cannabinoid compounds were concentration-dependent and observable between 1 and 10 µM. These effects were not dependent on cannabinoid or cannabinoid-like receptors. Both 4'-F-CBD and HU-910 were also capable of restraining the inflammogenic activity of Pam3CSK4, a lipopeptide that activates TLR2, and of BzATP, a prototypic agonist of P2X7 purinergic receptors, suggesting that these two cannabinoids could exert immunosuppressive effects against a variety of inflammatory stimuli. Using LPS-stimulated microglia and astrocytes, we established that the immunosuppressive action of 4'-F-CBD and HU-910 resulted from the inhibition of ROS produced by NADPH oxidase and subsequent repression of NF-κB-dependent signaling events. Our results suggest that 4'-F-CBD and HU-910 may have therapeutic utility in pathological conditions where neuroinflammatory processes are prominent.

Rescue of Dopamine Neurons from Iron-Dependent Ferroptosis by Doxycycline and Demeclocycline and Their Non-Antibiotic Derivatives.

Several studies have reported that the tetracycline (TC) class antibiotic doxycycline (DOX) is effective against Parkinson's disease (PD) pathomechanisms. The aim of the present work was three-fold: (i) Establish a model system to better characterize neuroprotection by DOX; (ii) Compare the rescue effect of DOX to that of other TC antibiotics; (iii) Discover novel neuroprotective TCs having reduced antibiotic activity. For that, we used cultures of mouse midbrain dopamine (DA) neurons and experimental conditions that model iron-mediated oxidative damage, a key mechanism in PD pathobiology. We found that DOX and the other TC antibiotic, demeclocycline (DMC), provided sustained protection to DA neurons enduring iron-mediated insults, whereas chlortetracycline and non-TC class antibiotics did not. Most interestingly, non-antibiotic derivatives of DOX and DMC, i.e., DDOX and DDMC, respectively, were also robustly protective for DA neurons. Interestingly, DOX, DDOX, DMC, and DDMC remained protective for DA neurons until advanced stages of neurodegeneration, and the rescue effects of TCs were observable regardless of the degree of maturity of midbrain cultures. Live imaging studies with the fluorogenic probes DHR-123 and TMRM revealed that protective TCs operated by preventing intracellular oxidative stress and mitochondrial membrane depolarization, i.e., cellular perturbations occurring in this model system as the ultimate consequence of ferroptosis-mediated lipid peroxidation. If oxidative/mitochondrial insults were generated acutely, DOX, DDOX, DMC, and DDMC were no longer neuroprotective, suggesting that these compounds are mostly effective when neuronal damage is chronic and of low-intensity. Overall, our data suggest that TC derivatives, particularly those lacking antibiotic activity, might be of potential therapeutic utility to combat low-level oxidative insults that develop chronically in the course of PD neurodegeneration.

Doxycycline attenuates l-DOPA-induced dyskinesia through an anti-inflammatory effect in a hemiparkinsonian mouse model.

The pharmacological manipulation of neuroinflammation appears to be a promising strategy to alleviate l-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD). Doxycycline (Doxy), a semisynthetic brain-penetrant tetracycline antibiotic having interesting anti-inflammatory properties, we addressed the possibility that this compound could resolve LID in l-DOPA-treated C57BL/6 mice presenting either moderate or intermediate lesions of the mesostriatal dopaminergic pathway generated by intrastriatal injections of 6-OHDA. Doxy, when given subcutaneously before l-DOPA at doses of 20 mg kg-1 and 40 mg kg-1, led to significant LID reduction in mice with moderate and intermediate dopaminergic lesions, respectively. Importantly, Doxy did not reduce locomotor activity improved by l-DOPA. To address the molecular mechanism of Doxy, we sacrificed mice with mild lesions 1) to perform the immunodetection of tyrosine hydroxylase (TH) and Fos-B and 2) to evaluate a panel of inflammation markers in the striatum, such as cyclooxygenase-2 and its downstream product Prostaglandin E2 along with the cytokines TNF-α, IL-1ß and IL-6. TH-immunodetection revealed that vehicle and Doxy-treated mice had similar striatal lesions, excluding that LID improvement by Doxy could result from neurorestorative effects. Importantly, LID inhibition by Doxy was associated with decreased Fos-B and COX-2 expression and reduced levels of PGE2, TNF-α, and IL-1ß in the dorsolateral striatum of dyskinetic mice. We conclude 1) that Doxy has the potential to prevent LID regardless of the intensity of dopaminergic lesioning and 2) that the anti-inflammatory effects of Doxy probably account for LID attenuation. Overall, the present results further indicate that Doxy might represent an attractive and alternative treatment for LID in PD.

Annonaceae Consumption Worsens Disease Severity and Cognitive Deficits in Degenerative Parkinsonism.

BACKGROUND: High consumption of Annona muricata fruit has been previously identified as a risk factor for atypical parkinsonism in the French Caribbean islands. OBJECTIVE: We tested whether consumption of Annonaceae products could worsen the clinical phenotype of patients with any form of degenerative parkinsonism. METHODS: We analyzed neurological data from 180 Caribbean parkinsonian patients and specifically looked for dose effects of lifelong, cumulative Annonaceae consumption on cognitive performance. Using unsupervised clustering, we identified one cluster with mild/moderate symptoms (N = 102) and one with severe symptoms including cognitive impairment (N = 78). RESULTS: We showed that even low cumulative consumption of fruits/juices (>0.2 fruit-years) or any consumption of herbal tea from Annonaceae worsen disease severity and cognitive deficits in degenerative parkinsonism including Parkinson's disease (OR fruits-juices: 3.76 [95% CI: 1.13-15.18]; OR herbal tea: 2.91 [95% CI: 1.34-6.56]). CONCLUSION: We suggest that more restrictive public health preventive recommendations should be made regarding the consumption of Annonaceae products. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Chemically-Modified Tetracycline COL-3 and Its Parent Compound Doxycycline Prevent Microglial Inflammatory Responses by Reducing Glucose-Mediated Oxidative Stress.

We used mouse microglial cells in culture activated by lipopolysaccharide (LPS) or α-synuclein amyloid aggregates (αSa) to study the anti-inflammatory effects of COL-3, a tetracycline derivative without antimicrobial activity. Under LPS or αSa stimulation, COL-3 (10, 20 µM) efficiently repressed the induction of the microglial activation marker protein Iba-1 and the stimulated-release of the pro-inflammatory cytokine TNF-α. COL-3's inhibitory effects on TNF-α were reproduced by the tetracycline antibiotic doxycycline (DOX; 50 µM), the glucocorticoid dexamethasone, and apocynin (APO), an inhibitor of the superoxide-producing enzyme NADPH oxidase. This last observation suggested that COL-3 and DOX might also operate themselves by restraining oxidative stress-mediated signaling events. Quantitative measurement of intracellular reactive oxygen species (ROS) levels revealed that COL-3 and DOX were indeed as effective as APO in reducing oxidative stress and TNF-α release in activated microglia. ROS inhibition with COL-3 or DOX occurred together with a reduction of microglial glucose accumulation and NADPH synthesis. This suggested that COL-3 and DOX might reduce microglial oxidative burst activity by limiting the glucose-dependent synthesis of NADPH, the requisite substrate for NADPH oxidase. Coherent with this possibility, the glycolysis inhibitor 2-deoxy-D-glucose reproduced the immunosuppressive action of COL-3 and DOX in activated microglia. Overall, we propose that COL-3 and its parent compound DOX exert anti-inflammatory effects in microglial cells by inhibiting glucose-dependent ROS production. These effects might be strengthened by the intrinsic antioxidant properties of DOX and COL-3 in a self-reinforcing manner.

CMT-3 targets different α-synuclein aggregates mitigating their toxic and inflammogenic effects.

Parkinson's disease (PD) is a neurodegenerative disorder for which only symptomatic treatments are available. Repurposing drugs that target α-synuclein aggregation, considered one of the main drivers of PD progression, could accelerate the development of disease-modifying therapies. In this work, we focused on chemically modified tetracycline 3 (CMT-3), a derivative with reduced antibiotic activity that crosses the blood-brain barrier and is pharmacologically safe. We found that CMT-3 inhibited α-synuclein amyloid aggregation and led to the formation of non-toxic molecular species, unlike minocycline. Furthermore, CMT-3 disassembled preformed α-synuclein amyloid fibrils into smaller fragments that were unable to seed in subsequent aggregation reactions. Most interestingly, disaggregated species were non-toxic and less inflammogenic on brain microglial cells. Finally, we modelled the interactions between CMT-3 and α-synuclein aggregates by molecular simulations. In this way, we propose a mechanism for fibril disassembly. Our results place CMT-3 as a potential disease modifier for PD and possibly other synucleinopathies.

Contributive Role of TNF-α to L-DOPA-Induced Dyskinesia in a Unilateral 6-OHDA Lesion Model of Parkinson's Disease.

Our present objective was to better characterize the mechanisms that regulate striatal neuroinflammation in mice developing L-DOPA-induced dyskinesia (LID). For that, we used 6-hydroxydopamine (6-OHDA)-lesioned mice rendered dyskinetic by repeated intraperitoneal injections of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) and quantified ensuing neuroinflammatory changes in the dopamine-denervated dorsal striatum. LID development was associated with a prominent astrocytic response, and a more moderate microglial cell reaction restricted to this striatal area. The glial response was associated with elevations in two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß. Treatment with the phytocannabinoid cannabidiol and the transient receptor potential vanilloid-1 (TRPV-1) channel antagonist capsazepine diminished LID intensity and decreased TNF-α levels without impacting other inflammation markers. To possibly reproduce the neuroinflammatory component of LID, we exposed astrocyte and microglial cells in culture to candidate molecules that might operate as inflammatory cues during LID development, i.e., L-DOPA, dopamine, or glutamate. Neither L-DOPA nor dopamine produced an inflammatory response in glial cell cultures. However, glutamate enhanced TNF-α secretion and GFAP expression in astrocyte cultures and promoted Iba-1 expression in microglial cultures. Of interest, the antidyskinetic treatment with cannabidiol + capsazepine reduced TNF-α release in glutamate-activated astrocytes. TNF-α, on its own, promoted the synaptic release of glutamate in cortical neuronal cultures, whereas cannabidiol + capsazepine prevented this effect. Therefore, we may assume that the release of TNF-α by glutamate-activated astrocytes may contribute to LID by exacerbating corticostriatal glutamatergic inputs excitability and maintaining astrocytes in an activated state through a self-reinforcing mechanism.

Neuroprotection of dopamine neurons by xenon against low-level excitotoxic insults is not reproduced by other noble gases.

Using midbrain cultures, we previously demonstrated that the noble gas xenon is robustly protective for dopamine (DA) neurons exposed to L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate uptake used to generate sustained, low-level excitotoxic insults. DA cell rescue was observed in conditions where the control atmosphere for cell culture was substituted with a gas mix, comprising the same amount of oxygen (20%) and carbon dioxide (5%) but 75% of xenon instead of nitrogen. In the present study, we first aimed to determine whether DA cell rescue against PDC remains detectable when concentrations of xenon are progressively reduced in the cell culture atmosphere. Besides, we also sought to compare the effect of xenon to that of other noble gases, including helium, neon and krypton. Our results show that the protective effect of xenon for DA neurons was concentration-dependent with an IC50 estimated at about 44%. We also established that none of the other noble gases tested in this study protected DA neurons from PDC-mediated insults. Xenon's effectiveness was most probably due to its unique capacity to block NMDA glutamate receptors. Besides, mathematical modeling of gas diffusion in the culture medium revealed that the concentration reached by xenon at the cell layer level is the highest of all noble gases when neurodegeneration is underway. Altogether, our data suggest that xenon may be of potential therapeutic value in Parkinson disease, a chronic neurodegenerative condition where DA neurons appear vulnerable to slow excitotoxicity.

Human diaphragm atrophy in amyotrophic lateral sclerosis is not predicted by routine respiratory measures.

Amyotrophic lateral sclerosis (ALS) patients show progressive respiratory muscle weakness leading to death from respiratory failure. However, there are no data on diaphragm histological changes in ALS patients and how they correlate with routine respiratory measurements.We collected 39 diaphragm biopsies concomitantly with laparoscopic insertion of intradiaphragmatic electrodes during a randomised controlled trial evaluating early diaphragm pacing in ALS (https://clinicaltrials.gov; NCT01583088). Myofibre type, size and distribution were evaluated by immunofluorescence microscopy and correlated with spirometry, respiratory muscle strength and phrenic nerve conduction parameters. The relationship between these variables and diaphragm atrophy was assessed using multivariate regression models.All patients exhibited significant slow- and fast-twitch diaphragmatic atrophy. Vital capacity (VC), maximal inspiratory pressure, sniff nasal inspiratory pressure (SNIP) and twitch transdiaphragmatic pressure did not correlate with the severity of diaphragm atrophy. Inspiratory capacity (IC) correlated modestly with slow-twitch myofibre atrophy. Supine fall in VC correlated weakly with fast-twitch myofibre atrophy. Multivariate analysis showed that IC, SNIP and functional residual capacity were independent predictors of slow-twitch diaphragmatic atrophy, but not fast-twitch atrophy.Routine respiratory tests are poor predictors of diaphragm structural changes. Improved detection of diaphragm atrophy is essential for clinical practice and for management of trials specifically targeting diaphragm muscle function.

Succinobucol, a Non-Statin Hypocholesterolemic Drug, Prevents Premotor Symptoms and Nigrostriatal Neurodegeneration in an Experimental Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by non-motor and motor disabilities. This study investigated whether succinobucol (SUC) could mitigate nigrostriatal injury caused by intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in mice. Moreover, the effects of SUC against MPTP-induced behavioral impairments and neurochemical changes were also evaluated. The quantification of tyrosine hydroxylase-positive (TH+) cells was also performed in primary mesencephalic cultures to evaluate the effects of SUC against 1-methyl-4-phenylpyridinium (MPP+) toxicity in vitro. C57BL/6 mice were treated with SUC (10 mg/kg/day, intragastric (i.g.)) for 30 days, and thereafter, animals received MPTP infusion (1 mg/nostril) and SUC treatment continued for additional 15 days. MPTP-infused animals displayed significant non-motor symptoms including olfactory and short-term memory deficits evaluated in the olfactory discrimination, social recognition, and water maze tasks. These behavioral impairments were accompanied by inhibition of mitochondrial NADH dehydrogenase activity (complex I), as well as significant decrease of TH and dopamine transporter (DAT) immunoreactivity in the substantia nigra pars compacta and striatum. Although SUC treatment did not rescue NADH dehydrogenase activity inhibition, it was able to blunt MPTP-induced behavioral impairments and prevented the decrease in TH and DAT immunoreactivities in substantia nigra (SN) and striatum. SUC also suppressed striatal astroglial activation and increased interleukin-6 levels in MPTP-intoxicated mice. Furthermore, SUC significantly prevented the loss of TH+ neurons induced by MPP+ in primary mesencephalic cultures. These results provide new evidence that SUC treatment counteracts early non-motor symptoms and neurodegeneration/neuroinflammation in the nigrostriatal pathway induced by intranasal MPTP administration in mice by modulating events downstream to the mitochondrial NADH dehydrogenase inhibition.

Acceleration of conduction velocity linked to clustering of nodal components precedes myelination.

High-density accumulation of voltage-gated sodium (Nav) channels at nodes of Ranvier ensures rapid saltatory conduction along myelinated axons. To gain insight into mechanisms of node assembly in the CNS, we focused on early steps of nodal protein clustering. We show in hippocampal cultures that prenodes (i.e., clusters of Nav channels colocalizing with the scaffold protein ankyrinG and nodal cell adhesion molecules) are detected before myelin deposition along axons. These clusters can be induced on purified neurons by addition of oligodendroglial-secreted factor(s), whereas ankyrinG silencing prevents their formation. The Nav isoforms Nav1.1, Nav1.2, and Nav1.6 are detected at prenodes, with Nav1.6 progressively replacing Nav1.2 over time in hippocampal neurons cultured with oligodendrocytes and astrocytes. However, the oligodendrocyte-secreted factor(s) can induce the clustering of Nav1.1 and Nav1.2 but not of Nav1.6 on purified neurons. We observed that prenodes are restricted to GABAergic neurons, whereas clustering of nodal proteins only occurs concomitantly with myelin ensheathment on pyramidal neurons, implying separate mechanisms of assembly among different neuronal subpopulations. To address the functional significance of these early clusters, we used single-axon electrophysiological recordings in vitro and showed that prenode formation is sufficient to accelerate the speed of axonal conduction before myelination. Finally, we provide evidence that prenodal clusters are also detected in vivo before myelination, further strengthening their physiological relevance.

Neuroprotective effects of a brain permeant 6-aminoquinoxaline derivative in cell culture conditions that model the loss of dopaminergic neurons in Parkinson disease.

Parkinson disease is a neurodegenerative disorder of aging, characterized by disabling motor symptoms resulting from the loss of midbrain dopaminergic neurons and the decrease of dopamine in the striatum. Current therapies are directed at treating the symptoms but there is presently no cure for the disease. In order to discover neuroprotective compounds with a therapeutical potential, our research team has established original and highly regioselective methods for the synthesis of 2,3-disubstituted 6-aminoquinoxalines. To evaluate the neuroprotective activity of these molecules, we used midbrain cultures and various experimental conditions that promote dopaminergic cell loss. Among a series of 11 molecules, only compound MPAQ (2-methyl-3-phenyl-6-aminoquinoxaline) afforded substantial protection in a paradigm where dopaminergic neurons die spontaneously and progressively as they mature. Prediction of blood-brain barrier permeation by Quantitative Structure-Activity Relationship studies (QSARs) suggested that MPAQ was able to reach the brain parenchyma with sufficient efficacy. HPLC-MS/MS quantification in brain homogenates and MALDI-TOF mass spectrometry imaging on brain tissue sections performed in MPAQ-treated mice allowed us to confirm this prediction and to demonstrate, by MALDI-TOF mass spectrometry imaging, that MPAQ was localized in areas containing vulnerable neurons and/or their terminals. Of interest, MPAQ also rescued dopaminergic neurons, which (i) acquired dependency on the trophic peptide GDNF for their survival or (ii) underwent oxidative stress-mediated insults mediated by catalytically active iron. In summary, MPAQ possesses an interesting pharmacological profile as it penetrates the brain parenchyma and counteracts mechanisms possibly contributive to dopaminergic cell death in Parkinson disease.

Flavaglines as potent anticancer and cytoprotective agents.

Flavaglines represent a family of plant natural products that display potent anticancer, cardioprotective, and neuroprotective activities. Novel flavagline derivatives were synthesized and examined for their cytotoxicity on a panel of human cancer cell lines, their cardioprotection against doxorubicin-induced apoptosis in cardiomyocytes, and their neuroprotection in culture models of Parkinson's disease and cisplatin-induced neurotoxicity. The structural requirements of flavaglines for cardio- and neuroprotection were for the first time unraveled and appeared to be slightly different from those for cytotoxicity on cancer cells. We provide also the first evidence that flavaglines may alleviate cisplatin-induced neurotoxicity, suggesting a prophylactic potential of these compounds to prevent this frequently encountered adverse effect of cancer chemotherapies.