minocycline and Amyotrophic-Lateral-Sclerosis

The recent failure of the clinical trial of ninocycline outlines if the mechanism of development of the death neuronal in the sporadic amyotrophic lateral sclerosis (SALS) is different to that happens in models of transgenic mice with human mutations related with SOD (TgALS).. Differences on profile and intensity exist among the oxidative stress mechanisms between TgALS and SALS. Whereas the origin of apoptosis pathway in TgALS comes from the mithocondria and drives to caspase 9 with previous Bid and citocrome C discharge, in SALS, if apoptosis exists, that could proceed through activation of FAS pathway by means of cathepsin B, or alpha-TNF, for microglía activation or from cell cytosol. In FAS pathway, TNF-alpha acts receptor ligands what drives to caspase 8 activation, although cathepsine B could act directly. Considering that the minocyicline decreases Citocrome discharge, reducing executors caspases expression proceeding from intrinsic pathway, is justified its effectiveness in TgALS, but could not be explained if apoptosis in SALS was developed primarily on FAS pathway.. Better knowledge of how cellular death occurs in SALS, could allow to suggest therapeutic options, and could permit to discriminate drugs that, showing effectiveness in TgALS, could not be beneficials in SALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Cell Death; Disease Models, Animal; Humans; Mice; Mice, Transgenic; Minocycline; Neurons; Oxidative Stress

Although genetic mutations that are responsible for most of the inherited neuromuscular diseases have been identified, the molecular and cellular mechanisms that cause muscle and nerve depletion are not well understood and therapies are lacking. Histological studies of many neuromuscular diseases indicated that loss of motor-nerve and/or skeletal-muscle function might be due to excessive cell death by apoptosis. Recent studies have confirmed this possibility by showing that pathology in mouse models of amyotrophic lateral sclerosis, congenital muscular dystrophy, oculopharyngeal muscular dystrophy and collagen-VI deficiency, but not Duchenne muscular dystrophy, is significantly ameliorated by genetic or pharmacological interventions that have been designed to inhibit apoptosis. Thus, apoptosis greatly contributes to pathology in mouse models of several neuromuscular diseases, and appropriate anti-apoptosis therapy might therefore be beneficial for the corresponding human diseases.

Topics: Agrin; Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Apoptosis; Apoptosis Regulatory Proteins; Clinical Trials as Topic; Disease Models, Animal; Doxycycline; Gene Expression Regulation; Genetic Therapy; Humans; Laminin; Mice; Minocycline; Muscular Dystrophies; Muscular Dystrophy, Oculopharyngeal; Mutation; Poly(A)-Binding Protein II; Superoxide Dismutase; Superoxide Dismutase-1

Topics: Acute Disease; Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Apoptosis; Caspases; Cytochrome c Group; Humans; Huntington Disease; Mice; Minocycline; Neurodegenerative Diseases

Combining agents with different mechanisms of action may be necessary for meaningful results in treating ALS. The combinations of minocycline-creatine and celecoxib-creatine have additive effects in the murine model. New trial designs are needed to efficiently screen the growing number of potential neuroprotective agents. Our objective was to assess two drug combinations in ALS using a novel phase II trial design. We conducted a randomized, double-blind selection trial in sequential pools of 60 patients. Participants received minocycline (100 mg)-creatine (10 g) twice daily or celecoxib (400 mg)-creatine (10 g) twice daily for six months. The primary objective was treatment selection based on which combination best slowed deterioration in the ALS Functional Rating Scale-Revised (ALSFRS-R); the trial could be stopped after one pool if the difference between the two arms was adequately large. At trial conclusion, each arm was compared to a historical control group in a futility analysis. Safety measures were also examined. After the first patient pool, the mean six-month decline in ALSFRS-R was 5.27 (SD=5.54) in the celecoxib-creatine group and 6.47 (SD=9.14) in the minocycline-creatine group. The corresponding decline was 5.82 (SD=6.77) in the historical controls. The difference between the two sample means exceeded the stopping criterion. The null hypothesis of superiority was not rejected in the futility analysis. Skin rash occurred more frequently in the celecoxib-creatine group. In conclusion, the celecoxib-creatine combination was selected as preferable to the minocycline-creatine combination for further evaluation. This phase II design was efficient, leading to treatment selection after just 60 patients, and can be used in other phase II trials to assess different agents.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Celecoxib; Creatine; Double-Blind Method; Drug Eruptions; Drug Therapy, Combination; Female; Humans; Male; Middle Aged; Minocycline; Neuroprotective Agents; Patient Selection; Pyrazoles; Sulfonamides; Treatment Outcome

Minocycline has anti-apoptotic and anti-inflammatory effects in vitro, and extends survival in mouse models of some neurological conditions. Several trials are planned or are in progress to assess whether minocycline slows human neurodegeneration. We aimed to test the efficacy of minocycline as a treatment for amyotrophic lateral sclerosis (ALS).. We did a multicentre, randomised placebo-controlled phase III trial. After a 4-month lead-in phase, 412 patients were randomly assigned to receive placebo or minocycline in escalating doses of up to 400 mg/day for 9 months. The primary outcome measure was the difference in rate of change in the revised ALS functional rating scale (ALSFRS-R). Secondary outcome measures were forced vital capacity (FVC), manual muscle testing (MMT), quality of life, survival, and safety. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00047723.. ALSFRS-R score deterioration was faster in the minocycline group than in the placebo group (-1.30 vs -1.04 units/month, 95% CI for difference -0.44 to -0.08; p=0.005). Patients on minocycline also had non-significant tendencies towards faster decline in FVC (-3.48 vs -3.01, -1.03 to 0.11; p=0.11) and MMT score (-0.30 vs -0.26, -0.08 to 0.01; p=0.11), and greater mortality during the 9-month treatment phase (hazard ratio=1.32, 95% CI 0.83 to 2.10; p=0.23) than did patients on placebo. Quality-of-life scores did not differ between the treatment groups. Non-serious gastrointestinal and neurological adverse events were more common in the minocycline group than in the placebo group, but these events were not significantly related to the decline in ALSFRS-R score.. Our finding that minocycline has a harmful effect on patients with ALS has implications for trials of minocycline in patients with other neurological disorders, and for how potential neuroprotective agents are screened for use in patients with ALS.

Topics: Aged; Amyotrophic Lateral Sclerosis; Anti-Bacterial Agents; Confidence Intervals; Double-Blind Method; Female; Humans; Male; Middle Aged; Minocycline; Muscle, Skeletal; Outcome Assessment, Health Care; Psychomotor Performance; Quality of Life; Survival Analysis; Vital Capacity

Two double-blind, randomized, placebo-controlled feasibility trials of minocycline in ALS were conducted. In Trial 1, 19 subjects received 200 mg/day or placebo for 6 months; there were no significant differences in adverse events (AE). In Trial 2, 23 subjects received up to 400 mg/day in an 8-month crossover trial. The mean tolerated dose was 387 mg/day, there was a trend toward more gastrointestinal AE (p = 0.057), and blood urea nitrogen and liver enzymes became elevated (p < 0.05). Using these data, the authors have designed and launched a phase III trial.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Chemical and Drug Induced Liver Injury; Double-Blind Method; Drug Therapy, Combination; Female; Gastrointestinal Diseases; Hand Strength; Humans; Kidney Diseases; Male; Middle Aged; Minocycline; Neuroprotective Agents; Respiratory Muscles; Riluzole; Treatment Outcome

Neurodegenerative diseases are still a challenge for effective treatments. The high cost of approved drugs, severity of side effects, injection site pain, and restrictions on drug delivery to the Central Nervous System (CNS) can overshadow the management of these diseases. Due to the chronic and progressive evolution of neurodegenerative disorders and since there is still no cure for them, new therapeutic strategies such as the combination of several drugs or the use of existing drugs with new therapeutic applications are valuable strategies. Tetracyclines are traditionally classified as antibiotics. However, in this class of drugs, doxycycline and minocycline exhibit also anti-inflammatory effects by inhibiting microglia/macrophages. Hence, they have been studied as potential agents for the treatment of neurodegenerative diseases. The results of in vitro and in vivo studies confirm the effective role of these two drugs as anti-inflammatory agents in experimentally induced models of neurodegenerative diseases. In clinical studies, satisfactory results have been obtained in Multiple sclerosis (MS) but not yet in other disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), or Amyotrophic lateral sclerosis (ALS). In recent years, researchers have developed and evaluated nanoparticulate drug delivery systems to improve the clinical efficacy of these two tetracyclines for their potential application in neurodegenerative diseases. This study reviews the neuroprotective roles of minocycline and doxycycline in four of the main neurodegenerative disorders: AD, PD, ALS and MS. Moreover, the potential applications of nanoparticulate delivery systems developed for both tetracyclines are also reviewed.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Anti-Bacterial Agents; Doxycycline; Drug Delivery Systems; Humans; Minocycline; Nanoparticle Drug Delivery System; Neurodegenerative Diseases; Parkinson Disease

Due to the anti-inflammatory, antioxidant and anti-apoptotic properties of minocycline, clinical trials have evaluated the potential of this drug to treat several psychiatric and neurological disorders, including major depressive disorder, schizophrenia, bipolar disorder, stroke and amyotrophic lateral sclerosis. This protocol proposes a systematic review (and potential meta-analysis) that aims to identify and critically evaluate randomised controlled trials of minocycline for treating psychiatric and neurological disorders.. PubMed, Embase, Cochrane Central Register of Controlled Clinical Trials, PsycINFO and Cumulative Index to Nursing and Allied Health Literature (CINAHL) will be used to identify randomised controlled trials that used minocycline to treat psychiatric and neurological disorders. Double-blind, randomised, controlled, clinical trials of participants aged 18 years or older and written in English will be included in the review. Data will be extracted by two independent reviewers. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines will be followed and the Cochrane Collaboration's 'Risk of Bias' tool will be used to assess the risk of bias in all studies included in the systematic review. The Grading of Recommendations, Assessment, Development and Evaluation system will be used to access the overall quality of the level of evidence of the studies. If sufficient evidence is identified, a meta-analysis will be conducted using the standardised mean difference approach and reported with 95% CIs. Heterogeneity of evidence will be evaluated using the I. This systematic review will evaluate only published data; therefore, ethical approval is not required. The systematic review will be published in a peer-reviewed journal and presented at relevant research conferences.. CRD42020153292.

Topics: Amyotrophic Lateral Sclerosis; Anti-Inflammatory Agents; Antioxidants; Humans; Mental Disorders; Meta-Analysis as Topic; Minocycline; Systematic Reviews as Topic

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics.

Topics: Algorithms; Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Artifacts; Computer Simulation; Disease Progression; Electromyography; Female; Humans; Imaging, Three-Dimensional; Lipids; Male; Mice; Mice, Transgenic; Minocycline; Motor Neurons; Myelin Sheath; Nerve Degeneration; Peripheral Nerves; Sciatic Nerve; Spectrum Analysis, Raman; Superoxide Dismutase-1; Transgenes

In a recent study we found that cerebrospinal fluids (CSFs) from amyotrophic lateral sclerosis (ALS) patients caused 20-30% loss of cell viability in primary cultures of rat embryo motor cortex neurons. We also found that the antioxidant resveratrol protected against such damaging effects and that, surprisingly, riluzole antagonized its protecting effects. Here we have extended this study to the interactions of riluzole with 3 other recognized neuroprotective agents, namely memantine, minocycline and lithium. We found: (1) by itself riluzole exerted neurotoxic effects at concentrations of 3-30 µM; this cell damage was similar to that elicited by 30 µM glutamate and a 10% dilution of ALS/CSF; (2) memantine (0.1-30 µM), minocycline (0.03-1 µM) and lithium (1-80 µg/ml) afforded 10-30% protection against ALS/CSF-elicited neurotoxicity, and (3) at 1-10 µM, riluzole antagonized the protection afforded by the 3 agents. These results strongly support the view that at the riluzole concentrations reached in the brain of patients, the neurotoxic effects of this drug could be masking the potential neuroprotective actions of new compounds being tested in clinical trials. Therefore, in the light of the present results, the inclusion of a group of patients free of riluzole treatment may be mandatory in future clinical trials performed in ALS patients with novel neuroprotective compounds.

Topics: Amyotrophic Lateral Sclerosis; Animals; Cell Survival; Excitatory Amino Acid Antagonists; Humans; Lithium; Memantine; Minocycline; Neurons; Neuroprotective Agents; Rats; Riluzole

Minocycline is commonly used to inhibit microglial activation. It is widely accepted that activated microglia exert dual functions, that is, pro-inflammatory (M1) and anti-inflammatory (M2) functions. The in vivo status of activated microglia is probably on a continuum between these two extreme states. However, the mechanisms regulating microglial polarity remain elusive. Here, we addressed this question focusing on minocycline. We used SOD1(G93A) mice as a model, which exhibit the motor neuron-specific neurodegenerative disease, amyotrophic lateral sclerosis. Administration of minocycline attenuated the induction of the expression of M1 microglia markers during the progressive phase, whereas it did not affect the transient enhancement of expression of M2 microglia markers during the early pathogenesis phase. This selective inhibitory effect was confirmed using primary cultured microglia stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, which induced M1 or M2 polarization, respectively. Furthermore, minocycline inhibited the upregulation of NF-κB in the LPS-stimulated primary cultured microglia and in the spinal cord of SOD1(G93A) mice. On the other hand, IL-4 did not induce upregulation of NF-κB. This study indicates that minocycline selectively inhibits the microglia polarization to a proinflammatory state, and provides a basis for understanding pathogeneses of many diseases accompanied by microglial activation.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; B7-2 Antigen; Calcium-Binding Proteins; Cells, Cultured; Disease Models, Animal; Humans; Inflammation; Interleukin-4; Lipopolysaccharides; Mice; Mice, Transgenic; Microfilament Proteins; Microglia; Minocycline; NF-kappa B; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Survival Rate; Up-Regulation

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Antioxidants; Ceftriaxone; Disease Models, Animal; Disease Progression; Drug Evaluation, Preclinical; Drug Therapy, Combination; Humans; Mice; Mice, Transgenic; Minocycline; Research Design; Superoxide Dismutase; Treatment Outcome; Vitamin E

Several reports have demonstrated that attenuation of microglial activation by minocycline, an antimicrobial drug with anti-inflammatory properties, delays disease progression in a mouse model of ALS. However, the negative results obtained in recent clinical trials raised some questions regarding the role of inflammatory response and glial cells as a therapeutic target in ALS. To investigate this controversy we took advantage of a mouse model for live imaging of neuroinflammatory responses in ALS (GFAP-luc/ SOD1(G93A) reporter mouse) and analyzed in real time the effects of minocycline treatment initiated at different stages of the disease. To our surprise, unlike neuroprotection that is conferred when minocycline is administered pre-symptomatically, treatment with minocycline initiated after the disease onset significantly altered glial responses and exaggerated neuroinflammation. Further analysis revealed that the late minocycline treatment was associated with significant induction of the end-stage GFAP-biophotonic signals, expression levels of connexin 43, a major protein of astrocytic gap junction and markers of microglial activation, such as Iba1 and CD68. The results of our study suggest that when administered at later stages of disease, once microglial cells are chronically reactive, minocycline may not have anti-inflammatory properties, and contrary to expectations, may alter astrocyte reactivity and increase microgliosis. Finally, our results further suggest the existence of close interactions/communication between activated microglia and astrocytes in late stage ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Female; Male; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mice, Transgenic; Microglia; Minocycline; Random Allocation; Superoxide Dismutase; Superoxide Dismutase-1; Treatment Outcome

Improving quality of life (QoL) is a major goal in ALS palliative care. Previous studies performed on the general ALS population showed no relationship between QoL and disease progression. ALS subjects participating in clinical trials may differ from those in the general ALS population. We explored the relationship between QoL and disease progression in 412 subjects enrolled in a minocycline trial. We examined correlations between Single Item McGill Quality of Life Scale (MQoL-SIS) score and disease duration, ALS Functional Rating Scale Revised (ALSFRS-R) score, FVC, and survival rate. We also analyzed how NIV and PEG affect QoL. Within subjects, MQoL-SIS scores correlated with ALSFRS-R and FVC (p<0.001). MQoL-SIS declined over time (p<0.001) and correlated with the decline of ALSFRS-R (p<0.001). MQoL-SIS tended to improve after initiation of NIV (p=0.07). There was a significant reduction in the rate of MQoL-SIS decline (p<0.001) after initiation of PEG. Subjects with slower QoL decline survived seven months longer than those with faster QoL decline (p<0.01). Our study demonstrated that QoL does decline with advancing ALS in subjects who participated in a minocycline trial, that the slope of QoL predicts survival, and that both NIV and PEG have beneficial impacts on QoL.

Topics: Amyotrophic Lateral Sclerosis; Anti-Bacterial Agents; Clinical Trials, Phase III as Topic; Disease Progression; Humans; Minocycline; Outcome Assessment, Health Care; Palliative Care; Predictive Value of Tests; Quality of Life; Randomized Controlled Trials as Topic; Respiratory Insufficiency; Survival Analysis

Magnetic resonance spectroscopy (MRS) allows to monitor brain metabolites noninvasively in amyotrophic lateral sclerosis (ALS). The objective of this study was to use MRS to monitor the effect of minocycline treatment (200 mg/day) over a short period (6 weeks) on the brain metabolites in the precentral gyrus and brainstem in newly diagnosed ALS patients.. Ten ALS patients (not on riluzole treatment) were recruited and submitted to single-voxel proton MRS longitudinal examinations (1) before minocycline treatment, (2) 3 weeks and (3) 6 weeks after initiation of treatment.. Results did not show the expected decrease of N-acetylaspartate/creatine (NAA/Cr) in the precentral gyrus, and an increased NAA/Cr ratio in the brainstem suggested neuronal recovery. The myo-inositol (mI)/Cr ratio was unchanged in the precentral gyrus, but increased in the brainstem, indicating a glial reaction.. MRS results suggest that minocycline treatment could be beneficial in the early stages of ALS.

Topics: Amyotrophic Lateral Sclerosis; Anti-Bacterial Agents; Biomarkers; Brain; Female; Humans; Magnetic Resonance Imaging; Male; Microglia; Middle Aged; Minocycline; Neurons; Tissue Distribution

amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by the loss of motorneurons. The only drug approved is riluzole. Minocycline is an antibiotic with numerous neuroprotective properties. riluzole and minocycline were given to an animal model of ALS and had beneficial effect on the disease. The combination was then tested in humans in phase II and phase III studies with less beneficial effects and a faster decline of the disease in the group treated with minocycline. In a previous study, we showed that riluzole is transported out of the brain by the P-glycoprotein at the blood-brain barrier level.. in this work, we studied in CF1 mice, the plasmatic and cerebral pharmacokinetics of riluzole combined or not with minocycline.. our results showed that the kinetics of riluzole are not linear with dose, but that cerebral AUC0-infinity increase proportionally with plasmatic AUC0-infinity. At the dose of 10 mg/kg, the cerebral AUC0-infinity /plasmatic AUC0-infinity ratio was 4.6 in mdr1a (-/-) mice and 2.4 in mdr1a (+/+) mice. The combination of minocycline (170 mg/kg) and riluzole (10 mg/kg) induced a 2 fold increase in the cerebral AUC0-infinity of riluzole and induced a neuromuscular toxicity in mice. This effect of minocycline was not found at low concentration (10 mg/kg of minocycline).. if our results are confirmed in humans, riluzole cerebral concentrations could be predicted by plasmatic concentrations. Furthermore, the combination of high doses of minocycline with riluzole could induce neurological toxicity that lead to deceiving results in ALS clinical studies.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Area Under Curve; ATP Binding Cassette Transporter, Subfamily B; Blood-Brain Barrier; Brain; Dose-Response Relationship, Drug; Drug Interactions; Female; Mice; Mice, Knockout; Minocycline; Neuroprotective Agents; Neurotoxicity Syndromes; Riluzole; Tissue Distribution

Topics: Amyotrophic Lateral Sclerosis; Biomarkers; Causality; Genetic Therapy; Humans; Minocycline; Research

A recent publication of the results of a clinical trial of minocycline in 412 ALS patient has aroused considerable controversy in the ALS scientific community. As on previous occasions, the results obtained in the laboratory are not reproduced in clinical practice. The reasons for this new disappointment in translational medicine are analysed by applying the successes obtained in the experimental animal model for ALS to humans. The most frequently suggested causes for explaining these continuous failures are unawareness of the correct dosage to be used, the ideal duration of the clinical trial in phase III, sample size, the search for a primary outcome for measurement other than survival, the need for biomarkers giving information on the progression of the disease and whether this is modified by the introduction of the drug for study. Debate focuses on whether the transgenic mouse model of ALS which expresses SOD1 mutations which we have been using for more than a decade is an exact reflection of the clinical profile and the physiopathogenic mechanisms present in patients with spo- radic ALS. There is the possibility that depending on the dose administered, minocycline can be a neuroprotector or a neurotoxin. In other words, at a dose of 200 mg/day, this drug behaves like <> and like <> at doses of 400 mg. For the authors of the trial, this possibility does not seem to be the cause of the disappointing results obtained. However, they acknowledge that one of the limitations of their study was that it was impossible to compare the effects of minocycline in the patient after receiving 200 or 400 mg. For many other researchers running ongoing clinical trials in both ALS and other neurological diseases, the dose of 200 mg/day is chosen as ideal for testing the effectiveness of minocycline in patients. The strategy of administering the maximum dose of a drug to be tested may give rise to misleading results. We agree with the opinion of other authors, who say that minocycline should be given a second chance.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Clinical Trials as Topic; Disease Models, Animal; Humans; Mice; Mice, Transgenic; Minocycline; Neuroprotective Agents; Neurotoxins; Treatment Outcome

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Disease Models, Animal; Drug Therapy, Combination; Humans; Mice; Mice, Knockout; Minocycline; Randomized Controlled Trials as Topic; Superoxide Dismutase; Superoxide Dismutase-1; Treatment Failure

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Drug Therapy, Combination; Humans; Mice; Minocycline; Randomized Controlled Trials as Topic

Topics: Amyotrophic Lateral Sclerosis; Animals; Controlled Clinical Trials as Topic; Humans; Minocycline

Topics: Aged; Amyotrophic Lateral Sclerosis; Anti-Bacterial Agents; Dental Enamel; Forearm; Humans; Male; Minocycline; Pigmentation Disorders; Tooth Discoloration

There is increasing evidence that apoptosis or a similar programmed cell death pathway is the mechanism of cell death responsible for motor neurone degeneration in amyotrophic lateral sclerosis. Knowledge of the relative importance of different caspases in the cell death process is at present incomplete. In addition, there is little information on the critical point of the death pathway when the process of dying becomes irreversible. In this study, using the well-established NSC34 motor neurone-like cell line stably transfected with empty vector, normal or mutant human Cu-Zn superoxide dismutase (SOD1), we have characterized the activation of the caspase cascade in detail, revealing that the activation of caspases-9, -3 and -8 are important in motor neurone death and that the presence of mutant SOD1 causes increased activation of components of the apoptotic cascade under both basal culture conditions and following oxidative stress induced by serum withdrawal. Activation of the caspases identified in the cellular model has been confirmed in the G93A SOD1 transgenic mice. Furthermore, investigation of the effects of anti-apoptotic neuroprotective agents including specific caspase inhibitors, minocycline and nifedipine, have supported the importance of the mitochondrion-dependent apoptotic pathway in the death process and revealed that the upstream caspase cascade needs to be inhibited if useful neuro-protection is to be achieved.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Apoptosis; Blotting, Western; Calcium Channel Blockers; Caspases; Cells, Cultured; Disease Models, Animal; Enzyme Activation; Humans; Mice; Mice, Transgenic; Minocycline; Motor Neurons; Nifedipine; Oxidative Stress; Superoxide Dismutase; Superoxide Dismutase-1; Transfection

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.

Topics: Acetylcysteine; Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Blotting, Western; Cell Survival; Chelating Agents; Chlorides; Copper; Detergents; Disease Models, Animal; Ditiocarb; Fibroblasts; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Minocycline; Mutation; Oxidative Stress; Paraquat; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Solubility; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Zinc Compounds

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Caregivers; Humans; Interpersonal Relations; Middle Aged; Minocycline; Neuroprotective Agents; Quality of Life; Riluzole

Topics: Amines; Amyotrophic Lateral Sclerosis; Calcium Channel Blockers; Ciliary Neurotrophic Factor; Clinical Trials as Topic; Cooperative Behavior; Cyclohexanecarboxylic Acids; Cyclophosphamide; Gabapentin; gamma-Aminobutyric Acid; Humans; Minocycline; Societies, Medical; Treatment Outcome

The known neuroprotective effects of minocycline and creatine in animal models of amyotrophic lateral sclerosis (ALS) led us to examine whether the combination of these agents would result in increased neuroprotection. As previously reported, we confirmed in ALS mice that either minocycline or creatine treatment results in improvement in motor performance and extended survival. We report that combination of minocycline and creatine resulted in additive neuroprotection, suggesting this to be a novel potential strategy for the treatment of ALS. To our knowledge, this is the first report demonstrating additive neuroprotection of a combinatorial approach in a mouse model of ALS. Adding relevancy to our findings, minocycline and creatine, are relatively safe, cross the blood-brain barrier, and are currently available for human evaluation.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Creatine; Disease Models, Animal; Drug Therapy, Combination; Mice; Mice, Mutant Strains; Minocycline; Neuroprotective Agents; Superoxide Dismutase; Superoxide Dismutase-1; Survival Analysis

There is growing evidence for involvement of members of the cyclin-dependent kinase (Cdk) family in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults. After our recent report that a deregulation of Cdk5 activity by p25 may contribute to pathogenesis of amyotrophic lateral sclerosis (ALS), we further examined the possible involvement of other Cdks in mice expressing a mutant form of superoxide dismutase (SOD1(G37R)) linked to ALS. No substantial changes in Cdk2 or Cdk6 distribution and kinase activities were detected in spinal motor neurons from SOD1(G37R) mice when compared with normal mice. Of particular interest was the upregulation and mislocalization of Cdk4, a regulator of the G1-S checkpoint of the cell cycle, in motor neurons of SOD1(G37R) mice. The increase of Cdk4 activity in SOD1(G37R) mice was associated with an increase in nuclear Cdk4, cyclin D1, its coactivator, and with the abnormal phosphorylation of the retinoblastoma (Rb) protein at Cdk phosphorylation sites. Pharmacological treatment of SOD1(G37R) mice with minocycline, a compound that attenuates microgliosis and slows down disease, lessened the dysregulation of Cdk5/Cdk4 and the phosphorylation of Rb. Interestingly, phospho-Rb was immunoprecipitated with anti-Cdk4 but not with anti-Cdk5 antibodies, suggesting a key role for Cdk4 in the phosphorylation of Rb. Remarkably, the overexpression of a transgene coding for human neurofilament H, a phosphorylation sink for deregulated Cdk5 activity by p25, resulted in a reduction in levels of nuclear Cdk4 and Rb phosphorylation. These results indicate that a cell cycle signaling at the neuronal G1-S checkpoint subsequent to Cdk5 deregulation may constitute a critical step of the neuronal death pathway in ALS caused by mutant SOD1.

Topics: Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Cell Cycle Proteins; Cell Death; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Disease Progression; G1 Phase; Humans; Mice; Mice, Neurologic Mutants; Mice, Transgenic; Minocycline; Motor Neurons; Neurofilament Proteins; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein; S Phase; Signal Transduction; Superoxide Dismutase; Transgenes; Up-Regulation

There is currently no effective pharmacological treatment for amyotrophic lateral sclerosis (ALS). Because evidence suggests that multiple pathways may contribute to ALS pathogenesis, we tested in a mouse model of ALS (SOD1(G37R) mice) a combination approach consisting of three drugs for distinct targets in the complex pathway to neuronal death: minocycline, an antimicrobial agent that inhibits microglial activation, riluzole, a glutamate antagonist, and nimodipine, a voltage-gated calcium channel blocker. The efficacy of this three-drug cocktail was remarkable when administered in the diet from late presymptomatic stage (8-9 months). It delayed the onset of disease, slowed the loss of muscle strength, and increased the average longevity of SOD1(G37R) mice by 6 weeks. The protective effect of the treatment was corroborated by the reduced immunodetection signals for markers of gliosis and neurodegeneration in the spinal cord of SOD1(G37R) mice. These results indicate that such three-drug combination may represent an effective strategy for ALS treatment.

Topics: Age of Onset; Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Axons; Calcium Channel Blockers; Caspase 3; Caspases; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Disease Models, Animal; Drug Therapy, Combination; Gliosis; Mice; Mice, Transgenic; Microglia; Minocycline; Motor Neurons; Muscle Contraction; Neuroprotective Agents; Nimodipine; Riluzole; Superoxide Dismutase; Superoxide Dismutase-1

There is currently no effective pharmacological treatment for amyotrophic lateral sclerosis (ALS). Because recent evidence suggests that secondary inflammation and caspase activation may contribute to neurodegeneration in ALS, we tested the effects of minocycline, a second-generation tetracycline with anti-inflammatory properties, in mice expressing a mutant superoxide dismutase (SOD1(G37R)) linked to human ALS. Administration of minocycline into the diet, beginning at late presymptomatic stage (7 or 9 months of age), delayed the onset of motor neuron degeneration, muscle strength decline, and it increased the longevity of SOD1(G37R) mice by approximately 5 weeks for approximately 70% of tested mice. Moreover, less activation of microglia was detected at early symptomatic stage (46 weeks) and at the end stage of disease in the spinal cord of SOD1(G37R) mice treated with minocycline. These results indicate that minocycline, which is clinically well tolerated, may represent a novel and effective drug for treatment of ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Axons; Disease Models, Animal; Disease Progression; Mice; Mice, Transgenic; Microglia; Minocycline; Superoxide Dismutase; Superoxide Dismutase-1

Minocycline mediates neuroprotection in experimental models of neurodegeneration. It inhibits the activity of caspase-1, caspase-3, inducible form of nitric oxide synthetase (iNOS) and p38 mitogen-activated protein kinase (MAPK). Although minocycline does not directly inhibit these enzymes, the effects may result from interference with upstream mechanisms resulting in their secondary activation. Because the above-mentioned factors are important in amyotrophic lateral sclerosis (ALS), we tested minocycline in mice with ALS. Here we report that minocycline delays disease onset and extends survival in ALS mice. Given the broad efficacy of minocycline, understanding its mechanisms of action is of great importance. We find that minocycline inhibits mitochondrial permeability-transition-mediated cytochrome c release. Minocycline-mediated inhibition of cytochrome c release is demonstrated in vivo, in cells, and in isolated mitochondria. Understanding the mechanism of action of minocycline will assist in the development and testing of more powerful and effective analogues. Because of the safety record of minocycline, and its ability to penetrate the blood-brain barrier, this drug may be a novel therapy for ALS.

Topics: Age of Onset; Amyotrophic Lateral Sclerosis; Animals; Caspases; Cell Death; Cells, Cultured; Cerebral Cortex; Cytochrome c Group; Disease Progression; Enzyme Activation; Humans; Infarction, Middle Cerebral Artery; Ischemia; Mice; Mice, Inbred C57BL; Minocycline; Mitochondria; Mitochondrial Swelling; N-Methylaspartate; Permeability; Rats; Survival Rate; Tumor Cells, Cultured

Microglial activation is thought to contribute to the progression of selective motor neuron death during amyotrophic lateral sclerosis (ALS). As minocycline has been shown to inhibit microglial activation, the therapeutic efficacy of this tetracycline derivative in the G93A mice model for familial ALS was tested. This drug with proven safety delayed disease onset and dose-dependently extended the survival of the G93A mice. At 120 days of age, minocycline protected mice from loss of motor neurons and from vacuolization. These results demonstrate that interference with immuno-inflammatory responses has a beneficial effect in the ALS mice model, suggesting this to be a potential new strategy to treat ALS.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Anti-Bacterial Agents; Cell Survival; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Female; Inflammation; Male; Mice; Mice, Transgenic; Microglia; Minocycline; Motor Activity; Motor Neurons; Neuroprotective Agents; Spinal Cord; Survival Rate; Treatment Outcome