Morphological effects of chronic excitotoxicity depend on autophagy activation failure

It is well known that exposure to excitatory amino acid in specific experimental conditions might produce a defect in the autophagy pathway. Such an effect was observed in motor neurons exposed chronically to glutamate agonists. In particular, it is well known that glutamate induces motor neuron death and this is supposed to play a key role in the physiopathology of motor neuron loss in amyotrophic lateral sclerosis (ALS). Similarly, a defective recruitment of autophagy was recently documented in ALS. In the present study, we used primary motor neuron cultures to analyzed whether AMPA receptor stimulation via kainic acid produces activation of autophagy and whether this is defective compared with what it is required to rescue motor neurons. In particular we found that exposure of motor neurons to kainic acid produces intracellular alterations associated with defective autophagy. The ultrastructural alterations consist of increased motor neurons size, damaged mitochondria, protein accumulation, large cytoplasmic vacuoles placed in perinuclear positions These cellular alterations is reminiscent of ALS, which in turn is characterized by a defective autophagy. Once we confirmed that excitotoxicity recruits autophagy, which remains defective to clear altered proteins and organelles, we provided a pharmacological stimulation of such a pathway in order to ameliorate motor neuron survival. In this experimental conditions, We observe that pharmacological activation of the autophagy machinery is able to counteract kainic acid-mediated motor neuron damage

mTOR Modulates Methamphetamine-Induced Toxicity through Cell Clearing Systems

Methamphetamine (METH) is abused worldwide, and it represents a threat for public health. METH exposure induces a variety of detrimental effects. In fact, METH produces a number of oxidative species, which lead to lipid peroxidation, protein misfolding, and nuclear damage. Cell clearing pathways such as ubiquitin-proteasome (UP) and autophagy (ATG) are involved in METH-induced oxidative damage. Although these pathways were traditionally considered to operate as separate metabolic systems, recent studies demonstrate their interconnection at the functional and biochemical level. Very recently, the convergence between UP and ATG was evidenced within a single organelle named autophagoproteasome (APP), which is suppressed by mTOR activation. In the present research study, the occurrence of APP during METH toxicity was analyzed. In fact, coimmunoprecipitation indicates a binding between LC3 and P20S particles, which also recruit p62 and alpha-synuclein. The amount of METH-induced toxicity correlates with APP levels. Specific markers for ATG and UP, such as LC3 and P20S in the cytosol, and within METH-induced vacuoles, were measured at different doses and time intervals following METH administration either alone or combined with mTOR modulators. Western blotting, coimmunoprecipitation, light microscopy, confocal microscopy, plain transmission electron microscopy, and immunogold staining were used to document the effects of mTOR modulation on METH toxicity and the merging of UP with ATG markers within APPs. METH-induced cell death is prevented by mTOR inhibition, while it is worsened by mTOR activation, which correlates with the amount of autophagoproteasomes. The present data, which apply to METH toxicity, are also relevant to provide a novel insight into cell clearing pathways to counteract several kinds of oxidative damage

High-intensity exercise training induces morphological and biochemical changes in skeletal muscles

IN THE PRESENT STUDY WE INVESTIGATED THE EFFECT OF TWO DIFFERENT EXERCISE PROTOCOLS ON FIBRE COMPOSITION AND METABOLISM OF TWO SPECIFIC MUSCLES OF MICE: the quadriceps and the gastrocnemius. Mice were run daily on a motorized treadmill, at a velocity corresponding to 60% or 90% of the maximal running velocity. Blood lactate and body weight were measured during exercise training. We found that at the end of training the body weight significantly increased in high-intensity exercise mice compared to the control group (P=0.0268), whereas it decreased in low-intensity exercise mice compared to controls (P=0.30). In contrast, the food intake was greater in both trained mice compared to controls (P < 0.0001 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively). These effects were accompanied by a progressive reduction in blood lactate levels at the end of training in both the exercised mice compared with controls (P=0.03 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively); in particular, blood lactate levels after high-intensity exercise were significantly lower than those measured in low-intensity exercise mice (P=0.0044). Immunoblotting analysis demonstrated that high-intensity exercise training produced a significant increase in the expression of mitochondrial enzymes contained within gastrocnemius and quadriceps muscles. These changes were associated with an increase in the amount of slow fibres in both these muscles of high-intensity exercise mice, as revealed by the counts of slow fibres stained with specific antibodies (P < 0.0001 for the gastrocnemius; P=0.0002 for the quadriceps). Our results demonstrate that high-intensity exercise, in addition to metabolic changes consisting of a decrease in blood lactate and body weight, induces an increase in the mitochondrial enzymes and slow fibres in different skeletal muscles of mice, which indicates an exercise-induced increase in the aerobic metabolism

Lamina X of the spinal cord in motor neuron disease

A number of plastic events were described in the spinal cord in the course of amyotrophic lateral sclerosis (ALS). These consist of various morphological effects, involving neurons, glia, and inflammatory cells, as well. Among plastic changes, an increase in neuronal progenitor cells (NPC) occurs within ependymal cells layer of lamina X. This stem cell-like activity is known to be weak in baseline conditions but it is known to increase significantly during spinal cord disorders, when it preferentially generates glial cells, due to the strong gliogenic effect of the spinal cord “milieu”. In the present work, we used immunohistochemistry and electron microscopy to analyze cell number within lamina X at the end stage of disease in the G93A mouse model of ALS in baseline conditions and following chronic lithium administration. These cells were identified by using GFAP, bIII-tubulin, NeuN, and calbindin- D28K immunostaining. In the absence of lithium we observed an increase of lamina X cells in ALS mice with a glial phenotype, while in G93A mice treated with lithium these cells differentiate towards neuronal-like phenotype. These effects of lithium are concomitant with slowed disease progression and are reminiscent of the neurogenetic effects described in the sub-ependymal ventricular zone of the hippocampus. The present data confirm the scarce NPC activity in the intact spinal cord which is enhanced by disease conditions; in the presence of chronic lithium, such increased NPCs differentiate towards a neuron-like rather than a glial phenotype

Morphological characterization of a single knock out double transgenic mouse model of spinal muscle atrophy

Spinal muscular atrophy (SMA) is a neurogenetic autosomal recessive disorder characterized by degeneration of lower motor neurons associated with muscle atrophy and paralysis. Due to a lack of an in depth knowledge on the molecular mechanisms and fine neuropathology of SMA, validation of appropriate animal models is key in fostering SMA research. Recent studies set up an animal model showing long survival and slow disease progression. This model is knocked out for mouse SMN (Smn−/−) gene and carries a human mutation of the SMN1 gene (SMN1A2G), along with human SMN2 gene. In the present study we used this knockout double transgenic mouse as a SMA III model, to characterize the spinal cord pathology along with motor deficit at prolonged survival times (18 months). This long time interval (i.e. up to 535 days) was never analyzed before especially concerning specific motor tasks. We found that the delayed disease progression was likely to maintain fair motor activity despite a dramatic loss of large motor neurons (44.77%). At this stage, spared motor neurons showed significant cell body enlargement. Moreover, similar to what was described in patients affected by SMA we found neuronal heterotopy in the anterior white matter. Motor neuron degeneration was accompanied by the loss of SMN protein in the spinal cord. In summary, the present study validates over a long time period a SMA III mouse model showing neuropathology reminiscent of human patients and provide a useful experimental model to probe novel therapeutic strategies

A Comparative Study on the Efficacy of NLRP3 Inflammasome Signaling Inhibitors in a Pre-clinical Model of Bowel Inflammation

Nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome is pivotal in maintaining intestinal homeostasis and sustaining enteric immune responses in the setting of inflammatory bowel diseases. Drugs acting as NLRP3 blockers could represent innovative strategies for treatment of bowel inflammation. This study was performed in rats with dinitrobenzenesulfonic acid (DNBS)-induced colitis, to investigate how the direct blockade of NLRP3 inflammasome with an irreversible inhibitor (INF39) compares with Ac-YVAD-cmk (YVAD, caspase-1 inhibitor) and anakinra (IL-1β receptor antagonist), acting downstream on NLRP3 signaling. Animals with DNBS-colitis received YVAD (3 mg/kg) or anakinra (100 mg/Kg) intraperitoneally, and INF39 (25 mg/kg) or dexamethasone (DEX, 1 mg/kg) orally for 6 days, starting on the same day of colitis induction. Under colitis, there was a body weight decrease, which was attenuated by YVAD, anakinra or INF39, but not DEX. All test drugs counteracted the increase in spleen weight. The colonic shortening and morphological colonic alterations associated with colitis were counteracted by INF39, anakinra and DEX, while YVAD was without effects. Tissue increments of myeloperoxidase, tumor necrosis factor and interleukin-1β were more effectively counteracted by INF39 and DEX, than YVAD and anakinra. These findings indicate that: (1) direct inhibition of NLRP3 inflammasome with INF39 is more effective than caspase-1 inhibition or IL-1β receptor blockade in reducing systemic and bowel inflammatory alterations; (2) direct NLRP3 inhibition can be a suitable strategy for treatment of bowel inflammation

Sub-cellular motor neuron analysis in a model of spinal muscle atrophy

Spinal muscular atrophy (SMA) is a neurogenetic autosomal recessive disorder characterized by degeneration of lower motor neurons associated with muscle atrophy and paralysis. The disease course including onset and severity depends by reduced amounts of the survival motor neuron (SMN) protein. Such a protein is increased when the enzyme glycogen synthase kinase-3beta (GSK3beta) is inhibited. In the present study we used a knockout double transgenic mouse (Smn−/−; SMN1A2G; SMN2) modelling SMAIII to dissect the spinal cord pathology at ultrastructural analysis at prolonged survival time (18 months). We analysed the subcellular structure of spinal cord motor neurons both in baseline conditions and following the administration of a GSK3beta inhibitor. We found that motor neurons increased their diameter confirming our previous light microscopy data. The amount of immunogold labelled SMN particles was dramatically reduced in the whole cell body incuding nucleus and cytoplasm. Remarkably, at nuclear level we could detect marked reduction of the SMN protein with Cajal-like bodies thus mimicking the human disease. In mice receiving long-term lithium administration the level of the SMN protein were massively increase way more than other SMAIII mice and significantly exceeding the levels counted in controls. When compared with control mice administered long-term lithium SMN levels in SMA III mice were overlapping with healthy animals, at large. The effects of lithium on ultrastructural morphology of motor neurons extended to the preservation of mitochondrial compartment which was slightly affected in motor neurons from SMA III mice. These data confirm the essential role of GSK3beta inhibition in increasing the amount of the SMN protein and provide a novel action for an old drug which increases SMN level exceeding any other compound tested so far in this motor neuron pathology. At the same time the beneficial effects of lithium on mitochondrial morphology are confirmed. As an appendix to the present study we wish to mention the ubiquitous nature of these effects which were replicated in non-motor neuron cell lines. Apart from the significance in cell biology this latter observation provide the basis to analyze the effects of a lithium treatment on affected patients using peripheral or skin-derived cell cultures. This work was supported by an educational grant from CUCC