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Aberrations in intracellular calcium (Ca 2+ ) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca 2+ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca 2+ cycle (ERMCC). The ER supplies Ca 2+ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca 2+ at the cellular level remains to be thoroughly investigated. Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca 2+ homeostasis in the presence of G93A hSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca 2+ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca 2+ clearance following a) AMPAR activation by kainate and b) IP 3 R–mediated ER Ca 2+ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca 2+ . Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca 2+ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca 2+ homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: Dietary restriction (DR) and rapamycin (Rapa) have been shown to increase the lifespan of a variety of organisms leading to the speculation that these interventions increase lifespan through related mechanisms. However, both these interventions have a detrimental effect in the G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). Our previous work indicated that different ALS SOD1 mutant mouse models differ in disease pathogenesis; therefore in this study we measured the effect of DR and Rapa in a second ALS mutant mouse model (the H46R/H48Q mutant). Interestingly, in mice expressing this mutant SOD1 protein, DR significantly delays disease onset and extends lifespan, while Rapa has no effect. These findings suggest that: (1) the effect of DR in ALS is not mediated through pathways common with Rapa, (2) the deleterious effect of DR and Rapa in the G93A ALS mouse model may not be universal to disease caused by all SOD1 mutations, and (3) the results reinforce our previous conclusions that the pathogenic mechanisms in G93A and H46R/H48Q mice are distinct. [Copyright &y& Elsevier]

Abstract: Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1week old pups, and after an additional week in vitro, some were exposed to either a low level (30μM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3weeks, or a higher level (50μM) for 48h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury. [Copyright &y& Elsevier]

Abstract: Defect of metallothionein-III (MT-III) has been reported to be a contributor to the progression of amyotrophic lateral sclerosis (ALS). We explored the expression and effects of MT-III on the motor neurons of spinal cords of ALS model mice (G93A Cu/Zn superoxide dismutase (SOD-1) mutant-transgenic (Tg) mice) using a retrograde viral delivery system. Once-weekly injection of the adenovirus encoding LacZ or MT-III gene was started at the age of 20 weeks, which was the mean age of ALS onset. Gene expression was detected in the motor neurons of the lumbar spinal cord. At 160 days of age (14 days after injection), the mean numbers of Nissl-stained α neurons were 15.42±5.32, 16.50±1.35, and 24.75±4.01 in 5-μm sections of the lumbar hemispinal cord from the untreated group, LacZ group, and MT-III group, respectively. The mean durations of illness were 15.20±5.30 days, 10.33±4.27 days, and 25.71±7.67 days in the untreated group, LacZ group, and MT-III group, respectively. The mean life spans were 163.20±7.72 days, 159.50±3.27 days, and 178.14±12.97 days in the untreated group, LacZ group, and MT-III group, respectively. We demonstrated that MT-III prevents the loss of motor neurons of ALS model mice and prolongs the life span, even when the administration is started at the time of onset. [Copyright &y& Elsevier]

Abstract: Oxidative damage, neuroinflammation, and mitochondrial dysfunction contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS), and these pathologic processes are tightly regulated by the Nrf2/ARE (NF-E2-related factor 2/antioxidant response element) signaling program. Therefore, modulation of the Nrf2/ARE pathway is an attractive therapeutic target for neurodegenerative diseases such as ALS. We examined two triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) ethylamide and CDDO trifluoroethylamide (CDDO-TFEA), that potently activate Nrf2/ARE in a cell culture model of ALS and in the G93A SOD1 mouse model of ALS. Treatment of NSC-34 cells stably expressing mutant G93A SOD1 with CDDO-TFEA upregulated Nrf2 expression and resulted in translocation of Nrf2 into the nucleus. Western blot analysis showed an increase in the expression of Nrf2/ARE-regulated proteins. When treatment started at a “presymptomatic age” of 30days, both of these compounds significantly attenuated weight loss, enhanced motor performance, and extended the survival of G93A SOD1 mice. Treatment started at a “symptomatic age,” as assessed by impaired motor performance, was neuroprotective and slowed disease progression. These findings provide further evidence that compounds that activate the Nrf2/ARE signaling pathway may be useful in the treatment of ALS. [Copyright &y& Elsevier]

Abstract: Amyotrophic Lateral Sclerosis (ALS) is a devastating adult-onset motor neuron disorder with marginal therapeutic options. The disease is characterized by progressive degeneration of motor neurons in spinal cord and motor cortex. Transgenic mice carrying the G93A mutation of the superoxide dismutase 1 (SOD1) gene develop a neurodegenerative disease closely mimicking human ALS. Several methods are currently used to record disease onset and progression of the animals in preclinical studies. For the interpretation of these preclinical trials, it is important to assess neurological function as sensitively as possible. In the present study, five different parameters (rotarod performance, weight, footprint analysis for both step length and runtime and the general condition of the mice scored from 1 to 5) were compared with respect to their significance to detect symptom onset and to monitor disease progression in transgenic G93A ALS mice. The rotarod and footprint analyses were performed weekly while the weight was recorded up to three times a week at later time points. General condition was assessed daily. First deficits were detected by rotarod testing and step length analyses. General condition score and weight showed first changes two weeks later. For preclinical testing of novel drug treatments rotarod and footprint analysis for step length therefore seem to be the most effective methods to detect symptom onset and potential treatment induced improvements. [Copyright &y& Elsevier]

Abstract: Abnormal mitochondrial function was reported in patients and models for amyotrophic lateral sclerosis (ALS). It is therefore important to set up sensitive tools for the monitoring of active agents that enhance energy metabolism delay onset, and extend lifespan of transgenic G93A-SOD1 ALS mice. In this report, primary motor cortex slices from G93A mice at different stages of disease were studied, using NAD(P)H autofluorescence post-synaptic signals following ultraviolet stimuli, as a probe to evaluate mitochondrial function. We observed consistent age-related alterations of responses in G93A primary motor cortex slices versus controls. We conclude that NAD(P)H autofluorescence post-synaptic signal is a highly sensitive real-time technique to detect mitochondrial function failure in primary cortex from living tissues. [Copyright &y& Elsevier]

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease. [Display omitted] • Gprin3 and Colgalt2 bacTRAP mice label distinct subtypes of layer 5b neurons in M1 • Gprin3 cells degenerate in a preclinical model of ALS, whereas Colgalt2 cells do not • Regulation of bioenergetics-related genes is a common disease response of L5b cells • Gprin3 genetic properties imply higher energy demand and oxidative stress sensitivity Moya et al. use bacTRAP mouse lines to characterize two highly related subpopulations of layer 5b projection neurons in motor cortex that are differentially susceptible to neurodegeneration in the SOD1-G93A mouse model of ALS. They identify the regulation of genes involved in bioenergetics as a key factor regulating susceptibility. [ABSTRACT FROM AUTHOR]

Abstract: Autophagy, like the ubiquitin–proteasome system, is considered to play an important role in preventing the accumulation of abnormal proteins. Rat microtubule-associated protein 1 light chain 3 (LC3) is important for autophagy, and the conversion from LC3-I into LC3-II is accepted as a simple method for monitoring autophagy. We examined a SOD1G93A transgenic mouse model for amyotrophic lateral sclerosis (ALS) to consider a possible relationship between autophagy and ALS. In our study we analyzed LC3 and mammalian target of rapamycin (mTOR), a suppressor of autophagy, by immunoassays. The level of LC3-II, which is known to be correlated with the extent of autophagosome formation, was increased in SOD1G93A transgenic mice at symptomatic stage compared with non-transgenic or human wild-type SOD1 transgenic animals. Moreover, the ratio of phosphorylated mTOR/Ser2448 immunopositive motor neurons to total motor neurons was decreased in SOD1G93A-Tg mice. The present data show the possibility of increased autophagy in an animal model for ALS. And autophagy may be partially regulated by an mTOR signaling pathway in these animals. [Copyright &y& Elsevier]

The incidence of amyotrophic lateral sclerosis (ALS) is higher among men than women but rises in women after the menopause. Estrogens may play a protective role. Treatment with estrogens has been shown to be neuroprotective in models of several neurodegenerative diseases. We therefore determined the effect of ovariectomy on female G93A mSOD1 transgenic mice, and the effect of subsequent treatment with 17β-estradiol (E2). Ovariectomy led to a significant acceleration of disease progression of the mice, and high-dose E2 treatment significantly delayed disease progression of ovariectomized G93A mSOD1 transgenic mice. We conclude that treatment with E2 may also delay disease progression of post-menopausal women with ALS. [Copyright &y& Elsevier]