Protein disulfide isomerase in ALS mouse glia links protein misfolding with NADPH oxidase-catalyzed superoxide production.

Protein disulfide isomerase (PDI) is an oxidoreductase assisting oxidative protein folding in the endoplasmic reticulum of all types of cells, including neurons and glia. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), up-regulation of PDI is an important part of unfolded protein response (UPR) that is thought to represent an adaption reaction and thereby protect the neurons. Importantly, studies on animal models of familial ALS with mutant Cu/Zn superoxide dismutase 1 (SOD1) have shown that the mutant SOD1 in astrocytes or microglia strongly regulates the progression of the disease. Here, we found an early up-regulation of PDI in microglia of transgenic (tg) mutant SOD1 mice, indicating that in addition to neurons, UPR takes place in glial cells in ALS. The observation was supported by the finding that also the expression of a UPR marker GADD34 (growth arrest and DNA damage-inducible protein) was induced in the spinal cord glia of tg mutant SOD1 mice. Because mutant SOD1 can cause sustained activation of NADPH oxidase (NOX), we investigated the role of PDI in UPR-induced NOX activation in microglia. In BV-2 microglia, UPR resulted in NOX activation with increased production of superoxide and increased release of tumor necrosis factor-α. The phenomenon was recapitulated in primary rat microglia, murine macrophages and human monocytes. Importantly, pharmacological inhibition of PDI or its down-regulation by short interfering RNAs prevented NOX activation in microglia and subsequent production of superoxide. Thus, results strongly demonstrate that UPR, caused by protein misfolding, may lead to PDI-dependent NOX activation and contribute to neurotoxicity in neurodegenerative diseases including ALS.

Immunomodulation by interleukin-33 is protective in stroke through modulation of inflammation.

Cerebral stroke induces massive Th1-shifted inflammation both in the brain and the periphery, contributing to the outcome of stroke. A Th1-type response is neurotoxic whereas a Th2-type response is accompanied by secretion of anti-inflammatory cytokines, such as interleukin-4 (IL-4). Interleukin-33 (IL-33) is a cytokine known to induce a shift towards the Th2-type immune response, polarize macrophages/microglia towards the M2-type, and induce production of anti-inflammatory cytokines. We found that the plasma levels of the inhibitory IL-33 receptor, sST2, are increased in human stroke and correlate with a worsened stroke outcome, suggesting an insufficient IL-33-driven Th2-type response. In mouse, peripheral administration of IL-33 reduced stroke-induced cell death and improved the sensitivity of the contralateral front paw at 5days post injury. The IL-33-treated mice had increased levels of IL-4 in the spleen and in the peri-ischemic area of the cortex. Neutralization of IL-4 by administration of an IL-4 antibody partially prevented the IL-33-mediated protection. IL-33 treatment also reduced astrocytic activation in the peri-ischemic area and increased the number of Arginase-1 immunopositive microglia/macrophages at the lesion site. In human T-cells, IL-33 treatment induced IL-4 secretion, and the conditioned media from IL-33-exposed T-cells reduced astrocytic activation. This study demonstrates that IL-33 is protective against ischemic insult by induction of IL-4 secretion and may represent a novel therapeutic approach for the treatment of stroke.

In depth behavioral phenotyping unravels complex motor disturbances in Cstb-/- mouse, a model for progressive myoclonus epilepsy type 1.

Progressive myoclonus epilepsy type 1 (EPM1) is an autosomal recessively inherited childhood-adolescence onset neurodegenerative disease caused by mutations in the cystatin B (CSTB gene). The key clinical manifestation in EPM1 is progressive, stimulus-sensitive, in particular action-induced myoclonus. The cystatin B-deficient mouse model, Cstb-/-, has been described to present with myoclonic seizures and progressive ataxia. Here we describe results from in-depth behavioral phenotyping of the Cstb-/- mouse model in pure isogenic 129S2/SvHsd background covering ages from 1.5 to 6 months. We developed a method for software-assisted detection of myoclonus from video recordings of the Cstb-/- mice. Additionally, we observed that the mice were hyperactive and showed reduced startle response, problems in motor coordination and lack of inhibition. We were, however, not able to demonstrate an ataxic phenotype in them. This detailed behavioral phenotyping of the Cstb-/- mice reveals new aspects of this mouse model. The nature of the motor problems in the Cstb-/- mice seems to be more complex and more resembling the human phenotype than initially described.

Production of monocytic cells from bone marrow stem cells: therapeutic usage in Alzheimer's disease.

Accumulation of amyloid ß (Aß) is a major hallmark in Alzheimer's disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aß burden in mouse models of AD, alleviating the AD pathology. BMM have been shown to be more efficient phagocytes in AD than the endogenous brain microglia. Because BMM have a natural tendency to infiltrate into the injured area, they could be regarded as optimal candidates for cell-based therapy in AD. In this study, we describe a method to obtain monocytic cells from BM-derived haematopoietic stem cells (HSC). Mouse or human HSC were isolated and differentiated in the presence of macrophage colony stimulating factor (MCSF). The cells were characterized by assessing the expression profile of monocyte markers and cytokine response to inflammatory stimulus. The phagocytic capacity was determined with Aß uptake assay in vitro and Aß degradation assay of natively formed Aß deposits ex vivo and in a transgenic APdE9 mouse model of AD in vivo. HSC were lentivirally transduced with enhanced green fluorescent protein (eGFP) to determine the effect of gene modification on the potential of HSC-derived cells for therapeutic purposes. HSC-derived monocytic cells (HSCM) displayed inflammatory responses comparable to microglia and peripheral monocytes. We also show that HSCM contributed to Aß reduction and could be genetically modified without compromising their function. These monocytic cells could be obtained from human BM or mobilized peripheral blood HSC, indicating a potential therapeutic relevance for AD.

Granulocyte colony stimulating factor attenuates inflammation in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery. METHODS: Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro. RESULTS: Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS. CONCLUSIONS: GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS.

Restoration of histone acetylation ameliorates disease and metabolic abnormalities in a FUS mouse model.

Dysregulation of epigenetic mechanisms is emerging as a central event in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). In many models of neurodegeneration, global histone acetylation is decreased in the affected neuronal tissues. Histone acetylation is controlled by the antagonistic actions of two protein families -the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). Drugs inhibiting HDAC activity are already used in the clinic as anti-cancer agents. The aim of this study was to explore the therapeutic potential of HDAC inhibition in the context of ALS. We discovered that transgenic mice overexpressing wild-type FUS ("Tg FUS+/+"), which recapitulate many aspects of human ALS, showed reduced global histone acetylation and alterations in metabolic gene expression, resulting in a dysregulated metabolic homeostasis. Chronic treatment of Tg FUS+/+ mice with ACY-738, a potent HDAC inhibitor that can cross the blood-brain barrier, ameliorated the motor phenotype and substantially extended the life span of the Tg FUS+/+ mice. At the molecular level, ACY-738 restored global histone acetylation and metabolic gene expression, thereby re-establishing metabolite levels in the spinal cord. Taken together, our findings link epigenetic alterations to metabolic dysregulation in ALS pathology, and highlight ACY-738 as a potential therapeutic strategy to treat this devastating disease.

Long-term interleukin-33 treatment delays disease onset and alleviates astrocytic activation in a transgenic mouse model of amyotrophic lateral sclerosis.

Inflammation is a prominent feature of the neuropathology of amyotrophic lateral sclerosis (ALS). Emerging evidence suggests that inflammatory cascades contributing to the disease progression are not restricted to the central nervous system (CNS) but also occur peripherally. Indeed, alterations in T cell responses and their secreted cytokines have been detected in ALS patients and in animal models of ALS. One key cytokine responsible for the shift in T cell responses is interleukin-33 (IL-33), which stimulates innate type 2 immune cells to produce a large amount of Th2 cytokines that are possibly beneficial in the recovery processes of CNS injuries. Since the levels of IL-33 have been shown to be decreased in patients affected with ALS, we sought to determine whether a long-term recombinant IL-33 treatment of a transgenic mouse model of ALS expressing G93A-superoxide dismutase 1 (SOD1-G93A) alters the disease progression and ameliorates the ALS-like disease pathology. SOD1-G93A mice were treated with intraperitoneal injections of IL-33 and effects on disease onset and inflammatory status were determined. Spinal cord (SC) neurons, astrocytes and T-cells were exposed to IL-33 to evaluate the cell specific responses to IL-33. Treatment of SOD1-G93A mice with IL-33 delayed the disease onset in female mice, decreased the proportion of CD4+ and CD8 + T cell populations in the spleen and lymph nodes, and alleviated astrocytic activation in the ventral horn of the lumbar SC. Male SOD1-G93A mice were unresponsive to the treatment. In vitro studies showed that IL-33 is most likely not acting directly on neurons and astrocytes, but rather conveying its effects through peripheral T-cells. Our results suggest that strategies directed to the peripheral immune system may have therapeutic potential in ALS. The effect of gender dimorphisms to the treatment efficacy needs to be taken into consideration when designing new therapeutic strategies for CNS diseases.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

CuII(atsm) Attenuates Neuroinflammation.

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer's disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation. Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex CuII(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro. Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of CuII(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). CuII(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes. Conclusion: The beneficial effects of CuII(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.

Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy.

Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene.

The role of oxidative stress in degeneration of the neuromuscular junction in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motoneurons and degradation of the neuromuscular junctions (NMJ). Consistent with the dying-back hypothesis of motoneuron degeneration the decline in synaptic function initiates from the presynaptic terminals in ALS. Oxidative stress is a major contributory factor to ALS pathology and affects the presynaptic transmitter releasing machinery. Indeed, in ALS mouse models nerve terminals are sensitive to reactive oxygen species (ROS) suggesting that oxidative stress, along with compromised mitochondria and increased intracellular Ca(2+) amplifies the presynaptic decline in NMJ. This initial dysfunction is followed by a neurodegeneration induced by inflammatory agents and loss of trophic support. To develop effective therapeutic approaches against ALS, it is important to identify the mechanisms underlying the initial pathological events. Given the role of oxidative stress in ALS, targeted antioxidant treatments could be a promising therapeutic approach. However, the complex nature of ALS and failure of monotherapies suggest that an antioxidant therapy should be accompanied by anti-inflammatory interventions to enhance the restoration of the redox balance.

Deregulation of biometal homeostasis: the missing link for neuronal ceroid lipofuscinoses?

Neuronal ceroid lipofuscinoses (NCLs), a group of genetically distinct fatal neurodegenerative disorders with no treatment or cure, are clinically characterised by progressive motor and visual decline leading to premature death. While the underlying pathological mechanisms are yet to be precisely determined, the diseases share several common features including inflammation, lysosomal lipofuscin deposits and lipid abnormalities. An important hallmark of most common neurodegenerative disorders including Alzheimer's, Parkinson's and motor neuron diseases is deregulation of biologically active metal homeostasis. Metals such as zinc, copper and iron are critical enzyme cofactors and are important for synaptic transmission in the brain, but can mediate oxidative neurotoxicity when homeostatic regulatory mechanisms fail. We previously demonstrated biometal accumulation and altered biometal transporter expression in 3 animal models of CLN6 NCL disease. In this study we investigated the hypothesis that altered biometal homeostasis may be a feature of NCLs in general using 3 additional animal models of CLN1, CLN3 and CLN5 disease. We demonstrated significant accumulation of the biometals zinc, copper, manganese, iron and cobalt in these mice. Patterns of biometal accumulation in each model preceded significant neurodegeneration, and paralleled the relative severity of disease previously described for each model. Additionally, we observed deregulation of transcripts encoding the anti-oxidant protein, metallothionein (Mt), indicative of disruptions to biometal homeostasis. These results demonstrate that altered biometal homeostasis is a key feature of at least 4 genetically distinct forms of NCL disease.

Gender-Specific Mechanism of Synaptic Impairment and Its Prevention by GCSF in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motoneurons which progresses differentially in males and females for unknown reason. Here we measured gender differences in pre- and post-synaptic parameters of the neuromuscular transmission in a mutant G93A-SOD1 mouse model of ALS. Using intracellular microelectrode technique we recorded miniature and evoked end-plate potentials (MEPPs and EPPs) in the diaphragm muscle of G93A-SOD1 mice at early symptomatic stage. While no evident alterations in the amplitude of MEPPs was observed in male or female G93A-SOD1 mice, G93A-SOD1 mice displayed dramatically reduced probability of spontaneous acetylcholine release. In contrast, the EPPs evoked by single nerve stimulation had unchanged amplitude and quantal content. In males, but not females, this was accompanied by reduced readily releasable transmitter pool. Transmitter release in both sexes was sensitive to the inhibitory action of reactive oxygen species (ROS), but the production of ROS was increased in the spinal cords of male but not female G93A-SOD1 mice. Treatment with granulocyte colony stimulating factor (GCSF), which we previously found to be beneficial in males, attenuated the increased ROS production indicating involvement of the antioxidant mechanisms and improved ALS-induced synaptic dysfunctions only in males being ineffective in females. Consistent with our findings at synaptic level, GCSF did not change the survival rate or motor performance of female ALS mice. In summary, neuromuscular transmission in ALS mice is impaired at early symptomatic stage when a dramatic presynaptic decline of spontaneous release occurs. Beneficial effects of GCSF treatment on survival in males may be explained by GCSF-improved presynaptic functions in male G93A-SOD1 mice. Development of efficient treatment strategies for ALS may need to be directed in a gender-specific manner.