Martina Svensson

Autophagy is increased in laminin α2 chain-deficient muscle and its inhibition improves muscle morphology in a mouse model of MDC1A

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

Effects of Physical Exercise on Neuroinflammation, Neuroplasticity, Neurodegeneration, and Behavior What We Can Learn From Animal Models in Clinical Settings

Physical exercise is a cornerstone in the management of many neurodegenerative disorders, such as Parkinson’s disease, dementia, and stroke. However, much of its beneficial effects on improving motor functions and cognition as well as decreasing neurodegeneration and neuroinflammation are not yet well understood. The obvious limitations of studying the protective mechanisms behind exercise, for example, brain plasticity and neurodegeneration, could be overcome by generating novel animal models of neurodegenerative disorders. In this narrative review, we discuss the beneficial effects of exercise performed in animal models of neurodegenerative disorders and how the results from animal studies can be used in clinical settings. From preclinical studies, the positive effects of exercise have been related to increased levels of neurotrophic factors, elevated expression of anti-inflammatory cytokines, and reduced levels of pro-inflammatory cytokines and activated microglia. It is clear that parameters influencing the effect of exercise, such as intensity, still remain to be investigated in animal studies in order to find the optimal program that can be translated into exercise interventions for patients with neurodegenerative diseases.

Genetic ablation of soluble tumor necrosis factor with preservation of membrane tumor necrosis factor is associated with neuroprotection after focal cerebral ischemia

Microglia respond to focal cerebral ischemia by increasing their production of the neuromodulatory cytokine tumor necrosis factor, which exists both as membrane-anchored tumor necrosis factor and as cleaved soluble tumor necrosis factor forms. We previously demonstrated that tumor necrosis factor knockout mice display increased lesion volume after focal cerebral ischemia, suggesting that tumor necrosis factor is neuroprotective in experimental stroke. Here, we extend our studies to show that mice with intact membrane-anchored tumor necrosis factor, but no soluble tumor necrosis factor, display reduced infarct volumes at one and five days after stroke. This was associated with improved functional outcome after experimental stroke. No changes were found in the mRNA levels of tumor necrosis factor and tumor necrosis factor-related genes (TNFR1, TNFR2, TACE), pro-inflammatory cytokines (IL-1β, IL-6) or chemokines (CXCL1, CXCL10, CCL2); however, protein expression of TNF, IL-1β, IL-6 and CXCL1 was reduced in membrane-anchored tumor necrosis factorΔ/Δ compared to membrane-anchored tumor necrosis factorwt/wt mice one day after experimental stroke. This was paralleled by reduced MHCII expression and a reduction in macrophage infiltration in the ipsilateral cortex of membrane-anchored tumor necrosis factorΔ/Δ mice. Collectively, these findings indicate that membrane-anchored tumor necrosis factor mediates the protective effects of tumor necrosis factor signaling in experimental stroke, and therapeutic strategies specifically targeting soluble tumor necrosis factor could be beneficial in clinical stroke therapy.

Forced treadmill exercise can induce stress and increase neuronal damage in a mouse model of global cerebral ischemia

Physical exercise is known to be a beneficial factor by increasing the cellular stress tolerance. In ischemic stroke, physical exercise is suggested to both limit the brain injury and facilitate behavioral recovery. In this study we investigated the effect of physical exercise on brain damage following global cerebral ischemia in mice. We aimed to study the effects of 4.5 weeks of forced treadmill running prior to ischemia on neuronal damage, neuroinflammation and its effect on general stress by measuring corticosterone in feces. We subjected C57bl/6 mice (n = 63) to either treadmill running or a sedentary program prior to induction of global ischemia. Anxious, depressive, and cognitive behaviors were analyzed. Stress levels were analyzed using a corticosterone ELISA. Inflammatory and neurological outcomes were analyzed using immunohistochemistry, multiplex electrochemoluminescence ELISA and Western blot. To our surprise, we found that forced treadmill running induced a stress response, with increased anxiety in the Open Field test and increased levels of corticosterone. In accordance, mice subjected to forced exercise prior to ischemia developed larger neuronal damage in the hippocampus and showed higher cytokine levels in the brain and blood compared to non-exercised mice. The extent of neuronal damage correlated with increased corticosterone levels. To compare forced treadmill with voluntary wheel running, we used a different set of mice that exercised freely on running wheels. These mice did not show any anxiety or increased corticosterone levels. Altogether, our results indicate that exercise pre-conditioning may not be beneficial if the animals are forced to run as it can induce a detrimental stress response.

Fumarate decreases edema volume and improves functional outcome after experimental stroke

Background Oxidative stress and inflammation exacerbate tissue damage in the brain after ischemic stroke. Dimethyl‐fumarate (DMF) and its metabolite monomethyl‐fumarate (MMF) are known to stimulate anti‐oxidant pathways and modulate inflammatory responses. Considering these dual effects of fumarates, we examined the effect of MMF treatment after ischemic stroke in mice. Methods Permanent middle cerebral artery occlusion (pMCAO) was performed using adult, male C57BL/6 mice. Thirty minutes after pMCAO, 20 mg/kg MMF was administered intravenously. Outcomes were evaluated 6, 24 and 48 h after pMCAO. First, we examined whether a bolus of MMF was capable of changing expression of kelch‐like erythroid cell‐derived protein with CNC homology‐associated protein 1 (Keap1) and nuclear factor erythroid 2‐related factor (Nrf)2 in the infarcted brain. Next, we studied the effect of MMF on functional recovery. To explore mechanisms potentially influencing functional changes, we examined infarct volumes, edema formation, the expression of heat shock protein (Hsp)72, hydroxycarboxylic acid receptor 2 (Hcar2), and inducible nitric oxide synthase (iNOS) in the infarcted brain using real‐time PCR and Western blotting. Concentrations of a panel of pro‐ and anti‐inflammatory cytokines (IFN&ggr;, IL‐1&bgr;, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐12p70, TNF) were examined in both the infarcted brain tissue and plasma samples 6, 24 and 48 h after pMCAO using multiplex electrochemoluminiscence analysis. Results Administration of MMF increased the protein level of Nrf2 6 h after pMCAO, and improved functional outcome at 24 and 48 h after pMCAO. MMF treatment did not influence infarct size, however reduced edema volume at both 24 and 48 h after pMCAO. MMF treatment resulted in increased Hsp72 expression in the brain 6 h after pMCAO. Hcar2 mRNA levels increased significantly 24 h after pMCAO, but were not different between saline‐ and MMF‐treated mice. MMF treatment also increased the level of the anti‐inflammatory cytokine IL‐10 in the brain and plasma 6 h after pMCAO, and additionally reduced the level of the pro‐inflammatory cytokine IL‐12p70 in the brain at 24 and 48 h after pMCAO. Conclusions A single intravenous bolus of MMF improved sensory‐motor function after ischemic stroke, reduced edema formation, and increased the levels of the neuroprotective protein Hsp72 in the brain. The early increase in IL‐10 and reduction in IL‐12p70 in the brain combined with changes in systemic cytokine levels may also contribute to the functional recovery after pMCAO. HighlightsMonomethyl‐fumarate treatment decreased edema volume after stroke.Monomethyl‐fumarate treatment improved sensory‐motor function after stroke.Monomethyl‐fumarate treatment increased Hsp72 levels after stroke.Monomethyl‐fumarate treatment increased neuroprotective IL‐10 levels after stroke.Monomethyl‐fumarate treatment decreased IL‐12p70 levels after stroke.

BID Mediates Oxygen-Glucose Deprivation-Induced Neuronal Injury in Organotypic Hippocampal Slice Cultures and Modulates Tissue Inflammation in a Transient Focal Cerebral Ischemia Model without Changing Lesion Volume

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic protein involved in death receptor-induced and mitochondria-mediated apoptosis. Recently, it has also been suggested that BID is involved in the regulation of inflammatory responses in the central nervous system. We found that BID deficiency protected organotypic hippocampal slice cultures in vitro from neuronal injury induced by oxygen-glucose deprivation. In vivo, BID-knockout (KO) mice and wild type (WT) mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) to induce focal cerebral ischemia, and allowed to recover for 24 h. Infarct volumes and functional outcome were assessed and the inflammatory response was evaluated using immunofluorescence, Western blotting, quantitative PCR (qPCR) and Mesoscale multiplex analysis. We observed no difference in the infarct volume or neurological outcome between BID-KO and WT mice. The inflammatory response was reduced by BID deficiency as indicated by a change in microglial/leukocyte response. In conclusion, our data suggest that BID deficiency is neuroprotective in an in vitro model and modulates the inflammatory response to focal cerebral ischemia in vivo. However, this is not translated into a robust neuroprotection in vivo.

Potent pro-inflammatory and profibrotic molecules, osteopontin and galectin-3, are not major disease modulators of laminin α2 chaindeficient muscular dystrophy

A large number of human diseases are caused by chronic tissue injury with fibrosis potentially leading to organ failure. There is a need for more effective anti-fibrotic therapies. Congenital muscular dystrophy type 1A (MDC1A) is a devastating form of muscular dystrophy caused by laminin α2 chain-deficiency. It is characterized with early inflammation and build-up of fibrotic lesions, both in patients and MDC1A mouse models (e.g. dy3K/dy3K). Despite the enormous impact of inflammation on tissue remodelling in disease, the inflammatory response in MDC1A has been poorly described. Consequently, a comprehensive understanding of secondary mechanisms (impaired regeneration, enhanced fibrosis) leading to deterioration of muscle phenotype in MDC1A is missing. We have monitored inflammatory processes in dy3K/dy3K muscle and created mice deficient in laminin α2 chain and osteopontin or galectin-3, two pro-inflammatory and pro-fibrotic molecules drastically increased in dystrophic muscle. Surprisingly, deletion of osteopontin worsened the phenotype of dy3K/dy3K mice and loss of galectin-3 did not reduce muscle pathology. Our results indicate that osteopontin could even be a beneficial immunomodulator in MDC1A. This knowledge is essential for the design of future therapeutic interventions for muscular dystrophies that aim at targeting inflammation, especially that osteopontin inhibition has been suggested for Duchenne muscular dystrophy therapy.

Focal, but not global, cerebral ischaemia causes loss of myenteric neurons and upregulation of vasoactive intestinal peptide in mouse ileum

Reduced blood flow to the brain induces cerebral ischaemia, potentially causing central injury and peripheral complications including gastrointestinal (GI) dysfunction. The pathophysiology behind GI symptoms is suspected to be neuropathy in the enteric nervous system (ENS), which is essential in regulating GI function. This study investigates if enteric neuropathy occurs after cerebral ischaemia, by analysing neuronal survival and relative numbers of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) expressing neurons in mouse ileum after three types of cerebral ischaemia. Focal cerebral ischaemia, modelled by permanent middle cerebral artery occlusion (pMCAO) and global cerebral ischaemia, modelled with either transient occlusion of both common carotid arteries followed by reperfusion (GCIR) or chronic cerebral hypoperfusion (CCH) was performed on C56BL/6 mice. Sham‐operated mice for each ischaemia model served as control. Ileum was collected after 1–17 weeks, depending on model, and analysed using morphometry and immunocytochemistry. For each group, intestinal mucosa and muscle layer thicknesses, neuronal numbers and relative proportions of neurons immunoreactive (IR) for nNOS or VIP were estimated. No alterations in mucosa or muscle layer thicknesses were noted in any of the groups. Loss of myenteric neurons and an increased number of VIP‐IR submucous neurons were found in mouse ileum 7 days after pMCAO. None of the global ischaemia models showed any alterations in neuronal survival or relative numbers of VIP‐ and nNOS‐IR neurons. We conclude that focal cerebral ischaemia and global cerebral ischaemia influence enteric neuronal survival differently. This is suggested to reflect differences in peripheral neuro‐immune responses.