Anke Witting

The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells.

Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.

Astrocytes in culture produce anandamide and other acylethanolamides

Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-γ-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.

Phagocytic clearance of apoptotic neurons by Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition.

Abstract: Microglia, the tissue macrophages of the brain, play a crucial role in recognition and phagocytic removal of apoptotic neurons. The microglial receptors for recognition of apoptotic neurons are not yet characterized. Here we established a co‐culture model of primary microglia and cerebellar granule neurons to examine the receptor systems involved in recognition/uptake of apoptotic neurons. Treatment with 100 μM S‐nitrosocysteine induced apoptosis of cerebellar neurons as indicated by nuclear condensation and phosphatidylserine exposure to the exoplasmic leaflet of the plasma membrane. Microglial cells were added to neurons 2 h after apoptosis induction and co‐cultured for 6 h in the presence of ligands that inhibit recognition by binding to respective receptors. Binding/phagocytosis was determined after combined 4′,6‐diamidino‐2‐phenylindole/propidium iodide (for apoptotic/necrotic neurons) and lectin staining (for microglia). Uptake of apoptotic neurons was reduced by N‐acetylglucosamine or galactose, suggesting that recognition involves asialoglycoprotein‐like lectins. Furthermore, the inhibition of microglial binding/uptake of apoptotic neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As microglia selectively bind lipid vesicles enriched in phosphatidylserine and O‐phospho‐L‐serine interfered with the uptake of apoptotic neurons, an involvement of phosphatidylserine receptor is rather likely. Apoptotic neurons do not release soluble signals that serve to attract or activate microglia. Collectively, these results suggest that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia.

Endocannabinoids accumulate in spinal cord of SOD1G93A transgenic mice

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in the super oxide dismutase 1 (SOD1) gene and transgenic mice for these mutations recapitulate many features of this devastating neurodegenerative disease. Here we show that the amount of anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), two endocannabinoids that have neuroprotective properties, increase in spinal cord of SOD1G93A transgenic mice. This increase occurs in the lumbar section of spinal cords, the first section to undergo neurodegeneration, and is significant before overt motor impairment. Our results show that chronic neurodegeneration induced by a genetic mutation increases endocannabinoid production possibly as part of an endogenous defense mechanism.

Cannabinol delays symptom onset in SOD1 (G93A) transgenic mice without affecting survival

Therapeutic options for amyotrophic lateral sclerosis (ALS), the most common adult‐onset motor neuron disorder, remain limited. Emerging evidence from clinical studies and transgenic mouse models of ALS suggests that cannabinoids, the bioactive ingredients of marijuana (Cannabis sativa) might have some therapeutic benefit in this disease. However, Δ9‐tetrahydrocannabinol (Δ9‐THC), the predominant cannabinoid in marijuana, induces mind‐altering effects and is partially addictive, compromising its clinical usefulness. We therefore tested whether cannabinol (CBN), a non‐psychotropic cannabinoid, influences disease progression and survival in the SOD1 (G93A) mouse model of ALS. CBN was delivered via subcutaneously implanted osmotic mini‐pumps (5 mg/kg/day) over a period of up to 12 weeks. We found that this treatment significantly delays disease onset by more than two weeks while survival was not affected. Further research is necessary to determine whether non‐psychotropic cannabinoids might be useful in ameliorating symptoms in ALS.

Microglia Cell Culture: A Primer for the Novice

Microglial cells are the resident immune cells of the central nervous system. Progress in the recent decade has clearly established that microglial cells participate or even actively drive neurological disease. Much of our current knowledge has been generated by investigating microglial cells in cell culture. The aim of this chapter is to give the uninitiated a basic and adaptable protocol for the culturing of microglial cells. We discuss the challenges of microglial cell culture and provide a collection of tips which reflect our 25+ years of collective experience.

Fumaric Acid Esters Stimulate Astrocytic VEGF Expression through HIF-1α and Nrf2

Fumaric acid esters (FAE) are oral analogs of fumarate that have recently been shown to decrease relapse rate and disease progression in multiple sclerosis (MS), prompting to investigate their protective potential in other neurological diseases such as amyotrophic lateral sclerosis (ALS). Despite efficacy in MS, mechanisms of action of FAEs are still largely unknown. FAEs are known to activate the transcription factor Nrf2 and downstream anti-oxidant responses through the succination of Nrf2 inhibitor KEAP1. However, fumarate is also a known inhibitor of prolyl-hydroxylases domain enzymes (PhD), and PhD inhibition might lead to stabilization of the HIF-1α transcription factor under normoxic conditions and subsequent activation of a pseudo hypoxic response. Whether Nrf2 activation is associated with HIF-1α stabilization in response to FAEs in cell types relevant to MS or ALS remains unknown. Here, we show that FAEs elicit HIF-1α accumulation, and VEGF release as its expected consequence, in astrocytes but not in other cell types of the central nervous system. Reporter assays demonstrated that increased astrocytic VEGF release in response to FAEs was dependent upon both HIF-1α and Nrf2 activation. Last, astrocytes of transgenic mice expressing SOD1(G93A), an animal model of ALS, displayed reduced VEGF release in response to FAEs. These studies show that FAEs elicit different signaling pathways in cell types from the central nervous system, in particular a pseudo-hypoxic response in astrocytes. Disease relevant mutations might affect this response.

Mutual exacerbation of PGC-1α deregulation and α-synuclein oligomerization

Aggregation of α‐synuclein (α‐syn) and α‐syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC‐1α in mitochondria, no studies have addressed whether PGC‐1α directly influences oligomerization of α‐syn or whether α‐syn oligomers impact PGC‐1α expression.

High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington's disease expansion mutation carriers shows normal mitochondrial respiratory function

Alterations in mitochondrial respiration are an important hallmark of Huntington’s disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.

B30 Integrated mitochondrial function in human fine-needle muscle biopsies of huntington’s disease mutation carriers and in tissues of HdhQ111 mice

The neurodegenerative genetic disorder of Huntington’s disease (HD) is characterised by mitochondrial impairments of the respiratory chain. The ubiquitous expression of the disease causing mutant huntingtin gene raises the question to which extent changes in mitochondrial respiration are evident in the human skeletal muscle. In addition characterisation of mitochondrial respiration in the muscle might allow conclusions about the respiratory status in the brain. The integrated respiratory chain function of the human quadriceps vastus lateralis was measured by high-resolution respirometry in fine-needle biopsies of four pre-symptomatic HD mutation carriers and seven controls. The respiratory parameters indicated a trend towards a reduction in the respiratory control ratio (RCR) of the HD carriers. In parallel, murine cortex, liver, soleus muscle and heart of male HD knock-in mice (HdhQ111), were examined by the same method. Significant changes of the respiration were restricted to the liver and the cortex. In addition mitochondrial DNA copy number and citrate synthase activity were determined to quantify the mitochondrial mass, showing no differences. From the murine tissues mRNA levels of key enzymes characterised the mitochondrial metabolic pathways. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that differences in respiratory parameters of pre-symptomatic human muscle biopsies are rather limited, which is confirmed by the analysis of murine skeletal muscle tissue. The murine cortex and liver turned out to show respiratory changes in the HdhQ111 mouse model, which indicates that respiratory capacities are different between tissues. Acknowledgement The work is supported by the research training group cellular and molecular mechanisms in ageing (CEMMA, GRK1789) which is funded by the DFG and the seed fund grant of the EINSTEIN study no 584/14 from the EHDN.