Lesley Probert

Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis.

We have generated transgenic mouse lines carrying and expressing wild‐type and 3′‐modified human tumour necrosis factor (hTNF‐alpha, cachectin) transgenes. We show that correct, endotoxin‐responsive and macrophage‐specific hTNF gene expression can be established in transgenic mice and we present evidence that the 3′‐region of the hTNF gene may be involved in macrophage‐specific transcription. Transgenic mice carrying 3′‐modified hTNF transgenes shows deregulated patterns of expression and interestingly develop chronic inflammatory polyarthritis. Treatment of these arthritic mice with a monoclonal antibody against human TNF completely prevents development of this disease. Our results indicate a direct involvement of TNF in the pathogenesis of arthritis. Transgenic mice which predictably develop arthritis represent a novel genetic model by which the pathogenesis and treatment of this disease in humans may be further investigated.

Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice models for multiple sclerosis with primary oligodendrogliopathy

The scientific dogma that multiple sclerosis (MS) is a disease caused by a single pathogenic mechanism has been challenged recently by the heterogeneity observed in MS lesions and the realization that not all patterns of demyelination can be modeled by autoimmune-triggered mechanisms. To evaluate the contribution of local tumor necrosis factor (TNF) ligand/receptor signaling pathways to MS immunopathogenesis we have analyzed disease pathology in central nervous system-expressing TNF transgenic mice, with or without p55 or p75TNF receptors, using combined in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling and cell identification techniques. We demonstrate that local production of TNF by central nervous system glia potently and selectively induces oligodendrocyte apoptosis and myelin vacuolation in the context of an intact blood-brain barrier and absence of immune cell infiltration into the central nervous system parenchyma. Interestingly, primary demyelination then develops in a classical manner in the presence of large numbers of recruited phagocytic macrophages, possibly the result of concomitant pro-inflammatory effects of TNF in the central nervous system, and lesions progress into acute or chronic MS-type plaques with axonal damage, focal blood-brain barrier disruption, and considerable oligodendrocyte loss. Both the cytotoxic and inflammatory effects of TNF were abrogated in mice genetically deficient for the p55TNF receptor demonstrating a dominant role for p55TNF receptor-signaling pathways in TNF-mediated pathology. These results demonstrate that aberrant local TNF/p55TNF receptor signaling in the central nervous system can have a potentially major role in the aetiopathogenesis of MS demyelination, particularly in MS subtypes in which oligodendrocyte death is a primary pathological feature, and provide new models for studying the basic mechanisms underlying oligodendrocyte and myelin loss.

TNF-α transgenic and knockout models of CNS inflammation and degeneration

Tumour necrosis factor-alpha (TNF-alpha) plays a central role in inflammatory events including those taking place in the central nervous system (CNS), and has been implicated as a key pathogenic mediator in several human inflammatory, infectious and autoimmune CNS disorders. Using transgenic and gene knockout mice we have investigated the role of deregulated TNF-alpha production in the CNS. We show that the overexpression of wild-type murine or human TNF-alpha transgenes by resident CNS astrocytes or neurons in sufficient to trigger a neurological disorder characterised by ataxia, seizures and paresis, with histopathological features of chronic CNS inflammation and white matter degeneration. Furthermore, we show that transmembrane human TNF-alpha is sufficient to trigger CNS inflammation and degeneration when overexpressed by astrocytes but not by neurons, indicating that target cells mediating the neuroinflammatory activities of TNF-alpha localise in the vicinity of astrocytes rather than neurons. Our results establish that both soluble and transmembrane molecular forms of TNF-alpha can play critical roles in vivo in the pathogenesis of CNS inflammation and demyelination, and validate TNF-alpha transgenic and mutant mice as important models for the further study of related human CNS diseases.

Increased Citrullination of Histone H3 in Multiple Sclerosis Brain and Animal Models of Demyelination: A Role for Tumor Necrosis Factor-Induced Peptidylarginine Deiminase 4 Translocation

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-α (TNF-α) protein. The elevated TNF-α in MS NAWM was not associated with CD3+ or CD8+ lymphocytes, nor was it associated with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-α may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-α treatment induced its nuclear translocation. To address the involvement of TNF-α in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-α also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.

Ischemic Neuronal Damage

Knowledge of the molecular mechanisms that underlie neuron death following stroke is important to allow the development of effective neuroprotective strategies. Since studies in human stroke are extremely limited due to the inability of collecting post mortem tissue at time points after the onset of stroke where neuronal death occurs, brain ischemia research focuses on information derived from animal models of ischemic injury. The two principal models for human stroke are induced in rodents either by global or focal ischemia. In both cases, blood flow disruptions limit the delivery of oxygen and glucose to neurons causing ATP reduction and energy depletion, initiating excitotoxic mechanisms that are deleterious for neurons. These include activation of glutamate receptors and release of excess glutamate in the extracellular space inducing neuron depolarisation and dramatic increase of intracellular calcium that in turn activates multiple intracellular death pathways. The notion that excitotoxicity leads only to neuron necrosis has been abandoned, as ultrastructural and biochemical analysis have shown signs of apoptotic and autophagic cell death in ischemic neurons and this has been further confirmed in neurons subjected to in vitro ischemia models. Both in vitro and in vivo studies, targeting a single death mechanism either by the inhibition of death-inducing molecules or the overexpression of antiapoptotic components in neurons, have shown tremendous neuroprotective potential. Despite their effectiveness in preclinical studies, a large number of neuroprotectants have failed in clinical trials for stroke suggesting that we still lack essential knowledge on the triggers and mediators of ischemic neuron death. In this review evidence will be presented on how ischemic injury occurs, what death mechanisms are activated and how these can be manipulated to induce neuroprotection.

Learning abilities, NGF and BDNF brain levels in two lines of TNF-α transgenic mice, one characterized by neurological disorders, the other phenotypically normal

In this study we used two lines of transgenic mice overexpressing tumor necrosis factor alpha (TNF-alpha) in the central nervous system (CNS), one characterized by reactive gliosis, inflammatory demyelination and neurological deficits (Tg6074) the other showing no neurological or phenotypical alterations (TgK3) to investigate the effect of TNF-alpha on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) levels and learning abilities. The results showed that the amount of NGF in the brain of Tg6074 and TgK3 transgenic mice is low in the hippocampus and in the spinal cord, increases in the hypothalamus of Tg6074 and showed no significant changes in the cortex. BDNF levels were low in the hippocampus and spinal cord of TgK3. BDNF increased in the hypothalamus of TgK3 and Tg6074 while in the cortex, BDNF increased only in Tg6074 mice. Transgenic mice also had memory impairments as revealed by the Morris Water Maze test. These findings indicate that TNF-alpha significantly influences BDNF and NGF synthesis, most probably in a dose-dependent manner. Learning abilities were also differently affected by overexpression of TNF-alpha, but were not associated with inflammatory activity. The possible functional implications of our findings are discussed.

TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.

TNF accelerates the onset but does not alter the incidence and severity of myelin basic protein-induced experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) induction in TNF gene‐targeted mice has resulted in conflicting reports in part due to the strong association of TNF with the MHC locus. To define the participation of TNF in EAE development, we back‐crossed TNF‐deficient mice (H‐2b) into the SJL/J strain and directly compared them to H‐2b congenic SJL or inbred SJL/J mice. Induction of EAE with myelin basic protein (MBP) revealed that H‐2b congenic SJL mice are fully susceptible, indicating that the H‐2b haplotype does not affect disease susceptibility. Using H‐2b congenic SJL mice we show here that TNF deficiency modifies the normal course of EAE by considerably delaying the onset for approximately 5 days, suggesting that TNF is required for the normal initiation of MBP‐induced EAE. However, TNF‐deficient mice eventually developed severe EAE with perivascular inflammation and primary demyelination similar to wild‐type controls, indicating that TNF is not essential during these processes. Taken together, these results indicate that although TNF is not required for the progression of MBP‐induced EAE, it contributes positively by advancing the onset of disease.

Exclusive tumor necrosis factor (TNF) signaling by the p75TNF receptor triggers inflammatory ischemia in the CNS of transgenic mice

Tumor necrosis factor (TNF) is up-regulated in a variety of central nervous system (CNS) diseases with diverse etiology and pathologic manifestation. TNF mediates multiple biological activities through two membrane receptors, the p55 and p75 TNF receptors (TNFRs). We have shown previously that human transmembrane TNF (tmTNF)/p55TNFR signaling in transgenic mice triggers oligodendrocyte apoptosis, endothelial cell activation, parenchymal inflammation, and primary demyelinating lesions similar to those of acute multiple sclerosis. To address the role of the p75TNFR in the CNS, we have generated “humanized” mice that express human tmTNF in astrocytes and a physiologically regulated human p75TNFR transgene, in the absence of the endogenous (murine) p55TNFR. Human tmTNF/p75TNFR transgenic mice develop CNS vascular pathology, characterized by endothelial cell activation, meningeal inflammation, and vessel fibrosis. There is no evidence of oligodendrocyte apoptosis or primary demyelination in these mice. Late in disease, vasculitis can result in vessel occlusion and secondary, multifocal CNS ischemic injury. These results identify a proinflammatory role for the p75TNFR at the level of the CNS vascular endothelium, which correlates with the expression pattern of this receptor in the CNS, and indicate that the differential expression patterns of the two TNFRs within the CNS play a significant role in shaping the outcome of TNF signaling during neuroimmune interactions.

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4+ T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8+ T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4+ T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.