Tony Frugier

In Situ Detection of Inflammatory Mediators in Post Mortem Human Brain Tissue after Traumatic Injury

Little is known about the molecular events following severe traumatic brain injury (TBI) in humans and to date there are no efficient therapies for the treatment of patients. In this study, the first of its kind in human tissue, a total of 21 post mortem trauma brain samples were analyzed. The inflammatory response within the brain tissue was explored by measuring the expression of various inflammatory cytokines at the mRNA and protein levels. These mediators were interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study shows for the first time in human brain tissue that 1) pro-inflammatory mediator protein levels are significantly increased in situ following acute brain injury while anti-inflammatory cytokines protein levels remain unchanged; 2) the cerebral inflammatory response begins within minutes of acute TBI, much earlier than previously thought; 3) IL-6, IL-8, TNF-alpha, and IL-1beta mRNA levels are significantly increased following injury; 4) the rise in cytokine protein level coincides with increased levels of their mRNAs suggesting that traumatic injury elicits an immediate cerebral inflammatory response. Altogether these data confirm and extend previous observations on the release of cytokines in the cerebrospinal fluid of severe TBI patients. Finally, this study highlights the need to characterize the cell source of cytokines and elucidate their mode of action.

The molecular bases of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common recessive autosomal disorder characterized by degeneration of motor neurons of the spinal cord. SMA is caused by mutations of the survival of motor neuron gene that encodes a multifunctional protein, and mouse models have been generated. These advances represent starting points towards an understanding of the pathophysiology of this disease and the design of therapeutic strategies in SMA.

Spinal muscular atrophy: Recent advances and future prospects

Spinal muscular atrophies (SMA) are characterized by degeneration of lower motor neurons associated with muscle paralysis and atrophy. Childhood SMA is a frequent recessive autosomal disorder and represents one of the most common genetic causes of death in childhood. Mutations of the SMN1 gene are responsible for SMA. The knowledge of the genetic basis of SMA, a better understanding of SMN function, and the recent generation of SMA mouse models represent major advances in the field of SMA. These are starting points towards understanding the pathophysiology of SMA and developing therapeutic strategies for this devastating neurodegenerative disease, for which no curative treatment is known so far.

A new large animal model of CLN5 neuronal ceroid lipofuscinosis in Borderdale sheep is caused by a nucleotide substitution at a consensus splice site (c.571 + 1G >>> A) leading to excision of exon 3

Batten disease (neuronal ceroid lipofuscinoses, NCLs) are a group of inherited childhood diseases that result in severe brain atrophy, blindness and seizures, leading to premature death. To date, eight different genes have been identified, each associated with a different form. Linkage analysis indicated a CLN5 form in a colony of affected New Zealand Borderdale sheep. Sequencing studies established the disease-causing mutation to be a substitution at a consensus splice site (c.571+1G>A), leading to the excision of exon 3 and a truncated putative protein. A molecular diagnostic test has been developed based on the excision of exon 3. Sequence alignments support the gene product being a soluble lysosomal protein. Western blotting of isolated storage bodies indicates the specific storage of subunit c of mitochondrial ATP synthase. This flock is being expanded as a large animal model for mechanistic studies and trial therapies.

CCL2 modulates cytokine production in cultured mouse astrocytes

BackgroundThe chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1) is upregulated in patients and rodent models of traumatic brain injury (TBI), contributing to post-traumatic neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that Ccl2-/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, Ccl2-deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2s potential ability to modulate immunoactivation of astrocytes in vitro.MethodsPrimary astrocytes were isolated from neonatal wild-type and Ccl2-deficient mice. Established astrocyte cultures were stimulated with various concentrations of lipopolysaccharide (LPS) and interleukin (IL)-1β for up to 24 hours. Separate experiments involved pre-incubation with mouse recombinant (r)CCL2 prior to IL-1β stimulation in wild-type cells. Following stimulation, cytokine secretion was measured in culture supernatant by immunoassays, whilst cytokine gene expression was quantified by real-time reverse transcriptase polymerase chain reaction.ResultsLPS (0.1-100 μg/ml; 8 h) induced the significantly greater secretion of five key cytokines and chemokines in Ccl2-/- astrocytes compared to wild-type cells. Consistently, IL-6 mRNA levels were 2-fold higher in Ccl2-deficient cells. IL-1β (10 and 50 ng/ml; 2-24 h) also resulted in exacerbated IL-6 production from Ccl2-/- cultures. Despite this, treatment of wild-type cultures with rCCL2 alone (50-500 ng/ml) did not induce cytokine/chemokine production by astrocytes. However, pre-incubation of wild-type astrocytes with rCCL2 (250 ng/ml, 12 h) prior to stimulation with IL-1β (10 ng/ml, 8 h) significantly reduced IL-6 protein and gene expression.ConclusionsOur data indicate that astrocytes are likely responsible for the exacerbated cytokine response seen in vivo post-injury in the absence of CCL2. Furthermore, evidence that CCL2 inhibits cytokine production by astrocytes following IL-1β stimulation, suggests a novel, immunomodulatory role for this chemokine in acute neuroinflammation. Further investigation is required to determine the physiological relevance of this phenomenon, which may have implications for therapeutics targeting CCL2-mediated leukocyte infiltration following TBI.

Ceruloplasmin and β-amyloid precursor protein confer neuroprotection in traumatic brain injury and lower neuronal iron

Traumatic brain injury (TBI) is in part complicated by pro-oxidant iron elevation independent of brain hemorrhage. Ceruloplasmin (CP) and β-amyloid protein precursor (APP) are known neuroprotective proteins that reduce oxidative damage through iron regulation. We surveyed iron, CP, and APP in brain tissue from control and TBI-affected patients who were stratified according to time of death following injury. We observed CP and APP induction after TBI accompanying iron accumulation. Elevated APP and CP expression was also observed in a mouse model of focal cortical contusion injury concomitant with iron elevation. To determine if changes in APP or CP were neuroprotective we employed the same TBI model on APP(-/-) and CP(-/-) mice and found that both exhibited exaggerated infarct volume and iron accumulation postinjury. Evidence supports a regulatory role of both proteins in defence against iron-induced oxidative damage after TBI, which presents as a tractable therapeutic target.

Modulation of LPA Receptor Expression in the Human Brain Following Neurotrauma

Lysophosphatidic acid (LPA) is involved in physiological and pathological states, including in neural development and inflammation. We assessed the expression pattern of the LPA receptors 1-3 and of LPA-producing enzyme autotaxin in post-mortem human brain tissue, both in normal individuals and in individuals who died following traumatic brain injury. We found that LPA receptors and autotaxin are weakly expressed in the normal control adult brain. Quantitative PCR for the LPA receptors and autotaxin mRNA showed an increase of LPAR2 and a decrease of autotaxin mRNA expression in the cortex following brain injury. Immunohistochemical analysis showed that LPAR1 colocalized with astrocytes and that LPAR2 is present on the ependymal cells lining the lateral ventricle in the brain samples from individuals who died following severe head injury. This work shows for the first time that key components of the LPA pathway are modulated following TBI in humans.

Transforming growth factor beta2 haploinsufficient mice develop age-related nigrostriatal dopamine deficits.

The transforming growth factor-betas (TGF-betas) regulate the induction of dopaminergic neurons and are elevated in the CSF of Parkinsons patients. We report here that mice with TGF-beta2 haploinsufficiency (TGF-beta2+/-) have subclinical defects in the dopaminergic neurons of their substantia nigra pars compacta. At 6 weeks of age, the TGF-beta2+/- mice had 12% fewer dopaminergic neurons than wild-type littermates. No additional loss of neurons occurred during the next 5 months, although striatal dopamine declined to 70% of normal. The level of 3,4-dihydroxphenylacetic acid was normal in the TGF-beta2+/- mice, indicating that a compensatory mechanism maintains dopamine stimulation of their striatum. The TGF-beta2+/- mice had normal sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, despite having reduced levels of monoamine oxidase-B. These results raise the possibility that people with naturally low levels of TGF-beta2 may have less functional reserve in their nigrostriatal pathway, causing them to be at increased risk of developing Parkinson disease.

Expression and activation of EphA4 in the human brain after traumatic injury.

Abstract Glial scars that consist predominantly of reactive astrocytes create a major barrier to neuronal regeneration after traumatic brain injury (TBI). In experimental TBI, Eph receptors and their ephrin ligands are upregulated on reactive astrocytes at injury sites and inhibit axonal regeneration, but very little is known about Eph receptors in the human brain after TBI. A better understanding of the functions of glial cells and their interactions with inflammatory cells and injured axons will allow the development of treatment strategies that may promote regeneration. We analyzed EphA4 expression and activation in postmortem brain tissue from 19 patients who died after acute closed head injury and had evidence of diffuse axonal injury and 8 controls. We also examined downstream pathways that are mediated by EphA4 in human astrocyte cell cultures. Our results indicate that, after TBI in humans, EphA4 expression is upregulated and is associated with reactive astrocytes. The expression was increased shortly after the injury and remained activated for several days. EphA4 activation induced under inflammatory conditions in vitro was inhibited using unclustered EphA4 ligand. These results suggest that blocking EphA4 activation may represent a therapeutic approach for TBI and other types of brain injuries in humans.

Activation of the kynurenine pathway and increased production of the excitotoxin quinolinic acid following traumatic brain injury in humans.

During inflammation, the kynurenine pathway (KP) metabolises the essential amino acid tryptophan (TRP) potentially contributing to excitotoxicity via the release of quinolinic acid (QUIN) and 3-hydroxykynurenine (3HK). Despite the importance of excitotoxicity in the development of secondary brain damage, investigations on the KP in TBI are scarce. In this study, we comprehensively characterised changes in KP activation by measuring numerous metabolites in cerebrospinal fluid (CSF) from TBI patients and assessing the expression of key KP enzymes in brain tissue from TBI victims. Acute QUIN levels were further correlated with outcome scores to explore its prognostic value in TBI recovery.MethodsTwenty-eight patients with severe TBI (GCS ≤ 8, three patients had initial GCS = 9–10, but rapidly deteriorated to ≤8) were recruited. CSF was collected from admission to day 5 post-injury. TRP, kynurenine (KYN), kynurenic acid (KYNA), QUIN, anthranilic acid (AA) and 3-hydroxyanthranilic acid (3HAA) were measured in CSF. The Glasgow Outcome Scale Extended (GOSE) score was assessed at 6 months post-TBI. Post-mortem brains were obtained from the Australian Neurotrauma Tissue and Fluid Bank and used in qPCR for quantitating expression of KP enzymes (indoleamine 2,3-dioxygenase-1 (IDO1), kynurenase (KYNase), kynurenine amino transferase-II (KAT-II), kynurenine 3-monooxygenase (KMO), 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyl transferase (QPRTase) and IDO1 immunohistochemistry.ResultsIn CSF, KYN, KYNA and QUIN were elevated whereas TRP, AA and 3HAA remained unchanged. The ratios of QUIN:KYN, QUIN:KYNA, KYNA:KYN and 3HAA:AA revealed that QUIN levels were significantly higher than KYN and KYNA, supporting increased neurotoxicity. Amplified IDO1 and KYNase mRNA expression was demonstrated on post-mortem brains, and enhanced IDO1 protein coincided with overt tissue damage. QUIN levels in CSF were significantly higher in patients with unfavourable outcome and inversely correlated with GOSE scores.ConclusionTBI induced a striking activation of the KP pathway with sustained increase of QUIN. The exceeding production of QUIN together with increased IDO1 activation and mRNA expression in brain-injured areas suggests that TBI selectively induces a robust stimulation of the neurotoxic branch of the KP pathway. QUIN’s detrimental roles are supported by its association to adverse outcome potentially becoming an early prognostic factor post-TBI.