The contribution of the meningeal immune interface to neuroinflammation in traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of disability and mortality worldwide, particularly among the elderly, yet our mechanistic understanding of what renders the post-traumatic brain vulnerable to poor outcomes, and susceptible to neurological disease, is incomplete. It is well established that dysregulated and sustained immune responses elicit negative consequences after TBI; however, our understanding of the neuroimmune interface that facilitates crosstalk between central and peripheral immune reservoirs is in its infancy. The meninges serve as the interface between the brain and the immune system, facilitating important bi-directional roles in both healthy and disease settings. It has been previously shown that disruption of this system exacerbates neuroinflammation in age-related neurodegenerative disorders such as Alzheimer's disease; however, we have an incomplete understanding of how the meningeal compartment influences immune responses after TBI. In this manuscript, we will offer a detailed overview of the holistic nature of neuroinflammatory responses in TBI, including hallmark features observed across clinical and animal models. We will highlight the structure and function of the meningeal lymphatic system, including its role in immuno-surveillance and immune responses within the meninges and the brain. We will provide a comprehensive update on our current knowledge of meningeal-derived responses across the spectrum of TBI, and identify new avenues for neuroimmune modulation within the neurotrauma field.

Traumatic brain injury induces an adaptive immune response in the meningeal transcriptome that is amplified by aging.

Traumatic Brain Injury (TBI) is a major cause of disability and mortality, particularly among the elderly, yet our mechanistic understanding of how age renders the post-traumatic brain vulnerable to poor clinical outcomes and susceptible to neurological disease remains poorly understood. It is well established that dysregulated and sustained immune responses contribute to negative outcomes after TBI, however our understanding of the interactions between central and peripheral immune reservoirs is still unclear. The meninges serve as the interface between the brain and the immune system, facilitating important bi-directional roles in healthy and disease settings. It has been previously shown that disruption of this system exacerbates inflammation in age related neurodegenerative disorders such as Alzheimer's disease, however we have an incomplete understanding of how the meningeal compartment influences immune responses after TBI. Here, we examine the meningeal tissue and its response to brain injury in young (3-months) and aged (18-months) mice. Utilizing a bioinformatic approach, high-throughput RNA sequencing demonstrates alterations in the meningeal transcriptome at sub-acute (7-days) and chronic (1 month) timepoints after injury. We find that age alone chronically exacerbates immunoglobulin production and B cell responses. After TBI, adaptive immune response genes are up-regulated in a temporal manner, with genes involved in T cell responses elevated sub-acutely, followed by increases in B cell related genes at chronic time points after injury. Pro-inflammatory cytokines are also implicated as contributing to the immune response in the meninges, with ingenuity pathway analysis identifying interferons as master regulators in aged mice compared to young mice following TBI. Collectively these data demonstrate the temporal series of meningeal specific signatures, providing insights into how age leads to worse neuroinflammatory outcomes in TBI.

Delayed treatment with ceftriaxone reverses the enhanced sensitivity of TBI mice to chemically-induced seizures.

The pathophysiological changes that occur after traumatic brain injury (TBI) can lead to the development of post-traumatic epilepsy, a life-long complication of brain trauma. The etiology of post-traumatic epilepsy remains unknown, but TBI brains exhibit an abnormal excitatory / inhibitory balance. In this study, we examine how brain injury alters susceptibility to chemically-induced seizures in C57Bl/6J mice, and if pharmacological enhancement of glutamate transporters can reduce chronic post-traumatic seizures. We found that controlled cortical impact (CCI) mice display delayed susceptibility to pentylenetetrazol (PTZ)-induced seizures. While CCI mice have no change in seizure susceptibility at 7d post-injury (dpi), at 70dpi they have reduced latency to PTZ-induced seizure onset, higher seizure frequency and longer seizure duration. Quantification of glutamate transporter mRNA showed that levels of Scl1a2 and Scl1a3 mRNA were increased at 7dpi, but significantly decreased at 70dpi. To test if increased levels of glutamate transporters can ameliorate delayed-onset seizure susceptibility in TBI mice, we exposed a new cohort of mice to CCI and administered ceftriaxone (200mg/kg/day) for 14d from 55-70dpi. We found that ceftriaxone significantly increased Scl1a2 and Scl1a3 in CCI mouse brain at 70dpi, and prevented the susceptibility of CCI mice to PTZ-induced seizures. This study demonstrates cortical impact can induce a delayed-onset seizure phenotype in mice. Delayed (55dpi) ceftriaxone treatment enhances glutamate transporter mRNA in the CCI brain, and reduces PTZ-induced seizures in CCI mice.

Sequential Isolation of Microglia and Astrocytes from Young and Aged Adult Mouse Brains for Downstream Transcriptomic Analysis.

In aging, the brain is more vulnerable to injury and neurodegenerative disease, but the mechanisms responsible are largely unknown. Evidence now suggests that neuroinflammation, mediated by resident brain astrocyte and microglia populations, are key players in the generation of inflammatory responses and may influence both age related processes and the initiation/progression of neurodegeneration. Consequently, targeting these cell types individually and collectively may aid in the development of novel disease-modifying therapies. We have optimized and characterized a protocol for the effective sequential isolation of both microglia and astrocytes from the adult mouse brain in young and aged mice. We demonstrate a technique for the sequential isolation of these immune cells by using magnetic beads technology, optimized to increase yield and limit potential artifacts in downstream transcriptomic applications, including RNA-sequencing pipelines. This technique is versatile, cost-effective, and reliable for the study of responses within the same biological context, simultaneously being advantageous in reducing mice numbers required to assess cellular responses in normal and age-related pathological conditions.

The Effect of Traumatic Brain Injury on Sleep Architecture and Circadian Rhythms in Mice-A Comparison of High-Frequency Head Impact and Controlled Cortical Injury.

Traumatic brain injury (TBI) is a significant risk factor for the development of sleep and circadian rhythm impairments. In this study we compare the circadian rhythms and sleep patterns in the high-frequency head impact (HFHI) and controlled cortical impact (CCI) mouse models of TBI. These mouse models have different injury mechanisms key differences of pathology in brain regions controlling circadian rhythms and EEG wave generation. We found that both HFHI and CCI caused dysregulation in the diurnal expression of core circadian genes (Bmal1, Clock, Per1,2, Cry1,2) at 24 h post-TBI. CCI mice had reduced locomotor activity on running wheels in the first 7 d post-TBI; however, both CCI and HFHI mice were able to maintain circadian behavior cycles even in the absence of light cues. We used implantable EEG to measure sleep cycles and brain activity and found that there were no differences in the time spent awake, in NREM or REM sleep in either TBI model. However, in the sleep states, CCI mice have reduced delta power in NREM sleep and reduced theta power in REM sleep at 7 d post-TBI. Our data reveal that different types of brain trauma can result in distinct patterns of circadian and sleep disruptions and can be used to better understand the etiology of sleep disorders after TBI.

High-frequency head impact causes chronic synaptic adaptation and long-term cognitive impairment in mice.

Repeated head impact exposure can cause memory and behavioral impairments. Here, we report that exposure to non-damaging, but high frequency, head impacts can alter brain function in mice through synaptic adaptation. High frequency head impact mice develop chronic cognitive impairments in the absence of traditional brain trauma pathology, and transcriptomic profiling of mouse and human chronic traumatic encephalopathy brain reveal that synapses are strongly affected by head impact. Electrophysiological analysis shows that high frequency head impacts cause chronic modification of the AMPA/NMDA ratio in neurons that underlie the changes to cognition. To demonstrate that synaptic adaptation is caused by head impact-induced glutamate release, we pretreated mice with memantine prior to head impact. Memantine prevents the development of the key transcriptomic and electrophysiological signatures of high frequency head impact, and averts cognitive dysfunction. These data reveal synapses as a target of high frequency head impact in human and mouse brain, and that this physiological adaptation in response to head impact is sufficient to induce chronic cognitive impairment in mice.

Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury.

BACKGROUND: Traumatic Brain Injury (TBI) is a major cause of disability and mortality, to which there is currently no comprehensive treatment. Blood Brain Barrier (BBB) dysfunction is well documented in human TBI patients, yet the molecular mechanisms that underlie this neurovascular unit (NVU) pathology remains unclear. The apolipoprotein-E (apoE) protein has been implicated in controlling BBB integrity in an isoform dependent manner, via suppression of Cyclophilin A (CypA)-Matrix metallopeptidase-9 (MMP-9) signaling cascades, however the contribution of this pathway in TBI-induced BBB permeability is not fully investigated. METHODS: We exposed C57Bl/6 mice to controlled cortical impact and assessed NVU and BBB permeability responses up to 21 days post-injury. We pharmacologically probed the role of the CypA-MMP-9 pathway in BBB permeability after TBI using Cyclosporin A (CsA, 20 mg/kg). Finally, as the apoE4 protein is known to be functionally deficient compared to the apoE3 protein, we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI. RESULTS: In C57Bl/6 mice there was an inverse relationship between soluble apoE and BBB permeability, such that damaged BBB stabilizes as apoE levels increase in the days following TBI. TBI mice displayed acute pericyte loss, increased MMP-9 production and activity, and reduced tight-junction expression. Treatment with the CypA antagonist CsA in C57Bl/6 mice attenuates MMP-9 responses and enhances BBB repair after injury, demonstrating that MMP-9 plays an important role in the timing of spontaneous BBB repair after TBI. We also show that apoe mRNA is present in both astrocytes and pericytes after TBI. We report that APOE3 and APOE4 mice have similar acute BBB responses to TBI, but APOE3 mice display faster spontaneous BBB repair than APOE4 mice. Isolated microvessel analysis reveals delayed pericyte repopulation, augmented and sustained MMP-9 expression at the NVU, and impaired stabilization of Zonula Occludens-1, Occludin and Claudin-5 expression at tight junctions in APOE4 mice after TBI compared to APOE3 mice. CONCLUSIONS: These data confirm apoE as an important modulator of spontaneous BBB stabilization following TBI, and highlights the APOE4 allele as a risk factor for poor outcome after TBI.

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Reduced cortical excitatory synapse number in APOE4 mice is associated with increased calcineurin activity.

Synaptic loss is a symptom of Alzheimer's disease (AD) that is associated with the onset of cognitive decline and the loss of executive function. The strongest genetic risk factor for AD is the APOE4 allele, which results in both a greater risk of developing AD as well as an earlier age of onset of AD. Dendritic spines, the anatomical substrate of the excitatory synapse, are reduced in the cortex of humanized APOE4 mice but the reason for this synaptic decline is unknown. Calcineurin, a calcium/calmodulin dependent phosphatase, is a mediator of dendritic spine retraction. We used humanized APOE mice to examine how APOE genotype altered calcineurin activity and found that APOE4 mice have 35% higher cortical calcineurin activity compared with APOE3 mice. This occurred in the absence of any increase in calcineurin protein levels or mRNA expression. The elevation in calcineurin was associated with 10% fewer dendritic spine number in layer II/III of the cortex. Treatment with the calcineurin inhibitor FK506 reduced calcineurin activity by 64% and resulted in normalization of dendritic spine numbers in APOE4 mice. In conclusion, we found that the APOE4 gene in mice was associated with elevated calcineurin activity and fewer dendritic spine numbers compared with APOE3 mice. Importantly, calcineurin in APOE4 remained sensitive to pharmacological inhibition and spine density can be rescued by treatment with FK506.

Microbial Immuno-Communication in Neurodegenerative Diseases.

Neuro-inflammation is a critical process by which the brain coordinates chemokine-regulated cellular recruitment, cytokine release, and cell-mediated removal of pathogenic material to protect against infection or brain injury. Dysregulation of this immune response is involved in multiple neurodegenerative disorders, however the precise contribution of neuro-inflammation to the exacerbation and progression of these diseases remains unclear. Evidence now suggests that commensal micro-organisms populating the host and their metabolites, collectively termed the microbiome, regulate innate immunity by influencing peripheral immune cell populations, and modulating microglial phenotype. Recent preclinical studies now demonstrate that perturbations in the host microbiome can induce alterations in pathological phenotypes associated with numerous neurodegenerative diseases. How perturbations in the host microbiome and subsequently altered peripheral immune status are communicated to the brain to influence neuro-inflammatory processes in these neurodegenerative disease settings is far from understood. This review provides insight into the regulation of neuro-inflammatory processes by the host microbiome in the context of neurodegenerative disease and highlights the potential importance of the blood-brain barrier and blood-cerebrospinal fluid-brain barrier, functioning as "immune barriers," to communicate host immune status to the brain. Understanding the mechanisms by which the commensal microbiome communicates with the brain to influence neuro-inflammatory processes will be critical in the development of microbially-targeted therapeutics in the potential treatment of neurodegenerative disorders.

Type-I interferons mediate the neuroinflammatory response and neurotoxicity induced by rotenone.

Evidence from post-mortem human brains, animal studies and cell culture models has implicated neuroinflammation in the aetiology of chronic neuropathologies including Alzheimer's and Parkinson's diseases. Although the neuroinflammatory response is considered detrimental in contributing to these pathologies, the underlying mechanisms are still not well understood. The type-I interferons (IFNs) have been well characterised in the periphery and are known to initiate/modulate the immune response. Recently, they have been implicated in ageing and we have also demonstrated increased type-I IFN expression in post-mortem human Alzheimer's and Parkinson's disease brains. We hypothesise that the type-I IFNs are key drivers of the damaging, self-perpetuating pro-inflammatory response that contributes to these chronic neuropathologies. In support of this, we have recently confirmed in models of Alzheimer's and Parkinson's disease that mice lacking the type-I IFN receptor (IFNAR1), display an attenuated neuroinflammatory response with subsequent neuroprotection. To further investigate type-I IFN-mediated neuroinflammation and the specific CNS cell types involved, this study treated primary cultured wild-type and IFNAR1-/- neurons or mixed glia with the mitochondrial complex I inhibitor, rotenone. Wild-type neurons and glia treated with 3 nM and 25 nM rotenone, respectively, exhibited a pro-inflammatory response, including increased type-I IFN expression that was attenuated in cells lacking IFNAR1. Reduced type-I IFN signalling in IFNAR1-/- neurons also conferred protection against caspase-3-mediated rotenone-induced cell death. Further, this reduced pro-inflammatory response in the IFNAR1-/- glia subsequently diminished their neurotoxic effects to wild-type neurons. In support of this, we confirmed that therapeutically targeting the type-I IFN glial response to rotenone through a specific IFNAR1 blocking monoclonal antibody was neuroprotective. Our data has confirmed that both neurons and glia contribute to the pro-inflammatory response induced by rotenone with attenuation of this response beneficial in reducing neuronal cell death. Read the Editorial Comment for this article on page 9.

Type-1 interferons contribute to the neuroinflammatory response and disease progression of the MPTP mouse model of Parkinson's disease.

Type-1 interferons (IFNs) are pleiotropic cytokines with a critical role in the initiation and regulation of the pro-inflammatory response. However, the contribution of the type-1 IFNs to CNS disorders, specifically chronic neuropathologies such as Parkinson's disease is still unknown. Here, we report increased type-1 IFN signaling in both post mortem human Parkinson's disease samples and in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model. In response to MPTP, mice lacking the type-1 IFN receptor (IFNAR1(-/-) ) displayed decreased type-1 IFN signaling, an attenuated pro-inflammatory response and reduced loss of dopaminergic neurons. The neuroprotective potential of targeting the type-1 IFN pathway was confirmed by reduced neuroinflammation and DA cell death in mice treated with a blocking monoclonal IFNAR1 (MAR-1) antibody. The MPTP/MAR-1 treated mice also displayed increased striatal dopamine levels and improved behavioural outcomes compared to their MPTP/IgG controls. These data, implicate for the first time, a deleterious role for the type-1 IFNs as key modulators of the early neuroinflammatory response and therefore the neuronal cell death in Parkinson's disease. GLIA 2016;64:1590-1604.

Neuroinflammation and oxidative stress: co-conspirators in the pathology of Parkinson's disease.

Parkinson's disease (PD) is a complex disease, with genetics and environment contributing to the disease onset. Recent studies of causative PD genes have confirmed the involvement of cellular mechanisms engaged in mitochondrial and UPS dysfunction, oxidative stress and apoptosis in the progressive degeneration of the dopaminergic neurons in PD. In addition, clinical, epidemiological and experimental evidence has implicated neuroinflammation in the disease progression. This review will discuss neuroinflammation in PD, with particular focus on the genetic and toxin-based models of the disease. These studies have confirmed elevated oxidative stress and the pro-inflammatory response occurs early in the disease and these processes contribute to and/or exacerbate the nigro-striatal degeneration. In addition, the experimental models discussed here have also provided strong evidence that these pathways are an important link between the familial and sporadic causes of PD. The potential application of anti-inflammatory interventions in limiting the dopaminergic neuronal cell death in these models is discussed with evidence suggesting that the further investigation of their use as part of multi-targeted clinical trials is warranted.