Acute and chronic effects of systemic inflammation on P301S tau mouse model of neurodegeneration

Systemic inflammation is thought to be an important driver in chronic neurodegeneration. During systemic infection, the inflammatory status of the periphery is communicated to the brain and conserved sickness behaviours initiated. However, in the context of dementia the same inflammatory stimulus might trigger delirium. Delirium is a severe, transient neuropsychiatric condition characterised by altered levels of arousal, inattention, cognitive deficits and psychoses. Delirium and systemic inflammation exacerbate the trajectory of pre-existing dementia, and are associated with increased risk of future dementia. Accumulating experimental studies suggest microglia are “primed” by chronic neurodegeneration, such that a subsequent inflammatory insult – central or systemic – induces an increased inflammatory response which manifests as exaggerated sickness behaviours. To date there have been no studies of microglial priming in the context of pure tau pathology, without amyloid pathology, and none investigating acute sickness behaviour in such a model. The overarching aim of this thesis is to address this gap in the literature and further our understanding of the interactions between systemic inflammation, neuroinflammation and neurodegeneration in the context of tauopathy. The P301S mouse over-expresses human mutant tau protein under the Thy1.2 promoter. It develops hyperphosphorylated and insoluble tau accumulations and progressive neuronal loss. Consequently, P301S mice develop progressive hind limb paralysis. This study identified the horizontal bar task, a test of motor control and coordination, conducted at weekly intervals from 8-22 weeks of age, as a non-invasive measure of disease progression. In addition, a detailed temporal profile of pathological hallmarks at 8, 9, 10, 11, 12, 16 and 20 weeks of age was determined. Key results presented here demonstrate progressive, superficial neuronal loss in the cortex of P301S mice, with associated astrogliosis and surprisingly this occurs in the absence of apparent cortical microgliosis. In stark contrast, there is progressive microgliosis in the spinal cord of P301S mice. On this background, lipopolysaccharide (LPS), a chemical moiety found on the outer surface of gram-negative bacteria, was used to mimic a systemic bacterial infection. P301S mice and C57BL/6 control mice were injected, at 10 or 16 weeks of age, intraperitoneally with 500 μg/kg LPS or saline and were monitored in the following hours and weeks. Acutely, P301S mice showed signs of an exaggerated, longer lasting sickness response. Importantly, exaggerated acute symptoms extended beyond those typically associated with sickness behaviour; LPS induced an exaggerated acute impairment of horizontal bar performance in P301S mice and not C57BL/6 mice – a function which is known to be impaired in P301S mice later in disease. Impairments were age-dependent in terms of timing of injection. These data suggest an interaction between acute infection and existing CNS vulnerability leading to acute neurological dysfunction that is not a feature observed in sickness in a normal animal. LPS-injected P301S mice also showed, again age-dependent, increased rate of decline in motor performance compared with controls. There was no evidence of microglial priming in P301S mice. LPS caused an acute increase in AT8-positive phospho-tau however this did not persist until end stage. At 22 weeks of age there was significant disease-associated cortical neuronal loss in the vehicle-injected P301S mice, and additional superficial cortical neuronal loss in LPS-injected P301S mice and control mice. There was significant IBA1-positive microgliosis in the spinal cord of P301S mice at end stage which was further increased in LPS-injected P301S mice. Taken together these data indicate a clear and clinically relevant interaction between systemic inflammation and tau-associated neuropathology with acute and long-term functional consequences. In the absence of evidence of microglial priming, future work will explore potential mechanisms

Therapeutic inhibition of the complement system in diseases of the central nervous system

The complement system plays critical roles in development, homeostasis, and regeneration in the central nervous system (CNS) throughout life; however, complement dysregulation in the CNS can lead to damage and disease. Complement proteins, regulators, and receptors are widely expressed throughout the CNS and, in many cases, are upregulated in disease. Genetic and epidemiological studies, cerebrospinal fluid (CSF) and plasma biomarker measurements and pathological analysis of post-mortem tissues have all implicated complement in multiple CNS diseases including multiple sclerosis (MS), neuromyelitis optica (NMO), neurotrauma, stroke, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Given this body of evidence implicating complement in diverse brain diseases, manipulating complement in the brain is an attractive prospect; however, the blood-brain barrier (BBB), critical to protect the brain from potentially harmful agents in the circulation, is also impermeable to current complement-targeting therapeutics, making drug design much more challenging. For example, antibody therapeutics administered systemically are essentially excluded from the brain. Recent protocols have utilized “Trojan horse” techniques to transport therapeutics across the BBB or used osmotic shock or ultrasound to temporarily disrupt the BBB. Most research to date exploring the impact of complement inhibition on CNS diseases has been in animal models, and some of these studies have generated convincing data; for example, in models of MS, NMO, and stroke. There have been a few recent clinical trials of available anti-complement drugs in CNS diseases associated with BBB impairment, for example the use of the anti-C5 monoclonal antibody (mAb) eculizumab in NMO, but for most CNS diseases there have been no human trials of anti-complement therapies. Here we will review the evidence implicating complement in diverse CNS disorders, from acute, such as traumatic brain or spine injury, to chronic, including demyelinating, neuroinflammatory, and neurodegenerative diseases. We will discuss the particular problems of drug access into the CNS and explore ways in which anti-complement therapies might be tailored for CNS disease

HspB5 Activates a Neuroprotective Glial Cell Response in Experimental Tauopathy

Progressive neuronal death during tauopathies is associated with aggregation of modified, truncated or mutant forms of tau protein. Such aggregates are neurotoxic, promote spreading of tau aggregation, and trigger release of pro-inflammatory factors by glial cells. Counteracting such pathogenic effects of tau by simultaneously inhibiting protein aggregation as well as pro-inflammatory glial cell responses would be of significant therapeutic interest. Here, we examined the use of the small heat-shock protein HspB5 for this purpose. As a molecular chaperone, HspB5 counteracts aggregation of a wide range of abnormal proteins. As a TLR2 agonist, it selectively activates protective responses by CD14-expressing myeloid cells including microglia. We show that intracerebral infusion of HspB5 in transgenic mice with selective neuronal expression of mutant human P301S tau has significant neuroprotective effects in the superficial, frontal cortical layers. Underlying these effects at least in part, HspB5 induces several potent neuroprotective mediators in both astrocytes and microglia including neurotrophic factors and increased potential for removal of glutamate. Together, these findings highlight the potentially broad therapeutic potential of HspB5 in neurodegenerative proteinopathies

Complement factor I deficiency: a potentially treatable cause of fulminant cerebral inflammation

Objective To raise awareness of complement factor I (CFI) deficiency as a potentially treatable cause of severe cerebral inflammation. Methods Case report with neuroradiology, neuropathology, and functional data describing the mutation with review of literature. Results We present a case of acute, fulminant, destructive cerebral edema in a previously well 11-year-old, demonstrating massive activation of complement pathways on neuropathology and compound heterozygote status for 2 pathogenic mutations in CFI which result in normal levels but completely abrogate function. Conclusions Our case adds to a very small number of extant reports of this phenomenon associated with a spectrum of inflammatory histopathologies including hemorrhagic leukoencephalopathy and clinical presentations resembling severe acute disseminated encephalomyelitis. CFI deficiency can result in uncontrolled activation of the complement pathways in the brain resulting in devastating cerebral inflammation. The deficit is latent, but the catastrophic dysregulation of the complement system may be the result of a C3 acute phase response. Diagnoses to date have been retrospective. Diagnosis requires a high index of suspicion and clinician awareness of the limitations of first-line clinical tests of complement activity and activation. Simple measurement of circulating CFI levels, as here, may fail to diagnose functional deficiency with absent CFI activity. These diagnostic challenges may mean that the CFI deficiency is being systematically under-recognized as a cause of fulminant cerebral inflammation. Complement inhibitory therapies (such as eculizumab) offer new potential treatment, underlining the importance of prompt recognition, and real-time whole exome sequencing may play an important future role

Genetic insights into the impact of complement in Alzheimer's disease

The presence of complement activation products at sites of pathology in post-mortem Alzheimer’s disease (AD) brains is well known. Recent evidence from genome-wide association studies (GWAS), combined with the demonstration that complement activation is pivotal in synapse loss in AD, strongly implicates complement in disease aetiology. Genetic variations in complement genes are widespread. While most variants individually have only minor effects on complement homeostasis, the combined effects of variants in multiple complement genes, referred to as the “complotype”, can have major effects. In some diseases, the complotype highlights specific parts of the complement pathway involved in disease, thereby pointing towards a mechanism; however, this is not the case with AD. Here we review the complement GWAS hits; CR1 encoding complement receptor 1 (CR1), CLU encoding clusterin, and a suggestive association of C1S encoding the enzyme C1s, and discuss difficulties in attributing the AD association in these genes to complement function. A better understanding of complement genetics in AD might facilitate predictive genetic screening tests and enable the development of simple diagnostic tools and guide the future use of anti-complement drugs, of which several are currently in development for central nervous system disorders

Novel monoclonal antibodies against mouse C1q: characterisation and development of a quantitative ELISA for mouse C1q

Recent studies have identified roles for complement in synaptic pruning, both physiological during development and pathological in Alzheimer’s disease (AD). These reports suggest that C1q initiates complement activation on synapses and C3 fragments then tag them for removal by microglia. There is an urgent need to characterise these processes in rodent AD models; this requires the development of reagents and methods for detection and quantification of rodent C1q in fluids and pathological tissues. These will enable better evaluation of the role of C1q in disease and its value as disease biomarker. We describe the generation in C1q-deficient mice of novel monoclonal antibodies against mouse and rat C1q that enabled development of a sensitive, specific, and quantitative ELISA for mouse and rat C1q capable of measuring C1q in biological fluids and tissue extracts. Serum C1q levels were measured in wild-type (WT), C1q knockout (KO), C3 KO, C7 KO, Crry KO, and 3xTg and APPNL-G-F AD model mice through ageing. C1q levels significantly decreased in WT, APPNL-G-F, and C7 KO mice with ageing. C1q levels were reduced in APPNL-G-F compared to WT at all ages and in 3xTg at 12 months; C3 KO and C7 KO, but not Crry KO mice, also demonstrated significantly lower C1q levels compared to matched WT. In brain homogenates, C1q levels increased with age in both WT and APPNL-G-F mice. This robust and adaptable assay for quantification of mouse and rat C1q provides a vital tool for investigating the expression of C1q in rodent models of AD and other complement-driven pathologies

Complement receptor 1 is expressed on brain cells and in the human brain

Genome wide association studies (GWAS) have highlighted the importance of the complement cascade in pathogenesis of Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among the top GWAS hits. The long variant of CR1 is associated with increased risk for AD; however, roles of CR1 in brain health and disease are poorly understood. A critical confounder is that brain expression of CR1 is controversial; failure to demonstrate brain expression has provoked the suggestion that peripherally expressed CR1 influences AD risk. We took a multi‐pronged approach to establish whether CR1 is expressed in brain. Expression of CR1 at the protein and mRNA level was assessed in human microglial lines, induced pluripotent stem cell (iPSC)‐derived microglia from two sources and brain tissue from AD and control donors. CR1 protein was detected in microglial lines and iPSC‐derived microglia expressing different CR1 variants when immunostained with a validated panel of CR1‐specific antibodies; cell extracts were positive for CR1 protein and mRNA. CR1 protein was detected in control and AD brains, co‐localizing with astrocytes and microglia, and expression was significantly increased in AD compared to controls. CR1 mRNA expression was detected in all AD and control brain samples tested; expression was significantly increased in AD. The data unequivocally demonstrate that the CR1 transcript and protein are expressed in human microglia ex vivo and on microglia and astrocytes in situ in the human brain; the findings support the hypothesis that CR1 variants affect AD risk by directly impacting glial functions

The impact of complement genes on the risk of late-onset Alzheimer's disease

Late-onset Alzheimer’s disease (LOAD), the most common cause of dementia, and a huge global health challenge, is a neurodegenerative disease of uncertain aetiology. To deliver effective diagnostics and therapeutics, understanding the molecular basis of the disease is essential. Contemporary large genome-wide association studies (GWAS) have identified over seventy novel genetic susceptibility loci for LOAD. Most are implicated in microglial or inflammatory pathways, bringing inflammation to the fore as a candidate pathological pathway. Among the most significant GWAS hits are three complement genes: CLU, encoding the fluid-phase complement inhibitor clusterin; CR1 encoding complement receptor 1 (CR1); and recently, C1S encoding the complement enzyme C1s. Complement activation is a critical driver of inflammation; changes in complement genes may impact risk by altering the inflammatory status in the brain. To assess complement gene association with LOAD risk, we manually created a comprehensive complement gene list and tested these in gene-set analysis with LOAD summary statistics. We confirmed associations of CLU and CR1 genes with LOAD but showed no significant associations for the complement gene-set when excluding CLU and CR1. No significant association with other complement genes, including C1S, was seen in the IGAP dataset; however, these may emerge from larger datasets

Dendritic spine loss in epileptogenic Type II focal cortical dysplasia: Role of enhanced classical complement pathway activation

Dendritic spines are the postsynaptic sites for most excitatory glutamatergic synapses. We previously demonstrated a severe spine loss and synaptic reorganization in human neocortices presenting Type II focal cortical dysplasia (FCD), a developmental malformation and frequent cause of drug‐resistant focal epilepsy. We extend the findings, investigating the potential role of complement components C1q and C3 in synaptic pruning imbalance. Data from Type II FCD were compared with those obtained in focal epilepsies with different etiologies. Neocortical tissues were collected from 20 subjects, mainly adults with a mean age at surgery of 31 years, admitted to epilepsy surgery with a neuropathological diagnosis of: cryptogenic, temporal lobe epilepsy with hippocampal sclerosis, and Type IIa/b FCD. Dendritic spine density quantitation, evaluated in a previous paper using Golgi impregnation, was available in a subgroup. Immunohistochemistry, in situ hybridization, electron microscopy, and organotypic cultures were utilized to study complement/microglial activation patterns. FCD Type II samples presenting dendritic spine loss were characterized by an activation of the classical complement pathway and microglial reactivity. In the same samples, a close relationship between microglial cells and dendritic segments/synapses was found. These features were consistently observed in Type IIb FCD and in 1 of 3 Type IIa cases. In other patient groups and in perilesional areas outside the dysplasia, not presenting spine loss, these features were not observed. In vitro treatment with complement proteins of organotypic slices of cortical tissue with no sign of FCD induced a reduction in dendritic spine density. These data suggest that dysregulation of the complement system plays a role in microglia‐mediated spine loss. This mechanism, known to be involved in the removal of redundant synapses during development, is likely reactivated in Type II FCD, particularly in Type IIb; local treatment with anticomplement drugs could in principle modify the course of disease in these patients