Myeloid and lymphoid expression of C9orf72 regulates IL-17A signaling in mice.

A mutation in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Patients with ALS or FTD often develop autoimmunity and inflammation that precedes or coincides with the onset of neurological symptoms, but the underlying mechanisms are poorly understood. Here, we knocked out murine C9orf72 in seven hematopoietic progenitor compartments by conditional mutagenesis and found that myeloid lineage C9orf72 prevents splenomegaly, loss of tolerance, and premature mortality. Furthermore, we demonstrated that C9orf72 plays a role in lymphoid cells to prevent interleukin-17A (IL-17A) production and neutrophilia. Mass cytometry identified early and sustained elevation of the costimulatory molecule CD80 expressed on C9orf72-deficient mouse macrophages, monocytes, and microglia. Enrichment of CD80 was similarly observed in human spinal cord microglia from patients with C9ORF72-mediated ALS compared with non-ALS controls. Single-cell RNA sequencing of murine spinal cord, brain cortex, and spleen demonstrated coordinated induction of gene modules related to antigen processing and presentation and antiviral immunity in C9orf72-deficient endothelial cells, microglia, and macrophages. Mechanistically, C9ORF72 repressed the trafficking of CD80 to the cell surface in response to Toll-like receptor agonists, interferon-γ, and IL-17A. Deletion of Il17a in C9orf72-deficient mice prevented CD80 enrichment in the spinal cord, reduced neutrophilia, and reduced gut T helper type 17 cells. Last, systemic delivery of an IL-17A neutralizing antibody augmented motor performance and suppressed neuroinflammation in C9orf72-deficient mice. Altogether, we show that C9orf72 orchestrates myeloid costimulatory potency and provide support for IL-17A as a therapeutic target for neuroinflammation associated with ALS or FTD.

Divergent functional outcomes of NLRP3 blockade downstream of multi-inflammasome activation: therapeutic implications for ALS.

NOD-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome modulation has emerged as a potential therapeutic approach targeting inflammation amplified by pyroptotic innate immune cell death. In diseases characterized by non-cell autonomous neurodegeneration including amyotrophic lateral sclerosis (ALS), the activation of several inflammasomes has been reported. Since functional redundancy can exist among inflammasome pathways, here we investigate the effects of NLRP3 inhibition on NLRP3, NLR family CARD Domain Containing 4 (NLRC4) and non-canonical pathways to understand whether NLRP3 blockade alone can mitigate pro-inflammatory cytokine release and pyroptotic cell death in contexts where single or multiple inflammasome pathways independent of NLRP3 are activated. In this study we do not limit our insights into inflammasome biology by solely relying on the THP-1 monocytic line under the LPS/nigericin-mediated NLRP3 pathway activation paradigm. We assess therapeutic potential and limitations of NLRP3 inhibition in multi-inflammasome activation contexts utilizing various human cellular systems including cell lines expressing gain of function (GoF) mutations for several inflammasomes, primary human monocytes, macrophages, healthy and Amyotrophic Lateral Sclerosis (ALS) patient induced pluripotent stem cells (iPSC)-derived microglia (iMGL) stimulated for canonical and non-canonical inflammasome pathways. We demonstrate that NLRP3 inhibition can modulate the NLRC4 and non-canonical inflammasome pathways; however, these effects differ between immortalized, human primary innate immune cells, and iMGL. We extend our investigation in more complex systems characterized by activation of multiple inflammasomes such as the SOD1G93A mouse model. Through deep immune phenotyping by single-cell mass cytometry we demonstrate that acute NLRP3 inhibition does not ameliorate spinal cord inflammation in this model. Taken together, our data suggests that NLRP3 inhibition alone may not be sufficient to address dynamic and complex neuroinflammatory pathobiological mechanisms including dysregulation of multiple inflammasome pathways in neurodegenerative disease such as ALS.

Innate immune activation and aberrant function in the R6/2 mouse model and Huntington's disease iPSC-derived microglia.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disease caused by CAG repeats in exon 1 of the HTT gene. A hallmark of HD along with other psychiatric and neurodegenerative diseases is alteration in the neuronal circuitry and synaptic loss. Microglia and peripheral innate immune activation have been reported in pre-symptomatic HD patients; however, what "activation" signifies for microglial and immune function in HD and how it impacts synaptic health remains unclear. In this study we sought to fill these gaps by capturing immune phenotypes and functional activation states of microglia and peripheral immunity in the R6/2 model of HD at pre-symptomatic, symptomatic and end stages of disease. These included characterizations of microglial phenotypes at single cell resolution, morphology, aberrant functions such as surveillance and phagocytosis and their impact on synaptic loss in vitro and ex vivo in R6/2 mouse brain tissue slices. To further understand how relevant the observed aberrant microglial behaviors are to human disease, transcriptomic analysis was performed using HD patient nuclear sequencing data and functional assessments were conducted using induced pluripotent stem cell (iPSC)-derived microglia. Our results show temporal changes in brain infiltration of peripheral lymphoid and myeloid cells, increases in microglial activation markers and phagocytic functions at the pre-symptomatic stages of disease. Increases in microglial surveillance and synaptic uptake parallel significant reduction of spine density in R6/2 mice. These findings were mirrored by an upregulation of gene signatures in the endocytic and migratory pathways in disease-associated microglial subsets in human HD brains, as well as increased phagocytic and migratory functions of iPSC-derived HD microglia. These results collectively suggest that targeting key and specific microglial functions related to synaptic surveillance and pruning may be therapeutically beneficial in attenuating cognitive decline and psychiatric aspects of HD.

C9orf72 suppresses systemic and neural inflammation induced by gut bacteria.

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.

Inhibition of Bruton's Tyrosine Kinase Modulates Microglial Phagocytosis: Therapeutic Implications for Alzheimer's Disease.

Bruton's tyrosine kinase (BTK), a critical component of B cell receptor signaling, has recently been implicated in regulation of the peripheral innate immune response. However, the role of BTK in microglia, the resident innate immune cells of the central nervous system, and its involvement in the pathobiology of neurodegenerative disease has not been explored. Here we found that BTK is a key regulator of microglial phagocytosis. Using potent BTK inhibitors and small interfering RNA (siRNA) against BTK, we observed that blockade of BTK activity decreased activation of phospholipase gamma 2, a recently identified genetic risk factor in Alzheimer's disease (AD), and reduced phagocytosis in rodent microglia and human monocyte-derived macrophages. Inhibition of BTK signaling also decreased microglial uptake of synaptosomes but did not have major impacts on other key microglial functions such as migration and cytokine release. Similarly, blocking BTK function ex vivo in acute brain slices reduced microglial phagocytosis and maintained numbers of resting microglia. In brain tissues from the 5xFAD mouse model of AD, levels of microglial BTK were elevated while in two gene expression datasets of post-mortem AD patient brain tissues, upregulation of BTK transcript was observed. Our study provides novel insights into the role of BTK in regulating microglial phagocytosis and uptake of synaptic structures and suggests that inhibiting microglial BTK may improve cognition in AD by preventing microglial activation and synaptic loss. Graphical Abstract Microglial-mediated synapse loss has been implicated in AD pathogenesis. Inhibition of BTK decreases activation of PLCγ2, a genetic risk factor in AD, and reduces microglial phagocytosis and uptake of synaptic structures. As such BTK inhibition may represent a therapeutic route to prevent microglial activation and synapse loss in AD.

Age-related changes in eye morphology and aqueous humor dynamics in DBA/2J mice using contrast-enhanced ocular MRI.

PURPOSE: Here, we are testing the hypothesis that dynamic contrast enhanced MRI (DCE-MRI) is a useful approach for non-invasively evaluating age-related changes in aqueous humor outflow and its contribution to elevated intraocular pressure in the DBA/2J model of pigmentary glaucoma. METHODS: A rodent-specific 7 T MRI was used to assess eye anatomy (anterior chamber (AC) and vitreous body (VB) morphology, eye size, lens size) and aqueous humor dynamics (via intravenous administration of Gd-DTPA and Gd-BOPTA contrast agents) in C57BL/6 and DBA/2J mice at 3 and 9 months of age. RESULTS: Gd-MRI was used to demonstrate an anterior solute pathway into the mouse AC. Topical latanoprost treatment in C57BL/6J mice reduced Gd-BOPTA accumulation in the AC. Age-related increases in AC area, AC depth and eye size were observed in DBA/2J mice compared to C57BL/6J mice. The rate of Gd-DTPA accumulation and peak Gd-DTPA intensity was lowest in 9-month old DBA/2J mice compared to 3-month old DBA/2J mice and C57BL/6J mice at both ages. Leakage of Gd-DTPA posteriorly into the VB was also observed in 9-month old DBA/2J mice. CONCLUSIONS: These studies support the idea that age-related changes in aqueous humor outflow contribute to elevated intraocular pressure (IOP) in the DBA/2J model of pigmentary glaucoma. Gd-MRI is a valuable tool for better understanding of mechanisms and dynamics of aqueous humor circulation in normal and glaucomatous mouse eyes or following topical administration of medicines to reduce IOP.

Neuropolypathology as a result of severe traumatic brain injury?

A history of brain trauma has long been acknowledged as increasing an individual's risk of developing dementia in later life. The underlying mechanisms that belie this pre-disposition are, however, very poorly understood. Here, we report a clinical-neuropathological correlation of a man who presented at the age of 66 with a progressive complex atypical dementia with early and prominent neurobehavioral symptoms. His neurological condition continued to decline up to his death at the age of 74. During the compilation of his clinical history, it was established that the subject had experienced a single severe traumatic brain injury (TBI) aged 12 years in 1954 resulting in loss of consciousness, hospitalization, and coma for a number of days after which he was deemed to have recovered. Following post-mortem neuropathological analysis, numerous distinct neuropathologies were observed in various brain regions and these included i) widespread Braak stage VI neurofibrillary tangle formation, ii) widespread α-synuclein positive Lewy bodies and Lewy neurites and iii) diffuse amyloid plaques and severe cerebral amyloid angiopathy (CAA). Added to this, a comprehensive analysis of blood-brain barrier (BBB) integrity, known to be disrupted during and after TBI, showed iv) distinct BBB breakdown with extravasated IgG and activated microglia present. This report represents an interesting documented case of neuropolypathology that may be associated with prior history of severe TBI. We propose one testable theory that a history of brain trauma may be a potential trigger for late onset dementia due to damage and unresolved functioning of the cerebral microvasculature.
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A systems-level framework for drug discovery identifies Csf1R as an anti-epileptic drug target.

The identification of drug targets is highly challenging, particularly for diseases of the brain. To address this problem, we developed and experimentally validated a general computational framework for drug target discovery that combines gene regulatory information with causal reasoning ("Causal Reasoning Analytical Framework for Target discovery"-CRAFT). Using a systems genetics approach and starting from gene expression data from the target tissue, CRAFT provides a predictive framework for identifying cell membrane receptors with a direction-specified influence over disease-related gene expression profiles. As proof of concept, we applied CRAFT to epilepsy and predicted the tyrosine kinase receptor Csf1R as a potential therapeutic target. The predicted effect of Csf1R blockade in attenuating epilepsy seizures was validated in three pre-clinical models of epilepsy. These results highlight CRAFT as a systems-level framework for target discovery and suggest Csf1R blockade as a novel therapeutic strategy in epilepsy. CRAFT is applicable to disease settings other than epilepsy.

Blood-Brain Barrier Dysfunction as a Hallmark Pathology in Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative condition associated with repetitive mild traumatic brain injury. In recent years, attention has focused on emerging evidence linking the development of CTE to concussive injuries in athletes and military personnel; however, the underlying molecular pathobiology of CTE remains unclear. Here, we provide evidence that the blood-brain barrier (BBB) is disrupted in regions of dense perivascular p-Tau accumulation in a case of CTE. Immunoreactivity patterns of the BBB-associated tight junction components claudin-5 and zonula occludens-1 were markedly discontinuous or absent in regions of perivascular p-Tau deposition; there was also immunohistochemical evidence of a BBB in these foci. Because the patient was diagnosed premortem clinically as having progressive supranuclear palsy (PSP), we also compromised that the CTE alterations appear to be distinct from those in the brain of a patient with PSP. This report represents the first description of BBB dysfunction in a pathologically proven CTE case and suggests a vascular component in the postconcussion cascade of events that may ultimately lead to development of a progressive degenerative disorder. BBB dysfunction may represent a correlate of neural dysfunction in live subjects suspected of being at risk for development of CTE.

Autoregulated paracellular clearance of amyloid-ß across the blood-brain barrier.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting neural tissue from damaging blood-borne agents. The barrier is characterized by endothelial tight junctions that limit passive paracellular diffusion of polar solutes and macromolecules from blood to brain. Decreased brain clearance of the neurotoxic amyloid-ß (Aß) peptide is a central event in the pathogenesis of Alzheimer's disease (AD). Whereas transport of Aß across the BBB can occur via transcellular endothelial receptors, the paracellular movement of Aß has not been described. We show that soluble human Aß(1-40) monomers can diffuse across the paracellular pathway of the BBB in tandem with a decrease in the tight junction proteins claudin-5 and occludin in the cerebral vascular endothelium. In a murine model of AD (Tg2576), plasma Aß(1-40) levels were significantly increased, brain Aß(1-40) levels were decreased, and cognitive function was enhanced when both claudin-5 and occludin were suppressed. Furthermore, Aß can cause a transient down-regulation of claudin-5 and occludin, allowing for its own paracellular clearance across the BBB. Our results show, for the first time, the involvement of the paracellular pathway in autoregulated Aß movement across the BBB and identify both claudin-5 and occludin as potential therapeutic targets for AD. These findings also indicate that controlled modulation of tight junction components at the BBB can enhance the clearance of Aß from the brain.

The dynamic blood-brain barrier.

With the endothelium as its central unit, the blood-brain barrier (BBB) is a complex multicellular structure separating the central nervous system (CNS) from the systemic circulation. Disruption of the BBB has now been implicated in a multitude of acute and chronic CNS disorders indicating the potentially devastating effects of BBB breakdown on brain function. However, the healthy BBB is not an impermeable wall, but rather a communication 'centre', responding to and passing signals between the CNS and blood. New studies are identifying BBB-specific transport pathways that tightly regulate the entry and exit of molecules to and from the brain. They are revealing a highly plastic barrier in which dynamic changes in BBB components like paracellular tight junction complexes can contribute to BBB maintenance. Here, we provide a succinct overview of the current state-of-play in BBB research and summarize novel findings into BBB regulation in homeostatic regulation of the brain.

IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common form of central retinal blindness globally. Distinct processes of the innate immune system, specifically activation of the NLRP3 inflammasome, have been shown to play a central role in the development of both "dry" and neovascular ("wet") forms of the disease. We show that the inflammatory cytokine interleukin-18 (IL-18) can regulate choroidal neovascularization formation in mice. We observed that exogenous administration of mature recombinant IL-18 has no effect on retinal pigment epithelial (RPE) cell viability, but that overexpression of pro-IL-18 or pro-IL-1ß alone can cause RPE cell swelling and subsequent atrophy, a process that can be inhibited by the promotion of autophagy. A direct comparison of local and systemic administration of mature recombinant IL-18 with current anti-VEGF (vascular endothelial growth factor)-based therapeutic strategies shows that IL-18 treatment works effectively alone and more effectively in combination with anti-VEGF therapy and represents a novel therapeutic strategy for the treatment of wet AMD.

Targeted suppression of claudin-5 decreases cerebral oedema and improves cognitive outcome following traumatic brain injury.

Traumatic brain injury is the leading cause of death in children and young adults globally. Malignant cerebral oedema has a major role in the pathophysiology that evolves after severe traumatic brain injury. Added to this is the significant morbidity and mortality from cerebral oedema associated with acute stroke, hypoxic ischemic coma, neurological cancers and brain infection. Therapeutic strategies to prevent cerebral oedema are limited and, if brain swelling persists, the risks of permanent brain damage or mortality are greatly exacerbated. Here we show that a temporary and size-selective modulation of the blood-brain barrier allows enhanced movement of water from the brain to the blood and significantly impacts on brain swelling. We also show cognitive improvement in mice with focal cerebral oedema following administration in these animals of short interfering RNA directed against claudin-5. These observations may have profound consequences for early intervention in cases of traumatic brain injury, or indeed any neurological condition where cerebral oedema is the hallmark pathology.

From RNA interference technology to effective therapy: how far have we come and how far to go?

Over a decade has passed since the first description of RNAi in animals--the fundamental endogenous process by which small dsRNAs mediate sequence-specific gene silencing. This discovery has radically transformed our understanding of gene regulation and function and spawned a whole new biotechnology industry focused on developing RNAi-based therapeutic approaches to a variety of human diseases that have otherwise proved challenging to conventional therapies. While RNAi technologies hold great promise as a powerful medical tool, successful delivery of RNAi agents and effective measurement of their uptake are major challenges in translating RNAi therapies to the clinic. Exciting developments in the field have also been tempered by safety concerns surrounding the immunogenic potential of this gene silencing technology and the potential side effects associated with exploiting a crucial biological pathway for therapeutic benefit. This article examines the progress of RNAi therapeutics including advances in delivery and safety, and recent findings from several Phase I-III clinical trials. The emergence of a novel application of RNAi in enhancing the delivery of low-molecular weight drugs to neuronal tissues will also be presented to provide an outlook on the future of RNAi technologies.

Prevention of autosomal dominant retinitis pigmentosa by systemic drug therapy targeting heat shock protein 90 (Hsp90).

Retinitis pigmentosa (RP) is the most prevalent cause of registered visual handicap among working aged populations of developed countries. Up to 40% of autosomal dominant cases of disease are caused by mutations within the rhodopsin, RDS-peripherin and inosine 5'-monophosphate dehydrogenase type 1 (IMPDH1) genes, at least 30 mutations within which give rise to proteins that cause disease pathology by misfolding and aggregation. Given the genetic complexity of this disease, therapies that simultaneously target multiple mutations are of substantial logistic and economic significance. We show here, in a murine model of autosomal dominant RP (RP10) involving expression of an Arg224Pro mutation within the IMPDH1 gene, that treatment with the low-molecular-weight drug, 17-allylamino-17-demethoxygeldanamycin (17-AAG), an ansamycin antibiotic that binds to heat shock protein Hsp90, activating a heat shock response in mammalian cells, protects photoreceptors against degeneration induced by aggregating mutant IMPDH1 protein, systemic delivery of this low-molecular-weight drug to the retina being facilitated by RNA interference-mediated modulation of the inner-blood retina barrier. 17-AAG has an orphan drug status and is in current clinical use for the treatment of non-ocular diseases. These data show that a single low-molecular-weight drug has the potential to suppress a wide range of mutant proteins causing RP.