Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.

Author Correction: Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

In Vivo Imaging of CNS Injury and Disease.

In vivo optical imaging has emerged as a powerful tool with which to study cellular responses to injury and disease in the mammalian CNS. Important new insights have emerged regarding axonal degeneration and regeneration, glial responses and neuroinflammation, changes in the neurovascular unit, and, more recently, neural transplantations. Accompanying a 2017 SfN Mini-Symposium, here, we discuss selected recent advances in understanding the neuronal, glial, and other cellular responses to CNS injury and disease with in vivo imaging of the rodent brain or spinal cord. We anticipate that in vivo optical imaging will continue to be at the forefront of breakthrough discoveries of fundamental mechanisms and therapies for CNS injury and disease.

Energy metabolism and energy-sensing pathways in mammalian embryonic and adult stem cell fate.

Metabolism is influenced by age, food intake, and conditions such as diabetes and obesity. How do physiological or pathological metabolic changes influence stem cells, which are crucial for tissue homeostasis? This Commentary reviews recent evidence that stem cells have different metabolic demands than differentiated cells, and that the molecular mechanisms that control stem cell self-renewal and differentiation are functionally connected to the metabolic state of the cell and the surrounding stem cell niche. Furthermore, we present how energy-sensing signaling molecules and metabolism regulators are implicated in the regulation of stem cell self-renewal and differentiation. Finally, we discuss the emerging literature on the metabolism of induced pluripotent stem cells and how manipulating metabolic pathways might aid cellular reprogramming. Determining how energy metabolism regulates stem cell fate should shed light on the decline in tissue regeneration that occurs during aging and facilitate the development of therapies for degenerative or metabolic diseases.

Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction, and impairs mitophagy as evidenced by the accumulation of the PINK1 substrate pS65-Ubiquitin (pUb). We discovered MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes its active complex, resulting in increased rates of mitophagy. Treatment with MTK458 mediates clearance of accumulated pUb and α-synuclein pathology in α-synuclein pathology models in vitro and in vivo. Our findings from preclinical PD models suggest that pharmacological activation of PINK1 warrants further clinical evaluation as a therapeutic strategy for disease modification in PD.

Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction and impairs mitophagy, driving accumulation of the PINK1 substrate pS65-Ubiquitin (pUb) in primary neurons and in vivo. We synthesized MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes an active heterocomplex, thereby increasing mitophagy. MTK458 mediates clearance of α-synuclein pathology in PFF seeding models in vitro and in vivo and reduces pUb. We developed an ultrasensitive assay to quantify pUb levels in plasma and observed an increase in pUb in PD subjects that correlates with disease progression, paralleling our observations in PD models. Our combined findings from preclinical PD models and patient biofluids suggest that pharmacological activation of PINK1 is worthy of further study as a therapeutic strategy for disease modification in PD. Highlights: Discovery of a plasma Parkinson's Disease biomarker candidate, pS65-Ubiquitin (pUb)Plasma pUb levels correlate with disease status and progression in PD patients.Identification of a potent, brain penetrant PINK1 activator, MTK458MTK458 selectively activates PINK1 by stimulating dimerization and stabilization of the PINK1/TOM complexMTK458 drives clearance of α-synuclein pathology and normalizes pUb in in vivo Parkinson's models.

Microglial Gi-dependent dynamics regulate brain network hyperexcitability.

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.

Fibrinogen Induces Microglia-Mediated Spine Elimination and Cognitive Impairment in an Alzheimer's Disease Model.

Cerebrovascular alterations are a key feature of Alzheimer's disease (AD) pathogenesis. However, whether vascular damage contributes to synaptic dysfunction and how it synergizes with amyloid pathology to cause neuroinflammation and cognitive decline remain poorly understood. Here, we show that the blood protein fibrinogen induces spine elimination and promotes cognitive deficits mediated by CD11b-CD18 microglia activation. 3D molecular labeling in cleared mouse and human AD brains combined with repetitive in vivo two-photon imaging showed focal fibrinogen deposits associated with loss of dendritic spines independent of amyloid plaques. Fibrinogen-induced spine elimination was prevented by inhibiting reactive oxygen species (ROS) generation or genetic ablation of CD11b. Genetic elimination of the fibrinogen binding motif to CD11b reduced neuroinflammation, synaptic deficits, and cognitive decline in the 5XFAD mouse model of AD. Thus, fibrinogen-induced spine elimination and cognitive decline via CD11b link cerebrovascular damage with immune-mediated neurodegeneration and may have important implications in AD and related conditions.

Pericytes: The Brain's Very First Responders?

Infection can negatively impact brain functions. Here, Duan et al. (2018) show that specific PDGFRß-expressing cell subtypes of the neurovascular unit release the chemokine CCL2 rapidly after systemic infection, leading to increased neural excitability.

Breaking boundaries-coagulation and fibrinolysis at the neurovascular interface.

Blood proteins at the neurovascular unit (NVU) are emerging as important molecular determinants of communication between the brain and the immune system. Over the past two decades, roles for the plasminogen activation (PA)/plasmin system in fibrinolysis have been extended from peripheral dissolution of blood clots to the regulation of central nervous system (CNS) functions in physiology and disease. In this review, we discuss how fibrin and its proteolytic degradation affect neuroinflammatory, degenerative and repair processes. In particular, we focus on novel functions of fibrin-the final product of the coagulation cascade and the main substrate of plasmin-in the activation of immune responses and trafficking of immune cells into the brain. We also comment on the suitability of the coagulation and fibrinolytic systems as potential biomarkers and drug targets in diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD) and stroke. Studying coagulation and fibrinolysis as major molecular pathways that regulate cellular functions at the NVU has the potential to lead to the development of novel strategies for the detection and treatment of neurologic diseases.

Hypoxia Inducible Factor-1α in Astrocytes and/or Myeloid Cells Is Not Required for the Development of Autoimmune Demyelinating Disease

Hypoxia-like tissue alterations, characterized by the upregulation of hypoxia-inducible factor-1α (HIF-1α), have been described in the normal appearing white matter and pre-demyelinating lesions of multiple sclerosis (MS) patients. As HIF-1α regulates the transcription of a wide set of genes involved in neuroprotection and neuroinflammation, HIF-1α expression may contribute to the pathogenesis of inflammatory demyelination. To test this hypothesis, we analyzed the effect of cell-specific genetic ablation or overexpression of HIF-1α on the onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. HIF-1α was mainly expressed in astrocytes and microglia/macrophages in the mouse spinal cord at the peak of EAE. However, genetic ablation of HIF-1α in astrocytes and/or myeloid cells did not ameliorate clinical symptoms. Furthermore, conditional knock-out of Von Hippel Lindau, a negative regulator of HIF-1α stabilization, failed to exacerbate the clinical course of EAE. In accordance with clinical symptoms, genetic ablation or overexpression of HIF-1α did not change the extent of spinal cord inflammation and demyelination. Overall, our data indicate that despite dramatic upregulation of HIF-1α in astrocytes and myeloid cells in EAE, HIF-1α expression in these two cell types is not required for the development of inflammatory demyelination. Despite numerous reports indicating HIF-1α expression in glia, neurons, and inflammatory cells in the CNS of MS patients, the cell-specific contribution of HIF-1α to disease pathogenesis remains unclear. Here we show that although HIF-1α is dramatically upregulated in astrocytes and myeloid cells in EAE, cell-specific depletion of HIF-1α in these two cell types surprisingly does not affect the development of neuroinflammatory disease. Together with two recently published studies showing a role for oligodendrocyte-specific HIF-1α in myelination and T-cell-specific HIF-1α in EAE, our results demonstrate a tightly regulated cellular specificity for HIF-1α contribution in nervous system pathogenesis.

Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-ß signaling and astrocyte functions.

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-ß signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

Expansion of oligodendrocyte progenitor cells following SIRT1 inactivation in the adult brain.

Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults, new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs), but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized, the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new OPCs in the adult mouse brain, in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally, generating fully myelinating oligodendrocytes. Remarkably, SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling, in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases, such as multiple sclerosis.

Energy metabolism in adult neural stem cell fate.

The adult mammalian brain contains a population of neural stem cells that can give rise to neurons, astrocytes, and oligodendrocytes and are thought to be involved in certain forms of memory, behavior, and brain injury repair. Neural stem cell properties, such as self-renewal and multipotency, are modulated by both cell-intrinsic and cell-extrinsic factors. Emerging evidence suggests that energy metabolism is an important regulator of neural stem cell function. Molecules and signaling pathways that sense and influence energy metabolism, including insulin/insulin-like growth factor I (IGF-1)-FoxO and insulin/IGF-1-mTOR signaling, AMP-activated protein kinase (AMPK), SIRT1, and hypoxia-inducible factors, are now implicated in neural stem cell biology. Furthermore, these signaling modules are likely to cooperate with other pathways involved in stem cell maintenance and differentiation. This review summarizes the current understanding of how cellular and systemic energy metabolism regulate neural stem cell fate. The known consequences of dietary restriction, exercise, aging, and pathologies with deregulated energy metabolism for neural stem cells and their differentiated progeny will also be discussed. A better understanding of how neural stem cells are influenced by changes in energy availability will help unravel the complex nature of neural stem cell biology in both the normal and diseased state.

The microRNA cluster miR-106b~25 regulates adult neural stem/progenitor cell proliferation and neuronal differentiation.

In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93, and miR-25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice. We find that knocking down miR-25 decreases NSPC proliferation, whereas ectopically expressing miR-25 promotes NSPC proliferation. Expressing the entire miR-106b~25 cluster in NSPCs also increases their ability to generate new neurons. Interestingly, miR-25 has a number of potential target mRNAs involved in insulin/insulin-like growth factor-1 (IGF) signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR-106b~25 is bound by FoxO3, a member of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF signaling. These results suggest that miR-106b~25 regulates NSPC function and is part of a network involving the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.

FoxO3 regulates neural stem cell homeostasis.

In the nervous system, neural stem cells (NSCs) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3(-/-) mice have fewer NSCs in vivo than wild-type counterparts. NSCs isolated from adult FoxO3(-/-) mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSCs suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSCs might have important implications for counteracting brain aging in long-lived species.

The cloning and characterization of a second brain enzyme with NAAG peptidase activity.

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.