Olga Chechneva

A TSPO ligand is protective in a mouse model of multiple sclerosis

Local production of neurosteroids such as progesterone and allopregnanolone confers neuroprotection in central nervous system (CNS) inflammatory diseases. The mitochondrial translocator protein (TSPO) performs a rate‐limiting step in the conversion of cholesterol to pregnenolone and its steroid derivatives. Previous studies have shown that TSPO is upregulated in microglia and astroglia during neural inflammation, and radiolabelled TSPO ligands such as PK11195 have been used to image and localize injury in the CNS. Recent studies have shown that modulating TSPO activity with pharmacological ligands such as etifoxine can initiate the production of neurosteroids locally in the injured CNS. In this study, we examined the effects of etifoxine, a clinically available anxiolytic drug, in the development and progression of mouse experimental autoimmune encephalomyelitis (EAE), an experimental model for multiple sclerosis (MS). Our results showed that etifoxine attenuated EAE severity when administered before the development of clinical signs and also improved symptomatic recovery when administered at the peak of the disease. In both cases, recovery was correlated with diminished inflammatory pathology in the lumbar spinal cord. Modulation of TSPO activity by etifoxine led to less peripheral immune cell infiltration of the spinal cord, and increased oligodendroglial regeneration after inflammatory demyelination in EAE. Our results suggest that a TSPO ligand, e.g. etifoxine, could be a potential new therapeutic option for MS with benefits that could be comparable to the administration of systemic steroids but potentially avoiding the detrimental side effects of long‐term direct use of steroids.

hESC-derived Olig2+ progenitors generate a subtype of astroglia with protective effects against ischaemic brain injury

Human pluripotent stem cells (hPSCs) have been differentiated to astroglia, but the utilization of hPSC-derived astroglia as cell therapy for neurological diseases has not been well studied. Astroglia are heterogeneous, and not all astroglia are equivalent in promoting neural repair. A prerequisite for cell therapy is to derive defined cell populations with superior therapeutic effects. Here we use an Olig2-GFP human embryonic stem cell (hESC) reporter to demonstrate that hESC-derived Olig2+ progenitors generate a subtype of previously uncharacterized astroglia (Olig2PC-Astros). These Olig2PC-Astros differ substantially from astroglia differentiated from Olig2-negative hESC-derived neural progenitor cells (NPC-Astros), particularly in their neuroprotective properties. When grafted into brains subjected to global ischaemia, Olig2PC-Astros exhibit superior neuroprotective effects and improved behavioural outcome compared to NPC-Astros. Thus, this new paradigm of human astroglial differentiation is useful for studying the heterogeneity of human astroglia, and the unique Olig2PC-Astros may constitute a new cell therapy for treating cerebral ischaemia and other neurological diseases.

PARP-1 Deficiency Increases the Severity of Disease in a Mouse Model of Multiple Sclerosis

Poly(ADP-ribose) polymerase-1 (PARP-1) has been implicated in the pathogenesis of several central nervous system (CNS) disorders. However, the role of PARP-1 in autoimmune CNS injury remains poorly understood. Therefore, we studied experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis in mice with a targeted deletion of PARP-1. We identified inherent physiological abnormalities in the circulating and splenic immune composition between PARP-1−/− and wild type (WT) mice. Upon EAE induction, PARP-1−/− mice had an earlier onset and developed a more severe EAE compared with WT cohorts. Splenic response was significantly higher in PARP-1−/− mice largely because of B cell expansion. Although formation of Th1 and Th17 effector T lymphocytes was unaffected, PARP-1−/− mice had significantly earlier CD4+ T lymphocyte and macrophage infiltration into the CNS during EAE. However, we did not detect significant differences in cytokine profiles between PARP-1−/− and WT spinal cords at the peak of EAE. Expression analysis of different PARP isozymes in EAE spinal cords showed that PARP-1 was down-regulated in WT mice and that PARP-3 but not PARP-2 was dramatically up-regulated in both PARP-1−/− and WT mice, suggesting that these PARP isozymes could have distinct roles in different CNS pathologies. Together, our results indicate that PARP-1 plays an important role in regulating the physiological immune composition and in immune modulation during EAE; our finding identifies a new aspect of immune regulation by PARPs in autoimmune CNS pathology.

Differentiating human stem cells into neurons and glial cells for neural repair.

Research on the biology of adult stem cells, embryonic stem cells and induced pluripotent stem cells, as well as cell-based strategies for treating nervous system disorders has begun to create the hope that these cells may be used for therapy in humans after injury or disease. In animal models of neurological diseases, transplantation of stem cells or their derivatives can improve function not only due to direct replacement of lost neurons or glia, but also by providing trophic support. Despite intense research efforts to translate these studies from the bench to bedside, critical problems remain at several steps in this process. Recent technological advancements in both the derivation of stem cells and their directed differentiation to lineage-committed progenitors have brought us closer to therapeutic applications. Several preclinical studies have already explored the behavior of transplanted cells with respect to proliferation, migration, differentiation and survival, especially in complex pathological disease environments. In this review, we examine the current status, progress, pitfalls, and potential of these stem cell technologies, focusing on directed differentiation of human stem cells into various neural lineages, including dopaminergic neurons, motor neurons, oligodendroglia, microglia, and astroglia, and on advancements in cell-based regenerative strategies for neural repair and criteria for successful therapeutic applications.

A Smoothened receptor agonist is neuroprotective and promotes regeneration after ischemic brain injury.

Ischemic stroke occurs as a result of blood supply interruption to the brain causing tissue degeneration, patient disabilities or death. Currently, treatment of ischemic stroke is limited to thrombolytic therapy with a narrow time window of administration. The sonic hedgehog (Shh) signaling pathway has a fundamental role in the central nervous system development, but its impact on neural cell survival and tissue regeneration/repair after ischemic stroke has not been well investigated. Here we report the neuroprotective properties of a small-molecule agonist of the Shh co-receptor Smoothened, purmorphamine (PUR), in the middle cerebral artery occlusion model of ischemic stroke. We found that intravenous administration of PUR at 6 h after injury was neuroprotective and restored neurological deficit after stroke. PUR promoted a transient upregulation of tissue-type plasminogen activator in injured neurons, which was associated with a reduction of apoptotic cell death in the ischemic cortex. We also observed a decrease in blood–brain barrier permeability after PUR treatment. At 14 d postinjury, attenuation of inflammation and reactive astrogliosis was found in PUR-treated animals. PUR increased the number of newly generated neurons in the peri-infarct and infarct area and promoted neovascularization in the ischemic zone. Notably, PUR treatment did not significantly alter the ischemia-induced level of Gli1, a Shh target gene of tumorigenic potential. Thus our study reports a novel pharmacological approach for postischemic treatment using a small-molecule Shh agonist, providing new insights into hedgehog signaling-mediated mechanisms of neuroprotection and regeneration after stroke.

p38α (MAPK14) critically regulates the immunological response and the production of specific cytokines and chemokines in astrocytes

In CNS lesions, “reactive astrocytes” form a prominent cellular response. However, the nature of this astrocyte immune activity is not well understood. In order to study astrocytic immune responses to inflammation and injury, we generated mice with conditional deletion of p38α (MAPK14) in GFAP+ astrocytes. We studied the role of p38α signaling in astrocyte immune activation both in vitro and in vivo, and simultaneously examined the effects of astrocyte activation in CNS inflammation. Our results showed that specific subsets of cytokines (TNFα, IL-6) and chemokines (CCL2, CCL4, CXCL1, CXCL2, CXCL10) are critically regulated by p38α signaling in astrocytes. In an in vivo CNS inflammation model of intracerebral injection of LPS, we observed markedly attenuated astrogliosis in conditional GFAPcre p38α−/− mice. However, GFAPcre p38α−/− mice showed marked upregulation of CCL2, CCL3, CCL4, CXCL2, CXCL10, TNFα, and IL-1β compared to p38αfl/fl cohorts, suggesting that in vivo responses to LPS after GFAPcre p38α deletion are complex and involve interactions between multiple cell types. This finding was supported by a prominent increase in macrophage/microglia and neutrophil recruitment in GFAPcre p38α−/− mice compared to p38αfl/fl controls. Together, these studies provide important insights into the critical role of p38α signaling in astrocyte immune activation.

The hGFAP-driven conditional TSPO knockout is protective in a mouse model of multiple sclerosis

The mitochondrial translocator protein (TSPO) has been implicated in CNS diseases. Here, we sought to determine the specific role of TSPO in experimental autoimmune encephalomyelitis (EAE), the most studied animal model of multiple sclerosis (MS). To fundamentally elucidate the functions of TSPO, we first developed a viable TSPO knockout mouse. A conditional TSPO knockout mouse was generated by utilizing the Cre-Lox system. We generated a TSPO floxed mouse, and then crossed this mouse with a Cre recombinase expressing mouse driven by the human glial fibrillary acidic protein (hGFAP) promoter. The resultant mouse was a neural linage line specific TSPO knockout. The loss of TSPO in the CNS did not result in overt developmental defects or phenotypes. The TSPO−/− mouse showed a decrease in GFAP expression, correlating with a decrease in astrogliosis in response to neural injury during EAE. This decrease in astrogliosis was also witnessed in the lessening of severity of EAE clinical scoring, indicating an in vivo functional role for TSPO in suppressing EAE. The TSPO−/− mouse could be a useful tool in better understanding the role of TSPO in CNS disease, and our results implicate TSPO as a potential therapeutic target in MS.

Empowering sonic hedgehog to rescue brain cells after ischemic stroke

Ischemic stroke occurs when blood supply to the brain is interrupted. This can cause irreversible injury to the central nervous system (CNS) tissue. Each year in the United States almost 800,000 people experience a new or recurrent stroke. 15% of stroke patients die shortly after insult and only 10% recover completely, leaving the majority of surviving stroke patients with disabilities. Tissue-type plasminogen activator (tPA) is the only available therapy for stroke but its clinical use is limited because of associated danger of intracranial hemorrhage. Therefore, there is an emergent need for stroke therapeutics that are safe and effective when administered at a later time point after insult.

Mitochondrial translocator protein(TSPO),astrocytes and neuroinflammation

Mitochondrial translocator protein (TSPO) has been a somewhat mysterious player in searching for its true biological functions. TSPO is involved in mitochondrial cholesterol transport and steroids biosynthesis and predominantly expressed in steroid-synthesizing tissues including the central nervous system (CNS). However, recent findings using conditional knockout animal models contradict the long-held view that TSPO plays an essential role in steroidogenesis (Banati et al., 2014; Morohaku et al., 2014). In the CNS, TSPO is upregulated in microglia and reactive astrocytes during injury, serving as a suitable diagnostic biomarker for neuroinflammation. TSPO has been under investigation as a drug target for inflammatory conditions, and several TSPO ligands have been reported to confer neuroprotection in various CNS disorders, including multiple sclerosis (MS) (Da Pozzo et al., 2015).

p38α in CD11c+ cells drives sex-specific differences during progression of autoimmune demyelination

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disorder. Most people with MS show a relapsing-remitting disease course that over time transitions into progressive decline of neurologic function. The mechanisms underlying disease progression in MS remain poorly understood. Here we demonstrate that reduction in CD11c+ microglia promotes dispersed coalescent parenchymal infiltration and clinical progression in chronic mild experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We found sex-dependent differences in EAE progression mediated by p38α signaling, a key regulator of inflammation. CD11c promoter-driven knockout of p38α (KO) reduced CD11c+ microglia proliferation in female mice and promoted CNS infiltration and transition of chronic mild to severe disease. In protected KO males, immune cells were trapped within perivascular spaces and prevented from crossing the glia limitans closely surrounded by dendritic cell-like CD11c+ microglia. Together, our study provides the first evidence that in chronic mild EAE, CD11c+ microglia interact with astrocytes to control CNS immune cell parenchymal infiltration.Multiple sclerosis (MS) is an autoimmune demyelinating disorder developing with higher prevalence in women than men, while accelerated MS progression is evident in men. Mechanisms underlying sexual dimorphism in MS remain unclear. Here, we found that although female and male mice with conditional knockout of p38α in CD11c+ cells initially developed attenuated clinical symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as has been shown earlier, female mice underwent spontaneous disease progression after day 30 post EAE induction. EAE progression in females was associated with T lymphocyte and macrophage infiltration into the CNS parenchyma. In contrast, male mice with CD11c cell specific p38α deficiency were protected from disease progression as immune cells were trapped within perivascular spaces without entering parenchyma. We identified male-specific p38α signaling in CD11c+CD11b+Iba1+ cells to control immune cell infiltration and progression of autoimmune demyelination. These findings advance our understanding of sex-biased mechanisms in MS relapse and progression and provide new rationale for the development of sex-specific MS therapies.