The Promise of Niacin in Neurology.

Niacin (vitamin B3) is an essential nutrient that treats pellagra, and prior to the advent of statins, niacin was commonly used to counter dyslipidemia. Recent evidence has posited niacin as a promising therapeutic for several neurological disorders. In this review, we discuss the biochemistry of niacin, including its homeostatic roles in NAD+ supplementation and metabolism. Niacin also has roles outside of metabolism, largely through engaging hydroxycarboxylic acid receptor 2 (Hcar2). These receptor-mediated activities of niacin include regulation of immune responses, phagocytosis of myelin debris after demyelination or of amyloid beta in models of Alzheimer's disease, and cholesterol efflux from cells. We describe the neurological disorders in which niacin has been investigated or has been proposed as a candidate medication. These are multiple sclerosis, Alzheimer's disease, Parkinson's disease, glioblastoma and amyotrophic lateral sclerosis. Finally, we explore the proposed mechanisms through which niacin may ameliorate neuropathology. While several questions remain, the prospect of niacin as a therapeutic to alleviate neurological impairment is promising.

Detecting monocyte trafficking in an animal model of glioblastoma using R2* and quantitative susceptibility mapping.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in glioblastoma (GBM) disease progression has received increasing attention. Recent advances have shown that TAMs can be re-programmed to exert a pro-inflammatory, anti-tumor effect to control GBMs. However, imaging methods capable of differentiating tumor progression from immunotherapy treatment effects have been lacking, making timely assessment of treatment response difficult. We showed that tracking monocytes using iron oxide nanoparticle (USPIO) with MRI can be a sensitive imaging method to detect therapy response directed at the innate immune system. METHODS: We implanted syngeneic mouse glioma stem cells into C57/BL6 mice and treated the animals with either niacin (a stimulator of innate immunity) or vehicle. Animals were imaged using an anatomical MRI sequence, R2* mapping, and quantitative susceptibility mapping (QSM) before and after USPIO injection. RESULTS: Compared to vehicles, niacin-treated animals showed significantly higher susceptibility and R2*, representing USPIO and monocyte infiltration into the tumor. We observed a significant reduction in tumor size in the niacin-treated group 7 days later. We validated our MRI results with flow cytometry and immunofluoresence, which showed that niacin decreased pro-inflammatory Ly6C high monocytes in the blood but increased CD16/32 pro-inflammatory macrophages within the tumor, consistent with migration of these pro-inflammatory innate immune cells from the blood to the tumor. CONCLUSION: MRI with USPIO injection can detect therapeutic responses of innate immune stimulating agents before changes in tumor size have occurred, providing a potential complementary imaging technique to monitor cancer immunotherapies. MANUSCRIPT HIGHLIGHT: We show that iron oxide nanoparticles (USPIOs) can be used to label innate immune cells and detect the trafficking of pro-inflammatory monocytes into the glioblastoma. This preceded changes in tumor size, making it a more sensitive imaging technique.

A Distinct Hibiscus sabdariffa Extract Prevents Iron Neurotoxicity, a Driver of Multiple Sclerosis Pathology.

Iron deposition in the brain begins early in multiple sclerosis (MS) and continues unabated. Ferrous iron is toxic to neurons, yet the therapies used in MS do not counter iron neurotoxicity. Extracts of Hibiscus sabdariffa (HS) are used in many cultures for medicinal purposes. We collected a distinct HS extract and found that it abolished the killing of neurons by iron in culture; medications used in MS were ineffective when similarly tested. Neuroprotection by HS was not due to iron chelation or anthocyanin content. In free radical scavenging assays, HS was equipotent to alpha lipoic acid, an anti-oxidant being tested in MS. However, alpha lipoic acid was only modestly protective against iron-mediated killing. Moreover, a subfraction of HS without radical scavenging activity negated iron toxicity, whereas a commercial hibiscus preparation with anti-oxidant activity could not. The idea that HS might have altered properties within neurons to confer neuroprotection is supported by its amelioration of toxicity caused by other toxins: beta-amyloid, rotenone and staurosporine. Finally, in a mouse model of MS, HS reduced disability scores and ameliorated the loss of axons in the spinal cord. HS holds therapeutic potential to counter iron neurotoxicity, an unmet need that drives the progression of disability in MS.

Remyelination trial failures: Repercussions of ignoring neurorehabilitation and exercise in repair.

Several putative neurorestorative therapies in multiple sclerosis (MS) have recently failed; this includes high-dose biotin, bexarotene, a retinoic acid receptor gamma agonist, and opicinumab (anti-LINGO-1). Are these failures biological or due to poor trial design? We argue that the failure to include exercise in these trials and selecting participants without the capacity for repair may explain these disappointing results. We propose the need for mapping the biological mechanisms of recovery within trials, understanding the critical window when remyelination/repair occurs in terms of targeting interventions at the right time and selecting subjects who are capable of repair. We also make the case for testing combinations that include other pro-repair interventions such as exercise, Nrf2 inducers and possibly neurostimulation. The MS community can't afford for any more treatments to fail because of poor trial design and ignoring biology.

Studying the microglia response to oxidized phosphatidylcholine in primary mouse neuron culture and mouse spinal cord.

Oxidized phosphatidylcholine (OxPC) found in multiple sclerosis brain lesions mediates neurodegeneration. Microglia are prominent responders to the OxPC insult, and thus, studying their protective or noxious functions is important to help halt neurodegeneration. Here, we present protocols including cell isolation and culture, animal surgeries, as well as tissue processing and isolation to study the microglia response to OxPC-mediated neurodegeneration in vitro and in vivo. For complete details on the use and execution of this protocol, please refer to Dong et al. (2021).

Chondroitin sulfate proteoglycans as novel drivers of leucocyte infiltration in multiple sclerosis.

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with leucocytes in inflammatory perivascular cuffs in all four patients. We conclude that the accumulation of chondroitin sulfate proteoglycans in the perivascular cuff in multiple sclerosis and experimental autoimmune encephalomyelitis boosts the activity and migration of leucocytes across the glia limitans into the central nervous system parenchyma. Thus, chondroitin sulfate proteoglycans represent a new class of molecules to overcome in order to reduce the inflammatory cascades and clinical severity of multiple sclerosis.

Systematic screening of generic drugs for progressive multiple sclerosis identifies clomipramine as a promising therapeutic.

The treatment of progressive multiple sclerosis (MS) is unsatisfactory. One reason is that the drivers of disease, which include iron-mediated neurotoxicity, lymphocyte activity, and oxidative stress, are not simultaneously targeted. Here we present a systematic screen to identify generic, orally available medications that target features of progressive MS. Of 249 medications that cross the blood-brain barrier, 35 prevent iron-mediated neurotoxicity in culture. Of these, several antipsychotics and antidepressants strongly reduce T-cell proliferation and oxidative stress. We focus on the antidepressant clomipramine and found that it additionally inhibits B-lymphocyte activity. In mice with experimental autoimmune encephalomyelitis, a model of MS, clomipramine ameliorates clinical signs of acute and chronic phases. Histologically, clomipramine reduces inflammation and microglial activation, and preserves axonal integrity. In summary, we present a systematic approach to identify generic medications for progressive multiple sclerosis with the potential to advance rapidly into clinical trials, and we highlight clomipramine for further development.

Remyelination therapies: a new direction and challenge in multiple sclerosis.

Multiple sclerosis is characterized by inflammatory activity that results in destruction of the myelin sheaths that enwrap axons. The currently available medications for multiple sclerosis are predominantly immune-modulating and do not directly promote repair. White matter regeneration, or remyelination, is a new and exciting potential approach to treating multiple sclerosis, as remyelination repairs the damaged regions of the central nervous system. A wealth of new strategies in animal models that promote remyelination, including the repopulation of oligodendrocytes that produce myelin, has led to several clinical trials to test new reparative therapies. In this Review, we highlight the biology of, and obstacles to, remyelination. We address new strategies to improve remyelination in preclinical models, highlight the therapies that are currently undergoing clinical trials and discuss the challenges of objectively measuring remyelination in trials of repair in multiple sclerosis.

Therapeutic activation of macrophages and microglia to suppress brain tumor-initiating cells.

Brain tumor initiating cells (BTICs) contribute to the genesis and recurrence of gliomas. We examined whether the microglia and macrophages that are abundant in gliomas alter BTIC growth. We found that microglia derived from non-glioma human subjects markedly mitigated the sphere-forming capacity of glioma patient-derived BTICs in culture by inducing the expression of genes that control cell cycle arrest and differentiation. This sphere-reducing effect was mimicked by macrophages, but not by neurons or astrocytes. Using a drug screen, we validated amphotericin B (AmpB) as an activator of monocytoid cells and found that AmpB enhanced the microglial reduction of BTIC spheres. In mice harboring intracranial mouse or patient-derived BTICs, daily systemic treatment with non-toxic doses of AmpB substantially prolonged life. Notably, microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of autologous BTICs, but this was rectified by AmpB. These results provide new insights into the treatment of gliomas.

Screening for inhibitors of microglia to reduce neuroinflammation.

BACKGROUND: Despite the significant role microglia play in the pathology of multiple sclerosis (MS), medications that act within the central nervous system (CNS) to inhibit microglia have not yet been identified as treatment options. OBJECTIVE: We screened 1040 compounds with the aim of identifying inhibitors of microglia to reduce neuroinflammation. METHODS: The NINDs collection of 1040 compounds, where most are therapeutic medications, was tested at 10 µM final concentration on lipopolysaccharide (LPS)-activated human microglia. An ELISA was run on the media to measure the level of TNF-α as an indicator of microglia activity. For compounds that reduce LPS-activated TNF-α levels by over 50%, considered as a potential inhibitor of interest, toxicity tests were conducted to exclude non-specific cytotoxicity. Promising compounds were subjected to further analyses, including toxicity to other CNS cell types, and multiplex assays. RESULTS: Of 1040 compounds tested, 123 reduced TNF-α levels of LPS-activated microglia by over 50%. However, most of these were cytotoxic to microglia at the concentration tested while 54 were assessed to be non-toxic. Of the latter, spironolactone was selected for further analyses. Spironolactone reduced TNF-α levels of activated microglia by 50-60% at 10 µM, and this concentration did not kill microglia, neurons or astrocytes. In multiplex assays, spironolactone reduced several molecules in activated microglia. Finally, during the screening, we identified 9 compounds that elevated further the TNF-α levels in LPS-activated microglia. CONCLUSION: Many of the non-toxic compounds identified in this screen as inhibitors of microglia, including spironolactone, may be explored as viable therapeutic options in MS.

Susceptibility-weighted imaging in the experimental autoimmune encephalomyelitis model of multiple sclerosis indicates elevated deoxyhemoglobin, iron deposition and demyelination.

BACKGROUND: Susceptibility-weighted imaging (SWI) is an iron-sensitive magnetic resonance imaging (MRI) method that has shown iron-related lesions in multiple sclerosis (MS) patients. The contribution of deoxyhemoglobin to the signals seen in SWI has not been well characterized in MS. OBJECTIVES: To determine if SWI lesions (seen as focal hypointensities) exist in the experimental autoimmune encephalomyelitis (EAE) animal model of MS, and to determine whether the lesions relate to iron deposits, inflammation, demyelination, and/or deoxyhemoglobin in the vasculature. METHODS: We performed SWI on the lumbar spinal cord and cerebellum of EAE and control mice (both complete Freund's adjuvant/pertussis toxin (CFA/PTX)-immunized and naive). We also performed SWI on mice before and after perfusion (to remove blood from vessels). SWI lesions were counted and their locations were compared to histology for iron, myelin and inflammation. RESULTS: SWI lesions were found to exist in the EAE model. Many lesions seen by SWI were not present after perfusion, especially at the grey/white matter boundary of the lumbar spinal cord and in the cerebellum, indicating that these lesion signals were associated with deoxyhemoglobin present in the lumen of vessels. We also observed SWI lesions in the white matter of the lumbar spinal cord that corresponded to iron deposition, inflammation and demyelination. In the cerebellum, SWI lesions were present in white matter tracts, where we found histological evidence of inflammatory perivascular cuffs. CONCLUSIONS: SWI lesions exist in EAE mice. Many lesions seen in SWI were a result of deoxyhemoglobin in the blood, and so may indicate areas of hypoxia. A smaller number of SWI lesions coincided with parenchymal iron, demyelination, and/or inflammation.

Relative importance of proteinase-activated receptor-1 versus matrix metalloproteinases in intracerebral hemorrhage-mediated neurotoxicity in mice.

BACKGROUND AND PURPOSE: To reduce bleeding and damage to central nervous system tissue in intracerebral hemorrhage, the coagulant effect of thrombin is essential. However, thrombin itself can kill neurons in intracerebral hemorrhage as can the matrix metalloproteinases (MMPs), which are also elevated in this condition, in part due to thrombin-mediated activation of MMPs. It is thus important to understand and block the neurotoxic effects of thrombin without inhibiting its therapeutic outcomes. In this study, we have investigated the relative roles of proteinase activated receptor-1, a thrombin receptor, and MMPs in brain injury induced by thrombin or blood. METHODS: Mice were subjected to stereotactic intracerebral injections of saline, thrombin, and autologous blood, with or without hirudin, a thrombin inhibitor, or GM6001, an MMP inhibitor. Twenty-four hours later, tissue sections were obtained to evaluate the area of brain damage and extent of dying neurons. Data from wild-type mice were compared with results obtained with proteinase activated receptor-1 null mice. RESULTS: In blood-induced damage to the brain parenchyma, both hirudin and GM6001 significantly reduced injury to a comparable extent (>40%) implicating both thrombin and MMPs in neurotoxicity. In proteinase activated receptor-1 null mice, blood-induced brain damage was reduced by 22.6% relative to wild-type animals; by comparison, the blood-induced brain damage was reduced by 48.3% using GM6001. CONCLUSIONS: The neurotoxicity of blood in intracerebral hemorrhage involves both proteinase activated receptor-1 and MMP activation, with the latter appearing more prominent in causing death.

The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility.

Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.