Stem Cell Therapies for Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal degeneration. MS patients typically present with a relapsing-remitting (RR) disease course, manifesting as sporadic attacks of neurological symptoms including ataxia, fatigue, and sensory impairment. While there are several effective disease-modifying therapies able to address the inflammatory relapses associated with RRMS, most patients will inevitably advance to a progressive disease course marked by a gradual and irreversible accrual of disabilities. Therapeutic intervention in progressive MS (PMS) suffers from a lack of well-characterized biological targets and, hence, a dearth of successful drugs. The few medications approved for the treatment of PMS are typically limited in their efficacy to active forms of the disease, have little impact on slowing degeneration, and fail to promote repair. In looking to address these unmet needs, the multifactorial therapeutic benefits of stem cell therapies are particularly compelling. Ostensibly providing neurotrophic support, immunomodulation and cell replacement, stem cell transplantation holds substantial promise in combatting the complex pathology of chronic neuroinflammation. Herein, we explore the current state of preclinical and clinical evidence supporting the use of stem cells in treating PMS and we discuss prospective hurdles impeding their translation into revolutionary regenerative medicines.

Physiological and Pathological Factors Affecting Drug Delivery to the Brain by Nanoparticles.

The prevalence of neurological/neurodegenerative diseases, such as Alzheimer's disease is known to be increasing due to an aging population and is anticipated to further grow in the decades ahead. The treatment of brain diseases is challenging partly due to the inaccessibility of therapeutic agents to the brain. An increasingly important observation is that the physiology of the brain alters during many brain diseases, and aging adds even more to the complexity of the disease. There is a notion that the permeability of the blood-brain barrier (BBB) increases with aging or disease, however, the body has a defense mechanism that still retains the separation of the brain from harmful chemicals in the blood. This makes drug delivery to the diseased brain, even more challenging and complex task. Here, the physiological changes to the diseased brain and aged brain are covered in the context of drug delivery to the brain using nanoparticles. Also, recent and novel approaches are discussed for the delivery of therapeutic agents to the diseased brain using nanoparticle based or magnetic resonance imaging guided systems. Furthermore, the complement activation, toxicity, and immunogenicity of brain targeting nanoparticles as well as novel in vitro BBB models are discussed.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Succinate Receptor 1: An Emerging Regulator of Myeloid Cell Function in Inflammation.

The rapidly evolving area of immunometabolism has shed new light on the fundamental properties of products and intermediates of cellular metabolism (metabolites), highlighting their key signaling roles in cell-to-cell communication. Recent evidence identifies the succinate-succinate receptor 1 (SUCNR1) axis as an essential regulator of tissue homeostasis. Succinate signaling via SUCNR1 guides divergent responses in immune cells, which are tissue and context dependent. Herein, we explore the main cellular pathways regulated by the succinate-SUCNR1 axis and focus on the biology of SUCNR1 and its roles influencing the function of myeloid cells. Hence, we identify new therapeutic targets and putative therapeutic approaches aimed at resolving detrimental myeloid cell responses in tissues, including those occurring in the persistently inflamed central nervous system (CNS).

Combination of In Situ Lcn2 pRNA-RNAi Nanotherapeutics and iNSC Transplantation Ameliorates Experimental SCI in Mice.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by different cellular and molecular mechanisms that interplay in exacerbating the progression of the pathology. No fully restorative therapies are yet available, and it is thus becoming recognized that combinatorial approaches aimed at addressing different aspects of SCI will likely results in greater functional outcomes. Here we employed packaging RNA-mediated RNA interference (pRNA-RNAi) nanotherapeutics to downregulate in situ the expression of lipocalin 2 (Lcn2), a known mediator of neuroinflammation and autocrine mediator of reactive astrogliosis, and to create a more amenable niche for the subsequent transplantation of induced neural stem cells (iNSCs). To our knowledge, this is the first approach that takes advantage of the modular and multifunctional pRNA three-way junction platform in the SCI niche, while also exploiting the therapeutic potential of immune-compatible and feasible iNSC transplants. We show the combination of such treatments in a mouse model of contusion thoracic SCI leads to significant improvement of locomotor function, albeit not better than single pRNA-RNAi treatment, and results in synergistic histopathological effects, such as reduction of glial scar volume, diminished pro-inflammatory response, and promotion of neuronal survival. Our results provide evidence for a novel combinatorial approach for treating SCI.

Promises and Limitations of Neural Stem Cell Therapies for Progressive Multiple Sclerosis.

Multiple disease-modifying medications with regulatory approval to treat multiple sclerosis (MS) are unable to prevent inflammatory tissue damage in the central nervous system (CNS), and none directly promote repair. Thus, there is an unmet clinical need for therapies that can arrest and reverse the persistent accumulation of disabilities associated with progressive forms of MS (P-MS). Preclinical research has revealed an unexpected ability of neural stem cell (NSC) therapies to provide neurotrophic support and inhibit detrimental host immune responses in vivo following transplantation into the chronically inflamed CNS. We discuss NSC transplantation as a promising therapy for P-MS, elaborate on the necessities of clinical trial validation and formalized usage guidelines, and caution about unscrupulous 'clinics' marketing unproven therapies to patients.

Parkinson's disease, aging and adult neurogenesis: Wnt/ß-catenin signalling as the key to unlock the mystery of endogenous brain repair.

A common hallmark of age-dependent neurodegenerative diseases is an impairment of adult neurogenesis. Wingless-type mouse mammary tumor virus integration site (Wnt)/ß-catenin (WßC) signalling is a vital pathway for dopaminergic (DAergic) neurogenesis and an essential signalling system during embryonic development and aging, the most critical risk factor for Parkinson's disease (PD). To date, there is no known cause or cure for PD. Here we focus on the potential to reawaken the impaired neurogenic niches to rejuvenate and repair the aged PD brain. Specifically, we highlight WßC-signalling in the plasticity of the subventricular zone (SVZ), the largest germinal region in the mature brain innervated by nigrostriatal DAergic terminals, and the mesencephalic aqueduct-periventricular region (Aq-PVR) Wnt-sensitive niche, which is in proximity to the SNpc and harbors neural stem progenitor cells (NSCs) with DAergic potential. The hallmark of the WßC pathway is the cytosolic accumulation of ß-catenin, which enters the nucleus and associates with T cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors, leading to the transcription of Wnt target genes. Here, we underscore the dynamic interplay between DAergic innervation and astroglial-derived factors regulating WßC-dependent transcription of key genes orchestrating NSC proliferation, survival, migration and differentiation. Aging, inflammation and oxidative stress synergize with neurotoxin exposure in "turning off" the WßC neurogenic switch via down-regulation of the nuclear factor erythroid-2-related factor 2/Wnt-regulated signalosome, a key player in the maintenance of antioxidant self-defense mechanisms and NSC homeostasis. Harnessing WßC-signalling in the aged PD brain can thus restore neurogenesis, rejuvenate the microenvironment, and promote neurorescue and regeneration.

Explicating Exosomes: Reclassifying the Rising Stars of Intercellular Communication.

There is growing interest surrounding the diagnostic and therapeutic potential of exosomes, but a definitive description of these extracellular vesicles remains elusive. In this issue, Jeppesen et al. characterize exosomes following a strict isolation protocol and in so doing challenge several of the accepted properties of these agents of intercellular communication.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

Quantitative high-throughput screening identifies cytoprotective molecules that enhance SUMO conjugation via the inhibition of SUMO-specific protease (SENP)2.

The development of novel neuroprotective treatments for acute stroke has been fraught with failures, which supports the view of ischemic brain damage as a highly complex multifactorial process. Post-translational modifications such as small ubiquitin-like modifier (SUMO)ylation have emerged as critical molecular regulatory mechanisms in states of both homeostasis and ischemic stress, as evidenced by our previous work. Accordingly, the clinical significance of the selective control of the global SUMOylation process has become apparent in studies of ischemic pathobiology and pathophysiology. Herein, we describe a process capable of identifying and characterizing small molecules with the potential of targeting the SUMO system through inhibition of SUMO deconjugation in an effort to develop novel stroke therapies.-Bernstock, J. D., Ye, D., Smith, J. A., Lee, Y.-J., Gessler, F. A., Yasgar, A., Kouznetsova, J., Jadhav, A., Wang, Z., Pluchino, S., Zheng, W., Simeonov, A., Hallenbeck, J. M., Yang, W. Quantitative high-throughput screening identifies cytoprotective molecules that enhance SUMO-conjugation via the inhibition of SUMO-specific protease (SENP)2.

Supramolecular approach to enantioselective DNA recognition using enantiomerically resolved cationic 4-amino-1,8-naphthalimide-based Tröger's bases.

The synthesis and photophysical studies of two cationic Tröger's base (TB)-derived bis-naphthalimides 1 and 2 and the TB derivative 6, characterized by X-ray crystallography, are presented. The enantiomers of 1 and 2 are separated by cation-exchange chromatography on Sephadex C25 using sodium (-)-dibenzoyl-l-tartarate as the chiral mobile phase. The binding of enantiomers with salmon testes (st)-DNA and synthetic polynucleotides are studied by a variety of spectroscopic methods including UV/vis absorbance, circular dichroism, linear dichroism, and ethidium bromide displacement assays, which demonstrated binding of these compounds to the DNA grooves with very high affinity (K ∼ 10(6) M(-1)) and preferential binding of (-)-enantiomer. In all cases, binding to DNA resulted in a significant stabilization of the double-helical structure of DNA against thermal denaturation. Compound (±)-2 and its enantiomers possessed significantly higher binding affinity for double-stranded DNA compared to 1, possibly due to the presence of the methyl group, which allows favorable hydrophobic and van der Waals interactions with DNA. The TB derivatives exhibited marked preference for AT rich sequences, where the binding affinities follow the order (-)-enantiomer > (±) > (+)-enantiomer. The compounds exhibited significant photocleavage of plasmid DNA upon visible light irradiation and are rapidly internalized into malignant cell lines.

Extracellular membrane vesicles and immune regulation in the brain.

The brain is characterized by a complex and integrated network of interacting cells in which cell-to-cell communication is critical for proper development and function. Initially considered as an immune privileged site, the brain is now regarded as an immune specialized system. Accumulating evidence reveals the presence of immune components in the brain, as well as extensive bidirectional communication that takes place between the nervous and the immune system both under homeostatic and pathological conditions. In recent years the secretion of extracellular membrane vesicles (EMVs) has been described as a new and evolutionary well-conserved mechanism of cell-to-cell communication, with EMVs influencing the microenvironment through the traffic of bioactive molecules that include proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Increasing evidence suggests that EMVs are a promising candidate to study cross-boundary cell-to-cell communication pathways. Herein we review the role of EMVs secreted by neural cells in modulating the immune response(s) within the brain under physiological and pathological circumstances.

Structure determination of an intercalating ruthenium dipyridophenazine complex which kinks DNA by semiintercalation of a tetraazaphenanthrene ligand.

We describe a crystal structure, at atomic resolution (1.1 Å, 100 K), of a ruthenium polypyridyl complex bound to duplex DNA, in which one ligand acts as a wedge in the minor groove, resulting in the 51° kinking of the double helix. The complex cation Λ-[Ru(1,4,5,8-tetraazaphenanthrene)(2)(dipyridophenazine)](2+) crystallizes in a 11 ratio with the oligonucleotide d(TCGGCGCCGA) in the presence of barium ions. Each complex binds to one duplex by intercalation of the dipyridophenazine ligand and also by semiintercalation of one of the orthogonal tetraazaphenanthrene ligands into a second symmetrically equivalent duplex. The result is noncovalent cross-linking and marked kinking of DNA.

Excited state dependent electron transfer of a rhenium-dipyridophenazine complex intercalated between the base pairs of DNA: a time-resolved UV-visible and IR absorption investigation into the photophysics of fac-[Re(CO)3(F2dppz)(py)]+ bound to either [poly(dA-dT)]2 or [poly(dG-dC)]2.

The transient species formed following excitation of fac-[Re(CO)(3)(F(2)dppz)(py)](+) (F(2)dppz = 11,12-difluorodipyrido[3,2-a:2',3'-c]phenazine) bound to double-stranded polynucleotides [poly(dA-dT)](2) or [poly(dG-dC)](2) have been studied by transient visible and infra-red spectroscopy in both the picosecond and nanosecond time domains. The latter technique has been used to monitor both the metal complex and the DNA by monitoring the regions 1900-2100 and 1500-1750 cm(-1) respectively. These data provide direct evidence for electron transfer from guanine to the excited state of the metal complex, which proceeds both on a sub-picosecond time scale and with a lifetime of 35 ps, possibly due to the involvement of two excited states. No electron transfer is found for the [poly(dA-dT)](2) complex, although characteristic changes are seen in the DNA-region TRIR consistent with changes in the binding of the bases in the intercalation site upon excitation of the dppz-complex.

Excited state behaviour of substituted dipyridophenazine Cr(III) complexes in the presence of nucleic acids.

The photophysics and photochemistry of [Cr(phen)(2)(dppz)](3+) and its 11,12-substituted derivatives [Cr(phen)(2)(X(2)dppz)](3+) {X = Me or F} have been studied in the presence of purine nucleotides or DNA using steady state and time-resolved absorption and luminescence spectroscopy. 5'-Adenosine monophosphate (5'-AMP) shows only a weak interaction with the excited states of each complex. By contrast they are efficiently quenched by 5'-guanosine monophosphate (5'-GMP), consistent with photo-induced electron transfer. Laser flash photolysis spectroscopy in the presence of 5'-GMP suggests that both forward and back electron-transfers are rapid. All complexes also display a strong affinity for DNA and evidence for both static and dynamic quenching mechanisms is provided.

Substituted dipyridophenazine complexes of Cr(III): synthesis, enantiomeric resolution and binding interactions with calf thymus DNA.

[Cr(phen)(2)(X(2)dppz)](3+) {X = H, Me, or F} have been synthesised, characterised, and chromatographically resolved into their constituent Delta and Lambda enantiomers. The DNA-binding interactions of each of the racemic complexes were investigated, with the results of linear dichroism, thermal denaturation, and emission quenching studies indicative of intercalative binding to CT-DNA with a significant electrostatic contribution. UV/Vis absorption titrations suggest strong DNA binding by each of the racemic complexes, with the methylated analogue [Cr(phen)(2)(Me(2)dppz)](3+) exhibiting the largest equilibrium binding constant. Emission quenching and UV-Vis titrations of the enantiomers of [Cr(phen)(2)(dppz)](3+) imply similar binding affinities for the Delta and Lambda isomers, although significant differences between the circular dichroism spectra of the enantiomers in the presence of DNA connote differences in binding orientation and/or conformation between the two.

Dinuclear ruthenium(ii) complexes with flexible bridges as non-duplex DNA binding agents.

The stereoisomers of a series of dinuclear ruthenium(ii) complexes [{Ru(phen)(2)}(2)(micro-BL)](4+) (phen = 1,10-phenanthroline) with flexible bridging ligands (BL) bb2 {1,2-bis[4(4'-methyl-2,2'-bipyridyl)]ethane}, bb5 {1,5-bis[4(4'-methyl-2,2'-bipyridyl)]pentane}, bb7 {1,7-bis[4(4'-methyl-2,2'-bipyridyl)]heptane}, and bb10 {1,10-bis[4(4'-methyl-2,2'-bipyridyl)]decane} have been synthesised. Their binding to a control dodecanucleotide, d(CCGGAATTCCGG)(2), and a tridecanucleotide, d(CCGAGAATTCCGG)(2), which contains a single adenine bulge have been studied using fluorescence displacement assays involving intercalating and groove-binding dyes, equilibrium dialysis and binding affinity chromatography. The fluorescence intercalator displacement (FID) assay indicated that LambdaLambda-[{Ru(phen)(2)}(2)(micro-bb7)](4+) had the greatest binding affinity with all the oligonucleotides, whereas an analogous fluorescence technique using a minor-groove binding dye, equilibrium dialysis and affinity binding chromatography showed that DeltaDelta-[{Ru(phen)(2)}(2)(micro-bb7)](4+) had the strongest binding. An (1)H NMR study of the binding of the DeltaDelta-enantiomer of [{Ru(phen)(2)}(2)(micro-bb7)](4+) to d(CCGAGAATTCCGG)(2) confirmed the selectivity of the metal complex for the bulge site and provided the basis for an energy-minimised binding model of the dinuclear ruthenium complex with the single adenine bulge containing trinucleotide. The binding model demonstrated the ability of the flexibly-linked complex to follow the curvature of the DNA minor groove.

DNA affinity binding studies using a fluorescent dye displacement technique: the dichotomy of the binding site.

We have observed a number of discrepancies and contradictions in the use of a fluorescent intercalator displacement assay in surveying the binding affinities of dinuclear polypyridyl ruthenium(II) complexes with DNA. By a modification of the assay using the fluorescent minor-groove binder 4',6-diamidino-2-phenylindole, rather than intercalating dyes (ethidium bromide or thiazole orange), results were obtained for all complexes studied which were consistent with relative affinities and stereoselectivities observed with other techniques, including NMR, affinity chromatography and equilibrium dialysis. It is believed that the difference in binding mode between the minor groove-binding Ru(II) complexes and the intercalating fluorescent dyes they are displacing may contribute to these discrepancies.

Dinuclear ruthenium(II) complexes as potential probes for RNA bulge sites.

1H NMR spectroscopy and molecular modelling have been used to investigate the binding of the DeltaDelta-and LambdaLambda-enantiomers of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; bpm = 2,2'-bipyrimidine] to an RNA tridecanucleotide duplex containing a single-base bulge [r(CCGAGAAUUCCGG)2]], and the corresponding control dodecanucleotide [r(CCGGAAUUCCGG)2]. Both enantiomers bound the control RNA sequence weakly. From upfield shifts of the metal complex H3 and H3' protons throughout the titration of the control dodecanucleotide with DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+, a binding constant of 1 x 10(3) M(-1) was determined. In NOESY spectra of the control sequence with added DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+, NOEs were only observed to protons from the terminal base-pair residues. No significant changes in chemical shift were observed for either the metal complex or RNA protons upon addition of the LambdaLambda-enantiomer to the control dodecanucleotide. The DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ complex bound the bulge-containing RNA with a significantly greater affinity (6 x 10(4) M(-1)) than the non-bulge control RNA duplex. Competition binding experiments indicated that the LambdaLambda-isomer bound the tridecanucleotide with similar affinity to the DeltaDelta-enantiomer. Addition of DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ to the bulge-containing tridecanucleotide induced selective changes in chemical shift for the base H8 and sugar H1' resonances from the adenine bulge residue, and resonances from nucleotide residues adjacent to the bulge site. Intermolecular NOEs observed in NOESY spectra of the tridecanucleotide with added DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ confirmed the selective binding of the ruthenium complex at the bulge site. Preliminary binding models, consistent with the NMR data, showed that the ruthenium complex could effectively associate in the RNA minor groove at the bulge site.