Drew L. Sellers

Postinjury Niches Induce Temporal Shifts in Progenitor Fates to Direct Lesion Repair after Spinal Cord Injury

Progenitors that express NG2-proteoglycan are the predominant self-renewing cells within the CNS. NG2 progenitors replenish oligodendrocyte populations within the intact stem cell niche, and cycling NG2 cells are among the first cells to react to CNS insults. We investigated the role of NG2 progenitors after spinal cord injury and how bone morphogen protein signals remodel the progressive postinjury (PI) niche. Progeny labeled by an NG2-specific reporter virus undergo a coordinated shift in differentiation profile. NG2 progeny born 24 h PI produce scar-forming astrocytes and transient populations of novel phagocytic astrocytes shown to contain denatured myelin within cathepsin-D-labeled endosomes, but NG2 progenitors born 7 d PI differentiate into oligodendrocytes and express myelin on processes that wrap axons. Analysis of spinal cord mRNA shows a temporal shift in the niche transcriptome of ligands that affect PI remodeling and direct progenitor differentiation. We conclude that NG2 progeny are diverse lineages that obey progressive cues after trauma to replenish the injured niche.

Dual Responsive, Stabilized Nanoparticles for Efficient In Vivo Plasmid Delivery†

Nucleic acid-based therapeutics, including plasmid DNA (pDNA) and small interfering RNA (siRNA), have been considered highly promising strategies to treat a gamut of diseases.[1] Successful nucleic acid delivery relies on the development of safe and efficient delivery vectors. Viral vectors have dominated as the delivery vehicles used in clinical trials but their progress has been hampered by their immunogenicity, safety risks and high manufacturing cost.[2] Therefore, the use of non-viral vectors such as cationic polymer-based vectors that offer advantages over viral vectors in these aspects has attracted broad attention.[3-4]

Remyelination reporter reveals prolonged refinement of spontaneously regenerated myelin

Neurological diseases and trauma often cause demyelination, resulting in the disruption of axonal function and integrity. Endogenous remyelination promotes recovery, but the process is not well understood because no method exists to definitively distinguish regenerated from preexisting myelin. To date, remyelinated segments have been defined as anything abnormally short and thin, without empirical data to corroborate these morphological assumptions. To definitively identify regenerated myelin, we used a transgenic mouse with an inducible membrane-bound reporter and targeted Cre recombinase expression to a subset of glial progenitor cells after spinal cord injury, yielding remarkably clear visualization of spontaneously regenerated myelin in vivo. Early after injury, the mean length of sheaths regenerated by Schwann cells and oligodendrocytes (OLs) was significantly shorter than control, uninjured myelin, confirming past assumptions. However, OL-regenerated sheaths elongated progressively over 6 mo to approach control values. Moreover, OL-regenerated myelin thickness was not significantly different from control myelin at most time points after injury. Thus, many newly formed OL sheaths were neither thinner nor shorter than control myelin, vitiating accepted dogmas of what constitutes regenerated myelin. We conclude that remyelination, once thought to be static, is dynamic and elongates independently of axonal growth, in contrast to stretch-based mechanisms proposed in development. Further, without clear identification, past assessments have underestimated the extent and quality of regenerated myelin.

Melittin-grafted HPMA-oligolysine based copolymers for gene delivery.

Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-l-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendent groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~100-200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain.

Adult spinal cord progenitor cells are repelled by netrin-1 in the embryonic and injured adult spinal cord.

Adult neural progenitor cells (aNPCs) exhibit limited migration in vivo with the exception of the rostral migratory stream and injury-induced movement. Surprisingly little is known regarding those signals regulating attraction or inhibition of the aNPC. These studies demonstrate that aNPCs respond principally to a repulsive cue expressed at the embryonic floor plate (FP) and also the injured adult CNS. Adult spinal cord progenitor cells (aSCPs) were seeded onto organotypic slice preparations of the intact embryonic or injured adult spinal cord. Cell migration assays combined with genetic and molecular perturbation of FP-derived migration cues or aSCP receptors establish netrin-1 (Ntn-1) but not Slit-2, Shh, or Ephrin-B3 as the primary FP-derived repellant. When slices were prepared from injured spinal cord, aSCP migration away from the injury core was Ntn-1-dependent. These studies establish Ntn-1 as a critical regulator of aSCP migration in the intact and injured CNS.

Fmr1 Transcript Isoforms: Association with Polyribosomes; Regional and Developmental Expression in Mouse Brain

The primary transcript of the mammalian Fragile X Mental Retardation-1 gene (Fmr1), like many transcripts in the central nervous system, is alternatively spliced to yield mRNAs encoding multiple proteins, which can possess quite different biochemical properties. Despite the fact that the relative levels of the 12 Fmr1 transcript isoforms examined here vary by as much as two orders of magnitude amongst themselves in both adult and embryonic mouse brain, all are associated with polyribosomes, consistent with translation into the corresponding isoforms of the protein product, FMRP (Fragile X Mental Retardation Protein). Employing the RiboTag methodology developed in our laboratory, the relative proportions of the 7 most abundant transcript isoforms were measured specifically in neurons and found to be similar to those identified in whole brain. Measurements of isoform profiles across 11 regions of adult brain yielded similar distributions, with the exceptions of the hippocampus and the olfactory bulb. These two regions differ from most of the brain in relative amounts of transcripts encoding an alternate form of one of the KH RNA binding domains. A possible relationship between patterns of expression in the hippocampus and olfactory bulb and the presence of neuroblasts in these two regions is suggested by the isoform patterns in early embryonic brain and in cultured neural progenitor cells. These results demonstrate that the relative levels of the Fmr1 isoforms are modulated according to developmental stage, highlighting the complex ramifications of losing all the protein isoforms in individuals with Fragile X Syndrome. It should also be noted that, of the eight most prominent FMRP isoforms (1–3, 6–9 and 12) in mouse, only two have the major site of phosphorylation at Ser-499, which is thought to be involved in some of the regulatory interactions of this protein.

Instructive niches: environmental instructions that confound NG2 proteoglycan expression and the fate-restriction of CNS progenitors

Cellullar deficits are replenished within the central nervous system (CNS) by progenitors to maintain integrity and recover function after injury. NG2 proteoglycan‐expressing progenitors replenish oligodendrocyte populations, but the nature of NG2 proteoglycan may not indicate a restricted population of progenitors. After injury, restorative spatiotemporal cues have the potential ability to regulate divergent fate‐choices for NG2 progenitors, and NG2 progenitors are known to produce multiple cell types in vitro. Recent data suggest that NG2 expression is attenuated while protein levels remain high within injurious tissue; thus, NG2 expression is not static but transiently controlled in response to a dynamic interplay of environmental cues. Therefore, NG2 proteoglycan expression could label newly generated cells or be inherited by resident cell populations that produce oligodendrocytes for remyelination, astrocytes that provide trophic support and other cells that contribute to CNS function.

Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding

Identifying single-cell types in the mouse brain The recent development of single-cell genomic techniques allows us to profile gene expression at the single-cell level easily, although many of these methods have limited throughput. Rosenberg et al. describe a strategy called split-pool ligation-based transcriptome sequencing, or SPLiT-seq, which uses combinatorial barcoding to profile single-cell transcriptomes without requiring the physical isolation of each cell. The authors used their method to profile >100,000 single-cell transcriptomes from mouse brains and spinal cords at 2 and 11 days after birth. Comparisons with in situ hybridization data on RNA expression from Allen Institute atlases linked these transcriptomes with spatial mapping, from which developmental lineages could be identified. Science, this issue p. 176 Single-cell analyses with SPLiT-seq (split-pool ligation-based transcriptome sequencing) elucidate development of the mouse nervous system. To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing, and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. More than 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation. Pseudotime analysis revealed transcriptional programs driving four developmental lineages, providing a snapshot of early postnatal development in the murine central nervous system. SPLiT-seq provides a path toward comprehensive single-cell transcriptomic analysis of other similarly complex multicellular systems.

Poly(lactic-co-glycolic) acid microspheres encapsulated in Pluronic F-127 prolong hirudin delivery and improve functional recovery from a demyelination lesion

Components of the blood have been proposed as potential therapeutic targets for improving cellular regeneration after injury and neurodegenerative disease. In this work, thrombin is shown to increase endogenous neural progenitor proliferation in the intact murine spinal cord. A local injection of heparin before a spinal cord injury reduces cell proliferation and astrogliogenesis associated with scarring. We sought to create depot-formulations of PLGA microsphere and Pluronic F-127 for sustained local delivery of two thrombin inhibitors, heparin and hirudin. Each hydrogel depot-formulation showed delayed drug release compared to microspheres or hydrogel alone. Animals with a lateral demyelination lesion showed a reduction in CD68+ macrophages when treated with hirudin-loaded PLGA/F-127 gels compared to control and heparin-treated animals. Moreover, hirudin-loaded materials showed an accelerated recovery in coordinated stepping and increased oligodendrocyte densities. Together, these data demonstrate that controlled delivery of hirudin accelerates functional recovery from a demyelination lesion in the spinal cord.

Evolution of a designed protein assembly encapsulating its own RNA genome

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.