Catherine Czeisler

Self-Assembling Nanofibers Inhibit Glial Scar Formation and Promote Axon Elongation after Spinal Cord Injury

Peptide amphiphile (PA) molecules that self-assemble in vivo into supramolecular nanofibers were used as a therapy in a mouse model of spinal cord injury (SCI). Because self-assembly of these molecules is triggered by the ionic strength of the in vivo environment, nanoscale structures can be created within the extracellular spaces of the spinal cord by simply injecting a liquid. The molecules are designed to form cylindrical nanofibers that display to cells in the spinal cord the laminin epitope IKVAV at nearly van der Waals density. IKVAV PA nanofibers are known to inhibit glial differentiation of cultured neural stem cells and to promote neurite outgrowth from cultured neurons. In this work, in vivo treatment with the PA after SCI reduced astrogliosis, reduced cell death, and increased the number of oligodendroglia at the site of injury. Furthermore, the nanofibers promoted regeneration of both descending motor fibers and ascending sensory fibers through the lesion site. Treatment with the PA also resulted in significant behavioral improvement. These observations demonstrate that it is possible to inhibit glial scar formation and to facilitate regeneration after SCI using bioactive three-dimensional nanostructures displaying high densities of neuroactive epitopes on their surfaces.

Noggin Expands Neural Stem Cells in the Adult Hippocampus

New neurons are added to the adult hippocampus throughout life and contribute to cognitive functions, including learning and memory. It remains unclear whether ongoing neurogenesis arises from self-renewing neural stem cells (NSCs) or from multipotential progenitor cells that cannot self-renew in the hippocampus. This is primarily based on observations that neural precursors derived from the subventricular zone (SVZ) can be passaged long term, whereas hippocampal subgranular zone (SGZ) precursors are rapidly depleted by passaging. We demonstrate here that high levels of bone morphogenetic protein (BMP) signaling occur in hippocampal but not SVZ precursors in vitro, and blocking BMP signaling with Noggin is sufficient to foster hippocampal cell self-renewal, proliferation, and multipotentiality using single-cell clonal analysis. Moreover, NSC maintenance requires continual Noggin exposure, which implicates BMPs as crucial regulators of NSC aging. In vivo, Noggin is expressed in the adult dentate gyrus and limits BMP signaling in proliferative cells of the SGZ. Transgenic Noggin overexpression in the SGZ increases multiple precursor cell populations but proportionally increases the glial fibrillary acidic protein-positive cell population at the expense of other precursors, suggesting that Noggin acts on NSCs in vivo. To confirm this, we used a dual thymidine analog paradigm to repeatedly label slowly dividing cells over a long duration. We find that small populations of label-retaining cells exist in the SGZ and that Noggin overexpression increases their numbers. Thus, we propose that the adult hippocampus contains a population of NSCs, which can be expanded both in vitro and in vivo by blocking BMP signaling.

Self‐assembling peptide amphiphile promotes plasticity of serotonergic fibers following spinal cord injury

Injection into the injured spinal cord of peptide amphiphile (PA) molecules that self‐assemble and display the laminin epitope IKVAV at high density improved functional recovery after spinal cord injury (SCI) in two different species, rat and mouse, and in two different injury models, contusion and compression. The improvement required the IKVAV epitope and was not observed with the injection of an amphiphile displaying a nonbioactive sequence. To explore the mechanisms underlying these improvements, the number of serotonergic fibers in the lesioned spinal cord was compared in animals receiving the IKVAV‐PA, a nonbioactive PA (PA control), or sham injection. Serotonergic fibers were distributed equally in all three groups rostral to the injury but showed a significantly higher density caudal to the injury site in the IKVAV PA‐injected group. Furthermore, this difference was not present in the subacute phase following injury but appeared in the chronically injured cord. The IKVAV PA‐injected groups also trended higher both in the total number neurons adjacent to the lesion and in the number of long propriospinal tract connections from the thoracic to the lumbar cord. IKVAV PA injection did not alter myelin thickness, total axon number caudal to the lesion, axon size distribution, or total axon area. Serotonin can promote stepping even in complete transection models, so the improved function produced by the IKVAV PA treatment may reflect the increased serotonergic innervation caudal to the lesion in addition to the previously demonstrated regeneration of motor and sensory axons through the lesion.

Modification of gelation kinetics in bioactive peptide amphiphiles

Peptide amphiphiles (PAs) previously designed in our laboratory are known to self-assemble into nanofibers that exhibit bioactivity both in vitro and in vivo. Self-assembly can be triggered by charge neutralization or salt-mediated screening of charged residues in their peptide sequences, and the resulting nanofibers can form macroscopic gels at concentrations as low as 0.5% by weight. Controlling the kinetics of gelation while retaining the bioactivity of nanofibers could be critical in tailoring these materials for specific clinical applications. We report here on a series of PAs with different rates of gelation resulting from changes in their peptide sequence without changing the bioactive segment. The pre-existence of hydrogen-bonded aggregates in the solution state of more hydrophobic PAs appears to accelerate gelation kinetics. Mutation of the peptide sequence to include more hydrophilic and bulky amino acids suppresses formation of these nuclei and effectively slows down gelation through self-assembly of the nanofiber network. The ability to modify gelation kinetics in self-assembling systems without disrupting bioactivity could be important for injectable therapies in regenerative medicine.

An anteroposterior wave of vascular inhibitor downregulation signals aortae fusion along the embryonic midline axis.

Paracrine signals, both positive and negative, regulate the positioning and remodeling of embryonic blood vessels. In the embryos of mammals and birds, the first major remodeling event is the fusion of bilateral dorsal aortae at the midline to form the dorsal aorta. Although the original bilaterality of the dorsal aortae occurs as the result of inhibitory factors (antagonists of BMP signaling) secreted from the midline by the notochord, it is unknown how fusion is later signaled. Here, we report that dorsal aortae fusion is tightly regulated by a change in signaling by the notochord along the anteroposterior axis. During aortae fusion, the notochord ceases to exert its negative influence on vessel formation. This is achieved by a transcriptional downregulation of negative regulators while positive regulators are maintained at pre-fusion levels. In particular, Chordin, the most abundant BMP antagonist expressed in the notochord prior to fusion, undergoes a dramatic downregulation in an anterior to posterior wave. With inhibitory signals diminished and sustained expression of the positive factors SHH and VEGF at the midline, fusion of the dorsal aortae is signaled. These results demonstrate a novel mechanism by which major modifications of the vascular pattern can occur through modulation of vascular inhibitors without changes in the levels of positive vascular regulators.

Dysregulation of locus coeruleus development in congenital central hypoventilation syndrome

Human congenital central hypoventilation syndrome (CCHS), resulting from mutations in transcription factor PHOX2B, manifests with impaired responses to hypoxemia and hypercapnia especially during sleep. To identify brainstem structures developmentally affected in CCHS, we analyzed two postmortem neonatal-lethal cases with confirmed polyalanine repeat expansion (PARM) or Non-PARM (PHOX2B∆8) mutation of PHOX2B. Both human cases showed neuronal losses within the locus coeruleus (LC), which is important for central noradrenergic signaling. Using a conditionally active transgenic mouse model of the PHOX2B∆8 mutation, we found that early embryonic expression (<E10.5) caused failure of LC neuronal specification and perinatal respiratory lethality. In contrast, later onset (E11.5) of PHOX2B∆8 expression was not deleterious to LC development and perinatal respiratory lethality was rescued, despite failure of chemosensor retrotrapezoid nucleus formation. Our findings indicate that early-onset mutant PHOX2B expression inhibits LC neuronal development in CCHS. They further suggest that such mutations result in dysregulation of central noradrenergic signaling, and therefore, potential for early pharmacologic intervention in humans with CCHS.

Upregulation of glial clusterin in brains of patients with AIDs

Since clusterin (CLU) production in reactive astrocytes may be neuroprotective, we examined its distribution in AIDS brains where brain injury and reactive astrocytosis are common. The relative area and number of CLU-positive astrocytes, as well as their percent total of all white matter glia, significantly increased in AIDS brains with and without HIV encephalitis (P<0.05). Proliferation markers were absent. In contrast, the relative area and number of GFAP-positive astrocytes and their percent of all white matter glia, increased in some cases but the mean increases were not significant. Clusterin is sensitive marker of glial reactivity in AIDS brains and its enhanced expression was not dependent on increases in GFAP.

Chronic systemic administration of tumor necrosis factor alpha and HIV gp120: effects on adult rodent brain and blood-brain barrier.

Since tumor necrosis factor alpha (TNF-alpha) and HIV gpl20 glycoprotein are both neurotoxic, the possibility that systemic sources of these two agents mediate AIDS-associated blood-brain barrier (BBB) breakdown and brain damage was tested in two murine models: (1) intramuscular implantation of a TNF-alpha-transfected tumor in nu/nu mice and (2) daily subcutaneous injections of HIV gpl20 in BALB/c mice. The BBB remained intact; brain damage was not found, and apoptotic cell numbers did not increase. These results show that normal adult brain and BBB is unaffected by exposure to TNF-alpha or HIV gpl20 and suggest that severity of brain disease is not directly affected by systemic levels of these compounds.

Cerebellar cortical lamination and foliation require cyclin A2

The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers

UNLABELLED Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogrammings clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. FROM THE CLINICAL EDITOR In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.