William T. Hendriks

Schwann cell to axon transfer of ribosomes: toward a novel understanding of the role of glia in the nervous system.

Schwann cells play pivotal roles in the development and maintenance of the peripheral nervous system. Here, we show that intact sciatic nerve axons of mice contain a small population of ribosomes, which increases by several orders of magnitude when axons are desomatized (severed from their cell bodies). We furthermore demonstrate, using the Wallerian degeneration slow mouse as a model, that Schwann cells transfer polyribosomes to desomatized axons. These data indicate that Schwann cells have the propensity to control axonal protein synthesis by supplying ribosomes on local basis.

Phosphatidic acid mediates demyelination in Lpin1 mutant mice

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function.

Differential effects of lentiviral vector-mediated overexpression of nerve growth factor and glial cell line-derived neurotrophic factor on regenerating sensory and motor axons in the transected peripheral nerve

Even after reconstructive surgery, major functional impairments remain in the majority of patients with peripheral nerve injuries. The application of novel emerging therapeutic strategies, such as lentiviral (LV) vectors, may help to stimulate peripheral nerve regeneration at a molecular level. In the experiments described here, we examined the effect of LV vector‐mediated overexpression of nerve growth factor (NGF) and glial cell line‐derived neurotrophic factor (GDNF) on regeneration of the rat peripheral nerve in a transection/repair model in vivo. We showed that LV vectors can be used to locally elevate levels of NGF and GDNF in the injured rat peripheral nerve and this has profound and differential effects on regenerating sensory and motor neurons. For sensory neurons, increased levels of NGF and GDNF do not affect the number of regenerated neurons 1 cm distal to a lesion at 4 weeks post‐lesion but do cause changes in the expression of markers for different populations of nociceptive neurons. These changes are accompanied by significant alterations in the recovery of nociceptive function. For motoneurons, overexpression of GDNF causes trapping of regenerating axons, impairing both long‐distance axonal outgrowth and reinnervation of target muscles, whereas NGF has no effect on these parameters. These observations show the feasibility of combining surgical repair of the transected nerve with the application of viral vectors. Furthermore, they show a difference between the regenerative responses of motor and sensory neurons to locally increased levels of NGF and GDNF.

Neuroregenerative effects of lentiviral vector-mediated GDNF expression in reimplanted ventral roots

Traumatic avulsion of spinal nerve roots causes complete paralysis of the affected limb. Reimplantation of avulsed roots results in only limited functional recovery in humans, specifically of distal targets. Therefore, root avulsion causes serious and permanent disability. Here, we show in a rat model that lentiviral vector-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) in reimplanted nerve roots completely prevents motoneuron atrophy after ventral root avulsion and stimulates regeneration of axons into reimplanted roots. However, over the course of 16 weeks neuroma-like structures are formed in the reimplanted roots, and regenerating axons are trapped at sites with high levels of GDNF expression. A high local concentration of GDNF therefore impairs long distance regeneration. These observations show the feasibility of combining neurosurgical repair of avulsed roots with gene-therapeutic approaches. Our data also point to the importance of developing viral vectors that allow regulated expression of neurotrophic factors.

Morphological Evidence for a Transport of Ribosomes from Schwann Cells to Regenerating Axons

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector‐mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.

Genome Editing in Human Pluripotent Stem Cells: Approaches, Pitfalls, and Solutions

Human pluripotent stem cells (hPSCs) with knockout or mutant alleles can be generated using custom-engineered nucleases. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases are the most commonly employed technologies for editing hPSC genomes. In this Protocol Review, we provide a brief overview of custom-engineered nucleases in the context of gene editing in hPSCs with a focus on the application of TALENs and CRISPR/Cas9. We will highlight the advantages and disadvantages of each method and discuss theoretical and technical considerations for experimental design.

Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS)

Background Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts. Methodology/Principal Findings Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP. Conclusions/Significance Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.

Gene Transfer to the Spinal Cord Neural Scar with Lentiviral Vectors: Predominant Transgene Expression in Astrocytes but Not in Meningeal Cells

Viral vector–mediated overexpression of neurotrophins in cells constituting the neural scar may represent a powerful approach to rendering scar tissue of a central nervous system (CNS) lesion permissive for neuronal regrowth. In this study a lentiviral vector encoding green fluorescent protein (LV‐GFP) was injected in and around the neural scar 2 weeks after a dorsal column lesion in the rat spinal cord in order to analyze transduction characteristics of the neural scar after 4, 7, and 14 days. GFP expression was found at all points after injection and increased from 4 to 7 days, with no apparent difference observed between 7 and 14 days. The core of the lesion was virtually devoid of GFP signal despite direct vector injections in this area. The colocalization of GFP with specific cell markers (GFAP, vimentin, Raldh2, NeuN, OX‐42, ED‐1, and NG‐2) indicated that the predominant cells transduced in the rim of the lesion were astrocytes, with neurons, microglia, oligodendrocyte precursors, and macrophages transduced to a lesser extent. None of the Raldh2‐positive meningeal cells, present in the core of the scar, expressed GFP. In vitro meningeal cells were readily transduced, indicating that in vivo the formation of an extracellular matrix might prevent LV particles from transducing cells in the core of the scar. Because astrocytes are important cellular constituents of the glial scar after CNS injury, transduction of astrocytes with LV vectors encoding neurotrophic factors like BDNF or NT‐3 may be used to enhance regeneration of severed axonal tracts through or along boundaries of a CNS lesion.

The importance of transgene and cell type on the regeneration of adult retinal ganglion cell axons within reconstituted bridging grafts

When grafted onto the cut optic nerve, chimeric peripheral nerve (PN) sheaths reconstituted with adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Regrowth can be further enhanced by using PN containing SCs transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of ciliary neurotrophic factor (CNTF). To determine whether other neurotrophic factors or different cell types also enhance RGC regrowth in this bridging model, we tested the effectiveness of (1) adult SCs transduced with brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), and (2) fibroblasts (FBs) genetically modified to express CNTF. SCs transduced with LV-BDNF and LV-GDNF secreted measurable and bioactive amounts of each of these proteins, but reconstituted grafts containing LV-BDNF or LV-GDNF transduced SCs did not enhance RGC survival or axonal regrowth. LV-BDNF modified grafts did, however, contain many pan-neurofilament immunolabeled axons, many of which were also immunoreactive for calcitonin gene-related peptide (CGRP) and were presumably of peripheral sensory origin. Nor-adrenergic and cholinergic axons were also seen in these grafts. There were far fewer axons in LV-GDNF engineered grafts. Reconstituted PN sheaths containing FBs that had been modified to express CNTF did not promote RGC viability or regeneration, and PN reconstituted with a mixed population of SCs and CNTF expressing FBs were less effective than SCs alone. These data show that both the type of neurotrophic factor and the cell types that express these factors are crucial elements when designing bridging substrates to promote long-distance regeneration in the injured CNS.

TALEN‐ and CRISPR/Cas9‐Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid‐Based Transfection

Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications.