Jan van Minnen

A glia-derived acetylcholine-binding protein that modulates synaptic transmission

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Differential Transport and Local Translation of Cytoskeletal, Injury-Response, and Neurodegeneration Protein mRNAs in Axons

Recent studies have begun to focus on the signals that regulate axonal protein synthesis and the functional significance of localized protein synthesis. However, identification of proteins that are synthesized in mammalian axons has been mainly based on predictions. Here, we used axons purified from cultures of injury-conditioned adult dorsal root ganglion (DRG) neurons and proteomics methodology to identify axonally synthesized proteins. Reverse transcription (RT)-PCR from axonal preparations was used to confirm that the mRNA for each identified protein extended into the DRG axons. Proteins and the encoding mRNAs for the cytoskeletal proteins β-actin, peripherin, vimentin, γ-tropomyosin 3, and cofilin 1 were present in the axonal preparations. In addition to the cytoskeletal elements, several heat shock proteins (HSP27, HSP60, HSP70, grp75, αB crystallin), resident endoplasmic reticulum (ER) proteins (calreticulin, grp78/BiP, ERp29), proteins associated with neurodegenerative diseases (ubiquitin C-terminal hydrolase L1, rat ortholog of human DJ-1/Park7, γ-synuclein, superoxide dismutase 1), anti-oxidant proteins (peroxiredoxins 1 and 6), and metabolic proteins (e.g., phosphoglycerate kinase 1 (PGK 1), α enolase, aldolase C/Zebrin II) were included among the axonally synthesized proteins. Detection of the mRNAs encoding each of the axonally synthesized proteins identified by mass spectrometry in the axonal compartment indicates that the DRG axons have the potential to synthesize a complex population of proteins. Local treatment of the DRG axons with NGF or BDNF increased levels of cytoskeletal mRNAs into the axonal compartment by twofold to fivefold but had no effect on levels of the other axonal mRNAs studied. Neurotrophins selectively increased transport of β-actin, peripherin, and vimentin mRNAs from the cell body into the axons rather than changing transcription or mRNA survival in the axonal compartment.

Axonal and presynaptic protein synthesis: new insights into the biology of the neuron.

The presence of a local mRNA translation system in axons and terminals was proposed almost 40 years ago. Over the ensuing period, an impressive body of evidence has grown to support this proposal -- yet the nerve cell body is still considered to be the only source of axonal and presynaptic proteins. To dispel this lingering neglect, we now present the wealth of recent observations bearing on this central idea, and consider their impact on our understanding of the biology of the neuron. We demonstrate that extrasomatic translation sites, which are now well recognized in dendrites, are also present in axonal and presynaptic compartments.

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.

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity

Subcellular localization of mRNAs is regulated by RNA–protein interactions. Here, we show that introduction of a reporter mRNA with the 3′UTR of β‐actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β‐actin mRNA, and introducing GFP with the 3′UTR of β‐actin mRNA depletes axons of endogenous β‐actin and GAP‐43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β‐actin and GAP‐43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

Local synthesis of actin-binding protein beta-thymosin regulates neurite outgrowth

Local protein synthesis plays an essential role in the regulation of various aspects of axonal and dendritic function in adult neurons. At present, however, there is no direct evidence that local protein translation is functionally contributing to neuronal outgrowth. Here, we identified the mRNA encoding the actin-binding protein β-thymosin as one of the most abundant transcripts in neurites of outgrowing neurons in culture. β-Thymosin mRNA is not evenly distributed in neurites, but appears to accumulate at distinct sites such as turning points and growth cones. Using double-stranded RNA knockdown, we show that reducing β-thymosin mRNA levels results in a significant increase in neurite outgrowth, both in neurites of intact cells and in isolated neurites. Together, our data demonstrate that local synthesis of β-thymosin is functionally involved in regulating neuronal outgrowth.

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.

An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy.

SUMMARY Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, GarsC201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The GarsC201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

Identification of molluscan nicotinic acetylcholine receptor (nAChR) subunits involved in formation of cation- and anion-selective nAChRs.

Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates.

Long-term proteasome dysfunction in the mouse brain by expression of aberrant ubiquitin

Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimers disease (AD) and polyglutamine diseases such as Huntingtons disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex. Moreover, by proteomic analysis, we identified expression changes in proteins involved in energy metabolism or organization of the cytoskeleton. These changes show a striking resemblance to the proteomic profiles of both AD brain and several AD mouse models. Moreover, UBB(+1) transgenic mice show a deficit in contextual memory in both water maze and fear conditioning paradigms. Although UBB(+1) partially inhibits the UPS in the cortex, these mice do not have an overt neurological phenotype. These mouse models do not replicate the full spectrum of AD-related changes, yet provide a tool to understand how the UPS is involved in AD pathological changes and in memory formation.