A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons.

Although intradendritic protein synthesis has been documented in adult neurons, the question of whether axons actively synthesize proteins remains controversial. Adult sensory neurons that are conditioned by axonal crush can rapidly extend processes in vitro by regulating the translation of existing mRNAs (Twiss et al., 2000). These regenerating processes contain axonal but not dendritic proteins. Here we show that these axonal processes of adult sensory neurons cultured after conditioning injury contain ribosomal proteins, translational initiation factors, and rRNA. Pure preparations of regenerating axons separated from the DRG cell bodies can actively synthesize proteins in vitro and contain ribosome-bound beta-actin and neurofilament mRNAs. Blocking protein synthesis in these regenerating sensory axons causes a rapid retraction of their growth cones when communication with the cell body is blocked by axotomy or colchicine treatment. These findings indicate that axons of adult mammalian neurons can synthesize proteins and suggest that, under some circumstances, intra-axonal translation contributes to structural integrity of the growth cone in regenerating axons. By immunofluorescence, translation factors, ribosomal proteins, and rRNA were also detected in motor axons of ventral spinal roots analyzed after 7 d in vivo after a peripheral axonal crush injury. Thus, adult motor neurons are also likely capable of intra-axonal protein synthesis in vivo after axonal injury.

Translational control of ribosomal protein L4 mRNA is required for rapid neurite regeneration.

Under some circumstances neurons can be primed to rapidly regenerate injured neuritic processes independent of new gene expression. Such transcription-independent neurite extension occurs in adult rat sensory neurons cultured after sciatic nerve crush and in NGF-differentiated PC12 cells whose neurites have been mechanically sheared. In the PC12 cells, neurite regeneration occurs by means of translational control of mRNAs which were transcribed prior to neurite injury. The survival of such translationally regulated mRNAs is relatively short in the differentiated PC12 cells (< or =10 h). By subtractive hybridization, we have isolated a short-lived mRNA from differentiated PC12 cells. This mRNA, which encodes the ribosomal protein L4, is translationally regulated during neurite regeneration in PC12 cells. Antisense oligonucleotides to L4 mRNA inhibit neurite regeneration from the differentiated PC12 cells as well as axonal elongation from conditioned sensory neurons, indicating that ongoing translation of L4 mRNA is needed for these forms of rapid transcription-independent neurite growth. Taken together, these data point to the importance of translational regulation of existing neuronal mRNAs in the regenerative responses to neuronal injury. Although there are other examples of neuronal translational control, there are no other known neuronal proteins whose levels are regulated predominantly by translational rather than transcriptional control.

Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA.

The role of the low affinity neurotrophin receptor p75LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75LNTR in nerve growth factor (NGF) signal transduction. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75LNTR enhances this response. p75LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75LNTR, but also the ratio of p75LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75LNTR and no Trk. Thus, p75LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA.

p75(NGFR) and TrkA receptors collaborate to rapidly activate a p75(NGFR)-associated protein kinase.

The role of the low affinity nerve growth factor receptor (p75(NGFR)) in NGF-mediated signaling is not yet understood. Here we show by co-immunoprecipitation that NGF activates a protein kinase that is directly associated with p75(NGFR) in dorsal root ganglion (DRG) cells and PC12 cells in culture. Two proteins of 120 and 104 kDa constitute the majority of this activity. In PC12 cells, TrkA activation was necessary to elicit p75(NGFR)-associated kinase activity. Although NGF binding to p75(NGFR) was not necessary for kinase activation, it accelerated the activation of the kinase at low NGF concentrations. Deletion analysis showed that a 43 amino acid region in the cytoplasmic domain of p75(NGFR) was responsible for this effect. These findings show that p75(NGFR) accelerates TrkA-mediated signaling and, in addition, demonstrate that p75NGFR and TrkA collaborate to activate a previously undescribed p75(NGFR)-associated protein kinase.

Nerve growth factor promotes neurite regeneration in PC12 cells by translational control.

When PC12 cells are primed with nerve growth factor (NGF) for periods of > or = 1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short-lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regeneration.

Concomitant branching enzyme and phosphorylase deficiencies. An unusual glycogenosis with extensive neuronal polyglucosan storage.

A baby girl was born hypotonic and was respirator-dependent until death at 43 days of age. A muscle biopsy revealed PAS-positive, diastase-resistant sarcoplasmic inclusions with a vaguely fibrillar structure by electron microscopy. Biochemical studies at autopsy disclosed complete absence of branching enzyme in skeletal muscle and heart, and a deficiency of phosphorylase activity in skeletal muscle with a modest reduction in myocardium. Storage material was present in glia and perikarya of neurons, increasing in amount in the rostrocaudal direction, involving most severely the motor neurons in the brain stem and spinal cord, dorsal root ganglia and myenteric plexi. Inclusions were also present in most organs, especially liver and skeletal muscle. Ultrastructurally, the inclusions ranged from granular aggregates of membrane-bound material concentrated in the region of Golgi apparatus to large filamentous bodies similar to polyglucosan bodies. This baby differs from other patients with infantile glycogenosis IV by the severity and onset of symptoms at birth, involvement of neuronal perikarya and widespread extraneural deposits. The combined deficiencies of branching enzyme and phosphorylase may have accounted for the unique clinical and neuropathological findings.

Neonatal encephalopathy with neuronal vacuolar degeneration.

A 6-month-old female with no family history of neurologic disease was born with dysmorphic features and hypotonia the cause of which could not be determined despite extensive laboratory evaluation for a primary metabolic disorder. Neuroimaging studies disclosed progressive cerebral atrophy and ventricular dilatation. Post-mortem examination of the brain revealed micrencephaly and severe neuronal cytoplasmic vacuolation notably in the thalamus and inferior olivary nuclei. The neurons of the cortex, cerebellum, and brain stem were also vacuolated, but to a lesser extent. The white matter was predominantly intact. While superficially reminsicent to the cases of spongy glio-neuronal dystrophy of Jellinger and Seitelberger [(1970) Acta Neuropathol (Berl) 16:125-40], the severity of the neuronal vacuolation without neuronal loss and absence of seizures in this patient suggests that this condition represents a distinct pathologic entity.

Inherited syndrome of infantile olivopontocerebellar atrophy, micronodular cirrhosis, and renal tubular microcysts: review of the literature and a report of an additional case.

An 8-month-old male infant who presented in the neonatal period with failure to thrive, bilateral pleural and pericardial effusions, and hepatic insufficiency characterized by elevated liver functions tests and hypoalbuminemia was found at autopsy to have an unusual combination of olivopontocerebellar atrophy (OPCA), micronodular cirrhosis, and renal tubular microcysts. Metabolic evaluation was significant only for elevated urine dicarboxylic acids. In the brain, sections from the cerebellum showed marked atrophy of folia most severe in the vermal and paravermal regions. In addition, mild neuronal loss was present in the basis pontis and inferior olivary nuclei accompanied by gliosis. Residual Purkinje cells in the cerebellar hemispheres exhibited greatly expanded and swollen arbors, which ultrastructurally were found to contain densely packed membranous cytoplasmic body-like inclusions that had the appearance of unwinding, lamellar coils. Review of the literature shows that this constellation of findings has been associated with carbohydrate-deficient transferrin. This biochemical marker along with the distinctive clinical presentation and pathological features clearly delineates a unique subset of OPCA.

Globular glial fibrillary acidic protein-reactive cytoplasmic inclusions in ependymoma: an immunoelectron-microscopic study.

We report of a 27-month-old boy with a recurrent infratentorial ependymoma; the initial resection was at 14 months of age. Both resection specimens were histologically similar. In addition to neoplastic ependymal cells forming perivascular pseudo-rosettes, a second population of cells with identical nuclear morphology displayed large hyaline, refractile cytoplasmic inclusion, causing these cells to superficially resemble gemistocytic astrocytes. These inclusions demonstrated strong glial fibrillary acidic protein (GFAP) immunoreactivity. Ultrastructurally, the inclusions appeared as fenestrated irregular-shaped bodies with jagged edges and were made up of electron-dense granular material. Although these inclusions superficially resembled Rosenthal fibers, immunoperoxidase stains for ubiquitin and alpha B-crystallin were negative. Immunoelectron microscopy showed that these unusual non-filamentous inclusions were diffusely GFAP positive.

Selective death of immature neurons in methylmalonic acidemia of the neonate: a case report.

We report the clinical and post-mortem findings in a 4-day-old female infant with methylmalonic acidemia (MMA) prematurely delivered at 36 weeks gestation. Complementation analysis revealed a defect in methylmalonyl-CoA mutase apoenzyme activity. There was selective cell death of immature neurons involving germinal matrix, migrating neuroblasts, and both external and internal granular cell layers of the cerebellum and dentate gyrus of the hippocampus. Karyorrhexis was also seen in the lymphoid follicles of spleen, lymph nodes and in the thymus. Additionally, the spinal nerve roots demonstrated optically empty spaces in their myelin sheaths due to adaxonal separation of their myelin lamellae.

Pigmented intracerebral neuroepithelial cyst: report of a case.

A 35-year-old man with seizure disorder was found to have a parietal lobe neuroepithelial cyst. The lining of the cyst was made up of ependymal cells containing neuromelanin, which was confirmed ultrastructurally. This appears to be the first report of a pigmented neuroepithelial cyst in the brain parenchyma.