George Zanazzi

Neuregulin-1 Type III Determines the Ensheathment Fate of Axons

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

Role of the Cell Wall Phenolic Glycolipid-1 in the Peripheral Nerve Predilection of Mycobacterium leprae

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Clustering of neuronal sodium channels requires contact with myelinating Schwann cells

Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the α subunit of the sodium channel and against ankyrinG, a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells.

Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier.

Voltage-dependent sodium (Na(+)) channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na(+) channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, we treated myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc had no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibited Na(+) channel and ankyrin G accumulation at the node. Nr-Fc bound directly to neurons and clustered and coprecipitated neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM.

The molecular architecture of ribbon presynaptic terminals

The primary receptor neurons of the auditory, vestibular, and visual systems encode a broad range of sensory information by modulating the tonic release of the neurotransmitter glutamate in response to graded changes in membrane potential. The output synapses of these neurons are marked by structures called synaptic ribbons, which tether a pool of releasable synaptic vesicles at the active zone where glutamate release occurs in response to calcium influx through L-type channels. Ribbons are composed primarily of the protein, RIBEYE, which is unique to ribbon synapses, but cytomatrix proteins that regulate the vesicle cycle in conventional terminals, such as Piccolo and Bassoon, also are found at ribbons. Conventional and ribbon terminals differ, however, in the size, molecular composition, and mobilization of their synaptic vesicle pools. Calcium-binding proteins and plasma membrane calcium pumps, together with endomembrane pumps and channels, play important roles in calcium handling at ribbon synapses. Taken together, emerging evidence suggests that several molecular and cellular specializations work in concert to support the sustained exocytosis of glutamate that is a hallmark of ribbon synapses. Consistent with its functional importance, abnormalities in a variety of functional aspects of the ribbon presynaptic terminal underlie several forms of auditory neuropathy and retinopathy.

Differential Expression of Proteoglycans at Central and Peripheral Nodes of Ranvier

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage‐gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan‐3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan‐3. We have examined the molecular composition of the PNS nodes in syndecan‐3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan‐3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.

Neurotrimin expression during cerebellar development suggests roles in axon fasciculation and synaptogenesis

We investigated the temporal expression of the neural cell adhesion molecule, neurotrimin, in the rat cerebellum and the brainstem from birth to adulthood using immunoreactive labeling. A wave of expression accompanied the development of projection pathways extending from brainstem nuclei (pons/inferior olive) through the cerebellar peduncles into the arbor vitae and disappeared with myelination by P14. Immuno-EM revealed expression of neurotrimin on the surface of unmyelinated axons but not on astrocytes or oligodendroglia. With the development of the molecular and internal granular layers, intense labeling occurred on the surface of parallel fiber bundles, granule cells and mossy fibers. With synaptogenesis, each excitatory junction was labeled by the immunoreaction. By P21, neurotrimin reactivity decreased on the surfaces of neuronal somata, dendrites and axons but remained at excitatory synaptic contact sites in both the molecular and granular layers. The spatial-temporal expression pattern of neurotrimin suggests that this adhesion molecule plays a role in axonal fasciculation of specific cerebellar systems and may also be involved in the formation of excitatory synapses and their stabilization into adulthood.

Aggressive resection at the infiltrative margins of glioblastoma facilitated by intraoperative fluorescein guidance

OBJECTIVE Extent of resection is an important prognostic factor in patients undergoing surgery for glioblastoma (GBM). Recent evidence suggests that intravenously administered fluorescein sodium associates with tumor tissue, facilitating safe maximal resection of GBM. In this study, the authors evaluate the safety and utility of intraoperative fluorescein guidance for the prediction of histopathological alteration both in the contrast-enhancing (CE) regions, where this relationship has been established, and into the non-CE (NCE), diffusely infiltrated margins. METHODS Thirty-two patients received fluorescein sodium (3 mg/kg) intravenously prior to resection. Fluorescence was intraoperatively visualized using a Zeiss Pentero surgical microscope equipped with a YELLOW 560 filter. Stereotactically localized biopsy specimens were acquired from CE and NCE regions based on preoperative MRI in conjunction with neuronavigation. The fluorescence intensity of these specimens was subjectively classified in real time with subsequent quantitative image analysis, histopathological evaluation of localized biopsy specimens, and radiological volumetric assessment of the extent of resection. RESULTS Bright fluorescence was observed in all GBMs and localized to the CE regions and portions of the NCE margins of the tumors, thus serving as a visual guide during resection. Gross-total resection (GTR) was achieved in 84% of the patients with an average resected volume of 95%, and this rate was higher among patients for whom GTR was the surgical goal (GTR achieved in 93.1% of patients, average resected volume of 99.7%). Intraoperative fluorescein staining correlated with histopathological alteration in both CE and NCE regions, with positive predictive values by subjective fluorescence evaluation greater than 96% in NCE regions. CONCLUSIONS Intraoperative administration of fluorescein provides an easily visualized marker for glioma pathology in both CE and NCE regions of GBM. These findings support the use of fluorescein as a microsurgical adjunct for guiding GBM resection to facilitate safe maximal removal.

Stabilization of Spontaneous Neurotransmitter Release at Ribbon Synapses by Ribbon-Specific Subtypes of Complexin

Ribbon synapses of tonically releasing sensory neurons must provide a large pool of releasable vesicles for sustained release, while minimizing spontaneous release in the absence of stimulation. Complexins are presynaptic proteins that may accomplish this dual task at conventional synapses by interacting with the molecular machinery of synaptic vesicle fusion at the active zone to retard spontaneous vesicle exocytosis yet facilitate release evoked by depolarization. However, ribbon synapses of photoreceptor cells and bipolar neurons in the retina express distinct complexin subtypes, perhaps reflecting the special requirements of these synapses for tonic release. To investigate the role of ribbon-specific complexins in transmitter release, we combined presynaptic voltage clamp, fluorescence imaging, electron microscopy, and behavioral assays of photoreceptive function in zebrafish. Acute interference with complexin function using a peptide derived from the SNARE-binding domain increased spontaneous synaptic vesicle fusion at ribbon synapses of retinal bipolar neurons without affecting release triggered by depolarization. Knockdown of complexin by injection of an antisense morpholino into zebrafish embryos prevented photoreceptor-driven migration of pigment in skin melanophores and caused the pigment distribution to remain in the dark-adapted state even when embryos were exposed to light. This suggests that loss of complexin function elevated spontaneous release in illuminated photoreceptors sufficiently to mimic the higher release rate normally associated with darkness, thus interfering with visual signaling. We conclude that visual system-specific complexins are required for proper illumination-dependent modulation of the rate of neurotransmitter release at visual system ribbon synapses.

The safety of resection for primary central nervous system lymphoma: a single institution retrospective analysis.

Surgical resection is not the standard of care for primary central nervous system lymphoma (PCNSL), as historical studies have demonstrated unfavorable complication rates and limited benefits. Some recent studies suggest that resection may provide a therapeutic benefit, yet the safety of these procedures has not been systematically investigated in the setting of modern neurosurgery. We examined the safety of surgical resection for PCNSL. We retrospectively analyzed all patients with PCNSL treated at Columbia University Medical Center between 2000 and 2015 to assess complications rates following biopsy or resection using the Glioma Outcomes Project system. We identified predictors of complications and selection for resection. Well-validated scales were used to quantify patients’ baseline clinical characteristics, including functional status, comorbid disease burden, and cardiac risk. The overall complication rate was 17.2% after resection, and 28.2% after biopsy. Cardiac risk (p = 0.047, OR 1.72 [1.01, 2.95]), and comorbid diagnoses (p = 0.004, OR 3.05 [1.42, 6.57]) predicted complications on multivariable regression. Patients who underwent resection had better KPS scores (median 70 v. 80, p = 0.0068, ∆ 10 [0.0, 10.00]), and were less likely to have multiple (46.5% v. 27.6%, p = 0.030, OR 1.42 [1.05, 1.92]) or deep lesions (70.4% v. 39.7%, p = 0.001, OR 1.83 [1.26, 2.65]). Age (p = 0.048, OR 0.75 per 10-year increase [0.56, 1.00]) and deep lesions (p = 0.003, OR 0.29 [0.13, 0.65]) influenced selection for resection on multivariable regression. Surgical resection of PCNSL is safe for select patients, with complication rates comparable to rates for other intracranial neoplasms. Whether there is a clinical benefit to resection cannot be concluded.