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This article describes primary data and resources available from the Boston Adolescent Neuroimaging of Depression and Anxiety (BANDA) study, a novel arm of the Human Connectome Project (HCP). Data were collected from 215 adolescents (14-17 years old), 152 of whom had current diagnoses of anxiety and/or depressive disorders at study intake. Data include cross-sectional structural (T1- and T2-weighted), functional (resting state and three tasks), and diffusion-weighted magnetic resonance images. Both unprocessed and HCP minimally-preprocessed imaging data are available within the data release packages. Adolescent and parent clinical interview data, as well as cognitive and neuropsychological data are also included within these packages. Release packages additionally provide data collected from self-report measures assessing key features of adolescent psychopathology, including: anxious and depressive symptom dimensions, behavioral inhibition/activation, exposure to stressful life events, and risk behaviors. Finally, the release packages include 6- and 12-month longitudinal data acquired from clinical measures. Data are publicly accessible through the National Institute of Mental Health Data Archive (ID: #2505)., (© 2024. The Author(s).)

The Human Connectome Project (HCP) was launched in 2010 as an ambitious effort to accelerate advances in human neuroimaging, particularly for measures of brain connectivity; apply these advances to study a large number of healthy young adults; and freely share the data and tools with the scientific community. NIH awarded grants to two consortia; this retrospective focuses on the "WU-Minn-Ox" HCP consortium centered at Washington University, the University of Minnesota, and University of Oxford. In just over 6 years, the WU-Minn-Ox consortium succeeded in its core objectives by: 1) improving MR scanner hardware, pulse sequence design, and image reconstruction methods, 2) acquiring and analyzing multimodal MRI and MEG data of unprecedented quality together with behavioral measures from more than 1100 HCP participants, and 3) freely sharing the data (via the ConnectomeDB database) and associated analysis and visualization tools. To date, more than 27 Petabytes of data have been shared, and 1538 papers acknowledging HCP data use have been published. The "HCP-style" neuroimaging paradigm has emerged as a set of best-practice strategies for optimizing data acquisition and analysis. This article reviews the history of the HCP, including comments on key events and decisions associated with major project components. We discuss several scientific advances using HCP data, including improved cortical parcellations, analyses of connectivity based on functional and diffusion MRI, and analyses of brain-behavior relationships. We also touch upon our efforts to develop and share a variety of associated data processing and analysis tools along with detailed documentation, tutorials, and an educational course to train the next generation of neuroimagers. We conclude with a look forward at opportunities and challenges facing the human neuroimaging field from the perspective of the HCP consortium., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest in relation to this manuscript., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The original Human Connectome Project yielded a rich data set on structural and functional connectivity in a large sample of healthy young adults using improved methods of data acquisition, analysis, and sharing. More recent efforts are extending this approach to include infants, children, older adults, and brain disorders. This paper introduces and describes the Human Connectome Project in Aging (HCP-A), which is currently recruiting 1200 + healthy adults aged 36 to 100+, with a subset of 600 + participants returning for longitudinal assessment. Four acquisition sites using matched Siemens Prisma 3T MRI scanners with centralized quality control and data analysis are enrolling participants. Data are acquired across multimodal imaging and behavioral domains with a focus on factors known to be altered in advanced aging. MRI acquisitions include structural (whole brain and high resolution hippocampal) plus multiband resting state functional (rfMRI), task fMRI (tfMRI), diffusion MRI (dMRI), and arterial spin labeling (ASL). Behavioral characterization includes cognitive (such as processing speed and episodic memory), psychiatric, metabolic, and socioeconomic measures as well as assessment of systemic health (with a focus on menopause via hormonal assays). This dataset will provide a unique resource for examining how brain organization and connectivity changes across typical aging, and how these differences relate to key characteristics of aging including alterations in hormonal status and declining memory and general cognition. A primary goal of the HCP-A is to make these data freely available to the scientific community, supported by the Connectome Coordination Facility (CCF) platform for data quality assurance, preprocessing and basic analysis, and shared via the NIMH Data Archive (NDA). Here we provide the rationale for our study design and sufficient details of the resource for scientists to plan future analyses of these data. A companion paper describes the related Human Connectome Project in Development (HCP-D, Somerville et al., 2018), and the image acquisition protocol common to both studies (Harms et al., 2018)., (Copyright © 2018. Published by Elsevier Inc.)

Recent technological and analytical progress in brain imaging has enabled the examination of brain organization and connectivity at unprecedented levels of detail. The Human Connectome Project in Development (HCP-D) is exploiting these tools to chart developmental changes in brain connectivity. When complete, the HCP-D will comprise approximately ∼1750 open access datasets from 1300 + healthy human participants, ages 5-21 years, acquired at four sites across the USA. The participants are from diverse geographical, ethnic, and socioeconomic backgrounds. While most participants are tested once, others take part in a three-wave longitudinal component focused on the pubertal period (ages 9-17 years). Brain imaging sessions are acquired on a 3 T Siemens Prisma platform and include structural, functional (resting state and task-based), diffusion, and perfusion imaging, physiological monitoring, and a battery of cognitive tasks and self-reports. For minors, parents additionally complete a battery of instruments to characterize cognitive and emotional development, and environmental variables relevant to development. Participants provide biological samples of blood, saliva, and hair, enabling assays of pubertal hormones, health markers, and banked DNA samples. This paper outlines the overarching aims of the project, the approach taken to acquire maximally informative data while minimizing participant burden, preliminary analyses, and discussion of the intended uses and limitations of the dataset., (Copyright © 2018 Elsevier Inc. All rights reserved.)

ConnectomeDB is a database for housing and disseminating data about human brain structure, function, and connectivity, along with associated behavioral and demographic data. It is the main archive and dissemination platform for data collected under the WU-Minn consortium Human Connectome Project. Additional connectome-style study data is and will be made available in the database under current and future projects, including the Connectome Coordination Facility. The database currently includes multiple modalities of magnetic resonance imaging (MRI) and magnetoencephalograpy (MEG) data along with associated behavioral data. MRI modalities include structural, task, resting state and diffusion. MEG modalities include resting state and task. Imaging data includes unprocessed, minimally preprocessed and analysis data. Imaging data and much of the behavioral data are publicly available, subject to acceptance of data use terms, while access to some sensitive behavioral data is restricted to qualified investigators under a more stringent set of terms. ConnectomeDB is the public side of the WU-Minn HCP database platform. As such, it is geared towards public distribution, with a web-based user interface designed to guide users to the optimal set of data for their needs and a robust backend mechanism based on the commercial Aspera fasp service to enable high speed downloads. HCP data is also available via direct shipment of hard drives and Amazon S3., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Human Connectome Project (HCP) has developed protocols, standard operating and quality control procedures, and a suite of informatics tools to enable high throughput data collection, data sharing, automated data processing and analysis, and data mining and visualization. Quality control procedures include methods to maintain data collection consistency over time, to measure head motion, and to establish quantitative modality-specific overall quality assessments. Database services developed as customizations of the XNAT imaging informatics platform support both internal daily operations and open access data sharing. The Connectome Workbench visualization environment enables user interaction with HCP data and is increasingly integrated with the HCP's database services. Here we describe the current state of these procedures and tools and their application in the ongoing HCP study., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The virulence of Gram-positive bacteria is enhanced by toxins like the Streptococcus pyogenes β-NAD(+) glycohydrolase known as SPN. SPN-producing strains of S. pyogenes additionally express the protein immunity factor for SPN (IFS), which forms an inhibitory complex with SPN. We have determined crystal structures of the SPN-IFS complex and IFS alone, revealing that SPN is structurally related to ADP-ribosyl transferases but lacks the canonical binding site for protein substrates. SPN is instead a highly efficient glycohydrolase with the potential to deplete cellular levels of β-NAD(+). The protective effect of IFS involves an extensive interaction with the SPN active site that blocks access to β-NAD(+). The conformation of IFS changes upon binding to SPN, with repacking of an extended C-terminal α helix into a compact shape. IFS is an attractive target for the development of novel bacteriocidal compounds functioning by blocking the bacterium's self-immunity to the SPN toxin., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Type 1 pili mediate binding, invasion, and biofilm formation of uropathogenic Escherichia coli (UPEC) in the host urothelium during urinary tract infection (UTI) via the adhesin FimH. In this study, we characterized the molecular basis of functional differences between FimH of the UPEC isolate UTI89 and the Klebsiella pneumoniae cystitis isolate TOP52. Type 1 pili characteristically mediate mannose-sensitive hemagglutination of guinea pig erythrocytes. Although the adhesin domain of K. pneumoniae TOP52 FimH (FimH(52)) is highly homologous to that of E. coli, with an identical mannose binding pocket and surrounding hydrophobic ridge, it lacks the ability to agglutinate guinea pig erythrocytes. In addition, FimH-dependent biofilm formation in K. pneumoniae is inhibited by heptyl mannose, but not methyl mannose, suggesting the need for contacts outside of the mannose binding pocket. The binding specificity differences observed for FimH(52) resulted in significant functional differences seen in the pathogenesis of K. pneumoniae UTI compared to E. coli UTI. Infections in a murine model of UTI demonstrated that although the K. pneumoniae strain TOP52 required FimH(52) for invasion and IBC formation in the bladder, FimH(52) was not essential for early colonization. This work reveals that a limited amount of sequence variation between the FimH of E. coli and K. pneumoniae results in significant differences in function and ability to colonize the urinary tract.

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 "secondary bridge" between the copper- and zinc-binding sites is disrupted, and the "electrostatic loop" and "zinc loop" elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1-SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.

Regeneration of goldfish optic nerve axons is accompanied by a major increase in axonally transported proteoglycans (21). To identify specific proteoglycans increased during regeneration, we have used proteoglycan preparations from regenerating goldfish optic tracts to produce monoclonal antibodies. Western blot analysis shows a 28-kD antigen reacting with our 1G4/G5 antibody is present in optic tract 21 days after nerve crush, but absent in nonregenerating tract. Treatment with chondroitinase AC removes IG4/G5 immunostaining of the 28-kD molecule. An anti-CS antibody further confirmed this molecule as a chondroitin sulfate proteoglycan. A slightly smaller core protein following chondroitinase AC treatment indicates a low level of glycosylation. The N-terminal amino acid sequence of its core protein is not similar to any known proteoglycans. The CSPG sediments through 1.4 M sucrose, indicative of an extracellular matrix localization. It is expressed during the outgrowth of regenerating axons. Cut nerve and retinal explant studies demonstrate that the 1G4/G5 CSPG is not axonal, suggesting a glial localization.

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 A resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.

Mutations in the SOD1 gene cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human FALS patients and in transgenic mice expressing these mutant proteins, aggregates containing FALS SOD1 are observed. Accumulation of SOD1 aggregates is believed to interfere with axonal transport, protein degradation and anti-apoptotic functions of the neuronal cellular machinery. Here we show that metal-deficient, pathogenic SOD1 mutant proteins crystallize in three different crystal forms, all of which reveal higher-order assemblies of aligned beta-sheets. Amyloid-like filaments and water-filled nanotubes arise through extensive interactions between loop and beta-barrel elements of neighboring mutant SOD1 molecules. In all cases, non-native conformational changes permit a gain of interaction between dimers that leads to higher-order arrays. Normal beta-sheet-containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions. Loss of this protection through conformational rearrangement in the metal-deficient enzyme could be a toxic property common to mutants of SOD1 linked to FALS.

Regenerating goldfish retinal explants cultured on poly-L-lysine overlaid with membranes isolated from 21-day regenerating 1/3 anterior optic tectum (Ant. OTec) exhibited extensive defasciculated neurite outgrowth. Heparatinase treatment of membranes caused the complete inhibition of neurite outgrowth on that substrate. Western blot analysis showed that the OTec membranes contain a 300 kDa heparan sulfate proteoglycan. Explants cultured on 21-day regenerating 1/3 Ant. OTec membranes in the presence of 1 mM beta-xyloside, an axonal proteoglycan synthesis inhibitor, showed a significant reduction in the number of neurites per explant and in the average neurite length. Taken all together, the present results provide evidence that a 300-kDa membrane HSPG present in the Ant. OTec is necessary for axonal outgrowth and that axonal PGs are involved in modulating outgrowth on 21-day regenerating 1/3 Ant. OTec membranes.

During development, a subpopulation (approximately 30%) of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), dies following removal of the cochlea. It is clear that neuronal activity coming from the auditory nerve provides trophic support critical for cell survival in the NM. Several aspects of the intracellular signaling cascades that regulate apoptosis have been defined for naturally occurring, or programmed cell death, in neurons. These intracellular cascades involve the extrusion of cytochrome-c from the mitochondria into the cytosol and the subsequent activation of proteolytic caspase cascades, which ultimately act on substrates that lead to the death of the cell. In contrast, the intracellular signaling cascades responsible for deafferentation-induced cell death are not fully understood. In the present series of experiments, the potential extrusion of cytochrome-c from the mitochondria into the cytosol, and the activation of caspases were examined in the NM following deafferentation. Cytochrome-c immunoreactivity increased within 6 h following deafferentation and persisted for at least 3-5 days following surgery. However, cytochrome-c was not detectable within immunoprecipitates obtained from cytosolic fractions of deafferented NM neurons. This suggests that the increased immunoreactivity of cytochrome-c is related to mitochondrial proliferation. As a positive control, cytochrome-c was detected in cytosolic fractions of deafferented NM neurons treated with kainic acid, a substance known to cause cytochrome-c release into the cytosol. In addition, immunoreactivity for downstream active caspase-9 did increase following cochlea ablation. This increase was observed within 3 h following cochlea removal, but was not observed 4 days following surgery, a time point after the dying population of NM neurons have already degenerated. Together, these findings suggest that deafferentation of NM neurons results in caspase activation, but this activation may be cytochrome-c independent.

Regenerating goldfish retinal explants were cultured on poly-L-lysine (control) or poly-L-lysine overlaid with membranes isolated from non-regenerating optic tract (OTr) and 10- or 21-day regenerating OTr. Non-regenerating OTr substrate inhibited all neurite growth while 10- and 21-day regenerating OTr substrates significantly increased the average neurite number per explant and average neurite length compared to controls. These results indicate an upregulation of neurite growth permissive properties of regenerating OTr membranes.

Our previous studies demonstrated that optic nerve regeneration in the goldfish is accompanied by significantly enhanced axonal transport of glycosaminoglycans (GAGs), with a particularly large increase in the transport of chondroitin 4-sulfate (C4S). We have further shown that inhibition of proteoglycan (PG) synthesis and transport with beta-xyloside impedes axonal outgrowth from regenerating retinal explants. These results suggest a role for PGs that contain C4S in the regeneration process. To begin to address the possible functions of C4S GAGs during regeneration of goldfish retinal axons, we evaluated the effect of exogenous C4S on axonal outgrowth from regenerating retina, explanted 7-14 days after optic nerve crush. Our results indicate that exogenous C4S added to the culture medium potentiates axonal outgrowth on both polylysine- and LN-containing substrata. At low C4S concentrations, this potentiation is more marked on substrata containing both polylysine and LN than on polylysine alone. These results obtained in vitro suggest that soluble C4S, whether arising after axonal transport and externalization or after release from nonneuronal cells, is a positive modulator of regenerative axonal outgrowth in vivo.

Previous studies have shown that goldfish retinal axons regenerating in vivo transport increased radioactivity in the glycosaminoglycan (GAG) components of proteoglycans (PGs). During this enhanced transport, the ratio of chondroitin sulfate (CS) to heparan sulfate (HS) was 60/40. In the present investigation, PG synthesis was inhibited during in vitro axon growth from regenerating goldfish retinal explants. Explants growing on either poly-L-lysine (PLYS) or poly-L-lysine + laminin (PLYS + LN) incorporated 35SO4 into proteoglycan-bound CS and HS in an approximate 2/1 ratio. Addition of 4-methylumbelliferyl beta-D-xyloside (beta-xyloside) to the culture medium reduced the sulfate radioactivity in proteoglycan-bound CS and HS by 89 and 71%, respectively, on PLYS and by 89 and 72% on PLYS + LN. Morphological evaluation of explants revealed that beta-xyloside treatment reduced both the number of retinal axons per explant and their growth rate on PLYS; on PLYS + LN this treatment reduced the number of axons, but had no effect on growth rate. This study suggests that retinal ganglion cell PGs containing CS and/or HS GAG chains are required for both the initiation and the maintenance of axonal outgrowth on artificial polycationic substrata such as PLYS, but only for the initiation of outgrowth on laminin.

Structural characteristics of glycosaminoglycans (GAGs) derived from axonally transported proteoglycans (PGs) were compared in 21 days regenerating and intact goldfish optic tracts. Twenty one days following unilateral optic nerve crushes, fish received intraocular injections of 35SO4. Eight hours post injection, tracts were removed and the 35SO4-labeled GAGs, chondroitin sulfate (CS) and heparan sulfate (HS), isolated. The HS from regenerating optic tracts had a DEAE elution profile indicative of decreased charge density, while heparitinase treatment of HS followed by Sephadex G50 analysis of the resulting fragments showed a change in the elution pattern, suggesting reduced overall sulfation. HPLC analysis of HS disaccharides revealed a difference in the sulfation pattern of regenerating tract HS, characterized by the reduced presence of tri-sulfated disaccharides. Other structural features, such as the sizes of CS and HS, and the sulfation of CS, showed no changes during regeneration. These results indicate that changes in the structure of axonally transported HS accompany regeneration of goldfish optic axons.

We report further analysis of axonally transported proteoglycans in soluble and membranous subfractions of goldfish optic tectum. Distribution of transported 35SO4 radioactivity was 35.2% soluble, 63.4% Triton-NaCl extractable and 1.4% unextracted. Proteoglycans isolated on DEAE cellulose were treated with chondroitinase AC or nitrous acid and remaining heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) were sized on Sepharose CL-6B. Kav values and estimated molecular weights were: Soluble CSPG-0.36 (160 kDa), Triton-NaCl extracted CSPG-.031 (200 kDa), Soluble HSPG-0.37 (150 kDa), Triton-NaCl extracted HSPG-0.37 (150 kDa). For constituent CS and HS chains the Kav values and estimated molecular weights on CL-6B were: Soluble CS-0.55 (15 kDa), Triton-NaCl extracted CS-0.55 (15 kDa), Soluble HS-0.59 (13 kDa) and Triton-NaCl extracted HS-0.65 (9 kDa). CS was shown to be sulfated exclusively at carbon 4 for both soluble and Triton NaCl extracted fractions.

Axonally transported proteoglycans were differentially solubilized by a sequence of extractions designed to infer their relationship to nerve terminal membranes. Groups of goldfish were injected unilaterally with 35SO4 and contralateral optic tecta containing axonally transported molecules were removed 16 h later. Tecta were homogenized in isotonic buffer and centrifuged at 100,000 g for 60 min to create a "total supernatant" fraction. Subsequent homogenizations followed by recentrifugation were with hypotonic buffer (lysis extract), 1 M NaCl, Triton X-100 or alternatively Triton-1 M NaCl. Populations of proteoglycans in each extract were isolated on DEAE ion exchange columns and evaluated for content of glycosaminoglycans (GAGs). Results show the distribution of transported proteoglycans to be 26.3% total soluble, 13.7% lysis extract, 13.8% NaCl extract, 12.2% Triton extract, and 46.2% Triton-NaCl extract. Proteoglycans from all fractions contained heparan sulfate as the predominant GAG, with lesser amounts of chondroitin (4 or 6) sulfate. The possible localizations of transported proteoglycans suggested by the extraction results are discussed.

Studies on isolated synaptic plasma membranes (SPM) have detected little if any heparan sulfate or other glycosaminoglycans (GAGs), while more recent studies employing proteoglycan antibodies have localized heparan sulfate proteoglycan in presynaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane, we have investigated the possible presence of axonally transported GAGs in SPM isolated from the goldfish optic tectum. SPMs isolated from tecta following rapid axonal transport of 35SO4 labeled molecules down the optic nerve, showed specific radioactivity approximately two-fold higher than the starting homogenate. Treatment of the transport labeled SPM with the enzyme heparitinase liberated 21% of the radioactivity, indicating the presence of a significant fraction of transported label in heparan sulfate. In a separate series of experiments a GAG fraction was isolated from transport labeled SPM and was found to consist of heparan sulfate containing 28% of transported radioactivity. Chondroitin (4 or 6) sulfate, which undergoes axonal transport in the goldfish optic system, was not found associated with SPM. Taken together the results support immunological evidence for the presence of heparan sulfate proteoglycans in presynaptic plasma membrane.

Previous studies of the olfactory nerve, mainly in higher vertebrates, have indicated that axonal injury causes total degeneration of the mature neurons, followed by replacement of new neuronal cells arising from undifferentiated mucosal cells. A similar regeneration process was confirmed in the garfish olfactory system. Regeneration of the nerve, crushed 1.5 cm from the cell bodies, is found to produce three distinct populations of regenerating fibers. The first traverses the crush site 1 wk postoperative and progresses along the nerve at a rate of 5.8 +/- 0.3 mm/d for the leading fibers of the group. The second group of fibers traverses the crush site after 2 wk postcrush and advances at a rate of 2.1 +/- 0.1 mm/d for the leading fibers. The rate of growth of this group of fibers remains constant for 60 d but subsequently falls to 1.6 +/- 0.2 for the leading population of fibers. The leading fibers in the third group of regenerating axons traverse the crush site after 4 wk and advance at a constant rate of 0.8 +/- 0.2 mm/d. The multiple populations of regenerating fibers with differing rates of growth are discussed in the context of precursor cell maturity at the time of nerve injury and possible conditioning effects of the lesion upon these cells. Electron microscopy indicates that the number of axons decreases extensively after crush. The first two phases of regenerating axons represent a total of between 6 and 10% of the original axonal population and are typically characterized by small fascicles of axons surrounded by Schwann cells and large amounts of collagenous material. The third phase of fibers represents between 50 and 70% of the original axonal population.

This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase-generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration-related increase in the proportion of [3H]glucosamine label in lower-molecular-weight Con A-binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose-rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A-binding glycopeptides are enriched in membrane subfractions, particularly in a high-density fraction that morphologically resembles isolated cell surface coat. Regeneration-related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport-labeled glycoproteins disclosed growth-correlated increases in radioactivity associated with 180-200K, 105-115K, and 80-90K components, while a 150-160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50-140K range.

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.

The study examined the subcellular distribution of [3H]glucosamine-labeled glycoconjugates undergoing axonal transport in 100,000 x g soluble and two membranous subfractions of the garfish olfactory nerve. Analysis was made of intact glycoconjugates and of glycopeptides and glycosaminoglycans derived from these molecules by limit protease digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed labeling of a variety of high-molecular-weight molecules with a lower molecular weight distribution in the soluble fraction than in the membranous fractions. Following protease digestion, nearly two-thirds of transported radioactivity in glycopeptides was recovered in the plasma membrane-enriched subfraction, with the remainder equally divided between soluble and higher density membrane fraction. Comparison of the distribution of glycopeptide radioactivity and chemically assayed hexosamine revealed transport labeling of a large variety of different-sized neutral and acidic glycopeptides in all subfractions. Transport labeling of most glycoprotein carbohydrate chains was in proportion of their hexosamine content. Transported glycosaminoglycan label was most heavily concentrated in the plasma membrane fraction, whereas hexosamine was most concentrated in the higher density membrane fraction. The labeling pattern suggested both transported and nontransported pools of these molecules. The specific glycosaminoglycans chondroitin sulfate and heparan sulfate were recovered in all subfractions, whereas hyaluronic acid was confined to the soluble fraction.

A floating fraction that layers on top of 0.25 sucrose has been obtained during the preparation of myelin from intact and 9 day degenerating goldfish optic tracts. The proportion of total tract protein isolated in floating fraction rises from 6.6% to 11.0% during degeneration. This increase is paralleled by a morphologically observed splitting of myelin lamellae. Floating fraction contains all of the major myelin proteins but shows a 40% increase in the proportion of basic protein and a 2-3 fold decrease in the proportion of IP proteins (intermediate molecular weight glycoproteins) and a 36 Kd (X) protein. The lipid to protein ratio is slightly higher in floating fraction than myelin. Lipid composition is characterized by 1/2-1/3 the myelin levels of galactolipids and twofold increased levels of triglycerides and cholesterol esters. Electron microscopy of floating fraction shows a mixture of myelin fragments with few lamellae and single membrane fragments. Taken together the results indicate that floating fraction in the degenerating goldfish optic tract is at least partially derived from the breakdown of myelin.

[3H]fucose labeled glycoproteins which are rapidly transported in the goldfish optic nerve have been isolated with purified preparations of goldfish optic tectal myelin. SDS acrylamide gels of myelin proteins isolated one day after intraocular injection of fucose show a broad distribution of radioactivity among high molecular weight proteins. At progressively longer times after injection there is a shift in the distribution of radioactivity with the buildup of a peak of label with electrophoretic mobility corresponding to a molecular weight of approximately 50,000. There is no corresponding peak of radioactivity in gels of total tectal membranes. Gels of myelin labeled through intracranial injections of fucose also show no single prominent peak of radioactivity. Results are discussed with reference to possible mechanisms for the shift in radioactivity patterns on gels and the possible functional significance of the myelin-associated glycoproteins.

Metabolic turnover of axonally transported glycoproteins has been examined in membranous and soluble subfractions of goldfish optic tectum following intraocular injection of [3H]fucose. Radioactivity in total transported glycoproteins reached a maximum in the tectum after 24-30 hr, then declined with a half-life of approximately 20 days. Radioactivity in the total membranous subfraction declined with a similar half-life of 20-21 days while radioactivity in the soluble fraction showed a significantly shorter half-life of approximately seven days. Various sized glycopeptides derived from the membranous subfraction showed differential rates of loss of radioactivity with the lower molecular weight nondialyzable molecules displaying the most rapid turnover. In contrast, the glycopeptides derived from the soluble fraction showed relatively uniform rates of turnover. The results are discussed in the context of metabolic compartmentalization between membranous and soluble glycoproteins and among the carbohydrate chains of the membranous molecules.

The size and concanavalin A-binding characteristics of glycopeptides derived from axonally transported glycoproteins were studied in regenerating garfish olfactory nerve. A regeneration related increase was observed in the proportion of total glycopeptide radioactivity associated with the lower molecular weight, dialyzable fraction. There was also a 3-5 fold increase in the axonal transport of low molecular weight concanavalin. A-binding glycopeptides in regenerating nerve. These results suggest a shift to an enhanced synthesis and axonal transport of glycoproteins containing low molecular weight, concanavalin A-binding carbohydrate chains in regenerating nerve.

Glycosaminoglycans (GAGs) derived from axonally transported proteoglycans were evaluated in regenerating goldfish optic tracts. Fish were administered unilateral optic nerve crushes and stored for 21 days at 21 degrees C. Retinas were labeled by bilateral injection of 35SO4 and sulfated macromolecules axonally transported to the optic tracts were analyzed 8 h postinjection. Results show regenerating tracts contain 6.7-fold more transported 35SO4 in GAGs than their contralateral intact counterparts. Further analysis revealed that regenerating tract GAGs were comprised of 60% chondroitin (4 or 6) sulfate (CS) and 40% heparan sulfate (HS), while unoperated tract GAGs contained 26% CS and 74% HS. These results indicate that there is a large regeneration related increase in the axonal transport of proteoglycans and particular enrichment of transported molecules containing CS chains. The findings can be viewed in the context of recent implication of axonal proteoglycans in processes of fiber outgrowth, adhesion and induction of glial mitosis.

[3H]Proline derived radioactivity which is slowly transported in the goldfish optic nerve has been isolated in association with purified preparations of optic tectal myelin. Acrylamide gel analysis reveals that most of the slowly transported radioactivity is distributed among the high molecular weight myelin proteins with a major peak comigrating with a band (approximately 42,000 daltons) which is prominent in goldfish brain myelin. A similar peak is not seen in gels of whole synaptosome protein. Relatively small but measureable amounts of radioactivity comigrate with the basic and proteolipid proteins. A low density myelin subfraction is 3-5 fold lower in specific radioactivity (disint./min/mug protein) than a corresponding high density fraction and shows particularly low specific radioactivities in the basic and proteolipid bands. The results are discussed with reference to the possible adherence of specific axonal membrane fragments as well as other potential sources of myelin label.

The study addressed the question of whether 35SO4 labeled molecules that have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a soluble fraction (soluble after centrifugation at 105,000 g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000 g) and a final 105,000 g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatography was resolved into a fraction of sulfated glycoproteins eluting at 0-0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32-0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated 35SO4, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the results support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.

Previous EM autoradiographic studies have shown that injection of [3H]proline ([3H]Pro) into the cat dorsal column nuclei (DCN) results in heavy labeling of macroglia but negligible labeling of DCN neurons. [3H]Leucine ([3H]Leu), in contrast, was extensively incorporated into both neurons and glia. We now report preliminary assessment of differences in molecular labeling patterns produced by the two amino acid precursors in DCN injection sites (24 h following injection; equal amounts of [3H]Pro and [3H]Leu of equal specific radioactivity). [3H]Pro, despite its lack of incorporation into neurons, labeled DCN to a significantly greater extent than [3H]Leu (30.0 dpm/micrograms protein/microCi for [3H]Pro vs 11.7 dpm/micrograms protein/microCi for [3H]Leu). Greater than 90% of the radioactivity from both precursors was recovered in protein as opposed to TCA soluble or ethanol soluble molecules. Of the [3H]Pro- or [3H]Leu-derived radioactivity recovered in protein, greater than 94% was found to remain in the original precursor form. Fluorographic analysis of SDS acrylamide gels showed labeling of a wide variety of individual proteins with either amino acid. However, particular molecular weight classes were relatively more heavily labeled with either [3H]Leu (47, 63, 77 kDa) or [3H]Pro (22, 45, 50, 66, 80 kDa). Overall the results indicate that the difference in cellular distribution of incorporated [3H]Pro and [3H]Leu, as observed by EM autoradiography is a reflection of the extent of labeling and specific labeling pattern of proteins isolated from the tissue.

When sulfur-35-labeled sodium sulfate is injected intraocularly in the goldfish, labeled sulfated mucopolysaccharides rapidly appear in the contra-lateral optic tectum of the brain, demonstrating the axonal flow of sulfated mucopolysaccharides. The transport rate is the same as that observed for proteins labeled after intraocular injection of tritiated proline. Treatment of the sulfur-35-labeled material with precipitants and enzymes reveals the presence of substances with properties similar to those of heparan sulfate (the major component) and chondroitin sulfate. Dermatan sulfate was not detected.