Effective therapeutic strategies for mucopolysaccharidosis type I (MPSI) rely on mannose-6-phosphate receptor-mediated uptake of extracellular alpha-l-iduronidase (IDUA), the missing lysosomal enzyme in this disease, by deficient cells. Intravenously infused recombinant human IDUA does not reach the central nervous system, whereas neuropathology and neurological manifestations are prominent in Hurler syndrome, the most severe and most frequent form of MPSI. The creation of a single intracerebral source of IDUA by gene therapy was proved efficient to deliver enzyme throughout the brain of MPSI mice. IDUA spreading far beyond areas where the enzyme was synthesized suggested transport along neuronal processes. To examine the mechanisms of IDUA spreading in the brain, we constructed a chimeric protein in which GFP is fused at the C-terminus of IDUA. The fusion protein was expressed in rat primary neurons using lentivirus vectors. Fluorescent IDUA retained full catalytic activity including on natural substrates, interacted with mannose-6-phosphate receptors and was appropriately addressed to lysosomes. Fluorescent vesicles were broadly distributed over neuronal soma and processes. Time-lapse fluorescent video-microscopy showed that 54% of fluorescent vesicles exhibited either retrograde or anterograde displacements along neurites. Most moving organelles showed complex movements with frequent direction changes and arrests. Motility depended on microtubule integrity. Efficient axono-dendritic transport of IDUA provides a rationale for gene therapy based on the release of therapeutic enzyme at discrete locations within the central nervous system of patients with severe form of MPSI.

Psychiatric symptoms of schizophrenia suggest alteration of cerebral neurons. However, the physical basis of the schizophrenia symptoms has not been delineated at the cellular level. Here, we report nanometer-scale three-dimensional analysis of brain tissues of schizophrenia and control cases. Structures of cerebral tissues of the anterior cingulate cortex were visualized with synchrotron radiation nanotomography. Tissue constituents visualized in the three-dimensional images were traced to build Cartesian coordinate models of tissue constituents, such as neurons and blood vessels. The obtained Cartesian coordinates were used for calculating curvature and torsion of neurites in order to analyze their geometry. Results of the geometric analyses indicated that the curvature of neurites is significantly different between schizophrenia and control cases. The mean curvature of distal neurites of the schizophrenia cases was ~1.5 times higher than that of the controls. The schizophrenia case with the highest neurite curvature carried a frame shift mutation in the GLO1 gene, suggesting that oxidative stress due to the GLO1 mutation caused the structural alteration of the neurites. The differences in the neurite curvature result in differences in the spatial trajectory and hence alter neuronal circuits. It has been shown that the anterior cingulate cortex analyzed in this study has emotional and cognitive functions. We suggest that the structural alteration of neurons in the schizophrenia cases should reflect psychiatric symptoms of schizophrenia.

The particular location of myenteric neurons, sandwiched between the 2 muscle layers of the gut, implies that their somata and neurites undergo mechanical stress during gastrointestinal motility. Existence of mechanosensitive enteric neurons (MEN) is undoubted but many of their basic features remain to be studied. In this study, we used ultra-fast neuroimaging to record activity of primary cultured myenteric neurons of guinea pig and human intestine after von Frey hair evoked deformation of neurites and somata. Independent component analysis was applied to reconstruct neuronal morphology and follow neuronal signals. Of the cultured neurons 45% (114 out of 256, 30 guinea pigs) responded to neurite probing with a burst spike frequency of 13.4 Hz. Action potentials generated at the stimulation site invaded the soma and other neurites. Mechanosensitive sites were expressed across large areas of neurites. Many mechanosensitive neurites appeared to have afferent and efferent functions as those that responded to deformation also conducted spikes coming from the soma. Mechanosensitive neurites were also activated by nicotine application. This supported the concept of multifunctional MEN. 14% of the neurons (13 out of 96, 18 guinea pigs) responded to soma deformation with burst spike discharge of 17.9 Hz. Firing of MEN adapted rapidly (RAMEN), slowly (SAMEN), or ultra-slowly (USAMEN). The majority of MEN showed SAMEN behavior although significantly more RAMEN occurred after neurite probing. Cultured myenteric neurons from human intestine had similar properties. Compared to MEN, dorsal root ganglion neurons were activated by neurite but not by soma deformation with slow adaptation of firing. We demonstrated that MEN exhibit specific features very likely reflecting adaptation to their specialized functions in the gut.

Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites ('bundled NFs'). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered.

Chromosome 9 open reading frame 72 (C9orf72) is an evolutionarily conserved protein with unknown function, expressed at high levels in the brain. An expanded hexanucleotide GGGGCC repeat located in the first intron of the C9orf72 gene represents the most common genetic cause of familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Previous studies by immunohistochemistry with two different anti-C9orf72 antibodies named sc-138763 and HPA023873 showed that C9orf72 is expressed chiefly in the cytoplasm of neurons, and is concentrated in the synaptic terminals in the brains of FTD/ALS with or without C9orf72 repeat expansion as well as those of controls. At present, a pathological role of C9orf72 in the process of neurodegeneration remains unknown.

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.

Olfactory sensory neurons (OSNs) send their axons to distinct glomeruli in the olfactory bulb. On the way to their target, outgrowing axons are guided, fasciculated, and resorted before they extend in homotypic bundles to the glomerulus. The molecular mechanisms underlying these complex processes supposedly involve multiple intrinsic and extrinsic cues. Although the contribution of typical guidance molecules has been proposed, a detailed understanding of the olfactory wiring process remains elusive. By using in vitro cultures of the olfactory epithelium (OE) from gene-targeted mice, which allowed visualization of mature OSN and their axons, the impact of distinct molecular and cellular cues on defined OSN populations could be studied. The differentiating factor retinoic acid induced a heterogeneous response pattern of OMP expression and axon elongation. Cocultures with forebrain explants revealed that tissue from the presumptive olfactory bulb of embryonic stage E14 exhibited nonpermissive, repellent effects on outgrowing neurites, whereas precultured bulb tissue strongly attracted them, even from distantly located OE explants. A selective attraction of fibers from OSNs expressing defined odorant receptor types to distinct bulb explants was observed. These data indicate a differential reaction of OSNs to their target tissue.

Chronic traumatic encephalopathy is a neurodegenerative disease associated with repeated traumatic brain injury (TBI). Chronic traumatic encephalopathy is a tauopathy, in which cognitive decline is accompanied by the accumulation of neurofibrillary tangles of the protein tau in patients' brains. We recently found that mechanical force alone can induce tau mislocalization to dendritic spines and loss of synaptic function in in vitro neuronal cultures with random cell organization. However, in the brain, neurons are highly aligned, so here we aimed to determine how neuronal organization influences early-stage tauopathy caused by mechanical injury. Using microfabricated cell culture constructs to control the growth of neurites and an in vitro simulated TBI device to apply controlled mechanical deformation, we found that neuronal orientation with respect to the direction of a uniaxial high-strain-rate stretch injury influences the degree of tau pathology in injured neurons. We found that a mechanical stretch applied parallel to the neurite alignment induces greater mislocalization of tau proteins to dendritic spines than does a stretch with the same strain applied perpendicular to the neurites. Synaptic function, characterized by the amplitude of miniature excitatory postsynaptic currents, was similarly decreased in neurons with neurites aligned parallel to stretch, whereas in neurons aligned perpendicular to stretch, it had little to no functional loss. Experimental injury parameters (strain, strain rate, direction of stretch) were combined with a standard viscoelastic solid model to show that in our in vitro model, neurite work density during stretch correlates with tau mislocalization. These findings suggest that in a TBI, the magnitude of brain deformation is not wholly predictive of neurodegenerative consequences of TBI but that deformation relative to local neuronal architecture and the neurite mechanical energy during injury are better metrics for predicting trauma-induced tauopathy.

PC12 cells are a well-known model of parasympathetic neurons. They have also been used to study the dynamics of heterologously expressed fragile X mental retardation (FMRP) granule trafficking down neurites. Here, we demonstrate that undifferentiated and differentiated PC12 cells harbor endogenous FMRP-containing granules. These granules are not stress granules because they do not associate with an authentic stress granule marker protein T-cell internal antigen 1 (TIA-1). Treatment with sodium arsenite induces stress granule formation in undifferentiated and differentiated PC12 cells. In NGF-treated cells, FMRP-containing stress granules are observed in the soma, neurites and growth cones by co-immunostaining with anti-TIA-1 antibody. These data demonstrate that all three microdomains respond similarly to oxidative stress. Nevertheless, we find significantly less co-localization of FMRP and TIA-1 and FMRP and its homologs in the neurites of differentiated PC12 cells treated with sodium arsenite than in the soma or growth cones. The heterogeneity of these granules suggests that FMRP has multiple roles in neurites.

Using a polyclonal antibody directed against somatostatin, normal somatostatin-positive neurons and fibers were seen in the amygdala and periamygdaloid cortex of both young and aged macaques. In addition, immunoreactive structures, identical in appearance to neurites demonstrated by silver impregnation methods, were seen in the amygdala of one aged monkey that exhibited numerous senile plaques. Some of these immunoreactive neurites were associated with deposits of amyloid, as seen with thioflavin-T stains, suggesting that these were neurites of senile plaques. This study provides direct evidence for abnormalities in peptidergic neurons in brains of aged non-human primates.

The WldS mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying WldS -mediated axonal protection are unclear, although many studies have attributed WldS neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury.

Mechanical forces actively shape cells during development, but little is known about their roles during neuronal morphogenesis. Developmental neurite pruning, a critical circuit specification mechanism, often involves neurite abscission at predetermined sites by unknown mechanisms. Pruning of Drosophila sensory neuron dendrites during metamorphosis is triggered by the hormone ecdysone, which induces local disassembly of the dendritic cytoskeleton. Subsequently, dendrites are severed at positions close to the soma by an unknown mechanism. We found that ecdysone signaling causes the dendrites to become mechanically fragile. Severing occurs during periods of increased pupal morphogenetic tissue movements, which exert mechanical forces on the destabilized dendrites. Tissue movements and dendrite severing peak during pupal ecdysis, a period of strong abdominal contractions, and abolishing ecdysis causes non-cell autonomous dendrite pruning defects. Thus, our data establish mechanical tearing as a novel mechanism during neurite pruning.

Few models allow the study of neurite damage in the human central nervous system. We used here dopaminergic LUHMES neurons to establish a culture system that allows for (i) the observation of highly enriched neurites, (ii) the preparation of the neurite fraction for biochemical studies, and (iii) the measurement of neurite markers and metabolites after axotomy. LUHMES-based spheroids, plated in culture dishes, extended neurites of several thousand µm length, while all somata remained aggregated. These cultures allowed an easy microscopic observation of live or fixed neurites. Neurite-only cultures (NOC) were produced by cutting out the still-aggregated somata. The potential application of such cultures was exemplified by determinations of their protein and RNA contents. For instance, the mitochondrial TOM20 protein was highly abundant, while nuclear histone H3 was absent. Similarly, mitochondrial-encoded RNAs were found at relatively high levels, while the mRNA for a histone or the neuronal nuclear marker NeuN (RBFOX3) were relatively depleted in NOC. Another potential use of NOC is the study of neurite degeneration. For this purpose, an algorithm to quantify neurite integrity was developed. Using this tool, we found that the addition of nicotinamide drastically reduced neurite degeneration. Also, the chelation of Ca2+ in NOC delayed the degeneration, while inhibitors of calpains had no effect. Thus, NOC proved to be suitable for biochemical analysis and for studying degeneration processes after a defined cut injury.

Alzheimer's disease (AD) is characterized by the presence of neuritic plaques in which dystrophic neurites (DNs) are typical constituents. We recently showed that DNs labeled by antibodies to the tubular endoplasmic reticulum (ER) protein reticulon-3 (RTN3) are enriched with clustered tubular ER. However, multi-vesicle bodies are also found in DNs, suggesting that different populations of DNs exist in brains of AD patients. To understand how different DNs evolve to surround core amyloid plaques, we monitored the growth of DNs in AD mouse brains (5xFAD and APP/PS1ΔE9 mice) by multiple approaches, including two-dimensional and three-dimensional (3D) electron microscopy (EM). We discovered that a pre-autophagosome protein ATG9A was enriched in DNs when a plaque was just beginning to develop. ATG9A-positive DNs were often closer to the core amyloid plaque, whereas RTN3 immunoreactive DNs were mostly located in the outer layers of ATG9A-positive DNs. Proteins such as RAB7 and LC3 appeared in DNs at later stages during plaque growth, likely accumulated as a part of large autophagy vesicles, and were distributed relatively furthest from the core amyloid plaque. Reconstructing the 3D structure of different morphologies of DNs revealed that DNs in AD mouse brains were constituted in three layers that are distinct by enriching different types of vesicles, as validated by immune-EM methods. Collectively, our results provide the first evidence that DNs evolve from dysfunctions of pre-autophagosomes, tubular ER, mature autophagosomes, and the ubiquitin proteasome system during plaque growth.

Localized translation in neurites helps regulate synaptic strength and development. Dysregulation of local translation is associated with many neurological disorders. However, due to technical limitations, study of this phenomenon has largely been limited to brain regions with laminar organization of dendrites such as the hippocampus or cerebellum. It has not been examined in the cortex, a region of importance for most neurological disorders, where dendrites of each neuronal population are densely intermingled with cell bodies of others. Therefore, we have developed a novel method, SynapTRAP, which combines synaptoneurosomal fractionation with translating ribosome affinity purification to identify ribosome-bound mRNA in processes of genetically defined cell types. We demonstrate SynapTRAP's efficacy and report local translation in the cortex of mice, where we identify a subset of mRNAs that are translated in dendrites by neuronal ribosomes. These mRNAs have disproportionately longer lengths, enrichment for FMRP binding and G-quartets, and their genes are under greater evolutionary constraint in humans. In addition, we show that alternative splicing likely regulates this phenomenon. Overall, SynapTRAP allows for rapid isolation of cell-type-specific localized translation and is applicable to classes of previously inaccessible neuronal and non-neuronal cells in vivoSIGNIFICANCE STATEMENT Instructions for making proteins are found in the genome, housed within the nucleus of each cell. These are then copied as RNA and exported to manufacture new proteins. However, in the brain, memory is thought to be encoded by strengthening individual connections (synapses) between neurons far from the nucleus. Thus, to efficiently make new proteins specifically where they are needed, neurons can transport RNAs to sites near synapses to locally produce proteins. Importantly, several mutations that cause autism disrupt this process. It has been assumed this process occurs in all brain regions, but has never been measured in the cortex. We applied a newly developed method measure to study, for the first time, local translation in cortical neurons.

Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. Here, we describe an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. We found that somatosensory neurons in Drosophila and zebrafish induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons.

Automating the process of neurite tracing from light microscopy stacks of images is essential for large-scale or high-throughput quantitative studies of neural circuits. While the general layout of labeled neurites can be captured by many automated tracing algorithms, it is often not possible to differentiate reliably between the processes belonging to different cells. The reason is that some neurites in the stack may appear broken due to imperfect labeling, while others may appear fused due to the limited resolution of optical microscopy. Trained neuroanatomists routinely resolve such topological ambiguities during manual tracing tasks by combining information about distances between branches, branch orientations, intensities, calibers, tortuosities, colors, as well as the presence of spines or boutons. Likewise, to evaluate different topological scenarios automatically, we developed a machine learning approach that combines many of the above mentioned features. A specifically designed confidence measure was used to actively train the algorithm during user-assisted tracing procedure. Active learning significantly reduces the training time and makes it possible to obtain less than 1% generalization error rates by providing few training examples. To evaluate the overall performance of the algorithm a number of image stacks were reconstructed automatically, as well as manually by several trained users, making it possible to compare the automated traces to the baseline inter-user variability. Several geometrical and topological features of the traces were selected for the comparisons. These features include the total trace length, the total numbers of branch and terminal points, the affinity of corresponding traces, and the distances between corresponding branch and terminal points. Our results show that when the density of labeled neurites is sufficiently low, automated traces are not significantly different from manual reconstructions obtained by trained users.

The morphology and neurochemistry of beta-amyloid (A beta) plaque-associated dystrophic neurites present in TgCRND8 and Tg2576 mice was demonstrated to be strikingly similar to that observed in pathologically aged human cases, but not in Alzheimer's disease (AD) cases. Specifically, pathologically aged cases and both transgenic mouse lines exhibited alpha-internexin- and neurofilament-triplet-labelled ring- and bulb-like dystrophic neurites, but no classical hyperphosphorylated-tau dystrophic neurite pathology. In contrast, AD cases demonstrated abundant classical hyperphosphorylated-tau-labelled dystrophic neurites, but no neurofilament-triplet-labelled ring-like dystrophic neurites. Importantly, quantitation demonstrated that the A beta plaques in TgCRND8 mice were highly axonopathic, and localised displacement or clipping of apical dendrite segments was also associated with A beta plaques in both transgenic mouse models. These results suggest that neuronal pathology in these mice represent an accurate and valuable model for understanding, and developing treatments for, the early brain changes of AD.

Sensory neurons with common functions are often nonrandomly arranged and form dendritic territories that show little overlap, or tiling. Repulsive homotypic interactions underlie such patterns in cell organization in invertebrate neurons. It is unclear how dendro-dendritic repulsive interactions can produce a nonrandom distribution of cells and their spatial territories in mammalian retinal horizontal cells, as mature horizontal cell dendrites overlap substantially. By imaging developing mouse horizontal cells, we found that these cells transiently elaborate vertical neurites that form nonoverlapping columnar territories on reaching their final laminar positions. Targeted cell ablation revealed that the vertical neurites engage in homotypic interactions that result in tiling of neighboring cells before the establishment of their dendritic fields. This developmental tiling of transient neurites correlates with the emergence of a nonrandom distribution of the cells and could represent a mechanism that organizes neighbor relationships and territories of neurons before circuit assembly.

This study examined the distribution of labile and stable microtubules (MTs) during axonal neurite elaboration in NB2a/d1 cells using immunocytochemical markers of unmodified (tyrosinated; Tyr), modified (detyrosinated [Glu] and acetylated [Acet]) and total tubulin. Prominent total and Tyr tubulin immunoreactivity was relatively evenly distributed throughout axonal neurites. By contrast, Acet or Glu immunoreactivity was relatively concentrated within the proximal region of the neurite. Ultrastructural analyses demonstrated an array of longitudinal MTs that apparently span the entire neurite length. The observed differential localization of modified tubulin subunits in axonal neurites of these cells may therefore derive from selective stabilization of proximal regions of full-length axonal MTs. This was substantiated by the observation of Acet immunoreactivity on 30-50% of MTs within the most proximal axonal region, along with a proximal-distal decline to < or =5% of Acet immunoreactive MTs, in immunoelectron microscopy (immuno-EM) analyses. Microinjected biotinylated subunits were initially detected in assembled form within soma and proximal neurites, indicative of ongoing tubulin subunit incorporation into MTs within, and/or MT translocation into, proximal neurites. Because acetylation and detyrosination are functions of MT age, their concentration in this region despite deposition and/or transport of biotinylated tubulin suggests that a subset of axonal MTs undergoes subunit turnover and/or translocation at rates vastly slower than that of the majority of axonal MTs. Selective stabilization of the proximal region of a subset of axonal MTs may serve to construct a relatively stationary scaffold against which other axonal elements could translocate to more distal axonal regions for continued axonal outgrowth.