Tracey Dickson

Microfluidic culture platform for studying neuronal response to mild to very mild axonal stretch injury

Publisher: AIP Publishing

Date: 07-2014

DOI: 10.1063/1.4891098

Abstract: A new model for studying localised axonal stretch injury is presented, using a microfluidic device to selectively culture axons on a thin, flexible poly (dimethylsiloxane) membrane which can be deflected upward to stretch the axons. A very mild (0.5% strain) or mild stretch injury (5% strain) was applied to primary cortical neurons after 7 days growth in vitro. The extent of distal degeneration was quantified using the degenerative index (DI, the ratio of fragmented axon area to total axon area) of axons fixed at 24 h and 72 h post injury (PI), and immunolabelled for the axon specific, microtubule associated protein-tau. At 24 h PI following very mild injuries (0.5%), the majority of the axons remained intact and healthy with no significant difference in DI when compared to the control, but at 72 h PI, the DI increased significantly (DI = 0.11 ± 0.03). Remarkably, dendritic beading in the somal compartment was observed at 24 h PI, indicative of dying back degeneration. When the injury level was increased (5% stretch, mild injury), microtubule fragmentation along the injured axons was observed, with a significant increase in DI at 24 h PI (DI = 0.17 ± 0.02) and 72 h PI (DI = 0.18 ± 0.01), relative to uninjured axons. The responses observed for both mild and very mild injuries are similar to those observed in the in vivo models of traumatic brain injury, suggesting that this model can be used to study neuronal trauma and will provide new insights into the cellular and molecular alterations characterizing the neuronal response to discrete axonal injury.

Axonal shearing in mature cortical neurons induces attempted regeneration and the reestablishment of neurite polarity

Publisher: Elsevier BV

Date: 11-2009

DOI: 10.1016/J.BRAINRES.2009.08.059

Abstract: While functional recovery after injury is limited, it has become evident that the mature central nervous system does retain some ability to regenerate. This study investigated the intrinsic capacity of relatively mature cortical neurons (21 days in vitro) to respond to axonal loss. Neurons, growing as clusters on poly-L-lysine, were completely sheared of axons through chemical and mechanical disruption and transferred to either an intact astrocyte monolayer or a substrate of poly-L-lysine. Injured neurons exhibited a regenerative sprouting response that was independent of neuronal cell ision or neural progenitors, as demonstrated by negative bromodeoxyuridine (BrdU) and the neuronal precursor intermediate filament nestin, labeling. At 24 h after injury, neurons had extended appropriately polarized neurites, demonstrated by compartmentalized microtubule-associated proteins MAP2 and tau immunolabeling. Newly sprouting axons were tipped by growth cones however, growth cones on the tips of sprouting axons (mean area, 26.32 +/- 2.20 microm) were significantly (p<0.05) smaller than their developmental counterparts (mean area, 48.64 +/- 5.9 microm), independent of substrate. Furthermore, live imaging indicated that regenerating neurons exhibited distinct axonal dynamics, with a significant (p<0.05) reduction (70%) in pausing, considered vital for interstitial branching and pathfinding, relative to developmental growth cones. This study indicates that mature cultured cortical pyramidal and interneurons have the intrinsic potential to survive, extend processes, and reestablish neurite polarity following significant physical damage. These results may aid in defining the cellular basis of neuronal structural plasticity and defining the role of astrocyte reactivity in the response to trauma.

Calretinin and Neuropeptide Y interneurons are differentially altered in the motor cortex of the SOD1G93A mouse model of ALS

Publisher: Springer Science and Business Media LLC

Date: 15-03-2017

DOI: 10.1038/SREP44461

Abstract: Increasing evidence indicates an excitatory/inhibitory imbalance may have a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Impaired inhibitory circuitry is consistently reported in the motor cortex of both familial and sporadic patients, closely associated with cortical hyperexcitability and ALS onset. Inhibitory network dysfunction is presumably mediated by intra-cortical inhibitory interneurons, however, the exact cell types responsible are yet to be identified. In this study we demonstrate dynamic changes in the number of calretinin- (CR) and neuropeptide Y-expressing (NPY) interneurons in the motor cortex of the familial hSOD1 G93A ALS mouse model, suggesting their potential involvement in motor neuron circuitry defects. We show that the density of NPY-populations is significantly decreased by ~17% at symptom onset (8 weeks), and by end-stage disease (20 weeks) is significantly increased by ~30%. Conversely, the density of CR-populations is progressively reduced during later symptomatic stages (~31%) to end-stage (~36%), while CR-expressing interneurons also show alteration of neurite branching patterns at symptom onset. We conclude that a differential capacity for interneurons exists in the ALS motor cortex, which may not be a static phenomenon, but involves early dynamic changes throughout disease, implicating specific inhibitory circuitry.

Characterisation of a type IV pilus produced by Aeromonas caviae

Publisher: Microbiology Society

Date: 06-1998

DOI: 10.1099/00222615-47-6-527

Abstract: A pilus produced by a clinical isolate of Aeromonas caviae (strain CA195) was purified and partially characterised. The Mr of the pilin was estimated to be 23 kDa by SDS-PAGE. Its N-terminal amino-acid sequence showed that it was closely related to 'bundle-forming' type IV pili purified from other Aeromonas spp. associated with gastro-enteritis and considered to be important intestinal colonisation factors. Bundle-forming pili, often with a polar location, were seen on the surface of strain CA195 which was highly adherent to HEp-2 cells. Removal of surface structures by mechanical means decreased adhesion (by > or = 50%) suggesting that these pili played some role in HEp-2 cell binding. This pilus type could prove an important marker for enteropathogenic A. caviae which appear to lack other putative virulence factors.

Pathogenic or protective? Neuropeptide Y in amyotrophic lateral sclerosis

Publisher: Wiley

Date: 08-2021

DOI: 10.1111/JNC.15125

Abstract: Neuropeptide Y (NPY) is an endogenous peptide of the central and enteric nervous systems which has gained significant interest as a potential neuroprotective agent for treatment of neurodegenerative disease. Amyotrophic lateral sclerosis (ALS) is an aggressive and fatal neurodegenerative disease characterized by motor deficits and motor neuron loss. In ALS, recent evidence from ALS patients and animal models has indicated that NPY may have a role in the disease pathogenesis. Increased NPY levels were found to correlate with disease progression in ALS patients. Similarly, NPY expression is increased in the motor cortex of ALS mice by end stages of the disease. Although the functional consequence of increased NPY levels in ALS is currently unknown, NPY has been shown to exert a erse range of neuroprotective roles in other neurodegenerative diseases through modulation of potassium channel activity, increased production of neurotrophins, inhibition of endoplasmic reticulum stress and autophagy, reduction of excitotoxicity, oxidative stress, neuroinflammation and hyperexcitability. Several of these mechanisms and signalling pathways are heavily implicated in the pathogenesis of ALS. Therefore, in this review, we discuss possible effects of NPY and NPY‐receptor signalling in the ALS disease context, as determining NPY’s contribution to, or impact on, ALS disease mechanisms will be essential for future studies investigating the NPY system as a therapeutic strategy in this devastating disease. image

The microtubule-modulating drug Epothilone D alters dendritic spine morphology in a mouse model of mild traumatic brain injury

Publisher: Frontiers Media SA

Date: 30-07-2018

DOI: 10.3389/FNCEL.2018.00223

Microfluidic primary culture model of the lower motor neuron–neuromuscular junction circuit

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.JNEUMETH.2013.06.002

Abstract: Modelling the complex process of neuromuscular signalling is key to understanding not only normal circuit function but also importantly the mechanisms underpinning a range of degenerative diseases. We describe a novel in vitro model of the lower motor neuron-neuromuscular junction circuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle. This culture model is designed to spatially mimic the unique anatomical and cellular interactions of this circuit in compartmented microfluidic devices, such that the glial cells are located with motor neuron cell bodies in the cell body chamber and motor neuron axons extend to a distal chamber containing skeletal muscle cells whilst simultaneously allowing targeted intervention. This model is suitable for use in conjunction with a range of downstream experimental approaches and could also be modified to utilise other cellular sources including appropriate immortal cell lines, cells derived from transgenic models of disease and also patient derived stem cells.

Intranasal neuropeptide Y1 receptor antagonism improves motor deficits in symptomatic SOD1 ALS mice

Publisher: Wiley

Date: 29-08-2023

DOI: 10.1002/ACN3.51885

Abstract: Neuropeptide Y (NPY) is a 36 amino acid peptide widely considered to provide neuroprotection in a range of neurodegenerative diseases. In the fatal motor neuron disease amyotrophic lateral sclerosis (ALS), recent evidence supports a link between NPY and ALS disease processes. The goal of this study was to determine the therapeutic potential and role of NPY in ALS, harnessing the brain‐targeted intranasal delivery of the peptide, previously utilised to correct motor and cognitive phenotypes in other neurological conditions. To confirm the association with clinical disease characteristics, NPY expression was quantified in post‐mortem motor cortex tissue of ALS patients and age‐matched controls. The effect of NPY on ALS cortical pathophysiology was investigated using slice electrophysiology and multi‐electrode array recordings of SOD1 G93A cortical cultures in vitro. The impact of NPY on ALS disease trajectory was investigated by treating SOD1 G93A mice intranasally with NPY and selective NPY receptor agonists and antagonists from pre‐symptomatic and symptomatic phases of disease. In the human post‐mortem ALS motor cortex, we observe a significant increase in NPY expression, which is not present in the somatosensory cortex. In vitro, we demonstrate that NPY can ameliorate ALS hyperexcitability, while brain‐targeted nasal delivery of NPY and a selective NPY Y1 receptor antagonist modified survival and motor deficits specifically within the symptomatic phase of the disease in the ALS SOD1 G93A mouse. Taken together, these findings highlight the capacity for non‐invasive brain‐targeted interventions in ALS and support antagonism of NPY Y1Rs as a novel strategy to improve ALS motor function.

Cytoplasmic cytochrome c immunolabelling in dystrophic neurites in Alzheimer’s disease

Publisher: Springer Science and Business Media LLC

Date: 20-07-2006

DOI: 10.1007/S00401-006-0107-3

Abstract: Cytochrome c has a well-established role in electron transfer and as a mediator of apoptotic cell death. The cortical and intracellular localisation of cytochrome c immunoreactivity was examined in Alzheimer's disease and control cases. No differences in the cortical labelling pattern or the density of cytochrome c-positive cells in neocortical layer V were present between control and Alzheimer's disease cases. Punctate cytochrome c labelling was present in a subset of neocortical neurons, including clusters of intensely labelled pyramidal neurons that were not specifically associated with beta-amyloid plaques. With respect to Alzheimer's disease associated pathology, only 6.7 +/- 1.4% of neurons showing neurofibrillary tangle formation demonstrated punctate cytochrome c immunoreactivity. These results suggest that cytochrome c may label a subset of pyramidal neurons that is susceptible, yet relatively resistant, to Alzheimer's disease pathology. A low percentage of neurofilament triplet protein medium, tau and chromogranin A labelled dystrophic neurites were also cytochrome c-positive. There was also a trend towards an increase in the percentage of cytochrome c immunoreactive dystrophic neurites in pathologically aged control cases compared to Alzheimer's disease cases, suggesting that cytochrome c may be an early and transient epitope within dystrophic neurites. In contrast to the punctate cytochrome c labelling observed in cortical cells, cytoplasmic cytochrome c labelling was observed within dystrophic neurites. Although cytochrome c release is indicative of the activation of the intrinsic apoptotic pathway, cytoplasmic cytochrome c may also indicate mitochondrial damage or dysfunction.

Acute reactive and regenerative changes in mature cortical axons following injury

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 12-02-2007

DOI: 10.1097/WNR.0B013E3280143CDB

ERMs colocalize transiently with L1 during neocortical axon outgrowth

Publisher: Wiley

Date: 29-07-2003

DOI: 10.1002/CNE.10809

Abstract: L1 is a member of the Ig superfamily of cell adhesion molecules (CAMs) that functions in many aspects of neuronal development including axonal outgrowth and neuronal migration. These functions require coordination between L1 and the actin cytoskeleton. Because CAMs and the cytoskeleton do not bind directly, membrane-cytoskeletal linkers (MCLs) such as ankyrin are thought to be crucial to their interactions, but data from a knockout mouse suggest that ankyrin is not necessary for the earliest events attributed to L1 function. Recent findings in hippoc al cell culture show that members of the ERM family of proteins (ezrin, radixin, and moesin) can also serve as MCLs between L1 and actin in neurons. Here, we demonstrate that ERM proteins are expressed in extending neuronal processes in the intermediate zone of the developing cortex, a region that is densely packed with migrating neurons and growing axons. ERMs and L1 are codistributed extensively over a transient time course that coincides with rapid axon growth and cortical expansion. This codistribution is strong at embryonic day 17 and 19 but diminishes by postnatal day 0, at which time ankyrin-L1 codistribution increases dramatically. These findings suggest that in the developing neocortex, ERMs are the predominant MCL for L1 during migration and axon extension, neither of which requires ankyrin function. Furthermore, these data suggest that there is a developmentally regulated switch in MCL function in the developing brain.

Cytoskeletal alterations differentiate presenilin-1 and sporadic Alzheimer’s disease

Publisher: Springer Science and Business Media LLC

Date: 18-11-2008

DOI: 10.1007/S00401-008-0458-Z

Abstract: Most cases of Alzheimer's disease (AD) are sporadic in nature, although rarer familial AD (FAD) cases have provided important insights into major pathological disease mechanisms. Mutations in the presenilin 1 gene (PS1) are responsible for the majority of FAD cases, causing an earlier age of onset and more rapid progression to end-stage disease than seen in sporadic AD. We have investigated the cytoskeletal alterations in neuritic AD pathology in a cohort of FAD cases in comparison to sporadic AD and pathologically aged cases. Tau-immunoreactive neurofibrillary tangle (NFT) loads were similar between PS1 FAD and sporadic AD cases. Similarly, plaque loads, both beta-amyloid (Abeta) and thioflavine S, in PS1 FAD and sporadic AD cases were not significantly different however, in pathologically aged cases, they were significantly lower than those in PS1 cases, but were not different from sporadic AD cases. The 'cotton wool' plaque characteristic of PS1 cases did not demonstrate a high density of dystrophic neurites compared to other Abeta plaque types, but did demonstrate a localised mass effect on the neuropil. Despite minimal differences in plaque and NFT loads, immunolabelling demonstrated clear phenotypic differences in the NFTs and dystrophic neurites in PS1 FAD cases. Presenilin-1 cases exhibited significantly (P < 0.05) more tau-positive NFTs that were immunolabelled by the antibody SMI312 (anti-phosphorylated NF protein and phosphorylated tau) than sporadic AD cases. Presenilin-1 cases also exhibited numerous ring-like NF-positive and elongated tau-labelled dystrophic neurites, whereas these dystrophic neurite types were only abundant at the very early (pathologically aged cases) or very late stages of sporadic AD progression, respectively. These differences in cytoskeletal pathology in PS1 cases suggest an accelerated rate of neuritic pathology development, potentially due to mutant PS1 influencing multiple pathogenic pathways.

In vivo and in vitro experimental axotomy mimics a subtype of dystrophic neurite associated with beta-amyloid plaque formation in Alzheimer's disease

Publisher: Elsevier BV

Date: 05-2000

DOI: 10.1016/S0197-4580(00)82624-X

Vaccination Strategies for Alzheimer???s Disease

Publisher: Springer Science and Business Media LLC

Date: 2007

DOI: 10.2165/00002512-200724020-00003

Abstract: The pathological hallmarks of Alzheimer's disease (AD) include beta-amyloid (Abeta) plaques, dystrophic neurites and neurofibrillary pathology, which eventually result in the degeneration of neurons and subsequent dementia. In 1999, international interest in a new therapeutic approach to the treatment of AD was ignited following transgenic mouse studies that indicated that it might be possible to immunise against the pathological alterations in Abeta that lead to aggregation of this protein in the brain. A subsequent phase I human trial for safety, tolerability and immunogenicity using an active immunisation strategy against Abeta had a positive outcome. However, phase IIA human trials involving active immunisation were halted following the diagnosis of aseptic meningoencephalitis in 6% of immunised subjects. Research into immunisation strategies involving transgenic AD mouse models has subsequently been refocused to determine the mechanisms by which plaque clearance and reduced memory deficits are attained, and to establish safer therapeutic approaches that may reduce potentially harmful brain inflammation. The vigour of international research on immunotherapy for AD provides significant hope for a strong therapeutic lead for the escalating number of in iduals who will develop this otherwise incurable condition.

Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors

Publisher: Springer Science and Business Media LLC

Date: 05-2004

DOI: 10.1007/S00018-004-4026-Y

Abstract: Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.

Initial calcium release from intracellular stores followed by calcium dysregulation is linked to secondary axotomy following transient axonal stretch injury

Publisher: Wiley

Date: 02-02-2010

DOI: 10.1111/J.1471-4159.2009.06531.X

Abstract: Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.

Microfluidic Device for Studying Traumatic Brain Injury

Publisher: Springer New York

Date: 2017

DOI: 10.1007/978-1-4939-7024-7_10

Does β-amyloid plaque formation cause structural injury to neuronal processes?

Publisher: Springer Science and Business Media LLC

Date: 2005

DOI: 10.1007/BF03033772

Epothilone D inhibits microglia-mediated spread of alpha-synuclein aggregates

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1016/J.MCN.2018.04.006

Abstract: Multiple System Atrophy (MSA) is a progressive neurodegenerative disease characterized by chronic neuroinflammation and widespread α-synuclein (α-syn) cytoplasmic inclusions. Neuroinflammation associated with microglial cells is typically located in brain regions with α-syn deposits. The potential link between microglial cell migration and the transport of pathological α-syn protein in MSA was investigated. Qualitative analysis via immunofluorescence of MSA cases (n = 4) revealed microglial cells bearing α-syn inclusions distal from oligodendrocytes bearing α-syn cytoplasmic inclusions, as well as close interactions between microglia and oligodendrocytes bearing α-syn, suggestive of a potential transfer mechanism between microglia and α-syn bearing cells in MSA and the possibility of microglia acting as a mobile vehicle to spread α-syn between anatomically connected brain regions. Further In vitro experiments using microglial-like differentiated THP-1 cells were conducted to investigate if microglial cells could act as potential transporters of α-syn. Monomeric or aggregated α-syn was immobilized at the centre of glass coverslips and treated with either cell free medium, undifferentiated THP-1 cells or microglial-like phorbol-12-myristate-13-acetate differentiated THP-1 cells (48 h n = 3). A significant difference in residual immobilized α-syn density was observed between cell free controls and differentiated (p = 0.016) as well as undifferentiated and differentiated THP-1 cells (p = 0.032) when analysed by quantitative immunofluorescence. Furthermore, a significantly greater proportion of differentiated cells were observed bearing α-syn aggregates distal from the immobilized protein than their non-differentiated counterparts (p = 0.025). Similar results were observed with Highly Aggressive Proliferating Immortalised (HAPI) microglial cells, with cells exposed to aggregated α-syn yielding lower residual immobilized α-syn (p = 0.004) and a higher proportion of α-syn positive distal cells (p = 0.001) than cells exposed to monomeric α-syn. Co-treatment of THP-1 groups with the tubulin depolymerisation inhibitor, Epothilone D (EpoD 10 nM), was conducted to investigate if inhibition of microtubule activity had an effect on cell migration and residual immobilized α-syn density. There was a significant increase in both residual immobilized α-syn between EpoD treated and non-treated differentiated cells exposed to monomeric (p = 0.037) and aggregated (p = 0.018) α-syn, but not with undifferentiated cells. Differentiated THP-1 cells exposed to immobilized aggregated α-syn showed a significant difference in the proportion of distal aggregate bearing cells between EpoD treated and untreated (p = 0.027). The results suggest microglia could play a role in α-syn transport in MSA, a role which could potentially be inhibited therapeutically by EpoD.

Mild Axonal Stretch InjuryIn VitroInduces a Progressive Series of Neurofilament Alterations Ultimately Leading to Delayed Axotomy

Publisher: Mary Ann Liebert Inc

Date: 10-2005

DOI: 10.1089/NEU.2005.22.1081

Abstract: We report a new model of transient axonal stretch injury involving pressurized fluid deflection of bundles of axons, resulting in a transient 1-6% increase in original axon length to investigate the slow progression of axonal alterations that are characteristic of diffuse axonal injury (DAI). We found no discernable difference in axon bundle morphology or cytoskeletal neurofilament protein arrangement between unstretched and stretched axonal bundles at 24 h post-injury. However, by 48 h post-injury, there was a stereotypical response of stretched axons involving characteristic neurofilament alterations that bear similarities to in vivo neuronal responses associated with DAI that have been reported previously. For instance, neurofilament protein immunoreactivity (SMI-312) was increased in axons contained within 51% of all injured axon bundles at 48 h compared to surrounding unstretched axon bundles, suggestive of neurofilament compaction. Furthermore, axonal bundle derangement occurred in 25% of injured axon bundles, with in idual fibres segregating from each other and becoming undulating and wavy. By 72 h post-stretch, 70% of injured axon bundles underwent secondary axotomy, becoming completely severed at the site of initial stretch injury. While these results suggest a temporal series of stereotypical responses of axons to injury, we were able to distinguish very clear differences between mildly (100-103% increase in original axonal length) injured and strongly injured (106%+) axons. For instance, mildly injured axons developed increased neurofilament immunoreactivtity (SMI-312) within 48 h, and the marked development of ring-like neurofilament immunoreactive structures within axonal bundles, which were rarely axotomized. Conversely, at more severe strain levels increased neurofilament immunoreactivity was less apparent, while axons often became distorted and disorganised within axonal bundles and eventually became completely disconnected. Almost no ring-like neurofilament structures were observed in these severely injured axonal bundles. This suggests that axons do not respond in a stereotypical manner to a transient stretch insult, and indeed that variable degrees of stretch injury activate different responses within axons, with dramatically different outcomes. Hence, it is possible that the cytoskeletal characteristics that we have used in this study may be useful parameters for discriminating between mildly and severely injured axons following TBI.

Stainless Steel Pinholes for Fast Fabrication of High-Performance Microchip Electrophoresis Devices by CO₂ Laser Ablation

Publisher: American Chemical Society (ACS)

Date: 15-10-2013

DOI: 10.1021/AC402631G

Abstract: With the introduction of hobby laser engravers/cutters, the use of CO2 laser micromachining on poly(methyl methacrylate) (PMMA) has the potential for flexible, low cost, rapid prototyping of microfluidic devices. Unfortunately, the feature size created by most entry-level CO2 laser micromachining systems is too large to become a functional tool in analytical microfluidics. In this paper, we report a novel method to reduce the feature size of microchannels and the bulges formed at the rim of the channel during CO2 laser micromachining by passing the laser beam through a stainless steel pinhole. Without the pinhole, the channel width was typically 300 μm wide. However, when 50 or 35 μm diameter pinholes were used, channel widths of 60 and 25 μm, respectively, could be obtained. The height of the bulge deposited directly next to the channel was reduced to less than 0.8 μm with the pinhole during ablation. Separations of fluorescent dyes on devices ablated with and without the pinhole were compared. On devices fabricated with the pinhole, the number of theoretical plates/m was 2.2-fold higher compared to devices fabricated without the pinhole, and efficiencies comparable to embossed PMMA and laser ablated glass chips were obtained. A mass-produced commercial hobby laser (retailing at ∼$2500), when equipped with a $500 pinhole, represents a rapid and low-cost approach to the rapid fabrication of rigid plastic microchips including the narrow microchannels required for microchip electrophoresis.

Disruption of the Ubiquitin Proteasome System following Axonal Stretch Injury Accelerates Progression to Secondary Axotomy

Publisher: Mary Ann Liebert Inc

Date: 05-2009

DOI: 10.1089/NEU.2008.0669

Abstract: The ubiquitin proteasome system (UPS) plays a vital role in the regulation of protein degradation. Ubiquitination of proteins has been implicated in the pathological cascade associated with neuronal degeneration in both neurodegenerative disease and following acquired central nervous system (CNS) injury. In the present study, we have investigated the role of the UPS following mild to moderate in vitro axonal stretch injury to mature primary cortical neurons, a model of the evolving axonal pathology characteristic of diffuse axonal injury following brain trauma. Transient axonal stretch injury in this model does not involve primary axotomy. However, delayed accumulation of ubiquitin in neuritic swellings at 48 h post-injury (PI) was present in axonal bundles, followed by approximately 60% of axonal bundles progressing to secondary axotomy at 72 h PI. This delayed accumulation of ubiquitin was temporally and spatially associated with cytoskeletal damage. Pharmacological inhibition of the UPS with both MG132 and lactacystin prior to axonal injury resulted in a significant (p < 0.05) increase in the number of axonal bundles progressing to secondary axotomy at 48 and 72 h PI. These results demonstrate that, following mild to moderate transient axonal stretch injury, UPS activity may assist structural reorganization within axons, potentially impeding secondary axotomy. Protein ubiquitination in the axon may therefore have a protective role relative to the diffuse axonal changes that follow traumatic brain injury.

Alpha-Synuclein protects neurons from apoptosis downstream of free-radical production through modulation of the MAPK signalling pathway

Publisher: Springer Science and Business Media LLC

Date: 31-08-2013

DOI: 10.1007/S12640-012-9352-5

Abstract: α-Synuclein is a pre-synaptic chaperone and its accumulation contributes to differential cell loss in Parkinson's disease. Cytoplasmic expression of α-synuclein can directly modulate apoptotic pathways and contribute to cell survival, whereas induced over-expression of the protein causes oxidative stress through mitochondrial and cytosolic free-radical production. This study aimed to clarify the contribution of endogenous α-synuclein to oxidative stress and its association with cell death. Primary cortical neurons were derived from α-synuclein knock-out (Snca-/-) and wild-type (C57BL/6 WT) mice and treated with in vitro models of oxidative-stress, complex I inhibition and excitotoxicity. Mitochondrial free radical production was determined in isolated mitochondria derived from each mouse strain. Snca-/- derived cortical cultures were more susceptible (P < 0.05) to oxidative-stress, but not excitotoxicity. This result was determined by significant increases in cell death (Propidium-Iodide staining) after 6 h treatment in Snca-/- (45 % ± 2.7 SEM), relative to WT (33 % ± 3.9 SEM) cultures. α-Synuclein also confers significant (P < 0.05) resistance to low-dose (5 nM) rotenone toxicity, with a twofold reduction in cell death in WT, compared with Snca-/- cortical neurons. The expression of α-synuclein had no effect on cortical glutathione levels, or the production of reactive oxygen intermediates in isolated mitochondria. These data indicate that endogenous levels of α-synuclein confer resistance to oxidative stress downstream of free radical production and scavenging. The current data suggest that α-synuclein prevents cytochrome c release and apoptosis through inhibition of the MAPK signalling pathway.

Excitotoxicity mediated by non‐NMDA receptors causes distal axonopathy in long‐term cultured spinal motor neurons

Publisher: Wiley

Date: 10-2007

DOI: 10.1111/J.1460-9568.2007.05845.X

Abstract: Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long-term culture. Excitotoxicity, mediated principally via non-N-methyl-D-aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.

Axonopathy and cytoskeletal disruption in degenerative diseases of the central nervous system

Publisher: Elsevier BV

Date: 10-2009

DOI: 10.1016/J.BRAINRESBULL.2009.08.004

Abstract: There has been growing interest in the axon as the initial focus of pathological change in a number of neurodegenerative diseases of the central nervous system. This review concentrates on three major neurodegenerative conditions--amyotrophic lateral sclerosis, multiple sclerosis and Alzheimer's disease--with emphasis on key cellular changes that may underlie early axonal dysfunction and pathology and, potentially, the degeneration of neurons. In particular, this review will address recent data that indicate that the main pathological stimuli for these conditions, though often not definitively determined, result in an initial perturbation of the axon and its cytoskeleton, which then results in slow neuronal degeneration and loss of connectivity. The identification of a degenerative process initiated in the axon may provide new therapeutic targets for early intervention to inhibit the grim outcomes related to the progression of these diseases.

Mislocalisation of TDP‐43 to the cytoplasm causes cortical hyperexcitability and reduced excitatory neurotransmission in the motor cortex

Publisher: Wiley

Date: 09-11-2020

DOI: 10.1111/JNC.15214

Inhibitory dysfunction in amyotrophic lateral sclerosis: future therapeutic opportunities

Publisher: Future Medicine Ltd

Date: 12-2015

DOI: 10.2217/NMT.15.49

Abstract: In amyotrophic lateral sclerosis, motor neuron hyperexcitability and inhibitory dysfunction is emerging as a potential causative link in the dysfunction and degeneration of the motoneuronal circuitry that characterizes the disease. Interneurons, as key regulators of excitability, may mediate much of this imbalance, yet we know little about the way in which inhibitory deficits perturb excitability. In this review, we explore inhibitory control of excitability and the potential contribution of altered inhibition to amyotrophic lateral sclerosis disease processes and vulnerabilities, identifying important windows of therapeutic opportunity and potential interventions, specifically targeting inhibitory control at key disease stages.

A Novel In Vitro Primary Culture Model of the Lower Motor Neuron–Neuromuscular Junction Circuit

Publisher: Springer New York

Date: 2015

DOI: 10.1007/978-1-4939-2510-0_11

Amyloid β precursor protein regulates neuron survival and maturation in the adult mouse brain

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.MCN.2016.09.002

Abstract: The amyloid-β precursor protein (APP) is a transmembrane protein that is widely expressed within the central nervous system (CNS). While the pathogenic dysfunction of this protein has been extensively studied in the context of Alzheimer's disease, its normal function is poorly understood, and reports have often appeared contradictory. In this study we have examined the role of APP in regulating neurogenesis in the adult mouse brain by comparing neural stem cell proliferation, as well as new neuron number and morphology between APP knockout mice and C57bl6 controls. Short-term EdU administration revealed that the number of proliferating EdU

Denervation of the Olfactory Bulb Leads to Decreased A&#946; Plaque Load in a Transgenic Mouse Model of Alzheimer&#8217; s Disease

Publisher: Bentham Science Publishers Ltd.

Date: 08-2013

DOI: 10.2174/15672050113109990147

Abstract: The aggregation of beta-amyloid (Aβ) into plaques in the extracellular compartment of the brain is a pathological hallmark of Alzheimer' s disease (AD). Although the pathways for misprocessing of Aβ leading to plaque formation are not well understood, they may be related to synapse turnover and neuron activity. In this study, we have utilised transgenic mice co-expressing mutations in the amyloid precursor protein and presenilin 1 genes (APP/PS1) to determine how long-term denervation of the olfactory bulb, a CNS area affected early by AD-like pathology, may affect Aβ plaque formation. The olfactory bulb of pre-symptomatic mice was denervated by ablating the olfactory epithelium unilaterally with Triton X-100 solution. Mice were subjected to nasal washes for a total of 4 or 8 times, at 3-week intervals either with 1% Triton X-100 solution or phosphate buffered saline (sham denervation). Denervation of the olfactory bulb resulted in a statistically significant (p<0.05) decrease in amyloid plaque load in the ipsilateral olfactory bulb, and bilaterally also in the neocortex and hippoc us at 8-9 months age. Amyloid precursor protein was predominantly expressed by mitral cells in the olfactory bulb, which are normally postsynaptic to olfactory axons. The number of APP positive mitral cells was significantly increased in the denervated olfactory bulb of wild type but not of the APP/PS1 mice, which consistently showed high immunoreactivity for APP pre- and post-denervation. In summary, our results show that Aβ plaque deposition in the central nervous system can be modified transsynaptically by deafferentation.

Neuron–glia interactions underlie ALS-like axonal cytoskeletal pathology

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.NEUROBIOLAGING.2009.04.004

Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating disorder involving loss of movement due to degeneration of motor neurons. Studies suggest that in ALS axonal dysfunction precedes the death of motor neurons. Pathologically, ALS is characterized by neurofilamentous swellings (spheroids) within the axons of motor neurons. However, the causes of this axonopathy and possible resulting axonal dysfunction are not known. Using a novel model of cultured mouse motor neurons, we have determined that these neurons are susceptible to proximal axonopathy, which is related to the glial environment. This axonopathy showed remarkable similarity, both morphologically and neurochemically, to spheroids that develop over months in SOD1(G93A) transgenic mice. Focal ubiquitination, as well as perturbations of neurofilaments and microtubules, occurred in the axonal spheroid-like swellings in vitro, and visualization of mitochondrial dynamics demonstrated that axonopathy resulted in impaired axonal transport. These data provide strong evidence for the involvement of non-neuronal cells in axonal dysfunction in ALS. This cell culture model may be of benefit for the development of therapeutic interventions directed at axonal preservation.

Mild Traumatic Brain Injury Leads to Decreased Inhibition and a Differential Response of Calretinin Positive Interneurons in the Injured Cortex

Publisher: Mary Ann Liebert Inc

Date: 09-2017

DOI: 10.1089/NEU.2017.4977

Abstract: It is clear that even mild forms of traumatic brain injury (TBI) can have lasting cognitive effects however, the specific cellular changes responsible for the functional deficits remain poorly understood. Previous studies suggest that not all neurons respond in the same way and that changes to neuronal architecture may be subtype specific. The current study aimed to characterize the response of interneurons to TBI. To model TBI in vitro, the neurites of primary cortical neurons were transected at 15 days in vitro. In response, calretinin

Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse

Publisher: Public Library of Science (PLoS)

Date: 04-05-2017

DOI: 10.1371/JOURNAL.PONE.0176997

Association of Metallothionein-III with Oligodendroglial Cytoplasmic Inclusions in Multiple System Atrophy

Publisher: Springer Science and Business Media LLC

Date: 29-12-2009

DOI: 10.1007/S12640-009-9146-6

Abstract: Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterised by Parkinsonian and autonomic symptoms and by widespread intracytoplasmic inclusion bodies in oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are comprised of 9-10 nm filaments rich in the protein alpha-synuclein, also found in neuronal inclusion bodies associated with Parkinson's disease. Metallothioneins (MTs) are a class of low-molecular weight (6-7 kDa), cysteine-rich metal-binding proteins the expression of which is induced by heavy metals, glucocorticoids, cytokines and oxidative stress. Recent studies have shown a role for the ubiquitously expressed MT-I/II isoforms in the brain following a variety of stresses, whereas, the function of the brain-specific MT isoform, MT-III, is less clear. MT-III and MT-I/II immunostaining of post-mortem tissue in MSA and normal control human brains showed that the number of MT-III-positive cells is significantly increased in MSA in visual cortex, whereas MT-I/II isoforms showed no significant difference in the distribution of immunopositive cells in MSA compared to normal tissue. GCIs were immunopositive for MT-III, but were immunonegative for the MT-I/II isoforms. Immunofluorescence double labelling showed the co-localisation of alpha-synuclein and MT-III in GCIs in MSA tissue. In isolated GCIs, transmission electron microscopy demonstrated MT-III immunogold labelling of the amorphous material surrounding alpha-synuclein filaments in GCIs. High-molecular weight MT-III species in addition to MT-III monomer were detected in GCIs by Western analysis of the detergent-solubilised proteins of purified GCIs. These results show that MT-III, but not MT-I/II, is a specific component of GCIs, present in abnormal aggregated forms external to the alpha-synuclein filaments.

Binding partners L1 cell adhesion molecule and the ezrin‐radixin‐moesin (ERM) proteins are involved in development and the regenerative response to injury of hippocampal and cortical neurons

Publisher: Wiley

Date: 09-2004

DOI: 10.1111/J.1460-9568.2004.03620.X

Intrinsic Regenerative Ability of Mature CNS Neurons

Publisher: SAGE Publications

Date: 08-2004

DOI: 10.1177/1073858404263511

Abstract: A prevailing view in neuroscience is that the mature CNS has relatively little capacity to respond adaptively to injury. Recent data indicating a high degree of structural plasticity in the adult brain provides an impetus to reexamine how central neurons react to trauma. An analysis of both in vivo and in vitro experimental studies demonstrates that certain brain neurons may have an intrinsic ability to respond to structural injury by an attempt at regenerative sprouting. Indeed, aberrant sprouting following neuronal injury may be the cause of epilepsy following brain trauma and may underlie the neuronal changes stimulated by plaque formation in Alzheimer’s disease. An understanding of the stereotypical reaction to injury of different CNS neurons, as well as the role of nonneuronal cells, may provide new avenues for therapeutic intervention for a range of neurodegenerative diseases and “acquired” forms of CNS injury.

Degeneration of axons in spinal white matter in G93A mSOD1 mouse characterized by NFL and alpha-internexin immunoreactivity

Publisher: Elsevier BV

Date: 07-2012

DOI: 10.1016/J.BRAINRES.2012.05.018

Abstract: Axonal degeneration is a prominent feature of amyotrophic lateral sclerosis (ALS) both in lower motor nerves as well as descending white matter axons in the spinal cord of human patients. Although the pathology of lower motor axonal degeneration has been described in both human ALS and related transgenic animal models, few studies have examined the pathological features of descending axon degeneration, particularly in mouse models of ALS. We have examined the degeneration of white matter tracts in the G93A mutant superoxide dismutase-1 (mSOD1+) mouse spinal cord white matter from 12 weeks of age to end-stage disease. In a G93A mSOD1 mouse model where green fluorescent protein was expressed in neurons (mSOD1+/GFP+), degeneration of white matter tracts was present from the ventral to dorsolateral funiculi. This pattern of axonal pathology occurred from 16 weeks of age. However, the dorsal funiculus, the site of the major corticospinal tract in mice, showed relatively less degeneration. Immunohistochemical analysis demonstrated that the neurofilament light chain (NFL) and neuronal intermediate filament protein alpha-internexin accumulated in axon swellings in the spinal white matter. Increased levels of alpha-internexin protein, in mSOD1+ mouse spinal cord tissue, were demonstrated by Western blotting. In contrast, degenerating axons did not show obvious accumulations of neurofilament medium and heavy chain proteins (NFM and NFH). These data suggest that white matter degeneration in this mouse model of ALS is widespread and involves a specific molecular signature, particularly the accumulation of NFL and alpha-internexin proteins.

Characterization of Cortical Neuronal and Glial Alterations during Culture of Organotypic Whole Brain Slices from Neonatal and Mature Mice

Publisher: Public Library of Science (PLoS)

Date: 15-07-2011

DOI: 10.1371/JOURNAL.PONE.0022040

Neuroprotective upregulation of endogenous alpha-synuclein precedes ubiquitination in cultured dopaminergic neurons

Publisher: Springer Science and Business Media LLC

Date: 09-07-2010

DOI: 10.1007/S12640-010-9207-X

Abstract: α-Synuclein is the major protein component of Lewy bodies--the pathological hallmark of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Its accumulation into intracellular aggregates is implicated in the process of Lewy body formation. However, its roles in both normal function, and disease, remain controversial. Using a novel model of chronic oxidative stress in cultured dopaminergic and cortical neurons, we report that endogenous α-synuclein is upregulated in response to low dose toxicity. This response is conserved between subpopulations of cortical and dopaminergic neurons, and confers relative resistance to apoptosis following secondary insult. Additional acute oxidative stress leads to intracellular accumulation of α-synuclein. These punctate deposits colocalize with ubiquitin, which is central to proteosome-mediated protein degeneration, and is the second major component of Lewy bodies. The current results imply that differential levels of α-synuclein expression may influence neuronal vulnerability in chronic neurodegenerative diseases. They further support a 'two hit' hypothesis for Lewy body formation, whereby mild stress causes a protective upregulation of α-synuclein. However, such increased levels of α-synuclein may drive its accumulation, following additional toxic insult. Finally, these results support a common mechanism for degeneration of dopaminergic and cortical neurons, affected in PD, and DLB, respectively.

Neuronal Response To Physical Injury And Its Relationship To The Pathology Of Alzheimer's Disease

Publisher: Wiley

Date: 17-07-2000

DOI: 10.1046/J.1440-1681.2000.03292.X

Abstract: 1. Central nerve cells undergo a stereotyped regenerative response following physical injury. 2. This reaction involves adaptive changes within the axon and cell body of origin, directed at sprouting and synaptogenesis. 3. Intimately associated with the regenerative response are specific alterations to cytoskeletal proteins, including the neurofilament (NF) triplet. 4. The morphological and neurochemical alterations to NF within axons following injury are reminiscent of plaque-associated dystrophic neurites (DN) in early Alzheimer's disease (AD). 5. Associated changes in perikaryal NF resemble Alzheimer neurofibrillary tangle pathology, while growth-associated sprouting markers are localized to the abnormal neurites of AD. 6. The present review postulates that beta-amyloid plaques in AD cause physical damage to local nerve cell processes and it is the chronic stimulation of the stereotyped response to injury that results in the end-stage pathology and neurodegeneration associated with AD.

The microtubule-stabilizing drug Epothilone D increases axonal sprouting following transection injury in vitro

Publisher: Elsevier BV

Date: 05-2015

DOI: 10.1016/J.MCN.2015.02.006

Abstract: Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are considered a hallmark feature of the degeneration that occurs after traumatic brain injury (TBI). Therefore, microtubule-stabilizing drugs are attractive potential therapeutics for use following TBI. The best-known drug in this category is Paclitaxel, a widely used anti-cancer drug that has produced promising outcomes when employed in the treatment of various animal models of nervous system trauma. However, Paclitaxel is not ideal for the treatment of patients with TBI due to its limited blood-brain barrier (BBB) permeability. Herein we have characterized the effect of the brain penetrant microtubule-stabilizing agent Epothilone D (Epo D) on post-injury axonal sprouting in an in vitro model of CNS trauma. Epo D was found to modulate axonal sprout number in a dose dependent manner, increasing the number of axonal sprouts generated post-injury. Elevated sprouting was observed when analyzing the total population of injured neurons, as well as in selective analysis of Thy1-YFP-labeled excitatory neurons. However, we found no effect of Epo D on axonal sprout length or outgrowth speed. These findings indicate that Epo D specifically affects injury-induced axonal sprout generation, but not net growth. Our investigation demonstrates that primary cultures of cortical neurons are tolerant of Epo D exposure, and that Epo D significantly increases their regenerative response following structural injury. Therefore Epo D may be a potent therapeutic for enhancing regeneration following CNS injury. This article is part of a Special Issue entitled 'Traumatic Brain Injury'.

Cortical Murine Neurons Lacking the Neurofilament Light Chain Protein Have an Attenuated Response to InjuryIn Vitro

Publisher: Mary Ann Liebert Inc

Date: 15-11-2013

DOI: 10.1089/NEU.2013.2850

Abstract: Neurofilaments (NFs) have been proposed to have a significant role in attempted axonal regeneration following a variety of forms of injury. The NF triplet proteins of the central nervous system are comprised of light (NF-L), medium (NF-M) and heavy (NF-H) chains and are part of the type IV intermediate filament family. We sought to define the role of NF-L in the neuronal response to trauma and regeneration by examining the effect of total absence of the NF-L protein on neuronal maturation and response to axotomy. This study utilized an in vitro model comprising relatively mature cortical murine neurons derived from either wild-type embryonic (E15) mice or mice with a genetic knockout of NF-L (NF-L KO). Whilst NF-L KO neurons developed to relative maturity at a comparable rate to wild-type control neurons, NF-L KO neurons demonstrated relatively increased expression of α-internexin and decreased expression of NF-M. Further, we demonstrate that α-internexin co-immunoprecipitates with the NF binding protein NDel1 in NFL-KO cortical neurons in vitro. Following localized axotomy, NF-L KO neurons demonstrated reduced amyloid precursor protein accumulation in damaged neurites as well as a significant reduction in the number of axons regenerating (4.79+/-0.58 sprouts) in comparison to control preparations (10.47+/-1.11 sprouts) (p<0.05). These studies indicate that NFs comprising NF-L have a dynamic role in the reactive and regenerative changes in axons following injury.

The morphological phenotype of β-amyloid plaques and associated neuritic changes in Alzheimer’s disease

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1016/S0306-4522(01)00169-5

Abstract: We have utilised laser confocal microscopy to categorise beta-amyloid plaque types that are associated with preclinical and end-stage Alzheimer's disease and to define the neurochemistry of dystrophic neurites associated with various forms of plaques. Plaques with a spherical profile were defined as either diffuse, fibrillar or dense-cored using Thioflavin S staining or immunolabelling for beta-amyloid. Confocal analysis demonstrated that fibrillar plaques had a central mass of beta-amyloid with compact spoke-like extensions leading to a confluent outer rim. Dense-cored plaques had a compacted central mass surrounded by an outer sphere of beta-amyloid. Diffuse plaques lacked a morphologically identifiable substructure, resembling a ball of homogeneous labelling. The relative proportion of diffuse, fibrillar and dense-cored plaques was 53, 22 and 25% in preclinical and 31, 49 and 20% in end-stage Alzheimer's disease cases, respectively. Plaque-associated dystrophic neurites in preclinical cases were immunolabeled for neurofilament proteins whereas, in end-stage cases, these abnormal neurites were variably labelled for tau and/or neurofilaments. Double labelling demonstrated that the proportion of diffuse, fibrillar and dense-cored plaques that were neuritic was 12, 47 and 82% and 24, 82 and 76% in preclinical and end-stage cases, respectively. Most dystrophic neurites in Alzheimer's disease cases were labelled for either neurofilaments or tau, however, confocal analysis determined that 30% of neurofilament-labelled bulb-like or elongated neurites had a core of tau immunoreactivity. These results indicate that all morphologically defined beta-amyloid plaque variants were present in both early and late stages of Alzheimer's disease. However, progression to clinical dementia was associated with both a shift to a higher proportion of fibrillar plaques that induced local neuritic alterations and a transformation of cytoskeletal proteins within associated abnormal neuronal processes. There data indicate key pathological changes that may be subject to therapeutic intervention to slow the progression of Alzheimer's disease.

α-Internexin immunoreactivity reflects variable neuronal vulnerability in Alzheimer's disease and supports the role of the β-amyloid plaques in inducing neuronal injury

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1016/J.NBD.2004.10.001

Abstract: This study investigated the role of alpha-internexin in the neuronal alterations associated with beta-amyloid plaque formation in Alzheimer's disease (AD). Cortical neurons could be defined by their variable content of neurofilament (NF) triplet and alpha-internexin proteins, with a distinct population of supragranular pyramidal cells containing alpha-internexin alone. Both NF triplet and alpha-internexin were localized to reactive axonal structures in physically damaged neurons in experimental trauma models. Similarly, NF triplet and alpha-internexin immunoreactive neurites were localized to plaques densely packed with beta-amyloid fibrils in preclinical AD cases, indicating that certain plaques may cause structural injury or impediment of local axonal transport. However, alpha-internexin, and not NF triplet, ring-like reactive neurites were present in end-stage AD cases, indicating the relatively late involvement of neurons that selectively contain alpha-internexin. These results implicate the expression of specific intermediate filament proteins in a distinct hierarchy of differential neuronal vulnerability to AD.

Rho kinase activates ezrin‐radixin‐moesin (ERM) proteins and mediates their function in cortical neuron growth, morphology and motility in vitro

Publisher: Wiley

Date: 30-10-2006

DOI: 10.1002/JNR.21102

Abstract: The ezrin-radixin-moesin (ERM) family of proteins contribute to cytoskeletal processes underlying many vital cellular functions. Their previously elucidated roles in non-neuronal cells are an indication of their potential importance in CNS neurons. The specific mechanisms of their activation are unknown, but are likely to depend on factors such as the cell type and biological context. For ERM proteins to become active they must be phosphorylated at a specific C-terminal threonine residue. In non-neuronal cells, several kinases, including the Rho GTPase family member Rho kinase, have been identified as capable of phosphorylating the C-terminal threonine. In these experiments we have investigated specifically the potential role of Rho kinase mediated ERM activation in cortical neurons, utilizing a new pharmacologic inhibitor of Rho kinase and quantitative analysis of aspects of neuronal functions potentially mediated by ERM proteins. Rho kinase inhibition significantly suppressed aspects of neuronal development including neurite initiation and outgrowth, as well as growth cone morphology, with a concomitant loss of phosphorylated ERM immunolabeling in areas associated with neuronal growth. The ability of the Rho kinase inhibitor to decrease the amount of pERM protein was shown by immunoblotting. Post-injury responses were negatively affected by Rho kinase inhibition, namely by a significant decrease in the number of regenerative neurites. We investigated a novel role for ERM proteins in neuron migration using a post-injury motility assay, where Rho kinase inhibition resulted in significant and drastic reduction in neuron motility and phosphorylated ERM immunolabeling. Thus, Rho kinase is an important activator of ERMs in mediating specific neuronal functions.

No difference in expression of apoptosis-related proteins and apoptotic morphology in control, pathologically aged and Alzheimer's disease cases

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.NBD.2005.11.010

Abstract: Apoptotic-like changes in the neocortex of control, pathologically aged and Alzheimer's disease (AD) cases were investigated. There was no increase in labeling or change in localization of labeling that distinguished between these cases for active caspase-3, -8, -9, Bax, Bcl-2 or TRADD. Bax, Bcl-2 and TRADD mRNA levels also differed little between case types, although there were small but significant decreases in Bax mRNA levels in AD compared to control cases and Bcl-2 mRNA in AD cases compared to pathologically aged and control cases. There was no difference in the percentage of apoptotic-like nuclei between these cases, except for a small but significant decrease in the inferior temporal gyrus of AD cases relative to controls. Nuclei observed within or adjacent to beta-amyloid plaques were rarely abnormal, and neurons bearing neurofibrillary tangles (NFTs) did not have abnormal nuclei. Apoptosis may not play a major role in the pathogenesis of neuronal degeneration of AD.

Reduced excitability and increased neurite complexity of cortical interneurons in a familial mouse model of amyotrophic lateral sclerosis

Publisher: Frontiers Media SA

Date: 28-09-2018

DOI: 10.3389/FNCEL.2018.00328

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex

Publisher: Elsevier BV

Date: 09-2002

DOI: 10.1016/S0891-0618(02)00043-1

Abstract: The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Alpha-synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.EXPNEUROL.2005.10.018

Abstract: Chronic oxidative stress has been linked to the neurodegenerative changes characteristic of Parkinson's disease, particularly alpha-synuclein accumulation and aggregation. However, it remains contentious whether these alpha-synuclein changes are cytotoxic or neuroprotective. The current study utilised long-term primary neural culture techniques with antioxidant free media to study the cellular response to chronic oxidative stress. Cells maintained in antioxidant free media were exquisitely more vulnerable to acute exposure to hydrogen peroxide, yet exposure of up to 10 days in antioxidant free media did not lead to morphological alterations in neurones or glia. However, a subpopulation of neurones demonstrated a significant increase in the level of alpha-synuclein expressed within the cell body and at synaptic sites. This subset of neurones was also more resistant to apoptotic changes following exposure to antioxidant free media relative to other neurones. These data indicate that increased alpha-synuclein content is associated with neuroprotection from relatively low levels of oxidative stress.

Altered synapses and gliotransmission in Alzheimer's disease and AD model mice

Publisher: Elsevier BV

Date: 10-2013

DOI: 10.1016/J.NEUROBIOLAGING.2013.04.010

Abstract: Amyloid-β (Aβ) plaque accumulation in Alzheimer's disease (AD) is associated with glutamatergic synapse loss, but less is known about its effect on inhibitory synapses. Here, we demonstrate that vesicular γ-aminobutyric acid (GABA) transporter (VGAT) presynaptic bouton density is unaffected in human preclinical and end-stage AD and in APP/PS1 transgenic (TG) mice. Conversely, excitatory vesicular glutamate transporter 1 (VGlut1) boutons are significantly reduced in end-stage AD cases and less reduced in preclinical AD cases and TGs. Aged TGs also show reduced protein levels of VGlut1 and synaptophysin but not VGAT or glutamate decarboxylase (GAD). These findings indicate that GABAergic synapses are preserved in human AD and mouse TGs. Synaptosomes isolated from plaque-rich TG cortex had significantly higher GAD activity than those from plaque-free cerebellum or the cortex of wild-type littermates. Using tissue fractionation, this increased activity was localized to glial synaptosomes, suggesting that Aβ plaques stimulate increased astrocyte GABA synthesis.

Cellular dynamics underlying regeneration of damaged axons differs from initial axon development

Publisher: Wiley

Date: 29-08-2007

DOI: 10.1111/J.1460-9568.2007.05750.X

Abstract: While long-distance regeneration may be limited in mammalian species, it is becoming apparent that damaged mature neurons retain some capacity for attempted regeneration and that the adult CNS is not entirely inhibitory to axon growth. Our investigations show that there are critical intrinsic features of postinjury axonal regeneration that differ from initial axon development, and that these distinct differences may account for the limited and inappropriate regenerative response that currently characterizes the mature CNS. We compared the neurochemical and dynamic characteristics of developing axons to relatively mature regenerating axons, utilizing an in vitro model of axonal transection to long-term cultured rat cortical neurons. Immunolabelling studies revealed that regenerating and developing axons have a similar localization of cytoskeletal proteins, but the tips of regenerating axons, although morphologically similar, were smaller with reduced fillopodial extension, relative to developmental growth cones. Live imaging demonstrated that regenerating axons exhibited significantly less outgrowth than developmental neurites. Furthermore, growth cones of regenerating axons had a significant reduction in pausing, considered vital for interstitial branching and pathfinding, than did developmental growth cones. In addition, unlike developing axons, the regenerating axons were unresponsive to the growth factors BDNF and GDNF. Thus, although similar in their cytoskeletal composition, the growth cones of regenerative sprouts differed from their developmental counterparts in their size, their dynamic behaviour and their ability to respond to critical growth factors. These intrinsic differences may account for the inability of post-traumatic locally sprouting axons to make accurate pathway decisions and successfully respond to trauma.

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

Publisher: Rockefeller University Press

Date: 17-06-2002

DOI: 10.1083/JCB.200111076

Abstract: Ayeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane–cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippoc al neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippoc al neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM–actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis – vulnerability of lower motor neurons to proximal excitotoxicity

Publisher: The Company of Biologists

Date: 03-2015

DOI: 10.1242/DMM.018606

Abstract: There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of in iduals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.

Localization of glutamate receptors in developing cortical neurons in culture and relationship to susceptibility to excitotoxicity

Publisher: Wiley

Date: 2006

DOI: 10.1002/CNE.21053

Abstract: Overactivation of glutamate receptors leading to excitotoxicity has been implicated in the neurodegenerative alterations of a range of central nervous system (CNS) disorders. We have investigated the cell-type-specific changes in glutamate receptor localization in developing cortical neurons in culture, as well as the relationship between glutamate receptor subunit distribution with synapse formation and susceptibility to excitotoxicity. Glutamate receptor subunit clustering was present prior to the formation of synapses. However, different receptor types showed distinctive temporal patterns of subunit clustering, localization to spines, and apposition to presynaptic terminals. N-methyl-D-aspartate (NMDA) receptor subunit immunolabelling was present in puncta along dendrites prior to the formation of synapses, with relatively little localization to spines. Vulnerability to NMDA receptor-mediated excitotoxicity occurred before receptor subunits became localized in apposition to presynaptic terminals. Clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors occurred concurrently with development of vulnerability to excitotoxicity and was related to localization of AMPA receptors at synapses and in spines. Different AMPA receptor subunits demonstrated cell-type-specific localization as well as distribution to spines, dendrites, and extrasynaptic subunit clusters. A subclass of neurons demonstrated substantial perineuronal synaptic innervation, and these neurons expressed relatively high levels of GluR1 and/or GluR4 at receptor puncta, indicating the presence of calcium-permeable AMPA receptors and suggesting alternative synaptic signalling mechanisms and vulnerability to excitotoxicity. These data demonstrate the relationship between glutamate receptor subunit expression and localization with synaptogenesis and development of neuronal susceptibility to excitotoxicity. These data also suggest that excitotoxicity can be mediated through extrasynaptic receptor subunit complexes along dendrites.

Identification and characterization of a population of motile neurons in long-term cortical culture

Publisher: Wiley

Date: 2007

DOI: 10.1002/CM.20182

Abstract: The specific phenotypes and progression to maturity of primary cortical neurons in long-term culture correlate well with neurons in vivo. Utilizing a model of neuronal injury in long-term cultures at 21 days in vitro (DIV), we have identified a distinct population of neurons that translocate into the injury site. 5-bromo-2'-deoxyUridine (BrdU) incorporation studies demonstrated that neurons with the capacity to translocate were 21 days old. However, this motile ability is not consistent with the traditional view of the maturation and structural stability of neurons in long-term culture. Therefore, we examined the neurons' cytoskeletal profile using immunocytochemistry, to establish relative stage of maturation and phenotype. Expression of marker proteins including beta-III-tubulin, alpha-internexin, NF-L and NF-M, tau and L1 indicated the neurons were differentiated, and in some cases polarized. The neurons did not immunolabel with NF-H or MAP2, which might suggest they had not reached the level of maturity of other neurons in culture. They did not express the microtubule-associated migration marker doublecortin (DCX). Cytoskeletal disrupting agents were used to further investigate the role of the microtubule cytoskeleton in translocation, and microtubule destabilization significantly enhanced aspects of their motility. Finally, molecular guidance cues affected their motility in a similar manner to that reported for both axon guidance and early neuron migration. Therefore, this study has identified and characterized a population of motile neurons in vitro that have the capacity to migrate into a site of injury. These studies provide new information on the structurally dynamic features of subsets of neurons.

Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury

Publisher: Wiley

Date: 2007

DOI: 10.1002/DNEU.20552

Abstract: Following central nervous system trauma, diffuse axonal injury and secondary axotomy result from a cascade of cellular alterations including cytoskeletal and mitochondrial disruption. We have examined the link between intracellular changes following mild/moderate axonal stretch injury and secondary axotomy in rat cortical neurons cultured to relative maturity (21 days in vitro). Axon bundles were transiently stretched to a strain level between 103% and 106% using controlled pressurized fluid. Double-immunohistochemical analysis of neurofilaments, neuronal spectrin, alpha-internexin, cytochrome-c, and ubiquitin was conducted at 24-, 48-, 72-, and 96-h postinjury. Stretch injury resulted in delayed cytoskeletal damage, maximal at 48-h postinjury. Accumulation of cytochrome-c and ubiquitin was also evident at 48 h following injury and colocalized to axonal regions of cytoskeletal disruption. Pretreatment of cultures with cyclosporin-A, an inhibitor of calcineurin and the mitochondrial membrane transitional pore, reduced the degree of cytoskeletal damage in stretch-injured axonal bundles. At 48-h postinjury, 20% of untreated cultures demonstrated secondary axotomy, whereas cyclosporin A-treated axon bundles remained intact. By 72-h postinjury, 50% of control preparations and 7% of cyclosporin A-treated axonal bundles had progressed to secondary axotomy, respectively. Statistical analyses demonstrated a significant (p < 0.05) reduction in secondary axotomy between treated and untreated cultures. In summary, these results suggest that cyclosporin-A reduces progressive cytoskeletal damage and secondary axotomy following transient axonal stretch injury in vitro.

Relationship between apolipoprotein E and the amyloid deposits and dystrophic neurites of Alzheimer's disease

Publisher: Wiley

Date: 12-1997

DOI: 10.1111/J.1365-2990.1997.TB01325.X

Abstract: Although the inheritance of certain apolipoprotein E (ApoE) alleles has been recognized as a genetic risk factor for Alzheimer's disease, the role of ApoE in the pathology underlying this disease is unclear. Several reports have emphasized the association of ApoE with either beta-amyloid plaque formation or the development of neurofibrillary pathology. Utilization of multiple label immunohistochemical methods enabled us to examine directly the localization of ApoE immunoreactivity relative to beta-amyloid plaques, dystrophic neurites and neurofibrillary tangles. In Alzheimer's disease cases, beta-amyloid plaques showing high ApoE immunoreactivity were localized to layers II, III and V of the neocortex. In layer I, beta-amyloid plaques were unlabelled for ApoE relative to beta-amyloid. Dense core plaques labelled for beta-amyloid often had only the central portions labelled for ApoE. Conversely, ApoE labelled spherical structures within some plaques were not immunoreactive for beta-amyloid or dystrophic neurite markers. Unlike beta-amyloid labelled plaques, all ApoE immunoreactive plaques were associated with dystrophic neurites. In preclinical Alzheimer's disease cases, most plaques were double labelled for beta-amyloid and ApoE. ApoE did not label dystrophic neurites or the early stages of neurofibrillary tangle formation, indicating that ApoE may not be directly involved in neurofibrillary pathology. The specific presence of ApoE in plaques associated with dystrophic neurites in demented patients suggests that ApoE may contribute toward a higher degree of beta-amyloid fibrillogenesis, enhancing the ability of certain plaques to cause damage to surrounding axons.

Cellular dynamics underlying regeneration of damaged axons differs from initial axon development

Publisher: Wiley

Date: 29-08-2007

DOI: 10.1111/J.1460-9568.2007.05750.X

Abstract: While long-distance regeneration may be limited in mammalian species, it is becoming apparent that damaged mature neurons retain some capacity for attempted regeneration and that the adult CNS is not entirely inhibitory to axon growth. Our investigations show that there are critical intrinsic features of postinjury axonal regeneration that differ from initial axon development, and that these distinct differences may account for the limited and inappropriate regenerative response that currently characterizes the mature CNS. We compared the neurochemical and dynamic characteristics of developing axons to relatively mature regenerating axons, utilizing an in vitro model of axonal transection to long-term cultured rat cortical neurons. Immunolabelling studies revealed that regenerating and developing axons have a similar localization of cytoskeletal proteins, but the tips of regenerating axons, although morphologically similar, were smaller with reduced fillopodial extension, relative to developmental growth cones. Live imaging demonstrated that regenerating axons exhibited significantly less outgrowth than developmental neurites. Furthermore, growth cones of regenerating axons had a significant reduction in pausing, considered vital for interstitial branching and pathfinding, than did developmental growth cones. In addition, unlike developing axons, the regenerating axons were unresponsive to the growth factors BDNF and GDNF. Thus, although similar in their cytoskeletal composition, the growth cones of regenerative sprouts differed from their developmental counterparts in their size, their dynamic behaviour and their ability to respond to critical growth factors. These intrinsic differences may account for the inability of post-traumatic locally sprouting axons to make accurate pathway decisions and successfully respond to trauma.

Synapse Dysfunction of Layer V Pyramidal Neurons Precedes Neurodegeneration in a Mouse Model of TDP-43 Proteinopathies

Publisher: Oxford University Press (OUP)

Date: 05-08-2016

DOI: 10.1093/CERCOR/BHW185

Abstract: TDP-43 is a major protein component of pathological neuronal inclusions that are present in frontotemporal dementia and amyotrophic lateral sclerosis. We report that TDP-43 plays an important role in dendritic spine formation in the cortex. The density of spines on YFP+ pyramidal neurons in both the motor and somatosensory cortex of Thy1-YFP mice, increased significantly from postnatal day 30 (P30), to peak at P60, before being pruned by P90. By comparison, dendritic spine density was significantly reduced in the motor cortex of Thy1-YFP::TDP-43A315T transgenic mice prior to symptom onset (P60), and in the motor and somatosensory cortex at symptom onset (P90). Morphological spine-type analysis revealed that there was a significant impairment in the development of basal mushroom spines in the motor cortex of Thy1-YFP::TDP-43A315T mice compared to Thy1-YFP control. Furthermore, reductions in spine density corresponded to mislocalisation of TDP-43 immunoreactivity and lowered efficacy of synaptic transmission as determined by electrophysiology at P60. We conclude that mutated TDP-43 has a significant pathological effect at the dendritic spine that is associated with attenuated neural transmission.

Focal demyelination in Alzheimer’s disease and transgenic mouse models

Publisher: Springer Science and Business Media LLC

Date: 03-03-2010

DOI: 10.1007/S00401-010-0657-2

Abstract: We have investigated alterations in myelin associated with Abeta plaques, a major pathological hallmark of Alzheimer's disease (AD), in human tissue and relevant transgenic mice models. Using quantitative morphological techniques, we determined that fibrillar Abeta pathology in the grey matter of the neocortex was associated with focal demyelination in human presenilin-1 familial, sporadic and preclinical AD cases, as well as in two mouse transgenic models of AD, compared with age-matched control tissue. This demyelination was most pronounced at the core of Abeta plaques. Furthermore, we found a focal loss of oligodendrocytes in sporadic and preclinical AD cases associated with Abeta plaque cores. In human and transgenic mice alike, plaque-free neocortical regions showed no significant demyelination or oligodendrocyte loss compared with controls. Dystrophic neurites associated with the plaques were also demyelinated. We suggest that such plaque-associated focal demyelination of the cortical grey matter might impair cortical processing, and may also be associated with aberrant axonal sprouting that underlies dystrophic neurite formation.

Sequence of Cellular Changes Following Localized Axotomy to Cortical Neurons in Glia-Free Culture

Publisher: Mary Ann Liebert Inc

Date: 11-2000

DOI: 10.1089/NEU.2000.17.1095

Abstract: This study utilizes an in vitro model of localized physical injury to axons to examine the specific responses of neocortical neurons to trauma in isolation from glia cell types. The neuronal response to axotomy was closely linked with nerve cell maturity. Cultures grown for 14 days in vitro showed no accumulation of either neurofilaments or, the axonal sprouting marker, GAP43, within injured axons following injury. In older cultures (21 days in vitro), however, temporally distinct axonal changes were evident following transection of axonal bundles. At 12 h postinjury, these included extensive accumulation of neurofilaments into ring-like structures within the cut stumps and an increase in punctate GAP43 labelling throughout the damaged area. At 24 h postinjury, bulb-like accumulations of neurofilaments were also present within the transected axons. Finally at 3 days postinjury, distinct GAP43 and neurofilament immunolabeled axons, and GAP43 immunopositive growth cones, emanated from the cut stump. These results indicate that injured axons of mature neurons undergo a defined series of reactive changes, ultimately culminating in a sprouting response, which occur independently of the presence or effects of glial cell populations.

The cause of neuronal degeneration in Alzheimer's disease

Publisher: Elsevier BV

Date: 02-2000

DOI: 10.1016/S0301-0082(99)00023-4

Abstract: Alzheimer's disease is associated with a specific pattern of pathological changes in the brain that result in neurodegeneration and the progressive development of dementia. Pathological hallmarks common to the disease include beta-amyloid plaques, dystrophic neurites associated with plaques and neurofibrillary tangles within nerve cell bodies. The exact relationship between these pathological features has been elusive, although it is clear that beta-amyloid plaques precede neurofibrillary tangles in neocortical areas. Examination of the brains of in iduals in the preclinical stage of the disease have shown that the earliest form of neuronal pathology associated with beta-amyloid plaques resembles the cellular changes that follow structural injury to axons. Thus, the development of beta-amyloid plaques in the brain may cause physical damage to axons, and the abnormally prolonged stimulation of the neuronal response to this kind of injury ultimately results in the profound cytoskeletal alterations that underlie neurofibrillary pathology and neurodegeneration. Therapeutically, inhibition of the neuronal reaction to physical trauma may be a useful neuroprotective strategy in the earliest stages of Alzheimer's disease.

Chronic Excitotoxin-Induced Axon Degeneration in a Compartmented Neuronal Culture Model

Publisher: SAGE Publications

Date: 2012

DOI: 10.1042/AN20110031

Abstract: Glutamate excitotoxicity is a major pathogenic process implicated in many neurodegenerative conditions, including AD (Alzheimer's disease) and following traumatic brain injury. Occurring predominantly from over-stimulation of ionotropic glutamate receptors located along dendrites, excitotoxic axonal degeneration may also occur in white matter tracts. Recent identification of axonal glutamate receptor subunits within axonal nanocomplexes raises the possibility of direct excitotoxic effects on axons. In idual neuronal responses to excitotoxicity are highly dependent on the complement of glutamate receptors expressed by the cell, and the localization of the functional receptors. To enable isolation of distal axons and targeted excitotoxicity, murine cortical neuron cultures were prepared in compartmented microfluidic devices, such that distal axons were isolated from neuronal cell bodies. Within the compartmented culture system, cortical neurons developed to relative maturity at 11 DIV (days in vitro) as demonstrated by the formation of dendritic spines and clustering of the presynaptic protein synaptophysin. The isolated distal axons retained growth cone structures in the absence of synaptic targets, and expressed glutamate receptor subunits. Glutamate treatment (100 μM) to the cell body chamber resulted in widespread degeneration within this chamber and degeneration of distal axons in the other chamber. Glutamate application to the distal axon chamber triggered a lesser degree of axonal degeneration without degenerative changes in the untreated somal chamber. These data indicate that in addition to current mechanisms of indirect axonal excitotoxicity, the distal axon may be a primary target for excitotoxicity in neurodegenerative conditions.

Direct determination of the proportion of intra- and extra-cellular neocortical neurofibrillary tangles in Alzheimer’s disease

Publisher: Elsevier BV

Date: 05-2003

DOI: 10.1016/S0006-8993(03)02429-6

Abstract: We investigated the cellular localisation of neurofibrillary tangles in Alzheimer's disease. All tau-positive tangles were stained for thioflavine S, while approximately 84% of thioflavine S-stained tangles were tau-immunolabelled. Approximately 58-62% and 73-76% of thioflavine S- and tau-labelled tangles, respectively, were present within cortical neurons labelled for microtubule-associated protein-2. Thus, most neocortical tangles in Alzheimer's disease are intracellular and may not be the principal cause of neocortical cell loss.

ERM proteins regulate growth cone responses to Sema3A

Publisher: Wiley

Date: 23-07-2008

DOI: 10.1002/CNE.21799

Amyotrophic lateral sclerosis mutant TDP-43 may cause synaptic dysfunction through altered dendritic spine function

Publisher: The Company of Biologists

Date: 2019

DOI: 10.1242/DMM.038109

Abstract: Altered cortical excitability and synapse dysfunction are early pathogenic events in amyotrophic lateral sclerosis (ALS) patients and animal models. Recent studies propose an important role for TAR DNA binding protein 43 (TDP-43), a protein whose mislocalization and aggregation are key pathological features of ALS, at the neuronal synapse. However, the relationship between ALS-linked TDP-43 mutations, excitability, and synaptic function is not fully understood. Here, we investigate the role of ALS-linked mutant TDP-43 in synapse formation by examining the morphological, immunocytochemical and excitability profile of transgenic mouse primary cortical pyramidal neurons over-expressing human TDP-43A315T. In TDP-43A315T cortical neurons, dendritic spine density was significantly reduced compared to wild type (WT) controls. TDP-43A315T over-expression increased the total amount of the AMPA glutamate receptor subunit GluR1, yet the localization of GluR1 to the dendritic spine was reduced. These post-synaptic changes were coupled with a decrease in the amount of the pre-synaptic marker synaptophysin colocalized with dendritic spines. Interestingly, action potential generation was reduced in TDP-43A315T pyramidal neurons. This work reveals a crucial effect of the over-expression mutation TDP-43A315T on the formation of synaptic structures and the recruitment of GluR1 to the synaptic membrane. This pathogenic effect may be mediated by cytoplasmic mislocalization of TDP-43A315T. Loss of synaptic GluR1, and reduced excitability within pyramidal neurons, implicates hypoexcitability and attenuated synaptic function in the pathogenic decline of neuronal function in TDP-43-associated ALS. Further studies into the mechanisms underlying AMPA receptor-mediated excitability changes within the ALS cortical circuitry may yield novel therapeutic targets for treatment of this devastating disease.

Neurochemical Diversity of Dystrophic Neurites in the Early and Late Stages of Alzheimer's Disease

Publisher: Elsevier BV

Date: 03-1999

DOI: 10.1006/EXNR.1998.7010

Abstract: We examined the neurochemical and morphological ersity of abnormal neurites associated with beta-amyloid plaque formation in the early and late stages of Alzheimer's disease. Preclinical Alzheimer's disease was characterised by the presence of abnormal neurites containing either neurofilament or chromogranin A immunoreactivity. All clustered dystrophic neurites in these cases were associated with beta-amyloid plaques. Neurofilament immunoreactive dystrophic neurites in preclinical Alzheimer's disease could be further subclassified into bulb- and ring-like structures, and these abnormal neurites contained both phosphorylated and dephosphorylated neurofilament epitopes. Dystrophic neurites in Alzheimer's disease could be sub ided into predominantly neurofilament, tau, or chromogranin A immunolabeled forms. Some neurofilament immunoreactive neurites had a core region labeled for tau. The neurofilaments of the dystrophic neurites in Alzheimer's disease had the same complement of phosphorylation- and dephosphorylation-dependent epitopes as observed in preclinical cases. Therefore, an abnormal accumulation of variably phosphorylated neurofilaments represent the earliest cytoskeletal alteration associated with dystrophic neurite formation. Furthermore, these data indicate that dystrophic neurites may "mature" through neurofilament-abundant forms to the neurites containing the profoundly altered filaments labeled for tau. The precise morphological and neurochemical changes associated with dystrophic neurite formation suggests that beta-amyloid plaques are causing physical damage to surrounding axons. The resultant axonal sprouting and profound cytoskeletal alterations would follow the chronic stimulation of the stereotypical reaction to such physical trauma.

The metabolomics of alpha-synuclein (SNCA) gene deletion and mutation in mouse brain

Publisher: Springer Science and Business Media LLC

Date: 28-06-2013

DOI: 10.1007/S11306-013-0561-6

Neurites containing the neurofilament-triplet proteins are selectively vulnerable to cytoskeletal pathology in Alzheimer’s disease and transgenic mouse models

Publisher: Frontiers Media SA

Date: 2013

DOI: 10.3389/FNANA.2013.00030

Axonal degeneration, distal collateral branching and neuromuscular junction architecture alterations occur prior to symptom onset in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.JCHEMNEU.2016.03.003

Abstract: Degeneration of the distal axon and neuromuscular junction (NMJ) is considered a key and early feature of the pathology that accompanies motor neuron loss in people with amyotrophic lateral sclerosis (ALS). The mutant SOD1(G93A) mouse replicates many features of the disease, however the sequence of events resulting in degeneration of the neuromuscular circuitry remains unknown. Furthermore, despite widespread degenerative neuronal pathology throughout the spinal cord in this model, hindlimb motor function is lost before forelimb function. We investigated axons and NMJs in the hindlimb (gastrocnemius) and forelimb (extensor) muscles in the high copy number mutant SOD1(G93A)xYFP (yellow fluorescent protein) mouse. We found that distal axonal and NMJ alterations were present prior to previously reported functional symptom onset in this strain. Indeed, increased branch complexity as well as colocalisation between pre- and post-synaptic markers indicated widespread early axonal and NMJ alterations in the hindlimb. Immunohistochemical analysis demonstrated that the colocalisation of the scaffolding proteins nestin, LRP-4, dystrophin and rapsyn were diminished before post-synaptic receptors in the gastrocnemius, and the degree of loss differed between proteins. Analysis of the forelimb muscle revealed axonal and NMJ degeneration at a late, post symptomatic stage, as well as novel differences in NMJ morphology, with reduced complexity. Furthermore, post-synaptic scaffolding proteins were preserved in the forelimb compared with the hindlimb. Analysis of protein levels indicated an increase in LRP-4, dystrophin and rapsyn in post symptomatic skeletal muscle that may suggest ongoing attempts at repair. This study indicates that axonal and NMJ degeneration in the SOD1 model of ALS is a complex and evolving sequence of events. We provide evidence that YFP can detect morphological and plastic alterations in the SOD1(G93A) mouse, and that the pre- and post-synaptic integrity of the NMJ plays an important role in the pathogenic mechanisms of ALS.

Dystrophic neurites in TgCRND8 and Tg2576 mice mimic human pathological brain aging

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1016/J.NEUROBIOLAGING.2007.09.003

Abstract: 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.

Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice

Publisher: Wiley

Date: 04-04-2013

DOI: 10.1002/CNE.23261

Abstract: The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age-matched wild-type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF-P, NF-DP), the medium NF subunit (NFM), and the intermediate filament α-internexin (INT) at P4. At 5 months, NF-DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF-P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron-specific β-tubulin and microtubule-associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas β-actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF-DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin-immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs.

Inducing Chronic Excitotoxicity in the Mouse Spinal Cord to Investigate Lower Motor Neuron Degeneration

Publisher: Frontiers Media SA

Date: 02-03-2016

DOI: 10.3389/FNINS.2016.00076

Focal Damage to the Adult Rat Neocortex Induces Wound Healing Accompanied by Axonal Sprouting and Dendritic Structural Plasticity

Publisher: Oxford University Press (OUP)

Date: 28-05-2010

DOI: 10.1093/CERCOR/BHQ091

Abstract: Accumulating evidence indicates that damage to the adult mammalian brain evokes an array of adaptive cellular responses and may retain a capacity for structural plasticity. We have investigated the cellular and architectural alterations following focal experimental brain injury, as well as the specific capacity for structural remodeling of neuronal processes in a subset of cortical interneurons. Focal acute injury was induced by transient insertion of a needle into the neocortex of anesthetized adult male Hooded-Wistar rats and thy1 green fluorescent protein (GFP) mice. Immunohistochemical, electron microscopy, and bromodeoxyuridine cell proliferation studies demonstrated an active and evolving response of the brain to injury, indicating astrocytic but not neuronal proliferation. Immunolabeling for the neuron-specific markers phosphorylated neurofilaments, α-internexin and calretinin at 7 days post injury (DPI) indicated phosphorylated neurofilaments and α-internexin but not calretinin immunopositive axonal sprouts within the injury site. However, quantitative studies indicated a significant realignment of horizontally projecting dendrites of calretinin-labeled interneurons at 14 DPI. This remodeling was specific to calretinin immunopositive interneurons and did not occur in a subpopulation of pyramidal neurons expressing GFP in the injured mouse cortex. These data show that subclasses of cortical interneurons are capable of adaptive structural remodeling.

University Of Tasmania

Location: Australia

View Organisation

Synaptic Alterations In ALS: A Novel Therapeutic Target?

Start Date: 2014

End Date: 2014

Funder: Motor Neurone Disease Research Institute of Australia Inc

Dendritic Spine Alterations In TDP-43 Aggregated Frontotemporal Dementia: A Novel Therapeutic Target?

Start Date: 2014

End Date: 2014

Funder: Brain Foundation

SYNAPTIC DYSFUNCTION: AN EARLY MECHANISM OF TDP-43 PATHOGENESIS IN ALS?

Start Date: 2015

End Date: 2015

Funder: Motor Neurone Disease Research Institute of Australia Inc

Investigating Synaptic Dysfunction In ALS

Start Date: 2016

End Date: 2016

Funder: Motor Neurone Disease Research Institute of Australia Inc

Spectramax M2 Multimode ELISA Plate Reader With Softmax Pro Software

Start Date: 2005

End Date: 2005

Funder: National Health & Medical Research Council

Establishment Of A Confocal/multiphoton Microscope For Imaging Of Living Systems

Start Date: 2008

End Date: 2008

Funder: Australian Research Council

Determining The Mechanism Underlying Post-operative Cognitive Decline

Start Date: 2012

End Date: 2012

Funder: Royal Hobart Hospital Research Foundation

Establishing A State-of-the-art 'Live Cell Recording And Imaging Facility' At Menzies Research Institute Tasmania

Start Date: 2013

End Date: 2013

Funder: Rebecca L Cooper Medical Research Foundation

Axonal Protection In ALS

Start Date: 2013

End Date: 2013

Funder: Motor Neurone Disease Research Institute of Australia Inc

Microfluidic Technology To Help Understand Physical Damage To Brain Cells

Start Date: 2015

End Date: 2017

Funder: Australian Research Council

Preclinical Development Of Neuropeptide Y For The Treatment Of Motor Neuron Disease

Start Date: 2017

End Date: 2020

Funder: FightMND

Neural Regulation Of Skeletal Muscle Insulin Sensitivity

Start Date: 2012

End Date: 2012

Funder: The Select Foundation

Discovery Projects - Grant ID: DP200103193

Start Date: 11-2020

End Date: 12-2024

Amount: $545,563.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150100998

Start Date: 2015

End Date: 12-2020

Amount: $415,500.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882701

Start Date: 2008

End Date: 12-2008

Amount: $300,000.00

Funder: Australian Research Council