Anna E. King

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

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

Chronic oxidative stress has been linked to the neurodegenerative changes characteristic of Parkinsons 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.

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

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 α‐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. J. Comp. Neurol. 498:277–294, 2006.

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

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 Alzheimers 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.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo

PURPOSE Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Glutamate induces rapid loss of axonal neurofilament proteins from cortical neurons in vitro

One of the primary hallmarks of glutamate excitotoxicity is degradation of the neuronal cytoskeleton. Using a tissue culture approach, we have investigated the relationship between excitotoxicity and cytoskeletal degradation within axons, with particular reference to the axon specific neurofilament proteins. Neurofilaments were rapidly lost from axons over a 24-h period in response to excitotoxic insult (as observed by immunocytochemistry and western blotting), while other axonal cytoskeletal markers (such as betaIII-tubulin) remained intact. Treatment with kainic acid and NMDA, or complementary experiments using the pharmacological glutamate receptors blockers CNQX (kainate/AMPA receptor antagonist) and MK-801 (NMDA receptor antagonist), demonstrated that neurofilament degeneration was mediated primarily by NMDA receptor activity. This work suggests that excitotoxicity triggers a progressive pathway of cytoskeletal degeneration within axons, initially characterised by the loss of neurofilament proteins.

Excitotoxicity in ALS: Overstimulation, or overreaction?

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease that results in motor dysfunction and death, generally from respiratory failure. 90% of ALS cases are sporadic with no known cause. Familial cases have been linked with mutations in several disparate classes of genes, including those involved in DNA/RNA metabolism, protein misfolding, oxidative stress and the cytoskeleton, leading to the proposition that ALS could be a multi-factorial disease. However, alterations in excitability have been reported in all types of ALS cases, and may be a common disease mechanism predisposing neurons to degeneration. Excitotoxicity has long been suspected as a mediator in the disease process, and may arise from changes in synaptic inputs, or alterations in the excitability of the neurons being stimulated. Although the glutamatergic system is widely recognised as a therapeutic avenue with the potential to extend lifespan and delay disease onset, the causes of altered excitability in ALS are currently unclear and warrant further investigation. This article reviews current evidence of alterations to excitatory and inhibitory signalling in the cortex and spinal cord, and in the intrinsic excitability of motor neurons, in ALS.

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

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.

Diffuse axonal injury in brain trauma: insights from alterations in neurofilaments

Traumatic brain injury (TBI) from penetrating or closed forces to the cranium can result in a range of forms of neural damage, which culminate in mortality or impart mild to significant neurological disability. In this regard, diffuse axonal injury (DAI) is a major neuronal pathophenotype of TBI and is associated with a complex set of cytoskeletal changes. The neurofilament triplet proteins are key structural cytoskeletal elements, which may also be important contributors to the tensile strength of axons. This has significant implications with respect to how axons may respond to TBI. It is not known, however, whether neurofilament compaction and the cytoskeletal changes that evolve following axonal injury represent a component of a protective mechanism following damage, or whether they serve to augment degeneration and progression to secondary axotomy. Here we review the structure and role of neurofilament proteins in normal neuronal function. We also discuss the processes that characterize DAI and the resultant alterations in neurofilaments, highlighting potential clues to a possible protective or degenerative influence of specific neurofilament alterations within injured neurons. The potential utility of neurofilament assays as biomarkers for axonal injury is also discussed. Insights into the complex alterations in neurofilaments will contribute to future efforts in developing therapeutic strategies to prevent, ameliorate or reverse neuronal degeneration in the central nervous system (CNS) following traumatic injury.

Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology

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.