Ja Chuckowree

Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (∼0.8–12 mm2). The surgical procedure can be completed within ∼1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed.

Cytoskeletal and morphological alterations underlying axonal sprouting after localized transection of cortical neuron axons in vitro

We examined the cytoskeletal dynamics that characterize neurite sprouting after axonal injury to cortical neurons maintained in culture for several weeks and compared these with initial neurite development. Cultured neocortical neurons, derived from embryonic day 18 rats, were examined at 3 d in vitro (DIV) and at various time points after axotomy at 21 DIV. The postinjury neuritic response was highly dynamic, progressing through an initial phase of retraction, followed by substantial axonal sprouting within 4–6 hr. Postinjury sprouts were motile and slender with expanded growth cone-like end structures. Microtubule markers were localized to sprout shafts and the proximal regions of putative growth cones and filamentous actin was distributed throughout growth cones, whereas neurofilament proteins were restricted to sprout shafts. A similar distribution of cytoskeletal proteins was present in developing neurites at 3 DIV. Exposure of developing and mature, injured cultures to the microtubule stabilizing agent taxol (10 μg/ml) caused growth inhibition, process distension, the transformation of growth cones into bulbous structures, and abnormal neurite directionality. Microtubule and neurofilament segregation occurred after taxol exposure in developing neurites and postinjury sprouts. Exposure to the microtubule destabilizing agent nocodazole (100 μg/ml) resulted in substantial morphological alteration of developing neurons and inhibited neurite growth and postinjury axonal sprouting. Our results indicate that the axons of cortical neurons have an intrinsic ability to sprout after transection, and similar cytoskeletal dynamics underlie neurite development and postinjury axonal sprouting.

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.

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

This study investigated the role of α-internexin in the neuronal alterations associated with β-amyloid plaque formation in Alzheimers disease (AD). Cortical neurons could be defined by their variable content of neurofilament (NF) triplet and α-internexin proteins, with a distinct population of supragranular pyramidal cells containing α-internexin alone. Both NF triplet and α-internexin were localized to reactive axonal structures in physically damaged neurons in experimental trauma models. Similarly, NF triplet and α-internexin immunoreactive neurites were localized to plaques densely packed with β-amyloid fibrils in preclinical AD cases, indicating that certain plaques may cause structural injury or impediment of local axonal transport. However, α-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 α-internexin. These results implicate the expression of specific intermediate filament proteins in a distinct hierarchy of differential neuronal vulnerability to AD.

Intrinsic Regenerative Ability of Mature CNS Neurons

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.

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

The precise role of β-amyloid plaque formation in the cascade of brain cell changes that lead to neurodegeneration and dementia in Alzheimer’s disease has been unclear. Studies have indicated that neuronal processes surrounding and within plaques undergo a series of biochemical and morphological alterations. Morphological alterations include reactive, degenerative and sprouting-related ’dystrophic’ neuritic structures, derived principally from axons, which involve specific changes in cytoskeletal proteins such as tau and NF triplet proteins. More compact and fibrous plaques are associated with more extensive neuritic pathology than non-fibrillar, diffuse β-amyloid deposits. Cortical apical dendritic processes are either ‘clipped’ by plaque formation or are bent around more compact plaques. Examination of cases of ‘pathological’ brain ageing, which may represent a preclinical form of Alzheimer’s disease, demonstrated that the earliest neuritic pathology associated with plaques was similar to the reactive changes that follow structural injury to axons.In vivo andin vitro experimental models of structural injury to axons produce identical reactive changes that subsequently lead to an attempt at regenerative sprouting by damaged axons. Thus, β-amyloid plaque formation may cause structural injury to axons that is subsequently followed by an aberrant sprouting response that presages neurodegeneration and dementia. Identification of the key neuronal alterations underlying the pathology of Alzheimer’s disease may provide new avenues for therapeutic intervention.

Regulation of the ITGA2 gene by epigenetic mechanisms in prostate cancer

Integrin alpha2 beta1 (α2β1) plays an integral role in tumour cell invasion, metastasis and angiogenesis, and altered expression of the receptor has been linked to tumour prognosis in several solid tumours. However, the relationship is complex, with both increased and decreased expression associated with different stages of tumour metastases in several tumour types. The ITGA2 gene, which codes for the α2 subunit, was examined to investigate whether a large CpG island associated with its promoter region is involved in the differential expression of ITGA2 observed in prostate cancer.

Epothilone D accelerates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Degeneration of the distal neuromuscular circuitry is a hallmark pathology of Amyotrophic Lateral Sclerosis (ALS). The potential for microtubule dysfunction to be a critical pathophysiological mechanism in the destruction of this circuitry is increasingly being appreciated. Stabilization of microtubules to improve neuronal integrity and pathology has been shown to be a particularly favourable approach in other neurodegenerative diseases. We present evidence here that treatment with the microtubule‐targeting compound Epothilone D (EpoD) both positively and negatively affects the spinal neuromuscular circuitry in the SOD1G93A mouse model of ALS.

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

Microtubule dynamics underpin a plethora of roles involved in the intricate development, structure, function, and maintenance of the central nervous system. Within the injured brain, microtubules are vulnerable to misalignment and dissolution in neurons and have been implicated in injury-induced glial responses and adaptive neuroplasticity in the aftermath of injury. Unfortunately, there is a current lack of therapeutic options for treating traumatic brain injury (TBI). Thus, using a clinically relevant model of mild TBI, lateral fluid percussion injury (FPI) in adult male Thy1-YFPH mice, we investigated the potential therapeutic effects of the brain-penetrant microtubule-stabilizing agent, epothilone D. At 7 days following a single mild lateral FPI the ipsilateral hemisphere was characterized by mild astroglial activation and a stereotypical and widespread pattern of axonal damage in the internal and external capsule white matter tracts. These alterations occurred in the absence of other overt signs of trauma: there were no alterations in cortical thickness or in the number of cortical projection neurons, axons or dendrites expressing YFP. Interestingly, a single low dose of epothilone D administered immediately following FPI (and sham-operation) caused significant alterations in the dendritic spines of layer 5 cortical projection neurons, while the astroglial response and axonal pathology were unaffected. Specifically, spine length was significantly decreased, whereas the density of mushroom spines was significantly increased following epothilone D treatment. Together, these findings have implications for the use of microtubule stabilizing agents in manipulating injury-induced synaptic plasticity and indicate that further study into the viability of microtubule stabilization as a therapeutic strategy in combating TBI is warranted.