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

OBJECTIVE: 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. METHODS: 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 SOD1G93A cortical cultures in vitro. The impact of NPY on ALS disease trajectory was investigated by treating SOD1G93A mice intranasally with NPY and selective NPY receptor agonists and antagonists from pre-symptomatic and symptomatic phases of disease. RESULTS: 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 SOD1G93A mouse. INTERPRETATION: 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.

Estrogen Enhances Dendrite Spine Function and Recovers Deficits in Neuroplasticity in the prpTDP-43A315T Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) attacks the corticomotor system, with motor cortex function affected early in disease. Younger females have a lower relative risk of succumbing to ALS than males and older females, implicating a role for female sex hormones in disease progression. However, the mechanisms driving this dimorphic incidence are still largely unknown. We endeavoured to determine if estrogen mitigates disease progression and pathogenesis, focussing upon the dendritic spine as a site of action. Using two-photon live imaging we identify, in the prpTDP-43A315T mouse model of ALS, that dendritic spines in the male motor cortex have a reduced capacity for remodelling than their wild-type controls. In contrast, females show higher capacity for remodelling, with peak plasticity corresponding to highest estrogen levels during the estrous cycle. Estrogen manipulation through ovariectomies and estrogen replacement with 17ß estradiol in vivo was found to significantly alter spine density and mitigate disease severity. Collectively, these findings reveal that synpatic plasticity is reduced in ALS, which can be amelioriated with estrogen, in conjuction with improved disease outcomes.

Differential NPY-Y1 Receptor Density in the Motor Cortex of ALS Patients and Familial Model of ALS.

Destabilization of faciliatory and inhibitory circuits is an important feature of corticomotor pathology in amyotrophic lateral sclerosis (ALS). While GABAergic inputs to upper motor neurons are reduced in models of the disease, less understood is the involvement of peptidergic inputs to upper motor neurons in ALS. The neuropeptide Y (NPY) system has been shown to confer neuroprotection against numerous pathogenic mechanisms implicated in ALS. However, little is known about how the NPY system functions in the motor system. Herein, we investigate post-synaptic NPY signaling on upper motor neurons in the rodent and human motor cortex, and on cortical neuron populations in vitro. Using immunohistochemistry, we show the increased density of NPY-Y1 receptors on the soma of SMI32-positive upper motor neurons in post-mortem ALS cases and SOD1G93A excitatory cortical neurons in vitro. Analysis of receptor density on Thy1-YFP-H-positive upper motor neurons in wild-type and SOD1G93A mouse tissue revealed that the distribution of NPY-Y1 receptors was changed on the apical processes at early-symptomatic and late-symptomatic disease stages. Together, our data demonstrate the differential density of NPY-Y1 receptors on upper motor neurons in a familial model of ALS and in ALS cases, indicating a novel pathway that may be targeted to modulate upper motor neuron activity.

The pathologic outcomes and efficacy of epothilone treatment following traumatic brain injury is determined by age.

Traumatic brain injury (TBI) can affect individuals at any age, with the potential of causing lasting neurologic consequences. The lack of effective therapeutic solutions and recommendations for patients that acquire a TBI can be attributed, at least in part, to an inability to confidently predict long-term outcomes following TBI, and how the response of the brain differs across the life span. The purpose of this study was to determine how age specifically affects TBI outcomes in a preclinical model. Male Thy1-YFPH mice, that express yellow fluorescent protein in the cytosol of a subset of Layer V pyramidal neurons in the neocortex, were subjected to a lateral fluid percussion injury over the right parietal cortex at distinct time points throughout the life span (1.5, 3, and 12 months of age). We found that the degree of neuronal injury, astrogliosis, and microglial activation differed depending on the age of the animal when the injury occurred. Furthermore, age affected the initial injury response and how it resolved over time. Using the microtubule stabilizing agent Epothilone D, to potentially protect against these pathologic outcomes, we found that the neuronal response was different depending on age. This study clearly shows that age must be taken into account in neurologic studies and preclinical trials involving TBI, and that future therapeutic interventions must be tailored to age.

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.

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

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+ interneurons underwent significant neurite remodeling around the injury site. By examining the response of pyramidal neurons, GAD67-GFP+ interneurons, and calretinin+ interneurons to the injury, we found that this response was specific to the calretinin+ cells. To determine whether calretinin+ interneurons respond in this way to a clinically relevant in vivo model of mild diffuse and focal injury, we subjected mice to the lateral fluid percussion injury model. We found that calretinin+ interneuron density was unaltered by this mild injury, but consistent with our in vitro data, these neurons underwent morphological alterations in their dendrites. These alterations evolved over a 28-day period, and calretinin+ interneurons in the injured mice had a reduction in mean dendrite length and reduced number of secondary dendrites than those in the sham-injured controls by 7 days post-injury. Further, these structural alterations were accompanied by a reduction in the frequency of miniature inhibitory post-synaptic currents in layer V pyramidal neurons. These data suggest that even a mild TBI can lead to an overall change in the excitatory/inhibitory balance of the cortex that may play an important role in the longer-term behavioral pathology associated with mild TBI.

A Case for Microtubule Vulnerability in Amyotrophic Lateral Sclerosis: Altered Dynamics During Disease.

Amyotrophic lateral sclerosis (ALS) is an aggressive multifactorial disease converging on a common pathology: the degeneration of motor neurons (MNs), their axons and neuromuscular synapses. This vulnerability and dysfunction of MNs highlights the dependency of these large cells on their intracellular machinery. Neuronal microtubules (MTs) are intracellular structures that facilitate a myriad of vital neuronal functions, including activity dependent axonal transport. In ALS, it is becoming increasingly apparent that MTs are likely to be a critical component of this disease. Not only are disruptions in this intracellular machinery present in the vast majority of seemingly sporadic cases, recent research has revealed that mutation to a microtubule protein, the tubulin isoform TUBA4A, is sufficient to cause a familial, albeit rare, form of disease. In both sporadic and familial disease, studies have provided evidence that microtubule mediated deficits in axonal transport are the tipping point for MN survivability. Axonal transport deficits would lead to abnormal mitochondrial recycling, decreased vesicle and mRNA transport and limited signaling of key survival factors from the neurons peripheral synapses, causing the characteristic peripheral "die back". This disruption to microtubule dependant transport in ALS has been shown to result from alterations in the phenomenon of microtubule dynamic instability: the rapid growth and shrinkage of microtubule polymers. This is accomplished primarily due to aberrant alterations to microtubule associated proteins (MAPs) that regulate microtubule stability. Indeed, the current literature would argue that microtubule stability, particularly alterations in their dynamics, may be the initial driving force behind many familial and sporadic insults in ALS. Pharmacological stabilization of the microtubule network offers an attractive therapeutic strategy in ALS; indeed it has shown promise in many neurological disorders, ALS included. However, the pathophysiological involvement of MTs and their functions is still poorly understood in ALS. Future investigations will hopefully uncover further therapeutic targets that may aid in combating this awful disease.

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

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'.

Focal damage to the adult rat neocortex induces wound healing accompanied by axonal sprouting and dendritic structural plasticity.

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.

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 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.

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

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.

Mild axonal stretch injury in vitro induces a progressive series of neurofilament alterations ultimately leading to delayed axotomy.

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 individual 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.

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

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.

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

The precise role of beta-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 beta-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 and in 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, beta-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.

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.

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 microg/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 microg/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.