Amyotrophic lateral sclerosis is a devastating neurodegenerative disease that, at present, has no effective cure. Evidence of increased circulating glutamate and hyperexcitability of the motor cortex in patients with amyotrophic lateral sclerosis have provided an empirical support base for the 'dying forward' excitotoxicity hypothesis. The hypothesis postulates that increased activation of upper motor neurons spreads pathology to lower motor neurons in the spinal cord in the form of excessive glutamate release, which triggers excitotoxic processes. Many clinical trials have focused on therapies that target excitotoxicity via dampening neuronal activation, but not all are effective. As such, there is a growing tension between the rising tide of evidence for the 'dying forward' excitotoxicity hypothesis and the failure of therapies that target neuronal activation. One possible solution to these contradictory outcomes is that our interpretation of the current evidence requires revision in the context of appreciating the complexity of the nervous system and the limitations of the neurobiological assays we use to study it. In this review we provide an evaluation of evidence relevant to the 'dying forward' excitotoxicity hypothesis and by doing so, identify key gaps in our knowledge that need to be addressed. We hope to provide a road map from hyperexcitability to excitotoxicity so that we can better develop therapies for patients suffering from amyotrophic lateral sclerosis. We conclude that studies of upper motor neuron activity and their synaptic output will play a decisive role in the future of amyotrophic lateral sclerosis therapy.

Alterations in upper motor neuron excitability are one of the earliest phenomena clinically detected in ALS, and in 97 % of cases, the RNA/DNA binding protein, TDP-43, is mislocalised in upper and lower motor neurons. While these are two major pathological hallmarks in disease, our understanding of where disease pathology begins, and how it spreads through the corticomotor system, is incomplete. This project used a model where mislocalised TDP-43 was expressed in the motor cortex, to determine if localised cortical pathology could result in widespread corticomotor system degeneration. Mislocalised TDP-43 caused layer V excitatory neurons in the motor cortex to become hyperexcitable after 20 days of expression. Following cortical hyperexcitability, a spread of pathogenic changes through the corticomotor system was observed. By 30 days expression, there was a significant decrease in lower motor neuron number in the lumbar spinal cord. However, cell loss occurred selectively, with a significant loss in lumbar regions 1-3, and not lumbar regions 4-6. This regional vulnerability was associated with alterations in pre-synaptic excitatory and inhibitory proteins. Excitatory inputs (VGluT2) were increased in all lumbar regions, while inhibitory inputs (GAD65/67) were increased in lumbar regions 4-6 only. This data indicates that mislocalised TDP-43 in upper motor neurons can cause lower motor neuron degeneration. Furthermore, cortical pathology increased excitatory inputs to the spinal cord, to which local circuitry compensated with an upregulation of inhibition. These findings reveal how TDP-43 mediated pathology may spread through corticofugal tracts in ALS and identify a potential pathway for therapeutic intervention.

Amyotrophic Lateral Sclerosis (ALS) is an incurable disease characterized by relentlessly progressive degeneration of the corticomotor system. Cortical hyperexcitability has been identified as an early pre-symptomatic biomarker of ALS. This suggests that hyperexcitability occurs upstream in the ALS pathological cascade and may even be part of the mechanism that drives development of symptoms or loss of motor neurons in the spinal cord. However, many studies also indicate a loss to the synaptic machinery that mediates synaptic input which raises the question of which is the driver of disease, and which is a homeostatic response. Herein, we used an inducible mouse model of TDP-43 mediated ALS that permits for the construction of detailed phenotypic timelines. Our work comprehensively describes the relationship between intrinsic hyperexcitability and altered synaptic input onto motor cortical layer 5 pyramidal neurons over time. As a result, we have constructed the most complete timeline of electrophysiological changes following induction of TDP-43 dysfunction in the motor cortex. We report that intrinsic hyperexcitability of layer 5 pyramidal neurons precedes changes to excitatory synaptic connections, which manifest as an overall loss of inputs onto layer 5 pyramidal neurons. This finding highlights the importance of hyperexcitability as a primary mechanism of ALS and re-contextualizes synaptic changes as possibly representing secondary adaptive responses. Recognition of the relationship between intrinsic hyperexcitability and reduced excitatory synaptic input has important implications for the development of useful therapies against ALS. Novel strategies will need to be developed that target neuronal output by managing excitability against synapses separately.

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.

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 17β 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.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease pathologically characterised by mislocalisation of the RNA binding protein TAR-DNA binding protein 43 (TDP-43) from the nucleus to the cytoplasm. Changes to neuronal excitability and synapse dysfunction in the motor cortex are early pathological changes occurring in people with ALS and mouse models of disease. To investigate the effect of mislocalized TDP-43 on the function of motor cortex neurons we utilised mouse models that express either human wild-type (TDP-43WT) or nuclear localization sequence-deficient TDP-43 (TDP-43ΔNLS) on an inducible promoter that is restricted to the forebrain. Pathophysiology was investigated through immunohistochemistry and whole-cell patch-clamp electrophysiology. Thirty days expression TDP-43ΔNLS in adult mice (60 days of age) does not cause any changes in the number of NeuN positive nor CTIP2 positive neurons in the motor cortex. However at this time-point the expression of TDP-43ΔNLS drives intrinsic hyperexcitability in layer V excitatory neurons of the motor cortex. This hyperexcitability occurs concomitantly with a decrease in excitatory synaptic input to these cells. This pathophysiology is not present when TDP-43WT expression is driven, demonstrating that the localisation of TDP-43 to the cytoplasm is crucial for the altered excitability phenotype. This study has important implications for the mechanisms of toxicity of one of the most notorious proteins linked to ALS, TDP-43. We provide the first evidence that TDP-43 mislocalization causes aberrant synaptic function and a hyperexcitability phenotype in the motor cortex, linking some of the earliest dysfunctions to arise in people with ALS to mislocalisation of TDP-43.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease pathologically characterised by mislocalisation of the RNA-binding protein TAR-DNA-binding protein 43 (TDP-43) from the nucleus to the cytoplasm. Changes to neuronal excitability and synapse dysfunction in the motor cortex are early pathological changes occurring in people with ALS and mouse models of disease. To investigate the effect of mislocalised TDP-43 on the function of motor cortex neurons we utilised mouse models that express either human wild-type (TDP-43(WT)) or nuclear localisation sequence-deficient TDP-43 (TDP-43(Delta NLS)) on an inducible promoter that enriches expression to forebrain neurons. Pathophysiology was investigated through immunohistochemistry and whole-cell patch-clamp electrophysiology. Thirty days expression of TDP-43(Delta NLS) in adult mice did not cause any changes in the number of CTIP2-positive neurons in the motor cortex. However, at this time-point, the expression of TDP-43(Delta NLS) drives intrinsic hyperexcitability in layer V excitatory neurons of the motor cortex. This hyperexcitability occurs concomitantly with a decrease in excitatory synaptic input to these cells and fluctuations in both directions of ionotropic glutamate receptors. This pathophysiology is not present with TDP-43(WT) expression, demonstrating that the localisation of TDP-43 to the cytoplasm is crucial for the altered excitability phenotype. This study has important implications for the mechanisms of toxicity of one of the most notorious proteins linked to ALS, TDP-43. We provide the first evidence that TDP-43 mislocalisation causes aberrant synaptic function and a hyperexcitability phenotype in the motor cortex, linking some of the earliest dysfunctions to arise in people with ALS to mislocalisation of TDP-43.

Amyotrophic lateral sclerosis (ALS) is defined by the destruction of upper- and lower motor neurons. Post-mortem, nearly all ALS cases are positive for cytoplasmic aggregates containing the DNA/RNA binding protein TDP-43. Recent studies indicate that this pathogenic mislocalization of TDP-43 may participate in generating hyperexcitability of the upper motor neurons, the earliest detectable change in ALS patients, yet the mechanisms driving this remain unclear. We investigated how mislocalisation of TDP-43 could initiate network dysfunction in ALS. We employed a tetracycline inducible system to express either human wildtype TDP-43 (TDP-43WT) or human TDP-43 that cannot enter the nucleus (TDP-43ΔNLS) in excitatory neurons (Camk2α promoter), crossed Thy1-YFPH mice to visualize dendritic spines, the major site of excitatory synapses. In comparison to both TDP-43WT and controls, TDP-43ΔNLS drove a robust loss in spine density in all the dendrite regions of the upper motor neurons, most affecting thin spines. This indicates that TDP-43 is involved in the generation of network dysfunction in ALS likely through impacting the formation or durability of excitatory synapses. These findings are relevant to the vast majority of ALS cases, and provides further evidence that upper motor neurons may need to be protected from TDP-43 mediated synaptic excitatory changes early in disease.

There is increasing interest in the role of epigenetic alterations in Alzheimer's disease (AD). The epigenome of every cell type is distinct, yet data regarding epigenetic change in specific cell types in aging and AD is limited. We investigated histone tail modifications in neuronal subtypes in wild-type and APP/PS1 mice at 3 (pre-pathology), 6 (pathology-onset) and 12 (pathology-rich) months of age. In neurofilament (NF)-positive pyramidal neurons (vulnerable to AD pathology), and in calretinin-labeled interneurons (resistant to AD pathology) there were no global alterations in histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 27 acetylation (H3K27ac) or histone 3 lysine 27 trimethylation (H3K27me3) in APP/PS1 compared to wild-type mice at any age. Interestingly, age-related changes in the presence of H3K27ac and H3K27me3 were detected in NF-labeled pyramidal neurons and calretinin-positive interneurons, respectively. These data suggest that the global levels of histone modifications change with age, whilst amyloid plaque deposition and its sequelae do not result in global alterations of H3K4me3, H3K27ac and H3K27me3 in NF-positive pyramidal neurons or calretinin-labeled interneurons.