Integrated analysis of transcriptomic and proteomic alterations in mouse models of ALS/FTD identify early metabolic adaptions with similarities to mitochondrial dysfunction disorders.

OBJECTIVE: Sporadic and familial amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease that results in loss of motor neurons and, in some patients, associates with frontotemporal dementia (FTD). Apart from the accumulation of proteinaceous deposits, emerging literature indicates that aberrant mitochondrial bioenergetics may contribute to the onset and progression of ALS/FTD. Here we sought to investigate the pathophysiological signatures of mitochondrial dysfunction associated with ALS/FTD. METHODS: By means of label-free mass spectrometry (MS) and mRNA sequencing (mRNA-seq), we report pre-symptomatic changes in the cortices of TDP-43 and FUS mutant mouse models. Using tissues from transgenic mouse models of mitochondrial diseases as a reference, we performed comparative analyses and extracted unique and common mitochondrial signatures that revealed neuroprotective compensatory mechanisms in response to early damage. RESULTS: In this regard, upregulation of both Acyl-CoA Synthetase Long-Chain Family Member 3 (ACSL3) and mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) were the most representative change in pre-symptomatic ALS/FTD tissues, suggesting that fatty acid beta-oxidation and mitochondrial protein translation are mechanisms of adaptation in response to ALS/FTD pathology. CONCLUSIONS: Together, our unbiased integrative analyses unveil novel molecular components that may influence mitochondrial homeostasis in the earliest phase of ALS.

5'ValCAC tRNA fragment generated as part of a protective angiogenin response provides prognostic value in amyotrophic lateral sclerosis.

Loss-of-function mutations in the ribonuclease angiogenin are associated with amyotrophic lateral sclerosis. Angiogenin has been shown to cleave transfer RNAs during stress to produce 'transfer-derived stress-induced RNAs'. Stress-induced tRNA cleavage is preserved from single-celled organisms to humans indicating it represents part of a highly conserved stress response. However, to date, the role of tRNA cleavage in amyotrophic lateral sclerosis remains to be fully elucidated. To this end, we performed small RNA sequencing on a human astrocytoma cell line to identify the complete repertoire of tRNA fragments generated by angiogenin. We found that only a specific subset of tRNAs is cleaved by angiogenin and identified 5'ValCAC transfer-derived stress-induced RNA to be secreted from neural cells. 5'ValCAC was quantified in spinal cord and serum from SOD1G93A amyotrophic lateral sclerosis mouse models where we found it to be significantly elevated at symptom onset correlating with increased angiogenin expression, imbalanced protein translation initiation factors and slower disease progression. In amyotrophic lateral sclerosis patient serum samples, we found 5'ValCAC to be significantly higher in patients with slow disease progression, and interestingly, we find 5'ValCAC to hold prognostic value for amyotrophic lateral sclerosis patients. Here, we report that angiogenin cleaves a specific subset of tRNAs and provide evidence for 5'ValCAC as a prognostic biomarker in amyotrophic lateral sclerosis. We propose that increased serum 5'ValCAC levels indicate an enhanced angiogenin-mediated stress response within motor neurons that correlates with increased survival. These data suggest that the previously reported beneficial effects of angiogenin in SOD1G93A mice may result from elevated levels of 5'ValCAC transfer RNA fragment.

Vascular regression precedes motor neuron loss in the FUS (1-359) ALS mouse model.

Amyotrophic lateral sclerosis (ALS) presents a poorly understood pathogenesis. Evidence from patients and mutant SOD1 mouse models suggests vascular damage may precede or aggravate motor dysfunction in ALS. We have previously shown angiogenin (ANG) treatment enhances motor neuron survival, delays motor dysfunction and prevents vascular regression in the SOD1G93A ALS model. However, the existence of vascular defects at different stages of disease progression remains to be established in other ALS models. Here, we assessed vascular integrity in vivo throughout different disease stages, and investigated whether ANG treatment reverses vascular regression and prolongs motor neuron survival in the FUS (1-359) mouse model of ALS. Lumbar spinal cord tissue was collected from FUS (1-359) and non-transgenic control mice at postnatal day (P)50, P90 and P120. We found a significant decrease in vascular network density in lumbar spinal cords from FUS (1-359) mice by day 90, at which point motor neuron numbers were unaffected. ANG treatment did not affect survival or counter vascular regression. Endogenous Ang1 and Vegf expression were unchanged at P50 and P90; however, we found a significant decrease in miRNA 126 at P50, indicating vascular integrity in FUS mice may be compromised via an alternative pathway. Our study demonstrates that vascular regression occurs before motor neuron degeneration in FUS (1-359) mice, and highlights that heterogeneity in responses to novel ALS therapeutics can already be detected in preclinical mouse models of ALS.This article has an associated First Person interview with the joint first authors of the paper.

Pleiotropic activity of systemically delivered angiogenin in the SOD1G93A mouse model.

Loss-of-function mutations in the angiogenin (ANG) gene have been identified in familial and sporadic ALS patients. Previous work from our group identified human ANG (huANG) to protect motoneurons in vitro, and provided proof-of-concept that daily intraperitoneal (i.p.) huANG injections post-symptom onset increased lifespan and delayed disease progression in SOD1G93A mice. huANG's mechanism of action remains less well understood. Here, we implemented a preclinical in vivo design to validate our previous results, provide pharmacokinetic and protein distribution data after systemic administration, and explore potential pleiotropic activities of huANG in vivo. SOD1G93A mice (n = 45) and non-transgenic controls (n = 31) were sex- age- and litter-matched according to the 2010 European ALS/MND group guidelines, and treated with huANG (1 µg, i.p., 3 times/week) or vehicle from 90 days on. huANG treatment increased survival and delayed motor dysfunction as assessed by rotarod in SOD1G93A mice. Increased huANG serum levels were detectable 2 and 24 h after i.p. injection equally in transgenic and non-transgenic mice. Exogenous huANG localized to spinal cord astrocytes, supporting a glia-mediated, paracrine mechanism of action; uptake into endothelial cells was also observed. 1 µg huANG or vehicle were administered from 90 to 115 days of age for histological analysis. Vehicle-treated SOD1G93A mice showed decreased motoneuron numbers and vascular length per ventral horn area, while huANG treatment resulted in improved vascular network maintenance and motoneuron survival. Our data suggest huANG represents a new class of pleiotropic ALS therapeutic that acts on the spinal cord vasculature and glia to delay motoneuron degeneration and disease progression.

Riluzole does not improve lifespan or motor function in three ALS mouse models.

BACKGROUND: Riluzole is the most widespread therapeutic for treatment of the progressive degenerative disease amyotrophic lateral sclerosis (ALS). Riluzole gained FDA approval in 1995 before the development of ALS mouse models. We assessed riluzole in three transgenic ALS mouse models: the SOD1G93A model, the TDP-43A315T model, and the recently developed FUS (1-359) model. METHODS: Age, sex and litter-matched mice were treated with riluzole (22 mg/kg) in drinking water or vehicle (DMSO) from symptom onset. Lifespan was assessed and motor function tests were carried out twice weekly to determine whether riluzole slowed disease progression. RESULTS: Riluzole treatment had no significant benefit on lifespan in any of the ALS mouse models tested. Riluzole had no significant impact on decline in motor performance in the FUS (1-359) and SOD1G93A transgenic mice as assessed by Rotarod and stride length analysis. CONCLUSIONS: Riluzole is widely prescribed for ALS patients despite questions surrounding its efficacy. Our data suggest that if riluzole was identified as a therapeutic candidate today it would not progress past pre-clinical assessment. This raises questions about the standards used in pre-clinical assessment of therapeutic candidates for the treatment of ALS.

A high-fat jelly diet restores bioenergetic balance and extends lifespan in the presence of motor dysfunction and lumbar spinal cord motor neuron loss in TDP-43A315T mutant C57BL6/J mice.

Transgenic transactivation response DNA-binding protein 43 (TDP-43) mice expressing the A315T mutation under control of the murine prion promoter progressively develop motor function deficits and are considered a new model for the study of amyotrophic lateral sclerosis (ALS); however, premature sudden death resulting from intestinal obstruction halts disease phenotype progression in 100% of C57BL6/J congenic TDP-43(A315T) mice. Similar to our recent results in SOD1(G93A) mice, TDP-43(A315T) mice fed a standard pellet diet showed increased 5' adenosine monophosphate-activated protein kinase (AMPK) activation at postnatal day (P)80, indicating elevated energetic stress during disease progression. We therefore investigated the effects of a high-fat jelly diet on bioenergetic status and lifespan in TDP-43(A315T) mice. In contrast to standard pellet-fed mice, mice fed high-fat jelly showed no difference in AMPK activation up to P120 and decreased phosphorylation of acetly-CoA carboxylase (ACC) at early-stage time points. Exposure to a high-fat jelly diet prevented sudden death and extended survival, allowing development of a motor neuron disease phenotype with significantly decreased body weight from P80 onward that was characterised by deficits in Rotarod abilities and stride length measurements. Development of this phenotype was associated with a significant motor neuron loss as assessed by Nissl staining in the lumbar spinal cord. Our work suggests that a high-fat jelly diet improves the pre-clinical utility of the TDP-43(A315T) model by extending lifespan and allowing the motor neuron disease phenotype to progress, and indicates the potential benefit of this diet in TDP-43-associated ALS.

Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

Bcl-2 homology domain 3-only proteins Puma and Bim mediate the vulnerability of CA1 hippocampal neurons to proteasome inhibition in vivo.

Bcl-2 homology domain 3 (BH3)-only proteins are pro-apoptotic Bcl-2 family members that play important roles in upstream cell death signalling during apoptosis. Proteasomal stress has been shown to contribute to the pathology of cerebral ischaemia and many neurodegenerative disorders. Here we explored the contribution of BH3-only proteins in mediating proteasome-inhibition-induced apoptosis in the murine brain in vivo. Stereotactic intrahippocampal microinjection of the selective proteasome inhibitor epoxomicin (2.5 nmol) induced a delayed apoptosis within only the CA1 hippocampal neurons and not neurons within the CA3 or dentate gyrus regions, a selective vulnerability similar to that seen during ischaemia. This injury developed over a time-course of 3 days and was characterized by positive terminal deoxynucleotidyl transferase dUTP nick end labelling staining and nuclear condensation. Previous work from our laboratory has identified the BH3-only protein p53-upregulated mediator of apoptosis (Puma) as mediating proteasome-inhibition-induced apoptosis in cultured neural cells. Genetic deletion of puma reduced the number of terminal deoxynucleotidyl transferase dUTP nick end labelling-positive cells within the CA1 following epoxomicin microinjection but it did not provide a complete protection. Subsequent studies identified the BH3-only protein Bim as also being upregulated during proteasome inhibition in organotypic hippocampal slice cultures and after epoxomicin treatment in vivo. Interestingly, the genetic deletion of bim also afforded significant neuroprotection, although this protection was less pronounced. In summary, we demonstrate that the BH3-only proteins Puma and Bim mediate the delayed apoptosis of CA1 hippocampal neurons induced by proteasome inhibition in vivo, and that either BH3-only protein can only partly compensate for the deficiency of the other.

Characterization of Puma-dependent and Puma-independent neuronal cell death pathways following prolonged proteasomal inhibition.

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model.

Differential expression patterns of Puma and Hsp70 following proteasomal stress in the hippocampus are key determinants of neuronal vulnerability.

Proteasomal stress is believed to contribute to the pathology of ischemic brain injury and several neurodegenerative disorders, but can activate both cytoprotective and cell death-inducing pathways. Here we have utilized the complex environment of organotypic hippocampal slice cultures (OHSCs) to investigate the stress responses activated in different neuronal populations following proteasome inhibition. Incubation of OHSCs with the specific proteasome inhibitors, epoxomicin or bortezomib led to a selective injury of the CA1 pyramidal neurons although similarly increased levels of poly-ubiquitinylated proteins were detected throughout all regions of the hippocampus. Micro-dissection, quantitative PCR and immunohistochemical analyses of epoxomicin-treated OHSCs identified a selective activation of cytoprotective genes in non-vulnerable regions, and a selective activation of p53 target genes within the CA1. Genetic deletion of the pro-apoptotic p53 target gene, p53-upregulated modulator of apoptosis (puma), significantly reduced injury within the CA1 following proteasomal inhibition. Activation of cytoprotective genes by treatment with inducers of heat shock protein 70 inhibited the selective activation of p53 signaling within the CA1 and protected CA1 neurons from epoxomicin-induced cell death. In summary, we demonstrate that the reciprocal activation of p53/p53-upregulated modulator of apoptosis and heat shock protein 70 signalling determines the selective vulnerability of neurons to proteasome inhibition.

Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.

The functional significance of neuronal death for pathogenesis of epilepsy and the underlying molecular mechanisms thereof remain incompletely understood. The p53 transcription factor has been implicated in seizure damage, but its target genes and the influence of cell death under its control on epilepsy development are unknown. In the present study, we report that status epilepticus (SE) triggered by intra-amygdala kainic acid in mice causes rapid p53 accumulation and subsequent hippocampal damage. Expression of p53-up-regulated mediator of apoptosis (Puma), a proapoptotic Bcl-2 homology domain 3-only protein under p53 control, was increased within a few hours of SE. Induction of Puma was blocked by pharmacologic inhibition of p53, and hippocampal damage was also reduced. Puma induction was also blocked in p53-deficient mice subject to SE. Compared to Puma-expressing mice, Puma-deficient mice had significantly smaller hippocampal lesions after SE. Long-term, continuous telemetric EEG monitoring revealed a approximately 60% reduction in the frequency of epileptic seizures in the Puma-deficient mice compared to Puma-expressing mice. These are the first data showing genetic deletion of a proapoptotic protein acting acutely to influence neuronal death subsequently alters the phenotype of epilepsy in the long-term, supporting the concept that apoptotic pathway activation is a trigger of epileptogenesis.-Engel, T., Murphy, B. M., Hatazaki, S., Jimenez-Mateos, E. M., Concannon, C. G., Woods, I., Prehn, J. H. M., Henshall, D. C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.

Effects of transient focal cerebral ischemia in mice deficient in puma.

Bcl-2 homology domain 3 (BH3)-only pro-apoptotic proteins may play an important role in upstream cell death signaling pathways underlying ischemic brain injury. Puma is a potent BH3-only protein that can be induced via p53, FoxO3a and endoplasmic reticulum stress pathways and is upregulated by global cerebral ischemia. To more completely define the contribution of Puma to ischemic brain injury we measured the expressional response of Puma to transient focal cerebral ischemia in mice and also compared infarct volumes in puma-deficient versus puma-expressing mice. Real-time quantitative PCR determined puma mRNA levels were significantly increased 8h after 90min middle cerebral artery (MCA) occlusion in the ipsilateral cortex, while expression remained unchanged contralaterally. Puma protein levels were also increased in the ischemic cortex over the same period. However, cortical and striatal infarct volumes were not significantly different between puma-deficient and puma-expressing mice at 24h, and no differences between genotypes were found for post-ischemic neurological deficit scores. These data demonstrate that focal cerebral ischemia is associated with puma induction but suggest that Puma does not contribute significantly to lesion development in the present model.

NMDA receptor-mediated excitotoxic neuronal apoptosis in vitro and in vivo occurs in an ER stress and PUMA independent manner.

Disruption of endoplasmic reticulum (ER) Ca2+ homeostasis and ER dysfunction have been suggested to contribute to excitotoxic and ischaemic neuronal injury. Previously, we have characterized the neural transcriptome following ER stress and identified the BH3-only protein, p53 up-regulated mediator of apoptosis (PUMA), as a central mediator of ER stress toxicity. In this study, we investigated the effects of excitotoxic injury on ER Ca2+ levels and induction of ER stress responses in models of glutamate- and NMDA-induced excitotoxic apoptosis. While exposure to the ER stressor tunicamycin induced an ER stress response in cerebellar granule neurons, transcriptional activation of targets of the ER stress response, including PUMA, were absent following glutamate-induced apoptosis. Confocal imaging revealed no long-term changes in the ER Ca2+ level in response to glutamate. Murine cortical neurons and organotypic hippocampal slice cultures from PUMA+/+ and PUMA-/- animals provided no evidence of ER stress and did not differ in their sensitivity to NMDA. Finally, NMDA-induced excitotoxic apoptosis in vivo was not associated with ER stress, nor did deficiency in PUMA alleviate the injury induced. Our data suggest that NMDA receptor-mediated excitotoxic apoptosis occurs in vitro and in vivo in an ER stress and PUMA independent manner.

Deletion of the BH3-only protein puma protects motoneurons from ER stress-induced apoptosis and delays motoneuron loss in ALS mice.

BH3-only proteins couple diverse stress signals to the evolutionarily conserved mitochondrial apoptosis pathway. Previously, we reported that the activation of the BH3-only protein p53-up-regulated mediator of apoptosis (Puma) was necessary and sufficient for endoplasmic reticulum (ER) stress- and proteasome inhibition-induced apoptosis in neuroblastoma and other cancer cells. Defects in protein quality control have also been suggested to be a key event in ALS, a fatal neurodegenerative condition characterized by motoneuron degeneration. Using the SOD1(G93A) mouse model as well as human post mortem samples from ALS patients, we show evidence for increased ER stress and defects in protein degradation in motoneurons during disease progression. Before symptom onset, we detected a significant up-regulation of Puma in motoneurons of SOD1(G93A) mice. Genetic deletion of puma significantly improved motoneuron survival and delayed disease onset and motor dysfunction in SOD1(G93A) mice. However, it had no significant effect on lifespan, suggesting that other ER stress-related cell-death proteins or other factors, such as excitotoxicity, necrosis, or inflammatory injury, may contribute at later disease stages. Indeed, further experiments using cultured motoneurons revealed that genetic deletion of puma protected motoneurons against ER stress-induced apoptosis but showed no effect against excitotoxic injury. These findings demonstrate that a single BH3-only protein, the ER stress-associated protein Puma, plays an important role during the early stages of chronic neurodegeneration in vivo.