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Excitatory Suppression and the Extension of Lifespan

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Excitatory Suppression and the Extension of Lifespan

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Finally, Zullo et al. wanted to confirm that the SPR3/4 contribute to the extended lifespans of DAF2 mutants through the suppression of neural excitation.
GCaMP calcium imaging demonstrates that DAF2 mutants have significantly lower levels of excitatory signaling when compared to WTs and triple mutants (who are closer to WT levels).
Furthermore, administering the anticonvulsant ivermectin (Ive, which reduces excitation - see above) restored DAF2-extended lifespans in the triple mutants. This provides strong evidence for the claim that a critical lifespan extending mechanism of SPR3/4 is the suppression of excitation.

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Mutant Worm Transcriptomes Resemble Human Profiles

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Mutant Worm Transcriptomes Resemble Human Profiles

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The authors performed RNA-seq on WT worms and those with mutations in SPR3, SPR4, DAF2, and a SPR3;SPR4;DAF2 triple mutant. They single out the comparison between the triple and DAF2-mutants. Specifically, they found that the genes that were downregulated in the DAF2 mutant but upregulated in the triple mutant pertained to neural excitation, signaling, and synaptic function. Indeed, the top downregulated genes greatly resemble the profile derived from human samples highlighted above.
This suggests that the aging-associated repression of excitatory genes is a conserved feature of REST and its orthologues.

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Neuronal SPRs are Needed for DAF2-Longevity

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Neuronal SPRs are Needed for DAF2-Longevity

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The authors wanted to elaborate on the relationship between DAF2 and SPR by modulating neuronal-SPR function.
They created a C. elegans line in which RNAi was abolished by deletion of the RNA SID-1 transporter except in neurons where  sid-1 transgene expression was driven by a neuron-specific promoter. This line was the crossed into the daf-2 mutant background and RNAi-knockdown on SPR3 and SPR4 was performed.
Once more, downregulating SPR3/4 function weakens the lifespan extending effects of reduced DAF2 function.
Specifically, however, it is the neural SPR activity that negates such extension, further establishing REST as a potential hub for neural and metabolic activity.

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Zullo et al. confirmed earlier findings by demonstrating that reducing DAF2 function (via RNAi) significantly extends wild-type (WT) lifespans.
They further found SPR3/4 mutations diminish the lifespan extending effects of DAF2-RNAi with the triple mutants (DAF2, SPR3, SPR4) having the shortest extension. The authors suggest this indicates orthologous-REST regulates lifespan by modulating IIS activity in worms and that they are needed for any health benefits of reduced IIS.

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In several species, dietary restriction and reduced IIS signaling has been demonstrated as a robust, reliable method of extending lifespan. IIS polymorphisms have been shown to not only extend lifespan in mice, worms, and flies, but also improve cognitive performance and memory in mice models with neurodegeneration. Therefore, there is a significant relationship between IIS and nervous system function.
This may be due to the fact that reduced IIS and dietary restriction are thought to involve an increase in global inhibitory tone and confer anticonvulsant properties.
In worms (C. elegans), DAF2 is the IIS-receptor orthologue. DAF2 loss-of-function mutations have been shown to delay aging-associated health issues and extend lifespan by up to 50%.
Zullo et al. studied worms that had reduced DAF2 function by externally-mediated RNAi and also strains of worms that had a LOF DAF2 allele (e1370).

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Expression of the Construct Reduced Excitation and Extended Lifespan

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Expression of the Construct Reduced Excitation and Extended Lifespan

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REST Orthologues Regulate Longevity in C. elegans

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REST Orthologues Regulate Longevity in C. elegans

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There is no direct mammalian REST homolog in C. elegans, however, there is a SPR-4 orthologue and an SPR-3 isoform. The authors equivocate between their usage, but tend towards examining SPR-4.
They induced endogenous expression of SPR4 using a construct consisting of dCas9 fused with a VP16 transcriptional activator (dCas9::VP64) which was then introduced into the worms with four sgRNAs targeting the SPR4 promoter.

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Bidirectionality of Excitation and Lifespan

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Bidirectionality of Excitation and Lifespan

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The authors additionally used RNA-mediated inhibition (RNAi) to target the GABA vesicular transporter, unc-47, which resulted in a robust increase in excitation and significantly reduced lifespan.
The authors thus claim that there is a bidirectional relationship between lifespan and neurotransmission insofar as reducing excitation extends lifespan while increasing excitation shortens it.

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Zullo et al. have performed a comprehensive set of experiments that compellingly make the case that a key feature of aging nervous systems is an excessive increase in neural excitation and that the repression of this increase can have untold health benefits.
A significant point of intervention for such repression may like in the aging-associated upregulation of REST (and orthologues) which is a master regulator essential for preserving cognitive health in late life and delaying neurodegeneration. REST's therapeutic effects likely unfold through its suppression of (hyper)excitation. Moreover, REST could also serve as a target to investigate the overlap between neural activity and metabolic pathways by virtue of its negation of IIS function.

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The authors conducted 3 broad sets of experiments on: humans, C. elegans, and mice. The bulk of the work focuses on characterising the human and mice results by understanding how excitation and REST (orthologues) regulate lifespan and longevity in the worms.

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The Regulation of Lifespan by REST and Excitation

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The Regulation of Lifespan by REST and Excitation

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Previous work by the authors had hinted towards the potential significance of REST in aging and against neurodegeneration. Independent research also revealed REST's unique mechanisms of tempering excessive excitation. Furthermore, such excitation may be a conserved feature of the aging nervous system.
Therefore, the authors conducted an exploratory study that sought to characterise the putative relationship between REST, excitation, and aging.

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Finally, work has demonstrated that REST uniquely downregulates intrinsic and synaptic mechanisms of signaling. That is, REST has been found to donwregulate sodium voltage channels (intrinsic) and structural proteins like synaptotagmin-1 and SNAP25 (synaptic).
Pecoraro-Bisogni et al., (2018) claim that this is the first protein found to influence both mechanistic pathways and may serve an essential role in maintaining excitatory-inhibitory homeostasis.

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As the figures demonstrate, REST is upregulated in age compared to younger individuals. Furthermore, nuclear-REST levels are abolished or severely diminished in a variety of neurodegenerative conditions. Since REST is a transcriptional factor, its nuclear presence is necessary for proper functionality.
Lu et al. also modulated REST in: neurodegeneration-model mice, C. elegans, and in vitro cultures. They consistently found that downregulating REST would significantly increase the rate of neurodegeneration and negatively affect survival outcomes.

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Previous work by the Yankner lab ( Lu et al., 2014, see figures) demonstrated that the RE-1 silencing transcription factor (REST) may be the most upregulated transcription factor with age. REST is a master transcriptional repressor that is essential for neurodevelopment through the epigenetic repression of non-neural genes. It is significantly downregulated post-development but is thought to be upregulated through senescent stimuli for neuroprotective functions. One of these functions may be the suppression of aging-associated (hyper)excitation

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In addition to aging, excessive excitation is a key feature of several neuropsychiatric, neurodevelopmental, and neurodegenerative conditions.
However, since neurodegenerative conditions are concurrent with the aging process, hyperexcitation can become a significantly greater problem when it manifests in later life.
Hyperactivation is a characteristic feature of mild cognitive impairment (MCI). Evidence suggests (see Dickerson and Sperling, 2008) that the degree of hyperactivation/excitation observed in MCI correlates directly with the severity of neurodegeneration in any subsequent disease.
Therefore, the maintenance of physiological levels of excitation are imperative to preserving cognitive health and longevity.

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A large body of work claims that the aging process alone, that is with minimal external intervention, results in excessive increases in excitation. For instance, Fischer et al. (2020, see figure) found that through aging alone, murine hippocampal networks became significantly more excitable than those of younger mice.
Moreover, several studies in primates and humans suggest that aging-associated processes that skew neuronal homeostasis towards excitation may be involved in comorbidities concomitant with aging like hearing and memory loss. This would make sense as increased excitation can cause excitotoxicity and impair information processing. Therefore, if the hippocampus is beset by hyperexcitation, it is reasonable to conclude that memory formation and recall would also be impaired.

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Why do some of us live long lives with intact cognitions while others succumb to neurodegeneration? Although research has yielded some clues in the form of fitness, diet, and genetics, the question remains a mystery.
However, we have begun to dissect some aging-associated effects on the brain that are conserved across multiple species.
Critically, research suggests that decreased excitation and insulin/insulin-like signaling (IIS) and increased REST may be critical to living long, cognitively-healthy lives.

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C. elegans is a well-established model system for aging, benefiting from a simple nervous system, short lifespans, and translatable science.
Zullo et al., monitored levels of excitation in the worms by recording fluorescence intensity produced by GCaMP calcium imaging in glutamatergic neurons. They confirmed the conserved nature of aging and excitation by recording that without any external interventions, the levels of excitation naturally increased over the lifespan.

They subsequently administered a variety of compounds that modulate excitation.
Nemadipine is an inhibitor of calcium channels and reduces excitation. Ivermectin is a chloride channel agonist that suppresses excitation. However, Ivermectin was only beneficial at certain dosages as shown in the figures.
Both drugs significantly extended lifespan and preserved worm motility suggesting that global inhibition of excitation extends lifespan in C. elegans

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Figure1: Human study from frontal cortex of 3 cognitively healthy aging cohorts. (a). Microarray data for the first cohort. (b). GO term analysis for 3 cohorts.

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Human study: Transcriptome profiles of cognitively normal aging cohorts

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Human study: Transcriptome profiles of cognitively normal aging cohorts

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RNA-seq and microarray identified a list of downregulated genes associated with extended longevity.

Meta-analysis of gene ontology (GO) term showed strong relation of the downregulated genes to excitatory synaptic transmission.

Conclusion: Extended longevity was significantly associated with downregulation of neural excitatory genes.

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Figure 5: immunocytochemical labelling for FOXO1 and neural marker MAP2 in Rest-/- and control mice (left); Quantification of FOXO1 nuclear level (right).

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FOXO1 of REST cKO mice: FOXO1 is regulated by REST and neural excitation

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FOXO1 of REST cKO mice: FOXO1 is regulated by REST and neural excitation

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The FOXO1 expression level in the brain of REST cKO mice and control mice were examined.

Age-dependent activation of FOXO1 was abolished in REST cKO mice. REST is needed for regulation of the forkhead transcriptional factors.

Inhibition of glutamate signal that was induced to neural cultures of mice led to an upregulation of FOXO1 level. Neural excitation regulates FOXO1 expression in mouse neurons.

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PROS: multiple model organisms + human data

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PROS: multiple model organisms + human data

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Genome-wide study of REST expression in human is badly needed for similar studies.

Coherent human data is important for clinical application of the lifespan studies.

Different transgenic manipulations were used on different model organisms, giving robust conclusions.

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Dallagnol et al. 2017: Physical activity upregulated REST expression in the hippocampus of aged animals.

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Cons: More background information needed for human cohort data.

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Cons: More background information needed for human cohort data.

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Zullo et al. note that the human data was collected from postmortem samples and provide no further information. Understandably, data privacy is a concern, however, it would have been informative to know some background information about the respective humans. Research has identified that REST expression is influenced by multiple factors in either healthy or pathological individuals, including mental stress (Singh-Taylor et al., 2018) and physical exercise (Dallagnol et al., 2017). Such information could have further strengthened their results as we do not know if the humans with the most upregulated REST and longest lifespans lived healthy, stress-free lives or not. Therefore, more background for the cognitively intact elders should be collected to find out other potential factors affecting longevity and REST expression.

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Future directions: REST, neural excitation and FOXO family genes

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Future directions: REST, neural excitation and FOXO family genes

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The paper revealed the regulatory effect of REST on forkhead transcription factors (e.g.FOXO1), which converges with insulin-IGF signaling pathway. In the future, the connection can be further investigated between IGF and REST, integrating metabolism with lifespan and neural activity.
Shown by the experiments in C.elegans, daf16 is important to SPR3/4 function and longevity, In the future, the authors may investigate whether FOXO1 influence the expression of REST and the lifespan of mice.

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Research has identified different REST expression patterns in humans and mice. REST was upregulated in ageing humans but downregulated in aging mice. This not only points to evolutionary developmental divergences between species, but stresses the importance of performing redundant experiments on multiple systems to collect translatable data. This study accordingly hints at the limitations of using mice as model systems to study aging.

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Undefined brain E/I balance: Both global excitation and inhibition are complex and multidimensional. Global hyperexcitation may induce regional inhibition.

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CONS: Complexity underlining global hyperexcitation should be considered.

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CONS: Complexity underlining global hyperexcitation should be considered.

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The mechanisms of REST to regulate synaptic genes and reduce neural excitation need further investigation. One remaining question of the study is, does the regulatory effect of REST on global neural excitation, or its repression of specific brain areas and neuron subtypes, leads to extension of lifespan? As global hyperexcitation is a complex and multidimensional phenomenon , the underlining mechanisms connecting it to the REST expression and individual longevity need further investigation. Indeed, the authors only quantify excitation in terms of fluorescence intensity. This results in us only having a relative understanding of the effects of excitation on lifespan.

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Challenges: Ethical issues of lifespan study

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Challenges: Ethical issues of lifespan study

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Controversy about the unnatural characteristics of life extension.

Issues about who should have the access to life-extending technology.

Potential risks and side-effects from applying genetic modifications on healthy individuals.

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 The activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.

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Future directions: REST expression in pathological conditions?

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Future directions: REST expression in pathological conditions?

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The authors only examine the REST expression profiles in cognitively intact individuals. As more and more research showed a connection between REST and multiple neurological diseases, the shifted expression of REST under pathological conditions can be investigated under multiple brain pathologies including Alzheimer's Disease.
Work by Ashton et al. (2017) demonstrates how REST levels are inversely correlated with the severity of MCI with more amnestic and severe versions resulting in lower peripherap REST levels. Furthermore, they found that performing mindfulness increases REST levels compared to controls. Therefore, it may be worthwhile to investigate the trajectory of REST in healthy and pathological aging and the precise associated mechanisms that influence REST levels.

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1.       Ashton, N. J., et al. "Plasma REST: a novel candidate biomarker of Alzheimer's disease is modified by psychological intervention in an at-risk population". (2017).  Translational psychiatry, 7(6), e1148.

2.       Dallagnol, Karine Mathilde Campestrini, et al. "Running for REST: physical activity attenuates neuroinflammation in the hippocampus of aged mice." Brain, behavior, and immunity 61 (2017): 31-35.

3.       Dickerson, B. C., & Sperling, R. A. (2008). Functional abnormalities of the medial temporal lobe memory system in mild cognitive impairment and Alzheimer's disease: insights from functional MRI studies. Neuropsychologia, 46(6), 1624-1635.

4.       Fischer, C., et al. "Prevention of age-associated neuronal hyperexcitability with improved learning and attention upon knockout or antagonism of LPAR2." (2021).  Cellular and molecular life sciences : CMLS, 78(3), 1029–1050.

5.       Hahn, O., et al. "Dietary restriction protects from age-associated DNA methylation and induces epigenetic reprogramming of lipid metabolism." (2017). Genome biology, 18(1), 56. https://doi.org/10.1186/s13059-017-1187-1

6.       Lu, Tao, et al. "REST and stress resistance in ageing and Alzheimer’s disease." Nature 507.7493 (2014): 448-454.

7.       McGann, James C., et al. "The genome-wide binding profile for human RE1 Silencing Transcription factor unveils a unique genetic circuitry in hippocampus." Journal of Neuroscience 41.31 (2021): 6582-6595.

8.       Partridge, Brad, et al. "Ethical, social, and personal implications of extended human lifespan identified by members of the public." Rejuvenation research 12.5 (2009): 351-357.

9.       Pecoraro-Bisogni, F., et al. "REST-Dependent Presynaptic Homeostasis Induced by Chronic Neuronal Hyperactivity." (2018). Molecular neurobiology, 55(6), 4959–4972.

10.    Singh-Taylor, Akanksha, et al. "NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience." Molecular psychiatry 23.3 (2018): 648-657.

11.    Sohal, Vikaas S., and John LR Rubenstein. "Excitation-inhibition balance as a framework for investigating mechanisms in neuropsychiatric disorders." Molecular psychiatry 24.9 (2019): 1248-1257.

12.    Tavernarakis, Nektarios. "Moderation of neural excitation promotes longevity." (2019): 338-340.

13.    Zullo, Joseph M., et al. "Regulation of lifespan by neural excitation and REST." Nature 574.7778 (2019): 359-364.

Background image from: wallpaperscraft.com

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Figure 2: PET-CT scanning revealed global hyperexcitation in Rest-/- mice compared to littermate controls.

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REST cKO mice: Rest as a regulator of neuroexcitation

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REST cKO mice: Rest as a regulator of neuroexcitation

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Neuron-specific conditional knockout of REST in aged mouse brain led to globally hyperexcitation.

Nestin-Cre:Rest lx/lx (Rest-/-) mice VS Rest lx/lx (control) mice

Neural activity detected by Positron emission tomography (PET-CT) scanning