With increased life expectancy, age-associated cognitive decline becomes a growing concern, even in the absence of recognizable neurodegenerative disease. The integrated stress response (ISR) is activated during aging and contributes to age-related brain phenotypes. We demonstrate that treatment with the drug-like small-molecule ISR inhibitor ISRIB reverses ISR activation in the brain, as indicated by decreased levels of activating transcription factor 4 (ATF4) and phosphorylated eukaryotic translation initiation factor eIF2. Furthermore, ISRIB treatment reverses spatial memory deficits and ameliorates working memory in old mice. At the cellular level in the hippocampus, ISR inhibition (i) rescues intrinsic neuronal electrophysiological properties, (ii) restores spine density and (iii) reduces immune profiles, specifically interferon and T cell-mediated responses. Thus, pharmacological interference with the ISR emerges as a promising intervention strategy for combating age-related cognitive decline in otherwise healthy individuals.
Introduction
“Of the capacities that people hope will remain intact as they get older, perhaps the most treasured is to stay mentally sharp” (Aging, 2015).
The impact of age on cognitive performance represents an important quality-of-life and societal concern, especially given our prolonged life expectancy. While often discussed in the context of disease, decreases in executive function as well as learning and memory decrements in older, healthy individuals are common (Connelly et al., 1991; Anderson et al., 1998; Kramer et al., 1999; Cepeda et al., 2001). According to the US Department of Commerce, the aging population is estimated by 2050 to reach 83.7 million individuals above 65 years of age in the US; this represents a rapidly growing healthcare and economic concern (An Aging Nation, 2014).
Age-related decline in memory has been recapitulated in preclinical studies with old rodents (Chou et al., 2018; Yousef et al., 2019; Villeda et al., 2011; Castellano et al., 2017). Specifically, prior studies have identified deficits in spatial memory (Villeda et al., 2011; Villeda et al., 2014), working and episodic memory (Yousef et al., 2019; Castellano et al., 2017) and recognition memory (Cabral-Miranda et al., 2020), when comparing young, adult mice with older sex-matched animals. The hippocampus is the brain region associated with learning and memory formation and is particularly vulnerable to age-related changes in humans and rodents (Disterhoft and Oh, 2007; McKiernan and Marrone, 2017; Oh et al., 2010; Rizzo et al., 2014). Deficits in a number of cellular processes have been suggested as underlying causes based on correlative evidence, including protein synthesis (Schimanski and Barnes, 2010), metabolism (Azzu and Valencak, 2017), inflammation (Franceschi et al., 2000), and immune responses (Villeda et al., 2011; Villeda et al., 2014; Baruch et al., 2014; Dulken et al., 2019). While providing a wealth of parameters to assess, by and large the causal molecular underpinnings of age-related memory decline have remained unclear.
The principle that blocking protein synthesis prevents long-term memory storage was discovered many years ago (Flexner et al., 1962). With age there is a marked decline of protein synthesis in the brain that correlates with defects in proper protein folding (Cabral-Miranda et al., 2020; López-Otín et al., 2013; Ingvar et al., 1985; Smith et al., 1995). Accumulation of misfolded proteins can activate the integrated stress response (ISR) (Harding et al., 2003), an evolutionary conserved pathway that decreases protein synthesis. In this way, the ISR may have a causative role in age-related cognitive decline. We previously discovered that interference with the drug-like small-molecule inhibitor (integrated stress response inhibitor, or ISRIB) rescued traumatic brain injury-induced behavioral and cognitive deficits (Chou et al., 2017; Krukowski et al., 2020; Costa-Mattioli and Walter, 2020), suggesting that this pharmacological tool may be useful in testing this notion.
Increasing age leads to structural and functional changes in hippocampal neurons. Specifically, in old animals there is an increase in neuronal hyperpolarization after spiking activity (‘afterhyperpolarization’, or AHP) that decreases intrinsic neuronal excitability and correlates with memory deficits (Disterhoft and Oh, 2007; McKiernan and Marrone, 2017; Oh et al., 2010; Rizzo et al., 2014; Kaczorowski and Disterhoft, 2009). Aging also manifests itself with synaptic excitability changes in the hippocampus that correlate with a reduction in the bulbous membrane projections that form the postsynaptic specializations of excitatory synapses, termed dendritic spines (von Bohlen und Halbach et al., 2006; Xu et al., 2018). Morphological changes in dendritic spine density are critical for spatial learning and memory (Bloss et al., 2011; Yasumatsu et al., 2008). Whether these age-related neuronal changes can be modified or are linked with ISR activation has yet to be determined.
In addition to neuronal changes, ISR activation can modify immune responses via alterations in cytokine production (Onat et al., 2019). Indeed, maladaptive immune responses have been linked with cognitive decline in the old brain (Yousef et al., 2019; Villeda et al., 2011; Villeda et al., 2014; Baruch et al., 2014). Initial studies focused on age-associated cytokine responses, including interferon (IFN)-mediated cognitive changes (Baruch et al., 2014; Deczkowska et al., 2017). Type-I IFN responses can induce age-related phenotypes in rodents. Furthermore, the adaptive immune system (T-cell infiltration into the old brain) can regulate neuronal function via IFN-γ production (Dulken et al., 2019), suggesting the possibility that age-induced maladaptive immune responses and the ISR are linked. Here we explore the possibility of ISR inhibition by ISRIB as a potential strategy for modifying age-induced neuronal, immune, and cognitive dysfunction.
Results
ISRIB resets the ISR in the brain of old mice
ISR activation leads to global reduction in protein synthesis but also to translational up-regulation of a select subset of mRNAs whose translation is controlled by small upstream open-reading frames in their 5′-UTRs (Hinnebusch et al., 2016; Sonenberg and Hinnebusch, 2009). One well-studied ISR-upregulated target protein is ATF4 (activating transcription factor 4) (Chen et al., 2003; Pasini et al., 2015). We recently showed ISRIB administration reversed mild head trauma-induced elevation in ATF4 protein (Krukowski et al., 2020). Using the same ISRIB treatment paradigm of daily injections on 3 consecutive days (Chou et al., 2017; Krukowski et al., 2020), we found decreased age-associated ATF4 protein levels in mouse brain lysates when compared to vehicle-treated controls during ISRIB administration
(Figure 1—figure supplement 1). ATF4 levels 18 days after cessation of ISRIB treatment showed persistent reduction in age-induced ATF4 protein levels that were indistinguishable from young mice (Figure 1A,B, Figure 1—figure supplement 2A).Figure 1 with 2 supplements Download asset Open asset
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