Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression.SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

Aims: Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD.Results: Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APPSwe/PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9–10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains.Innovation: Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity.Conclusions: We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269–1280.

Anabolic and catabolic signaling mediated via mTOR and AMPK (AMP-activated kinase) have to be intrinsically coupled to mitochondrial functions for maintaining homeostasis and mitigate cellular/organismal stress. Although glutamine is known to activate mTOR, whether and how differential mitochondrial utilization of glutamine impinges on mTOR signaling has been less explored. Mitochondrial SIRT4, which unlike other sirtuins is induced in a fed state, is known to inhibit catabolic signaling/pathways through the AMPK-PGC1α/SIRT1–peroxisome proliferator-activated receptor α (PPARα) axis and negatively regulate glutamine metabolism via the tricarboxylic acid cycle. However, physiological significance of SIRT4 functions during a fed state is still unknown. Here, we establish SIRT4 as key anabolic factor that activates TORC1 signaling and regulates lipogenesis, autophagy, and cell proliferation. Mechanistically, we demonstrate that the ability of SIRT4 to inhibit anaplerotic conversion of glutamine to α-ketoglutarate potentiates TORC1. Interestingly, we also show that mitochondrial glutamine sparing or utilization is critical for differentially regulating TORC1 under fed and fasted conditions. Moreover, we conclusively show that differential expression of SIRT4 during fed and fasted states is vital for coupling mitochondrial energetics and glutamine utilization with anabolic pathways. These significant findings also illustrate that SIRT4 integrates nutrient inputs with mitochondrial retrograde signals to maintain a balance between anabolic and catabolic pathways.

Background Accumulation of free fatty acids (FFAs) in hepatocytes is a hallmark of liver dysfunction and non-alcoholic fatty liver disease (NAFLD). Excessive deposition of FFAs alters lipid metabolism pathways increasing the oxidative stress and mitochondrial dysfunction. Attenuating hepatic lipid accumulation, oxidative stress, and improving mitochondrial function could provide potential targets in preventing progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). Earlier studies with Picrorhiza kurroa extract have shown reduction in hepatic damage and fatty acid infiltration in several experimental models and also clinically in viral hepatitis. Thus, the effect of P. kurroa's phytoactive, picroside II, needed mechanistic investigation in appropriate in vitro liver cell model. Objective(s) To study the effect of picroside II on FFAs accumulation, oxidative stress and mitochondrial function with silibinin as a positive control in in vitro NAFLD model. Materials and methods HepG2 cells were incubated with FFAs-1000μM in presence and absence of Picroside II-10 μM for 20 hours. Results HepG2 cells incubated with FFAs-1000μM lead to increased lipid accumulation. Picroside II-10μM attenuated FFAs-induced lipid accumulation (33%), loss of mitochondrial membrane potential (ΔΨm), ATP depletion, and production of reactive oxygen species (ROS). A concomitant increase in cytochrome C at transcription and protein levels was observed. An increase in expression of MnSOD, catalase, and higher levels of tGSH and GSH:GSSG ratios underlie the ROS salvaging activity of picroside II. Conclusion Picroside II significantly attenuated FFAs-induced-lipotoxicity. The reduction in ROS, increased antioxidant enzymes, and improvement in mitochondrial function underlie the mechanisms of action of picroside II. These findings suggest a need to develop an investigational drug profile of picroside II for NAFLD as a therapeutic strategy. This could be evaluated through the fast-track path of reverse pharmacology.

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