Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions.

Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy.

Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.

Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6-8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1-2 h.

Consequences of the Lack of TNFR1 in Ouabain Response in the Hippocampus of C57BL/6J Mice.

Ouabain is a cardiac glycoside that has a protective effect against neuroinflammation at low doses through Na+/K+-ATPase signaling and that can activate tumor necrosis factor (TNF) in the brain. TNF plays an essential role in neuroinflammation and regulates glutamate receptors by acting on two different receptors (tumor necrosis factor receptor 1 [TNFR1] and TNFR2) that have distinct functions and expression. The activation of constitutively and ubiquitously expressed TNFR1 leads to the expression of pro-inflammatory cytokines. Thus, this study aimed to elucidate the effects of ouabain in a TNFR1 knockout (KO) mouse model. Interestingly, the hippocampus of TNFR1 KO mice showed a basal increase in both TNFR2 membrane expression and brain-derived neurotrophic factor (BDNF) release, suggesting a compensatory mechanism. Moreover, ouabain activated TNF-α-converting enzyme/a disintegrin and metalloprotease 17 (TACE/ADAM17), decreased N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) expression, and induced anxiety-like behavior in both genotype animals, independent of the presence of TNFR1. However, ouabain induced an increase in interleukin (IL)-1ß in the hippocampus, a decrease in IL-6 in serum, and an increase in NMDA receptor subunit 1 (NR1) only in wild-type (WT) mice, indicating that TNFR1 or TNFR2 expression may be important for some effects of ouabain. Collectively, our results indicate a connection between ouabain signaling and TNFR1, with the effect of ouabain partially dependent on TNFR1.

Inverse sex-based expression profiles of PTEN and Klotho in mice.

Sex differences are considered predictive factors in the development of several neurological diseases, which are also known to coincide with impaired phosphoinositide 3-kinase (PI3K)-AKT pathway activity, an essential signaling cascade involved in the control of several cellular functions such as autophagy and apoptosis. Here, under physiological conditions, we show important sex differences in the underlying balancing mechanisms that lead to similar AKT activity levels and autophagy and apoptosis processes in the two sexes. We demonstrate inverse sex-based expression of PTEN and Klotho, two important proteins that are known to negatively regulate the AKT pathway, and inverse sex-dependent levels of mTOR and FoxO3a activity. Taken together, our findings indicate that inverse sex-based regulation may be one of the underlying balancing mechanisms that differ between the sexes and a possible cause of sex-based autophagic and apoptotic responses to triggering situations that can lead to a sex-based predisposition to some neurological diseases.

Insulin and Autophagy in Neurodegeneration.

Crosstalk in the pathophysiological processes underpinning metabolic diseases and neurodegenerative disorders have been the subject of extensive investigation, in which insulin signaling and autophagy impairment demonstrate to be a common factor in both conditions. Although it is still somewhat conflicting, pharmacological and genetic strategies that regulate these pathways may be a promising approach for aggregate protein clearancing and consequently the delaying of onset or progression of the disease. However, as the response due to this modulation seems to be time-dependent, finding the right regulation of autophagy may be a potential target for drug development for neurodegenerative diseases. In this way, this review focuses on the role of insulin signaling/resistance and autophagy in some neurodegenerative diseases, discussing pharmacological and non-pharmacological interventions in these diseases.