Xianhua Wang

Here we present FHIRM-TPM 3.0, a 2.6 g miniature two-photon microscope capable of multicolor deep-brain imaging in freely behaving mice. The system was integrated with a broadband anti-resonant hollow-core fiber featuring low transmission loss, minimal dispersion from 700 nm to 1,060 nm and high tolerance of laser power. By correcting chromatic and spherical aberrations and optimizing the fluorescence collection aperture, we achieved cortical neuronal imaging at depths exceeding 820 μm and, using a GRIN lens, hippocampal Ca²⁺ imaging at single dendritic spine resolution. Moreover, we engineered three interchangeable parfocal objectives, allowing for a tenfold scalable field of view up to 1 × 0.8 mm², with lateral resolutions ranging from 0.68 μm to 1.46 μm. By multicolor imaging at excitation wavelengths of 780 nm, 920 nm and 1,030 nm, we investigated mitochondrial and cytosolic Ca²⁺ activities relative to the deposition of amyloid plaques in the cortex of awake APP/PS1 transgenic mice. Thus, FHIRM-TPM 3.0 provides a versatile imaging system suitable for diverse brain imaging scenarios.

Reactive thiols of proteinaceous cysteines are vital to cell biology by serving as sensor, effector and buffer of environmental redox fluctuations. Being the major source, as well as the prime target, of reactive oxygen species (ROS), mitochondria confront great challenges in preserving their thiol pool. Here we show that ROS modulator 1 (ROMO1), a small inner mitochondrial membrane protein, plays a role in protecting the mitochondrial cysteinome. ROMO1 is redox sensitive and reactive and overexpression can prevent deleterious oxidation of proteinaceous thiols. ROMO1 upregulation leads to a reductive shift of the mitochondrial cysteinome, exerting beneficial effects on mitochondria, such as promoting energy metabolism and Ca²⁺ uniport while inhibiting vicious membrane permeability transition. Importantly, ROMO1 overexpression reverses mitochondrial cysteinome oxidations in multiple organs and slows functional decline in aged male mice. These findings unravel a redox regulatory mechanism of the mitochondrial cysteinome and mark ROMO1 as a potential target for combating oxidative stress and improving healthspan.

Mitochondrial dysfunction is strongly associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron degeneration. However, it remains obscure whether mitochondrial abnormities are sufficient to drive ALS development. Here, we show that selective depletion of reactive oxygen species modulator 1 (ROMO1), an inner mitochondrial membrane-delimited protein, in cholinergic neurons leads to adult-onset, progressive locomotor deficits in mice that closely resemble ALS pathology. ROMO1 ablation in cholinergic neurons induces ALS-like neuromuscular degeneration, as evidenced by age-dependent motor neuron loss, axon degeneration, disrupted cholinergic transmission, neuromuscular junction denervation, and the resultant muscle atrophy. Notably, ROMO1 loss induces early and progressive mitochondrial cristae deformation in motor neurons, preceding the onset of ALS-like syndromes. Our findings support that mitochondrial impairment in vulnerable motor neurons is a sufficient contributor to ALS etiology, positioning mitochondria as a potential therapeutic target.

Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state switches during natural sleep-wake cycles remains unclear. To address this question, we used miniature two-photon microscopy (mTPM) to acquire time-lapse high-resolution microglia images of the somatosensory cortex, along with EEG/EMG recordings and behavioral video, in freely-behaving mice. We uncovered fast and robust brain state-dependent changes in microglia surveillance, occurring in parallel with sleep dynamics and early-onset phagocytic microglial contraction during sleep deprivation stress. We also detected local norepinephrine fluctuation occurring in a sleep state-dependent manner. We showed that the locus coeruleus-norepinephrine system, which is crucial to sleep homeostasis, is required for both sleep state-dependent and stress-induced microglial responses and β₂-adrenergic receptor signaling plays a significant role in this process. These results provide direct evidence that microglial surveillance is exquisitely tuned to signals and stressors that regulate sleep dynamics and homeostasis so as to adjust its varied roles to complement those of neurons in the brain. In vivo imaging with mTPM in freely behaving animals, as demonstrated here, opens a new avenue for future investigation of microglia dynamics and sleep biology in freely behaving animals.

Serving as the power plant and signaling hub of a cell, mitochondria contain their own genome which encodes proteins essential for energy metabolism and forms DNA–protein assemblies called nucleoids. Mitochondrial DNA (mtDNA) exists in multiple copies within each cell ranging from hundreds to tens of thousands. Maintaining mtDNA homeostasis is vital for healthy cells, and its dysregulation causes multiple human diseases. However, the players involved in regulating mtDNA maintenance are largely unknown though the core components of its replication machinery have been characterized. Here, we identify C17orf80, a functionally uncharacterized protein, as a critical player in maintaining mtDNA homeostasis. C17orf80 primarily localizes to mitochondrial nucleoid foci and exhibits robust double-stranded DNA binding activity throughout the mitochondrial genome, thus constituting a bona fide new mitochondrial nucleoid protein. It controls mtDNA levels by promoting mtDNA replication and plays important roles in mitochondrial metabolism and cell proliferation. Our findings provide a potential target for therapeutics of human diseases associated with defective mtDNA control.

Intracellular pH (pH_i) homeostasis is crucial for cellular functions and signal transduction across all kingdoms of life. In particular, bacterial pH_i homeostasis is important for physiology, ecology, and pathogenesis. Here we report an exquisite bacterial acid-resistance (AR) mechanism in which proton leak elicits a pre-emptive AR response. A single bacterial cell undergoes quantal electrochemical excitation, termed “BacFlash”, which consists of membrane depolarization, transient pH_i rise, and bursting production of reactive oxygen species. BacFlash ignition is dictated by acid stress in the form of proton leak across the plasma membrane and the rate of BacFlash occurrence is reversely correlated with the pH_i buffering capacity. Through genome-wide screening, we further identify the ATP synthase F_o complex subunit a as the putative proton sensor for BacFlash biogenesis. Importantly, persistent BacFlash hyperactivity activates transcription of a panel of key AR genes and predisposes the cells to survive imminent extreme acid stress. These findings demonstrate a prototypical coupling between electrochemical excitation and nucleoid gene expression in prokaryotes.

Fatty acids are the most major substrate source for adult cardiac energy generation. Prohibitin 2 (PHB2), a highly conserved protein located in mitochondrial inner membrane, plays key roles in cellular energy metabolic homeostasis. However, its functions in regulating cardiac fatty acid metabolism have remained largely unknown. Our study demonstrates that cardiac-specific knockout of Phb2 leads to accumulation of lipid droplets and causes heart failure. Mechanistically, ablation of PHB2 impairs cardiac fatty acid oxidation (FAO) through downregulating carnitine palmitoyltransferase1b (CPT1b), a rate-limiting enzyme of cardiac mitochondrial FAO. Moreover, overexpression of CPT1b alleviates impaired FAO in PHB2-deficient cardiomyocytes. Thus, our study provides direct evidence for the link between PHB2 and cardiac fatty acid metabolism. Our study points out that PHB2 is a potential FAO regulator in cardiac mitochondrial inner membrane, as well as the connection between PHB2 and CPT1b and their relationships to cardiac pathology especially to cardiac fatty acid metabolic disorder.

Mitochondria are fundamental organelles for cellular and systemic metabolism, and their dysfunction has been implicated in the development of diverse metabolic diseases. Boosted mitochondrial metabolism might be able to protect against metabolic stress and prevent metabolic disorders. Here we show that NADH:ubiquinone oxidoreductase (NDU)‐FAB1, also known as mitochondrial acyl carrier protein, acts as a novel enhancer of mitochondrial metabolism and protects against obesity and insulin resistance. Mechanistically, NDUFAB1 coordinately enhances lipoylation and activation of pyruvate dehydrogenase mediated by the mitochondrial fatty acid synthesis pathway and increases the assembly of respiratory complexes and supercomplexes. Skeletal muscle‐specific ablation of NDUFAB1 causes systemic disruption of glucose homeostasis and defective insulin signaling, leading to growth arrest and early death within 5 postnatal days. In contrast, NDUFAB1 overexpression effectively protects mice against obesity and insulin resistance when the animals are challenged with a high‐fat diet. Our findings indicate that NDUFAB1 could be a novel mitochondrial target to prevent obesity and insulin resistance by enhancing mitochondrial metabolism.—Zhang, R., Hou, T., Cheng, H., Wang, X. NDUFAB1 protects against obesity and insulin resistance by enhancing mitochondrial metabolism. FASEB J. 33, 13310–13322 (2019). www.fasebj.org