αII Spectrin Forms a Periodic Cytoskeleton at the Axon Initial Segment and Is Required for Nervous System Function.

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse ß subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and ßIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of αII spectrin causes profound reductions in all ß spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system.SIGNIFICANCE STATEMENT Spectrin cytoskeletons play diverse roles in neurons, including assembly of excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. However, the molecular composition and structure of these cytoskeletons remain poorly understood. Here, we show that αII spectrin partners with ßIV spectrin to form a periodic cytoskeleton at the AIS. Using a new αII spectrin conditional knock-out mouse, we show that αII spectrin is required for AIS assembly, neuronal excitability, cortical lamination, and to protect against neurodegeneration. These results demonstrate the broad importance of spectrin cytoskeletons for nervous system function and development and have important implications for nervous system injuries and diseases because disruption of the spectrin cytoskeleton is a common molecular pathology.

Continuous assessment of metabolic activity of mitochondria using resonance Raman microspectroscopy.

Dysfunctional mitochondrial activity can lead to a variety of different diseases. As such, there exists a need to quantify changes in mitochondria function as it relates to these specific diseased states. Here, we present the use of resonance Raman (RR) spectroscopy as a tool to determine changes in isolated mitochondrial activity. RR spectroscopy, using 532 nm as the excitation source, specifically provides information on the reduction and oxidation (RedOx) state of cytochrome c, which is determined by the activity of protein complexes in the electron transport chain (ETC). In this model, injection of the substrate succinate into the mitochondrial sample is used to drive the ETC, which causes a subsequent change in cytochrome c RedOx state. This change in RedOx state is tracked by RR spectroscopy. This tool gives real-time information on the rise and fall of the amount of reduced cytochrome c within the mitochondrial sample, providing a method for rapid assessment of mitochondrial metabolism that has broad applications in both basic science and medical research.