• Mechanistic crosstalk between neuronal energy metabolism and neuroregeneration after CNS injury

CNS neurons require a large amount of energy (ATP) to maintain their physiological functions. Mitochondria produce ATP through oxidative phosphorylation, which are the main energy source in neurons. Due to highly specialized structure and limited diffusion capacity of ATP, neurons require a set of rapid and efficient mechanisms to maintain the energy homeostasis at distal ends of neuronal axons. In recent years, more and more studies have shown that abnormal energy metabolism within axons is correlated with nerve injury and pathogenesis of various neurodegenerative diseases. Therefore, the study on energy supply and maintenance within axons is becoming a new hot spot in neurobiological research and clinical therapy.

The first work led by the applicant is the study of the molecular mechanism between neuronal axon energy metabolism and neuroregeneration after acute CNS injury (such as stroke, and SCI, etc.). With high disability and fatality rates, stroke and SCI bring huge loss to the patients and families, and cause serious social burdens. Unfortunately, mechanisms of stroke recovery and SCI regeneration are extremely complicated, and there is a lack of clinical therapeutic strategies and targets for precise drug intervention. Recent studies have shown that once mature neurons are acutely damaged, the impaired axonal mitochondrial integrity and transport will cause the extreme energy crisis at damaged area, and eventually lead to axon degeneration and neuron death. Thus, it is important to study how mature neurons respond to injury stimuli, restore energy metabolism, and maintain physiological functions.

To answer the scientific questions raised above, the applicant combined several advanced techniques, including large-scale sequencing analysis, in vivo and in vitro fluorescent imaging, and SCI mouse model, and discovered AKT-PAK5 axis as a novel signaling in response to axon injury in mature neurons. By enhancing the bi-directional transport of axonal mitochondria, AKT-PAK5 axis facilitates the rapid recovery of energy homeostasis after axon injury. The applicant mainly 1) established acute stress models that simulate cerebral ischemia and axon injury in vitro by using microfluidic devices. Combined with translatome sequencing analysis and Puro-PLA assay, the axonal synthesis of the neuron-specific kinase PAK5 was found significantly increased after two different injury stimuli; 2) monitored axonal ATP levels by the fluorescent probe GO-ATeam2 and revealed that activating AKT-PAK5 axis quickly restore the energy homeostasis in injured axons; 3) revealed that activating AKT-PAK5 signal enhances bi-directional transport of axonal mitochondria in three different systems such as mature cortical neurons, dorsal root ganglion (DRG) neurons, and ex vivo sciatic nerves from aging mice; 4) found that in two different in vitro injury models, AKT-PAK5 axis activation promotes mitochondrial transport in the damaged axons, which accelerates the delivery of healthy mitochondria to the damaged area to produce sufficient ATP to maintain local energy supply, and ensures the survival and regeneration of axons after injury; 5) collaborated with Prof. Xiao-Ming Xu’s lab in Indiana University, and found that activating PAK5 via AAV injection also promotes axonal regeneration after SCI in mice. This study reveals for the first time that AKT-PAK5 axis, as a mature neuron axonal signal in response to injury stimuli, regulates the axonal mitochondrial transport to ensure energy homeostasis within axons. This study also provides new therapeutic targets and clinical strategies for promoting neuron survival and regeneration after cerebral ischemia and SCI. This work was recently published in Current Biology (2021), and highly appraised by preview article named Neurobiology: Resetting the axon’s batteries. The applicant also joined in the writing of an invited review article named Mitochondrial trafficking and axonal energy maintenance in neuronal degeneration and regeneration by Neuron journal (under preparation, 2021).

• Neuron-glia communications and pathogenesis of neurodegenerative disorders

Neurodegenerative diseases are a series of cognitive impairment caused by the loss of neuronal function and structure. With high morbidity and disability, neurodegenerative diseases pose a great threat to human health and life quality. However, multiple types of causes and pathogenesis in neurodegenerative diseases make it difficult to develop the clinical strategies and drugs with precise targets. Human brain is highly organized by approximately 86 billion neurons and 84 billion glial cells. As an essential part of this complex system, glial cells also play important roles in axonal energy metabolism. Oligodendrocytes are one of the crucial types in glial cells, which provide myelination sheaths for the axons in CNS, although their regulatory mechanism in axonal energy metabolism under physiological and pathological conditions is still unclear. Previous studies have found that axonal mitochondrial dysfunction and axon demyelination are the common features in the early onset of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Multiple Sclerosis (MS), indicating the potential correlation among axon degeneration, impaired energy supply and oligodendrocyte dysfunction. Investigating their crosstalk will uncover the molecular mechanism of the occurrence and development of neurodegenerative diseases, and provide new targets and strategies for clinical therapy.

Combining neuron-oligodendrocyte co-culture system, fluorescent probe imaging, and mitochondrial energetics analysis­­, the applicant uncovered a myelination-independent neuron-oligodendrocyte signaling that maintains axonal energy metabolism. 1) Axonal mitochondrial ATP production is significantly increased after co-culturing with oligodendrocytes; 2) Oligodendrocytes-derived exosomes contain energy metabolism signals and could be shuttled into neuronal axons; 3) SIRT families-contained exosomes effectively regulate the acetylation status of mitochondrial proteins ANT1/2, thereby promoting mitochondrial function and energy metabolism within axons; 4) Purified  oligodendrocyte-released exosomes rescue the mitochondrial integrity within axon bundles after injecting into the spinal cords of mitochondrial deficit mice.

This work uncovers how oligodendrocytes facilitate axonal energy metabolism and communicate with neurons in the physiological condition. In addition, as a new type of bio-carrier, exosomes are currently used in the pre-clinical trials against cancer and nerve injury, but not neurodegenerative diseases. This study also provides a theoretical basis for exosomes as a potential therapeutic strategy against neurodegenerative diseases.

This work is now accepted by the top neuroscience journal Neuron (2021).

• Role of cytoskeletal protein network in tumorigenesis and embryonic nervous system development

In mammalian cells, centrosomes play as microtubule organization centers, which ensure the stability of cell structure and the progress of mitosis. Primary cilia are the immobile organelles composed of microtubules and accessory structural proteins, which protrude from the cell as antenna-like structures, and sense different signaling to maintain tissue homeostasis and development. Recent studies have shown that deficits in the biogenesis and function of centrosome and cilia can lead to genetic diseases such as impaired brain development, polycystic kidneys, and retinal degeneration. Furthermore, the abnormal centrosome and cilia number is also regarded as one of the important clinical indications in various types of cancer. Compared with the research on other organelles, the studies on centrosome and cilia is still at a very preliminary stage. Although several proteins located in the centrosome and cilia have been identified, it is still unclear how these proteins assemble into highly organized structures to function and support embryonic development. The research on these fundamental questions are crucial to guide the diagnosis of genetic diseases and cancer.

To answer these questions, the applicant applied: 1) CRISPR-Cas9 and high-throughput microscopy to screen the candidates related to centrosome and cilia function, 2) mass spectrometry analysis to identify protein interaction and related modifications, 3) super-resolution microscopy, and 4) gene-editing mouse models. Three centrosome and cilia-related protein complexes function in 1) the assembly of the centriole proximal connecting linker, 2) the assembly and function of the subdistal appendages of the mother centrioles, and 3) the facilitation of ciliogenesis and embryonic development were identified. Based on these achievements, the applicant published four first/co-first authorship SCI articles, with a total of more than 160 citations.

The applicant found that LRRC45-CCDC102B protein complex is located at the proximal ends of the two centrioles. Super-resolution microscope 3D-SIM and electron microscope images showed that this complex could form a fibrous structure. At the beginning of mitosis, LRRC45-CCDC102B complex is removed from the centrosome to ensure bipolar spindle formation. Impaired removal of this complex will lead to several cancer phenotypes such as mitosis delay, abnormal spindle formation, and uneven chromatin separation. As a potential cancer cell marker, the localization and dysfunction of this complex has clinical significance for cancer screening and diagnosis. This work was published in Cell Reports (2013) and Journal of Cell Science (2018). Next, the applicant successfully identified two unreported subdistal appendages proteins, CCDC68 and CCDC120, through mass spectroscopy analysis, and proposed a hierarchical assembly model of subdistal appendages through ODF2, CCDC68, CCDC120, Ninein, and CEP170 recruitment. This work revealed the mechanisms of centrosomes in anchoring microtubules and cell morphology maintenance, and was also an interpretation to the fundamental cell biology questions. The work was published in Nature Communications (2017), a top comprehensive journal. Finally, the applicant successfully screened out MPP9, a negative regulator of ciliogenesis, by combining high-content microscopy and gene-editing technique. Through mass spectrometry analysis, the applicant found that MPP9 can form protein complex with CEP97 and CP110 located on the distal ends of the centrioles. During serum starvation, MPP9-CEP97-CP110 complex is removed from the distal ends of the mother centrioles, which ultimately promotes the cilia formation. In addition, mpp9 knockout mice appeared typical ciliopathy phenotypes such as hydrocephalus and renal tubule malformations during the developmental stage, and the percentage of ciliated cells was also abnormally increased. As a potential cause of ciliopathy, mpp9 gene mutation could be applied as a new target for the newborns genetic screening. This work was published in Nature Communications (2018), a top comprehensive journal, and recommended by Faculty Opinions (F1000Prime) https://facultyopinions.com/prime/734317253.