Microglia are generally considered the resident immune cells in the central nervous system (CNS) that regulate the primary events of neuroinflammatory responses. Microglia also play key roles in repair and neurodegeneration of the CNS after injury. Recent studies showed that trains of bipolar/rod-shaped microglia align end-to-end along the CNS injury site during the initial recovery phase. However, the cellular characteristics of bipolar/rod-shaped microglia remain largely unknown. Here, we established a highly reproducible in vitro culture model system to enrich and characterize bipolar/rod-shaped microglia by simply generating multiple scratches on a poly-d-lysine/laminin-coated culture dish. Trains of bipolar/rod-shaped microglia formed and aligned along the scratches in a manner that morphologically resembled microglial trains observed in injured brain. These bipolar/rod-shaped microglia were highly proliferative and expressed various M1/M2 markers. Further analysis revealed that these bipolar/rod-shaped microglia quickly transformed into amoeboid microglia within 30 minutes of lipopolysaccharide treatment, leading to the upregulation of pro-inflammatory cytokine gene expression and the activation of Jak/Stat. In summary, our culture system provides a model to further characterize this highly dynamic cell type. We suggest that bipolar/rod-shaped microglia are crucial for repairing the damaged CNS and that the molecular mechanisms underlying their morphological changes may serve as therapeutic biomarkers.

Axon regeneration is an energy-demanding process that requires active mitochondrial transport. In contrast to the central nervous system (CNS), axonal mitochondrial transport in regenerating axons of the peripheral nervous system (PNS) increases within hours and sustains for weeks after injury. Yet, little is known about targeting mitochondria in nervous system repair. Here, we report the induction of sustained axon regeneration, neural activities in the superior colliculus (SC), and visual function recovery after optic nerve crush (ONC) by M1, a small molecule that promotes mitochondrial fusion and transport. We demonstrated that M1 enhanced mitochondrial dynamics in cultured neurons and accelerated in vivo axon regeneration in the PNS. Ex vivo time-lapse imaging and kymograph analysis showed that M1 greatly increased mitochondrial length, axonal mitochondrial motility, and transport velocity in peripheral axons of the sciatic nerves. Following ONC, M1 increased the number of axons regenerating through the optic chiasm into multiple subcortical areas and promoted the recovery of local field potentials in the SC after optogenetic stimulation of retinal ganglion cells, resulting in complete recovery of the pupillary light reflex, and restoration of the response to looming visual stimuli was detected. M1 increased the gene expression of mitochondrial fusion proteins and major axonal transport machinery in both the PNS and CNS neurons without inducing inflammatory responses. The knockdown of two key mitochondrial genes, Opa1 or Mfn2 , abolished the growth-promoting effects of M1 after ONC, suggesting that maintaining a highly dynamic mitochondrial population in axons is required for successful CNS axon regeneration.

Aldose reductase (AR) is implicated in the development of a number of diabetic complications, but the underlying mechanisms remain to be fully elucidated. We performed this study to determine whether and how AR might influence hepatic peroxisome proliferator-activated receptor α (PPARα) activity and lipid metabolism. Our results in mouse hepatocyte AML12 cells show that AR overexpression caused strong suppression of PPARα/δ activity (74%, p < 0.001) together with significant down-regulation of mRNA expression for acetyl-CoA oxidase and carnitine palmitoyltransferase-1. These suppressive effects were attenuated by the selective AR inhibitor zopolrestat. Furthermore, AR overexpression greatly increased the levels of phosphorylated PPARα and ERK1/2. Moreover, AR-induced suppression of PPARα activity was attenuated by treatment with an inhibitor for ERK1/2 but not that for phosphoinositide 3-kinase, p38, or JNK. Importantly, similar effects were observed for cells exposed to 25 mm glucose. In streptozotocin-diabetic mice, AR inhibitor treatment or genetic deficiency of AR resulted in significant dephosphorylation of both PPARα and ERK1/2. With the dephosphorylation of PPARα, hepatic acetyl-CoA oxidase and apolipoprotein C-III mRNA expression was greatly affected and that was associated with substantial reductions in blood triglyceride and nonesterified fatty acid levels. These data indicate that AR plays an important role in the regulation of hepatic PPARα phosphorylation and activity and lipid homeostasis. A significant portion of the AR-induced modulation is achieved through ERK1/2 signaling.

Microglia are immune cells in the central nervous system (CNS) that contribute to primary innate immune responses. The morphology of microglia is closely associated with their functional activities. The majority of microglial studies have focused on the ramified or amoeboid morphology; however, bipolar/rod-shaped microglia have recently received much attention. Bipolar/rod-shaped microglia form trains with end-to-end alignment in injured brains and retinae, which is proposed as an important mechanism in CNS repair. We previously established a cell culture model system to enrich bipolar/rod-shaped microglia simply by growing primary microglia on scratched poly-D-lysine (PDL)/laminin-coated surfaces. Here, we investigated the role of laminin in morphological changes of microglia. Bipolar/rod-shaped microglia trains were transiently formed on scratched surfaces without PDL/laminin coating, but the microglia alignment disappeared after 3 days in culture. Amoeboid microglia digested the surrounding laminin, and the gene and protein expression of laminin-cleaving genes Adam9 and Ctss was up-regulated. Interestingly, lipopolysaccharide (LPS)-induced transformation from bipolar/rod-shaped into amoeboid microglia increased the expression of Adam9 and Ctss, and the expression of these genes in LPS-treated amoeboid-enriched cultures remained unchanged. These results indicate a strong association between laminin and morphological transformation of microglia, shedding new light on the role of bipolar/rod-shaped microglia in CNS repair.

In the cerebellar cortex, Purkinje cells (PCs) receive signals from different inputs through their extensively branched dendrites and serve as an integration centre. Defects in the dendritic development of PCs thus disrupt cerebellar circuitry and cause ataxia. Here we report that specific inactivation of both Lhx1 and Lhx5 in postnatal PCs results in ataxic mutant mice with abnormal dendritic development. The PCs in the mutants have reduced expression of Espin, an F-actin cytoskeleton regulator. We show that Espin expression is transcriptionally activated by Lhx1/5. Downregulation of Espin leads to F-actin mislocalization, thereby impairing dendritogenesis and dendritic spine maturation in the PCs. The mutant PCs therefore fail to form proper synapses and show aberrant electrophysiological properties. By overexpressing Espin, we can successfully rescue the defects in the mutant PCs. Our findings suggest that Lhx1/5, through regulating Espin expression, control dendritogenesis and spine morphogenesis in postnatal PCs.