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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.

The cerebellum, the region of the brain primarily responsible for motor coordination and balance, also contributes to non-motor functions, such as cognition, speech, and language comprehension. Maldevelopment and dysfunction of the cerebellum lead to cerebellar ataxia and may even be associated with autism, depression, and cognitive deficits. Hence, normal development of the cerebellum and its neuronal circuitry is critical for the cerebellum to function properly. Although nine major types of cerebellar neurons have been identified in the cerebellar cortex to date, the exact functions of each type are not fully understood due to a lack of cell-specific markers in neurons that renders cell-specific labeling and functional study by genetic manipulation unfeasible. The availability of cell-specific markers is thus vital for understanding the role of each neuronal type in the cerebellum and for elucidating the interactions between cell types within both the developing and mature cerebellum. This review discusses various technical approaches and recent progress in the search for cell-specific markers for cerebellar neurons.

The laboratory mouse is the most commonly used mammalian model for biomedical research. An enormous number of mouse models, such as gene knockout, knockin, and overexpression transgenic mice, have been created over the years. A common practice to maintain a genetically modified mouse line is backcrossing with standard inbred mice over several generations. However, the choice of inbred mouse for backcrossing is critical to phenotypic characterization because phenotypic variabilities are often observed between mice with different genetic backgrounds. In this review, the major features of commonly used inbred mouse lines are discussed. The aim is to provide information for appropriate selection of inbred mouse lines for genetic and behavioral studies.

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.

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 alpha (PPARalpha) activity and lipid metabolism. Our results in mouse hepatocyte AML12 cells show that AR overexpression caused strong suppression of PPARalpha/delta 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 PPARalpha and ERK1/2. Moreover, AR-induced suppression of PPARalpha 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 PPARalpha and ERK1/2. With the dephosphorylation of PPARalpha, 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 PPARalpha phosphorylation and activity and lipid homeostasis. A significant portion of the AR-induced modulation is achieved through ERK1/2 signaling.

Hirschsprung’s disease, or congenital megacolon, is the most common gastrointestinal motility disorder in newborns. The prominent feature of Hirschsprung’s disease is an abnormal dilatation of the distal colon resulting from a regional absence or reduction of enteric ganglion cells. It has been known that all intrinsic enteric ganglion cells are derived from neural crest cells, which migrate along defined pathways from the neural tube (embryonic central nervous system) to the gut during embryonic development. Recent studies on avian embryos have also indicated that neural crest cells at the sacral level contribute a significant number of enteric neurons to the hindgut, the region of the gut where aganglionosis is usually detected in Hirschsprung’s disease. In the present study, we aimed to identify anomalies in the early migration of sacral neural crest cells in the Dominant megacolon(Dom) mouse mutant, a model for Hirschsprung’s disease. A combination of whole embryo culture, in situ cell labeling and histochemical staining was used to follow the early sacral neural crest cell migration. In the wild-type embryos, when sacral neural crest cells caudal to the 24th somite were labeled at embryonic day 10.0 (E10.0), labeled cells were found in the mesenchyme on the two sides of neural tube and many of them resided in the region of dorsal root ganglia at E11.0. Some of them were also found in the region around the dorsal aorta. In embryos heterozygous and homozygous for Dom, similar distribution and migratory pattern were found, indicating that the early migration of sacral neural crest cells was not affected in the mutant. Our results hence implicated that anomalies in the early sacral crest cell migration are unlikely to be a cause of aganglionosis in the hindgut of the Dom mutant.

Interstitial cells of Cajal (ICCs) in the gastrointestinal tract are a group of cells interacting with enteric neurons and smooth muscle cells. They serve as mediators of neurotransmission and pacemakers of the peristaltic movement of the gut. Previous investigations on patients with Hirschsprung’s disease and mice with aganglionic megacolon showed contrasting results on the relationship between the abnormalities of ICCs and enteric neurons. In order to ascertain whether the development of ICCs is also perturbed in the aganglionic segment of the gut, we used Dominant megacolon (Dom) mice as an animal model and an antibody specific to c-kit (a tyrosine kinase receptor expressed in ICCs) to examine the spatial distribution of ICCs in the developing and adult colon. Our results showed that in the wild-type adult colon, ICCs were bipolar in shape in the smooth muscle layer and dense networks of ICCs were observed in the myenteric and deep muscular plexuses, whereas in the aganglionic colon of Dom heterozygous mice, no ICCs could be found in the smooth muscle layer and disrupted patterns of ICC networks were observed in the myenteric and deep muscular plexuses. On 14.5 days of development, the levels of expression of c-kit and its ligand (stem cell factor) were similar in the wild-type, heterozygous and homozygous Dom embryos. However, by 18.5 days, the numbers of ICCs were greatly reduced in the myenteric and submucosal plexuses of the terminal colon of the heterozygous embryos comparing with the wild-type counterparts. Based on our results, we postulate that the abnormal development of ICCs may disrupt the neurotransmission and pacemaker activity of the gut, leading to interrupted peristalsis.

During early embryonic development, cell migration is one of the most important morphogenetic processes. Neural crest cells arise from the dorsal part of the neural tube and migrate along different pathways to numerous locations where they differentiate into a variety of tissues. In the mouse, studies of neural crest cell migration have been difficult partly because of the absence of specific markers which can label neural crest cells throughout their migration from their origin to the site of differentiation. Nevertheless, the use of different experimental strategies involving extrinsic, intrinsic or genetic cell markers has already led to a good understanding of this migration. In our studies, extrinsic markers such as wheat germ agglutinin-gold conjugates and DiI and genetic markers including Hoxb2-lacZ and green fluorescent protein have been employed in tracing migrating neural crest cells. The labelling procedures and the strength and weaknesses of the tracing methods are reviewed herein.

The lysosome is a membrane-bound organelle involved in the turnover of various intracellular and extracellular macromolecules. These are degraded by acidic hydrolases in the lumen of lysosome. The lysosomal membrane is important not only in retaining the acidic hydrolases to protect cells against cytosolic proteolysis, but it also facilitates protein trafficking though organelle fusion. In this study, we report on a novel lysosomal membrane protein transmembrane 6 superfamily 1 (Tm6sf1). Expression of Tm6sf1-DsRed fusion proteins in HEK293A cells displayed punctate or ringlike vesicles, which colocalized with conventional lysosome markers including LAMP1/2, RAB7, and Rnf167. Using fluorescence time-lapse live cell imaging, we demonstrated the fusion of Tm6sf1 vesicles with lysosomes and the integration of Tm6sf1 into the lysosomal membrane. We also examined the expression of Tm6sf1 in mouse tissues and found immunopositive signals in major organs such as the cerebellum, kidney, and intestine. These data suggest that Tm6sf1 is a widely expressed lysosomal transmembrane protein and can be used as a novel marker of lysosome.

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.