Song Lu

Cerebellar defects in Pdss2 conditional knockout mice during embryonic development and in adulthood

PDSS2 is a gene that encodes one of the two subunits of trans-prenyl diphosphate synthase that is essential for ubiquinone biosynthesis. It is known that mutations in PDSS2 can cause primary ubiquinone deficiency in humans and a similar disease in mice. Cerebellum is the most often affected organ in ubiquinone deficiency, and cerebellar atrophy has been diagnosed in many infants with this disease. In this study, two Pdss2 conditional knockout mouse lines directed by Pax2-cre and Pcp2-cre were generated to investigate the effect of ubiquinone deficiency on cerebellum during embryonic development and in adulthood, respectively. The Pdss2(f/-); Pax2-cre mouse recapitulates some symptoms of ubiquinone deficiency in infants, including severe cerebellum hypoplasia and lipid accumulation in skeletal muscles at birth. During early cerebellum development (E12.5-14.5), Pdss2 knockout initially causes the delay of radial glial cell growth and neuron progenitor migration, so the growth of mutant cerebellum is retarded. During later development (E15.5-P0), increased ectopic apoptosis of neuroblasts and impaired cell proliferation result in the progression of cerebellum hypoplasia in the mutant. Thus, the mutant cerebellum contains fewer neurons at birth, and the cells are disorganized. The developmental defect of mutant cerebellum does not result from reduced Fgf8 expression before E12.5. Electron microscopy reveals mitochondrial defects and increased autophagic-like vacuolization that may arise in response to abnormal mitochondria in the mutant cerebellum. Nevertheless, the mutant mice die soon after birth probably due to cleft palate and micrognathia, which may result from Pdss2 knockout caused by ectopic Pax2-cre expression in the first branchial arch. On the other hand, the Pdss2(f/-); Pcp2-cre mouse is healthy at birth but gradually loses cerebellar Purkinje cells and develops ataxia-like symptoms at 9.5 months; thus this conditional knockout mouse may serve as a model for ubiquinone deficiency in adult patients. In conclusion, this study provides two mouse models of Pdss2 based ubiquinone deficiency. During cerebellum development, Pdss2 knockout results in severe cerebellum hypoplasia by impairing cell migration and eliciting ectopic apoptosis, whereas Pdss2 knockout in Purkinje cells at postnatal stages leads to the development of cerebellar ataxia.

LRRK2 R1441G mice are more liable to dopamine depletion and locomotor inactivity

Mutations in leucine‐rich repeat kinase 2 (LRRK2) pose a significant genetic risk in familial and sporadic Parkinsons disease (PD). R1441 mutation (R1441G/C) in its GTPase domain is found in familial PD. How LRRK2 interacts with synaptic proteins, and its role in dopamine (DA) homeostasis and synaptic vesicle recycling remain unclear.

Inducible Activation of ERK5 MAP Kinase Enhances Adult Neurogenesis in the Olfactory Bulb and Improves Olfactory Function

Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury.

Methods to measure olfactory behavior in mice

Mice rely on the sense of olfaction to detect food sources, recognize social and mating partners, and avoid predators. Many behaviors of mice, including learning and memory, social interaction, fear, and anxiety are closely associated with their function of olfaction, and behavior tasks designed to evaluate those brain functions may use odors as cues. Accurate assessment of olfaction is not only essential for the study of olfactory system but also critical for proper interpretation of various mouse behaviors, especially learning and memory, emotionality and affect, and sociality. Here we describe a series of behavior experiments that offer multidimensional and quantitative assessments for mouse olfactory function, including olfactory habituation, discrimination, odor preference, odor detection sensitivity, and olfactory memory, with respect to both social and nonsocial odors.

Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system.

Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10-9-10-7M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.

The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb.

The type 3 adenylyl cyclase (AC3) is localized to olfactory cilia in the main olfactory epithelium (MOE) and primary cilia in the adult mouse brain. Although AC3 has been strongly implicated in odor perception and olfactory sensory neuron (OSN) targeting, its role in granule cells (GCs), the most abundant interneurons in the main olfactory bulb (MOB), remains largely unknown. Here, we report that the deletion of AC3 leads to a significant reduction in the size of the MOB as well as the level of adult neurogenesis. The cell proliferation and cell cycle in the subventricular zone (SVZ), however, are not suppressed in AC3−/− mice. Furthermore, AC3 deletion elevates the apoptosis of GCs and disrupts the maturation of newly formed GCs. Collectively, our results identify a fundamental role for AC3 in the development of adult-born GCs in the MOB.

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

Individual Variation in Conditional β Cell Ablation Mice Contributes Significant Biases in Evaluating β Cell Functional Recovery

Despite the βDTA (Ins2-rtTA; Tet-DTA) mice have been developed as a valuable tool to study β cell regeneration, their individual variation in therapeutic efficacy has not been characterized. Here, we demonstrated that the βDTA mice exhibited significant variations in both spontaneous and acquired β cell regeneration. We found that doxycycline (DOX)-induced β cell death was sufficient to cause polydipsia, translating even subtle difference in drinking habit into large variations in actual DOX intake among individuals within the same group. Accumulating evidence shows that transient expression of VEGFA enhances β cell functional recovery after injury. Therefore, we utilized the chemically modified mRNA (modRNA) technology to enable transient yet efficient VEGFA expression in the pancreas after DOX-induced β cell death. Surprisingly, under optimized DOX dose permissive of β cell regeneration, VEGFA modRNA only demonstrated marginal benefits on β cell functional recovery with large individual variations. We also revealed that the therapeutic efficacy of VEGFA modRNA on β cell regeneration was dependent on the degree of β cell loss induced by the accumulated DOX intake. Therefore, our results highlight a significant contribution of individual variation in the βDTA model and call for attention in evaluating potential efficacy of therapeutic agents in β cell regeneration studies.