Disease-Associated Q159X Mutant Prion Protein Is Sufficient to Cause Fatal Degenerative Disease in Mice.

PRNP Q160X is one of the five dominantly inheritable nonsense mutations causing familial prion diseases. Till now, it remains unclear how this type of nonsense mutations causes familial prion diseases with unique clinical and pathological characteristics. Human prion protein (PrP) Q160X mutation is equivalent to Q159X in mouse PrP, which produces the mutant fragment PrP1-158. Through intracerebroventricular injection of recombinant adeno-associated virus in newborn mice, we successfully overexpressed mouse PrP1-158-FLAG in the central nervous system. Interestingly, high level PrP1-158-FLAG expression in the brain caused death in these mice with an average survival time of 60 ± 9.1 days. Toxicity correlated with levels of PrP1-158-FLAG but was independent of endogenous PrP. Histopathological analyses showed microgliosis and astrogliosis in mouse brains expressing PrP1-158-FLAG and most of PrP1-158-FLAG staining appeared intracellular. Biochemical characterization revealed that the majority of PrP1-158-FLAG were insoluble and a significant part of PrP1-158-FLAG appeared to contain an un-cleaved signal peptide that may contribute to its cytoplasmic localization. Importantly, an ~10-kDa proteinase K-resistant PrP fragment was detected, which was the same as those observed in patients suffering from this type of prion diseases. To our knowledge, this is the first animal study of familial prion disease caused by Q159X that recapitulates key features of human disease. It will be a valuable tool for investigating the pathogenic mechanisms underlying familial prion diseases caused by nonsense mutations.

Recombinant Mammalian Prions: The "Correctly" Misfolded Prion Protein Conformers.

Generating a prion with exogenously produced recombinant prion protein is widely accepted as the ultimate proof of the prion hypothesis. Over the years, a plethora of misfolded recPrP conformers have been generated, but despite their seeding capability, many of them have failed to elicit a fatal neurodegenerative disorder in wild-type animals like a naturally occurring prion. The application of the protein misfolding cyclic amplification technique and the inclusion of non-protein cofactors in the reaction mixture have led to the generation of authentic recombinant prions that fully recapitulate the characteristics of native prions. Together, these studies reveal that recPrP can stably exist in a variety of misfolded conformations and when inoculated into wild-type animals, misfolded recPrP conformers cause a wide range of outcomes, from being completely innocuous to lethal. Since all these recPrP conformers possess seeding capabilities, these results clearly suggest that seeding activity alone is not equivalent to prion activity. Instead, authentic prions are those PrP conformers that are not only heritable (the ability to seed the conversion of normal PrP) but also pathogenic (the ability to cause fatal neurodegeneration). The knowledge gained from the studies of the recombinant prion is important for us to understand the pathogenesis of prion disease and the roles of misfolded proteins in other neurodegenerative disorders.

The effect of P85 on neuronal proliferation and differentiation during development of mouse cerebral cortex.

Proliferation of neural stem cells and differentiation of newly generated cells are crucial steps during the development of mammalian neocortex, which are able to generate suitable number of neurons and glial cells to ensure normal formation of cortex. Any disturbance in these processes leads to structural and functional abnormalities of cerebral cortex, such as epilepsy or intellectual disability. Numerous molecules involved in the development of disorders of the nervous system have been discovered in the recent years. The PI3K/AKT signaling pathway has been shown to be widely involved in the corticogenesis. Recently we could show that overexpression of regulatory subunit P85 of PI3K disrupts neuronal migration. However, it remains unclear whether the regulatory subunit P85 plays a role in the proliferation of neural stem cells and differentiation of newly generated cells during mouse brain development. Here, by using in utero electroporation and immunohistochemistry, we show that overexpression of P85 inhibited proliferation of neural progenitor cells and neuronal differentiation. By using 5-bromo-2-deoxyuridine (BrdU) labeling, we reveal that overexpression of P85 extended the cell cycle duration, which may result in developmental retardation during mouse corticogenesis.

P85 regulates neuronal migration through affecting neuronal morphology during mouse corticogenesis.

In mammalian developing embryonic cortex, projection neurons migrate from the ventricular zone to the cortical plate, guided by radial glial cells with a transformation between bipolar and multipolar morphology. Previous studies have demonstrated that the PI3K-Akt-mTOR signal plays a critical role in brain development. However, the function of P85 in cortical development is still unclear. In the present study, we found that overexpression of P85 impaired cortical neuronal migration. Using in utero electroporation, we revealed that the length of the leading process in P85 overexpressed neurons became shorter than that in the control group but with more branches. Using markers for new-born neurons, we further found that overexpression of P85 did not affect the ultimate fate of these cortical neurons. These findings indicated that the P85 subunit plays an essential role in neuronal migration and neuronal morphology during mouse corticogenesis.

The function of sperm-associated antigen 6 in neuronal proliferation and differentiation.

Sperm-associated antigen 6 (SPAG6) is initially found in human testis and is essential for sperm motility and male fertility. Later studies indicate that it also express in the chick Central Nervous System and human embryonic stem cells. However, the function of Spag6 in cortical development is still largely unclear. Using in utero electroporation, we showed that overexpression of Spag6 induced the transfected cells excluded from the proliferation zone of the mouse cortex. Ki67 Co-labeling and BrdU incorporation experiment suggested that overexpression of Spag6 inhibited proliferation of neural progenitor cells. Furthermore, we demonstrated that Spag6-overexpressing cells preferred to differentiated into neurons, which could be labeled by Brn2, rather than GFAP positive astrocytes. Taken together, our data indicate that Spag6 plays an essential role in the process of neuronal proliferation and differentiation.

Spag6 Negatively Regulates Neuronal Migration During Mouse Brain Development.

Sperm-associated antigen 6 (Spag6) is a Chlamydomonas reinhardtii PF16 homologous gene detected in the human testis and is crucial for sperm motility. Neuronal migration is a dynamic process requiring coordinated cytoskeletal remodeling, and Spag6 is co-localized with microtubules in Chinese hamster ovary cells and COS-1 cells. However, the role of Spag6 in neuronal migration remains unclear. Here, we demonstrated that Spag6 was continuously expressed in the developing cerebral cortex. Using in utero electroporation (IUE), we found that overexpression of Spag6 delayed the rate of neuronal migration, rather than affecting the ultimate fate of cortical neurons. Furthermore, overexpression of Spag6 caused a significant decrease in neurite number and length of cortical neurons. Our results indicated that Spag6 controlled neuronal migration as well as neurite branching and elongation.

The mouse radial spoke protein 3 is a nucleocytoplasmic shuttling protein that promotes neurogenesis.

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of radial spoke, which is important for flagellar motility. The mammalian homolog of the Chlamydomonas RSP3 protein is found to be a mammalian protein kinase A-anchoring protein that binds ERK1/2. Here we show that mouse RSP3 is a nucleocytoplasmic shuttling protein. The full-length RSP3-EGFP fusion protein is mainly located in the cytoplasm of Chinese hamster ovary cells. However, by using deletion mutants of RSP3, we identified two nuclear localization signals and a nuclear export signal in RSP3. Moreover, using in utero electroporation, we found that overexpression of RSP3 in the developing cerebral cortex promotes neurogenesis. The layer II/III of the neocortex was much thicker in the RSP3-transfected region than that of the untransfected region in the neocortex. We also show that RSP3 is specifically located in the primary cilia of the radial glial cells, where it acts as a signaling mediator that regulates neurogenesis. Thus, our results suggest that RSP3 is a nucleocytoplasmic shuttling protein and plays an essential role in neurogenesis.

Subcellular localization and function of mouse radial spoke protein 3 in mammalian cells and central nervous system.

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of the radial spoke. The mammalian homologue of the Chlamydomonas RSP3 gene is mainly expressed in testis and developing central nervous system (CNS). However, the subcellular localization and function of mammalian RSP3 in the developing brain and mammalian cells remain poorly understood. Here we show that the mouse RSP3 accumulates at the perinuclear region of Chinese hamster ovary (CHO) and 293T cells. Detailed analysis shows that the mouse RSP3 is not co-localized with the endoplasmic reticulum or Golgi apparatus markers in CHO cells. Using in utero electroporation, we found that over-expression of mammalian RSP3 increases the percentage of neurons reaching the upper cortical plate. In vivo analysis shows that the mouse RSP3 mainly accumulates in the proximal cytoplasmic dilation of the leading process of the migrating cortical neurons. Furthermore, we find that the mammalian RSP3 concentrates in the ependymal cilia as a component of the cilia. Thus, our data provide the first evidence for the subcellular localization and function of mammalian RSP3 in mammalian cells and developing CNS.

Effective suppression of acrylamide neurotoxicity by lithium in mouse.

The primary objective of this investigation was to assess the neuroprotective efficacy of lithium in an acrylamide (ACR)-induced neuropathy model in mice. In this study, Kunming male mice were administered ACR (25 mg/kg bw, i.p. once a day) with or without lithium (25 mg/kg bw, i.p. once a day) for 2 weeks. All ACR-administered mice exhibited severe symptoms of neuropathy. We found that treatment with lithium effectively alleviated behavioral deficits in animals elicited by acrylamide. Interestingly, the reduction of hippocampal neurogenesis resulting from ACR injection was promoted by administration of lithium. Further, lithium treatment significantly offset ACR-induced depletion in p-GSK-3ß (Ser9) levels in hippocampus. Collectively our findings suggest the propensity of lithium to attenuate ACR-induced neuropathy. Further studies are necessary to understand the precise molecular mechanism by which the lithium attenuates neuropathy. Nevertheless, our data clearly demonstrate the beneficial effects of lithium on ACR-induced neuropathy in mice and suggest its possible therapeutic application as an adjuvant in the management of other forms of neuropathy in humans.

Expression of macrophage migration inhibitory factor in the mouse neocortex and posterior piriform cortices during postnatal development.

Macrophage migration inhibitory factor (MIF) functions as a pleiotropic protein, participating in a vast array of cellular and biological processes. Abnormal expression of MIF has been implicated in many neurological diseases, including Parkinson's disease, epilepsy, Alzheimer's Disease, stroke, and neuropathic pain. However, the expression patterns of mif transcript and MIF protein from the early postnatal period through adulthood in the mouse brain are still poorly understood. We therefore investigated the temporal and spatial expression of MIF in the mouse neocortex during postnatal development in detail and partially in posterior piriform cortices (pPC). As determined by quantitative real-time PCR (qPCR), mif transcript gradually increased during development, with the highest level noted at postnatal day 30 (P30) followed by a sharp decline at P75. In contrast, Western blotting results showed that MIF increased constantly from P7 to P75. The highest level of MIF was at P75, while the lowest level of MIF was at P7. Immunofluorescence histochemistry revealed that MIF-immunoreactive (ir) cells were within the entire depth of the developed neocortex, and MIF was heterogeneously distributed among cortical cells, especially at P7, P14, P30, and P75; MIF was abundant in the pyramidal layer within pPC. Double immunostaining showed that all the mature neurons were MIF-ir and all the intensely stained MIF-ir cells were parvalbumin positive (Pv +) at adult. Moreover, it was demonstrated that MIF protein localized in the perikaryon, processes, presynaptic structures, and the nucleus in neurons. Taken together, the developmentally regulated expression and the subcellular localization of MIF should form a platform for an analysis of MIF neurodevelopmental biology and MIF-related nerve diseases.