Joanna C. Bakowska

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury

Fibroblast growth factor-2 (FGF-2) promotes proliferation of neuroprogenitor cells in culture and is up-regulated within brain after injury. Using mice genetically deficient in FGF-2 (FGF-2−/− mice), we addressed the importance of endogenously generated FGF-2 on neurogenesis within the hippocampus, a structure involved in spatial, declarative, and contextual memory, after seizures or ischemic injury. BrdUrd incorporation was used to mark dividing neuroprogenitor cells and NeuN expression to monitor their differentiation into neurons. In the wild-type strain, hippocampal FGF-2 increased after either kainic acid injection or middle cerebral artery occlusion, and the numbers of BrdUrd/NeuN-positive cells significantly increased on days 9 and 16 as compared with the controls. In FGF-2−/− mice, BrdUrd labeling was attenuated after kainic acid or middle cerebral artery occlusion, as was the number of neural cells colabeled with both BrdUrd and NeuN. After FGF-2−/− mice were injected intraventricularly with a herpes simplex virus-1 amplicon vector carrying FGF-2 gene, the number of BrdUrd-labeled cells increased significantly to values equivalent to wild-type littermates after kainate seizures. These results indicate that endogenously synthesized FGF-2 is necessary and sufficient to stimulate proliferation and differentiation of neuroprogenitor cells in the adult hippocampus after brain insult.

Gene therapy in the CNS

Gene therapy for neurological disorder is currently an experimental concept. The goals for clinical utilization are the relief of symptoms, slowing of disease progression, and correction of genetic abnormalities. Experimental studies are realizing these goals in the development of gene therapies in animal models. Discoveries of the molecular basis of neurological disease and advances in gene transfer systems have allowed focal and global delivery of therapeutic genes for a wide variety of CNS disorders. Limitations are still apparent, such as stability and regulation of transgene expression, and safety of both vector and expressed transgene. In addition, the brain adds several challenges not seen in peripheral gene therapy paradigms, such as post-mitotic cells, heterogeneity of cell types and circuits, and limited access. Moreover, it is likely that several modes of gene delivery will be necessary for successful gene therapies of the CNS. Collaborative efforts between clinicians and basic researchers will likely yield effective gene therapy in the CNS.

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.

Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets

BackgroundSpartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.ResultsIn this study, we show that spartin is not a substrate for AIP4 activity and that spartins binding to AIP4 significantly increases self-ubiquitination of this E3 ligase, indicating that spartin disrupts the AIP4 autoinhibitory intramolecular interaction. Correspondingly, spartin has a seven times higher binding affinity to the WW region of AIP4 than the binding of the WW region has to the catalytic homologues of the E6-associated protein C-terminus (HECT) domain, as measured by enzyme-linked immunosorbent assay. We also show that spartin recruits AIP4 to lipid droplets and promotes ubiquitination of lipid droplet-associated protein, adipophilin, which regulates turnover of lipid droplets.ConclusionsOur findings demonstrate that spartin acts as an adaptor protein that activates and recruits AIP4 E3 ubiquitin ligase to lipid droplets and by this means regulates the level of ubiquitination of adipophilin and potentially other lipid-associated proteins. We propose that this is one of the mechanisms by which spartin regulates lipid droplet turnover and might contribute to the pathology of SPG20.

SPG20 protein spartin is recruited to midbodies by ESCRT-III protein Ist1 and participates in cytokinesis.

This study demonstrates that the SPG20 hereditary spastic paraplegia protein spartin interacts with the ESCRT-III protein Ist1. This interaction is required for completion of the abscission phase of cytokinesis.

The distribution of mRNA for the short form of the prolactin receptor in the forebrain of the female rat.

Prolactin exerts its diverse effects on peripheral tissue and on the brain via receptors that have two forms, a short form and a long form. The distribution of the mRNA for both forms of the receptor has been examined in brain and peripheral tissue regions using methods based on regional dissection. Although the cell-specific distribution of the long form of the prolactin receptor has been examined using in situ hybridization in the rat brain, the cell-specific distribution of the short form has not been described. In this study we mapped the distribution of neurons and other cells expressing the short from of the receptor transcript in the forebrain, ovary, and uterus of the female rat by using in situ hybridization with a 33P-labeled cRNA probe specific for the short form of the prolactin receptor mRNA (PRL-SR mRNA). Neurons expressing the PRL-SR mRNA were located predominantly in the preoptic area and hypothalamus as well as in certain limbic structures. Specific nuclei included the anteroventral periventricular nucleus, paraventricular and supraoptic nucleus, medial preoptic area, suprachiasmatic nucleus, and ventromedial and arcuate nuclei of the hypothalamus, as well as the bed nucleus of stria terminalis and the medial amygdala. Scattered neurons expressing PRL-SR mRNA were also found in the cortex, habenula, zona incerta, and thalamus. Cells in the choroid plexus expressed high levels of PRL-SR mRNA, as did the luteal cells of the corpus luteum and the epithelial cells of the uterine glands. These data confirm previous reports and extend our knowledge of the distribution of the short form of the receptor to the cellular level. The neuroanatomic distribution of neurons expressing PRL-SR mRNA suggests that they may influence the mediation and coordination of prolactin-regulated endocrine and behavioral events.

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep+ hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep+, rep− (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep+ hybrid amplicon vector than with rep− or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep+ hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep− or standard amplicon vectors resulted in random integrations of the entire rep− hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

Lack of Spartin Protein in Troyer Syndrome A Loss-of-Function Disease Mechanism?

BACKGROUND Hereditary spastic paraplegias (SPG1-SPG33) are characterized by progressive spastic weakness of the lower limbs. A nucleotide deletion (1110delA) in the (SPG20; OMIM 275900) spartin gene is the origin of autosomal recessive Troyer syndrome. This mutation is predicted to cause premature termination of the spartin protein. However, it remains unknown whether this truncated spartin protein is absent or is present and partially functional in patients. OBJECTIVE To determine whether the truncated spartin protein is present or absent in cells derived from patients with Troyer syndrome. DESIGN Case report. SETTING Academic research. PATIENTS We describe a new family with Troyer syndrome due to the 1110delA mutation. MAIN OUTCOME MEASURES We cultured primary fibroblasts and generated lymphoblasts from affected individuals, carriers, and control subjects and subjected these cells to immunoblot analyses. RESULTS Spartin protein is undetectable in several cell lines derived from patients with Troyer syndrome. CONCLUSIONS Our data suggest that Troyer syndrome results from complete loss of spartin protein rather than from the predicted partly functional fragment. This may reflect increased protein degradation or impaired translation.

Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown. Here we demonstrate that mixed-lineage kinase 3 (MLK3), a MAP3K family member, phosphorylates Pin1 on a Ser138 site to increase its catalytic activity and nuclear translocation. This phosphorylation event drives the cell cycle and promotes cyclin D1 stability and centrosome amplification. Notably, Pin1 pSer138 is significantly up-regulated in breast tumors and is localized in the nucleus. These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role in regulating the cell cycle, centrosome numbers, and oncogenesis.

Spg20−/− mice reveal multimodal functions for Troyer syndrome protein spartin in lipid droplet maintenance, cytokinesis and BMP signaling

Hereditary spastic paraplegias (HSPs; SPG1-48) are inherited neurological disorders characterized by lower extremity spasticity and weakness. Loss-of-function mutations in the SPG20 gene encoding spartin cause autosomal recessive Troyer syndrome (SPG20), which has additional features of short stature, cognitive deficits and distal amyotrophy. To identify cellular impairments underlying Troyer syndrome, we generated Spg20-/- mice, which exhibit progressive gait defects. Although gross central nervous system pathology appeared largely normal, cerebral cortical neurons cultured from neonatal Spg20-/- mice exhibited increased axon branching, a phenotype suppressed by reintroducing spartin and which required its interaction with the endosomal sorting complex required for transport (ESCRT)-III protein IST1. Analysis of the bone morphogenetic protein (BMP) signaling pathway in Spg20-/- embryonic fibroblasts indicated that Smad1/5 phosphorylation is modestly elevated, possibly due to alterations in BMP receptor trafficking. Cytokinesis was impaired in embryonic fibroblasts cultured from Spg20-/- mice, and binucleated chondrocytes were prominent in epiphyseal growth plates of bones in Spg20-/- mice, perhaps explaining the short stature of patients. Finally, adipose tissue from Spg20-/- female mice exhibited increased lipid droplet (LD) numbers and alterations in perilipin levels, supporting a role for spartin in LD maintenance. Taken together, our results support multimodal functions for spartin that provide important insights into HSP pathogenesis.