Synergistic effects of the SAPK/JNK and the proteasome pathway on glial fibrillary acidic protein (GFAP) accumulation in Alexander disease

Protein aggregates in astrocytes that contain glial fibrillary acidic protein (GFAP), small heat shock proteins, and ubiquitinated proteins are termed Rosenthal fibers and characterize Alexander disease, a leukodystrophy caused by heterozygous mutations in GFAP. The mechanisms responsible for the massive accumulation of GFAP in Alexander disease remain unclear. In this study, we show that overexpression of both wild type and R239C mutant human GFAP led to cytoplasmic inclusions. GFAP accumulation also led to a decrease of proteasome activity and an activation of the MLK2-JNK pathway. In turn, the expression of activated mixed lineage kinases (MLKs) induced JNK activation and increased GFAP accumulation, whereas blocking the JNK pathway decreased GFAP accumulation. Activated MLK also inhibited proteasome function. A direct inhibition of proteasome function pharmacologically further activated JNK. Our data suggest a synergistic interplay between the proteasome and the SAPK/JNK pathway in the context of GFAP accumulation. Feedback interactions among GFAP accumulation, SAPK/JNK activation, and proteasomal hypofunction cooperate to produce further protein accumulation and cellular stress responses.     

Cellular therapies in motor neuron diseases

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are prototypical motor neuron diseases that result in progressive weakness as a result of motor neuron dysfunction and death. Though much work has been done in both diseases to identify the cellular mechanisms of motor neuron dysfunction, once motor neurons have died, one of potential therapies to restore function would be through the use of cellular transplantation. In this review, we discuss potential strategies whereby cellular therapies, including the use of stem cells, neural progenitors and cells engineered to secrete trophic factors, may be used in motor neuron diseases. We review pre-clinical data in rodents with each of these approaches and discuss advances and regulatory issues regarding the use of cellular therapies in human motor neuron diseases.     

A computational model of oxygen transport in skeletal muscle for sprouting and splitting modes of angiogenesis

Oxygen transport from capillary networks in muscle at a high oxygen consumption rate was simulated using a computational model to assess the relative efficacies of sprouting and splitting modes of angiogenesis. Efficacy was characterized by the volumetric fraction of hypoxic tissue and overall heterogeneity of oxygen distribution at steady state. Oxygen transport was simulated for a three-dimensional vascular network using parameters for rat extensor digitorum longus (EDL) muscle when oxygen consumption by tissue reached 6, 12, and 18 times basal consumption. First, a control network was generated by using straight non-anastomosed capillaries to establish baseline capillarity. Two networks were then constructed simulating either abluminal lateral sprouting or intraluminal splitting angiogenesis such that capillary surface area was equal in both networks. The sprouting network was constructed by placing anastomosed capillaries between straight capillaries of the control network with a higher probability of placement near hypoxic tissue. The splitting network was constructed by splitting capillaries from the control network into two branches at randomly chosen branching points. Under conditions of moderate oxygen consumption (6 times basal), only minor differences in oxygen delivery resulted between the sprouting and splitting networks. At higher consumption levels (12 and 18 times basal), the splitting network had the lowest volume of hypoxic tissue of the three networks. However, when total blood flow in all three networks was made equal, the sprouting network had the lowest volume of hypoxic tissue. This study also shows that under the steady-state conditions the effect of myoglobin (Mb) on oxygen transport was small.     

Functional characterization of the putative Aspergillus nidulans poly(ADP-ribose) polymerase homolog PrpA

Poly(ADP-ribose) polymerase (PARP) is a highly conserved enzyme involved in multiple aspects of animal and plant cell physiology. For example, PARP is thought to be intimately involved in the early signaling events that trigger the DNA damage response. However, the genetic dissection of PARP function has been hindered by the presence of multiple homologs in most animal and plant species. Here, we present the first functional characterization of a putative PARP homolog (PrpA) in a microbial system (Aspergillus nidulans). PrpA belongs to a group of PARP homologs that includes representatives from filamentous fungi and protists. The genetic analysis of prpA demonstrates that it is an essential gene whose role in the DNA damage response is sensitive to gene dosage. Notably, temporal patterns of prpA expression and PrpA-GFP nuclear localization suggest that PrpA acts early in the A. nidulans DNA damage response. Additional studies implicate PrpA in farnesol-induced cell death and in the initiation of asexual development. Collectively, our results provide a gateway for probing the diverse functions of PARP in a sophisticated microbial genetic system.     

Influence of gonadal sex hormones on behavioral components of the reproductive hierarchy in naked mole-rats

Naked mole-rats (Heterocephalus glaber) are fossorial, eusocial rodents that live in colonies which typically include 60-80 individuals. Generally, only one of the females and 1-3 of the males in a colony are reproductives. The reproductives engage in mutual genital nuzzling behavior that is rarely exhibited by subordinates (non-reproductives). Thus, genital nuzzling may represent a mechanism of bonding and/or specific recognition between reproductive individuals. We investigated whether gonadal hormones are involved in the maintenance of genital nuzzling behavior and mating behaviors in isolated pairs of mole-rats and also in established breeding pairs of mole-rats within colonies. We also explored whether sex hormone deprivation would alter the strict partner preference for performance of nuzzling within colonies. Our results indicate (a) considerable variation between pairs in the frequency of nuzzling, (b) a reduction in the frequency of nuzzling following castration of the male and restoration of the 'baseline' frequency after replacement of testosterone in castrated males, (c) the failure of either castration or combined castration and ovariectomy to eliminate genital nuzzling in established pairs, and (d) the exhibition of nuzzling behavior by some of the subordinates in all three experimental colonies beginning several weeks after gonadectomy of both of the reproductives. No cases of lordosis behavior were seen during the approximately 109 h of behavioral observations. This is not surprising, since female mole-rats have an approximately 30-day ovulatory cycle, and lordosis only occurs during a peri-ovulatory period of a few hours. A total of 44 cases of mounting behavior were recorded; all these involved breeding males in colonies or males from isolated pairs, and all occurred when males were either gonad-intact or castrated with testosterone replacement. Thus, in contrast to nuzzling behavior, male sex behavior appeared to be eliminated during androgen deprivation.     

Assembling an intermediate filament network by dynamic cotranslation

We have been able to observe the dynamic interactions between a specific messenger RNA (mRNA) and its protein product in vivo by studying the synthesis and assembly of peripherin intermediate filaments (IFs). The results show that peripherin mRNA-containing particles (messenger ribonucleoproteins [mRNPs]) move mainly along microtubules (MT). These mRNPs are translationally silent, initiating translation when they cease moving. Many peripherin mRNPs contain multiple mRNAs, possibly amplifying the total amount of protein synthesized within these "translation factories." This mRNA clustering is dependent on MT, regulatory sequences within the RNA and the nascent protein. Peripherin is cotranslationally assembled into insoluble, nonfilamentous particles that are precursors to the long IF that form extensive cytoskeletal networks. The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation.     

A reporter-assisted mutagenesis screen using alpha 1-tubulin-GFP transgenic zebrafish uncovers missteps during neuronal development and axonogenesis

Alpha1-tubulin expression occurs in a neural-specific, temporally regulated, and regeneration-inducible fashion in zebrafish. A GFP reporter driven by the alpha1-tubulin promoter in transgenic zebrafish acts as a stable, in vivo molecular tag that follows neuronal development from birth/specification through postmitotic differentiation to axonal outgrowth and synaptogenesis. We exploited this transgenic system in a reporter expression-dependent (morphology-independent) mutagenesis screen to identify disruptions in genetic loci essential for neuronogenesis and axon elaboration, which would manifest as visually appreciable perturbations in GFP fluorescence. Thirty-two such recessive mutations were obtained, a subset of which was screened through a secondary RNA quantification-based assay to eliminate housekeeping gene defects. Three representative loci, when characterized in detail, were found to exhibit missteps in discrete, sequential stages of embryonic neuronal development. Mutation in sookshma panneurally diminishes the neural precursor pool by affecting cell proliferation in the developing embryo while patterning along the neuraxis remains unperturbed. Disruption of drishti on the other hand ameliorates the mitotic neural population by affecting cell cycle exit of progenitors and stalling their progression to the postmitotic neuronal stage, without impairing subsequent cell fate determination or differentiation. Finally, dhruva is required during neuronal differentiation for axonal branching and terminal innervation in spinal motoaxons and the retinotectal projection. Molecular identification of these loci and analysis of the remaining mutational repertoire will offer unique insights into the genetic inputs that go on to make a mature, differentiated neuron.