Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis.

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.

Two Metabolic Fuels, Glucose and Lactate, Differentially Modulate Exocytotic Glutamate Release from Cultured Astrocytes.

Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis. Astrocytes synthesize glutamate de novo owing to the pyruvate entry to the citric/tricarboxylic acid cycle via pyruvate carboxylase, an astrocyte specific enzyme. Pyruvate can be sourced from two metabolic fuels, glucose and lactate. Thus, we investigated the role of these energy/carbon sources in exocytotic glutamate release from astrocytes. Purified astrocyte cultures were acutely incubated (1 h) in glucose and/or lactate-containing media. Astrocytes were mechanically stimulated, a procedure known to increase intracellular Ca2+ levels and cause exocytotic glutamate release, the dynamics of which were monitored using single cell fluorescence microscopy. Our data indicate that glucose, either taken-up from the extracellular space or mobilized from the intracellular glycogen storage, sustained glutamate release, while the availability of lactate significantly reduced the release of glutamate from astrocytes. Based on further pharmacological manipulation during imaging along with tandem mass spectrometry (proteomics) analysis, lactate alone, but not in the hybrid fuel, caused metabolic changes consistent with an increased synthesis of fatty acids. Proteomics analysis further unveiled complex changes in protein profiles, which were condition-dependent and generally included changes in levels of cytoskeletal proteins, proteins of secretory organelle/vesicle traffic and recycling at the plasma membrane in aglycemic, lactate or hybrid-fueled astrocytes. These findings support the notion that the availability of energy sources and metabolic milieu play a significant role in gliotransmission.

RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. METHODS: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. RESULTS: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. CONCLUSIONS: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT.

Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

Pharmacokinetic profile and quantitation of protection against soman poisoning by the antinicotinic compound MB327 in the guinea-pig.

Current organophosphorus nerve agent medical countermeasures do not directly address the nicotinic effects of poisoning. A series of antinicotinic bispyridinium compounds has been synthesized in our laboratory and screened in vitro. Their actions can include open-channel block at the nicotinic receptor which may contribute to their efficacy. The current lead compound from these studies, MB327 1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) as either the diiodide (I2) or dimethanesulfonate (DMS) has been examined in vivo for efficacy against nerve agent poisoning. MB327 I2 (0-113mgkg(-1)) or the oxime HI-6 DMS (0-100mgkg(- 1)), in combination with atropine and avizafone (each at 3mgkg(-1)) was administered to guinea-pigs 1min following soman poisoning. Treatment increased the LD50 of soman in a dose-dependent manner. The increase was statistically significant (p<0.01) at the 33.9mgkg(-1) (MB327) or 30mgkg(-1) (HI-6) dose with a comparable degree of protection obtained for both compounds. Following administration of 10mgkg(-1) (i.m.), MB327 DMS reached plasma Cmax of 22µM at 12min with an elimination t1/2 of 22min. In an adverse effect study, in the absence of nerve agent poisoning, a dose of 100mgkg(-1) or higher of MB327 DMS was lethal to the guinea-pigs. A lower dose of MB327 DMS (30mgkg(-1)) caused flaccid paralysis accompanied by respiratory impairment. Respiration normalised by 30min, although the animals remained incapacitated to 4h. MB327 or related compounds may be of utility in treatment of nerve agent poisoning as a component of therapy with atropine, anticonvulsant and oxime, or alternatively as an infusion under medical supervision.

Splice site, frameshift, and chimeric GFAP mutations in Alexander disease.

Alexander disease (AxD) is a usually fatal astrogliopathy primarily caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in astrocytes. We describe three patients with unique characteristics, and whose mutations have implications for AxD diagnosis and studies of intermediate filaments. Patient 1 is the first reported case with a noncoding mutation. The patient has a splice site change producing an in-frame deletion of exon 4 in about 10% of the transcripts. Patient 2 has an insertion and deletion at the extreme end of the coding region, resulting in a short frameshift. In addition, the mutation was found in buccal DNA but not in blood DNA, making this patient the first reported chimera. Patient 3 has a single-base deletion near the C-terminal end of the protein, producing a short frameshift. These findings recommend inclusion of intronic splice site regions in genetic testing for AxD, indicate that alteration of only a small fraction of GFAP can produce disease, and provide caution against tagging intermediate filaments at their C-terminal end for cell biological investigations.

Archetypal and new families with Alexander disease and novel mutations in GFAP.

OBJECTIVE: To describe genetic analyses of the 2 most thoroughly studied, historically seminal multigenerational families with Alexander disease described prior to the identification of GFAP as the related gene, as well as 1 newly discovered family. DESIGN: Clinical histories were obtained and DNA was analyzed from blood, cheek epithelial cells, or fixed paraffin-embedded surgical samples. SUBJECTS: Affected and unaffected adult members of 3 families and affected children were included. MAIN OUTCOME MEASURES: Mutations in GFAP and behavior of mutant protein in cellular transfection assays. RESULTS: Family A contains 4 siblings in whom we found a novel p.Ser247Pro mutation that was paternally inherited. The phenotypes of these siblings include 1 unaffected adult, 1 individual with juvenile-onset disease, and 2 individuals with adult-onset disease. Family B spans 4 generations, including the first described patient with adult-onset disease originally reported in 1968. Analysis of members of the later generations revealed a novel p.Asp417Ala mutation. Family C contains 3 generations. We detected a novel p.Gln426Leu mutation that, to our knowledge, is the farthest C-terminal mutation known. CONCLUSIONS: These families display clear evidence of variable phenotypes but do not support recessive inheritance. While germline mosaicism cannot be excluded for 1 family (A), we propose that for genetic counseling purposes the risk of germline mosaicism should be described as less than 1%.

Origins of helix-coil switching in a light-sensitive peptide.

Intramolecular cross-linking of peptides by the light-sensitive compound diiodoacetamideazobenzene has been shown to permit reversible photocontrol of the helix-coil transition. Cross-linking between Cys residues spaced at i and i + 7 positions with the trans form of the linker was found to produce a decreased helix content compared to that of the non-cross-linked peptide. Photoisomerization to the cis form of the linker led to substantially higher helix content than in the non-cross-linked peptide. Detailed conformational analysis of the system leads to the conclusion that photocontrol of helix content does not involve specific interactions between the linker and the peptide. Instead, the change in peptide helix content caused by photoisomerization can be predicted by comparing the length ranges of the cis and trans forms of the linker with the expected distance distribution of the Cys attachment points in the intrinsic conformational ensemble of the peptide. The analysis presented here should help to guide the use of these and related linkers for the conformational control of a variety of peptide and protein systems.

Achieving photo-control of protein conformation and activity: producing a photo-controlled leucine zipper.

We have recently developed a technique that has great potential in producing proteins with photo-control of conformation and consequently activity (J. R. Kumita, O. S. Smart and G. A. Woolley, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 3803-3808). The method is based on incorporating two cysteine residues into the sequence of a polypeptide. An azobenzene derivative is subsequently used to produce an intramolecular cross-link between the cysteine sulfhydryl groups. In previous work photo-isomerisation of the azobenzene moiety has been used to control the helicity of a monomeric peptide. In the experiments described here this method has been applied to the coiled coil leucine zipper peptide GCN4-p1. The aim was to produce a variant of GCN4-p1 whose helicity and consequently dimerisation is under direct photo-control. We have produced a modified GCN4-p1 incorporating two cysteine residues. The mutations introduced are shown to interfere with the ability of the uncross-linked peptide to form a coiled coil. After the peptide was cross-linked with the azobenzene derivative more normal coiled-coil behaviour was restored. Irradiation of the peptide producing a conformational change in the azobenzene cross-linker was accompanied by an increase in the helicity of the peptide. The work presented here highlights the potential of the use of photo-isomerisable cross-linkers to control protein activity through induced conformational change. In addition, the methodology has the potential to provide a fast trigger for the initiation of protein conformational changes.

Photo-control of peptide helix content by an azobenzene cross-linker: steric interactions with underlying residues are not critical.

Photo-control of protein conformation could prove useful for probing function in diverse biological systems. Recently, we reported photo-switching of helix content in a short peptide containing an azobenzene cross-linker between cysteine residues at positions i and i + 7 in the sequence. In the original sequence, underlying residues at positions i + 3 and i + 4 were made bulky as preliminary modelling suggested that this would enhance photo-control of helix content. To test this hypothesis, peptides with Val, Aib; Ile, Aib; and Ala, Ala at positions i + 3 and i + 4 were synthesized, cross-linked and characterized. Before cross-linking, the peptides show distinct conformational behaviours: two with differing helix/coil mixtures whereas the other has a circular dichroism (CD) spectrum characteristic of beta-sheet and a tendency to aggregate. However, upon cross-linking the peptides have very similar CD spectra: predominantly random coil in the dark but predominantly helical upon irradiation. These results refute the original hypothesis. Steric interactions between the linker and underlying residues do not appear to be critical for photo-switching behaviour. When the cross-linking bridge is lengthened by replacing the i, i + 7 cysteine residues with homocysteine, a lower degree of photo-control of helicity is observed. Furthermore, a non-cross-linking version of the azobenzene reagent is shown not to produce any photo-control of helicity. We conclude that the intramolecular cross-link is essential for photo-switching and that it should be applicable to a wide range of peptides and proteins.

Using an azobenzene cross-linker to either increase or decrease peptide helix content upon trans-to-cis photoisomerization.

Reversible photocontrol of peptide and protein conformation could prove to be a powerful tool for probing function in diverse biological systems. Here, we report reversible photoswitching of the helix content in short peptides containing an azobenzene cross-linker between cysteine residues at positions i, i + 4, or i, i + 11 in the sequence. Trans-to-cis photoisomerization significantly increases the helix content in the i, i + 4 case and significantly decreases the helix content in the i, i + 11 case. These cross-linker designs significantly expand the possibilities for photocontrol of peptide and protein structure.