Comparison of Type 1 D-3-phosphoglycerate dehydrogenases reveals unique regulation in pathogenic Mycobacteria.

D-3-phosphoglycerate dehydrogenases (PGDH) from all organisms catalyze the conversion of D-3-phosphoglycerate to phosphohydroxypyruvate as the first step in the biosynthesis of l-serine. This investigation compares the properties of Type 1 PGDHs from seven different species and demonstrates that conserved residues in the ACT and ASB domains of some allow l-serine to act as a feedback inhibitor at low micromolar concentrations. In addition, the serine sensitivity is dependent on the presence of phosphate ions. These residues are most highly conserved among PGDHs from the actinomycetales family, but only certain pathogenic mycobacteria appear to have the full complement of residues required for high sensitivity to serine. These basic residues are also responsible for the presence of dual pH optima in the acidic region that is also phosphate dependent. Analytical ultracentrifugation analysis demonstrates that the dual pH optima do not require changes in oligomeric state. This study also demonstrates that substrate inhibition is a common feature of Type 1 PGDHs and that it is suppressed by phosphate, indicating that phosphate likely interacts at both the catalytic and regulatory sites. The unique features resulting from the complement of basic residues conserved in pathogenic mycobacteria may impart important metabolic advantages to these organisms.

SAK3 confers neuroprotection in the neurodegeneration model of X-linked Dystonia-Parkinsonism.

Background X-linked Dystonia-Parkinsonism(XDP) is an adult-onset neurodegenerative disorder that results in the loss of striatal medium spiny neurons (MSNs). XDP is associated with disease-specific mutations in and around the TAF1 gene. This study highlights the utility of directly reprogrammed MSNs from fibroblasts of affected XDP individuals as a platform that captures cellular and epigenetic phenotypes associated with XDP-related neurodegeneration. In addition, the current study demonstrates the neuroprotective effect of SAK3 currently tested in other neurodegenerative diseases. Methods XDP fibroblasts from three independent patients as well as age- and sex-matched control fibroblasts were used to generate MSNs by direct neuronal reprogramming using miRNA-9/9*-124 and thetranscription factors CTIP2 , DLX1 -P2A- DLX2 , and MYT1L . Neuronal death, DNA damage, and mitochondrial health assays were carried out to assess the neurodegenerative state of directly reprogrammed MSNs from XDP patients (XDP-MSNs). RNA sequencing and ATAC sequencing were performed to infer changes in the transcriptomic and chromatin landscapesof XDP-MSNs compared to those of control MSNs (Ctrl-MSNs). Results Our results show that XDP patient fibroblasts can be successfully reprogrammed into MSNs and XDP-MSNs display several degenerative phenotypes, including neuronal death, DNA damage, and mitochondrial dysfunction, compared to Ctrl-MSNs reprogrammed from age- and sex-matched control individuals' fibroblasts. In addition, XDP-MSNs showed increased vulnerability to TNFα -toxicity compared to Ctrl-MSNs. To dissect the altered cellular state in XDP-MSNs, we conducted transcriptomic and chromatin accessibility analyses using RNA- and ATAC-seq. Our results indicate that pathways related to neuronal function, calcium signaling, and genes related to other neurodegenerative diseases are commonly altered in XDP-MSNs from multiple patients. Interestingly, we found that SAK3, a T-type calcium channel activator, that may have therapeutic values in other neurodegenerative disorders, protected XDP-MSNs from neuronal death. Notably, we found that SAK3-mediated alleviation of neurodegeneration in XDP-MSNs was accompanied by gene expression changes toward Ctrl-MSNs.

Longitudinal modeling of human neuronal aging reveals the contribution of the RCAN1-TFEB pathway to Huntington's disease neurodegeneration.

Aging is a common risk factor in neurodegenerative disorders. Investigating neuronal aging in an isogenic background stands to facilitate analysis of the interplay between neuronal aging and neurodegeneration. Here we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs) in Huntington's disease identified pathways involving RCAN1, a negative regulator of calcineurin. Notably, RCAN1 protein increased with age in reprogrammed MSNs as well as in human postmortem striatum and RCAN1 knockdown rescued patient-derived MSNs of Huntington's disease from degeneration. RCAN1 knockdown enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, leading to TFEB's nuclear localization by dephosphorylation. Furthermore, G2-115, an analog of glibenclamide with autophagy-enhancing activities, reduced the RCAN1-calcineurin interaction, phenocopying the effect of RCAN1 knockdown. Our results demonstrate that targeting RCAN1 genetically or pharmacologically can increase neuronal resilience in Huntington's disease.

Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming.

Late-onset Alzheimer's disease (LOAD) is the most common form of Alzheimer's disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-ß (Aß) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)-based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aß-dependent neurodegeneration, and treatment with ß- or γ-secretase inhibitors before (but not subsequent to) Aß deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aß deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

Longitudinal modeling of human neuronal aging identifies RCAN1-TFEB pathway contributing to neurodegeneration of Huntington's disease.

Aging is a common risk factor in neurodegenerative disorders and the ability to investigate aging of neurons in an isogenic background would facilitate discovering the interplay between neuronal aging and onset of neurodegeneration. Here, we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs), a primary neuronal subtype affected in Huntington's disease (HD), identified pathways associated with RCAN1, a negative regulator of calcineurin. Notably, RCAN1 undergoes age-dependent increase at the protein level detected in reprogrammed MSNs as well as in human postmortem striatum. In patient-derived MSNs of adult-onset HD (HD-MSNs), counteracting RCAN1 by gene knockdown (KD) rescued HD-MSNs from degeneration. The protective effect of RCAN1 KD was associated with enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, which in turn dephosphorylates and promotes nuclear localization of TFEB transcription factor. Furthermore, we reveal that G2-115 compound, an analog of glibenclamide with autophagy-enhancing activities, reduces the RCAN1-Calcineurin interaction, phenocopying the effect of RCAN1 KD. Our results demonstrate that RCAN1 is a potential genetic or pharmacological target whose reduction-of-function increases neuronal resilience to neurodegeneration in HD through chromatin reconfiguration.

Endogenous recapitulation of Alzheimer's disease neuropathology through human 3D direct neuronal reprogramming.

Alzheimer's disease (AD) is a neurodegenerative disorder that primarily affects elderly individuals, and is characterized by hallmark neuronal pathologies including extracellular amyloid-ß (Aß) plaque deposition, intracellular tau tangles, and neuronal death. However, recapitulating these age-associated neuronal pathologies in patient-derived neurons has remained a significant challenge, especially for late-onset AD (LOAD), the most common form of the disorder. Here, we applied the high efficiency microRNA-mediated direct neuronal reprogramming of fibroblasts from AD patients to generate cortical neurons in three-dimensional (3D) Matrigel and self-assembled neuronal spheroids. Our findings indicate that neurons and spheroids reprogrammed from both autosomal dominant AD (ADAD) and LOAD patients exhibited AD-like phenotypes linked to neurons, including extracellular Aß deposition, dystrophic neurites with hyperphosphorylated, K63-ubiquitin-positive, seed-competent tau, and spontaneous neuronal death in culture. Moreover, treatment with ß- or γ-secretase inhibitors in LOAD patient-derived neurons and spheroids before Aß deposit formation significantly lowered Aß deposition, as well as tauopathy and neurodegeneration. However, the same treatment after the cells already formed Aß deposits only had a mild effect. Additionally, inhibiting the synthesis of age-associated retrotransposable elements (RTEs) by treating LOAD neurons and spheroids with the reverse transcriptase inhibitor, lamivudine, alleviated AD neuropathology. Overall, our results demonstrate that direct neuronal reprogramming of AD patient fibroblasts in a 3D environment can capture age-related neuropathology and reflect the interplay between Aß accumulation, tau dysregulation, and neuronal death. Moreover, miRNA-based 3D neuronal conversion provides a human-relevant AD model that can be used to identify compounds that can potentially ameliorate AD-associated pathologies and neurodegeneration.

Allosteric activation and contrasting properties of L-serine dehydratase types 1 and 2.

Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between L-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with L-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of L-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.

Age-related Huntington's disease progression modeled in directly reprogrammed patient-derived striatal neurons highlights impaired autophagy.

Huntington's disease (HD) is an inherited neurodegenerative disorder with adult-onset clinical symptoms, but the mechanism by which aging drives the onset of neurodegeneration in patients with HD remains unclear. In this study we examined striatal medium spiny neurons (MSNs) directly reprogrammed from fibroblasts of patients with HD to model the age-dependent onset of pathology. We found that pronounced neuronal death occurred selectively in reprogrammed MSNs from symptomatic patients with HD (HD-MSNs) compared to MSNs derived from younger, pre-symptomatic patients (pre-HD-MSNs) and control MSNs from age-matched healthy individuals. We observed age-associated alterations in chromatin accessibility between HD-MSNs and pre-HD-MSNs and identified miR-29b-3p, whose age-associated upregulation promotes HD-MSN degeneration by impairing autophagic function through human-specific targeting of the STAT3 3' untranslated region. Reducing miR-29b-3p or chemically promoting autophagy increased the resilience of HD-MSNs against neurodegeneration. Our results demonstrate miRNA upregulation with aging in HD as a detrimental process driving MSN degeneration and potential approaches for enhancing autophagy and resilience of HD-MSNs.

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila.

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and ß domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron-sulfur center while the ß domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 ß domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex.

Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs.

Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9∗-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade.

STAT5 mediates antiapoptotic effects of methylprednisolone on oligodendrocytes.

Methylprednisolone (MP), a synthetic glucocorticoid agonist, is widely used for the clinical therapy of white matter diseases in the nervous system, such as spinal cord injury and multiple sclerosis. In addition to its potent anti-inflammatory and antioxidant properties, we recently discovered a selective antiapoptotic effect of MP on oligodendrocytes via the activation of the glucocorticoid receptor (GR) and the upregulation of bcl-X(L), a splicing isoform of the bcl-x gene. Based on published findings of the functional interactions between GR and STAT5, a transcription factor from the family of signal transducers and activators of transcription (STAT), we examined whether the glucocorticoid signaling pathway interacts with STAT5 to upregulate bcl-X(L) and protect oligodendrocytes. We show herein that (1) the GR and STAT5 complex is present on the STAT5-binding site of the bcl-x promoter region in oligodendrocytes; (2) the overexpression of an activated form of STAT5 prevents alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced oligodendrocyte cell death; and (3) this prevention is lost when the STAT5 gene is knocked down. Thus, our results provide one molecular mechanism underlying the postinjury protective effects of oligodendrocytes by stress hormones.

Huntington's Disease Pathogenesis Is Modified In Vivo by Alfy/Wdfy3 and Selective Macroautophagy.

Despite being an autosomal dominant disorder caused by a known coding mutation in the gene HTT, Huntington's disease (HD) patients with similar trinucleotide repeat mutations can have an age of onset that varies by decades. One likely contributing factor is the genetic heterogeneity of patients that might modify their vulnerability to disease. We report that although the heterozygous depletion of the autophagy adaptor protein Alfy/Wdfy3 has no consequence in control mice, it significantly accelerates age of onset and progression of HD pathogenesis. Alfy is required in the adult brain for the autophagy-dependent clearance of proteinaceous deposits, and its depletion in mice and neurons derived from patient fibroblasts accelerates the aberrant accumulation of this pathological hallmark shared across adult-onset neurodegenerative diseases. These findings indicate that selectively compromising the ability to eliminate aggregated proteins is a pathogenic driver, and the selective elimination of aggregates may confer disease resistance.

Methylprednisolone protects oligodendrocytes but not neurons after spinal cord injury.

Methylprednisolone (MP) is used to treat a variety of neurological disorders involving white matter injury, including multiple sclerosis, acute disseminated encephalomyelitis, and spinal cord injury (SCI). Although its mechanism of action has been attributed to anti-inflammatory or antioxidant properties, we examined the possibility that MP may have direct neuroprotective activities. Neurons and oligodendrocytes treated with AMPA or staurosporine died within 24 h after treatment. MP attenuated oligodendrocyte death in a dose-dependent manner; however, neurons were not rescued by the same doses of MP. This protective effect was reversed by the glucocorticoid receptor (GR) antagonist (11, 17)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (RU486) and small interfering RNA directed against GR, suggesting a receptor-dependent mechanism. MP reversed AMPA-induced decreases in the expression of anti-apoptotic Bcl-x(L), caspase-3 activation, and DNA laddering, suggesting anti-apoptotic activity in oligodendrocytes. To examine whether MP demonstrated this selective protection in vivo, neuronal and oligodendrocyte survival was assessed in rats subjected to spinal cord injury (SCI); groups of rats were treated with or without MP in the presence or absence of RU486. Eight days after SCI, MP significantly increased oligodendrocytes (CC-1-immunoreactive cells) after SCI, but neuronal (neuronal-specific nuclear protein-immunoreactive cells) number remained unchanged; RU486 reversed this protective effect. MP also inhibited SCI-induced decreases in Bcl-x(L) and caspase-3 activation. Consistent with these findings, the volume of demyelination, assessed by Luxol fast blue staining, was attenuated by MP and reversed by RU486. These results suggest that MP selectively inhibits oligodendrocyte but not neuronal cell death via a receptor-mediated action and may be a mechanism for its limited protective effect after SCI.

Role of the anion-binding site in catalysis and regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis displays substantial substrate inhibition in the direction of NADH oxidation by its physiological substrate, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site. While addition of substrate at the active site is ordered, with HPAP binding before NADH, NADH can compete with the substrate for binding to the allosteric site and thereby eliminate the substrate inhibition. Fluorescence resonance energy transfer analysis of mutants with specific tryptophan residues converted to phenylalanine residues demonstrates that the main interaction of NADH with the enzyme, in the absence of substrate, is at the allosteric anion-binding site. This is further confirmed by mutations of basic residues at the anion-binding site which also demonstrates that these residues are necessary for inhibition by l-serine when it binds to the regulatory domain. This may indicate that a ligand must be bound to the anion-binding site for l-serine inhibition, providing a potential mechanism for low levels of activity in the presence of high levels of inhibitor.

Transient kinetic analysis of the interaction of L-serine with Escherichia coli D-3-phosphoglycerate dehydrogenase reveals the mechanism of V-type regulation and the order of effector binding.

Pre-steady state stopped-flow analysis of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) reveals that the physiological inhibitor, l-serine, exerts its effect on at least two steps in the kinetic mechanism, but to very different degrees. First, there is a small but significant effect on the dissociation constant of NADH, the first substrate to bind in the ordered mechanism. The effect of serine is mainly on the binding off rate, increasing the K(d) to 5 and 23 muM from 0.6 and 9 muM, respectively, for the two sets of sites in the enzyme. A more profound effect is seen after the second substrate is added. Serine reduces the amplitude of the signal without a significant effect on the observed rate constants for binding. The serine concentration that reduces the amplitude by 50% is equal to the K(0.5) for serine inhibition. The data are consistent with the conclusion that serine binding eliminates a conformational change subsequent to substrate binding by formation of a dead-end quaternary complex consisting of enzyme, coenzyme, substrate, and effector. Thus, the mechanistic basis for V-type regulation in this enzyme is a reduction in the population of active species rather than a differential decrease in the velocity of active species. Pre-steady state analysis of binding of serine to a mutant PGDH (W139F/E360W) demonstrates that each serine binding interface produces an integrated fluorescent signal. The observed rate data are complex but conform to a model in which serine can bind to two forms of the enzyme with different affinities. The integrated signal from each interface allows the amplitude data to clearly define the order of binding to each site, and modeling the amplitude data with species distribution equations clearly demonstrates an alternate interface binding mechanism and the direction of binding cooperativity.

Amyloid-beta peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway.

Amyloid-beta peptide (Abeta) accumulation in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Abeta induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Abeta-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Abeta and ceramide induced OLG death. In addition, Abeta activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Abeta cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Abeta-induced OLG death. Glutathione (GSH) precursors inhibited Abeta activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Abeta-induced death. These results suggest that Abeta induces OLG death by activating the nSMase-ceramide cascade via an oxidative mechanism.

Protein phosphatase 2A regulates bim expression via the Akt/FKHRL1 signaling pathway in amyloid-beta peptide-induced cerebrovascular endothelial cell death.

Amyloid-beta peptide (Abeta)-induced death in cerebral endothelial cells (CECs) is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. Mitochondria-dependent apoptosis engages Bcl-2 family proteins, especially the BH3-only homologues, which play a key role in initiating the apoptotic cascade. Here, we report that the expression of bim, but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy (CAA), suggesting a pivotal role for Bim in Abeta-induced cerebrovascular degeneration in vivo. A similar expression profile was observed in Abeta-treated CECs. Furthermore, Abeta induction of bim expression involved a pro-apoptotic transcription factor, FKHRL1. FKHRL1 bound to a consensus sequence in the bim promoter region and was activated by Abeta before bim expression. FKHRL1 activity was negatively regulated by phosphorylation catalyzed by Akt, an anti-apoptotic kinase. Akt upregulation by adenoviral gene transfer inhibited Abeta-induced FKHRL1 activation and bim induction. In addition, Abeta increased the activity of protein phosphatase 2A (PP2A), a ceramide-activated protein phosphatase. Suppression of PP2A activity by RNA interference or a specific inhibitor, okadaic acid, effectively suppressed Abeta-induced Akt inactivation and FKHRL1 activation, leading to an attenuation of bim expression and cell death in CECs. Coimmunoprecipitation experiments revealed that Abeta enhanced the binding of the PP2A regulatory subunit PP2ACalphabeta to Akt. These results implicate PP2A as an early regulator of Abeta-induced bim expression and CEC apoptosis via the Akt/FKHRL1 signaling pathway. We raise the possibility that this pathway may play a role in cerebrovascular degeneration in CAA.

Antisense RNA to inducible nitric oxide synthase reduces cytokine-mediated brain endothelial cell death.

We test whether inhibition of inducible nitric oxide synthase (iNOS) can exert a cytoprotective effect on cerebral endothelial cells upon stimulation by pro-inflammatory cytokines. Mouse brain endothelial cells were stably transfected to express an antisense RNA against iNOS driven by an endothelium-specific von Willebrand factor (vWF) promoter. Upon stimulation with tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma), antisense transfectants showed less iNOS enzymatic activity with less nitric oxide (NO) when compared to the sense control cells. Correspondingly, the antisense cells showed a reduced LDH release and less cytosolic content of oligonucleosomes. These findings establish a cell-specific antisense strategy and confirm the cytotoxic role of iNOS expression in cultured cerebral endothelial cells.

Matrix metalloproteinase-9 in cerebral-amyloid-angiopathy-related hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) is one of the most recognized complications of cerebral amyloid angiopathy (CAA), but little is known about the molecular pathogenesis of this life-threatening complication. In this review, we present preliminary evidence which suggests that the extracellular-matrix-degrading protease, matrix metalloproteinase-9 (MMP-9), may play a role in the development of spontaneous ICH resulting from CAA. The amyloid-beta peptide (Abeta) induced the synthesis, cellular release, and activation of MMP-9 in murine cerebral endothelial cells (CECs), resulting in increased extracellular matrix (ECM) degradation. Furthermore, in a mouse model of CAA (APPsw transgenic mice), MMP-9 immunoreactivity was observed in amyloid-laden cerebral vessels in aged APPsw mice but not in young APPsw or aged wild-type mice. More extensive MMP-9 immunostaining was present in amyloid-laden vessels with evidence of microhemorrhage. These results suggest that increased vascular MMP-9 expression, stimulated by Abeta, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage (ICH) in patients with CAA.

A novel mechanism for substrate inhibition in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase undergoes significant inhibition of activity with increasing concentrations of its substrate, hydroxypyruvic acid phosphate. The enzyme also displays an unusual dual pH optimum. A significant decrease in the K(i) for substrate inhibition at pH values corresponding to the valley between these optima is responsible for this phenomena. The change in K(i) has an average pK of approximately 5.8 and involves two functional groups that are protonated and two functional groups that are unprotonated for optimal substrate inhibition to occur. Mutagenesis of positively charged amino acid residues at a putative anion binding site previously revealed by the x-ray structure, produces significant changes in the pH-dependent profile of substrate inhibition. Several single residue mutations eliminate the dual pH optima by reducing substrate inhibition between pH 5 and 7 and a triple mutation was identified that eliminates the substrate inhibition altogether. The mutagenesis data support the conclusion that the anion binding site represents a new allosteric site for the control of enzyme activity and functions in a novel mechanism for substrate inhibition.