Design, synthesis, and characterization of novel system xC- transport inhibitors: inhibition of microglial glutamate release and neurotoxicity.

Neuroinflammation appears to involve some degree of excitotoxicity promulgated by microglia, which release glutamate via the system xC- (SxC-) cystine-glutamate antiporter. With the aim of mitigating this source of neuronal stress and toxicity, we have developed a panel of inhibitors of the SxC- antiporter. The compounds were based on L-tyrosine, as elements of its structure align with those of glutamate, a primary physiological substrate of the SxC- antiporter. In addition to 3,5-dibromotyrosine, ten compounds were synthesized via amidation of that parent molecule with a selection of acyl halides. These agents were tested for the ability to inhibit release of glutamate from microglia activated with lipopolysaccharide (LPS), an activity exhibited by eight of the compounds. To confirm that the compounds were inhibitors of SxC-, two of them were further tested for the ability to inhibit cystine uptake. Finally, these agents were shown to protect primary cortical neurons from the toxicity exhibited by activated microglia. These agents may hold promise in reducing the neurodegenerative effects of neuroinflammation in conditions, such as encephalitis, traumatic brain injury, stroke, or neurodegenerative diseases.

Thiadiazolidinone (TDZD) Analogs Inhibit Aggregation-Mediated Pathology in Diverse Neurodegeneration Models, and Extend C. elegans Life- and Healthspan.

Chronic, low-grade inflammation has been implicated in aging and age-dependent conditions, including Alzheimer's disease, cardiomyopathy, and cancer. One of the age-associated processes underlying chronic inflammation is protein aggregation, which is implicated in neuroinflammation and a broad spectrum of neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's diseases. We screened a panel of bioactive thiadiazolidinones (TDZDs) from our in-house library for rescue of protein aggregation in human-cell and C. elegans models of neurodegeneration. Among the tested TDZD analogs, PNR886 and PNR962 were most effective, significantly reducing both the number and intensity of Alzheimer-like tau and amyloid aggregates in human cell-culture models of pathogenic aggregation. A C. elegans strain expressing human Aß1-42 in muscle, leading to AD-like amyloidopathy, developed fewer and smaller aggregates after PNR886 or PNR962 treatment. Moreover, age-progressive paralysis was reduced 90% by PNR886 and 75% by PNR962, and "healthspan" (the median duration of spontaneous motility) was extended 29% and 62%, respectively. These TDZD analogs also extended wild-type C. elegans lifespan by 15-30% (p < 0.001), placing them among the most effective life-extension drugs. Because the lead drug in this family, TDZD-8, inhibits GSK3ß, we used molecular-dynamic tools to assess whether these analogs may also target GSK3ß. In silico modeling predicted that PNR886 or PNR962 would bind to the same allosteric pocket of inactive GSK3ß as TDZD-8, employing the same pharmacophore but attaching with greater avidity. PNR886 and PNR962 are thus compelling candidate drugs for treatment of tau- and amyloid-associated neurodegenerative diseases such as AD, potentially also reducing all-cause mortality.

Glucose transport in the regulation of T-cell activation: the journey may be as important as the destination.

A shift in the energy-metabolism balance from oxidative phosphorylation to glycolysis is observed in several phenomena, from oncogenesis to differentiation. And this shift is not merely an indicator of the phenotypic change-an increase in glucose delivery often drives the adaption. At first blush, it seems that any route of entry should be equivalent, as long as sufficient quantities are supplied. However, an extensive study comparing the Th17 and Th1 subtypes of T cells now suggests that similar glucose transporters may not be interchangeable. Manipulation of individual transporters, or the downstream metabolites of their substrates, may afford dampening of autoimmunity potential with some degree of precision.

Serine Racemase Expression Differentiates Aging from Alzheimer's Brain.

Aging is an inevitable process characterized by progressive loss of physiological integrity and increased susceptibility to cancer, diabetes, cardiovascular, and neurodegenerative diseases; aging is the primary risk factor for Alzheimer's disease (AD), the most common cause of dementia. AD is characterized by brain pathology, including extracellular deposition of amyloid aggregation and intracellular accumulation of neurofibrillary tangles composed of hyperphosphorylated tau protein. In addition, losses of synapses and a wide range of neurons are pivotal pathologies in the AD brain. Accumulating evidence demonstrates hypoactivation of hippocampal neural networks in the aging brain, whereas AD-related mild cognitive impairment (AD-MCI) begins with hyperactivation, followed by a diminution of hippocampal activity as AD develops. The biphasic trends of the activity of the hippocampal neural network are consistent with the alteration of N-methyl-D-aspartate receptor (NMDA-R) activity from aging to prodromal (AD-MCI) to mid-/late stage AD. D-serine, a product of racemization catalyzed by serine racemase (SR), is an important co-agonist of the NMDA-R which is involved in synaptic events including neurotransmission, synaptogenesis, long-term potentiation (LTP), development, and excitotoxicity. SR and D-serine are decreased in the hippocampus of the aging brain, correlating with impairment of cognitive function. By contrast, SR is increased in AD brain, which is associated with a greater degree of cognitive dysfunction. Emerging studies suggest that D-serine levels in the brain or in cerebral spinal fluid from AD patients are higher than in age-matched controls, but the results are inconsistent. Very recently, serum D-serine levels in AD were reported to correlate with sex and clinical dementia rating (CDR) stage. This review will discuss alterations of NMDA-R and SR in aging and AD brain, and the mechanisms underlying the differential regulation of SR will be probed. Collectively, we propose that SR may be a molecular switch that distinguishes the effects of aging from those of AD on the brain.

Functional assessments through novel proteomics approaches: Application to insulin/IGF signaling in neurodegenerative disease'.

BACKGROUND: Events that instigate disease may involve biochemical events distinct from changes in the steady-state levels of proteins. Even chronic degenerative disorders appear to involve changes such as post-translational modifications. NEW METHOD: We have begun a series of proteomics analyses on proteins that have been fractionated by functional status. Because Alzheimer's disease (AD) is associated with metabolic perturbations such as Type-2 diabetes, fractionation hinged on binding to phosphatidylinositol trisphosphate (PIP3), key to insulin/insulin-like growth factor signaling. We compared mice on normal chow to counterparts subjected to diet-induced obesity (DIO) or to mice expressing human Aß1-42 from a transgene. RESULTS: The prevailing phenotypic finding in either experimental group was loss of PIP3 binding. Of the 1228 proteins that showed valid PIP3 binding in any group of mice, 55% exhibited a significant quantitative difference in the number of spectral counts as a function of DIO, 63% as function of the Aß transgene, and 79% as a function of either variable. There was remarkable overlap among the proteins altered in the two experimental groups, and pathway analysis indicated effects on proteostasis, apoptosis, and synaptic vesicles. COMPARISON WITH EXISTING METHODS: Most proteomics approaches only identify differences in the steady-state levels of proteins. Our overlay of a functional distinction permits new levels of discovery that may achieve novel insights into physiology in an unbiased and inclusive manner. CONCLUSIONS: Proteomics analyses have revolutionized the discovery phase of biomedical research but are conventionally limited in scope. The creative use of fractionation prior to proteomic discovery is likely to provide important insights into AD and related disorders.

Apolipoprotein E4 inhibits autophagy gene products through direct, specific binding to CLEAR motifs.

INTRODUCTION: Alzheimer apolipoprotein E (APOE) ɛ4/ɛ4 carriers have earlier disease onset and more protein aggregates than patients with other APOE genotypes. Autophagy opposes aggregation, and important autophagy genes are coordinately regulated by transcription factor EB (TFEB) binding to "coordinated lysosomal expression and regulation" (CLEAR) DNA motifs. METHODS: Autophagic gene expression was assessed in brains of controls and Alzheimer's disease (AD) patients parsed by APOE genotype and in a glioblastoma cell line expressing either apoE3 or apoE4. Computational modeling assessed interactions between apoE and mutated apoE with CLEAR or modified DNA. RESULTS: Three TFEB-regulated mRNA transcripts-SQSTM, MAP1LC3B, and LAMP2-were lower in AD ɛ4/ɛ4 than in AD ɛ3/ɛ3 brains. Computational modeling predicted avid specific binding of apoE4 to CLEAR motifs. ApoE was found in cellular nuclei, and in vitro binding assays suggest competition between apoE4 and TFEB at CLEAR sites. CONCLUSION: ApoE4-CLEAR interactions may account for suppressed autophagy in APOE ɛ4/ɛ4 carriers and, in this way, contribute to earlier AD onset.

Protein Kinases and Parkinson's Disease.

Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson's disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson's disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson's disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules.

NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions. No component of the NFκB pathway was permissive for endogenous NFκB activity, and none stimulated the activity of exogenous RelA. Surprisingly, however, the latter was inhibited by cotransfection of NFκB-inducing kinase (NIK). Fluorescence imaging of RelA indicated that co-expression of NIK sequestered RelA in the cytoplasm, similar to the effect of IκBα. NIK-knockout mice showed elevated expression of an NFκB-reporter construct in neurons in vivo. Cortical neurons cultured from NIK-knockout mice showed elevated expression of an NFκB-reporter transgene. Consistent with data from other cell types, a C-terminal fragment of NIK suppressed RelA activity in astrocytes as well as neurons. Therefore, the inhibitory ability of the NIK C-terminus was unbiased with regard to cell type. However, inhibition of NFκB by full-length NIK is a novel outcome that appears to be specific to CNS neurons. This has implications for unique aspects of transcription in the CNS, perhaps relevant to aspects of development, neuroplasticity, and neuroinflammation. Full-length NIK was found to inhibit (down arrow) transcriptional activation of NFκB in neurons, while it elevated (up arrow) activity in astrocytes. Deletion constructs corresponding to the N-terminus or C-terminus also inhibited NFκB in neurons, while only the C-terminus did so in astrocytes. One possible explanation is that the inhibition in neurons occurs via two different mechanisms, including the potential for a neuron-specific protein (e.g., one of the 14-3-3 class) to create a novel complex in neurons, whereas the C-terminus may interact directly with NFκB. [Structure of NIK is based on Liu J., Sudom A., Min X., Cao Z., Gao X., Ayres M., Lee F., Cao P., Johnstone S., Plotnikova O., Walker N., Chen G., and Wang Z. (2012) Structure of the nuclear factor κB-inducing kinase (NIK) kinase domain reveals a constitutively active conformation. J Biol Chem. 287, 27326-27334); N-terminal lobe is oriented at top].

Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3ß signaling pathway.

We recently found that sAPPα decreases amyloid-beta generation by directly associating with ß-site amyloid precursor protein (APP)-converting enzyme 1 (BACE1), thereby modulating APP processing. Because inhibition of BACE1 decreases glycogen synthase kinase 3 beta (GSK3ß)-mediated Alzheimer's disease (AD)-like tau phosphorylation in AD patient-derived neurons, we determined whether sAPPα also reduces GSK3ß-mediated tau phosphorylation. We initially found increased levels of inhibitory phosphorylation of GSK3ß (Ser9) in primary neurons from sAPPα over-expressing mice. Further, recombinant human sAPPα evoked the same phenomenon in SH-SY5Y cells. Further, in SH-SY5Y cells over-expressing BACE1, and HeLa cells over-expressing human tau, sAPPα reduced GSK3ß activity and tau phosphorylation. Importantly, the reductions in GSK3ß activity and tau phosphorylation elicited by sAPPα were prevented by BACE1 but not γ-secretase inhibition. In accord, AD mice over-expressing human sAPPα had less GSK3ß activity and tau phosphorylation compared with controls. These results implicate a direct relationship between APP ß-processing and GSK3ß-mediated tau phosphorylation and further define the central role of sAPPα in APP autoregulation and AD pathogenesis.

Nox4-generated superoxide drives angiotensin II-induced neural stem cell proliferation.

Reactive oxygen species (ROS) have been reported to affect neural stem cell self-renewal and therefore may be important for normal development and may influence neurodegenerative processes when ROS activity is elevated. To determine if increasing production of superoxide, via activation of NADPH oxidase (Nox), increases neural stem cell proliferation, 100 nM angiotensin II (Ang II) - a strong stimulator of Nox - was applied to cultures of a murine neural stem cell line, C17.2. Twelve hours following a single treatment with Ang II, there was a doubling of the number of neural stem cells. This increase in neural stem cell numbers was preceded by a gradual elevation of superoxide levels (detected by dihydroethidium fluorescence) from the steady state at 0, 5, and 30 min and gradually increasing from 1 h to the maximum at 12 h, and returning to baseline at 24 h. Ang II-dependent proliferation was blocked by the antioxidant N-acetyl-L-cysteine. Confocal microscopy revealed the presence of two sources of intracellular ROS in C17.2 cells: (i) mitochondrial and (ii) extramitochondrial; the latter indicative of the involvement of one or more specific isoforms of Nox. Of the Nox family, mRNA expression for one member, Nox4, is abundant in neural stem cell cultures, and Ang II treatment resulted in elevation of the relative levels of Nox4 protein. SiRNA targeting of Nox4 mRNA reduced both the constitutive and Ang II-induced Nox4 protein levels and attenuated Ang II-driven increases in superoxide levels and stem cell proliferation. Our findings are consistent with our hypothesis that Ang II-induced proliferation of neural stem cells occurs via Nox4-generated superoxide, suggesting that an Ang II/Nox4 axis is an important regulator of neural stem cell self-renewal and as such may fine-tune normal, stress- or disease-modifying neurogenesis.

Metabotropic glutamate receptors inhibit microglial glutamate release.

Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action.

Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis.

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.

Roles of quaternary structure and cysteine residues in the activity of human serine racemase.

BACKGROUND: D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors. It is chiefly synthesized in the CNS by serine racemase (SR). Regulation of SR activity is still poorly understood. As step toward developing reagents and methods for investigating SR in vitro, we analyzed structure-function relationships of a recombinant enzyme of human sequence. RESULTS: Michaelis-Menten kinetic analysis indicated a KM value of 14 mM and Vmax value of 3.66 µmol·mg⁻¹·hr⁻¹ when L-serine was used as a substrate for purified SR. Gel-filtration chromatography and protein cross-linking experiments revealed that dimer is the major oligomeric form of recombinant SR in aqueous solution, though the proportions of monomer, tetramer, and larger aggregates differed somewhat with the specific buffer used. These buffers also altered activity in a manner correlating with the relative abundance of dimer. Activity assays showed that the dimeric gel-filtration fraction held the highest activity. Chemical reduction with DTT increased the activity of SR by elevating Vmax; cystamine, a reagent that blocks sulfhydryl groups, abolished SR activity. Gel-filtration chromatography and western blot analysis indicated that DTT enhanced the recovery of noncovalent SR dimer. CONCLUSIONS: These data suggest that SR is most active as a noncovalent dimer containing one or more free sulfhydryls in the enzyme's active center or a modulatory site. Buffer composition and reduction/oxidation status during preparation can dramatically impact interpretations of SR activity. These findings also highlight the possibility that SR is sensitive to oxidative stress in vivo.

Apolipoprotein E expression is elevated by interleukin 1 and other interleukin 1-induced factors.

BACKGROUND: We have previously outlined functional interactions, including feedback cycles, between several of the gene products implicated in the pathogenesis of Alzheimer's disease. A number of Alzheimer-related stressors induce neuronal expression of apolipoprotein E (ApoE), ß-amyloid precursor protein (ßAPP), and fragments of the latter such as amyloid ß-peptide (Aß) and secreted APP (sAPP). These stressors include interleukin-1 (IL-1)-mediated neuroinflammation and glutamate-mediated excitotoxicity. Such circumstances are especially powerful when they transpire in the context of an APOE ε4 allele. METHODS: Semi-quantitative immunofluorescence imaging was used to analyze rat brains implanted with IL-1ß slow-release pellets, sham pellets, or no pellets. Primary neuronal or NT2 cell cultures were treated with IL-1ß, glutamate, Aß, or sAPP; relative levels of ApoE mRNA and protein were measured by RT-PCR, qRT-PCR, and western immunoblot analysis. Cultures were also treated with inhibitors of multi-lineage kinases--in particular MAPK-p38 (SB203580), ERK (U0126), or JNK (SP600125)--prior to exposure of cultures to IL-1ß, Aß, sAPP, or glutamate. RESULTS: Immunofluorescence of tissue sections from pellet-implanted rats showed that IL-1ß induces expression of ßAPP, IL-1α, and ApoE; the latter was confirmed by western blot analysis. These protein changes were mirrored by increases in their mRNAs, as well as in those encoding IL-1ß, IL-1ß-converting enzyme (ICE), and tumor necrosis factor (TNF). IL-1ß also increased ApoE expression in neuronal cultures. It stimulated release of sAPP and glutamate in these cultures too, and both of these agents--as well as Aß--stimulated ApoE expression themselves, suggesting that they may contribute to the effect of IL-1ß on ApoE levels. Inhibitors of MAPK-p38, ERK, and JNK inhibited ApoE induction by all these agents except glutamate, which was sensitive only to inhibitors of ERK and JNK. CONCLUSION: Conditions of glial activation and hyperexcitation can elevate proinflammatory cytokines, ApoE, glutamate, ßAPP, and its secreted fragments. Because each of these factors promotes glial activation and neuronal hyperexcitation, these relationships have the potential to sustain self-propagating neurodegenerative cycles that could culminate in a progressive neurodegenerative disorder such as Alzheimer's disease.

Unique aspects of transcriptional regulation in neurons--nuances in NFkappaB and Sp1-related factors.

The unique physiology and function of neurons create differences in their cellular physiology, including their regulation of gene expression. We began several years ago exploring the relationships between the NFkappaB transcription factor, neuronal survival, and glutamate receptor activation in telencephalic neurons. These studies led us to conclude that this population of cells is nearly incapable of activating the NFkappaB that is nonetheless expressed at reasonable levels. A subset of the kappaB cis elements are instead bound by members of the Sp1 family in neurons. Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation. These findings and their implications for neuronal differentiation--as well as potential dedifferentiation during degenerative processes--are discussed here.

Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation.

When activated by proinflammatory stimuli, microglia release substantial levels of glutamate, and mounting evidence suggests this contributes to neuronal damage during neuroinflammation. Prior studies indicated a role for the Xc exchange system, an amino acid transporter that antiports glutamate for cystine. Because cystine is used for synthesis of glutathione (GSH) synthesis, we hypothesized that glutamate release is an indirect consequence of GSH depletion by the respiratory burst, which produces superoxide from NADPH oxidase. Microglial glutamate release triggered by lipopolysaccharide was blocked by diphenylene iodonium chloride and apocynin, inhibitors of NADPH oxidase. This glutamate release was also blocked by vitamin E and elicited by lipid peroxidation products 4-hydroxynonenal and acrolein, suggesting that lipid peroxidation makes crucial demands on GSH. Although NADPH oxidase inhibitors also suppressed nitrite accumulation, vitamin E did not; moreover, glutamate release was largely unaffected by nitric oxide donors, inhibitors of nitric oxide synthase, or changes in gene expression. These findings indicate that a considerable degree of the neurodegenerative consequences of neuroinflammation may result from conversion of oxidative stress to excitotoxic stress. This phenomenon entails a biochemical chain of events initiated by a programmed oxidative stress and resultant mass-action amino acid transport. Indeed, some of the neuroprotective effects of antioxidants may be due to interference with these events rather than direct protection against neuronal oxidation.

Glutamate receptor activation evokes calpain-mediated degradation of Sp3 and Sp4, the prominent Sp-family transcription factors in neurons.

Sp-family transcription factors (Sp1, Sp3 and Sp4) contain a zinc-finger domain that binds to DNA sequences rich in G-C/T. As assayed by RT-PCR analysis of mRNA, western-blot analysis, immunofluorescence, and antibody-dependent "supershift" of DNA-binding assays, the prominent Sp-family factors in cerebral neurons were identified as Sp3 and Sp4. By contrast, glial cells were found to express Sp1 and Sp3. We previously showed that the pattern of G-C/T binding activity of Sp-family factors is rapidly and specifically altered by the calcium influx accompanying activation of glutamate receptors. Here, we demonstrate that Sp-factor activity is also lost after a cerebral ischemia/reperfusion injury in vivo. Consistent with its calcium-dependent nature, we found that glutamate's effect on Sp-family factors could be blocked by inhibitors of calpains, neutral cysteine proteases activated by calcium. Purified calpain I cleaved Sp3 and Sp4 into products that retained G-C/T-binding activity, consistent with species observed in glutamate-treated neurons. These data provide details of an impact of glutamate-receptor activation on molecular events connected to gene expression.

Differential transcriptional control of the superoxide dismutase-2 kappaB element in neurons and astrocytes.

In addition to their conventional G-C/T target sequences, Sp1 family transcription factors (Sp-factors) can interact with a subset of the target sequences for NFkappaB. Due to the low level of bona fide NFkappaB activity in most resting cells, this interaction between Sp-factors and kappaB-sites could play important roles in cell function. Here we used mutagenesis of a canonical kappaB element from the immunoglobulin and HIV promoters to identify the GC-rich sequences at each end required for Sp-factor targeting. Through screening of multiple kappaB elements, a sequence element located in the second intron of superoxide dismutase-2 (SOD2) was identified as a good candidate for both NFkappaB and Sp-factor binding. In neurons, the prominent proteins interacting with this site were Sp3 and Sp4, whereas Sp1, Sp3, and NFkappaB were associated with this site in astroglia. The neuronal Sp-factors repressed transcriptional activity through this kappaB-site. In contrast, astroglial Sp-factors activated promoter activity through the same element. NFkappaB contributed to control of the SOD2 kappaB element only in astrocytes. These findings imply that cell-type specificity of transcription in the central nervous system, particularly with regard to kappaB elements, may include two unique aspects of neurons: 1) a recalcitrant NFkappaB and 2) the substitution of Sp4 for Sp1.

sAPPalpha enhances the transdifferentiation of adult bone marrow progenitor cells to neuronal phenotypes.

The remediation of neurodegeneration and cognitive decline in Alzheimer's Disease (AD) remains a challenge to basic scientists and clinicians. It has been suggested that adult bone marrow stem cells can transdifferentiate into different neuronal phenotypes. Here we demonstrate that the alpha-secretase-cleaved fragment of the amyloid precursor protein (sAPPalpha), a potent neurotrophic factor, potentiates the nerve growth factor (NGF)/retinoic acid (RA) induced transdifferentiation of bone marrow-derived adult progenitor cells (MAPCs) into neural progenitor cells and, more specifically, enhances their terminal differentiation into a cholinergic-like neuronal phenotype. The addition of sAPPalpha to NGF/RA-stimulated MAPCs resulted in their conversion to neuronal-like cells as evidenced by the extension of neurites and the appearance of immature synaptic complexes. MAPCs differentiated in the presence of sAPPalpha and NGF/RA exhibited a 40% to as much as 75% increase in neuronal proteins including NeuN, beta-tubulin III, NFM, and synaptophysin, compared to MAPCs differentiated by NGF/RA alone. This process was accompanied by an increase in the levels of choline acetyltransferase, a marker of cholinergic neurons, compared to those of GABAergic and dopaminergic neuronal subtypes. MAPCs immunopositive for sAPPalpha were identified within the septohippocampal system of transgenic PS/APP mice injected intravenously with sAPPalpha-transfected MAPCs and found in close proximity to the cerebral vasculature. Given that in AD cholinergic neurons are severely vulnerable to neurodegeneration and that the levels of sAPPalpha are significantly reduced, these findings suggest the combined use of sAPPalpha and MAPCs offers a new and potentially powerful therapeutic strategy for AD treatment.

An unconventional hypothesis of oxidation in Alzheimer's disease: intersections with excitotoxicity.

There are two major lines of investigation from which a connection has been traditionally drawn between chemical oxidation and Alzheimer's disease. First, a major risk factor for AD is age, and oxidative stress has long been a component of general hypotheses about biological aging. The second line of reasoning is a corollary of the Amyloid Hypothesis, the assumption that the amyloid beta-peptide (A-beta) which comprises AD's pathognomic plaques is a key mediator of the neurodegeneration occurring in this disorder. Under many experimental conditions, A-beta has been shown to evoke oxidative damage to tissues, cells, and biomolecules; even the redox properties of the peptide itself have been hotly debated. These two modalities of conjecture intersect under the Inflammatory Hypothesis of AD, as inflammation produces oxidation, old age is associated with elevation in inflammatory events, and A-beta can further exacerbate such inflammatory reactions in brain cells. This review discusses these arguments about the pathogenesis of AD and how they might be generalized to other neurodegenerative conditions. But, additional speculation is offered in the form of an inclusionary mechanism that may be specific and novel enough to qualify as a third line of theory; namely, the possibility that inflammatory reactions in microglia--activated by A-beta or other factors among the "usual suspects"--initiate programmed oxidation that is converted to the neuron-specific stress of excitotoxicity.