Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines.

In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.

On the etiology of Crohn disease.

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease.

Huntington disease is a dominantly inherited, untreatable neurological disorder featuring a progressive loss of striatal output neurons that results in dyskinesia, cognitive decline, and, ultimately, death. Neurotrophic factors have recently been shown to be protective in several animal models of neurodegenerative disease, raising the possibility that such substances might also sustain the survival of compromised striatal output neurons. We determined whether intracerebral administration of brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, or ciliary neurotrophic factor could protect striatal output neurons in a rodent model of Huntington disease. Whereas treatment with brain-derived neurotrophic factor, nerve growth factor, or neurotrophin-3 provided no protection of striatal output neurons from death induced by intrastriatal injection of quinolinic acid, an N-methyl-D-aspartate glutamate receptor agonist, treatment with ciliary neurotrophic factor afforded marked protection against this neurodegenerative insult.

The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

Dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase have been found in members of certain families with familial amyotrophic lateral sclerosis (ALS). To better understand the contribution of SOD1 mutations in the pathogenesis of familial ALS, we developed transgenic mice expressing one of the mutations found in familial ALS. These animals display clinical and pathological features closely resembling human ALS. Early changes observed in these animals were intra-axonal and dendritic vacuoles due to dilatation of the endoplasmic reticulum and vacuolar degeneration of mitochondria. We have reported that the Golgi apparatus of spinal cord motor neurons in patients with sporadic ALS is fragmented and atrophic. In this study we show that spinal cord motor neurons of transgenic mice for an SOD1 mutation display a lesion of the Golgi apparatus identical to that found in humans with sporadic ALS. In these mice, the stacks of the cisternae of the fragmented Golgi apparatus are shorter than in the normal organelle, and there is a reduction in Golgi-associated vesicles and adjacent cisternae of the rough endoplasmic reticulum. Furthermore, the fragmentation of the Golgi apparatus occurs in an early, presymptomatic stage and usually precedes the development of the vacuolar changes. Transgenic mice overexpressing the wild-type human superoxide dismutase are normal. In familial ALS, an early lesion of the Golgi apparatus of motor neurons may have adverse functional effects, because newly synthesized proteins destined for fast axoplasmic transport pass through the Golgi apparatus.

Checkpoints in the progression of autoimmune disease: lessons from diabetes models.

In the last few years, data from experiments employing transgenic models of autoimmune disease have strengthened a particular concept of autoimmunity: disease results not so much from cracks in tolerance induction systems, leading to the generation of anti-self repertoire, as from the breakdown of secondary systems that keep these cells in check. T cells with anti-self specificities are readily found in disease-free individuals but ignore target tissues. This is also the case in some transgenic models, in spite of overwhelming numbers of autoreactive cells. In other instances, local infiltration and inflammation result, but they are well tolerated for long periods of time and do not terminally destroy target tissue. We review the possible molecular and cellular mechanisms that underlie these situations, with a particular emphasis on the destruction of pancreatic beta cells in transgenic models of insulin-dependent disease.

Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease amyloid, binds A beta and stimulates A beta aggregation.

NACP, a 140-amino acid presynaptic protein, is the precursor of NAC [the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease (AD) amyloid], a peptide isolated from and immunologically localized to brain amyloid of patients afflicted with AD. NACP produced in Escherichia coli bound to A beta peptides, the major component of AD amyloid. NACP bound to A beta 1-38 and A beta 25-35 immobilized on nitrocellulose but did not bind to A beta 1-28 on the filter under the same conditions. NACP binding to A beta 1-38 was abolished by addition of A beta 25-35 but not by A beta 1-28, suggesting that the hydrophobic region of the A beta peptide is critical to this binding. NACP-112, a shorter splice variant of NACP containing the NAC sequence, bound to A beta, but NACP delta, a deletion mutant of NACP lacking the NAC domain, did not bind A beta 1-38. Furthermore, binding between NACP-112 and A beta 1-38 was decreased by addition of peptide Y, a peptide that covers the last 15 residues of NAC. In an aqueous solution, A beta 1-38 aggregation was observed when NACP was also present in an incubation mixture at a ratio of 1:125 (NACP/A beta), whereas A beta 1-38 alone or NACP alone did not aggregate under the same conditions, suggesting that the formation of a complex between A beta and NACP may promote aggregation of A beta. Thus, NACP can bind A beta peptides through the specific sequence and can promote A beta aggregation, raising the possibility that NACP may play a role in the development of AD amyloid.

Genetic dissection of Alzheimer disease, a heterogeneous disorder.

The genetics of Alzheimer disease (AD) are complex and not completely understood. Mutations in the amyloid precursor protein gene (APP) can cause early-onset autosomal dominant AD. In vitro studies indicate that cells expressing mutant APPs overproduce pathogenic forms of the A beta peptide, the major component of AD amyloid. However, mutations in the APP gene are responsible for 5% or less of all early-onset familial AD. A locus on chromosome 14 is responsible for AD in other early-onset AD families and represents the most severe form of the disease in terms of age of onset and rate of decline. Attempts to identify the AD3 gene by positional cloning methods are underway. At least one additional early-onset AD locus remains to be located. In late-onset AD, the apolipoprotein E gene allele epsilon 4 is a risk factor for AD. This allele appears to act as a dose-dependent age-of-onset modifier. The epsilon 2 allele of this gene may be protective. Other late-onset susceptibility factors remain to be identified.

Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis.

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.

Electroencephalogram based Causality Graph Analysis in Behavior Tasks of Parkinson’s Disease Patients

Electroencephalographic (EEG) signals of the human brains represent electrical activities for a number of channels recorded over a the scalp. The main purpose of this thesis is to investigate the interactions and causality of different parts of a brain using EEG signals recorded during a performance subjects of verbal fluency tasks. Subjects who have Parkinson's Disease (PD) have difficulties with mental tasks, such as switching between one behavior task and another. The behavior tasks include phonemic fluency, semantic fluency, category semantic fluency and reading fluency. This method uses verbal generation skills, activating different Broca's areas of the Brodmann's areas (BA44 and BA45). Advanced signal processing techniques are used in order to determine the activated frequency bands in the granger causality for verbal fluency tasks. The graph learning technique for channel strength is used to characterize the complex graph of Granger causality. Also, the support vector machine (SVM) method is used for training a classifier between two subjects with PD and two healthy controls. Neural data from the study was recorded at the Colorado Neurological Institute (CNI). The study reveals significant difference between PD subjects and healthy controls in terms of brain connectivities in the Broca's Area BA44 and BA45 corresponding to EEG electrodes. The results in this thesis also demonstrate the possibility to classify based on the flow of information and causality in the brain of verbal fluency tasks. These methods have the potential to be applied in the future to identify pathological information flow and causality of neurological diseases.

Inventory of seeds and plants imported / U.S. Department of Agriculture, Bureau of Plant Industry.

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An introduction to the chemistry of plant products by Paul Haas and T. G. Hill.

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