Kosagi Sharaf Jagannatha Rao

Copper- and iron-induced differential fibril formation in \alpha-synuclein: TEM study

alpha-Synuclein filaments are the central component of intracytoplasmic inclusion bodies characteristic of Parkinsons disease (PD) and related disorders. Metals are the significant etiological factors in PD, and their interaction with alpha-synuclein affect dramatically the kinetics of fibrillation. Currently, we have investigated the influence of Cu(II) and Fe(III) on alpha-synuclein fibril formation. Cu(II) and Fe(III) selectively and differentially induced the formation of discrete alpha-synuclein fibrillar species. Transmission electron microscopy was used to monitor the aggregation state of alpha-synuclein (wild-type, A30P, A53T, and E46K) after 60h with stirring at 37 degrees C in the presence and absence of metal ions. Cu(II) has induced thin long network-like fibrils with the wild-type of alpha-synuclein, while the mutant, showed amorphous aggregates with no fibrillar forms. Fe(III) induced short and thick fibrils with both wild and mutant forms and were similar to alpha-synuclein fibrils incubated without metal ion. The present study illustrates the metal-specific fibril morphology, and has relevance in understanding the role of metals in neurodegeneration.

Increased expression of the γ‐secretase components presenilin‐1 and nicastrin in activated astrocytes and microglia following traumatic brain injury

γ‐Secretase is an aspartyl protease composed of four proteins: presenilin (PS), nicastrin (Nct), APH1, and PEN2. These proteins assemble into a membrane complex that cleaves a variety of substrates within the transmembrane domain. The γ‐secretase cleavage products play an important role in various biological processes such as embryonic development and Alzheimers disease (AD). The major role of γ‐secretase in brain pathology has been linked to AD and to the production of the amyloid β‐peptide. However, little is known about the possible role of γ‐secretase following acute brain insult. Here we examined by immunostaining the expression patterns of two γ‐secretase components, PS1 and Nct, in three paradigms of brain insult in mice: closed head injury, intracerebroventricular injection of LPS, and brain stabbing. Our results show that in naïve and sham‐injured brains expression of PS1 and Nct is restricted mainly to neurons. However, following insult, the expression of both proteins is also observed in nonneuronal cells, consisting of activated astrocytes and microglia. Furthermore, the proteins are coexpressed within the same astrocytes and microglia, implying that these cells exhibit an enhanced γ‐secretase activity following brain damage. In view of the important role played by astrocytes and microglia in brain disorders, our findings suggest that γ‐secretase may participate in brain damage and repair processes by regulating astrocyte and microglia activation and/or function.

Modulation in Acetylcholinesterase of Rat Brain by Pyrethroids In Vivo and an In Vitro Kinetic Study

Abstract: The modulation in acetylcholinesterase (AChE) of rat brain by two pyrethroids—permethrin (PM) and cypermethrin (CPM)—was studied both in vivo and in vitro. PM inhibited AChE activity in all regions of the rat brain (cerebral cortex, cerebellum, corpora striata, brainstem, hippocampus, and hypothalamus) at 4, 8, and 12 h after gastric intubation, whereas CPM elevated the enzyme activity in vivo. Substrate‐dependent enzyme kinetic studies have shown that PM and CPM behave as mixed‐type inhibitors, as evidenced by alterations in both Michaelis‐Menten constant (Km) and maximal velocity (Vmax) values. This indicates that both PM and CPM and substrate acetylcholine interact at hydrophobic subsites and may be able to bind simultaneously to the enzyme.

Alteration of superhelical state of DNA by aluminium (Al)

The effect of aluminium (Al) on the supercoiled state of pUC18 DNA was studied by ethidium bromide fluorescence and agarose gel electrophoresis. Al at physiologically relevant concentrations relaxed the intact supercoiled DNA as well as the topoisomers induced by chloroquine. EDTA prevented the unwinding effect of Al on supercoiled DNA. Al did not alter the mobility of linear DNA in agarose gels. The implications of this finding in neurological disorders are discussed.

Anti-amyloidogenic property of leaf aqueous extract of Caesalpinia crista.

Amyloid beta (Abeta) is the major etiological factor implicated in Alzheimers disease (AD). Abeta(42) self-assembles to form oligomers and fibrils via multiple aggregation process. The recent studies aimed to decrease Abeta levels or prevention of Abeta aggregation which are the major targets for therapeutic intervention. Natural products as alternatives for AD drug discovery are a current trend. We evidenced that Caesalpinia crista leaf aqueous extract has anti-amyloidogenic potential. The studies on pharmacological properties of C. crista are very limited. Our study focused on ability of C. crista leaf aqueous extract on the prevention of (i) the formation of oligomers and aggregates from monomers (Phase I: Abeta(42)+extract co-incubation); (ii) the formation of fibrils from oligomers (Phase II: extract added after oligomers formation); and (iii) dis-aggregation of pre-formed fibrils (Phase III: aqueous extract added to matured fibrils and incubated for 9 days). The aggregation kinetics was monitored using thioflavin-T assay and transmission electron microscopy (TEM). The results showed that C. crista aqueous extract could able to inhibit the Abeta(42) aggregation from monomers and oligomers and also able to dis-aggregate the pre-formed fibrils. The study provides an insight on finding new natural products for AD therapeutics.

New evidences on Tau-DNA interactions and relevance to neurodegeneration §

Tau is mainly distributed in cytoplasm and also found to be localized in the nucleus. There is limited data on DNA binding potential of Tau. We provide novel evidence on nicking of DNA by Tau. Tau nicks the supercoiled DNA leading to open circular and linear forms. The metal ion magnesium (a co-factor for endonuclease) enhanced the Tau DNA nicking ability, while an endonuclease specific inhibitor, aurinetricarboxylic acid (ATA) inhibited the Tau DNA nicking ability. Further, we also evidenced that Tau induces B-C-A mixed conformational transition in DNA and also changes DNA stability. Tau-scDNA complex is more sensitive to DNAse I digestion indicating stability changes in DNA caused by Tau. These findings indicate that Tau alters DNA helicity and integrity and also nicks the DNA. The relevance of these novel intriguing findings regarding the role Tau in neuronal dysfunction is discussed.

Role of advanced glycation on aggregation and DNA binding properties of α-synuclein.

Parkinsons disease (PD) is a neurodegenerative disease with multiple etiologies. Advanced glycation end products (AGEs) accumulate in the aging brain and could be one of the reasons for age-related diseases like PD. Oxidative stress also leads to the formation of AGEs and may be involved in neurodegeneration by altering the properties of proteins. α-Synuclein is involved in pathogenesis of PD and there are limited studies on the role of AGE-α-synuclein in neurodegeneration. We studied the aggregation and DNA binding ability of AGE-α-synuclein in vitro. α-Synuclein is glycated using methylglyoxal and formation of AGE-α-synuclein is characterized using fluorescence studies, intrinsic tyrosine fluorescence, and fructosamine estimation. The results indicated that AGE-α-synuclein aggregates into smaller globular-like aggregates compared to fibrils formed with native α-synuclein. Further, it is found that AGE-α-synuclein induced conformational changes in scDNA from B-form to B-C-A mixed conformation. Additionally, AGE-α-synuclein altered DNA integrity as evidenced by the melting temperature, ethidium bromide, and DNAse I sensitivity studies. AGE-α-synuclein converted biphasic Tm to higher monophasic Tm. The Tm of AGE-α-synuclein-scDNA complex is more than that of native α-synuclein-scDNA complex, indicating that AGE-α-synuclein stabilized the uncoiled scDNA. AGE-α-synuclein could stabilize the uncoiled scDNA, as shown by the decrease in the number of ethidium bromide binding molecules per base pair of DNA. DNAse I sensitive studies indicated that both AGE-α-synuclein-scDNA and α-synuclein-scDNA are resistant to DNAse I digestion. The relevance of these findings to neuronal cell death is discussed.

In vitro Evidence that an Aqueous Extract of Centella asiatica Modulates α-Synuclein Aggregation Dynamics

α-Synuclein aggregation is one of the major etiological factors implicated in Parkinsons disease (PD). The prevention of aggregation of α-synuclein is a potential therapeutic intervention for preventing PD. The discovery of natural products as alternative drugs to treat PD and related disorders is a current trend. The aqueous extract of Centella asiatica (CA) is traditionally used as a brain tonic and CA is known to improve cognition and memory. There are limited data on the role of CA in modulating amyloid-β (Aβ) levels in the brain and in Aβ aggregation. Our study focuses on CA as a modulator of the α-synuclein aggregation pattern in vitro. Our investigation is focused on: (i) whether the CA leaf aqueous extract prevents the formation of aggregates from monomers (Phase I: α-synuclein + extract co-incubation); (ii) whether the CA aqueous extract prevents the formation of fibrils from oligomers (Phase II: extract added after oligomers formation); and (iii) whether the CA aqueous extract disintegrates the pre-formed fibrils (Phase III: extract added to mature fibrils and incubated for 9 days). The aggregation kinetics are studied using a thioflavin-T assay, circular dichroism, and transmission electron microscopy. The results showed that the CA aqueous extract completely inhibited the α-synuclein aggregation from monomers. Further, CA extract significantly inhibited the formation of oligomer to aggregates and favored the disintegration of the preformed fibrils. The study provides an insight in finding new natural products for future PD therapeutics.

Effect of aluminium (Al) on brain mitochondrial monoamine oxidase-A (MAO-A) activity--an in vitro kinetic study.

Effect of aluminium (Al) on rat brain mitochondrial monoamine oxidase-A (MAO-A) was studiedin vitro at three different pH (4.0, 7.4 and 9.0) values. The results have shown that Al is a non-competitive inhibitor for MAO-A. The data also showed that MAO-A inhibition by Al varies with free Al3+ concentration and different forms of Al under different pH conditions. Al altered the maximum velocity (Vmax) of MAO-A but did not affect substrate-enzyme affinity (Km). Al formed a strong chelation with the substrate (Kynuramine) (1:1).

Aβ(42) induced MRI changes in aged rabbit brain resembles AD brain.

Alzheimers disease is the most common form of dementia and is structurally characterized by brain atrophy and loss of brain volume. Aβ is one of the widely accepted causative factors of AD. Aβ deposition is positively correlated with brain atrophy in AD. In the present study, structural brain imaging techniques such as Magnetic Resonance Imaging (MRI) were used to measure neuroanatomical alterations in Alzheimers disease brain. MRI is a non-invasive method to study brain structure. The objective of the present study was to elucidate the role of Aβ on brain structure in the aged rabbit brain. Among 20 aged rabbits, one batch (n=10) rabbits was injected chronically with Aβ(1-42) and another batch (n=10) with saline. The MRI was conducted before Aβ(1-42)/saline injection and after 45 days of Aβ(1-42)/saline injection. All the aged rabbits underwent MRI analysis and were euthanized after 45 days. The MRI results showed a significant reduction in thickness of frontal lobe, hippocampus, midbrain, temporal lobe and increases in the lateral ventricle volume. We also conducted an MRI study on AD (n=10) and normal (n=10) cases and analyzed for the thicknesses of frontal lobe, hippocampus, midbrain, temporal lobe and lateral ventricle lobe. We found significant reductions in thickness of the frontal lobe and the hippocampus. However, no significant reduction in the thickness of midbrain, temporal lobe or increase in the lateral ventricle volume was observed compared to normal. Correlations in brain atrophy changes between rabbit brain and human AD brain were found for frontal lobe and hippocampal regions. In contrast, other regions such as midbrain, temporal lobe, and lateral ventricles were not correlated with rabbit brain atrophy changes in the corresponding regions. The relevance of these changes in AD is discussed.