Synopsis

Synopsis of the thesis submitted for the award of Ph.D. degree (Biochemistry) to the University of Mysore, Mysore, ndia. !it"e of thesis#
STUDIES ON DNA HELICITY, STABILITY AND -SYNUCLEIN-DNA INTERACTIONS IN RELEVANCE TO PARKINSONS DISEASE

$andidate# Muralidhar L. H !d
Parkinsons disease (PD) is a progressive, neurodegenerative disorder that is characterized by the severe motor symptoms, including uncontrollable tremor, postural imbalance, slowness of movement and rigidity. he main pathological hallmark of PD is a pronounced loss of dopamine!producing neurons in the substantia nigra ("#), which results in a drastic decrease in dopamine in the striatum, to which these neurons pro$ect. he etiology of PD has not been fully understood. %nproven hypotheses have included environmental to&ins including metals, pesticides etc and genetic factors. 't is proposed that a cross!talk of environmental and genetic factors may be playing a role in causing PD. ( variety of mechanisms that are believed to cause accelerated cell death have also been suggested, including o&idative stress, e&citoto&icity and mitochondrial dysfunction. Pathologically, PD is characterized by the loss of the pigmented dopaminergic neurons from the substantia nigra pars compacta, the presence of e&tracellular melanin (a dark pigment), released from degenerating neurons, reactive gliosis (increase in numbers of glial or support cells), and pink!staining cellular inclusions known as Lewy Bodies.

he e&act causes and molecular pathogenesis of PD is largely undefined. )owever, accumulating evidence suggest the involvement of protein conformational deficit (!synuclein to&icity), metal to&icity, o&idative stress, D#( instability etc. *e

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focused on the D#( topology and stability and !synuclein!D#( interactions in the present investigation. he ob$ectives of the present investigation were+ ,. o study the helicity and stability (topology) of genomic D#( isolated from selected regions of Parkinson disease affected human post!mortem brain ( Chapter -) -. o study the mechanism of D#( binding and nicking property !synuclein (Chapter .) .. o study the conformation/aggregation of !synuclein in presence of D#( and understanding the mechanism using the chaperon property of naturally occurring osmolytes (Chapter 0) 0. o map trace elemental homeostasis in the serum samples Parkinson1s patients (Chapter 2). he research work carried towards achieving these ob$ectives makes the sub$ect matter of the thesis. he thesis is divided in to five chapters.

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his chapter begins with a general account of PD, followed by an overview of current literature on symptoms, causes, disease diagnosis, treatments available and the molecular mechanism of PD pathogenesis. he role of !synuclein in PD has been

discussed in detail. he chapter also highlights the aims and scope of the present study. *e also provided a hypothesis on the comple&ity of !synuclein to&icity in PD.

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3ecently few studies showed that o&idative stress, D#( damage, chromatin condensation, and altered e&pression of genes are also associated with neurodegeneration in PD like (lzheimer1s disease ((D). 4ne of the conse5uences of

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redo& imbalance is apoptosis and/or necrosis (programmed vs passive cell death) which are associated with neurodegeneration in PD. "tudies have also shown that the levels of the nucleoside, 6!hydro&y !-!deo&yguanosine (6!4)d7), a product of free radical attack on D#( were generally increased and differentially distributed in PD brains with highest levels in caudate, putamen, "# and cerebral corte&. 8urther, our laboratory evidenced that the genomic D#( undergoes a helicity change in (lzheimers disease ((D) from 9!form to left handed :!D#(. hese observations suggest that topological changes in the genetic material may be involved in the pathogenesis of PD also. "o far no studies have been reported on the D#( conformation in PD affected brain cells. I# $hi' ) r') &$i. , we studied the stability, integrity and topology of D#( isolated from five clinically and neuropathologically confirmed PD cases and si& age! matched controls. 7enomic D#( was isolated from eight regions in the human brain, namely frontal, temporal and occipital corte&, hippocampus, caudate/ putamen, thalamus, cerebellum and midbrain collected at post!mortem from cases of PD and controls and were analyzed for single and double strand breaks in D#(, and their conformations and topology. he results showed that D#( from midbrain in PD accumulated significant number of strand breaks than age!matched controls. ;audate nucleus/ putamen, thalamus and hippocampus also showed more D#( fragmentation compared to control brains. ;ircular dichroism studies showed that D#( conformation was altered with imprecise base stacking in midbrain, caudate nucleus/ putamen, thalamus and hippocampus in PD. )owever, D#( from frontal, temporal and occipital corte&, cerebellum was not affected significantly in PD group as compared to controls. his study provides a new data on stability, damage and conformations of D#( in different regions in brains of PD patients.

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he pathological hallmark of PD is the formation of insoluble protein aggregates known as Lewy bodies. he ma$or constituent of these fibrillar structures is -

'*#u&l i#, a ,0< amino acid protein with a basic amino terminal and an acidic carbo&y terminal. here is little information available about the neurobiology of !synuclein

under normal and neurodegenerative conditions. 'n amyloid pla5ues of PD and other

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neurodegenerative disorders, !synuclein has cross beta conformation while it adopts ! helical conformation in presence of phospholipids and lipid membranes. he formation of !heli& is suggested as a protective mechanism against formation of beta sheet and aggregated structures. he ma$or efforts of drug development are focused on the prevention or delaying of the protein aggregation and formation of pla5ues and recently emphasis has been given to stabilizing the non-toxic form of the protein . 3ecent observations showed that !synuclein is localized in the chromatin region of nuclei in the brain. =oreover, the presence of the ma$ority of the lysine residues in the #! terminal region of !synuclein suggests a possible D#( binding role for !synuclein. 3 d (%#'$ra$ d i# $h )r ' #$ '$ud* , two new and novel properties of ! synuclein. 8irst, we showed that !synuclein binds to D#( and alters the conformation of D#(. "econd, !synuclein was shown to have D#( nicking activity and it behaves like a nuclease enzyme. hese are new evidences in literature on !synuclein binding to D#(. 't was also observed that the nicking activity involves the formation of only single strand breaks. )owever, during long term incubations with !synuclein, double strand breaks were formed chopping the D#( into small pieces indicating that ! synuclein preferentially nicks only single stranded D#(. 8urther, the ability of known nuclease inhibitors, (urintricarbo&ylic acid (( () and Diethyl pyrocarbonate (D>P;) to abolish D#( nicking activity of !synuclein reveals that histidine residue at 2< th position in !synuclein se5uence plays a crucial role in the nicking activity. *e further showed that conformational change or oligomerisation of !synuclein would enhance the nicking activity. his indicates that the oligomers of !synuclein are more to&ic in terms of D#( nicking than monomers and aggregates. his work was awarded I#$ r#a$i%#al Al4h i( r' 5 ll%1'hi) and was presented in the ?th 'nternational =eeting on (lzheimers disease and 3elated Disorders held in Philadelphia, %"( in @uly, -<<0. *e discussed the potential implications of the above in vitro findings to neurodegenerative changes associated with PD. *e proposed that the D#( binding property of !synuclein characterized in the present study may have a significant effect

Synopsis

on nuclear!translocated !synuclein functioning. 'n particular, !synuclein may interact with histone!free D#( segments and induce nicking.

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"tructurally, purified !synuclein is a natively unfolded protein. his lack of

folding has been shown to correlate with the specific combinations of low overall hydrophobicity and large net charge. In vitro !synuclein readily assembles into fibrils, with morphologies and staining characteristics similar to those of fibrils e&tracted from PD affected brain. 't has been assumed that !synuclein may e&ist in two structurally different isoforms in vivo+ a heli&!rich, membrane!bound form and a disordered, cytosolic form, with the membrane!bound !synuclein generating nuclei that seed the aggregation of the more abundant cytosolic form. I# $hi' &ha)$ r, 1 $ri d $% understand the ability of different D#( to induce conformational changes in !synuclein. *e have used the effect of chaperonic properties of five osmolytes viz. glycerol, betaine, taurine, =(4, sarcosine for their ability to induce folding in !synuclein, as a model system to understand the D#( induced conformational changes in !synuclein. *e provided a comprehensive picture of D#( binding effect on !synuclein fibrillation using different D#(s such as double and single stranded D#(, ( and 7; se5uence specific D#(, of different sizes, genomic D#( etc. *e showed that only those D#( which induce a partial folding in !synuclein (7;A rich D#() promote its aggregation, while, sscD#( forms !heli& conformation in !synuclein and also inhibit aggregation to a considerable e&tent. 't was also observed that among the osmolytes used in the present study, glycerol, =(4, betaine and taurine induced partial folding in !synuclein and enhanced the fibrillation kinetics. he ability of D#( and osmolytes in inducing conformational transition in ! synuclein, indicates that two factors are critical in modulating !synuclein folding+ (i) >lectrostatic interaction as in the case of D#(, and (ii) )ydrophobic interactions as in

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the case of osmolytes. *e feel that the property of sscD#( in inducing !helical conformation in !synuclein and inhibiting the fibrillation may be of significance in engineering DNA-&hi) +a' d $h ra) u$i& a))r%a&h ' to PD and other amyloid disorders. 8urther, from the fact that other amyloidogenic peptides like ( , tau and prions also have D#( binding property, it appears that the D#( binding is a unifying property of amyloidogenic peptides implicated in various neurodegenerative disorders.

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r la$i%#'hi)' i# )a$i #$' 1i$h PD

=etals are well established to be risk factors for PD, though the significance of metals in the aetiopathogenesis of PD is still unknown. "everal studies have shown decreased copper, increased zinc and iron in the substantia nigra and increased copper concentrations in the cerebrospinal fluid of PD affected patients. hough much work has been done on metal homeostasis in PD brain, limited data is available on trace elemental levels and their inter!relationships in serum samples of Parkinson1s patients. =oreover, most of the available information is limited to few selected elements and there is no study, which e&amines inter!elemental relationships with regard to severity of PD. he aim of this study was to assess the serum levels of ,- elements (#a, B, 8e, (l, ;u, :n, ;a, =g, =n, "i, P and ") in patients with early and severe PD compared with a control population and also to understand the possible relevance of inter! elemental relationships to the progression of PD. Th r 'ul$' 'h%1 d a definite pattern of variation among certain elements in early and severe PD compared to control. 'n both early and severe PD serum, (l and " (pC<.<2) were decreased compared to control. 8e (pC<.<,) and :n (pC<.<2) were significantly low in severe PD, while B, =g, ;u (pC<.<,) and P (pC<.<2) were high in early and severe PD compared to control. he data revealed a clear imbalance in the elemental interrelationship in both early and severe PD serum compared to control as shown by the direct and inverse correlations. hese results suggested that a definite here disturbance in the elemental homeostasis occurs during the progression of PD.

has been a controversy regarding metal levels in PD serum and also possible role of metals as risk factors for PD. 't is not clear whether alteration in metal homeostasis is a

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cause or conse5uence of disease pathology. "o far, there is no detailed or comprehensive database on metal homeostasis and inter!relationships. he available reports only indicated the changes in levels of one or two individual elements, but fall short to correlate the element!to!element inter!relationship pattern with the disease progression. 'n this perspective, the present study provided a comprehensive database on concentrations of as many as ,- elements (ma$ority of essential and few important to&ic elements) in PD serum in comparison with control groups. 3 ,ur$h r ad.%&a$ d from the above results that irrespective of metals being primary risk factors or conse5uences of disease mechanism, a moderate change in a single metal ion will upset the whole elemental homeostasis pool resulting in the significant imbalance in elemental levels in the body system (serum, ;"8 and brain). he effect of increase or decrease of a single metal is not restricted to the presenting metal alone, it will affect the total elemental and charge distribution pattern in the system. he thesis ends with a comprehensive " ummary and Conclusions of the present study.

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,. he study provided a new data on D#( stability, integrity and topology of genomic D#( isolated from eight different brain regions from PD affected human brain samples (Chapter -). -. *e evidenced two new and novel properties of !synuclein+ i) D#( binding and ii) D#( nicking. *e showed that !synuclein behaves as an endonuclease. hese have been very intriguing observations which e&posed a new to&ic role of ! synuclein. he observations lead to a new debate on whether direct !synuclein induced D#( damage has any role to play in D#( fragmentation observed in PD brain and hence open new avenues of research in this direction ( Chapter .).

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*e showed that single stranded circular D#( induces a stable, aggregation resistant, !helical conformation in natively random!coil !synuclein. his

observation is very important in investigating the possibility of using D#(!chip based therapy for PD. *e also showed that 7;A rich D#( forms a partially folded intermediate conformation in !synuclein and this conformation has high aggregation propensity. %sing chaperonic properties of !synuclein as model system we showed that increase in hydrophobicity and decrease in the net charge are critical in !synuclein conformational transition and fibrillation ( Chapter 0).

0.

*e generated a comprehensive database on the levels of ,- elements (both essential and to&ic) in serum samples of PD affected patients and showed that there is a definite disturbance in inter!element homeostasis pattern in PD serum (Chapter 2).

6Muralidhar L. H !d 7 ;andidate

6Dr. K.S. 8a!a##a$ha Ra%7 3esearch "upervisor