MITOCHONDRIAL RESPIRATORY CHAIN FUNCTION IN PD

It has been proposed that late-onset neurodegenerative diseases m ay be caused by mitochondria! respiratory chain (MRC) abnormalities. In support of this theory it has been shown th at MRC function decreases w ith age, and th at the brain is highly dependent on oxidative phosphorylation for ATP generation (Wallace,

1 9 9 2 ). Abnormal MRC function has been associated w ith a number of neurodegenerative diseases including Alzheim er's disease, Huntington's disease, dystonia and Parkinson's disease.

Following the discovery that the Parkinson-causing neurotoxin MPP^ is a specific inhibitor of mitochondrial complex I (Nicklas e t al, 1 9 8 5 ), interest has particularly focused on the function of the MRC in idiopathic Parkinson's disease. MRC function has been studied in the PD brain, skeletal muscle platelets and lym phocytes, as detailed below.

1 .4 .1 . MRC function in the PD brain

In the largest study published to date, MRC function w as measured in substantia nigra brain homogenate from 2 2 PD patients and 17 controls (Mann e t ai,

1 9 9 2 a ). The activity of complexes ll/lll and IV w ere normal, but there w as a 3 7 % decrease in mean complex I activity in the PD substantia nigra. Patient and control groups w ere matched for age, death to refrigeration and death to autopsy tim es, and there w as no correlation betw een complex I activity and duration of disease, L-dopa treatm ent or tim e from death to freezing. The observed complex I defect has been shown to be disease specific (Schapira e t ai, 1 9 9 0 b ), in that complex I activity w as normal in the substantia nigra of patients w ho had died w ith multiple system atrophy (M SA ). This disease is characterised by cell death in many areas of the brain, including the substantia nigra, w here cell death is observed at levels equal to or greater than th at seen in PD. Normal complex I activity in the substantia nigra of M S A patients therefore provides evidence that the complex I defect observed in PD is not simply the result of neuronal degeneration. Patients w ith M SA are prescribed L-dopa, providing more evidence that the complex I defect in the PD brain is not simply caused by L-dopa therapy. The complex I deficiency has been shown to be anatom ically specific to the substantia nigra in the PD brain (Schapira e t ai,

the caudate nucleus, cerebral cortex or the medial or lateral globus pallidus in four PD patients and five controls, or in the tegm entum (ten samples), cerebellum (sixteen samples) or posterior putamen (tw elve samples; Dr JM Cooper, personal communication). These results closely resemble the effects of MPTP in term s of anatomical selectivity (to the substantia nigra) and biochemical specificity (to a deficiency of complex I), suggesting that the cause of cell death in the idiopathic disease may be similar to that in the MPTP model.

Only tw o other studies of mitochondrial function in the PD brain have been published. Mizuno et a! (1 9 9 0 ) detected a slight but significant decrease in mean complex III activity, and normal complex I activity, in the striatum of five PD patients. Janetzky e t a! (1 9 9 4 ) detected a 3 3 % decrease in the citrate synthase corrected activity of rotenone-sensitive NADH cytochrome c reductase (an assay representing complex I activity) in the substantia nigra of seven PD patients compared to seven controls, matched for age and post-mortem parameters.

Two studies have reported on the composition of complex I in the PD brain by Western blotting w ith complex I antibodies. Schapira e t a! (1 9 9 0 a ) using substantia nigra homogenates probed w ith antibodies to the complex I holoenzyme and specific for the 3 0 , 2 4 and 13kDa subunits, could detect no differences in molecular w eight or staining intensity of any complex I polypeptide. Mizuno e t a! (1 9 8 9 ) detected moderate to marked decreases in the staining intensity of polypeptides of 3 0 , 2 5 , and 24kD a in four out of five PD striatum samples compared to controls. Hattori e t a! (1 9 9 1 ) have used immunohistochemical techniques to quantify the amount of complex I in PD substantia nigra sections compared to controls. Using antisera to the complex I holoenzyme, they observed that neurons w ith reduced staining, w ere far more abundant in the eight PD brain sections than in the control sections.

1 .4 .2 . MRC function in PD skeletal muscle

To date there have been seven reports of MRC function in PD skeletal muscle. Bindoff e t a! (1 9 9 1 ) detected statistically significant generalised defects of complexes I, II and IV in five PD patients the mean activities being decreased by 4 0 , 4 9 and 4 0 % respectively compared to controls. Shoffner e t a! (1 9 9 1 ) compared six PD patients and eleven controls. Two of the patients had only complex I defects, tw o had complex I, II, III and IV defects, one had complex II, III and IV defects and one showed normal MRC function. Relative to the control

m ean, the activities of complex I in the four patients showing a complex I defect w ere 9 2 , 9 0 , 8 6 , and 6 2 % decreased. N akagaw a-H attori e t a! (1 9 9 2 ) using mitochondria prepared from post-mortem samples from four PD patients detected 4 9 , 2 6 and 2 1 % decreases in the mean activity of com plexes I, II and III respectively (only the difference in complex I activity w as statistically significant). The activity of complex IV w as not significantly different in patients and controls. Mann e t a! (1 9 9 2 a ) compared MRC function in nine PD patients and six controls by both spectrophotometry and polarography. No differences w ere detected. Anderson e t a! (1 9 9 3 ) compared MRC function in seven PD patients and six controls by spectrophotom etry and polarography. Although some MRC enzyme activities w ere decreased in the patient group, none of the results w ere statistically significant, which may have been due to the extrem ely large spread of data in the control group. Cardellach e t a! (1 9 9 3 ) detected statistically significant decreases of 2 6 % and 6 8 % in the mean activities of complexes I and IV respectively in muscle mitochondria from eight PD patients compared to ten controls. DiDonato e t a! (1 9 9 3 ) measured MRC enzyme activity in skeletal muscle crude homogenates and purified mitochondria from sixteen PD patients and eight controls (homogenates), and six patients and six controls (purified mitochondria). There w ere no significant differences in the citrate synthase (CS) corrected activities of any of the MRC enzymes betw een the tw o groups, measured in crude homogenates or purified mitochondria.

From the above studies it is clear that the presence of a MRC abnormality in the skeletal muscle of PD patients is controversial. Evidence from ^^P magnetic resonance spectroscopy (MRS) studies provides evidence of normal mitochondrial function in PD skeletal muscle (Taylor e t al, 1 9 9 4 ). In a group of mitochondrial myopathy patients, w ith defined biochemical complex I defects, many of the parameters measured were abnormal; during exercise, stores of high energy phosphate w ere depleted more rapidly, and during recovery, phosphocreatine and free ADP concentrations returned to basal levels more slowly than in controls. In the PD patients however, none of these parameters w ere statistically significantly different from controls. Although these results do not rule out the presence of small complex I deficiencies in PD skeletal muscle, they do suggest that any defect present is not as great as th at in the mitochondrial myopathy patients, whose mean complex I activity w as 6 0 % less than controls. Other evidence against the presence of an MRC defect in PD skeletal muscle is the absence of increased blood lactate levels post-exercise in PD patients (Nakagaw a-H attori e t ai, 1 9 9 2 ), and the lack of significant morphological abnormalities in PD skeletal muscle biopsies (Bindoff e t ai, 1 9 9 1 ; Shoffner e t ai, 1 9 9 1 ; Mann e t ai, 1 9 9 2 a ).

1 .4 .3 . MRC function in PD platelets

The main role of platelets in the circulation is the form ation of blood clots in response to vascular damage. They adhere to damaged vascular epithelia, and w hen appropriately stimulated aggregate into clumps sealing the damaged vessel. Platelets are discoid cells, 2-5 //m in diameter, and normal blood contains 1 5 0 -3 5 0 ,0 0 0 platelets per jj\. They have a short life span surviving in the circulation for only eight to tw elve days. Platelets are released from cytoplasmic projections of the m egakaryocyte stem cells of bone marrow. They contain lysosomes, mitochondria, glycogen granules, dense granules and alpha granules w hich store substances involved in the aggregation process, but have no nucleus. It has been estim ated that they only contain four mitochondria per cell, and only one copy of m tD N A per mitochondrion (Shuster e t al, 1 9 8 8 ). Platelets have been considered a useful model system for central serotonergic neurons (Da Prada e t ai, 1 9 8 8 ); they have an active transport system for serotonin (5- hydroxytryptam ine or 5-H T ), which is stored in dense bodies. Their use as a model system for dopaminergic neurons is less well established, although dopamine shows some affinity for the 5-H T uptake and storage systems.

In 1 9 8 9 Parker e t a! published the first report of MRC function in PD platelets. Mitochondrial fractions w ere prepared on Percoll density gradients after plateletphoresis from ten PD patients and eight controls. A specific 5 5 % decrease in the activity of complex I in the PD group w as detected. Since platelets are a peripheral tissue, this led to the possibility that the detection of a complex I defect in platelets could be used as a diagnostic test for PD. Further studies of PD platelet MRC function are detailed in Chapters 3 and 4.

1 .4 .4 . M tD N A and PD

Mitochondrial DNA codes for 13 functional polypeptides, all of which are constituent subunits of the complexes of the mitochondrial respiratory chain. Since seven of the 41 subunits of complex I are coded for by m tD N A (Chomyn e t ai, 1 9 8 5 , 1 9 8 6 ), any complex I defect in PD may be due to a mutation in a complex I gene within m tD N A . As PD is not m aternally inherited, a m tDN A mutation is unlikely to be the sole cause of the disease (Zweig e t ai, 1 9 9 2 ) (although m tD N A deletions appear to be sporadic and are not m aternally inherited). However, m tD N A mutations may be one of many factors contributing to the pathogenesis of PD.

1 .4 .4 .1 . M tDNA deletions and PD

The presence of large amounts of deleted m tD N A in the PD substantia nigra (Schapira e t al, 1 9 9 0 c ; Lestienne a t ai, 1 9 9 0 ) or skeletal muscle (Shoffner a t ai, 1 9 9 1 ; N akagaw a-Hattori a t ai, 1 9 9 2 ) has been ruled out by Southern blotting techniques. Initial reports, using the more sensitive polymerase chain reaction (PCR), detected an increased level of m tD N A in the PD brain carrying the 5kb "common" deletion (Ikebe a t ai, 1 9 9 0 ; O zaw a a t ai, 1 9 9 0 ). This deletion is commonly found in the skeletal muscle of patients w ith mitochondrial myopathies (Holt a t ai, 1 9 8 8 ) and covers the genes encoding four complex I subunits (N D 3, 4L, 4 and 5), one complex IV subunit (COIN), tw o complex V subunits (A6 and A6L) and five tRNAs. Correcting for differences in the efficiency of amplification of normal and deleted m tD N A by PCR, Mann a t a! (1 9 9 2 b ) detected similarly low levels (0 .0 1 % ) of deleted m tD N A in both PD and age-m atched control substantia nigra samples. They conclude that the common deletion is not specific to the PD brain, but is rather an age-related phenomenon. This result has subsequently been confirmed by Di Donato a t a! (1 9 9 3 ), and the same phenomenon has been observed in platelet m tD N A from PD patients and controls (Sandy a t ai, 1 9 9 3 ). Interestingly, DiDonato a t a! (1 9 9 3 ) detected higher concentrations (at least a factor of three) of deleted m tD N A in the substantia nigra than in any of the nine other brain regions studied, in a single PD and a single control case.

1 .4 .4 .2 . M tDNA point mutations and PD

Mitochondrial DNA polymorphisms have been detected in individual PD patients in many studies (Lestienne a t ai, 1 9 9 0 ; Schapira a t ai, 1 9 9 0 c ; Johns a t at, 1 9 9 1 ; O zaw a a t ai, 1 9 9 1 ) although the relevance of these to disease pathogenicity is not known. Recently, Shoffner a t a! (1 9 9 3 ) detected the presence of a homoplasmic point mutation in the mitochondrial tR N A °‘" gene in a large group of PD patients. The mutation is a transition (A to G) of a conserved nucleotide (n t4 3 3 6 ) in the gene, which connects the amino acid acceptor stem w ith the T rC stem of tRNA^'" and is presumed to a ffe c t its function. The m utation w as detected in 5 .3 % (2 /3 8 ) of PD samples and in 0 .7 % (1 2 /1 6 9 1 ) of controls; approximately eight times more common in PD patients than controls. The increased frequency of this mutation w as not how ever specific to PD, and w as also detected in 3 .2 % (2 /6 2 ) of Alzheim er's disease (AD) samples. The authors state that the presence of the mutation in a small proportion of controls

could reflect the prevalence of the presymptomatic stage of these diseases in the general population.

A mutation in a mitochondrial tRNA gene would be expected to a ffe c t the translation of all mitochondrial mRNAs, and since mitochondrial polypeptides are constituents of complexes I, III and IV, this tRNA^'" mutation would be expected to a ffect the activities of all of these complexes. Two mitochondrial diseases caused by mitochondrial tRNA m utations are MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged red fibres). The MELAS syndrome is associated w ith a mitochondrial tRNA^®“ m utation, and the MERRF syndrome w ith a mitochondrial tRNA^''® m utation. Interestingly, in both syndromes a complex I defect in skeletal muscle mitochondria is most often the principal biochemical abnormality (review; Cooper and Clark, 1 9 9 4 ). Therefore, it is possible for a tR N A gene mutation to primarily cause a deficiency of complex I.

1 .4 .5 . MRC function in neurodegenerative diseases other than PD

Alzheim er's disease (AD) pathology includes degeneration of the cerebral cortex, particularly the temporal cortex. Reichmann e t a! (1 9 9 3 ) have reported a 4 0 % decrease in the activity of complex ll/lll in the AD temporal cortex. Cytochrome oxidase (complex IV) activity has been reported as slightly (1 7 % ) decreased (Kish e t al, 1 9 9 2 ), or normal (Cooper e t ai, 1 9 9 3 ) in the AD temporal cortex , or as 5 0 % decreased in AD platelet mitochondria (Parker e t ai, 1 9 9 0 b ).

Huntington's disease (HD) is characterised pathologically by degeneration in the caudate nucleus and putamen. Mann e f a/ (1 9 9 0 ) have reported a 7 7 % decrease in the activity of complex ll/lll in the HD caudate nucleus, and Parker e t a! (1 9 9 0 a ) have reported a 7 2 % decrease in the activity of complex I in HD platelet mitochondria.

Dystonia is a m ovem ent disorder believed to be caused by dysfunction of the basal ganglia. Benecke e t a! (1 9 9 2 ) have reported 6 2 % and 3 7 % reductions in the mean activity of complex I in the platelet mitochondria of individuals w ith either segmental/generalised dystonia or focal dystonia respectively. Only the reduction in segmental/generalised dystonia complex I activity compared to controls reached statistical significance ( p < 0 .0 0 1 ) .

1 .4 .6 . Mitochondria and ageing

The most important risk factor for many neurodegenerative diseases, including PD, is age. If abnormal mitochondrial function plays a role in the aetiology of PD, decreasing mitochondrial function w ith age may help to explain the late onset of the disease.

1 .4 .6 .1 . Ageing and MRC function

Mitochondrial respiratory chain function has been found to decrease w ith age in human liver (Yen e t a!, 1 9 8 9 ) and skeletal muscle (Trounce a t a/, 1 9 8 9 ; Cooper a t a/, 1 9 9 2 ), and tw o studies have shown a decrease in MRC function w ith age in the primate brain. Bowling a t a! (1 9 9 3 ) found a significant negative correlation betw een complex I and IV activity and age in the frontoparietal cortex of 20 rhesus monkeys, and Di Monte a t a! (1 9 9 3 ) found reduced respiratory chain activity w ith NAD-linked, but not FAD-linked substrates, in the striatum from aged squirrel monkeys.

1 .4 .6 .2 . Ageing and mtPNA

Mitochondrial DNA m utations have been shown to accum ulate w ith age in human liver (Yen a t a/, 1 9 9 1 ) and skeletal muscle (Cooper a t at, 1 9 9 2 ). The levels of the 5kb "common" deletion in human skeletal muscle increased w ith age to a maximum proportion of 1 in 5 0 0 0 (Cooper at al, 1 9 9 2 ). Such a low level of deleted molecules is unlikely to account for any decrease in respiratory chain function w ith age. However it is possible that a number of different deletions and point mutations of m tD N A accum ulate w ith age and together account for the decreased respiratory chain function.

Generalised oxidative damage to cellular DNA, measured as the presence of the oxidised nucleoside 8-hydroxy-2'-deoxyguanosine (OH^dG), increases w ith age in the human brain (Mecocci at al, 1 9 9 3 ). This damage w as predominantly detected in m tD N A , and may therefore be expected to result in reduced function of the respiratory chain w ith age.