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Hideaki Nagase, Ph.D.
Kennedy Institute of Rheumatology-Centre for Degenerative Diseases, University of Oxford, UK

Osteoarthritis: diagnosis, treatment and challenges

Hideaki Nagase¹, Ngee Han Lim¹, George Bou-Gharios¹, Ernst Meinjohanns²  and Morten Meldal³

1. Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, London, W6 8LH  UK
2. Carlsberg Laboratory, Copenhagen, Denmark,
3. Nano-Science Center, Department of Chemistry, University of Copenhagen, Denmark

Osteoarthritis (OA) is the most prevalent age-related degenerative joint disease. With the expanding ageing population, it imposes a major socio-economic burden on society.  A key feature of OA is a gradual loss of articular cartilage and deformation of bone, resulting in the impairment of joint function. Currently, there is no effective disease-modifying treatment except joint replacement surgery. There are many possible causes of cartilage loss (e.g. mechanical load, injury, reactive oxygen species, aging, etc.) and etiological factors (obesity, genetics), but the degradation of cartilage is primarily caused by elevated levels of active metalloproteinases.  It is therefore attractive to consider proteinase inhibitors as potential therapeutics. However, there are several hurdles to overcome, namely early diagnosis and continuous monitoring of the efficacy of inhibitor therapeutics. We are therefore aiming at developing non-invasive probes to detect cartilage degrading metalloproteinase activities.

We have designed in vivo imaging probes to detect MMP-13 (collagenase 3) activity that participates in OA by degrade cartilage collagen II and MMP-12 (macrophage elastase) activity involved in inflammatory arthritis. These activity-based probes consist of a peptide that is selectively cleaved by the target proteinase, a near-infrared fluorophore and a quencher. The probe’s signal multiplies upon proteolysis.  They were first used to follow the respective enzyme activity in vivo in the mouse model of collagen-induced arthritis and we found MMP-12 activity probe (MMP12AP) activation peaked at 5 days after onset of the disease, whereas MMP13AP activation was observed at 10-15 days. The in vivo activation of these probes was inhibited by specific low molecule inhibitors.  We proceeded to test both probes in the mouse model of OA induced by the surgical destabilization of medial meniscus of the knee joints.  In this model, degradation of knee cartilage is first detected histologically 6 weeks after surgery with significant erosion detectable at 8 weeks. Little activation of MMP12AP was detected, which was expected, as macrophage migration is not obvious in OA. MMP13AP, on the other hand, was significantly activated in the operated knee at 6 weeks compared with the non-operated contralateral knee, but there were no significant differences between the operated and sham-operated knees.  At 8 weeks, however, the signals in the operated knees were significantly higher than both the contralateral and sham-operated controls. Activation of aggrecanases and MMP-13 are observed before structural changes of cartilage. We are therefore currently improving the MMP-13 probe for earlier detection by attaching it to polymers that are retained in  cartilage.

K. P. Mohanakumar, Ph.D.
Chief Scientist, Cell Biology & Physiology Division, Indian Institute of Chemical Biology, Kolkata

Neuroprotective and neurodestructive effects of Ayurvedic drug constituents: Parkinson’s disease

The present study reports the good and the bad entities in an Indian traditional medicine used for treating Parkinson’s disease (PD). A prospective clinical trial on the effectiveness of Ayurvedic medication in a population of PD patients revealed significant benefits, which has been attributed to L-DOPA present in the herbs [1]. Later studies revealed better benefits with one of the herbs alone, compared to pure L-DOPA in a clinical trial conducted in UK [2], and in several studies conducted on animal models of PD in independent laboratories world over [3-5]. We have adapted strategies to segregate molecules from the herb, and then carefully removed L-DOPA contained therein, and tested each of these sub-fractions for anti-PD activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone and 6-hydroxydopamine -induced parkinsonian animal models, and transgenic mitochondrial cybrids. We report here two classes of molecules contained in the herb, one of which possessed severe pro-parkinsonian (phenolic amine derivatives) and the other having excellent anti-parkinsonian potential (substituted tetrahydroisoquinoline derivatives). The former has been shown to cause severe dopamine depletion in the striatum of rodents, when administered acutely or chronically. It also caused significant behavioral aberrations, leading to anxiety and depression [6]. The latter class of molecules administered in PD animal model [7], caused reversal of behavioral dysfunctions and significant attenuation of striatal dopamine loss. These effects were comparable or better than the effects of the anti-PD drugs, selegiline or L-DOPA. The mechanism of action of the molecule has been found to be novel, at the postsynaptic receptor signaling level, as well as cellular α-synuclein oligomerization and specifically at mitochondria. The molecule helped in maintaining mitochondrial ETC complex activity and stabilized cellular respiration, and mitochondrial fusion-fission machinery with specific effect on the dynamin related protein 1. Although there existed significant medical benefits that could be derived to patients due to the synergistic actions of several molecules present in a traditional preparation, accumulated data in our hands suggest complicated mechanisms of actions of Ayurvedic medication. Our results also provide great hope for extracting, synthesizing and optimizing the activity of anti-parkinsonian molecules present in traditional Ayurvedic herbs, and for designing novel drugs with novel mechanisms of action.

1. N, Nagashayana, P Sankarankutty, MRV Nampoothiri, PK Mohan and KP Mohanakumar, J Neurol Sci. 176, 124-7, 2000.
2. Katzenschlager R, Evans A, Manson A, Patsalos PN, Ratnaraj N, Watt H, Timmermann L, Van der Giessen R, Lees AJ. J Neurol Neurosurg Psychiatry.75, 1672-7, 2004.
3. Manyam BV, Dhanasekaran M, Hare TA. Phytother Res. 18, 706-12, 2004.
4. Kasture S, Pontis S, Pinna A, Schintu N, Spina L, Longoni R, Simola N, Ballero M, Morelli M. Neurotox Res. 15, 111-22, 2009.
5. Lieu CA, Kunselman AR, Manyam BV, Venkiteswaran K, Subramanian T. Parkinsonism Relat Disord.16, 458-65, 2010.
6. T Sengupta and KP Mohanakumar, Neurochem Int. 57, 637-46, 2010.
7. T Sengupta, J Vinayagam, N Nagashayana, B Gowda, P Jaisankar and KP Mohanakumar, Neurochem Res 36, 177-86, 2011

D. Narasimha Rao, Ph.D.
Professor, Dept of Biochemistry, Indian Institute of Science, Bangalore, India

Genomics of Restriction-Modification Systems

Restriction endonucleases occur ubiquitously among procaryotic organisms. Up to 1% of the genome of procaryotic organisms is taken up by the genes for these enzymes. Their principal biological function is the protection of the host genome against foreign DNA, in particular bacteriophage DNA. Restriction-modification (R-M) systems are composed of pairs of opposing enzyme activities: an endonuclease and a DNA methyltransferase (MTase). The endonucleases recognise specific sequences and catalyse cleavage of double-stranded DNA. The modification MTases catalyse the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, R-M systems are generally classified into three groups. In general R-M systems restrict unmodified DNA, but there are other systems that specifically recognise and cut modified DNA. More than 3500 restriction enzymes have been discovered so far. With the identification and sequencing of a number of R-M systems from bacterial genomes, an increasing number of these have been found that do not seem to fit into the conventional classification.

It is well documented that restriction enzyme genes always lie close to their cognate methyltransferase genes. Analysis of the bacterial and archaeal genome sequences shows that MTase genes are more common than one would have expected on the basis of previous biochemical screening. Frequently, they clearly form part of a R-M system, because the adjacent open reading frames (ORFs) show similarity to known restriction enzyme genes. Very often, though, the adjacent ORFs have no homologs in the GenBank and become candidates either for restriction enzymes with novel specificities or for new examples of previously uncloned specificities. Sequence-dependent modification and restriction forms the foundation of defense against foreign DNAs and thus RM systems may serve as a tool of defense for bacterial cells. RM systems however, sometimes behave as discrete units of life, and any threat to their maintenance, such as a challenge by a competing genetic element can lead to cell death through restriction breakage in the genome, thus providing these systems with a competitive advantage. The RM systems can behave as mobile-genetic elements and have undergone extensive horizontal transfer between genomes causing genome rearrangements. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.

The similarities and differences in the different mechanisms used by restriction enzymes will be discussed. Although it is not clear whether the majority of R-M systems are required for the maintenance of the integrity of the genome or whether they are spreading as selfish genetic elements, they are key players in the “genomic metabolism” of procaryotic organisms. As such they deserve the attention of biologists in general. Finally, restriction enzymes are the work horses of molecular biology. Understanding their enzymology will be advantageous to those who use these enzymes, and essential for those who are devoted to the ambitious goal of changing the properties of these enzymes, and thereby make them even more useful.