Parkinson's disease, Precision Medicine, and LRRK2

Ten years ago, in 2008, I participated in a meeting in New York with the management of eight of the world’s largest pharmaceutical companies. The Michael J. Fox Foundation had asked us to discuss investment in leucine-rich repeat kinase (LRRK2 – pronounced ‘Lark 2’) inhibitors for Parkinson’s disease (PD), and whether such a development might halt or even prevent it. Typically, it takes a billion dollars to take a novel drug to market. At that time ‘precision medicine’ for PD was a relatively new concept, as was neuroprotection for the US Food and Drug Administration i.e. to halt or prevent disease and by lifelong administration of a therapeutic targeted to a genetic cause. While I was elated to be part of this development it was sobering to hear the arguments that ensued. Nevertheless, genetic discoveries are revolutionary, and they continue to provide novel approaches to predict and prevent PD.

LRRK2 was identified using a classical genetic linkage approach. In 2002 a region of chromosome 12p12 was reported to segregate ‘identical-by-descent’ with clinical parkinsonism in the Japanese Sagamihara family1,. We found similar results for chromosome 12 in two Caucasian kindreds, Family A (German-Canadian) and Family D (Western Nebraska)2, and soon after in Norwegian pedigrees3. In these families Parkinson’s disease (PD) is inherited in a dominant fashion i.e. approximately half of every generation eventually becomes affected. Although fourteen percent of patients have an affected first-degree relative (a parent, sibling or child) with PD4, it is uncommon to find more than two affected subjects in a family. Genetic research requires informed consent to ask questions about family history, to perform clinical exams, take blood for DNA extraction, and potentially ask permission for studies of brain pathology post-mortem. For the advances made we are indebted to those who have participated.

In 2004, we described the precise LRRK2 mutations that resulted in PD, namely LRRK2 R1441C (Families D and 469), Y1699C (Family A) and I2020T (Family 32)5. The work was done with Dr. Zbigniew Wszolek at Mayo Clinic, and as part of an international team. Concomitantly, a group at the US National Institutes of Health reported LRRK2 R1396G in four Basque Spanish families and Y1654C in an English kindred6. Although incorrectly labelled, LRRK2 R1396G is actually R1441G, and Y1654C is Y1699C, these families provided more evidence for pathogenicity. LRRK2 I2020T was also found to cause disease in the Sagamihara kindred7.

Notably, six affected subjects of Family A and D, and six members of the Japanese Sagamihara family donated their brains to research8,9. All were found to have mid-brain neurodegeneration with profound neuronal loss in the substantia nigra, typical of sporadic PD. However, only about a quarter of these patients had mid-brain Lewy body disease (‘alpha-synuclein immunopositive’ aggregates). Many patients had neurofibrillary tangles (‘tau immunopositive’ aggregates) or alternatively ‘ubiquitin immunopositive’ aggregates. It was amazing the brain pathology was so different (termed ‘pleomorphic’) among individuals, despite clinically similar presentations, and even within each family with the same disease-causing LRRK2 mutation8. Until recently, a diagnostic requirement for ‘definite’ PD had required the presence of mid-brain Lewy body disease so this observation was really contentious. Nevertheless, as many as ~20% of autopsy-confirmed cases with “probable” PD may not have mid-brain Lewy body pathology, (although clinically these patient may have been quite typical)10. To this date, and with >37 brains examined, only half of all patients with LRRK2 mutations and clinical PD develop mid-brain Lewy body pathology11.

With Dr. Jan Aasly’s help, then Head of the Department of Neurology at St. Olav’s, Trondheim, we discovered LRRK2 G2019S in Norwegian families3. The mutation also segregated with PD in a dominant fashion12, and through many generations of affected subjects dating as far back as the 15th century18. While we also reported affected families in Poland, Ireland, Spain and the US3, LRRK2 G2019S proved to be most frequent in ‘seemingly sporadic’ PD south and east of the Mediterranean. The frequency was highest in Ashkenazi Jews in Israel (and New York) and Arab-Berber populations in North Africa (Algeria and Tunisia), where it is found in 13% and 30% of their PD, respectively13,14. I write ‘seemingly sporadic’ as in all these families and populations – whether from Trondheim in Norway, Tel Aviv in Israel or Tunis in Tunisia – LRRK2 G2019S originates from the same ancestral founder, in effect, these patients are all distant cousins3,15. Arguably, the LRRK2 G2019S mutation has been dated to ~1,000 BC, a time when Mediterranean sea trade was most active between Phoenician ports in North Africa (Tunisia, Algeria and Morocco), Europe (Spain and Italy) and the Levant (Lebanon, Syria, Israel)16. In the present day populations of Israel and Tunisia, 1.9% of Ashkenazi and Arab-Berber subjects have LRRK2 G2019S due to a combination of genetic selection and drift. Ancient Norse trade, that included long-term settlements in Carthage ~1000 AD, may explain LRRK2 G2019S families in Norway. In contrast, the absence of LRRK2 G2019S in Central Europe, in Austrian and German patients, may reflect migration during World War II and the holocaust.

Although its origin is lost in history the LRRK2 G2019S diaspora is arguably the greatest single genetic cause of PD. Why most individuals appear to have “sporadic” rather than familial disease remains unclear. From meta-analysis the lifetime probability of LRRK2 G2019S heterozygotes becoming affected (termed penetrance) is ~30% (28% at 59, 51% at 69 and 74% at 78 years)17. However, there are families in which LRRK2 G2019S PD is clearly inherited, as that is how it was originally identified12,18,19. In 2005, I started working with Dr. Faycel Hentati, the Director of the National Institute of Neurology in Tunis, North Africa. There is a pandemic of PD in Arab-Berbers, the predominant population in North Africa, and to visit the Clinic and families in their homes has been a humbling experience, given their plight. Their generosity to help describe the clinical syndrome and its penetrance, and to help find genetic modifiers that influence when subjects with LRRK2 G2019S will become affected, has been remarkable19–21. Many other LRRK2 genetic variants contribute to risk, albeit more modestly. For example, LRRK2 G2385R, specific to Asian populations from Korea to Taiwan, is found in 6-8% of patients and doubles the risk of PD22,23. There are also LRRK2 genetic variants that are ‘protective’ i.e. inversely associated with PD24.

Molecular model of LRRK2’s kinase. The position of the G2019S mutation is shown as a yellow ball, at one ‘hinge’ of the activation segment. The effect of the mutation, of having a serine (S) at this position, is to keep the ‘activation segment’ (shown in red) always ajar. Normally this may close and open to regulate access to the pocket beneath, to allow LRRK2 substrates access to be phosphorylated (P). An ATP molecule that donates the phosphate (P) is shown in light blue. Other domains of the LRRK2 kinase are shown in pink and dark blue for contrast.

Molecular model of LRRK2’s kinase. The position of the G2019S mutation is shown as a yellow ball, at one ‘hinge’ of the activation segment. The effect of the mutation, of having a serine (S) at this position, is to keep the ‘activation segment’ (shown in red) always ajar. Normally this may close and open to regulate access to the pocket beneath, to allow LRRK2 substrates access to be phosphorylated (P). An ATP molecule that donates the phosphate (P) is shown in light blue. Other domains of the LRRK2 kinase are shown in pink and dark blue for contrast.

LRRK2 G2019S directly affects the ‘activation segment’ of LRRK2’s kinase (Figure). In effect this mutation ‘always keeps the door ajar’, allowing LRRK2 to phosphorylate other proteins (substrates), even when it should be inactive3. This notion has been supported with each substrate identified - first auto-phosphorylation of LRRK2 itself, and most recently in phosphorylation of many members of the Ras GTPase superfamily25. Thus a competitive but specific inhibitor of LRRK2 kinase activation, even with modest efficacy, might prevent disease in those susceptible.

To develop a safe and affordable drug, and without off-target side-effects for a lifetime of use, is our challenge. Several classes of LRRK2 inhibitors have now been identified, but which will meet this criteria is unclear26. Recent studies have shown LRRK2 kinase inhibition more often destabilizes the protein, lowering its expression, and may result in undesirable side-effects in lung and kidney. More research funding is needed in three areas: i) to identify and measure “PD-relevant” outcomes of LRRK2 kinase inhibition; ii) to develop preclinical models in which such effects can be easily and reliably assessed, and; iii) to prospectively identify and follow the natural evolution of disease in those patients and families with LRRK2 genetic variability, most likely to benefit from such a therapeutic approach.

“PD-relevant” outcomes of LRRK2 dysfunction may be contested but in neurons many agree mutant effects are subtle, chronic and initially synaptic27. Animal models that faithfully recapitulate the etiology and physiology of germline LRRK2 mutations in humans have been successfully developed, with measureable differences28,29. While drug screening requires more simple, innovative and informative assays of LRRK2 function, several avenues hold promise and not least in human “inducible-pluripotent stem cell’ derived dopaminergic neurons30. LRRK2 G2019S is also appreciated to cause PD in >50,000 Arab-Berber patients, and Tunisia, among other North African nations, definitely has the infrastructure, expertise and desire to develop neuroprotection, anticipating such drugs might be made affordable.

Now we know genetic causes of PD, the associated biologic mechanisms, and the patients most likely to benefit, “neuroprotection” to halt or prevent symptoms can be realized. LRRK2-directed therapeutics are likely to have broad efficacy as neuroprotective agents and beyond patients with specific mutations24. A similar approach has been taken for patients with PD and glucocerebrosidase (GBA) mutations by Sanofi-Genzyme who recently launched as Phase II clinical trial of their ceramide substrate inhibitor (GZ/SAR402671)31. Several clinical trials have also been initiated to clear or prevent Lewy body formation by lowering alpha-synuclein expression32. While neuropathologic examination of LRRK2 patients suggests Lewy bodies may be neither cause nor consequence of the disease process, clearing those protein aggregates is likely to be of benefit to many patients who develop that pathology.

  1. Funayama, M. et al. A new locus for Parkinson’s Disease (PARK8) maps to chromosome 12p11.2-q13.1. Ann. Neurol. 51, 296–301 (2002).
  2. Zimprich, A. et al. The PARK8 locus in autosomal dominant parkinsonism: confirmation of linkage and further delineation of the disease-containing interval. Am. J. Hum. Genet. 74, 11–19 (2004).
  3. Kachergus, J. et al. Identification of a novel LRRK2 mutation linked to autosomal dominant parkinsonism: evidence of a common founder across European populations. Am. J. Hum. Genet. 76, 672–80 (2005).
  4. Rocca, W. A. et al. Familial aggregation of Parkinson’s disease: The Mayo Clinic family study. Ann. Neurol. 56, 495–502 (2004).
  5. Zimprich, A. et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44, 601–7 (2004).
  6. Paisán-ruíz, C. et al. Dardarin Mutations in PARK8 PD Cloning of the Gene Containing Mutations that Cause PARK8-Linked Parkinson’s Disease Dardarin Mutations in PARK8 PD. Neuron 44, 595–600 (2004).
  7. Funayama, M. et al. An LRRK2 mutation as a cause for the Parkinsonism in the original PARK8 family. Ann. Neurol. 57, 918–921 (2005).
  8. Wszolek, Z. K. et al. Autosomal dominant parkinsonism associated with variable synuclein and tau pathology. Neurology 62, 1619–22 (2004).
  9. Hasegawa, K. et al. Familial parkinsonism: Study of original Sagamihara PARK8 (I2020T) kindred with variable clinicopathologic outcomes. Park. Relat. Disord. 15, 300–306 (2009).
  10. Hughes, A. J., Daniel, S. E., Kilford, L. & Lees, A. J. Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: A clinico-pathological study of 100 cases. J. Neurol. Neurosurg. Psychiatry 55, 181–184 (1992).
  11. Kalia LV, Lang AE, Hazrati LN et al. Clinical correlations with Lewy body pathology in LRRK2-related Parkinson disease. JAMA Neurol. 72, 100-5 (2015).
  12. Aasly, J. O. et al. Clinical features of LRRK2-associated Parkinson’s disease in Central Norway. Ann. Neurol. 57, 762–765 (2005).
  13. Lesage, S. et al. LRRK2 G2019S as a Cause of Parkinson’s Disease in North African Arabs. N. Engl. J. Med. 354, 422–423 (2006).
  14. Ozelius, L. J. et al. LRRK2 G2019S as a Cause of Parkinson’s Disease in Ashkenazi Jews. N. Engl. J. Med. 354, 424–425 (2006).
  15. Lesage, S. et al. Parkinson’s disease-related LRRK2 G2019S mutation results from independent mutational events in humans. Hum. Mol. Genet. 19, 1998–2004 (2010).
  16. Farrer, M. J., Gibson, R. & Hentati, F. The ancestry of LRRK2 Gly2019Ser parkinsonism - Authors’ reply. The Lancet Neurology 7, 770–771 (2008).
  17. Marder, K. et al. Age-specific penetrance of LRRK2 G2019S in the Michael J. Fox Ashkenazi Jewish LRRK2 Consortium. Neurology 85, 89–95 (2015).
  18. Johansen, K. K., Hasselberg, K., White, L. R., Farrer, M. J. & Aasly, J. O. Genealogical studies in LRRK2-associated Parkinson’s disease in central Norway. Parkinsonism Relat. Disord. 16, 527–530 (2010).
  19. Trinh, J. et al. DNM3 and genetic modifiers of age of onset in LRRK2 Gly2019Ser parkinsonism: a genome-wide linkage and association study. Lancet Neurol. 15, 1248–1256 (2016).
  20. Hulihan, M. M. et al. LRRK2 Gly2019Ser penetrance in Arab–Berber patients from Tunisia: a case-control genetic study. Lancet Neurol. 7, 591–594 (2008).
  21. Trinh, J. et al. A comparative study of Parkinson’s disease and leucine-rich repeat kinase 2 p.G2019S parkinsonism. Neurobiol. Aging 35, 1125–1131 (2014).
  22. Farrer, M. J. et al. Lrrk2 G2385R is an ancestral risk factor for Parkinson’s disease in Asia. Park. Relat. Disord. 13, 89–92 (2007).
  23. Xie, C. L. et al. The association between the LRRK2 G2385R variant and the risk of Parkinson’s disease: A meta-analysis based on 23 case-control studies. Neurological Sciences 35, 1495–1504 (2014).
  24. Ross, O. A. et al. Association of LRRK2 exonic variants with susceptibility to Parkinson’s disease: a case–control study. Lancet Neurol. 10, 898–908 (2011).
  25. Steger, M. et al. Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. Elife 5, (2016).
  26. West, A. B. Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease.  Experimental Neurology 298, 236–245 (2017).
  27. Volta, M., Milnerwood, A. J. & Farrer, M. J. Insights from late-onset familial parkinsonism on the pathogenesis of idiopathic Parkinson’s disease. Lancet Neurol. 14, 1054–1064 (2015).
  28. Yue, M. et al. Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice. Neurobiol. Dis. 78, 172–95 (2015).
  29. Volta, M. et al. Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice. Elife 6, (2017).
  30. Beevers, J. E., Caffrey, T. M. & Wade-Martins, R. Induced pluripotent stem cell (iPSC)-derived dopaminergic models of Parkinson’s disease. Biochem Soc Trans 41, 1503–1508 (2013).
  31. Sanofi Initiates Phase 2 Clinical Trial to Evaluate Therapy for Genetic Form of Parkinson’s Disease | Sanofi Genzyme News. Available at: http://news.genzyme.com/press-release/sanofi-initiates-phase-2-clinical-trial-evaluate-therapy-genetic-form-parkinsons-disea. (Accessed: 8th January 2018)
  32. Olanow, C. W. & Kordower, J. H. Targeting α-Synuclein as a therapy for Parkinson’s disease: The battle begins. Mov. Disord. 32, 203–207 (2017)

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Matthew Farrer, PhD presented at the 1st World Parkinson Congress in Washington DC; the 2nd World Parkinson Congress in Glasgow, Scotland; and the 3rd World Parkinson Congress in Montreal, Canada. He is a Professor in the Department of Medical Genetics at the University of British Columbia, the Canada Excellence Research Chair, and the Don Rix BC Leadership Chair in Genetic Medicine.

Ideas and opinions expressed in this post reflect that of the authors solely. They do not reflect the opinions or positions of the World Parkinson Coalition®