The Necessity of a Radical Change in the Interpretation

1. The Necessity of a Radical Change in the Interpretation and Cure of Alzheimer's Disease, Parkinson's Disease and Age-Related Macular Degeneration Giacinto Libertini, Graziamaria Corbi, Nicola Ferrara Federico II Univ., Dept. of Translational Medical Sciences, Naples, Italy International Association of Gerontology and Geriatrics European Region (IAGG-ER) Congress – 23-25 May 2019, Gothenburg, Sweden

2. There are three diseases of the central nervous system that are particularly relevant for frequency and severity of their manifestations [1, 2]: Parkinson's disease (PD) Alzheimer's disease (AD) Age-related macular degeneration (AMD) [1] Jankovic J, et al (2015) Bradley's Neurology in Clinical Practice, 7th edition, Elsevier [2] Lim JI (2012) Age-Related Macular Degeneration, CRC Press 2/20

3. These troubles are considered as distinct diseases. However, there are some characteristics that make them similar: 1) Accumulation of particular substances: 2) Death of specific neurons with the consequent symptoms; 3) Treatments that block or relieve only certain complications or symptoms; 4) No treatment able to stop or reverse the progression of the diseases [6, 7] [1] Sadigh-Eteghad S, et al (2006) Amyloid-beta: a crucial factor in AD. Med Princ Pract 24:1-10 [2] Galpern WR, Lang AE (2006) Interface between tauopathies and synucleinopathies: a tale of two proteins. Ann Neurol 59:449-58 [3] Schulz-Schaeffer WJ (2010) The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies, Parkinson’s disease and Parkinson’s disease dementia. Acta Neuropathol 120:131-3 [4] Crouch RK, et al (2015) A2E and Lipofuscin. Prog Mol Biol Transl Sci 134:449-63. [5] Ermilov VV, Nesterova AA (2016) β-amyloidopathy in the Pathogenesis of Age-Related Macular Degeneration in Correlation with Neurodegenerative Diseases. Adv Exp Med Biol 854:119-25 [6] Jankovic J, et al (2015) Bradley's Neurology in Clinical Practice, 7th Edition, Elsevier [7] Mitchell P, et al (2018) Age-related macular degeneration. Lancet 392:1147-59 3/20

4. The search for a treatment of these troubles is based on some hypothetical assumptions about their pathogenesis [1, 2]: Unknown or hypothetical primary alterations Accumulation of specific substances (β-amyloid protein and tau protein for AD; alpha-synuclein for PD; A2E, lipofuscin and β-amyloid protein for AMD) Neuronal suffering and death Disease manifestations Because of its seriousness and great social impact, Alzheimer's disease has deserved a particular attention and there has been for years a colossal commitment to the search for effective treatments. [1] Lim JI (2012) Age-Related Macular Degeneration, CRC Press [2] Jankovic J, et al (2015) Bradley's Neurology in Clinical Practice, 7th Edition, Elsevier 4/20

5. For AD, the researches have tried the removal of the β-amyloid and tau substances with the aim of eliminating the hypothesized consequences [1]. However, even drugs and monoclonal antibodies with proved capacity of drastically reducing these substances do not show evidence of clinical efficacy [1-4]. No clinical results despite the colossal investments of the pharmaceutical industry [1, 2] means that this is perhaps the greatest failure of pharmaceutical research. [1]Cummings JL, et al (2014) Alzheimer’s disease drug-development pipeline: few candidates, frequent failures. Alzheimer's Research & Therapy 6:37 [2] Abbott A (2008) The plaque plan. Nature 456:161-164 [3] Panza F, et al (2014) Is there still any hope for amyloid-based immunotherapy for Alzheimer's disease? Curr Opin Psychiatry 27:128-37 [4] Montoliu-Gaya L, Villegas S (2016) Aβ-Immunotherapeutic strategies: a wide range of approaches for Alzheimer's disease treatment. Expert Rev Mol Med 18:e13 5/20

6. When a scientific way turns out to be a failure, it is better to evaluate if its assumptions are erroneous and so a different path is necessary. The doubts regarding the aforesaid assumptions are increasing [1, 2] and other hypotheses have been proposed [3], but none appears satisfying. A possible alternative way is the topic of this presentation. This alternative path is based on radically different assumptions [4-6] that will be briefly mentioned here. [1] Herrup K (2015) The case for rejecting the amyloid cascade hypothesis. Nat Neurosci 18:794-9. [2] Karran E, De Strooper (2016) The amyloid cascade hypothesis: are we poised for success or failure? J Neurochem 139 S2:237-52 [3] Li H, et al (2018) Amyloid, tau, pathogen infection and antimicrobial protection in Alzheimer's disease - conformist, nonconformist, and realistic prospects for AD pathogenesis. Transl Neurodegener 7:34. [4] Fossel MB (2004) Cells, Aging and Human Disease. Oxford University Press, New York [5] Libertini G (2009) The Role of Telomere-Telomerase System in Age-Related Fitness Decline, a Tameable Process. In: Mancini L (ed) Telomeres: Function, Shortening and Lengthening. Nova Science Publ., New York [6] Libertini G, Ferrara N (2016) Possible Interventions to Modify Aging. Biochem (Mosc) 81:1413-28 6/20

7. The different assumptions Aging (here precisely defined as age-related increasing mortality or declining fitness) is not the effect of random accumulation of many degenerative processes [1]. On the contrary, aging is a type of phenoptosis (“programmed death of the organism”) [2], i.e., a physiological phenomenon [3] favoured by supra-individual selection [1, 4]. Being a physiological phenomenon, aging must have and has a phylogeny [5], genetically determined and modulated mechanisms [3], and a pathology [3]. [1] Libertini G (2015) Non-programmed versus programmed aging paradigm. Curr Aging Sci 8:56-68 [2] Skulachev VP (1997) Aging is a specific biological function ... Biochem (Mosc) 62:1191-5 [3] Libertini G (2014) The Programmed Aging Paradigm: How We Get Old. Biochem (Mosc) 79:1004-16 [4] Mitteldorf J, Martins AC (2014) Programmed life span in the context of evolvability. Am Nat 184:289-302 [5] Libertini G (2015) Phylogeny of Aging and Related Phenoptotic Phenomena, Biochem (Mosc) 80:1529-46 7/20

8. The core of aging mechanisms is the telomere-subtelomere-telomerase system At each replication, the telomere shortens. Telomerase, if active, restores the previous length [1]. If telomerase is inactive or partially active, the restoration is absent or partial, the telomere remains shortened, and the heterochromatin hood covering the telomere slides over the subtelomere sequence and represses it [2]. [1] Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 51:405-13 [2] Fossel MB (2004) Cells, Aging and Human Disease. Oxford University Press, New York 8/20

9. The progressive repression of the subtelomere determines: (1) gradual impairment of cell functions (gradual cell senescence) [1, 2]; (2) increasing probability of activation [3] of the cell senescence program[4] (replicative senescence + gradual cell senescence at the highest level) [1, 2]. [1] Fossel MB (2004) Cells, Aging and Human Disease. Oxford University Press, New York [2] Libertini G (2014) Programmed aging paradigm: how we get old. Biochem (Mosc) 79:1004-16 [3] Blackburn EH (2000) Telomere states and cell fates. Nature 408:53-6 [4] Ben-Porath I, Weinberg R (2005) The signals and pathways activating cellular senescence. Int J Biochem Cell Biol 37:961-6 9/20

10. As most cells show turnover (i.e., cell losses due to Programmed Cell Death [PCD] are balanced by the duplication of specific stem cells [1, 2]), the slackening of cell duplication capacity gradually slows down and jeopardizes cell turnover [3]. Moreover, there is an increasing number of cells in gradual cell senescence or in cell senescence, i.e., with altered functions [3]. Sufficient replacement by stem cells Detachment of cells from skin, hair and mucosae, apoptosis, etc. (PCD) Insufficient replacement by stem cells Increasing number of cells in gradual cell senescence and in cell senescence [1] Reed JC (1999) Dysregulation of Apoptosis in Cancer. J Clin. Oncol 17:2941-53 [2] Alberts B, et al (eds) (2013) Essential Cell Biology, 4th ed. Garland Science, New York [3] Libertini G (2014) Programmed aging paradigm: how we get old. Biochem (Mosc) 79: 1004-16 10/20

11. This explains well aging manifestations in continuously renewing tissues but would seem totally unsuitable to explain the alterations and the decline of perennial cells like most neurons [1]. Yes! ? The explanatory key can be easily obtained by considering the most studied neuronal tissue, i.e., the retina [1, 2]. [1] Libertini G (2014) Programmed aging paradigm: how we get old. Biochem (Mosc) 79: 1004-16 [2] Libertini G, Ferrara N (2016) Aging of perennial cells and organ parts according to the programmed aging paradigm. Age (Dordr) 38:35. doi: 10.1007/s11357-016-9895-0 11/20

12. The retina is part of the central nervous system. The photoreceptors (cones and rods) are highly differentiated neurons. Approximately 10% of the photoreceptor membranes on which there is photopsin are phagocytized daily by retina pigmented epithelium (RPE) cells. These cells are highly differentiated gliocytes and are subject to cell turnover. Each RPE cell serves about fifty photoreceptors [1, 2]. [1] Fine SL, et al (2000) Age-related macular degeneration. N Engl J Med 342:483-92 [2] Jager RD, et al (2008) Age-related macular degeneration. N Engl J Med 358:2606-17 12/20

13. When RPE cell turnover slows below critical levels, each RPE cell expands and serves more photoreceptors but with less efficiency. A further slowing down of cell turnover causes the formation of holes in the RPE layer and the consequent death of the photoreceptors served [1]. Decline of RPE cell turnover and of their functions Suffering and death of the photoreceptors served Age-related macular degeneration (AMD) No No Inflammation and other secondary manifestations Accumulation of A2E, lipofuscin andβ-amyloid protein This is evidenced clinically earlier with the disappearance of vision in the macula, the most important part of the retina, hence the name age-related macular degeneration [2]. So, the accumulation of some substances should be considered a consequence of the loss of RPE function and not the cause of photoreceptor death. [1] Berger JW, et al (1999) Age-related macular degeneration, Mosby, USA [2] Fine SL, et al (2000) Age-related macular degeneration. N Engl J Med 342:483-92 13/20

14. There are various indications that induce to propose a similar genesis for AD, PD and AMD. In these diseases there are differences for: • - the specialized type of gliocytes whose turnover slackens; • - the substances that accumulate due to the decline of these gliocytes; • the symptoms resulting from the death of the neurons no longer served; • but, in essence, AMD, AD and PD appear as different manifestations of the same mechanism [1]. [1] Libertini G, Ferrara N (2016) Aging of perennial cells and organ parts according to the programmed aging paradigm. Age (Dordr) 38:35. doi: 10.1007/s11357-016-9895-0 14/20

15. Scheme of the aging of perennial cells and of the consequent diseases Limits imposed by telomere-subtelomere-telomerase system Progressive general decline of duplication capacity Decline of satellite gliocytes turnover and of their functions Suffering and death of the neurons served Clinical diseases (AD, PD, AMD, etc.) No No Inflammation and other secondary manifestations Accumulation of substances not removed or not metabolized by glyocytes [1] Libertini G, Ferrara N (2016) Aging of perennial cells and organ parts according to the programmed aging paradigm. Age(Dordr) 38:35. doi 10.1007/s11357-016-9895-0 15/20

16. Why are most neurons not subject to turnover? Each neuron is connected to many other neurons (about one thousand connections on average) [1]. These links are indispensable for the functions performed by the neuron. The replacement of a neuron could hardly restore the pre-existing connections with the other neurons and the function would be compromised. So, the organism follows a different strategy: it does not replace the neuron but uses satellite (trophic) cells to renew the parts of the neuron [2]. On the contrary, neurons with simple links (e.g., olfactory receptor cells ) show cell turnover [3]. [1] Williams RW, Herrup K (1988) The control of neuron number. Annu Rev Neurosc 11:423-53 [2] Libertini G, Ferrara N (2016) Aging of perennial cells and organ parts according to the programmed aging paradigm. Age(Dordr) 38:35. doi 10.1007/s11357-016-9895-0 [3] Bermingham-McDonogh O, Reh TA (2011) Regulated reprogramming in the regeneration of sensory receptor cells. Neuron 71:389-405 16/20

17. For an effective strategy for the treatment of AD, PD, and AMD, some concepts are an essential premise [1]: (A) Aging is a physiological process of which gradual cell senescence and cell senescence are fundamental mechanisms; (B) The main consequences of aging mechanisms are: --- progressive slowing down of cell turnover and the depletion of functional cells; --- increasing number of cells with functions altered by gradual cell senescence and by cell senescence; (C) The decline of specific neuronal trophic gliocytes causes AD, PD, and AMD. [1] Libertini G, Ferrara N (2016) Aging of perennial cells and organ parts according to the programmed aging paradigm. Age(Dordr) 38:35. doi 10.1007/s11357-016-9895-0 17/20

18. This means that AD, PD and AMD (and other troubles) are part of the physiological process of aging. However, it is also true that: - there are particular early or anomalous cases of such disorders (e.g., caused by genetic alterations) that must be considered as specific diseases; - Factors similar to those that cause premature aging of cells / tissues with turnover (e.g., diabetes, hypertension, alcohol abuse, etc.) also cause acceleration and worsening of these disorders. In this case the prevention is possible and the use of suitable drugs (statins, ACE inhibitors, sartans) appears to be effective [1]. Table 1 (partial from [1])– Relations between some troubles and some “risk factors” or “protective drugs” [1] Libertini G, Corbi G, Cellurale M, Ferrara N (2019) Age-related dysfunctions: evidence and relationship with some risk factors and protective drugs. A systematic review. In preparation 18/20

19. The strategy to combat these disorders must aim at restoring normal physiology and turnover of satellite gliocytes, similarly to what can be proposed for cells with turnover [1]. The main method should be the reactivation of the telomerase [1]. An important experiment showed that in mice the reactivation of the telomerase as well as significantly improving the conditions of the tissues with turnover also improved the functionality of the nervous system [2]. Auxiliary strategy should be the elimination of senescent cells (which are not eliminated by telomerase reactivation [3]). [1] Libertini G, Ferrara N (2016) Possible Interventions to Modify Aging. Biochem (Mosc) 81:1413-28 [2] Bernardes de Jesus B, et al (2012) Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. EMBO Mol Med 4:691-704 [3] Libertini G, Ferrara N, Rengo G, Corbi G (2018) Elimination of Senescent Cells: Prospects According to the Subtelomere-Telomere Theory. Biochem (Mosc) 83:1477-88 19/20

20. Experiments with these objectives as well as having favourable effects on three serious diseases would be an important signal on the way to a complete control of the aging of all types of cells, i.e., of aging in general. Thanks for your attention!