Retinal Degenerative Diseases Xix John Ash All Chapter
Retinal Degenerative Diseases Xix John Ash All Chapter
Retinal Degenerative Diseases Xix John Ash All Chapter
John Ash
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Advances in Experimental Medicine and Biology 1415
Retinal
Degenerative
Diseases XIX
Mechanisms and Experimental Therapy
Advances in Experimental Medicine
and Biology
Volume 1415
Series Editors
Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives
d’Aquitaine, CNRS and University of Bordeaux, Pessac Cedex, France
Haidong Dong, Departments of Urology and Immunology, Mayo Clinic
Rochester, MN, USA
Heinfried H. Radeke, Institute of Pharmacology and Toxicology, Clinic of
the Goethe University Frankfurt Main, Frankfurt am Main, Hessen,
Germany
Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical
Center, Tehran University of Medical Sciences, Tehran, Iran
Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital
Munich, Germany
Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of
Cardiovascular Sciences, School of Life Science, Shanghai University
Shanghai, China
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John D. Ash • Eric Pierce
Robert E. Anderson
Catherine Bowes Rickman
Joe G. Hollyfield • Christian Grimm
Editors
Retinal Degenerative
Diseases XIX
Mechanisms and Experimental
Therapy
Editors
John D. Ash Eric Pierce
Department of Ophthalmology Ocular Genomics Institute
University of Pittsburgh School Department of Ophthalmology
of Medicine Massachusetts Eye and Ear Infirmary
Pittsburgh, PA, USA Harvard Medical School
Boston, MA, USA
Robert E. Anderson
Health Sciences Center Catherine Bowes Rickman
University of Oklahoma Health Department of Ophthalmology
Sciences Center Duke Medical Center
Oklahoma City, OK, USA Durham, NC, USA
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The editors are pleased dedicate this publication to the memory
of our long-time friend and colleague, Alan M. Laties. Except
for the most recent years, Alan attended each of these biennial
retinal degeneration meetings since they began in 1984. Early
on Alan recognized the importance of our attempt to provide a
continuing international platform for discussions and scientific
exchange to take place among investigators focused on retinal
degeneration research. Through his scientific leadership at the
Foundation Fighting Blindness (formerly the Retinitis
Pigmentosa Foundation), we received the first meeting grant to
partially cover some of the expenses of the RD meeting held in
San Francisco in 1988. The Foundation has provided
continuing support for each of the subsequent meetings in the
form of travel grant support for young investigators.
Born in Beverly, Massachusetts, the son of Russian immigrants,
he attended Harvard College (BA, 1954) and completed
medical school at Baylor College of Medicine (MD, 1959),
followed by a residency in ophthalmology in the Hospital of the
University of Pennsylvania (1961–63). A United States Public
Health Service Special Research Fellowship supported his
vi
ix
x Preface
xi
xii Contents
Part VI Inflammation
Part X Neuroprotection
Part XI Photoreceptors
Lysine Ubiquitylation Drives Rhodopsin Protein Turnover���������������� 493
Allen P. F. Chen, Leon Chea, Eun-Jin Lee,
and Jonathan H. Lin
Silico Prediction of MYO1C-Rhodopsin Interactions
In
and Its Significance in Protein Localization
and Visual Function �������������������������������������������������������������������������������� 499
Glenn P. Lobo, Rakesh Radhakrishnan, Matthias Leung,
Andrew Gruesen, Hans-Joachim Knölker, Frederik J. van Kuijk,
and Sandra R. Montezuma
A Ciliary Branched Actin Network Drives
Photoreceptor Disc Morphogenesis�������������������������������������������������������� 507
William J. Spencer and Vadim Y. Arshavsky
Part XII RPE
Revisiting the Daily Timing of POS Phagocytosis�������������������������������� 515
Antonio E. Paniagua, Harjas S. Sabharwal, Kausalya Kethu,
Andrew W. Chang, and David S. Williams
Inhibition of Bacterial Peptidoglycan Cytopathy
by Retina Pigment Epithelial PGRP2 Amidase������������������������������������ 521
Marlyn P. Langford, Laura A. Perilloux-Lyons,
and A. Scott Kavanaugh
Understanding Ischemic Retinopathies: The Role
of Succinate and Its Receptor in Retinal
Pigment Epithelium �������������������������������������������������������������������������������� 527
Bilge Esin Ozturk
The Amphipathic Helix in Visual Cycle Proteins: A Review���������������� 533
Sheetal Uppal, Eugenia Poliakov, Susan Gentleman,
and T. Michael Redmond
The Retinal Pigment Epithelium: Cells That Know the Beat!������������ 539
Elora M. Vanoni and Emeline F. Nandrot
Index�������������������������������������������������������������������������������������������������������� 583
Part I
Age-related Macular Degeneration
High-Resolution Imaging Mass
Spectrometry of Human Donor Eye:
Photoreceptors Cells and Basal
Laminar Deposit of Age-Related
Macular Degeneration
Fig. 1 MALDI IMS signals consistent with localization MALDI IMS images and H&E-stained tissue images
to photoreceptor and RPE compartments. (a) Schematic overlaid in perifoveal retina displaying signals from mul-
diagram of outer retina, excerpted from Fig. 1a. Blue, tiple lipid classes that localize to subcellular compart-
pink, yellow, and green bands indicate layers formed by ments of the photoreceptor cells. (b) Overlay showing
highly compartmentalized and vertically aligned photore- four separate signals. (c) Localized to ONL. (d) Localized
ceptors and RPE cells in panels b, c. Layers: OPL outer to photoreceptor inner and outer segments. (e) Localized
plexiform layer, ONL outer nuclear layer, ELM external to mitochondria-rich photoreceptor inner segments. (f)
limiting membrane, RPE retinal pigment epithelium, BrM Localized to RPE apical processes
Bruch’s membrane, R rod, C cone photoreceptors. (b–f)
6 D. M. G. Anderson et al.
Fig. 2 Imaging mass spectrometry (IMS) for molecularly pigmented debris (yellow) and dysmorphic RPE overly-
informed optical coherence tomography (OCT) and ing BLamD. Insert, BLamD with basal mound (arrow).
tissue-level target discovery. Asterisk, foveal pit; RPE, Basal mounds contain soft druse material. Layers: GCL
retinal pigment epithelium. Color-coded arrowheads rep- ganglion cell, INL inner nuclear, HFL Henle fiber, ONL
resent corresponding structures across modalities, in a outer nuclear. (c) Autofluorescent, pigmented debris (yel-
93-year-old human donor eye. (a) OCT B-scan shows low) and dysmorphic RPE (green). (d) IMS reveals an m/z
subretinal hyperreflective material (yellow) and an RPE signal at 799.673, restricted to BLamD and not detected in
elevation (green). (b) H&E stained cryosection shows the RPE
ease. Understanding molecular processes occur- 9. Chen Y, Jester JV, Anderson DM, Marchitti SA, Schey
KL, Thompson DC, et al. Corneal haze phenotype
ring in BLamD in early AMD is important as in Aldh3a1-null mice: in vivo confocal microscopy
they are early-identified histologic risk factors and tissue imaging mass spectrometry. Chem Biol
for AMD progression [18] and are just now being Interact. 2017;276:9–14.
recognized clinically [19, 20]. 10. Anderson DM, Ablonczy Z, Koutalos Y, Spraggins
J, Crouch RK, Caprioli RM, et al. High reso-
lution MALDI imaging mass spectrometry of
Acknowledgments This project was supported by the retinal tissue lipids. J Am Soc Mass Spectrom.
National Institutes of Health P41 GM103391 (R.M.C.) 2014;25(8):1394–403.
and R01 EY027948 (C.A.C.). Support was also received 11. Anderson DMG, Ablonczy Z, Koutalos Y,
by a Research to Prevent Blindness Catalyst Award for Hanneken AM, Spraggins JM, Calcutt MW, et al.
Innovative Research Approaches for Age-Related Macular Bis(monoacylglycero)phosphate lipids in the retinal
Degeneration to K.L.S. pigment epithelium implicate lysosomal/endosomal
dysfunction in a model of Stargardt disease and
human retinas. Sci Rep. 2017;7(1):17352.
12. Patterson NH, Tuck M, Van de Plas R, Caprioli
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The Noncanonical Role
of Complement Factor H in Retinal
Pigment Epithelium (RPE) Cells
and Implications for Age-Related
Macular Degeneration (AMD)
the individual’s quality of life but also poses a 402H of FH causes uncontrolled complement
significant burden on healthcare systems world- activation in vitro and in vivo [9]. Moreover,
wide [3]. The early stages of the disease are char- complement factors accumulate in drusen [10],
acterized by the presence of enlarging drusen in and increased complement system activation has
the retina. With disease progression, early AMD been detected in AMD patients, independent of
advance to late stages: wet or dry AMD. Wet their genotype [11].
AMD is defined by the presence of neovascular- Due to the strong implications of complement
ization in the retina, while dry AMD is limited to system activation in AMD pathology, various
progressive RPE and retinal atrophy, called geo- complement inhibitors have been tested in clini-
graphic atrophy (GA), without newly formed cal trials. However, most agents that entered
vessels. Around 10% of the cases constitute wet phase II were discontinued due to a reported lack
AMD, and intravitreal anti-VEGF is effective in of efficacy. Interestingly, in some clinical trials, a
treating most cases. However, only recently, group of subjects showed improvements, while
one approved treatment has become available for others did not [12]. Apparently, one of the sus-
dry AMD in the US [2, 8]. pected reasons for the observed lack of efficacy is
The main pathogenetic driver for AMD is a lack of patient’s stratification and identification
advanced age. Specific genetic factors can pre- of those patients that are likely to benefit from
dispose for AMD or protect from it. Unhealthy complement inhibition. Moreover, several stud-
lifestyle habits, like smoking or malnutrition, ies point to a lack of understanding of the role of
can further increase the risk for AMD. The com- FH in AMD pathology, especially in RPE cells,
bination of those risk factors leads to pathologi- which are degenerating in AMD.
cal changes in the retina, involving a plethora of
cellular mechanisms promoting disease pro-
gression [4]. 3 Noncanonical Role
of Complement Factor H
in RPE Cells
2 Complement System in AMD
RPE cells exert several functions vital for retinal
Among the processes involved in AMD, the com- homeostasis, which are impaired in AMD [13].
plement system is associated to AMD at different First of all, RPE cells are responsible for phago-
levels. First of all, many single nucleotide poly- cytosis of shed photoreceptor outer segments
morphisms (SNPs) associated with AMD cluster (POS) and recycling of visual pigments, needed
into genes of the alternative pathway of the com- for a proper visual cycle. This process is energy
plement system with the Complement Factor H intensive, requiring constant mitochondrial res-
(CFH) gene carrying the second highest risk for piration and glycolysis within RPE cells. Due to
AMD [5]. The most common risk haplotype a lack of the inner retina vasculature in the mac-
causes an amino acid substitution from tyrosine ula, RPE cells are vital to ascertain nutrient sup-
to histidine at position 402 (called Y402H) in ply from the choriocapillaris. Any pathological
proteins coded by CFH, which are full length FH disruption or perturbance of metabolic activity
and a truncated version, FHL-1 [6, 7]. The com- of RPE cells would affect the amount of nutri-
plement system is a part of the innate immune ents that can reach the neuroretina. Indeed, the
system designed to recognize and mediate the so-called bioenergetics crisis of RPE cells has
removal of pathogens or waste material [8]. The been described for AMD pathology [14].
eye is an immune-privileged organ, meaning that Moreover, RPE cells quench short UV light as
inflammation needs to be strictly limited, and well as the continuous influx of peroxidized lip-
therefore uncontrolled complement activation is ids from phagocytosed photoreceptor outer seg-
deleterious [8]. FH is a major negative regulator ments and thus need a high degree of antioxidant
of the complement system, and the AMD variant capacity as well as perfect functioning of their
The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and… 11
mitochondria to protect the retina from exces- [15] or in the presence of the CFH 402H variant
sive oxidative stress. [20]. Moreover, accumulation of lipids was
Our recent work demonstrates that FH is shown also in cfh Y402H mouse models of AMD,
endogenously produced by RPE cells. Within especially when subjected to high cholesterol
RPE cells, FH is acting locally supporting retinal diet [21].
homeostasis [15, 16]. This function of FH is con- The mechanism of action of intracellular FH
text dependent, and we suggest that locally pro- is not fully elucidated. Based on the data gener-
duced FH exerts specific functions at the ated in iPSC-RPE cells carrying the CFH 402H
RPE/retina interface. Most of these newly dis- variant, it has been speculated that more than one
covered functions (summarized in Fig. 1) affect a signaling pathway mediates the effects of FH
balanced regulation of immune competence and [19]. Specifically, it has been postulated that the
surveillance as well as regulating oxidative stress NF-kB pathway and mTOR pathway are likely
response and energy metabolism, mechanisms involved, in concomitance with autophagy and
which are part of AMD pathology [8]. proteasome activity dysregulation, which ulti-
FH plays a protective role against oxidative mately lead to mitochondrial damage [19].
stress in RPE cells. Exogenously applied recom- In our model of CFH-silenced hTERT-RPE1
binant FH protects RPE cells from damage cells, we could verify these hypotheses. Indeed,
induced by the exposure to lipid oxidation prod- we found that in absence of endogenous FH, both
ucts [17, 18]. In parallel, loss of endogenous FH the NF-kB and mTOR pathway are upregulated,
in RPE cells, via CFH silencing, also led to with NF-kB triggering pro-inflammatory cyto-
increased vulnerability to oxidative stress, kine expression [22] and the mTOR pathway
increased levels of oxidized lipids, and reduced modulating mitochondrial respiration, but not
viability [15]. Interestingly, addition of recombi- glycolysis [23]. The mTOR pathway is a major
nant FH in CFH-silenced RPE cells did not cause regulator of autophagy [24]. Dysfunction in the
any rescuing effects [15]. This finding indicates autophagy-lysosomal axis in iPSC-RPE cells
that, although exogenous FH is protective, a with CFH high risk was reported but is not regu-
proper intracellular FH function must be present lated by this pathway. Its dysfunction rather
to ascertain physiological balance and resilience depends on the accumulation of complement
to stress. This phenomenon may be explained by activation products [25]. Based on our analyses
impaired mitochondrial function seen in iPSC- of the intracellular FH protein interactions that
RPE cells carrying the CFH Y402H polymor- form a functional interactome in RPE cells, we
phism [19] and CFH-silenced hTERT-RPE1 cells suggest a regulatory role of FH in interaction
[15]. In both models, major processes involved in with the ubiquitin proteasome system (UPS) and
energy production, glycolysis, and mitochondrial RB1/E2F signaling, adding a new level of com-
respiration are impaired. In addition, a misbal- plexity in the understanding of FH mechanism of
ance in the degree of mitochondria turnover action [23]. Interestingly, and based on studies in
seems to be involved, with an exaggerated other cell types than RPE, it has already been
increase in mitophagy [15, 19]. Moreover, it has suggested that FH has a noncanonical intracellu-
been shown in iPSC-RPE carrying CFH 402H lar function, independent from complement acti-
variant that mitochondria are enlarged and accu- vation. In clear renal cell carcinoma cells and
mulating [20]. Mitochondria are essential for a kidney endothelial cells, FH knockdown impairs
correct response to oxidative stress; therefore, cellular homeostasis, at least partially via NF-kB
damage or dysfunction of these organelles is del- activation [26, 27].
eterious in such an oxidative milieu like the ret- The synopsis of these findings indicates that
ina. Accumulation of oxidized lipids has been understanding the function of FH in a specific
reported in RPE cells under FH dysregulation cell and tissue context is key. In the context of
and induced oxidative stress, when FH function AMD, it is important to advance our understand-
was impaired as a consequence of CFH silencing ing on how RPE cells interact with the neuroret-
12 A. Armento et al.
ina once intracellular FH dysregulation or a CFH ment pathway inhibitor strategy to treat AMD
402H high risk variant perturbs intracellular progression may not suffice. Rational re-balancing
homeostasis within these cells. In this regard, we strategies in a form of combinatorial therapies
have recently established a novel coculture model may be needed to target several drivers of AMD
of RPE and neuroretina. Using this system, we molecular pathology in a systems-oriented fash-
show that CFH-silenced RPE cells promote reti- ion simultaneously. More specifically, the increase
nal degeneration, which could not be rescued by of alternative complement pathway activity at the
the addition of exogenous FH. Moreover, we Bruch’s membrane/RPE interface may have to be
showed that the damage occurred at the level of considered concomitantly with pro-inflammatory,
mitochondrial activity and lipid oxidation in the oxidative stress- related perturbation and meta-
photoreceptors, rather than a canonical inflam- bolic disbalance of RPE and neuroretina to
mation or complement activation [16]. achieve effective future therapeutic regimes for
dry AMD. Last but not least, a very thorough
stratification of patient groups may be needed to
4 Future Perspective move to an individualized and rational therapy for
dry AMD of the future.
Recent works of several groups, including ours,
emphasize a noncanonical and intracellular role
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Macular Pigment Carotenoids
and Bisretinoid A2E
Abstract Keywords
Lutein (L), zeaxanthin (Z), and meso- Abca4 · A2E · Antioxidant · Bisretinoids ·
zeaxanthin (MZ) are the three macular pig- Carotenoids · Lipofuscin · Macular pigments
ments (MP) carotenoids that uniquely · Photo-oxidation · Retina · Retinal pigment
accumulate in the macula lutea region of the epithelium
human retina. L and Z are obtained by humans
through dietary intake. The third MP, MZ, is
rarely present in diet, and its abundance in the 1 Introduction
human fovea is due to the metabolic conver-
sion of dietary L by the retinal pigment epithe- Carotenoids are natural pigments synthesized by
lium’s RPE65 enzyme. The major functions of photosynthetic bacteria, algae, yeast, and plants.
MP in ocular health are to filter high-intensity, L, Z, and MZ are oxygenated carotenoids (xan-
phototoxic blue light and to act as effective thophylls) obtained by humans through dietary
antioxidants for scavenging free radicals. The intake of leafy greens, fruits, and vegetables.
pyridinium bisretinoid, N-retinylidene-N- Among the 15 major dietary carotenoids detected
retinylethanolamine (A2E), contributes to in human serum, these three xanthophyll carot-
drusen formation in dry age-related macular enoids selectively accumulate with a unique dis-
degeneration (AMD) and to the autofluores- tribution in the macular region of human retina
cent flecks in autosomal recessive Stargardt [1]. The MP’s total concentration is about 1 mM
disease (STGD1). Retinal carotenoids attenu- at the fovea, and its concentration declines to less
ate A2E formation and can directly and indi- than 10 μM in the peripheral retina [2]. The ratio
rectly alleviate A2E-mediated oxidative of L:Z:MZ in the peripheral retina is about 3:1:0,
damage. In this chapter, we review these more and the concentration of total carotenoids rises
recently recognized interconnections between 100-fold in the macula lutea with a change in the
MP carotenoids and A2E bisretinoids. ratio of L:Z:MZ of about 1:1:1, as measured by
high-performance liquid chromatography
(HPLC) [3]. More recently, high-resolution con-
R. Arunkumar · P. S. Bernstein (*) focal resonance Raman microscopy imaging of
Department of Ophthalmology and Visual Sciences, the human retina from our laboratory has showed
John A. Moran Eye Center, University of Utah that the ratio of Z + MZ:L can be greater than 9:1
School of Medicine, Salt Lake City, UT, USA at the foveal center, while L is more diffusely dis-
e-mail: [email protected]
tributed across the macula at a relatively lower aided by conjugated double-bond structure with
concentration [4]. Retinal carotenoids mainly the hydrophilic group on the ionone ring [2]. The
localize to the outer plexiform (Henle fiber) layer absorption maximum of L is around 445 nm, and
and the inner plexiform layer, with axial exten- Z is around 450 nm, corresponding with the haz-
sion from the inner limiting membrane to the ardous blue light range of 430–500 nm.
outer limiting membrane. The specificity in the Depending on the retinal carotenoid concentra-
distribution of retinal carotenoids in the human tion, they are estimated to absorb 40–90% of
retina may be due to the selective uptake of reti- incident short-wavelength, high-energy photo-
nal carotenoids by carotenoid binding proteins toxic blue light [8].
StARD3 (L binding protein) and GSTP1 (Z and Retinal carotenoids with conjugated double
MZ binding protein). The other major factors that bond (C=C) structures are associated with greater
influence the MP carotenoid distributions and singlet oxygen quenching. Z has 11 conjugated
concentrations between individuals are the double bonds and is 50% better at quenching sin-
amount of oral intake of carotenoids and their glet oxygen relative to L with its 10-conjugated
bioavailability [5]. L is present in higher concen- double bonds [9]. MZ, a stereoisomer of Z, with
trations in the diet, but it is converted to MZ by an identical 11-conjugated double-bond structure
the RPE65 enzyme, and the preferential accumu- would be expected to possess the same antioxi-
lation of Z and MZ at the foveal center may be dant properties as Z, but it exhibited better singlet
due to their higher antioxidant potential relative oxygen quenching properties relative to L and Z
to L. The fovea is prone to photo-damage caused [9]. Furthermore, the antioxidant activity of all
by light-induced oxidative stress from reactive three retinal carotenoids as a mixture showed bet-
oxygen species, and singlet oxygen can be gener- ter singlet oxygen quenching ability than any of
ated by photo-oxidation of A2E and other bisreti- the individual MP carotenoids. Retinal carot-
noid components of lipofuscin [6]. A2E acts as a enoids not only quench singlet oxygen, but they
photosensitizer in the presence of short- also quench superoxide anion radicals and
wavelength blue light and oxygen that generate hydroxyl radicals, the major causative agents of
reactive oxygen caused by photo-oxidation and lipid peroxidation and light-induced damage [10,
photo-degradation, resulting in retinal degenera- 11]. Generally, retinal carotenoids are better
tion and apoptosis of photoreceptor and RPE hydroxyl radical scavengers than superoxide
cells [7]. Supplementation with retinal carot- anion scavengers, and Z exhibits better hydroxyl
enoids potentially attenuates harmful blue light radical scavenging activity than lutein [10]. MP
and effectively scavenges singlet oxygen and carotenoids protect the retina, which is vulnera-
reactive free radicals in ocular tissues and in bio- ble to oxidative damage caused by exposure to
chemical assays. light, high concentration of oxygen, abundant
photosensitizers, and high concentrations of
polyunsaturated fatty acids (PUFAs).
2 Structure and Function
of the Retinal Carotenoids
3 A2E Bisretinoids
Retinal carotenoids are characterized by the pres-
ence of hydroxyl (O-H) functional groups In the visual cycle, the activation of rhodopsin by
attached at the 3 and 3′ positions of terminal ion- a photon of light during phototransduction results
one rings connected by an isoprenoid backbone in the isomerization of 11-cis-retinal to all-trans-
structure (Fig. 1). The presence of hydroxyl retinal in photoreceptor outer segments and the
groups and the conjugated double bonds struc- release of all-trans-retinal, which is subsequently
ture determine the light-absorbing properties and reduced to all-trans-retinol and transported to the
antioxidant activities of retinal carotenoids. The RPE cells. In the RPE, the all-trans-retinol is
light filtering function of retinal carotenoids is either stored as a fatty acid ester or is converted
Macular Pigment Carotenoids and Bisretinoid A2E 17
OH
HO
Lutein
OH
HO
Zeaxanthin OH
N
OH
A2E
HO
Meso-Zeaxanthin
Fig. 1 Structures of the macular carotenoids: lutein (L), zeaxanthin (Z), meso-zeaxanthin (MZ), and N-retinylidene-N-
retinylethanolamine (A2E)
back to an 11-cis-retinoid that is transported back RPE, A2PE is further hydrolyzed to A2E by
to the photoreceptor outer segment, which can phospholipase D inside RPE phagosomes.
bind to opsin to produce a regenerated visual pig- Mutation or dysfunction in the ABCA4 gene
ment. Some all-trans-retinal in the photoreceptor leads to excessive accumulation of A2E and other
outer segments reacts with phosphatidylethanol- bisretinoids in RPE lipofuscin, resulting in reti-
amine (PE) to form N-retinylidene-PE (NRPE), nal cell death and vision loss. A2E is reported to
which is transported or “flipped” by the ATP- accumulate with aging and dry AMD and in
binding cassette, subfamily A, member 4 STGD1 [13].
(ABCA4) protein present in photoreceptor outer A2E (C42H58ON; molecular weight, 592) is a
segments. ABCA4 translocates NRPE across the well-characterized component of lipofuscin. It
lipid bilayer from the luminal side to the cyto- exhibits absorbance in both the UV and visible
plasmic side of the disc membrane. NRPE regions of the spectrum [A2E: absorbance max-
released on the cytoplasmic side is hydrolyzed ima (λmax) are 440 and 340 nm, and iso-A2E’s
into all-trans-retinal and reduced to all-trans- λmax are 430 and 340 nm]. A2E’s hydrophobic
retinol by retinol dehydrogenases (RDHs) [12]. If side arms and positively charged hydrophilic
ABCA4 does not effectively translocate NRPE, it head group confer an amphiphilic structure
reacts with one more molecule of all-trans-retinal (Fig. 1). A2E has a detergent-like structure that
to form a toxic bisretinoid, dihydropyridinium- may influence membrane properties and inhibit
A2PE. Dihydropyridinium-A2PE is further the lysosomal degradation of lipids. A2E induces
oxidized either to A2-dihydropyridine-PE
loss of membrane integrity and perturbs mem-
(A2-DHP-PE) by eliminating one hydrogen or to brane stability, and it inhibits cytochrome C oxy-
phosphatidylethanolamine-pyridinium bisreti- genase and the ATP-driven proton pump. A2E
noid (A2PE) by eliminating two hydrogens in the photo-oxidation products can activate the com-
acidic and oxidizing environment (Fig. 2). During plement system and cause inflammation and
phagocytosis of photoreceptor outer segments by DNA damage [12, 13].
18 R. Arunkumar and P. S. Bernstein
Fig. 2 The visual cycle and bisretinoid formation in pho- hydrogens generates A2PE, and loss of one hydrogen gen-
toreceptor outer segments and RPE. When a photon of erates A2-DHP-PE; phosphate hydrolysis of the latter
light is captured by the visual chromophore, its 11-cis- produces A2-DHP-E. A2E, lysoA2PE, and A2-GPE are
retinal (11-cis-RAL) Schiff base chromophore photoi- produced from A2PE. Reaction of the all-trans-RAL
somerizes to all-trans-retinal (all-trans-RAL). The dimer with PE with the formation of a Schiff base linkage
released all-trans-RAL from opsin is reduced to all-trans- generates all-trans-retinal dimer-PE (atRALdi-PE), and
retinol (all-trans-ROL) by retinol dehydrogenases phosphate hydrolysis of the latter yields all-trans-retinal
(RDHs). Alternatively, some all-trans-RAL reacts with dimer-ethanolamine (atRALdi-E). All-trans-ROL is
phosphatidylethanolamine (PE) in the photoreceptor outer transported into RPE cells where all-trans-ROL is esteri-
segments to form N-retinylidene-PE (NRPE), which is fied to fatty acids to make all-trans retinyl esters (all-
transported by ABCA4. The bisretinoid synthesis path- trans-RE) by the enzyme lecithin retinol acyl transferase
way (red) is initiated when NRPE, rather than hydrolyzing (LRAT). All-trans-RE is converted to 11-cis retinol
to all-trans-ROL and PE, reacts with a second molecule of (11-cis-ROL) by the RPE65 isomerohydrolase. 11-cis
retinaldehyde. A multistep pathway leads to the formation retinol dehydrogenase (11cRDH) oxidizes 11-cis-ROL to
of the intermediate dihydropyridinium-A2PE. Auto- 11-cis-RAL which enters the photoreceptor outer seg-
oxidation of dihydropyridinium-A2PE with loss of two ments for the regeneration of opsin
in human tissue [2]. MMPs typically have an The specific interplay of pathophysiological
80-amino acid propeptide, a 170-amino acid cat- events that converge to AMD and related degen-
alytic metalloproteinase domain, a variable- eration mechanisms remain to be fully elucidated
length linker peptide (hinge region), and a [11]. Nevertheless, several studies have been
200-amino acid hemopexin (Hpx) domain. A highlighting the alterations in the regulation of
detailed description of MMPs structure and an ECM, by dysregulation of the activity of MMPs
overview of their substrate preferences and their and TIMPs, as one of the main mechanisms
association with extracellular matrix (ECM) com- involved in the development of AMD [12]. This
ponents and inhibitors can be found in other modulation of ECM turnover is associated with
excellent reports [2–4]. the other molecular mechanisms involved in the
The MMPs are usually secreted as zymogens pathophysiology of the disease since some stud-
by a variety of cells into the extracellular space ies have shown that both oxidative stress and acti-
[3] or stay tethered to their plasma membrane [5]. vation of the complement system can modulate
In humans, there are six membrane-type (MT)- the activity of ECM components [13, 14].
MMPs [5]. In the generation of active MMPs, Additionally, the formation of drusen, the hall-
enzymatic cleavages of the zymogens are needed. mark of AMD, is also believed to be associated
Active MMP-2, for example, is made from pro- with a dysregulation of the ECM components,
MMP-2 by MMP-14 cleavage, while pro-MMP-9 especially in the RPE and Bruch’s membrane
is cleaved by MMP-3 [5, 6]. (BM) [15]. These alterations in ECM modulation
MMPs are mainly classified based on their caused by dysregulation of MMP/TIMP complex
substrate preference and domain organization, can also be influenced not only by environmental
being grouped into gelatinases (MMP-2, MMP- but also by genetic factors. For example, poly-
9), collagenases (MMP-1, MMP-8, MMP-13), morphisms in MMP-2 (rs243865) and in MMP-9
stromelysins (MMP-3, MMP-10), MT-MMPs (rs3918424 and rs3918241) are associated with
(MMP-14, MMP-15, MMP-16, MMP-17), and increased risk of AMD, mainly in younger
others [7]. Under normal conditions, both MMPs patients (<65 years) [16–18]. Other MMP-2
and the tissue inhibitors of metalloproteinases polymorphisms (rs243865, rs243866, and
(TIMPs) have an important role in the regulation rs2287074) are associated with a lower likeli-
of the turnover of ECM [2, 8]. A dysregulation of hood of AMD development [19, 20]. There are
these components can both contribute to patho- also some TIMPs polymorphisms associated
logical ECM remodeling and serve as a nexus for with AMD. Despite some polymorphisms in
a variety of pathways involved in age-related TIMP-2 (rs817909) and in TIMP-3 (rs5754227)
macular degeneration (AMD) pathogenesis. appear to have a protective effect, other polymor-
phisms in TIMP-3 (rs713685 and rs743751) are
more frequent in AMD patients [18, 21, 22]. As a
2 MMPs Are Implicated in AMD result, these findings highlight evidence that
changes in ECM turnover are essential mecha-
AMD is a retinal degenerative disease that is a nisms implicated in the development of AMD
leading cause of central vision loss in the elderly. and may help explain the susceptibility to the dis-
This disease, in its early stages, is characterized ease’s late stages.
by the accumulation of drusen that causes pro-
gressive degeneration of the photoreceptors and
retinal pigment epithelium (RPE). The disease 2.1 MMPs in Dry AMD
can progress to geographic atrophy (GA), an
advanced form of dry AMD, and/or choroidal BM is a five-layered ECM located at the inter-
neovascularization (CNV), also known as wet face of the retina and choriocapillaris. BM plays
AMD [9, 10]. an important role in cell-cell communication and
Disturbed Matrix Metalloproteinases Activity in Age-Related Macular Degeneration 23
regulates the exchange of oxygen and nutrients oxygen supply to the outer retina. The oxidative
between the choroid and the outer retina [23]. and inflammatory processes that occur in late
However, BM undergoes a number of age-related stages of AMD might promote a pro-angiogenic
alterations that may have a role in AMD patho- environment, resulting in CNV, which is the hall-
genesis. Especially noteworthy, the accumulation mark of wet AMD. Neovessels from choroid,
of cellular debris and ECM proteins leads BM to also referred as CNV, develop beneath the RPE
thicken and to the formation of drusen [24]. or penetrate into the subretinal space in patients
Several studies have been demonstrated that with wet AMD [38, 39].
aging causes structural and functional changes in Similar to GA and BM thickness, in this case
the BM due to a decrease in the activity of the also dysregulation of ECM turnover appears to
gelatinase components of the MMP system. In have a close relation with CNV. Increased activ-
this case, increased levels of high molecular ity of MMPs can degrade some components of
weight components of the MMPs pathway BM, essential to the growth of new vessels [40,
(HMW1 and HMW2) reduce the pool of the acti- 41]. Several studies have shown that patients with
vated gelatinases MMP-2 and MMP-9 and con- wet AMD present increased levels of MMP-9 in
tribute to reduced matrix degradation and aqueous humor, vitreous, and plasma [42–44].
thickness of BM [25–27]. Another study revealed This increase in the levels of MMP-9 contributes
that higher TIMP-3 concentrations in the drusen to CNV since this MMP can increase VEGF lev-
block MMPs activity, lowering proteolytic activ- els (pro-angiogenic factor) by decreasing PEDF
ity within the drusen [28]. Oxidative stress levels (anti-angiogenic factor), both secreted by
reduces MMP-2 activity and leads to the forma- the RPE [45]. MMP-2, MMP-7, and MMP-13
tion of sub-RPE deposits that can be involved in have likewise shown a rise in their expression and
the formation of drusen during AMD develop- levels in patients with CNV lesions [18, 46, 47].
ment [29]. Moreover, cellular and animal models of CNV
Nevertheless, several other studies also lesions reported increased expression of MMP-2
detected increased levels of other MMPs in and MMP-9, which were involved in the forma-
patients with GA. MMP-7 levels were found to tion of experimental CNV. Additionally, the inhi-
be lower in these patients, but MMP-9 and bition of these two gelatinases appears to be a
TIMP-1 levels were higher [30, 31]. In vitro and good strategy to treat CNV, since their elimina-
in vivo models of dry AMD led to increased tion is associated with decreased CNV lesion size
expression, secretion, and activation of MMP-1, [48–50]. Some studies have also been underlin-
MMP-3, and MMP-9 and are associated with ing the alterations in TIMPs activity as an impor-
decreased RPE cell viability [32–34]. Moreover, tant mechanism underlying CNV. On both wet
MMP-9’s role in the breakdown of the RPE bar- AMD patients and animal models, decreased lev-
rier has been highlighted, as silencing of this els of TIMPs (TIMP-2 and TIMP-3) contribute to
MMP can improve the barrier integrity [35, 36]. CNV susceptibility [18, 51].
Regarding genetic alterations, only a few stud- Regarding genetic alterations, several studies
ies have related polymorphisms specifically with identified various polymorphisms on both MMPs
dry AMD. Liutkeviciene et al. detected that a and TIMPs that are related to CNV and wet
polymorphism in the MMP-2 gene (rd243865) is AMD. Microsatellites in the promoter of MMP-
associated with the development of hard drusen 9, along with other polymorphisms on the
in AMD patients [37]. MMP-9 gene (rs142450006, rs3918424), have
been associated with increased risk of CNV pro-
gression and wet AMD development [17, 52, 53].
2.2 MMPs in Wet AMD Also polymorphisms on MMP-1 (rs1799750),
MMP-2 (rs243865), MMP-7 (rs11568818), and
The choroid is a network of blood vessels, located MMP-20 (rs10895322) are associated with
below the BM that is critical for nutrients and increased CNV lesion size and development of
24 B. Martins and R. Fernandes
wet AMD [54–56]. Finally, a polymorphism in 8. Agren MS, Auf dem Keller U. Matrix metallo-
proteinases: how much can they do? Int J Mol Sci.
the TIMP-3 gene (rs9621532) has a protective 2020;21(8):2678.
effect and is associated with decreased risk of 9. Mitchell P, Liew G, Gopinath B, Wong
wet AMD [57]. TY. Age-related macular degeneration. Lancet.
2018;392(10153):1147–59.
10. Al-Zamil WM, Yassin SA. Recent developments
in age-related macular degeneration: a review. Clin
3 Concluding Remarks Interv Aging. 2017;12:1313–30.
11. Ambati J, Fowler BJ. Mechanisms of age-related
Due to their ability to break down ECM constitu- macular degeneration. Neuron. 2012;75(1):26–39.
12. Peng H, Hulleman JD. Prospective application of
ents, MMPs are relevant components in many activity-based proteomic profiling in vision research-
pathological processes of AMD. Specifically, potential unique insights into ocular protease biology
they play an important role in the accumulation and pathology. Int J Mol Sci. 2019;20(16):3855.
of drusen and degeneration of photoreceptors/RPE 13. Klettner A. Oxidative stress induced cellular sig-
naling in RPE cells. Front Biosci (Schol Ed).
in dry AMD and pathological vessel growth in 2012;4:392–411.
wet AMD. TIMPs control changes in their 14. Fernandez-Godino R, Pierce EA. C3a triggers for-
expression and activity. To better understand the mation of sub-retinal pigment epithelium depos-
role of each MMP in AMD pathogenesis, further its via the ubiquitin proteasome pathway. Sci Rep.
2018;8(1):9679.
research is needed. 15. Luibl V, Isas JM, Kayed R, Glabe CG, Langen R,
Chen J. Drusen deposits associated with aging and
Acknowledgments Funding provided by the Global age-related macular degeneration contain nonfibrillar
Ophthalmology Awards Program (GOAP), a Bayer spon- amyloid oligomers. J Clin Invest. 2006;116(2):378–85.
sored initiative committed to supporting ophthalmic 16. Liutkeviciene R, Lesauskaite V, Zaliaduonyte-
research across the world. Thanks are also due to FCT Peksiene D, et al. Role of MMP-2 (-1306 C/T)
(Portugal) and Strategic Projects UIDB/04539/2020 and polymorphism in age-related macular degeneration.
UIDP/04539/2020 (CIBB), COMPETE-FEDER (POCI- Ophthalmic Genet. 2016;37(2):170–6.
01-0145-FEDER-007440), and Centro 2020 Regional 17. Liutkeviciene R, Lesauskaite V, Sinkunaite-
Operational Program: BRAINHEALTH 2020 Marsalkiene G, et al. The role of matrix metallo-
(CENTRO-01-0145- FEDER-000008). The authors also proteinases polymorphisms in age-related macular
acknowledge FCT for the doctoral research grant degeneration. Ophthalmic Genet. 2015;36(2):149–55.
2020.04811.BD (to B.M.). 18. Oszajca K, Szemraj M, Szemraj J, Jurowski
P. Association analysis of genetic polymorphisms and
expression levels of selected genes involved in extra-
cellular matrix turnover and angiogenesis with the
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Current Views on Chr10q26
Contribution to Age-Related
Macular Degeneration
Abstract 1 Introduction
tory [28, 36], dietary choices [30, 37–40], race 2.1 Reported Tissue, Cellular
and ethnicity [41–43], gender [21], and smoking and Subcellular Expression
[25, 44–46]. In addition, genetic factors explain of ARMS2/ARMS2
46–71% of the variation in overall disease sever-
ity [47]. To date, a total of 52 independently asso- The expression of ARMS2 was detected in pla-
ciated common and rare gene variants cental tissue [55], at low levels in retinal samples
(p < 5 × 10−8), distributed across 34 loci, have [56, 73] and later, ubiquitously, in human tissues
been associated with AMD risk [48–51]. [77]. The Bgee gene expression database (v14.2)
Genome-wide association (GWA) and family- currently lists 62 distinct human tissues with
based linkage studies identified genetic variants varying ARMS2 mRNA levels [78].
in the complement factor H (CFH) gene on Kanda et al. expressed human retinal ARMS2
Chr1q32 and two tightly linked genes—ARMS2 mRNA in COS-1, ARPE-19, and JEG cells.
and HTRA1—on Chr10q26 as major contributors Subcellular fractionation, followed by co-staining
to AMD risk [52–65]. Together, these increase with MitoTracker and cytochrome C oxidase
AMD predisposition by more than 40-fold [66]. subunit IV, located ARMS2 to the mitochondrial
While significant progress has been made toward (Mt) outer membrane [73]. Subsequently, another
understanding the role of CFH in AMD risk [67– group co-stained ARMS2 with retinoschisin (an
70], uncertainty remains regarding the gene(s) extracellular protein that stains PR inner seg-
responsible at the Chr10q26 locus. Strong link- ments) and Mt cytochrome c oxidase subunit II in
age disequilibrium between ARMS2 and HTRA1 human retinal sections. ARMS2 localized in the
genes has made it difficult to distinguish individ- Mt-enriched ellipsoid region of the inner seg-
ual gene effects by statistical methods [71]. ments of both rod and cone PR cells [79].
Moreover, PLEKHA1 is also found in linkage However, conflicting results were presented by
disequilibrium with ARMS2 [55]. Several studies Wang et al. [80]. Using immunofluorescence
suggest that ARMS2/HTRA1 genetic variations (IF), verified by immunoblotting, endogenous
are more strongly associated with AMD risk than ARMS2 in ARPE-19 cells and overexpressed
PLEKHA1 [56, 72, 73]. In this chapter, we review GFP-tagged and HIS-tagged exogenous ARMS2
existing information about ARMS2 and HTRA1 in COS7 cells were found to localize in the cyto-
and their association with AMD. sol and not in Mt or any other organelle [80]. The
group further performed in silico analyses to
identify the protein features required for Mt tar-
2
ARMS2 geting and concluded that ARMS2 lacked canon-
ical mitochondrial targeting sequences [80].
The ARMS2 gene, age-related maculopathy sus- Kortvely et al. transfected HEK293 cells with
ceptibility 2, is only present in higher primates plasmid constructs coding for normal or risk vari-
[74]. ARMS2 encodes an 11-kDa protein [75]; ant ARMS2. Immunostaining findings suggested
however the function, localization, and native that ARMS2 is a secreted protein and a compo-
structure of the protein have not been firmly nent of the extracellular matrix (ECM), found
established. An in silico approach to predict the mainly in choroid pillars of human eye [81].
structure of ARMS2 concluded that it has 15 Using a yeast two-hybrid system, followed by
putative phosphorylation sites, four putative gly- coprecipitation, ARMS2 was found to bind sev-
cation sites of ε-amino groups of lysine, two eral ECM proteins [81]. Further, ARMS2 colo-
putative O-linked glycation sites, and three calized with the endoplasmic reticulum (ER) in
potential binding sites [76]. The alanine at posi- transfected ARPE-19 cells expressing ARMS2.
tion 69 is predicted to be a functional residue for Adding a small N-terminal tag to ARMS2 did not
protein binding. have any effect, whereas the same tag at the
Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration 29
C-terminus abolished ER colocalization, indicat- to human apoptotic and necrotic cells and initiate
ing that the C-terminus is integral to proper properdin-mediated complement activation and
targeting [81]. A follow-up study concluded that C3b surface opsonization for phagocytosis [75].
ARMS2 is secreted via an unconventional, Golgi- Two additional variants of ARMS2 have been
independent pathway. Blocking the canonical ER reported: an insertion-deletion (indel) polymor-
to Golgi transport did not affect ARMS2 secre- phism (NM_001099667.1:c.*372_815del443
tion. Instead, ARMS2 was shown to colocalize ins54) in the 3′untranslated region (3′-UTR) and
with calnexin-positive and protein disulfide a coding nonsense polymorphism (R38X) [79].
isomerase-negative vesicle-like structures and, The ARMS2 indel lies within the HTRA1 cis-
with GRASP65, a marker for unconventional regulatory region, upstream of its transcription
protein secretion. Exon 2 of ARMS2 codes for start site [83, 84], and is suggested to regulate
eight amino acids (-SIIHTAAR) at the HTRA1 expression. In heterozygous retinal and
C-terminus. By generating a set of mutants, the placental samples from human donors, Fritsche
two isoleucines were found to be indispensable et al. showed that this indel mutation destabilizes
for correct targeting. The intracellular distribu- the ARMS2 transcript by removing the polyade-
tion of the A69S risk allele was similar to the nylation signal and inserting a 54-bp element
non-risk allele, with both protein variants secreted known to mediate rapid mRNA turnover [79].
into the culture medium [82]. The group validated their findings by developing
Micklisch et al. found endogenous ARMS2 recombinant ARMS2 isoforms, quantifying heter-
gene expression in human blood-derived mono- ologous ARMS2 expression in vitro and conclud-
cytes and in human-induced pluripotent stem ing that the indel polymorphism in ARMS2 leads
cell-derived microglia (iPSdM) by PCR and laser to reduced mRNA levels [83].
scanning microscopy. Contrary to the study by The ARMS2 variant identified by Fritsche
Kortvely et al. which showed translation of the et al., rs2736911(C>T:R38X), is predicted to
ARMS2 A69S variant, this study reported that result in a premature stop codon (R38X) and a
ARMS2 protein was absent in patients homozy- truncated protein [79]. Yang et al. observed a
gous for the ARMS2 AMD risk variant A69S or 50% decrease in mRNA in patients heterozygous
the non-risk variant R38X [75]. for R38X [85], presumably due to nonsense-
mediated mRNA decay. They reasoned that the
loss of ARMS2 is insufficient to explain AMD
2.2
ARMS2 Risk Variants and Their susceptibility as both the indel and R38X variants
Putative Role in AMD result in a decrease in ARMS2, but only the for-
mer confers risk to AMD, while the latter is
The missense variant, rs10490924 (G>T:A69S) mildly protective [85, 86]. This led to a hypothe-
[73, 80, 82], encoding an alanine-to-serine sub- sis of dual causality, in which concomitant down-
stitution at position 69 (A69S) is the most fre- regulation of ARMS2 and upregulation of HTRA1
quently observed ARMS2 risk allele in the human explains the disease [85]. Similar results were
population. Little is known about the effect of obtained in another study that showed reduced
this mutation on ARMS2 protein structure and ARMS2 expression in human postmortem retinal
function. One study reports that the A69S varia- and RPE samples, heterozygous for R38X [83].
tion in ARMS2 creates a new putative phosphory- However, a subsequent investigation failed to
lation site that breaks a predicted α-helix [73]. It detect a rs2736911-dependent alteration in
has been suggested that the A69S change affects ARMS2 expression [87].
ARMS2’s function in either Mt [73, 79] or its Since ARMS2 is not found in mice, to gain a
role in the cytoskeleton in COS7cells [80]. A better understanding of ARMS2 function, two
recent study implicated ARMS2 as a surface com- transgenic mouse models expressing human
plement regulator [75]. Recombinant ARMS2, ARMS2 and ARMS2 A69S were developed. The
expressed in Pichia pastoris, was shown to bind constructs used a ubiquitous cytomegalovirus
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The Project Gutenberg eBook of Stay off the
Moon!
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Language: English
By RAYMOND F. JONES
Illustrated by FINLAY
Sam turned back to look at the robot machine and the swarming
technicians. "Yes, you could," he said. "All of us have gone through
heartbreak time and again the past five years, watching them blow
up, or fall back and burn in the atmosphere because the motors didn't
ignite. Or seeing them get all the way to the moon and have some
five-dollar transistor conk out. But we always have at it again. You
will, too. You're new, but you're one of us now. You never back out
when you've come this far."
Watching the Prospector, Jim knew Sam was right. It had taken some
persuasion to bring him to this point, however. Until a couple of years
ago he had believed he would be content with ivory-tower plastics
research for the rest of his life. The persuasion had been applied
when Mary's brother, Allan Wright, had made the astronaut team.
Allan and Jim had grown up together. There was no other person Jim
felt closer to except Mary and their two children. Allan had dreamed
of space when they were kids, and when he was fifteen, he said, "I'm
going out there. I don't know how. But, somehow, I'm going out there."
Now, he had been selected to captain the first Apollo voyage. He had
been born for that purpose, he said.
But while he was still in the general pool of astronauts he had opened
his campaign to get Jim into the space program. "They need the best
brains they can get," Allan said. "You haven't got any right to sit in a
musty old plastics lab while guys with half your ability try to get us into
space. NASA will take you tomorrow!"
Jim didn't try to tell him that his plastics lab wasn't exactly musty, or
that he didn't think of himself as one of the best brains in the country.
But Mary sided with Allan; she was almost as excited about space as
he was. In the end, Jim went to NASA. Within days, he had been
assigned to head the development of the Prospector chemical
mechanization.
It had been something of a jolt to pull up all the roots he had so
carefully put down for him and his family, and move to the hectic,
bustling, space-frontier community of the Center. But he wasn't sorry.
It put something new in the blood, something men had never known
before.
Space!
The great Saturn lifted slowly, on a vast blossom of fire, with snowy
lox streaming down its sides. Then it was gone, a twinkle of fire high
above, among the stars. That was all.
Mary and Jim Cochran continued to stare at the fading twinkle, and
finally they turned away. Allan had obtained permission to be in the
blockhouse during the firing. It hadn't been necessary for Jim to be
there. He didn't want to know the instant-by-instant telemetry reports
which told whether or not the flight was successful. Sam or Allan
would call him when they knew. That would be soon enough for him.
"Let's drive down to the beach and watch the moon from there," said
Mary. "We can't just turn around and go home, like—like nothing had
happened."
Jim smiled in the darkness. Mary was as eager as he was for the
success of the flight. And she didn't have his fear of failure, that kept
him from wanting to know the maybe-yes, maybe-no indications that
the telemetry would first show.
"Sure," he said, "that's a good place to watch it."
The moon.
They were still in the blockhouse, but the tension was relaxed. They
were talking and watching the meters and cathode ray tubes without
the strain and fear of failure. Jim knew the answer even before Sam
and Allan walked up to him and slapped him on the back.
"Where the devil did you go?" said Sam. "I thought you were going to
be right behind me when we fired, and you weren't here at all!"
"It's like your first baby, you want to be there, and you don't. Was it a
good shot?"
"Was it a good shot?" Allan's face became ecstatic. "We've never had
a better one. On course all the way!"
The Project Director, Emil Hennesey was behind them. His face was
bleak. "I expected you to be here for the firing, Cochran," he said. "It
seems to display little interest in your end of the project that you didn't
feel it necessary to show up."
Jim looked at him steadily and shrugged without answer. Hennesey
was one guy whose presence on the space team Jim couldn't figure
out. He was an ex-Major, and he had no capacity for dreaming. Men,
machines, transistors, rockets—they were all the same thing, merely
objects to be made to obey.
"You are aware of your next sequence of duties, I trust?" said
Hennesey.
Jim nodded curtly. "I'll be ready."
Sixty-six hours to the moon. That's what it takes with marginal escape
velocity and free-fall conditions. But it was really five hundred
thousand years and sixty-six hours, Jim thought. Surely there hadn't
been a single hour in all that time when someone, somewhere on
earth had not felt the longing to solve the secret mystery of the moon.
Now they were about to find the answer. But what would they have
when they found it? They would know that the surface dust of the
moon consisted of certain percentages of silicates and oxides. They
would know that the under layers were composed of rocks, maybe of
granite or limestone or basalt. They would determine how much of
each.
And then it would be over. The quest of the ages would be answered
with a few simple statements that could be obtained in any high-
school chemistry lab—if the lab were on the moon.
Jim Cochran felt there had to be more to it than that.
Why do dogs howl at the moon on winter nights?
Why do men say that madness of the mind is lunacy?
Why must planting be done in the right phase of the moon?
Little sleep was had by any of the crew during the next two nights,
even though the instrument stage of the ship was now completely
inert except for occasional telemetry signals that were fed to the
computers for course checking and correction. The ship was simply
falling on its own momentum.
Six hours before moonfall, activities in the tracking center accelerated
and the tension increased. There was no question of hitting the
moon; the landing had to be made safe for the cargo of instruments.
Jim Cochran watched the operators during this period. He told
himself he didn't understand it, but he had actually learned a great
deal of electronics during the past two years. He had had to in order
to design and operate a chemical laboratory 240,000 miles away.
The television screens came on, showing the pock-marked surface of
the moon as the ship orbited. The thrill and the fear of the great
unknown began to rise in Jim's throat. By the silence in the room, he
was sure the others sensed it, too.
Abruptly, the braking command was given and the ship began to fall
out of orbit towards the planned landing in the Sea of Rains. On the
screens, the images swelled as the ship plummeted faster. In one
corner could be seen the spring-loaded extension legs, like those of
some great spider. It seemed impossible that these could cushion the
violent shock of landing.
The sudden surge of a retro rocket and the blast of moondust blinded
the television eye, but there was a sense of crazy, rocking, rolling
motion. Then the eye went dead.
Jim almost cried out. The ship couldn't have crashed.
There were hours of testing and calibration yet to be done before the
Prospector could be used for its primary mission. Hundreds of
electronic circuits had to be checked to see that they survived the
takeoff and landing without becoming distorted or inoperative.
Jim went home for the rest of the night. When he returned the next
morning Sam reported that all circuits were go, and the Prospector
was his.
He had operated the laboratory in the Prospector many times, either
on a mock-up or from this control panel while the Prospector was in
the hangar. But he couldn't keep the faint tremor from his hand as he
reached for the first control that would manipulate the machine on the
moon.
The drill had been extended to operating position, but the head had
not yet been energized. Jim touched it to the fine dust of the floor of
the Sea of Rains. The drill went quickly to a depth of eighteen inches
in the dust before it struck something firmer.
"That kills the theory about eighty feet of that stuff, anyway," said Sam
as he read the instruments.
Jim was not interested in depth at this time. He fed some of the
surface material into the laboratory and set the controls to run the
preprogrammed analysis. They waited minutes; then the analysis
began to appear in cryptic symbols on a paper tape.
Jim glanced at it and frowned.
"What's the matter? Isn't it working right?" Sam asked anxiously.
Jim hesitated. "It indicates the presence of several silicates, some
carbonates, and a high percentage of oxides. These are mostly of
sodium, calcium, and iron, as you might expect. But there's
something wrong with your calibration. The atomic and molecular
characteristics aren't coming through right."
"The boys ran checks on the standard samples aboard the
Prospector last night," said Sam. "The results tallied exactly. I'll show
you the tapes."
Jim waited, puzzled, while Sam brought up the check tapes. When he
saw them, he shook his head. "There's a standard calcium carbonate
sample carried aboard the Prospector. Here's a calcium carbonate
picked off the surface. You can see the difference yourself. The
nominal analysis is the same, but the atomic weights and the energy
levels are just slightly different. That doesn't make sense unless your
circuits are out of calibration."
"Let's run another standard sample," said Sam.
Within a few minutes the calibration check had been repeated. Jim
held up the tape. Sam peered over his shoulder. "Just like the first
one," said Sam. "Nothing's wrong with the circuits. Maybe you've got
some new stuff there, that's never been identified before."
"That's hardly possible," said Jim. "There aren't any new elements in
the places where sodium and calcium and silica are supposed to be.
Yet, I don't understand how this can be. If the atomic weights are
different, and the energy levels are different, they have to be different
elements. It doesn't make sense."
"Well, why don't we push on," said Sam, "that is, if you've completed
the surface sampling in this spot. Some samples at lower depths may
give other indications."
Jim agreed. He drove the drill deeper into the face of the moon. At
ten-foot intervals he removed samples and ran them through the
analyzer. The results were the same down to the hundred-foot level.
All results showed common chemical elements with slightly variant
atomic characteristics.
Which made them different chemical elements!
After six hours, Jim stood up from the console and shook his head
wearily. "It's no good, Sam. There's something wrong that I can't put
my finger on. If it isn't in the circuits, I don't know where it is. But
these readings just aren't right. There's no use going deeper until we
find out where the error is."
Sam's face was somber. "There just isn't any error. There can't be.
Unless it was made by whoever put the moon together—"
"Please make a complete check of every analyzer and telemetry
circuit tonight, and we'll try again tomorrow. I want to think about this."
He thought about it, and he dreamed about it. And along about three
o'clock in the morning he sat bolt upright in bed and stared at the dim
moonlight on the opposite wall of the bedroom.
It wasn't possible, he told himself audibly. It just wasn't possible!
Mary stirred and leaned on one elbow beside him. "What's the matter.
Are you having nightmares?"
"Yeah—yeah, I guess I am. I'll be back in a minute, honey." He got up
and padded to the door. "I've got to make a phone call."
"At this time of night?"
But Jim was gone. He turned on the hall light and dialed Sam's
number. After a long time Sam answered sleepily.
"Wake up!" said Jim. "I've just figured it out!"
"Who the devil—? Oh, it's you, Jim. Figured out what? Do you know
what time it is."
"Do you know the results of your calibration re-check?"
"How would I know that? I've got the night crew on it, but I didn't ask
them to report to me in the middle of the night. Go back to bed, and
let's talk about it in the morning."
"They're not going to find anything wrong, Sam."
"I could have told you that."
"But the elements of the moon are different—and there's only one
explanation."
"What?"
"Think about it a minute, Sam. We take a spectrograph of the sun,
and we find the same elements that are here on earth. We turn it on
Alpha Centauri and find the same thing. We turn it to the farthest
stars we can find that give enough light to record by. Always the
same. Calcium is calcium, whether it's on the earth or on a star a half
billion light years away."
"So?" Sam's voice was tired, and he sounded as if he was listening
only because Jim was too good a friend to tell to go to hell for calling
in the middle of the night.
"So? So what?" Sam repeated.
"So we go to the moon," said Jim, "and all of a sudden calcium isn't
calcium, and the sodium on the moon isn't the same as the sodium
on earth and on the sun and on Alpha Centauri and the stars a half
billion light years away. Don't you see what that means!"
"No, I guess not," said Sam dully. "Maybe in the morning—"
"It means the moon just doesn't belong, Sam! It means the moon is
completely foreign to anything in the Solar System, in the whole
galaxy—in any galaxy we have been able to analyze. It means the
moon has come from somewhere else, from a region of space where
atoms and electrons are not even the same as atoms and electrons
here. It must be a somewhere that's so far away it's beyond the edge
of space as we know it!"
"I'll get dressed and come over," said Sam.
Mary made chocolate and toast, and they sat around the kitchen
table thinking and talking of the awesome implications of Jim's theory.
"If what you say is true," said Sam, "it might be that the slightest
contact with any substance of the moon would be sheer poison to a
human being. A returning vessel could never be permitted to enter
the earth's atmosphere, and decontamination would become one of
the major branches of science."
"That's entirely possible. It would complicate enormously the
problems of establishing a moon-base. A speck of moondust inside
the base might be as lethal as an unshielded reactor."
Mary was looking out the kitchen window toward the thin crescent of
moon that was setting over the city. She thought of Allan, who would
soon be voyaging to that alien world. "It's like a trap up there in the
sky. We should never have tried to reach it."
"No—it's not like that at all," said Jim vigorously. "We'll solve whatever
problems we find there. But think of it! We don't have to build a ship
capable of crossing billions of light years of space to see what's out
there. Something from out there has come to us and parked right in
our own front yard. We have a thousand times more reason to go
now!"
Sam toyed with his toast and dunked it in the chocolate. "I think you
ought to keep it quiet—until we really know, don't you."
"Why? I'll run some more tests, sure. I'll plug every loophole there can
possibly be. But unless I find something new I'm going to announce it.
Why shouldn't I?"
"I don't think Hennesey will like it, for one thing. Too sensational.
Even when we actually land there and confirm your analyses by on-
the-spot checks—it's still only a theory that the moon doesn't belong
to this galaxy. You'll never be able to prove that."
"What we've found already is proof enough!"
"Not for Hennesey. He'll ask to see the shipping manifest by which
the moon was transferred here. You know Hennesey."
"Sure, I know Hennesey," said Jim bitterly. "And he doesn't count. Not
in something like this. This is big, and important, and I'm going to
announce it. I want the credit for discovering it. It'll be called
Cochran's Theory, and some University will offer me at least an
honorary Ph.D. Is that bad?"
Sam shook his head. "Of course it's not bad. But I wonder what the
public reaction will be like, and how about Congress—especially if
this business about possible poisoning from moondust turns out to be
correct? There might be a lot of pressure to cut funds and maybe
cancel the whole moon project."
"If moondust is lethal, there's no better time to be warned against it
than right now!"