Alzheimer's Disease is Responsible for Progressive Age Dependent

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Alzheimer’s Disease is Responsible for Progressive Age Dependent Differential Expression of Various Protein Cascades in Retina of Mice Javed Iqbal, Kaoyuan Zhang, Na Jin, Yuxi Zhao, Liu Xukun, Qiong Liu, Jiazuan Ni, and Liming Shen ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00710 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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ACS Chemical Neuroscience

Alzheimer’s Disease is Responsible for Progressive Age Dependent Differential Expression of Various Protein Cascades in Retina of Mice Javed Iqbal1, Kaoyuan Zhang1,2, Na Jin1, Yuxi Zhao1, Xukun Liu1, Qiong Liu1*, Jiazuan Ni1, Liming Shen1* 1 College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, P. R. China 2 Department of Dermatology, Peking University Shenzhen Hospital, Guangdong, China

*Corresponding Author: Qiong Liu and Liming Shen, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, P. R. China E-mail: [email protected], [email protected] Phone: +86-755-26012653

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Abstract Alzheimer’s disease (AD) is a devastating neurodegenerative disease associated with cognitive impairments and memory loss usually in aged people. Since last few years, it has been detected in the retina of eye manifesting the systematic spread of the disease. This might be used for biomarker discovery for early detection and treatment of the disease. Here, we have described the proteomic alterations in retina of 2, 4 and 6 months old 3×Tg-AD mice by using iTRAQ (isobaric tags for relative and absolute quantification) proteomics technology. Out of total identified proteins, 121 (71 up and 50 down-regulated), 79 (51 up and 28 down-regulated) and 153 (37 up and 116 down-regulated) significantly differentially expressed proteins (DEPs) are found in 2, 4 and 6 month’s mice retina, respectively. Seventeen (17) DEPs are found common in these three groups with consistent expression behavior or opposite expression in the three groups. Bioinformatics analysis of these DEPs highlighted their involvement in vital AD related biological phenomenon. To further prompt the results, four proteins from 2M group, 3 from 4M and 4 from 6M age group are successfully validated with Western blot analysis. This study confirms the retinal involvement of AD in the form of proteomic differences and further explains the protein based molecular mechanisms which might be a step towards biomarker discovery for early detection and treatment of the disease. Keywords: Alzheimer’s disease, Retina, iTRAQ proteomics, Biomarker discovery, Aging

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Introduction Man has been trying to enhance his living conditions and life expectancy since a long time. He has been trying to overcome the fatal diseases specially the neurodegenerative diseases as being directly associated with the nervous system. Being second most devastating neurodegenerative disease, Alzheimer’s disease (AD) has gained much attention. There are 46 million people directly affected by AD and this number is expected to rise up to 131.5 million by the year 20501. AD has been found a main cause of cognitive impairments and dementia in the world 2. The major pathology of the disease involves the aggregation and propagation of misfolded amyloid β-protein (Aβ) and hyperphosphorylated tau protein (pTau) present in neurofibrillary tangles (NFTs) in various parts of the nervous system 3, 4. For effective treatment of the disease, its early detection is necessary but it is impossible to be used widely by brain imaging tools which target A or tau pathology5. This is due to the difficulties in screening of large populations and prediction of disease progression6, 7. It is thus urgent for researchers to find new biomarkers in peripheral fluids of the body which can help for early detection of the disease. Previous studies uncovered the involvement of AD pathology in retina of eyes which is considered as an extension of the central nervous system8-11. This was supported by the fact that AD patients with mild cognitive impairments also suffer ocular abnormalities and visual disorders11. Pathological analysis of the AD affected retinal tissues shows retinal ganglion cell (RGC) degeneration, reduction of blood flow and vascular alterations, nerve fiber layer (NFL) thinning, astrogliosis, and abnormal electroretinogram patterns11,

12.

These show similar

pathological characteristics as observed in brain. This is due to the sharing of similar structural features like microglia, neurons, and microvasculature with similar morphological and physiological properties, astroglia, and a blood barrier11-14. Another important feature is the optic nerve which establishes a link between the brain and the retina and might be a source of transportation of amyloid precursor proteins (APP) in the retina15. Several studies confirmed the presence of in the retina of humans except few16, 17. They have detected these by using specific tissue processing and immunostaining techniques with screening of large retinal parts which were previously investigated16-19. Recently, in AD patients, sufficient 3



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accumulation of Aβ was observed around and inside the degenerating melanopsin-containing RGCs (mRGCs). These mRGCs are kind of photoreceptors which are used to drive certain circadian photoentrainment20. The sleep disturbances in these patients might be due to the existence of Aβ which causes degeneration of these photoreceptors21. Similarly, various investigations have reported the deposits of Aβ, vascular pTau and Aβ, along with paired helical filament-tau (PHF-tau) in sporadic and transgenic animal models of AD. These findings might be associated with RGC degeneration, local inflammation (i.e., microglial activation), and impairments of retinal structure and function16-18. Proteomics analysis of early AD retinopathy revealed that retinal neurodegeneration and brain share the common pathways22 Above literature ensures that retina is deeply affected by AD and it can be used for early detection of AD and its treatment. None of the study reported the protein based molecular mechanisms of retinal AD pathology which will not only be helpful to understand the molecular mechanisms of the disease but also will help to identify and establish biomarkers for its earlier detection. This study has been designed to fulfill the gap and uncover the proteomics changes in mice retina. iTRAQ (isobaric tags for relative and absolute quantification) based proteomics technology has been applied to study the progressive proteomics changes in retina of 3×Tg-AD mice (triple transgenic AD mice) while they were of 2M, 4M and 6M old. Proteomics has been applied to the study of diseases and toxicological mechanisms22-24. The 3 × Tg-AD mice are derived from mixed 129/C57BL6 mice harboring APPswe, PS1M146V and TauP301L mutants and can develop age-dependent and progressive neuropathology including senile plaque and NFTs, thus mimic AD progression in humans25. These show pathological hallmarks for AD and are based on Aβ deposits in extracellular spaces of the cells before the tangle formation. These mice are deficient in synaptic plasticity along with long term potentiation which appear before the Aβ deposition and tangle formation26, 27. This study will not only elaborate the progressive proteomics changes in retina with the age of mice but will also help to provide raw data for future biomarkers studies for early detection of the disease. Results To find out the proteomics changes in retina with progression in age, we have taken three age groups (2M, 4M and 6M) of 3×Tg-AD mice along with non-Tg respective control groups. After mass spectrometry data analysis, the results are divided into 4 groups. 4


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Figure 1. A-C Differentially expressed protein in A- 2M old mice, B- 4M old mice, C- 6M old mice, D- Venn diagram showing the total proteins along with common protein in the three groups, E-Heat map of common proteins in three groups with expression intensities. 2 M AD/WT group Out of total 2284 identified proteins, 121 retinal proteins (supporting table S1) were found significantly different in expression from the control group. Out of these, 71 proteins were found up-regulated while 50 were down-regulated (Figure 1A). Omicsbean based bioinformatics analysis of these differentially expressed proteins shows the involvement of these proteins in vital functions related with amyloid pathology and optics of the eye Figure 2 A, B &C. Twenty enriched biological processes (BP) found in Gene ontology (GO) analysis of the DEPs include multicellular organismal process, anatomical structure development, lens development in 5



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camera-type eye, sensory organ development, system development, eye development, lens fiber cell differentiation, camera-type eye development, sensory perception of light stimulus, lens fiber cell development, visual perception, and cellular protein complex assembly. To evaluate the structural stability of the retinal tissue of 2M old 3×Tg-AD mice, we have obtained the cellular components through GO bioinformatics analysis. Ten enriched component processes are shown in Figure 2 A while twenty enriched processes are given in Figure 2C, which include supramolecular fiber, extracellular region part, organelle part, myelin sheath, extracellular vesicle, extracellular organelle, protein complex, extracellular membrane-bounded organelle, vesicle, membrane-bounded vesicle, contractile fiber, cytoskeleton, and cytoskeletal part as important components. Some of molecular processes (10 enriched processes) are given in Figure 2A for these DEPs. These help to understand the involvement of these proteins in variety of vital molecular functions being disturbed during AD. Integrated pathway maps of the DEPs are expressed with the KEGG (Kyoto Encyclopedia of Genes and Genomes) bioinformatics tool (Figure 3A). The important pathways include oxidative phosphorylation, cGMP–PKG signaling pathway, phagosome, regulation of actin cytoskeleton, adherence junction, leukocyte transendothelial migration, PPAR signalling pathway, carbohydrate

digestion

and

absorption,

mineral

absorption,

synaptic

vesicle

cycle,

phototransduction, Parkinson’s disease, amyotrophic lateral sclerosis(ALS), and Huntington’s disease. Protein-protein interaction network (Figure 3B) elucidate that the DEPs are interlinked with functional interactions highlighting the disturbance of various biological phenomenon i.e., synaptic vesicle cycle, oxidative phosphorylation, and various neurodegenerative diseases, etc. To further prompt the MS quantification results, four proteins i.e., Beta-crystalline B2, fatty acid binding protein, adipocyte, glial fibrillay acidic proteins and phakinin were validated by Western blot analysis. These proteins show significant changes as shown in MS analysis, hence validating the MS results (Figure 4).

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Figure 2. Bioinformatics analysis of DEPs in 2M age mice: A- ten enriched GO functions for BP, CC, and M. B-Biological processes. C- Cellular components.

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Figure 3. Bioinformatics analysis of DEPs in 2M age mice: A- KEGG pathway analysis. BProtein-protein interactions.

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Figure 4. Western blot validation of four key proteins. A. Glial fibrillay acidic proteins (GFAP), B. Fatty acid binding protein, adipocyte (FABP4). C. Beta-crystalline B2 (CRYBB2), D. Phakinin (BFSP2). Fiji ImgeJ software was used to quantify the proteins in individual bands while data was analyzed with Graphpad software. The bands were normalized with relative band density of GAPDH in individual blots accordingly. n=3. 4M age group (AD/WT) To find out the effect of AD on proteomics changes with increasing age, we have taken 4M old 3×Tg-AD mice and performed iTRAQ proteomic analysis. Mass spectrometric analysis identified total of 2284 proteins among which 79 protein (supporting table S1) were significantly differentially expressed with 51 up and 28 down-regulated proteins (Figure 1B). GO of 10 enriched biological processes are shown in Figure 5A while 20 important enriched biological processes for functional network of these proteins (Figure 5B) include the involvement of these 9



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proteins in multicellular organismal process, lens development in camera type eye, muscle system process, multicellular organismal movement, sensory organ development, muscle contraction, musculoskeletal movement, eye

Figure 5. Bioinformatics analysis of DEPs in 4M age mice: A- ten enriched GO functions for BP, CC, and MF.B-Biological processes. C- Cellular components.

10


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Figure 6. Bioinformatics analysis of DEPs in 2M age mice: A- ten KEGG pathway analysis. BProtein-protein interactions. development, lens fiber cell differentiation, camera type eye development, striated muscle contraction, lense fiber cell development, skeletal muscle contraction, visual perception, and voluntary skeletal muscle contraction. Involvement of these DEPs in structural cascades of retina, cellular component analysis was performed (Figure 5A,C) which include, supramolecular fiber, extracellular region part, myelin sheath, extracellular vesicle, cytoplasm, extracellular exome, cytoplasmic part, contractile fiber, cytoskeleton, myofibril, contractile fiber part, actin cytoskeleton, cytoskeletal part, sarcomere, myofilament, striated muscle thin filament, and i band. 10 enriched molecular processes are shown in Figure 5A highlighting the involvement of these DEPs in molecular cascades during AD. KEGG analysis (Figure 6A) revealed the involved 11



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pathways which include, arginine and proline metabolism, leukocyte transendothelial migration, PPAR signaling pathway, collecting duct acid secretion, systemic lupus erythematosus, and Parkinson’s disease. PPI analysis (Figure 6B) revealed the interaction network of the KEGG pathways highlighting the association of these pathways. Three proteins i.e., glial fibrillary acidic protein, fatty acid binding protein, adipocyte and Beta-crystalline proteins were successfully validated by western blot analysis showing significant expressional changes as described by MS analysis Figure 7.

Figure 7. Western blot results of respective proteins along with GAPDH as a loading control (n=3). A. Glial fibrillay acidic proteins (GFAP). B. Fatty acid binding protein, adipocyte (FABP4). C. Beta-crystalline B2 (CRYBB2). Fiji ImgeJ was used to quantify the proteins in individual bands while data was analysed with Graphpad software. The bands were normalized with relative band density of GAPDH in individual blots accordingly. 12


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6M age group (AD/WT) Out of total 2284 identified proteins, we have found 153 DEPs (supporting table S1) with 37 up and 116 down-regulated proteins in 6M old 3×Tg-AD mice as shown in Figure1C. Biological processes based on GO analysis (Figure 8 A&B with 10 and 20 enriched processes respectively ) revealed functional cascades of retina which include, generation of precursor metabolites, oxidation-reduction process, small molecule metabolic process, tricarboxylic acid cycle, energy derivation by oxidation, electron transport chain, cellular respiration, nucleoside monophosphate metabolic process, nucleoside triphosphate metabolic process, respiratory electron transport chain, nucleotide metabolic process, purine nucleoside monophosphate metabolic process, ribonucleoside monophosphate metabolic process, ribonucleoside triphosphate metabolic process, purine nucleoside monophosphate metabolic process, aerobic respiration, purine ribonucleoside monophosphate metabolic process, purine ribonucleoside triphosphate metabolic process, citrate metabolic process, and ATP metabolic process. Cellular components (Figure 8 A&C) include, supramolecular fiber, myelin sheath, respiratory chain, envelop; mitochondrial membrane part, organelle inner membrane, mitochondrial protein complex, inner mitochondrial membrane protein complex, mitochondrial respiratory chain, mitochondrial part, mitochondrial inner membrane, cytoplasmic part, respiratory chain complex, organelle envelop, mitochondrion, mitochondrial envelop, contractile fiber, mitochondrial membrane, and myofibril. Additionally, figure 8A is showing 10 enriched molecular processes performed by these proteins. KEGG analysis (Figure 9A) revealed the different functional units integrating in the form of map. These include, 2-Oxocarboxylic acid metabolism, biosynthesis of amino acids, citrate cycle (TCA cycle),

pyruvate

metabolism,

propanoate

metabolism,

butanoate

metabolism,

glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, fatty acid degradation, fatty acid elongation, synthesis and degradation of ketone, valine, leucine and isoleusine degradation, tryptophan metabolism, lysine degradation, arginine and proline metabolism, beta-Alanine metabolism, D-Glutamine and D-Glutamate metabolism, calcium signaling pathway, HIF-1 signaling pathway, cGMP-PKG signaling pathway, tight junction, PPAR signaling pathway, adrenergic signaling in cardiomyocytes, phototransduction, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and arginine biosynthesis. PPI network analysis (Figure 9B) established the interaction network of KEGG pathways elucidating 13



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the association of these functional cascades. Finally, Beta-crystalline B2, fatty acid binding protein, adipocyte, and glial fibrillay acidic proteins were successfully validated by Western blot analysis in accordance with the MS results (Figure 10).

Figure 8. Bioinformatics analysis of DEPs in 6M age mice: A- ten enriched GO functions for BP, CC, and MF. B-Biological processes. C- Cellular components.

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Figure 9. Bioinformatics analysis of DEPs in 2M age mice: A- KEGG pathway analysis. BProtein-protein interactions network.

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Figure 10. Western blot results of respective proteins along with GAPDH as a loading control (n=3) A. Glial fibrillay acidic proteins (GFAP). B. Fatty acid binding protein, adipocyte (FABP4). C. Beta-crystalline B2 (CRYBB2). D. Phakinin (BFSP2). Fiji ImgeJ software was used to quantify the proteins in individual bands while data was analyzed with Graphpad software. The bands were normalized with relative band density of GAPDH in individual blots accordingly. Expression trend in 2M, 4M and 6M age groups (Common proteins) To observe the expression behavior of proteins in the 3 age groups, we have carefully analyzed the DEPs in these groups. The results revealed that 17 DEPs are found common in the three groups with consistent similar expression or opposite expression from one group to another group (Figure 1 D, E). Eight proteins with gene names Dbi, Cryaa, Cryab, Cryba1, Crybb2, Bfsp1, Bfsp2 and Anxa3 showed consistent upregulation while none of the protein showed 16


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consistent downregulation in the three age groups. Seven proteins i.e., Actn2, Ckmt2, Fabp4, Myl1, Mylpf, Ndufv1, Tnnc2 showed consistent upregulation in 2M and 4M groups while downregulation in 6M age group. Only one protein (Alb) was up-regulated in 2M group while it showed downregulation in 4M and 6M age groups. One protein (Gfap) showed downregulation in 2M and 4M age groups while its expression was insignificant in the third 6M age group. Collectively, in 2M group, 20 proteins were found up-regulated, 3 proteins down-regulated and 7 proteins with insignificant expression. In 4M age group, 20 proteins were up-regulated, 3 were down-regulated and 7 proteins were noted with insignificant expression. In 6M age group, 16 proteins were found down-regulated, 8 up-regulated and 6 proteins with insignificant expression. Discussion We have already reported the proteomic changes in cortex and hippocampus of 3×Tg-AD mice highlighting the role of various vital proteins in AD24, 28 along with some therapeutic studies. This study is an extension of previously published literature in support of AD manifestations in retina 11, 16, 18-20 and demonstrates the proteomic alterations in the retina of 3×Tg-AD mice of 2M, 4M and 6M age groups. Several proteins were found whose expression levels were dysregulated in AD conditions in the three age groups. These proteins were found associated with various important functions in the retina. AD as a heterogeneous disease with multiple cognitive subtypes involves wide range of functions including language, memory, attention, executive and visuospatial activities29, 30. One of the prominent variant which induces visual symptoms and known as visual variant AD (VVAD) usually defined as a localized pathology in the parieto-occipital region31. The eye and brain interconnection is an extension of the CNS and it suggests to observe the manifestations of neurodegenerative diseases in eyes. It should be considered that in embryological development, a similar origin is defined for the eyes and brain. Anterior neural tube as a region that later gives rise to the fore brain forms the eyes and specification of the eye field and post-neural induction leads to ocular development 32. In the eye, the characteristics of neurons in retina are comparable to the counterpart neurons of brain in many ways. As essential neuronal features, retinal neurons have a soma dendrites, and an axon,

33and

can be stained by using many usual neuronal

markers34-36. As well, they arrange a network for processing the complex information similar to 17



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those in the cerebrum37-39. Over 60 types of neuron are found in the retina40 which they are essential for information processing. One of the five main types of neurons of retina is known as photoreceptors and the other four types are bipolar, horizontal, amacrine, and ganglion cells. After exiting by light, photoreceptors conduct the signals in a neuronal fashion and are classified into two main subtypes including cones and rods. This information is transmitted by bipolar cells to retinal ganglion cells (RGCs). Horizontal cells provide inhibitory feedback by connecting to photoreceptors as well as bipolar cells, to both refine and adjust the light signal and Amacrine cells can act as intermediaries between bipolar and ganglion cells and also modify direct signals between the two. In retinal networks, amacrine, horizontal, and bipolar cells behave similar to inter-neurons, and the RGCs act as projection neurons. The retina contains the similar classes of neurotransmitters, such as glutamate and GABA (γ-aminobutyric acid), which are important for processing the retinal information41-43. Recently, researchers introduced a group of retinal cells subtype, known as intrinsical photosensitive retinal ganglion cells (ipRGCs) which respond to light through expression of the photopigment melanopsin in the absence of rod and cone photoreceptor input44. Thus, due to the close relationship of eye to the brain, it is not surprising that neurodegenerative diseases such as AD affect the eye structure. Visual disorders have been well recorded in AD patients, and there are relevant evidence to show the ocular pathology as part of AD45,

46.

Therefore, AD model provides an opportunity to utilize minimal invasive methods to evaluate the pathological characteristics in the brain – through the transparent medium of the eye. Deposits of retinal Aβ in patients with AD were frequently focused in the mid- and far-periphery of the superior quadrants, which could be detected in living patients using retinal curcumin imaging. These results together with superior retinal neuronal loss may clarify the recently published reports about the loss of axons and thinning in NFL in these patients20, 47, 48, noticed mostly in the same retina are as that amyloid pathology was prominent. Likewise, it was described an extensive loss of neurons, commonly in the superior quadrant and as well as the mid- and far-peripheries49. The meaningful reduction in the NFL thickness in the superior quadrant is consistent with previous findings on loss of mRGC and accumulation of Aβ in the same retinal areas20. This enhancement in Aβ pathology in the superior quadrant, along with intense NFL thinning and mRGCs loss, also known as ipRGCs including melanopsin and implicated in controlling the circadian rhythm

20.

It may help to explain the reasons of sleep 18


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disturbances and visual field dysfunctions in AD patients even at early phases of disease14, 20, 21. Moreover, the presence of Aβ deposits in regions with abundant rod cells can contribute in visual motion impairments and low-contrast sensitivity reported in patients with AD50,

51.

This

discussion explains the main causes of protein changes in retina of mice model of AD. According to the results of this study, four dysregulated proteins were found in 2M old 3×Tg-AD mice which were related to oxidative stress and also light-dependent procedures in retina. These proteins are Gstm1, Gnat1, Sag, and Vsnl1. None of such proteins are found in 4M old 3×Tg-AD mice while Gnat1, Sag, Rbp3, and Rho were confirmed to dysregulate in 6M old 3×Tg-AD mice. Sag family members have been found to be involved in desensitization of G-protein coupled receptor (GPCR), internalization and GPCR-activated activation of mediated mitogen-protein kinase (MAPK) pathways with close association with the rhodopsin. Rod arrestin (also called Santigen, SAG) were characterized as the first member in the family

52.

It has a key role in

quenching the light which stimulates phototransduction cascade in rod photoreceptors through binding to light-phosphorylated-activated, rhodopsin

53, 54.

Lack of Sag expression as a result of

gene targeting knockout technology leads to prolonged photoresponses and boosts susceptibility to light damage in photoreceptors of rod cells55, 56. Previous researches demonstrated that Sag also contribute in the molecular pathway for apoptosis of light-induced photoreceptor in Drosophila through the creation of stable rhodopsin-arrestin complexes that are recruited to the cytoplasmic compartments through clathrin-dependent endocytosis 57-60. The retina is especially vulnerable to oxidative stress because of its high proportion of polyunsaturated fatty acids with high levels of oxygen consumption, and contact with visible light61, 62. Localization of Gstm1 in the macula as a zeaxanthin-binding protein proposes that Gstm1 have a key role in modulating the antioxidants levels in the macula. It has already been shown that levels of Gstm1 are reduced in human AMD retina compared to normal subjects (control)63. Vsnl1 protein expressed in E. coli displays Ca 2+ binding function. These findings proved that Vsnl1 protein is a photoreceptor-specific Ca2+ binding protein and may participate in the phototransduction of cone cells 64. The Rbp3 is a huge glycoprotein identified to bind retinoids and found primarily in the interphotoreceptor matrix of the retina between the photoreceptor cells and retinal pigment epithelium. It is suggested to transfer retinoids between the retinal pigment epithelium and the photoreceptors, hence, playing an important role in the visual process65. 19



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Due to deep relation between retina and optic nerve, certain dysregulated proteins linked with synaptic functions were detected. These include Stx1b, Cadps, Stxbp1, Syt1, Ctnna2, Dbnl, Fabp5 in 2M 3×Tg-AD mice while Pdlim5, Syt1, and Cplx4 were found in 6M AD mice. None of the significant synaptic protein was found in 4M 3×Tg-AD mice. Synapses are considered to be an early areas of pathology/dysfunction in AD cases66, and loss of synapses is the best etiology which correlate with cognitive dysfunctions in AD patients 67. For many years, it has been recognized that Aβ peptide, one of the basic substance in pathologic feature of AD formed by cleavage of the β- and γ-secretase from amyloid precursor protein (APP), can induce functional and morphological changes to synapses and synaptic plasticity 66, 68, 69.

It has already been explored that syntaxin (Stx)

is adequate to drive spontaneous Ca 2+-

independent fusion of synaptic vesicles containing v-SNAREs70. Stx bind synaptotagmin in a Ca 2+-dependent way and interact with voltage dependent Ca 2+and K+ channels through the domain of C-terminal H3. During depolarization of the presynaptic axonal buttons, the interaction of direct syntaxin-channel is appropriate molecular mechanism for proximity between the fusion machinery and the Ca2+ entry gates71. Stx1b may play a critical role in regulating the docking and fusion of synaptic vesicles, perhaps via interaction with GTP-binding proteins. As well, it is important for neurotransmission and binds syntaxin, a well known component of fusion machinery of synaptic vesicles, in a 1:1 ratio72, 73. Ctnna2 may act as synaptic as well as structural protein when forms a complex with cadherins74, 75. Along with its role in shaping cells and plasticity of dendritic spines, Dbnl might participate in synaptic function regulation. Indeed, the electrophysiological data of this study exposed that overexpression of Dbnl in cultured mature neurons of hippocampus inhibits synaptic transmission and increases excitation resulting in the normal excitatory/inhibitory (E/I) balance changes in favor of excitation76, with ageing in

the

brain

of

mouse,

77.

Levels of Fabp5 have been revealed to reduce

possibly

contributing

to

age-related

decline

in synaptic functions78. It has been shown that Cplx4 participates as a positive regulator in exocytosis of synaptic vesicle, and binds selectively to the complex of neuronal SNARE. A twofold function has been introduced for Cplx4 which can act as either a promoter or an inhibitor in the vesicle fusion. This dual-functionality is dependent upon synaptic function such as a depolarizing stimulus arriving at the synapse. By contributing as a fusion clamp as fusion 20


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inhibitor, and a promoter during depolarization, levels of Cplx4 regulate the size of vesicle pool such as that of the ready releasable pool, essential for short term response alterations79. We have found 12 proteins (5 up-regulated and 7 down-regulated) related to metabolic cascades which were found differentially expressed in 2M old 3×Tg-AD mice. These include Atp1b1, Atp6v1h, Slc25a5, Atp6v0d1, Hk1, Ndufv1, Etfa, Atp5o, Pygl, Ckmt2, Vdac2, and Atp1a3. While 5 differentially expressed proteins (all up-regulated) were detected in 4M old 3×Tg-AD mice. These proteins include Cox5b, Ndufv1, Hadha, Ckm, and Ckmt2. The third 6M age group gave 56 differentially expressed proteins which were all down-regulated except two proteins i.e., Hexokinase-1 and V-type proton ATPase subunit 1 which were up-regulated. Other downregulated proteins include Ckm, Ak1, Ndufb6, Mtco2, Cox4i1, Slc25a4, Actn2, Acaa2, Hadh, Hadha, Cycs, Atp5h, Cox5a, Sdhb, Pdhb, Ogdh, Ndufb10, Idh3a, Idh2, Aco2, Atp5b, Cyc1, Ndufs5, Acat1, Ndufs3, Dlat, Mdh2, Pfkm, Sucla2, Pygm, Cs, Ndufa13, Acadm, Atp5f1, Acadvl, Sdha, Etfa, Got2, Ndufa10, Uqcrc1, Fh, Etfdh, Uqcrc2, Pdha1, Cox6b1, Vdac1, Ndufv1, Ndufs2, Atp5o, Slc25a12, Oxct1, Uqcrfs1, and Ndufs1. Down-regulation of all these proteins leads to low respiratory rate resulting in reduced ATP outcomes in AD mice. Several investigations have documented that AD and other cognitive impairments are associated with lower rates of metabolism and energy production. According to the literature, low amount of glucose consumption couples with lower metabolic energy outcomes in the brains of AD patients80-87. The axonal growth is a delicate mechanism required for contacts between neurons. This process of axon is necessary to create a connection network relying on axonal path finding, which represents guidance to the growth of target. A wide range of diffusible guidance molecules contribute in the correct signaling path of the growing axon. Dihydropyrimidinase-related proteins appear to be involved in the outgrowth of axonal process regulated by extracellular signals88. Dpysl2 is extensively investigated for its important role in path finding and neuronal migration s89-91. Igsf8 may involve in the regulation of the neurite outgrowth and the neural network maintenance in the brain of adults92. Sncb is a synuclein protein and was primarily found in the brain tissue and mainly observed in presynaptic terminals. It may play a critical role in neuroplasticity and it is predominantly expressed in different regions such as cerebellum, neocortex, striatum, hippocampus, and thalamus93. Twenty six (26) differentially expressed proteins were found in 2M old 3×Tg-AD mice which are related to structural cascades of the retina and optic nerve. These include Gfap, Plp1, Cnp, 21



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Nefl, Ina, Nefm, Nefh, Tuba1a, Tuba4a, Map4, Map1b, Clta, Tppp, Dsp, Myl2, Actn2, Vcl, Myoz1, Tnnc2, Myl1, Myl6b, Krt15, Pfn1, Lgsn, Synm, and Sep5. In 4M old mice, 13 proteins were found differentially expressed. They include Gfap, Spta1, Ctnna1, Actn2, Myom3, Mpz, Actn3, Tnnc2, Tnnt3, Synm, Lgsn, Myh4, and Sep5. Similarly in 6M old 3×Tg-AD mice, we have found 5 proteins related to structural cascades. These include Actn3, Myom1, Krt14, Vim, and Krt10. It has already been recorded that AD induces alterations in the amyloid and tau-derived cytoskeleton which are deeply related to the loss of synapstic functions in AD94. One of the necessary hallmarks of AD is NFTs which are created as a result of tau protein hyperphospohorylation.

Tau

is

important

in

microtubules

stabilization

and

its

hyperphosphorylation which eventually leads to destabilization and collapse in microtubules of cytoskeletal structure, resulting in neuronal apoptosis95. Equally, neurodegeneration is a main phenomenon during AD in brain and related structures. Loss of neurons and other tissues may lead to dysregulation of various structural proteins as is depicted in the present study results. We have retrieved 17 proteins which are associated with eye lens structure and maintenance in 2M old 3×Tg-AD mice. These proteins include Crygs, Ig gamma-2A chain C region secreted form, Crygn, Cryba4, Crybb1, Cryab, Crygb, Crybb3, Cryba1, Cryba2, Bfsp2, Crybb2, Cryaa, Crygd, Crygc, Bfsp1, Cryge, and Cryga. None of the protein related to lens is detected in 4M old 3×Tg-AD mice while 7 proteins i.e., Cryaa, Crygc, Bfsp1, Cryab, Bfsp2, Cryba1, and Crybb2 are detected in 6 M old 3×Tg-AD mice. Most of the proteins in our results belong to the superfamily crystallines. Since, it has been reported that lens damage may upgrade the nerve regeneration93, crystalline has been an area of neural research. So far, it has been demonstrated that crystallin β b2 (crybb2) may be a neuritepromoting factor96. The basic function of crystallins at least in the lens of the eye is probably to enhance the refractive index while not obstructing light97. Nevertheless, this is not their only activities. It is clear that crystallins may have regulatory and metabolic actions, both within the lens and other parts of the body98. Overexpression of crystallin family members act as a mechanism of cellular response against different types of cellular stressors, such as osmotic stress, cellular damages, as well as bacterial infections in the various tissues99, 100. The current study suggest that up-regulation of crystallins, especially the α-crystallin family member, provides protective effects against the apoptosis in neurons during AD 101. It was proved that the 22


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expression of crystallin proteins in the retina changes following trauma maybe due to its protective roles102-104. Two lens-specific intermediate filament-like proteins, filensin and phakinin are expressed only after the initiation of fiber cells to differentiate. Both proteins are known to have a unique structure as a cytoskeletal element that is denoted to as the beaded filament (BF)

105.

In short, we can say that dysregulation of all these proteins in the retina and

optic nerve of the three 3×Tg-AD mice age groups might bring changes in eyes of the AD patients. In these common proteins, protein expression pattern of 2M and 4M age groups is similar, except for ighg1 and Alb, which are up-regulated in 2M and 4M groups. Among these, nine proteins (Ckmt2, Myl1, Fabp4, Actn2, Tnnc2, Mylpf, Ndufv1, Ighg1, and Alb) showed opposite expression behavior and were down-regulated in the 6M group. Our results show highest number of differentially expressed proteins (153) in 6M age group as being the most aged group as compared to the other two groups. This agrees with the previous findings which revealed that pathological hallmarks for AD are found to be increased with the aging106. For validation of quantitative results, four proteins from 2M and 6M age groups while three proteins from 4M age group with important functions and consistent changes in two or three groups were validated by Western blot analysis. Among these, BFSP2 is reported to be involved in stabilization of lens fiber cell cytoskeleton107 and CRYBB2 is the dominant structural components of eye lens107 . FABP4 has been reported to be involved in metabolism of lipids108. GFAP is the other common protein which act as a astrocytes -specific marker protein and has been associated to AD109. It is shown to be down-regulated in 2 and 4M age groups while its expression is insignificant in 6M age group. This shows a trend towards normalization which might show overexpression in more aged animals as reported in previous findings110. Although the study is well explained but still it has few bottleneck. Validation of large number of proteins with multiple techniques will make the results more authentic. Conclusion This study is an extension of previous findings which describe the presence of AD pathological signs in retina. These abnormally expressed proteins in retina and optic nerve are involved in many vital functions and pathways, including metabolic pathways, structural cascades, retinal processes, synaptic and neuronal proteins, and eye lens associated phenomenon. This abnormal expression behavior of proteins would disturb the above mentioned functions and pathways 23



Page 24 of 36

which in return will affect the normal function of the eye. Further studies on these reported differentially expressed proteins will not only help to understand the molecular mechanism of the disease but may also help to establish biomarkers for early detection of the disease through the eyes. Materials and Methods Animal Modeling For animal modeling and sample preparation, the method described in our previous study24, 111 was followed. Briefly, both of the control wild type mice (non-transgenic (non-Tg) and experimental mice (3×Tg-AD) of the 3 age groups were all acquired from animal house of the Shenzhen University, China. These were maintained under a 12 h light/12 h dark cycle with continuous access to food and water. All protocols were implemented in accordance with NIH guidelines (NIH publication No. 85-23, revised 1985) for animal care and welfare. The mice were divided into three groups (6 mice in each group) i.e., 2M old 3×Tg-ADmice, 4M old 3×TgAD mice and 6M old 3×Tg-AD along with their age matched control. The mice were killed and eyes were rapidly excised on an ice-cold plate with the optic nerve intact. Retinal tissues were carefully separated along with the optic nerve from each group of mice for proteomics analysis. The tissues were lysed in lysis buffer (7M urea, 2M thioureia, 4% (w/v) CHAPS, 40mM dithiothreitol (DTT), and 40mM Tris base), sonicated 10 times for 5s with 10s pause in an icewater bath, and centrifuged at 14,000 rpm, at 4°C for 30min. The supernatant was taken and stored at −80°C until use. The protein concentrations were determined and optimized by Bradford assay. Trypsin Digestion and iTRAQ Labeling The protein digestion and iTRAQ labeling were performed as reported earlier.

28, 112.

Briefly,

100µg of processed tissue proteins were reduced with 10mM DTT (Sigma-Aldrich Co., St. Louis, USA) for an hour at 70°C. After processing, the proteins were alkylated by 50mM iodoacetamide for 15 minutes in the dark at room temperature. Later the samples were desalted and buffer-exchanged several times with 100μL 0.5M triethylammonium bicarbonate (TEAB, AB Sciex, Foster City, CA, USA) by Amicon® Ultra Centrifugal Filters (10kDa cut-off; 24


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Millipore, Billerica, MA, USA). The proteins were then digested with trypsin (Promega, Madison, WI, USA) at a ratio of 1 to 30 at 37°C overnight. The digested peptides were then labeled with the iTRAQ reagents (AB Sciex) as follows: the non-Tg control groups were labeled with iTRAQ 115, 116, and 117 for 2M, 4M and 6M respectively, while the 3×Tg-AD groups with iTRAQ 118, 119 and 121. As such, the labeled samples were mixed and lyophilized. The labeled peptides were then subjected to a high pH reverse-phase (RP) chromatography (Durashell, C18, 250 mm × 4.6 mm, 5 μm; Bonna-Agela Technologies Inc., Wilmington, DE, USA) and the excess label and salts were removed by the Agilent high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA). Finally, the peptides were eluted and combined into 12 batches, lyophilized and stored at -80°C.

25



Page 26 of 36

Figure 11 Schematic presentation of the whole experiment.

26


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NanoLC-MS/MS

(Mass

Spectrometry)

Analysis,

Database

Search

and

iTRAQ

Quantification The twelve (12) pooled batches were reconstituted in 30μL of 2% acetonitrile and 0.1% formic acid, among which 8μL of each fraction was submitted to a Triple TOF 5600 system fitted with a NanosprayIII source (AB Sciex). The data was obtained with a 2.4-kV ion spray voltage, 30-psi curtain gas, 5-psi nebulizer gas, and an interface heater temperature of 150°C. The scan scope for TOF-MS is 350-1500 and for MS/MS is from 400 to 1250. The automatic collision energy and automatic MS/MS accumulation was employed to activate smart information-dependent acquisition (IDA). The ProteinPilot v4.5 (AB Sciex) connected with the Paragon Algorithm against the uniprot ‘complete proteome’ mouse proteins database was used to identify and quantify the proteins. To minimize false positive identification results, a minimum unused score of 1.3 (equivalent to 95% confidence) and false discovery rate (FDR) less than 1% were necessary for all reported proteins. Hence, on the basis of 95% confidence level, at least one unique peptide per protein group was required for identifying proteins, and two quantified peptides were required for quantifying proteins113. Finally, a 1.2-fold cut-off value was implemented to determine the up and down-regulated proteins in addition to a p-value of less than 0.05 in at least two technical replicates24. The whole work scheme is shown in Figure 11. Bioinformatics Analysis Bioinformatics analysis of the differentially expressed proteins was performed by OMICSBEAN database (http://www.omicsbean.cn)24, 28, 114. Functional analysis, disease analysis and proteinprotein interaction (PPI) networks were all analyzed by this web-based tool OMICSBEAN. Western Blot Analysis Functionally important proteins were selected from each group to further validate the results by Western Blot analysis. For validation of quantitative results, Western blot analysis of functionally important proteins was performed. Samples were prepared by extracting the proteins after homogenizing the retinal tissues. Proteins were estimated by using Bradford reagent and optimized. Samples were boiled for 5 min at 100°C after adding equal volume of SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) buffer. To separate the proteins, 27



Page 28 of 36

samples were run on 10% SDS-PAGE (gel electrophoresis) for 90 min. Later the separated proteins were allowed to transfer on nitrocellulose membrane at 4°C for about 2 hrs and then these membranes were blocked in 5% skim milk with gentle shaking. It was followed by washing with TBST buffer (tris-buffered saline and Tween 20) and incubation with primary antibodies at 4°C for overnight with gentle shaking. The membranes were washed 3 times with TBST buffer each time for 10 min and incubated with secondary antibody (anti-rabbit horse radish-peroxidase conjugated for two hours. The membranes were washed again and visualized by incubating the blots with ECL reagent (Amersham, NJ) followed by exposing to the GelDocXR System (Bio-Rad, Hercules, CA, USA)115. Statistical analysis GraphPad Prism 7 software was used for statistical analyse. All data were expressed as the mean ± SEM and considered statistically significant at a level of p

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