SMOC1 colocalizes with Alzheimer’s disease neuropathology and delays Aβ aggregation

doi:10.21203/rs.3.rs-5229472/v1

Download PDF

Article

SMOC1 colocalizes with Alzheimer’s disease neuropathology and delays Aβ aggregation

https://doi.org/10.21203/rs.3.rs-5229472/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

SMOC1 has emerged as one of the most significant and consistent new biomarkers of early Alzheimer’s disease (AD). Recent studies show that SMOC1 is one of the earliest changing proteins in AD, with levels in the cerebrospinal fluid increasing many years before symptom onset. Despite this clear association with disease, little is known about the role of SMOC1 in AD or its function in the brain. Therefore, the aim of this study was to examine the distribution of SMOC1 in human AD brain tissue and to determine if SMOC1 influenced amyloid beta (Aβ) aggregation. The distribution of SMOC1 in human brain tissue was assessed in 3 brain regions (temporal cortex, hippocampus, frontal cortex) using immunohistochemistry in a cohort of 73 cases encompassing advanced AD, mild cognitive impairment (MCI), preclinical AD and cognitively normal controls. The Aβ- and phosphorylated tau-interaction with SMOC1 was assessed in control, MCI and advanced AD human brain tissue using co-immunoprecipitation, and the influence of SMOC1 on Aβ aggregation kinetics was assessed using Thioflavin T assays and electron microscopy. SMOC1 strongly colocalized with a subpopulation of amyloid plaques in AD (43.8±2.4%), MCI (32.8±5.4%) and preclinical AD (28.3±6.4%). SMOC1 levels in the brain strongly correlated with plaque load, irrespective of disease stage. SMOC1 also colocalized with a subpopulation of phosphorylated tau aggregates in AD (9.6±2.6%). Co-immunoprecipitation studies showed that SMOC1 strongly interacted with Aβ in human MCI and AD brain tissue and with phosphorylated tau in human AD brain tissue. Thioflavin T aggregation assays showed that SMOC1 significantly delayed Aβ aggregation in a dose-dependent manner, and electron microscopy confirmed that the Aβ fibrils generated in the presence of SMOC1 had an altered morphology. Overall, our results emphasize the importance of SMOC1 in the onset and progression of AD and suggest that SMOC1 may influence pathology development in AD.

SMOC1

Alzheimer’s Disease

beta amyloid

tau

tangles

plaques

preclinical

mild cognitive impairment

immunohistochemistry

thioflavin T

electron microscopy

SPARC-related modular calcium-binding protein 1 (SMOC1) has been shown to be one of the most dysregulated proteins in Alzheimer’s disease (AD) brain tissue, cerebrospinal fluid (CSF) and plasma in 23 proteomic analyses to date [9, 10, 22, 28, 29, 32, 51, 54, 66, 67, 87, 88, 96, 98, 99, 102, 104, 113, 116, 118, 120, 121, 128]. SMOC1 is significantly increased in human AD brain tissue, starting at the earliest preclinical stages and progressively increasing throughout disease [9, 22, 54, 66–68]. SMOC1 is significantly increased in the CSF of AD patients when compared to cognitively normal controls [28, 31, 32, 50, 51, 54, 96, 109, 113, 116, 118, 128] or other neurodegenerative diseases [30, 32, 77, 98, 107, 112, 121]. SMOC1 levels are significantly increased in the CSF 29 years before symptom onset in autosomal dominant AD [64, 108], making it one of the earliest altered proteins in AD [50]. SMOC1 is also one of the most enriched proteins in amyloid plaques in multiple subtypes of AD including advanced AD, early onset AD, Down syndrome with AD and preclinical AD [36, 123]. SMOC1 enrichment is observed in both sporadic and autosomal dominant AD in the brain [109], CSF [64, 108, 109, 113], and plasma [108]. SMOC1 is also significantly enriched in cerebral amyloid angiopathy (CAA) in comparison to blood vessels without CAA, suggesting that the SMOC1 association with amyloid beta (Aβ) is not limited to plaques [76, 121]. Proteomic studies have established that SMOC1 levels in the human brain and CSF strongly correlate with Aβ levels [10, 50, 51, 96, 98, 102, 113, 116, 118, 128] and SMOC1 has been identified as a strong AD-specific biomarker candidate either in isolation [50, 118, 128] or within a protein panel [50, 51, 87, 109]. SMOC1 is also significantly increased in blood serum [44] and plasma [49, 109] in AD, suggesting it could be attractive blood biomarker for AD. When brain tissue, CSF and blood plasma are combined, SMOC1 is the highest ranked biomarker of AD [116].

Despite the consistently reported link between SMOC1 and AD, little is known about the role of SMOC1 in the brain or in AD. SMOC1 is a secretory calcium binding protein present throughout the brain, blood vessels and pancreas [25, 46, 114]. SMOC1 is predominantly expressed by oligodendrocyte precursor cells (OPCs), with expression also observed in inhibitory vasoactive intestinal peptide interneurons, and oligodendrocytes at comparatively lower levels [11, 20, 61, 85, 105, 127]. SMOC1 contains two EF-hand calcium binding sites [72, 114] which facilitate calcium-dependent binding to several proteins [19, 92], many of which are implicated in AD pathogenesis [60, 74, 89, 122]. SMOC1 plays a critical role in ocular, limb, and reproductive tract development [2, 95, 97], with genetic mutations causing Waardenburg Anophthalmia Syndrome [2, 80, 101]. SMOC1 has additionally been implicated in Chronic Kidney Disease [62], coronary artery disease [63], type II diabetes [33, 90] and various cancers [5–7, 117, 124].

Given the limited prior analysis of the distribution and role of SMOC1 in AD, the aims of this study were to perform a detailed neuropathological study of SMOC1 changes in human brain tissue throughout the spectrum of AD, to determine if SMOC1 interacted with Aβ or phosphorylated tau in human AD brain tissue, and to determine if SMOC1 influenced aggregation of Aβ.

Human Tissue Samples

Human brain tissue was acquired from Rush University (USA), the New York University Alzheimer’s Disease Center (USA), and the Sydney Brain Bank (Australia), which all provide human brain tissue from ethically approved longitudinally assessed regional brain donor programs on neurodegenerative diseases. Brain tissue was acquired under protocols with Institutional Review Board (IRB) approval at NYU Grossman School of Medicine, Rush University and the Southeastern Sydney and Illawarra Local Health District and the Universities of New South Wales and Sydney, Australia. In all cases, written informed consent for research was obtained from the patient or legal guardian, and the material used had appropriate ethical approval for use in this project. All patients’ data and samples were coded and handled according to NIH and NHMRC guidelines to protect patients’ identities.

Inferior temporal cortex tissue was obtained from Rush University, which was from cases part of the Religious Orders Study (ROS) and Memory and Aging Project (MAP) cohorts [13]. Clinical assessment and neuropathology were performed at Rush University [16, 79, 106]. Cases were stratified into control, preclinical AD, MCI, and advanced AD experimental groups using a combination of both clinical and neuropathological criteria. Cases were initially stratified by the clinical cognitive final consensus diagnosis that was generated by a neurologist with expertise in dementia by a review of all available cognitive data that was blinded to post-mortem data. The following neuropathological inclusion criteria was then used to refine case selection in each group: neuropathological ABC score of A0-1/B0-2/C0-1 for control, A2-3/B1-2/C2-3 for MCI, and A3/B3/C3 for AD. Control cases were further stratified into low-pathology controls and preclinical AD cases by staining for Aβ and confirmation of a moderate Aβ plaque load in the temporal cortex. Cases were prioritized to exclude those with high TDP-43 and Lewy body pathology. Tissue included in this study from this cohort included 8 µm formalin-fixed paraffin-embedded (FFPE) sections of the inferior temporal cortex from control (n = 12), preclinical AD (n = 10), MCI (n = 12), and AD cases (n = 12).

Hippocampal tissue used in this study was obtained from New York University Alzheimer’s Disease Center (USA). This tissue included 8 µm FFPE sections containing the hippocampus from n = 7 AD and n = 4 cognitively unimpaired age-matched controls. Superior frontal cortex tissue was obtained from the Sydney Brain Bank (Australia). This included 8 µm FFPE sections from n = 10 AD and n = 7 cognitively unimpaired age-matched control cases and fresh frozen tissue from n = 7 AD, n = 5 MCI, and n = 6 cognitively unimpaired age-matched controls. Case-specific details are summarized in Table 1.

Table 1

Case Information
Case ID	Brain Region	Age	Sex	PMI	ABC Score	Western Blot Characterisation	SMOC1 IP	SMOC1 / Aβ (4G8) IHC	SMOC1 / pAβ IHC	SMOC1 / pTau (AT8) IHC	Cell Type IHC
Control 1	SFC	93	F	21	A1 B0 C0	x		x
Control 2	SFC	79	M	8	A0 B1 C0	x	x	x
Control 3	SFC	89	F	23	A1 B1 C0	x	x	x
Control 4	SFC	84	M	36	A1 B0 C0	x
Control 5	SFC	89	M	27	A0 B2 C0	x
Control 6	SFC	97	F	25	A1 B2 C0	x	x
MCI 1	SFC	84	F	6	A3 B2 C2	x
MCI 2	SFC	88	F	26	A3 B2 C2	x	x
MCI 3	SFC	89	M	33	A3 B2 C1	x	x
MCI 4	SFC	84	F	34	A3 B2 C2	x	x
MCI 5	SFC	95	F	17	A3 B2 C2	x
AD 1	SFC	80	F	32	A3 B3 C3	x
AD 2	SFC	86	M	9	A3 B3 C3	x	x
AD 3	SFC	85	F	10	A3 B3 C3	x	x
AD 4	SFC	75	F	14	A3 B3 C3	x		x
AD 5	SFC	92	M	21	A3 B3 C3	x	x
AD 6	SFC	70	M	23	A3 B3 C3	x		x
AD 7	SFC	73	M	26	A3 B3 C3	x		x
Control 7	SFC	68	M	11	A0 B0 C0			x
Control 8	SFC	90	F	58	A1 B0 C0			x
Control 9	SFC	84	M	9	A1 B1 C1			x
Control 10	SFC	93	F	15	A2 B1 C1			x
AD 8	SFC	66	M	9	A3 B3 C3			x
AD 9	SFC	91	F	6	A3 B3 C2			x
AD 10	SFC	64	F	18	A3 B3 C3			x
AD 11	SFC	70	M	8	A3 B3 C2			x
AD 12	SFC	74	M	35	A3 B3 C3			x
AD 13	SFC	69	M	19	A3 B3 C3			x
Control 11	ITC	82	M	7	A1 B1 C0			x
Control 12	ITC	90	F	26	A0 B1 C0			x
Control 13	ITC	87	M	4	A0 B1 C0			x
Control 14	ITC	95	F	5	A1 B1 C0			x
Control 15	ITC	76	F	6	A1 B0 C0			x
Control 16	ITC	91	F	15	A0 B2 C0			x
Control 17	ITC	80	M	17	A0 B1 C0			x
Control 18	ITC	81	F	5	A1 B1 C1			x
Control 19	ITC	93	F	3	A0 B2 C0			x
Control 20	ITC	83	F	1	A1 B1 C0			x
Control 21	ITC	91	M	7	A1 B2 C0			x
Control 22	ITC	84	M	11	A1 B1 C0			x
Preclinical 1	ITC	89	F	27	A2 B2 C2			x
Preclinical 2	ITC	80	M	27	A2 B2 C2			x
Preclinical 3	ITC	85	F	5	A2 B2 C2			x			x
Preclinical 4	ITC	83	M	6	A2 B1 C1			x
Preclinical 5	ITC	85	F	6	A2 B2 C2			x
Preclinical 6	ITC	78	M	10	A1 B1 C1			x
Preclinical 7	ITC	85	F	6	A1 B2 C1			x
Preclinical 8	ITC	94	F	12	A2 B1 C1			x
Preclinical 9	ITC	86	M	18	A1 B2 C2			x
Preclinical 10	ITC	70	M	21	A2 B0 C2			x
MCI 6	ITC	84	F	6	A2 B2 C1			x
MCI 7	ITC	94	F	12	A2 B1 C2			x
MCI 8	ITC	95	F	ND	A2 B2 C2			x
MCI 9	ITC	84	M	5	A3 B2 C2			x
MCI 10	ITC	85	M	21	A2 B2 C2			x
MCI 11	ITC	89	M	5	A2 B2 C2			x
MCI 12	ITC	75	M	16	A2 B2 C2			x
MCI 13	ITC	91	F	13	A2 B2 C3			x
MCI 14	ITC	85	F	18	A2 B2 C2			x
MCI 15	ITC	88	F	6	A2 B3 C3			x
MCI 16	ITC	98	F	7	A2 B2 C2			x
MCI 17	ITC	89	F	20	A1 B2 C2			x
AD 14	ITC	83	F	2	A3 B3 C3			x
AD 15	ITC	81	F	6	A3 B3 C3			x
AD 16	ITC	84	F	17	A3 B3 C3			x			x
AD 17	ITC	90	F	8	A3 B3 C3			x
AD 18	ITC	88	F	26	A3 B3 C3			x
AD 19	ITC	93	M	6	A3 B3 C3			x
AD 20	ITC	87	M	9	A3 B3 C3			x
AD 21	ITC	95	F	8	A3 B3 C3			x			x
AD 22	ITC	96	F	14	A3 B3 C3			x
AD 23	ITC	87	M	4	A3 B3 C3			x			x
AD 24	ITC	96	M	5	A3 B3 C3			x			x
AD 25	ITC	72	F	3	A3 B3 C3			x
Control 23	H	77	M	ND	A0 B0 C0			x		x
Control 24	H	59	M	ND	A1 B0 C0			x		x
Control 25	H	71	F	ND	ND			x		x
Control 26	H	81	F	ND	ND			x		x
AD 26	H	89	F	ND	A3 B3 C3			x	x	x
AD 27	H	79	F	ND	A3 B3 C3			x	x	x
AD 28	H	73	M	ND	A3 B3 C3			x	x	x
AD 29	H	72	F	ND	A3 B3 C3			x	x	x
AD 30	H	85	F	ND	A3 B3 C3			x		x
AD 31	H	92	F	ND	A3 B3 C3			x	x	x
AD 32	H	84	F	ND	A3 B3 C3			x		x

IP; immunoprecipitation, IHC; immunohistochemistry, PMI; postmortem interval, SFC; superior frontal cortex, ITC; inferior temporal cortex, H; hippocampus, ND; not determined.

Immunohistochemistry for Image Analysis

FFPE tissue sections underwent fluorescent immunohistochemistry using the method described in [37]. Briefly, sections were deparaffinized and rehydrated through a series of xylene and ethanol washes. Antigen retrieval was achieved using 99% formic acid for 7 min followed by boiling in citrate buffer for 21 min (0.05 mM sodium citrate, 0.05% Tween-20, pH 6). Sections were blocked in 10% normal goat serum and incubated with anti-SMOC1 (abcam, ab200219, 1:100) combined with either anti-Aβ (BioLegend, 4G8, 800701, 1:1000), anti-pyroglutamated Aβ (BioLegend, 822301, 1:250) or anti-pTau (ThermoFisher, AT8, MN1020, 1:500) in 4% normal goat serum overnight at 4^oC. AlexaFluor488-, AlexaFluor647-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, 1:500) and Hoechst 33342 (Sigma, B2261, 1:1000) were applied for 2 h at room temperature prior to coverslipping with Antifade ProLong Glass (Invitrogen, P36984). Whole slide images were acquired using an Olympus VS200 Slide Scanner at 10x (SMOC1/4G8) or 20x (SMOC1/AT8) magnification. For SMOC1/pyroglutamated Aβ, whole slides were captured across multiple images on a Leica Thunder Fluorescence Microscope at 10x magnification with 10% stitching. Representative 60x images were captured on a Nikon C2 Confocal microscope. Empty channel 568 was captured to allow for autofluorescence subtraction.

Immunohistochemistry for Cell Type Investigation

FFPE tissue sections underwent deparaffinization and rehydration as above, prior to boiling in citrate buffer for 21 min (10 mM sodium citrate, 0.05% Tween-20, pH 6). Sections were blocked in 10% normal horse serum and incubated with anti-SMOC1 (abcam, ab200219, 1:100) and anti-Aβ (BioLegend, 4G8, 800701, 1:1000), combined with either anti-PDGFRa (R&D Systems, AF-307, 1:250), anti-Olig2 (R&D Systems, AF2418, 1:500), anti-GFAP (Novus Biologicals, NOVNBP1-05198, 1:1500), anti-Iba1 (abcam, ab5076, 1:500) or anti-NeuN (Merck, ABN91, 1:500) in 4% normal horse serum overnight at 4°C. CF488- (Sigma, SAB4600036, 1:1000), AlexaFluor594- (Jackson, 703-585-155, 1:1500), AlexaFluor647- (Thermo, A32849, 1:1000), AlexaFluor750- (abcam, ab175739, 1:500) conjugated secondary antibodies and Hoechst 33342 (Sigma, B2261, 1:1000) were applied for 2 h at room temperature prior to coverslipping with Antifade ProLong Glass (Invitrogen, P36984). Whole slide images were acquired using an Olympus VS200 Slide Scanner at 40x magnification. Representative 60x images were captured on a Nikon C2 Confocal microscope.

SMOC1/4G8 Immunohistochemistry Analysis

SMOC1 load in amyloid plaques was assessed in temporal cortex, frontal cortex and hippocampal sections. Gray matter regions of each frontal cortex and temporal cortex section were manually annotated in QuPath (v0.4.4). For hippocampal sections, the hippocampus was defined as the combined area containing CA1-4 and subiculum. A pixel classifier was trained in QuPath to recognize amyloid plaques and was used to generate a mask of all plaques in grey matter or hippocampus for each section. Images were exported as .tif images with corresponding plaque masks. Plaque masks were applied to images in ImageJ2 (v2.14.0), and empty channel 568 subtracted from 488 to minimize autofluorescent signal. For each cohort, a SMOC1 threshold was determined based on the negative control, and thresholded SMOC1 signal measured within masked regions.

SMOC1/AT8 Immunohistochemistry Analysis

SMOC1 load in AT8-immunoreactive pTau lesions was assessed in hippocampal sections. Region annotation, image export and background subtraction was performed in QuPath as above for the hippocampus (CA1-4 and subiculum). AT8-positive signal was masked in ImageJ2, and SMOC1 signal within masked regions was thresholded and measured.

SMOC1/pyroglutamated Aβ Immunohistochemistry Analysis

SMOC1 load in pyroglutamated Aβ (pAβ) lesions was assessed in hippocampal sections. Hippocampus regions (CA1-4 and subiculum) were manually annotated in ImageJ2. pAβ-positive signal was masked in ImageJ2, and SMOC1 signal within masked regions thresholded and measured.

Tissue Homogenization

Tissue homogenization was performed as per [38]. Briefly, fresh frozen frontal cortex tissue was pulverized and dounce homogenized in 20% w/v ice-cold homogenization buffer (50 mM HEPES pH 7.0, 250 mM sucrose, 1 mM EDTA, protease and phosphatase inhibitor cocktails). Total brain homogenates were aliquoted and stored at -80°C.

Co-Immunoprecipitation

2 µg of SMOC1 antibody (abcam, ab200219) or rabbit IgG control antibody (Invitrogen, 02-6102) was added to 300 µg of total brain homogenate and brought up to 350 µL with homogenization buffer. Samples were incubated for 24 h at 4°C with rotation (25 rpm) to allow antibody binding. Samples were then incubated with Dynabeads (1.5 mg/sample) and incubated on a rotator for 24 h at 4°C. Elution was conducted by adding 20 µL of 1x LDS Sample Buffer to beads and incubating for 15 min (70°C, 1000 rpm). The co-IP product was transferred to a clean tube and stored at -20°C until use.

Western Blot Analysis

Relative protein content of co-IP products and brain homogenates were visualized by Western Blot. Co-IP products were blotted using equal volumes per well. Brain homogenate total protein concentration was determined using Pierce BCA and normalized with deionized water. Samples were denatured by boiling at 95°C for 5 min with DTT and LDS Sample Buffer. Proteins were resolved on 4–12% or 12% NuPage Bis-Tris gels and transferred to PVDF membranes. Membranes were blocked in 5% skim milk (Aβ, Rb pTau, PHF-1) or 5% skim milk with 5% NGS (SMOC1) in TBST and then incubated with anti-Aβ (CST 8243, 1:1000 dilution), anti-SMOC1 (abcam 200219, 1:1000) or anti-PHF-1 (Peter Davies [48], 1:1000) overnight at 4°C. Secondary HRP-linked IgG (Cytiva NA931V, NA934V, 1:25,000) was applied for 2 h and ECL signal recorded on an iBright CL1500 (Invitrogen).

Recombinant Aβ₄₂ and SMOC1 Protein Preparation

Aβ₄₂ was prepared as per [110]. Briefly, recombinant lyophilized Aβ₄₂ (rPeptide, A-1163-1) was resuspended in cold 50 mM NaOH to a concentration of 1 mg/ml, sonicated for 5 minutes in an ice bath, and passed through a 0.22 µm filter (10,000 x g, 1min) to remove larger aggregates. Filtrate concentration was determined via Nanodrop (A280, ε = 1490cm^− 1M^− 1) prior to storage at -20°C for no longer than one month. To remove any large aggregates formed during thawing, thawed Aβ₄₂ aliquots underwent an additional round of sonication and filtration, and concentration was re-measured as above.

Lyophilized recombinant His-tagged SMOC1 (abcam, ab276453) was resuspended in ice-cold PBS and centrifuged at 16,000 x g for 10 minutes at 4°C to remove large aggregates. The supernatant was collected and dialyzed overnight against fresh PBS to eliminate contaminants. Protein concentration was determined via Nanodrop (A280, ε = 39765cm^− 1M^− 1) prior to storage at -20°C.

Aβ₄₂ Thioflavin-T Assays

Thioflavin-T (ThT) assays were set up by initially adding neutralization buffer (100 mM phosphate buffer, pH 6.5) to each well of a 96-well half-area non-binding microplate (Corning 3881). SMOC1 or BSA at 0.125–1.25 µM was added prior to addition of Aβ₄₂ at a final concentration of 2.5 µM. Control samples with SMOC1 or BSA alone at 1.25 µM were included to assess baseline ThT fluorescence. Plates were immediately transferred to a POLARstar Omega microplate reader (BMG Labtech) at 37°C. ThT fluorescence was measured at Ex/Em 440/480 nm for 50 h under quiescent conditions. Lag times were calculated as per [26, 91]. Post-assay, samples were processed for SDS-PAGE and TEM analysis.

Preparation of Mature Aβ₄₂ Fibrils

Aβ₄₂ in 50 mM NaOH was neutralized (100 mM phosphate buffer, pH 6.5) to a final concentration of 20 uM and incubated for 32 h at 37°C. Following incubation, fibrils were diluted to a final concentration of 10 µM in the presence or absence of SMOC1 or BSA at a molar ratio of 10:1. SMOC1 and BSA alone at 1 µM were included as additional controls. All samples were incubated for 2 h at RT prior to SDS-PAGE and TEM analysis.

SDS-PAGE

A portion of each ThT assay sample was reserved (‘Total’) with the remaining solution centrifuged at 4°C (80,000 x g, 20 min). The supernatant was collected (‘Soluble fraction’) and the pellet resuspended in 8 M urea (‘Pellet fraction’). Samples were denatured by boiling at 95°C for 5 min with DTT and Novex Tricine SDS Sample Buffer (ThermoFisher, LC1676). Proteins were resolved on NuPAGE 10–20% Tricine Protein Gels, stained with Sypro-Ruby protein gel stain and imaged using an iBright CL1500 (Invitrogen).

Transmission Electron Microscopy (TEM)

5 µL of each sample was applied to a glow-discharged, standard Formvar on carbon film copper TEM grids (ProScitech, GSFC100CU) for 2 min. Excess liquid was removed with filter paper, and grids washed three times with water. Each grid was incubated with 10 µl of 10 nm Ni-NTA-Nanogold (Nanoprobes, 2084-3ML, 1:50) followed by 3x buffer washes (20 mM Tris, pH 7.6, 150 mM NaCl, 8 mM imidazole) as per the manufacturer’s instructions. Grids were stained with 2% uranyl acetate solution for 2 min and air-dried overnight. Replicate samples were imaged by TEM and showed consistent morphology. For analysis of fibril length, n ≥ 20 TEM images per sample were collected using an FEI Tecnai T12 at 120 kV and fibril lengths calculated using ImageJ.

Statistical Analysis

All results were analyzed in GraphPad Prism (v10.0.3). Each dataset was assessed for Gaussian distribution using a Shapiro-Wilk normality test. For immunohistochemistry analysis, normally distributed datasets were analyzed using Brown-Forsyth and Welch ANOVA tests with Dunnett’s T3 post-hoc, or Pearson correlation where appropriate. Non-normal datasets were analyzed using Kruskal-Wallis test with Dunn’s post-hoc, nonparametric Spearman correlation or Mann-Whitney two-tailed U test where applicable. Statistics were not performed on pAβ/4G8 staining comparisons due to differences in image analysis methods. Thioflavin T assay results were fitted with Boltzmann sigmoidal curves and analyzed using an ordinary two-way ANOVA with Tukey’s post-hoc. Aβ₄₂ fibril lengths were analyzed by a Kruskal-Wallis test with Dunn’s post-hoc.

SMOC1 colocalizes with a subset of Aβ plaques in early and advanced AD

Our first aim was to determine how early SMOC1 accumulates in the brain in AD. To determine this, we performed immunohistochemistry on temporal cortex FFPE sections from n = 12 AD, n = 12 MCI, n = 10 preclinical AD and n = 12 age- and sex-matched control cases to determine when SMOC1 accumulates in AD, and if SMOC1 colocalizes with amyloid plaques in early stages of AD.

To first determine the extent of pathology within our disease cohort, amyloid plaque load was assessed in temporal cortex grey matter. As expected, plaque load was significantly higher at all disease stages compared to control (p < 0.02). Preclinical AD and MCI showed similar levels of amyloid pathology (cortical loads of 2.0 ± 0.2% and 2.3 ± 0.3% respectively, p > 0.999). Amyloid plaque load was significantly increased in AD cases (5.1 ± 0.5%) compared to control (p < 0.001) and preclinical AD (p = 0.038) but not MCI (p = 0.061), with increased variation between cases being observed as disease progressed (Fig. 1a).

A trained classifier was used to identify all amyloid plaques in each section and SMOC1 immunoreactivity within amyloid plaques was measured. We found no significant differences in SMOC1-positive plaques loads between AD stages (p > 0.263): 28.3 ± 6.4% of plaques in preclinical AD, 32.8 ± 5.4% of plaques in MCI, and 43.8 ± 2.4% of plaques in AD (Fig. 1b, c). Interestingly, SMOC1 also colocalized with a similar subpopulation (27.2 ± 9.0%) of the small number of amyloid plaques present in control cases (amyloid load: 0.003 ± 0.001%). The inter-patient variation in SMOC1 colocalization in amyloid plaques decreased with advancing disease stage, suggesting that SMOC1 colocalization in plaques may plateau as disease progresses.

Given the variation observed in amyloid plaque load in our cohort, particularly in AD, we next evaluated SMOC1 colocalization in amyloid plaques independent of disease stage. This analysis showed that total SMOC1 immunoreactivity in amyloid plaques strongly correlated with plaque load, irrespective of disease stage (Spearman’s ⍴ = 0.890, p < 0.0001) (Fig. 1d). The intensity of SMOC1 staining within amyloid plaques showed an inverse relationship to plaque load (Spearman’s ⍴ = -0.570, p = 0.0004) (Fig. 1e), indicating that SMOC1 expression in plaques is most concentrated when initial plaque pathology appears.

Having shown that SMOC1 colocalized with plaques in all stages of AD within the temporal cortex, we next aimed to determine if SMOC1 similarly colocalized with amyloid plaques in other brain regions, as this has not been previously explored. To do this, we performed an immunohistochemistry study using hippocampal (n = 7 AD, n = 4 control) and frontal cortex sections (n = 9 AD, n = 7 control) from a separate cohort of cases. Despite significantly different total amyloid plaque loads in the temporal cortex and hippocampus (p < 0.0001) (Fig. 2a), a similar degree of SMOC1 colocalization with amyloid plaques was observed (43.8 ± 2.4% and 46.9 ± 6.8% respectively, p = 0.962) (Fig. 2b). In contrast, SMOC1 colocalized with a significantly smaller proportion of amyloid plaques in the frontal cortex (10.4 ± 1.7%) compared to the temporal cortex (p < 0.0001) or hippocampus (p = 0.0036). Similarly to the temporal cortex, SMOC1 colocalization and average intensity in amyloid plaques strongly correlated with plaque load in the hippocampus (Spearman’s ⍴ = 0.964, p < 0.0001; Pearson’s r = -0.949, p = 0.0039) (Fig. 2c) and frontal cortex (Spearman’s ⍴ = 0.892, p < 0.0001; Pearson’s r = -0.724, p = 0.0179) (Fig. 2d).

In all brain regions assessed, SMOC1 immunoreactive plaques showed a range of morphologies, including diffuse, cotton-wool, lake-like and dense-core, suggesting that these different morphologies did not define the subpopulation of plaques immunoreactive for SMOC1 (Fig. 2e-f). Plaques of the same morphology displayed a range of SMOC1 immunoreactivity, with SMOC1 colocalization variably evident in none, part, or all of plaques with the same morphology. Cored plaques positive for SMOC1 consistently contained SMOC1 in the core, but did not always show SMOC1 positivity in the corona of the plaque. The pattern of SMOC1 immunoreactivity within plaques was also diverse within individuals, with some plaques exhibiting highly specific colocalization between SMOC1 and Aβ (Fig. 2e), while other plaques showed SMOC1 immunoreactivity in the gaps between amyloid fibrils (Fig. 2f). Plaques exhibiting SMOC1 in fibril gaps were less prevalent in frontal cortex, possibly contributing to the lower proportion of SMOC1 colocalization in amyloid plaques observed in this region.

SMOC1 colocalization with Aβ plaques is not driven by pyroglutamated Aβ

We were next interested to explore why only a subset of amyloid plaques showed SMOC1 immunoreactivity. As mentioned above, SMOC1 colocalization in amyloid plaques was not unique to plaques with a particular morphology. SMOC1 immunoreactivity in plaques did not correlate to plaque size (r = 0.2460, p = 0.1748) (Supplementary Fig. 1), indicating that SMOC1 specificity was not determined by the size of amyloid plaque. Based on our previous preliminary observations [36], we next hypothesized that SMOC1 specifically colocalized with plaques containing the pyroglutamated form of Aβ at Glu3. To determine if this was the case, double immunofluorescence for SMOC1 and pAβ was conducted on n = 5 AD hippocampal sections. Surprisingly, while some pAβ plaques were SMOC1 positive, many pAβ + plaques were devoid of SMOC1 staining (Fig. 3a). As expected, plaque load for pAβ was lower than 4G8 (Fig. 3b), however SMOC1 colocalized with a considerably lower proportion of pAβ immunoreactive plaques (16.6 ± 5.7%) in comparison to 4G8 immunoreactive plaques (46.9 ± 6.8%)) (Fig. 3c). It is therefore unlikely that pAβ is the determining factor for SMOC1 plaque colocalization.

SMOC1 immunoreactivity is observed in blood vessels, spongiform and small cells

As detailed above, the most predominant SMOC1 immunoreactivity in all sections was observed in amyloid plaques. However, a diverse array of additional staining patterns was also observed with lower SMOC1 immunoreactivity. For example, a low level of diffuse SMOC1 staining was evident in white matter in all cases. SMOC1 staining was also observed in most blood vessels, localized to vessel walls throughout the parenchyma and leptomeninges (Fig. 4a). SMOC1 immunoreactivity in blood vessels was notably increased when CAA was present (Fig. 4b), consistent with our recent findings [76]. SMOC1 clearly stained spongiform that was variably present in our cohort (Fig. 4c). Interestingly, some localized accumulations of SMOC1 were also observed in the hippocampus that had a similar morphology to amyloid plaques, despite the lack of Aβ present (Fig. 4d). These were observable in all hippocampal AD sections in low numbers and were predominantly localized to CA1-subiculum. Small cells showing SMOC1 positivity in the soma were also observable within both the grey and white matter (Fig. 4e). These were evident at all stages of AD but were most apparent in cases with low plaque load and were infrequently observed in control cases. Where amyloid pathology was present, cells were often observed adjacent to amyloid plaques (Fig. 4f). Occasionally cellular processes could be observed extending into plaques, with cell bodies containing small Aβ puncta (Fig. 4g).

SMOC1 is predominantly expressed by Olig2-negative Oligodendrocyte Precursor Cells

Given the proximity of SMOC1-positive cells to amyloid plaques, we sought to identify which cell type was expressing SMOC1 in AD. Temporal cortex sections containing consistent SMOC1 cellular staining and abundant amyloid plaques were co-stained for SMOC1, Aβ, and markers for OPCs (PDGFRα), oligodendrocytes (Olig2), astrocytes (GFAP), microglia (Iba1) and neurons (NeuN). Co-localization of SMOC1 and PDGFRα was frequently observed (Fig. 5a) indicating that SMOC1 expression is highest in OPCs, as expected from RNAseq datasets [23, 40, 47, 61, 83–85, 105]. Interestingly, SMOC1-positive cells were negative for Olig2 (Fig. 5b), indicating that SMOC1 expression in AD may be specific to a subpopulation of OPCs negative for this marker [41, 42]. No colocalization was observed for astrocytes (Fig. 5c) or neurons (Fig. 5d). Iba1 + microglia occasionally showed SMOC1 immunoreactivity (Fig. 5e), however this was observed infrequently and to a lesser extent than OPCs.

SMOC1 colocalizes with tau

SMOC1 also appeared to colocalize with a subpopulation of neurofibrillary tangles in all regions (Fig. 6a). This was unexpected given that SMOC1 has not been indicated as a tau interactor in previous proteomic studies [38, 55]. To confirm these observations and determine the prevalence of SMOC1 colocalization with phosphorylated tau, n = 4 control and n = 7 AD hippocampal FFPE sections were co-stained for SMOC1 and AT8 (pTau^S202,T205). The hippocampus was chosen due to the abundance of tau pathology in advanced AD [17]. pTau load in AD was confirmed to be significantly higher than controls (0.8 ± 0.2%, p = 0.0061) (Fig. 6b). SMOC1 colocalized with a subpopulation of pTau (9.6 ± 2.6%) in hippocampal AD tissue. Interestingly, although minimal pTau was observed in control cases (0.01%±0.006% load), SMOC1 colocalized with a higher proportion of pTau in controls than in AD (23.7%±6.1% compared to 9.6%±2.6%) (Fig. 6c). The extent of SMOC1 immunoreactivity in pTau aggregates correlated strongly with total pTau load (r = 0.955, p = < 0.0001) (Fig. 6d). SMOC1 was observed to colocalize with all types of pTau morphologies present in AD tissue including neurofibrillary tangles, dystrophic neurites and neuritic plaques (Fig. 6e-g), with the majority of colocalization found within NFTs (77.3 ± 3.8%).

SMOC1 interacts with Aβ and pTau in human AD brain

Given the strong colocalization of SMOC1 immunoreactivity observed with both Aβ and pTau pathology, we hypothesized that this colocalization resulted from a direct interaction between SMOC1 and Aβ or pTau in AD. To test this hypothesis, we used co-immunoprecipitation to determine if SMOC1 interacted with Aβ and PHF-1 immunoreactive pTau in human frontal cortex homogenate. Characterization of total SMOC1, Aβ and PHF-1 was performed by immunoblotting n = 6 control, n = 5 MCI, and n = 7 AD cases (Fig. 7a). n = 3 control, MCI and AD cases were selected for immunoprecipitation to encompass the wide range of pathology load in our cohort. Immunoprecipitation of SMOC1 showed a strong interaction with both Aβ and PHF-1-immunoreactive pTau, that was not observed after pulldown with an IgG control antibody (Fig. 7b). Enrichment of both Aβ and PHF-1 immunoreactive pTau reflected total load in each case. Interestingly, SMOC1 appeared to interact with both monomers and dimers of Aβ, even in MCI cases when total Aβ load was comparatively low.

SMOC1 inhibits Aβ₄₂ fibril formation and alters fibril morphology

We next aimed to determine if SMOC1 influenced Aβ₄₂ fibril formation in vitro. Thioflavin T assays were performed in triplicate at 1:0.05, 1:0.1 and 1:0.5 molar ratios of monomeric Aβ₄₂ to SMOC1 or BSA control. SMOC1 significantly delayed Aβ₄₂ fibril formation at all concentrations tested in a dose-response manner. The lag phase to fibril assembly for Aβ₄₂ alone under these conditions was 2.8 ± 1.2 h. Addition of SMOC1 resulted in a lag phase of 12.9 ± 5.0 hours for an Aβ₄₂:SMOC1 ratio of 1:0.05 (p = 0.019), 20.4 ± 9.0 hours for a ratio of 1:0.1 (p = 0.0007), and 29.6 ± 5.1 hours at a ratio of 1:0.5 (p < 0.0001) (Fig. 8a, b). We did not observe a similar lag effect after addition of the BSA control compared to Aβ₄₂ alone (2.8 ± 1.2 h) at any concentration.

We were next interested to determine if SMOC1 altered Aβ₄₂ fibril morphology. To assess this, ThT assay samples were collected at assay conclusion and probed with Ni-NTA-NanoGold particles to identify the presence of the His-tagged SMOC1 protein. TEM image analysis revealed that Aβ₄₂ fibrils were significantly longer when formed in the presence of SMOC1 (0.55 ± 0.03 µm) compared to BSA (0.34 ± 0.02 µm, p = 0.02) at a 1:0.5 molar ratio (Fig. 8c). The presence of SMOC1 in a 10x lower molar ratio resulted in a slight lengthening of fibrils (0.39 ± 0.03 µm), indicating a dose-dependent effect of SMOC1 upon Aβ₄₂ fibril length. Interestingly, fibrils formed in SMOC1 conditions exhibited a corkscrew-like morphology, which was distinct from the fibrils formed by pure Aβ₄₂ or in the presence of BSA (Fig. 8d, e). Ni-NTA-NanoGold particles were not observed bound to the fibrils, suggesting that SMOC1 is not stably bound to Aβ₄₂ fibrils at the assay endpoint. Spherical structures resembling Aβ₄₂ oligomers were also observable in the SMOC1 + Aβ₄₂ condition, suggesting that SMOC1 may delay Aβ fibril formation by interacting with on- or off-pathway oligomers. To confirm if SMOC1 was bound to Aβ₄₂ fibrils, ThT assay samples were pelleted and the total, soluble and pellet fractions analyzed via SDS-PAGE. SMOC1 was observed primarily in the soluble fraction (Fig. 8f), confirming that the majority of SMOC1 was not bound to Aβ₄₂ fibrils at assay endpoint. A similar result was observed when SMOC1 was added to mature, preformed Aβ₄₂ fibrils (Supplementary Fig. 2), further supporting that SMOC1 does not bind stably to preformed Aβ₄₂ fibrils.

We have shown that SMOC1 consistently colocalizes with a subpopulation of amyloid plaques in preclinical AD, MCI and advanced AD. SMOC1 interacts with Aβ in AD human brain tissue, and delays Aβ aggregation in vitro. Additionally, SMOC1 interacts with phosphorylated tau and colocalizes with a subpopulation of tau pathology in human AD brain tissue. Together, our findings provide new evidence about the important role of SMOC1 in AD and provide new context for why SMOC1 is consistently reported as a leading fluid biomarker for early AD in proteomic studies [44, 49–51, 87, 109, 116, 118, 128].

The reason why SMOC1 is increased in AD is still unknown. One possibility is increased expression by OPCs in response to local Aβ pathology. SMOC1 is secreted by OPCs [47], which have been reported to become senescent around amyloid plaques in AD [126] and adopt a ‘reactive’ phenotype in an AD model [115] or in response to acute injury [3, 53, 82]. This could potentially result in altered expression profiles of OPC proteins including SMOC1; however, significant alterations in other OPC proteins in addition to SMOC1 would also be expected, which is not reflected in proteomic studies of human AD brain tissue [36–38, 66]. Instead, we hypothesize that the appearance of pathological Aβ species in the brain is the stimulus for increased SMOC1 expression. This is supported by our finding that SMOC1 accumulation in brain strongly correlated to plaque load, irrespective of disease stage, which is consistent with the highly significant correlation between SMOC1 and Aβ levels reported in brain tissue, CSF and plasma in AD [10, 49–51, 64, 96, 98, 102, 113, 116, 118, 128]. Increased SMOC1 expression is only reported in other neurodegenerative diseases when Aβ is present (e.g. Lewy Body Dementia) and correlates to Aβ pathology load rather than cognitive decline in AD [64, 65, 94, 118, 119, 128], suggesting that the SMOC1 increase is linked specifically to Aβ rather than AD pathogenesis more broadly. While further study is required to determine the mechanistic role of SMOC1 in AD, preliminary results from an independent group suggest that it may be neuroprotective as SMOC1 overexpression in a transgenic mouse model of AD significantly reduced amyloid plaque count without any accompanying neurotoxicity [4].

Interestingly, the increase of SMOC1 precisely coincides with the decrease of Aβ in the CSF of pre-symptomatic autosomal dominant AD, presumably reflecting Aβ sequestration into plaques in the brain [64]. These findings emphasize the strong association between SMOC1 and Aβ protein levels through AD progression, however it is unclear why SMOC1 and Aβ protein levels change in opposing directions in the CSF. This is particularly notable given that SMOC1 increases proportionally to Aβ load in the AD brain [9, 10, 22, 54, 66–68]. Supporting this proteomic data, our co-immunoprecipitation results suggest a direct interaction between SMOC1 and Aβ in human brain tissue, and our immunostaining results clearly show that SMOC1 is sequestered in plaques in the brain. A possible explanation for the discrepancy between brain and CSF expression may be that only a fraction of the increased SMOC1 produced in AD binds to Aβ in plaques, while the remaining pool of soluble SMOC1 that does not complex with fibrillar Aβ is continuously cleared from the brain, thus accounting for the increased levels of SMOC1 in the CSF and plasma. Additional future studies are required to determine if this is the case.

We were interested to observe that SMOC1 only colocalized with a subpopulation of amyloid plaques in our study, consistent with previous observations [36, 98]. The frontal cortex showed significantly less colocalization of SMOC1 with Aβ plaques compared to the hippocampus and temporal cortex, perhaps reflective of lower levels of total OPCs within this region [84]. In all regions, the SMOC1-immunopositive plaque subpopulation was not defined by a particular morphology such as diffuse, cored, or neuritic plaques, and was not defined by layer-specific cortical location. In our previous study we had hypothesized that this SMOC1-immunopositive plaque subpopulation could be defined by the presence of pyroglutamated Aβ [35], however, our study here using double immunofluorescence showed this was not the defining characteristic of this plaque subpopulation. Looking forward, it would be interesting to determine if this plaque population was defined by presence of other types of Aβ (e.g. Aβ40, Aβ42, Aβ38, phosphorylated Aβ etc) or alternative factors such as localized association with OPCs.

The SMOC1 colocalization with tau pathology was surprising, as SMOC1 has not previously been detected in tau interactome analyses [38, 69, 111], and has not been reported in immunostaining studies examining SMOC1 in human AD brain tissue [10, 36, 98]. The interaction between SMOC1 and tau in AD has likely been overlooked in previous studies due to the comparatively lower abundance of SMOC1 in tau pathology in comparison to plaques. Indeed, our results show only a low level (~ 10%) of SMOC1 colocalization with disease-associated phosphorylated tau observable in neurofibrillary tangles, dystrophic neurites and neuritic plaques. Despite this relatively low colocalization, our co-immunoprecipitation results show a strong interaction with phosphorylated tau in human AD brain tissue. As we only examined the interaction between SMOC1 and PHF-1 immunoreactive tau in co-IP studies and AT8-immunoreactive tau in immunofluorescence studies, it is unclear if the observed interaction is specific to these types of tau, or if a broader interaction with multiple subspecies of phosphorylated tau may be occurring.

The calcium-binding actions of SMOC1 may indicate its role in AD. While the role of calcium-binding proteins in AD is not fully understood, calcium dyshomeostasis is an early feature of AD and other calcium-binding proteins have protective actions in AD [1, 12, 14, 15, 24, 34, 39, 43, 45, 70, 71, 73, 75, 78, 81].

Proteins with EF-hand calcium binding domains similar to SMOC1 such as calbindin, parvalbumin, S100B, and calretinin have all been implicated in AD [8, 18, 21, 27, 52, 56–59, 78, 86, 93, 100, 103, 125]. Parallels between these proteins and SMOC1 may be used to inform on the actions of SMOC1 in AD. Of particular interest is S100B, which also inhibits Aβ₄₂ fibril formation in vitro [27], binds Aβ in a calcium-dependent manner [103], and can rescue Aβ₄₂-induced toxicity in SHSY5Y cells [27], supporting a neuroprotective role of calcium-binding proteins. The effect of calcium on the SMOC1-Aβ₄₂ interaction was not investigated here, but would be interesting to explore in future studies examining the role of SMOC1 in AD.

To conclude, we have shown that SMOC1 interacts with both Aβ and phosphorylated tau in AD and is significantly associated with the three major neuropathological hallmarks of AD: plaques, tangles and CAA. We hypothesize that SMOC1 is significantly increased in the brain in response to Aβ aggregation in the first stages of AD, which would provide context as to why SMOC1 has been consistently identified as an early AD biomarker. The ability of SMOC1 to inhibit Aβ aggregation suggests that SMOC1 could be a new potential therapeutic target for AD. However, future studies are essential to closely explore the role of SMOC1 in AD, particularly studies that examine if SMOC1 is neuroprotective in AD.

Competing Interests

The authors declare no competing interests.

Funding

This study was supported by funding from Bluesand Foundation to E.D., University of Sydney Centre for Drug Discovery Innovation EMCR Grant to E.D., Alzheimer’s Association (Blas Frangione Early Career Award) to E.D., Faculty of Medicine and Health, University of Sydney (FMH EMCR Emerging Star Grant) to E.D., Australian Research Council Discovery Project (DP200102463) to M.S., NIH (P01AG060882 and P30AG066512) to T.W., NIH (R01AG017917 [MAP], P30AG010161 [ROS/ADRC], P30AG072975 [ROS/ADRC]) to J. S.

Author Contributions

E.D. conceived and designed this study. K.B. was responsible for analysis, immunohistochemistry, co-immunoprecipitation, and figure generation. C.J was responsible for Thioflavin T assays and electron microscopy. D.L performed initial immunohistochemistry and semi-quantification. E.D., T.K., and M.S. provided expert advice on the interpretation of data. J.S., T.W., G.H., performed neuropathological characterization and provided human tissue. E.D. and K.B. wrote the manuscript with input from co-authors. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare no competing interests.

Acknowledgements

The authors acknowledge the technical and scientific assistance of Sydney Microscopy & Microanalysis, the University of Sydney node of Microscopy Australia. We also acknowledge and the patients and families for their generous brain donations to the Sydney Brain Bank (which is supported by Neuroscience Research Australia), the New York University Alzheimer’s Disease Centre and Rush University Medical Centre.

Calcium Hypothesis of Alzheimer's disease and brain aging: A framework for integrating new evidence into a comprehensive theory of pathogenesis. Alzheimers Dement 13: 178-182.e117 Doi 10.1016/j.jalz.2016.12.006
Abouzeid H, Boisset G, Favez T, Youssef M, Marzouk I, Shakankiry N, Bayoumi N, Descombes P, Agosti C, Munier FLet al (2011) Mutations in the SPARC-Related Modular Calcium-Binding Protein 1 Gene, SMOC1, Cause Waardenburg Anophthalmia Syndrome. The American Journal of Human Genetics 88: 92-98 Doi https://doi.org/10.1016/j.ajhg.2010.12.002
Adams KL, Gallo V (2018) The diversity and disparity of the glial scar. Nature neuroscience 21: 9-15
Afroz KF, Levites Y, Ryu D, Ran Y, Gadhavi JD, Liu X, Seyfried N, Golde T (2024) SPARC-related modular calcium-binding proteins (SMOC1 and 2) as a modulator of amyloid pathology in Alzheimer’s Disease. Alzheimer's Association International Conference, City
Ao X, Jiang M, Zhou J, Liang H, Xia H, Chen G (2019) lincRNA‑p21 inhibits the progression of non‑small cell lung cancer via targeting miR‑17‑5p. Oncol Rep 41: 789-800 Doi 10.3892/or.2018.6900
Aoki H, Takasawa A, Yamamoto E, Niinuma T, Yamano H-o, Harada T, Kubo T, Yorozu A, Kitajima H, Ishiguro Ket al (2024) Downregulation of SMOC1 is associated with progression of colorectal traditional serrated adenomas. BMC Gastroenterology 24: 91 Doi 10.1186/s12876-024-03175-1
Aoki H, Yamamoto E, Takasawa A, Niinuma T, Yamano H-O, Harada T, Matsushita H-O, Yoshikawa K, Takagi R, Harada Eet al (2017) Epigenetic silencing of SMOC1 in traditional serrated adenoma and colorectal cancer. Oncotarget 9:
Arai H, Emson P, Mountjoy C, Carassco L, Heizmann C (1987) Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia. Brain research 418: 164-169
Askenazi M, Kavanagh T, Pires G, Ueberheide B, Wisniewski T, Drummond E (2023) Compilation of reported protein changes in the brain in Alzheimer's disease. Nature communications 14: 4466 Doi 10.1038/s41467-023-40208-x
Bai B, Wang X, Li Y, Chen PC, Yu K, Dey KK, Yarbro JM, Han X, Lutz BM, Rao Set al (2020) Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer's Disease Progression. Neuron 105: 975-991 e977 Doi 10.1016/j.neuron.2019.12.015
Bakken TE, Jorstad NL, Hu Q, Lake BB, Tian W, Kalmbach BE, Crow M, Hodge RD, Krienen FM, Sorensen SAet al (2021) Comparative cellular analysis of motor cortex in human, marmoset and mouse. Nature 598: 111-119 Doi 10.1038/s41586-021-03465-8
Baracaldo-Santamaría D, Avendaño-Lopez SS, Ariza-Salamanca DF, Rodriguez-Giraldo M, Calderon-Ospina CA, González-Reyes RE, Nava-Mesa MO (2023) Role of Calcium Modulation in the Pathophysiology and Treatment of Alzheimer’s Disease. International Journal of Molecular Sciences 24: 9067
Bennett DA, Buchman AS, Boyle PA, Barnes LL, Wilson RS, Schneider JA (2018) Religious orders study and rush memory and aging project. Journal of Alzheimer's disease 64: S161-S189
Bezprozvanny I, Mattson MP (2008) Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease. Trends in Neurosciences 31: 454-463 Doi https://doi.org/10.1016/j.tins.2008.06.005
Bezprozvanny IB (2010) Calcium signaling and neurodegeneration. Acta Naturae 2: 72-82
Boyle PA, Yu L, Nag S, Leurgans S, Wilson RS, Bennett DA, Schneider JA (2015) Cerebral amyloid angiopathy and cognitive outcomes in community-based older persons. Neurology 85: 1930-1936
Braak H, Braak E (1995) Staging of alzheimer's disease-related neurofibrillary changes. Neurobiology of Aging 16: 271-278 Doi https://doi.org/10.1016/0197-4580(95)00021-6
Brady DR, Mufson EJ (1997) Parvalbumin-immunoreactive neurons in the hippocampal formation of Alzheimer's diseased brain. Neuroscience 80: 1113-1125 Doi https://doi.org/10.1016/S0306-4522(97)00068-7
Brellier F, Ruggiero S, Zwolanek D, Martina E, Hess D, Brown-Luedi M, Hartmann U, Koch M, Merlo A, Lino Met al (2011) SMOC1 is a tenascin-C interacting protein over-expressed in brain tumors. Matrix Biol 30: 225-233 Doi 10.1016/j.matbio.2011.02.001
Bryois J, Calini D, Macnair W, Foo L, Urich E, Ortmann W, Iglesias VA, Selvaraj S, Nutma E, Marzin Met al (2022) Cell-type-specific cis-eQTLs in eight human brain cell types identify novel risk genes for psychiatric and neurological disorders. Nature Neuroscience 25: 1104-1112 Doi 10.1038/s41593-022-01128-z
Bu J, Sathyendra V, Nagykery N, Geula C (2003) Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex. Experimental Neurology 182: 220-231 Doi https://doi.org/10.1016/S0014-4886(03)00094-3
Carlyle BC, Kandigian SE, Kreuzer J, Das S, Trombetta BA, Kuo Y, Bennett DA, Schneider JA, Petyuk VA, Kitchen RRet al (2021) Synaptic proteins associated with cognitive performance and neuropathology in older humans revealed by multiplexed fractionated proteomics. Neurobiol Aging 105: 99-114 Doi 10.1016/j.neurobiolaging.2021.04.012
Chamling X, Kallman A, Fang W, Berlinicke CA, Mertz JL, Devkota P, Pantoja IEM, Smith MD, Ji Z, Chang Cet al (2021) Single-cell transcriptomic reveals molecular diversity and developmental heterogeneity of human stem cell-derived oligodendrocyte lineage cells. Nature Communications 12: 652 Doi 10.1038/s41467-021-20892-3
Chandran R, Kumar M, Kesavan L, Jacob RS, Gunasekaran S, Lakshmi S, Sadasivan C, Omkumar RV (2019) Cellular calcium signaling in the aging brain. Journal of Chemical Neuroanatomy 95: 95-114 Doi https://doi.org/10.1016/j.jchemneu.2017.11.008
Choi Y-A, Lim J, Kim KM, Acharya B, Cho J-Y, Bae Y-C, Shin H-I, Kim S-Y, Park EK (2010) Secretome Analysis of Human BMSCs and Identification of SMOC1 as an Important ECM Protein in Osteoblast Differentiation. Journal of Proteome Research 9: 2946-2956 Doi 10.1021/pr901110q
Cox D, Selig E, Griffin MD, Carver JA, Ecroyd H (2016) Small Heat-shock Proteins Prevent alpha-Synuclein Aggregation via Transient Interactions and Their Efficacy Is Affected by the Rate of Aggregation. J Biol Chem 291: 22618-22629 Doi 10.1074/jbc.M116.739250
Cristóvão JS, Morris VK, Cardoso I, Leal SS, Martínez J, Botelho HM, Göbl C, David R, Kierdorf K, Alemi Met al (2018) The neuronal S100B protein is a calcium-tuned suppressor of amyloid-β aggregation. Science Advances 4: eaaq1702 Doi doi:10.1126/sciadv.aaq1702
Cruchaga C, Ali M, Shen Y, Do A, Wang L, Western D, Liu M, Beric A, Budde J, Gentsch Jet al (2024) Multi-cohort cerebrospinal fluid proteomics identifies robust molecular signatures for asymptomatic and symptomatic Alzheimer's disease. Res Sq: Doi 10.21203/rs.3.rs-3631708/v1
Dai J, Johnson ECB, Dammer EB, Duong DM, Gearing M, Lah JJ, Levey AI, Wingo TS, Seyfried NT (2018) Effects of APOE Genotype on Brain Proteomic Network and Cell Type Changes in Alzheimer's Disease. Front Mol Neurosci 11: 454 Doi 10.3389/fnmol.2018.00454
Dammer EB, Ping L, Duong DM, Modeste ES, Seyfried NT, Lah JJ, Levey AI, Johnson ECB (2022) Multi-Platform Proteomic Analysis of Alzheimer’s Disease Cerebrospinal Fluid and Plasma Reveals Network Biomarkers Associated with Proteostasis and the Matrisome. bioRxiv: 2022.2006.2020.494087 Doi 10.1101/2022.06.20.494087
Dayon L, Nunez Galindo A, Wojcik J, Cominetti O, Corthesy J, Oikonomidi A, Henry H, Kussmann M, Migliavacca E, Severin Iet al (2018) Alzheimer disease pathology and the cerebrospinal fluid proteome. Alzheimers Res Ther 10: 66 Doi 10.1186/s13195-018-0397-4
de Geus MB, Leslie SN, Lam T, Wang W, Roux-Dalvai F, Droit A, Kivisakk P, Nairn AC, Arnold SE, Carlyle BC (2023) Mass spectrometry in cerebrospinal fluid uncovers association of glycolysis biomarkers with Alzheimer’s disease in a large clinical sample. Scientific Reports 13: 22406 Doi 10.1038/s41598-023-49440-3
Delgado Lagos F, Elgheznawy A, Kyselova A, Meyer Zu Heringdorf D, Ratiu C, Randriamboavonjy V, Mann AW, Fisslthaler B, Siragusa M, Fleming I (2021) Secreted modular calcium-binding protein 1 binds and activates thrombin to account for platelet hyperreactivity in diabetes. Blood 137: 1641-1651 Doi 10.1182/blood.2020009405
Demuro A, Mina E, Kayed R, Milton SC, Parker I, Glabe CG (2005) Calcium Dysregulation and Membrane Disruption as a Ubiquitous Neurotoxic Mechanism of Soluble Amyloid Oligomers. Journal of Biological Chemistry 280: 17294-17300
Drummond E, Kavanagh T, Pires G, Marta-Ariza M, Kanshin E, Nayak S, Faustin A, Berdah V, Ueberheide B, Wisniewski T (2022) The amyloid plaque proteome in early onset Alzheimer's disease and Down syndrome. Acta neuropathologica communications 10: 53 Doi 10.1186/s40478-022-01356-1
Drummond E, Kavanagh T, Pires G, Marta-Ariza M, Kanshin E, Nayak S, Faustin A, Berdah V, Ueberheide B, Wisniewski T (2022) The amyloid plaque proteome in early onset Alzheimer’s disease and Down syndrome. Acta Neuropathologica Communications 10: 53 Doi 10.1186/s40478-022-01356-1
Drummond E, Nayak S, Faustin A, Pires G, Hickman RA, Askenazi M, Cohen M, Haldiman T, Kim C, Han Xet al (2017) Proteomic differences in amyloid plaques in rapidly progressive and sporadic Alzheimer's disease. Acta Neuropathol 133: 933-954 Doi 10.1007/s00401-017-1691-0
Drummond E, Pires G, MacMurray C, Askenazi M, Nayak S, Bourdon M, Safar J, Ueberheide B, Wisniewski T (2020) Phosphorylated tau interactome in the human Alzheimer's disease brain. Brain 143: 2803-2817 Doi 10.1093/brain/awaa223
Fairless R, Williams SK, Diem R (2019) Calcium-Binding Proteins as Determinants of Central Nervous System Neuronal Vulnerability to Disease. International Journal of Molecular Sciences 20: 2146
Falcão AM, van Bruggen D, Marques S, Meijer M, Jäkel S, Agirre E, Samudyata, Floriddia EM, Vanichkina DP, ffrench-Constant Cet al (2018) Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis. Nature Medicine 24: 1837-1844 Doi 10.1038/s41591-018-0236-y
Fang LP, Bai X (2023) Implications of Olig2 silencing in oligodendrocyte precursor cells. Neural Regen Res 18: 2649-2650 Doi 10.4103/1673-5374.373666
Fang LP, Liu Q, Meyer E, Welle A, Huang W, Scheller A, Kirchhoff F, Bai X (2023) A subset of OPCs do not express Olig2 during development which can be increased in the adult by brain injuries and complex motor learning. Glia 71: 415-430 Doi 10.1002/glia.24284
Ferreira IL, Bajouco LM, Mota SI, Auberson YP, Oliveira CR, Rego AC (2012) Amyloid beta peptide 1–42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures. Cell Calcium 51: 95-106 Doi https://doi.org/10.1016/j.ceca.2011.11.008
Frick EA, Emilsson V, Jonmundsson T, Steindorsdottir AE, Johnson ECB, Puerta R, Dammer EB, Shantaraman A, Cano A, Boada Met al (2024) Serum proteomics reveal APOE-ε4-dependent and APOE-ε4-independent protein signatures in Alzheimer’s disease. Nature Aging: Doi 10.1038/s43587-024-00693-1
Ge M, Zhang J, Chen S, Huang Y, Chen W, He L, Zhang Y (2022) Role of Calcium Homeostasis in Alzheimer's Disease. Neuropsychiatr Dis Treat 18: 487-498 Doi 10.2147/ndt.S350939
Gersdorff N, Muller M, Schall A, Miosge N (2006) Secreted modular calcium-binding protein-1 localization during mouse embryogenesis. Histochem Cell Biol 126: 705-712 Doi 10.1007/s00418-006-0200-7
Godoy MI, Pandey V, Wohlschlegel JA, Zhang Y (2024) Secretome analysis of oligodendrocytes and precursors reveals their roles as contributors to the extracellular matrix and potential regulators of inflammation. bioRxiv: 2024.2007.2022.604699 Doi 10.1101/2024.07.22.604699
Greenberg SG, Davies P, Schein JD, Binder LI (1992) Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau. J Biol Chem 267: 564-569
Guo Q, Ping L, Dammer EB, Duong DM, Yin L, Xu K, Shantaraman A, Fox EJ, Johnson ECB, Roberts BRet al (2023) Global analysis of the heparin-enriched plasma proteome captures matrisome-associated proteins in Alzheimer’s disease. bioRxiv: 2023.2011.2006.565824 Doi 10.1101/2023.11.06.565824
Guo Y, Chen S-D, You J, Huang S-Y, Chen Y-L, Zhang Y, Wang L-B, He X-Y, Deng Y-T, Zhang Y-Ret al (2024) Multiplex cerebrospinal fluid proteomics identifies biomarkers for diagnosis and prediction of Alzheimer’s disease. Nature Human Behaviour: Doi 10.1038/s41562-024-01924-6
Haque R, Watson CM, Liu J, Carter EK, Duong DM, Lah JJ, Wingo AP, Roberts BR, Johnson ECB, Saykin AJet al (2023) A protein panel in cerebrospinal fluid for diagnostic and predictive assessment of Alzheimer's disease. Sci Transl Med 15: eadg4122 Doi 10.1126/scitranslmed.adg4122
Heizmann CW, Braun K (1992) Changes in Ca(2+)-binding proteins in human neurodegenerative disorders. Trends Neurosci 15: 259-264 Doi 10.1016/0166-2236(92)90067-i
Hesp ZC, Yoseph RY, Suzuki R, Jukkola P, Wilson C, Nishiyama A, McTigue DM (2018) Proliferating NG2-cell-dependent angiogenesis and scar formation alter axon growth and functional recovery after spinal cord injury in mice. Journal of Neuroscience 38: 1366-1382
Higginbotham L, Ping L, Dammer EB, Duong DM, Zhou M, Gearing M, Hurst C, Glass JD, Factor SA, Johnson ECBet al (2020) Integrated proteomics reveals brain-based cerebrospinal fluid biomarkers in asymptomatic and symptomatic Alzheimer's disease. Sci Adv 6: Doi 10.1126/sciadv.aaz9360
Hondius DC, Koopmans F, Leistner C, Pita-Illobre D, Peferoen-Baert RM, Marbus F, Paliukhovich I, Li KW, Rozemuller AJM, Hoozemans JJMet al (2021) The proteome of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer’s disease. Acta Neuropathol 141: 341-358 Doi 10.1007/s00401-020-02261-4
Iacopino AM, Christakos S (1990) Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. Proceedings of the National Academy of Sciences 87: 4078-4082
Ichimiya Y, Emson PC, Mountjoy CQ, Lawson DEM, Heizmann CW (1988) Loss of calbindin-28K immunoreactive neurones from the cortex in Alzheimer-type dementia. Brain research 475: 156-159
Inaguma Y, Shinohara H, Inagaki T, Kato K (1992) Immunoreactive parvalbumin concentrations in parahippocampal gyrus decrease in patients with Alzheimer's disease. Journal of the Neurological Sciences 110: 57-61 Doi https://doi.org/10.1016/0022-510X(92)90009-A
Iritani S, Niizato K, Emson PC (2001) Relationship of calbindin D28K-immunoreactive cells and neuropathological changes in the hippocampal formation of Alzheimer's disease. Neuropathology 21: 162-167 Doi https://doi.org/10.1046/j.1440-1789.2001.00393.x
Iwamoto N, Nishiyama E, Ohwada J, Arai H (1994) Demonstration of CRP immunoreactivity in brains of Alzheimer's disease: immunohistochemical study using formic acid pretreatment of tissue sections. Neurosci Lett 177: 23-26 Doi 10.1016/0304-3940(94)90035-3
Jäkel S, Agirre E, Mendanha Falcão A, van Bruggen D, Lee KW, Knuesel I, Malhotra D, ffrench-Constant C, Williams A, Castelo-Branco G (2019) Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature 566: 543-547 Doi 10.1038/s41586-019-0903-2
Jalal D, Sanford B, Renner B, Ten Eyck P, Laskowski J, Cooper J, Sun M, Zakharia Y, Spitz D, Dokun Aet al (2021) Detection of pro angiogenic and inflammatory biomarkers in patients with CKD. Scientific Reports 11: 8786 Doi 10.1038/s41598-021-87710-0
Ji Y, Yan T, Zhu S, Wu R, Zhu M, Zhang Y, Guo C, Yao K (2021) The Integrative Analysis of Competitive Endogenous RNA Regulatory Networks in Coronary Artery Disease. Frontiers in Cardiovascular Medicine 8: Doi 10.3389/fcvm.2021.647953
Johnson ECB, Bian S, Haque RU, Carter EK, Watson CM, Gordon BA, Ping L, Duong DM, Epstein MP, McDade Eet al (2023) Cerebrospinal fluid proteomics define the natural history of autosomal dominant Alzheimer's disease. Nat Med 29: 1979-1988 Doi 10.1038/s41591-023-02476-4
Johnson ECB, Carter EK, Dammer EB, Duong DM, Gerasimov ES, Liu Y, Liu J, Betarbet R, Ping L, Yin Let al (2021) Large-Scale Deep Multi-Layer Analysis of Alzheimer’s Disease Brain Reveals Strong Proteomic Disease-Related Changes Not Observed at the RNA Level. bioRxiv: 2021.2004.2005.438450 Doi 10.1101/2021.04.05.438450
Johnson ECB, Carter EK, Dammer EB, Duong DM, Gerasimov ES, Liu Y, Liu J, Betarbet R, Ping L, Yin Let al (2022) Large-scale deep multi-layer analysis of Alzheimer’s disease brain reveals strong proteomic disease-related changes not observed at the RNA level. Nature Neuroscience 25: 213-225 Doi 10.1038/s41593-021-00999-y
Johnson ECB, Dammer EB, Duong DM, Yin L, Thambisetty M, Troncoso JC, Lah JJ, Levey AI, Seyfried NT (2018) Deep proteomic network analysis of Alzheimer's disease brain reveals alterations in RNA binding proteins and RNA splicing associated with disease. Mol Neurodegener 13: 52 Doi 10.1186/s13024-018-0282-4
Kamalian A, Ho SG, Patel M, Lewis A, Bakker A, Albert M, O’Brien RJ, Moghekar A, Lutz MW (2023) Exploratory Assessment of Proteomic Network Changes in Cerebrospinal Fluid of Mild Cognitive Impairment Patients: A Pilot Study. Biomolecules 13: 1094
Kavanagh T, Halder A, Drummond E (2022) Tau interactome and RNA binding proteins in neurodegenerative diseases. Molecular neurodegeneration 17: 66 Doi 10.1186/s13024-022-00572-6
Kawahara M, Kuroda Y, Arispe N, Rojas E (2000) Alzheimer's β-Amyloid, Human Islet Amylin, and Prion Protein Fragment Evoke Intracellular Free Calcium Elevations by a Common Mechanism in a Hypothalamic GnRH Neuronal Cell Line *. Journal of Biological Chemistry 275: 14077-14083 Doi 10.1074/jbc.275.19.14077
Khachaturian ZS (1994) Calcium hypothesis of Alzheimer's disease and brain aging. Ann N Y Acad Sci 747: 1-11 Doi 10.1111/j.1749-6632.1994.tb44398.x
Klemencic M, Novinec M, Maier S, Hartmann U, Lenarcic B (2013) The heparin-binding activity of secreted modular calcium-binding protein 1 (SMOC-1) modulates its cell adhesion properties. PLoS One 8: e56839 Doi 10.1371/journal.pone.0056839
Kuchibhotla KV, Goldman ST, Lattarulo CR, Wu HY, Hyman BT, Bacskai BJ (2008) Abeta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks. Neuron 59: 214-225 Doi 10.1016/j.neuron.2008.06.008
Kuo HK, Yen CJ, Chang CH, Kuo CK, Chen JH, Sorond F (2005) Relation of C-reactive protein to stroke, cognitive disorders, and depression in the general population: systematic review and meta-analysis. Lancet Neurol 4: 371-380 Doi 10.1016/S1474-4422(05)70099-5
LaFerla FM (2002) Calcium dyshomeostasis and intracellular signalling in alzheimer's disease. Nature Reviews Neuroscience 3: 862-872 Doi 10.1038/nrn960
Leitner D, Kavanagh T, Kanshin E, Balcomb K, Pires G, Thierry M, Suazo JI, Schneider J, Ueberheide B, Drummond Eet al (2024) Differences in the cerebral amyloid angiopathy proteome in Alzheimer’s disease and mild cognitive impairment. Acta Neuropathol 148: 9 Doi 10.1007/s00401-024-02767-1
Li Y, Chen Z, Wang Q, Lv X, Cheng Z, Wu Y, Tang F, Shen Y, Gao F (2023) Identification of hub proteins in cerebrospinal fluid as potential biomarkers of Alzheimer’s disease by integrated bioinformatics. Journal of Neurology 270: 1487-1500 Doi 10.1007/s00415-022-11476-2
Lia A, Sansevero G, Chiavegato A, Sbrissa M, Pendin D, Mariotti L, Pozzan T, Berardi N, Carmignoto G, Fasolato Cet al (2023) Rescue of astrocyte activity by the calcium sensor STIM1 restores long-term synaptic plasticity in female mice modelling Alzheimer’s disease. Nature Communications 14: 1590 Doi 10.1038/s41467-023-37240-2
Love S, Chalmers K, Ince P, Esiri M, Attems J, Jellinger K, Yamada M, McCarron M, Minett T, Matthews F (2014) Development, appraisal, validation and implementation of a consensus protocol for the assessment of cerebral amyloid angiopathy in post-mortem brain tissue. American journal of neurodegenerative disease 3: 19
Mancini C, Zonta A, Botta G, Breda Klobus A, Valbonesi S, Pasini B, Giorgio E, Viora E, Brusco A, Brussino A (2019) A fetal case of microphthalmia and limb anomalies with abnormal neuronal migration associated with SMOC1 biallelic variants. European Journal of Medical Genetics 62: 103578 Doi https://doi.org/10.1016/j.ejmg.2018.11.012
Marambaud P, Dreses-Werringloer U, Vingtdeux V (2009) Calcium signaling in neurodegeneration. Molecular Neurodegeneration 4: 20 Doi 10.1186/1750-1326-4-20
Marin MA, Carmichael ST (2018) Stroke in CNS white matter: Models and mechanisms. Neuroscience letters 684: 193-199
Marques S, Zeisel A, Codeluppi S, van Bruggen D, Mendanha Falcão A, Xiao L, Li H, Häring M, Hochgerner H, Romanov RAet al (2016) Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352: 1326-1329 Doi 10.1126/science.aaf6463
Mathys H, Boix CA, Akay LA, Xia Z, Davila-Velderrain J, Ng AP, Jiang X, Abdelhady G, Galani K, Mantero Jet al (2024) Single-cell multiregion dissection of Alzheimer’s disease. Nature 632: 858-868 Doi 10.1038/s41586-024-07606-7
Mathys H, Peng Z, Boix CA, Victor MB, Leary N, Babu S, Abdelhady G, Jiang X, Ng AP, Ghafari Ket al (2023) Single-cell atlas reveals correlates of high cognitive function, dementia, and resilience to Alzheimer’s disease pathology. Cell 186: 4365-4385.e4327 Doi https://doi.org/10.1016/j.cell.2023.08.039
McLachlan DC, Wong L, Bergeron C, Baimbridge K (1987) Calmodulin and calbindin D28K in Alzheimer disease. Alzheimer Disease & Associated Disorders 1: 171-179
Meng F, Zhang X, Alzheimer’s Disease Neuroimaging I (2024) Unveiling Potential Biomarkers in Cerebrospinal Fluid for Amyloid Pathological Positivity in Non-Demented Individuals. The Journal of Prevention of Alzheimer's Disease: Doi 10.14283/jpad.2024.129
Merrihew GE, Park J, Plubell D, Searle BC, Keene CD, Larson EB, Bateman R, Perrin RJ, Chhatwal JP, Farlow MRet al (2023) A peptide-centric quantitative proteomics dataset for the phenotypic assessment of Alzheimer’s disease. Scientific Data 10: 206 Doi 10.1038/s41597-023-02057-7
Mi Z, Halfter W, Abrahamson EE, Klunk WE, Mathis CA, Mufson EJ, Ikonomovic MD (2016) Tenascin-C Is Associated with Cored Amyloid-β Plaques in Alzheimer Disease and Pathology Burdened Cognitively Normal Elderly. Journal of Neuropathology & Experimental Neurology 75: 868-876 Doi 10.1093/jnen/nlw062
Montgomery MK, Bayliss J, Devereux C, Bezawork-Geleta A, Roberts D, Huang C, Schittenhelm RB, Ryan A, Townley SL, Selth LAet al (2020) SMOC1 is a glucose-responsive hepatokine and therapeutic target for glycemic control. Science translational medicine 12: Doi 10.1126/scitranslmed.aaz8048
Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, Vyas S, Uversky VN, Fink AL (2001) Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry 40: 6036-6046 Doi 10.1021/bi002555c
Novinec M, Kovacic L, Skrlj N, Turk V, Lenarcic B (2008) Recombinant human SMOCs produced by in vitro refolding: calcium-binding properties and interactions with serum proteins. Protein Expr Purif 62: 75-82 Doi 10.1016/j.pep.2008.07.009
O’Day DH, Eshak K, Myre MA (2015) Calmodulin Binding Proteins and Alzheimer’s Disease. Journal of Alzheimer's Disease 46: 553-569 Doi 10.3233/JAD-142772
Oh HS-H, Urey DY, Karlsson L, Zhu Z, Shen Y, Farinas A, Timsina J, Guldner IH, Morshed N, Yang Cet al (2024) Synapse protein signatures in cerebrospinal fluid and plasma predict cognitive maintenance versus decline in Alzheimer’s disease. bioRxiv: 2024.2007.2022.604680 Doi 10.1101/2024.07.22.604680
Okada I, Hamanoue H, Terada K, Tohma T, Megarbane A, Chouery E, Abou-Ghoch J, Jalkh N, Cogulu O, Ozkinay Fet al (2011) SMOC1 is essential for ocular and limb development in humans and mice. American journal of human genetics 88: 30-41 Doi 10.1016/j.ajhg.2010.11.012
Panyard DJ, McKetney J, Deming YK, Morrow AR, Ennis GE, Jonaitis EM, Van Hulle CA, Yang C, Sung YJ, Ali Met al (2023) Large-scale proteome and metabolome analysis of CSF implicates altered glucose and carbon metabolism and succinylcarnitine in Alzheimer's disease. Alzheimer's & Dementia 19: 5447-5470 Doi https://doi.org/10.1002/alz.13130
Pazin DE, Albrecht KH (2009) Developmental expression of Smoc1 and Smoc2 suggests potential roles in fetal gonad and reproductive tract differentiation. Dev Dyn 238: 2877-2890 Doi 10.1002/dvdy.22124
Pichet Binette A, Gaiteri C, Wennström M, Kumar A, Hristovska I, Spotorno N, Salvadó G, Strandberg O, Mathys H, Tsai L-Het al (2024) Proteomic changes in Alzheimer disease associated with progressive Aβ plaque and tau tangle pathologies. Nature Neuroscience: Doi 10.1038/s41593-024-01737-w
Ping L, Kundinger SR, Duong DM, Yin L, Gearing M, Lah JJ, Levey AI, Seyfried NT (2020) Global quantitative analysis of the human brain proteome and phosphoproteome in Alzheimer's disease. Sci Data 7: 315 Doi 10.1038/s41597-020-00650-8
Popugaeva E, Pchitskaya E, Bezprozvanny I (2017) Dysregulation of neuronal calcium homeostasis in Alzheimer's disease – A therapeutic opportunity? Biochemical and Biophysical Research Communications 483: 998-1004 Doi https://doi.org/10.1016/j.bbrc.2016.09.053
Rainger J, van Beusekom E, Ramsay JK, McKie L, Al-Gazali L, Pallotta R, Saponari A, Branney P, Fisher M, Morrison Het al (2011) Loss of the BMP antagonist, SMOC-1, causes Ophthalmo-acromelic (Waardenburg Anophthalmia) syndrome in humans and mice. PLoS Genet 7: e1002114 Doi 10.1371/journal.pgen.1002114
Roberts JA, Varma VR, Candia J, Tanaka T, Ferrucci L, Bennett DA, Thambisetty M (2023) Unbiased proteomics and multivariable regularized regression techniques identify SMOC1, NOG, APCS, and NTN1 in an Alzheimer’s disease brain proteomic signature. npj Aging 9: 18 Doi 10.1038/s41514-023-00112-6
Rodrigues FEP, Figueira AJ, Gomes CM, Machuqueiro M (2021) Computational Analysis of the Interactions between the S100B Extracellular Chaperone and Its Amyloid β Peptide Client. International Journal of Molecular Sciences 22: 3629
Sathe G, Albert M, Darrow J, Saito A, Troncoso J, Pandey A, Moghekar A (2020) Quantitative proteomic analysis of the frontal cortex in Alzheimer's disease. J Neurochem: Doi 10.1111/jnc.15116
Schirmer L, Velmeshev D, Holmqvist S, Kaufmann M, Werneburg S, Jung D, Vistnes S, Stockley JH, Young A, Steindel Met al (2019) Neuronal vulnerability and multilineage diversity in multiple sclerosis. Nature 573: 75-82 Doi 10.1038/s41586-019-1404-z
Schneider JA, Arvanitakis Z, Bang W, Bennett DA (2007) Mixed brain pathologies account for most dementia cases in community-dwelling older persons. Neurology 69: 2197-2204
Shantaraman A, Dammer EB, Ugochukwu O, Duong DM, Yin L, Carter EK, Gearing M, Chen-Plotkin A, Lee EB, Trojanowski JQet al (2024) Network Proteomics of the Lewy Body Dementia Brain Reveals Presynaptic Signatures Distinct from Alzheimer’s Disease. bioRxiv: 2024.2001.2023.576728 Doi 10.1101/2024.01.23.576728
Shen Y, Ali M, Timsina J, Wang C, Do A, Western D, Liu M, Gorijala P, Budde J, Liu Het al (2024) Systematic proteomics in Autosomal dominant Alzheimer’s disease reveals decades-early changes of CSF proteins in neuronal death, and immune pathways. medRxiv: 2024.2001.2012.24301242 Doi 10.1101/2024.01.12.24301242
Sung YJ, Yang C, Norton J, Johnson M, Fagan A, Bateman RJ, Perrin RJ, Morris JC, Farlow MR, Chhatwal JPet al (2023) Proteomics of brain, CSF, and plasma identifies molecular signatures for distinguishing sporadic and genetic Alzheimer’s disease. Science Translational Medicine 15: eabq5923 Doi doi:10.1126/scitranslmed.abq5923
Taylor AIP, Davis PJ, Aubrey LD, White JBR, Parton ZN, Staniforth RA (2023) Simple, Reliable Protocol for High-Yield Solubilization of Seedless Amyloid-β Monomer. ACS Chemical Neuroscience 14: 53-71 Doi 10.1021/acschemneuro.2c00411
Thierry M, Ponce J, Marta-Ariza M, Askenazi M, Faustin A, Leitner D, Pires G, Kanshin E, Drummond E, Ueberheide Bet al (2024) The influence of APOE(epsilon4) on the pTau interactome in sporadic Alzheimer's disease. Acta neuropathologica 147: 91 Doi 10.1007/s00401-024-02744-8
Trautwig AN, Fox EJ, Dammer EB, Shantaraman A, Ping L, Duong DM, Levey AI, Lah JJ, Fournier CN, McEachin ZTet al (2024) Network Analysis of the Cerebrospinal Fluid Proteome Reveals Shared and Unique Differences Between Sporadic and Familial Forms of Amyotrophic Lateral Sclerosis. bioRxiv: 2024.2002.2029.582840 Doi 10.1101/2024.02.29.582840
van der Ende EL, In 't Veld S, Hanskamp I, van der Lee S, Dijkstra JIR, Hok AHYS, Blujdea ER, van Swieten JC, Irwin DJ, Chen-Plotkin Aet al (2023) CSF proteomics in autosomal dominant Alzheimer's disease highlights parallels with sporadic disease. Brain 146: 4495-4507 Doi 10.1093/brain/awad213
Vannahme C, Smyth N, Miosge N, Gosling S, Frie C, Paulsson M, Maurer P, Hartmann U (2002) Characterization of SMOC-1, a novel modular calcium-binding protein in basement membranes. The Journal of biological chemistry 277: 37977-37986 Doi 10.1074/jbc.M203830200
Vanzulli I, Papanikolaou M, De-La-Rocha IC, Pieropan F, Rivera AD, Gomez-Nicola D, Verkhratsky A, Rodríguez JJ, Butt AM (2020) Disruption of oligodendrocyte progenitor cells is an early sign of pathology in the triple transgenic mouse model of Alzheimer's disease. Neurobiology of Aging 94: 130-139 Doi https://doi.org/10.1016/j.neurobiolaging.2020.05.016
Wang H, Dey KK, Chen PC, Li Y, Niu M, Cho JH, Wang X, Bai B, Jiao Y, Chepyala SRet al (2020) Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer's disease. Molecular neurodegeneration 15: 43 Doi 10.1186/s13024-020-00384-6
Wang J, Xia S, Zhao J, Gong C, Xi Q, Sun W (2021) Prognostic Potential of Secreted Modular Calcium-Binding Protein 1 in Low-Grade Glioma. Front Mol Biosci 8: 666623 Doi 10.3389/fmolb.2021.666623
Watson CM, Dammer EB, Ping L, Duong DM, Modeste E, Carter EK, Johnson ECB, Levey AI, Lah JJ, Roberts BRet al (2023) Quantitative Mass Spectrometry Analysis of Cerebrospinal Fluid Protein Biomarkers in Alzheimer's Disease. Sci Data 10: 261 Doi 10.1038/s41597-023-02158-3
Wingo AP, Dammer EB, Breen MS, Logsdon BA, Duong DM, Troncosco JC, Thambisetty M, Beach TG, Serrano GE, Reiman EMet al (2019) Large-scale proteomic analysis of human brain identifies proteins associated with cognitive trajectory in advanced age. Nat Commun 10: 1619 Doi 10.1038/s41467-019-09613-z
Wingo AP, Fan W, Duong DM, Gerasimov ES, Dammer EB, Liu Y, Harerimana NV, White B, Thambisetty M, Troncoso JCet al (2020) Shared proteomic effects of cerebral atherosclerosis and Alzheimer's disease on the human brain. Nat Neurosci 23: 696-700 Doi 10.1038/s41593-020-0635-5
Wojtas AM, Dammer EB, Guo Q, Ping L, Shantaraman A, Duong DM, Yin L, Fox EJ, Seifar F, Lee EBet al (2024) Proteomic changes in the human cerebrovasculature in Alzheimer's disease and related tauopathies linked to peripheral biomarkers in plasma and cerebrospinal fluid. Alzheimer's & Dementia 20: 4043-4065 Doi https://doi.org/10.1002/alz.13821
Xie K, Liu Y, Hao W, Walter S, Penke B, Hartmann T, Schachner M, Fassbender K (2013) Tenascin-C deficiency ameliorates Alzheimer's disease-related pathology in mice. Neurobiol Aging 34: 2389-2398 Doi 10.1016/j.neurobiolaging.2013.04.013
Xiong F, Ge W, Ma C (2019) Quantitative proteomics reveals distinct composition of amyloid plaques in Alzheimer's disease. Alzheimer's & dementia : the journal of the Alzheimer's Association 15: 429-440 Doi 10.1016/j.jalz.2018.10.006
Yu H, Wang M, Wang X, Jiang X (2023) Immune-related matrisomes are potential biomarkers to predict the prognosis and immune microenvironment of glioma patients. FEBS Open Bio 13: 307-322 Doi 10.1002/2211-5463.13541
Zallo F, Gardenal E, Verkhratsky A, Rodríguez JJ (2018) Loss of calretinin and parvalbumin positive interneurones in the hippocampal CA1 of aged Alzheimer’s disease mice. Neuroscience Letters 681: 19-25 Doi https://doi.org/10.1016/j.neulet.2018.05.027
Zhang P, Kishimoto Y, Grammatikakis I, Gottimukkala K, Cutler RG, Zhang S, Abdelmohsen K, Bohr VA, Misra Sen J, Gorospe Met al (2019) Senolytic therapy alleviates Aβ-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer’s disease model. Nature Neuroscience 22: 719-728 Doi 10.1038/s41593-019-0372-9
Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guarnieri P, Caneda C, Ruderisch Net al (2014) An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci 34: 11929-11947 Doi 10.1523/jneurosci.1860-14.2014
Zhou M, Haque RU, Dammer EB, Duong DM, Ping L, Johnson ECB, Lah JJ, Levey AI, Seyfried NT (2020) Targeted mass spectrometry to quantify brain-derived cerebrospinal fluid biomarkers in Alzheimer's disease. Clin Proteomics 17: 19 Doi 10.1186/s12014-020-09285-8

No competing interests reported.

SuppFig1.tiff
Supplementary Figure 1: SMOC1 distribution in plaques is not determined by plaque size. SMOC1 immunofluorescence in plaques did not correlate with amyloid plaque size. Nonparametric Spearman correlation of n= 10 preclinical AD, n = 12 MCI and n = 12 temporal cortex sections. Dotted lines represent 95% confidence intervals.
SuppFig2.tif
Supplementary Figure 2: SMOC1 does not bind to mature Ab₄₂ fibrils. (a) SMOC1 remains in the soluble fraction after incubation with mature Ab₄₂ fibrils. (b) Electron microscopy of Ab₄₂ fibrils show no binding of NanoGold to fibrils, and no change of fibril morphology with SMOC1 addition. Scale bar = 500 nm.
SuppFig3.png
Supplementary Figure 3: SMOC1 antibody validation. (a) Three SMOC1 antibodies were tested for Western Blot application on SMOC1-tGFP overexpressing SHSY5Y lysates. ab200219 (abcam) was determined to give the clearest signal compared to PA5-31392 (ThermoFisher) or PA5-113408 (ThermoFisher). All antibodies were tested at 1:500 in 2.5% skim milk. (b) ab200219 (abcam) was tested with blocking buffers of 2.5% skim milk, 2.5% skim milk + 2.5% NGS, and 2.5% BSA on human brain homogenate (1:500). The combination buffer of skim milk + NGS was determined to give the least non-specific signal. (c) To confirm antibody specificity, oligodendrocyte MO3.13 and neuroblastoma SHSY5Y cells were transfected with HA-tagged SMOC1 plasmid, incubated for 48 hours and lysates collected. SMOC1 detection was then examined in lysates, human brain homogenate, and SMOC1 recombinant peptides (shorter exposure). ab20219 detected a strong 62 kDa band in the SMOC1 peptide and SMOC1-overexpression conditions, confirming antibody specificity. (d-f) Three SMOC1 antibodies were tested for immunofluorescence in human AD FFPE brain tissue. All antibodies were tested at 1:100 on the same case. ab200219 (abcam) was determined to give the best immunofluorescent signal in plaques (arrows) (d) compared to PA5-31392 (ThermoFisher) (e) or WH0064093M3 (Sigma-Aldrich) (f). Scale bars = 200µm (overviews), 20µm (inserts). NGS; normal goat serum, BSA; bovine serum albumin.
SuppFig4.tiff
Supplementary Figure 4: High magnification visualisation of SMOC1 colocalization with Ab. Plaques were imaged on a Nikon C2 at 100x magnification over 6µm depth (0.1µm steps). Images were deconvoluted using Huygens Professional and visualized in ImageJ. SMOC1 immunofluorescence (green) was observed to closely colocalize with amyloid fibrils (red) within plaques. Scale bars = 20µm.

Download PDF

Editorial decision: Revision requested
24 Oct, 2024
Reviews received at journal
23 Oct, 2024
Reviews received at journal
15 Oct, 2024
Reviewers agreed at journal
11 Oct, 2024
Reviewers agreed at journal
11 Oct, 2024
Reviewers invited by journal
09 Oct, 2024
Editor assigned by journal
09 Oct, 2024
Submission checks completed at journal
09 Oct, 2024
First submitted to journal
09 Oct, 2024

You are reading this latest preprint version

SMOC1 colocalizes with Alzheimer’s disease neuropathology and delays Aβ aggregation

Status:

Version 1

Abstract

Figures

Introduction

Methods

Human Tissue Samples

Immunohistochemistry for Image Analysis

Immunohistochemistry for Cell Type Investigation

SMOC1/4G8 Immunohistochemistry Analysis

SMOC1/AT8 Immunohistochemistry Analysis

SMOC1/pyroglutamated Aβ Immunohistochemistry Analysis

Tissue Homogenization

Co-Immunoprecipitation

Western Blot Analysis

Recombinant Aβ₄₂ and SMOC1 Protein Preparation

Aβ₄₂ Thioflavin-T Assays

Preparation of Mature Aβ₄₂ Fibrils

SDS-PAGE

Transmission Electron Microscopy (TEM)

Statistical Analysis

Results

SMOC1 colocalizes with a subset of Aβ plaques in early and advanced AD

SMOC1 colocalization with Aβ plaques is not driven by pyroglutamated Aβ

SMOC1 immunoreactivity is observed in blood vessels, spongiform and small cells

SMOC1 is predominantly expressed by Olig2-negative Oligodendrocyte Precursor Cells

SMOC1 colocalizes with tau

SMOC1 interacts with Aβ and pTau in human AD brain

SMOC1 inhibits Aβ₄₂ fibril formation and alters fibril morphology

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

SMOC1 colocalizes with Alzheimer’s disease neuropathology and delays Aβ aggregation

Status:

Version 1

Abstract

Figures

Introduction

Methods

Human Tissue Samples

Immunohistochemistry for Image Analysis

Immunohistochemistry for Cell Type Investigation

SMOC1/4G8 Immunohistochemistry Analysis

SMOC1/AT8 Immunohistochemistry Analysis

SMOC1/pyroglutamated Aβ Immunohistochemistry Analysis

Tissue Homogenization

Co-Immunoprecipitation

Western Blot Analysis

Recombinant Aβ42 and SMOC1 Protein Preparation

Aβ42 Thioflavin-T Assays

Preparation of Mature Aβ42 Fibrils

SDS-PAGE

Transmission Electron Microscopy (TEM)

Statistical Analysis

Results

SMOC1 colocalizes with a subset of Aβ plaques in early and advanced AD

SMOC1 colocalization with Aβ plaques is not driven by pyroglutamated Aβ

SMOC1 immunoreactivity is observed in blood vessels, spongiform and small cells

SMOC1 is predominantly expressed by Olig2-negative Oligodendrocyte Precursor Cells

SMOC1 colocalizes with tau

SMOC1 interacts with Aβ and pTau in human AD brain

SMOC1 inhibits Aβ42 fibril formation and alters fibril morphology

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Recombinant Aβ₄₂ and SMOC1 Protein Preparation

Aβ₄₂ Thioflavin-T Assays

Preparation of Mature Aβ₄₂ Fibrils

SMOC1 inhibits Aβ₄₂ fibril formation and alters fibril morphology