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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Exp Neurol. 2020 Feb 28;328:113261. doi: 10.1016/j.expneurol.2020.113261

Thiol-mediated and catecholamine-enhanced multimerization of a cerebrovascular disease enriched fragment of NOTCH3

Kelly Z Young 1,2,*, Naw May P Cartee 1,*, Magdalena I Ivanova 1, Michael M Wang 1,2,3
PMCID: PMC7146869  NIHMSID: NIHMS1575973  PMID: 32119934

Abstract

Cerebral small vessel disease is a common condition linked to dementia and stroke. As an age-dependent brain pathology, cerebral SVD may share molecular processes with core neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Many neurodegenerative diseases feature abnormal protein accumulation and aberrant protein folding, resulting in multimerization of specific proteins. We investigated if a small NOTCH3 N-terminal fragment (NTF) that co-registers with pathologically affected cells in the inherited SVD, CADASIL, is capable of multimerization. We also characterized endogenous small molecule vascular enhancers and inhibitors of multimerization. NTF multimerizes spontaneously and also forms conjugates with vascular catecholamines, including dopamine and norepinephrine, which avidly promote multimerization of the protein. Inhibition of catecholamine-dependent multimerization by vitamin C and reversal by reducing agents implicate an essential role of oxidation in NTF multimerization. Antibodies that react with degenerating arteries in CADASIL tissue preferentially bind to multimerized forms of NTF. These studies suggest that multimerization of proteins in the aging brain is not restricted to neuronal molecules and may participate in age-dependent vascular pathology.

Keywords: CADASIL, NOTCH3, proteinopathies, catecholamines, dopamine, norepinephrine, small vessel disease, protein oligomerization, cysteine residues, vascular dementia

Introduction

Cerebrovascular disorders are among the most vexing diseases, as they are common and result in significant permanent disability. In addition, the indirect effects of cerebrovascular dysfunction, as in the case of small vessel disease, include increased incidence and severity of neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. The molecular causes of cerebrovascular disease have only recently been investigated in detail and remain incompletely understood.

A large contingent of neurological diseases is thought to result from proteinopathies. For example, Alzheimer’s disease (AD) features misfolded tau and multimerized amyloid beta protein (A-beta) (Goedert et al., 2010). Parkinson’s disease (PD) is associated with abnormal conformation, accumulation, and multimerization of synuclein (Goedert et al., 2010). Creutzfeld-Jacob Disease, the prototypical propagating proteinopathy, results from pathological, self-perpetuating conformational alterations in PrP (Prusiner, 1982). Protein inclusions are notable features in a wide array of additional neurological disorders including Huntington’s disease, frontotemporal dementia, and amyotrophic lateral sclerosis. In all of these disorders, protein abnormalities localize in or around neurons; this proximity to the pathology is felt by some to implicate a direct effect of proteinopathy on cell viability.

Several vascular diseases of the brain share similarities to proteinopathies seen in neuronal disorders. The sporadic and genetic cerebral small vessel diseases (eg. CADASIL; Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) exhibit dramatic protein accumulation in the vascular media of penetrating arteries (Dong et al., 2012; Joutel et al., 2000; Monet-Leprêtre et al., 2013; Zhang et al., 2014a; 2015). Protein which accumulates in a stereotypical location (the vascular media) includes altered conformations of NOTCH3 which are not part of normal vessels and may result from progressive redox alterations (Zhang et al., 2014a). In addition, in CADASIL caused by NOTCH3 mutations, extracellular smooth muscle molecular aggregates indent the smooth muscle plasma membrane; these bodies called granular osmiophilic material (GOM) appear to be composed of aggregated proteins and other poorly characterized material (Ruchoux et al., 1995). It is unclear what provokes multimerization of proteins in the vessel wall.

Work in Parkinson’s disease has demonstrated profound toxicity of dopamine, a neurotransmitter that is made in large quantities in the substantia nigra, the region of the brain that is preferentially affected in PD (Damier et al., 1999; Fearnley and Lees, 1991). The role of dopamine (DA) in pathogenesis remains debated, though considerable work has demonstrated that it can cause cell oxidative stress. Recent work has implicated oxidized dopamine as an intracellular toxin in nigral neurons. Reactive oxygen and nitrogen species generated by DA and dihydroxyphenylalanine (DOPA) oxidation is thought to lead to neuronal damage and death (Asanuma et al., 2003). Additionally, DA and DOPA can exert cytotoxicity through the generation of highly reactive quinones. DA and DOPA quinones can form conjugates with the sulfhydrl group of cysteines to become 5-cysteinyl catechols on proteins and ultimately alter protein function (Fornstedt et al., 1986; Graham, 1978; Ito and K. Fujita, 1982). DA has been shown to covalently conjugate with proteins, such as alpha-synuclein (Conway et al., 2001) and tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis (Kuhn et al., 1999; Xu et al., 1998). In addition, DA has also been shown to cause changes in proteins that include multimerization of proteins, such as alpha-synuclein in a mouse model of Parkinson’s (Mor et al., 2017), Disrupted in schizophrenia 1 (DISC1) (Trossbach et al., 2016); prion protein (da Luz et al., 2015), among others. Outside of the nervous system, the catecholamine DOPA has been shown to participate in extremely strong adhesion reactions in marine animals (Nicklisch and Waite, 2012). Many reports have demonstrated prevention of DA and DOPA induced neuronal damage by antioxidants, including glutathione (GSH), ascorbate, and free cysteines both in vivo and in vitro (Hastings et al., 1996; Kuhn et al., 1999; Lai and Yu, 1997; LaVoie and Hastings, 1999).

Catecholamines, including DA and norepinephrine (NE), are also expressed in the vasculature. NE has vasoconstrictive functions in brain vessels (Toda and Y. Fujita, 1973), while DA has been described in activity dependent vascular flow regulation (Krimer et al., 1998). Catecholamines directly affect the vascular smooth muscle, causing contraction and consequent vasoconstriction that has effects on cerebral blood flow.

Here, we investigate the propensity of a fragment of the vascular pathological protein NOTCH3 (NTF; N-terminal fragment) to oligomerize. We further investigate the role of neurotransmitters in oligomerization of NOTCH3 fragments.

Materials and Methods

Chemicals and reagents. Unless noted, chemicals were obtained from Sigma

Monoclonal antibodies against NOTCH3

Rabbit monoclonal antibodies were generated similar to polyclonal antibodies as described in (Zhang et al., 2014b). Animals were immunized with peptides from the N-terminal EGF-like repeat of human NOTCH3: ACLCPPGWGERCQLED. Splenic lymphocytes from immunized rabbits were fused to form hybridomas that were cloned and screened for secretion of antibodies exhibiting binding to the immunizing peptides (performed by Epitomics). Avidly binding antibodies were further selected based on ability to detect protein in the media of vessels from CADASIL patients. Two of these antibodies, UMI-D and UMI-F, exhibited similar characteristics and were used extensively in this study (see Results).

Protein analysis

NTF was synthesized by ThermoScientific. Lyophilized NTF was reconstituted in dH2O at 1ug/uL and aliquoted and stored at −20°C. All protein treatments were performed in PBS with supplemental chemicals, as noted in specific experiments. At the end of each treatment, proteins were denatured in sample buffer with or without 5% beta-mercaptoethanol and boiled at 100°C for 5 minutes. All samples were separated on standard 8–16% tris-glycine or 10–20% tricine gels (ThermoFisher and Bio-Rad) and electroblotted to nitrocellulose using the iBlot 2 system (Invitrogen). Western Blot analysis was performed with antibodies as indicated, followed by incubation with infrared fluorophore labeled secondary antibodies (Rockland). Bands were detected using a Licor Odyssey infrared scanner. To visualize total protein, standard Coomassie Blue (Bio-Rad) staining and silver staining (ThermoScientific) protocols were used. For relative analysis of NTF proteins, multimerized NTF was compared to monomer NTF by antibody detection and normalized to multimerized/monomer NTF detected by Coomassie Blue staining that was quantified by IR fluorescence.

Mass Spectrometry

Synthetized NTF was incubated with chemical treatments in dH2O. Samples were diluted to 5pmol/uL in 20% acetonitrile and 0.1% formic acid and infused into Orbitrap Fusion Tribid mass spectrometer at a flow rate of 4uL/min. The precursor mass (MS1) scans were collected at a resolution of 120,000 for a full minute. The MS1 data was deconvoluted using the Xtract function of QualBrowser (ThermoScientific, ZXCalibur v3.0.63) to determine the monoisotopic intact mass of the NTF peptides.

Results

Spontaneous multimerization of NOTCH3 N-terminal fragment (NTF)

We have described the non-enzymatic fragmentation of NOTCH3 in CADASIL arteries at autopsy (Young et al., 2020). This cleavage event is predicted to generate a 41 amino acid fragment (NOTCH3 N-terminal fragment; NTF). A synthetic peptide corresponding to this sequence was analyzed for all subsequent studies. This peptide migrates close to the predicted molecular mass on 10–20% SDS-tricine gels (ThermoFisher) which were used to visualize the small proteins. In pilot studies, we observed laddering of NTF after incubations of the protein at 37°C. We therefore investigated the effect of prolonged incubation of NTF on the migration of NTF protein. Fig 1A shows progressive laddering with increased time of incubation of NTF in PBS at 37°C when proteins were run on nonreducing gels and probed with antibodies that recognize the C-terminus of NTF (UMI-D and UMI-F). At very long incubation times, little protein was found in gels, perhaps due to the high molecular mass of the formed complexes. Addition of reducing agents completely reversed the laddering of the peptide (Fig 1B). NTF demonstrated similar spontaneous multimerization following incubation at 37°C in dH2O and 10mM Sodium Phosphate Buffer, pH=7.5 (data not shown).

Figure 1. N-terminal fragment (NTF) of NOTCH3 undergoes spontaneous thiol-mediated multimerization.

Figure 1.

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Synthetic NTF was incubated at 37°C for 0, 1, 4, 16, 24, 32, 30, 38, or 56 hours. Samples were boiled in nonreducing sample buffer (A) and reducing sample buffer (B) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF monomers and multimers were detected using UMI-D antibody. Multimerization of NTF was observed in nonreducing conditions at 16 hours and continued up to 56 hours. Reduction with BME eliminated time-dependent multimers. Slight non-thiol mediated oligomerization was noted at baseline. NTF and a form of NTF with all cysteines mutated to serine (6S) were incubated at 37°C for 0, 1, or 3 days. NTF and 6S samples were run under nonreducing (C) and reducing (D) conditions on an 8–16% tris-glycine gel (ThermoFisher) and probed with UMI-F. NTF formed time-dependent multimers while 6S did not. Slight non-thiol mediated oligomerization of 6S was also noted at baseline. All experiments were performed at least four times with similar results.

In control experiments, we tested whether NTF without cysteines (6S; 6 Cys to Ser mutations) was capable of multimerization into large molecular weight populations. We incubated NTF and 6S for varying lengths of time in PBS at 37°C and electrophoresed on an 8–16% tris glycine gel (ThermoFisher). In contrast to NTF, the 6S peptide formed small BME resistant oligomers that did not evolve into large molecular weight complexes (Figs 1C and 1D).

To identify the effect of pH on NTF multimerization, we incubated NTF in PBS at varying pH for 24 hours at 37°C and ran the samples on an 8–16% tris glycine gel (ThermoFisher). Although there appeared to be a slight increase in multimerization in acidic conditions, we found that multimerization was comparable in the different pH buffers tested (Fig 2). Addition of reducing agent, beta-mercaptoethanol, reversed the multimerization due to prolonged incubation.

Figure 2. The effect of pH on spontaneous NTF multimerization.

Figure 2.

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Paired synthetic NTF was exposed to buffers at specific pH for 0 or 1 day (24 hours) at 37°C, boiled in nonreducing (A) and reducing (B) sample buffer, and electrophoresed on 8–16% tris-glycine gels (ThermoFisher). Western analysis was performed using UMI-D to detect NTF. There was a slight increase in multimerization at modestly acidic conditions. We performed this experiment at least three times and obtained similar results.

Chemical modulation of spontaneous multimerization of NTF

Physiological reducing agents were tested for activity against multimerization. NTF was incubated with a range of reducing agent concentrations for 24 hours at 37°C and electrophoresed on 8–16% tris glycine gels (ThermoFisher and BioRad). Both glutathione (GSH) and homocysteine (HC) inhibited multimer formation at supraphysiologic concentrations (Fig 3 A–B). 10mM GSH or 1mM HC was sufficient for partial inhibition NTF multimerization, whereas 100mM GSH or 10mM HC was sufficient for complete inhibition.

Figure 3. The effect of physiological reducing agents on spontaneous multimer formation.

Figure 3.

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NTF was incubated for 24 hours at 37°C in the presence of varying concentrations of physiological reducing agents, glutathione (A; GSH) and homocysteine (B; HC). Samples were boiled in nonreducing sample buffer and run on 8–16% tris-glycine gels (ThermoFisher and Bio-Rad). Western analysis was performed using UMI-D to detect NTF. 100mM glutathione or 10mM homocysteine was sufficient to prevent multimerization of NTF. All experiments were performed at least three times with similar results.

Because DA is a catecholamine transmitter implicated in vascular regulation in the brain that likely comes in contact with smooth muscles cells (the site of NOTCH3 protein synthesis), we tested if DA could chemically interact with NOTCH3. First, we tested whether DA alters the multimerization of NTF. Neurotransmitter concentrations, such as those of dopamine, are known to fluctuate within the intact brain. Current models suggest that DA neurons typically exhibit two modes of spike firing: tonic single spike basal activity and burst spike firing(Floresco et al., 2003; Grace, 1991). Measurement of tonic firing of DA neurons in rats demonstrated extracellular DA levels of 5–40nM within the striatal region (Floresco et al., 2003; Keefe et al., 1993; Parsons and Justice, 1992; Wightman and Robinson, 2002), while burst spike firing triggers transient high amplitude DA levels within targeted areas, ranging from hundreds of uM to mM (Goto et al., 2007; Wanat et al., 2009).Concentration of DA Is likely highest in vesicles, yielding roughly 33,000 molecules of dopamine per vesicle or an estimated ~0.1M intraluminal dopamine (Omiatek et al., 2013). In Fig 4A, we mixed a physiologically relevant range of DA with NTF and show a time-dependent increase in NTF multimer formation in the presence of DA, with progressively longer incubations resulting in large shifts in multimer sizes visualized on 10–20% SDS tricine gels (ThermoFisher). This was also dose dependent (Fig 4C). The DA induced oligomers of NTF were only partially sensitive to chemical reduction (Fig 4B; Fig 4D). Although most of the complexes were reduced to lower molecular weights, at longer incubations or higher concentrations of DA, larger complexes persisted.

Figure 4. Dose-dependent and time-dependent effects of dopamine on NTF multimerization.

Figure 4.

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NTF was incubated with 0.8mM dopamine for varying lengths of time up to 16 hours at 37°C. Samples were boiled in nonreducing sample buffer (A) and reducing sample buffer containing beta-mercaptoethanol (B) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF was also incubated with varying concentrations of dopamine for 4 hours at 37°C and run on a gel under nonreducing (C) and reducing (D) conditions. Western analysis was performed using UMI-D to detect NTF. Dopamine enhanced multimerization in a time-dependent and dose-dependent manner. Dopamineenhanced NTF multimers were only partially resistant to reducing agents. Experiments were performed at least four times with similar results.

Another catecholamine present in cerebral arteries, norepinephrine (NE), was then assessed for NOTCH3 interactions. Estimates of basal extracellular NE levels range from 14–35nM in rats(Abercrombie et al., 1988; Harley et al., 1996). The average rate sympathetic nerve norepinephrine is produced is 3–4ug/min in healthy humans(Esler et al., 1988). The overall rate of spillover of norepinephrine to plasma in healthy subjects is of the order of 0.3ug/min(Esler et al., 1988). Exposing NTF to NE at similar concentrations to those found at physiological levels, while keeping the doses the same as those for DA, resulted in dose and time dependent multimerization that was partially sensitive to BME (Fig 5A–D). The related catecholamine epinephrine (Epi) showed similar activities on NTF (Fig 6A–D). In comparison, at similar concentrations used above, tyramine, a non-catecholamine with strong similarity to DA and NE, stimulated multimerization (Fig 7A–D) that was completely reversible with reducing agents.

Figure 5. Dose-dependent and time-dependent effects of norepinephrine on NTF multimerization.

Figure 5.

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NTF was incubated with 0.8mM norepinephrine for varying lengths of time at 37°C. Samples were then boiled in nonreducing (A) or reducing sample buffer (B) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF was also incubated with increasing concentrations of norepinephrine for 4 hours at 37°C and electrophoresed under both nonreducing (C) and reducing (D) conditions. Monoclonal antibody UMI-D was used to detect NTF. Norepinephrine enhanced multimerization in a time-dependent and dose-dependent manner. Norepinephrine-enhanced NTF multimers were partially resistant to reversal in the presence of reducing agents. Each experiment was performed at least four times with similar results.

Figure 6. Dose-dependent and time-dependent effects of epinephrine on NTF multimerization.

Figure 6.

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NTF was incubated with 0.8mM epinephrine for increasing lengths of time at 37°C. Samples were boiled in nonreducing sample buffer (A) and reducing sample buffer containing beta-mercaptoethanol (B) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF was also incubated with varying concentrations of epinephrine for 4 hours at 37°C and run on a 10–20% tricine gel under nonreducing (C) and reducing (D) conditions. Monoclonal antibody UMI-D was used to detect NTF by western analysis. Epinephrine enhanced multimerization in a time-dependent and dose-dependent manner. The majority of epinephrine-enhanced NTF multimers were susceptible to reduction by chemical reducing agents. Each experiment was repeated at least four times with similar results.

Figure 7. Dose-dependent and time-dependent effects of tyramine on NTF multimerization.

Figure 7.

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NTF was incubated with 0.8mM tyramine for varying lengths of time up to 16 hours at 37°C. Samples were boiled in nonreducing sample buffer (A) and reducing sample buffer containing beta-mercaptoethanol (B) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF was also incubated with varying concentrations of tyramine for 4 hours at 37°C and run on a gel under nonreducing (C) and reducing (D) conditions. Western analysis was performed using antibody UMI-D to detect NTF. Tyramine enhanced multimerization in a time-dependent and dose-dependent manner. Tyramine enhanced multimers were reversible by reducing agents, such as BME. All experiments were performed at least four times with similar results.

To determine the effect of pH on catecholamine-enhanced multimerization of NTF, we incubated NTF with 0.8mM dopamine for 4 hours at 37°C in PBS buffers with varying pH and electrophoresed on 10–20% SDS tricine gels (ThermoFisher). We identified the greatest amounts of multimer formation at alkaline pH (Fig 8A). These complexes were partially reversible in presence of reducing agent (Fig 8B).

Figure 8. The effect of pH on dopamine-enhanced NTF multimerization.

Figure 8.

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Paired synthetic NTF samples were exposed to buffers at specific pH in the presence of 0 or 0.8mM dopamine for 4 hours at 37°C, boiled in nonreducing (A) and reducing (B) sample buffer, and electrophoresed on 10–20% tricine gels (ThermoFisher). Western analysis was performed using UMI-D to detect NTF. There was an increase in multimerization at alkaline conditions. We performed these experiments at least four times and obtained similar results.

Catecholamine-enhanced multimerization of NTF (Fig 9A–B) was also compared to the control peptide lacking cysteines, 6S (Fig 9C–D). NTF and 6S protein was incubated in the presence of 0.8mM DA, NE, Epi, or Tyr for 4 hours at 37°C and electrophoresed on 10–20% SDS tricine gels. DA, NE and Epi consistently enhanced multimerization of NTF, and DA and NE consistently enhanced multimerization of 6S. However, unlike catecholamine enhanced multimerization of NTF (Fig 9B), multimerized 6S was completely resistant to degradation by reducing agent (Fig 9D).

Figure 9. Effect of cysteines on catecholamine-enhanced NTF multimerization.

Figure 9

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NTF (A, B) and a form of NTF with all cysteines mutated to serine (6S; C, D) were incubated with and without 0.8mM dopamine (DA), 0.8mM norepinephrine (NE), 0.8mM epinephrine (Epi) and 0.8mM tyramine (Tyr) at 37°C. Samples were boiled in nonreducing sample buffer (A, C) and reducing sample buffer (B, D) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF monomers and multimers were detected using UMI-D antibody. In the presence of catecholamines, both NTF and 6S generated more multimers. Most NTF multimers reversed with addition of reducing agent, while 6S multimers were largely resistant to reducing agents. Slight non-thiol mediated NTF and 6S oligomers were noted at baseline. Experiments were performed at least three times with similar results.

The mixtures of NTF and DA or NE were analyzed by mass spectroscopy in order to understand the molecular effects of catecholamines. Expected and measured masses are displayed in Table 1 for DA and Table 2 for NE. A heterogeneous mixture of products was discovered (Fig 10). For both DA (Fig 10A) and NE (Fig 10B), major peaks were identified that were consistent with covalent addition of one, two, or three catecholamines to NTF.

Table 1.

NTF was incubated with or without dopamine and evaluated by mass spectrometry. Expected and measured masses are shown for dopamine conjugation to NTF. Measured mass refers to the major peak, but multiple oxidation products of each product were observed.

NTF with Dopamine (153.18 Da)
# Conjugates Expected mass Measured mass Difference
0 4257.88 4256.87 1.01
1 4410.05 4407.93 2.12
2 4563.23 4560.01 3.22
3 4716.41 4712.08 4.33
4 4869.59 4863.14 6.45
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Table 2.

NTF was incubated with or without norepinephrine and evaluated by mass spectrometry. Expected and measured masses are shown for norepinephrine conjugation to NTF. Measured mass refers to the major peak, but multiple oxidation products of each product were observed.

NTF with Norepinephrine (169.18 Da)
# Conjugates Expected mass Measured mass Difference
0 4257.88 4254.86 3.02
1 4424.04 4421.92 2.12
2 4593.22 4589.98 3.24
3 4762.4 4758.05 4.35
4 4931.58 4926.11 5.47
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Figure 10. Visualizing dopamine and norepinephrine conjugated NTF by mass spectrometry.

Figure 10.

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NTF and dopamine (A) or norepinephrine (B) were incubated for 16 hours at 37°C and diluted to 5pmol/uL NTF in 0.2mM dopamine or 0.2mM norepinephrine in dH2O for top down mass spectrometry analysis. Major peaks consistent with covalent addition of one, two, or three dopamine molecules to NTF were identified (A). Covalent addition of one, two, or three norepinephrine molecules to NTF were also identified (B). Minor peaks corresponding to multiple oxidation products were also identified for each of the products.

Differences between the observed NTF masses in the presence of DA or NE and the expected masses in adducts may result from oxidation of NTF.

Synthetic catechols and NTF oligomerization

NTF was incubated with increasing concentrations of DA, Dihydroxybenzylamine (3, 4-DHBA), and Pyrocatechol violet (PCV) for 3 hours at 37°C and electrophoresed on a 10–20% SDS tricine gel (ThermoFisher). 3, 4-DHBA, which retains the catechol ring but has a smaller primary amine side chain compares to dopamine, was also able to induce multimerization, though this was weaker than DA. PCV, another catechol, was tested for ability to affect multimerization. PCV stimulated multimerization at a higher degree than the biogenic catecholamines (Fig 11A). Partial reversal of catecholamine-enhanced multimerization was seen in presence of reducing agents (Fig 11B).

Figure 11. Synthetic catechols and NTF oligomerization.

Figure 11.

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Catechol containing chemicals dopamine (DA), 2,4-dihydroxybenzylamine (3,4-DHBA), and pyrocatechol violet (PCV) were tested for reactivity with NTF. NTF was incubated with increasing doses of DA, 3,4-DHBA, and PCV for 3 hours at 37°C. Samples were boiled in nonreducing sample buffer (A) or reducing sample buffer (B) and electrophoresed on a 10–20% tricine gel (ThermoFisher). NTF monomers and multimers were detected using UMI-D antibody. DA and 3,2-DHBA increased NTF multimerization in a dose dependent manner. Presence of PCV also increased multimerization, as detected by UMI-D. Addition of beta-mercaptoethanol reversed most catechol-enhanced multimerization. All experiments were performed at least three times with similar results.

The weaker effect of epinephrine on oligomerization suggested that the primary amine group of the biogenic catecholamine may participate in multimerization. To test whether amines could affect the multimerization reaction, we co-incubated DA and NTF with a series of amines. Spermidine, tyramine, and dihyrdoxybenzylamine (which was a weaker multimerization agent than DA) did not demonstrate blockade of NTF multimerization when the amines were added before or together with DA (not shown).

On the other hand, the addition of anti-oxidant agents such as ascorbate (Fig 12A) or reduced glutathione (GSH) were effective in blocking NTF multimerization (Fig 12B). Vitamin C and GSH were mixed with DA and NTF and incubated overnight at 37°C. Samples were then run on a 10–20% SDS tricine gel (ThermoFisher). 12.8mM Vitamin C and 3.2mM GSH was sufficient to partially inhibit NTF multimerization.

Figure 12. Identifying inhibitors of catecholamine-enhanced multimer formation.

Figure 12.

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NTF was incubated overnight at 37°C in the presence of 0.8mM dopamine and increasing concentrations of either vitamin C (A; Vit C) or glutathione (B; GSH). Samples were boiled in nonreducing sample buffer and ran on 10–20% tricine gels (ThermoFisher). Western analysis was performed using UMI-D to detect NTF. 12.8mM Vit C or 3.2mM GSH partially prevented catecholamine-enhanced multimerization of NTF, while 12.8mM GSH completely inhibited NTF multimerization. Experiments were performed at least four times with similar results.

UMI-D and UMI-F antibodies preferentially recognize oligomerized NTF

In an earlier study, we show that UMI-D and UMI-F recognizes the C-terminus of the NTF sequence and targets pathological vessels in CADASIL (Young et al., 2020). These two monoclonal antibodies thus recognize pathological forms of NTF. Characterization of the forms of NTF that are recognized by these antibodies may shed light on the nature of NTF in CADASIL. We formally tested if UMI-D and UMI-F react preferentially with oligomerized NTF. Synthetic NTF peptides were analyzed by Western blotting against UMI-D (Fig 13A) and UMI-F (Fig 13B). UMI-D and UMI-F exhibit higher binding to multimeric forms of the peptide compared to monomers detected by Coomassie Blue staining (Fig 13C) and silver staining (not shown). Quantification reveals that UMI-D and UMI-F immunoreactivity to multimers was on average 17.4 fold higher and 16 fold higher respectively, compared to monomer reactivity (Fig 13D). The UMI-D and UMI-F antibody preference for multimerized NTF was consistent for at least three independent experiments. This suggests that the forms of NTF recognized by UMI-D and UMI-F in CADASIL vessels include protein multimers.

Figure 13. UMI-D and UMI-F preferentially recognize multimerized NTF.

Figure 13.

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NTF was incubated for either 0 or 1 day at 37°C and run under nonreducing conditions on 8–16% tris-glycine gels (ThermoFisher). Western analysis of NTF multimers was performed using UMI-D (A) and UMI-F (B) which preferentially label degenerating vessels in CADASIL. Additional gel was stained with Coomassie Blue (C), demonstrating majority of the NTF at the monomer level. Silver stained gel (not shown) demonstrated similar findings as Coomassie Blue staining. Immunoreactivity was quantified for antibodies and Coomassie staining using Li-Cor Odyssey software. Analysis involved comparing the ratio of multimer to monomer NTF detected using antibodies to the ratio detected using Coomassie Blue (D). Three independent experiments demonstrated antibody preference for multimerized NTF compared to Coomassie staining (UMI-D: 17.4x, p=.003; UMI-F: 16.017x, p=.0002).

Discussion

Age-dependent disruption of protein stability hallmarks the vast majority of neurodegenerative disorders. Vascular diseases, such as small vessel disease of the brain, are strongly age-dependent and feature arterial protein accumulation; yet proteopathic changes in vascular diseases have not garnered the same degree of attention as protein destabilization disorders such as AD and PD. Here, we provide evidence that a peptide found in the SVD CADASIL undergoes changes that are analogous to those found in the core neurodegenerative diseases. In particular, we find that a peptide fragment of NOTCH3, NTF, oligomerizes and that protein aggregates are promoted by catecholamines. Our studies suggest that overlapping molecular mechanisms may drive neurodegenerative disorders and cerebral small vessel disease.

Spontaneous multimerization of a pathological NOTCH3 fragment

Multimerization of proteins has been demonstrated in AD and PD, and other neurodegenerative diseases. In AD, the 42 amino acid fragment, A-beta, spontaneously forms oligomeric aggregates that are believed to mediate disease pathology (Bitan et al., 2003; Lee et al., 2017; Sengupta et al., 2016). Tau, another protein involved in AD and a spectrum of other pathologies, forms oligomers as well. The different oligomeric forms of Tau are capable of templating normal Tau into pathological forms that could perpetuate within brain tissue (Castillo-Carranza et al., 2014; Gerson and Kayed, 2013; Lasagna-Reeves et al., 2012).

We have previously demonstrated that NOTCH3 undergoes N-terminal fragmentation to generate NTF in CADASIL tissue (Young et al., 2020). Here, we find that this fragment, NTF, also forms oligomers upon prolonged incubation without catecholamines. In the case of NTF, the oligomerization is mediated by disulfide bonding. This fits well with the molecular genetics of CADASIL in which abnormalities in disulfide pairing of thiols are thought to play a role in disease progression. We propose that NTF, which is spontaneously formed by cleavage of NOTCH3 and facilitated by mutations in cysteine residues, is capable of forming intermolecular disulfide bonds leading to larger molecular weight complexes (Young et al., 2020). The facile disassembly of complexes by reducing agents suggests that disulfide bonding drives multimerization. Of the multimerizing proteins described to date, only Tau has been suggested to utilize cysteines for multimerization (Sahara et al., 2013; 2007). Since the mechanism of NTF oligomerization (in the absence of catecholamines) appears to be via formation of disulfide linkages between strings of molecules, it is possible that NTF forms complex branched structures.

Previous work has shown that NOTCH3 is capable of forming complexes with itself and to other proteins (Duering et al., 2011; Meng et al., 2012; Monet-Leprêtre et al., 2013; Zhang et al., 2015). It is possible that should NTF form complexes with itself and NOTCH3, that large heterogeneous complexes with poor solubility could form in vivo in CADASIL vessels. Further work will need to be done to explore whether NTF nucleation of heteromeric complexes via disulfides could contribute to pathological structures such as GOM.

Potent enhancement of multimerization by catecholamines

Physiologically, catechols are regulators of blood flow in the brain. Pathologically, catechols, in particular dopamine, have been implicated in proteinopathies such as Parkinson’s disease, where it potentially facilitates multimerization of synuclein. However, the potential involvement of catechols in affecting the homeostasis of brain vascular proteins implicated in cerebrovascular diseases has not been explored. Here, we show that 1) catecholamines selectively destabilize NTF, resulting in oligomerization; 2) catecholamines dopamine and norepinephrine are more effective than other catechols in multimerizing NTF; 3) agents capable of binding or inactivating quinones inhibit the multimerization reaction; 4) unlike spontaneously formed NTF oligomers, catecholamine-stimulated NTF multimers acquire reducing agent-resistance.

One possible route by which catecholamines react with proteins includes a pathway that involves 1) oxidation of catechols to quinones; 2) reaction of quinones with thiols of reduced cysteines; 3) non-enzymatic conjugation of quinones to one another. The partial sensitivity to reducing agents suggests that the reaction is chemically complex, and also involves disulfide bond formation, which, under our current understanding, cannot be accounted for by catecholamine chemistry. Under alkaline conditions, there is likely increased oxidation of DA (Graham, 1978). The increase in multimerization mediated by DA in alkaline conditions (Fig 8) provides support for the idea that the redox state of DA is involved in multimerization.

Observational studies suggest that small vessel disease correlates with accelerated motor changes in Parkinson’s disease(Schwartz et al., 2018). Currently, a common therapy for patients with Parkinson’s includes administration of dopamine. Because we found that catecholamines such as dopamine accelerate multimerization of a NOTCH3 fragment abundant in inherited small vessel disease, the relationship between catecholamines and NTF should be kept in mind as future treatments and therapies are being developed.

Need for future studies

There are many biochemical and biophysical techniques to characterize protein multimerization in addition to running nonreducing SDS PAGE gels. For example, dynamic light scattering, native mass spectrometry, matrix-assisted laser desorption/ionization, have been widely utilized to identify cross-linking of proteins. Furthermore, in Alzheimer’s disease, a-beta oligomers and fibrils are readily detectable by transmission electron microscopy and thioflavin-T, thioflavin-S, and congo red assays. Further studies will be needed to better characterize biophysical properties of NTF multimers in CADASIL and to determine whether NTF multimers are similar to other proteins enriched in neurodegeneration.

One limitation of this study is that in vitro assessments of multimerization use short time spans and higher concentrations of peptide and catecholamines. It is unclear if the same concentrations are present in brain vessels, in which catecholamines are likely lower than in neurons. Animal models in which NTF is expressed in the context of vascular catecholamines will be useful to address this. Although antibodies to NTF show a strong preference for multimerized NTF, further studies are needed to assess whether multimerized NTF is found in human CADASIL tissue.

Several factors were found to modify NTF oligomerization. In addition to pH, protein concentration, and catecholamine concentration, there could be many effectors involved in the modulation of NTF multimerization. For example, in Alzheimer’s disease and prion proteins, it is thought that aggregated proteins or seeds can induce the aggregation of normal proteins(Harper and Peter T Lansbury, 2003). Additional studies are needed to determine if similar mechanisms play a role in NTF multimerization.

In addition, it is not year clear whether there are consequences of NTF multimerization. The large molecular weight of the complexes in gels suggests a high valency which has the potential to complex multiple proteins in aggregates that are seen in small vessel disease. There is precedent for NOTCH3 forming complexes with itself and to other proteins (Duering et al., 2011; Meng et al., 2012; Monet-Leprêtre et al., 2013; Zhang et al., 2015). As such, heteromeric complexes involving other vascular proteins could increase the potential number of pathological molecules generated by this process.

Furthermore, studies will need to be conducted to determine if accumulation of multimerized NTF contributes to pathophysiologic changes at the cellular and organismal levels. One possibility is that multimerized NTF accumulates, complexes with other proteins, and leads to vascular smooth muscle cell toxicity and death, ultimately contributing to the characteristic vascular changes in human CADASIL disease. If so, prevention and/or neutralization of multimerization of NTF may represent a therapeutic target. In fact, recent studies have demonstrated the neutralizing potential of NOTCH3 ectodomain monoclonal antibody therapies in preclinical mouse models of CADASIL(Ghezali et al., 2018).

Conclusions

In summary, our results support the spontaneous thiol mediated oligomerization of NTF and enhancement of this process by catecholamines. The latter suggest that catecholamines not only affect neurons in the brain, but confer instability of pathological proteins in blood vessels. Given that both neurodegenerative conditions and vascular diseases of the brain are common and frequently co-existent in patients, developing antidotes to catecholamine-mediated proteinopathies should be further explored.

Highlights.

  • A NOTCH3 protein cleavage product (N-terminal fragment; NTF) undergoes spontaneous, cysteine-mediated multimerization in vitro

  • Catecholamines interact with NOTCH3, facilitating protein multimerization in vitro

  • Catechol-like components are incorporated into NTF multimers

  • Inhibitors and reversal agents of NTF multimerization include compounds that block oxidation, suggesting the role of protein oxidation state in NTF multimerization

Acknowledgments

We thank Dr. Venkatesha Basrur and the Proteomic Resource Facility at the Department of Pathology, University of Michigan, for help with the mass spectrometry experimentation and analysis. We thank Soo Jung Lee for helpful discussions and for technical assistance with protein studies. We also thank Simon G. Keep for his assistance with proofreading the manuscript.

Funding Sources

This study was funded by the National Institutes of Health (NS099783, NS099160, HL108842, T32-HL125242, and T32 GM007863) and the U.S. Department of Veterans Affairs (BX000375 and BX003824).

Abbreviations

CADASIL

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy

AD

Alzheimer’s disease

A-beta

Amyloid Beta Protein

PD

Parkinson’s disease

DA

Dopamine

DOPA

Dihydroxyphenylalanine

GSH

Glutathione

NE

Norepinephrine

NTF

N-terminal fragment of NOTCH3

HC

Homocysteine

3,4-DHBA

Dihydroxybenzylamine

PCV

Pyrocatechol violet

Epi

Epinephrine

SVD

Small Vessel Disease

PBS

Phosphate Buffered Saline

Footnotes

Declaration of Interests: none.

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