Essential requirement for nicastrin in marginal zone and B-1 development

Jin Huk Choia,b,1,2, Jonghee Hanc,1, Panayotis C. Theodoropoulosa,d, Xue Zhonga, Jianhui Wanga, Dawson Medlera, Sara Ludwiga, Xiaoming Zhana, Xiaohong Lia, Miao Tanga, Thomas Gallaghera, Gang Yuc, and Bruce Beutlera,2

aCenter for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390; bDepartment of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; cDepartment of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; and dDepartment of Internal Medicine, Physician Scientist Training Program, Washington University in St. Louis, Barnes Jewish Hospital, St. Louis, MO 63110

Contributed by Bruce Beutler, January 7, 2020 (sent for review September 24, 2019; reviewed by Douglas J. Hilton and Ellen V. Rothenberg) γ-secretase is an intramembrane protease complex that catalyzes peritoneal cavity, and are maintained by self-renewal throughout the proteolytic cleavage of amyloid precursor protein and Notch. the life of the organism (10). It is well established that the is Impaired γ-secretase function is associated with the development also required for B-1 (especially B-1a) cell development (11); of Alzheimer’s disease and familial acne inversa in humans. In a however, the underlying mechanism(s) that mediate B-1 cell dif- forward genetic screen of mice with N-ethyl-N-nitrosourea-induced ferentiation remain largely unknown. mutations for defects in adaptive immunity, we identified animals The γ-secretase protease complex cleaves multiple type I mem- within a single pedigree exhibiting both hypopigmentation of brane proteins, including amyloid precursor protein (APP) and the fur and diminished -independent (TI) antibody responses. Notch. APP undergoes proteolytic processing by either α-or The causative mutation was in Ncstn, an essential gene encoding β-secretase to release soluble APP ectodomains into the extra- the protein nicastrin (NCSTN), a member of the γ-secretase complex cellular space. Then γ-secretase cleaves the remaining membrane- that functions to recruit substrates for proteolysis. The missense anchored APP C-terminal fragments (APP-CTFs) and generates mutation severely limits the glycosylation of NCSTN to its mature p3 (the byproduct of α-andγ-secretase cleavages) or amyloid β form and impairs the integrity of the γ-secretase complex as well as peptides (the byproduct of β- and/or γ-secretase cleavage) together its catalytic activity toward its substrate Notch, a critical regulator of with the APP intracellular domain (12). Notch plays essential roles B cell and T cell development. Strikingly, however, this missense in thymic T cell lineage commitment (13), as well as in specifica- IMMUNOLOGY AND INFLAMMATION mutation affects B cell development but not thymocyte or T cell tion of MZ B cell versus B-2 cell fate (14), and it undergoes a development. The Ncstn allele uncovered in these studies reveals series of proteolytic cleavages by ADAM family metalloproteases an essential requirement for NCSTN during the type 2 transitional- and γ-secretase to generate the Notch intracellular domain (NICD) marginal zone precursor stage and peritoneal B-1 B cell develop- (15). The γ-secretase complex consists of four core subunits: ment, the TI antibody response, fur pigmentation, and intestinal presenilin (PS), PS enhancer 2 (PEN-2), anterior pharynx-defective homeostasis in mice. 1 (APH-1), and nicastrin (16). Nicastrin is a type I membrane protein with a large extracellular domain (17) that functions as nicastrin | marginal zone B cells | B-1 B cells | T cell-independent antibody response Significance

cell responses to are classified as T cell-dependent Follicular B cells generally require T cell help to contribute to B(TD) or T cell-independent (TI) based on their need for adaptive immunity, producing several isotypes of immuno- T cell help in antibody production. Antigens eliciting a TD an- globulins that carry out distinct effector functions. Two spe- tibody response are proteins that are processed and presented to cialized B cell subsets, marginal zone (MZ) and B-1 B cells, arise helper T cells in the context of MHC II molecules. The TD from different developmental steps and have different func- antibody responses are mediated by follicular B cells (also known tions. They become activated to produce antibodies without as B-2 cells, the major B cell subset in the body) and are long- T cell help and are the major sources of plasma IgM. We identi- lasting to deploy high-affinity antibodies of multiple isotypes. In fied a viable missense allele of Ncstn in a forward genetic contrast, TI antigens, such as bacterial capsular polysaccharides screen. We discovered that the development of MZ and B-1 and viral capsids, stimulate antibody responses that do not require B cells, TI antibody response, fur pigmentation, and intestinal MHC II-restricted T cell help (1). The TI antibody response is homeostasis critically depend on the glycosylation status of mediated by the marginal zone (MZ) and B-1 B cell populations, NCSTN and the catalytic activity of γ-secretase toward its sub- which expand on immunization in extrafollicular sites (2–4) and strate Notch, a critical receptor in numerous developmental confer protective immunity by producing -specific IgM decisions. without somatic hypermutation (4–7). Thus, TI responses give rise to less specific but more immediate protection compared with Author contributions: J.H.C., J.H., P.C.T., G.Y., and B.B. designed research; J.H.C., J.H., TD antibody responses. P.C.T., X. Zhong, and J.W. performed research; D.M., S.L., X. Zhan, X.L., and M.T. contrib- uted new reagents/analytic tools; J.H.C., J.H., P.C.T., G.Y., and B.B. analyzed data; B-2 cells are continuously replenished from precursors in bone and J.H.C., J.H., T.G., G.Y., and B.B. wrote the paper. marrow, where they undergo both positive and negative selection. Reviewers: D.J.H., Institute of Medical Research; and E.V.R., California Institute of Immature B cells in migrate to the spleen, where Technology. they differentiate through two transitional stages and become The authors declare no competing interest. mature naïve B-2 cells (8) or, alternatively, MZ B cells. Their fate Published under the PNAS license. is determined during the transitional stages and depends on signals 1J.H.C. and J.H. contributed equally to this work. κ from the B cell receptor, B cell activating factor, nuclear factor 2To whom correspondence may be addressed. Email: [email protected] or light chain enhancer of activated B cells, and Notch2, as well as [email protected]. signals involved in anatomical retention of MZ B cells in the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ spleen (9). In contrast, B-1 cells are generated mainly from fetal doi:10.1073/pnas.1916645117/-/DCSupplemental. liver progenitors rather than bone marrow precursors, reside in the

www.pnas.org/cgi/doi/10.1073/pnas.1916645117 PNAS Latest Articles | 1of8 Downloaded by guest on September 27, 2021 a γ-secretase substrate receptor (18). Activation of the γ-secretase screens, we identified several mice from a single pedigree complex requires extensive N-linked glycosylation of nicastrin, exhibiting hypopigmentation of the fur (Fig. 1A) and diminished which helps stabilize the protein (19). Mutations in γ-secretase TI antibody responses to (4-hydroxy-3-nitrophenyl) acetyl-Ficoll complex proteins and impaired catalytic activity of the complex (NP-Ficoll) compared with wild-type C57BL/6J mice (Fig. 1B). have been implicated in Alzheimer’s disease (AD) (20), fa- The mice exhibited comparable TD antibody responses to alumi- milial type acne inversa (21), hypopigmentation (22, 23), and num hydroxide-precipitated ovalbumin (OVA/alum; Fig. 1C). The thymic hypoplasia (24); however, little is known about the role phenotype, named truffle, was transmitted as a recessive trait. By and function of the γ-secretase complex in B cell-mediated automated meiotic mapping (25), the truffle phenotype was cor- immunity. Here we describe the effect of a severely hypomor- related with a missense mutation in Ncstn (Fig. 1D), resulting in a phic but viable missense mutation of Ncstn on MZ B cell and valine (V)-to-glycine (G) substitution at position 439 (V439G) in B-1 B cell development. the DYIGS and peptidase homologous (DAP) domain of the NCSTN protein (Fig. 1E)(18),whichwaspredictedtobedam- Results aging by PolyPhen-2 (score = 1.000) (26). Identification of a Viable Ncstn Missense Mutation. To identify genes To verify causation, CRISPR/Cas9-mediated gene targeting was required for the development and function of adaptive immu- used to generate a single nucleotide replacement allele (A→Cat nity, we carried out a forward genetic screen in third-generation chr1_172,070,009), causing the same amino acid change as that (G3) C57BL/6J mice bred to carry homozygous and heterozy- caused by the ENU-induced mutation (V439G). Consistent with gous mutations induced by N-ethyl-N-nitrosourea (ENU) in their truffle mice, NcstnV439G/V439G mice showed hypopigmentation of the great-grandsires (G0). During the course of adaptive immunity fur (Fig. 1F) and diminished TI and normal TD antibody responses,

A B CD 4 4 10 m)

m) Ncstn Wild-type truffle n -10

0 8 p=5.35 ×10 3 3 ) 45 10 6 @ -log

2 D 2 4 truffle truffle G (O 1 1 -value ( 2 P

0

NP IgM (OD0 @ 450 n 0

OVA Ig 123 45679 8 10 11131817 19 X WT G3 WT G3 Genomic location E SP DAP domain TM

132 134 273 498 669 691708 V439G (truffle) FGH I J V439G/ 4 4 110 ** 8 ** +/+ V439G m) *** n 0 cm)

100 ( 7

3 45 3 @

2 D 2 90 6 al body weight i G (O

M(OD@450nm) 1 1 80 5 Colon length %init

NP Ig 0 0 70 4 V439G/ OVA Ig +/+ V439G/ +/+ V439G/ +/+ V439G/ +/+ V439G V439G V439G V439G

+/+ males (n=18) KL+/+ females (n=22) 50 V439G/V439G males (n=29) V439G/V439G females (n=40)

40 + / + 30 l prolapse 20

10 %Recta V439G V439G/ 0 02479121416 Age (months)

Fig. 1. The truffle phenotype. (A)Photographofamaletruffle mouse and wild-type littermate. (B) Decreased TI NP-specific antibodies in truffle mice (highlighted in gray dots) at 6 d after NP-Ficoll immunization. Data are from 247 G3 mice in a screen group that includes the truffle pedigree. Wild-type (C57BL/6J) mice immunized with the same antigen served as controls. Data are presented as absorbance at 450 nm. (C) Normal TD OVA-specific anti-

bodies in truffle mice at 14 d after immunization with OVA/alum. (D) Manhattan plot. −Log10 P values (y-axis) plotted against the chromosomal positions of mutations (x-axis) identified in the affected pedigree. (E) Protein domains of mouse NCSTN (708-aa long). The location of the truffle mutation, which results in substitution of valine 439 to glycine (V439G) in Ncstn, is highlighted in red; SP, signal peptide; DAP, DYIGS and peptidase homologous; TM, transmembrane. (F) Photograph of a male mouse with CRISPR/Cas9-induced V439G substitution (NcstnV439G/V439G)andawild-typelittermateatage8wk. (G and H)TI(G)orTD(H) antibody responses in mice with the indicated genotypes for Ncstn following immunization with NP-Ficoll or OVA/alum, re- spectively. Data are presented as absorbance at 450 nm. (I and J) Weight loss (I)andcolonlength(J) analysis of mice with indicated genotypes on day 10 of treatment with 1.3% DSS. (K) Incidence of spontaneous rectal prolapse in wild-type and NcstnV439G/V439G mice. (L) Macroscopic view of rectal prolapse in NcstnV439G/V439G mice. Each symbol represents an individual mouse. P values were determined by Student’s t test. Results are representative of two (G and H)orthree(I and J) independent experiments with 4 to 20 mice per genotype. Error bars indicate SD.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1916645117 Choi et al. Downloaded by guest on September 27, 2021 respectively, to NP-Ficoll and OVA/alum immunization (Fig. 1 G corner of the scatterplot compared with that of wild-type litter- and H) compared with wild-type littermates. These data conclusively mates (Fig. 2L). In the spleen, newly formed B cells that have truffle established a causative relationship between the Ncstn muta- migrated from the bone marrow undergo distinct transitional B tion and the observed phenotype in NcstnV439G/V439G mice. cell stages (type 1 to 3 transitional B; T1 to T3) (9, 30). T2 B cells Previous studies demonstrated the biological significance of are direct precursors of follicular B cells, but a subset of these cells, γ-secretase protease-mediated Notch signaling in the mainte- termed T2 to MZ precursors (T2-MZP), are known as precursors nance of intestinal homeostasis and host defense functions in of MZ B cells (31). MZ B cells and B-1 B cells, which predominate mice (27–29). To assess the effect of a viable Ncstn missense in the splenic MZ and peritoneal cavities, respectively, each con- mutation on intestinal homeostasis, NcstnV439G/V439G mice and tribute a massive wave of serum IgM production in response to wild-type littermates were given 1.3% dextran sodium sulfate immunization with TI antigens (4). (DSS) in their drinking water for 7 d, and body weight was Importantly, a previous study demonstrated that conditional measured on days 0 and 10 after initiation of DSS treatment. deletion of Notch2, which encodes one of the substrate proteins The NcstnV439G/V439G mice developed severe colitis, losing 13.9% for the γ-secretase complex, results in complete arrest of MZ B of their initial body weight by day 10 of treatment, compared with cell development in mice (14). In contrast to Notch2, Notch1 has an average 3.5% of initial body weight lost by wild-type littermates an indispensable function in early T cell development in mice (Fig. 1I). The NcstnV439G/V439G mice treated with DSS also dis- (13). We observed a normal number of T1 and T2 transitional B played greater shortening of the colon compared with wild-type cells in the of NcstnV439G/V439G mice compared with littermates (Fig. 1J). In the absence of DSS, NcstnV439G/V439G mice wild-type littermates (Fig. 2J),butthedevelopmentofT2-MZP often developed rectal prolapse. A higher frequency of prolapse cells (Fig. 2 J and P) and MZP-MZ B cells (Fig. 2 J, Q,andR) was observed among females compared with age-matched males was significantly impaired. Furthermore, the frequency (Fig. (37.5% vs. 10.3%) (Fig. 1 K and L). Spontaneous rectal prolapse 2S)andnumbers(Fig.2J) of peritoneal B-1 B cells, including was very uncommon in wild-type mice (Fig. 1 K and L). These data the B-1a subpopulation (Fig. 2T), were significantly reduced in demonstrate that hypopigmentation of fur, impaired TI antibody NcstnV439G/V439G mice compared with wild-type littermates. response, and diminished intestinal homeostasis all result from a The proportion of the B-1b subpopulation in peritoneal B-1B viable Ncstn missense mutation in mice. cells was increased (Fig. 2T), but the total number was significantly reduced in NcstnV439G/V439G mice compared with wild-type litter- Impaired Immune Cell Development Caused by a Viable Ncstn Mutation mates (Fig. 2J). Consequently, significantly decreased serum IgM in Mice. To further characterize the immunologic defect caused by U V levels (Fig. 2 ), but normal IgG1 levels (Fig. 2 ) were detected in IMMUNOLOGY AND INFLAMMATION Ncstn the mutation, we immunophenotyped mice by complete NcstnV439G/V439G mice compared with wild-type littermates. Col- blood count (CBC) testing and flow cytometry analysis of lym- lectively, these data suggest that decreased peritoneal B-1 B cells phoid and myeloid cells in blood, spleen, , bone marrow, and near-complete arrest of splenic MZ B cell development and peritoneum. Previous studies demonstrated that an inducible caused by a viable Ncstn mutation result in impaired TI antibody Mx1-Cre Vav-Cre Ncstn ( ) or a hematopoietic-specific ( ) deletion of responses in NcstnV439G/V439G mice. caused thymic hypoplasia, an enlarged spleen, and aberrant ac- cumulation of granulocyte-monocyte progenitors (GMPs), leuko- A Cell-Intrinsic Failure of Marginal Zone and B-1 B Cell Development. cytosis, and monocytosis (24). Unlike the phenotype observed in To determine the cellular origins of the Ncstn-associated defects hematopoietic-specific NCSTN-deficient mice, gross examination in MZ B cell and B-1 B cell development, we reconstituted ir- of lymphoid organs showed that the NcstnV439G/V439G mice had no radiated wild-type (CD45.1) or NcstnV439G/V439G mice (CD45.2) + + sign of thymic hypoplasia (Fig. 2A) or an enlarged splenic en- with unmixed Ncstn / (CD45.2), NcstnV439G/V439G (CD45.2), or largement (Fig. 2B). We also examined the hematopoietic stem wild-type (CD45.1) bone marrow cells. The bone marrow cells and progenitor cell (HSPC) populations in the bone marrow. from NcstnV439G/V439G donors were unable to repopulate MZ B ThemissensemutationinNcstn did not appreciably affect the cells and B-1 B cells in wild-type recipients (CD45.1) as effi- total HSPC population, including long-term-hematopoietic ciently as cells derived from wild-type donors (Fig. 3 A and B). In stem cells (HSCs), short-term HSCs, multipotent progenitors, contrast, bone marrow cells from wild-type donors (CD45.1) common lymphoid progenitors, common myeloid progenitors, were able to fully repopulate MZ B cells and B-1 B cells in megakaryocyte–erythrocyte progenitors, or GMPs (Fig. 2C). NcstnV439G/V439G recipients (CD45.2) (Fig. 3 A and B). In ad- Furthermore, CBC testing (Fig. 2D) and cell counts (Fig. 2E) dition, irradiated NcstnV439G/V439G recipients reconstituted with showed that the NcstnV439G/V439G mice had comparable num- + wild-type bone marrow mounted TI antibody responses compa- bers of white blood cells, monocytes, , and CD11b + rable to those of irradiated wild-type recipients engrafted with or CD11c myeloid cells compared with wild-type littermates. wild-type bone marrow (Fig. 3C). However, irradiated wild-type In addition, the development of thymocytes was not impaired recipients engrafted with NcstnV439G/V439G bone marrow showed in NcstnV439G/V439G mice compared with wild-type littermates C + significantly decreased TI antibody responses (Fig. 3 ). This (Fig. 2 F and G). No significant difference was found in CD3 suggests a specific cell-autonomous effect of a viable Ncstn T cell number; however, NcstnV439G/V439G mice had slightly + mutation on MZ B cell and B-1 B cell development and TI fewer CD8 T cells in the spleen compared with wild-type lit- antibody responses. We note that the B-1 B cells examined here H + + termates (Fig. 2 ). Therefore, the CD4 -to-CD8 Tcellratio originated in the bone marrow (32) and might not necessarily NcstnV439G/V439G I was increased in mice (Fig. 2 ). Flow cytometry reflect the phenotype of those originating from fetal liver. analysis of bone marrow showed that NcstnV439G/V439G mice had comparable numbers of B cell progenitors in the prepro-B stage, Effect of a Viable Ncstn Mutation on Mature and Functional γ-Secretase pro-B to pre-B transition, immature stage, and transitional B stage Complex Formation. To investigate the effect of the NcstnV439G/V439G (Fig. 2 J–L). The frequency and numbers of mature recirculating B mutation at a molecular level, we isolated total cell lysates (TCLs) cells in the bone marrow appeared normal in NcstnV439G/V439G from thymus, spleen, lymph nodes, brain, skin, splenic pan T cells, mice compared with wild-type littermates (Fig. 2 J and L); how- and splenic pan B cells from NcstnV439G/V439G mice and wild-type ever, the surface IgD and IgM expression on mature recirculating littermates. TCLs were subjected to Western blot analysis to check BcellsofNcstnV439G/V439G mice was decreased (Fig. 2 M and N). expression levels of γ-secretase complex proteins. The predicted As a result, the mature recirculating B cell populations in the bone molecularmassofNCSTNis∼80 kDa (17). In cells, NCSTN exists marrow of NcstnV439G/V439G mice has shifted to the lower left in higher (150 kDa) and intermediate (110 kDa) molecular

Choi et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 27, 2021 A BDC E 10 8 Gated on LSK+ GatedonLK+ 15 10 6 r een) u 7

10 pl

m 10 s e (

+/+ f r +/+ 10 6 L of blood) 5 10 5 μ pe 2 s l l 5 e 10 +/+ 1 lnumber +/+ l V439G/ C V439G/V439G e V439G/V439G Cells (K/ V439G/ V439G 10 4 0 C 10 4 s s b c V439G C P P P P e il 1 1 SK S SC L t h 1 1 L C M WBC cy D H -H MP C MEP GM o rop D T- n t C C L ST u Mo Ne

F G H 8 I +/+ V439G/V439G ) 10 8 10 +/+ 4 **

s +/+ 5 V439G/V439G SP4: 8±1% DP: 85±2% 8±1% 84±2% V439G/V439G 10 10 7 ymu 3

4 h t

10 ( 6 10 * 3 2 10 10 5 7 10 DN: SP8: 10 4 1 0 CD4:CD8 ratio 4±1% 3±1% 5±2% 3±1% CD4 3 Cell number (spleen) Cell10 number 0 0103 104 0103 104 CD8 DN1 DN2 DN3 DN4 DP SP4 SP8 CD3 CD4 CD8 +/+ V439G/ V439G

J 10 8 Bone marrow Spleen Peritoneum K +/+ V439G/V439G 5

** 10

** Pre-B 6 ** 4 10 10 13.9±4.3% 16.6±5.2% s ** ** 103 Pre-pro B

Cell ** ** 9.8±3.2% 10.0±4.2% 10 4 ** +/+ 0 V439G/V439G Pro-B: 75.6±5.4% 72.2±6.9% 10 2 BP-1 3 4 5 3 4 5 l 01010 10 01010 10 ro o re re re 1 P P O a b p Pr P u T T2 T3 Z MZ F B1 1 CD24 - tu iona t M B B1 e a it a -MZ r m M 2 P ns T Im a Tr L +/+ V439G/V439G M 6000 *** N 800 ** O +/+ V439G/V439G 5 Mature B: 22±3% 16±4% T1: 38±5%T2: 32±7% 23±8% 28±3% 4 Transitional B 10 10 600 4.4±2.3% 5.4 4 4000 FI 10 3 ± 10 Pre-B 1.1% 400 MM 3

57±3% 58±3% g 10 IgD MFI 2000 I 200 0 0 T3: 10±2% 31±5% Immature B: 7±2% 6±3% IgM 3 4 3 4 0 0 IgM 01010 01010 0103 104 105 0103 104 105 IgD +/+ V439G/ +/+ V439G/ CD23 V439G V439G Gated on PQ+/+ V439G/V439G T2-MZP +/+ V439G/V439G R +/+ V439G/V439G 5 5 5 10 MZ MZ 10 MZB 10 8.7±3.0% FOB 0.4±0.2% 4 T2-MZP 70±4% 7±3% 4 10 12.5±3.9% 0.39±0.2% 4 10 70.7± 86.3± 10 3 2.4% 3.9% 103 10 3 10 MZP MZP 2 28±5% 92±3% 10 0 0 1 10 CD21 CD21 IgD 0103 104 105 0103 104 105 0103 104 105 0103 104 105 0103 104 105 0103 104 105 IgM CD23 CD23

ST+/+ V439G/V439G Gated on B1 +/+ V439G/V439G U 200 *** V 3

5 5 g/ml) 10 10  B2 B2 150 4 2

4 M( 9.8±3.2% 7.4±2.4% 10 10 B1a B1a g

62±4% 24±5% 100 IgG1 (mg/ml)

3 l 3 al I 10 10 B1 B1 B1b B1b ot 1 19.0± 4.98± 30±5% 55±9% 50 0 4.5% 2.3% 0 mt CD5 B220 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 0 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Seru +/+ V439G/ Serum tota +/+ V439G/ CD19 CD43 V439G V439G

Fig. 2. Impaired development caused by a Ncstn missense mutation in mice. (A and B) Representative photographs of thymus (A) and spleen (B). (C) Total numbers of stem and progenitor cell subsets per femur in the bone marrow of NcstnV439G/V439G mice and wild-type littermates as determined by flow cytometry. The FACS gating strategy for HSC subsets is described in detail in SI Appendix, Fig. S1. LSK+, Lineage-Sca-1+c-Kit+;LK+, lineage-Sca-1-c-Kit+.(D) + + Whole blood cell counts in 12-wk-old NcstnV439G/V439G mice and wild-type littermates. (E) Numbers of CD11b and CD11c myeloid cells in the spleen of 12-wk-old NcstnV439G/V439G mice and wild-type littermates. (F and G) Flow cytometry scatterplots of thymocytes (F) and total numbers of thymocyte subsets per thymus (G) isolated from 12-wk-old NcstnV439G/V439G mice and wild-type littermates. Numbers adjacent to outlined areas or in quadrants indicate the per- centage of cells in each. Thymocytes were analyzed by flow cytometry for CD4, CD8, CD25, and CD44 surface markers. The FACS gating strategy for thymic T cell development is described in detail in SI Appendix, Fig. S2.(H and I) Numbers (H) and ratio (I) of splenic T cells in 12-wk-old NcstnV439G/V439G mice and wild- type littermates. (J) Numbers of B cell subsets in bone marrow, spleen, and peritoneal cavity of 12-wk-old NcstnV439G/V439G mice and wild-type littermates. (K, L, and O–T) Representative FACS plots showing B cell development in the bone marrow (K and L), spleen (O–R), and peritoneal cavity (S and T) from 12-wk-old NcstnV439G/V439G mice and wild-type littermates. The FACS gating strategy for B cell development is described in detail in SI Appendix, Fig. S3.(M and N) Surface IgD (M) and IgM (N) expression on peripheral blood B cells in 12-wk-old NcstnV439G/V439G mice and wild-type littermates. (U and V) Serum IgM (U)and IgG1 (V) concentration in unimmunized mice at 12 wk of age with indicated genotypes for Ncstn. Each symbol represents an individual mouse. P values were determined by Student’s t test. Data are representative of three independent experiments with 4 to 11 mice per genotype. Error bars indicate SD.

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1916645117 Choi et al. Downloaded by guest on September 27, 2021 +/+BM>WT A B WT BM > V439G/V439G WT BM (CD45.1) V439G/V439G V439G/V439G BM > WT +/+ BM (CD45.2) > V439G/V439G BM (CD45.2) 12 100 > WT (CD45.1) (CD45.2) > WT (CD45.1) **

5 (%) 10 *** 90

98.6±1.2% 1.14±0.7% 99.0±0.4% B 4 8 10 80

3 0.14±0.12% 97.8±2.0% 0.21±0.1% 70 10 4

0 Follicular B (%) 60 Marginal zone

CD45.2 0 50 0103 104 105 0103 104 105 0103 104 105 CD45.1 20 *** 60 5 MZB (%) FOB (%) 10 5.72% 6.59% 0.20% 15 C 79.6% 81.7% 86.8% C 4 40 10 P

3 in 10 s sinP

10 l el 2 ell 20 10 5 B1 c 1 B2 c 10 CD45.2 CD45.1 CD45.2 0 0 CD21 0103 104 105 0103 104 105 0103 104 105 CD23 3 +/+BM>WT C WT BM > V439G/V439G 105 B2 CD45.2 B2 CD45.1 B2 CD45.2 V439G/V439G BM > WT 42.4% 40.9% 34.9% 2 104 IgM

3 P

10 N 1

B1 B1 B1 D@450nm)

15.7% 13.3% 3.13% O 0 ( 0 B220 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 23456 CD19 Serum dilution (log10)

Fig. 3. A cell-intrinsic failure of MZ and B1 B cell development. (A) Repopulation of donor-derived in the peripheral blood (Top), MZ and follicular B cells in the spleen (Middle), and B1 cells in the peritoneal cavity (Bottom) of recipients at 10 wk after reconstitution of bone marrow isolated from mice with indicated genotypes. Numbers adjacent to outlined areas or in quadrants indicate percent cells in each population. (B) The frequencies of B cell subsets in the bone IMMUNOLOGY AND INFLAMMATION marrow chimeras as determined by flow cytometry. (C) TI antibody responses after immunization with NP-Ficoll in bone marrow chimeras at 10 wk after re- constitution. Data are presented as absorbance at 450 nm. Each symbol represents an individual mouse. P values were determined by one-way analysis of variance with Dunnett’s test for multiple comparisons. Data are representative of two independent experiments with four to nine mice per genotype. Error bars indicate SD.

weight forms due to glycosylation events (33). It has been shown To further characterize mutant NCSTN, we subjected TCLs to that a functional γ-secretase complex has the 150-kDa glycosylated glycosidase analysis. Peptide:N-glycosidase F (PNGase F) treat- form of NCSTN (34). Immunoblot analysis revealed significant ment, which removes all N-linked oligosaccharides regardless of reduction in the quantity of the 150-kDa form of NCSTN in TCLs complexity, fully converted wild-type and V439G NCSTN into an isolated from NcstnV439G/V439G mice compared with those from ∼70-kDa nonglycosylated form (highlighted by purple arrowheads wild-type littermates (highlighted by the red arrowhead in Fig. 4A). in Fig. 4B). This suggests that both wild-type and mutant NCSTN

A B C IP: IgG IP: FLAG D EV EV EV EV NCSTN-V439G NCSTN-V439G NCSTN-V439G NCSTN-V439G NCSTN-WT NCSTN-WT Lymph pLVX-FLAG- NCSTN-WT NCSTN-WT Thymus Spleen nodes Brain Skin B cells T cells pLVX-FLAG- Ncstn -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- EV NCSTN-WT NCSTN-V439G NCSTN-WT NCSTN-V439G EV

G Splenocytes Ncstn -/- -/- -/- -/- -/- -/- 9G 9G 9 MW 720 39G 39G 3 MW 150 IP 4 43 439G 4 439G 43 (kDa) V V V V4 Ncstn +/+ V439G/V439G (kDa) / /V / IB: FLAG 480 G/V G G/V G 9 9G/ 100 39G 3 39 39 39G/ MW 39 + + + + + 4 43 4 (kDa) / 242 +/ V +/ V4 +/ V4 +/+ V4 V4 + V +/ +/+ V IB: PS1-NTF MW 25 150 146 (kDa) IB: NT NT Endo H PNGase F PNGase F IB: Endo H 15 IB: PEN2 NCSTN 150 66 IB: NCSTN PS1-NTF IB: PEN2 IB: APH1 100 150 Input Notch1-N100-FLAG Notch2-N100-FLAG 15 IB: FLAG IB: 100 IB: 100 E APP-CTF NCSTN 10 37 75 Ncstn V439G/ V439G +/+ V439G +/+ +/+ +/+ V439G/ V439G V439G/ V439G/ V439G 25 IB: IB: PS1-NTF GSI + + - - + + -- PS1-NTF MW 25 N100 (kDa) 15 IB: IB: NICD 50 37 15 α-tubulin GAPDH IB: PEN2 IB: FLAG

Fig. 4. Effect of a Ncstn mutation in mature and functional γ-secretase complex formation. (A) Immunoblot analysis of NCSTN, APP-CTF, and PS1-NTF in TCLs of + + thymus, spleen, lymph nodes, brain, skin, splenic B220 ,andCD3 T cells from NcstnV439G/V439G mice and wild-type littermates. The red arrowhead indicates the mature form of NCSTN; blue arrowhead, the intermediate form of NCSTN. (B) Glycosidase treatments of NCSTN in splenocytes isolated from NcstnV439G/V439G mice or wild-type littermates. Endo H and PNGase F treatments show the sensitivity of the various bands to deglycosylation. The purple arrowhead indicatesnon- − − glycosylated NCSTN. (C) TCLs isolated from Ncstn / MEFs stably expressing FLAG-tagged wild-type, mutant (V439G) NCSTN, or empty vector (EV) were immu- noprecipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against FLAG, PS1, and PEN2. The red arrowhead indicates the mature form of NCSTN; blue arrowhead, the intermediate form of NCSTN; black arrowhead, full-length PS1; green arrowhead, PS1-NTF. (D) BN-PAGE analysis of γ-secretase subunits (nicastrin, PS1, PEN2, and APH1) from an equivalent quantity of total membrane proteins from Ncstn−/− MEFs stably expressing FLAG-tagged wild-type, mutant (V439G) NCSTN, or EV control. (E) γ-secretase activity was measured by mixing a FLAG-tagged mouse Notch1 or 2 substrate (N1- or N2-N100) with mi- crosomal proteins isolated from NcstnV439G/V439G mice and wild-type littermates in the presence or absence of the γ-secretase inhibitor DAPT in vitro. N1-N100 and N2-N100 are membrane-tethered Notch fragments resulting from ectodomain shedding of Notch1 and 2, respectively. Both N1- and N2-N100 and their γ-secretase- cleaved product, NICD, were visualized by immunoblotting using an anti-FLAG antibody. Data are representative of three independent experiments.

Choi et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 27, 2021 have N-linked glycosylation. While most N-linked glycans in mature and it controls melanocyte differentiation in hair follicles (22, NCSTN molecules are complex oligosaccharides, some are high- 36), controls hematopoiesis (13, 14), and promotes proliferation mannose residues. When TCLs were treated with endoglycosidase of intestinal crypts (27–29). Through its signaling cascade, H (Endo H), which cleaves only high mannose residues, the γ-secretase—mediated cleavage of Notch releases the NICD, 150- and 110-kDa forms of wild-type NCSTN downshifted to which moves to the nucleus to regulate gene expression. Con- the 110-kDa and 70-kDa forms, respectively. However, Endo H sistent with several phenotypes observed in Notch-deficient mice, treatmentofmutantNCSTNdownshiftedthe110-kDaformto we found that the V439G substitution leads to hypopigmentation the 70-kDa form (Fig. 4B). This result suggests that unlike in of fur and spontaneous colitis in mice, manifested by rectal prolapse. wild-type NCSTN, most of the N-linkedsugarsinmutant Given the widespread effect of altering NCSTN glycosylation in NCSTN are high-mannose residues. Furthermore, TCLs from multiple tissues, we consider it likely that impaired proteolytic NcstnV439G/V439G mice contained an increased quantity of α-or cleavage of Notch also occurs in the skin and intestinal epithelium β-secretase–cleaved APP CTF and a decreased quantity of the of NcstnV439G/V439G mice. Our data suggest that the NCSTN active PS1 species N-terminal fragment (PS1-NTF) compared V439G-containing γ-secretase complex retains some proteolytic with that in wild-type littermates (Fig. 4A). function toward Notch substrates. Assembly of NCSTN with other γ-secretase components oc- It has been reported that there is no heterozygous effect of f/− curs in the ER and glycosylation occurs during transit through Notch1 deletion (Notch1 ;Mx1-Cre) in T cell development (13). the Golgi apparatus (12). To assess the effect of the V439G sub- Notch2 Notch2f/+;Mx1 −/− In contrast, haploinsufficiency of in B cells ( stitution in these cellular processes, we generated Ncstn cell or CD19-Cre) significantly impairs MZ B cell development, with lines stably expressing FLAG-tagged wild-type or mutant NCSTN. mutants displaying between one-sixth and one-fourth the number +/+ As expected, a significant reduction in the higher molecular weight of MZ B cells observed in the control mice (Notch2 ;Mx1-Cre) form (150 kDa) of NCSTN was observed in TCLs extracted from NcstnV439G −/− (14). The phenotypic effects of homozygous suggest Ncstn cells expressing mutant NCSTN compared with cells that these mice have less Notch1/2 activity than has been observed expressing wild-type NCSTN (indicated by the red arrowhead in in heterozygous Notch1 or Notch2 mutants. C Fig. 4 ). In addition, decreased levels of PEN2 and PS1 endo- Comparing the activity of wild-type and mutant NCSTN in the proteolytic fragments (PS1-NTF; indicated by the green arrow- γ-secretase complex revealed that Notch1 processing is more C Ncstn−/− head in Fig. 4 ) were also observed in cells expressing resistant to the effect of mutant NCSTN than Notch2 processing. mutant NCSTN compared with cells expressing wild-type NCSTN. Therefore, Notch2 processing appears to be reduced to a point Since the glycosylation status of NCSTN influences mature at which it causes a defect in MZ B cell and B-1 B cell devel- γ -secretase complex assembly (35), we determined the effect of the opment, while there is sufficient processing of Notch1 to permit γ mutation in binding with other -secretase components. Coim- normal T cell differentiation in NcstnV439G/V439G mice. Com- munoprecipitation assays revealed that mutant NCSTN, which plete ablation of Ncstn causes prenatal lethality in mice due to exists predominantly in an intermediate molecular weight form, is deficient Notch signaling (24). In contrast, offspring with the γ able to interact with other -secretase components, including PS1 NcstnV439G/V439G genotype were born at expected Mendelian C + + and PEN2 (Fig. 4 ). frequencies (P = 0.2698, χ2 test; n = 100 mice: 27 / ,55V439G/+, We next determined whether the mutation affects mature V439G/V439G γ and 18 ). We cannot yet explain why the missense -secretase complex integrity and function. Blue native-PAGE Ncstn allele is viable in mice, but we postulate that residual (BN-PAGE) analysis of the membrane fraction isolated from γ Ncstn−/− -secretase complex activity in these mice is sufficient for cells expressing wild-type or mutant NCSTN indicated critical developmental processes. that the mutant NCSTN can form the mature γ-secretase com- γ ∼ Missense mutations in the PS1/2 subunit of the -secretase plex ( 480 kDa). However, the level of the mature complex is complex cause early-onset familial AD (FAD). The mechanism significantly decreased in cells expressing mutant NCSTN, sug- by which these mutations lead to AD remains controversial (20). gesting that the mutation impairs the integrity of the mature γ D While a prevailing view is that they are gain-of-function muta- -secretase complex (Fig. 4 ). Furthermore, the mutation resul- tions that cause γ-secretase–mediated overproduction of toxic ted in clear inhibition of γ-secretase activity toward Notch2 but β-amyloid peptides in AD brains, others believe that they are had a milder effect on its activity toward Notch1 (Fig. 4E). These loss-of-function mutations during AD pathogenesis. In this regard, results suggest that the glycosylation status of NCSTN influences the viable Ncstn missense allele provides a novel tool for testing the integrity and catalytic activity of the γ-secretase complex. whether partial loss of γ-secretase activity would suppress AD — Discussion phenotypes in mice that is, if PS missense FAD mutations are bona fide gain-of-function mutations. Alternatively, it would be of The role of the γ-secretase complex in regulating intramembrane interest to learn whether the Ncstn V439G mutation facilitates AD proteolysis has been extensively studied and is well recognized. development—that is, if PS FAD mutations contribute to AD via a However, the significance of NCSTN and other γ-secretase proteins in immunity remains unclear due to the lack of viable loss-of-function mechanism. Future research will explore these and animal models. Taking advantage of our forward genetic screen other questions pertinent to the understanding of AD pathogen- esis using this mouse model, including but not limited to the re- of mice with mutations induced by ENU, we have identified a γ previously undescribed γ-secretase–inactivating mutation and lationships among -secretase deficiency, impaired immunity, have demonstrated that the γ-secretase complex/Notch signaling amyloid pathology, neuronal death, and loss of memory. In view of the strong phenotypic effect and importance of NCSTN in diverse axis controls pigmentation, intestinal homeostasis, MZ B cell Ncstn and B-1 B cell development, and TI B cell responses in mice. cellular processes, this viable missense allele will be useful to These phenotypes observed in mice carrying a viable hypomorphic researchers in numerous fields. Ncstn missense mutation of may stem from reduced glycosylation Materials and Methods of NCSTN. Our data indicate that the glycosylation status of NCSTN critically influences the integrity of the γ-secretase com- Mice. C57BL/6J male mice age 8- to 10-wk purchased from The Jackson Laboratory were mutagenized with ENU, as described previously (37). plex, as well as the catalytic activity toward its substrate Notch. γ Mutagenized G0 males were bred to C57BL/6J females, and the resulting G1 The -secretase complex offers a valuable system for studies in males were crossed to C57BL/6J females to produce G2 mice. G2 females multiple fields, because the protein complex cleaves several type were backcrossed to their G1 sires to yield G3 mice, which were screened for I membrane proteins, including Notch, APP, and others. Notch phenotypes. Whole-exome sequencing and meiotic mapping were per- signaling is essential for cell-cell communication during development, formed as described previously (25).

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1916645117 Choi et al. Downloaded by guest on September 27, 2021 The NcstnV439G/V439G mice were generated using a CRISPR/Cas9 system as Cell Culture, Isolation of Primary Lymphocytes, Immunoprecipitation, and Immunoblot described previously (38, 39). Female C57BL/6J mice were superovulated by Analysis. FLAG-tagged wild-type or mutant (V439G) mouse NCSTN-expressing injection of 6.5 U of pregnant mare serum gonadotropin (Millipore Sigma), cell lines were generated in Ncstn−/− mouse embryonic fibroblasts (MEFs) (18). followed by injection of 6.5 U of human chorionic gonadotropin (Sigma-Aldrich) The Ncstn−/− MEFs were infected with lentivirus encoding either C-terminal 48 h later. The superovulated mice were subsequently mated overnight with FLAG-tagged wild-type or mutant (V439G) mouse Ncstn. At 48 h after infection, C57BL/6J male mice. The next day, fertilized eggs were collected from the puromycin at a final concentration of 2 μg/mL was added to create stable cell lines. oviducts, and in vitro-transcribed Cas9 mRNA (50 ng/μL) and Ncstn small Splenic pan B and T cells were purified using the EasySep Mouse Pan B and base-pairing guide RNA (50 ng/μL; 5′- GGCTCGAAACATCTCTGGCG-3′) were T Cell Isolation Kits (StemCell Technologies), respectively, according to the injected into the cytoplasm or pronucleus of the embryos. The injected manufacturer’s instructions. Purity exceeded 95% in all experiments as embryos were cultured in M16 medium (Sigma-Aldrich) at 37 °C in 5% CO2. tested by flow cytometry. For the production of mutant mice, two-cell stage embryos were transferred Immunoprecipitation was performed using anti-FLAG M2 agarose beads into the ampulla of the oviduct (10 to 20 embryos per oviduct) of pseudo- (Sigma-Aldrich) in wild-type or mutant (V439G) mouse NCSTN-expressing − − pregnant Hsd:ICR (CD-1) female mice (Harlan Laboratories). All experiments Ncstn / MEFs. Cells were harvested in CHAPSO lysis buffer (50 mM Tris·HCl in this study were approved by the University of Texas Southwestern pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% [vol/vol] CHAPSO, and protease in- Medical Center’s Institutional Animal Care and Use Committee. hibitors) for 45 min at 4 °C. TCLs and immunoprecipitates were analyzed using anti-FLAG M2 (Sigma-Aldrich), anti-HA (Cell Signaling Technology), anti-PEN2 Immunization and Enzyme-Linked Immunosorbent Assay. Eight- to 12-wk-old (Cell Signaling Technology), and anti-PS1 (A14; ref. 42) antibodies using stan- G3 mice or NcstnV439G/V439G and wild-type littermates were immunized dard procedures for immunoblot analysis as described below. with the TD antigen OVA/alum (200 μg, i.m.; Invivogen) on day 0 and with For Western blot analysis, cells were lysed in lysis buffer composed of 1% μ the TI antigen NP50-AECM-Ficoll (50 g, i.p.; Biosearch Technologies) on day (wt/vol) SDS (Thermo Fisher Scientific), 0.01% (wt/vol) Benzonase (Sigma- 8, as described previously (40). At 6 d after NP50-AECM-Ficoll immunization, Aldrich), and protease inhibitor mixture (Cell Signaling Technology) for blood was collected in MiniCollect tubes (Mercedes Medical) and centri- 30 min at 4 °C. Protein concentration was measured using a BCA assay (Pierce). fuged at 1,500 × g to separate the serum for enzyme-linked immunosorbent Here 10 μg of protein was separated on 4% to 12% Bris-Tris protein gels (Life assay (ELISA) analysis. Technologies), and proteins were transferred to nitrocellulose membranes ELISA analysis of antigen-specific IgG and IgM responses was performed as (Bio-Rad) for 45 min at 13 V. After blocking in Tris-buffered saline containing described previously (38, 39), Nunc MaxiSorp flat-bottom 96-well microplates 0.05% (vol/vol) Tween-20 (TBS-T) with 5% (wt/vol) nonfat dry milk (NFDM) at (Thermo Fisher Scientific) were coated with 5 μg/mL soluble OVA (Invivogen) room temperature for 1 h, the membrane was incubated overnight with or NP8-BSA (Biosearch Technologies) and incubated at 4 °C overnight. Plates primary antibodies anti-NCSTN (Cell Signaling Technology), anti-APP (Sigma- were washed four times with washing buffer (0.05% [vol/vol] Tween-20 in Aldrich), anti-APH1 (H2D2; ref. 43), anti-PS1 (A14; ref. 42), anti–α-tubulin PBS) using a BioTek microplate washer, then blocked with 1% (vol/vol) (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology) at BSA for 1 h at room temperature. Serum samples were serially diluted in 1% 4 °C in 5% (wt/vol) NFDM in TBS-T with gentle rocking. The membrane was (vol/vol) BSA, after which the 1:50 and 1:150 dilutions were added to the then incubated with secondary goat anti-rabbit or mouse IgG-HRP antibody IMMUNOLOGY AND INFLAMMATION prepared ELISA plates. After a 2-h incubation, the plates were washed eight (Thermo Fisher Scientific) for 1 h at room temperature with gentle rocking. times with washing buffer and then incubated with HRP-conjugated goat anti- The chemiluminescence signal was developed using the SuperSignal West mouse IgG or IgM for 1 h at room temperature. Plates were again washed Dura Extended-Duration Substrate Kit (Thermo Fisher Scientific) and detected eight times with washing buffer, then developed with KPL SureBlue TMB using the G:Box Chemi XX6 gel doc system (Syngene). Microwell Peroxidase Substrate and TMB Stop Solution (SeraCare). Absorbance was measured at 450 nm on a Synergy Neo2 Plate Reader (BioTek). Basal levels Isolation of Plasma Membrane and De-Glycosylation Assay. To analyze glyco- of anti-OVA IgG and anti-NP IgM were determined using preimmune serum. sylation patterns of wild-type and mutant (V439G) NCSTN, freshly isolated All ELISA data shown represent the 1:150 serum dilution. splenocytes were lysed in SDS lysis buffer, as described previously (35). TCLs For determination of serum antibody isotype levels (IgG1 and IgM), freshly were treated with Endo H (500 mU/mL; New England BioLabs) or PNGase isolated serum was subjected to sandwich ELISA analysis according to the (200 U/mL; New England BioLabs) according to the manufacturer’s instruc- manufacturer’s instructions (Invitrogen). tions and analyzed by immunoblotting using anti-FLAG M2 (Sigma-Aldrich) and anti-GAPDH (Cell Signaling Technology) antibodies. DSS-Induced Colitis Screen. For DSS-induced colitis induction (36,000 to 50,000 molecular weight; MP Biomedicals), 8- to 12-wk-old mice received 1.3% DSS in Blue Native-PAGE Analysis. Collected cells were washed with ice-cold PBS and the drinking water for 7 d, followed by 3 d off DSS, as described previously suspended in ice-cold Pipes buffer (50 mM Pipes pH 7.0, 250 mM sucrose, (41). Body weight was recorded daily and reported as a percentage relative 1 mM EGTA, and protease inhibitors). Cells were lysed by bioruptor sonication to the pretreatment body weight. in an ice water bath, and the nuclear fraction was removed by centrifugation at 600 × g for 10 min at 4 °C. The membrane fraction was isolated by ultra- Flow Cytometry. Bone marrow cells, thymocytes, splenocytes, peripheral blood, centrifugation at 100,000 × g for 1 h at 4 °C, and membrane proteins were or peritoneal cells were isolated, and red blood cell (RBC) lysis buffer was added extracted with a solubilization buffer (50 mM Pipes pH 7.0, 250 mM sucrose, to remove the RBCs. Cells were stained at a 1:200 dilution with mouse 1 mM EGTA, 1% digitonin, and protease inhibitors). The same amount of fluorochrome-conjugated monoclonal antibodies specific for the following soluble proteins was subjected to Native-PAGE using the NativePAGE Novex murine cell surface markers encompassing the major immune lineages: B220 Bis-Tris Gel System (Life Technologies). The gel was transferred to a PVDF (clone RA3-6B2; BD), CD19 (clone 1D3; BD), IgM (clone R6-60.2; BD), IgD (clone membrane and destained with water/methanol/acetic acid (5/4/1) for 30 min. 11-26c.2a; Biolegend), CD3e (clone 145-2C11; BD), CD4 (clone RM4-5; BD), CD5 The membrane was further used for Western blot analysis. (clone 53-7.3; BD), CD11c (clone HL3; BD), CD44 (clone IM7; BD), CD43 (clone S7; BD), CD25 (clone PC61; Biolegend), CD21/CD35 (clone 7E9; Biolegend), CD23 In Vitro γ-Secretase Activity Assay. To measure the effect of the V439G (clone B3B4; BD), Ly-51 (clone BP-1; BD), CD8α (clone 53-6.7; Biolegend), CD11b substitution in γ-secretase function, γ-secretase activity was assessed in vitro (clone M1/70; Biolegend), NK1.1 (clone PK136; BD), F4/80 (clone BM8.1; Tonbo), as described previously (18). In brief, brains isolated from wild-type and and CD62L (clone MEL-1; Tonbo), and in the presence of Fc shield (clone 2.4G2; NcstnV439G/V439G mice were suspended in ice-cold Pipes buffer (50 mM Pipes Tonbo) for 1 h at 4 °C. After staining, cells were washed twice in PBS and pH 7.0, 250 mM sucrose, 1 mM EGTA, and protease inhibitors) and gently analyzed by flow cytometry. Data were acquired on an LSRFortessa cell ana- sonicated. Pellets containing the nuclear fraction were removed by centri- lyzer (BD Biosciences) and analyzed with FlowJo software (Tree Star). fugation at 600 × g for 10 min at 4 °C. Cleared supernatants containing the microsome fraction were isolated by ultra-centrifugation at 100,000 × g for Bone Marrow Chimeras. Bone marrow chimeras were prepared as described 1 h at 4 °C and solubilized in 1% CHAPSO buffer (50 mM Pipes pH 7.0, previously (38, 39). Recipient mice were lethally irradiated with 13 Gy via 250 mM sucrose, 1 mM EGTA, and protease inhibitors). An equal wt/vol of gamma radiation (X-RAD 320 Precision X-Ray; Accela). Each mouse received microsomal protein was incubated with FLAG-tagged mouse-N1-N100 and an i.v. injection of 5 × 106 bone marrow cells derived from donor tibias and N2-N100 in the presence or absence of the γ-secretase inhibitor DAPT femurs. The mice were maintained on antibiotics for 4 wk after engraft- (100 μM) for 24 h at 37 °C. N1-N100 contains Val1711-Glu1809 of mouse ment. At 12 wk after bone marrow engraftment, the chimeras were eu- Notch-1 and is tagged with a C-terminal Flag/His (18). N2-N100 contains thanized to assess immune cell development in spleen, peripheral blood, Val1668-Pro1766 of mouse Notch-2 with a C-terminal Flag/His tag (this and peritoneal cavity, which were analyzed by flow cytometry. Chimerism study). The products were analyzed by immunoblotting using an anti-FLAG was assessed using congenic CD45 markers. antibody (Sigma-Aldrich).

Choi et al. PNAS Latest Articles | 7of8 Downloaded by guest on September 27, 2021 Statistical Analysis. The statistical significance of differences between groups Research Center (C57BL/6J-MtgxR3969Btlr/Mmmh; stock no. 040937-MU). was analyzed using GraphPad Prism by performing the indicated statistical Strain information is available at https://mutagenetix.utsouthwestern.edu/ tests. Differences in the raw values among groups were considered statisti- incidental/incidental_rec.cfm?mid=310864&rn=20&rl=41&so=&ac=1&r0=0&nr= cally significant at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not 100&scd=r3969. significant with P > 0.05. ACKNOWLEDGMENTS. This work was supported by the NIH (Grants R01 Data Availability. All data are contained in the main text and SI Appendix. AI125581, to B.B. and R01 NS079796 and RF1-AG064909, to G.Y.) and by the The truffle mouse strain is available from the Mutant Mouse Resource and Lyda Hill Foundation (B.B.).

1. J. J. Mond, A. Lees, C. M. Snapper, T cell-independent antigens type 2. Annu. Rev. 23. R. Wang, P. Tang, P. Wang, R. E. Boissy, H. Zheng, Regulation of tyrosinase trafficking Immunol. 13, 655–692 (1995). and processing by presenilins: Partial loss of function by familial Alzheimer’s disease ’ 2. C. García de Vinuesa, P. O Leary, D. M. Sze, K. M. Toellner, I. C. MacLennan, mutation. Proc. Natl. Acad. Sci. U.S.A. 103, 353–358 (2006). T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but 24. A. Klinakis et al., A novel tumour-suppressor function for the Notch pathway in – exponential growth is confined to extrafollicular foci. Eur. J. Immunol. 29, 1314 1323 myeloid leukaemia. Nature 473, 230–233 (2011). (1999). 25. T. Wang et al., Real-time resolution of point mutations that cause phenovariance in 3. F. Martin, J. F. Kearney, Marginal-zone B cells. Nat. Rev. Immunol. 2, 323–335 (2002). mice. Proc. Natl. Acad. Sci. U.S.A. 112, E440–E449 (2015). 4. F. Martin, A. M. Oliver, J. F. Kearney, Marginal zone and B1 B cells unite in the early 26. I. A. Adzhubei et al., A method and server for predicting damaging missense muta- response against T-independent blood-borne particulate antigens. Immunity 14, 617–629 tions. Nat. Methods 7, 248–249 (2010). (2001). 27. S. Fre et al., Notch signals control the fate of immature progenitor cells in the in- 5. K. R. Alugupalli et al., The resolution of relapsing fever borreliosis requires IgM and is – concurrent with expansion of B1b lymphocytes. J. Immunol. 170, 3819–3827 (2003). testine. Nature 435, 964 968 (2005). 6. K. R. Alugupalli et al., B1b lymphocytes confer T cell-independent long-lasting im- 28. Y. Obata et al., Epithelial cell-intrinsic Notch signaling plays an essential role in the – munity. Immunity 21, 379–390 (2004). maintenance of gut immune homeostasis. J. Immunol. 188, 2427 2436 (2012). 7. K. M. Haas, J. C. Poe, D. A. Steeber, T. F. Tedder, B-1a and B-1b cells exhibit distinct 29. J. H. van Es et al., Notch/gamma-secretase inhibition turns proliferative cells in in- developmental requirements and have unique functional roles in innate and adaptive testinal crypts and adenomas into goblet cells. Nature 435, 959–963 (2005). immunity to S. pneumoniae. Immunity 23,7–18 (2005). 30. T. Lopes-Carvalho, J. F. Kearney, Development and selection of marginal zone B cells. 8. D. Allman et al., Resolution of three nonproliferative immature splenic B cell subsets Immunol. Rev. 197, 192–205 (2004). reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167, 31. B. Srivastava, W. J. Quinn, 3rd, K. Hazard, J. Erikson, D. Allman, Characterization of 6834–6840 (2001). marginal zone B cell precursors. J. Exp. Med. 202, 1225–1234 (2005). 9. S. Pillai, A. Cariappa, The follicular versus marginal zone B lymphocyte cell fate de- 32. E. Montecino-Rodriguez, H. Leathers, K. Dorshkind, Identification of a B-1 B cell- – cision. Nat. Rev. Immunol. 9, 767 777 (2009). specified progenitor. Nat. Immunol. 7, 293–301 (2006). 10. K. Hayakawa, R. R. Hardy, L. A. Herzenberg, L. A. Herzenberg, Progenitors for Ly-1 B 33. W. T. Kimberly et al., Complex N-linked glycosylated nicastrin associates with active – cells are distinct from progenitors for other B cells. J. Exp. Med. 161, 1554 1568 gamma-secretase and undergoes tight cellular regulation. J. Biol. Chem. 277, (1985). 35113–35117 (2002). 11. H. Wardemann, T. Boehm, N. Dear, R. Carsetti, B-1a B cells that link the innate and 34. W. P. Esler et al., Activity-dependent isolation of the presenilin- gamma -secretase adaptive immune responses are lacking in the absence of the spleen. J. Exp. Med. 195, complex reveals nicastrin and a gamma substrate. Proc. Natl. Acad. Sci. U.S.A. 99, 771–780 (2002). – 12. D. R. Dries, G. Yu, Assembly, maturation, and trafficking of the gamma-secretase 2720 2725 (2002). complex in Alzheimer’s disease. Curr. Alzheimer Res. 5, 132–146 (2008). 35. D. S. Yang et al., Mature glycosylation and trafficking of nicastrin modulate its – 13. F. Radtke et al., Deficient T cell fate specification in mice with an induced inactivation binding to presenilins. J. Biol. Chem. 277, 28135 28142 (2002). of Notch1. Immunity 10, 547–558 (1999). 36. M. Moriyama et al., Notch signaling via Hes1 transcription factor maintains survival of 14. T. Saito et al., Notch2 is preferentially expressed in mature B cells and indispensable melanoblasts and melanocyte stem cells. J. Cell Biol. 173, 333–339 (2006). for marginal zone B lineage development. Immunity 18, 675–685 (2003). 37. P. Georgel, X. Du, K. Hoebe, B. Beutler, ENU mutagenesis in mice. Methods Mol. Biol. 15. B. De Strooper et al., A presenilin-1-dependent gamma-secretase-like protease me- 415,1–16 (2008). diates release of Notch intracellular domain. Nature 398, 518–522 (1999). 38. J. H. Choi et al., IgD class switching is initiated by microbiota and limited to mucosa- 16. R. Kopan, M. X. Ilagan, Gamma-secretase: Proteasome of the membrane? Nat. Rev. associated lymphoid tissue in mice. Proc. Natl. Acad. Sci. U.S.A. 114, E1196–E1204 – Mol. Cell Biol. 5, 499 504 (2004). (2017). 17. G. Yu et al., Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction 39. J. H. Choi et al., LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling. – and betaAPP processing. Nature 407,48 54 (2000). Science 364, eaau0812 (2019). 18. S. Shah et al., Nicastrin functions as a gamma-secretase-substrate receptor. Cell 122, 40. C. N. Arnold et al., A forward genetic screen reveals roles for Nfkbid, Zeb1, and 435–447 (2005). Ruvbl2 in . Proc. Natl. Acad. Sci. U.S.A. 109, 12286–12293 (2012). 19. K. Shirotani et al., Gamma-secretase activity is associated with a conformational 41. K. W. Wang et al., Enhanced susceptibility to chemically induced colitis caused by change of nicastrin. J. Biol. Chem. 278, 16474–16477 (2003). excessive endosomal TLR signaling in LRBA-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 20. R. J. Kelleher, 3rd, J. Shen, Presenilin-1 mutations and Alzheimer’s disease. Proc. Natl. – Acad. Sci. U.S.A. 114, 629–631 (2017). 116, 11380 11389 (2019). 21. B. Wang et al., Gamma-secretase gene mutations in familial acne inversa. Science 330, 42. G. Thinakaran et al., Endoproteolysis of presenilin 1 and accumulation of processed – 1065 (2010). derivatives in vivo. Neuron 17, 181 190 (1996). 22. G. Aubin-Houzelstein et al., Melanoblasts’ proper location and timed differentiation 43. S. F. Lee et al., Mammalian APH-1 interacts with presenilin and nicastrin and is re- depend on Notch/RBP-J signaling in postnatal hair follicles. J. Invest. Dermatol. 128, quired for intramembrane proteolysis of amyloid-beta precursor protein and Notch. 2686–2695 (2008). J. Biol. Chem. 277, 45013–45019 (2002).

8of8 | www.pnas.org/cgi/doi/10.1073/pnas.1916645117 Choi et al. Downloaded by guest on September 27, 2021