biology

Article
Early Animal Origin of BACE1 APP/Aβ Proteolytic Function
James A. Langeland, Lillian Baumann † , Eva M. DeYoung † , Raphaela Angelina Varella † , Nkatha Mwenda,
Alejandro Aguirre and D. Blaine Moore *

Department of Biology, Kalamazoo College, 1200 Academy Street, Kalamazoo, MI 49006, USA
* Correspondence: [email protected]
† These authors contributed equally to this work.

Simple Summary: One feature of Alzheimer’s disease is the accumulation β-amyloid (Aβ) in the
brain. Since the BACE1 protease is required to produce Aβ, and is a potential clinical target, it is of
interest to know when its enzymatic function evolved and for what purpose. Here, we show that
BACE1 likely evolved from a gene duplication event near the base of the animal clade, well before
the evolution of the APP/Aβ substrate.

Abstract: Alzheimer’s disease is characterized, in part, by the accumulation of β-amyloid (Aβ) in the
brain. Aβ is produced via the proteolysis of APP by BACE1 and γ-secretase. Since BACE1 is the rate-
limiting enzyme in the production of Aβ, and a target for therapeutics, it is of interest to know when its
proteolytic function evolved and for what purpose. Here, we take a functional evolutionary approach
to show that BACE1 likely evolved from a gene duplication event near the base of the animal clade
and that BACE1 APP/Aβ proteolytic function evolved during early animal diversification, hundreds
of millions of years before the evolution of the APP/Aβ substrate. Our examination of BACE1
APP/Aβ proteolytic function includes cnidarians, ctenophores, and choanoflagellates. The most
basal BACE1 ortholog is found in cnidarians, while ctenophores, placozoa, and choanoflagellates have
genes equally orthologous to BACE1 and BACE2. BACE1 from a cnidarian (Hydra) can cleave APP to
release Aβ, pushing back the date of the origin of its function to near the origin of animals. We tested
more divergent BACE1/2 genes from a ctenophore (Mnemiopsis) and a choanoflagellate (Monosiga),
and neither has this activity. These findings indicate that the specific proteolytic function of BACE1
Citation: Langeland, J.A.; Baumann,
evolved during the very earliest diversification of animals, most likely after a gene-duplication event.
L.; DeYoung, E.M.; Varella, R.A.;
Mwenda, N.; Aguirre, A.; Moore, D.B. Keywords: β-amyloid (Aβ); BACE; evolution; amyloid precursor protein (APP); animal
Early Animal Origin of BACE1
APP/Aβ Proteolytic Function. Biology
2024, 13, 320. https://doi.org/
10.3390/biology13050320 1. Introduction
Academic Editor: Yan Zhang Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that results in
progressive changes in cognition, behavior, and mood [1]. Many proteins are implicated,
Received: 6 March 2024 including Tau, but the precipitating event in AD appears to be the abnormal buildup of
Revised: 24 April 2024
extracellular plaques in the brain containing β-amyloid (Aβ) [1]. Aβ, a 4 kDa peptide, is
Accepted: 30 April 2024
derived from the amyloid precursor protein (APP) via proteolysis [2]. APP is expressed in
Published: 4 May 2024
all human tissues, with the APP695 isoform being most commonly expressed in neurons [3].
APP is proteolyzed by β-secretase, generating a c-terminal fragment with 99 amino acids
(C99), which is then cut by γ-secretase to produce Aβ, which is secreted into the extracellular
Copyright: © 2024 by the authors.
space [3].
Licensee MDPI, Basel, Switzerland. The β-secretase involved in the cleavage of APP to produce Aβ has been identified
This article is an open access article as BACE1 [4]. The BACE1 homologue BACE2 has been found to be irrelevant to Aβ
distributed under the terms and production [5]. BACE1 is the primary β-secretase in the brain and the rate-limiting enzyme
conditions of the Creative Commons in the production of Aβ, as previous studies have shown Aβ secretion to be inhibited in
Attribution (CC BY) license (https:// BACE1 knockout mice [6]. However, BACE1 knockout studies have shown that BACE1
creativecommons.org/licenses/by/ may have some essential functions. For example, BACE1 knockout mice were found
4.0/). to have delayed myelination and thinner myelin in central and in peripheral nerves [7].

Biology 2024, 13, 320. https://doi.org/10.3390/biology13050320 https://www.mdpi.com/journal/biology

Biology 2024, 13, 320 2 of 9

Additionally, mice lacking BACE1 have abnormal hippocampal synaptic plasticity and
cognitive performance [8], and BACE1 knockout mice had decreased grip strength and
increased sensitivity to pain [7]. As BACE1 may be a target for AD treatments, it is necessary
to understand the essential functions that would be impacted by such a treatment. Given
the undesirable side effects associated with BACE1 inhibitors such as Verubecestat and
Lanabecestat [9,10], it is of interest to more fully understand the implications of BACE1
inhibition. Knowing precisely when BACE1 evolved, and for what purpose, will be
important context for eventual therapeutic inhibition.
We have previously shown that BACE1 and Aβ evolved asynchronously [11]. Phyloge-
netic analysis showed that BACE1 arose near the origin of animals, while the Aβ substrate
evolved hundreds of millions of years later and cannot be found outside of vertebrates.
Furthermore, functional analysis showed that BACE1 from an animal (the cephalochordate
Branchiostoma floridae) that never evolved Aβ can nonetheless proteolyze the APP/Aβ
substrate. These findings indicate that BACE1 has deeply conserved and essential functions
that have nothing to do with APP/Aβ processing per se, but that were later co-opted into
this processing.
In the current study, we perform a functional evolutionary analysis of BACE1 APP/Aβ
proteolysis in taxa flanking the base of the animal clade. Animal origins remain opaque,
and early animal phylogeny is unresolved; while choanoflagellates are accepted as a sister
group of animals, the relationships of the non-bilaterian basal animal groups of porifera,
ctenophores, and cnidaria are not agreed upon [12–15]. Our aim is not to resolve these
relationships, but rather to probe where we can find BACE1 orthologs in these taxa and to
determine whether they can proteolyze APP/Aβ. Through our functional analysis, we find
that cnidaria are the most basal taxon to have a bona fide BACE1 ortholog, and through
our functional assay we find that this ortholog can proteolyze human APP to release Aβ.

2. Materials and Methods

2.1. Phylogenetic Analysis
Our previously described search approach [11] remained focused on basal animals and
yielded new sequences from cnidarians (Orbicella faveolata XP_020602753.1, Nematostella
vectensis EDO39359.1, Actinia tenebrosa XP_031568918.1, Stylophora pistillata XP_022793028.1
and Pocillopora damicornis XP_027053766.1), placozoans (Trichoplax adhaerens RDD46120;
RDD46118.1), ctenophores (Pleurobrachia bachei Neurobase sb|2666892|), sponges (Am-
phimedon queenslandica XP_003385244.1, Oopsacas minuta KAI6660898.1, Aphrocallistes vastus
CAC83293.1, Halisarca dujardinii QSX72298.1), and choanoflagellates (Salpingoeca rosetta
XP_004993482.1). We were also able to add to our previously identified Mnemiopsis partial
transcript (ML154145a) by fusing it with an overlapping partial sequence ML1541_cuf_154
(Mnemiopsis Genome Project Portal). Multiple sequence alignments and evolutionary
history inferences were produced using MEGA 11 [16]. The evolutionary history was
inferred by using the Maximum likelihood method and JTT matrix-based model [17], and a
bootstrap consensus tree was inferred from 100 replicates [18]. Branches corresponding
to partitions reproduced in less than 50% of bootstrap replicates were collapsed. Initial
tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining
and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model,
and then selecting the topology with the superior log likelihood value. A discrete Gamma
distribution was used to model evolutionary-rate differences among sites (5 categories
(+G, parameter = 2.1022)). For purposes of clarity, the final tree produced excludes some
non-informative sequences from our cnidarian, sponge, and ctenophore sampling. Tests for
positive selection around BACE active sites were carried out using the codon-aligned BACE
nucleotide files in the Datamonkey implementation of HyPhy. All sequences, alignments,
and tree files are available as Supplementary Materials.
Biology 2024, 13, 320 3 of 9

2.2. Expression Constructs

For each relevant BACE amino acid sequence (Hydra, Mnemiopsis, and Monosiga—
see [11], for sequence IDs), full-length cDNAs were synthesized with codon optimization for
CHO cells (GenScript, Piscataway, NJ, USA). These cDNAs were cloned into pcDNA3.1(+)
expression plasmids with either C-HA or C-GFP tags, and industrial-grade plasmid preps
were obtained (GenScript). Branchiostoma floridae BACE1 construct detail is provided in [11].

2.3. Cell Culture and Transfection

Chinese Hamster Ovary cells stably transfected with the 695 amino acid variety of
human APP (CHO 695) were maintained in minimum essential alpha media (αMEM;
Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 1% glutamine,
and 1% penicillin/streptomycin (Invitrogen). The cells were passaged as specified in
Balan et al. [19]. The cells were seeded into 6-well plates at a density of 1.25 × 104
prior to transient transfection. CHO 695 cells at 70% confluence were transfected with
GenePorter (Genlantis, San Diego, CA, USA) 24 h after plating, following manufacturer’s
instructions. In total, 2 µg DNA (GFP, Homo BACE1, Branchiostoma BACE1, Hydra BACE1,
Mnemiopsis BACE 1/2 or Monosiga BACE 1/2) and 10 µL transfection reagent were delivered
in 1.0 mL serum-free αMEM for five hours. Transfections were stopped by the addition of
1.0 mL serum-containing αMEM. The visual inspection of GFP controls verified successful
transfection. Successful localizations of BACE constructs were confirmed with GFP-tagged
versions of each BACE construct. Microscopy was performed on an inverted Nikon
DIAPHOT 200 compound light microscope (Melville, NY, USA).

2.4. Collection of Conditioned Media and ELISA

Conditioned media were harvested 16 h following transfection. A complete Mini
protease-inhibitor cocktail (Roche, Indianapolis, IN, USA) was used to prevent the degrada-
tion of secreted proteins. In total, 1.0 mL of media was transferred to microfuge tubes con-
taining protease inhibitors and incubated on ice. The tubes were centrifuged at 13,000 RPM
for 20 min at 4 ◦ C. The supernatant was transferred to new microfuge tubes and stored
at −80 ◦ C until analysis. Secreted Aβ 1–40 was measured via a human-specific ELISA kit
(Invitrogen) following the manufacturer’s instructions. The absorbance was measured at
450 nm on a µquant microplate spectrophotometer (Biotek, Winooski, VT, USA). The data
were normalized to total protein levels measured in cell lysates by the BCA protein assay
(Thermo Fisher Scientific, Waltham, MA, USA). See details on lysate preparation and BCA
protein assay in Balan et al. [19].

2.5. Statistics
The significance of ELISA data was determined by a one-way analysis of variance
(ANOVA) followed by Tukey’s post hoc testing.

3. Results
3.1. BACE Phylogenetic Analysis
We searched genomic databases of basal animal species for BACE1 orthologs. Our
phylogenetic analysis (Figure 1) shows that cnidaria (e.g. Hydra vulgaris) have a bona
fide BACE1 ortholog (see the gray box). They are the most basal group to have this
gene, albeit weakly supported with a 54% bootstrap value. We also find clear BACE
orthologs in ctenophores (e.g., Mnemiopsis leidyi) and placozoans (Trichoplax adhaerens), as
well as in choanoflagellates (e.g Monosiga bevicollis and Salpingoeca rosetta). Together with a
divergent Hydra BACE (termed BACE2 in the database), these more basal BACE genes form
a polytomy with the BACE1 and BACE2 clades. Sponges (e.g., Amphimedon in our tree) do
not appear to have BACE 1 genes; the closest sequence is a Cathepsin D, a related aspartyl
protease. A simple but highly tentative model to explain our tree would be that the BACE1
and BACE 2 gene families arose via gene duplication in the cnidarian+bilaterian ancestor.
We were unable to detect signals of positive selection in or around BACE active sites that
 choanoflagellates (e.g Monosiga bevicollis and Salpingoeca rosetta). Together with a diver-
gent Hydra BACE (termed BACE2 in the database), these more basal BACE genes form a
polytomy with the BACE1 and BACE2 clades. Sponges (e.g., Amphimedon in our tree) do
Biology 2024, 13, 320 not appear to have BACE 1 genes; the closest sequence is a Cathepsin D, a related aspartyl
4 of 9
protease. A simple but highly tentative model to explain our tree would be that the BACE1
and BACE 2 gene families arose via gene duplication in the cnidarian+bilaterian ancestor.
We were unable to detect signals of positive selection in or around BACE active sites that
may may
havehave
resulted from
resulted fromthis duplication.
this duplication. Given thatthe
Given that thephylogeny
phylogeny of the
of the basalbasal animal taxa
animal
themselves are unresolved,
taxa themselves it is not
are unresolved, it issurprising thatthat
not surprising thethe
base
baseofofour
ourBACE treeisisun-
BACE tree unresolved.
Whatresolved.
is clearWhat is clear
is that is thatare
cnidaria cnidaria are thebasal
the most most group
basal group to have
to have a bonafide
a bona fideBACE1
BACE1 gene.
gene.

Figure 1. Cnidarians are the most basal group with a BACE1 ortholog. Maximum-likelihood tree
Figure 1. Cnidarians are the most basal group with a BACE1 ortholog. Maximum-likelihood tree
of BACE sequences with nodes collapsed below the 50% bootstrap support. BACE1 and BACE2
of BACE sequences
sequences with
form clear nodes
clades withcollapsed below
cnidarians being thethe
most50%
basalbootstrap support.
group found BACE1
in our search and BACE2
to have
a BACE1 sequence (shown in the gray box). Other BACE-like sequences can be found
sequences form clear clades with cnidarians being the most basal group found in our search to have ain basal ani-
mal taxa such as ctenophores (e.g., Mnemiopsis) and placozoans (Tricoplax), as well as in the sister
BACE1 sequence (shown in the gray box). Other BACE-like sequences can be found in basal animal
group to animals, choanoflagellates (Monosiga). These sequences form a polytomy with the BACE1
taxa such as ctenophores
and BACE2 (e.g., Mnemiopsis)
groups, mirroring the polytomiesand
that placozoans (Tricoplax),atasthe
are found in phylogenies well as in the sister group
whole-organism
level. Aschoanoflagellates
to animals, a tentative model, we consider them
(Monosiga). to be sequences
These single-gene, form
pre-duplicate precursors
a polytomy withtothe
BACEBACE1 and
1 and BACE 2. Tricoplax appears to have undergone an independent duplication of this BACE1/2
BACE2 groups, mirroring the polytomies that are found in phylogenies at the whole-organism level.
precursor. Cathepsin D aspartyl proteases are the sister group to the BACE genes; we can find this
As a tentative
gene but notmodel, we consider
BACE genes in porifera them to be single-gene, pre-duplicate precursors to BACE 1 and
(Amphimedon).
BACE 2. Tricoplax appears to have undergone an independent duplication of this BACE1/2 precursor.
Cathepsin D aspartyl proteases are the sister group to the BACE genes; we can find this gene but not
BACE genes in porifera (Amphimedon).

3.2. Analysis of BACE Functional Activity

The functional activity of BACE proteins was examined in our well-described in vitro
model system, the CHO695 cell line, which stably expresses the 695-amino acid, a primarily
neuronal isoform of human APP (CHO695 cells; [11]). Using this system, we previously
showed that BACE1 functional activity towards APP/Aβ extends to the cephalochordate
subphylum [11]. To determine whether the BACE1 proteolysis of human APP is conserved
in cnidarians, CHO695 cells were transfected with Hydra BACE1. GFP transection was
used as a negative control, while Homo BACE1 was used as a positive control. BACE1
from the cephalochordate Branchiostoma (Branch.), which we have previously shown to
be a functional ortholog of Homo BACE1, was transfected for comparison and served
 model system, the CHO695 cell line, which stably expresses the 695-amino acid, a primar-
ily neuronal isoform of human APP (CHO695 cells; [11]). Using this system, we previously
showed that BACE1 functional activity towards APP/Aβ extends to the cephalochordate
subphylum [11]. To determine whether the BACE1 proteolysis of human APP is con-
Biology 2024, 13, 320 served in cnidarians, CHO695 cells were transfected with Hydra BACE1. GFP transection 5 of 9
was used as a negative control, while Homo BACE1 was used as a positive control. BACE1
from the cephalochordate Branchiostoma (Branch.), which we have previously shown to be
a functional ortholog of Homo BACE1, was transfected for comparison and served as a
as a positive control. Sixteen hours after transfection, conditioned media were collected
positive control. Sixteen hours after transfection, conditioned media were collected and
and analyzed with a 1–40 Aβ sandwich ELISA. A bicinchoninic acid (BCA) protein assay
analyzed with a 1–40 Aβ sandwich ELISA. A bicinchoninic acid (BCA) protein assay was
was
performedperformed on cell
on cell lysate lysate
samples samples
and used toand used to
normalize Aβnormalize
levels and Aβ levels
account forand
any account for
any minor variation in the total protein or cell number. A one-way
minor variation in the total protein or cell number. A one-way ANOVA revealed signifi- ANOVA revealed
significant differences in Aβ secretion (Figure 2A) [F(3,32) = 5.2398,
cant differences in Aβ secretion (Figure 2A) [F(3,32) = 5.2398, p < 0.01]. Tukey’s post hoc p < 0.01]. Tukey’s
post hoc testing showed a significant increase in Aβ secretion for
testing showed a significant increase in Aβ secretion for both Homo BACE1 and Branchi- both Homo BACE1
and Branchiostoma BACE1 (p < 0.05), and a significant increase in Hydra BACE1 (p < 0.01),
ostoma BACE1 (p < 0.05), and a significant increase in Hydra BACE1 (p < 0.01), confirming
ourconfirming
phylogeneticour
analysis and proving
phylogenetic cnidarian
analysis and BACE1
provingis cnidarian
a functional ortholog
BACE1 is of human
a functional ortholog
BACE1.
of human BACE1.

Biology 2024, 13, x FOR PEER REVIEW 6 of 10

B
Figure
Figure2. BACE1 functional
2. BACE1 activityactivity
functional towards towards
human APP/Aβ
humanis APP/Aβ
conservedisinconserved
cnidarians but not
in cnidarians but
in ctenophores or choanoflagellates. CHO 695 cells stably transfected with human APP were tran-
not in ctenophores or choanoflagellates. CHO 695 cells stably transfected with human APP were
siently transfected with the following cDNA expression constructs: GFP (negative control), Hydra
transiently
BACE1, transfected
Mnemiopsis BACE with theMonosiga
1/2, or following cDNA
BACE 1/2.expression
Homo BACE1 constructs: GFP (negative
and Branchiostoma control), Hydra
(Branch.)
BACE1,
BACE1 Mnemiopsis
constructs BACE
were used as positive Monosiga
1/2, orcontrols. BACE 1/2.
Conditioned Homo
media wereBACE1
harvested Branchiostoma
andafter 16 h, and (Branch.)
theBACE1
secretion of humanwere
constructs Aβ was
useddetermined viacontrols.
as positive ELISA. Data were normalized
Conditioned to total
media were protein and
harvested after 16 h, and
analyzed with one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05; ** p < 0.01). Data are
the secretion of human Aβ was determined via ELISA. Data were normalized to total protein and
representative of three independent transfection rounds. Homo, Branchiostoma, and Hydra BACE1 all
analyzed
elevated with one-way
Aβ secretion ANOVA
(A), while followed
Mnemiopsis by Tukey’s
and Monosiga BACEpost
1/2hoc (* p < 0.05; ** p < 0.01). Data are
test(B).
did not
representative of three independent transfection rounds. Homo, Branchiostoma, and Hydra BACE1 all
Next, Aβ
elevated we turned our
secretion attention
(A), to ctenophores
while Mnemiopsis and choanoflagellates
and Monosiga BACE 1/2 didtonot
determine
(B). if
more divergent BACE genes show functional activity towards APP/Aβ. We transfected
CHO695 cells with Mnemiopsis BACE 1/2 or Monosiga BACE 1/2, again using GFP and
Homo BACE1 as negative and positive controls. We note that despite exhaustive searches,
we were not able to obtain full-length sequences of either gene. Our Mnemiopsis sequence
does encompass the known full-length mature BACE1 (BACE1 is proteolyzed prior to
Biology 2024, 13, 320 6 of 9

Next, we turned our attention to ctenophores and choanoflagellates to determine

if more divergent BACE genes show functional activity towards APP/Aβ. We trans-
fected CHO695 cells with Mnemiopsis BACE 1/2 or Monosiga BACE 1/2, again using GFP
and Homo BACE1 as negative and positive controls. We note that despite exhaustive
searches, we were not able to obtain full-length sequences of either gene. Our Mnemiopsis
sequence does encompass the known full-length mature BACE1 (BACE1 is proteolyzed
prior to function) [4], but our Monosiga sequence is missing one of two known BACE1
active sites. Aβ concentration in conditioned media samples was determined via an
Aβ 1-40 ELISA, and total protein levels from cell lysate samples were used to normal-
ize secretion data. A one-way ANOVA revealed significant differences in Aβ secretion
when comparing GFP, Homo BACE1, Mnemiopsis BACE 1/2 (Figure 2B), and Monosiga
BACE 1/2 [F(3,45) = 4.0945; p < 0.05]. Tukey’s post hoc analysis showed that Homo BACE1
significantly elevated Aβ secretion (p < 0.05), while GFP control, Mnemiopsis BACE 1/2,
and Monosiga BACE 1/2 did not. We confirmed successful transfection and sub-cellular
localization via fluorescence microscopy using GFP-tagged versions of Mnemiopsis and
Monosiga BACE 1/2 proteins (see Supplemental Figure S1), indicating that a lack of Aβ
secretion is not due to a failure of expression or proper localization. Our results demonstrate
that BACE proteolysis towards APP/Aβ is not conserved in the divergent BACE1/2 gene
present in ctenophores, reinforcing the conclusion that it is BACE1-specific and found only
in the bilateria+cnidaria group. Our result with Monosiga is consistent with this conclusion
but must be taken with greater caution given that we know one of two active sites is
lacking. For completeness, future studies can include full-length choanoflagellate BACE
1/2 sequences and placozoa BACE1/2 sequences.

4. Discussion
The present study documents when BACE orthologs evolved the ability to proteolyze
APP/Aβ. Our earlier work demonstrated that APP-like proteins are found throughout
most animal taxa, but sequences homologous to Aβ only evolved within gnathostomes
(jawed vertebrates), and the β cut site is only conserved within sarcopterygians (lobe-finned
fishes). Further, we demonstrated the functional conservation of the BACE1 proteolysis
of APP/Aβ in a cephalochordate (e.g., Branchiostoma floridae). These new data, combining
phylogenetic and functional analyses, push the experimentally verified proteolysis further
back to cnidarians (e.g., Hydra vulgaris). Hydra never evolved the Aβ sequence, and
their evolutionary history predates the origin of Aβ by hundreds of millions of years.
Our demonstration that BACE1 from Branchiostoma and Hydra, which never evolved the
Aβ sequence, can proteolyze human APP and liberate Aβ indicates a very high level
of functional conservation since the origin of BACE1 and suggests BACE1 has deeply
conserved and essential functions that have nothing to do with APP processing. The fact
that BACE1/2 from ctenophores and choanoflagellates is unable to proteolyze human APP
indicates that functional BACE activity towards APP/Aβ is unique to the BACE1 clade.
Indeed, cnidaria are the most basal group to have BACE1, and the most basal group to have
a BACE gene capable of cleaving APP. While the base of our tree remains unresolved, the
combination of phylogenetic with functional analyses strongly suggests that this specific
proteolytic activity evolved in the ancestor of cnidarians+bilterians, likely after a gene
duplication (see Figure 3 for a summary).
Our data re-emphasize that BACE1 has deeply conserved and essential functions
that are independent of APP/Aβ proteolysis. BACE1 has multiple substrates besides
APP, and some of its potential non-amyloidogenic roles are beginning to emerge. Among
the more intriguing ones are the BACE1 proteolysis of NRG1 during myelination [20],
the cleavage of SEZ6 and the promotion of dendritic branching [21], and the proteolysis
of CHL1, resulting in semaphorin-regulated growth cone collapse [22]. These essential
developmental functions likely represent the ancestral role of BACE1 in animals, but further
work will be needed to demonstrate this.
 deed, cnidaria are the most basal group to have BACE1, and the most basal group to have
a BACE gene capable of cleaving APP. While the base of our tree remains unresolved, the
combination of phylogenetic with functional analyses strongly suggests that this specific
proteolytic activity evolved in the ancestor of cnidarians+bilterians, likely after a gene du-
Biology 2024, 13, 320 plication (see Figure 3 for a summary). 7 of 9

Figure 3. Relative timeline for BACE1 and Aβ evolution. Simplified animal phylogeny with key
Figure 3. Relative timeline for BACE1 and Aβ evolution. Simplified animal phylogeny with key
events supported in this paper and our previous paper [11]. Pre-duplicate BACE1/2 predates animal
events supported in this paper and our previous paper [11]. Pre-duplicate BACE1/2 predates ani-
origins and can be found in extant choanoflagellates, as well as in ctenophores and placozoans
mal origins and can be found in extant choanoflagellates, as well as in ctenophores and placozoans
(not
(notshown
shownfor forsimplicity).
simplicity).Cnidaria
Cnidariaarearethe
themost
most basal
basal group
group that
that definitively shows BACE1.
definitively shows BACE1. We We
demonstrate
demonstratethat thatthis
thisgene
genecan
canproteolyze
proteolyzeAPP/Aβ,
APP/Aβ,while
whileBACE1/2
BACE1/2 genes
genes cannot.
cannot. This
This proteolytic
proteolytic
ability
abilitythus
thuscorrelates
correlateswith
withthe
thelikely advent
likely adventofof
BACE1
BACE1 byby
gene
geneduplication.
duplication.Strikingly, thethe
Strikingly, ability of
ability
of BACE1
BACE1 to proteolyze
to proteolyze APP/Aβ
APP/Aβ predates
predates thethe actual
actual origin
origin of of
Aβ Aβinin vertebrates
vertebrates byby severalhundred
several hun-
dred million
million years. years.

Comparisons between BACE1

Our data re-emphasize and BACE2
that BACE1 showconserved
has deeply that the two andhomologous sequences
essential functions that
are
aredistinct in function
independent and regulation.
of APP/Aβ BACE2
proteolysis. BACE1 is has
expressed
multipleat substrates
low levels besides
in the brain
APP,but
and
issome
highlyof expressed in non-amyloidogenic
its potential peripheral tissues such
rolesas
areinbeginning
the pancreas, colon, and
to emerge. Among kidney
the [23].
more
BACE2 functions
intriguing include
ones are regulating
the BACE1 glucose
proteolysis ofhomeostasis
NRG1 during inmyelination
beta-islet cells
[20],and
thepigmen-
cleavage
tation in melanosomes [24]. Furthermore, BACE2 does not appear to
of SEZ6 and the promotion of dendritic branching [21], and the proteolysis of CHL1, proteolyze APP tore-
produce Aβ; instead, it cleaves within the Aβ sequence at the theta cut site
sulting in semaphorin-regulated growth cone collapse [22]. These essential developmental [25] and lowers
Aβ secretion from cultured cells [26].
Alzheimer’s disease is the fifth-leading cause of death among Americans 65 and older
and costs billions of dollars annually [27,28]. The development of therapeutics is critically
important, and BACE1 inhibition remains a preferred target for AD therapeutics despite
setbacks and adverse side effects [9,10]. Further elucidation of the basal animal species
in which BACE proteolytic function arose and its core biological functions and substrates
will help inform treatment strategies and their implications. In particular, it may prove
necessary to develop BACE modulators that selectively inhibit the beta-cleavage of APP. It
will also be of interest to determine the functional origin of the APP β-cleavage site. Aβ-
containing APPs extend deep into vertebrate phylogeny, but not beyond vertebrates [11].
Elucidating which taxa retain bona fide BACE1 substrates will help complete the picture of
BACE1 evolution vis-a-vis APP/Aβ proteolysis and will further contextualize our finding
that BACE1 proteolytic function evolved during the very earliest diversification of animals.

5. Conclusions
In conclusion, we have performed a detailed phylogenetic and functional biochemical
examination of the evolutionary history of BACE1, the rate-limiting protease responsible
for liberating the Aβ peptide from amyloid precursor protein in Alzheimer’s disease. Using
a combination of phylogenetic and functional cell-based biochemical analyses, we have
demonstrated that the most basal BACE1 ortholog is found in cnidarians, while ctenophores
and choanoflagellates have genes equally orthologous to BACE1 and BACE2. A cnidarian
BACE1 (Hydra vulgaris) can cleave human APP to release Aβ, but neither ctenophore nor
Biology 2024, 13, 320 8 of 9

choanoflagellate BACE1/2 has this activity. These findings indicate that the proteolytic
function of BACE1 evolved deep in the animal lineage, likely after a gene-duplication event.

Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/biology13050320/s1: Figure S1: Successful expression and similar
localization of BACE proteins.
Author Contributions: Conceptualization, J.A.L. and D.B.M.; Methodology, J.A.L., L.B., E.M.D.,
R.A.V., N.M., A.A. and D.B.M.; Investigation, J.A.L. and D.B.M.; Resources, J.A.L. and D.B.M.;
Data curation, J.A.L. and D.B.M.; Writing—Original draft, J.A.L. and D.B.M.; Writing—Review and
editing, J.A.L. and D.B.M.; Supervision, J.A.L. and D.B.M.; Project administration, J.A.L. and D.B.M.;
Funding acquisition, J.A.L. and D.B.M. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by a National Science Foundation grant for DBM and JAL
(NSF RUI 1817867), and by a summer research fellowship (for EMD) from the F.W. and Elsie L.
Heyl Foundation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
Acknowledgments: We gratefully thank Virginia M-Y Lee, University of Pennsylvania, for the gift of
the CHO 695 cell line.
Conflicts of Interest: The authors declare no conflicts of interest.

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