identified genes are more cohesive than would be
expected with candidates selected at random.
To identify proximal interactors, we expanded
the global network by including HumanNet
protein interaction database (9) and literature-curated interactions from STRING (10) to derive
an expanded global network (fig. S6). This network propagation method assigns a priority score
to each protein within the network (11). From this
expanded network, we extracted the expanded HSP
seeds network and found that 7 of the 15 newly
identified candidates have significant support in
the network (ARS1, DDHD2, ERLIN1, FLRT1,
KIF1C, PGAP1, and RAB3GAP2, FDR < 0.1). Genes
involved in biochemical pathways, such as NT5C2,
AMPD2, and ENTPD1, did not emerge from this
analysis, probably because of a lack of metabolic
network edges in the input networks. Proteins
that were not well characterized or represented in
public databases also did not show enrichment.
We next expanded the HSP seed + candidate
network to derive the HSPome (i.e., HSP seeds +
candidates + proximal interactors network), allowing a global view of HSP and flagging other potential genes that may be mutated in HSP patients. The
HSPome contains 589 proteins (i.e., potential HSP
candidates) (supplementary data 2 and table S5).
Implicated Causal Genes Suggest Modules of
Studies in HSP consistently report an ascending
axonal CST degeneration (12), but the processes
modulating this degeneration are not well defined.
Supporting the hypothesis that individual rare
mutations in distinct genes may converge on specific biological pathways, we identified major
modules involved in the pathophysiology of HSP.
Several HSP genes have previously implicated
endoplasmic reticulum (ER) biology (i.e., ATL1,
REEP1, RTN2, and SPAST) and the ER-associated
degradation (ERAD) pathway (i.e., ERLIN2)
(13–15). From the HSPome, we focused on this
ER subnetwork containing the newly identified
genes ARL6IP1 and ERLIN1 (fig. S7). ARL6IP1
encodes a tetraspan membrane protein localized to
the ER, composed of highly conserved hydropho-
bic hairpin domains implicated in the formation of
ER tubules (16). We overexpressed ARL6IP1 in
cells and noted dramatically altered ER shape (fig.
S7). The ERAD system controls protein quality
control, critical for cellular adaptation to stress and
survival. ERLIN1 encodes a prohibin-domain-
containing protein localized to the ER that forms
a ring-shaped complex with ERLIN2, further im-
plicating defective ERAD in HSP etiology.
We identified an endosomal and membrane-trafficking subnetwork composed of seeds and
candidates KIF1C, USP8, and WDR48, implicating the endosomal sorting complexes required
for transport (ESCRT) pathway (fig. S8). USP8
MOctrl2.5ng MOarl6ip11ng MOarl6ip12.5ng MOpgap110ng MOusp81ng MOusp82.5ng
MOctrl MOarl6ip1 MOpgap1 MOusp8
MOctrl2.5ngMOarl6ip12.5ng MOpgap110ng MOusp81ng MOusp82.5ng
MOctrl2.5ngMOarl6ip12.5ngMOpgap110ng MOusp81ng MOusp82.5ng
MOctrl2.5ng MOMO arl6ip11ng arl6ip12.5ng MOarl6ip15ng MOarl6ip110ng MOmars0.25ng MOmars0.5ng MOmars1ng MOmars2.5ng MOmars5ng MOmars10ng MOpgap110ng MOusp81ng MOusp82.5ng MOusp85ng MOusp810ng
Fig. 1. Functional validation of private HSP genes in zebrafish. (A)
Quantification of 24-hours-post-fertilization (hpf) embryos mortality (black)
and curly-tail (gray) phenotypes for noninjected (NI), scrambled, and morphants
(MO) at stated nanogram concentrations. Overt phenotypes were observed for
all MOs except MOpgap1. (B) Average touch-response distance (in arbitrary units,
A.U.) in 72-hpf larvae, showing blunted response for all MOs. (C) Immediate
touch-response trajectory of example larvae, each shaded uniquely. Mars2
MO was too severe to be tested, whereas others showed reduced response.
(D to F) Spontaneous locomotion at 6 days post fertilization. (D) Average
percent of time spent moving over a 30-min window showed a reduction
for all for at least one dose. (E) Average active period duration, showing
reduction for all. (F) Representative kymographs recording fish position
(black dot) over 30-min recording. MOs showed either short distance traveled
(MOarl6ip1) or reduced movements per recording (MOpgap1 and MOusp8). *P < 0.01
(t test). N > 2 experiments with n > 20 animals per experiment. Error bars
indicate standard error.