Proteostasis dysfunction plays a central role in Alzheimer's disease (AD), where aberrant accumulation of amyloid precursor protein (APP)-derived peptides, including APP-C99 and amyloid-β (Aβ), contributes to neurotoxicity. Previous work with an APP-C99 neuronal cell model revealed impaired proteasome activity, lysosomal dysfunction, and increased autophagic markers LC3 and p62.
Objective
To identify small molecule modulators of proteostasis that reduce Aβ-mediated toxicity and to evaluate their mechanism of action in both cellular and C. elegans models of AD.
Methods
We screened a library of small molecule proteostasis modulators in the APP-C99 cell model to identify compounds that reduce Aβ-mediated cell death. Hits were further analyzed for their effects on APP-C99/Aβ clearance, autophagy, and proteasomal function. Neuroprotective effects were validated in an AD C. elegans model.
Results
The USP14 deubiquitinase inhibitor IU1 increased cell survival by 40%, reduced APP-C99 and Aβ accumulation, restored proteasomal activity, LC3 and p62 levels to control. IU1 also decreased neuronal loss and improved survival and behavior in AD worms. Notably, autophagy activators, including mTOR inhibitors rapamycin, everolimus, and temsirolimus, worsened Aβ toxicity. Conversely, autophagy inhibitors such as Bafilomycin A and chloroquine reduced APP-C99/Aβ accumulation, enhanced proteasomal activity, decreased cell death, and improved neurodegeneration and behavior in the worm model.
Conclusions
This study reveals that, under Aβ-mediated proteostasis dysfunction, autophagy activation exacerbates toxicity, whereas proteasome activation via allosteric inhibition of USP14 using IU1 was neuroprotective. These findings provide evidence to suggest that targeting proteasome stimulation via pharmacological inhibition of USP14 offers a promising therapeutic strategy for AD.
KriskoARadmanM. Protein damage, ageing and age-related diseases. Open Biol2019; 9: 180249.
2.
BharadwajPRDubeyAKMastersCL, et al.Abeta aggregation and possible implications in Alzheimer's disease pathogenesis. J Cell Mol Med2009; 13: 412–421.
3.
GoutagnyRGuNCavanaghC, et al.Alterations in hippocampal network oscillations and theta-gamma coupling arise before Abeta overproduction in a mouse model of Alzheimer's disease. Eur J Neurosci2013; 37: 1896–1902.
4.
HammVHeraudCBottJB, et al.Differential contribution of APP metabolites to early cognitive deficits in a TgCRND8 mouse model of Alzheimer's disease. Sci Adv2017; 3: e1601068.
5.
LauritzenIPardossi-PiquardRBauerC, et al.The beta-secretase-derived C-terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple-transgenic mouse hippocampus. J Neurosci2012; 32: 16243–11655a.
6.
MitaniYYarimizuJSaitaK, et al.Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice. J Neurosci2012; 32: 2037–2050.
7.
NeveRLBoyceFMMcPhieDL, et al.Transgenic mice expressing APP-C100 in the brain. Neurobiol Aging1996; 17: 191–203.
8.
BolandBKumarALeeS, et al.Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. J Neurosci2008; 28: 6926–6937.
9.
BolandBYuWHCortiO, et al.Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing. Nat Rev Drug Discov2018; 17: 660–688.
10.
BharadwajPRVerdileGBarrRK, et al.Latrepirdine (dimebon) enhances autophagy and reduces intracellular GFP-Abeta42 levels in yeast. J Alzheimers Dis2012; 32: 949–967.
11.
SteeleJWLachenmayerMLJuS, et al.Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model. Mol Psychiatry2013; 18: 889–897.
12.
SarkarSRubinszteinDC. Small molecule enhancers of autophagy for neurodegenerative diseases. Mol Biosyst2008; 4: 895–901.
13.
SarkarS. Chemical screening platforms for autophagy drug discovery to identify therapeutic candidates for Huntington's disease and other neurodegenerative disorders. Drug Discov Today Technol2013; 10: e137–e144.
14.
SarkarSPerlsteinEOImarisioS, et al.Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol2007; 3: 331–338.
15.
CaoJZhongMBToroCA, et al.Endo-lysosomal pathway and ubiquitin-proteasome system dysfunction in Alzheimer's disease pathogenesis. Neurosci Lett2019; 703: 68–78.
16.
NixonRAWegielJKumarA, et al.Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol2005; 64: 113–122.
17.
Martinez-VicenteMCuervoAM. Autophagy and neurodegeneration: when the cleaning crew goes on strike. Lancet Neurol2007; 6: 352–361.
18.
RubinszteinDCGestwickiJEMurphyLO, et al.Potential therapeutic applications of autophagy. Nat Rev Drug Discov2007; 6: 304–312.
19.
ShintaniTKlionskyDJ. Autophagy in health and disease: a double-edged sword. Science2004; 306: 990–995.
20.
MputhiaZHoneETripathiT, et al.Autophagy modulation as a treatment of amyloid diseases. Molecules2019; 24: 3372.
21.
BharadwajPMartinsRN. PRKAG2 Gene expression is elevated and its protein levels are associated with increased amyloid-beta accumulation in the Alzheimer's disease brain. J Alzheimers Dis2020; 74: 441–448.
22.
BharadwajPRMartinsRN. Autophagy modulates Abeta accumulation and formation of aggregates in yeast. Mol Cel Neurosci2020; 104: 103466.
23.
BharadwajPRBatesKAPorterT, et al.Latrepirdine: molecular mechanisms underlying potential therapeutic roles in Alzheimer's and other neurodegenerative diseases. Transl Psychiatry2013; 3: e332.
24.
DomiseMSauveFDidierS, et al.Neuronal AMP-activated protein kinase hyper-activation induces synaptic loss by an autophagy-mediated process. Cell Death Dis2019; 10: 221.
25.
KrishnanSShresthaYJayatungaDPW, et al.Activate or inhibit? Implications of autophagy modulation as a therapeutic strategy for Alzheimer's disease. Int J Mol Sci2020; 21: 6739.
26.
NixonRA. The role of autophagy in neurodegenerative disease. Nat Med2013; 19: 983–997.
CiechanoverABrundinP. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron2003; 40: 427–446.
29.
RubinszteinDC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature2006; 443: 780–786.
30.
KellerJNHanniKBMarkesberyWR. Impaired proteasome function in Alzheimer's disease. J Neurochem2000; 75: 436–439.
31.
McNaughtKSOlanowCWHalliwellB, et al.Failure of the ubiquitin-proteasome system in Parkinson's disease. Nat Rev Neurosci2001; 2: 589–594.
32.
OrtegaZDiaz-HernandezMLucasJJ. Is the ubiquitin-proteasome system impaired in Huntington's disease?Cell Mol Life Sci2007; 64: 2245–2257.
33.
CecariniVBonfiliLAmiciM, et al.Amyloid peptides in different assembly states and related effects on isolated and cellular proteasomes. Brain Res2008; 1209: 8–18.
34.
GregoriLFuchsCFigueiredo-PereiraME, et al.Amyloid beta-protein inhibits ubiquitin-dependent protein degradation in vitro. J Biol Chem1995; 270: 19702–19708.
35.
LinderssonEBeedholmRHojrupP, et al.Proteasomal inhibition by alpha-synuclein filaments and oligomers. J Biol Chem2004; 279: 12924–12934.
36.
BenceNFSampatRMKopitoRR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science2001; 292: 1552–1555.
37.
OhSHongHSHwangE, et al.Amyloid peptide attenuates the proteasome activity in neuronal cells. Mech Ageing Dev2005; 126: 1292–1299.
38.
TsengBPGreenKNChanJL, et al.Abeta inhibits the proteasome and enhances amyloid and tau accumulation. Neurobiol Aging2008; 29: 1607–1618.
39.
KristiansenMDeriziotisPDimcheffDE, et al.Disease-associated prion protein oligomers inhibit the 26S proteasome. Mol Cell2007; 26: 175–188.
40.
CliffeRSangJCKundelF, et al.Filamentous aggregates are fragmented by the proteasome holoenzyme. Cell Rep2019; 26: 2140–2149.e3.
41.
YeYKlenermanDFinleyD. N-terminal ubiquitination of amyloidogenic proteins triggers removal of their oligomers by the proteasome holoenzyme. J Mol Biol2020; 432: 585–596.
42.
CurraisAQuehenbergerOArmandoAM, et al.Amyloid proteotoxicity initiates an inflammatory response blocked by cannabinoids. NPJ Aging Mech Dis2016; 2: 16012.
43.
SopherBLFukuchiKSmithAC, et al.Cytotoxicity mediated by conditional expression of a carboxyl-terminal derivative of the beta-amyloid precursor protein. Brain Res Mol Brain Res1994; 26: 207–217.
44.
HiltSLiuRMaezawaI, et al.Novel stilbene-nitroxyl hybrid compounds display discrete modulation of amyloid beta toxicity and structure. Front Chem2022; 10: 896386.
45.
GrayNEMorréJKelleyJ, et al.Caffeoylquinic acids in Centella asiatica protect against amyloid-β toxicity. J Alzheimers Dis2014; 40: 359–373.
46.
MaezawaIZouBDi LucenteJ, et al.The anti-amyloid-β and neuroprotective properties of a novel tricyclic pyrone molecule. J Alzheimers Dis2017; 58: 559–574.
47.
HiltSAltmanRKálaiT, et al.A bifunctional anti-amyloid blocks oxidative stress and the accumulation of intraneuronal amyloid-beta. Molecules2018; 23: 2010.
48.
HuangLMcClatchyDBMaherP, et al.Intracellular amyloid toxicity induces oxytosis/ferroptosis regulated cell death. Cell Death Dis2020; 11: 828.
49.
YedlapudiDXuLLuoD, et al.Targeting alpha synuclein and amyloid beta by a multifunctional, brain-penetrant dopamine D2/D3 agonist D-520: potential therapeutic application in Parkinson's disease with dementia. Sci Rep2019; 9: 19648.
50.
SoaresPRahamanMMaherP, et al.Protection against amyloid-β aggregation and ferroptosis/oxytosis toxicity by arylpyrazolones: Alzheimer's disease therapeutics. ACS Med Chem Lett2025; 16: 294–300.
51.
GriffinEFScopelSEStephenCA, et al.ApoE-associated modulation of neuroprotection from Aβ-mediated neurodegeneration in transgenic Caenorhabditis elegans. Dis Model Mech2019; 12: dmm037218.
52.
SopherBLFukuchiKKavanaghTJ, et al.Neurodegenerative mechanisms in Alzheimer disease. A role for oxidative damage in amyloid beta protein precursor-mediated cell death. Mol Chem Neuropathol1996; 29: 153–168.
53.
AriyathAKanthiJMPaul-PrasanthB. Differentiation potential of cultured extracellular DEAD-box helicase 4+oogonial stem cells from adult human ovaries into somatic lineages. Cells Tissues Organs2022; 211: 577–588.
54.
TaylorSCBerkelmanTYadavG, et al.A defined methodology for reliable quantification of western blot data. Mol Biotechnol2013; 55: 217–226.
55.
PorterTBharadwajPGrothD, et al.The effects of latrepirdine on amyloid-β aggregation and toxicity. J Alzheimer Dis2016; 50: 895–905.
56.
ByerlyLCassadaRCRussellRL. The life cycle of the nematode Caenorhabditis elegans. I. Wild-type growth and reproduction. Dev Biol1976; 51: 23–33.
57.
HsinHKenyonC. Signals from the reproductive system regulate the lifespan of C. elegans. Nature1999; 399: 362–366.
58.
HippMSKasturiPHartlFU. The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol2019; 20: 421–435.
59.
MputhiaZHoneETripathiT, et al.Autophagy modulation as a treatment of amyloid diseases. Molecules2019; 24: 3372.
60.
AnekondaTSQuinnJFHarrisC, et al.L-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for Alzheimer's disease. Neurobiol Dis2011; 41: 62–70.
61.
CopenhaverPFAnekondaTSMusasheD, et al.A translational continuum of model systems for evaluating treatment strategies in Alzheimer's disease: isradipine as a candidate drug. Dis Model Mech2011; 4: 634–648.
62.
MizushimaNYoshimoriT. How to interpret LC3 immunoblotting. Autophagy2007; 3: 542–545.
63.
MizushimaNYoshimoriTLevineB. Methods in mammalian autophagy research. Cell2010; 140: 313–326.
64.
KimJKunduMViolletB, et al.AMPK And mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol2011; 13: 132–141.
65.
RussellRCYuanHXGuanKL. Autophagy regulation by nutrient signaling. Cell Res2014; 24: 42–57.
66.
EganDFShackelfordDBMihaylovaMM, et al.Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science2011; 331: 456–461.
67.
SteeleJWLachenmayerMLJuS, et al.Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model. Mol Psychiatry2013; 18: 889–897.
68.
BharadwajPVerdileGBarrRK, et al.Latrepirdine (Dimebon) enhances autophagy and reduces intracellular GFP-Aβ42 levels in yeast. J Alzheimers Dis2012; 32: 949–967.
69.
LiLZhangSZhangX, et al.Autophagy enhancer carbamazepine alleviates memory deficits and cerebral amyloid-beta pathology in a mouse model of Alzheimer's disease. Curr Alzheimer Res2013; 10: 433–441.
70.
LeeBHLeeMJParkS, et al.Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature2010; 467: 179–184.
71.
BoselliMLeeBHRobertJ, et al.An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination in cultured neurons. J Biol Chem2017; 292: 19209–19225.
72.
HommaTIshibashiDNakagakiT, et al.Ubiquitin-specific protease 14 modulates degradation of cellular prion protein. Sci Rep2015; 5: 11028.
73.
JinYNChenPCWatsonJA, et al.Usp14 deficiency increases tau phosphorylation without altering tau degradation or causing tau-dependent deficits. PLoS One2012; 7: e47884.
74.
KimEParkSLeeJH, et al.Dual function of USP14 deubiquitinase in cellular proteasomal activity and autophagic flux. Cell Rep2018; 24: 732–743.
75.
KiprowskaMJStepanovaATodaroDR, et al.Neurotoxic mechanisms by which the USP14 inhibitor IU1 depletes ubiquitinated proteins and Tau in rat cerebral cortical neurons: relevance to Alzheimer's disease. Biochim Biophys Acta Mol Basis Dis2017; 1863: 1157–1170.
76.
SiwachAPatelHKhairnarA, et al.Molecular symphony of mitophagy: ubiquitin-specific protease-30 as a maestro for precision management of neurodegenerative diseases. CNS Neurosci Ther2025; 31: e70192.
77.
ChakrabortyJvon StockumSMarchesanE, et al.USP14 Inhibition corrects an in vivo model of impaired mitophagy. EMBO Mol Med2018; 10: e9014.
78.
XuDShanBSunH, et al.USP14 Regulates autophagy by suppressing K63 ubiquitination of Beclin 1. Genes Dev2016; 30: 1718–1730.
79.
XiaXHuangCLiaoY, et al.Inhibition of USP14 enhances the sensitivity of breast cancer to enzalutamide. J Exp Clin Cancer Res2019; 38: 220.
80.
YamamotoATagawaYYoshimoriT, et al.Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct1998; 23: 33–42.
81.
KuangHTanCYTianHZ, et al.Exploring the bi-directional relationship between autophagy and Alzheimer's disease. CNS Neurosci Ther2020; 26: 155–166.
82.
PivtoraikoVNHarringtonAJMaderBJ, et al.Low-dose bafilomycin attenuates neuronal cell death associated with autophagy-lysosome pathway dysfunction. J Neurochem2010; 114: 1193–1204.
83.
BustamanteHAGonzálezAECerda-TroncosoC, et al.Interplay between the autophagy-lysosomal pathway and the ubiquitin-proteasome system: a target for therapeutic development in Alzheimer's disease. Front Cell Neurosci2018; 12: 126.
84.
ChalfieMSulstonJEWhiteJG, et al.The neural circuit for touch sensitivity in Caenorhabditis elegans. J Neurosci1985; 5: 956–964.
85.
HilliardMABargmannCIBazzicalupoP. C. elegans responds to chemical repellents by integrating sensory inputs from the head and the tail. Curr Biol2002; 12: 730–734.
86.
YousefiSPerozzoRSchmidI, et al.Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol2006; 8: 1124–1132.
87.
YangDSKumarAStavridesP, et al.Neuronal apoptosis and autophagy cross talk in aging PS/APP mice, a model of Alzheimer's disease. Am J Pathol2008; 173: 665–681.
88.
DesclouxCGinetVRummelC, et al.Enhanced autophagy contributes to excitotoxic lesions in a rat model of preterm brain injury. Cell Death Dis2018; 9: 853.
89.
GinetVPuyalJClarkePG, et al.Enhancement of autophagic flux after neonatal cerebral hypoxia-ischemia and its region-specific relationship to apoptotic mechanisms. Am J Pathol2009; 175: 1962–1974.
90.
ScottRCJuhaszGNeufeldTP. Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol2007; 17: 1–11.
91.
LeeDJWHodzic KuerecAMaierAB. Targeting ageing with rapamycin and its derivatives in humans: a systematic review. Lancet Healthy Longev2024; 5: e152–e162.
92.
WuFWeiXWuY, et al.Chloroquine promotes the recovery of acute spinal cord injury by inhibiting autophagy-associated inflammation and endoplasmic reticulum stress. J Neurotrauma2018; 35: 1329–1344.
93.
JiangSZhaoYZhangT, et al.Galantamine inhibits beta-amyloid-induced cytostatic autophagy in PC12 cells through decreasing ROS production. Cell Prolif2018; 51: e12427.
94.
ShackaJJKlockeBJRothKA. Autophagy, bafilomycin and cell death: the “a-B-cs” of plecomacrolide-induced neuroprotection. Autophagy2006; 2: 228–230.
95.
ShackaJJKlockeBJShibataM, et al.Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons. Mol Pharmacol2006; 69: 1125–1136.
96.
GaoMMonianPPanQ, et al.Ferroptosis is an autophagic cell death process. Cell Res2016; 26: 1021–1032.
97.
KoikeMShibataMTadakoshiM, et al.Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury. Am J Pathol2008; 172: 454–469.
98.
CherraSJChuCT3rd. Autophagy in neuroprotection and neurodegeneration: a question of balance. Future Neurol2008; 3: 309–323.
99.
SamaraCSyntichakiPTavernarakisN. Autophagy is required for necrotic cell death in Caenorhabditis elegans. Cell Death Differ2008; 15: 105–112.
100.
QinBChenXWangF, et al.DUBs in Alzheimer's disease: mechanisms and therapeutic implications. Cell Death Discov2024; 10: 475.
101.
KurtishiARosenBPatilKS, et al.Cellular proteostasis in neurodegeneration. Mol Neurobiol2019; 56: 3676–3689.
102.
OrtunoDCarlisleHJMillerS. Does inactivation of USP14 enhance degradation of proteasomal substrates that are associated with neurodegenerative diseases?F1000Res2016; 5: 137.
103.
ChadchankarJKorboukhVConwayLC, et al.Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells. PLoS One2019; 14: e0225145.
104.
WangYJiangYDingS, et al.Small molecule inhibitors reveal allosteric regulation of USP14 via steric blockade. Cell Res2018; 28: 1186–1194.
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