Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90

Investigation of the Hsp90 co-chaperone, STI1, in cellular resilience and neurodegenerative diseases

In neurodegenerative diseases, certain proteins misfold and form toxic aggregates that cause brain matter atrophy, leading to decline in motor and/or cognitive functions. To maintain cellular proteostasis and survival, molecular chaperones regulate protein maturation and help to prevent aberrant protein aggregation. The molecular chaperone Hsp90 regulates hundreds of proteins and interestingly, several of those are misfolded in neurodegenerative diseases. Stress inducible-phosphoprotein-1 (STI1, STIP1), an Hsp90 co-chaperone, orchestrates client protein transfer between chaperones Hsp70 and Hsp90 through physical interactions with both chaperones. Notably, previous work in yeast, worms, and mouse neurons all showed that STI1 protects organisms against stressors and amyloid-like proteotoxicity in vitro. However, the physiological roles of STI1 during aging, and whether STI1 can modulate proteotoxicity and aggregation in mammals is unknown. In this dissertation, we explore whether decreased or increased STI1 levels in mice, can modulate aging and proteostasis responses to misfolded protein stress. Our hypothesis is that modifying intracellular and extracellular levels of STI1, in vivo, affects neuronal resilience during aging, disturbs Hsp90 chaperone machinery function, and modulates levels of protein misfolding and aggregation. We reveal that STI1 knockdown in mice reduces Hsp90 machinery function, and that mice present with age-dependent decline in neuronal resilience in the hippocampus, resulting in memory impairments. Unexpectedly, we find that overexpressing STI1 in an AD mouse model accelerates insoluble Aβ aggregation, resulting in greater levels of neurodegeneration and cognitive impairments. Likewise, STI1 overexpression augments α-synuclein accumulation in a mouse model of synucleinopathy, however, knocking down STI1 attenuated α-synuclein aggregation, improving motor performance. Notably, in both models of proteinopathies, STI1 colocalized with protein aggregates, likely modulating STI1 functions. Since our results suggest that reducing STI1 would be favourable for decreasing protein aggregation, we generated STI1 conditional knockdown mice, to establish whether reducing STI1 after development is tolerable, and indeed, we found that to be the case. Overall, in this thesis we provide a greater understanding of mammalian STI1 in neuronal resilience and protein misfolding in vivo and discover STI1 as a potential therapeutic target for treating protein misfolding diseases in the brain

Attenuated Late-Phase Arc Transcription in the Dentate Gyrus of Mice Lacking Egr3

The dentate gyrus (DG) engages in sustained Arc transcription for at least 8 hours following behavioral induction, and this time course may be functionally coupled to the unique role of the DG in hippocampus-dependent learning and memory. The factors that regulate long-term DG Arc expression, however, remain poorly understood. Animals lacking Egr(3) show less Arc expression following convulsive stimulation, but the effect of Egr3 ablation on behaviorally induced Arc remains unknown. To address this, Egr3(-/-) and wild-type (WT) mice explored novel spatial environments and were sacrificed either immediately or after 5, 60, 240, or 480 minutes, and Arc expression was quantified by fluorescence in situ hybridization. Although short-term (i.e., within 60 min) Arc expression was equivalent across genotypes, DG Arc expression was selectively reduced at 240 and 480 minutes in mice lacking Egr3. These data demonstrate the involvement of Egr3 in regulating the late protein-dependent phase of Arc expression in the DG.Natural Sciences and Engineering Research Council of Canada; Ontario Mental Health Foundation; US National Institute of Mental Health Award [MH097803]; Science Foundation of Arizona Bisgrove ScholarshipThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases

The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide—either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome. They form an important line of defense against misfolded proteins and are part of the cellular quality control system. The heat shock protein (Hsp) family, particularly Hsp70 and Hsp90, plays a major part in this process and it is well-known to regulate protein misfolding in a variety of diseases, including tau levels and toxicity in AD. However, the role of Hsp90 in regulating protein misfolding is not yet fully understood. For example, knockdown of Hsp90 and its co-chaperones in a Caenorhabditis elegans model of Aβ misfolding leads to increased toxicity. On the other hand, the use of Hsp90 inhibitors in AD mouse models reduces Aβ toxicity, and normalizes synaptic function. Stress-inducible phosphoprotein 1 (STI1), an intracellular co-chaperone, mediates the transfer of clients from Hsp70 to Hsp90. Importantly, STI1 has been shown to regulate aggregation of amyloid-like proteins in yeast. In addition to its intracellular function, STI1 can be secreted by diverse cell types, including astrocytes and microglia and function as a neurotrophic ligand by triggering signaling via the cellular prion protein (PrPC). Extracellular STI1 can prevent Aβ toxic signaling by (i) interfering with Aβ binding to PrPC and (ii) triggering pro-survival signaling cascades. Interestingly, decreased levels of STI1 in C. elegans can also increase toxicity in an amyloid model. In this review, we will discuss the role of intracellular and extracellular STI1 and the Hsp70/Hsp90 chaperone network in mechanisms underlying protein misfolding in neurodegenerative diseases, with particular focus on AD