Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In ...this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson’s disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
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•Reprogramming rejuvenates old donor fibroblasts by erasing age-related markers•Differentiation of old donor iPSCs is not sufficient to trigger memory of age•Progerin induces age-associated phenotypes in iPSC-derived fibroblasts and neurons•Progerin reveals late-onset disease phenotypes in iPSC-based models of genetic PD
The induction of aging-related features in human iPS-derived cells through expression of progerin addresses one of the major limitations of human iPS-based disease modeling and enables analysis of late-onset phenotypes in conditions such as Parkinson’s disease.
Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish ...optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.
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•Human ES cell-derived spinal motorneurons can support optogenetic control•Cocultures of human motorneurons and skeletal muscle twitch upon light stimulation•Functional connectivity is evident from imaging, physiology, and pharmacology•Patient-derived serum can model autoimmune neuromuscular disease
Steinbeck and colleagues use optogenetics in human spinal motorneurons to establish a functional neuromuscular junction with co-cultured skeletal muscle in vitro and then apply this system for phenotypic analysis of an autoimmune disease.
Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal ...models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
Parkinson's disease (PD) is characterized by the selective loss of dopamine neurons in the substantia nigra; however, the mechanism of neurodegeneration in PD remains unclear. A subset of familial PD ...is linked to mutations in PARK2 and PINK1, which lead to dysfunctional mitochondria-related proteins Parkin and PINK1, suggesting that pathways implicated in these monogenic forms could play a more general role in PD. We demonstrate that the identification of disease-related phenotypes in PD-patient-specific induced pluripotent stem cell (iPSC)-derived midbrain dopamine (mDA) neurons depends on the type of differentiation protocol utilized. In a floor-plate-based but not a neural-rosette-based directed differentiation strategy, iPSC-derived mDA neurons recapitulate PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. We propose that these form a pathogenic loop that contributes to disease. Our study illustrates the promise of iPSC technology for examining PD pathogenesis and identifying therapeutic targets.
•Disease modeling study with patient (monogenic)-derived iPSC for Parkinson's disease•Disease phenotypes exhibited by PD iPSC-derived midbrain DA neurons involved•Mitochondria, α-synuclein, selective vulnerability, and neurotransmitter regulation•These phenotypes may interact synergistically throughout PD progression
Shim, Studer, and colleagues demonstrate that using a floor-plate-based differentiation strategy, Parkinson's disease (PD) patient iPSC-derived mDA neurons recapitulate several PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. The authors further propose that these phenotypes form a pathogenic loop contributing to disease.
Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by ...developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.
► We generated mutant mice with a forebrain-specific
cereblon gene deletion. ► We examined the behavioral profile of these mutant mice. ► A decrease in
cereblon mRNA expression was confined to the ...forebrain. ► The
cereblon knock-out mice have deficits in fear conditioning. ► These mice model humans with a
cereblon mutation and an intellectual disability.
A nonsense mutation in the human
cereblon gene (
CRBN) causes a mild type of autosomal recessive non-syndromic intellectual disability (ID). Animal studies show that
crbn is a cytosolic protein with abundant expression in the hippocampus (HPC) and neocortex (CTX). Its diverse functions include the developmental regulation of ion channels at the neuronal synapse, the mediation of developmental programs by ubiquitination, and a target for herpes simplex type I virus in HPC neurons. To test the hypothesis that anomalous
CRBN expression leads to HPC-mediated memory and learning deficits, we generated germ-line
crbn knock-out mice (
crbn
−/−). We also inactivated
crbn in forebrain neurons in conditional knock-out mice in which
crbn exons 3 and 4 are deleted by
cre recombinase under the direction of the
Ca
2+
/calmodulin-dependent protein kinase II alpha promoter (
CamKII
cre/+,
crbn
−/−).
crbn mRNA levels were negligible in the HPC, CTX, and cerebellum (CRBM) of the
crbn
−/− mice. In contrast,
crbn mRNA levels were reduced 3- to 4-fold in the HPC, CTX but not in the CRBM in
CamKII
cre/+,
crbn
−/− mice as compared to wild type (
CamKII
cre/+,
crbn
+/+). Contextual fear conditioning showed a significant decrease in the percentage of freezing time in
CamKII
cre/+,
crbn
−/− and
crbn
−/− mice while motor function, exploratory motivation, and anxiety-related behaviors were normal. These findings suggest that
CamKII
cre/+,
crbn
−/− mice exhibit selective HPC-dependent deficits in associative learning and supports the use of these mice as
in vivo models to study the functional consequences of
CRBN aberrations on memory and learning in humans.
The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' ...given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.
The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are ...capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics.
Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and ...folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.
Environmental and genetic risk factors contribute to the selective degeneration of midbrain (mDA) neurons in Parkinson’s disease (PD) patients. Current PD treatments can only suppress symptoms as the ...disease continues to progress. To develop treatments that could slow or reverse disease, we must be able to precisely identify events that lead to neuronal death before cells die. However, attempts to detect PD and other neurodegenerative disorders in their early stages have been hindered by the inaccessibility of appropriate cell types and the absence of techniques to pinpoint these biochemical changes. Induced pluripotent stem cells (iPSCs) reprogrammed from patient skin fibroblasts have potential to be an unlimited source of disease-relevant neurons. Chemical probes for heat shock protein 90 (HSP90) have enabled the identification of stress-related proteins critical for cancer cell function. Here we use HSP90 biochemical sensors combined with proteomic and bioinformatics analyses to identify dysfunctional signaling networks in Parkinson’s disease (PD) iPSC-derived mDA neurons. We identify a stress-related HSP90 co-chaperone response, stratified in response to PD-related genetic versus toxic trigger. We define downstream consequences of these HSP90 complexes on the mDA neuron proteome. One pathway enriched in PARKIN mutant mDA neuron HSP90 complexes and attenuated following HSP90 inhibition was STAT3 signaling. Similarly, we identified HSP90 complexes selectively responding to PD-related toxic challenge including stress induced increase of phosphorylated tyrosine hydroxylase (TH). TH is the rate-limiting enzyme in dopamine synthesis and phosphorylation is thought to increase its activity. Indeed, we observed higher levels of intracellular dopamine in stressed mDA neurons. We showed that HSP90 inhibition reversed levels of phosphorylated TH and intracellular dopamine, as well as improved viability of mDA neurons under toxic stress conditions. Our study demonstrates the use of HSP90 chemical sensors to define PD-related genetic and toxic stress associated pathways that may represent novel therapeutic targets in PD. Combining patient-specific iPSC technology with biochemical sensors for HSP90 may represent a general approach to identify protein networks involved in neurodegenerative diseases.
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