Patent Application
20250085272
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The present disclosure relates to methods for modulating cellular aging and/or progression of neurodegenerative diseases (e.g., Alzheimer's disease). The present disclosure also relates to methods and systems for modeling aging related neurodegenerative diseases (e.g., Alzheimer's disease) in vitro.
Alzheimer's disease (AD) has been intensely studied since mutations in APP and PSEN were linked to AD over 25 years ago. Despite the enormous amount of research on this topic, there has been limited success in translating findings into therapies that impact disease outcomes in AD patients. The risk of developing AD increases markedly with age raising the question of how the aging process may contribute to the development of AD. Answering this question has been challenging as AD is a largely human-specific disease, and human aging occur over periods of several decades, a process difficult to capture in any experimental system. Therefore, there is great need to develop a platform that allows controlled manipulations to either age or rejuvenate human brain cells on demand.
The present disclosure relates to methods for modulating cellular aging and/or progression of neurodegenerative diseases (e.g., AD). The present disclosure also relates to methods and systems for modeling aging related neurodegenerative diseases (e.g., AD) in vitro.
In certain embodiments, the present disclosure provides methods of preparing an in vitro model of neurodegenerative disease comprising modulating protein neddylation in a population of neurons, wherein the population of neurons exhibit genetic mutation of at least one gene that is associated with neurodegenerative disease. The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), or Huntington's disease. In certain embodiments, modulating protein neddylation comprises exposing the population of neurons to a compound that modulates protein neddylation. In certain embodiments, the at least one compound that modulates protein neddylation is selected from the group consisting of MLN4924, TAS4464, CSN5i-3, ZM223, NACM-OPT, Keap1-Nrf2-IN-4, WS-383, VII-31, derivatives thereof, and combinations thereof. In certain embodiments, modulating protein neddylation comprises modifying expression of at least one gene which regulates protein neddylation pathways. In certain embodiments, the at least one gene which regulates protein neddylation pathways is selected from the group consisting of UBA3, NAE1, and combinations thereof. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of amyloid-beta peptide. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in a change in the ratio of Aβ40 to Aβ42 peptide.
In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the Amyloid-beta precursor protein (APP) gene. In certain embodiments, the mutation of the APP gene comprises K595N/M596L. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the presenilin-1 (PSEN) gene. In certain embodiments, the mutation of the PSEN gene comprises M146V. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of N-ethylmaleimide sensitive factor (NSF) aggregates. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the (LRRK2) gene. In certain embodiments, the mutation of the LRRK2 gene comprises G2019S.
In certain embodiments, the neurons are obtained from in vitro differentiation of stem cells. In certain embodiments, the stem cells are human stem cells. In certain embodiments, the human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, human primordial germ cell-like pluripotent stem cells, human epiblast stem cells, human F-class pluripotent stem cells, and combinations thereof. In certain embodiments, the neurons are cortical neurons.
In certain embodiments, the present disclosure provides methods of identifying genes associated with cellular aging and/or progression of neurodegenerative disease comprising: obtaining a first population of neurons; obtaining a second population of neurons, and modifying expression of a test gene in the second population of neurons; measuring functional activity of the second population of neurons relative to the first population of neurons; wherein the first population of neurons and the second population of neurons exhibit genetic mutation of at least one gene that is associated with neurodegenerative disease; and wherein a difference in the functional activity between the first population of neurons and the second population of neurons indicates that the test gene is associated with cellular aging and/or progression of neurodegenerative disease. In certain embodiments, the functional activity is cell viability. In certain embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), or Huntington's disease. In certain embodiments, modifying expression of the test gene modulates protein neddylation in the second population of neurons.
In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of amyloid-beta peptide. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in a change in the ratio of Aβ40 to Aβ42 peptide. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the Amyloid-beta precursor protein (APP) gene. In certain embodiments, the mutation of the APP gene comprises K595N/M596L. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the presenilin-1 (PSEN) gene. In certain embodiments, the mutation of the PSEN gene comprises M146V. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of N-ethylmaleimide sensitive factor (NSF) aggregates. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the (LRRK2) gene. In certain embodiments, the mutation of the LRRK2 gene comprises G2019S.
In certain embodiments, the neurons are obtained from in vitro differentiation of stem cells. In certain embodiments, the stem cells are human stem cells. In certain embodiments, the human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, human primordial germ cell-like pluripotent stem cells, human epiblast stem cells, human F-class pluripotent stem cells, and combinations thereof. In certain embodiments, the neurons are cortical neurons.
In certain embodiments, the present disclosure provides compositions for identifying genes associated with cellular aging and/or progression of neurodegenerative disease comprising a population of neurons, wherein the population of neurons exhibit genetic mutation at a test gene, wherein the population of neurons exhibit genetic mutation of at least one gene that is associated with neurodegenerative disease. In certain embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), or Huntington's disease. In certain embodiments, the genetic mutation at the test gene modulates protein neddylation.
In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of amyloid-beta peptide. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in a change in the ratio of Aβ40 to Aβ42 peptide. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the Amyloid-beta precursor protein (APP) gene. In certain embodiments, the mutation of the APP gene comprises K595N/M596L In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the presenilin-1 (PSEN) gene. In certain embodiments, the mutation of the PSEN gene comprises M146V. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of N-ethylmaleimide sensitive factor (NSF) aggregates. In certain embodiments, the at least one gene that is associated with neurodegenerative disease comprises the (LRRK2) gene. In certain embodiments, the mutation of the LRRK2 gene comprises G2019S.
In certain embodiments, the neurons are obtained from in vitro differentiation of stem cells. In certain embodiments, the stem cells are human stem cells. In certain embodiments, the human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, human primordial germ cell-like pluripotent stem cells, human epiblast stem cells, human F-class pluripotent stem cells, and combinations thereof. In certain embodiments, the neurons are cortical neurons.
The present disclosure relates to methods for modulating cellular aging and/or progression of neurodegenerative diseases (e.g., AD). The present disclosure also relates to methods and systems for modeling aging related neurodegenerative diseases (e.g., AD) in vitro.
In certain embodiments, the present disclosure is based on the discovery that neddylation pathway regulates both cellular age and AD-neurodegeneration. Specifically, the present disclosure identifies that blocking neddylation increased cellular senescence, protein aggregation and DNA damage and decreased heterochromatin in cortical neurons. Blocking neddylation also led to an increase in high molecular weight phosphorylated Tau that was specific to neurons with the APPswe/swe mutation. Finally, aged APPswe/swe neurons also showed a greater loss of viability than wild-type neurons
The present disclosure provides a genome screening platform to identify physiologically relevant regulators of cellular age and AD-neurodegeneration. The present disclosure defines hit genes as those whose loss of function selectively compromises the viability of Alzheimer's disease but not control isogenic neurons. The present disclosure identifies that AD-enhanced loss of viability resulted from the synergistic action of the AD genetic susceptibility with a screen-induced age-related vulnerability. The present disclosure shows that experimentally validated hit genes selectively compromised the viability of Alzheimer's disease neurons over isogenic control neurons but did not impact APP processing. Consistent with the hypothesis that age-related vulnerability can synergize with genetic susceptibility, 4 of the 6 experimentally validated hits showed a significant decrease in expression in aged human and mouse primary brain tissue compared to matched young samples. While the present disclosure focuses on UBA3 for further characterization, NAE1, the other member of the heterodimeric E1 ligase for Nedd8 was also a hit in the screen, and chemical inhibition of the neddylation pathway similarly resulted in an AD enhanced loss of viability. Consistent with age-related phenotype, blocking neddylation triggered known hallmarks of age including cellular senescence, DNA damage, loss of proteostasis and a global reduction in heterochromatin. Finally, blocking neddylation also led to an AD-specific increase in high molecular weight phosphorylated Tau and resulted in phospho-Tau positive inclusions.
The present disclosure demonstrates how cellular age and disease genetics can synergize to trigger late-onset disease phenotypes. The present disclosure uses developmentally defined cortical neurons generated by directed differentiation (rather than transcription-factor based iNeurons) to perform a whole genome CRISPR screen. In addition, the present disclosure takes one of the major challenges of stem cell models of neurodegenerative disease—namely that late onset phenotypes like neuronal loss have been challenging to model in vitro due to their embryonic nature of hPSC-derived cells—and uses this as the basis for a phenotypic “gain-of-disease” screen. The presently disclosed aged AD-PSC model can be used in screening for drugs that can prevent disease progression and neuronal loss. Finally, the present disclosure also has broad implications for human disease modelling, as it highlights the importance of generating cells of the appropriate “age” in addition to the correct developmental lineage and cellular identity.
For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:
The terms used in this disclosure generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold, or within 2-fold, of a value.
As used herein, the term “stem cell” refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. In certain embodiments, a stem cell can refer to an embryonic stem cell or an induced pluripotent stem cell (iPSC). A human stem cell refers to a stem cell that is derived from a human.
As used herein, the term “embryonic stem cell” refers to a primitive (undifferentiated) cell that is derived from preimplantation-stage embryo, capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers. A human embryonic stem cell refers to an embryonic stem cell that is from a human. As used herein, the term “human embryonic stem cell” or “hESC” refers to a type of pluripotent stem cells derived from early stage human embryos, up to and including the blastocyst stage, that is capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.
As used herein, the term “embryonic stem cell line” refers to a population of embryonic stem cells which have been cultured under in vitro conditions that allow proliferation without differentiation for up to days, months to years. For example, “embryonic stem cell” can refers to a primitive (undifferentiated) cell that is derived from preimplantation-stage embryo, capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers. A human embryonic stem cell refers to an embryonic stem cell that is from a human. As used herein, the term “human embryonic stem cell” or “hESC” refers to a type of pluripotent stem cells derived from early stage human embryos, up to and including the blastocyst stage, that is capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.
As used herein, the term “pluripotent” refers to an ability to develop into the three developmental germ layers of the organism including endoderm, mesoderm, and ectoderm.
As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell, similar to an embryonic stem cell, formed by the introduction of certain embryonic genes (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), herein incorporated by reference) into a somatic cell.
As used herein, the term “somatic cell” refers to any cell in the body other than gametes (egg or sperm); sometimes referred to as “adult” cells.
As used herein, the term “somatic (adult) stem cell” refers to a relatively rare undifferentiated cell found in many organs and differentiated tissues with a limited capacity for both self-renewal (in the laboratory) and differentiation. Such cells vary in their differentiation capacity, but it is usually limited to cell types in the organ of origin.
As used herein, the term “proliferation” refers to an increase in cell number.
As used herein, the term “undifferentiated” refers to a cell that has not yet developed into a specialized cell type.
As used herein, the term “differentiation” refers to a process whereby an unspecialized embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.
As used herein, the term “directed differentiation” refers to a manipulation of stem cell culture conditions to induce differentiation into a particular (for example, desired) cell type. In certain embodiments, the term “directed differentiation” in reference to a stem cell refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of a stem cell from the pluripotent state into a more mature or specialized cell fate (e.g., prefrontal cortex cells or neural crest cells, etc.).
As used herein, the term “inducing differentiation” in reference to a cell refers to changing the default cell type (genotype and/or phenotype) to a non-default cell type (genotype and/or phenotype). Thus, “inducing differentiation in a stem cell” refers to inducing the stem cell (e.g., human stem cell) to divide into progeny cells with characteristics that are different from the stem cell, such as genotype (e.g., change in gene expression as determined by genetic analysis such as a microarray) and/or phenotype (e.g., change in expression of a protein).
As used herein, the term “culture medium” refers to a liquid that covers cells in a culture vessel, such as a Petri plate, a multi-well plate, and the like, and contains nutrients to nourish and support the cells. Culture medium may also include growth factors added to produce desired changes in the cells.
An “effective amount” is an amount effective, at dosages and for periods of time necessary, that produces a desired effect, e.g., the desired therapeutic or prophylactic result.
As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.
As used herein, the term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.
As used herein, the term “expressing” in relation to a gene or protein refers to making an mRNA or protein which can be observed using assays such as microarray assays, antibody staining assays, and the like.
As used herein, the term “marker” or “cell marker” refers to gene or protein that identifies a particular cell or cell type, e.g., prefrontal cortex cells or neural crest cells. A marker for a cell may not be limited to one marker, markers may refer to a “pattern” of markers such that a designated group of markers may identity a cell or cell type from another cell or cell type.
The terms “detection” or “detecting” include any means of detecting, including direct and indirect detection.
As used herein, the term “derived from” or “established from” or “differentiated from” when made in reference to any cell disclosed herein refers to a cell that was obtained from (e.g., isolated, purified, etc.) a parent cell in a cell line, tissue (such as a dissociated embryo, or fluids using any manipulation, such as, without limitation, single cell isolation, cultured in vitro, treatment and/or mutagenesis using for example proteins, chemicals, radiation, infection with virus, transfection with DNA sequences, such as with a morphogen, etc., selection (such as by serial culture) of any cell that is contained in cultured parent cells. A derived cell can be selected from a mixed population by virtue of response to a growth factor, cytokine, selected progression of cytokine treatments, adhesiveness, lack of adhesiveness, sorting procedure, and the like.
As used herein, the term “signaling” in reference to a “signal transduction protein” refers to a protein that is activated or otherwise affected by ligand binding to a membrane receptor protein or some other stimulus. Examples of signal transduction proteins include, but are not limited to, a SMAD, transforming growth factor beta (TGFB), Activin, Nodal, bone morphogenic (BMP) and NFIA proteins. For many cell surface receptors or internal receptor proteins, ligand-receptor interactions are not directly linked to the cell's response. The ligand activated receptor can first interact with other proteins inside the cell before the ultimate physiological effect of the ligand on the cell's behavior is produced. Often, the behavior of a chain of several interacting cell proteins is altered following receptor activation or inhibition. The entire set of cell changes induced by receptor activation is called a signal transduction mechanism or signaling pathway.
As used herein, the term “signals” refer to internal and external factors that control changes in cell structure and function. They can be chemical or physical in nature.
As used herein, the term “ligands” refers to molecules and proteins that bind to receptors, e.g., transforming growth factor-beta (TFGβ), Activin, Nodal, bone morphogenic proteins (BMPs), etc.
As used herein, the term “inhibitor” refers to a compound or molecule (e.g., small molecule, peptide, peptidomimetic, natural compound, siRNA, anti-sense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, decreases, suppresses, eliminates, or blocks) the signaling function of the molecule or pathway. An inhibitor can be any compound or molecule that changes any activity of a named protein (signaling molecule, any molecule involved with the named signaling molecule, or a named associated molecule) (e.g., including, but not limited to, the signaling molecules described herein). Inhibitors are described in terms of competitive inhibition (binds to the active site in a manner as to exclude or reduce the binding of another known binding compound) and allosteric inhibition (binds to a protein in a manner to change the protein conformation in a manner which interferes with binding of a compound to that protein's active site) in addition to inhibition induced by binding to and affecting a molecule upstream from the named signaling molecule that in turn causes inhibition of the named molecule. An inhibitor can be a “direct inhibitor” that inhibits a signaling target or a signaling target pathway by actually contacting the signaling target.
“Activators”, as used herein, refer to compounds that increase, induce, stimulate, activate, facilitate, or enhance activation of a protein or molecule, or the signaling function of the protein, molecule or pathway.
As used herein, the term “derivative” refers to a chemical compound with a similar core structure.
An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.
As used herein, the term “disease” or “disorder” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
As used herein, the term “treating” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed cither for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.
The term “differentiation day” as used herein, refers to a time line having twenty-four-hour intervals (i.e., days) after a stem cell culture is contacted by differentiation molecules. For example, such molecules may include, but are not limited to, SMAD inhibitor molecules, BMP inhibitor molecules, WNT inhibitor molecules and BMP molecules. The day of contacting the culture with the molecules is referred to as differentiation day 1. For example, differentiation day 2 represents anytime between twenty-four and forty-eight hours after the stem cell culture had been contacted by a differentiation molecule.
As used herein, the term “gene” refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term “gene” also refers to a DNA sequence that encodes an RNA product. The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5′ and 3′ ends.
The term “multi-gene disorder” as used herein, refers to a disorder that results from the presence of mutations in two or more genes. In certain embodiments, patients having the same multi-gene disorder can harbor different single-gene mutations. In certain embodiments, a single patient having the multi-gene disorder can harbor mutations in multiple genes, and different patients having multi-gene disorder will likely harbor distinct combinations of mutations. Non-limiting examples of multi-gene disorders include autism, schizophrenia, intellectual disability, epilepsy, major depression, bipolar disorder, hyperlipidemia, autoimmune disease, multiple sclerosis, arthritis, lupus, inflammatory bowel disease, refractive error, cleft palate, hypertension, asthma, heart disease, type 2 diabetes, cancer, Alzheimer's disease and obesity.
The term “mutation” refers to a change in a nucleotide sequence (e.g., an insertion, deletion, inversion, duplication, or substitution of one or more nucleotides) of a gene. The term also encompasses the corresponding change in the complement of the nucleotide sequence, unless otherwise indicated.
The present disclosure relates to methods for modulating cellular aging and/or progression of neurodegenerative diseases (e.g., AD). In certain embodiments, the methods induce cellular aging. In certain embodiments, the methods promote progression of neurodegenerative diseases (e.g., AD). The present disclosure also relates to methods and systems for modeling aging related neurodegenerative diseases (e.g., AD) in vitro. In certain embodiments, the neurodegenerative disease is AD, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), or Huntington's disease.
In certain embodiments, the neurons can be obtained from in vitro differentiation of stem cells (e.g., human stem cells). In certain embodiments, the stem cell is a human stem cell. Non-limiting examples of human stem cells include human embryonic stem cells (hESC), human pluripotent stem cell (hPSC), human induced pluripotent stem cells (hiPSC), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. In certain embodiments, the human stem cell is a human pluripotent stem cell. In certain embodiments, the human stem cell is a human embryonic stem cell (hESC). In certain embodiments, the human stem cell is a human induced pluripotent stem cell (hiPSC). In certain embodiments, the stem cells are non-human stem cells, including, but not limited to, mammalian stem cells, primate stem cells, or stem cells from a rodent, a mouse, a rat, a dog, a cat, a horse, a pig, a cow, a sheep, etc. In certain embodiments, the neurons are cortical neurons.
In certain embodiments, the neurons exhibit genetic mutation of at least one gene that is associated with neurodegenerative disease. Non-limiting examples of such mutations include those utilized in models of AD, e.g., APP Swedish mutation K595N/M596L and PSEN M146V. Additional non-limiting examples include those utilized in models of Parkinson's disease, e.g., LRRK2 G2019S. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in increased production of amyloid-beta peptide. In certain embodiments, the mutation of at least one gene that is associated with neurodegenerative disease results in a change in the ratio of Aβ40 to Aβ42 peptide.
In certain embodiments, the methods disclosed herein comprise inhibiting protein neddylation pathway. In certain embodiments, inhibiting protein neddylation pathway comprises knocking out or knocking down genes (e.g., UBA3, NAE1) that regulate protein neddylation pathway.
In certain embodiments, modulating protein neddylation pathway comprises administering a neddylation inhibitor to cells. Non-limiting examples of compounds that modulate neddylation include MLN4924, TAS4464, CSN5i-3, ZM223, NACM-OPT, Keap1-Nrf2-IN-4, WS-383, VII-31 and derivatives thereof.
MLN4924 refers to IUPAC name [(1S,2S,4R)-4-[4-[[(1S)-2,3-Dihydro-1H-inden-1-yl]amino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl sulfamic acid ester. MLN4924 is an inhibitor of NEDD8 activating enzyme (NAE).
In certain embodiments, the neurons are contacted with between about 100 nM and about 10 μM, between about 100 nM and about 1 μM, or between about 100 nM and about 1 μM. In certain embodiments, the neurons are contacted with about 100 nm, about 1 μM, or about 10 μM neddylation inhibitor.
In certain embodiments, the neurons are contacted with the neddylation inhibitor for up to 10 days, up to 20 days, or up to 30 days, or up to 4 weeks, or up to 5 weeks, or up to 6 weeks. In certain embodiments, the neurons are contacted with the neddylation inhibitor for about 3 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 20 days, or about 30 days, or about 4 weeks, or about 5 weeks, or about 6 weeks.
The present disclosure relates to methods of identifying genes associated with cellular aging and/or progression of neurodegenerative disease. In certain embodiments, a target gene is selected based on differential expression in a model of neurodegenerative disease relative to healthy tissue. In certain embodiments, mutation is introduced at a target gene in a PSC model of neurodegenerative disease. In certain embodiments, functional activity is measured between the PSC model comprising further mutation at the target gene and compared to healthy tissue. In certain embodiments, the functional activity is cellular senescence, protein aggregation, DNA damage, decreased heterochromatin, and/or cell viability.
Any methods known in the art can be used to generate gene modifications in the PSC lines, e.g., hPSC lines. In certain embodiments, genome editing technique can be used to generate gene modifications in the PSC lines. For example, but not by way of limitation, a CRISPR/Cas9 system is employed to modify the genes. Clustered regularly-interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9), and trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9). The terms “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). CRISPR/Cas9 strategies can employ a plasmid to transfect the mammalian cell. The gRNA can be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in a cell. Multiple crRNA' s and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). The sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells. In certain embodiments, the CRISPR/Cas9 system comprising a Cas9 molecule, and a guide RNA (gRNA) comprising a targeting domain that is complementary with a target sequence of the targeted gene.
In certain embodiments, a zinc-finger nuclease (ZFN) system is employed for generating the gene modifications in the PSCs, e.g., hPSCs. The ZEN can act as restriction enzyme, which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific
DNA sequences which allows the zinc-finger nuclease to target desired sequences within genomes. The DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of base pairs. The most common method to generate new zinc-finger domain is to combine smaller zinc-finger “modules” of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type IIs restriction endonuclease Fokl. ZFN modulates the expression of proteins by producing double-strand breaks (DSBs) in the target DNA sequence, which will, in the absence of a homologous template, be repaired by non-homologous end-joining (NHEJ). Such repair may result in deletion or insertion of base-pairs, producing frame-shift and preventing the production of the harmful protein (Durai et ah, Nucleic Acids Res., 33 (18): 5978-90.) Multiple pairs of ZFNs can also be used to completely remove entire large segments of genomic sequence (Lee et ah, Genome Res., 20 (1): 81-9).
In certain embodiments, a transcription activator-like effector nuclease (TALEN) system is employed in generating the gene modifications in the PSCs, e.g., hPSCs. TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN systems operate on a similar principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome (Boch et ah, Nature Biotechnology;29(2): 135-6).
The genetic modification system disclosed herein can be delivered into the PSCs, e.g, hPSCs, using a retroviral vector, e.g, gamma-retroviral vectors, and lentiviral vectors. Combinations of retroviral vector and an appropriate packaging line are suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are suitable too, e.g, particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art. Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g, by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g, by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.
Other transducing viral vectors can also be used to generate gene modification in the PSCs, e.g., hPSCs, disclosed herein. In certain embodiments, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S. A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107: 77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).
Non-viral approaches can also be employed for generating gene modifications in the PSCs, e.g., hPSCs. For example, a nucleic acid molecule can be introduced into the PSC by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S. A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101: 512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of nucleic acid molecules into a cell.
A1. The foregoing method of A, wherein modulating protein neddylation comprises exposing the population of neurons to a compound that modulates protein neddylation.
The present disclosure will be better understood by reference to the following Example, which is provided as exemplary of the presently disclosed subject matter, and not by way of limitation.
Aging is the biggest risk factor for the development of AD, but it not known whether the aging process directly contributes to AD initiation or progression. Understanding the how cellular age is regulated is also important because pluripotent stem cell (PSC) derived neurons are transcriptionally and functionally young and this may limit their utility in modelling the late onset degenerative phase of AD. It was previously shown that age can be directly programmed into PSC derived neurons through the ectopic expression of Progerin raising the question of what genes and pathways regulate this process during normal aging. To address this, past studies either performed RNAseq on postmortem brain tissue from young versus old individuals or generated induced neurons via direct reprogramming from primary fibroblasts donated by individuals of varying ages. Although neurons generated via direct reprogramming may retain a fibroblast-related aging signature, it has been challenging to identify which changes have a deterministic impact on cellular age.
hESC culture and differentiation. Engineered human embryonic stem cells (H9; WA-09) were maintained on Vitronectin coated plates in E8 medium and passaged twice a week using EDTA. For differentiation to cortical neurons, the PSCs were dissociated to single cells using Accutase and replated onto Matrigel coated dishes at a density of 300,000 k/cm2 in E8 medium supplemented with ROCK inhibitor (Y-27632; 10 μM). The following day (DIV=0) culture medium was replaced with E6 containing SB431542 (10 μM), LDN193189 (100 nM) and XAV939 (2 μM). Differentiation media was changed daily and XAV939 removed after 3 days. At 10 DIV the media was changed to neurobasal supplemented with N2 and B27 and the monolayer was maintained for an additional 10 days. On day 20 after neural induction cells were dissociated using Accutase and replated onto poly-L-ornithine/fibronectin/laminin-coated plates. Neurons were maintained in Neurobasal medium supplemented with BDNF, ascorbic acid, GDNF, CAMP, L-glutamine and B-27 supplement. DAPT was also added to the culture media until 30 days DIV.
Cell line engineering. WA-09 were sequentially engineered to generate the cell lines for this study. First an inducible Cas9 construct was knocked into the AAVS1 locus as described in Gonzalez et al. but with a hygro-resistant Cas9 donor plasmid instead of the puromycin resistant donor. After checking for the correct insertion of the iCas9 construct the newly established iCas9 cell line was engineered to insert the APPswe/swe mutation as described in Paquet et al. The maintenance of an intact karyotype was confirmed after each engineering step.
Whole genome CRISPR Cas9 screen in PSC-derived neurons. The Brunello human CRISPR Knockout Pooled Library was used for this screen; this library includes 4 guide RNAs for 19,114 genes as well as 1000 non targeting controls. Stem cell culture, transduction, selection, and differentiation of the isogenic stem cell pair was done in parallel. To perform the screen PSCs were dissociated with Accutase and a total of 250 million cells per line were replated at a density of 150,000/cm2 in E8 medium with ROCK inhibitor (Y-27632; 10 μM). The whole genome lenti-guide RNA library was added during the replating step at an MOI of 0.3-0.5. The virus was removed 16-18 h post transduction and fresh E8 medium added to the culture plate. The following day transduced cells were selected by adding 0.4 μg/ml puromycin to the E8 medium. After selection, PSCs were dissociated with Accutase, the culture plates were pooled and a total of 116 million cells used for differentiation to ensure that 1000× guide representation was maintained throughout. PSCs were differentiated as described in the ‘hESC culture and differentiation’ subsection. After differentiation (DIV 20) cultures were dissociated using Accutase to generate a single cell suspension for each cell line and the cells were split to give triplicate samples for the screen with a total of 91 million cells per replicate. The T=0 control samples were harvested immediately whereas the endpoint samples were replated at a density of 200,000/cm2 in neurobasal supplemented with N2, B27 and ROCK inhibitor (Y-27632; 10 μM). Doxycycline (2 μg/ml) was added to half the culture plates to induce Cas9 expression. Cells were treated with doxycycline for a total of 48 h before switching to neural maintenance media with DAPT until DIV 30. From DIV 30 onwards half the culture media was replaced every 2-3 days. Neurons were harvested at DIV 65. To remove any dead cells from the culture the monolayer was washed 2× with PBS followed by a 5 min incubation in EDTA at RT. Then, the neuronal monolayer was scrapped off the culture dish, pelleted and snap frozen. Cell Pellets from pooled screen were lysed, and genomic DNA was extracted (Qiagen) and PCR amplified to add Illumina adapters and multiplexing barcodes. Amplicons were quantified by Qubit and Bioanalyzer (Agilent) and sequenced on Illumina HiSeq 2500.
Data analysis for Pooled CRISPR screen. Sequencing reads were aligned to the screened library and the CRISPR screen was analyzed using the MAGECK-MLE pipeline as previously described. To calculate the beta scores for each gene the representation of guide RNAs in the endpoint samples (DIV65+Dox) was compared to either the DIV20 or DIV65−Dox samples. Non targeting sgRNAs were used for normalization. Hit genes were identified by comparing the beta scores in the wild-type viability screen with the beta scores in the Appswe/swe viability screen. Genes with a beta score<0, FDR<0.3 and Pval<0.05 were considered viability genes. Essential genes met these criteria in both genotypes. Candidate age regulators met these criteria in the APPswe/swe genotype. The hit list was filtered to exclude genes that had a viability phenotype in the wild-type neurons (Beta scoreWT was greater than 1.5 standard deviations from the mean or <0 with an FDRWT<0.3 and PvalWT<0.05).
RNA extraction and qPCR. RNA was extracted using the Zymo RNA Micro Kit and total of lug of RNA was used to generate cDNA using iScript (BioRad). Realtime PCR was performed using SSoFAST EvaGreen Mix (BioRad) in a BioRad CFX96
Thermal Cycler. The manufacturers protocol was used for all steps.
Immunocytochemistry. Cells were fixed in 4% paraformaldehyde for 10 mins then permeabilized in PBS+0.3% Triton. Cells were blocked in 5% donkey or goat serum for 1 h. Primary antibody incubation was performed overnight. Primary antibodies used in this study were: p-ATM (S1981) (Thermo Fisher; MA1-2020), Cas9 (Cell Signaling Technologies; 14697S), COL1A1 (R&D; AF6220), FOXG1 (Takara; M227), Ki67 (Dako; M7240), MAP2 (Thermo Fisher; PA1-16751), NANOG (Cell Signaling Technologies; 4903S), PAX6 (Biolegend; 901301), TAU (Thermo Fisher; MN1000), p-TAU (S235) (Thermo Fisher; PA5-104785). For all image quantifications images were taken from 3 individual wells and averaged. This was repeated three times with neurons from independent differentiations. Volocity was used to count MAP2+ and Ki67+ cells with pyknotic. DAPIbright nuclei excluded from the cell count. FIJI was used for TAU and pATM quantifications.
Aβ ELISA. To quantify amyloid peptide production cell culture media was harvested after 48 h and the culture media was briefly centrifuged to remove any cellular debris. Quantification was performed using the Mesoscale Discovery Assay kit (K15200E-2) according to the manufacturer's instructions. A total of 25 μl of culture medium was assayed. For each condition, the reported values represent the average of 3-4 culture wells per differentiation/experiment. For normalization, protein was extracted and quantified from neurons of matched age/gene knockout. The mean change in total protein between −Dox and +Dox samples from 3 independent differentiations/experiments was used to normalize total Aβ measurements.
Western blotting. Samples for western blotting were harvested, pelleted and snap frozen. Cell pellets were resuspended in RIPA buffer supplemented with Halt protease and phosphatase inhibitors (ThermoFisher) followed by centrifugation to clarify the sample. Protein concentration was quantified using the Precision Red Advanced Protein Assay according to manufacturer's instructions and equal amounts of protein were mixed with NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent and heated to 72 degrees for 10 mins. A total of 5-20 μg of protein was separated on NuPAGE Novex 4-12% Bis Tris gels and transferred by wet blotting onto PVDF membranes. Membranes were blocked in 5% milk protein or 5% BSA when probing for the phospho-Tau. Primary antibodies used for this study were: β-ACTIN (Sigma, A2228-100UL), BAG1 (Santa Cruz, sc-33704), BAG3 (Abcam, ab47124), Cas9 (Cell Signaling Technologies; 14697S), GAPDH-HRP (Santa Cruz Biotech, sc-47724 HRP), LMNB1 (Abcam, ab16048), NAE1(Cell Signaling Technologies, 14321S), NEDD8 (Abcam, ab81264), p21 (Cell Signaling Technologies, 2947), TAU (Dako, A0024), p-TAU(S202/T205) (Thermo Fisher; MN1020), UBA3 (Abcam, ab124728). Band intensity was visualized using BioRad ChemiDoc XRS+molecular imager. After imaging the membrane was re-probed with either GAPDH or β-ACTIN antibodies for normalization. Band intensity was quantified using Fiji.
Viability assays. Viability assays were performed in 96 well plates using the PrestoBlue Cell Viability Reagent or CCK8. Presto blue reagent was diluted 1:10 in neural maintenance media and 85 μl was applied to each well. For the CCK8 assay the assay reagent was prepared as described by the manufacturer with 110 μl used per well. Culture plates were incubated with the assay reagent for 2 h at 37 degrees before assaying. For the secondary validation experiments, each well was normalized to the mean absorbance of the no doxycycline control wells and the technical replicates averaged to give a single value for each differentiation/experiment.
Secondary validation. Secondary validation was performed in array in 96 well plates. Cells for secondary validation were differentiated as described in the ‘hESC culture and differentiation’ subsection. At DIV 20 neurons were dissociated and plated at 150,000/cm2 in 96 wells. Three replicates were plated for each experiment/independent differentiation. For each guide RNA there were 4 conditions: WT neurons+guideRNA lentivirus, WT+guideRNA lentivirus+doxycycline, APPswe/swe neurons+guideRNA lentivirus, APPswe/swe neurons+guideRNA lentivirus+doxycycline. Unconcentrated virus was applied at a 1:30 dilution at DIV20 and DIV21. Doxycycline (2 ug/mL) was also added for the first 48 h. On DIV22 1 ug/mL puromycin was added to the cultures for 48 h to select for neurons transduced with the guideRNA of interest. Neurons were then maintained as previously described until DIV60 then assayed using the PrestoBlue Cell Viability Reagent as described in the ‘Viability assays’ subsection.
Generation of lentiGuide RNA viruses. GuideRNAs used for secondary validation were the top scoring guide RNA from the WGS. A list of guideRNA sequences used for this study can be found in Table 2. Guide RNAs were cloned into the lentiGuide-Puro plasmid (Addgene 52963) as described by the Zhang lab. For viral packaging, the lentiGuide-Puro plasmid and packaging plasmids (psPAX2; Addgene 12260 and pMD2.G; Addgene 12259) were transfected into 293T cells using X-tremeGENE HP (Sigma) in a 10:10:1 molar ratio, respectively. Virus particles were harvested after 48 h.
Flow Cytometry. Neuronal cultures were dissociated to single cell suspensions using Accutase (Innovative Cell Technologies) supplemented with Neuron Isolation Enzyme for Pierce™ (Thermo 88285) solution at 1:50. Single cell suspensions were stained with Zombie UV™ Fixable Viability Kit (Biolegend 423107) at 1:2500 in PBS for 15 minutes at room temperature, followed by fixation in 4% Paraformaldehyde for 10 minutes (4° C.). Cells stained with CellEvent Senescence Green (Thermo C10840) were done so at 1:250 in assay buffer for 2 hours at 37° C. For intracellular probes, cells were permeabilized in 0.5% triton-x for 10 minutes (4° C.), and blocked in 5% BSA for 10 minutes (4° C.). Cells were stained with H3k9me3-PE antibody (Cell Signaling Technologies #13969S) diluted 1:200, and Proteostat (Enzo Life Sciences ENZ-51023-KP050) diluted 1:2500, in 5% BSA in PBS for 30 minutes at 4° C. Cells were analyzed on the Cytek Aurora Flow Cytometer. Experiments were repeated with cells from 3 independent differentiations.
Statistics and Reproducibility. Exact number of replicates, statistical test used, and error bars are defined in the relevant figure legends. Independent replicates consisted of an independent differentiation for neurons or independent passage for stem cells.
To define genes whose loss of function can drive the aging process a functional screening approach was developed that could be performed at whole genome scale with a single readout. Biomarker studies have shown that individuals with AD show a decades long age-associated progression of AD pathologies beginning with disordered amyloid precursor protein (APP) processing followed by Tau mislocalization and aggregation and finally, neuronal loss and cognitive dysfunction. Therefore, neuronal death was selected as an age and AD-dependent cellular readout of the whole genome screen (WGS). An isogenic stem cell model of familial AD (fAD) that was amenable to whole genome CRISPR/Cas9 screening was generated by sequential genome engineering of human PSCs (
Both the Control and APPswe/swe engineered cell lines had a normal karyotype and showed equivalent induction of Cas9 upon the addition of doxycycline (
Further, it was confirmed that both lines could be differentiated into cortical neurons with equivalent efficiency (
Next, experiments were performed to characterize amyloid, Tau and neuronal loss phenotypes in both genotypes at baseline. Knock-in of the APPswe/swe mutation resulted in approximately 3-fold more total Aβ (Aβ38+Aβ40+Aβ42) than the isogenic control neurons without altering the ratio of Aβ40 to Aβ42 (
The workflow and experimental design of the paired whole genome CRISPR/Cas9 viability screen are summarized in
A scatter plot was used to visualize the beta scores for each gene in the two different genotypes (
For each of the hit categories, the top 1000 genes were selected and a KEGG pathway analysis was run at an FDR<0.01. There were only 4 genes whose loss of function resulted in an increase in guide RNA representation in both genotypes. KEGG analysis shows that 3 of these genes fall within the hedgehog signaling pathway (
Pathway analysis of genes that were significantly depleted in the APPswe/swe neurons and not in the WT neurons showed a significant overrepresentation of genes associated with both AD and Huntington's disease (
To perform secondary validation experiments and test many single guide RNAs simultaneously, the platform (
The top ranked gene (SUFU) with a positive beta score (
To select AD enhanced regulators of neuronal viability for further investigation, the list of genes that were significantly and specifically decreased in the AD neurons in both the T=0 control and endpoint control datasets were overlapped. This gave a total of 273 genes that were ranked according to the difference between the beta scores of the wild-type and APPswe/swe neurons (
It was hypothesized that AD enhanced loss of viability seen in
Existing RNA sequencing datasets were used to identify validated hit genes that are also significantly decreased in the aging human or mouse brain. It was postulated that genes that show age-dependent changes in expression are more likely to contribute to age-associated phenotypes. Of the 6 validated genes DNAJB11, UBA3, VPS36 and PPP1CB showed a significant decrease in both the aged mouse and human cortex. UBA3 was prioritized for further analysis because NAE1, the regulatory subunit of the Uba3-Nae1 E1 enzyme, was also a significant hit in the screen (
For further validation, western blots were performed to confirm that the guide RNAs used in this study resulted in a robust decrease in UBA3 or NAE1 protein in cortical neurons (
While UBA3 expression is decreased in the aged mouse and human brain (
The instant data indicates that genetic or pharmacological inhibition of neddylation can trigger cortical neuron degeneration in an AD-dependent manner, thereby capturing a late disease phenotype not captured in a standard hPSC-based AD model. This raises the question of whether other late-stage Tau phenotypes can be captured in pharmacologically aged AD neurons. pTau(S235) was the focus of the instant analyses because it is one of the first sites of phosphorylation that can identify symptomatic AD. Immunofluorescence indicated that overall p-Tau(S235) was not significantly increased in AD cortical neurons lacking UBA3 relative to total Tau levels. However, there was a significant increase in p-Tau(S235)+inclusions in the UBA3 KO APPswe/swe neurons (
Whether inhibiting neddylation can drive Tau-related changes that are characteristic not only to early disease progression such as p-Tau(S235) but also to later stages of disease progression such as p-Tau(S202/T205) was tested. pTau(T205) occurs later in disease progression and is associated with fibril formation. In particular, high molecular weight (HMW; >250 kDA) oligomeric hyperphosphorylated p-Tau (S202/T205) has been shown to promote the seeding of Tau aggregation. Finally, in iPSC derived neurons, decreased pTau (MAJ; 50 kDA) and increased HMW tau have been shown to correlate with cognitive decline and the presence of tangles in the postmortem brain. Western blot analysis for p-Tau (S202/T205) showed that inhibiting neddylation had no impact on levels of either the major(MAJ) or HMW forms of p-Tau (S202/T205) in wild-type cortical neurons (
Using a whole genome CRISPR screening approach, neddylation was identified as a regulator of neuronal aging and it was shown that cellular aging can synergize with AD-genetics to trigger late onset AD phenotypes in vitro. These findings indicate that cellular aging can have a causal impact on the progression of fAD and highlight the importance of developing therapeutic strategies to reverse cellular aging. In addition to the confirmation of the neddylation pathway in this study, several other genes were identified and validated whose loss of function had a more pronounced impact on the viability of APPswe/swe neurons than control neurons. Further characterization of hit genes may define a more complete set of genes and pathways capable of potentiating fAD disease. For example, two of the validated hit genes VPS36 and PPP1CB have also been linked to Tau propagation and phosphorylation respectively.
This study further has several implications for in vitro disease modelling efforts. A question is whether capturing age-related features in neurons will reveal late-stage phenotypes in other familial or sporadic AD-iPSC models or trigger age-related features and disease phenotypes in other neuronal lineages and late-onset neurodegenerative disorders such as Parkinson's disease or ALS. Finally, parallel efforts focus on the identification of strategies that drive neuronal maturation independent of neuronal age. It is conceivable such efforts to accelerate maturation could be combined with the presently disclosed induced aging platform to better capture synaptic or spine degeneration, phenotypes that for human neurons are currently limited largely to in vivo and postmortem studies. In conclusion, protein neddylation is identified as a physiologically relevant regulator of neuronal age and increased cellular age can contribute to the potentiation of AD phenotypes in in vitro human PSC models of disease.
Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and compositions of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, this present disclosures of which are incorporated herein by reference in their entireties for all purposes.
This patent application is a continuation of International Patent Application No. PCT/US2023/023722, filed May 26, 2023, which claims priority to U.S. provisional application 63/346,182 filed May 26, 2022, the contents of each of which is incorporated herein by reference in its entirety, and to each of which priority is claimed.
The present disclosure was made with government support under Grant Nos. 1R01AG056298 and 1R01AG054720 awarded by the National Institute of Health. The government has certain rights in the disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 63346182 | May 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/023722 | May 2023 | WO |
| Child | 18960311 | US |
