PKC pathway in Parkinson's disease

Abstract

Induced pluripotent stem cell (iPSC) technology enables the generation and study of living brain tissue associated with Parkinson's disease (PD) ex vivo. The use of cell lines derived from PD patients provides a powerful discovery system that links cellular phenotypes observed in vitro with actual clinical data. Differentiation of patient-derived iPSCs toward a dopaminergic (DA) neuronal fate revealed that these cells exhibit molecular and functional characteristics of DA neurons, which are observed to significantly degenerate in the substantia nigra of PD patients. Clinical symptoms driving the generation of other relevant cell types may also yield novel PD-specific phenotypes in vitro that may lead to new therapeutic avenues for patients with PD. Due to the patients' early onset and non-familial origin, differentiated neural tissue from these patients offers a significant opportunity to discover neuronal subtype-specific pathogenesis mechanisms and, importantly, to investigate the contribution of patients' genetic background in PD susceptibility.

Description

Technical Field

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. NS105703 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0002] Field of the Invention Methods and compositions related to the generation of midbrain neurons, including those related to Parkinson's disease, are described herein.

Background Art

[0003] Background Parkinson's disease (PD) is the second most commonly diagnosed neurodegenerative disorder and represents a significant economic burden among the current elderly population. The classically associated pathology in PD is characterized by the progressive loss of dopaminergic neurons (DaN) in the substantia nigra pars compacta, as well as the presence of cytoplasmic inclusions known as Lewy bodies and Lewy neurites. These inclusions are composed primarily of the protein α-synuclein. Mutations or triplications in the gene (SNCA) encoding α-synuclein are the cause of these specific familial PD cases. In its native state, α-synuclein is found in presynaptic terminals of neurons throughout the human brain and functions in vesicular transport, neurotransmitter release, and reuptake.

[0004] While many genes and proteins, such as α-synuclein, are associated with Parkinson's disease (PD), the inability to extract living neurons from patients and the lack of effective PD models leave unresolved questions regarding disease onset and progression. Reprogramming patient-derived cells into iPSCs allows for observation of disease progression and pathological phenotypes at the molecular level. Interestingly, previous iPSC studies in larger non-familial (sporadic) populations have not shown clear differences compared to those derived from control individuals. Therefore, there is a strong need in this field for iPSC disease models that represent the complex biological background underlying the pathology of Parkinson's disease.

[0005] Compositions and methods for modeling and treating Parkinson's disease are described herein. Importantly, by floor plate induction in a manner that faithfully reflects the generation and development of midbrain neurons, it is possible to identify cellular cues leading to neurodegeneration, including the complex pathogenesis behind sporadic PD cases that have not yet been fully utilized in iPSC models. Having established such a model, the inventors hereby identify previously unknown roles of α-synuclein and lysosomal degradation dysfunction as partly mediated by PKC. Targeting PKC with agonists improved measurable results, thereby proposing a novel therapeutic approach for Parkinson's disease. [Invention 1001] It is a treatment method, A method comprising administering a pharmaceutical composition comprising a therapeutically effective agent and a pharmaceutically acceptable carrier to a subject suffering from Parkinson's disease, thereby treating the subject. [Invention 1002] The method of the present invention 1001, wherein the therapeutically effective drug comprises a small molecule. [Invention 1003] The method of the present invention 1001, wherein the small molecule comprises a PKC activator, its analogues, and derivatives. [Invention 1004] The method of the present invention 1003, wherein the PKC activator comprises DAG, DAG lactone, phorbol, ingenol, indolactam, benzolactam, bryostatin, and carphostin. [Invention 1005] The method of the present invention 1003, wherein the PKC activator, its analogues and derivatives include ingenol derivatives. [Invention 1006] The method of the present invention 1005, wherein the ingenol derivative comprises ingenol-3-angerate. [Invention 1007] The method of the present invention 1003, comprising the PKC activator, its analogues and derivatives, and a PKC agonist. [Invention 1008] The method of the present invention 1001, wherein the therapeutically effective agent can modulate the activity of one or more of the following: α-synuclein, TFEB, ZKSCAN3, LAMP, GCase, tyrosine hydroxylase (TH), and dopamine. [Invention 1009] The method of the present invention 1001, wherein the therapeutically effective agent can modulate the expression of one or more of α-synuclein, TFEB, ZKSCAN3, LAMP, GCase, tyrosine hydroxylase (TH), and dopamine. [Invention 1010] The method of the present invention 1009, wherein the regulation of expression includes the level of expression of the transcript. [Invention 1011] The method of the present invention 1009, wherein the regulation of expression includes the protein expression level. [Invention 1012] The method of the present invention 1011, wherein the protein expression level includes a decrease in α-synuclein protein. [Invention 1013] The method of the present invention 1011, wherein the protein expression level includes an increase in TH protein. [Invention 1014] The method of the present invention 1001, wherein the therapeutically effective agent can promote lysosomal protein degradation. [Invention 1015] The method of the present invention 1001, wherein the therapeutically effective agent can improve the synchronous burst of electrical activity. [Invention 1016] The method of the present invention 1001, wherein the therapeutically effective agent can improve one or more of stepping, rotational asymmetry, and kinesia. [Invention 1017] A method for reversing or slowing the progression of Parkinson's disease, A method comprising administering a pharmaceutical composition containing a therapeutically effective agent and a pharmaceutically acceptable carrier to a subject suffering from Parkinson's disease, thereby reversing or delaying the progression of Parkinson's disease in the subject. [Invention 1018] The method of the present invention 1016, wherein the therapeutically effective agent comprises PKC activator, its analogues, and derivatives. [Invention 1019] The method of the present invention 1018, wherein the PKC activator, its analogues, and derivatives include ingenol-3-angelate. [Invention 1020] The method of the present invention 1017, wherein the aforementioned therapeutically effective agent can lower α-synuclein protein levels. [Invention 1021] The method of the present invention 1017, wherein the therapeutically effective agent can reduce the promotion of lysosomal protein degradation. [Invention 1022] The method of the present invention 1017, wherein the therapeutically effective drug can reverse or delay the degeneration of the substantia nigra. [Invention 1023] The method of the present invention 1017, wherein the therapeutically effective agent can maintain or promote dopamine levels.

Brief Description of the Drawings

[0007] A therapeutic method is described herein, comprising administering a pharmaceutical composition comprising a therapeutically effective agent and a pharmaceutically acceptable carrier to a subject suffering from Parkinson's disease, thereby treating the subject. In other embodiments, the therapeutically effective agent comprises a small molecule. In other embodiments, the small molecule comprises PKC activators, their analogs and derivatives. In other embodiments, the PKC activators comprise DAG, DAG lactone, phorbol, ingenol, indolactam, benzolactam, bryostatin, and carphostine. In other embodiments, the PKC activators, their analogs and derivatives comprise ingenol derivatives. In other embodiments, the ingenol derivatives comprise ingenol-3-angelate. In other embodiments, the PKC activators, their analogs and derivatives comprise PKC agonists. In other embodiments, the therapeutically effective agent can modulate the activity of one or more of the following: α-synuclein, TFEB, ZKSCAN3, LAMP, GCase, tyrosine hydroxylase (TH), and dopamine. In other embodiments, therapeutically effective agents can modulate the expression of one or more of α-synuclein, TFEB, ZKSCAN3, LAMP, GCase, tyrosine hydroxylase (TH), and dopamine. In other embodiments, the modulation of expression includes transcript expression levels. In other embodiments, the modulation of expression includes protein expression levels. In other embodiments, the protein expression level includes a decrease in α-synuclein protein. In other embodiments, the protein expression level includes an increase in TH protein. In other embodiments, therapeutically effective agents can promote lysosomal proteolysis. In other embodiments, therapeutically effective agents can improve synchronous bursts of electrical activity. In other embodiments, therapeutically effective agents can improve one or more of stepping, rotational asymmetry, and kinesia.

[0008] This specification describes a method for reversing or delaying the progression of Parkinson's disease, comprising administering a pharmaceutical composition comprising a therapeutically effective agent and a pharmaceutically acceptable carrier to a subject suffering from Parkinson's disease, thereby reversing or delaying the progression of Parkinson's disease in the subject. In other embodiments, the therapeutically effective agent includes PKC activator, its analogues and derivatives. In other embodiments, the PKC activator, its analogues and derivatives include ingenol-3-angelate. In other embodiments, the therapeutically effective agent can reduce α-synuclein protein levels. In other embodiments, the therapeutically effective agent can reduce the promotion of lysosomal proteolysis. In other embodiments, the therapeutically effective agent can reverse or delay substantia nigra degeneration. In other embodiments, the therapeutically effective agent can maintain or promote dopamine levels. [Modes for carrying out the invention]

[0009] Detailed description of the invention All references cited herein are incorporated by reference in the same way as if they were fully cited. Unless otherwise defined, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 4. th The ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provides a general guide to many of the terms used in this application.

[0010] Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein that can be used in carrying out the present invention. In fact, the present invention is by no means limited to the methods and materials described herein.

[0011] Parkinson's disease (PD) is one of the most common neurodegenerative diseases, characterized by tremors and inability to initiate movement. The core pathology involves the progressive loss of dopamine neurons in the substantia nigra, as well as the presence of cytoplasmic inclusions known as Lewy bodies and Lewy processes. These inclusions are largely composed of abnormally aggregated α-synuclein protein. Normally, α-synuclein is localized to the presynaptic terminals of neurons throughout the brain and functions in vesicular transport, as well as in the release and reuptake of neurotransmitters. Mutations or triplications of the α-synuclein gene (SNCA) are known to cause PD, suggesting that this protein is important for the onset and progression of the disease.

[0012] The unavailability of live dopamine neurons from patients and limitations in animal models hinder disease research and effective drug discovery. Reprogramming patient-derived cells into induced pluripotent stem cells (iPSCs) and then differentiating them into dopamine neurons provides a human tissue-specific model of Parkinson's disease (PD). During the reprogramming process, most of the epigenetic changes in the cells are completely erased. The new neurons generated from iPSCs are immature but still reflect the patient's genetic makeup. While this immaturity may be a weakness for modeling adult-onset disease, it is also a strength, as any disease-specific phenotype present in the cells will inevitably stem from the patient's genetic makeup and represent a very early stage of the disease process.

[0013] Extensive efforts have been made to elucidate the role of α-synuclein in the onset and progression of Parkinson's disease (PD) using iPSC-based modeling. Several studies have used iPSCs derived from PD patients with monogenic mutations, including SNCA tripletization and mutations in the LRRK2 and GBA1 genes. Dopaminergic neuron cultures from such monogenic iPSCs exhibit phenotypic abnormalities and demonstrate α-synuclein accumulation, although these hereditary cases account for less than 10% of all PD patients. However, iPSC models using the more frequently occurring sporadic PD population do not show α-synuclein accumulation compared to control cells.

[0014] Ten percent of PD patients are classified as having an early-onset form, where symptoms begin before the age of 50.26 More than 80% of these early-onset patients are called sporadic, and do not have a family history or known PD mutation. The inventors hypothesized that, as has been seen in early-onset diseases such as spinal muscular atrophy, early onset of disease symptoms may lead to a more severe phenotype in iPSC models.

[0015] As explained, reprogramming patient-derived cells into iPSCs allows for observation of disease progression and pathological phenotypes at the molecular level. Genetically identical iPSCs to the donor can be differentiated into DaNs, which provide an in vitro tissue-specific model of Parkinson's disease, possessing the genetic background known to be relevant to the clinical presentation in vivo. In recent years, considerable effort has been made to elucidate the role of α-synuclein in the onset and progression of PD using similar iPSC modeling techniques. Several studies have used iPSCs derived from PD patients with monogenic mutations, including SNCA tripletization and mutations in the LRRK2 and GBA1 genes. DA neurons derived from these iPSC lines exhibit some phenotypic abnormalities and demonstrate α-synuclein accumulation; however, familial monogenic mutations are present in only a small number of PD patients, and the pathophysiology of these cases does not readily correlate with the overall PD population. Interestingly, previous iPSC studies on larger non-familial (sporadic) populations have not shown clear differences compared to those derived from control individuals.

[0016] In this specification, the inventors generate iPSC strains from a cohort of patients with early-onset sporadic Parkinson's disease (EOSPD). The inventors hypothesized that these strains offer a promising opportunity to better understand sporadic PD, as patients with early-onset sporadic PD may have unknown genetic risk factors that could influence more aggressive disease morphologies. By comparing differentiated DaNs from either EOSPD patient strains or unaffected control strains, the inventors demonstrate that abnormal accumulation of α-synuclein protein is indeed specifically reproduced in the PD patient cohort. Molecular and physiological profiling of these tissues, including proteomic assays, whole transcriptome assays, and enzyme activity assays, reveals dysregulation of the degradation pathway and suggests previously unreported upregulation of phosphorylated PKC-α in EOSPD cultures. Finally, by targeting this pathway, the inventors observe a reversal of α-synuclein accumulation after treatment with the small molecule PEP005, both in vitro and in vivo. The iPSC-based models described herein provide evidence of the genetic origin of sporadic PD contributing to PD, and offer a platform for potential clinical diagnosis and the development of novel therapeutic targets for EOSPD patients.

[0017] A method is described herein comprising contacting a certain amount of blood cells with one or more vectors encoding reprogramming factors; delivering a certain amount of reprogramming factors into the blood cells; and culturing the blood cells in a reprogramming medium, wherein the amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further, the delivery of the reprogramming factors and culturing in the reprogramming medium generate induced pluripotent stem cells (iPSCs) derived from the blood cells. In other embodiments, the neurodegenerative disease is Parkinson's disease (PD). In other embodiments, the neurodegenerative disease is early-onset PD. In other embodiments, the neurodegenerative disease is familial PD. In other embodiments, the iPSCs are further cultured in fluid communication with one or more of astrocytes, microglia, and vascular cells. In other embodiments, one or more vectors are oriP / EBNA1 vectors. In other embodiments, the method comprises differentiating the iPSCs into neurons. In other embodiments, the method comprises differentiating the iPSCs into vascular cells. In various embodiments, the vascular cells are brain microvascular endothelial cells (BMECs). In other embodiments, the method includes differentiating iPSCs into astrocytes. In other embodiments, the method includes differentiating iPSCs into microglia. In other embodiments, the method includes differentiating iPSCs into neurons, including neurons of the forebrain, midbrain, and / or hindbrain. In various embodiments, the neurons are spinal motor neurons, dopaminergic neurons, or cholinergic neurons.Details of iPSC reprogramming can be found in Barrett, R. et al. Reliable Generation of Induced Pluripotent Stem Cells from Human Lymphoblastoid Cell Lines. Stem Cells Transl Med. 2014 Dec;3(12):1429-34, and in U.S. Provisional Applications 62 / 653,697, 62 / 755,282, 62 / 816,785, 62 / 664,888, 62 / 664,827, 62 / 816,795, 62 / 664,942, and 62 / 755,365, which are fully incorporated herein by reference.

[0018] A therapeutic method is described herein, comprising administering a pharmaceutical composition comprising a therapeutically effective agent and a pharmaceutically acceptable carrier to a subject suffering from Parkinson's disease, thereby treating the subject. In other embodiments, the therapeutically effective agent comprises a small molecule. In other embodiments, the small molecule comprises PKC activators, their analogs and derivatives. In other embodiments, the PKC activators comprise DAG, DAG lactone, phorbol, ingenol, indolactam, benzolactam, bryostatin, and carphostine. In other embodiments, the PKC activators, their analogs and derivatives comprise ingenol derivatives. In other embodiments, the ingenol derivatives comprise ingenol-3-angelate. In other embodiments, the PKC activators, their analogs and derivatives comprise PKC agonists. In other embodiments, the small molecules comprising PKC activators, their analogs and derivatives can modulate DAG signaling. In other embodiments, PKC activators, their analogs, and derivatives are specific to conventional, novel, and atypical PKC isoforms, such as PKC-alpha, PKC-beta1 / beta2, and PKC-gamma, among many isoforms known to those skilled in the art. In various embodiments, the therapeutically effective agent is administered intravenously at doses of 0.1-1, 1-5, 5-10, 10-25, 25-50, or 50 ug / kg or more. In various embodiments, the therapeutically effective agent is administered intravenously at a dose of 5 ug / kg. In various embodiments, the therapeutically effective agent is administered via direct cerebral infusion at doses of 0.1-1, 1-5, 5-10, 10-25, 25-50, or 50 ug / kg or more.

[0019] In other embodiments, therapeutically effective agents can modulate the activity of one or more of α-synuclein, TFEB, ZKSCAN3, LAMP, GCase, tyrosine hydroxylase (TH), and dopamine. In other embodiments, therapeutically effective agents can modulate the expression of one or more of α-synuclein, TFEB, ZKSCAN3, LAMP, GCase, tyrosine hydroxylase (TH), and dopamine. In other embodiments, the modulation of expression includes transcript expression levels. In other embodiments, the modulation of expression includes protein expression levels. In other embodiments, the protein expression level includes a decrease in α-synuclein protein. In other embodiments, the protein expression level includes an increase in TH protein. In other embodiments, therapeutically effective agents can promote lysosomal proteolysis. In other embodiments, therapeutically effective agents can improve synchronous bursts of electrical activity. In other embodiments, therapeutically effective agents can improve one or more of stepping, rotational asymmetry, and kinesia. In various embodiments, Parkinson's disease can be familial, sporadic, and further include early-onset sporadic Parkinson's disease.

[0020] This specification describes a method for preventing Parkinson's disease, comprising administering a prophylactic agent and a pharmaceutically acceptable carrier to a subject who has been prognosed to be at high risk of developing Parkinson's disease, thereby preventing Parkinson's disease. This specification also describes a method for reversing or delaying the progression of Parkinson's disease, comprising administering a pharmaceutical composition comprising a therapeutically effective agent and a pharmaceutically acceptable carrier to a subject suffering from Parkinson's disease, thereby reversing or delaying the progression of Parkinson's disease in the subject. In other embodiments, the prophylactic or therapeutically effective agent includes PKC activator, its analogues and derivatives. In other embodiments, the PKC activator, its analogues and derivatives include ingenol-3-angelate. In other embodiments, the prophylactic or therapeutically effective agent can reduce α-synuclein protein levels. In other embodiments, the prophylactic or therapeutically effective agent can reduce the promotion of lysosomal proteolysis. In other embodiments, the prophylactic or therapeutically effective agent can reverse or delay substantia nigra degeneration. In other embodiments, therapeutically effective agents can maintain or promote dopamine levels. In various embodiments, subjects who are prognosed to be at high risk of developing Parkinson's disease or who have Parkinson's disease include human subjects who have one or more mutations in EIFG1, PARK2, LRRK2, GBA, SNCA, PINK1, PARK7, VSP35, ATP13A2, or proliferation of the SNCA locus. In various embodiments, subjects who are prognosed to be at high risk of developing Parkinson's disease or who have Parkinson's disease include human subjects with reduced DAT signature in the striatum. In various embodiments, Parkinson's disease can be familial, sporadic, and further include early-onset sporadic Parkinson's disease. [Examples]

[0021] Example 1 Generation of iPSCs from patients with early-onset sporadic Parkinson's disease (EO-sPD) Three patients aged 30–39 years with early-onset sporadic Parkinson's disease and no reported family history of PD were selected for iPSC production (Figure 1). Based on analysis using the NeuroX platform, no monogenic mutations in EIFG1, PARK2, LRRK2, GBA, SNCA, PINK1, PARK7, VSP35, or ATP13A2, or amplification of the SNCA locus, were detected in the patient strains. All three patients showed a reduction in the striatal DAT (phenyltropane) signature, consistent with a diagnosis of PD (Figure 1). For comparison, three control strains were generated from normal individuals without neurological disease at the time of collection.

[0022] Peripheral blood mononuclear cells (PBMCs) were collected and subsequently reprogrammed into iPSCs using a non-integrated episomatic technique (Figure 1). All iPSC lines had a normal karyotype and expressed standard pluripotency markers.

[0023] Example 2 Efficient differentiation from iPSC to DAN using EOSPD iPSC Since a defining characteristic of Parkinson's disease (PD) is the specific loss of dopaminergic neurons in the substantia nigra, differentiating iPSCs along this cell lineage is of interest. iPSC lines from both PD patients and control patients were differentiated into dopaminergic neurons using the protocols described in Table 1, Figure 2A, and Figure 6.

[0024] In short, iPSC strains were subjected to a modified dual-SMAD inhibitor-based baseplate induction protocol. Exposure to LDN / SB, followed by SHH / palmorfamine / FGF8 and CHIR99021, and then removal of SB and addition of retinoic acid, supported the production of midbrain FP and DA neurons (see Figure 1d). Further maturation was performed in Neurobasal / B27 medium supplemented with AA, BDNF, GDNF, TGFβ3, and dbcAMP.

[0025] (Table 1) Differentiation protocol: Culture medium TIFF0007857731000001.tif140170TIFF0007857731000002.tif170170

[0026] On day 30, differentiated cells expressed dopamine neuron markers including TH, Nurr1, and GRIK2, with approximately 15% of cells expressing TH (Supplementary Figure 2a) (Figures 2b, c). Overall differentiation efficiency was compared across all six strains by counting the number of TH-expressing cells using flow cytometry (Figure 2c). Two of the PD strains showed a similar number of DA neurons as the control. However, differentiation of the 190iPD strain produced fewer TH-positive neurons, and these cells showed less expression of the floorplate precursor markers FOXA2 and LMX1A, but more expression of the mature neuron markers GRIK2 and NEFH.

[0027] To determine whether dopamine levels in developing neurons are altered by the TH enzyme, 30-day-old DANs were lysed and analyzed for dopamine production by HPLC. Differences in total dopamine existed between strains, again with less dopamine production in the 190iPD strain and more in the WP3i control strain. However, when normalized by the number of TH-expressing neurons, all strains produced dopamine at similar levels (Figure 2d, e). Multielectrode array recordings were performed over time during culture to determine the electrophysiological function and potential disease signatures of developing neurons. Spontaneous activity was observed at day 20 of differentiation, and by day 30, both PD cells and control cells exhibited synchronous bursts of activity. When activity was quantified across all strains, similar levels of spontaneous spikes were observed between diseased and control DaN cultures. In summary, these data indicate that iPSCs derived from EOSPD patients efficiently differentiated into functional dopaminergic neurons possessing similar neuronal activity to unaffected patient strains.

[0028] Example 3 α-synuclein specifically accumulates in EOSPD DAN. In all forms of Parkinson's disease, α-synuclein protein is known to accumulate abnormally in Lewy bodies, and this accumulation, through duplication or triplication of the SNCA gene, is known to cause PD. However, its precise role in sporadic PD remains unclear, and previous studies have not shown consistent differences in adult-onset sporadic PD. To determine whether α-synuclein protein accumulates in cultures originating from early-onset sporadic PD, six strains were differentiated for 30 days and investigated for soluble α-synuclein by Western blotting.

[0029] Surprisingly, all three EOSPD DAN lysates showed increased levels of α-synuclein protein compared to the control (Figure 2f, g). To verify α-synuclein accumulation, ELISA was performed on both the culture supernatant and cell lysates. α-synuclein concentrations in the supernatant were below the detection limit, and cell lysates supported a significant increase in α-synuclein protein in affected strains. Protein lysates from strains in the iPSC stage did not show an increase in α-synuclein, indicating that accumulation was specific to differentiated cultures.

[0030] To determine whether the increase in protein could be attributed to increased transcription of the SNCA gene, QPCR was performed on DAN cultures at day 30 (Figure 2h). These data showed that two of the EOSPD strains, 190iPD and 200iPD, exhibited increased SNCA expression compared to the control strain, while the third strain, 194iPD, did not, suggesting that increased transcription was not the sole cause of α-synuclein accumulation.

[0031] Example 4 Lysosomal proteins are abnormally regulated in EOSPD DAN. Since the increased transcription of the SNCA gene could not fully explain the accumulation of EOSPD-specific α-synuclein protein, we then attempted to determine other factors that might contribute to this effect through both RNA sequencing and proteomics of paired sample sets derived from the same culture well. Whole transcriptome RNA sequencing (RNA-Seq) detected 27,384 unique transcripts, while proteomics analysis yielded 2,478 proteins that met the reproducibility threshold. The Pearson correlation coefficient showed high consistency across sample replications.

[0032] Combination analysis of proteins and transcripts common to both proteomics and RNA-Seq datasets yielded 2437 identical genes across the two analytical modes (Figure 3a). Unsupervised principal component analysis (PCA) of the identical gene sets revealed a clear demarcation between PD cells and controls along PC1 from both transcriptome and proteomics datasets (Figure 3b). Analysis of the entire RNA-Seq dataset yielded similar PCA results. To determine the key pathways contributing to this segregation, all identical genes were ranked by PC1 gene weighting from both mRNA and proteomics PCA analyses. Next, individual GSEA analyses from each ranked list were merged to reveal common pathways that were significantly abnormally regulated between PD cells and control cells (Figure 3c). α-synuclein, as well as other synaptic vesicle genes involved in dopamine release such as synapsin (SYP), synaptic vesicle 2A (SV2A), and SNAP25, were significantly enriched in their respective terms, as well as in terms related to general synaptic mechanisms and functions, such as GO_EXOCYTIC_VESICLE (Figure 3c). Metabolic genes included in KEGG_OXIDATIVE_PHOSPHORYLATION were also significantly upregulated in the ESOPD strain. In addition, terms related to neurodegenerative diseases such as PD, Alzheimer's disease, and Huntington's disease were significantly upregulated in PD DAN, suggesting that important aspects of neurodegeneration were captured in the culture system (Figure 3c). The terms GO_LYSOSOMAL_LUMEN and GO_ENDOPLASMIC_RETICULUM_LUMEN, which were significantly downregulated, showed defects in protein formation and lysosomal protein degradation compared to unaffected controls (Figure 3f).

[0033] Example 5 The degradation of α-synuclein is impaired in PD DAN. The reduction in lysosomal proteins in EOSPD DANs allowed us to determine whether the accumulation of α-synuclein was a result of impaired degradation function. To test the overall degradation rate, we inhibited overall transcriptional function in DANs for 48 hours via cycloheximide treatment and quantified α-synuclein protein over time (Figure 4a, b). In the control strain 02i, α-synuclein was degraded during the 48-hour treatment, with a half-life of approximately 10 hours observed (Figure 4b). However, in the most severe EOSPD strain (190iPD), α-synuclein actually accumulated over the duration of this treatment. This striking dichotomy suggests an underlying defect in the specific degradation of α-synuclein. This is supported by similar degradation profiles between control cells and PD cells for other proteins such as TH (Figure 4a, c) or synaptophysin (Figure 4a, d).

[0034] Protein degradation is mainly divided into the proteasomal degradation pathway and the autophagy / lysosomal degradation pathway. To determine that proteasomal degradation is the cause of α-synuclein protein degradation, DaN cultures were treated with the proteasomal inhibitor MG132 for 24 hours. As a result, P53, a protein typically degraded via the proteasomal pathway, accumulated, but there was no substantial change in α-synuclein levels (Figure 4e). This result indicates that proteasomal degradation was not a significant factor in α-synuclein degradation in DAN cultures.

[0035] To determine the involvement of lysosomes in α-synuclein degradation, we investigated the activity of glucocerebrosidase or GCase and total LAMP1 protein. Consistent with proteomic analysis, we observed a decrease in LAMP1 levels in all three EOSPD strains (Figure 4f). GCase is a class of lysosomal hydrolases whose decreased activity has been reported in the peripheral blood of some PD patients. A significant decrease in GCase activity was observed in 30-day-old DaNs from EOSPD patients compared to controls (Figure 4f). Other researchers have found that decreased GCase activity in iPSC-derived DaNs is caused by an increase in oxidized dopamine. However, a similar increase in oxidized dopamine was not observed in our 30-day-old PD DaNs (Figure 4h). Considering the significant downregulation of lysosomal pathway proteins, these results provide evidence of lysosomal degradation dysfunction as a putative cause of α-synuclein accumulation in EOSPD DaNs.

[0036] Example 6 EOSPD phenotype can be rescued by regulating PKC signaling. The inventors selected three lysosomal agonists to test whether they could reduce the synuclein levels of their EOSPD DaN through activation of a lysosome-specific pathway. The compounds selected were PEP005, a PKC agonist and structural analogue of the HEP14 drug; SMER28, a small molecule TFEB agonist shown to reduce Huntington's and α-synuclein aggregates in the PC12 cell model; and trehalose, another biocompound shown to promote α-synuclein clearance. From day 27, DaN was treated with the above lysosomal agonists for 3 days. Treatment with PEP005 and SMER28, rather than trehalose, significantly reduced the amount of α-synuclein protein in DaN from the control strain (Figure 5a). However, in EOSPD DaN, only the PKC agonist PEP005 significantly reduced the synuclein levels. Interestingly, PEP005 treatment also resulted in an increase in the amount of TH enzyme present in both control and PD DaN (Figure 5a).

[0037] The intriguing combined effect of PEP005, which simultaneously reduces synuclein levels and increases TH expression in both control and PD DaNs, led us to investigate the mechanism of action of the drug. PEP005 is an established PKCδ agonist that produces a short burst of PKC phosphorylation followed by a more prolonged, potent decrease in phosphorylated PKC. At the end of this study, we observed an increase in basal levels of PKCα phosphorylation in untreated 190iPD DaNs (Figure 5a), and PEP005 treatment completely eliminated this signal in both control and PD DaNs (Figures 5a, c).

[0038] After observing an increase in baseline phosphorylated PKCa in the 190iPD strain, the inventors reviewed all additional DANs to determine if this observation was demonstrated across multiple strains. The inventors found higher levels of p-PKCa in the 30-day DANs from all three EOSPD strains (Figure 5b). The inventors also reviewed three additional newly induced EOSPD strains (172iPD, 183iPD, 192iPD), three additional controls (0771i control, 1034i control, 1185i control), and a typical-onset PD strain (78iPD, age 67 at onset, family history of PD) for both α-synuclein accumulation and increased p-PKCa (Figure 5g).

[0039] Elevated PKCa phosphorylation was not present in undifferentiated iPSCs, and there was no clear pattern in peripheral blood from individual patients exhibiting specificity to differentiated DaN. Elevated PKCa phosphorylation was clearly removed by adding 1 μM PEP005 to DaN from all iPSC strains for 3 days (Figure 5c). This removal correlated with a decrease in synuclein in all treated strains, but neither LAMP1 nor LC3 appeared to respond to PEP treatment in PD cells (Figure 5c), suggesting that the mechanism of action in PD cells may differ from the typical upregulation of lysosomal proteins. The time course of PEP treatment in control and PD DaN shows that both p-PKCa and α-synuclein are degraded in response to drug treatment within approximately 24 hours (Figure 5d). This same time course also shows a significant decrease in cleaved caspase 3 (CC3) present in PD cells. Gene expression data from paired samples along the same time course show that SNCA is downregulated 4 hours after PEP treatment (Figure 5e), and TH is upregulated approximately 8 hours after initial exposure (Figure 5f).

[0040] Example 7 In vivo reduction of α-synuclein in WT mice In vivo, PEP stimulates the degradation of synuclein. Dosage studies of 0.3, 3, and 30 μM PEP were injected into the ventricles of wild-type mice. Synuclein levels decreased and TH levels increased in the mouse striatum at 1 and 5 days post-injection.

[0041] Example 8 Consideration The inventors initiated this study to search for signs of parkinsonism in dopaminergic neurons differentiated from iPSCs of early-onset sporadic Parkinson's disease patients. The inventors reprogrammed PBMCs from three individuals among three randomly selected early-onset PD patients with no family history. The resulting iPSC strains were genetically normal and lacked many of the known single-gene PD mutations. The genomic chip assay used to assess this covers approximately 260,000 known SNPs associated with neurodegenerative disease. While highly improbable, it is possible that the three idiopathic individuals used to generate the PD iPSCs possessed all previously unknown single-gene mutations missed by NeuroX screening. In any case, the complex genetic nature underlying these EOSPD iPSCs resulted in α-synuclein accumulation in DaN at as young as 30 days of age. This is the first phenotype identified in iPSCs derived from sporadic Parkinson's disease patients.

[0042] Next, the inventors proceeded to complete a detailed analysis of these differentiated cells using both transcriptome and proteomics techniques. Transcriptome analysis revealed increased expression of numerous synaptic and exocytosis transcripts in PD cells. This increase in transcripts, when translated, directly leads to elevated protein levels in PD DaN, indicating an overgrowth of synaptic mechanisms. However, despite the presence of more synaptic mechanisms, neither MEA recording nor live calcium imaging showed any difference in activity between PD DaN and control DaN. Conversely, proteomics data showed a decrease in the amount of lysosomal lumen protein in PD DaN. This decrease was not reflected in the RNA of the same cells, which indicates a disruption of this signaling pathway. There is no response of producing more protein but producing more cells. This decrease in lysosomal protein was further confirmed by decreased GCase activity in PD DaN, decreased LAMP1 protein by Western blotting, and accumulation of α-synuclein under cycloheximide inhibition, all of which point to some deficiency in proteolysis in PD DaN. Furthermore, since inhibition of proteasomal degradation did not result in changes in α-synuclein levels, this deficiency appears to be specific to the lysosomal degradation pathway.

[0043] The inventors then selected a series of lysosomal agonists to correct this observed deficiency. Of the three agonists tested, only the PEP005 small molecule reduced α-synuclein levels in both control and PD DaN. Interestingly, PEP treatment also resulted in an increase in the amount of TH present in the treated DaN cultures of both control and PD. The dual effects observed here—a decrease in intracellular α-synuclein levels and an increase in TH—make PEP005 a very attractive candidate as a potential therapeutic agent.

[0044] PEP005 (ingenol-3-angelate) is an FDA-approved topical treatment for actinic keratosis that also possesses anti-leukemic activity and may play a role in the reactivation of latent HIV. Also known as ingenol-3-angelate and ingenol mebutate, it is the most studied ingenol derivative, first extracted from the sap of the Euphorbia peplus plant. This small molecule binds to the PKC C1 domain with subnanomolar affinity and does not exhibit selectivity for individual PKC isoforms in vitro, although the patterns of PKC isoform migration and downregulation induced by PEP005 can vary, sometimes in a cell line-dependent manner. In this study, we selected it as a structural analog derived from the same Euphorbia peplus plant as HEP14 (5β-O-angelate-20-deoxyingenol), a compound identified by Li and colleagues that acts as a TFEB agonist independently of the MTOR pathway.

[0045] In control cells treated with PEP005, the inventors observed an increase in the lysosomal protein LAMP1, consistent with the activation of the lysosomal master regulator TFEB, but this increase does not appear to be reproducible in drug-treated PD DaN. PEP is described as both an activator of pro-apoptotic PKCδ and an inhibitor of PKCα. PEP005 is described as inhibiting the proliferation of various cancer cell lines and primary acute myeloid leukemia (AML) cells. In leukemia cell lines and primary AML cells, PEP005 induces apoptosis by activating PKCδ, followed by sustained activation of ERK1 / 2.

[0046] In our DaN cultures, we did not observe strong PKCδ signaling, nor did we see the increase in LDH during drug therapy that might be expected if cell death was being induced. In fact, although we observed a decrease in the amount of active caspase-3 during drug therapy, this effect was most readily observed in PD DaN, where the initial levels of cleaved caspase-3 were high. Our differentiated neurons were mainly postmittal, while the toxicity of PEP is likely more specific to highly proliferative cells.

[0047] When the inventors investigated the mechanism of action of the PEP005 small molecule in DaN, they observed an increase in the level of phosphorylated PKCα in PD DaN. Synuclein is suggested to bind not only to and share homology with typical 14-3-3 proteins involved in TFEB activation, but also to PKCα, suggesting a correlation between the synuclein accumulation observed by the inventors, lysosome biosynthesis, and the PKC agonist PEP005. PKC links the activation of the TFEB transcription factor and the inactivation of the ZKSCAN3 transcription repressor via two parallel signaling cascades. Activated PKC inactivates GSK3, reducing TFEB phosphorylation, nuclear translocation, and activation, while PKC phosphorylates ZKSCAN3, inactivating it by translocating it out of the nucleus. Therefore, PKC activation mediates lysosomal adaptation to numerous extracellular cues, including clearance of aggregated proteins, thereby providing viable therapeutic options for diseases and disorders mediated by the lysosomal nexus, such as the mechanism of Parkinson's disease outlined herein.

[0048] While α-synuclein degradation is controversial, it appears that the majority of the degradation of monomeric WTα-synuclein in neuronal cell systems occurs via the lysosomal pathways of chaperone-mediated autophagy (CMA) and macroautophagy. Dysfunction of these degradation pathways may contribute to the pathogenesis of Parkinson's disease (PD). Herein, we demonstrate the feasibility of a strategy to promote enhanced clearance of excess α-synuclein by the endogenous degradation system, and it may also have the advantage of mitigating the abnormal effects that α-synuclein has on its function.

[0049] This study is the first to identify a molecular signature for sporadic Parkinson's disease in iPSCs from early-onset patients. The inventors found that these cells accumulate α-synuclein, exhibit dysregulation of lysosomal biosynthesis and function, and show more severely phosphorylated PKCα. These three biomarkers, taken together, provide a platform for screening novel therapeutics that may affect the underlying mechanisms of PD. The inventors then embarked on identifying novel drugs for PD that eliminate this signature and reduce intracellular α-synuclein in both control and PD cells. These findings suggest a specific and novel drug discovery pathway that offers an opportunity to finally treat some of the underlying mechanisms of PD.

[0050] Example 9 Further research The inventors generated the first iPSC strains from a cohort of early-onset sporadic Parkinson's disease (EOSPD) patients in whom no PD mutations were detected. Surprisingly, the inventors found that the majority of dopamine neuron cultures derived from iPSCs of EOSPD patients showed robust increases in α-synuclein and phosphorylated protein kinase C-α (p-PKCα), as well as decreases in key lysosomal proteins. Targeting these pathways with small molecules revealed that phorbol esters could reverse many of these disease-related phenotypes. The inventors' findings demonstrate that EOSPD has an undetermined genetic basis for the subsequent impairment of lysosomal degradation of α-synuclein. The fact that specific phorbol esters can reverse this loss highlights the potential of these drugs as novel therapeutics for EOSPD.

[0051] Further research was planned as follows:

[0052] iPSC line generation: iPSC lines were generated by nucleofecting parental cells with a non-integrated oriP / EBNA1 plasmid, and then episomal expression of reprogramming factors was performed on these cells, as previously described in collaboration with Cedars-Sinai iPSC Core.

[0053] iPSC maintenance, mDA neuron differentiation, and drug treatment: iPSCs were maintained in E8 medium on Matrigel and passaged every 5 days at a partition ratio of 1:6 to 1:12 using Versene as needed. Only iPSCs from generation 17 to 35 were used in this study. For differentiation, iPSCs were grown to approximately 80% confluence. Cells were homogenized with Accutase (Millipore / Sigma#SCR005) (5 min at 37°C) and 1 cm³ in E8 medium containing 5 μM Y27632 (StemGent). 2Cells were plated in 6-well plates coated with Matrigel (BD Biosciences) with 200,000 cells per well (for fully confluent monolayers). 24 hours after plating, the medium was changed to Stage 1 (50% DMEM / F12, 50% Neurobasal, N2, B27-Vitamin A, LDN-193189 (LDN), SB431542 (SB)). Stage 1 medium was changed daily for 3 days (3 mL per well). Next, the medium was switched to Stage 2 (50% DMEM / F12, 50% Neurobasal, N2, B27-Vitamin A, LDN, SB, Palmorphamine (PMN), CHIR99021 (CHIR), Sonic Hedgehog (SHH), Fibroblast Growth Factor 8 (FGF8)). Stage 2 medium was changed daily for 4 days (3 mL per well). Subsequently, the culture medium was switched to Stage 3 (50% DMEM / F12, 50% Neurobasal, N2, B27-Vitamin A, LDN, CHIR, all-trans retinoic acid (ATRA)). The Stage 3 medium was changed daily (3 mL per well) for 4 days. Finally, the culture medium was switched to Stage 4 (50% DMEM / F12, 50% Neurobasal, N2, B27-Vitamin A, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), dibutyryl cyclic AMP sodium salt (dbCAMP), L-ascorbic acid (AA), γ-secretase inhibitor (DAPT), CHIR, transforming growth factor beta-3 (TGF β3)). The Stage 4 medium was changed daily (3 mL per well) for 3 days. On day 15, cells were separated into single cells using Accutase (20 minutes at 37°C) and gently suspended. The dissociated cells were resuspended in maturation medium (50% DMEM / F12, 50% Neurobasal, N2, B27 + Vitamin A, BDNF, GDNF, dbCAMP, AA, DAPT, TGF3) and 5 μM Y27632, and irrigated at a rate of 200,000 / cm³ in a total volume of 1 mL. 2The cells were then re-seed into 6-well plates coated with Matrigel, or into L-glass coverslips coated with Matrigel in 24-well plates using 200,000 / 50 μL droplets. After allowing the cells to adhere at 37°C for 45 minutes, maturation medium was added to a final volume of 3 mL per well in 6-well plates and 1.5 mL per well in 24-well plates with coverslips. A complete medium change was performed 48 hours after seeding, and the medium was changed every 3 days until day 30. For drug treatment, maturation medium containing the indicated drug was given to the cells on day 27, and the cells were analyzed on day 30. The drugs were PEP005 (1 μM, Tocris, #4054), SMER-28 (5 μM, Tocris, #4297), trehalose (25 M, Sigma-Aldrich, #T0167), PMA (10 μM, Tocris, #1201), and prostratin (PRO) (5 μM, Tocris, #5739).

[0054] Table 1. Culture medium composition TIFF0007857731000003.tif35137

[0055] Table 2. Culture medium composition TIFF0007857731000004.tif98151

[0056] Analytical flow cytometry: 30-day cultures in 6-well plates were washed once with phosphate-buffered saline (PBS), then 1 mL of Accutase was added to each well, and the cells were incubated at 37°C for 25 minutes, or until the cells were completely suspended. The cells were washed with an additional 2 mL of maturation medium and gently crushed until no large clumps were visible, at which point they were pelleted by centrifugation (1500 RPM for 3 minutes). The cells were gently resuspended in 4% paraformaldehyde (PFA) in PBS and fixed at room temperature for 10–15 minutes. Fixed single cells were permeabilized with 1% Trition X-100 (Sigma-Aldrich) and stained with primary antibodies against TH (1:500, Immunostar, 22941), α-synuclein (1:1000, Abcam, ab138501), and MAP2ab (1:1000, Sigma-Aldrich, M1406), or the same dilutions of isotype controls (Cell Signaling IgG Isotype Control rabbit #3900S and mouse #5415S). Secondary antibodies (Alexa Fluor 488 and 594 anti-donkey mouse and anti-donkey rabbit, Invitrogen) were used at 1:500. Stained samples were quantified using an LSR Fortessa cytometer with BD FACSDiva software.

[0057] Dopamine detection: Cultures of mDA neurons were plated onto L glass coverslips and grown as described above. For total dopamine, the cultures were washed with artificial cerebrospinal fluid (aCSF) and immediately lysed in 200 μL of 0.2 M perchloric acid / 0.1 mM EDTA. The lysates were flash-frozen in LN2. To detect dopamine released on day 30, the culture was aspirated and the cells were washed twice with aCSF. After washing, 200 μL of aCSF was carefully plated onto each coverslip and incubated at 37°C for 15 minutes for collection. Next, 200 μL of high K2 was added. +aCSF was added to each coverslip, incubated for 15 minutes, and collected. Immediately after collection, 20 μL of 10x stabilizing buffer (2 M perchloric acid / 1 mM EDTA) was added to each sample. The stabilized samples were flash-frozen in LN2 and stored at -80°C until HPLC (high-performance liquid chromatography) analysis. Separation was performed using a 2.1 × 100 mm 3 μm reversed-phase Hypersil ODS column, with a mobile phase consisting of 75 mM sodium acetate, 0.75 mM sodium dodecanesulfonate, 2.5% acetonitrile, 12.5% ​​methanol, and 10 μM EDTA (pH=5.5) pumped in at a rate of 0.2 ml / min. Electrochemical detection of dopamine was performed using a glassy carbon electrode held at a potential of 0.29 V relative to an Ag / AgCl reference electrode, and a detection limit of 0.1 nM was obtained for an injection of 10 μl. The samples were analyzed in triplicate against known standard concentrations of dopamine.

[0058] Immunocytochemistry and Imaging: Cultures of mDA neurons were plated onto L glass coverslips and grown as described above. Neurons at day 30 were fixed in 4% PFA at room temperature for 10-15 minutes. The fixed coverslips were washed in PBS and permeabilized in 1% Triton X-100 in PBS at room temperature for 10 minutes. They were then stained overnight at 4°C in primary antibody solution (5% normal donkey serum, 0.125% Triton-X, PBS) with the following antibodies: TH (1:5000, Immunostar, #22941) and α-synuclein (1:500, Abcam, #ab138501). The samples were washed three times in PBS and stained with species-specific Alexa Fluor 488 or 594-conjugated secondary antibody (1:500, Invitrogen) at room temperature for 2 hours, followed by DAPI counterstaining. Confocal Z-stacked images were acquired using an A1 microscope (Nikon) equipped with 40x and 20x objective lenses, and rendered using maximum intensity projection with IMARIS software (Bitplane).

[0059] Western blotting: Cells were gently scraped from the plate, washed with PBS, centrifuged at 15,000 RPM for 1 minute, and the dry pellet was frozen at -80°C. The sample was then thawed and lysed using 1x NETN buffer supplemented with a phosphatase / protease inhibitor cocktail (MS-SAFE, Sigma-Aldrich) (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5 mM EDTA, and 0.5% NP-40). The lysate was sonicated in an automated cold bath ultrasonic cleaner for 20 minutes, alternating between 10-second pulses and 10-second pauses. The sample was centrifuged at 15,000 RPM at 4°C for 20 minutes. Total soluble protein concentration was measured using the Bradford assay (BIO-RAD). 100 μg or 50 μg of total protein extract was added to 4x Laemmli sample buffer (BIO-RAD, 161-0774), and the sample was boiled for 5 minutes. The sample was electrophoresed on 4–20% Mini-PROTEAN TGX Precast gel (BIO-RAD, 456-1094) and transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (BIO-RAD). After blocking the membrane with Odyssey barrier buffer (LI-COR), it was incubated with the primary antibody overnight at 4°C or at room temperature for 3 hours. After incubation with the dye-labeled secondary antibody at room temperature for 2 hours, the signal was visualized using the Odyssey Fc imaging system (LI-COR).The primary antibodies used were human α-synuclein (1:1000, Abcam, #ab138501), mouse α-synuclein (1:1000, Abcam, #ab212184), TH (1:2000, ImmunoStar, #22941), total PKCα (1:1000, Cell Signaling, #2056S), p-PKCα (1:1000, Cell Signaling, #9375S), LAMP1 (1:1000, Cell Signaling, #9091S), LCI / II (1:1000, Cell Signaling, #12741S), and cleaved caspase 3-CC3 (1:1000, Cell The antibodies used were Signaling (#9661S), synaptophysin (1:1000, Abcam, ab32127), P53 (1:2000, Santa Cruz, #sc-126), GAPDH (1:5000, Sigma-Aldrich, G8795), and β-actin (1:5000, Sigma-Aldrich, #A5441). The secondary antibodies were IRDye 680 RD goat anti-mouse and IRDye 800CW goat anti-rabbit at a 1:5,000 dilution (LI-COR 926-68070 and 926-32211, respectively). Expression plots of α-synuclein and p-PKCα were calculated by first normalizing the band with β-actin, and then normalizing with the O2i control signal present in each blot. Next, all values ​​were compared across at least three independent differentiations of each strain. The proportion of predicted probabilities was determined by calculating the ROC plot and area under the curve using the R package ROCR.

[0060] qPCR: Total cellular RNA was isolated using a Qiagen RNeasy Mini kit, followed by TRIzol reagent and DNase treatment. Total RNA (1 μg) was used for cDNA synthesis using the Quantitate Reverse Transcription Kit (Qiagen) for PCR. Real-time PCR was performed using SYBR Green Supermix (BIO-RAD). The expression levels of each gene were normalized by the corresponding GAPDH values ​​and shown as a multiplier of change relative to the control sample (ΔΔCt method).

[0061] NIRF detection of oxidized dopamine: The assay was performed as described. In short, neurons were scraped in cold PBS and centrifuged at 15,000 RPM for 1 minute. The cell pellet was frozen and then thawed and homogenized in 1x NETN lysis buffer containing a phosphatase / protease inhibitor cocktail. The lysate was sonicated in an ultrasonic cleaner for 10 minutes and rotated at 15,000 RPM for 15 minutes. The supernatant was removed and the insoluble pellet was resuspended in 18 MΩ deionized water. Total protein was measured using the Bradford assay, with 100 μg of protein taken in 20 L and dropped onto a Biodyne Nylon Transfer Membrane (Pall, #Pall-60209). The membrane was scanned using a 700-channel Odyssey infrared imaging system (LI-COR). The sample was quantified by obtaining integrated spot intensity using Odyssey infrared imaging software, version 3.1.

[0062] GCase activity: The assay was performed as described. In short, the sample was dissolved as described above and centrifuged at 15,000 rpm for 15 minutes at 4°C. 50 μg of total protein was incubated in activity assay buffer (0.25% (v / v) Triton X-100, 1 mM EDTA, citrate / phosphate buffer, pH 5.4) in 1% bovine serum albumin (BSA) containing a total volume of 200 μl of 1 mM 4-methylumbelliferyl β-glucopyranoside (4-MU, Sigma-Aldrich, #M3633). After incubation at 37°C for 40 minutes, the reaction was stopped by adding an equal volume of 1 M glycine, pH 12.5. A 100 μl replica was loaded into a white 96-well plate (Corning Assay plate), and fluorescence (excitation = 355 nm, fluorescence = 460 nm) was determined using a Molecular Devices SpectraMax i3 Multi-Mode microplate reader and SoftMax Pro software.

[0063] Transcriptomics: Three wells from each strain were differentiated as described above and divided into cell pellets for either mRNA sequencing or proteomics analysis. mRNA was isolated using the previously described method. In short, library construction was performed using the Illumina TruSeq Stranded mRNA Library Preparation Kit (Illumina). In short, total RNA samples were evaluated for concentration using a Qubit fluorometer (ThermoFisher) and for quality using a 2100 Bioanalyzer (Agilent Technologies). A maximum of 1 μg of total RNA per sample was used for poly-A mRNA selection. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Invitrogen) and random primers. The cDNA was further converted to double-stranded DNA (dsDNA), and the resulting dsDNA was enriched by PCR for library preparation. The PCR-amplified libraries were purified using Agincourt AMPure XP beads (Beckman Coulter). The concentration of the amplified library was measured using a Qubit fluorometer, and aliquots of the library were resolved using a Bioanalyzer. The sample libraries were multiplexed and sequenced on a NextSeq 500 platform (Illumina) using 75 bp single-ended sequencing. On average, approximately 20 million reads were generated from each sample.

[0064] A custom human GRCh38 (or mouse CRCm38) transcriptome reference downloaded from http: / / www.gencodegenes.org, including all protein-coding and long non-coding RNA genes based on human GENCODE version 23 (or Mouse GENCODE M8) annotations, was used to align raw reads obtained from RNA-Seq to the transcriptome using STAR (version 2.5.0)62 / RSEM (version 1.2.25)63 with default parameters. The expression level (TPM: transcripts per million) of each gene in all samples was normalized by sequencing depth. To determine the detected transcripts, a filter greater than 0.1 TPM in at least nine samples was used as a threshold for detecting unique transcripts (Cluster 3.0). PCA was performed on logarithmically transformed data using Cluster 3.0 software. All transcriptome data from this study are available in the GEO repository under GSE120746.

[0065] Proteomics: Frozen pellets were dissolved in 2% SDS + 10 mM TCEP (tris-2-carboxyethylphosphine) buffer and sonicated. Protein concentrations were determined using a bicinchoninic acid assay (BCA assay, Pierce, #23225), and 125 μg of protein was digested using a FASP Protein Digestion Kit (Expedeon). Each sample was digested overnight at 37°C with 125 μg of trypsin / lysC, shaking at 1000 rpm. Samples were desalted using an Oasis MCX μelution plate and eluted with 300 μl methanol / ammonium hydroxide. Samples were dried to a dry state using SpeedVac and resuspended in Biognosys iRT solution. Sample / iRT solution (4 μg) was loaded onto an Eksigent 415 LC connected to a 6600 TripleTOF (Sciex) operating in microflow mode. Peptides were preloaded onto a trap column (ChromXP C18CL 10×0.3mm 5μm 120Å) at a flow rate of 10 μL / min for 3 minutes and separated on an analytical column (ChromXP C18CL 150×0.3mm 3μm 120Å) at a temperature of 30°C and a flow rate of 5 μL / min. For DIA samples, peptides were separated using a linear AB gradient consisting of isocratic hold for 38 minutes with 3–30% A, 5 minutes with 30–40% B, 2 minutes with 40–85% B, 3 minutes with 85%, and re-equilibrium for 8 minutes with 3% A. Data were acquired using 400–1250 m / z with MS1 scans of 150 ms and MS2 scans of 25 ms with a 100 variable window. Source parameters were set to the following values: Gas 1 = 15, Gas 2 = 20, Curtain Gas = 25, Source Temperature = 100, and Voltage = 5500V. The DDA sample was electrophoresed using a linear AB gradient consisting of 60 minutes at 3–35% A, 2 minutes at 35–85% B, followed by a 5 minute isocratic hold at 85% with a 7 minute re-equilibrium at 3% A. For DDA acquisition, an MS1 ​​scan was acquired using a residence time of 250 milliseconds in the mass range of 400–1250 m / z, and the top 50 ions reaching a threshold of 100 counts per second were selected for fragmentation.Using rolling collision energy and collision energy spread, MS2 scans were acquired for ions in the +2 to +5 range with a residence time of 25 milliseconds in high-sensitivity mode with the dynamic acquisition option enabled. Ions were excluded from fragmentation after occurring once over a 15-second period. As previously outlined, the DIA files were compared to the DDA library using OpenSWATH. MS2 normalized transition level data were passed through MAP DIA software to obtain normalized peptide and protein level data. In addition, protein difference analysis was performed by MAP DIA. A CV filter was applied to remove any peptides with high variability within the triplicate, excluding peptides with a CV exceeding 20% ​​within each technical iteration. Next, the peptide level data were summed to obtain protein level data. This data was then used for downstream analyses, including principal component analysis, GSEA, and stringing. The mass spectrometry proteomics data are deposited in the ProteomeXchange Consortium via the PRIDE partner repository under dataset identifier PXD011326.

[0066] GSEA and STRING Analysis: Gene set enrichment analysis (GSEA) was performed as previously described.68 For concordance analysis of mRNA-Seq and proteomics data, concordant genes found in both datasets were analyzed individually using GSEA. Pre-ranked PC1 gene weighting from each independent PCA analysis was performed on the Gene Ontology (GO) and KEGG databases using the GSEA algorithm. Ranked pathway lists obtained from RNA-Seq and proteomics analyses were matched using R software and ranked by significance calculated by FDR. A predetermined list of differently expressed proteins from MAP DIA was used with the STRING protein-protein interaction online tool to obtain highly reliable protein-protein interactions and enrichment for the whole genome.

[0067] MEA Recording: Cells were plated onto 48-well microelectrode array (MEA) plates (Axion Biosystems) on day 15 of differentiation. Spontaneous activity was measured daily for 5 minutes using the Maestro MEA platform (Axion Biosystems). Waveform events were identified using adaptive spike threshold crossing with a standard deviation of electrode noise set to 6, and events were further sorted using Offline Sorter v.4 (Plexon). A minimum of 5 spikes per minute were used for inclusion in the analysis.

[0068] Patch Clamp: Whole-cell patch clamps were performed on approximately 30-day-old cultures plated on L glass coverslips. Cells were placed in phenol red-free brain phys medium (STEMCELL Technologies, 5790) at room temperature and maintained for up to 2 hours during acquisition. Glass pipettes were drawn using a Sutter Instruments P-1000 with a tip resistance of 4–5 MΩ. The internal solution (in mM) consisted of 112.5 K-gluconate, 4 NaCl, 17.5 KCl, 0.5 CaCl2, 1 MgCl2, 5 ATP, 1 NaGTP, 5 EGTA, and 10 HEPES. Voltage and current clamp recordings were performed using a Multiclamp 700B amplifier, Digidata 1300, and PClamp 10 acquisition software (Molecular Devices). Neurons with access resistance exceeding 30 MΩ, or neurons whose resistance changed above 4 MΩ during recording, were excluded. The resting membrane potential (RMP) was measured during current clamping by averaging continuous voltages that recorded 0 pA. Voltage-gated sodium and potassium currents were measured from a holding voltage of -70 mV, followed by stepwise measurements from -120 mV to 40 mV in 10 mV increments over 100 milliseconds. Induced action potentials were measured with current clamping, where the holding current was adjusted to maintain a constant -60 mV baseline voltage across the entire cell, and then 10 pA increment steps were applied over 500 milliseconds.

[0069] In silico modeling of the PKC-PEP005 protein complex: The putative binding of PEP005 to PKC was determined using homology modeling followed by docking studies. In short, the C1 domain of PKC complexed with 12-acetylphorbol was used as a template (PDB:1PTR)69. A model structure of PKC□ was developed by homology modeling using Rosetta70. Next, the binding of PEP005 was evaluated using Glide71 with a higher-level 3D model of PKC (out of five predicted structures). The optimal binding orientation of PEP005 was selected based on the Glide XP score. Other putative protein targets of PEP005 were investigated using DALI72 based on the 3D structure of the C1 domain of PKC.

[0070] In vivo evaluation of PEP005 activity: Wild-type C57BL / 6 mice (Jackson labs) were used, and all animal experiments were performed at Cedars-Sinai Medical Center according to IACUC 6462. PEP005 was diluted to 10, 1, or 0.1 mM in 0.9% sterile saline. The vehicle (DMSO) was diluted to 10 mM in 0.9% sterile saline. A single 2 μl injection was administered into the left striatum of the mice at the following coordinates: 0.7 mm AP and 2.5 mm ML from the front, and 3.5 mm DV from the dura mater. Animals were sacrificed 3 days after injection. For immunohistochemical (IHC) analysis, mice were perfused with 4% PFA / PBS, the whole brain was excised, and post-fixed overnight in 4% PFA at 4°C. The brain was then rinsed with PBS and stored at 4°C in 30% sucrose. Brain tissue was sectioned to 30 μm using a microtome and collected as suspension. Striatal sections were washed three times with PBS for 5 minutes each and quenched with 0.3% H2O2 for 30 minutes. Sections were washed three times with 0.005% TritonX-100 (PBS-T) in PBS for 5 minutes each, blocked at room temperature for 1 hour in a solution of 3% normal horse serum (NHS) and 2% BSA in PBS-T, and incubated overnight at room temperature in α-synuclein antibody (1:300, Abcam, #ab212184) in the blockage solution. Slides were washed three times with PBS-T for 5 minutes each and incubated with biotinylated anti-rabbit IgG (Vector, BA-1000) in the blockage solution. Next, sections were washed three times with PBS-T for 10 minutes each, incubated with Avidin Biotin Complex (Vector, VECTASTAIN ABC Kits (HRP), #AK5000) for 45 minutes, and the signal was visualized using DAB (3,3'-diaminobenzidine) (1:500, Vector SK4100). For Western blot analysis, mice were perfused with PBS and the left and right striatums were excised. Individual striatal hemispheres were immediately homogenized and lysed in 1x NETN buffer supplemented with a phosphatase / protease inhibitor cocktail. The lysates were sonicated by serial probe and then ultrasonic washing, and centrifuged at 15000 RPM for 20 minutes at 4°C.Total protein (50 μg) from each lysate was electrophoresed on 4-20% Mini-PROTEAN TGX Precast gels and transferred to a PVDF membrane. Mouse-specific α-synuclein (Abcam, #ab212184) and β-actin antibodies were used. Protein bands were quantified using LI-COR software to show the relative synuclein levels (α-synuclein / β-actin) on the opposite side (R) of the injected side (L).

[0071] Example 10 Generation of iPSCs from patients with early-onset sporadic Parkinson's disease The inventors first collected peripheral blood mononuclear cells from three EOSPD patients (30-39 years old) (190iPD, 194iPD, 200iPD) with no known family history of PD. These cells were reprogrammed into iPSCs using an established non-integrated episome technique. The iPSC lines expressed pluripotency markers and were karyotype normal. Analysis using the NeuroX platform did not detect any established single-gene mutations in the PD genes EIFG1, PARK2, LRRK2, GBA, SNCA, PINK1, PARK7, VSP35, or ATP13A2, nor any proliferation of the SNCA locus in the patient lines (data omitted). Patient presentations included tremor-dominant, akinesia-rigid, or mixed phenotypes, all of which exhibited asymmetric onset with corresponding asymmetry deficits in striatal DAT uptake that confirm the PD diagnosis. In addition, three control iPSC lines were generated from the blood or fibroblasts of individuals without neurological disorders at the time of collection (02i control, WP3i control, and 00i control).

[0072] Example 11 Efficient differentiation from iPSCs to mDA cultures iPSC lines from both EOSPD and control patients were differentiated into midbrain dopaminergic (mDA) neuron cultures using a modified 30-day protocol based on Kriks et al. 31. At day 30, differentiated cultures expressed mature neuronal markers (PAX6, NEFH), neuronal markers (TUB3 and MAP2), and, importantly, dopaminergic neuron markers including tyrosine hydroxylase (TH), Nurr1, DAT, and GIRK231-33. Immunostaining confirmed TH production in all six strains. Flow cytometry quantification showed similar numbers of TH-expressing cells between control and EOSPD strains; however, individual strain comparisons revealed that one EOSPD strain, 190iPD, produced significantly fewer TH-positive neurons compared to the 02i control strain. Differences between other strains were not significant. To determine whether EOSPD altered dopamine content and / or release, mDA culture extracts and effluents were analyzed by HPLC. When normalized by the number of TH-expressing neurons, all strains produced and released dopamine at similar levels. To determine the electrophysiological function of neurons during EOSPD development, patch-clamp and multi-electrode array (MEA) recordings were performed time-course during culture. Spontaneous activity from MEA recordings was observed at day 21 of differentiation, and by day 30, both EOSPD cells and control cells exhibited synchronous bursts of activity. Quantifying activity across all strains revealed a similar number of spontaneous spikes between diseased mDA cultures and control mDA cultures. Neurons patched at day 30 exhibited spontaneous activity with large voltage-gated sodium and potassium currents, eliciting a series of action potentials upon current injection, thereby indicating mature neurons. In summary, these data demonstrate that iPSCs derived from EOSPD patients efficiently differentiate into functionally dopaminergic neurons and possess similar neuronal profiles controlling the strains, suggesting that these phenotypic measures did not provide disease-specific signatures.

[0073] Example 12 α-synuclein specifically accumulates in EOSPD mDA cultures. Quantitative PCR was performed to determine whether α-synuclein was expressed differently in mDA cultures derived from EOSPD. Compared to control cultures, SNCA gene expression was not significantly increased in EOSPD mDA cultures. The inventors also attempted to confirm whether the accumulation of α-synuclein protein was similar. Interestingly, Western blot analysis showed that EOSPD mDA cultures had significantly increased α-synuclein protein levels compared to controls. Subsequent ELISA of cell lysates confirmed a significant increase in α-synuclein protein levels in affected strains compared to controls. Protein lysates from EOSPD strains in the iPSC stage did not show an increase in α-synuclein, indicating that the accumulation was specific to differentiated cultures. Taken together, these data demonstrate a transcription-independent accumulation phenotype of α-synuclein protein in mDA cultures derived from EOSPD patients.

[0074] Example 13 Lysosomal proteins are abnormally regulated in EOSPD mDA cultures. Next, the inventors attempted to determine what factors might contribute to this increase in α-synuclein through both RNA sequencing and proteomics on paired sample sets derived from the same culture well. Whole transcriptome RNA sequencing (RNA-Seq) detected 19,004 unique transcripts between EOSPD cultures and control mDA cultures, and data-independent acquired mass spectrometry (SWATH) proteomics analysis identified 2,478 unique proteins. Independent unsupervised principal component analysis (PCA) of both transcriptome and proteomics data revealed a clear demarcation between EOSPD cells and controls along principal component 1 (PC1).

[0075] Considering the similarities between transcriptome signatures and protein signatures, we compared two datasets along PC1 to identify both identical and non-identical cellular pathways that may contribute to α-synuclein accumulation in EOSPD mDA cultures. To enable direct comparison of pathways, PCA was repeated for 2440 genes and corresponding proteins present in both datasets. PC1-ranked genes and proteins from this identical list were analyzed in separate gene set enrichment analysis (GSEA) and compared by term significance. Performing GSEA on the entire RNA-Seq dataset yielded similar significant terms. α-synuclein, as well as other synaptic vesicle genes associated with dopamine release, such as synapsin (SYP), synaptic vesicle 2A (SV2A), and SNAP25, were included in the Go Presynapse terms, which were significantly upregulated in both RNA and protein. Metabolic genes included in KEGG oxidative phosphorylation were also significantly upregulated in the EOSPD strain, in both mRNA and protein. Protein data showed that terms associated with neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease were significantly upregulated in the EOSPD strain, suggesting that general aspects of neurodegeneration were captured.

[0076] Both the transcript and protein observed in the Go Endoplasmic Reticulum Lumen term were significantly downregulated, suggesting a deficiency in genes related to protein synthesis. Interestingly, lysosomal proteins observed in the Go Lysosomal Lumen term were significantly downregulated in protein form, but not in mRNA data. To confirm that the abnormally regulated pathway observed in PC1 was specific to the EOSPD strain, a separate differential expression analysis between the control and EOSPD strains was performed using proteomics data and input into a STRING pathway analysis. Here again, lysosomal proteins were found to be significantly reduced in the EOSPD strain. These data indicate that while normal transcription of the lysosomal mechanism was present in the EOSPD mDA culture, the amount of protein obtained was lower compared to the control mDA culture.

[0077] The reduction in lysosomal proteins in EOSPD mDA cultures suggested that α-synuclein accumulation may be a consequence of impaired degradation. To test the overall degradation rate, the entire transcriptional function was inhibited for 48 hours via cycloheximide treatment in the culture, and the protein was assessed and quantified over time. During the 48-hour treatment, α-synuclein was degraded in the 02i control strain, with an observed half-life of approximately 10 hours, slightly longer than the half-life previously reported in PC12 cells. In stark contrast, α-synuclein accumulated in the most severely affected EOSPD strain (190iPD) during cycloheximide treatment. However, for other dopamine neuron-related proteins such as TH and synaptophysin, affected and control strains showed similar proteolytic rates, suggesting that the degradation deficiency is specific to α-synuclein and that proteolytic degradation may be mainly divided into the proteasomal degradation pathway and the autophagy / lysosomal degradation pathway. To determine whether proteasomal degradation is the cause of α-synuclein protein degradation, mDA cultures were treated with the proteasome inhibitor MG132 for 24 hours. MG132 treatment resulted in the accumulation of P53, a protein canonically degraded via the proteasome. However, there was no significant increase in α-synuclein in either the control culture or the EOSPD culture, indicating that α-synuclein degradation was not mediated through the proteasome in this context. To determine the potential involvement of lysosomes in α-synuclein degradation,36,37, we then investigated lysosome-associated membrane protein 1 (LAMP1). A significant decrease in LAMP1 levels was detected in all three EOSPD strains, which was consistent with proteomic analysis. GCase is a lysosomal hydrolase that has been reported to have reduced activity in the peripheral blood of some PD patients. When relative GCase function was quantified using 1 mM 4-methylumbelliferyl β-glucophilanoside, the inventors found a significant decrease in activity in EOSPD mDA cultures compared to the control.In previous studies using iPSC lines from sporadic patients with non-early onset EOSPD, decreased GCase activity in differentiated neuronal cultures was attributed to increased oxidized dopamine at later culture time points (over 60 days).42 In contrast, at 30 days of culture, we observed no increase in oxidized dopamine. However, with longer growth periods (60 days), oxidized dopamine began to accumulate in EOSPD cultures. These results suggest that in this model, increased α-synuclein and lysosomal deficiency precede oxidized dopamine accumulation. This provides further evidence of dysfunctional lysosomal degradation as a putative cause of α-synuclein accumulation in EOSPD mDA cultures.

[0078] Example 14 PEP005 adjusts the EOSPD phenotype. The inventors then tested the potential for reducing α-synuclein levels through activation of lysosome-specific pathways using three lysosomal agonists.43 The compounds selected were PEP005, a PKC agonist and structural analogue of the HEP14 drug; SMER28, a small molecule autophagy promoter shown to reduce huntingtin and α-synuclein aggregates in the PC12 cell model; and trehalose, another compound shown to promote α-synuclein clearance in PC12 cells.46 mDA cultures from control strain 02i and EOSPD strain 190iPD were treated with each agonist for 3 days, starting at day 27 of differentiation. Interestingly, treatment with PEP005 and SMER28, rather than trehalose, reduced the amount of α-synuclein protein in the control cultures. In PD mDA cultures, PEP005 and trehalose reduced α-synuclein levels, but SMER28 did not. However, only the PKC agonist PEP005 reduced α-synuclein levels in both PD and control EOSPD mDA cultures. A surprising further finding was that PEP005 treatment resulted in an increase in the amount of TH enzyme present in both control and EOSPD mDA cultures. Immunostaining confirmed that individual mDA cultures treated with PEP005 showed enhanced TH and decreased α-synuclein. Due to quantification difficulties caused by high-density immunostained mDA cultures, flow cytometry was used instead to quantify TH. Cultures treated with PEP005 did indeed contain neurons with significantly higher levels of TH expression, but PEP005 did not result in an increase in the number of TH neurons. These data indicate that PEP005 increased TH expression levels in neurons already producing the enzyme, while simultaneously decreasing abnormally high levels of α-synuclein expression.

[0079] PEP005 has well-established activity against both PKC alpha (PKCα) and delta (PKCδ). Although the two isoforms are antagonistic to each other, the literature has reported that treatment with PEP005 produces a short burst of PKCδ phosphorylation followed by a more long-term, potent decrease in phosphorylated PKCδ. Surprisingly, we found that on day 30, the basal level of phosphorylated PKC-α (p-PKCα) was higher in 190iPD mDA cultures compared to controls, and that PEP005 treatment completely eliminated this signal in both control and EOSPD cultures. By evaluating all other strains used in this study, we confirmed that p-PKCα levels were higher in mDA cultures from all three EOSPD strains compared to the control strain, although the difference in total PKCα was not significant. Undifferentiated iPSCs did not show elevated p-PKCα levels, and no clear pattern was observed in the peripheral blood of individual patients (data omitted), demonstrating specificity for differentiated mDA cultures. In addition, this elevated p-PKCα was removed by adding 1 μM PEP005 to mDA cultures from all iPSC strains for 3 days. This removal was accompanied by a decrease in α-synuclein levels and an increase in LAMP1.

[0080] To further evaluate the response to PEP005, the time course of treatment in control and EOSPD mDA cultures was assessed, showing that both p-PKCα and α-synuclein were degraded in response to drug treatment within approximately 24 hours. Over the same time course, cleaved caspase 3 (CC3) was also significantly reduced in EOSPD mDA cultures, indicating no toxic response to the drug. Gene expression data from paired samples showed that SNCA expression was downregulated 4 hours after PEP005 treatment, and TH was upregulated approximately 8 hours after initial exposure, suggesting an antagonistic relationship between the two proteins.48–50

[0081] Example 15 Confirmation of EOSPD phenotype in additional patients The inventors then wished to confirm these findings in a wider range of controls and EOSPD patients. They derived two additional EOSPD strains (172iPD, 192iPD) and three control strains (0771i control, 1034i control, 1185i control) from the Lothian birth cohort, a group of individuals who reached 83 years of age without signs of neurodegeneration or cognitive decline.51 The inventors also evaluated a typical-onset PD strain (78iPD, symptom onset at 67 years of age and family history of PD) to determine whether these phenotypes could be identified across the entire PD population. iPSCs from all new strains were differentiated into mDA cultures and examined for both α-synuclein accumulation and p-PKCα increase at day 30. Supporting our previous findings, cells from the Lothian control did not accumulate α-synuclein or show an increase in p-PKCα, while cells from one of the two new EOSPD strains (172iPD) exhibited both phenotypes. Interestingly, mDA cultures from the conventional PD strain and mDA cultures from one of the new EOSPD strains showed neither α-synuclein accumulation nor an increase in p-PKCα. To compare Western blotting data from all strains, expression values ​​in each blot were normalized to the 02i control. While mDA cultures from control individuals revealed similar α-synuclein and p-PKCα expression, most EOSPD patients fell into distinct clusters. To determine the potential of detecting α-synuclein and p-PKCα expression as a predictive tool for EOSPD, we plotted normalized expression values ​​on receiver operating characteristic curves (ROCs). The predictive accuracy for distinguishing EOSPD from the control, as reflected in the area under the curve, was 0.84 for α-synuclein (red), 0.93 for LAMP1 (blue), and 0.96 for p-PKCα (orange), demonstrating that these phenotypic markers accurately differentiate patients in the inventor's cohort.

[0082] Example 16 Additional phorbol esters alter α-synuclein and TH levels in mDA cultures. To further investigate the mechanism of interaction between PEP005 and α-synuclein, the inventors tested two additional PKC agonists having similar chemical structures to PEP005: phorbol 13-12-myristate acetate (PMA) and prostratin (PRO). Both PMA and PRO, additional phorbol ester compounds, exhibited similar activity to PEP005, with treatment of mDA cultures resulting in a decrease in p-PKCα and α-synuclein and a corresponding increase in TH.

[0083] Example 17 PEP005 regulates α-synuclein independently of p-PKCα. To determine the optimal potency, the inventors conducted a series of dose-response studies examining the expression of p-PKCα and α-synuclein in response to both PEP005 and PRO. Evaluation of mDA cultures treated with PEP005 revealed a clear dose-response relationship for p-PKCα, the direct target of the drug. However, a robust decrease in α-synuclein was observed at low doses of PEP005, which did not alter p-PKCα levels. This suggests that the mode of action of PEP005 in reducing α-synuclein may be independent of p-PKCα. PRO reduced both p-PKCα and α-synuclein in a dose-dependent manner, but was less efficient than PEP005 in reducing α-synuclein levels, suggesting a lower affinity for interaction with the α-synuclein regulatory pathway.

[0084] Considering the differences in the responses of α-synuclein and p-PKCα to PEP005 at low doses, and the fact that further digging had a similar effect to PEP005 treatment, we performed in silico modeling of potential PEP005 binding sites to identify novel drug binding partners. As expected, we identified several similar affinity target sites for PEP005 to bind to PKCα, but interestingly, we also found binding sites on several additional proteins. One of the more interesting findings based on the 3D model was binding to the GTPase RAS with similar affinity to PKCα. The RAS directly influences cell proliferation via the mTORC pathway (MAPK pathway) and lysosome biosynthesis.

[0085] Example 18 PEP005 lowers alpha-synuclein levels in vivo. Finally, we wanted to evaluate whether PEP005 could also reduce α-synuclein levels in vivo. To do this, we injected PEP005 (2.15 ng, 21.5 ng, or 215 ng) into one side of the striatum of adult wild-type C57BL / 6 mice. Both Western blotting and immunohistochemical evaluation of the mouse striatum three days post-treatment showed that the 215 ng dose of PEP005 significantly reduced α-synuclein levels compared to the other side. These results demonstrate that PEP005 activity reduces α-synuclein levels in vivo.

[0086] Example 19 Consideration The causes of Parkinson's disease (PD) have been proposed to be either genetic, environmental, or some combination of both, and in almost all cases, abnormal accumulation of alpha-synuclein is involved. Focusing on early-onset sporadic patients (3–10% of the US PD population), we hereby identified a reliable molecular signature in iPSC-derived mDA cultures from 4 out of 5 patients in this study. Since iPSC conversion clears most epigenic memory, this molecular signature implies the existence of a strong genetic contribution to EOSPD, suggesting the involvement of a number of previously unknown pathogenic and modifying genes. Alpha-synuclein accumulation was not observed in adult-onset patients in the current study, nor in previous adult-onset sporadic PD studies. The inability of adult-onset PD cells to reproduce the EOSPD phenotype suggests that either further culture time is required to manifest these in vitro phenotypes, or alternatively, the accelerated nature of EOSPD provides a different subpopulation of PD cells with α-synuclein handling defects that are readily reproducible in vitro.

[0087] A large-scale genome-wide association study (GWAS) of sporadic patients identified allele variants associated with protein degradation and linked the lysosomal degradation pathway to disease pathogenesis. Our results support this study and many previous studies that point to lysosomal dysfunction as a major cause of PD. Although EOSPD patients did not carry lysosomal risk variants or show altered lysosomal gene expression at the transcriptional level, lysosomal proteins were significantly downregulated. The combination of downregulation of protein processing-related pathways, such as those observed in the endoplasmic reticulum lumen, and downregulation of lysosomal proteins suggests that lysosomal protein biosynthesis and / or stability may contribute to the accumulation of EOSPD-specific α-synuclein. By directly stimulating the lysosomal pathway, we were able to induce a decrease in intracellular α-synuclein protein. However, treatment with phorbol ester compounds not only induced a decrease in α-synuclein levels but also resulted in an increase in TH levels present in mDA cultures from both control and PD origins. Other studies have suggested an antagonistic relationship between α-synuclein and TH expression, but since the increase in TH was specific to PEP treatment, the interaction may be more complex than a simple direct correlation. Control mDA cultures treated with SMER-28 small molecule and PD mDA cultures treated with trehalose showed a decrease in α-synuclein protein levels, but unlike phorbol ester, neither decrease resulted in an increase in TH enzyme. These differing results suggest that the two pathways may converge around a common target but are otherwise independent.

[0088] The dual effects observed here—a decrease in intracellular α-synuclein levels and an increase in TH—make PEP005 a very attractive candidate as a potential therapeutic agent. PEP005 is an FDA-approved topical treatment for actinic keratosis that also possesses anti-leukemic activity and may play a role in the reactivation of latent HIV. In this study, we selected it as a structural analog derived from the same plant as the HEP14 compound, which acts as a TFEB agonist independently of the mTORC pathway. In control and PD cells treated with PEP005, we observed an increase in the lysosomal protein LAMP1, consistent with the activation of the lysosomal master regulator TFEB. Network analysis of proteomics data also suggested that TFEB is central to the downregulation of the lysosomal pathway in EOSPD mDA cultures. However, the rapid decrease in α-synuclein after PEP005 treatment both in vitro and in vivo may suggest that the transcription of the new lysosomal protein is not initially responsible for the immediate decrease in α-synuclein.

[0089] Investigation of the mechanism of action of the small molecule PEP005 revealed an elevation of p-PKCα levels specific to EOSPD mDA cultures. This is a novel signal in the PD literature, although it is associated with certain cancers, and the inventors pursued this signal as a biomarker and possible cause of α-synuclein accumulation observed in EOSPD mDA cultures. All three PKC agonists (three phorbol esters) tested by the inventors resulted in a decrease in p-PKCα, accompanied by a decrease in α-synuclein and an increase in TH levels. In addition, dose-response studies showed that PEP005 can alter α-synuclein levels without substantially affecting p-PKCα. These results suggest that some structure-specific interaction of the phorbol ester compounds drives the decrease in α-synuclein but not the decrease in p-PKCα.

[0090] The intrinsic patient specificity of this model presents opportunities for EOSPD diagnosis and treatment development. More patient strains are needed to fully evaluate the predictive accuracy of this approach to support EOSPD diagnosis. While the model accurately distinguished most EOSPD patients, one patient was not correctly identified. This in vitro phenotypic absence was consistent with a unique clinical feature: non-tremor-dominant symptoms. The diverse clinical features of this patient and the in vitro results suggest an alternative etiology or a unique set of modifying genes that may alter the timing or acquisition of EOSPD biomarkers.

[0091] This study is the first to identify the molecular signature of sporadic Parkinson's disease in iPSCs from early-onset patients. These cells accumulate α-synuclein, exhibit abnormal regulation of lysosomal biosynthesis and function, and also show increased p-PKCα, thus providing a biomarker that may allow prediction of whether young patients presenting motor symptoms have EOSPD, and thus a novel diagnostic tool for clinicians. Furthermore, this system presents a platform for screening novel therapeutic agents that may affect the underlying mechanisms of EOSPD. For example, the inventors have already identified a set of novel drugs that target this signature and reduce intracellular α-synuclein in both control and PD cells. These phorbol ester drugs, particularly PEP005, may treat the underlying causes of EOSPD and may reveal principles common to other neurodegenerative diseases.

[0092] Example 20 Treatment and administration The inventors note that in vitro plasma binding studies in multiple animal models (rats, rabbits, miniature pigs, and humans) demonstrate highly efficient plasma binding of PEP005 and additional phorbol compounds. This could complicate IV delivery.

[0093] In multiple IV bolus administration studies, the following was observed: Respiratory effects in rats - No biologically relevant responses were observed at doses up to 10 ug / kg. Cardiovascular effects in miniature pigs - Dose up to 5 ug / kg (the highest dose tested) did not produce adverse clinical or behavioral effects, nor did it produce significant cardiovascular changes.

[0094] Dose determination studies in rats and miniature pigs: Repeated administration for 7 days was tolerated at 15 ug / kg / day in rats and 5 ug / kg in pigs.

[0095] Repeated dose toxicity study in rats: Animals were administered up to 10 ug / kg / day by IV tail vein injection for 7 days. All doses were well tolerated. Repeated dose toxicity study in miniature pigs: Animals were administered up to 5 ug / kg / day by IV approximately once a week. Treatment with the 5 ug / kg dose resulted in decreased food intake the following day, but intake subsequently recovered.

[0096] A study to determine the PK profile of PEP in mice was conducted, administering 10 and 50 ug / kg via IV tail vein injection. One hour after the test, serum concentrations fell below the detection limit, indicating very high clearance (above the blood flow through the liver). This may also pose a problem for IV delivery.

[0097] Based on the above results, the inventors decided to directly test tail vein IV administration in the dose range of 10 ug / kg to determine whether it can produce a measurable effect on cerebral synuclein levels. However, given the high binding affinity and very fast clearance / metabolism rate, IV administration is unlikely to be an efficient delivery route. Another delivery route that the inventors may consider is direct injection into the brain.

[0098] The inventors needed to know how long the effects of a single dose of PEP lasted in order to determine the drug administration plan for future animal studies. This study demonstrates the duration of action of a 24-hour single dose of PEP005. Synuclein levels remained reduced for 168 hours, and pPKCa levels were still reduced even 240 hours after administration. Therefore, the inventors are considering administering the drug to animals approximately once a week.

[0099] The various methods and techniques described above provide numerous means of carrying out the present invention. Naturally, it should be understood that not all of the objectives or benefits described are necessarily achieved according to any particular embodiment described herein. Therefore, those skilled in the art will recognize that, for example, a method may be implemented in a manner that achieves or optimizes one benefit or group of benefits taught herein without necessarily achieving other objectives or benefits, as may be taught or suggested herein. Various advantageous and disadvantageous alternatives are described herein. It should be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while yet others specifically mitigate these disadvantageous features by including one, another, or several advantageous features.

[0100] Furthermore, those skilled in the art will recognize the applicability of various features of different embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents for each such element, feature, or step, can be combined and adapted by those skilled in the art to carry out the method according to the principles described herein. In various embodiments, some of the various elements, features, and steps will be specifically included, while others will be specifically excluded.

[0101] Although the present invention is disclosed in the context of specific embodiments and examples, it will be understood by those skilled in the art that embodiments of the present invention extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as modifications and equivalents thereof.

[0102] Many variations and alternatives are disclosed in embodiments of the present invention. Further variations and alternatives will be apparent to those skilled in the art. These variations include, but are not limited to, compositions and methods related to induced pluripotent stem cells (iPSCs), differentiated iPSCs including midbrain neurons, floor plate neurons, and dopaminergic neurons, techniques and compositions and the use of solutions used therein, and specific uses of products produced by teaching the present invention. Various embodiments of the present invention may specifically include or exclude any of these variations or elements.

[0103] In some embodiments, numerical values ​​representing properties such as the amount, concentration, and reaction conditions of components used to describe and claim specific embodiments of the present invention should be understood, in some cases, to be modified with the term "approximately." Therefore, in some embodiments, numerical parameters described in the written description and appended claims are approximations that may vary depending on the desired properties to be obtained by the particular embodiment. In some embodiments, numerical parameters should be interpreted by taking into account the number of significant digits reported and by applying common rounding techniques. While numerical ranges and parameters described for a broad range of some embodiments of the present invention are approximations, numerical values ​​described in specific embodiments are reported as accurately as possible. Numerical values ​​presented in some embodiments of the present invention may include certain errors that inevitably arise from the standard deviation found in each test measurement.

[0104] In some embodiments, the terms “a,” “an,” and “the” and similar references used in the context describing a particular embodiment of the Invention (in particular, in the specific context of the following claims) may be interpreted as encompassing both singular and plural forms. The enumeration of value ranges herein is intended simply as a simple way to refer individually to each separate value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein may be carried out in any preferred order unless otherwise indicated herein or unless otherwise clearly contradicted by other contexts. The use of any and all examples provided herein with respect to a particular embodiment, or exemplary language (e.g., “etc.”), is intended to better represent the Invention and does not limit the scope of the Invention as otherwise claimed. The language herein should not be interpreted as indicating any unclaimed element essential to the practice of the Invention.

[0105] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group member may be claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or removal, this Specified Publication shall be deemed to include the thus modified group herein and shall satisfy the written description of all Markush groups used in the appended claims.

[0106] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments will become apparent to those skilled in the art by reading the above description. It is intended that those skilled in the art may use such variations as needed, and the Invention can be carried out in ways other than those specifically described herein. Accordingly, many embodiments of the Invention include all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is incorporated into the Invention unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0107] Furthermore, numerous references are made throughout this specification to patents and printed publications. Each of the references and printed publications cited above is incorporated herein by reference in whole.

[0108] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be used may be within the scope of the invention. Therefore, alternative configurations of the present invention can be used in accordance with the teachings herein, not as examples but as examples. Accordingly, embodiments of the present invention are not limited to those strictly shown and described.

Claims

1. A pharmaceutical composition for modulating the EOSPD phenotype in subjects suffering from early-onset sporadic Parkinson's disease (EOSPD) by lowering the activity of α-synuclein and upwardly regulating the activity of tyrosine hydroxylase (TH), wherein the therapeutically effective agent comprises a phorbol ester compound or ingenol-3-angerate.

2. The pharmaceutical composition according to claim 1, wherein the phorbol ester compound is phorbol 13-12-myristate acetate (PMA) or prostratin (PRO).

3. The pharmaceutical composition according to claim 1, wherein the therapeutically effective agent lowers the expression of α-synuclein and upwardly regulates the expression of tyrosine hydroxylase (TH).

4. The pharmaceutical composition according to claim 3, wherein the regulation of expression includes the regulation of the transcript expression level.

5. The pharmaceutical composition according to claim 3, wherein the regulation of expression includes the regulation of the protein expression level.

6. The pharmaceutical composition according to claim 5, wherein the regulation of protein expression levels includes a decrease in α-synuclein protein and an increase in TH protein.

7. A pharmaceutical composition for modulating the phenotype of early-onset sporadic Parkinson's disease (EOSPD) in subjects suffering from EOSPD, wherein the therapeutically effective agent comprises a phorbol ester compound or ingenol-3-angerate, wherein the therapeutically effective agent comprises a phorbol ester compound or ingenol-3-angerate.

8. The pharmaceutical composition according to claim 7, wherein the phorbol ester compound is phorbol 13-12-myristate acetate (PMA) or prostratin (PRO).

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