Methods and compounds modifying mitochondrial function

Abstract

To provide a method for screening candidate agents for activity to reduce the MIRO1 level.SOLUTION: A method for screening is provided, the method comprising: (a) contacting a candidate agent with a sample obtained from a subject who is deficient in, or suspected of being deficient in, removal of MIRO1; (b) measuring an MIRO1 level in the sample; (c) comparing the MIRO1 level in the sample with a control MIRO1 level; and (d) assessing an activity of the candidate agent when the MIRO1 level in the sample is lower than the control MIRO1 level.SELECTED DRAWING: Figure 4

Description

[Background technology]

[0001] Neurons are metabolically active cells with high energy demands away from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that mitochondrial dysfunction diseases often have a neurodegenerative component. Recent findings highlight the particular dependence of neurons on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria.

[0002] Mitochondria participate in repeated cycles of fusion and fission, which serve to mix lipids and contents within the mitochondrial mass. Additionally, mitochondria are actively recruited to subcellular sites, such as neuronal axons and dendrites. Finally, the quality of the mitochondrial mass is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. Deficiencies in key aspects of mitochondrial dynamics, such as mitochondrial fusion, fission, transport, and mitophagy, are associated with neurodegenerative disorders. Several major neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and Huntington's disease, involve disruption of mitochondrial dynamics.

[0003] Mitochondrial movement is tightly regulated to maintain energy homeostasis and prevent oxidative stress. Mitochondrial movement ceases before the initiation of mitophagy, a key cellular mechanism by which depolarized mitochondria are degraded via autophagosomes and lysosomes. Cessation of movement can sequester damaged mitochondria, immobilizing them and preventing the reintroduction of damage to other healthy mitochondria.

[0004] Miro is a mitochondrial outer membrane (OMM) protein that anchors the microtubule motors kinesin and dynein to mitochondria (Glater EE, Megeath LJ, Stowers RS, Schwarz TL. Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent.The Journal of cell biology.2006;173:545-557, Koutsopoulos OS, Laine D, Osellame L, Chudakov DM, Parton RG, Frazier AE, Ryan MT.Human Miltons associate with mitochondria and induce microtubule-remodeling of mitochondrial networks.Biochimica et biophysica acta.2010;1803:564-574). This depolarization-induced mitochondrial arrest is achieved by removing Miro from the damaged mitochondrial surface (Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J, LaVoie MJ, Schwarz TL. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell. 2011;147:893-906). Miro is then degraded by the proteasome (Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D). D,Rice S,Steen J,LaVoie MJ,Schwarz TL.PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility.Cell.2011;147:893-906). Two PD-associated proteins, PINK1 (PTEN-induced putative kinase 1) and Parkin, have been shown to act in concert to target Miro for degradation. (Ashrafi G, Schlehe JS, LaVoie MJ, Schwarz TL. Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin. The Journal of cell biology. 2014;206:655-670. Liu S, Sawada T, Lee S, Yu W, Silverio G, Alapatt P, Millan I, Shen A, Saxton W, Kanao T, et al. Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria. PLoS Genetics. 2012;8:e1002537. Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J, LaVoie MJ, Schwarz TL. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell. 2011;147:893-906). Mutations in PINK1 or Parkin are associated with a rare form of recessive early-onset PD.

[0005] Altered mitochondrial transport is one of the pathogenic changes in major adult-onset neurodegenerative diseases. In mutant LRRK2GS2019 cells, the mitochondrial outer membrane protein Miro is stabilized and remains on damaged mitochondria longer than normal, prolonging active transport and inhibiting mitochondrial degradation (Hsieh et al., 2016). In sporadic PD subjects, Miro degradation and mitochondrial movement are also impaired. Prolonged retention of Miro and its subsequent downstream consequences may constitute a central component of PD pathogenesis.

[0006] Thus, there is a need for novel therapeutic approaches and methods to identify regulators of Miro retention and degradation that will promote the arrest of mitochondrial movement in damaged mitochondria and the restoration of mitochondrial dynamics in neurodegenerative diseases. Summary of the Invention

[0007] Methods and compositions are provided for monitoring, treating, and screening for neurodegenerative disorders, such as Parkinson's disease, in a subject. An embodiment of the method includes administering a MIRO1-reducing agent to a subject with Parkinson's disease. A companion diagnostic assay is also provided for determining whether a subject is suitable for treatment with the MIRO1-reducing agent and treating the subject accordingly.

[0008] In one aspect, a method for determining MIRO1 status in a subject is provided, comprising measuring MIRO1 response to mitochondrial depolarization using a biochemical assay, Western blotting, or ELISA to determine whether the subject is deficient in MIRO1 removal after depolarization, wherein subjects deficient in MIRO1 removal after depolarization are selected for treatment by administering a MIRO1-reducing agent. Determining MIRO1 status can include detecting MIRO1 levels in the subject and comparing the MIRO1 levels to control MIRO1 levels in a control subject. In some embodiments, detecting MIRO1 levels includes detecting MIRO1 in a tissue sample, such as skin fibroblasts, from the subject. In some embodiments, the method comprises an assay described in any one of the Examples.

[0009] Provided herein is a method for screening candidate agents for activity in reducing MIRO1 levels, the method comprising: (a) contacting the candidate agent with a sample from a subject in whom MIRO1 removal is deficient or suspected of being deficient; (b) measuring the MIRO1 level in the sample; (c) comparing the MIRO1 level in the sample with a control MIRO1 level; and (d) evaluating the activity of the candidate agent when the MIRO1 level in the sample is lower than the control MIRO1 level.

[0010] Provided herein is a method for treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein the Miro-reducing agent reduces Miro in sporadic Parkinson's disease cells having depolarized mitochondria by three standard deviations or more compared to the reduction in Miro in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria that have not been contacted with the Miro-reducing agent. The healthy cells can be healthy cells having depolarized mitochondria that have been contacted with a mitochondrial depolarizing agent.

[0011] Provided herein is a method for treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein the Miro-reducing agent reduces Miro in Parkinson's disease cells having depolarized mitochondria by more than three standard deviations compared to the reduction in Miro in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria that have not been contacted with the Miro-reducing agent.

[0012] Provided herein are methods for treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein (a) the Miro-reducing agent reduces Miro in Parkinson's disease cells having depolarized mitochondria by more than two standard deviations compared to the reduction in Miro in control Parkinson's disease cells having depolarized mitochondria that have not been contacted with the Miro-reducing agent, and (b) the Miro1-reducing agent reduces Miro in Parkinson's disease cells having non-depolarized mitochondria by less than one standard deviation compared to the reduction in Miro in control Parkinson's disease cells having non-depolarized mitochondria that have not been contacted with the Miro-reducing agent.

[0013] The Parkinson's disease cell can be a Parkinson's disease cell having depolarized mitochondria that have been contacted with a mitochondrial depolarizing agent. The Parkinson's disease cell can be a sporadic Parkinson's disease cell. The Parkinson's disease cell can be a fibroblast.

[0014] 1. A method for treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein the Miro-reducing agent is selected from the following: (a) plating fibroblasts from a sporadic Parkinson's disease patient into wells of an array; (b) 24 hours after step (a), adding a candidate agent to the test wells and not adding the candidate agent to the control wells; (c) 10 hours after step (b), adding FCCP to the test wells and the control wells; and (d) 14 hours after step (c), culturing the cells in the test wells and the control wells in ice-cold 9 ml of fibroblasts. (e) immunostaining the cells in the test wells and the control wells with anti-Miro and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), imaging by confocal microscopy, and measuring Miro intensity / cell for these images of the test wells and the control wells; and (f) measuring the Miro intensity / cell for these images of the test wells and the control wells. Provided herein are methods for reducing Miro in an assay where the candidate agent reduces Miro in the test wells by 3 standard deviations or more compared to the control wells, the candidate agent is a Miro reducer.

[0015] 1. A method of treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein the Miro-reducing agent is administered by the following steps: (a) plating fibroblasts from a sporadic Parkinson's disease patient into wells of an array; (b) 24 hours after step (a), adding a candidate agent to a first test well and not adding the candidate agent to a first control well; and (c) performing step (d) 14 hours after step (c), the cells in the first test well and the first control well are fixed with ice-cold 90% methanol; (e) the cells in the first test well and the first control well are immunostained with anti-Miro and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), imaged by confocal microscopy, and the Miro intensity / cell is measured for these images of the first test well and the first control well. According to the assay, Miro is reduced in sporadic Parkinson's disease cells, and the Miro-reducing agent is determined to be: (a1) fibroblasts from a sporadic Parkinson's disease patient are plated into wells of the array; (b1) 24 hours after step (a1), a candidate agent is added to the second test well and the second control well; (c1) 14 hours after step (b1), the cells in the second test well and the second control well are fixed with ice-cold 90% methanol; and (d1) immunostaining the cells in the second test well and the second control well with anti-Miro and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), imaging by confocal microscopy, and measuring Miro intensity / cell for these images of the second test well and the second control well, wherein the candidate agent does not reduce Miro in the sporadic Parkinson's disease cells according to an assay, wherein the candidate agent is a Miro reducer if the candidate agent reduces Miro in the first test well by more than two standard deviations compared to the first control well, and the candidate agent reduces Miro in the second test well by less than one standard deviation compared to the second control well.

[0016] In certain embodiments, the neurodegenerative disorder is Parkinson's disease (PD). In some such embodiments, PD is a familial form of PD, such as PTEN-induced putative kinase 1 (PINK-1)-associated PD, Parkin-associated PD, LRRK2-associated PD, alpha-synuclein (SNCA)-associated PD, E3 ligase (parkin)-associated PD, GBA-associated PD, ubiquitin carboxy-terminal hydrolase L1 (UCHL1)-associated PD, Parkinson protein 7 (PARK7, DJ-1)-associated PD, ATP13A2-associated PD, group VI phospholipase A2 (PLA2G6)-associated PD, or the like. PD, DnaJ (Hsp40) homolog, subfamily C, member 6 (DNAJC6, PARK19)-associated PD, eukaryotic translation initiation factor 4 gamma, 1 (EIF4G1, PARK18)-associated PD, F-box protein 7 (FBXO7)-associated PD, GRB10-interacting GYF protein 2 (GIGYF2)-associated PD, HtrA serine peptidase 2 (HTRA2)-associated PD, synaptojanin 1 (SYNJ1)-associated PD, and vacuolar protein sorting 35 homolog (VPS35)-associated PD. In other such embodiments, the PD is a sporadic form of Parkinson's disease, for example, associated with a sporadic mutation in one of the aforementioned genes. In certain such embodiments, the MIRO1-reducing agent is administered to the midbrain and / or putamen of the subject.

[0017] In some embodiments, an assay for determining a subject's MIRO1 status is provided. In some embodiments, the assay is performed on a population of patient cells, advantageously using fibroblasts, to identify the subject's MIRO1 phenotype. Miro1 localizes to the mitochondrial surface and mediates mitochondrial movement. Miro1 is removed from depolarized mitochondria and promotes mitochondrial clearance via mitophagy. Measurement of the Miro1 response to mitochondrial depolarization using biochemical assays, ELISA, etc., indicates that a high percentage of Parkinson's disease subjects have defective MIRO1 removal following depolarization.

[0018] As described in the Examples herein, MIRO1 status correlates with the presence of certain neurodegenerative disorders, for example, elevated levels of MIRO1 are associated with Parkinson's disease.

[0019] Miro-reducing agents can reduce intracellular calcium levels by 20% or more. The agent may have a molecular weight of 100 to 2000 daltons. The Miro-reducing agent may be an antibody, peptide, or protein, or a portion thereof. The Miro-reducing agent may bind to an EF-hand protein. The Miro-reducing agent may be a calcium channel blocker. The calcium channel blocker may be an L-type or N-type calcium channel blocker. The subject may have elevated Miro. The subject may be otherwise asymptomatic for the neurodegenerative disorder. The neurodegenerative disorder may be Parkinson's disease.

[0020]

[0003] Provided herein is a method for selecting a subject for treatment with a therapeutic agent for a neurodegenerative disorder, comprising: (a) collecting cells from the subject and evaluating a first control portion of the cells for pre-depolarization Miro levels within the cells; (b) contacting a second test portion of the cells with a depolarizing agent; and (c) evaluating a post-depolarization Miro level in the second test portion of the cells contacted with the depolarizing agent and comparing the Miro level to the pre-depolarization Miro level in the first control portion of the cells; wherein the subject is treated with the therapeutic agent for a neurodegenerative disorder if the post-depolarization Miro level in the second test portion of the cells is reduced by 40% or less compared to the pre-depolarization Miro level in the first control portion of the cells.

[0004] Provided herein is a method for selecting a subject for treatment with a therapeutic agent for a neurodegenerative disorder, wherein the subject is treated with the therapeutic agent for a neurodegenerative disorder if the post-depolarization Miro level in the second test portion of the cells is reduced by 10% to 50% compared to the pre-depolarization Miro level in the first control portion of the cells.

[0021] The neurodegenerative disorder may be Parkinson's disease. The subject may be asymptomatic for Parkinson's disease. The therapeutic agent may be selected from levodopa and a dopamine antagonist. The therapeutic agent may be a Miro reducer. The therapeutic agent may have a molecular weight of 100 to 2000 daltons. The therapeutic agent may comprise an antibody, peptide, or protein, or a portion thereof. The therapeutic agent may bind to an EF-hand protein. The therapeutic agent may be a calcium channel blocker. The calcium channel blocker may be an L-type or N-type calcium channel blocker.

[0022] Provided herein is a method for improving aging conditions, comprising administering a Miro-reducing agent to a subject in need thereof.The aging conditions can be selected from memory impairment, muscle degeneration, arthritis, cardiovascular disease, osteoporosis, glaucoma, dementia, macular degeneration, and cataracts.The Miro-reducing agent can be administered prophylactically.

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0024] Before the present methods and compositions are described, it is to be understood that aspects of this invention are not limited to the particular methods or compositions described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0026] [Figure 1A] FIG. 1 shows Miro1 response to CCCP in fibroblasts, a schematic representation of our readout. [Figure 1B] 1 shows Miro1 response to CCCP in fibroblasts, with examples of readouts using Healthy-1, PD-2, and Risk-2. [Figure 1C] 1 shows Miro1 response to CCCP in fibroblasts, with examples of readouts using Healthy-1, PD-2, and Risk-2. [Figure 1D] Figure 1 shows the Miro1 response to CCCP in fibroblasts. The heatmap shows relative mitochondrial protein levels. The intensity of each band in the mitochondrial fraction was normalized to the intensity of the mitochondrial-loaded control VDAC from the same blot and expressed as a percentage of the average of DMSO-treated healthy-1. This control was included in all experiments. The average values ​​are imported into the heatmap. n = 3–35. [Figure 1E] Miro1 response to CCCP in fibroblasts is shown. ELISA for Miro1 is shown. n=4 for each duplicate. Comparisons within the same subject are shown. *: P<0.05, **: P<0.01, ***: P<0.001 throughout. [Figure 1F] Miro1 response to CCCP in fibroblasts is shown. ELISA for Miro1 is shown. n=4 for each duplicate. Comparisons within the same subject are shown. *: P<0.05, **: P<0.01, ***: P<0.001 throughout. [Figure 2] Miro1 ratio (CCCP / DMSO) for different variables: A: healthy subjects and Parkinson's disease subjects ("PD"), B: male and female subjects, C: age at sampling (years), D: age at PD onset (years), E: PD progression (years with PD), F: UPDRS, G: Hoehn and Yahr scale for PD subjects, H: Mini-Mental State Examination in PD subjects. [Figure 3]A shows the relative β-actin or Miro1 levels (ng / ml) in the subject, B shows the absorbance at 450 nm versus β-actin levels, C shows the relative β-actin levels versus capture antibody, D shows the absorbance at 450 nm versus Miro1 levels, E shows the relative Miro1 levels versus capture antibody, F shows the absorbance at 450 nm versus Miro1 (ng / ml), and G shows Miro1 (ng / ml) versus capture antibody. [Figure 4] A schematic diagram of a custom-designed in vitro screen for Miro1 drug discovery is shown. A) Fibroblast cultures obtained from both healthy subjects and subjects with sporadic PD are shown. B) Fibroblast transfer by seeding fibroblasts into 384-well plates is shown. C) The steps for adding a compound library to fibroblasts in 384-well plates are shown. D) Immunocytochemistry (ICC) performance for fibroblasts treated with the compound library is shown. E) The procedure for detecting Miro1 protein levels under a confocal microscope, as well as exemplary images of DAPI and Miro1 in one individual well, are shown. F) A custom data analysis algorithm pipeline designed to identify compounds that alter Miro1 levels after mitochondrial depolarization. DETAILED DESCRIPTION OF THE INVENTION

[0027] In one aspect, the disclosure herein provides a method for diagnosing a neurodegenerative disorder, e.g., Parkinson's disease, comprising detecting Miro, e.g., Miro1, in a subject. In certain embodiments, the Miro1 level in a subject, e.g., the Miro1 level in a tissue sample from a subject, is detected using an assay described herein. To determine the presence or absence of a neurodegenerative disorder, the Miro1 level in a subject can be compared with a control Miro1 level in a control subject. For example, a Miro1 level higher than the control Miro1 level, e.g., 20% or more, or 30% or more, can indicate the presence of Parkinson's disease. If the presence of a neurodegenerative disorder is indicated, the subject can be treated for the neurodegenerative disorder, such as with a treatment known in the art or described herein.

[0028] In one aspect, the disclosure herein provides a method for treating a neurodegenerative disorder, such as Parkinson's disease. Provided are methods of treatment, comprising administering a Miro-reducing agent, e.g., a Miro1-reducing agent, to a subject in need thereof. In certain embodiments, the Miro-reducing agent is identified using the assays described herein. In certain embodiments, the Miro-reducing agent reduces Miro, e.g., Miro1, levels in cells, e.g., cells from a subject with or suspected of being deficient in Miro1 removal. In certain embodiments, the Miro-reducing agent reduces Miro, e.g., Miro1, levels in cells to a level consistent with Miro, e.g., Miro1, levels in control cells.

[0029] In certain embodiments, the present disclosure provides a method for treating a subject with a neurodegenerative disorder, such as Parkinson's disease, wherein the subject is diagnosed with Parkinson's disease using the methods described herein. Methods for treating neurodegenerative disorders with Miro-reducing agents are described below.

[0030] In certain embodiments, the present disclosure provides methods for identifying Miro1 reducers. In certain embodiments, the present disclosure provides methods for screening candidate agents for use in treating neurodegenerative disorders, such as Parkinson's disease, using the assays described herein.

[0031] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0032] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within this stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may also be encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can also be used to carry out or test the present invention, preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in connection with which the publication is cited.

[0034] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely cures the disease and / or the deleterious effects caused by the disease. "Treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., halting its development, or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, where treatment stabilizes or reduces undesirable clinical symptoms in a subject, is of particular interest. Such treatment is best administered before complete loss of function in the affected tissue. Preferably, the subject treatments are administered during, and in some cases after, the symptomatic stage of the disease.

[0035] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0036] It should be noted that, as used in this specification and the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0037] The terms "candidate agent," "test agent," "agent," "substance," and "compound" are used interchangeably herein. Candidate agents encompass numerous chemical classes that are typically synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules. Candidate agents include those found in large libraries of synthetic or natural compounds.

[0038] Mitochondrial Rho (Miro) is a small GTPase known for its functions in mitochondrial transport and homeostasis. Miro is a highly conserved protein across unicellular and multicellular eukaryotes. Miro1 and Miro2 are conserved single-pass integral membrane proteins with cytosol-exposed N-terminal regions and consist of two GTPase domains separated by a pair of canonical EF-hands. Miro also contains a C-terminal hydrophobic domain that enables membrane insertion. Miro GTPase localizes to the outer mitochondrial membrane and plays an important role in intracellular mitochondrial trafficking in metazoans, particularly over long distances along microtubules in neurons. Miro is also known to mediate intercellular transport of mitochondria between cells via nanotube tunneling. Miro mediates bidirectional mitochondrial movement along microtubules by engaging both kinesin and dynein. Miro associates with microtubule tracks via the Milton / Trak family of cargo adaptors. Miro also transports mitochondria along actin tracks using the mitochondrial actin motor Myo19. Miro is also involved in peroxisome distribution through binding to peroxin 26. In addition, Miro also plays a role in mitochondrial fusion and fission dynamics through the regulation of the mitochondrial dynamin Drp1 and its interaction with the mitochondrial fusion proteins mitofusins ​​1 and 2.

[0039] Miro turnover is regulated by the PTEN-induced putative kinase 1 (PINK1) / Parkin pathway. PINK1 phosphorylates Miro, thus promoting its interaction with the E3 ubiquitin ligase, Parkin. Parkin promotes Miro ubiquitination and degradation, which effectively stops the axonal transport of damaged mitochondria. On the other hand, PINK1-phosphorylated Miro also recruits Parkin to damaged mitochondria, subsequently tagging them for mitophagy destruction.

[0040] Miro is located in the outer mitochondrial membrane and anchors the microtubule motors kinesin and dynein to mitochondria. When mitochondrial damage is caused by mitochondrial depolarization, mitophagy is initiated in cells. Mitochondrial movement is halted before the onset of mitophagy, the cellular mechanism by which depolarized mitochondria are degraded via autophagosomes and lysosomes. The halt in movement can sequester damaged mitochondria, immobilizing them and preventing the reintroduction of damage to other healthy mitochondria. This depolarization Polarization-induced mitochondrial arrest is achieved by the removal of Miro from the surface of damaged mitochondria and subsequent degradation by the proteasome. However, in skin fibroblasts from both familial and sporadic PD subjects, a significant impairment in Miro degradation and subsequent clearance of damaged mitochondria is observed.

[0041] The Miro EF hand mediates bidirectional mitochondrial transport of Ca 2+ Mitochondrial transport is mediated by binding of Miro to KIF5. 2+ The binding either dissociates KIF5 from mitochondria or "turns off" KIF5 binding to microtubules. 2+ Binding induces a direct interaction of the motor domain with Miro, thus preventing the motor from binding to MTs.

[0042] As used herein, "Miro" can refer to any one or all of family members, isoforms, homologs, paralogs, mutants, alleles, variants, derivatives, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide strands, etc. Miro refers to the human sequence Miro, such as the complete amino acid sequences of the human Miro isoforms having Uniprot identifiers Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. A human Miro sequence can differ from a human Miro isoform with Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7, for example, by having a conserved mutation or a mutation in a non-conserved region, and Miro has substantially the same biological function as a human Miro isoform with Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7, such as binding to mitochondria. Miro is also referred to as RHOT1, mitochondrial Rho GTPase 1. Miro includes isoforms of Miro, such as Miro1 and Miro2.

[0043] A particular Miro sequence may be at least 90%, at least 95%, or even at least 96%, at least 97%, at least 98%, or at least 99% identical in amino acid sequence to a Miro with Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. 。In certain embodiments, the human Miro sequence may differ by more than 10 amino acid differences from the Miro sequence of Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. In certain embodiments, the human Miro may show no more than 5 amino acid differences, or even no more than 4 amino acid differences, no more than 3 amino acid differences, no more than 2 amino acid differences, or no more than 1 amino acid differences from the Miro sequence of Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. Percent identity can be determined as described herein.

[0044] An "isoform" of a protein may be, for example, a protein resulting from alternative splicing of a gene that expresses the protein, or a degradation product of the protein. "Sequence homology" refers to the nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. As used herein, "sequence identity" or "identity" refers to the residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window, with respect to two nucleic acid or amino acid sequences.

[0045] As used herein, "percent sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions, i.e., gaps, compared to a reference sequence that does not contain additions or deletions that can be used for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleotide or amino acid occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions occurring in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity.

[0046] Sequence comparison, such as for purposes of assessing identity, can be performed by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / , optionally using default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally using default settings), and the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / , optionally using default settings). Optimal alignment can be assessed using any appropriate parameters of the selected algorithm, including default parameters.

[0047] The "percent identity" between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined by comparing sequence information using, for example, advanced BLAST computer programs, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and is as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program can define identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine percent identity over the entire length of the sequences being compared. Default parameters may be provided to optimize searches with short query sequences using, for example, the blastp program. Programs also include those described in Wootton and Federhen, Computers and This can enable the use of an SEG filter to mask off segments of the query sequence, as determined by the SEG program in Chemistry 17:149-163 (1993). High sequence identity can include sequence identity within the range of about 80% to 99% sequence identity and integer values ​​therebetween.

[0048] A "homolog" can refer to any sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to another sequence. In certain embodiments, a homolog has 70%-99%, 80%-99%, 85%-99%, 90%-99%, or even 95%-99% sequence identity. In some cases, a homolog is a naturally occurring or naturally occurring sequence. In some cases, a homolog may have functional or structural equivalence to a domain, motif, or portion of a protein encoded by a native or naturally occurring sequence.

[0049] Homology comparison can be performed using a sequence comparison program. The computer program can calculate the percent homology (%) between two or more sequences, or can calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homology can be generated by any of several computer programs, such as BLAST or FASTA. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Other examples of software capable of performing sequence comparisons include, but are not limited to, the BLAST package (Ausubel et al., 1999, supra, Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410), and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, ibid., pp. 7-58 to 7-60).

[0050] Homology percentage can be calculated over a continuous sequence, that is, one sequence is aligned with the other sequence, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time.This is called "gapless" alignment.Usually, this gapless alignment can be performed over a relatively small number of residues.

[0051] In otherwise identical sequence pairs, a single insertion or deletion can cause the following amino acid or nucleotide residue to fall out of alignment, thus resulting in a significant decrease in percent homology when the entire alignment is performed. Thus, sequence comparison methods can be designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology or identity score. This can be achieved by inserting "gaps" in the sequence alignment that attempt to maximize local homology or identity.

[0052] Calculation of maximum % homology can use optimal alignment, taking into account gap penalties. BLAST 2 Sequences is another tool that can be used to compare protein and nucleotide sequences (FEMS Microbiol Lett. 1999 174(2):247-50, FEMS Microbiol Lett. 1999 177(1):187-8 and National Institutes of National Center for Health website (See Biotechnology Information website).

[0053] Homologous sequences can also have deletions, insertions, or substitutions of amino acid residues that result in functionally equivalent substances. Deliberate amino acid substitutions can be made based on similarities in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues), and it is therefore useful to group amino acids together by functional group. Amino acids can also be grouped together based on the properties of their side chains alone. Sets of conserved amino acids can be described in the form of Venn diagrams (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of amino acids"). ysis of residue conservation”Comput.Appl.Biosci.9:745-756)(Taylor WR(1986)“The classification of amino acid conservation”J.Theor.Biol.119;205-218).

[0054] A "Miro reducer," e.g., a MIRO1 reducer, refers to any agent that reduces the level of Miro protein or its homologs in cells with depolarized mitochondria. In an exemplary embodiment, a Miro reducer can reduce at least one biological activity of Miro protein in cells with depolarized mitochondria. Representative biological activities of Miro include, among others, promotion of mitochondrial transport, mitophagy, microtubule binding, mitochondrial fission and fusion. A Miro reducer can be, for example, a small molecule, peptide, aptamer, protein, or functional fragment of a protein. As used herein, a functional fragment of a protein refers to all or a portion of a molecular component of a protein that affects a specific function, such as protein binding, signal transduction, etc.

[0055] In some embodiments, the MIRO1-reducing agent inhibits the level or biological activity of MIRO1 by 20% or more, e.g., 30% or more, 40% or more, or 50% or more, and sometimes 60% or more, 70% or more, or 80% or more, e.g., 90%, 95%, or 100%, compared to an untreated control not contacted with the agent. The agent can be so verified by any convenient method in the art for detecting MIRO1 levels and / or activity in the presence versus absence of the MIRO1-reducing agent.

[0056] For example, the level and / or phosphorylation status of Miro proteins (Ser156, Thr298 or Thr299 of Miro1 and Miro2, e.g., Wang et al. al. Cell 2011, 147(4):893-906) can be detected, for example, by immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a phospho-specific or general antibody, where an increase in phosphorylation of Miro protein and / or a decrease in total Miro protein levels, or a decrease in phosphorylation of Khc after contact with the agent, can indicate that the agent treats Parkinson's disease. As another example, the level and / or ubiquitination of Miro protein can be detected, for example, by immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a ubiquitin-specific antibody, where an increase in ubiquitination after contact with a candidate agent indicates that the agent treats Parkinson's disease. As another example, the ability of a target mitochondrial protein to transport mitochondria within cells can be assessed, for example, by treating cultured cells (e.g., neurons) with a MIRO1-reducing agent and observing the transport of mitochondria within the cells, e.g., using live cell imaging techniques, compared to cells not treated with the MIRO1-reducing agent (see, e.g., Brickley and (See, e.g., Stephenson J. Biol Chem 286(20):18079-92(2011); Misko et al. J Neurosci 30(19):4232-40(2010); Russo GJ et al. J. Neurosci 29(17):5443-55(2009)). As another example, because the formation of a complex between Miro (e.g., Miro1 and 2), TRAK (e.g., TRAK1 and 2), and Khc is essential for mitochondrial transport within neurons (see, e.g., Brickley and Stephenson J. Biol Chem 286(20):18079-92(2011)), the effect of a MIRO1 reducer on Miro function can be assessed by assessing the ability of Miro, TRAK, and Khc to form a complex in the presence of a MIRO1 reducer. Such assessment can be performed using any technique for determining protein-protein interactions, including, but not limited to, co-immunoprecipitation and affinity purification techniques. In certain embodiments, the ability to identify familial PD mutations, e.g., PINK1 or LRRK2 mutations, is assessed. It is evaluated in cells that have the abnormality.

[0057] "Mitochondrial depolarization" is the process by which the mitochondrial membrane potential shifts from its resting potential to a depolarized, negative to positive state. Normal mild calcium influx from the cytosol into the mitochondrial matrix causes a transient depolarization that is compensated for by proton pumping. Chemically induced mitochondrial depolarization provides a suitable assay for determining the effects of damage to mitochondria.

[0058] Several drugs are known and have been used experimentally to induce mitochondrial depolarization and can be used for this purpose in the methods of the present invention. These drugs are generally a type of mitochondrial uncoupler or uncoupler that disrupts oxidative phosphorylation in mitochondria by dissociating the ATP synthesis reaction from the electron transport chain. As a result, mitochondria consume energy to generate proton-motive force, but the proton-motive force is lost before ATP synthase can recapture this energy and create ATP.

[0059] The concentration and exposure time of the agent are sufficient to uncouple or depolarize mitochondria. For example, CCCP concentrations of about 10-100 μM, e.g., about 20-80 μM, or about 30-50 μM, are sufficient. Other agent doses may provide activity comparable to these concentrations of CCCP. The cells are incubated for a period sufficient to depolarize mitochondria and initiate clearance, e.g., at least about 1 hour, at least about 2 hours, and usually no longer than about 24 hours, but may be about 1-24 hours, about 2-20 hours, about 3-18 hours, about 4-14 hours, about 5-10 hours, or about 4-8 hours.

[0060] Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide-3-chlorophenylhydrazone (CCCP) are mitochondrial uncouplers frequently used in research. These molecules are lipophilic, weak acids that act as protonophores. Due to their hydrophobic nature, these compounds readily cross biological membranes, allowing protons to traverse these membranes. Other molecules suitable for this purpose include 1,3-bis(3,5-dichlorophenyl)urea, dodecyltriphenylphosphonium, dinitrophenol, and BAM15 ((2-fluorophenyl)6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)amine), a mitochondrial-specific protonophore uncoupler with potency similar to FCCP or DNP. FR58P1 (bromoalkyl ester of a hydroquinone derivative) is another mitochondrial protonophore.

[0061] Other mitochondrial-specific uncouplers include MitoFluo, a conjugate of fluorescein with a triphenylphosphonium cation that acts as a fluorescent uncoupler that preferentially accumulates within mitochondria; dodecyltriphenylphosphonium (C12TPP), which acts as a fatty acid anion carrier and promotes fatty acid cycling across membranes and thus mitochondrial uncoupling; rhodamine 19 butyl ester C4R1, which acts as a mild mitochondrial uncoupler; and MitoPhotoDNP, a fusion of DNP with an o-nitrobenzyl group (a photoactivatable group) and a triphenylphosphonium. The local anesthetic bupivacaine can act partially as a protonophore, but also exhibits an inhibitory effect on state 3 respiration by altering mitochondrial proton pump stoichiometry. Ortho-carborane (1,2-C2B 10 H 12 Weak CH acids such as FCCP also have comprehensive uncoupling properties when used at concentrations comparable to FCCP (in the 10 μM range).

[0062] Affinity assays are often immunoassays, and are assays or analyses that rely on the binding of a target molecule, i.e., Miro1, to a receptor, antibody, or other macromolecule. This is a procedure. A detection method is used to determine the presence and extent of the binding complex formed. Many formats for such assays are known and used in the art and are suitable for detecting Miro1 degradation after mitochondrial uncoupling or depolarization. In some embodiments, the assay format is suitable for high-throughput analysis.

[0063] Suitable assay formats include immunoassays utilizing antibodies specific for Miro1. Suitable antibodies for this purpose are known and are commercially available, for example, as polyclonal or monoclonal compositions, including, for example, Invitrogen monoclonals CL1095, CL1083, Sigma Aldrich clone 4H4, Santa Cruz Biotechnology anti-Rho T1 antibody (A-8), and the like.

[0064] Assays of interest include, for example, Western blot, immunohistochemistry, immunoprecipitation, and in particular immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and enzyme immunoassay (EIA).

[0065] Enzyme-linked immunosorbent assays (ELISAs) are used to qualitatively and quantitatively analyze the presence or concentration of specific soluble antigens, such as Miro1, in liquid samples such as cell lysates. These assays generally utilize the ability of multiwell or other plates to bind antibodies that capture the cognate antigen. They typically use a colorimetric endpoint that can be detected via absorbance wavelength and quantified from a known standard curve of antigen or antibody dilution. The detection antibody is often labeled with an enzyme, such as horseradish peroxidase or alkaline phosphatase, or a fluorescent tag, or an electrochemiluminescent label, or an intermediate label such as biotin.

[0066] Common ELISA formats include sandwich ELISA, so named because the analyte is "sandwiched" between two different antibodies. The capture substrate in this format is a capture antibody, often a monoclonal antibody, because it increases assay specificity and reduces background noise. The analyte is detected by binding to the capture antibody and then to the detection antibody. In a variation of the sandwich ELISA assay called a single molecule assay (Simoa), beads are coated with the capture antibody, each bead binds to either one or zero target molecules, and each individual bead is detected with another antibody (detection antibody) and a labeled enzyme.

[0067] Other ELISA formats include indirect ELISA, in which the capture substrate is the specific antigen being tested, and the detection step is mediated by a primary antibody and an enzyme-conjugated secondary antibody that is reactive with the primary antibody. Thus, the primary antibody that recognizes the antigen is not labeled. In direct ELISA, the capture substrate is the specific antigen being tested, and an enzyme that catalyzes a color-changing reaction is conjugated to the antigen-detecting antibody.

[0068] Immuno-PCR (I-PCR) is a technique that combines the sensitivity of nucleic acid amplification by PCR with the specificity of antibody-based assays, resulting in increased sensitivity of detection.

[0069] Screening Methods In some embodiments of the present invention, a method is provided for screening candidate drugs for the activity of treating Parkinson's disease in subjects with Parkinson's disease.Therefore, it has been shown that mitochondrial transport proteins, such as Miro protein, transport kinase, and kinesin heavy chain, promote the development or progression of Parkinson's disease or its symptoms.Therefore, screening candidate drugs that inhibit the expression or activity of mitochondrial transport proteins in cells can be used to treat Parkinson's disease in subjects. In particular, agents are tested for their ability to decrease levels of Miro1 following mitochondrial depolarization, preferably without substantially decreasing basal Miro1 levels.

[0070] Test agents can be obtained using any of a number of approaches in combinatorial library methods known in the art, including biological libraries, spatially addressable parallel solid-phase or solution-phase libraries, synthetic library methods requiring deconvolution, "one bead one compound" library methods, and synthetic library methods using affinity chromatography selection.

[0071] In one embodiment, to identify an agent that alters Miro1 degradation, cells, for example, living cells or cell populations, can be contacted with the agent to be tested, or proteins can be directly contacted with the agent to be tested. The level (amount) of activity can be assessed directly or indirectly, for example, compared with the level of activity in a control in the absence of the agent to be tested. If the level of activity in the presence of the agent differs from the level of activity in the absence of the agent by a statistically significant amount, the agent is an agent that alters activity.

[0072] The present invention further relates to novel agents identified by the above-mentioned screening assay.Therefore, it is within the scope of the present invention to further use the agents identified as described herein in the treatment methods described herein.For example, the agents identified as described herein can be used to alter Miro1 degradation with the agents identified as described herein.

[0073] For example, in a screening assay for a biologically active agent, cells expressing a mitochondrial transport protein of interest are contacted with a candidate agent of interest, and the effect of the candidate agent on the expression or function of the mitochondrial transport protein is assessed by monitoring one or more mitochondrial-related parameters. In particular, the activity of the candidate agent can be assessed by determining the level of Miro1 after mitochondrial depolarization.

[0074] A parameter is a quantifiable component of a cell, a component that can be accurately measured, particularly in a high-throughput system. A parameter can be any cellular component or cell product, including cell surface determinants, receptors, proteins or their conformations or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids such as mRNA, DNA, etc., or moieties derived from such cellular components, or combinations thereof. Most parameters provide quantitative readouts, but in some cases, semi-quantitative or qualitative results are acceptable. Readouts can include a single determined value or can include the mean, median, or variance. Typically, a range of parameter readouts is obtained for each parameter from multiple identical assays. Variability is expected, and the range of values ​​for each set of test parameters is obtained using standard statistical methods, with common statistical methods used to provide a single value. Thus, for example, one such method may include contacting cells expressing a mitochondrial transport protein with a candidate agent and comparing the mitochondrial-related parameter with the mitochondrial-related parameter in cells expressing the mitochondrial transport protein but not contacted with the candidate agent, wherein a difference in the parameter in the cells contacted with the candidate agent indicates that the candidate agent treats Parkinson's disease.

[0075] An example of a mitochondrial-related parameter that can be quantified when screening drugs to treat Parkinson's disease is immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with phospho-specific and general antibodies. , the phosphorylation status of Miro protein (Ser156, Thr298, or Thr299 of Miro1 and Miro2), TRAK protein, or Khc (Ser residue; see, e.g., Lee and Hollenbeck J Biol Chem. 1995 270(10):5600-5), where an increase in phosphorylation of Miro protein and / or a decrease in total Miro levels, or a decrease in phosphorylation of Khc after contact with a candidate drug indicates that the candidate drug may treat Parkinson's disease. Another example of a parameter is the ubiquitination status of Miro protein, TRAK protein, or Khc, e.g., by immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a ubiquitin-specific antibody, where an increase in ubiquitination after contact with a candidate drug indicates that the drug treats Parkinson's disease. Another example is the rate at which mitochondria are transported around cells, which can be measured, for example, by live cell imaging techniques, and a decrease in the transport rate after contacting the cells with a candidate drug indicates that the drug treats Parkinson's disease. Another example is the length of mitochondria within cells, and a decrease in the length of mitochondria after contacting the cells with a candidate drug indicates that the drug treats Parkinson's disease. Other output parameters can include those that reflect the ability of Miro protein, TRAK protein, and khc to form a complex in the presence of a MIRO1 reducer (see, for example, Brickley and Stephenson J. Biol Chem 286(20):18079-92(2011)), which can be assessed, for example, by co-immunoprecipitation or affinity purification techniques, and a decrease in complex formation after contacting the cells with a candidate drug indicates that the drug treats Parkinson's disease. In some cases, one parameter is measured. In some cases, multiple parameters are measured.

[0076] All cells contain mitochondria, and therefore, any cell can be used in the subject screening method.In some cases, the cell is a cell type that is typically affected by Parkinson's disease, such as a muscle cell or a neuron, such as a motor neuron.In certain cases, the cell contains a gene mutation associated with Parkinson's disease, i.e., a mutation in a nuclear or mitochondrial gene.In some cases, the cell can be acutely cultured from a subject with Parkinson's disease.

[0077] Candidate drugs of interest are biologically active agents encompassing numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, and the like. An important aspect of the present invention is the evaluation of candidate drugs, selected therapeutic antibodies, and protein-based therapeutics, for desirable biological response functions. Candidate drugs contain functional groups necessary for structural interaction with proteins, particularly through hydrogen bonding, typically at least an amine, carbonyl, hydroxyl, or carboxyl group, and often at least two functional chemical groups. Candidate drugs often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate drugs are also found among biomolecules, including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0078] These include pharmacologically active drugs, genetically active molecules, and the like. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Examples of pharmaceutical agents suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY (1996), 9th Edition, in the following sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts, and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs; Hormones and Hormone Antagonists Antagonists); Vitamins; Dermatology; and Toxicology, all of which are incorporated herein by reference.

[0079] Test compounds include all of the above molecular classes and may also include samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources, such as plants. While many samples contain compounds in solution, solid samples that can be dissolved in a suitable solvent can also be assayed. Samples of interest include environmental samples, such as groundwater, seawater, and mining waste; biological samples, such as lysates prepared from crops and tissue samples; manufacturing samples, such as time courses during pharmaceutical preparation; and libraries of compounds prepared for analysis. Samples of interest include compounds to be evaluated for potential therapeutic value, i.e., drug candidates.

[0080] Antibodies and small molecules can be screened using a variety of methods, such as ELISA assays, to detect changes in MIRO1 levels in vitro and in vivo. These methods include, but are not limited to, methods that measure binding affinity to a target, biodistribution of the compound in animals or cells, or compound-mediated cytotoxicity. As a first test, an antibody or small molecule may be tested to establish an interaction with a target that signals changes in MIRO1 levels. After selective binding is established, candidate antibodies can be tested for appropriate activity in an in vivo model. These and other screening methods known in the art provide information about the compound's ability to bind to, modulate, or otherwise interact with a particular target, which is a measure of the compound's effectiveness. In some embodiments, the screening method comprises the assay described in any one of the Examples.

[0081] Diagnostic methods In various embodiments, subjects are screened to determine whether they are suffering from or susceptible to neurodegenerative disorders such as Parkinson's disease. Screening methods include behavioral, biophysical, biochemical, and imaging assays and observations, as well as questionnaires, to determine whether a subject is at risk for or suffering from the early stages of a neurodegenerative disorder (e.g., Parkinson's disease). Biophysical and behavioral observations, such as a subject's physical examination for overt symptoms of disease, can be evaluated independently or in combination with questionnaires and biochemical / imaging assays. Each individual assay can also be used independently or in combination with biophysical assessments or other tests known in the art and related to specific neurodegenerative disorders / diseases. Examples of biochemical assays include genetic screening for mutations and / or polymorphisms (e.g., SNP analysis, short tandem repeat analysis), biomarker-based assays, protein expression assays, immunohistochemical assays, or any combination thereof. Materials for biochemical assays can be sampled from all bodily fluids and tissues. Commonly used bodily fluids include, but are not limited to, blood, serum, plasma, saliva, urine, gastric and digestive fluids, tears, stool, semen, vaginal fluid, interstitial fluid from tumor tissue, and cerebrospinal fluid. Methods for obtaining bodily tissue and fluid samples include, but are not limited to, biopsy, buccal swab, nasal swab, rectal swab, skin fat extraction, or other collection techniques for obtaining biological materials or chemicals. In some embodiments, the sample is a tissue sample. For example, the tissue sample may be fibroblasts, such as skin fibroblasts.

[0082] The control value is measured from a control, i.e., a corresponding control sample from a subject without the disease. For example, in some embodiments, the MIRO1 level in skin fibroblasts from the subject is compared to the control MIRO1 level in control skin fibroblasts from a control subject.

[0083] Antibody and small molecule inhibitors can be screened using various methods for detecting target binding in vitro and in vivo, such as ELISA assay.These methods include, but are not limited to, measuring the binding affinity to target, the biodistribution of compound in animals or cells, or compound-mediated cytotoxicity.As a first test, antibody or small molecule can be tested for binding to target mitochondrial transport protein.After selective binding is established, candidate antibody can be tested for appropriate activity in in vivo model.These and other screening methods known in the art provide information about the ability of compound to bind to specific target, regulate target, or otherwise interact with target, which is a measure of the effectiveness of compound.

[0084] A method for determining MIRO1 status in a subject can include measuring MIRO1 response to mitochondrial depolarization using a biochemical assay, Western blotting, or ELISA to determine whether the subject is deficient in MIRO1 removal after depolarization, and selecting the subject for treatment with a MIRO1-reducing agent. Determining MIRO1 status can include detecting MIRO1 levels in the subject and comparing the MIRO1 levels to control MIRO1 levels in a control subject. In some embodiments, detecting MIRO1 levels includes detecting MIRO1 in a tissue sample, such as skin fibroblasts, from the subject. In some embodiments, the method includes an assay described in any one of the Examples.

[0085] The method can further include treating the subject with a MIRO1-reducing agent. In such examples, MIRO1 levels can be monitored before and / or after treatment with the MIRO1-reducing agent. In some embodiments, the MIRO1-reducing agent is administered to the midbrain and / or putamen of the subject. The treatment can include administering to the subject a therapeutically effective amount of one or more agents selected from the group consisting of levodopa, a dopamine agonist, an MAO-B inhibitor, amantadine, or an anticholinergic agent before, concurrently with, or after administering the inhibitor of a mitochondrial transport protein.

[0086] If MIRO1 status indicates the presence of Parkinson's disease, in some embodiments, the Parkinson's disease is PTEN-induced putative kinase 1 (PINK-1)-associated PD. In some cases, Parkinson's disease is associated with Parkin, leucine-rich repeat kinase 2, alpha-synuclein, Parkinson protein 7, 13A2 type ATPase, group VI phospholipase A2, DnaJ (Hsp40) homolog, subfamily C, member 6, eukaryotic It is associated with mutations in either translation initiation factor 4 gamma 1, F-box protein 7, GRB10-interacting GYF protein 2, HtrA serine peptidase 2, synaptojanin 1, or vacuolar protein sorting 35 homolog. Parkinson's disease can be sporadic.

[0087] The MIRO1-reducing agent can be administered alone or in combination with any pharmaceutically acceptable carrier or salt as known in the art and described below.

[0088] The method for detecting MIRO1 levels can be performed by any method known in the art. In some embodiments, the method for detecting MIRO1 levels comprises electrophoresis, chromatography, enzyme assay, binding assay, or a combination thereof. In some embodiments, the method for detecting MIRO1 levels comprises ELISA. In some embodiments, the method comprises the assay described in any one of the Examples.

[0089] As described herein, MIRO1 status correlates with the presence or increased risk of developing a particular neurodegenerative disorder. In some embodiments, elevated levels of MIRO1 are associated with a neurodegenerative disorder, such as Parkinson's disease. In some embodiments, MIRO1 levels in a subject are about 5% or more, about 10% or more, or about 20% or more, e.g., about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, or about 80% or more, e.g., about 90% or more, about 95% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 800% or more, about 1000% or more, about 2000% or more, or about 3000% or more, higher than control MIRO1 levels in a control subject. In some embodiments, MIRO1 levels in a subject are about 20% or more higher than control MIRO1 levels in a control subject. In some embodiments, the MIRO1 level in the subject is greater than or equal to about 30% as compared to the control MIRO1 level in a control subject.

[0090] The subject may be suffering from symptoms of a neurodegenerative disorder. In one aspect, the present invention provides a method for diagnosing a subject for a neurodegenerative disorder, comprising: (a) obtaining a MIRO1 level from the subject; and (b) comparing the MIRO1 level with information from a control, the information being predetermined to indicate the absence of a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Parkinson's disease.

[0091] Movement Disorders. Movement disorders are generally classified as having decreased or slowed movement (hypokinesia disorders) or increased movement (hyperkinetic disorders). The classic and most common hypokinetic disorder is Parkinson's disease. Hyperkinetic disorders include tremor, myoclonus, dystonia, chorea, and tics.

[0092] Atypical parkinsonism refers to a group of neurodegenerative disorders other than Parkinson's disease that share some characteristics of Parkinson's disease but have some distinct clinical features and pathologies. As shown herein, the Miro1 status of a subject assessed by the methods described herein distinguishes between PD and atypical parkinsonism. Atypical parkinsonism encompasses neurodegenerative disorders such as progressive supranuclear palsy, dementia with Lewy bodies, corticobasal ganglionic degeneration, and multiple system atrophy. Deficits suggestive of neurodegenerative disorders other than Parkinson's disease include gaze palsy, signs of corticospinal tract dysfunction (e.g., hyperreflexia), myoclonus, autonomic dysfunction (in early or severe cases), cerebellar ataxia, prominent dystonia, ideomotor apraxia (inability to mimic hand movements), early dementia, early falls, and wheelchair confinement.

[0093] Parkinson's disease. Parkinson's disease (PD), also known as idiopathic or primary parkinsonism, hypokinetic rigidity syndrome / HRS, or paralysis agitans, is a degenerative disorder of the central nervous system. The motor symptoms of Parkinson's disease result from the death of dopamine-producing neurons in the substantia nigra and putamen, regions of the midbrain; the cause of this cell death is unknown. Early in the disease course, the most obvious symptoms are movement-related and include tremors, rigidity, resting tremor, bradykinesia, postural instability, slow movements, and difficulty walking and gait. Later, dementia can lead to thought and behavioral problems, such as cognitive impairment, hallucinations, delusions, behavioral abnormalities, depression, and sleep and wake disorders. These commonly occur in more advanced stages of the disease, with depression being the most common psychiatric symptom. Other symptoms include sensory problems (loss of smell), sleep problems (sleep and wake disorders), emotional problems, constipation, hypotension, frequent urination, impotence, and sweating. Parkinson's disease is more common in older people, with most cases occurring after the age of 50.

[0094] Parkinson's disease can be familial or sporadic. Familial refers to the inheritance of genetic mutations from parent to child through gametes. Examples of genes that may be involved in Parkinson's disease include PTEN-induced putative kinase 1 (PINK-1), Parkin (also known as RBR E3 ubiquitin protein ligase, or PARK2), leucine-rich repeat kinase 2 (LRRK2), alpha-synuclein (SNCA, PARK4), ubiquitin carboxy-terminal hydrolase L1 (UCHL1), Parkinson's protein 7 (PARK7, DJ-1), type 13A2 ATPase (ATP13A2), group VI phospholipase A2 (PLA2G6), DnaJ (Hsp40) homolog, subfamily C, and melanoma. Several different inherited mutations have been associated with PD, including mutations in DNAJC6 (PARK19), eukaryotic translation initiation factor 4 gamma, 1 (EIF4G1, PARK18), F-box protein 7 (FBXO7), GBR10-interacting GYF protein 2 (GIGYF2), HtrA serine peptidase 2 (HTRA2), synaptojanin 1 (SYNJ1), or vacuolar protein sorting 35 homolog (VPS35). Sporadic disease means that the disease occurs sporadically, i.e., due to a sporadic mutation in one of the aforementioned genes.

[0095] In some cases, the subject does not suffer from symptoms of a neurodegenerative disorder. In one aspect, the present invention provides a method for detecting an increased risk of a neurodegenerative disorder in a subject, the method comprising: (a) obtaining a MIRO1 level from the subject; and (b) comparing the MIRO1 level with information from a control, the information being predetermined to indicate the absence of a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Parkinson's disease.

[0096] In some embodiments, a method for diagnosing a neurodegenerative disorder in a subject includes providing a sample from a subject suspected of having a neurodegenerative disorder, determining the level of MIRO1 in the sample, comparing the determined level of MIRO1 to a control level of MIRO1, detecting an elevated level of MIRO1 in the sample from the subject compared to the control level of MIRO1, and diagnosing the presence of a neurodegenerative disorder in the subject from the elevated level of MIRO1. After a neurodegenerative disorder is diagnosed, the subject can be treated using methods described herein or known in the art. In some embodiments, the method includes an assay described in any one of the Examples.

[0097] Methods for assaying Miro1 status Assays are provided for determining the Miro1 status of a cell or cell population. Miro1 is normally removed from damaged, e.g., depolarized, mitochondria and promotes mitochondrial clearance via mitophagy. Defects in this degradation can occur and can be detected using biochemical assays. Defective degradation is a hallmark of PD. or is strongly associated with a predisposition to PD. A high proportion of Parkinson's disease subjects are defective in Miro1 removal after depolarization. However, Miro1 is efficiently degraded in control cells upon depolarization. Detection of this PD-related defect is useful for diagnosing PD and prognosing susceptibility to PD, optionally including treating subjects diagnosed in this way, monitoring clinical responses to PD after treatment in the context of clinical trials, screening drugs for their effectiveness in reducing this defect in Miro1 degradation, etc. These methods allow for accurate, early, and clinically practical assessment of Parkinson's disease.

[0098] Analytical methods can be performed by examining cellular compositions for the presence of Miro1 polypeptide after mitochondrial uncoupling, including chemically induced depolarization. These assay methods can be performed using cells obtained from a subject, including, but not limited to, biological samples such as cell lines, fibroblasts, and peripheral blood lymphocytes. Assays are generally performed on viable, i.e., living, cells. Fibroblasts are a convenient source of cells from an individual and can be easily obtained from a subject through minimally invasive and painless procedures. Cultured cells may be derived from a patient or control sample and may be modified to generate genetically modified cells, cells differentiated in vitro, cells exposed to a potential therapeutic agent, etc. In some embodiments, assays are performed on a population of cells from a subject to determine the individual's Miro1 phenotype.

[0099] Furthermore, cells that have been genetically modified, for example, by transfection or transduction with recombinant genes or by antisense technology, can be used with the present invention to provide gene function gain or loss.Methods for generating genetically modified cells are known in the art, for example, see "Current Protocols in Molecular Biology", Ausubel et al., eds, John Wiley & Sons, New York, NY, 2000.Genetic modification can be knockout, in which homologous recombination usually results in deletion, which knocks out the expression of the target gene, or knockin, in which a gene sequence that is not normally present in the cell is stably introduced.

[0100] Various methods may be used in the present invention to achieve knockout, including site-specific recombination, expression of antisense or dominant-negative mutations, etc. In the case of gene targeting, knockout involves partial or complete loss of function in one or both alleles of an endogenous gene. Preferably, the expression of the target gene product is undetectable or insignificant in the analyzed cells. This can be achieved by introducing a disruption to the coding sequence, for example, by inserting one or more stop codons, inserting a DNA fragment, deleting the coding sequence, substituting a stop codon for the coding sequence, etc. In some cases, the introduced sequence is ultimately deleted from the genome, leaving a net change to the native sequence.

[0101] The cell sample may contain, for example, at least about 10 2 cells, at least about 10 3 cells, at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 The assay may include 100,000,000 cells, or more. Larger numbers of cells are optionally assayed in multiple aliquots. The cells are contacted with an agent that uncouples or depolarizes mitochondria. The concentration and exposure time of the agent are sufficient to uncouple or depolarize mitochondria. For example, CCCP at a concentration of about 10-100 μM, e.g., about 20-80 μM, or about 30-50 μM, is sufficient. Other agent doses may produce activity comparable to CCCP at these concentrations. The cells are incubated for a period of time sufficient to depolarize mitochondria and initiate clearance, e.g., at least about 1 hour, at least about 2 hours, and usually not more than about 24 hours, and may be about 1-24 hours, about 2-20 hours, about 3-18 hours, about 4-14 hours, about 5-10 hours, and preferably about 4-8 hours. could be.

[0102] In some embodiments, the agent is a mitochondrial-specific uncoupler, such as a protonophore. Suitable agents for this purpose include FCCP, CCCP, DNP, BAM15, and the like, known in the art and described herein. After mitochondrial depolarization, cells are lysed and assessed for Miro1 levels, and failure to degrade Miro1 relative to control indicates an association with PD. The failure can be expressed, for example, as the ratio of Miro1 levels in cell lysates subjected to a mitochondrial uncoupler compared to the same cells in the absence of the uncoupler. PD-associated cells may have at least two-fold, at least three-fold, at least four-fold, or even more Miro1 than normal cells.

[0103] A test sample from a subject is evaluated for the presence of changes in Miro1 degradation in response to mitochondrial uncoupling. As used herein, the term "change" in polypeptide level refers to a change in level compared to a control sample. The control sample is a sample corresponding to the test sample (e.g., derived from the same type of cells) and is derived from a subject who is not affected by susceptibility to PD. A change in the polypeptide level in the test sample indicates susceptibility to PD compared to the control sample. Protein levels can be determined by various methods, including enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence, etc.

[0104] For example, in one embodiment, an antibody capable of binding to a polypeptide (e.g., as described above) can be used for capture or detection. The antibody can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2), can be used. The term "labeled," with respect to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, an enzyme-linked assay, a radiolabeled antibody, etc., as known in the art.

[0105] Miro1 degradation can be monitored in various ways. For example, Western blot analysis using an antibody that specifically binds to Miro1 can be used to identify its presence in a test sample by tracking cellular fractions, which can be quantitative. In other embodiments, high-throughput immunoassays are preferred. Advantageously, Miro1 removal is detected in a test sample by immunoassays such as ELISA or other high-throughput affinity assays.

[0106] In some embodiments, the Miro1 assay described above is utilized in the diagnosis and clinical monitoring of movement disorders, including, but not limited to, Parkinson's disease. In some embodiments, the methods of the present invention are used to determine the effectiveness of a therapy for the treatment of a movement disorder, either in vitro, e.g., in a drug screening assay, or at the subject level, e.g., in the analysis of a group of subjects in a clinical trial format. Clinical trial embodiments may involve the comparison of two or more time points for a subject or group of subjects. Patient status is expected to differ between two time points as a result of the administration of a therapeutic agent, treatment regimen, or exposure to a disease-inducing agent to the subject undergoing treatment. The response of a subject with a movement disorder to therapy is assessed by detecting the ability of a cell sample from the subject, including, but not limited to, a fibroblast sample, to degrade Miro1 following mitochondrial damage, e.g., mitochondrial depolarization.

[0107] In some embodiments, the method includes identifying a subject as having PD or a predisposition to PD, e.g., by the criteria described above for Miro1 degradation, administering a dose of a therapeutic agent to the patient, and quantifying Miro1 degradation in response to mitochondrial depolarization in at least one patient sample.

[0108] In some embodiments, the methods of the present invention are used to determine the effectiveness of a therapy for the treatment of movement disorders, either at the subject level or in a group of subjects, for example, in a clinical trial format. Such embodiments typically involve the comparison of two time points for a subject or group of subjects. It is expected that the patient's condition will differ between the two time points as a result of the therapeutic agent, treatment regimen, or disease exposure of the subject being treated.

[0109] For regulatory approval of treatments that provide symptomatic benefit in subjects with Parkinson's disease, clinical trials have used double-blind, placebo-controlled, parallel-group designs with fixed or flexible dosing strategies. Various efficacy outcome measures (combinations of one or more subscales of the Unified Parkinson's Disease Rating Scale [UPDRS]) and the need for additional symptomatic treatment, such as the dopamine agonist levodopa, have been used to evaluate the effectiveness of treatment.

[0110] Exemplary assays for quantifying MIRO1 include those described in Hsieh CH, Li L, Vanhauwaert R, Nguyen KT, Davis MD, Bu G, et al. Miro1 Marks Parkinson's Disease Subset and Miro1 Reducer Rescues Neuron Loss in Parkinson's Models. Cell Metab. 2019;1131-1140, and Shaltouki A, Hsieh CH, Kim MJ, Wang X. Alpha-synuclein delays mitophagy and targeting Miro rescues neuron loss in Parkinson's models. Acta Neuropathol. 2018;136:607-620.

[0111] Treatment method Provided herein are methods for treating a neurodegenerative disorder, comprising administering a Miro-reducing agent to a subject in need thereof, wherein the Miro-reducing agent reduces Miro, e.g., Miro1, in Parkinson's disease cells having depolarized mitochondria to an amount equal to, or within 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the amount of Miro in control healthy cells having depolarized mitochondria that have not been contacted with the Miro-reducing agent.

[0112] Neurodegenerative disorders encompassed by the methods of the present invention include, but are not limited to, neurological disorders that share symptoms similar to those seen in Parkinson's disease-related disorders. In some cases, neurological disorders may exhibit symptoms similar to Parkinson's disease, atypical Parkinson's disease, or Parkinson's-plus disease. Examples include, but are not limited to, drug-induced parkinsonism, progressive supranuclear palsy, vascular parkinsonism, dementia with Lewy bodies, diffuse Lewy body disease, corticobasal degeneration, multiple system degeneration (Shy-Drager syndrome), Alzheimer's disease, Pick's disease, and progressive supranuclear palsy (Steel-Richardson syndrome). Other conditions encompassed by the methods of the present invention include age-related and other dementias, including vascular dementia, diffuse white matter disease (Binswanger's disease), dementia of endocrine or metabolic origin, dementia due to head trauma and diffuse brain injury, dementia pugilistica, and frontal lobe dementia, as well as conditions associated with memory loss. In some cases, neurological disorders may not respond well to dopaminergic treatment and may occur as a result of a variety of vascular, drug-related, infectious, toxic, structural, and other known secondary causes. Drug-induced parkinsonism may be associated with antipsychotic medications. and antiemetics, as well as drugs that block postsynaptic dopamine D2 receptors with high affinity, such as sodium valproate, antidepressants, reserpine, and tetrabenazine.

[0113] Various subjects are suitable for treatment with the drug identified by the method of the present disclosure.Suitable subjects include any subject that shows Parkinson's disease symptoms such as bradykinesia, repetitive movements, tremor, limb rigidity, walking and balance problems, eye muscle weakness and eye focus inability, weakness, loss of sensation, non-motor symptoms such as REM sleep behavior disorder, mood disorders and neuropsychiatric symptoms including cognitive changes, anxiety, lethargy, changes in thinking ability, attention or vigilance level and visual hallucinations, intellectual and functional deterioration, forgetfulness, personality changes, autonomic dysfunction affecting cardiovascular, respiratory, urinary, gastrointestinal and sudomotor functions, breathing and swallowing difficulties, inability to sweat, orthostatic hypotension, pain, constipation and loss of smell.In some cases, subjects may experience significant speech or language disorder, significant frontal lobe projection and freezing of gait.

[0114] In certain embodiments, a subject may not exhibit any obvious symptoms of Parkinson's disease. In some cases, a subject in need may exhibit increased susceptibility to infections, hypothermia, weak bones, joint stiffness, arthritis, hunched posture, slow movement, overall low energy, constipation, urinary incontinence, memory loss, slow thinking, slow reflexes, difficulty with balance, decreased vision, decreased peripheral vision, hearing loss, wrinkled skin, gray hair, weight loss, and loss of muscle tissue.

[0115] In some cases, the subject may be a subject diagnosed with Alzheimer's disease, a subject who has experienced one or more strokes, a subject who has suffered a traumatic head injury, an individual with high serum cholesterol levels, a subject with a protein disorder involving deposits in brain tissue, a subject who has experienced one or more cardiac events, a subject undergoing cardiac surgery, and a subject with multiple sclerosis.

[0116] In some cases, the subject may have amyotrophic lateral sclerosis, degenerative ataxia, corticobasal degeneration, ALS-Parkinson's-Dementia Complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / Spinocerebellar ataxia type 3 and olivopontocerebellar degeneration, Gilles de la Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinal muscular atrophy (Kennedy's disease), primary lateral sclerosis, The subject may exhibit symptoms associated with neurological disorders, including motor neuron diseases such as familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial spastic disorder, Worfert-Kugelberg-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, and prion diseases (including Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, and Kuhl- and fatal familial insomnia). The subject may also exhibit other neurodegenerative disorders resulting from cerebral ischemia or infarction, including embolic and thrombotic occlusion, and any type of intracranial hemorrhage (including, but not limited to, epidural, subdural, subarachnoid, and intracranial), and intracranial and intraspinal lesions (including, but not limited to, contusion, penetrating, shear, compression, and laceration). In some cases, Miro levels during mitochondrial depolarization in cells from a subject compared to Miro levels during mitochondrial depolarization in cells from a control healthy subject can be used as a diagnostic assay to identify subjects who may benefit from the prophylactic use of a Miro-reducing agent to prevent abnormalities in mitochondrial homeostasis. Mitochondrial homeostasis refers to the balance between the processes of mitochondrial biogenesis, mitophagy, transport, fission, fusion, and maintenance of mitochondrial function and other processes that are at least partially dependent on Miro function.

[0117] As used in this disclosure, mitochondrial depolarization refers to the process by which the voltage difference across the mitochondrial membrane is reduced from its steady-state level, causing the mitochondrial membrane potential to change from negative to positive in a depolarized direction from the resting potential. Mitochondrial depolarization is partially influenced by the mitochondrial permeability transition pore. In some cases, depolarization below a certain Δψm may indicate impaired mitochondrial function and initiate mitophagy. In some cases, mitochondrial depolarization may precede the translocation of proteins such as Parkin and Pink1. In some cases, cytochrome release may accompany mitochondrial depolarization. Mitochondrial depolarization can be monitored by, among others, rhodamine 123, Mitotracker Red, DiOC6, or tetramethylrhodamine methyl ester (TMRE), or methyl ester (TMRM), nonyl acridine orange (NAO), Saphranine O, melissianin 540, JC-1, or JC-9. Mitochondrial depolarization may involve a decrease in resting mitochondrial potential of up to 5%, up to 7%, up to 10%, up to 15%, up to 18%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 90%, up to 95% or up to 100% as measured by a quantitative assay.

[0118] Mitochondrial depolarization can be induced by depolarizing agents, including, among others, calcium dysregulation, ROS production, chemicals such as barbiturates, ginsenoside-Rh2, rotonen, complex I inhibitors, complex II inhibitors, complex III inhibitors, complex IV inhibitors, mycotoxins such as aurovertins A-E, leucinostatin A and B, venturicidin and ossamycin, efrapeptins, oligomycins A-D, vancomycin, antimycin, naturally occurring flavonoids, propranolol, local anesthetics, the herbicides paraquat, pyrethroids, DDT, parathion, diethylstilbestrol, some cationic dyes and organotin compounds, uncouplers such as substituted phenols, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), carbonyl cyanide methacrylate, and the like. These compounds may include fluorophenylhydrazones (CCCPs), trifluoromethylbenzimidazole, salicylanilide, and carbonyl cyanide phenylhydrazones, endogenous and exogenous free fatty acids (FFAs) and fatty acid-like compounds such as perfluorodecanoic acid, sulfuramide, and methyl-substituted hexadecanedioic acid, peptides such as adenine nucleotide translocase, ionophores such as gramicidins (gramicidins A, D, and S), nigericin, valinomycin, cationic uncouplers such as the cyanine dye tri-S-C4(5), Cu2-(o-phenanthroline)2 complex, and pentamidine, membrane-active peptides such as alamethicin and mastoparan, alternative electron acceptors such as adriamycin and paraquat, and various substituted naphthoquinones and nitrosamines.

[0119] In one aspect, the present disclosure provides a method for measuring Miro degradation in cells obtained from a subject diagnosed with sporadic or familial neurodegeneration under conditions in which the mitochondrial membrane permeability of the cells is altered. In some cases, the cells are fibroblasts derived from tissue obtained from a subject diagnosed with sporadic or familial neurodegeneration, and the method includes obtaining tissue, preferably a skin biopsy, from the patient, dissecting the skin biopsy into evenly sized pieces, transferring the dissected skin biopsy to a gelatin-coated tissue culture plate, and replacing the medium with complete DMEM / 20% FBS medium every 2-3 days until the fibroblasts reach confluence. In some cases, the fibroblasts are obtained from a subject diagnosed with sporadic or familial Parkinson's disease. In some cases, the neurodegenerative disorder is Parkinson's disease or a parkinsonian-like disorder. In some cases, the subject has the disease and is asymptomatic. In some cases, the subject has risk factors for the disease and is asymptomatic. As used herein, the term sporadic Parkinson's disease refers to a non-familial form of Parkinson's disease. In some cases, sporadic PD is caused by environmental factors. In some cases, subjects with sporadic PD may have genes associated with PD with low penetrance or late onset, which makes any familial occurrence less evident.

[0120] In some cases, the cells are subject-specific induced pluripotent stem cells (iPSCs) associated with Parkinson's disease or Parkinson-like disease. In one embodiment, the induced pluripotent stem cells are derived from human fibroblasts. In some cases, the subject may have a genetic or sporadic disease. In related embodiments, the induced pluripotent stem cells are produced without the use of retroviruses or lentiviruses. In certain embodiments, the induced pluripotent stem cells are produced by a method comprising the use of three factors, for example, OCT4, SOX2, and KLF4. In another embodiment, the induced pluripotent stem cells are further differentiated to adopt the fate of midbrain dopaminergic cells. In some cases, the induced pluripotent stem cells are differentiated to adopt the fate of about 20 days.

[0121] In some embodiments, the cells are neural cells derived from a subject with a diagnosis of Parkinson's disease or a Parkinson-like disorder, and the method includes obtaining fibroblasts from the subject, dedifferentiating the fibroblasts into pluripotent stem cells, and differentiating the stem cells toward a neural cell fate. In one embodiment, the fibroblasts are skin fibroblasts. In another embodiment, the stem cells differentiate toward a midbrain dopaminergic cell fate. In certain embodiments, dedifferentiation of the fibroblasts induces pluripotency.

[0122] In some embodiments, the subject has a genetic variation or mutation known to be associated with Parkinson's disease or Parkinson-like disease. In other embodiments, the genetic variation of interest is a copy number variation of the gene of interest. In certain embodiments, the cell line has three copies of the gene of interest. The genetic variation or mutation can be a deletion, insertion, complex multistate variant, deletion, substitution, transition, transversion, or duplication of one or more nucleotides in the gene of interest. In exemplary embodiments, the gene of interest is selected from PARK1 (SNCA or alpha-synuclein), PARK2 (parkin), PARK5 (UCHL1), PARK6 (PINK1), PARK7 (DJ-1), PARK8 (LRRK2), and PARK11 (GIGFY2). In certain embodiments, the gene of interest is PARK1 (SNCA or alpha-synuclein). In other specific embodiments, the gene of interest is PARK8 (LRRK2). In one embodiment, the subject has a homozygous mutation of LRRK2, and the mutation comprises a G2019S mutation. In some embodiments, the subject has Parkinson's disease. In certain embodiments, the subject has an idiopathic form of Parkinson's disease.

[0123] In some embodiments, the method includes providing cells having a sequence mutation or multiple copies of a gene of interest and inducing pluripotency, multipotency, or totipotency in the cells to create a cell line with the gene mutation of interest. The method may also include identifying a subject having a gene mutation of interest and obtaining one or more cells from the subject. Cells from the subject may be fibroblasts, tumor cells, bone marrow cells, stomach cells, blood cells (e.g., leukocytes, blood progenitor cells), hepatocytes, etc., or any convenient or relevant cell source obtained from the subject. The method for generating a cell line having a copy number mutation of a gene of interest may also include inducing differentiation of the cell line. The cell line may be differentiated into any cell type of interest, including endoderm, ectoderm, mesoderm, e.g., neural, e.g., neuronal, cell line, epithelial cell line, cardiac cell line, etc.

[0124] In some embodiments, the cell lines and methods for their use include cell lines with gene copy number variations, such as at least one copy, for example, two or three copies of the gene. In other embodiments, the copy number variation is one or more deletions, insertions, or complex multistate mutations of the gene of interest.

[0125] In some embodiments, the cell lines and methods of use thereof include cell lines that have a genetic mutation that is a mutation in a gene of interest. For example, the mutation can be a mutation in a gene of interest. In some embodiments, the mutation may be a deletion, substitution, transition, transversion, or duplication of one or more nucleotides. In some embodiments, the mutation is a point mutation.

[0126] In one aspect, the present disclosure provides a method of treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein the Miro-reducing agent is: (a) plating fibroblasts from a sporadic Parkinson's disease subject into wells of an array; (b) 24 hours after step (a), adding a candidate agent to the first test wells and no candidate agent to the first control wells; (c) 10 hours after step (b), adding FCCP to the first test well and the first control well; (d) 14 hours after step (c), fixing the cells in the first test well and the first control well with ice-cold 90% methanol; (e) immunostaining cells in the first test well and the first control well with anti-Miro1 and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), imaging by confocal microscopy, and measuring Miro1 intensity / cell for these images of the first test well and the first control well.

[0127] In some cases, the Miro-reducing agent reduces Miro1 in the first test well by up to 3, up to 4, up to 5, up to 6, or up to 7 standard deviations compared to the first control well. In some cases, the Miro-reducing agent reduces Miro1 in the first test well by about 2 to about 7, or about 2 to about 5, or about 2 to 4, or about 2 to 6 standard deviations compared to the first control well. In some cases, the candidate agent reduces Miro1 in the second test well by less than one or half standard deviations compared to the second control well, and the candidate agent is a Miro-reducing agent. In some cases, the Parkinson's disease cells are obtained from a subject with familial Parkinson's disease. In some cases, the Parkinson's disease cells exhibit elevated levels of Miro compared to cells from a healthy subject. In some cases, the Parkinson's disease cells exhibit levels of Miro comparable to those of a healthy subject. In some cases, the mitochondrial depolarizing agent is not FCCP. In some instances, the Miro reducer is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule, or an aptamer. In some instances, Miro is detected by ELISA or Western blotting, or fluorescent live imaging.

[0128] In one aspect, the disclosure provides a method of treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro reducer, wherein the Miro reducer reduces the following in sporadic Parkinson's disease cells: (a) fibroblasts from a subject with sporadic Parkinson's disease are plated into wells of the array; (b) 24 hours after step (a), adding a candidate agent to the first test wells and not adding a candidate agent to the first control wells; (c) 10 hours after step (b), adding FCCP to the first test well and the first control well; (d) 14 hours after step (c), fixing the cells in the first test well and the first control well with ice-cold 90% methanol; (e) immunostaining the cells in the first test well and the first control well with anti-Miro1 and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), imaging them by confocal microscopy, and measuring Miro1 intensity / cell for the images of the first test well and the first control well, thereby reducing Miro1 according to the assay. In one embodiment, the present disclosure provides a method for treating a neurodegenerative disorder in need thereof. Provided is a method comprising administering to a subject a Miro-reducing agent, wherein the Miro-reducing agent reduces Miro1 in sporadic Parkinson's disease cells having depolarized mitochondria by 2, 3, 4, 5, or 6 standard deviations or more compared to sporadic Parkinson's disease cells having depolarized mitochondria that are not treated with the Miro-reducing agent.

[0129] In some cases, the Miro reducer identified in the above assay is one of the following: (a1) Fibroblasts from a subject with sporadic Parkinson's disease are plated into wells of the array; (b1) 24 hours after step (a1), adding a candidate agent to a second test well and not to a second control well; (c1) 14 hours after step (b1), fixing the cells in the second test well and the second control well with ice-cold 90% methanol; (d1) Immunostaining cells in the second test well and the second control well with anti-Miro1 and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), imaging them by confocal microscopy, and measuring Miro1 intensity / cell for these images of the second test well and the second control well. In one embodiment, the present disclosure provides a method of treating a neurodegenerative disorder, comprising administering a Miro-reducing agent to a subject in need thereof, wherein the Miro-reducing agent reduces Miro1 in sporadic Parkinson's disease cells with depolarized mitochondria by 2, 3, 4, 5, or 6 standard deviations or more compared to sporadic Parkinson's disease cells with depolarized mitochondria not treated with the Miro-reducing agent, and reduces Miro in sporadic Parkinson's disease cells by less than 1 standard deviation compared to sporadic Parkinson's disease cells not treated with the Miro-reducing agent.

[0130] In one aspect, the invention described herein provides a method for treating a neurodegenerative disorder, comprising administering a Miro-reducing agent to a subject in need thereof, wherein the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria to an amount that is equal to or within 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62%, 65%, 67.5%, 70%, 72.5%, or 75% of the amount of Miro in control healthy cells having depolarized mitochondria that have not been contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro in Parkinson's disease cells to between 15%-25%, 15%-30%, 20%-30%, 20%-40%, 25%-40%, 25%-35%, 25%-45%, 30%-45%, 30%-50%, 35%-55%, 35%-60%, 35%-50%, 35%-55%, 40%-50%, 40%-55%, 40%-60%, 45%-65%, 45%-70%, or 45%-75% of the amount of Miro in control healthy cells having depolarized mitochondria that have not been contacted with the Miro-reducing agent. In some cases, the healthy cells having depolarized mitochondria are healthy cells that have been contacted with a mitochondrial depolarizing agent.

[0131] In one aspect, the disclosure provides a method of treating a neurodegenerative disorder, comprising administering to a subject in need thereof a Miro-reducing agent, wherein the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria compared to the reduction of Miro1 in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria by more than three standard deviations ... The Miro-reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by more than two standard deviations compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by more than four standard deviations compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by more than five standard deviations compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria not contacted with the Miro-reducing agent.

[0132] In one aspect, the disclosure provides a method of treating a neurodegenerative disorder, comprising administering a Miro-reducing agent to a subject in need thereof, wherein (a) the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria by more than two standard deviations compared to the reduction in Miro1 in control Parkinson's disease cells having depolarized mitochondria not contacted with the Miro-reducing agent, and (b) the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having non-depolarized mitochondria by less than one standard deviation compared to the reduction in Miro1 in control Parkinson's disease cells having non-depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria by more than three standard deviations compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria by more than four standard deviations compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria by more than five standard deviations compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria not contacted with the Miro-reducing agent. In some cases, the Parkinson's disease cells having depolarized mitochondria are Parkinson's disease cells contacted with a mitochondrial depolarizing agent. In some cases, the Parkinson's disease cells are sporadic Parkinson's disease cells. In some cases, the Parkinson's disease cells are familial Parkinson's disease cells. In some cases, the Parkinson's disease cells are fibroblasts. In some cases, Parkinson's disease cells exhibit elevated levels of Miro compared to cells from healthy subjects, and in some cases, Parkinson's disease cells exhibit levels of Miro comparable to healthy subjects.In some cases, the mitochondrial depolarizing agent is not FCCP. In some instances, the Miro reducer is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule, or an aptamer. In some cases, Miro is detected by ELISA, Western blotting, or fluorescent live imaging.

[0133] In some cases, a subject has a higher Miro level, e.g., a higher Miro1 level, than the corresponding Miro level in a control subject. For example, a subject may have about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, or about 1000% or more higher levels of Miro1 than the control subject. In some cases, a subject may have about 20% or more or about 30% or more higher levels of Miro1 compared to the Miro1 level in a control subject.

[0134] In some cases, the candidate agent may be a peptide, an antibody, a protein, a protein fragment, an aptamer, or the like. The screening assays of the present invention are typically small molecules or small molecules. In one embodiment, the screening assays of the present invention are high-throughput or ultra-high-throughput. For example, the screening assays of the present invention may be performed in a multi-well format, e.g., a 96-well format, a 384-well format, or a 1,536-well format, and are amenable to automation. In particular, each well of a microtiter plate can be used to perform a separate assay for a selected test agent. In some cases, the concentration or incubation time effect of a single test can be observed, and a single test agent can be tested every 5 to 10 wells. It is possible to assay many plates per day. Using this method, assay screens of up to approximately 6,000, 20,000, 50,000, or even more than 100,000 different compounds can be performed.

[0135] The candidate agent can be a protein or protein fragment, an antibody or antibody fragment, a small molecule, or an aptamer. The candidate agent can be in the form of a library of candidate agents, such as a combinatorial library or a randomized library, which provides a sufficient range of diversity. The candidate agent is optionally linked to a fusion partner, such as a targeting compound, a label or detectable moiety, a rescue compound, a dimerization compound, a stabilizing compound, an addressable compound, and other functional moieties. In some cases, the candidate agent is bound to the surface of an array. In some cases, the candidate agent is delivered to cells expressing Miro in a liquid sample.

[0136] In some cases, Parkinson's cells and healthy cells express Miro proteins linked to a label or reporter moiety. A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 Miro levels can be detected by using fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, or haptens, and proteins that can be made detectable by incorporating radioactive labels into the peptide or by using antibodies that specifically react with the peptide. In some cases, Miro levels can be detected by using fluorescence, luminescence, chemiluminescence, absorbance, and other optical methods. In some cases, the difference in Miro levels between Parkinson's disease and healthy cells is measured by a computer algorithm.

[0137] In some cases, cells can be engineered to express Miro proteins tagged with an enzyme reporter capable of generating a fluorescent signal or binding to small molecules that can be tagged with fluorescent moieties. In some examples, small molecules can be tagged with fluorescent reagents such as fluorescein, rhodamine, Texas Red, BODIPY, and other commercially available molecules (e.g., those available from Molecular Probes / Invitrogen and other suppliers) to generate various fluorescent readouts. In other examples, ligands and other probes can be directly tagged with fluorescein or another fluorophore to detect binding to cellular proteins, or tagged with enzymes such as alkaline phosphatase or horseradish peroxidase to allow indirect detection and localization of the signal. In other examples, Miro can be tagged with enzymes that fluoresce upon enzymatic cleavage or that can be used to generate fluorescent signals in live cells by using specific cell-permeable substrates that shift their fluorescence spectrum, resulting in a shift in the fluorophore absorption or emission wavelength. In another example, Miro may be tagged with an enzyme that can be used to generate a fluorescent signal in living cells by cleavage of the covalent bond of an emission-absorption matched fluorophore pair in a covalently linked form that maintains resonance energy transfer between the two fluorophores that is lost when the two fluorophores are separated.

[0138] Luminescent, fluorescent, or bioluminescent signals are easily detected and quantified using any one of a variety of automated and / or high-throughput measurement systems, including fluorescent multiwell plate readers, fluorescence-activated cell sorters (FACS), and automated cell-based imaging systems that provide spatial resolution of the signal. Various instrumentation systems have been developed to automate HCS, including automated fluorescence imaging and automated microscopy systems developed by Cellomics, Amersham, TTP, Q3DM, Evotec, Universal Imaging, and Zeiss. Fluorescence recovery after photobleaching (FRAP) and time-lapse fluorescence microscopy have also been used to study protein mobility in live cells.

[0139] In one aspect, the present invention also provides a method for identifying a diagnostic cellular phenotype, comprising comparing a set of cells from a subject with cells from a subject not suffering from Parkinson's disease, wherein the detected cellular phenotype is degradation of Miro upon mitochondrial depolarization. In some cases, the comparison of Miro levels is performed in silico. The cells may be fibroblasts. The cells may be cells differentiated from induced pluripotent stem cells or induced stem cells into neural stem cells or neurons. In one embodiment, the detected response is a change in mitochondrial function, mitochondrial fission, fusion, morphology, mitophagy, mitochondrial transport, intracellular calcium levels, or other cellular characteristics dependent on Miro function.

[0140] In one aspect, the present invention also includes a method for determining the risk of Parkinson's disease in a subject, comprising comparing at least one phenotype determined in a first set of cells from the subject with at least one phenotype determined in a second set of cells from a subject not suffering from Parkinson's disease and at least one phenotype determined in a third set of cells from a subject suffering from Parkinson's disease; and indicating that the subject is at high risk for Parkinson's disease if the at least one phenotype determined in the first set of cells is more similar to the at least one phenotype determined in the third set of cells than to the at least one phenotype determined in the second set of cells, wherein the first, second, and third sets of cells are induced pluripotent stem cells or cells differentiated from induced pluripotent stem cells, and the phenotype is degradation of Miro upon mitochondrial depolarization. In one embodiment, the detected response is a change in mitochondrial function, mitochondrial fission, fusion, morphology, mitophagy, mitochondrial transport, or other cellular characteristic dependent on Miro function. In some cases, the comparison is performed in silico. In some cases, Parkinson's disease cells exhibit elevated levels of Miro compared to cells from healthy subjects. In some cases, Parkinson's disease cells exhibit levels of Miro comparable to those of healthy subjects. In some cases, the mitochondrial depolarizing agent is not FCCP. In some examples, the Miro reducer is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule, or an aptamer. In some cases, Miro is detected by ELISA, Western blotting, or fluorescent live imaging.

[0141] In one aspect, the disclosure provides a method of selecting a subject for treatment with a therapeutic agent for a neurodegenerative disorder, comprising: (a) collecting cells from a subject and evaluating a first control portion of the cells for intracellular pre-depolarization Miro levels; (b) contacting a second test portion of the cell with a depolarizing agent; (c) assessing post-depolarization Miro levels in a second test portion of the cells contacted with a depolarizing agent and comparing the Miro levels to pre-depolarization Miro1 levels in the first control portion of the cells.

[0142] In some cases, the subject is treated with an agent for treating a neurodegenerative disorder when the Miro1 level after depolarization in the second test portion of the cell is reduced by 5% to 70%, 10% to 70%, 20% to 80%, 25% to 90%, 5% to 50%, or 10% to 50% compared to the Miro1 level before depolarization in the first control portion of the cell. In certain embodiments, the depolarizing agent reduces the Miro1 level after depolarization in the second test portion of the cell by 5% to up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, or up to 15% compared to the Miro1 level before depolarization in the first control portion of the cell.

[0143] In some cases, the neurodegenerative disorder is Parkinson's disease. In some cases, the cell is a fibroblast. In some cases, the cell is an induced pluripotent stem cell or a cell differentiated from an induced pluripotent stem cell. In some cases, the cell is a neuron differentiated from an induced pluripotent stem cell. In some cases, the neuron is a dopaminergic neuron. In some cases, the depolarizing agent is FCCP.

[0144] The present invention includes a method for identifying an agent that corrects a phenotype associated with Parkinson's disease or a predisposition to Parkinson's disease, comprising contacting a first cell population with a candidate agent, contacting a second cell population with a control agent, assaying the two populations, and identifying the candidate agent as correcting the phenotype if the first population is closer to a normal phenotype than the second population after treatment, wherein the phenotype is cellular degradation of Miro after mitochondrial depolarization and a reduction in Miro levels. In some cases, the candidate agent is a Miro-reducing agent. In some cases, cells in both populations have been treated with a mitochondrial depolarizing agent. In some cases, cells in both populations contain at least one endogenous allele associated with a neurodegenerative disorder or a predisposition to neurodegeneration. In some cases, Parkinson's disease cells exhibit elevated levels of Miro compared to cells from a healthy subject. In some cases, Parkinson's disease cells exhibit levels of Miro comparable to those of a healthy subject. In some cases, the mitochondrial depolarizing agent is not FCCP. In some instances, the Miro reducer is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule, or an aptamer. In some instances, Miro is detected by ELISA or Western blotting, or fluorescent live imaging.

[0145] In one aspect, the disclosure provides a method of screening candidate agents to identify a Miro reducer, comprising: (a) obtaining cells that are deficient in Miro1 clearance; (b) contacting a first test portion of the cells with a candidate agent and not contacting a second control portion of the cells with the candidate agent; (c) contacting the first test portion and the second control portion of the cells of step (b) with a depolarizing agent; (d) assessing Miro1 levels in the first test portion compared to the second control portion.

[0146] In some cases, a candidate agent is identified as a Miro reducer if it reduces the Miro level in the first test portion by 2 or more standard deviations, 3 or more standard deviations, 4 or more standard deviations, 5 or more standard deviations, 6 or more standard deviations, or 7 or more standard deviations compared to the Miro level in the second control portion. In some cases, the first and second test portions comprise cells obtained from a subject identified with or at risk for Parkinson's disease. In some cases, the cells are fibroblasts. In some cases, the cells are induced pluripotent stem cells or cells differentiated from pluripotent stem cells. In some cases, the cells are neurons differentiated from pluripotent stem cells. In one embodiment, the present disclosure provides a method for treating or preventing parkinsonism disorders by administering to a subject an effective amount of a Miro reducer identified by the screening methods described herein to treat or prevent Parkinson's disease. Provide the law.

[0147] In one aspect, the invention provides a method for reducing the risk of drug toxicity in a human subject with Parkinsonism, comprising contacting one or more cells generated from the subject with a dose of a pharmacological agent, assaying the contacted one or more differentiated cells for toxicity, and prescribing or administering the pharmacological agent to the subject if, and only if, the assay for toxicity in the contacted cells is negative. The cells may be fibroblasts from the subject, induced pluripotent cells generated from the subject, or cells differentiated from an induced pluripotent stem cell line, such as neurons.

[0148] In one aspect, the present disclosure provides a method for screening cell lines harboring a mutation in a gene of interest for a candidate drug for treating a neurodegenerative disorder. The method involves contacting cells from a subject suspected of or deficient in MIRO1 ablation with the candidate drug, observing a change or lack of change in the cells, and correlating the change or lack of change with the drug's ability to treat the disease. Such changes can be observed, for example, by staining for intracellular levels of Miro upon depolarization. The method may further include comparing the cell line or its progeny to a cell line lacking the mutation in the gene of interest, i.e., a normal cell line, or a cell line associated with the same disorder of interest but lacking the mutation in the gene of interest present in the first cell line. The cells may be fibroblasts from the subject, induced pluripotent cells generated from the subject, or cells differentiated from an induced pluripotent stem cell line, such as neurons.

[0149] Also provided herein is a method for studying the mechanisms of neurodegenerative disorders such as Parkinson's disease. This method involves contacting a cell line or its progeny produced by the methods described herein with a drug or condition that affects a cellular pathway of interest, and observing changes or lack of changes in the cells, thereby identifying molecular determinants of the disorder or disease. In some cases, the cellular pathway of interest is Miro degradation upon mitochondrial depolarization. In one embodiment, the cellular pathway is a change in mitochondrial function, mitochondrial fission, fusion, morphology, mitophagy, mitochondrial transport, or other cellular characteristics that depend on Miro function.

[0150] In some cases, Miro reducers bind to the EF-hand domain of Miro. In some cases, Miro reducers increase intracellular calcium in cells. In some cases, Miro reducers bind to the GTPase domain of Miro. In some cases, Miro reducers bind to the nucleotide-binding domain of Miro. In some cases, Miro reducers bind to the microtubule-binding region of Miro. In some cases, Miro reducers bind to the Pink1 phosphorylation site of Miro.

[0151] In some cases, Miro reducers are calcium channel blockers. Calcium channels are protein molecules that contain pores that extend through the membrane of a cell or organelle and reversibly open and close, allowing the passage of calcium into and out of the cell or organelle. 2+ They regulate the passage of ions. Known calcium channels include L-type, N-type, and R-type calcium channels.

[0152] In some cases, calcium channels are L-type channels. L-type channels have (1) a "high threshold" for activation, i.e., a strong depolarization of the cell membrane in which they are located is required to open such channels, and (2) a large "single-channel conductance," i.e., when open, each channel can transport Ca at a relatively high rate. 2+ (3) Ca 2+ More than Ba 2+ (4) calcium channels, such as nimodipine and nifedipine, They are characterized by their sensitivity to high-potency block by the dihydropyridine class of antagonists (typically, the IC50 values ​​for L-channel block by these agents are less than 1 μM). In most cases, calcium "action potentials" mediated by L-type channels under normal physiological conditions are relatively long in duration, typically 100 ms or longer. In some cases, MiR-reducing agents are L-type channel blockers.

[0153] In some cases, the calcium channel is an N-type channel. N-type channels are high-threshold channels best described as dihydropyridine-insensitive but blocked by interaction with the cone snail toxin omega-conotoxin. Qualitatively, as a class, N-type channels inactivate somewhat more rapidly than L-type channels. In this regard, there is overlap between the L-type and N-type channel classes, so differences in inactivation kinetics do not constitute a defining feature. In some cases, Miro reducers are N-type channels.

[0154] In some cases, the calcium channel is an R-type channel. R-type channels can be characterized as high-threshold calcium channels that are relatively resistant to blockade by dihydropyridines and omega-conotoxins. Such channels are found in a wide variety of neurons, and are particularly abundant in cerebellar Purkinje cells. R-type channels may play a role in synaptic transmission and other processes that depend on calcium entry but are insensitive to these blockers. In some cases, the Miro reducer is an R-type channel.

[0155] In some cases, the Miro reducer may be a calcium channel antagonist capable of reversibly blocking calcium channels. In some cases, the Miro reducer may function by non-permanently (i.e., non-covalently) binding to the protein molecules that make up such channels. In some cases, the Miro reducer may be a calcium channel antagonist that blocks calcium channels to a greater extent than it blocks neurotransmitter-activated channels, voltage-sensitive sodium channels, and potassium channels, i.e., its IC50 for calcium channels is lower than that for such neurotransmitter-activated channels, sodium channels, and potassium channels. In some cases, the Miro reducer may be capable of crossing the blood-brain barrier of a subject. In some cases, the Miro reducer may be used to treat neurodegenerative disorders associated with excessive calcium influx into neurons, including Parkinson's disease.

[0156] In some examples, the effect of the Miro reducing agent on intracellular calcium levels can be monitored using synthetic organic fluorescent dyes such as Fura-2, Fluo-3, Fluo-4, Indo-1, Calcium Green-1, Oregon Green BAPTA, and Rhod-2. In some examples, the effect of the Miro reducing agent on intracellular calcium levels can be monitored using an aequorin-based luminescent calcium indicator such as aequorin. In other examples, the effect of the Miro reducing agent on intracellular calcium levels can be monitored using fluorescent protein-based calcium indicators such as DsRed / inverse-pericam, YC2.1, G-Camp, YC3.1, G-Camp2, Synapcam, YC2.1, YC2.12, Camgaroo, G-CaMP2, G-CaMP2, YC3.12, CerTN-L15, and GCamp3.

[0157] Pharmaceutical Composition In some embodiments, a pharmaceutical composition comprising an effective dose of a Miro1 reducer is provided, which may be sufficient to achieve a therapeutic level of Miro1 of at least 1 μM, at least 5 μM, at least 10 μM, at least 20 μM, up to about 1 μM, up to about 500 μM, up to about 250 μM, up to about 100 μM, up to about 75 μM, or up to about 50 μM. Unit doses may be, for example, 1 μg / kg, 10 μg / kg, 100 μg / kg, 500 μg / kg, or 1 μg / kg. It may be μg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg or more.

[0158] The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized foreign pharmacopeia for use in animals, more specifically in humans. The term "carrier" or "vehicle" refers to a diluent, adjuvant, excipient, or vehicle administered with the Miro1-reducing agent. Such pharmaceutical carriers can be sterile liquids, such as lipids, e.g., liposomes, e.g., liposomal dendrimers, saline solutions in water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions are preferably used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat powdered milk, glycerol, propylene glycol, water, ethanol, etc. If necessary, the compositions may contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions may be formulated as suppositories with traditional binders and carriers such as triglycerides. The reducing agent may be formulated as a neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, which is incorporated herein by reference in its entirety.Such compositions will contain a therapeutically effective amount of the Miro1-reducing agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0159] Pharmaceutical compositions can also include any of a variety of stabilizing agents, such as antioxidants. When a pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with a variety of well-known compounds that improve the polypeptide's in vivo stability or otherwise improve its pharmacological properties (e.g., increase the polypeptide's half-life, reduce its toxicity, improve solubility, or uptake). Examples of such modifying or complexing agents include sulfate, gluconate, citrate, and phosphate. The polypeptides of the composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0160] Further guidance on suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th Edition (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0161] Ingredients used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least as pure as National Foods (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, that may be present during the synthesis or purification process.

[0162] The subject pharmaceutical compositions are typically sterile. Sterilization is readily achieved by filtration through sterile filtration membranes (e.g., 0.2 μm membranes). Therapeutic compositions are generally placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle. The pharmaceutical composition can be stored in unit or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10 mL vial is filled with 5 mL of a sterile-filtered 1% (w / v) aqueous solution of the compound, and the resulting mixture is lyophilized. A pharmaceutical composition containing a lyophilized Miro1 reducing agent is prepared by reconstituting the lyophilized compound, for example, using bacteriostatic water for injection.

[0163] The pharmaceutical composition can be formulated for intravenous, oral, implant, transmucosal, transdermal, intramuscular, intrathecal, or subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In other embodiments, the pharmaceutical composition is formulated for subcutaneous administration. The following delivery systems using some commonly used pharmaceutical carriers are merely representative of the many embodiments contemplated for administering the compositions of the present invention.

[0164] The components of the pharmaceutical composition can be supplied separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate.When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, sufficient sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0165] In some embodiments, the pharmaceutical composition is provided as a dry, sterile, lyophilized powder that can be reconstituted to the appropriate concentration for administration to a subject. In some embodiments, the pharmaceutical composition is provided as a water-free concentrate. In some embodiments, the pharmaceutical composition is provided as a dry, sterile, lyophilized powder in a unit dosage of at least 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, or at least 75 mg.

[0166] Administration method In the subject methods, the active agent can be administered to the subject using any convenient means capable of resulting in the desired reduction in impaired mitochondrial integrity and / or function, a reduction in any associated neurological disorders, and the like.

[0167] Therefore, the agent can be incorporated into various preparations for therapeutic administration.More specifically, the agent of the present invention can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[0168] In pharmaceutical dosage forms, the drugs may be administered in the form of their pharmaceutically acceptable salts. or they may be used alone or in suitable association and combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0169] For oral preparations, the agent may be used alone or in combination with suitable additives to form tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato starch, with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin, with disintegrating agents such as corn starch, potato starch or sodium carboxymethylcellulose, with lubricants such as talc or magnesium stearate, and, if desired, with diluents, buffers, wetting agents, preservatives and flavorings.

[0170] The drug can be formulated into a preparation for injection by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives can be used as necessary.

[0171] Medicaments can be utilized in aerosol formulations to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0172] Furthermore, the drug can be made into a suppository by mixing with various bases such as emulsifying bases or water-soluble bases.The compound of the present invention can be administered rectally via suppositories.Suppositories can contain vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0173] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, e.g., teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of a composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitor(s) in a composition as a solution in sterile water, saline, or another pharmaceutically acceptable carrier.

[0174] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the subject.

[0175] Other modes of administration can also be used with the subject invention. For example, the agents of the present invention can be formulated into suppositories, and in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition includes traditional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w / w), preferably about 1% to about 2%.

[0176] Intranasal formulations usually contain a vehicle that does not cause irritation to the nasal mucosa and does not significantly interfere with ciliary function. Diluents such as water, aqueous saline, or other known substances can be used with the present invention. Nasal formulations can also contain preservatives, such as, but not limited to, chlorobutanol and benzalkonium chloride. Surfactants are used to prevent targeting by the nasal mucosa. May be present to enhance absorption of the protein.

[0177] The agent of the present invention can be administered as an injection.Usually, injectable composition can be prepared as liquid solution or suspension, but also can be prepared in solid form suitable for solution or suspension in liquid vehicle before injection.Preparation can also be emulsified or active ingredient can be encapsulated in liposome vehicle.

[0178] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof.In addition, if necessary, vehicle can contain small amounts of auxiliary substances, such as wetting or emulsifying agents, or pH buffering agents.The actual method of preparing such dosage forms is known or obvious to those skilled in the art.For example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th Edition, 1985, Remington: The Science and Practice of Pharmacy, AR Gennaro, (2000) Lippincott, Williams & Wilkins.In any case, the composition or preparation to be administered contains an appropriate amount of drug to achieve the desired state in the treated subject.

[0179] Pharmaceutically acceptable excipients, for example, vehicles, adjuvants, carriers, or diluents, are readily available to the public. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0180] Administration route Conventional and pharmaceutically acceptable administration routes include intranasal, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical, intravenous, rectal, nasal, oral and other parenteral administration routes.Administration routes may be combined as needed or adjusted depending on the drug and / or desired effect.The composition may be administered in a single dose or multiple doses.

[0181] The agent can be administered to a subject using any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. Generally, routes of administration contemplated by the present invention include, but are not limited to, enteral, parenteral, or inhalation routes.

[0182] Parenteral administration routes other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, that is, any administration route other than through the digestive tract.Parenteral administration can be carried out to achieve systemic or local delivery of drugs.When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[0183] Agents can also be delivered to a subject by enteral administration. Enteral administration routes include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

[0184] Methods of administering drugs through the skin or mucosa include, but are not limited to, topical application of suitable pharmaceutical preparations, transdermal delivery, injection, and epidermal administration. For transdermal delivery, absorption enhancers or iontophoresis are preferred. Iontophoretic delivery can be achieved using commercially available "patches," which deliver the product continuously through intact skin via electrical pulses for periods of several days or longer.

[0185] kit Also provided are reagents, devices, and kits thereof for carrying out one or more of the above-described methods. The subject reagents, devices, and kits thereof can vary widely. Subject reagents and devices include those described above with respect to the method of treating Parkinson's disease in a subject.

[0186] The kit is provided with a unit dose of active agent, for example, oral or injection dose.In this kit, in addition to the container that contains the unit dose, the package insert of information that explains the use of the drug in the treatment of the target pathological condition and its associated benefits is provided.Preferred compound and unit dose are as described above.

[0187] In one embodiment, the kit includes a MIRO1-reducing agent and a pharmaceutically acceptable carrier. In certain embodiments, the MIRO1-reducing agent and the pharmaceutically acceptable carrier are packaged separately. For example, the MIRO1-reducing agent may be included in the kit in a dry form packaged in a container or vial, separate from the carrier. In other embodiments, the MIRO1-reducing agent is formulated in a pharmaceutically acceptable carrier.

[0188] In certain embodiments, the kit comprises at least one additional therapeutic agent, hi certain embodiments, the additional therapeutic agent is selected from the group consisting of levodopa, a dopamine agonist, an MAO-B inhibitor, amantadine, an anticholinergic, a PUM1 antagonist, an SR protein antagonist, a Parkin agonist, a PINK1 agonist, a 4E-BP1 agonist, a Drp1 agonist, an Atg1 agonist, a TauS2A agonist, an Rbf1 agonist, a Dp antagonist, an E2f1 antagonist, a Polo-like kinase 2 antagonist, and a Notch agonist.

[0189] In addition to the above components, the subject kits further include instructions for carrying out the subject methods of diagnosis or treatment (e.g., instructions regarding route of administration, dosage, dosing regimen, site of administration, etc.). These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, in the kit packaging, in a package insert, etc. Yet another means is a computer-readable medium on which the information is recorded, such as a diskette, CD, etc. Yet another form in which it may be present is a website address that can be used via the Internet to access the information at a remote location. Any convenient means may be present in the kit.

[0190] Crossing the blood-brain barrier The blood-brain barrier limits the uptake of many therapeutic agents from the systemic circulation into the brain and spinal cord. Molecules that cross the blood-brain barrier use two major mechanisms: free diffusion and facilitated transport. Due to the presence of the blood-brain barrier, achieving beneficial concentrations of a given therapeutic agent in the central nervous system (CNS) may require the use of drug delivery strategies. Delivery of therapeutic agents to the CNS can be achieved by several methods.

[0191] One method relies on neurosurgical techniques. In severely ill patients, such as accident victims or patients with various forms of dementia, surgical intervention is warranted despite the associated risks. For example, therapeutic agents can be delivered by direct physical introduction into the CNS, such as intraventricular or intrathecal injection of the drug. Intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Introduction methods can also be provided by rechargeable or biodegradable devices. Another approach is the disruption of the blood-brain barrier with substances that increase its permeability. Examples include drugs with low diffusibility, such as mannitol, drugs that increase cerebrovascular permeability, such as etoposide, or vasoactive drugs, such as leukotrienes. Examples include intra-arterial infusion of vasopressin. See Neuwelt and Rappoport (1984) Fed. Proc. 43:214-219, Baba et al. (1991) J. Cereb. Blood Flow Metab. 11:638-643, and Gennuso et al. (1993) Cancer Invest. 11:638-643.

[0192] Additionally, it may be desirable to administer pharmaceutical agents locally to the area in need of treatment, which may be achieved, for example, by local infusion during surgery, infusion, catheter, or implant, which may be a porous, non-porous, or gel-like material, including a membrane such as a silastic membrane, or a fiber.

[0193] Therapeutic compounds can also be delivered by using pharmacological techniques, including chemical modification or screening for analogs that cross the blood-brain barrier. Compounds can be modified to increase the hydrophobicity of the molecule, reduce the net charge or molecular weight of the molecule, or modify the molecule so that it resembles one that is normally transported through the blood-brain barrier. See Levin (1980) J. Med. Chem. 23:682-684, Pardridge (1991): Peptide Drug Delivery to the Brain, and Kostis et al. (1994) J. Clin. Pharmacol. 34:989-996.

[0194] Encapsulation of drugs in a hydrophobic environment, such as a liposome, is also effective for delivering drugs to the CNS. For example, WO 91 / 04014 describes a liposomal delivery system in which a drug is encapsulated within liposomes to which molecules that are normally transported across the blood-brain barrier have been added.

[0195] Another method for formulating drugs to pass through the blood-brain barrier is to encapsulate drugs in cyclodextrin.Any suitable cyclodextrin that passes through the blood-brain barrier can be used, including but not limited to J-cyclodextrin, K-cyclodextrin and their derivatives.Generally, refer to United States Patent No. 5,017,566, United States Patent No. 5,002,935 and United States Patent No. 4,983,586.This composition can also contain glycerol derivatives as described in United States Patent No. 5,153,179.

[0196] Delivery can also be achieved by conjugating therapeutic agents to transportable agents to obtain novel chimeric transportable therapeutic agents. For example, vasoactive intestinal peptide analogs (VIPa) exert their vasoactive effects only after conjugation to a monoclonal antibody (Mab) against a specific carrier molecule, the transferrin receptor, and this conjugation facilitated the uptake of the VIPa-Mab conjugate through the blood-brain barrier. See Pardridge (1991) and Bickel et al. (1993) Proc. Natl. Acad Sci. USA 90:2618-2622. Several other specific transport systems have been identified, including, but not limited to, those for transporting insulin or insulin-like growth factors I and II. Other suitable nonspecific carriers include, but are not limited to, pyridinium, fatty acid, inositol, cholesterol, and glucose derivatives. Certain prodrugs have been described in which, upon entry into the central nervous system, the drug is cleaved from the carrier to release the active drug. See U.S. Patent No. 5,017,566. [Example]

[0197] Example 1: Assay to identify Miro-reducing agents cell culture Human skin isolated from a PD patient (ND33424) and a healthy control (ND36091) Primary skin fibroblasts were cultured in cell culture medium DMEM (ThermoFisher, 11995-065) supplemented with 10% fetal bovine serum (Gemini Bio-Products, 900-108, heat-inactivated), 1x AntiAnti (ThermoFisher, 15240096), and 1x GlutaMax (ThermoFisher, 35050061), maintained in a 37°C, 5% CO2 incubator with a humidified atmosphere.

[0198] Western blot Fibroblasts were cultured in high-glucose DMEM (SH30243.01, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (F0926, Sigma-Aldrich, and 900-108, Gemini Bio Products) and maintained in a humidified atmosphere at 37°C in a 5% CO2 incubator. Medium was refreshed every 3–4 days and split every 7–8 days. CCCP (C2759, Sigma-Aldrich) was prepared fresh at 40 mM in DMSO and applied at 40 μM in fresh medium (1:1000 dilution). IP was performed as described (Hsieh et al., 2016).

[0199] For transfection in fibroblasts, the medium was replaced with Opti-MEM (Gibco) before transfection. 0.5 μg of DNA or 2 μl of Lipofectamine 2000 was diluted in Opti-MEM to a final volume of 50 μl in two separate tubes at room temperature (22°C). The contents of the two tubes were gently mixed and incubated at room temperature for 25 minutes before being added to the fibroblasts. After 6 hours of transfection, the Opti-MEM containing the DNA-Lipofectamine complex was replaced with regular medium. After 18 hours of transfection, the fibroblasts were live-imaged or treated with Miro1 reducing agent and / or CCCP.

[0200] Mitochondria were isolated from cultured human fibroblasts as previously described with minor modifications. Briefly, fibroblasts treated with CCCP in DMSO or the same volume of DMSO were lifted with a cell scraper and mechanically homogenized in a Dounce homogenizer in 750 μl of isolation buffer (200 mM sucrose, 10 mM TRIS / MOPS, pH 7.4). After centrifugation at 500 g for 10 min, the crude supernatant was spun at 10,000 g for 10 min to pellet intact mitochondria. The mitochondrial pellet was washed twice with isolation buffer. After this step, the supernatant was designated the "cytosolic fraction (Cyto)" and the pellet was resuspended in 50 μl of lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, and 1% Triton X-100-T8787, Sigma-Aldrich) containing 0.25 mM phenylmethanesulfonyl fluoride (P7626, Sigma-Aldrich) and protease inhibitors (Roche) and designated the "mitochondrial fraction (Mito)."

[0201] Samples were mixed 1:1 with 2x Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, 0.02% bromophenol blue, 700 mM 2-mercaptoethanol) and boiled for 5 min before loading onto SDS-PAGE (Mito:Cyto = 25:1). 10% polyacrylamide gels (acrylamide:bis-acrylamide = 29:1) and Tris-glycine-SDS buffer (24.8 mM Tris, 192 mM glycine, 0.1% SDS) were used for electrophoresis.

[0202] After electrophoresis, nitrocellulose membranes (1620115, Bio-Rad) were used for wet transfer using Tris-glycine buffer (24.8 mM Tris, 192 mM glycine) at 360 mA for 2 hours on ice. The transferred membranes were first blocked overnight at 4°C in phosphate-buffered saline (PBS) containing 5% nonfat milk and 0.1% Tween-20, and then incubated with the following primary antibodies at 1:1,000: Mouse anti-Miro1 (WH0055288M1, Sigma-Aldrich), rabbit anti-Miro1 (HPA010687, Sigma-Aldrich) at 1:1,000, rabbit anti-Miro2 (HPA012624, Sigma-Aldrich) at 1:800, rabbit anti-VDAC (4661S, Cell Signaling Technology) at 1:1,000, mouse anti-mitofusin 2 (H00009927-M01, Abnova) at 1:1,000, mouse anti-parkin (sc32282, Santa Cruz Biotechnology) at 1:500, rabbit anti-LRRK2 (NB300-268, Novus Biotechnology) at 1:500 The membranes were incubated overnight at 4°C in blocking buffer with rabbit anti-OPA1 (ab42364, Abcam) at 1:750, mouse anti-β-actin (A00702, Genscript) at 1:1,000, mouse anti-ubiquitin (A-104, Boston Biochem) at 1:500, or rabbit anti-GAPDH (5174S, Cell Signaling Technology) at 1:3,000. HRP-conjugated goat anti-mouse or rabbit IgG (Jackson ImmunoResearch Laboratories) was used at 1:5–10,000. West Dura ECL reagent (34075, GE Healthcare) was used for ECL immunoblotting. Membranes were exposed to UltraCruz autoradiography film (Santa Cruz Biotechnology) and developed on a Konica Minolta SRX-101A developer. For fluorescent Western blots, the blots were probed with 1:5,000 Cy5-conjugated goat anti-mouse IgG (PA45009, GE Healthcare) and scanned using a Molecular Dynamics Storm 860 Imager (Amersham BioSciences, Piscataway, NJ) in the linear range for fluorescence detection at 635 nm. A representative raw blot is shown in the Supplementary Table. Experiments were repeated at least three times.

[0203] Statistics of fibroblast Western blotting data. All experiments were performed in a blinded manner, and the identity of the cell lines was unblinded by either the present inventors (NINDS cell lines) or PPMI researchers (PPMI cell lines). Films were scanned or digital blots were exported as 16-bit tiff format. Protein band intensities were measured using ImageJ (version 1.48V, NIH). The intensity of each band in the mitochondrial fraction was normalized to the intensity of the mitochondrial-loaded control VDAC from the same blot and expressed as a percentage of the mean for DMSO-treated healthy-1. This control was included in all independent experiments. The mean values ​​± SEM for Miro1 are reported in Table S1A. The mean values ​​for Miro1, mitofusin 2, LRRK2, and parkin were imported into the heatmap in Figure 1D. VDAC band intensities were not significantly different among all fibroblast cell lines and conditions (p = 0.8490, one-way ANOVA with correction, post-hoc Tukey's test). n = 3–35 independent experiments. To compare normalized Miro1 band intensities within the same subject, a Mann-Whitney U test was performed (DMSO vs. CCCP), and P values ​​are reported in Table S1A. The number of subjects with P values ​​>0.05 and <0.05, respectively, was counted and used in the Fisher exact test in Table A. Linear regression analysis was used to determine correlations with the ratio of Miro1 intensity (mean intensity with CCCP / DMSO). One-way ANOVA with post hoc Tukey's test with correction was performed on band intensities at baseline (P >0.3509 for all markers for "Cyto" + "Mito"). Statistical analysis was two-tailed and performed using Prism software (version 5.01, GraphPad).

[0204] ELISA All experiments were performed blinded. 40 μM CCCP in DMSO or an equal volume of DMSO alone was applied to fibroblasts for 6 h, and then the cells were lysed in lysis buffer (100 mM Tris) containing a protease inhibitor cocktail (539134, Calbiochem). Cells were lysed in 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, and 0.5% sodium deoxycholate. Cell debris was removed by centrifugation at 17,000 g for 10 minutes at 4°C. Covered to prevent evaporation, microplates (MaxiSorp, NUNC) were coated overnight at room temperature with mouse anti-Miro1 (clone 4H4, WH0055288M1, Sigma-Aldrich), chicken anti-β-actin (LS-C82919, LifeSpan BioSciences), mouse anti-β-actin (A00702, Genscript), or mouse anti-ATP5β (ab14730, Abcam) in 0.1 M sodium carbonate-sodium bicarbonate buffer (3:7, pH 9.6) at 1:1,000. After washing the plate with wash buffer (0.05% Tween 20 in PBS, pH 7.3), nonspecific binding sites were blocked with 2% BSA in PBS (BP-1600-100, Fisher Scientific) for 1 hour. Next, 50 μl of the cell lysate prepared above, purified full-length Miro1 protein (0–900 ng / ml, ab163047, Abcam), or serial dilutions of cell lysates from fibroblasts (Healthy-1) or HEK cells (1 / 16–2x) were added and incubated for 2 hours at room temperature. After washing, the plates were incubated for 2 hours with biotinylated rabbit anti-Miro1 (ARP44818_P050, Aviva Systems Biology) at 1:1000 or biotinylated rabbit anti-β-actin (#5057S, Cell Signaling Technology) at 1:500 in 100 μl diluent (1% BSA in PBS, pH 7.3). The plates were then washed and incubated for 20 minutes with horseradish peroxidase-conjugated streptavidin (21130, Thermo Scientific) at 1:2000 in 100 μl diluent. The plates were washed again, and 100 μl of tetramethylbenzidine liquid substrate (SB01, Life Technologies) was added and incubated for an additional 20 minutes.The colorimetric reaction was stopped with 50 μl of 1 M H2SO4, and absorbance was read at 450 nm using a microplate reader (FlexStation 3, Molecular Devices). An experiment to generate a standard curve was included on each plate, and representative standard plots are shown in the figures. Each data point was obtained from four independent experiments with two technical replicates each time. A Mann-Whitney U test was performed to compare Miro1 signals within the same subject (DMSO vs. CCCP). Basal Miro1 signals were not significantly different among all cell lines (P > 0.1177, one-way ANOVA with correction followed by Tukey's test). The distribution of data points was presented as a boxplot (extreme values, quartiles, and median).

[0205] GTPases IPed Miro1 was eluted from the Protein A beads by incubation with 60 μl of 0.2 M glycine (pH 2.5) for 10 min, followed by centrifugation at 3000 g for 2 min. The supernatant (eluate) was collected and neutralized by adding an equal volume of Tris pH 8.0. The eluate was processed for colorimetric analysis of GTPase activity at room temperature using the GTPase Activity Kit (602-0120, Novus Biologicals) according to the manufacturer's instructions. 100 μl of the eluate was mixed with 100 μl of substrate / buffer mixture (20 μl of 0.5 M Tris buffer, 5 μl of 0.1 M MgCl2, 10 μl of 10 mM GTP, and 65 μl of ddH2O). 200 μl of inorganic phosphate (Pi) standards (0–50 μM) were prepared in water. Next, 50 μl of PiColorLock™ mixture was added to either the Pi standards or the samples. After 2 minutes, the stabilizer was added and mixed thoroughly. After 30 minutes, the absorbance was read at 650 nm by a microplate reader (FlexStation3, Molecular Devices). The assay was validated using purified Miro1 protein (ab163047, Abcam). Concentrations of purified Miro1 protein ranging from 0 to 900 ng / ml resulted in a 650 nm absorbance that reflected the released Pi. showed a linear dependence on luminosity (R 2 =0.9711, P=0.0021). Omitting GTP or Miro1 eliminated the signal (Pi<3 μM). Experiments were repeated twice.

[0206] qPCR Total RNA was extracted using TRIzol® (GIBCO) according to the manufacturer's instructions, with at least 10 6 Miro1 was extracted from 1000 cells. The concentration of total RNA was measured using Nanodrop. 1 μg of total RNA was then subjected to DNA digestion using DNase I (Ambion) followed immediately by reverse transcription using iScript Reverse Transcription Supermix (1708841, BIO-RAD). qPCR was performed using a StepOnePlus™ instrument (Thermo Fisher Scientific) and SYBR® Green Supermix (172-5270, BIO-RAD) according to the manufacturer's instructions. Human GAPDH was amplified as an internal standard. Expression levels were analyzed using StepOne™ software (version 2.2.2). The relative expression level of Miro1 was divided by the expression level of GAPDH from the same experiment. Each sample was analyzed in duplicate from four independent biological replicates. The following primers were used: GAPDH forward: 5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID NO: 1) GAPDH reverse: 5'-TCCACCACCCTGTTGCTGT-3' (SEQ ID NO: 2) Miro1 forward: 5'-GGGAGGAACCTCTTCTGGA-3' (SEQ ID NO: 3) Miro1 reverse: 5'-ATGAAGAAAGACGTGCGGAT-3' (SEQ ID NO: 4).

[0207] Screening Assay Protocol Fibroblasts were plated onto clear-bottom, black-walled 384-well plates (EK-30091, E&K Scientific, Santa Clara, CA) at 2000 cells / well using a Matrix Wellmate dispenser (Thermo Scientific, Sunnyvale, CA), and the plates were incubated at 37°C and 5% CO for 24 hours. Next, using a Caliper Life Sciences Staccato system with a Twister II robot and a fully automated liquid handling system, a Sciclone ALH3000 (Caliper Life Sciences, Alameda, CA, USA) integrated with a V&P Scientific pin tool, defined concentrations of chemical library compounds were added, and the plates were incubated at 37°C and 5% CO for 10 hours. Then, 20 μM FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Sigma-Aldrich, C2920) in cell culture medium was added to defined wells, and the plates were incubated at 37°C and 5% CO for an additional 14 hours. Cells were fixed with ice-cold 90% methanol (Fisher, 482332) for 20 min at -20°C, incubated with blocking buffer (10% normal goat serum (NGS) (ThermoFisher, 50062Z), 0.5% BSA (ThermoFisher, BP1600), 0.2% Triton X-100 (ThermoFisher, T8787) for 15 min at room temperature (RT), and then incubated with a 1:100 solution of anti-Miro1 (Sigma-Aldrich, HPA0101687) in blocking buffer overnight at 4°C. Samples were plated. Washed with 1x PBS (ThermoFisher, 10010-049) using a Washer multivalve (Bio-Tek, ELx405UV), incubated with 1:500 goat anti-rabbit IgG (H+L) cross-adsorbed, AlexaFluor488 (ThermoFisher, A11008) in blocking buffer for 2 hours at 25°C, washed again with 1x PBS, and finally incubated with 1x PBS. 1.0 μg / mL DAPI (ThermoFisher, D1306) in BS was added, and the plate was sealed using PlateLoc (Velocity11, 01867.001). Unless otherwise specified, all liquids were dispensed using a Multidrop384 (Titertek, 5840200). Fluorescent signals in the plate were automatically imaged using ImageXpress Micro (Molecular Devices, IXMicro), and the data were analyzed using MetaXpress Analysis (Molecular Devices). The percent Miro1 reduction was calculated as follows: % Miro1 positive cells / median = [(positive cell Miro integrated intensity / total nuclear Miro integrated intensity) - plate median of this same calculation (median % positive Miro / nuclear Miro in plate data)] / [plate median of this same calculation (median % positive Miro / nuclear Miro in plate data)]. Positive cells are cells with Miro1 immunostaining. A zero value indicates no difference from the median (inactive). A negative value indicates a decrease in Miro. A positive value indicates an increase in Miro. Screening and data analysis were performed in collaboration with the Stanford University High-Throughput Bioscience Centers (HTBC).

[0208] Hit confirmation - immunocytochemistry, confocal microscopy, image analysis Fibroblasts were plated at 400,000 cells / well in 6-well plates (VWR, 10861-554) on cover slips (Fisher Scientific, 22-293232), and the plates were incubated at 37°C and 5% CO2 for 24 hours. Next, newly ordered compounds dissolved in DMSO were added at the specified concentrations, and the plates were incubated at 37°C / 5% CO2 for 10 hours. FCCP (final concentration 20 μM, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Sigma-Aldrich, C2920) in cell culture medium was then added to the specified wells, and the plates were incubated at 37°C and 5% CO2 for an additional 14 hours. Cells were fixed with ice-cold 90% methanol (Fisher, 482332) for 20 min at -20°C, incubated with blocking buffer (10% normal goat serum (NGS) (ThermoFisher, 50062Z), 0.5% BSA (ThermoFisher, BP1600), 0.2% Triton X-100 (ThermoFisher, T8787)) for 15 min at 25°C, and then incubated with anti-Miro1 (Sigma-Aldrich, HPA0101687) at 1:100 in blocking buffer overnight at 4°C. Samples were washed three times with 1x PBS (ThermoFisher, 10010-049) and incubated with 1:500 goat anti-rabbit IgG (H+L) cross-adsorbed AlexaFluor 488 (ThermoFisher, A11008) in blocking buffer for 2 hours at 25°C. Then, washed three times again with 1x PBS, samples were mounted on glass slides with ProLong™ Glass Antifade Mountant with NucBlue™ Stain (hard setting, ThermoFisher, P36983) and allowed to harden overnight. Samples were imaged at 25°C with a 20x / NA 0.60 oil Plan-Apochromat objective on a Leica SPE laser scanning confocal microscope (JH Technologies) using identical imaging parameters across different genotypes. Images were analyzed using ImageJ (version 1.48, NIH) at Intensity 1000. Processing was performed using the Ratio Nuclei Cytoplasm Tool (http: / / dev.mri.cnrs.fr / projects / imagej-macros / wiki / Intensity_Ratio_Nuclei_Cytoplasm_Tool), and the mean cytoplasmic intensity (average intensity in the cytoplasmic region) was plotted.

[0209] Drugs from the NIH Clinical Collection Library were screened at a defined 10 μM concentration, with four biological replicates for each drug (377 compounds). Eleven of the 377 unique compounds inhibited Miro1 after FCCP treatment in all biological replicates. The compounds were found to reduce Miro1 protein levels by 3 standard deviations. These compounds are ranked in order of the extent of Miro1 reduction (Table 1A). Twelve small molecules that reduced Miro1 protein levels in three of four biological replicates were ranked in order of the extent of Miro1 reduction. The BioMol FDA library was screened in duplicate at different 5-fold doses (1.25, 2.5, 5, 10, and 20 μM) to identify three hits that partially but significantly reduced Miro1 protein levels after FCCP treatment in a dose-dependent manner. These hits are ranked in order of the extent of Miro1 reduction. Six compounds were found to reduce Miro1 protein levels by 20% or more (Table 1A). Furthermore, an additional 12 small molecules reduced Miro1 protein levels in three of four biological replicates (Table 1B). We then screened the Biomol FDA library in duplicate at five-fold doses (1.25, 2.5, 5, 10, and 20 μM). From 175 unique compounds, we identified three hits that partially but significantly reduced Miro1 protein levels after FCCP treatment in a dose-dependent manner. We ranked these three hits in order of the degree of Miro1 reduction (Table 1A). Overall, we identified 14 active compounds (2.5% primary hit rate) and 12 potential active compounds.

[0210] To reduce experimental biases, such as the freshness of compounds, experimental reagents, and imaging devices, the 14 hits identified in the high-throughput screening were individually validated. The 14 compounds were tested at their highest screening concentrations in at least four biological replicates using the same sporadic PD strain. Miro1 levels were also assessed without mitochondrial depolarization (DMSO), but compound treatment revealed drug effects on Miro1 on polarized, healthy mitochondria. When samples were imaged under a confocal microscope with identical settings, a negative control without primary antibody produced no signal under the imaging settings. Seven of the initial 14 hit compounds were found to consistently reduce Miro1 protein after FCCP treatment (Table 1A). One compound, fenbufen, appeared to be toxic (Table 1A). Of the seven confirmed hits, the compounds tranilast and benidipine HCl had no effect on basal Miro1 levels, whereas the compounds physostigmine, pravastatin sodium, lofepramine, temozolomide, and escitalopram oxalate reduced Miro1 protein already at baseline.

[0211] Example 2: Miro1 represents a subset of Parkinson's disease Identifying molecular targets and pharmacodynamic markers for Parkinson's disease (PD) would enable more effective clinical management and experimental therapies. Miro1 localizes to the mitochondrial surface and mediates mitochondrial movement. Miro1 is removed from depolarized mitochondria and promotes mitochondrial clearance via mitophagy. Here, we explore the clinical utility of Miro1 for detecting PD and measuring potential treatments. We measured the Miro1 response to mitochondrial depolarization using a biochemical assay in skin fibroblasts from a wide range of PD subjects and found that over 94% of subject fibroblast cell lines were unable to remove Miro1 after depolarization.

[0212] Miro1 is resistant to removal from depolarized mitochondria in skin fibroblasts from a large population of PD subjects. Skin fibroblasts can be easily obtained from subjects through a minimally invasive and painless procedure. We aimed to determine the frequency of the Miro1 phenotype in skin fibroblasts from a large cohort of both sporadic and familial PD subjects. We fractionated mitochondria after CCCP treatment, which depolarizes the mitochondrial membrane potential (ΔΨm). In wild-type controls, both Miro1 and mitofusin 2 were removed from damaged mitochondria as detected by Western blotting 6 hours after treatment (Figures 1A and 1B).

[0213] Table S1A. Related to Figure 1. Demographic information and Miro1 values ​​are shown. Demographic information and clinical scores are obtained from the consortium's online database. Not all information is available for all subjects. Certain analyses use data from subjects with available information. P values ​​are derived from comparisons of normalized Miro1 intensities within the same subject (CCCP vs. DMSO).

[0214] The OMM protein mitofusin 2 is a target of the PINK1-Parkin pathway for depolarization-induced degradation, but not of LRRK2. We included mitofusin 2 as a readout to compare phenotypic frequency in PD with Miro1. We screened a total of 71 PD and three at-risk fibroblast cell lines, including the entire PD fibroblast collection from the National Institute of Neurological Disorders and Stroke (NINDS) Human and Cellular Repository and the first published PD control cohort from the Parkinson's Progression Markers Initiative (PPMI). All subjects were diagnosed with PD and had no other signs of neurological disorder. At-risk subjects were asymptomatic family members younger than the proband and had the same genetic mutation (in LRRK2 or SNCA). 28 subjects had a family history. We included 22 controls, consisting of 12 age-, sex-, and race-matched healthy subjects recruited from the same cohort and 10 subjects with other neurological disorders, including Huntington's disease (HD) or Alzheimer's disease (AD) (Table A). We performed the assay in a blinded manner.

[0215] [Table 1]

[0216] Notably, we found a consistent impairment in Miro1 removal from the mitochondrial fraction 6 hours after CCCP treatment in 69 PD and risk cell lines (93.2%). In contrast, Miro1 was efficiently removed after depolarization in every single control subject (0%) (Table A). The phenotype was more strikingly demonstrated when the mean band intensities were imported into a heat map, and the lack of color change after treatment reflected the failure of Miro1 removal (Figure 1D). The lack of Miro1 color change occurred widely in PD subjects. A smaller number of PD cell lines also failed to remove mitofusin 2 after CCCP treatment (Figure 1D). Basal protein levels of Miro1 and mitofusin 2 were nearly equivalent among all cell lines (Figure 1D; P > 0.0906).

[0217] This phenotype of Miro1 removal was significantly associated with PD (P<0.00001). The ratio of Miro1 intensity (with CCCP / with DMSO) also correlated significantly with PD (P<0.0001; Figure 2A), but not with age (at sampling and onset) or gender (Figures 2B-2D). There was no significant correlation between the Miro1 ratio and disease progression (years with PD) or clinical symptoms in subjects using the Unified Parkinson's Disease Rating Scale (UPDRS), Hoehn and Yahr scale, or Mini-Mental State Examination (Mini-Mental State Examination) (Figures 2E-2H). We confirmed that cell passage number within the range of 5-19 did not affect the phenotype. Taken together, these observations indicate that failure to remove Miro1 from damaged mitochondria is a common cellular defect in a large population of PD subjects.

[0218] The LRRK2 and PINK1-Parkin pathways are extensively affected in PD fibroblasts. We previously identified two parallel molecular pathways, both essential for the removal of Miro1 from the OMM of depolarized mitochondria-LRRK2 and PINK1-Parkin axes. To investigate the mechanism underlying Miro1 accumulation on damaged mitochondria in PD fibroblasts, we tested the hypothesis that the accumulation is due to impaired LRRK2 or PINK1-Parkin pathways. We established that in wild-type control fibroblasts, mitochondrial depolarization by CCCP treatment for only 1 hour triggered the recruitment of cytosolic LRRK2 and Parkin to mitochondria and Miro1, prior to Miro1 removal at 6 hours (Figures 1A and 1C). Antibodies against LRRK2, Parkin, and Miro1 have been validated in human cells lacking the corresponding genes.

[0219] We used this readout to screen all 96 fibroblast cell lines and found a variety of phenotypes in the cells of interest. Some cell lines showed impaired recruitment of only LRRK2 to depolarized mitochondria, some showed impaired recruitment of only Parkin, and some showed defects in recruitment of both proteins. Because failure of LRRK2 translocation to damaged mitochondria prevents the subsequent removal of Miro1 but not Mitofusin 2, this result may explain the lower frequency of the Mitofusin 2 phenotype in these subjects (Figure 1D). We found that in seven PD cell lines, recruitment of both LRRK2 and Parkin to damaged mitochondria appeared normal compared to controls, while Miro1 removal was still impaired (Figure 1D), suggesting that additional mechanisms may play a role in these cells. Basal levels of LRRK2 and Parkin were comparable in all cell lines (P > 0.8684). Taken together, our results provide evidence that the LRRK2 and PINK1-Parkin pathways are greatly affected in fibroblasts of PD subjects, resulting in convergent downstream impairments to remove Miro1 from damaged mitochondria.

[0220] ELISA confirms the high frequency and specificity of the Miro1 phenotype in PD fibroblasts. We established an enzyme-linked immunosorbent assay (ELISA) to detect Miro1 responses to CCCP (Figure 3). This is useful for clinical testing. We examined the 14 PD / risk and 15 control cell lines used in Figure 1D. For each cell line, the Miro1 response to mitochondrial depolarization (Figure 3A) was consistent with the results obtained using mitochondrial fractions and Western blotting (Figure 1D, Table S1A). We used this ELISA to validate an additional independent cohort. We included 40 healthy controls and 12 PD subjects from the Stanford Alzheimer's Disease Research Center (ADRC) and the Coriell Institute (Table S1B). We found that Miro1 was efficiently degraded upon depolarization in all control cell lines but not in any of the PD subjects (Figure 1E). This finding validated the high frequency of Miro1 accumulation on depolarized mitochondria in the PD population (Figure 1, Table A). We also obtained cell lines from subjects with movement disorders exhibiting clinical presentations similar to PD, including four cases of sporadic dementia with Lewy bodies (DLB; Stanford), three cases of frontotemporal degeneration (FTD; Coriell and NINDS), two cases of sporadic corticobasal degeneration (CBD; Mayo Clinic), and three cases of sporadic progressive supranuclear palsy (PSP; Mayo Clinic) (Table S1B). Miro1 was effectively degraded after CCCP treatment in all cell lines (Figure 1F), demonstrating the specificity of Miro1 accumulation in PD. Establishing an ELISA to detect Miro1 may facilitate the clinical application of our findings.

[0221] Example 3: Effects of Mitochondrial Reducers on Mitochondrial Motility Dopaminergic neurons expressing LRRK2G2019 were grown on glass coverslips and plated in 35 mm Petri dishes containing Hibernate E low-fluorescence medium (BrainBits) on a heated stage at 37°C. Images were taken with a 63x water-immersion objective (NA 0.9) while exciting at 561 nm or 488 nm. The LRRK2GS2019-expressing neurons were divided into a test pool and a control pool. The test pool was treated with the Miro-reducing agent identified in Example 1, while the control pool was untreated. After 10 hours, both pools of neurons were treated with FCCP for 14 hours. Mitochondrial movement was measured.

[0222] Time-lapse movies were acquired consecutively at 3-5 second intervals before and after the addition of mitochondria reducing agent (10 μM). Axons longer than 50 μm were selected for recording. Movies were recorded for 120-300 minutes. 250 nM TMRM was applied for 30 minutes as needed. For quantification, kymographs were generated from the time-lapse movies using ImageJ, representing 100-second periods either immediately before or at different time points after the addition of antimycin A. Each kymograph was then imported into a macro written in Labview (NI, TX), and individual mito-dsRed points were traced using a mouse-driven cursor at the center of the mito-dsRed object. Matlab (The MathWorks, MA) was used to quantify the following parameters: 1) instantaneous velocity of each mitochondrion, 2) average velocity of those mitochondria in motion, 3) percent of time each mitochondrion was in motion, 4) stopping frequency, and 5) reversal frequency. Mitochondrial length and intensity were measured using ImageJ. Neurons in the test pool with depolarized mitochondria will exhibit reduced mitochondrial movement as measured by the parameters described above, while the control pool of neurons with depolarized mitochondria will exhibit mitochondrial movement equivalent to that before addition of the Miro reducer.

[0223] Example 4: Effects of Miro-reducing agents on Parkinson's disease in iPSC neurons LRRK2G2019S iPSC-derived neuronal cultures were treated with a range of concentrations of the mitochondrial stressor antimycin A for 6 hours to induce oxidative stress. LRRK2G2019S iPSC-derived neuronal cultures exhibited antimycin A dose-dependent neuronal loss, indicating vulnerability to mitochondrial stress. LRRK2G2019S neurons treated with Mitochondrial Reducer rescued the neuronal loss caused by 1 or 10 μM antimycin A, demonstrating protection against neuronal loss caused by mitochondrial stress.

[0224] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0225] [Table 2]

[0226] [Table 3]

[0227] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 896,450, filed September 5, 2019, which is incorporated herein in its entirety for all purposes.

Claims

1. 1. A method of screening a candidate agent for activity in decreasing MIRO1 levels, comprising:

10. A method for manufacturing a device according to claim 1, wherein the device is substantially as shown in the drawings and substantially as described in the specification. method.

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