Treatment of neurological diseases

Abstract

The invention is directed to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4Hdibenzo[de,g]quinoline-10,11-diol for the treatment of diseases mediated by protein expressions and gene regulations, as well as the use of personalized medicine approach.

Description

1959747.00063 TREATMENT OF NEUROLOGICAL DISEASES RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Patent Application No. 63 / 529,307, filed July 27, 2023, the entire content of which is incorporated herein by reference. FIELD

[0002] The invention is directed to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol for the treatment of diseases mediated by protein expression and gene regulation, as well as the use of personalized medicine approach. BACKGROUND

[0003] Therapeutic targeting of oxidative stress in amyotrophic lateral sclerosis (ALS) has not made much progress into clinical benefit for patients to date. This might be rationalized by the current use of antioxidant drugs that target the downstream biology. An alternative approach is to target the entire biological pathway by engaging the master regulator, the transcription factor nuclear factor erythroid 2–related factor 2 (NRF2), and thus, involving the activation of several neuroprotective pathways (Nguyen et al., Annu. Rev. Pharmacol. Toxicol., 2003. 43:233–60). Under normal conditions, NRF2 activation is maintained at low levels through negative regulation by the Kelch-like ECH-associated protein 1 (Keap1) complex. When exposed to oxidative stress, the cysteine residues on Keap1 are oxidized, dissociating the interaction between NRF2 and Keap1 (Wakabayashi et al., Proc Natl Acad Sci U S A., 200417;101(7):2040-5). This prevents ubiquitination of NRF2, allowing its translocation to the nucleus and driving the expression of detoxification and antioxidant enzymes via its interaction with the antioxidant response elements (AREs) on multiple cytoprotective genes.

[0004] The expression of NRF2 activates a “programmed cell life” response, up-regulating cytoprotective and antioxidant genes as well as genes related to glutathione (GSH) synthesis, nicotinamide adenine dinucleotide phosphate (NADPH) generation and, lipid and glucose / glycogen metabolism, which are neuroprotective. Downregulation of NRF2 has also been reported in both mouse models and human cases of sporadic ALS (sALS). Previous studies have demonstrated the beneficial effects of NRF2 activation on ALS models. In particular, the astrocytic-specific expression of NRF2 delayed disease onset and 1 507965132.11959747.00063 extended survival in SOD1 transgenic mouse models. In theory, NRF2 activating compounds could protect against astrocytic toxicity towards MNs, delaying disease progression in ALS patients.

[0005] Genome-wide expression profiling is a widely used tool to investigate the transcriptome. Changes in gene expression in ALS have been explored using microarray, a library to detect the expression of thousands of genes at the same time, or the sequencing of mRNA (Cooper-Knock et al. Neurology., 2012, 8(9), 518–530). While transcriptome analysis gives a good indication of differential gene expression (DGE), the significance of the data is limited since mRNA levels do not necessarily correlate with the level of protein translated. This variation can be attributed to protein degradation, oxidative stress, or the control of protein synthesis. Therefore, the profiling of the translatome, which involves the sequencing of the mRNAs recruited to the ribosomes for protein synthesis, should be more reflective of protein expression changes and the directionality of disease processes in ALS induced astrocytes (iAstrocytes).

[0006] There are three main methodologies of translatome profiling, including polysomal profiling, ribosomal profiling, and more recently, ribosome affinity purification techniques. Early methods to study the global translatome were carried out by comparing the ribosome- bound mRNA to the total mRNA present within the sample. Polysomal profiling is a classical technique of mRNA extraction that involves the separation of mRNAs depending on the number of bound ribosomes (polysomes) using a sucrose gradient. While polysome profiling was considered as the “gold standard” for many years, the use of a sucrose gradient required specialized and expensive equipment as well as additional precipitation steps, due to the fact that the heparin, a potent RNAse inhibitor, presents in the sucrose solution. Polysome fractions may also be contaminated with other high molecular weight complexes such as lipid rafts or pseudo-polysomes.

[0007] Ribosomal profiling is based on the sequencing of ribosome-protected fragments (RPFs) after RNase I treatment of the cell lysate. This methodology works on the basis that the average ribosome density per mRNA correlates with the level of protein synthesis. For translatome analysis, mRNA extraction and sequencing are performed in parallel to normalize RPFs to the total mRNA. This method has its own challenges, including a sucrose gradient like polysome profiling, a labor-intensive method and potential contamination with pseudoRPFs from the structured double-stranded region of RNA, since RNase I only degrades single-stranded RNA, leading to misinterpretation of the data. 2 507965132.11959747.00063

[0008] Ribosome affinity purification has become a popular tool to monitor gene expression in specific cell types such as neurons, due to the difficulty in the isolation of these cells without contamination from the surrounding cells or tissue. This method involves the construction of genetically modified cells / organisms which express affinity-tagged ribosomal subunits that can be controlled by a tissue-specific promoter. Tagged ribosomes are recovered by affinity selection, capturing ribosomes purely from the cells of interest. RNA is isolated from the captured ribosomes and measured through microarrays / RNA sequencing. While this method offers the best high-throughput prospects compared to polysome and ribosome profiling, this technology is still limited due to the lack of discrimination between actively translating and non-translating mRNA, which can be either mRNA indirectly bound to ribosomes or bound to another non-translating mRNA.

[0009] A novel method of translatome profiling has been developed by the present inventors, which allows for the isolation of RNA molecules that co-precipitate with ribosomes and are, therefore, likely to be undergoing translation into protein.

[0010] Genome-wide expression profiling can be used for personalized medicine. In the concept of personalized medicine, a sub-group of “responders” are selected based on specific criteria. A stringent cut-off is chosen to avoid noise from mild responders. On the other hand, “non-responders” are a group which does not fulfil the specific criteria and will be compared against the “responders” group. Biomarkers are used to identify the “responders” from the “non-responders” group, and the respective drug gains a more favorable risk-benefit ratio, allowing clinicians to make better treatment choices for their patients.

[0011] Personalized medicine has been applied extensively to certain types of cancer and it is beginning to be applied to the treatment of Alzheimer's disease and multiple sclerosis (MS) patients (Krzyszczyk et al., Technology, 2018, 6(3–4), 79–100; Hampel et al., The Journal of Prevention of Alzheimer’s Disease, 2016, 3(4), 243–259; Gafson et al., Multiple Sclerosis (Houndmills, Basingstoke, England) England, 2016, 23(3), 362–369). Over the past decade, there has been an accumulation of evidence that no two patient’s cancers will be the same, leading to variable patient responses to the traditional cancer therapies such as radiation and chemotherapy. The application of the personalized medicine approach has led to the development of specialized treatments for each subtype of cancer based upon patient genetic data, including transcriptomics, metabolomics, and proteomics. One example of this is the discovery of a mutation in anaplastic lymphoma kinase (ALK) that 3 507965132.11959747.00063 drives tumor formation in roughly 5% of non-small-cell lung cancers (Soda et al., Nature. 2017, 448(7153), 561–566). After the identification of this mutation, ALK blockers were developed (e.g., crizotinib and certinib) and given specifically to patients who test positive for the ALK mutation.

[0012] Multiple sclerosis (MS) is a syndrome with a large variation in the clinical phenotype, disease manifestation, as well as treatment response. With an increasing number of available treatments for MS patients, all with different mechanisms of action, range of efficacy and relative risk, a personal tailored approach needs to be adapted to make the best decision for each individual patient. In MS, clinical presentation alongside laboratory tests, imaging and CSF examinations are being used collectively to define the syndrome of each patient by rejecting possible alternative disorders and recognizing the distinct sub- syndrome of primary progressive MS. Neuromyelitis optica spectrum disorders (NMOSD) can be identified by serum antibodies against aquaporin4 (AQP4-IgG). It is important to be able to distinguish this disorder from MS as patients with NMOSD do not respond, or may worsen, with interferon (IFN) treatment.

[0013] In contrast, personalized medicine approach has not been applied to many other neurodegenerative diseases including ALS. Similar to MS, ALS is a heterogenous neurodegenerative disease that urgently needs new treatment. Understanding the heterogeneity of ALS involves the deconstruction of the biologically significant pathways that lead to disease and how patient-specific factors influence these pathways. Here we identify specific pathways of disease or transcriptional signatures that can aid in choosing the correct drug to give to a patient, as well as identifying new compounds that target disease in specific patients. We also identify a panel of biomarkers for drug response, by investigating how the significance of the differentially expressed genes (DEGs) in patient iAstrocytes compared to control changed after drug treatment, i.e., significantly dysregulated transcripts at baseline were no longer significantly dysregulated after treatment. To investigate changes in individual genes, a stringent statistical analysis was applied to decrease the probability of accepting false positive results. A false discovery rate (FDR) was applied to produce an adjusted p-value (p-adj). Specifically, p-adj<0.05 means that we accepted the probability that 5% of the already selected DEGs (p<0.05) were false positives. 4 507965132.11959747.00063 SUMMARY

[0014] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, the enantiomer of currently approved (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, is a weak dopamine antagonist and does not exhibit the side effects associated with dopamine agonism after administration. (6aS)-6-methyl- 5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, also known as S-(+)-10,11- dihydroxyaporphine, is depicted by the following chemical structure:

[0015] The present -6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol can significantly increase p62 proteins, increase LC3 proteins, reduce mitochondrial form factor, change biological pathways, and change the level of gene expression.

[0016] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be used in methods to increase p62 proteins, to increase LC3 proteins, to reduce mitochondrial form factor, to change biological pathways, and change the level of gene expression in a cell. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may further be used in methods for treating diseases mediated by p62 proteins, LC3 proteins, and mitochondrial form factor.

[0017] In one aspect, disclosed herein are methods of increasing the level of p62 proteins in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0018] In one aspect, disclosed herein are methods of increasing the level of LC3 proteins in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0019] In one embodiment, the LC3 protein is an LC3-I protein. In another embodiment, the LC3 protein is an LC3-II protein. 5 507965132.11959747.00063

[0020] In one aspect, disclosed herein are methods of reducing the level of mitochondrial form factor in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0021] In one aspect, disclosed herein are methods of changing biological pathways in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6-methyl- 5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0022] In one embodiment, the biological pathway represents the cellular response to organic cyclic compound, MAPK cascade, morphogenesis of an epithelial fold, cell adhesion, angiogenesis, inflammatory response, immune response, oxidation-reduction process, xenobiotic metabolic process, cholinergic synaptic transmission, or collagen fibril organization.

[0023] In one aspect, disclosed herein are methods of changing the level of gene expression in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0024] In one embodiment, the genes are selected from one or more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, 6 507965132.11959747.00063 MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0025] In another embodiment, the genes are selected from one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0026] As used herein, the term “effective amount” means an amount that will result in the desired effect or result, e.g., an amount that will result in increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, and / or changing the levels of gene expression.

[0027] In one embodiment, the method may be an in vitro method.

[0028] In another aspect, disclosed herein are methods of increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, and / or changing the levels of gene expression in a cell, comprising the step of contacting said cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol.

[0029] In one embodiment, the method may be an in vitro method.

[0030] In one embodiment, the cell in one of the above aspects, or other aspect herein, is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder and vagina. In a further embodiment, said brain cell is from a brain tissue selected from cerebrum (including cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including dentate nucleus, interposed nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including thalamus, hypothalamus, etc. and the posterior portion of the pituitary gland), and brain-stem (including pons, substantia nigra, medulla oblongata). In a further embodiment, said brain cell is selected from a neuron or glia cell (e.g., an astrocyte, oligodendrocyte, or microglia). In a further embodiment, said neuron is a sensory neuron, motor neuron, interneuron, or brain neuron. 7 507965132.11959747.00063

[0031] In one embodiment, the cell is an animal cell, e.g., mammalian cell. In a further embodiment, said cell in a human cell or non-human cell. In a further embodiment, said cell is in vitro, in vivo, or ex vivo.

[0032] In another embodiment, the cell is a diseased cell. In another embodiment, the cell is diseased cell from a patient suffering from a disease or disorder as defined below.

[0033] In another aspect, disclosed herein are methods of treating an animal having a disease or disorder that would benefit from increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, or changing the levels of gene expression, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to said animal.

[0034] In another aspect, disclosed herein is (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol for use in the treatment of a disease or disorder by increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, or changing the levels of gene expression.

[0035] In one embodiment, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol may be for use in the treatment of an animal having a disease or disorder characterized by increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, or changing the level of genes.

[0036] In one embodiment, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol may be comprised in a pharmaceutical composition.

[0037] In one embodiment, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol or pharmaceutical composition comprising (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for administration to the animal in an effective amount.

[0038] In one embodiment, said animal is a mammal. In another embodiment, said mammal is a human or a non-human mammal. In a further embodiment, said mammal is a human.

[0039] In another embodiment, said disease or disorder is caused by dysregulations of p62, LC3, mitochondrial form factor, biological pathways, and / or genes.

[0040] In another embodiment, the disease is a neurodegenerative disease. 8 507965132.11959747.00063

[0041] In another embodiment, said disease is selected from any one or more of: age-related macular degeneration, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) / motor neuron disease (MND), atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease, Crohn's disease, Duchenne's muscular dystrophy, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Huntington's disease (HD), Inclusion Body Myopathy (IBM)inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), Lou Gehrig's disease, multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, neurofibromatosis type I, Paget's disease of the bone (PDB), Parkinson’s disease (PD), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, valosin-containing protein (VCP)-related disorders, or Werdnig- Hoffmann disease, transient ischemic attack, ischaemia, cerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown-Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth Disease, macular degeneration, X-Linked bulbo-spinal atrophy (Kennedy’s disease), presenile dementia, depressive disorder, temporal lobe epilepsy, hereditary Leber optic atrophy, cerebrovascular accident, subarachnoid hemorrhage, and schizophrenia.

[0042] In one embodiment, the disease is amyotrophic lateral sclerosis (ALS).

[0043] In one embodiment, the disease is ALS caused by a mutation. In one embodiment, the disease is ALS caused by a mutation selected from: a C9orf72 mutation, a SOD1 mutation, or another rarer mutation causing or predisposing a subject, with the mutation, to ALS.

[0044] In another aspect, disclosed herein are in vitro methods of screening a candidate therapeutic agent(s) for its ability to treat an animal, the method comprising: (1) exposing the animal, e.g., a cell of the animal, to a candidate therapeutic; (2) after a period of time, comparing the levels of gene expression in the cells pre- and post- treatment; (3) based on the comparison result, determine if the animal can benefit from the treatment by the candidate therapeutic agent(s).

[0045] In some embodiments, the methods herein include comparing the levels of gene expression in the cells pre- and post- treatment wherein the candidate therapeutic is (6aS)- 9 507965132.11959747.00063 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, or a salt thereof. In some embodiments, the comparing includes comparing, or determining, the expression of, or significant change in expression of, p62 proteins, LC3 proteins, or mitochondrial form factor. In some embodiments, the comparing includes comparing, or determining, the expression of, or significant change in biological pathways or change in the level of gene expression.

[0046] In one embodiment, the method may be an in vitro method.

[0047] In one embodiment, the period of time to the cells is between 1-30 days, suitably between 1-15 days, suitably 1-5 days.

[0048] In one embodiment, the candidate therapeutic agent is (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0049] In one embodiment, said animal is a mammal. In another embodiment, said mammal is a human or a non-human mammal. In a further embodiment, said mammal is a human.

[0050] In one embodiment, the cell in one of the above aspects, or other aspect herein, is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder and vagina. In a further embodiment, said brain cell is from a brain tissue selected from cerebrum (including cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including dentate nucleus, interposed nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including thalamus, hypothalamus, etc. and the posterior portion of the pituitary gland), and brain-stem (including pons, substantia nigra, medulla oblongata). In a further embodiment, said brain cell is selected from a neuron or glia cell (e.g., an astrocyte, oligodendrocyte, or microglia). In a further embodiment, said cell is a peripheral blood mononuclear cell (PBMC).

[0051] In one embodiment, the cell is an animal cell, e.g., mammalian cell. In a further embodiment, said cell is a human cell or non-human cell. In a further embodiment, said cell is in vitro, in vivo, or ex vivo. 10 507965132.11959747.00063

[0052] In another embodiment, the cell is a diseased cell. In another embodiment, the cell is diseased cell from a patient suffering from a disease or disorder as defined below.

[0053] In one embodiment, the genes are selected from one or more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, ONECUT2.

[0054] In another embodiment, the genes are selected from one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, GRM4.

[0055] The foregoing and other features and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. Such description is meant to be illustrative, and not limiting, of the invention. Obvious variants of the disclosed thiazolidinediones in the text, including those described by the drawings and examples will be readily apparent to the person of ordinary skill in the art having the present disclosure, and such variants are considered to be a part of the current invention. 11 507965132.11959747.00063 BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In all figures disclosed herein, dexamethasone, rosiglitazone, pioglitazone and thiazolidinedione are abbreviated as Dex, Rosi, Pio, and TZD, respectively.

[0057] FIG. 1. Direct conversion of ALS patient fibroblasts into iNPCs. Fibroblasts are transduced using retroviral vectors containing the reprogramming factors Oct4, Sox2, Klf4, and c-Myc and supplemented with NPC medium and growth factors. Cells were grown until the 18-day mark where iNPCs were obtained.

[0058] FIG. 2A-B. Quantification of mouse motor neuron rescue in co-cultures with induced astrocytes from healthy controls and ALS patients (CTR: pooled data from three healthy controls; ALS patients with C9orf72 mutations: C9orf72_183, C9orf72_78, and C9orf72_201; ALS patients with SOD1 mutations: SOD1_210, SOD1_102, and SOD1_100; sporadic ALS patients: sALS_17, sALS_12, and sALS_009). The change of motor neuron survival using 5 μM or 10 μM andrographolide (FIG. 2A) and (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (labelled as Drug) (FIG. 2B) were compared to vehicle (DMSO).

[0059] FIG. 3A-B. Quantification of mouse motor neuron rescue in co-cultures with induced astrocytes from healthy controls and ALS patients (CTR: pooled data from three healthy controls; ALS patients with C9orf72 mutations: C9orf72_183, C9orf72_78, and C9orf72_201; ALS patients with SOD1 mutations: SOD1_210, SOD1_102, and SOD1_100; sporadic ALS patients: sALS_17, sALS_12, and sALS_009). The change of motor neuron survival using 5 μM or 10 μM monomethyl fumarate (FIG.3A) and riluzole (FIG.3B) were compared to vehicle (DMSO).

[0060] FIG.4. Quantification of mouse Hb9GFP+ motor neuron rescue in co-cultures with induced astrocytes by an increase in percentage of motor neuron survival 3 days after administration of riluzole, andrographolide and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol (labelled as Drug) at 10 µM compared to vehicle (DMSO). The human iAstrocytes were from the same various ALS patients: healthy controls (Control) and 3 different sporadic ALS patients (sALS, n=3); 3 different ALS patients with SOD1 mutations (SOD1, n=3) and 3 different ALS patients with C9orf72 mutations (C9orf, n=3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0061] FIG. 5. Representative images showing the expression of of p62 (568) and cytoplasmic marker CD44 (488), nuclei by Hoechst staining, in control and patient cells 12 507965132.11959747.00063 before and after treatment with NRF2 activator compounds (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and andrographolide. Scale bar 10µm.

[0062] FIG. 6A-C. Quantification of p62 expression in control and patient iAstrocytes before and after drug treatment. The percentage of p62-positive cells after treatment with (FIG. 6A) S[+]-apomorphine (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p<0.05, Column Factor p<0.0001), (FIG.6B) andrographolide (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p<0.0001, Column Factor p<0.001), and (FIG. 6C) MMF (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p=0.1945, Column Factor p<0.0001), individual significance is displayed on the graph. The control bar consists of 3050 & 155 pooled together.

[0063] FIG.7A-C. Quantification of p62 expression in the perinuclear region of control and patient iAstrocytes before and after drug treatment. The number of perinuclear spots after treatment with (FIG. 7A) (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline- 10,11-diol (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p<0.05, Column Factor p=0.1104), (FIG. 7B) andrographolide (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p<0.0001, Column Factor p=0.5064), and (FIG. 7C) MMF (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p=0.2456, Column Factor p<0.001). The control bar consists of 3050 & 155 pooled together.

[0064] FIG.8A-B. Quantification of p62 expression in the cytoplasm of control and patient iAstrocytes before and after riluzole treatment. (FIG. 8A) The percentage of p62-positive cells (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p=0.8922, Column Factor p<0.001). (FIG.8B) The number of perinuclear spots (two-way ANOVA, MC, n=3, technical repeats = 2, Row Factor p=0.2791, Column Factor p<0.01). The control bar consists of 3050 & 155 pooled together.

[0065] FIG. 9A-F. Quantification of autophagy markers p62 and LC3-I / LC3-II protein expression in control and C9ORF72 patient iAstrocytes before and after drug treatment. (FIG. 9A) Western blot of p62 expression in control and C9ORF72 iAstrocytes and quantification of p62 protein expression (mean ±SD, n=2) after treatment with (FIG. 9B) (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and andrographolide, and (FIG.9C) andrographolide. (FIG.9D) Western blot of LC3-I / LC3-II protein expression in control and C9ORF72 iAstrocytes and quantification of the ratio between LC3-I / LC-3II protein expression (mean ±SD, n=2) after treatment with (FIG.9E) 13 507965132.11959747.00063 (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and andrographolide, and (FIG. 9F) andrographolide. The control bars consist of 3050 & 155 pooled together.

[0066] FIG.10. Visualization of mitochondria (TMRM) and nuclei (Hoechst) in control and patient iAstrocytes before and after treatment with andrographolide. Scale bar 10µm.

[0067] FIG. 11A-C. Quantification of mitochondrial form factor in control and patient iAstrocytes before and after treatment with (FIG.11A) (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol (two-way ANOVA, MC, n=3, technical repeats = 3, Row Factor p<0.001, Column Factor p<0.0001), (FIG. 11B) MMF (two-way ANOVA, MC, n=3, technical repeats = 3, Row Factor p<0.05, Column Factor p<0.001), and (FIG. 11C). riluzole (two-way ANOVA, MC, n=3, technical repeats = 3, Row Factor p<0.01, Column Factor p<0.001). The control bar consists of 3050, 155 & 209 pooled together.

[0068] FIG. 12A-B. Visual representation of the differentially expressed genes (DEGs) shared between the control and different patient subgroups after (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment, generated from comparisons run in Table 1. (FIG. 12A) The total number of genes shared between groups. (FIG.12B) The number of up-regulated (upper number of each pair) and down-regulated (lower number of each pair) transcripts shared between subgroups. S = (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment; CRT = control; U = untreated.

[0069] FIG.13. Visual interpretation of the DEGs shared between (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol response comparisons demonstrated in Table 6. The numbers of upregulated genes are shown black and downregulated transcripts grey. Among 14 genes that were unique to the baseline S Resp. U vs CTR U comparison (11 up, 3 down), 10 of them were uniquely dysregulated in M102 responders at the baseline and corrected after treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol. S = (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol.

[0070] FIG. 14A-C. The normalized gene counts (TPM values) for patient responders and non-responders to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol before and after drug treatment. (FIG. 14A) Normalized gene counts for NECTIN3 (n=1), (FIG. 14B) MFF-DT (n=1), and (FIG. 14C) GRM4 (n=1). Control bar consists of 3050 & AG. 14 507965132.11959747.00063

[0071] FIG 15. Summary of the gene expression change in each biomarker identified within the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment response group. The normalized gene counts were plotted to determine how the transcript expression changed in each cell line after treatment with (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. Each one was given a score based on the response after treatment; a change in the direction of the control (+1), no change (0) or a change in the opposite direction of the control (-1), and the total value for each cell line was presented at the bottom. DETAILED DESCRIPTION

[0072] The term ‘(6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol’ means R-(-)-10,11-dihydroxyaporphine, including prodrug, salts, solvates, hydrates, and co-crystals thereof.

[0073] The term ‘(6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol’ means S-(+)-10,11-dihydroxyaporphine, including prodrug, salts, solvates, hydrates, and co-crystals thereof.

[0074] The term ‘6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol’ means (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, or (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, or racemic form of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, including prodrug, salts, solvates, hydrates, and co-crystals thereof.

[0075] As used herein, the terms ‘treat’, ‘treating’ or ‘treatment’ means to alleviate, reduce or abrogate one or more symptoms or characteristics of a disease and may be curative, palliative, prophylactic or slow the progression of the disease.

[0076] The term “effective amount” means an amount that will result in a desired effect or result, e.g., increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, or changing the level of genes. The term ‘therapeutically effective amount’ means an amount of (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, alone or combined with other active ingredients, that will elicit a desired biological or pharmacological response, e.g., effective to prevent, alleviate, or ameliorate symptoms of a disease or disorder; slow, halt or reverse 15 507965132.11959747.00063 an underlying disease process or progression; partially or fully restore cellular function; or prolong the survival of the subject being treated.

[0077] The term ‘patient’ or ‘subject’ includes mammals, including non-human animals and especially humans. In one embodiment the patient or subject is a human. In another embodiment the patient or subject is a human male. In another embodiment the patient or subject is a human female.

[0078] The term ‘significant’ or ‘significantly’ is determined by t-test at 0.05 level of significance.

[0079] The present disclosure relates to methods of using of (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to increase the level of p62, increase the level of LC3, reduce mitochondrial form factor, change biological pathways, or change the level of genes in a cell, tissue or animal.

[0080] The present disclosure further relates to methods of using (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the treatment, prevention, alleviation, or amelioration of a disease that is mediated by the level of p62, the level of LC3, mitochondrial form factor, biological pathways, or the levels of gene expression.

[0081] Accordingly, in one aspect, the present disclosure provides for a method of increasing the level of p62 proteins in a cell, comprising the step of contacting said cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol.

[0082] In one embodiment, the method may be an in vitro method.

[0083] In another embodiment, the p62 protein is a perinuclear p62 protein.

[0084] In a related aspect, the present disclosure provides for a method of increasing the level of LC3 protein in a cell, comprising the step of contacting said cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0085] In one embodiment, the method may be an in vitro method.

[0086] In another embodiment, the LC3 protein is an LC3-I protein or an LC3-II protein.

[0087] In another aspect, the present disclosure provides for a method of reducing the level of mitochondrial form factor in a cell, comprising a step of contacting the cell with an 16 507965132.11959747.00063 effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol.

[0088] In another aspect, the present disclosure provides for a method of changing biological pathways in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0089] In one embodiment, the biological pathway is cellular response to organic cyclic compound, MAPK cascade, morphogenesis of an epithelial fold, cell adhesion, angiogenesis, inflammatory response, immune response, oxidation-reduction process, xenobiotic metabolic process, cholinergic synaptic transmission, or collagen fibril organization

[0090] In another aspect, the present disclosure provides for a method of changing the levels of gene expression in a cell, comprising a step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0091] In one embodiment, the genes are selected from one or more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, ONECUT2. 17 507965132.11959747.00063

[0092] In another embodiment, the genes are selected from one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, GRM4.

[0093] In another aspect, the present disclosure provides for a method of: (a) increasing the level of p62, (b) increasing the level of LC3, (c) reducing mitochondrial form factor, (d) changing biological pathways, or (e) changing the level of genes in a cell, said method comprising the step of contacting said cell with an effective amount of (6aS)-6-methyl- 5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0094] In one embodiment, the method may be an in vitro method.

[0095] In one embodiment, the cell in one of the above aspects, or other aspect or embodiments herein, is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder and vagina. In a further embodiment, said brain cell is from a brain tissue selected from cerebrum (including cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including dentate nucleus, interposed nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including thalamus, hypothalamus, etc. and the posterior portion of the pituitary gland), and brain-stem (including midbrain, pons, substantia nigra, medulla oblongata). In a further embodiment, said brain cell is selected from a neuron or glia cell (e.g., an astrocyte, oligodendrocyte, or microglia). In a further embodiment, said neuron is a sensory neuron, motor neuron, interneuron, or brain neuron. In some embodiments, the brain cell is from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem. In some embodiments, the brain cell is selected from: neuron, astrocyte, oligodendrocyte, or microglia. In some embodiments, the neuron is a sensory neuron, motor neuron, interneuron, or brain neuron.

[0096] In some embodiments of the methods described herein, a cell is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, 18 507965132.11959747.00063 epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder, or vagina.

[0097] In one embodiment, the cell is an animal cell, e.g., mammalian cell. In a further embodiment, said cell in a human cell or non-human cell. In a further embodiment, said cell is a human cell. In a further embodiment, said cell is in vitro, in vivo, or ex vivo.

[0098] In another embodiment, the cell is a diseased cell. In another embodiment, the cell is diseased cell from a patient suffering from a disease or disorder disclosed herein.

[0099] In another aspect, the disclosure provides for a method of treating an animal having a disease or disorder would benefit from increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, or changing the level of genes, for example, where a symptom that is prevented, alleviated, or ameliorated, or a disease process or progression that slowed, halted or reversed, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline- 10,11-diol to said animal.

[0100] In another aspect, the disclosure provides for (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol for use in the treatment of a disease or disorder by increasing the level of p62, increasing the level of LC3, reducing mitochondrial form factor, changing biological pathways, or changing the levels of gene expression, for example, where a symptom that is prevented, alleviated, or ameliorated, or a disease process or progression that slowed, halted or reversed.

[0101] In one embodiment, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol may be comprised in a pharmaceutical composition.

[0102] In one embodiment, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol or pharmaceutical composition comprising (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for administration to the animal in an effective amount. 19 507965132.11959747.00063

[0103] In one embodiment, the animal is mammal. In a further embodiment, the mammal is a human. In another embodiment, the mammal is a non-human mammal.

[0104] In another embodiment, said disease or disorder is caused by the level of p62, the level of LC3, mitochondrial form factor, biological pathways, or the level of genes.

[0105] In some embodiments, the said disease or disorder is selected from any one or more of: aging-related tau astrogliopathy (ARTA), Alexander Disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), Critical Illness Myopathy (CIM), Primary Age- Related Tauopathy (PART), aortic medial amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, argyrophillic grain disease, ataxia telangiectasia, atrial fibrillation, Autosomal Dominant Hyper-IgE Syndrome, cardiac atrial amyloidosis, Bloom's syndrome, cardiovascular diseases, coronary artery disease, myocardial infarction, stroke, restenosis, arteriosclerosis, cataracts, cerebral amyloid angiopathy, Christianson syndrome, chronic traumatic encephalopathy, Cockayne's syndrome, corneal lactoferrin amyloidosis, corticobasal degeneration, Crohn's Disease, Cushing's disease, cutaneous lichen amyloidosis, cystic fibrosis, Dentatorubropallidoluysian Atrophy (DRPLA), dialysis amyloidosis, diffuse neurofibrillary tangles with calcification, Down syndrome, endotoxin shock, familial amyloidosis of the Finnish type, familial amyloidotic neuropathy, Familial British Dementia (FBD) , Familial Danish Dementia (FDD), familial dementia, fibrinogen amyloidosis, fragile X syndrome, Fragile X-associated Tremor / Ataxia Syndrome (FXTAS), Friedreich's ataxia, fronto-temporal degeneration, glaucoma, Glycogen Storage Disease type IV (Andersen Disease), Guadeloupean Parkinsonism, hereditary lattice corneal dystrophy, Huntington's disease, inclusion body myositis / myopathy, inflammation, inflammatory bowel disease, ischemic condition, ischemia / reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia, light chain or heavy chain amyloidosis, lysosomal storage diseases, aspartylglucosaminuria, Fabry's disease, Batten disease, Cystinosis, Farber, Fucosidosis, Galactasidosialidosis, Gaucher's disease Type 1, 2 or 3, Gml gangliosidosis, Hunter's disease, Hurler-Scheie's disease, Krabbe's disease, a-Mannosidosis, B-Mannosidosis, Maroteaux-Lamy's disease, Metachromatic Leukodystrophy, Morquio A syndrome, Morquio B syndrome, Mucolipidosis II, Mucolipidosis III, Neimann-Pick Disease Type A, B or C, Pompe's disease, Sandhoff disease, Sanfilippo syndrome Type A, B, C or D, Schindler disease, Schindler-Kanzaki disease, Sialidosis, Sly syndrome, Tay-Sach's disease, Wolman disease, lysozyme amyloidosis, Mallory bodies, medullary thyroid 20 507965132.11959747.00063 carcinoma, mitochondrial myopathies, multiple sclerosis, multiple system atrophy, myotonic dystrophy, myotonic dystrophy, neurodegeneration with brain iron accumulation, neurofibromatosis, neuronal ceroid lipofuscinosis, odontogenic (Pinborg) tumor amyloid, Parkinsonism-Dementia of Guam, Parkinson's disease, peptic ulcers, Pick's disease, pituitary prolactinoma, post-encephalitic Parkinsonism, prion diseases (Transmissible Spongiform Encephalopathies), including Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease, Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, Kuru, progressive supranuclear palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, seminal vesical amyloid, senile systemic amyloidoses, Serpinopathies, sickle cell disease, spinal and bulbar muscular atrophy (SBMA), spinocerebellar ataxias, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 8, spinocerebellar ataxia type 17), subacute sclerosing panencephalitis, tauopathies, type II diabetes, vascular dementia, or Werner syndrome.

[0106] In another embodiment, said disease is selected from any one or more of: age-related macular degeneration, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease Crohn's disease, Duchenne's paralysis, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Huntington's disease (HD), Inclusion Body Myopathy (IBM)inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), Lou Gehrig's disease, multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, Neurofibromatosis type I, Paget's disease of the bone (PDB), Parkinson disease (PD), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, Valosin-Containing Protein (VCP)-related disorders, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, cerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown-Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth Disease, macular degeneration, X-Linked Bulbo-Spinal Atrophy, presenile dementia, depressive disorder, temporal lobe epilepsy, Hereditary Leber Optic Atrophy, cerebrovascular accident, subarachnoid hemorrhage, and schizophrenia. 21 507965132.11959747.00063

[0107] In another embodiment, said disease is a neurological disease.

[0108] In one embodiment, the disease is a neurodegenerative disease or disorder.

[0109] In one embodiment, the disease is ALS.

[0110] In one embodiment, the disease is ALS caused by a mutation. In one embodiment, the disease is ALS caused by a mutation selected from: a C9orf72 mutation, a SOD1 mutation, or an another mutation known to cause of predispose to ALS.

[0111] In some embodiments, provided is a method of treating an animal having a disease or disorder that would benefit from changing biological pathways in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol, where the biological pathways are selected from any one or more of: cellular response to organic cyclic compound, MAPK cascade, morphogenesis of an epithelial fold, cell adhesion, angiogenesis, inflammatory response, immune response, oxidation-reduction process, xenobiotic metabolic process, cholinergic synaptic transmission, or collagen fibril organization.

[0112] In some embodiments, provided is a method of treating an animal having a disease or disorder that would benefit from changing the level of genes, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the genes are selected from any one of more of the following: HS6ST2- AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, 22 507965132.11959747.00063 TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0113] In some embodiments, provided is a method of treating an animal having a disease or disorder that would benefit from changing the level of genes, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the genes are selected from any one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0114] In some embodiments, provided is a method of treating an animal having a disease or disorder that would benefit from changing the level of genes, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the genes are selected from any one of more of the following: ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0115] In some embodiments, provided is a method of treating an animal having a disease or disorder, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol to said animal, where the animal is selected by the changes genes in a cell responding to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol, and the genes are selected from any one of more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, 23 507965132.11959747.00063 GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0116] In some embodiments, provided is a method of treating an animal having a disease or disorder, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the animal is selected by the changes genes responding in a cell to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol, and the genes are selected from any one of more of the following 14 genes: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0117] In some embodiments, provided is a method of treating an animal having a disease or disorder, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the animal is selected by the changes genes responding in a cell to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol, and the genes are selected from any one of more of the following 14 genes: ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0118] In some embodiments of the methods, the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol is administered at a dose of 0.12 mg / kg or higher.

[0119] In some embodiments of the methods, the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol is administered at a dose between 5-5000 mg / day.

[0120] In some embodiments of the methods, the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol is administered parenterally, enterally, or topically. 24 507965132.11959747.00063

[0121] In some embodiments of the methods, the 6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol is administered by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular, or subcutaneous administration.

[0122] Provided is the use of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol for the preparation of a medicament for treating a human having a disease of claim 40.

[0123] In some embodiments of the methods, the increasing, reducing, or changing is determined relative to a corresponding measurement prior to the step of contacting the cell with the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0124] In some embodiments of the methods, the cell is a cell of a human subject, and the human subject has Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson's disease (PD).

[0125] In another aspect, the disclosure provides for an in vitro method of screening a candidate therapeutic agent(s) for its ability to treat an animal, the method comprising: (1) exposing the animal to a candidate therapeutic; (2) after a period of time, comparing the levels of gene expression in the cells pre- and post- treatment; (3) based on the comparison result, determine if the animal can benefit from the treatment by the candidate therapeutic agent(s)

[0126] In one embodiment, the method may be an in vitro method.

[0127] In one embodiment, the period of time to the cells is between 1-30 days, suitably between 1-15 days, suitably 1-5 days.

[0128] In one embodiment, the candidate therapeutic agent is (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0129] In one embodiment, said animal is a mammal. In another embodiment, said mammal is a human or a non-human mammal. In a further embodiment, said mammal is a human.

[0130] In one embodiment, the cell in one of the above aspects, or other aspect herein, is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, 25 507965132.11959747.00063 parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder and vagina. In a further embodiment, said brain cell is from a brain tissue selected from cerebrum (including cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including dentate nucleus, interposed nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including thalamus, hypothalamus, etc. and the posterior portion of the pituitary gland), and brain-stem (including midbrain, pons, substantia nigra, medulla oblongata). In a further embodiment, said brain cell is selected from a neuron or glia cell (e.g., an astrocyte, oligodendrocyte, or microglia). In a further embodiment, said cell is a peripheral blood mononuclear cell (PBMC).

[0131] In one embodiment, the cell is an animal cell, e.g., mammalian cell. In a further embodiment, said cell is a human cell or non-human cell. In a further embodiment, said cell is in vitro, in vivo, or ex vivo.

[0132] In another embodiment, the cell is a diseased cell. In another embodiment, the cell is diseased cell from a patient suffering from a disease or disorder as defined below.

[0133] In another embodiment, the genes are selected from one or more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, 26 507965132.11959747.00063 MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, ONECUT2.

[0134] In another embodiment, the genes are selected from one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, GRM4.

[0135] In some embodiments, provided is a method, comprising: A) predicting efficacy of a treatment of a disease or disorder in a subject, wherein the treatment comprises a first administration of an active pharmaceutical agent to a first cell of the subject, the first cell having a first cell type, and optionally a second administration of the active pharmaceutical agent to a second cell of the subject, the second cell having the first cell type, and the active pharmaceutical agent has the formula or a pharmaceutically acceptable salt thereof; B)sample comprising a control cell from the subject prior to (e.g., less than a month prior to, e.g., less than a week prior to) the first administration, the control cell having the first cell type; C) collecting a second biological sample from the subject after (e.g., up to 1, 2, 3, or 30 days after) the first administration and prior to (e.g., less than a month prior to, e.g., less than a week prior to) the optional second administration, the second biological sample comprising the first cell; D) determining a first differential expression of one or more genes in the second biological sample compared to the first biological sample; and E) predicting efficacy of the treatment based on the first differential expression.

[0136] In some embodiments, provided is a method, comprising: A) monitoring a treatment of a disease or disorder in a subject, wherein the treatment comprises a first administration of an active pharmaceutical agent to a first cell of the subject, the first cell having a first cell type, and optionally a second administration of the 27 507965132.11959747.00063 active pharmaceutical agent to a second cell of the subject, the second cell having the first cell type, and the active pharmaceutical agent has the formula or a pharmaceutically acceptable salt thereof;sample comprising a control cell from the subject prior to (e.g., less than a month prior to, e.g., less than a week prior to) the first administration, the control cell having the first cell type; C) collecting a second biological sample from the subject after (e.g., up to 1, 2, 3, or 30 days after) the first administration and prior to (e.g., less than a month prior to, e.g., less than a week prior to) the optional second administration, the second biological sample comprising the first cell; D) determining a first differential expression of one or more genes in the second biological sample compared to the first biological sample; and E) monitoring the treatment based on the first differential expression.

[0137] In some embodiments, the disease or disorder is a neurological disease.

[0138] In some embodiments, the disease or disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD).

[0139] In some embodiments, the disease or disorder is selected from any one or more of: age-related macular degeneration, atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease, Crohn's disease, Duchenne's paralysis, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Inclusion Body Myopathy (IBM), inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, Neurofibromatosis type I, Paget's disease of the bone (PDB), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, Valosin-Containing Protein (VCP)-related disorders, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, cerebral hemorrhage, 28 507965132.11959747.00063 senile cataract, retinal ischemia, retinal vasculitis, Brown-Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot-Marie- Tooth Disease, macular degeneration, hereditary Leber optic atrophy (Kennedy’s disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Hereditary Leber Optic Atrophy, cerebrovascular accident, subarachnoid hemorrhage, or schizophrenia.

[0140] In some embodiments, the one or more genes are selected from HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0141] In some embodiments, the one or more genes are selected from ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0142] In some embodiments, the one or more genes are selected from ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0143] In some embodiments, the first differential expression of one or more genes identifies significantly dysregulated transcripts of the one or more genes in the first biological sample as compared to the second biological sample. 29 507965132.11959747.00063

[0144] In some embodiments, the method is an in vitro method.

[0145] In some embodiments, the first cell type is from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder, or vagina.

[0146] In some embodiments, the animal is a mammal. In some embodiments, the mammal is a non-human animal. In some embodiments, the mammal is a human.

[0147] In some embodiments, the active pharmaceutical agent is administered at a dose of 0.12 mg / kg or higher. In some embodiments, the active pharmaceutical agent is administered at a dose between 5–5000 mg / day. In some embodiments, the active pharmaceutical agent is administered parenterally, enterally, or topically. In some embodiments, the active pharmaceutical agent is administered by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular, or subcutaneous administration.

[0148] In some embodiments, method further comprises: F) monitoring the effectiveness of the treatment based on a second differential expression or a third differential expression; G) continuing the treatment with the second administration; H) collecting a third biological sample from the subject after the second administration, the third biological sample comprising the second cell; and I) determining the second differential expression of one or more genes compared to the first biological sample or compared to the third biological sample.

[0149] In some embodiments, the subject experiences an increase in the level of p62, an increase in the level of LC3, or a reduction of mitochondrial form factor as a result of the first administration, the second administration, or both.

[0150] In some embodiments, the first cell type is a brain cell from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem, optionally wherein the brain cell is an induced brain cell. 30 507965132.11959747.00063

[0151] In some embodiments, the first cell type is a brain cell selected from: neuron, astrocyte, oligodendrocyte, or microglia, optionally wherein the brain cell is an induced brain cell.

[0152] In some embodiments, the first cell type is a brain cell selected from: sensory neuron, motor neuron, interneuron, or brain neuron, optionally wherein the brain cell is an induced brain cell.

[0153] In some embodiments, the method is a method of treatment of the disease or disorder.

[0154] Provided is a method of treating Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD) in a subject in need thereof, comprising: 1) administering parenterally, enterally, or topically to the subject at a first dose of at least 0.12 mg / kg, and optionally at a dose between 5–5000 mg / day, an active pharmaceutical agent having the formula or a pharmaceutically acceptable salt thereof;of p62, LC3, or mitochondrial form factor in a biological sample comprising a cell, which may include a brain cell from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem, optionally wherein the brain cell is an induced brain cell, wherein the measuring comprises translatome profiling; and 3) treating the Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD) in the subject with a second dose that is greater than the first dose (e.g., a second dose of more than 0.12 mg / kg, and optionally at a dose from more than 5 to about 5000 mg / day) if the subject does not experience an increase in the level of p62, an increase in the level of LC3, or a reduction of mitochondrial form factor as a result of administration of the first dose.

[0155] In some embodiments of the methods herein, the measuring, determining, or sample collecting step(s), or analogously termed step(s) herein, is performed up to 1, 2, 3, or 30 days after an administration step(s) (e.g., a first or second administration of an API) and / or 31 507965132.11959747.00063 less than about one month or less than 5, 4, 3, 2, or 1 week prior to an administration step(s) (e.g., a first or second administration of an API). In some embodiments of the methods herein, administration of an API may refer to contacting a cell, whether an isolated cell (e.g., in vitro or ex vivo) or a cell in the subject’s body, with an API.

[0156] The pharmaceutical compositions disclosed herein comprise a therapeutically effective amount (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol and at least one pharmaceutically acceptable excipient. The term “excipient” refers to a pharmaceutically acceptable, inactive substance used as a carrier for the pharmaceutically active ingredient ((6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol), and includes antiadherents, binders, coatings, disintegrants, fillers, diluents, solvents, flavors, bulkants, colours, glidants, dispersing agents, wetting agents, lubricants, preservatives, sorbents and sweeteners. The choice of excipient(s) will depend on factors such as the particular mode of administration and the nature of the dosage form. Solutions or suspensions used for injection or infusion can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, including autoinjectors, or multiple dose vials made of glass or plastic.

[0157] A pharmaceutical formulation of the present disclosure may be in any pharmaceutical dosage form. The pharmaceutical formulation may be, for example, a tablet, capsule, nanoparticulate material, e.g., granulated particulate material or a powder, a lyophilized material for reconstitution, liquid solution, suspension, emulsion or other liquid form, injectable suspension, solution, emulsion, etc., suppository, or topical or transdermal preparation or patch. The pharmaceutical formulations generally contain about 1% to about 99% by weight of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol and 99% to 1% by weight of a suitable pharmaceutical excipient. In one embodiment, the dosage form is an oral dosage form. In another embodiment, the dosage form is a parenteral dosage form. In another embodiment, the dosage form is an enteral dosage form. In another embodiment, the dosage form is a topical 32 507965132.11959747.00063 dosage form. In one embodiment, the pharmaceutical dosage form is a unit dose. The term 'unit dose' refers to the amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol administered to a patient in a single dose.

[0158] In some embodiments, a pharmaceutical composition of the present disclosure is delivered to a subject via a parenteral route, an enteral route, or a topical route.

[0159] Examples of parental routes of the present disclosure include, without limitation, any one or more of the following: intra-abdominal, intra-amniotic, intra-arterial, intra- articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal, intracoronary, intracorporus, intracranial, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intraocular, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous (bolus or drip), intraventricular, intravesical, and / or subcutaneous.

[0160] Enteral routes of administration of the present disclosure include administration to the gastrointestinal tract via the mouth (oral), stomach (gastric), and rectum (rectal). Gastric administration typically involves the use of a tube through the nasal passage (NG tube) or a tube inserted directly into the stomach (PEG tube). Rectal administration typically involves rectal suppositories. Oral administration includes sublingual and buccal administration.

[0161] Topical administration includes administration to a body surface, such as skin or mucous membranes, including intranasal and pulmonary administration. Transdermal forms include cream, foam, gel, lotion or ointment. Intranasal and pulmonary forms include liquids and powders, e.g., liquid spray.

[0162] The dose may vary depending upon the dosage form employed, sensitivity of the patient, and the route of administration. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, 33 507965132.11959747.00063 age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to therapy.

[0163] In one embodiment, the daily dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol administered to a patient is selected from: up to 200 mg, 175 mg, 150 mg, 125 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 30 mg, 25 mg, 20 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, or up to 2 mg. In another embodiment, the daily dose is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 2,000 mg, 3,000 mg, 4,000 mg, or at least 5,000 mg. In another embodiment, the daily dose is 1-2 mg, 2-4 mg, 1-5 mg, 5-7.5 mg, 7.5-10 mg, 10-15mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30- 35 mg, 35-40 mg, 40-45 mg, or 45-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 5-200 mg, 5-300 mg, 5-400 mg, 5-500 mg, 5-600 mg, 5- 700 mg, 5-800 mg, 5-900 mg, 5-1,000 mg, 5-2,000 mg, 5-5,000 mg or more than 5,000 mg.

[0164] In another embodiment, a single dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol administered to a patient is selected from: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg ,150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg 490 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 2,000 mg, 3,000 mg, 4,000 mg, or 5,000 mg. In another embodiment, a single dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered to a patient is selected from: 1-2 mg, 2-4 mg, 1-5 mg, 5-7.5 mg, 7.5-10 mg, 10-15mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5- 25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50- 75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225- 250 mg, 250-275 mg, 275-300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, 475-500 mg, 500-1,000 mg, 1,000-2,000 mg, 34 507965132.11959747.00063 3,000-4,000 mg, 4,000-5,000 mg, or more than 5,000 mg. In one embodiment, the single dose is administered by a route selected from any one of: oral, buccal, or sublingual administration. In another embodiment, said single dose is administered by injection, e.g., subcutaneous, intramuscular, or intravenous. In another embodiment, said single dose is administered by inhalation or intranasal administration.

[0165] As a non-limited example, the dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol administered by subcutaneous injection may be about 3 to 5,000 mg per day to be administered in divided doses. A single dose of (6aS)-6-methyl- 5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered by subcutaneous injection may be about 1-6 mg, preferably about 1-4 mg, 1-3 mg, or 2 mg. Other embodiments include ranges of about 5-5,000 mg, preferably about 100-1,000 mg, 100- 500 mg, 200-400 mg, 250-350 mg, or 300 mg. Subcutaneous infusion may be preferable in those patients requiring division of injections into more than 10 doses daily. The continuous subcutaneous infusion dose may be 1 mg / hour daily and is generally increased according to response up to 4 mg / hour.

[0166] The fine particle dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol administered by pulmonary administration, e.g., inhalation using a pressurized metered dose inhaler (pMDI), dry powder inhaler (DPI), soft- mist inhaler, nebulizer, or other device, may be in the range of about, 0.5-15 mg, preferably about 0.5-8 mg or 2-6 mg. Other embodiments include ranges of about 5-5,000 mg, preferably about 100-1,000 mg, 100-500 mg, 200-400 mg, 250-350 mg, or 300 mg. The Nominal Dose (ND), i.e., the amount of drug metered in the receptacle (also known as the Metered Dose), of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol administered by pulmonary administration may be , for example, in the range of 0.5- 15 mg, 3-10 mg, 10-15mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg. Other embodiments include ranges of about 5-5,000 mg, preferably about 100-1,000 mg, 100-500 mg, 200-400 mg, 250-350 mg, or 300 mg. Long-acting pharmaceutical compositions may be administered, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times daily (preferably ≤ 10 times per day), every other day, every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. 35 507965132.11959747.00063 EXAMPLES

[0167] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0168] It is further to be understood that all values are approximate and are provided for description. All references cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference. Example 1: In vitro cell model derived from ALS patient fibroblasts

[0169] Over the last decade, in vitro modelling of neurodegeneration has undergone impressive development, mainly due to the reprogramming of adult human fibroblasts into induced pluripotent stem cells (iPSCs) and induced neural progenitor cells (iNPCs). In the ALS research field, this offers an opportunity to model familial and sporadic diseases in vitro.

[0170] NPCs harvested from postmortem spinal cord of ALS patients have already been successfully differentiated into motor neurons, astrocytes and oligodendrocytes. Deriving astrocytes using this method avoids inducing major epigenetic alterations. However, the availability of post-mortem samples is limited. In addition, the disadvantages of reprogramming astrocytes from human derived iPSCs include time-consuming protocols, as well as complex and highly variable maturation time of the astrocytes.

[0171] Therefore, a promising alterative to iPSC resources is the direct reprogramming of fibroblasts into astrocytes from an immuno-matched host. Instead of generating iPSCs, direct reprogramming involves the use of cell-lineage transcription factors to convert adult somatic cells into another cell type. This technology has been used to generate sub-specific neural lineages such as cholinergic, dopaminergic and motor neurons. Direct reprogramming technology was also used to derive astrocytes from ALS patient fibroblasts, and tripotent iNPCs from ALS patients and controls were generated within one month. When these cells were differentiated into astrocytes, they displayed similar toxicity towards motor neurons in co-cultures as autopsy-derived astrocytes, making them useful tools in the development of drug screens (FIG.1). 36 507965132.11959747.00063

[0172] Methodology:

[0173] iNPCs were generated from adult human fibroblasts from patients who had been diagnosed with ALS and from age-matched healthy controls, using an approach reported previously (Kim et al., PNAS, 2001. 108(19), 7838-7843; Meyer et al., PNAS, 2014. 111(2), 829–832). iNPCs are differentiated into iAstrocytes by culturing the progenitors in iAstrocyte medium for a total of 7 days with a medium change at Day 3.

[0174] Induced astrocytes from control or ALS patients were used in a co-culture assay to determine their effect on mouse motor neuron (MN) survival. Mouse embryonic stem cell- derived motor neurons expressing green fluorescence protein (GFP) under the control of the HB9 promoter were sorted and added to iAstrocytes from patients and controls. Meanwhile, andrographolide, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, monomethyl fumarate (MMF), and riluzole were screened in this co-culture system of patient iAstrocytes and wildtype mouse MNs. The survival of mouse MNs was monitored on Day 1 and 3 with confocal image acquisition.

[0175] Results:

[0176] The MN survival on Day 3 was evaluated as a percentage of survived MN cells observed on Day 1. As expected, iAstrocytes from a healthy control did not significantly change the survival of mouse MNs on Day 3. The introduction of all four drugs also did not change the survival of mouse MNs (FIGs.2A-B and 3A-B).

[0177] When iAstrocytes from three ALS patients with the C9orf72 mutation (i.e., patients C9orf72_183, C9orf72_201, and C9orf72_78) were co-cultured with mouse MNs, no more than 33% of the MN cells survived on Day 3, among all three ALS patients. However, the survival of MN cells on Day 3 was significantly improved, when andrographolide, (6aS)- 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and MMF were introduced to the culture. More specifically, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol improved the MN survival to up to 38%.

[0178] When iAstrocytes from ALS patients with SOD1 mutation (i.e., patients SOD1_210, SOD1_102, and SOD1_100) were co-cultured with mouse MNs, approximately 40% or less of the MN cells survived on Day 3. The survival of MN cells on Day 3 showed most significant improvement with the introduction of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol. 37 507965132.11959747.00063

[0179] When iAstrocytes from three ALS patients with sporadic ALS mutations (i.e., patients sALS_17, sALS_12, and sALS_009) were co-cultured with mouse MN, the survival of MN cells on Day 3 varied between 21 to 40%. In this study, the survival of MN cells on Day 3 was most significantly improved in the presence of andrographolide (FIGs, 2A-B, 3A-B, and 4). Example 2: Autophagy regulator expression – p62 and LC3 proteins

[0180] Immunocytochemistry was used to detect the percentage of cells positive for cytoplasmic p62 and the number of perinuclear p62 spots within the cytoplasm. In this staining, all ALS patient cells had a higher percentage of p62 positive cells as well as a higher number of perinuclear p62 spots than the control iAstrocytes. Therefore, we investigated if treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, andrographolide and MMF, or riluzole would influence the presence of p62 using the same protocol.

[0181] Immunocytochemistry images showed that p62 expression was increased in control and all patient iAstrocyte lines with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol and andrographolide treatment, specifically in the perinuclear region of the cell (FIG.5). When these images were quantified in terms of the percentage of p62 positive cells, there was a significant increase in the number of cells expressing the p62 protein 24 hours post (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treatment across control and patient cell lines (two-way ANOVA, multiple comparisons, n=3, p<0.05). The addition of MMF had no significant effect on p62 expression levels in control or patient iAstrocytes (FIGs.6A-C).

[0182] Similar to the percentage of p62 positive cells, there was also a significant increase in the number of perinuclear p62 spots across control and patient iAstrocyte lines when treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol; the highest increase was seen in the sALS iAstrocytes (two-way ANOVA, multiple comparisons, n=3, p<0.05). In contrast, MMF treatment had little effect on perinuclear p62 spots (FIGs.7A-C). When control and patient iAstrocytes were treated with riluzole, there was little change in both the percentage of p62 positive cells and the number of perinuclear p62 spots (FIGs.8A-B).

[0183] This increase in p62 expression after treatment with (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and andrographolide could be explained 38 507965132.11959747.00063 by the activation of the autophagy pathway. Therefore, the effect of these two compounds on the protein expression of autophagic markers LC3-I / LC3-II was investigated. Since autophagy deficits are commonly reported in C9ORF72-ALS, this genetic subgroup of cell lines was focused on.

[0184] Similar to the p62 staining results, western blotting confirmed higher levels of p62 protein in C9ORF72 iAstrocyte lines compared to the control and this protein level was increased further after treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol and andrographolide (FIGs. 9A-C). The LC3 protein is responsible for the formation of autophagosomes, vesicles that carry unwanted proteins sent for degradation. It exists in two forms, i.e., LC3-I which is located within the cytoplasm and LC3-II which is bound to the membrane of autophagosomes. The conversion of LC3-I to LC3-II is indicative of the initiation of autophagosome formation and therefore is a useful biomarker to detect autophagy. C9ORF72 iAstrocytes presented increased levels of LC3-I protein compared to the control cell lines (FIG.9D). This is a known mechanism that has been linked to C9ORF72 haploinsufficiency, resulting in an initial activation of autophagy, which is then affected by the defective interaction of the protein encoded by C9ORF72 with Rab1 and the ULK1 complex, thus preventing the formation of autophagosomes and allowing the accumulation of LC3-I in the cytoplasm. However, treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or andrographolide had no effect on the levels of LC3-I or LC3-II in control and C9ORF72 iAstrocytes (FIGs.9E-F). Example 3: Mitochondrial dynamics

[0185] Mitochondria were labelled and visualized with a fluorescent TMRM dye to investigate mitochondrial morphology in the patient iAstrocytes. Patient iAstrocytes demonstrated differences in mitochondrial dynamics in comparison to control lines; there was extensive mitochondrial fragmentation observed in SOD1 ND29505 and all patients lines had a significantly higher percentage of perinuclear mitochondria compared to the control. Afterwards, we investigated if the addition of antioxidant compounds or riluzole would influence changes in the mitochondrial dynamics of the cell.

[0186] Mitochondrial staining images showed that there was an intense fusion of the mitochondrial network after treatment with andrographolide; the mitochondrial network of 39 507965132.11959747.00063 sALS 17 after treatment with the compound appears to form thin, hairlike structures around the network (FIG.10).

[0187] When the staining images were quantified, there was a significant decrease in the mitochondrial form factor with andrographolide treatment, implying that the mitochondrial network had become more fused together and there was less mitochondrial branching (two- way ANOVA, multiple comparisons, n=3, p<0.0001). This network fusion was reflected by the significant increase in mitochondrial area after andrographolide treatment (two-way ANOVA, multiple comparisons, n=3, p<0.001). However, there was no significant difference in the percentage of perinuclear mitochondria after treatment with andrographolide, indicating that treatment does not have an effect on the localization of the mitochondria within the network.

[0188] Mitochondrial branching was also influenced by the other antioxidant compounds as well as riluzole; there was a reduction in mitochondrial form factor of varying significance with all compounds tested (FIGs. 11A-C, two-way ANOVA, multiple comparisons, n=3, FIG. 11A: (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol p<0.001; FIG. 11B: MMF p<0.05; and FIG. 11C: riluzole p<0.01). Example 4: RNA-sequencing to determine mechanism of action of antioxidant compounds

[0189] RNA extraction and quality control

[0190] Mechanism of actions of therapeutics were investigated to understand if they can produce a beneficial reduction of the astrocyte toxicity of a particular patient subgroup. Therefore, control and patient iAstrocytes were plated into 10 cm dishes as a monoculture and were treated with the compound diluted in 0.01% DMSO for 48 hours. The cells were lysed and the RNA was extracted using the translatome protocol.

[0191] The quality of the RNA samples, as well as the detection of any residual ribosomal RNA (rRNA), was assessed on a Picochip in an Agilent 2100 Bioanalyzer, while sample quantity was assessed using the Nanodrop system. The electropherograms were used to assess whether samples could be taken forward for sequencing, required another collection because of RNA degradation or needed purification from contaminating rRNA.

[0192] Transcriptional changes driven by (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treatment of patient astrocytes 40 507965132.11959747.00063

[0193] We have interrogated the gene expression changes driven by drug treatment in each patient subgroup to determine the mechanism of action of drugs. Interestingly, untreated and treated samples tend to cluster very closely on the PCA plot, with the exception of the C9ORF72 samples, which display a large shift on the main axis towards control samples after drug treatment. This suggests that (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treatment caused different expression changes in C9ORF72 patients compared to the other groups.

[0194] To investigate the mechanism of action of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol in the patient iAstrocytes, lists of differentially regulated transcripts between each patient iAstrocyte subgroup treated with the compound and each patient iAstrocyte subgroup untreated were generated by running the comparisons in the table below (Table 1): Table 1. List of bioinformatic comparisons for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treated iAstrocytes and gene count. Bioinformatic comparison Total genes Upregulated Downregulated

[0195] A Venn diagram of shared genes between control and patient cell lines after (6aS)- 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment is shown in FIG.12A-B. CTR, SOD1 and sALS iAstrocyte lines displayed a similar number of unique DEGs after (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment (CTR = 204 DEGs, 119 up and 85 down; SOD1 = 213 DEGs, 148 up and 65 down; and sALS = 202 DEGs, 47 up and 155 down). In C9ORF72 patient iAstrocytes, on the other hand, only 128 genes were significantly altered after (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment (64 up and 64 down). SOD1 & sALS lines shared the most genes (23 DEGs, 22 up and 1 down) while less genes were shared amongst the different subgroups (9 DEGs, 6 up and 3 down); treatment with (6aS)- 41 507965132.11959747.00063 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol resulted in transcriptional upregulation in the majority of genes shared between patient and control iAstrocytes.

[0196] When the original gene lists were imported into the DAVID pathway analysis software, SOD1 iAstrocytes displayed the highest number of significantly altered GO terms (44 pathways), followed by sALS (33 pathways), and CTR and C9ORF72 iAstrocytes (19 and 12 pathways respectively). To identify drug-induced transcriptional changes common to all patient groups, we focused on the pathways shared between 2 or more iAstrocyte groups. All of the shared pathways between control and patient iAstrocyte subgroups after (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment are presented in Table 2. Table 2. List of GO pathways shared between ALS patient subgroups treated with (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. Gene Ontology Term CTR C9ORF72 SOD1 sALS l l l l42 507965132.11959747.00063

[0197] There were 2 pathways shared between CTR & SOD1 iAstrocytes, i.e., the cellular response to organic cyclic compound and the MAPK cascade. The first pathway refers to the change in activity of the cell in response to an organic cyclic compound, including movement, secretion, enzyme production or even further gene expression which is likely to reflect compound metabolism. This included a significant upregulation in the cytochrome P450 enzyme transcripts (CYP1A1: CTR p-value = 1.87E-74, log2FC = +6.99, SOD1 p-value = 2.18E-09, log2FC = +4.99; CYP1B1: CTR p-value = 5.81E-06, log2FC = +2.62, SOD1 p-value = 8.13E-27, log2FC = +2.59).

[0198] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment also induced DGE changes in transcripts within the MAP kinase signaling pathway, in particular, artemin, a secreted ligand belonging to both the GDNF and TGF-β family of proteins (ARTN: CTR p-value = 0.03, log2FC = +2.06, SOD1 p-value = 0.004, log2FC = +2.93), and interleukin-1 beta, an important mediator of the inflammatory response (IL1B: CTR p-value = 4.36E-05, log2FC = +2.08, SOD1 p-value = 1.21E-04, log2FC = +2.05).

[0199] However, there were many pathways that were shared between the groups of patient iAstrocytes; 2 pathways between all the patient groups and 6 pathways between SOD1 & sALS iAstrocytes. This was interesting as (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treatment caused different pathways to change in control and patient iAstrocytes, with the control lines minimally affected, while the patients saw a large gene expression change. This reflected what was seen in the co-culture experiments, indicating a patient-specific drug response.

[0200] All patient groups presented DEGs related to cell adhesion, however different transcripts were targeted within each group, contactin and protocadherins in C9ORF72, integrins in SOD1 and cadherins in sALS iAstrocytes. There was a significant increase in protocadherin-related transcripts in all patient groups compared to control iAstrocytes, however, after (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment, there was a reduction in protocadherin transcripts in C9ORF72 iAstrocytes only (PCDHA4: p-value = 7.90E-04, log2FC = -1.75; PCDHB15: p-value = 0.02, log2FC = - 2.02; PCDHGC4: p-value = 0.003, log2FC = -3.06). There was also a significant increase in CDH2 shared among all patient iAstrocytes, however, only sALS lines demonstrated a reduction in cadherin transcripts after (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treatment (CDH2: p-value = 0.003, log2FC = -1.92; 43 507965132.11959747.00063 CDH6: p-value = 7.51E-04, log2FC = -2.24). This suggests that (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment might have an effect on cellular migration and cell-cell contact but only in lines with a specific cellular environment.

[0201] SOD1 & sALS iAstrocytes displayed the highest number of shared transcripts after (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment, therefore it was unsurprising that they shared the most pathways. (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol appeared to target the inflammatory response within these cell types. SOD1 iAstrocytes displayed downregulation of the expression of X-C Motif Chemokine Receptor 1 (XCR1: p-value = 0.03, log2FC = -2.71), while there was an upregulation of the IFN-inducible T-cell alpha chemoattractant (CXCL11: p-value = 0.02, log2FC = +2.70). On the other hand, sALS iAstrocytes displayed downregulation of inflammatory chemoattractant molecules (CXCL1: p-value = 0.003, log2FC = -3.22; CXCL6; p-value = 0.02, log2FC = -3.87).

[0202] There were also DGE changes in the immune response after (6aS)-6-methyl- 5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment; there was an increased expression of interleukin 1 alpha, alongside interleukin-1 beta, in SOD1 iAstrocytes (IL1A: p-value = 0.03, log2FC = +2.53), while there was a downregulation of interleukin 32 (IL32: p-value = 0.03, log2FC = -2.07) and DGE in transcripts expressing major histocompatibility complex, class II proteins in sALS iAstrocytes (HLA-DOA: p- value = 0.03, log2FC = +1.58; HLA-DQB1: p-value = 0.005, log2FC = -3.85).

[0203] SOD1 and sALS iAstrocytes also shared DGE changes in oxidation processes after treatment, as expected from the known antioxidant properties of this drug. Both groups displayed a significant upregulation in transcripts related to aldehyde dehydrogenase enzymes, that are responsible for the detoxification of long-chain aldehydes as well as the metabolism of neurotransmitters (ALDH3A1: SOD1 p-value = 1.58E-11, log2FC = +2.09, sALS p-value = 1.47E-11, log2FC = +1.85), which is associated with NRF2. Interestingly, sALS iAstrocytes displayed a significant downregulation in another aldehyde dehydrogenase transcript involved in the synthesis of retinoic acid from retinaldehyde (ALDH1A2: p-value = 0.04, log2FC = -1.94), which is associated with MN death in ALS. Cytochrome P450 enzymes involved in the metabolism of retinoic acid were also differentially regulated in SOD1 and sALS iAstrocytes after treatment (CYP26B1: SOD1 p-value = 1.83E-05, log2FC = +1.50, CYP26A1: sALS p-value = 0.009, log2FC = -5.16). 44 507965132.11959747.00063

[0204] Treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline- 10,11-diol significantly increased the expression of arachidonate 15-lipoxygenase, an essential factor for ferroptosis, in SOD1 iAstrocytes (ALOX15B: p-value = 3.19E-07, log2FC = +3.12), as well as an increased expression in Prostaglandin-Endoperoxide Synthase 1 that may be involved in iron binding (PTGS1: p-value 4.49E-06, log2FC = +1.57), implying that treatment might have an effect on iron levels in SOD1 iAstrocytes, as was witnessed in spinal cord injury (SCI). sALS iAstrocytes displayed a significant downregulation in expression of peroxidasin, an enzyme that catalyses peroxidative reactions using hydrogen peroxide generated by NADPH oxidase enzymes (PXDN: p-value = 9.91E-05, log2FC = -3.57) which is a novel target of NRF2.

[0205] In summary, as hypothesised in the beginning, (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol treatment resulted in specific DGE changes depending upon the mutational status of the patient group. There was a mild change in gene expression related to cytochrome P450 enzymes and MAPK signalling shared between CTR and SOD1 patient lines. While all patient groups shared DGE changes in cell adhesion, only C9ORF72 iAstrocytes presented changes in protocadherin transcripts and there was DGE in cadherin genes in sALS lines only. SOD1 and sALS lines displayed the most DGE changes after treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol and these were related to pathways including inflammation, the immune response, oxidation, retinoic acid metabolism and iron binding. Example 5: Transcriptional ALS patient response at baseline

[0206] In the previous examples, the RNA-sequencing data were investigated to uncover the similarities and differences between the patient subgroups (SOD1, C9ORF72, and sALS) as well as the mechanism of action of drugs across the patient subgroups. In the current study, the RNA-sequencing samples have been collated based upon the response to drug, taken from the co-culture data, as identified in Table 3 below. 45 507965132.11959747.00063 Table 3. Patient responders to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, andrographolide and riluzole. Drug Patient responders (6aS)-6-methyl-5,6,6a,7- sALS 009

[0207] Table 3 above shows that each patient response group has a variety of genetic subgroups. It is crucial to understand if the response groups presented unique transcriptional features in comparison to controls, as this would be helpful to identify what was unique about these patients. Lists of DEGs between each patient iAstrocyte response group and the control iAstrocyte group were generated by running the comparisons in Table 4. Table 4. List of bioinformatic comparisons for patient responders at baseline. Bioinformatic comparisons Total genes Upregulated Downregulated46 507965132.11959747.00063

[0208] Since this analysis was looking into specific transcripts and not pathways, DEGs were selected based on a p-adj<0.05, meaning that we expected less than 5% of the significant transcripts to be false positives.

[0209] DEGs that were unique to each patient response group are identified using a multi- group comparison with Andrographolide and Riluzole. There were 11 DEGs unique to patient lines that responded to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol treatment (Table 5). These transcripts were involved in mitochondrial protein synthesis, cellular adhesion, and receptor tyrosine kinase signaling.

[0210] While little is known about the MRPS9 gene in regard to ALS, the influence of mutant SOD1 (mSOD1) on the mitochondria has been widely reported. Deviations from normal SOD1 levels increased the mitochondrial DNA (mtDNA) copy number as well as the impaired mitochondrial protein synthesis associated with mSOD1. The dipeptide repeat protein (DPRs) associated with C9ORF72-ALS were also found to preferentially bind to mitochondrial ribosomal proteins, compromising mitochondrial function. Meanwhile, both SOD1 and C9ORF72 iAstrocytes displayed differential mitochondrial dynamics and the significant reduction in mitochondrial form factor after (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment.

[0211] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment was shown to have an effect on transcripts associated with cell adhesion. NECTIN3 encodes a cellular adhesion molecule responsible for cell-cell contact at adherens junctions, the expression of which can be altered by stress. In neurons, these proteins are present at synaptic junctions, forming inter-neuronal connections and maintaining synapse formation and transmission. The expression of Nectin-3 was reportedly reduced in models of Alzheimer’s disease in relation to tauopathy, however the increased expression witnessed in the ALS patient iAstrocytes could be in relation to the stressed cellular environment. It was known that the expression of miR-29a was increased in the SOD1 mouse model, and therefore that this microRNA is potentially regulating COL5A3 expression in the SOD1 patient iAstrocytes.

[0212] Ephrin receptors are the largest protein family of receptor tyrosine kinases and are responsible for cell-cell interactions as well as the development of the nervous system, in particular neuronal cell migration and axon guidance. Loss of ephrin receptor signaling has 47 507965132.11959747.00063 been reported as protective in ALS, while deletion of EPHA3 was identified as a protective factor in a sALS patient population while inhibition of Epha4 signaling increased survival in a SOD1 mouse model. This indicated that the significant upregulation of EPHA3 within these ALS patient iAstrocytes was detrimental to the surrounding MNs. Example 6: ALS patient response of transcripts to drugs

[0213] To identify the transcripts that determine a “patient responder”-specific response to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol, gene expression changed after drug treatment was compared within the patient responder cell lines. Lists of differentially regulated transcripts between the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol iAstrocyte response group before and after treatment compared to the control iAstrocyte group were generated by running the comparisons in the table below (Table 6). 48 507965132.11959747.00063 Table 5. List of 11 transcripts unique to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol patient responders at baseline. Gene Name (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol Responders49 507965132.11959747.00063 EPH Receptor A3 Fold +7.463762783 (EPHA3) changeabe 5. Contnued. Gene Name Andrographolide Riluzole R d R d 6 4 2 850 507965132.11959747.00063 Glutamine Fold +2.915583083 +3.620472183 Amidotransferase Like change 3 6 5 551 507965132.11959747.00063 Table 6. List of bioinformatic comparisons for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol patient responders. Bioinformatic comparisons Responder (6aS)-6-methyl-566a7-tetrahydro-4H-dibenzo[deg]quinoline-1011-diol U vs

[0214] The highest number of significant DEGs was observed in the (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol responder treated (S Resp. T) vs (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol responder untreated (S Resp. U) comparison (161 genes), while the other comparisons displayed fewer transcripts; the S Resp. U vs control untreated (CTR U) comparison presented 44 DEGs, while S Resp. T vs CTR U displayed 73 genes. A full list of transcripts generated from the S Resp. T vs S Resp. U comparison are presented in Table 7. Table 7. List of 73 transcripts for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol responders after treatment (S Resp. T vs CTR U). Gene Name p-adj log2FC 4 8 1 3 11959747.00063 Gene Name p-adj log2FC ADAM metallopeptidase with thrombospondin type 1 motif 5 1.47E-04 +9.204241492 6 8 7 9 8 5 6 2 4 6 5 8 7 3 1 3 4 2 9 4 5 8 8 31959747.00063 Gene Name p-adj log2FC Secreted and transmembrane 1 (SECTM1) 0.005320126 +3.904561871 4 8 6 5 7 5 9 9 9 2 9 7 3 8 9 1 3 3 1 3 4 5 5 7 91959747.00063 Gene Name p-adj log2FC Uncharacterized LOC25845 (PP7080) 0.02564762 -2.082370978 6 5 9 7 5 5 2 1 2 2 2 1 1 9 5 4

[0215] The gene lists were imported into Venny to identify transcripts that were uniquely or commonly dysregulated in the 3 comparisons (FIG 13). There were 153 genes (111 up, 42 down) unique to the S Resp. T vs S Resp. U comparison. This comparison identified genes where there was a large significant change in expression after treatment, potentially due to the mechanism of action of the drug. However, this list of 153 genes did not share any transcripts with the S Resp. U vs CTR U comparison, implying that none of these transcripts were dysregulated at baseline.1959747.00063 Table 8. List of 153 transcripts for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol responders after treatment (S Resp. T vs S Resp. U). Gene Name p-adj log2FC HS6ST2-AS1 3.25E-08 3.358974899IL1B 7.09E-06 1.701955186 4.81E-06 1.686680378FXYD3 4.52E-09 1.626783581 CYP26B1 8.73E-06 1.608460702 CYP1B1-AS1 1.74E-08 1.604241463 CH25H 4.31E-05 1.550752827 COLEC12 1.31E-14 1.537477914 TIPARP 1.77E-06 1.360272657 KIAA1549L 2.30E-04 1.35385984 IFI30 1.20E-28 1.3425215871959747.00063 Gene Name p-adj log2FC NPTX1 1.59E-05 1.313292867 ZMIZ1-AS1 2.01E-04 1.311386771 ANPEP 4.31E-06 1.270863355 ARHGEF16 1.45E-05 1.2588397041959747.00063 Gene Name p-adj log2FC PRELP 2.24E-06 0.958965749 TGFBI 1.88E-04 0.957522709 FAM47E-STBD1 2.21E-05 0.957422812 TSPAN15 1.52E-06 0.9573003381959747.00063 Gene Name p-adj log2FC1959747.00063 Gene Name p-adj log2FC LMO4 2.65E-04 -0.4837233171959747.00063 Gene Name p-adj log2FC MDFI 3.60E-04 -1.099602671

[0216] The 38 DEGs unique to the S Resp. T vs CTR U comparison (28 up, 10 down) highlighted transcripts that were significantly changed after treatment but did not combat the original dysfunction in the patient cell lines, as these transcripts were not altered in the baseline comparison. The DEGs shared between S Resp. U vs CTR U and S Resp. T vs CTR U comparisons (27 transcripts, 27 up, 0 down) identified transcripts that were dysregulated at baseline and remained dysregulated without changing regulation, increase or decrease, after drug treatment. Therefore, these 14 genes that were unique to the S Resp. U vs CTR U comparison (11 up, 3 down) was further investigated, as these transcripts were significantly different to control lines prior to treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, however this significance was lost after treatment, thus indicating that (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment has corrected their dysregulation. Hence, these transcripts would be good candidates to identify a response gene signature.

[0217] To identify the biomarkers that discriminated between patient responders, we expected the gene expression biomarkers to be corrected after treatment. The baseline S Resp. U vs CTR U comparison in the previous example was a starting point and these transcripts either 1) changed direction and were still significantly dysregulated compared to controls or 2) went1959747.00063 back to baseline and were no longer significantly dysregulated anymore after treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0218] Among 14 genes that were unique to the baseline S Resp. U vs CTR U comparison (11 up, 3 down), 10 of them were uniquely dysregulated in M102 responders at the baseline and corrected after treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline- 10,11-diol (Table 5).

[0219] There were 10 transcripts that were unique to the S Resp. U vs CTR U comparison, indicating that after treatment, these transcripts were no longer significantly dysregulated in the patient responder lines. Interestingly, ICA1, MRPS9, NECTIN3, EPHA3, MFF-DT & ROCK1P1 were all previously investigated as significantly dysregulated transcripts unique to (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol responders at baseline (Table 5) compared to other responders groups. Additional transcripts identified in the S Resp. U vs CTR U comparison would have been significantly dysregulated in other response groups at baseline. The transcripts with the largest change in significance after (6aS)- 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment were MRPS9, MFF-DT, ROCK1P1 & GRM4 (Table 9). Table 9. List of genes dysregulated at baseline and corrected after (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment in the comparison responders untreated vs Control untreated (S Resp. U vs CTR U) Gene Name S Resp. U vs CTR S Resp. T vs CTR1959747.00063 Gene Name S Resp. U vs CTR S Resp. T vs CTR Nectin Cell Adhesion Fold +1.80096269 +1.445576666

[0220] To identify whether this change in significance after (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment was due to a response in just one or multiple patient responder lines, the normalized gene counts (TPM values) for each transcript before and after (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-1959747.00063 10,11-diol treatment in both patient responder and non-responder iAstrocytes were plotted (FIG.14). The graphs indicated that not all biomarkers were changed in all responder cell lines after treatment. Therefore, it is possible that a combination of these transcripts could be used as an appropriate biomarker or biomarker panel of drug response.

[0221] The patient cell lines were then given a score depending on the change in each biomarker after treatment with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11- diol (FIG 15). If there was a change towards the control after treatment, there was given a score of +1, a score of 0 for no change and a score of -1 if the gene expression change was against the control after treatment; the total scores were presented at the bottom of the table. The figure shows that patient responders typically scored between 2 and 6 when they were assessed against the whole gene panel, while the patient non-responder lines scored between 0 and -2. This confirmed the previous hypothesis that not every biomarker changed in every patient line, but selected gene biomarkers can be used as a panel to discriminate patient responders and non-responders. EMBODIMENTS

[0222] Embodiment 1. A method of increasing p62 proteins in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol.

[0223] Embodiment 2. The method of embodiment 1, wherein the p62 proteins are perinuclear P62 proteins.

[0224] Embodiment 3. A method of increasing LC3 proteins in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol.

[0225] Embodiment 4. The method of embodiment 3, wherein the LC3 proteins are LC3-I or LC3-II proteins.

[0226] Embodiment 5. A method of reducing mitochondrial form factor in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol.1959747.00063

[0227] Embodiment 6. A method of changing biological pathways in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol.

[0228] Embodiment 7. The method of embodiment 6, where the biological pathways are selected from any one or more of: cellular response to organic cyclic compound, MAPK cascade, morphogenesis of an epithelial fold, cell adhesion, angiogenesis, inflammatory response, immune response, oxidation-reduction process, xenobiotic metabolic process, cholinergic synaptic transmission, or collagen fibril organization.

[0229] Embodiment 8. A method of changing the levels of gene expression in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0230] Embodiment 9. The method of embodiment 8, where the genes are selected from any one or more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.1959747.00063

[0231] Embodiment 10. The method of embodiment 8, where the genes are selected from any one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0232] Embodiment 11. The method of any one of embodiments 1-10, wherein the cell is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder, or vagina.

[0233] Embodiment 12. The method of embodiment 11, wherein the brain cell is from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem.

[0234] Embodiment 13. The method of embodiment 12, wherein the brain cell is selected from: neuron, astrocyte, oligodendrocyte, or microglia.

[0235] Embodiment 14. The method of embodiment 13, wherein the neuron is a sensory neuron, motor neuron, interneuron, or brain neuron.

[0236] Embodiment 15. The method of any one of embodiments 1-14, wherein the cell is an animal cell

[0237] Embodiment 16. The method of embodiment 15, wherein the cell in a human cell

[0238] Embodiment 17. The method of any one of embodiments 1-16, wherein the cell is in vitro.

[0239] Embodiment 18. The method of any one of embodiments 1-16, wherein the cell is ex vivo.

[0240] Embodiment 19. The method of any one of embodiments 1-16, wherein the cell is in vivo.

[0241] Embodiment 20. The method of any one of embodiments 1-16, wherein the cell is a diseased cell.1959747.00063

[0242] Embodiment 21. The method of embodiment 20, wherein the diseased cell is from an animal having a disease or disorder selected from any one or more of: aging-related tau astrogliopathy (ARTA), Alexander Disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), Critical Illness Myopathy (CIM), Primary Age-Related Tauopathy (PART), aortic medial amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, argyrophillic grain disease, ataxia telangiectasia, atrial fibrillation, Autosomal Dominant Hyper-IgE Syndrome, cardiac atrial amyloidosis, Bloom's syndrome, cardiovascular diseases, coronary artery disease, myocardial infarction, stroke, restenosis, arteriosclerosis, cataracts, cerebral amyloid angiopathy, Christianson syndrome, chronic traumatic encephalopathy, Cockayne's syndrome, corneal lactoferrin amyloidosis, corticobasal degeneration, Crohn's Disease, Cushing's disease, cutaneous lichen amyloidosis, cystic fibrosis, Dentatorubropallidoluysian Atrophy (DRPLA), dialysis amyloidosis, diffuse neurofibrillary tangles with calcification, Down syndrome, endotoxin shock, familial amyloidosis of the Finnish type, familial amyloidotic neuropathy, Familial British Dementia (FBD) , Familial Danish Dementia (FDD), familial dementia, fibrinogen amyloidosis, fragile X syndrome, Fragile X-associated Tremor / Ataxia Syndrome (FXTAS), Friedreich's ataxia, fronto-temporal degeneration, glaucoma, Glycogen Storage Disease type IV (Andersen Disease), Guadeloupean Parkinsonism, hereditary lattice corneal dystrophy, Huntington's disease, inclusion body myositis / myopathy, inflammation, inflammatory bowel disease, ischemic condition, ischemia / reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia, light chain or heavy chain amyloidosis, lysosomal storage diseases, aspartylglucosaminuria, Fabry's disease, Batten disease, Cystinosis, Farber, Fucosidosis, Galactasidosialidosis, Gaucher's disease Type 1, 2 or 3, Gml gangliosidosis, Hunter's disease, Hurler-Scheie's disease, Krabbe's disease, a-Mannosidosis, B-Mannosidosis, Maroteaux-Lamy's disease, Metachromatic Leukodystrophy, Morquio A syndrome, Morquio B syndrome, Mucolipidosis II, Mucolipidosis III, Neimann-Pick Disease Type A, B or C, Pompe's disease, Sandhoff disease, Sanfilippo syndrome Type A, B, C or D, Schindler disease, Schindler-Kanzaki disease, Sialidosis, Sly syndrome, Tay-Sach's disease, Wolman disease, lysozyme amyloidosis, Mallory bodies, medullary thyroid carcinoma, mitochondrial myopathies, multiple sclerosis, multiple system atrophy, myotonic dystrophy, myotonic dystrophy, neurodegeneration with brain iron accumulation, neurofibromatosis,1959747.00063 neuronal ceroid lipofuscinosis, odontogenic (Pinborg) tumor amyloid, Parkinsonism-Dementia of Guam, Parkinson's disease, peptic ulcers, Pick’s disease, pituitary prolactinoma, post- encephalitic Parkinsonism, prion diseases (Transmissible Spongiform Encephalopathies), including Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease, Gerstmann- Straussler-Scheinker Syndrome, Fatal Familial Insomnia, Kuru, progressive supranuclear palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, seminal vesical amyloid, senile systemic amyloidoses, Serpinopathies, sickle cell disease, spinal and bulbar muscular atrophy (SBMA), spinocerebellar ataxias, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 8, spinocerebellar ataxia type 17), subacute sclerosing panencephalitis, tauopathies, type II diabetes, vascular dementia, or Werner syndrome.

[0243] Embodiment 22. A method of treating an animal having a disease or disorder that would benefit from increasing p62 proteins, increasing LC3 proteins, or reducing mitochondrial form factor, or a combination thereof, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal.

[0244] Embodiment 23. A method of treating an animal having a disease or disorder that would benefit from changing biological pathways in a cell, comprising the step of contacting the cell with an effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, where the biological pathways are selected from any one or more of: cellular response to organic cyclic compound, MAPK cascade, morphogenesis of an epithelial fold, cell adhesion, angiogenesis, inflammatory response, immune response, oxidation-reduction process, xenobiotic metabolic process, cholinergic synaptic transmission, or collagen fibril organization.

[0245] Embodiment 24. A method of treating an animal having a disease or disorder that would benefit from changing the level of genes, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal,1959747.00063 where the genes are selected from any one of more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1- AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0246] Embodiment 25. A method of treating an animal having a disease or disorder that would benefit from changing the level of genes, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the genes are selected from any one or more of the following: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0247] Embodiment 26. A method of treating an animal having a disease or disorder that would benefit from changing the level of genes, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the animal, where the genes are selected from any one of more of the following: ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.1959747.00063

[0248] Embodiment 27. The method of any one of embodiments 22-26, wherein the disease or disorder is selected from any one or more of: age-related macular degeneration, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease Crohn's disease, Duchenne's paralysis, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Huntington's disease (HD), Inclusion Body Myopathy (IBM)inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, Neurofibromatosis type I, Paget's disease of the bone (PDB), Parkinson disease (PD), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, Valosin-Containing Protein (VCP)-related disorders, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, cerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown- Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth Disease, macular degeneration, X-Linked bulbo-spinal muscular atrophy, presenile dementia, depressive disorder, temporal lobe epilepsy, hereditary Leber optic atrophy (Kennedy’s disease), cerebrovascular accident, subarachnoid hemorrhage, or schizophrenia.

[0249] Embodiment 28. The method of any one of embodiments 22-27, wherein the animal is a mammal.

[0250] Embodiment 29. The method of embodiment 28, wherein the mammal is a non-human animal.

[0251] Embodiment 30. The method of embodiment 28, wherein the mammal is a human.

[0252] Embodiment 31. The method of any one of embodiments 22-30, wherein the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered at a dose of 0.12 mg / kg or higher.

[0253] Embodiment 32. The method of any one of embodiments 22-30, wherein the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered at a dose between 5-5000 mg / day.1959747.00063

[0254] Embodiment 33. The method of any one of embodiments 22-30, wherein the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered parenterally, enterally, or topically.

[0255] Embodiment 34. The method of embodiment 33, wherein the 6-methyl-5,6,6a,7- tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular, or subcutaneous administration.

[0256] Embodiment 35. Use of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol for the preparation of a medicament for treating a human having a disease of embodiment 27.

[0257] Embodiment 36. A method of treating an animal having a disease or disorder, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol to said animal, where the animal is selected by the changes genes in a cell responding to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, and the genes are selected from any one of more of the following: HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3,1959747.00063 RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1- MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0258] Embodiment 37. A method of treating an animal having a disease or disorder, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol to the animal, where the animal is selected by the changes genes responding in a cell to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, and the genes are selected from any one of more of the following 14 genes: ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0259] Embodiment 38. A method of treating an animal having a disease or disorder, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol to the animal, where the animal is selected by the changes genes responding in a cell to the treatment of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol, and the genes are selected from any one of more of the following 14 genes: ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0260] Embodiment 39. The method of any one of embodiments 36-38, wherein said cell is a cell type or from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder, or vagina.

[0261] Embodiment 40. The method of any one of embodiments 36-39, wherein the disease or disorder is selected from any one or more of: age-related macular degeneration, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease Crohn's1959747.00063 disease, Duchenne's paralysis, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Huntington's disease (HD), Inclusion Body Myopathy (IBM), inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, Neurofibromatosis type I, Paget's disease of the bone (PDB), Parkinson disease (PD), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, Valosin-Containing Protein (VCP)-related disorders, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, cerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown- Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth Disease, macular degeneration, hereditary Leber optic atrophy (Kennedy’s disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Hereditary Leber Optic Atrophy, cerebrovascular accident, subarachnoid hemorrhage, or schizophrenia.

[0262] Embodiment 41. The method of any one of embodiments 36-40, wherein the animal is a mammal.

[0263] Embodiment 42. The method of embodiment 41, wherein the mammal is a non-human animal.

[0264] Embodiment 43. The method of embodiment 41, wherein the mammal is a human.

[0265] Embodiment 44. The method of any one of embodiments 36-43, wherein the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered at a dose of 0.12 mg / kg or higher.

[0266] Embodiment 45. The method of any one of embodiments 36-43, wherein the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered at a dose between 5-5000 mg / day.

[0267] Embodiment 46. The method of any one of embodiments 36-43, wherein the (6aS)-6- methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is administered parenterally, enterally, or topically.1959747.00063

[0268] Embodiment 47. The method of claim 46, wherein the 6-methyl-5,6,6a,7-tetrahydro- 4H-dibenzo[de,g]quinoline-10,11-diol is administered by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular, or subcutaneous administration.

[0269] Embodiment 48. Use of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline-10,11-diol for the preparation of a medicament for treating a human having a disease of embodiment 40.

[0270] Embodiment 49. The method of one of embodiments 1–21, wherein the increasing, reducing, or changing is determined relative to a corresponding measurement prior to the step of contacting the cell with the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline- 10,11-diol.

[0271] Embodiment 50. The method of embodiment 49, wherein the cell is a cell of a human subject, and the human subject has Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD).

[0272] Embodiment 51. A method, comprising: A) predicting efficacy of a treatment of a disease or disorder in a subject, wherein the treatment comprises a first administration of an active pharmaceutical agent to a first cell of the subject, the first cell having a first cell type, and optionally a second administration of the active pharmaceutical agent to a second cell of the subject, the second cell having the first cell type, and the active pharmaceutical agent has the formula or a pharmaceutically acceptable salt thereof; B)sample comprising a control cell from the subject prior to the first administration, the control cell having the first cell type; C) collecting a second biological sample from the subject after the first administration and prior to the optional second administration, the second biological sample comprising the first cell; D) determining a first differential expression of one or more genes in the second biological sample compared to the first biological sample; and E) predicting efficacy of the treatment based on the first differential expression.1959747.00063

[0273] Embodiment 53. A method, comprising: A) monitoring a treatment of a disease or disorder in a subject, wherein the treatment comprises a first administration of an active pharmaceutical agent to a first cell of the subject, the first cell having a first cell type, and optionally a second administration of the active pharmaceutical agent to a second cell of the subject, the second cell having the first cell type, and the active pharmaceutical agent has the formula or a pharmaceutically acceptable salt thereof; B)sample comprising a control cell from the subject prior to the first administration, the control cell having the first cell type; C) collecting a second biological sample from the subject after the first administration and prior to the optional second administration, the second biological sample comprising the first cell; D) determining a first differential expression of one or more genes in the second biological sample compared to the first biological sample; and E) monitoring the treatment based on the first differential expression.

[0274] Embodiment 53. The method of embodiments 51 or 52, wherein the disease or disorder is a neurological disease.

[0275] Embodiment 54. The method of embodiments 51 or 52, wherein the disease or disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD).

[0276] Embodiment 55. The method of embodiments 51 or 52, wherein the disease or disorder is selected from any one or more of: age-related macular degeneration, atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease, Crohn's disease, Duchenne's paralysis, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Inclusion Body Myopathy (IBM), inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, Neurofibromatosis type I, Paget's disease of the bone (PDB), primary lateral sclerosis (PLS),1959747.00063 progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, Valosin-Containing Protein (VCP)-related disorders, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, cerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown-Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot- Marie-Tooth Disease, macular degeneration, hereditary Leber optic atrophy (Kennedy’s disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Hereditary Leber Optic Atrophy, cerebrovascular accident, subarachnoid hemorrhage, or schizophrenia.

[0277] Embodiment 56. The method of one of embodiments 51–55, wherein the one or more genes are selected from HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1-AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189-UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

[0278] Embodiment 57. The method of one of embodiments 51–55, wherein the one or more genes are selected from ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.1959747.00063

[0279] Embodiment 58. The method of one of embodiments 51–55, wherein the one or more genes are selected from ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

[0280] Embodiment 59. The method of one of embodiments 51–58, wherein the first differential expression of one or more genes identifies significantly dysregulated transcripts of the one or more genes in the first biological sample as compared to the second biological sample.

[0281] Embodiment 60. The method of one of embodiments 51–59, wherein the method is an in vitro, ex vivo, or in vivo method.

[0282] Embodiment 61. The method of one of embodiments 51–60, wherein the first cell type is from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder, or vagina.

[0283] Embodiment 62. The method of any one of embodiments 51–61, wherein the animal is a mammal.

[0284] Embodiment 63. The method of embodiment 62, wherein the mammal is a non-human animal.

[0285] Embodiment 64. The method of embodiment 62, wherein the mammal is a human.

[0286] Embodiment 65. The method of one of embodiments 51–64, wherein the active pharmaceutical agent is administered at a dose of 0.12 mg / kg or higher.

[0287] Embodiment 66. The method of one of embodiments 51–65, wherein the active pharmaceutical agent is administered at a dose between 5–5000 mg / day.

[0288] Embodiment 67. The method of one of embodiments 51–66, wherein the active pharmaceutical agent is administered parenterally, enterally, or topically.1959747.00063

[0289] Embodiment 68. The method of one of embodiments 51–67, wherein the active pharmaceutical agent is administered by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular, or subcutaneous administration.

[0290] Embodiment 69. The method of one of embodiments 51–68, the method further comprising: F) monitoring the effectiveness of the treatment based on a second differential expression or a third differential expression; G) continuing the treatment with the second administration; H) collecting a third biological sample from the subject after the second administration, the third biological sample comprising the second cell; and I) determining the second differential expression of one or more genes compared to the first biological sample or compared to the third biological sample.

[0291] Embodiment 70. The method of one of embodiments 51–69, wherein the subject experiences an increase in the level of p62, an increase in the level of LC3, or a reduction of mitochondrial form factor, or a combination thereof, as a result of the first administration, the second administration, or both.

[0292] Embodiment 71. The method of one of embodiments 51–70, wherein the first cell type is a brain cell from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain- stem, optionally wherein the brain cell is an induced brain cell.

[0293] Embodiment 72. The method of one of embodiments 51–70, wherein the first cell type is a brain cell selected from: neuron, astrocyte, oligodendrocyte, or microglia, optionally wherein the brain cell is an induced brain cell.

[0294] Embodiment 73. The method of one of embodiments 51–70, wherein the first cell type is a brain cell selected from: sensory neuron, motor neuron, interneuron, or brain neuron, optionally wherein the brain cell is an induced brain cell.

[0295] Embodiment 74. The method of one of embodiments 51–73, wherein the method is a method of treatment of the disease or disorder.1959747.00063

[0296] Embodiment 75. A method of treating Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD) in a subject in need thereof, comprising: 1) administering parenterally, enterally, or topically to the subject at a first dose of at least 0.12 mg / kg, and optionally at a dose between 5–5000 mg / day, an active pharmaceutical agent having the formula or a pharmaceutically acceptable salt thereof; 2) measuringor mitochondrial form factor, or a combination thereof, in a biological sample comprising a cell, which may include a brain cell from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem, optionally wherein the brain cell is an induced brain cell, wherein the measuring comprises translatome profiling; and 3) treating the Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Lou Gehrig's disease, or Parkinson’s disease (PD) in the subject with a second dose that is greater than the first dose if the subject does not experience an increase in the level of p62, an increase in the level of LC3, or a reduction of mitochondrial form factor, or a combination thereof, as a result of administration of the first dose.

[0297] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the1959747.00063 numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0298] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0299] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0300] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible1959747.00063 variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0301] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0302] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0303] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

1959747.00063 CLAIMS What is claimed is:

1. A method, comprising: A) predicting efficacy of a treatment of a disease or disorder in a subject, wherein the treatment comprises a first administration of an active pharmaceutical agent to a first cell of the subject, the first cell having a first cell type, and optionally a second administration of the active pharmaceutical agent to a second cell of the subject, the second cell having the first cell type, and the active pharmaceutical agent has the formula or a pharmaceutically acceptable salt thereof; B)sample comprising a control cell from the subject prior to the first administration, the control cell having the first cell type; C) collecting a second biological sample from the subject after the first administration and prior to the optional second administration, the second biological sample comprising the first cell; D) determining a first differential expression of one or more genes in the second biological sample compared to the first biological sample; and E) predicting efficacy of the treatment based on the first differential expression.

2. A method, comprising: A) monitoring a treatment of a disease or disorder in a subject, wherein the treatment comprises a first administration of an active pharmaceutical agent to a first cell of the subject, the first cell having a first cell type, and optionally a second administration of the active pharmaceutical agent to a second cell of the subject, the second cell having the first cell type, and the active pharmaceutical agent has the formula1959747.00063 or a pharmaceutically acceptable salt thereof; B)sample comprising a control cell from the subject prior to the first administration, the control cell having the first cell type; C) collecting a second biological sample from the subject after the first administration and prior to the optional second administration, the second biological sample comprising the first cell; D) determining a first differential expression of one or more genes in the second biological sample compared to the first biological sample; and E) monitoring the treatment based on the first differential expression.

3. The method of claims 1 or 2, wherein the disease or disorder is a neurological disease.

4. The method of claims 1 or 2, wherein the disease or disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD).

5. The method of claims 1 or 2, wherein the disease or disorder is selected from any one or more of: age-related macular degeneration, atherosclerosis, autism spectrum disorder (ASD), benign focal amyotrophy, cerebral infarction, Creutzfeldt-Jakob disease, Crohn's disease, Duchenne's paralysis, Friedreich's ataxia, frontotemporal dementia (FTD), glaucoma, hereditary spastic paraplegia (HSP), Inclusion Body Myopathy (IBM), inflammatory bowel disease, ischemia, Kugelberg-Welander syndrome, Lewy body diseases (LBD), multiple sclerosis (MS), myocardial infarction, necrotizing enterocolitis, Neurofibromatosis type I, Paget's disease of the bone (PDB), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, Valosin- Containing Protein (VCP)-related disorders, Werdnig-Hoffmann disease, transient ischemic1959747.00063 attack, ischemia, cerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown- Vialetto-Van Laere syndrome, Eales Disease, meningitis and encephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth Disease, macular degeneration, hereditary Leber optic atrophy (Kennedy’s disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Hereditary Leber Optic Atrophy, cerebrovascular accident, subarachnoid hemorrhage, or schizophrenia.

6. The method of one of claims 1–5, wherein the one or more genes are selected from HS6ST2-AS1, CYP1B1, MEGF6, AHRR, CILP, TNS4, CACNA1S, IFNLR1, KSR2, OLFML2B, BCL11B, FGF18, GALNT5, NKAIN1, DAPP1, LINC00599, GPAT3, IL1B, PRAL, FXYD3, CYP26B1, CYP1B1-AS1, CH25H, COLEC12, TIPARP, KIAA1549L, IFI30, NPTX1, ZMIZ1- AS1, ANPEP, ARHGEF16, WFDC11, KIAA1549, SUSD3, H19, GFRA1, NRG1, NQO1, TCF7, DTX1, PRXL2A, COLGALT2, LUM, HSPB6, SLC16A10, COL24A1, KCND3, SLC1A2, WNT11, CLDN7, RNF128, PTGS1, ZNF385D, ADGRE2, LINC00639, ADGRD1, PRELP, TGFBI, FAM47E-STBD1, TSPAN15, SLC2A1, IER3, AJUBA, NTN4, CEMIP, CX3CL1, SLC16A3, STC2, THBD, IGFBP2, TNFRSF1B, ANXA3, NRP1, CD248, CDC42EP2, IGSF3, ETNPPL, APCDD1, GNAZ, S1PR2, STK26, RDH10, RRAD, TNFAIP2, IL15RA, ITGB4, CD14, CAMK2B, SYNJ2, PIR, FAM156A, TPRA1, F2R, RGL1, GATAD2B, SLC6A6, NEDD4L, MN1, ATF3, ADCY9, SASH1, SLC22A23, SLC7A5, ANXA11, ZNF70, CD58, IL6R, ATXN1, FHL2, GABRA2, CSGALNACT1, S100A4, BIN1, PAPSS2, LMO4, RAB31, EPM2A, TP53INP1, SESN3, LGI2, C14orf132, ENHO, GCSHP5, LRATD1, LGALS7B, MARCHF3, CRYAB, ID1, PLPP4, RPSAP58, EEF1A1P5, TPPP3, MBNL1-AS1, CCDC171, TMEM189- UBE2V1, AFF2, SCIN, ID3, RUBCNL, EFEMP1, MYRIP, MEST, MDFI, LY6D, RHBDL3, SAMD11, OR2S1P, HSPE1-MOB4, LGALS7, HPGD, SULT1E1, LINC00598, or ONECUT2.

7. The method of one of claims 1–5, wherein the one or more genes are selected from ICA1, MRPS9, NRIP3, NECTIN3, GATD3A, COL5A3, EPHA3, MFF_DT, IGFBP5, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.1959747.00063 8. The method of one of claims 1–5, wherein the one or more genes are selected from ICA1, MRPS9, NECTIN3, EPHA3, MFF_DT, ROCK1P1, LYPD6, DKK2, CAP2, or GRM4.

9. The method of one of claims 1–8, wherein the first differential expression of one or more genes identifies significantly dysregulated transcripts of the one or more genes in the first biological sample as compared to the second biological sample.

10. The method of one of claims 1–9, wherein the method is an in vitro or ex vivo method.

11. The method of one of claims 1–10, wherein the first cell type is from a tissue selected from any one or more of: adrenal gland, bone marrow, brain, breast, bronchus, caudate, cerebellum, cerebral cortex, cervix, uterine, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, heart muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, peripheral blood mononuclear cell (PBMC), placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, urinary bladder, or vagina.

12. The method of any one of claims 1–11, wherein the animal is a mammal.

13. The method of claim 12, wherein the mammal is a non-human animal.

14. The method of claim 12, wherein the mammal is a human.

15. The method of one of claims 1–14, wherein the active pharmaceutical agent is administered at a dose of 0.12 mg / kg or higher.1959747.00063 16. The method of one of claims 1–15, wherein the active pharmaceutical agent is administered at a dose between 5–5000 mg / day.

17. The method of one of claims 1–16, wherein the active pharmaceutical agent is administered parenterally, enterally, or topically.

18. The method of one of claims 1–17, wherein the active pharmaceutical agent is administered by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular, or subcutaneous administration.

19. The method of one of claims 1–18, the method further comprising: F) monitoring the effectiveness of the treatment based on a second differential expression or a third differential expression; G) continuing the treatment with the second administration; H) collecting a third biological sample from the subject after the second administration, the third biological sample comprising the second cell; and I) determining the second differential expression of one or more genes compared to the first biological sample or compared to the third biological sample.

20. The method of one of claims 1–19, wherein the subject experiences an increase in the level of p62, an increase in the level of LC3, or a reduction of mitochondrial form factor, or a combination thereof, as a result of the first administration, the second administration, or both.

21. The method of one of claims 1–20, wherein the first cell type is a brain cell from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem, optionally wherein the brain cell is an induced brain cell.1959747.00063 22. The method of one of claims 1–20, wherein the first cell type is a brain cell selected from: neuron, astrocyte, oligodendrocyte, or microglia, optionally wherein the brain cell is an induced brain cell.

23. The method of one of claims 1–20, wherein the first cell type is a brain cell selected from: sensory neuron, motor neuron, interneuron, or brain neuron, optionally wherein the brain cell is an induced brain cell.

24. The method of one of claims 1–23, wherein the method is a method of treatment of the disease or disorder.

25. A method of treating Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD) in a subject in need thereof, comprising: 1) administering parenterally, enterally, or topically to the subject at a first dose of at least 0.12 mg / kg, and optionally at a dose between 5–5000 mg / day, an active pharmaceutical agent having the formula or a pharmaceutically acceptable salt thereof; 2) measuringor mitochondrial form factor, or a combination thereof, in a biological sample comprising a cell, which may include a brain cell from a brain tissue selected from: cerebrum, cerebellum, diencephalon, or brain-stem, optionally wherein the brain cell is an induced brain cell, wherein the measuring comprises translatome profiling; and 3) treating the Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington's disease (HD), or Parkinson’s disease (PD) in the subject with a second dose that is greater than the first dose if the subject does not experience an increase in the level of p62, an increase in the level of LC3, or a reduction of mitochondrial form factor, or a combination thereof, as a result of administration of the first dose.

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