Patent Application
20240238254
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 24, 2022, is named 02240_561481_SL.txt and is 24,350 bytes in size.
The field of the currently claimed embodiments of this invention relate to methods of treating amyotrophic lateral sclerosis (ALS) including: selecting a therapeutic compound: treating an aberrant arachidonic acid (AA) metabolic pathway in an ALS cell.
Progressive loss of spinal motor neurons (sMN) is a hallmark of amyotrophic lateral sclerosis (ALS)1-3, which causes progressive paralysis and eventually death4. Around 10% of ALS cases are inherited, and over 20 genes are now known to cause familial ALS (fALS) when mutated such as superoxide dismutase 1 (SOD1)2, C9ORF725, TAR DNA binding protein (TDP43)6, fused in sarcoma (FUS)7, optineurin (OPTN)8, profilin1 (PFNI)9, matrin-3 (MATR3)10, Tubulin Alpha 4A (TUBA4A)11, TANK binding kinase1 (TBK1)12 and several others.13. Much of the current understanding of ALS pathogenesis has been made through investigations of animal models carrying mutations in genes such as SOD1, C9ORF72 and TDP4314,15, and these models provide opportunities to test therapeutic targets. However, 90% of ALS cases are sporadic (sALS) and caused by unknown factors13,16. Recently, human induced pluripotent stem cells (hiPSCs) have emerged as an alternative and complementary system to animal models17-21. One of the advantages of hiPSC systems is enabling the generation of personalized cellular models with patient-specific mutations and genetic backgrounds. Using this technique, ALS cellular models have been generated without complicated genetic modifications for fALS cases, as well as for sALS. These hiPSC-based ALS cellular models have been used to elucidate pathogenic molecular mechanisms in ALS22-26 by comparing ALS-specific sMN and healthy sMN, although the healthy control hiPSCs have variable genetic backgrounds. Recently, genetically corrected isogenic control hiPSCs have been proposed as an ideal control using newly developed gene editing technology25,27,28 because the isogenic hiPSCs could minimize genetic variations in multiple healthy control hiPSCs. Nevertheless, an isogenic control is not feasible in cases with multiple and/or unknown mutations or in sporadic cases. Unsurprisingly, some reports have suggested that the widely-used CRISPR-Cas9 system may cause inadvertent DNA changes that could result in unintended phenotypes irrelevant to disease29,30. Thus, the new concept of comparative disease modeling using hiPSCs may lead to new insights into underlying ALS disease mechanisms.
Clinical features of ALS patients show selective vulnerability depending upon the specific motor neuron subtypes. Even in late stages of ALS disease progression, ocular motor neuron (oMN) is functionally intact in most patients. While motor neuron subtypes in the spinal cord, hindbrain and cortex are gradually impaired or lost during ALS progression leading to disability of voluntary movement, eye movements controlled by oMNs in the midbrain remain relatively unaffected until the final stage31-34. These findings suggest that there might be an intrinsic resistant mechanism underlying oMN-specific tolerance against ALS pathogenesis, and comparative studies between sMN and oMN could help identify the mechanistic basis underlying selective susceptibility in ALS. To this end, oMN have been studied as an ALS-resistant cell population35-38. Kaplan and colleagues compared differentially expressed genes in oMNs and sMNs of wildtype (WT) postnatal mice and found that matrix metalloproteinase-9 (MMP-9) is a relevant gene for neurodegeneration in fast motor neurons of a SOD1 ALS mouse model35. Unfortunately, this has not yet been studied or tested with human ALS neuronal subtypes, primarily because isolation of oMNs is too challenging to perform in human patients. Allodi and colleagues also suggested that IGF2 (Insulin-like growth factor 2) has a protective role in SOD1 mouse model and ALS sMN in vitro, however, it is unknow whether it is also found in other mutations or sporadic cases of ALS. (Allodi 2019) Moreover, any given studies with already degenerated sMNs in ALS patients may not be adequate to uncover a primary cell intrinsic causality of ALS pathogenesis.
Many studies have attempted to understand different aspects of ALS pathogenesis and have identified mitochondrial dysfunction39, excitotoxicity40,41 and astrocyte induced non-cell autonomous effects42,43 as potential mechanisms. However, one understudied hypothesis is that ALS disease might be associated with alterations in lipid metabolism. Previous studies reported abnormal levels of ceramide44, cholestenoic acids45, cholesterol or low density lipoprotein (LDL)/high density lipoprotein (HDL) in biofluids of ALS patients46. However, these reports focused on the role of specific genes involved in lipid metabolism using a rodent model, or simply showed alterations of specific metabolites in patient cerebrospinal fluid (CSF). Although these publications have provided interesting data that allow the speculation of a relationship between ALS pathology and aberrant lipid metabolism, more dynamic understanding of lipid metabolic dysregulation in ALS-specific human sMNs must be performed to elucidate the metabolic causality of altered lipid metabolism in ALS disease.
All publications and patent applications identified herein are incorporated by reference in their entirety and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
An embodiment of the invention relates to a method of treating an amyotrophic lateral sclerosis (ALS) cell including: selecting a therapeutic compound: treating an aberrant arachidonic acid (AA) metabolic pathway in the ALS cell including contacting the ALS cell with the therapeutic compound.
An embodiment of the invention relates to a method of treating a subject with ALS including: selecting a therapeutic compound: and treating an aberrant arachidonic acid (AA) metabolic pathway in the subject including administering to the subject the therapeutic compound.
An embodiment of the invention relates to a method of differentiating a human stem cell to an ocular motor neuron (oMN) ALS-specific human cell type, including: culturing the human stem cell in a first media including recombinant sonic hedgehog signaling protein and purmorphamine for 9 days: culturing the human stem cell in a second media including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and Ascorbic Acid for at least 1 day. In such an embodiment, the second media does not include sonic hedgehog signaling protein or purmorphamine.
An embodiment of the invention relates to an ocular motor neuron (oMN) ALS-specific human cell generated from the method discussed above.
An embodiment of the invention relates to a method for identifying whether a metabolic pathway is dysregulated in a sMN ALS cell, including: isolating the sMN ALS cell: isolating an oMN ALS cell: isolating total RNA from the sMN cell: isolating total RNA from the oMN cell: and performing a differential gene expression assay from the total RNA from the sMN cell and from the total RNA from the oMN cell, the differential gene expression assay including comparing an expression level of a gene associated with the metabolic pathway from the sMN ALS cell with an expression level of the gene associated with the metabolic pathway from the oMN ALS cell: where a difference in the expression level of the gene associated with the metabolic pathway from the sMN ALS cell as compared to the expression level of the gene associated with the metabolic pathway from the oMN ALS cell is indicative of a dysregulation of the metabolic pathway.
Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.
An embodiment of the invention relates to a method of treating an amyotrophic lateral sclerosis (ALS) cell, including: selecting a therapeutic compound; treating an aberrant arachidonic acid (AA) metabolic pathway in the ALS cell including contacting the ALS cell with the therapeutic compound.
An embodiment of the invention relates to the method above, where the treating the aberrant arachidonic acid (AA) metabolic pathway results in a reduction of a cellular level of AA in the ALS cell.
An embodiment of the invention relates to the method above, where the therapeutic compound is an inhibitor of 5-lipoxygenase (5-LOX).
An embodiment of the invention relates to the method above, where the inhibitor of 5-LOX includes a redox-active compound, an iron ligand inhibitor, a non-redox-type inhibitor, a redox-type inhibitor, a Dual (COX/5-LOX) type inhibitor, or an iron ligand-type inhibitor.
An embodiment of the invention relates to the method above, where the inhibitor of 5-LOX includes a redox-active inhibitor.
An embodiment of the invention relates to the method above, where the inhibitor of 5-LOX includes caffeic acid (3,4-dihydroxy benenearcrylic acid), apigenin, BW755C, nordihydroguaretic acid, or a functional analog or derivative thereof.
An embodiment of the invention relates to a method of treating a subject with ALS, including: selecting a therapeutic compound: and treating an aberrant arachidonic acid (AA) metabolic pathway in the subject including administering to the subject the therapeutic compound.
An embodiment of the invention relates to the method above, where the therapeutic compound results in a reduction of a cellular level of AA in the spinal motor neuron cell of the subject.
An embodiment of the invention relates to the method above, where the therapeutic compound is an inhibitor of 5-lipoxygenase (5-LOX).
An embodiment of the invention relates to the method above, where the inhibitor of 5-LOX includes a redox-active compound, an iron ligand inhibitor, a non-redox-type inhibitor, a redox-type inhibitor, a Dual (COX/5-LOX) type inhibitor, or an iron ligand-type inhibitor.
An embodiment of the invention relates to the method above, where the inhibitor of 5-LOX includes a redox-active inhibitor.
As used throughout, the terms “5-LOX inhibitor” and “inhibitor of 5-LOX” are used interchangeably throughout. In general, the four classes of direct 5-lipoxygenase inhibitors encompass: i) redox-active compounds that interrupt the redox cycle of the enzyme, ii) iron ligand inhibitors that chelate the active site iron, iii) nonredox-type inhibitors that compete with arachidonic acid and iv) novel class inhibitors that may act in an allosteric manner.
According to some embodiments, redox-active 5-LOX inhibitors comprise lipophilic reducing agents including many natural plant-derived (e.g., nordihydroguaretic acid, caffeic acid, flavonoids, coumarins and several polyphenols) and synthetic compounds. The first synthetic 5-LOX inhibitors such as AA-861, L-656,224, phenidone or BW755C belong to this class. These drugs act by keeping the active site iron in the ferrous state, thereby, uncoupling the catalytic cycle of the enzyme.
According to some embodiments, iron ligand inhibitors represent hydroxamic acids or N-hydroxyurea derivatives that chelate the active site iron but also possess weak reducing properties. The hydroxamic acid BWA4C and the hydrolytic-stable N-hydroxyurea derivative zileuton are potent and orally active 5-LOX inhibitors. Some examples include Zileuton, ABT-761, and LDP-977 (CMI-977).
According to some embodiments, nonredox-type 5-LOX inhibitors compete with AA or LOOH for binding to 5-LOX. They are devoid of redox properties and encompass structurally diverse molecules. Representatives out of this class such as the orally active compounds ZD 2138, L-739,010 or CJ-13,610 as well as the thiopyranoindole L-699,333 are highly potent and selective for 5-LOX in cellular assays, with IC50 values in the low nanomolar range.
Some embodiments relate to the use of a 5-LOX inhibitor which binds to other relevant targets including COX enzymes, the PAF or the HI receptor (so-called dual inhibitors). One example of such a dual 5-LOX/COX pathway inhibitors includes licofelone.
According to some embodiments, 5-LOX inhibitors may include the polyphenolic (b)-3,4,3′,4′-tetrahydroxy-9,7′a-epoxy lignano-7 a,9-lactone, novel caffeoyl clusters (trimers or tetramers), NSAIDs that are covalently linked to an iron-chelating moiety, the urea derivative RBx 7796, substituted coumarins based on the structure of L-739,010, fluorophenyl-substituted coumarins where the thioaryl moiety carrying the hexafluorcarbinol is replaced by an amino-oxadiazol moiety, tetrahydropyrane-carboxamides (exemplified by CJ-13,610), tricyclic thiazole-based derivatives with a thiazolone core moiety, tetrahydronaphtol derivatives, sulfonamide-spiro(2H-1-benzopyrane-2,4-piperidin) derivatives, benzoxazole derivatives, Licofelone, macrolide conjugates, oflicofelone or related pyrrolizine and indolizine derivatives with macrocyclic antibiotics, celecoxib, etoricoxib, rofecoxib, novel diaryl-dithiolanes and isothiazoles, rofecoxib derivatives, 1,3-diarylprop-2-yn-ones with a C3 p-SO2Me COX-2 pharmacophore, 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran derivatives, phenylsulfonyl urenyl chalcone derivatives, 2-(4-aminophenyl)-3-(3,5-dihydroxylphenyl) propenoic acid, a-(n)-alkyl-substituted pir-inixic acid derivatives, indole-3-carboxylates, 2-amino-5-hydroxyindole-3-carboxylates, benzo[g]indole-3-carbox-ylates, sulfonimides based on the aryl-pyrrolizine scaffold oflicofelone, lipophilic phenolic compounds such as curcumin from turmeric, garcinol from the fruit rind of Guttiferae species, myrtucommulone from myrtle (Myrtus communis) and epigallocatechin-3-gallate from green tea (Camellia sinensis), hyperforin. Examples of 5-LOX inhibitors are not restricted to the list above, or to specific compounds disclosed herein.
An embodiment of the invention relates to a method of differentiating a human stem cell to an ocular motor neuron (oMN) ALS-specific human cell type, including: culturing the human stem cell in a first media including recombinant sonic hedgehog signaling protein and purmorphamine for 9 days: culturing the human stem cell in a second media including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and Ascorbic Acid for at least 1 day. In such an embodiment, the second media does not include sonic hedgehog signaling protein or purmorphamine.
An embodiment of the invention relates to the method above, where the human stem cell is an embryonic human stem cell or a human induced pluripotent stem cell.
An embodiment of the invention relates to the method above, where an expression of at least one oMN-specific gene is increased in the oMN ALS-specific human cell.
An embodiment of the invention relates to the method above, where the at least one oMN-specific gene is selected from the list consisting of ISL1, PHOX2A, NKX6.1, EN1, CHAT, PHOX2B, TBX20, FGF10, EYA1, EYA2, PLEXINA4, SEMA6D and MAP2.
An embodiment of the invention relates to a method of differentiating a human stem cell to an ocular motor neuron (oMN) ALS-specific human cell type, including: culturing the human stem cell in a first media including recombinant sonic hedgehog signaling protein and purmorphamine for 9 days: culturing the human stem cell in a second media including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and Ascorbic Acid for at least 1 day. In such an embodiment, the second media does not include sonic hedgehog signaling protein or purmorphamine. Table 1 discloses example media types, compounds, and incubation periods of such a method.
An embodiment of the invention relates to an ocular motor neuron (oMN) ALS-specific human cell generated from the methods discussed above.
An embodiment of the invention relates to a method for identifying whether a metabolic pathway is dysregulated in a sMN ALS cell, including: isolating the sMN ALS cell; isolating an oMN ALS cell; isolating total RNA from the sMN cell; isolating total RNA from the oMN cell; and performing a differential gene expression assay from the total RNA from the sMN cell and from the total RNA from the oMN cell, the differential gene expression assay including comparing an expression level of a gene associated with the metabolic pathway from the sMN ALS cell with an expression level of the gene associated with the metabolic pathway from the oMN ALS cell: where a difference in the expression level of the gene associated with the metabolic pathway from the sMN ALS cell as compared to the expression level of the gene associated with the metabolic pathway from the oMN ALS cell is indicative of a dysregulation of the metabolic pathway.
An embodiment of the invention relates to the method above, where the sMN ALS cell is differentiated from a human stem cell.
An embodiment of the invention relates to the method above, where the oMN ALS cell is differentiated from a human stem cell.
An embodiment of the invention relates to the method above, further including determining whether a metabolite associated with the metabolic pathway is dysregulated in the sMN ALS cell, including: isolating the metabolite from the sMN cell: isolating the metabolite from the oMN cell: determining the relative abundance of the metabolite from the sMN cell: determining the relative abundance of the metabolite from the oMN cell; and comparing the relative abundance of the metabolite from the sMN cell with the relative abundance of the metabolite from the oMN cell. In such an embodiment, a difference in the relative abundance of the metabolite from the sMN cell as compared to the relative abundance of the metabolite from the oMN cell indicative of a dysregulation of the metabolic pathway.
The following describes some embodiments of the current invention more specifically. The general concepts of this invention are not limited to these particular embodiments.
In the following example, data from studies showing the identification of cell intrinsic factor(s) that cause differential susceptibility between sMN and oMN subtypes in ALS are presented. Transcriptomics and metabolomics profiling was used to generate the data. The results reveal aberrant lipid metabolism in ALS patient-derived sMN populations. One of major dysregulated metabolism pathways is the off-controlled arachidonic acid (AA) metabolism, whose pharmacological modulation unexpectedly increased the survival rates of HB9::GFP+ ALS sMNs and partially reversed ALS-related phenotypes in a Drosophila and SOD1G944 mouse model. These studies provide new insights into ALS pathogenic mechanism and potential therapeutic targets for both fALS and sALS.
PHOX2B genetic reporter system enriched hESC and iPSC-derived oMN-like molecular patterns
The transcriptional remodeling of oMN during development is different from that of sMNs in the spinal cord47. Initially, Nkx6.1+ basal plate cells in the midbrain give rise to oMN precursors and differentiated Phox2a/b-expressing oMNs to form the ocular motor nucleus in the midbrain ventrolateral region48-51. First, expression of several markers for oMNs in embryonic mouse midbrains were evaluated by immunohistochemistry (
To isolate pure oMN-like cells from ALS hiPSC lines, a PHOX2B::GFP reporter from healthy control and ALS hiPSC lines (SOD1A4V and (9ORF72 with 500 GGGGCC hexanucleotide repeats SEQ ID NO: 81) was generated using the CRISPR-Cas9 system (
HB9::GFP neurons represent ALS hiPSC-derived sMN cells
To compare overall transcriptomic patterns between oMN-like and sMN subtypes, a sMN-specific genetic reporter system was developed from ALS hiPSCs. HB9 is known to be a specific transcriptional marker for sMN specification in the spinal cord61,62. Indeed, whole mount staining of Hb9::GFP63 and Isl1::GFP64 transgenic embryos clearly showed that projection of Isl1::GFP+ cell populations was identical to Hb9::GFP+ cells in the spinal cord, but not oculomotor neuronal projection in the midbrain65,66 (
Additionally, it was determined that there are increased expression levels of different HOX subfamily genes (HOXA2, 5, 7 and 10, detected by qRT-PCR) in the HB9::GFP+ cells, but not in PHOX2B::GFP+ cells, supporting the regional specificity of both neuronal populations (
Transcriptomic differences in lipid metabolism between PHOX2B::GFP+ oMN-like and HB9::GFP+ sMN populations in C9ORF72 and SOD1A4V ALS lines were revealed by comparative transcriptome profiling
In order to identify intrinsic properties that explain selective vulnerability in sMN subtypes, unbiased transcriptome analysis was performed using post-sorted PHOX2B: GFP+ oMN-like and HB9::GFP+ sMN cells derived from C9ORF72 and SOD1A4V ALS lines (
Firstly, for further confirmation of the cellular identities of PHOX2B::GFP+ oMN-like and HB9::GFP+ sMNs, the RNA-sequencing data were compared with a published data set68 where transcriptomic differences between oMN and sMN were shown based on the other published data35,69, including oMN markers (PHOX2A, PHOX2B, TBX20, EN1, FGF10, EYA1, EYA2, PLXNA4 and SEMA6D) and sMN markers (HB9, FOXP1, SEMA4A, HOXA2, HOXA3, HOXA4, HOXA5, HOXB4, HOXB5, HOXB6, HOXB7, HOXC4 and HOXC5) (
Unbiased metabolomics analysis corroborated transcriptional discrepancies in ALS-specific PHOX2B::GFP+ oMN-like and HB9::GFP+ sMNs cells
To verify the RNA sequencing data and pinpoint target pathways that are involved in cell type specific phenotypic vulnerability, studies were taken to identify metabolic differences by performing liquid chromatography mass spectrometry (LC/MS) using post-sorted PHOX2B::GFP+ cells and HB9::GFP+ cells of both SOD1A4V and C9ORF72 ALS hiPSC lines (total 4 groups: SOD1A4V HB9::GFP+, SOD1A4V PHOX2B::GFP+, C9ORF72 HB9::GFP+, and C9ORF72 PHOX2B::GFP+ cells with 3 technical replicates) (
Activation of arachidonic acid pathway is a common metabolic signature of sMN cells with various genetic backgrounds
After the unbiased multi-omics analysis using two ALS lines, it was asked whether this phenotype is common in different mutations and sporadic ALS. Thus, a more stringent experimental plan was set to analyze the ‘unsorted’ crude sMN culture of multiple ALS hiPSC lines (
To pinpoint specific pathways that are associated with ALS sMN pathology, commonly over-represented or nearly absent metabolites were first classified by statistical analysis and it was determined that 29 metabolites belonged to the highly represented group and 22 metabolites belonged to the downregulated group that were common in multiple ALS (C9ORF72, 6 lines: SOD1, 3 lines: TDP 43, 3 lines: sporadic, 5 lines; each line had 3 independent technical replicates) (
Based upon the metabolomics findings, absence of AA861 analog and the previous metabolomics data of ALS patient plasma, it was speculated that the loss of regulation in biosynthesis of AA may be one of the causes of lipid metabolism dysregulation common in ALS sMN, corroborated with the multi-omics analysis by indicating a significantly presented ‘Arachidonic acid metabolism’ with significant Expected Score (˜9.12 by MetaboAnalyst v.4.0)(
Arachidonic acid pathway alterations cause ALS phenotypes
It is well known that the cellular level of AA is critical to cell viability and linked to disease phenotypes such as hypertension, cancer and leukemia75-77. To examine the functional contribution of AA metabolism to sMN pathology, a set of functional analogs of AA861(C21H26O3), a known 5-LOX inhibitor78-80, were tested including caffeic acid (CA: 3,4-dihydroxy benenearcrylic acid), apigenin, BW755C and nordihydroguaretic acid in optimized condition (
To confirm the rescuing effects of CA treatment seen in the in vitro hESC and hiPSC sMN model, a Drosophila model of C9ORF72-ALS that overexpresses 30 G4C2 repeats81 (SEQ ID NO:82) was employed. Surprisingly, significant dose-dependent recovery of eye degeneration phenotypes, progeny rate and survival rate were observed in this model CA (Caffeic acid), NDGA (Nordihydroguaretic acid) and Api. (Apigenin) (
Next, the integrity of motor neuron and astrogliosis was analyzed using histological assessments at early symptomatic stage (16 wks, Exp 2) and late symptomatic stage (20 wks, Exp 3). The number of motor neuron (larger than 25 μm) in the ventral horn of spinal cord was significantly reduced in control SOD1G93A mice (18.0±3.5) compared to wild-type mice (30.0±2.8) at early symptomatic stage, however, the number was significantly increased by the administration of caffeic acid (22.8±4.3). The significant difference was also maintained at late symptomatic stage (13.9±1.8 in control vs 17.5±3.8 in caffeic acid) (
Next, it was tested how dysregulation of the AA pathway mediates ALS pathogenesis using CA treatment, an inhibitor of 5-lipoxygenase (5-LOX)73,74, can restore the dysregulated levels of AA and related glycophopholipid species. Levels of AA and glycophospholipids were quantified before and after CA treatment in sMN culture of the 4 ALS lines using a focused metabolomics analysis. When comparing vehicle (ethanol) and CA (25 μg/ml) treated ALS samples (C9ORF72, 6 lines: SOD1, 3 lines: TDP43, 3 lines: sporadic, 5 lines sMN culture), significantly decreased levels of AA and increased levels of glycophospholipid species in CA-treated ALS samples were observed (
Here, through unbiased comparative multi-omics analyses of two distinct motor neuron subtypes (oMN-like PHOX2B::GFP+ and HB9::GFP+ sMNs), it was determined that various pathways in lipid metabolism, especially the AA metabolic pathway, are dysregulated in SOD1A4V and mutant (9ORF72 HB9::GFP+ populations but normal in PHOX2B::GFP+ populations. These findings were also validated by a targeted metabolomics study between healthy and ALS (SOD1A4V, C9ORF72. TDP 439343R and sporadic) neuronal populations. Importantly, the ALS phenotypes are rescuable, both in vitro and in vivo, by chemical regulation of the AA metabolism, showing an untapped translational potential of findings described in this study.
Previously, the several groups suggested therapeutic target molecules35,46,82, but these transcriptome analysis results did not confirm those findings (data not shown). One possible reason is species and/or mutation differences between the SOD1G93A mouse strain and the SOD1A4V hiPSC-derived sMNs. For example, one previous study identified MMP-9 gene in a comparison of oMNs and sMNs in WT mice35, but the comparative analysis was conducted on ALS-specific human oMN-like and sMNs in SOD1A4V and C9ORF72 mutations. The comparative analysis between human oMN-like PHOX2B::GFP+ and HB9::GFP+ sMNs revealed significant changes in lipid metabolism pathways in ALS sMN populations. Interestingly, the expression levels of DEGs within the lipid metabolism pathways (
Consistent with these findings, accumulating evidence has also shown potential connections between ALS pathogenesis and aberrant lipid mechanisms44-46,86, but there are few proven detailed mechanistic studies. The present study is the first report providing systematic profiling of ALS patient motor neuron cells by recapitulating both SOD1A4V and C9ORF72 HB9::GFP+ sMN versus PHOX2B::GFP+ oMN-like cells and reveal the causative contribution of lipid metabolism dysregulation to ALS pathogenesis by employing RNA sequencing and metabolomics analysis. These data were also consistent with another set of analysis for targeted metabolomics between healthy donor and ALS (SOD1A4V, C9ORF72, TDP43Q343R and sporadic) patient-derived sMN culture. Based on the multi-omics data analysis, one of the aberrantly regulated lipid metabolism pathways in ALS-derived sMN populations is the AA pathway. It was also identified that C21H26O3, an AA861 structural analog, is almost undetectable in sMN of ALS hiPSC lines (SOD1A4V, C9ORF72. TDP43Q343R and sporadic) by metabolomics analysis. Indeed, AA861 is a well-known natural inhibitor of 5-lipoxygenase (5-LOX) that metabolizes AA into other metabolites, which is consistent with the multi-omics data. Interestingly, AA levels are closely associated with apoptosis, suggesting that metabolic pathways regulating AA levels might be a therapeutic target for ALS75,76,79. For example, other group showed that AA modulation by PLA2 (phospholipase A2) inhibitor has a protective effect in SOD1 mouse model (ref, Ouchi). Therefore, it was hypothesized that pharmacological modulation of the AA pathway could restore the levels of AA as well as ALS-relevant phenotypes. Indeed, treatment of several 5-LOX inhibitors was sufficient to restore the decreased levels of ALS-derived HB9::GFP+ cells (
Previously, another group reported that increased levels of ceramide were identified in CSF of ALS patients, and pharmacological inhibition of the sphingolipid synthesis pathway by ISP-1 could inhibit spinal motor neuron death in vitro44. To find relevance of other lipid pathways in this study, two additional candidate metabolites were tested that are not detected in the four ALS line derived sMNs (Ajamaline and Creatine) and two chemical compounds that can compensate for altered levels of metabolites (R-Deprenyl hydrochloride and ISP-1 for decreasing the dys-regulated levels of Putrescine and Ceramide (d18:1/16:0), respectively) (
Taken together, the data herein demonstrate that substantially dysregulated lipid metabolism pathways are common in 17 different ALS hiPSC-derived sMN cultures, and pharmacological modulation of AA metabolism shows protective effects in an in vitro human sMN model and a Drosophila and SOD1G93A mouse model. The current study provides a new framework for disease modeling by comparing affected and non-affected cell types from a disease hiPSC line, leading to the unraveling of metabolic aberrations in ALS sMN and identification of potential drug candidates.
Generation of Reporter Lines in hESC/iPSC by CRISPR-Cas9)
CRISPR-Cas9 knock-in strategy was performed as previously described89. Feeder-free H9 hESCs, 01582 hiPSCs (PHOX2B::GFP)56, and C9ORF72 and SOD1A4V iPSC lines (PHOX2B::GFP and HB9::GFP) were dissociated using Accutase (Innovative Cell Technologies Inc.). Cells (2×106) were resuspended in nucleofection solution V (Lonza) with 4 μg of hCas9—gRNA plasmid (gRNA #1 and #2 were used for HB9::GFP) and 4 μg of dsDNA donor plasmid. The nucleofection was performed by Nucleofector™ II according to manufacturer's instruction (B-16, Lonza), then nucleofected cells were plated on puromycin resistant MEFs (DR4, Global Stem) in hES medium (DMEM/F12 (Invitrogen) containing 20% knockout serum replacement (KSR, Gibco), 0.1 mM MEM-NEAA (Gibco), 1 mM L-glutamine (Gibco), 55 uM β-mercaptoethanol (Gibco), 4 ng/ml FGF2 (Gibco)) with 10 μM Y-27632 (Cayman Chemical). After 3 or 4 days, knock-in cells were selected by treatment with 0.5 μg/ml puromycin (MilliporeSigma) in hES medium. After selection, puromycin resistant colonies were verified for GFP expression by FACS analysis using each differentiation protocol.
For the PHOX2B::GFP reporter line, plasmids were used as previously described56. For the HB9::GFP reporter line, left arm 1512 bp and right arm 900 bp were designed from stop codon of the human HB9 locus. Each arm was generated by PCR using (H9) hESC genomic DNA and inserted into OCT4-2A-eGFP-PGK-Puro donor vector backbone (Addgene #31938)90 between BamHI and NheI for left arm and AscI and NotI for right arm. The gRNA sequence was designed by Zhang lab gRNA design resource89 and subcloned into gRNA vector (Addgene #48138) as previously described91. All insert sequences were verified by DNA sequencing (JHU synthesis & sequencing facility).
hESC/iPSC Culture and Differentiation
H9 hESCs, 01582 (GM01582), 2623 (GM02623), 24C (GM00024C) iPSCs (derived from each fibroblasts, Coriell Institute) and OCT4::GFP in hESC (H9), PHOX2B::GFP in hESC (H9) and 01582 iPSC, C9ORF72, SOD1A4V for PHOX2B::GFP and HB9::GFP (ALS patient fibroblasts (JH078[C9ORF72]92 and GO013 [SOD1A4V]93 were collected at Johns Hopkins hospital with patient consent)94. TDP43Q343R (gift from Nicolas J. Maragakis), Sporadic (gift from Nicolas J. Maragakis) iPSC were cultured (passages 12-60) on inactivated mouse embryonic fibroblasts (MEF, Applied Stem Cell) with hES medium at 37° C. and 5% CO2 in a humidified incubator as described previously 18. For neuronal differentiation, LSB (LDN193189+SB431542) protocol was used as described previously95,96 and adapted for each neuronal differentiation.
Cells were dissociated using Accutase after incubation for 20 min at 37° C. and suspended with buffer containing 40 μg/ml DNase I (Roche Applied Science). For GFP analysis, BD FACS Calibur (Becton Dickinson) and FlowJo software (Tree Star Inc.) were used. To purify GFP+ populations for each reporter line, a MoFlo high-speed sorter (Dako Cytomation) in the Johns Hopkins School of Public Health Flow Cytometry Core Facility and a BD FACSJazz sorter in the Stem Cell Core Facility of Johns Hopkins Medicine (Institute for Cell Engineering) were used.
For Immunohistochemistry, E12.5 midbrain was dissected and fixed with 4% paraformaldehyde (PFA) overnight. After fixation, tissues were washed with PBS and incubated with 30% sucrose for cryosection as described previously97. The following antibodies were used as a primary antibody: rabbit anti-TH (Pel-Freez Biologicals), mouse anti-Isl1 (DSHB) and rabbit anti-Phox2b (gift from Jean-Francois Brunet)98. For Isl1 staining, a mouse on mouse kit (Vector Laboratory) was used. For immunocytochemistry, cells were fixed with 4% PFA and stained with mouse anti-ISL1 (DSHB), mouse anti-NKX6.1 (DSHB), rabbit anti-TUJ1 (BioLegend) and rabbit anti-PHOX2B (gift from Jean-Francois Brunet). For FACS analysis, cells were fixed with 4% PFA and stained with mouse anti-HB9 (DSHB) and mouse anti-ISL1 (DSHB) as described previously99. For 7AAD analysis by FACS, staining was conducted based on commercial instruction (BD #559925). Appropriate Alexa Fluor 488, 568 and 647 (Life Technologies) labeled secondary antibodies were used with DAPI (Roche Applied Science) for nuclear staining. All images were visualized with fluorescence microscopy (Eclipse TE2000-E, Nikon).
qRT-PCR Analysis and Primer Information
Total RNA was extracted using TRIzol Reagent (Life Technologies) and reverse transcribed using High Capacity cDNA Reverse Transcription kit (Applied Biosystem). qRT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) and Mastercycler ep Realplex2 (Eppendorf) with the primers shown below. All primers were designed using GenScript primer design software.
SMN differentiation was performed as previously described67. For general sMN culture media, neurobasal medium (Gibco) containing B27 (Gibco), N2 (Gibco), and 2 mM L-glutamine was used as a normal medium. For compound testing, neurobasal medium with N2 was used as a conditioned medium using caffeic acid (Sigma, C0625), R-Deprenyl hydrochloride (Sigma, M003), Ajamaline (MP Biomedicals, 4360-12-7), Creatine (Sigma, 1150320) and ISP-1 (Sigma, M1177), BW755C (Tocris, 105910), Nordihydroguaiaretic acid (Sigma, 74540), Apigenin (Fisher Scientific, 50908414), U-73122 (Thermo, 126810). For Arachidonic acid testing, Arachidonic acid (Cayman, 506-32-1) was treated in normal media. For mitomycin C treatment, 1 μg/ml of mitomycin C was treated in differentiating oMN or sMN cells for 1 hr at D17 and analyzed after 2 days (D19) by FACS. For fold change value, non-treated % of GFP+ were considered as a control and fold change values were normalized upon % of GFP expression of non-treated cells by FACS. For fold change value, non-treated cells were considered as a control and fold change values were normalized upon GFP expression of non-treated cells by FACS.
Total RNA was extracted using miRNeasy mini kit (Qiagen) as per manufacturer's instructions. RNA concentration and purity were assessed by Nanodrop (Thermo Fisher), and RNA integrity was assessed using the Agilent Bioanalyzer. cDNA Libraries were prepared for mRNA-enriched sequencing using TruSeq Stranded mRNA kit (Illumina). This was followed by normalization to 6 nM and pooling of libraries, followed by single end 75 bp sequencing on the Illumina HighSeq 4000. RNA-seq reads were aligned to the human reference genome (gencode.v27.primary_assembly.annotation.gtf downloaded from Gencode) using STAR aligner100. Differential gene expression analysis was performed using EdgeR101 and Limma-Voom102. The Limma-Voom package was used for data normalization and generation of differential expression gene matrices. Genes with |fold change (FC)|>=2 and adjusted p-value <0.05 were considered as differentially expressed genes: 4,016 up-regulated and 3,749 down-regulated DEGs were identified. To reduce the number of differentially expressed genes, the Treat method103 was used to calculate p-values from empirical Bayes moderated t-statistics with minimum log-FC requirement. The number of differentially expressed genes was reduced to a total of 1,806 DEGs for comparison of HB9::GFP+ versus PHOX2B::GFP+. To identify enriched transcriptomic signatures out of the large number of DEGs between HB9::GFP+ and PHOX2B::GFP+, a non-parametric method was used, gene set enrichment analysis (GSEA), where the enrichment score reflects the degree to which a gene set is over-represented at the top or bottom of a ranked list of genes104. Enrichment analysis and visualization were performed using clusterProfiler R package105 and biological process terms from Gene Ontology (GO) with gene set size between 15 and 300 genes. To validate the oMN-like and sMN populations, the datasets were compared with previous mouse dataset from a previous literature where transcriptomic signature of oMN and sMN was well established68 (GEO dataset id: GSE118620). The mouse dataset was aligned to mouse reference genome (gencode. vM24.primary_assembly.annotation.gtf downloaded from Gencode) and further analyzed in the same way as thehuman dataset is analyzed. For healthy control of hESC and hiPSC lines, oMN-like cells were differentiated from hESC/hiPSC PHOX2B::GFP lines, and sMN cells from hESC/hiPSC lines were stained by HB9 antibody (DSHB) (ref, Rubin). A pooled library of 12 samples was subjected to Illumina platform (NovaSeq6000 S4) in 150 bp paired-end mode. Raw data (FASTQ files) were imported into Altanalyze v2.1.4 software, which uses the embedded software Kallisto and Ensembl 72 annotations. Two QC-failed control samples were excluded from the analyses. Processed expression files, including transcript-level expression values (TPMs) summed at the gene-level and read counts, were used in R (v4.0.3) to generate volcano plot, principal component (PC) plot, heatmaps, and dot plots shown in
Liquid chromatography mass spectrometry (LC-MS) differentiation and detection of each metabolite (C9ORF72 PHOX2B::GFP+, SOD1A4V PHOX2B::GFP+, C9ORF72 HB9::GFP+, SOD1A4V HB9::GFP+, un-sorting of C9ORF72, SOD1A4V, TDP43Q343R, Sporadic and control line derived sMN) were performed with an Agilent Accurate Mass 6230 TOF coupled with an Agilent 1290 Liquid Chromatography system using a Cogent Diamond Hydride Type C column (Microsolve Technologies, Long Branch, NJ, USA) with solvents and configuration as previously described106. An isocratic pump was used for continuous infusion of a reference mass solution to allow mass axis calibration. Detected ions were classified as metabolites based on unique accurate mass-retention time identifiers for masses showing the expected distribution of accompanying isotopologues. Metabolites were analyzed using Agilent Qualitative Analysis B.07.00 and Profinder B.08.00 software (Agilent Technologies, Santa Clara, CA, USA) with a mass tolerance of <0.005 Da. Standards of authentic chemicals of known amounts were mixed with bacterial lysates and analyzed to generate the standard curves used to quantify metabolite levels. All data obtained by metabolomics profiling were the average of at least two independent triplicates. Bioinformatics analysis was carried out using MetaboAnalyst v.4.0 (www.metaboanalyst.ca), which is a web-based available software for processing metabolomics data, and pathway mapping was performed on the basis of annotated Human metabolic pathways available in the Kyoto Encyclopedia of Genes and Genomes pathway database. Metabolomics data were analyzed by statistical analysis. The clustered heat map and hierarchical clustering trees were generated using Cluster 3.0 and Java Tree View 1.0. A univariate statistical analysis involving an unpaired t-test was used to identify significant differences in the abundances of metabolites between each group.
Hb9::GFP and ISL1::GFP mice were described previously107,108. All experiments used protocols approved by the Animal Care and Ethics Committees of the Gwangju Institute of Science and Technology (GIST) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For efficacy assessment of the caffeic acid, B6.Cg-Tg(SOD1G93A)1Gur/J mice (Jackson Laboratory, Bar Harbor, ME) was used after in vitro fertilization (Macrogen, Seoul, Korea) and all the protocol was approved by the Institutional Animal Care and Use Committees of Dong-A University.
Caffeic acid (30) mg/kg) dissolved in PBS containing 10% ethanol or vehicle (PBS containing 10% ethanol) were orally administered 5 days/week from 60 days to 120 days of age. Three independent experiments were performed to evaluate the efficacy of caffeic acid. A total of 24 mice in each group (female=12, male=12) were used for evaluation of survival and behavioral assessments (Exp 1), and the same number of mice were used for histologic analyses (Exps 2 and 3). In the Exp 1, mice were monitored for neurological disease progression according to guidelines for preclinical animal research in ALS/MND (Ludolph AC et al, 2010). The neurological score was followed as Score 0.5 as disease onset (first signs of tremor and hind-limb splay defects) and the end stage (Score 4) was determined as loss of righting reflex within 30s. Neurological scoring was monitored daily and mice at the end stage were euthanized. Kaplan-Meier curves was used to analyze age of onset and survival using Graphpad Prism7 (GraphPad Software, San Diego, CA). Motor coordination and muscle integrity were assessed weekly using a Rotarod apparatus and grip strength device (Panlab Harvard Apparatus, Barcelona, Spain). Tissue analyses were performed in the Exp 2 (n=14 for each group) and Exp 3 (n=10 for each group). They were anesthetized with euthanized isofluran at 16 wks (Exp 2) and 20 wks (Exp 3) of age then perfused with 4% paraformaldehyde in PBS. L4-L5 segments of spinal cord were serially cut with the cryostat into 20 μm sections then stained with 0.1% (w/v) cresyl violet stain solution. Motor neurons with larger than 25 μm of dimeter in Lamina IX of the ventral horn were counted using Image J software (National Institutes of Health, Bethesda, USA) program, and 10) sections per sample (n=10 for each group) were averaged. Activated astrocytes and microglia in the spinal cord were detected using anti-GFAP Ab and rabbit anti-Iba1 Ab (Cell Signaling Technology, Beverly, MA) for 10 sections per sample (n=10 for each group). The integrated density of fraction area in the ventral horn were measured using Image J software for quantification of activated astrocytes and microglia. Neuromuscular junction was analyzed in gastrocnemius muscle (30 μm) with anti-a-bungarotoxin Ab to label AChR and anti-neurofilament H/synapsin Ab (Cell Signaling Technology) to label axon terminals. The innervated pretzel structures merged with two fluorescence were counted.
For whole mount immunostaining, E11.5 embryos were fixed in 4% PFA, permeabilized in PBS-T (1% Triton X-100 in PBS), blocked using blocking buffer (1% heat inactivated goat serum, 1% Triton X-100 in PBS) at 4° C. Embryos were incubated 3-5 days at 4° C. with rabbit anti-GFP (Invitrogen) primary antibody in blocking buffer. Fluorophore-conjugated secondary antibody (Invitrogen) was incubated for 1 day at 4° C., and images were captured using a Zeiss confocal microscope109.
Flies were maintained on a cornmeal-molasses-yeast medium at room temperature (22° C.) with 60-65% humidity. The following Drosophila lines were obtained from the Bloomington Stock Center: elav-GAL4, GMR-GAL4, and OK371-GAL4. The UAS-(G4C2)3 and UAS-(G4C2)30 lines were obtained from Dr. Peng Jin's laboratory81.
UAS-(G4C2)30 flies recombined with GMR-Gal4 were selected as male parental flies for crossing (♀w1118×♂GMR-Gal4; UAS-(G4C2)30/CyO). Overexpressing 30 hexanucleotide repeat (HRE) in all photoreceptors using GMR-Gal4 causes eye degeneration in adult flies during aging. Eye degeneration scores were examined based on Dr. Paul Taylor's study110. Data of eye degeneration was quantified for the presence of: supernumerary inter-ommatidial bristles (IOBs), IOBs with abnormal orientation, necrotic patches, a decrease in size, retinal collapse, fusion of ommatidia, disorganization of ommatidial array and loss of pigmentation in adult male progeny. Points were added if: there was complete loss of IOBs (+1), more than 3 small or 1 large necrotic patch (+1), retinal collapse extended to the midline of the eye (+1) or beyond (+2), loss of ommatidial structure in less than 50% (+1) or more than 50% (+2) of the eye, and if pigmentation loss resulted in change of eye color from red to orange (+1) or pale orange/white (+2).
UAS-(G4C2)30 flies recombined with OK371-Gal4 were selected as male parental flies for crossing (♀w1118×♂OK371-Gal4: UAS-(G4C2)30/TM6B, GAL80). Overexpressing 30 HRE in fly motor neurons using OK371-Gal4 causes lethality due to paralysis, preventing eclosion of the adult from the pupal case. According to Mendelian inheritance, the theoretical ratio of progenies with 30 HRE expressions from the above crossing is 50%. A total of 100 adult flies were collected in each group. Survival rate was calculated as the ratio of the flies with 30 HRE that survive to adulthood to total adult flies and then divided by theoretical ratio 50%.
Each value is from at least 3 different experiments of multiple batches and reported as mean±SEM. Statistical differences between samples were analyzed by unpaired Student's t-test in GraphPad Prism 7 and indicated the p-value level in each legend.
The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. Moreover, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly stated above. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
This application claims priority to U.S. Provisional Application No. 63/192,284, filed May 24, 2021 and U.S. Provisional Application No. 63/278,779, filed Nov. 12, 2021, the contents of which are incorporated herein by reference in their entirety.
This invention was made with government support under R01NS093213 and AI143870 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/030773 | 5/24/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63192284 | May 2021 | US | |
| 63278779 | Nov 2021 | US |
