PIKFYVE inhibition mitigates disease in models of diverse forms of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.

Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD.

GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, we identified a number of genetic modifiers of poly(GR) toxicity. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.

Endocannabinoids affect innate immunity of Muller glia during HIV-1 Tat cytotoxicity.

In the retina, increased inflammatory response can cause visual impairment during HIV infection in spite of successful anti-retroviral therapy (HAART). The HIV-1 Tat protein is implicated in neurodegeneration by eliciting a cytokine response in cells of the CNS, including glia. The current study investigated whether innate immune response in human retinal Muller glia could be immune-modulated to combat inflammation. Endocannabinoids, N-arachidonoylethanolamide and 2-arachidonoylglycerol are used to alleviate Tat-induced cytotoxicity and rescue retinal cells. The neuroprotective mechanism involved suppression in production of pro-inflammatory and increase of anti-inflammatory cytokines, mainly through the MAPK pathway. The MAPK regulation was primarily by MKP-1. Both endocannabinoids regulated cytokine production by affecting at the transcriptional level the NF-κB complex, including IRAK1BP1 and TAB2. Stability of cytokine mRNA is likely to have been influenced through tristetraprolin. These findings have direct relevance in conditions like immune-recovery uveitis where anti-retroviral therapy has helped immune reconstitution. In such conditions drugs to combat overwhelming inflammatory response would need to supplement HAART. Endocannabinoids and their agonists may be thought of as neurotherapeutic during certain conditions of HIV-1 induced inflammation.

The exocyst subunit EXOC2 regulates the toxicity of expanded GGGGCC repeats in C9ORF72-ALS/FTD.

GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology, we deleted EXOC2, which encodes an essential exocyst subunit, in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2, suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to, surprisingly, the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA, making it a potential therapeutic target in C9ORF72-ALS/FTD.

Traumatic injury causes selective degeneration and TDP-43 mislocalization in human iPSC-derived C9orf72-associated ALS/FTD motor neurons.

A hexanucleotide repeat expansion (HRE) in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, patients with the HRE exhibit a wide disparity in clinical presentation and age of symptom onset suggesting an interplay between genetic background and environmental stressors. Neurotrauma as a result of traumatic brain or spinal cord injury has been shown to increase the risk of ALS/FTD in epidemiological studies. Here, we combine patient-specific induced pluripotent stem cells (iPSCs) with a custom-built device to deliver biofidelic stretch trauma to C9orf72 patient and isogenic control motor neurons (MNs) in vitro. We find that mutant but not control MNs exhibit selective degeneration after a single incident of severe trauma, which can be partially rescued by pretreatment with a C9orf72 antisense oligonucleotide. A single incident of mild trauma does not cause degeneration but leads to cytoplasmic accumulation of TDP-43 in C9orf72 MNs. This mislocalization, which only occurs briefly in isogenic controls, is eventually restored in C9orf72 MNs after 6 days. Lastly, repeated mild trauma ablates the ability of patient MNs to recover. These findings highlight alterations in TDP-43 dynamics in C9orf72 ALS/FTD patient MNs following traumatic injury and demonstrate that neurotrauma compounds neuropathology in C9orf72 ALS/FTD. More broadly, our work establishes an in vitro platform that can be used to interrogate the mechanistic interactions between ALS/FTD and neurotrauma.

Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons.

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide repeat expansion disorders.

Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD.

GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, we found that several antimicrobial peptide genes, such as metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

Endocannabinoids alleviate proinflammatory conditions by modulating innate immune response in muller glia during inflammation.

Muller cells play a prominent role in inflammatory conditions of the retina. They are part of the retinal innate immune response. The endocannabinoid system functions as an immune modulator in both the peripheral immune system as well as the central nervous system. We hypothesized that the neuroprotective ability of exogenous endocannabinoids in the retina is partially mediated through Muller glia. This study reports that exposure to endocannabinoids in activated but not resting primary human Muller glia inhibit production of several proinflammatory cytokines, while elevating anti-inflammatory mediators. Cytokine generation in activated Muller glia is regulated by endocannabinoids through the mitogen-activated protein kinase (MAPK) family at multiple signaling stages. Anandamide (AEA) acts to control MAPK phosphorylation through MKP-1. Both AEA and 2-arachidonoylglycerol (2-AG) inhibit the transcription factor NF-κB and increases the regulatory protein, IL1-R-associated kinase 1-binding protein 1. Endocannabinoids also increase expression of Tristetraprolin in activated Muller cells, which is implicated in affecting AU-rich proinflammatory cytokine mRNA. We demonstrate that exogenous application of AEA and 2-AG aid in retinal cell survival under inflammatory conditions by creating an anti-inflammatory milieu. Endocannabinoids or synthetic cannabinoid therapy may therefore orchestrate a molecular switch to bias the innate immune system suchthat the balance of pro- and anti-inflammatory cytokine generation creates a prosurvival milieu.

The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Poly(GR) and poly(GA) in cerebrospinal fluid as potential biomarkers for C9ORF72-ALS/FTD.

GGGGCC repeat expansion in C9ORF72, which can be translated in both sense and antisense directions into five dipeptide repeat (DPR) proteins, including poly(GP), poly(GR), and poly(GA), is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we developed sensitive assays that can detect poly(GA) and poly(GR) in the cerebrospinal fluid (CSF) of patients with C9ORF72 mutations. CSF poly(GA) and poly(GR) levels did not correlate with age at disease onset, disease duration, or rate of decline of ALS Functional Rating Scale, and the average levels of these DPR proteins were similar in symptomatic and pre-symptomatic patients with C9ORF72 mutations. However, in a patient with C9ORF72-ALS who was treated with antisense oligonucleotide (ASO) targeting the aberrant C9ORF72 transcript, CSF poly(GA) and poly(GR) levels decreased approximately 50% within 6 weeks, indicating they may serve as sensitive fluid-based biomarkers in studies directed against the production of GGGGCC repeat RNAs or DPR proteins.

CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.

A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.

A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation.

A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.

Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD.

Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. Here, we show in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies.

Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD.

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo.

The GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not known which dysregulated molecular pathways are primarily responsible for disease initiation or progression. We established an inducible mouse model of poly(GR) toxicity in which (GR)80 gradually accumulates in cortical excitatory neurons. Low-level poly(GR) expression induced FTD/ALS-associated synaptic dysfunction and behavioral abnormalities, as well as age-dependent neuronal cell loss, microgliosis and DNA damage, probably caused in part by early defects in mitochondrial function. Poly(GR) bound preferentially to the mitochondrial complex V component ATP5A1 and enhanced its ubiquitination and degradation, consistent with reduced ATP5A1 protein level in both (GR)80 mouse neurons and patient brains. Moreover, inducing ectopic Atp5a1 expression in poly(GR)-expressing neurons or reducing poly(GR) level in adult mice after disease onset rescued poly(GR)-induced neurotoxicity. Thus, poly(GR)-induced mitochondrial defects are a major driver of disease initiation in C9ORF72-related ALS/FTD.

Differential immune mechanism to HIV-1 Tat variants and its regulation by AEA [corrected].

In the retina, Müller glia is a dominant player of immune response. The HIV-1 transactivator viral protein (Tat) induces production of several neurotoxic cytokines in retinal cells. We show that HIV-1 clades Tat B and C act differentially on Müller glia, which is reflected in apoptosis, activation of cell death pathway components and pro-inflammatory cytokines. The harsher immune-mediated pathology of Tat B, as opposed to milder effects of Tat C, manifests at several signal transduction pathways, notably, MAPK, STAT, SOCS, the NFκB signalosome, and TTP. In activated cells, anandamide (AEA), acting as an immune-modulator, suppresses Tat B effect through MKP-1 but Tat C action via MEK-1. AEA lowers nuclear NF-κB and TAB2 for both variants while elevating IRAK1BP1 in activated Müller glia. Müller glia exposed to Tat shows enhanced PBMC attachment. Tat-induced increase in leukocyte adhesion to Müller cells can be mitigated by AEA, involving both CB receptors. This study identifies multiple signalling components that drive immune-mediated pathology and contribute to disease severity in HIV clades. We show that the protective effects of AEA occur at various stages in cytokine generation and are clade-dependant.

Detergent resistant membrane fractions are involved in calcium signaling in Müller glial cells of retina.

Compartmentalization of the plasma membrane into lipid microdomains promotes efficient cellular processes by increasing local molecular concentrations. Calcium signaling, either as transients or propagating waves require integration of complex macromolecular machinery. Calcium waves represent a form of intercellular signaling in the central nervous system and the retina. We hypothesized that the mechanism for calcium waves would require effector proteins to aggregate at the plasma membrane in lipid microdomains. The current study shows that in Müller glia of the retina, proteins involved in calcium signaling aggregate in detergent resistant membranes identifying rafts and respond by redistributing on stimulation. We have investigated Purinoreceptor-1 (P2Y1), Ryanodine receptor (RyR), and Phospholipase C (PLC-ß1). P2Y1, RyR and PLC-ß1, redistribute from caveolin-1 and flotillin-1 positive fractions on stimulation with the agonists, ATP, 2MeS-ATP and Thapsigargin, an inhibitor of sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA). Redistribution is absent on treatment with cyclopiazonic acid, another SERCA inhibitor. Disruption of rafts by removing cholesterol cause proteins involved in this machinery to redistribute and change agonist-induced calcium signaling. Cholesterol depletion from raft lead to increase in time to peak of calcium levels in agonist-evoked calcium signals in all instances, as seen by live imaging. This study emphasizes the necessity of a sub-population of proteins to cluster in specialized lipid domains. The requirement for such an organization at the raft-like microdomains may have implications on intercellular communication in the retina. Such concerted interaction at the rafts can regulate calcium dynamics and could add another layer of complexity to calcium signaling in cells.

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.