In vivo genome editing using novel AAV-PHP variants rescues motor function deficits and extends survival in a SOD1-ALS mouse model.

CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, denervation at neuromuscular junction (NMJ) and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression, demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our approach uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared to similar approaches and can also serve to accelerate drug target validation.

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

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.

Discovery of Phospholipase D Inhibitors with Improved Drug-like Properties and Central Nervous System Penetrance.

Phospholipase D (PLD) is a phospholipase enzyme responsible for hydrolyzing phosphatidylcholine into the lipid signaling molecule, phosphatidic acid, and choline. From a therapeutic perspective, PLD has been implicated in human cancer progression as well as a target for neurodegenerative diseases, including Alzheimer's. Moreover, knockdown of PLD rescues the ALS phenotype in multiple Drosophila models of ALS (amyotrophic lateral sclerosis) and displays modest motor benefits in an SOD1 ALS mouse model. To further validate whether inhibiting PLD is beneficial for the treatment of ALS, a brain penetrant small molecule inhibitor with suitable PK properties to test in an ALS animal model is needed. Using a combination of ligand-based drug discovery and structure-based design, a dual PLD1/PLD2 inhibitor was discovered that is single digit nanomolar in the Calu-1 cell assay and has suitable PK properties for in vivo studies. To capture the in vivo measurement of PLD inhibition, a transphosphatidylation pharmacodynamic LC-MS assay was developed, in which a dual PLD1/PLD2 inhibitor was found to reduce PLD activity by 15-20-fold.

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.

Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target.

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a devastating neurodegenerative disorder lacking effective treatments. ALS pathology is linked to mutations in >20 different genes indicating a complex underlying genetic architecture that is effectively unknown. Here, in an attempt to identify genes and pathways for potential therapeutic intervention and explore the genetic circuitry underlying Drosophila models of ALS, we carry out two independent genome-wide screens for modifiers of degenerative phenotypes associated with the expression of transgenic constructs carrying familial ALS-causing alleles of FUS (hFUSR521C) and TDP-43 (hTDP-43M337V). We uncover a complex array of genes affecting either or both of the two strains, and investigate their activities in additional ALS models. Our studies indicate the pathway that governs phospholipase D activity as a major modifier of ALS-related phenotypes, a notion supported by data we generated in mice and others collected in humans.

Antibody Therapy Targeting RAN Proteins Rescues C9 ALS/FTD Phenotypes in C9orf72 Mouse Model.

The intronic C9orf72 G4C2 expansion, the most common genetic cause of ALS and FTD, produces sense- and antisense-expansion RNAs and six dipeptide repeat-associated, non-ATG (RAN) proteins, but their roles in disease are unclear. We generated high-affinity human antibodies targeting GA or GP RAN proteins. These antibodies cross the blood-brain barrier and co-localize with intracellular RAN aggregates in C9-ALS/FTD BAC mice. In cells, α-GA1 interacts with TRIM21, and α-GA1 treatment reduced GA levels, increased GA turnover, and decreased RAN toxicity and co-aggregation of proteasome and autophagy proteins to GA aggregates. In C9-BAC mice, α-GA1 reduced GA as well as GP and GR proteins, improved behavioral deficits, decreased neuroinflammation and neurodegeneration, and increased survival. Glycosylation of the Fc region of α-GA1 is important for cell entry and efficacy. These data demonstrate that RAN proteins drive C9-ALS/FTD in C9-BAC transgenic mice and establish a novel therapeutic approach for C9orf72 ALS/FTD and other RAN-protein diseases.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.

Exploring the genetics and non-cell autonomous mechanisms underlying ALS/FTLD.

Although amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, was first described in 1874, a flurry of genetic discoveries in the last 10 years has markedly increased our understanding of this disease. These findings have not only enhanced our knowledge of mechanisms leading to ALS, but also have revealed that ALS shares many genetic causes with another neurodegenerative disease, frontotemporal lobar dementia (FTLD). In this review, we survey how recent genetic studies have bridged our mechanistic understanding of these two related diseases and how the genetics behind ALS and FTLD point to complex disorders, implicating non-neuronal cell types in disease pathophysiology. The involvement of non-neuronal cell types is consistent with a non-cell autonomous component in these diseases. This is further supported by studies that identified a critical role of immune-associated genes within ALS/FTLD and other neurodegenerative disorders. The molecular functions of these genes support an emerging concept that various non-autonomous functions are involved in neurodegeneration. Further insights into such a mechanism(s) will ultimately lead to a better understanding of potential routes of therapeutic intervention. Facts ALS and FTLD are severe neurodegenerative disorders on the same disease spectrum. Multiple cellular processes including dysregulation of RNA homeostasis, imbalance of proteostasis, contribute to ALS/FTLD pathogenesis. Aberrant function in non-neuronal cell types, including microglia, contributes to ALS/FTLD. Strong neuroimmune and neuroinflammatory components are associated with ALS/FTLD patients. Open Questions Why can patients with similar mutations have different disease manifestations, i.e., why do C9ORF72 mutations lead to motor neuron loss in some patients while others exhibit loss of neurons in the frontotemporal lobe? Do ALS causal mutations result in microglial dysfunction and contribute to ALS/FTLD pathology? How do microglia normally act to mitigate neurodegeneration in ALS/FTLD? To what extent do cellular signaling pathways mediate non-cell autonomous communications between distinct central nervous system (CNS) cell types during disease? Is it possible to therapeutically target specific cell types in the CNS?

Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules.

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.

Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription.

Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.

Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy.

The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.

The Notch signalling system: recent insights into the complexity of a conserved pathway.

Notch signalling links the fate of one cell to that of an immediate neighbour and consequently controls differentiation, proliferation and apoptotic events in multiple metazoan tissues. Perturbations in this pathway activity have been linked to several human genetic disorders and cancers. Recent genome-scale studies in Drosophila melanogaster have revealed an extraordinarily complex network of genes that can affect Notch activity. This highly interconnected network contrasts our traditional view of the Notch pathway as a simple linear sequence of events. Although we now have an unprecedented insight into the way in which such a fundamental signalling mechanism is controlled by the genome, we are faced with serious challenges in analysing the underlying molecular mechanisms of Notch signal control.

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling.

Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model we developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, we functionally linked the fibroblast growth factor (FGF) signaling pathway to the Drosophila homologue of SMN, Smn. Here, we characterize this relationship and demonstrate that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, we show that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities.

Nodal points and complexity of Notch-Ras signal integration.

Metazoans use a handful of highly conserved signaling pathways to create a signaling backbone that governs development. How these few signals have such a versatile action likely depends upon the larger-scale network they form through integration, as exemplified by cross-talk between the Notch and receptor tyrosine kinase (RTK) pathways. We examined the transcriptional output of Notch-RTK cross-talk during Drosophila development and present in vivo data supporting a role for selected mutually regulated genes in signal integration. Interestingly, Notch-RTK integration did not lead to general antagonism of either pathway, as is commonly believed. Instead, integration had a combinatorial effect on specific cross-regulated targets, which unexpectedly included numerous core components of the RTK and other major signaling pathways (TGF-beta, Hh, Jak/Stat, nuclear receptor and Wnt). We find the majority of Ras-responsive genes are also Notch-responsive, suggesting Notch may function to specify the response to Ras activation.

Modeling spinal muscular atrophy in Drosophila.

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets.

Investigating the genetic circuitry of mastermind in Drosophila, a notch signal effector.

Notch signaling regulates multiple developmental processes and is implicated in various human diseases. Through use of the Notch transcriptional co-activator mastermind, we conducted a screen for Notch signal modifiers using the Exelixis collection of insertional mutations, which affects approximately 50% of the Drosophila genome, recovering 160 genes never before associated with Notch, extending the previous roster of genes that interact functionally with the Notch pathway and mastermind. As the molecular identity for most recovered genes is known, gene ontology (GO) analysis was applied, grouping genes according to functional classifications. We identify novel Notch-associated GO categories, uncover nodes of integration between Notch and other signaling pathways, and unveil groups of modifiers that suggest the existence of Notch-independent mastermind functions, including a conserved ability to regulate Wnt signaling.

Crossing paths with Notch in the hyper-network.

The development of complex and diverse metazoan morphologies is coordinated by a surprisingly small number of evolutionarily conserved signaling mechanisms. These signals can act in parallel but often appear to function as an integrated hyper-network. The nodes defining this complex molecular circuitry are poorly understood, but the biological significance of pathway cross-talk is profound. The importance of such large-scale signal integration is exemplified by Notch and its ability to cross-talk with all the major pathways to influence cell differentiation, proliferation, survival and migration. The Notch pathway is, thus, a useful paradigm to illustrate the complexity of pathway cross-talk: its pervasiveness, context dependency, and importance in development and disease.