Scalable, optically-responsive human neuromuscular junction model reveals convergent mechanisms of synaptic dysfunction in familial ALS.

Neuromuscular junctions (NMJs) are specialized synapses that mediate communication between motor neurons and skeletal muscles and are essential for movement. The degeneration of this system can lead to symptoms observed in neuromuscular and motor neuron diseases. Studying these synapses and their degeneration has proven challenging. Prior NMJ studies heavily relied upon the use of mouse, chick, or isolated primary human cells, which have demonstrated limited fidelity for disease modeling. To enable the study of NMJ dysfunction and model genetic diseases, we, and others, have developed methods to generate human NMJs from pluripotent stem cells (PSCs), embryonic stem cells, and induced pluripotent stem cells. However, published studies have highlighted technical limitations associated with these complex in vitro NMJ models. In this study, we developed a robust PSC-derived motor neuron and skeletal muscle co-culture method, and demonstrated its sensitivity in modeling motor neuron disease. Our method spontaneously and reproducibly forms human NMJs. We developed multiwell-multielectrode array (MEA) parameters to quantify the activity of PSC-derived skeletal muscles, as well as measured the electrophysiological activity of functional human PSC-derived NMJs. We further leveraged our method to morphologically and functionally assess NMJs from the familial amyotrophic lateral sclerosis (fALS) PSCs, C9orf72 hexanucleotide (G4C2)n repeat expansion (HRE), SOD1 A5V , and TDP43 G298S to define the reproducibility and sensitivity of our system. We observed a significant decrease in the numbers and activity of PSC-derived NMJs developed from the different ALS lines compared to their respective controls. Furthermore, we evaluated a therapeutic candidate undergoing clinical trials and observed a variant-dependent rescue of functionality of NMJs. Our newly developed method provides a platform for the systematic investigation of genetic causes of NMJ neurodegeneration and highlights the need for therapeutic avenues to consider patient genotype.

Skeletal muscle TFEB signaling promotes central nervous system function and reduces neuroinflammation during aging and neurodegenerative disease.

Skeletal muscle has recently arisen as a regulator of central nervous system (CNS) function and aging, secreting bioactive molecules known as myokines with metabolism-modifying functions in targeted tissues, including the CNS. Here, we report the generation of a transgenic mouse with enhanced skeletal muscle lysosomal and mitochondrial function via targeted overexpression of transcription factor E-B (TFEB). We discovered that the resulting geroprotective effects in skeletal muscle reduce neuroinflammation and the accumulation of tau-associated pathological hallmarks in a mouse model of tauopathy. Muscle-specific TFEB overexpression significantly ameliorates proteotoxicity, reduces neuroinflammation, and promotes transcriptional remodeling of the aged CNS, preserving cognition and memory in aged mice. Our results implicate the maintenance of skeletal muscle function throughout aging in direct regulation of CNS health and disease and suggest that skeletal muscle originating factors may act as therapeutic targets against age-associated neurodegenerative disorders.

Generation of advanced cerebellar organoids for neurogenesis and neuronal network development.

Neurons within the cerebellum form temporal-spatial connections through the cerebellum, and the entire brain. Organoid models provide an opportunity to model the early differentiation of the developing human cerebellum, which is difficult to study in vivo, and affords the opportunity to study neurodegenerative and neurodevelopmental diseases of the cerebellum. Previous cerebellar organoid models focused on early neuron generation and single cell activity. Here, we modify previous protocols to generate more mature cerebellar organoids that allow for the establishment of several classes of mature neurons during cerebellar differentiation and development, including the establishment of neural networks during whole-organoid maturation. This will provide a means to study the generation of several more mature cerebellar cell types, including Purkinje cells, granule cells and interneurons expression as well as neuronal communication for biomedical, clinical and pharmaceutical applications.

Multiplexed microfluidic chip for cell co-culture.

Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually-addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1 h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.

Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration.

Tight regulation of mRNA isoform expression is essential for neuronal development, maintenance, and function; however, the repertoire of proteins that govern isoform composition and abundance remains incomplete. Here, we show that the RNA kinase CLP1 regulates mRNA isoform expression through suppression of proximal cleavage and polyadenylation. We found that human stem-cell-derived motor neurons without CLP1 or with the disease-associated CLP1 p.R140H variant had distinct patterns of RNA-polymerase-II-associated cleavage and polyadenylation complex proteins that correlated with polyadenylation site usage. These changes resulted in imbalanced mRNA isoform expression of long genes important for neuronal function that were recapitulated in vivo. Strikingly, we observed the same pattern of reduced mRNA isoform diversity in 3' end sequencing data from brain tissues of patients with neurodegenerative disease. Together, our results identify a previously uncharacterized role for CLP1 in mRNA 3' end formation and reveal an mRNA misprocessing signature in neurodegeneration that may suggest a common mechanism of disease.

Implications of mRNA translation dysregulation for neurological disorders.

The translation of information encoded in the DNA into functional proteins is one of the tenets of cellular biology. Cell survival and function depend on the tightly controlled processes of transcription and translation. Growing evidence suggests that dysregulation in mRNA translation plays an important role in the pathogenesis of several neurodevelopmental diseases, such as autism spectrum disorder (ASD) and fragile X syndrome (FXS) as well as neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In this review, we provide an overview of mRNA translation and its modes of regulation that have been implicated in neurological disease.

An epilepsy-associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model.

ACTL6B is a component of the neuronal BRG1/brm-associated factor (nBAF) complex, which is required for chromatin remodeling in postmitotic neurons. We recently reported biallelic pathogenic variants in ACTL6B in patients diagnosed with early infantile epileptic encephalopathy, subtype 76 (EIEE-76), presenting with severe, global developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal central nervous system myelination. However, the pathophysiological mechanisms underlying their phenotype is unknown. Here, we investigate the molecular pathogenesis of ACTL6B p.(Val421_Cys425del) using in silico 3D protein modeling predictions and patient-specific induced pluripotent stem cell-derived neurons. We found neurons derived from EIEE-76 patients showed impaired accumulation of ACTL6B compared to unaffected relatives, caused by reduced protein stability. Furthermore, EIEE-76 patient-derived neurons had dysregulated nBAF target gene expression, including genes important for neuronal development and disease. Multielectrode array system analysis unveiled elevated electrophysiological activity of EIEE-76 patients-derived neurons, consistent with the patient phenotype. Taken together, our findings validate a new model for EIEE-76 and reveal how reduced ACTL6B expression affects neuronal function.

Direct Generation of Human Cortical Organoids from Primary Cells.

The study of variations in human neurodevelopment and cognition is limited by the availability of experimental models. While animal models only partially recapitulate the human brain development, genetics, and heterogeneity, human-induced pluripotent stem cells can provide an attractive experimental alternative. However, cellular reprogramming and further differentiation techniques are costly and time-consuming and therefore, studies using this approach are often limited to a small number of samples. In this study, we describe a rapid and cost-effective method to reprogram somatic cells and the direct generation of cortical organoids in a 96-well format. Our data are a proof-of-principle that a large cohort of samples can be generated for experimental assessment of the human neural development.

Selective modulation of the androgen receptor AF2 domain rescues degeneration in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.

Adenylyl cyclase activating polypeptide reduces phosphorylation and toxicity of the polyglutamine-expanded androgen receptor in spinobulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases, which are fatal neurodegenerative disorders mainly caused by protein-mediated toxic gain-of-function mechanisms and characterized by deposition of misfolded proteins in the form of aggregates. The neurotoxicity of the polyQ proteins can be modified by phosphorylation at specific sites, thereby providing the rationale for the development of disease-specific treatments. We sought to identify signaling pathways that modulate polyQ-AR phosphorylation for therapy development. We report that cyclin-dependent kinase 2 (CDK2) phosphorylates polyQ-AR specifically at Ser96 Phosphorylation of polyQ-AR by CDK2 increased protein stabilization and toxicity and is negatively regulated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway. To translate these findings into therapy, we developed an analog of pituitary adenylyl cyclase activating polypeptide (PACAP), a potent activator of the AC/PKA pathway. Chronic intranasal administration of the PACAP analog to knock-in SBMA mice reduced Ser96 phosphorylation, promoted polyQ-AR degradation, and ameliorated disease outcome. These results provide proof of principle that noninvasive therapy based on the use of PACAP analogs is a therapeutic option for SBMA.

PPAR-δ is repressed in Huntington's disease, is required for normal neuronal function and can be targeted therapeutically.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which encodes a polyglutamine tract in the HTT protein. We found that peroxisome proliferator-activated receptor delta (PPAR-δ) interacts with HTT and that mutant HTT represses PPAR-δ-mediated transactivation. Increased PPAR-δ transactivation ameliorated mitochondrial dysfunction and improved cell survival of neurons from mouse models of HD. Expression of dominant-negative PPAR-δ in the central nervous system of mice was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPAR-δ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR-δ using the agonist KD3010 improved motor function, reduced neurodegeneration and increased survival. PPAR-δ activation also reduced HTT-induced neurotoxicity in vitro and in medium spiny-like neurons generated from stem cells derived from individuals with HD, indicating that PPAR-δ activation may be beneficial in HD and related disorders.

Nemo-like kinase is a novel regulator of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a progressive neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR) protein. Despite extensive research, the exact pathogenic mechanisms underlying SBMA remain elusive. In this study, we present evidence that Nemo-like kinase (NLK) promotes disease pathogenesis across multiple SBMA model systems. Most remarkably, loss of one copy of Nlk rescues SBMA phenotypes in mice, including extending lifespan. We also investigated the molecular mechanisms by which NLK exerts its effects in SBMA. Specifically, we have found that NLK can phosphorylate the mutant polyglutamine-expanded AR, enhance its aggregation, and promote AR-dependent gene transcription by regulating AR-cofactor interactions. Furthermore, NLK modulates the toxicity of a mutant AR fragment via a mechanism that is independent of AR-mediated gene transcription. Our findings uncover a crucial role for NLK in controlling SBMA toxicity and reveal a novel avenue for therapy development in SBMA.

Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA.

Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise when dysregulated. Here we consider X-linked spinal and bulbar muscular atrophy (SBMA), a repeat disorder caused by polyglutamine-expanded androgen receptor (polyQ-AR). We found that polyQ-AR reduced long-term protein turnover and impaired autophagic flux in motor neuron-like cells. Ultrastructural analysis of SBMA mice revealed a block in autophagy pathway progression. We examined the transcriptional regulation of autophagy and observed a functionally significant physical interaction between transcription factor EB (TFEB) and AR. Normal AR promoted, but polyQ-AR interfered with, TFEB transactivation. To evaluate physiological relevance, we reprogrammed patient fibroblasts to induced pluripotent stem cells and then to neuronal precursor cells (NPCs). We compared multiple SBMA NPC lines and documented the metabolic and autophagic flux defects that could be rescued by TFEB. Our results indicate that polyQ-AR diminishes TFEB function to impair autophagy and promote SBMA pathogenesis.