Cryptic exon inclusion is a molecular signature of LATE-NC in aging brains.

The aggregation, mislocalization, and phosphorylation of TDP-43 are pathologic hallmarks of several neurodegenerative diseases and provide a defining criterion for the neuropathologic diagnosis of Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). LATE neuropathologic changes (LATE-NC) are often comorbid with other neurodegenerative pathologies including Alzheimer's disease neuropathologic changes (ADNC). We examined whether TDP-43 regulated cryptic exons accumulate in the hippocampus of neuropathologically confirmed LATE-NC cases. We found that several cryptic RNAs are robustly expressed in LATE-NC cases with or without comorbid ADNC and correlate with pTDP-43 abundance; however, the accumulation of cryptic RNAs is more robust in LATE-NC with comorbid ADNC. Additionally, cryptic RNAs can robustly distinguish LATE-NC from healthy controls and AD cases. These findings expand our current understanding and provide novel potential biomarkers for LATE pathogenesis.

Rab11 regulates autophagy at dendritic spines in an mTOR- and NMDA-dependent manner.

Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment and learning and memory. Autophagy is a mechanism that allows the cell to degrade its unnecessary or dysfunctional components. Autophagosomes appear at dendritic spines in response to plasticity-inducing stimuli. Autophagy defects contribute to altered dendritic spine development, autistic-like behavior in mice, and neurological disease. While several studies have explored the involvement of autophagy in synaptic plasticity, the initial steps of the emergence of autophagosomes at the postsynapse remain unknown. Here, we demonstrate a postsynaptic association of autophagy-related protein 9A (Atg9A), known to be involved in the early stages of autophagosome formation, with Rab11, a small GTPase that regulates endosomal trafficking. Rab11 activity was necessary to maintain Atg9A-positive structures at dendritic spines. Inhibition of mTOR increased Rab11 and Atg9A interaction and increased the emergence of LC3 positive vesicles, an autophagosome membrane-associated protein marker, in dendritic spines when coupled to NMDA receptor stimulation. Dendritic spines with newly formed LC3+ vesicles were more resistant to NMDA-induced morphologic change. Rab11 DN overexpression suppressed appearance of LC3+ vesicles. Collectively, these results suggest that initiation of autophagy in dendritic spines depends on neuronal activity and Rab11a-dependent Atg9A interaction that is regulated by mTOR activity.

Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells.

The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells. High-content imaging reveals self-organized bundles of aligned muscle fibers surrounded by innervating motor neurons that form functional neuromuscular junctions. Optogenetic activation and pharmacological interventions show that the spinal neurons actively instruct the synchronous skeletal muscle contraction. The generation of a soNMJ model from spinal muscular atrophy patient-specific iPSCs reveals that the number of NMJs and muscle contraction is severely affected, resembling the patient's pathology. In the future, the soNMJ model could be used for high-throughput studies in disease modeling and drug development. Thus, this model will allow us to address unmet needs in the neuromuscular disease field.

Cross-species analysis identifies mitochondrial dysregulation as a functional consequence of the schizophrenia-associated 3q29 deletion.

The 1.6-megabase deletion at chromosome 3q29 (3q29Del) is the strongest identified genetic risk factor for schizophrenia, but the effects of this variant on neurodevelopment are not well understood. We interrogated the developing neural transcriptome in two experimental model systems with complementary advantages: isogenic human cortical organoids and isocortex from the 3q29Del mouse model. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 and 12 months, as well as perinatal mouse isocortex, all at single-cell resolution. Systematic pathway analysis implicated dysregulation of mitochondrial function and energy metabolism. These molecular signatures were supported by analysis of oxidative phosphorylation protein complex expression in mouse brain and assays of mitochondrial function in engineered cell lines, which revealed a lack of metabolic flexibility and a contribution of the 3q29 gene PAK2. Together, these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species.

Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes.

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs.

Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2α-dependent ISR in C9ORF72 FTD/ALS.

GGGGCC (G4C2) hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C4G2) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C4G2 repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C4G2 RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of C9ORF72 FTD/ALS patients. Finally, only antisense C4G2, but not sense G4C2, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C4G2 repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination.

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

Cross-species transcriptomic analysis identifies mitochondrial dysregulation as a functional consequence of the schizophrenia-associated 3q29 deletion.

Recent advances in the genetics of schizophrenia (SCZ) have identified rare variants that confer high disease risk, including a 1.6 Mb deletion at chromosome 3q29 with a staggeringly large effect size (O.R. > 40). Understanding the impact of the 3q29 deletion (3q29Del) on the developing CNS may therefore lead to insights about the pathobiology of schizophrenia. To gain clues about the molecular and cellular perturbations caused by the 3q29 deletion, we interrogated transcriptomic effects in two experimental model systems with complementary advantages: isogenic human forebrain cortical organoids and isocortex from the 3q29Del mouse model. We first created isogenic lines by engineering the full 3q29Del into an induced pluripotent stem cell line from a neurotypical individual. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 months and 12 months, as well as day p7 perinatal mouse isocortex, all at single cell resolution. Differential expression analysis by genotype in each cell-type cluster revealed that more than half of the differentially expressed genes identified in mouse cortex were also differentially expressed in human cortical organoids, and strong correlations were observed in mouse-human differential gene expression across most major cell-types. We systematically filtered differentially expressed genes to identify changes occurring in both model systems. Pathway analysis on this filtered gene set implicated dysregulation of mitochondrial function and energy metabolism, although the direction of the effect was dependent on developmental timepoint. Transcriptomic changes were validated at the protein level by analysis of oxidative phosphorylation protein complexes in mouse brain tissue. Assays of mitochondrial function in human heterologous cells further confirmed robust mitochondrial dysregulation in 3q29Del cells, and these effects are partially recapitulated by ablation of the 3q29Del gene PAK2 . Taken together these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species. These results converge with data from other rare SCZ-associated variants as well as idiopathic schizophrenia, suggesting that mitochondrial dysfunction may be a significant but overlooked contributing factor to the development of psychotic disorders. This cross-species scRNA-seq analysis of the SCZ-associated 3q29 deletion reveals that this copy number variant may produce early and persistent changes in cellular metabolism that are relevant to human neurodevelopment.

Development of single-molecule ubiquitination mediated fluorescence complementation to visualize protein ubiquitination dynamics in dendrites.

The ubiquitination/proteasome system is important for the spatiotemporal control of protein synthesis and degradation at synapses, while dysregulation may underlie autism spectrum disorders (ASDs). However, methods allowing direct visualization of the subcellular localization and temporal dynamics of protein ubiquitination are lacking. Here we report the development of Single-Molecule Ubiquitin Mediated Fluorescence Complementation (SM-UbFC) as a method to visualize and quantify the dynamics of protein ubiquitination in dendrites of live neurons in culture. Using SM-UbFC, we demonstrate that the rate of PSD-95 ubiquitination is elevated in dendrites of FMR1 KO neurons compared with wild-type controls. We further demonstrate the rapid ubiquitination of the fragile X messenger ribonucleoprotein, FMRP, and the AMPA receptor subunit, GluA1, which are known to be key events in the regulation of synaptic protein synthesis and plasticity. SM-UbFC will be useful for future studies on the regulation of synaptic protein homeostasis.

Altered Behavioral Responses Show GABA Sensitivity in Muscleblind-Like 2-Deficient Mice: Implications for CNS Symptoms in Myotonic Dystrophy.

Considerable evidence from mouse models and human postmortem brain suggests loss of Muscleblind-like protein 2 (MBNL2) function in brain is a major driver of CNS symptoms in Myotonic dystrophy type 1 (DM1). Increased hypersomnia, fatigue, and surgical complications associated with general anesthesia suggest possible sensitivity to GABAergic inhibition in DM1. To test the hypothesis that MBNL2 depletion leads to behavioral sensitivity to GABAA receptor (GABAA-R) modulation, Mbnl2 knock-out (KO) and wild-type (WT) littermates were treated with the anesthetic sevoflurane, the benzodiazepine diazepam, the imidazopyridine zolpidem, and the benzodiazepine rescue agent, flumazenil (Ro 15-1788), and assessed for various behavioral metrics. Mbnl2 KO mice exhibited delayed recovery following sevoflurane, delayed emergence and recovery from zolpidem, and enhanced sleep time at baseline that was modulated by flumazenil. A significantly higher proportion of Mbnl2 KO mice also loss their righting reflex [loss of righting reflex (LORR)] from a standard diazepam dose. We further examined whether MBNL2 depletion affects total GABAA-R mRNA subunit levels and validated RNA-sequencing data of mis-spliced Gabrg2, whose isoform ratios are known to regulate GABA sensitivity and associated behaviors. While no other GABAA-R subunit mRNA levels tested were altered in Mbnl2 KO mouse prefrontal cortex, Gabrg2S/L mRNA ratio levels were significantly altered. Taken together, our findings indicate that loss of MBNL2 function affects GABAergic function in a mouse model of myotonic dystrophy (DM1).

ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function.

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.

Mechanisms Driving the Emergence of Neuronal Hyperexcitability in Fragile X Syndrome.

Hyperexcitability is a shared neurophysiological phenotype across various genetic neurodevelopmental disorders, including Fragile X syndrome (FXS). Several patient symptoms are associated with hyperexcitability, but a puzzling feature is that their onset is often delayed until their second and third year of life. It remains unclear how and why hyperexcitability emerges in neurodevelopmental disorders. FXS is caused by the loss of FMRP, an RNA-binding protein which has many critical roles including protein synthesis-dependent and independent regulation of ion channels and receptors, as well as global regulation of protein synthesis. Here, we discussed recent literature uncovering novel mechanisms that may drive the progressive onset of hyperexcitability in the FXS brain. We discussed in detail how recent publications have highlighted defects in homeostatic plasticity, providing new insight on the FXS brain and suggest pharmacotherapeutic strategies in FXS and other neurodevelopmental disorders.

Metabolic effects of the schizophrenia-associated 3q29 deletion.

The 1.6 Mb 3q29 deletion is associated with developmental and psychiatric phenotypes, including a 40-fold increased risk for schizophrenia. Reduced birth weight and a high prevalence of feeding disorders in patients suggest underlying metabolic dysregulation. We investigated 3q29 deletion-induced metabolic changes using our previously generated heterozygous B6.Del16+/Bdh1-Tfrc mouse model. Animals were provided either standard chow (STD) or high-fat diet (HFD). Growth curves were performed on HFD mice to assess weight change (n = 30-50/group). Indirect calorimetry and untargeted metabolomics were performed on STD and HFD mice to evaluate metabolic phenotypes (n = 8-14/group). A behavioral battery was performed on STD and HFD mice to assess behavior change after the HFD challenge (n = 5-13/group). We found that B6.Del16+/Bdh1-Tfrc animals preferentially use dietary lipids as an energy source. Untargeted metabolomics of liver tissue showed a strong sex-dependent effect of the 3q29 deletion on fat metabolism. A HFD partially rescued the 3q29 deletion-associated weight deficit in females, but not males. Untargeted metabolomics of liver tissue after HFD revealed persistent fat metabolism alterations in females. The HFD did not affect B6.Del16+/Bdh1-Tfrc behavioral phenotypes, suggesting that 3q29 deletion-associated metabolic and behavioral outcomes are uncoupled. Our data suggest that dietary interventions to improve weight phenotypes in 3q29 deletion syndrome patients are unlikely to exacerbate behavioral manifestations. Our study also highlights the importance of assessing sex in metabolic studies and suggests that mechanisms underlying 3q29 deletion-associated metabolic phenotypes are sex-specific.

Diversity on location.

The RNA binding protein FMRP regulates the synthesis of synaptic and nuclear proteins within different compartments of a neuron.

Caregiver Perspectives on a Child's Diagnosis of 3q29 Deletion: "We Can't Just Wish This Thing Away".

OBJECTIVE: Genetic diagnoses are increasingly common in cases of intellectual disability and developmental delay. Although ascertainment of a relatively common, well-studied variant may provide guidance related to treatments and developmental expectations, it is less clear how the diagnosis of a rare variant affects caregivers, especially when the phenotype may include later-onset manifestations such as psychosis. In this study, we sought to identify caregiver concerns in the first qualitative study to assess the psychosocial impact of diagnosis on caregivers of individuals with 3q29 deletion syndrome (3q29Del), which is associated with a 40-fold increase in risk for psychosis. METHODS: Participants were recruited from the national 3q29Del registry housed at Emory University (3q29deletion.org). Fifteen participants completed a semistructured phone interview during which they were asked about their experiences before, during, and after their child received a diagnosis of 3q29Del. Interview responses were analyzed using the general inductive approach, and overarching themes were identified. RESULTS: We identified the following overarching themes: difficult "diagnostic odyssey," mixed feelings about diagnosis, frustration with degree of uncertainty, and importance of resources. Importantly, our data suggest that future risk for psychosis is often not disclosed by medical professionals, consistent with the experience of individuals with 22q11.2 deletion syndrome. CONCLUSIONS: These results highlight potential gaps in how caregivers are informed of risk for adult-onset conditions and indicate key caregiver concerns for consideration in the diagnosis of 3q29Del.

Mitochondrial Structure and Polarity in Dendrites and the Axon Initial Segment Are Regulated by Homeostatic Plasticity and Dysregulated in Fragile X Syndrome.

Mitochondrial dysfunction has long been overlooked in neurodevelopmental disorders, but recent studies have provided new links to genetic forms of autism, including Rett syndrome and fragile X syndrome (FXS). Mitochondria show plasticity in morphology and function in response to neuronal activity, and previous research has reported impairments in mitochondrial morphology and function in disease. We and others have previously reported abnormalities in distinct types of homeostatic plasticity in FXS. It remains unknown if or how activity deprivation triggering homeostatic plasticity affects mitochondria in axons and/or dendrites and whether impairments occur in neurodevelopmental disorders. Here, we test the hypothesis that mitochondria are structurally and functionally modified in a compartment-specific manner during homeostatic plasticity using a model of activity deprivation in cortical neurons from wild-type mice and that this plasticity-induced regulation is altered in Fmr1-knockout (KO) neurons. We uncovered dendrite-specific regulation of the mitochondrial surface area, whereas axon initial segment (AIS) mitochondria show changes in polarity; both responses are lost in the Fmr1 KO. Taken together, our results demonstrate impairments in mitochondrial plasticity in FXS, which has not previously been reported. These results suggest that mitochondrial dysregulation in FXS could contribute to abnormal neuronal plasticity, with broader implications to other neurodevelopmental disorders and therapeutic strategies.

A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies.

Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that can regulate the translation of specific mRNAs. In this study, we developed an FXS human forebrain organoid model and observed that the loss of FMRP led to dysregulated neurogenesis, neuronal maturation and neuronal excitability. Bulk and single-cell gene expression analyses of FXS forebrain organoids revealed that the loss of FMRP altered gene expression in a cell-type-specific manner. The developmental deficits in FXS forebrain organoids could be rescued by inhibiting the phosphoinositide 3-kinase pathway but not the metabotropic glutamate pathway disrupted in the FXS mouse model. We identified a large number of human-specific mRNAs bound by FMRP. One of these human-specific FMRP targets, CHD2, contributed to the altered gene expression in FXS organoids. Collectively, our study revealed molecular, cellular and electrophysiological abnormalities associated with the loss of FMRP during human brain development.

Convergent and distributed effects of the 3q29 deletion on the human neural transcriptome.

The 3q29 deletion (3q29Del) confers high risk for schizophrenia and other neurodevelopmental and psychiatric disorders. However, no single gene in this interval is definitively associated with disease, prompting the hypothesis that neuropsychiatric sequelae emerge upon loss of multiple functionally-connected genes. 3q29 genes are unevenly annotated and the impact of 3q29Del on the human neural transcriptome is unknown. To systematically formulate unbiased hypotheses about molecular mechanisms linking 3q29Del to neuropsychiatric illness, we conducted a systems-level network analysis of the non-pathological adult human cortical transcriptome and generated evidence-based predictions that relate 3q29 genes to novel functions and disease associations. The 21 protein-coding genes located in the interval segregated into seven clusters of highly co-expressed genes, demonstrating both convergent and distributed effects of 3q29Del across the interrogated transcriptomic landscape. Pathway analysis of these clusters indicated involvement in nervous-system functions, including synaptic signaling and organization, as well as core cellular functions, including transcriptional regulation, posttranslational modifications, chromatin remodeling, and mitochondrial metabolism. Top network-neighbors of 3q29 genes showed significant overlap with known schizophrenia, autism, and intellectual disability-risk genes, suggesting that 3q29Del biology is relevant to idiopathic disease. Leveraging "guilt by association", we propose nine 3q29 genes, including one hub gene, as prioritized drivers of neuropsychiatric risk. These results provide testable hypotheses for experimental analysis on causal drivers and mechanisms of the largest known genetic risk factor for schizophrenia and highlight the study of normal function in non-pathological postmortem tissue to further our understanding of psychiatric genetics, especially for rare syndromes like 3q29Del, where access to neural tissue from carriers is unavailable or limited.