Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism.

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language, and cognitive deficits. A mouse model of this syndromic ASD (Mef2c-Het) recapitulates many of the MEF2C haploinsufficiency syndrome-linked behaviors, including communication deficits. We show here that Mef2c-Het mice of both sexes exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types; and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments.SIGNIFICANCE STATEMENT This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD-related behaviors, including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, subcellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.

Functional analysis of VPS41-mediated neuroprotection in Caenorhabditis elegans and mammalian models of Parkinson's disease.

Disruption of the lysosomal system has emerged as a key cellular pathway in the neurotoxicity of α-synuclein (α-syn) and the progression of Parkinson's disease (PD). A large-scale RNA interference (RNAi) screen using Caenorhabditis elegans identified VPS-41, a multidomain protein involved in lysosomal protein trafficking, as a modifier of α-syn accumulation and dopaminergic neuron degeneration (Hamamichi et al., 2008). Previous studies have shown a conserved neuroprotective function of human VPS41 (hVPS41) against PD-relevant toxins in mammalian cells and C. elegans neurons (Ruan et al., 2010). Here, we report that both the AP-3 (heterotetrameric adaptor protein complex) interaction domain and clathrin heavy-chain repeat domain are required for protecting C. elegans dopaminergic neurons from α-syn-induced neurodegeneration, as well as to prevent α-syn inclusion formation in an H4 human neuroglioma cell model. Using mutant C. elegans and neuron-specific RNAi, we revealed that hVPS41 requires both a functional AP-3 (heterotetrameric adaptor protein complex) and HOPS (homotypic fusion and vacuole protein sorting)-tethering complex to elicit neuroprotection. Interestingly, two nonsynonymous single-nucleotide polymorphisms found within the AP-3 interacting domain of hVPS41 attenuated the neuroprotective property, suggestive of putative susceptibility factors for PD. Furthermore, we observed a decrease in α-syn protein level when hVPS41 was overexpressed in human neuroglioma cells. Thus, the neuroprotective capacity of hVPS41 may be a consequence of enhanced clearance of misfolded and aggregated proteins, including toxic α-syn species. These data reveal the importance of lysosomal trafficking in maintaining cellular homeostasis in the presence of enhanced α-syn expression and toxicity. Our results support hVPS41 as a potential novel therapeutic target for the treatment of synucleinopathies like PD.

Caenorhabditis elegans as a model system for identifying effectors of α-synuclein misfolding and dopaminergic cell death associated with Parkinson's disease.

Protein misfolding and aggregation are key pathological features observed in numerous neurodegenerative diseases, including the misfolding of α-synuclein (α-syn) in Parkinson's disease (PD) and ß-amyloid in Alzheimer's disease. While this phenomenon is widely observed, the etiology and progression of these diseases is not fully understood. Furthermore, there is a lack of therapeutic treatments directed at halting the progression and neurodegeneration associated with these diseases. This demands a need for an inexpensive, easy to manipulate multicellular organism to conduct both genetic and chemical screens within to identify factors that may play a pivotal role in the pathology of these diseases. Herein, we describe methodology involved in identifying genetic modifiers of α-syn misfolding and toxicity in the nematode roundworm, Caenorhabditis elegans. Transgenic nematodes engineered to express human α-syn in the body wall muscles or dopaminergic (DA) neurons result in formation of cytoplasmic puncta or DA neurodegeneration, respectively. Using these models, we describe the use of RNA interference (RNAi) and transgenic gene expression to functionally elucidate potential therapeutic gene targets that alter α-syn misfolding and DA neurotoxicity.

C. elegans as a model organism to investigate molecular pathways involved with Parkinson's disease.

Parkinson's disease (PD) is an age-related movement disorder resulting, in part, from selective loss of dopaminergic neurons. Both invertebrate and mammalian models have been developed to study the cellular mechanisms altered during disease progression; nevertheless there are limitations within each model. Mammalian models remain invaluable in studying PD, but are expensive and time consuming. Here, we review genetic and environmental factors associated with PD, and describe how the nematode roundworm, Caenorhabditis elegans, has been used as a model organism for studying various aspects of this neurodegenerative disease. Both genetic and chemical screens have been conducted in C. elegans to identify molecular pathways, proteins, and small molecules that can impact PD pathology. Lastly, we highlight future areas of investigation, in the context of emerging fields in biology, where the nematode can be exploited to provide mechanistic insights and potential strategies to accelerate the path toward possible therapeutic intervention for PD.

Low-dose bafilomycin attenuates neuronal cell death associated with autophagy-lysosome pathway dysfunction.

We have shown previously that the plecomacrolide antibiotics bafilomycin A1 and B1 significantly attenuate cerebellar granule neuron death resulting from agents that disrupt lysosome function. To further characterize bafilomycin-mediated cytoprotection, we examined its ability to attenuate the death of naïve and differentiated neuronal SH-SY5Y human neuroblastoma cells from agents that induce lysosome dysfunction in vitro, and from in vivo dopaminergic neuron death in C. elegans. Low-dose bafilomycin significantly attenuated SH-SY5Y cell death resulting from treatment with chloroquine, hydroxychloroquine amodiaquine and staurosporine. Bafilomycin also attenuated the chloroquine-induced reduction in processing of cathepsin D, the principal lysosomal aspartic acid protease, to its mature 'active' form. Chloroquine induced autophagic vacuole accumulation and inhibited autophagic flux, effects that were attenuated upon treatment with bafilomycin and were associated with a significant decrease in chloroquine-induced accumulation of detergent-insoluble alpha-synuclein oligomers. In addition, bafilomycin significantly and dose-dependently attenuated dopaminergic neuron death in C. elegans resulting from in vivo over-expression of human wild-type alpha-synuclein. Together, our findings suggest that low-dose bafilomycin is cytoprotective in part through its maintenance of the autophagy-lysosome pathway, and underscores its therapeutic potential for treating Parkinson's disease and other neurodegenerative diseases that exhibit disruption of protein degradation pathways and accumulation of toxic protein species.

VPS41, a protein involved in lysosomal trafficking, is protective in Caenorhabditis elegans and mammalian cellular models of Parkinson's disease.

VPS41 is a protein identified as a potential therapeutic target for Parkinson's disease (PD) as a result of a high-throughput RNAi screen in Caenorhabditis elegans. VPS41 has a plausible mechanistic link to the pathogenesis of PD, as in yeast it is known to participate in trafficking of proteins to the lysosomal system and several recent lines of evidence have pointed to the importance of lysosomal system dysfunction in the neurotoxicity of alpha-synuclein (alpha-syn). We found that expression of the human form of VPS41 (hVPS41) prevents dopamine (DA) neuron loss induced by alpha-syn overexpression and 6-hydroxydopamine (6-OHDA) neurotoxicity in C. elegans. In SH-SY5Y neuroblastoma cell lines stably transfected with hVPS41, we determined that presence of this protein conferred protection against the neurotoxins 6-OHDA and rotenone. Overexpression of hVPS41 did not alter the mitochondrial membrane depolarization induced by these neurotoxins. hVPS41 did, however, block downstream events in the apoptotic cascade including activation of caspase-9 and caspase-3, and PARP cleavage. We also observed that hVPS41 reduced the accumulation of insoluble high-molecular weight forms of alpha-syn in SH-SY5Y cells after treatment with rotenone. These data show that hVPS41 is protective against both alpha-syn and neurotoxic-mediated injury in invertebrate and cellular models of PD. These protective functions may be related to enhanced clearance of misfolded or aggregated protein, including alpha-syn. Our studies indicate that hVPS41 may be a useful target for developing therapeutic strategies for human PD.

An essential role for MEF2C in the cortical response to loss of sleep in mice.

Neuronal activity and gene expression in response to the loss of sleep can provide a window into the enigma of sleep function. Sleep loss is associated with brain differential gene expression, an increase in pyramidal cell mEPSC frequency and amplitude, and a characteristic rebound and resolution of slow wave sleep-slow wave activity (SWS-SWA). However, the molecular mechanism(s) mediating the sleep-loss response are not well understood. We show that sleep-loss regulates MEF2C phosphorylation, a key mechanism regulating MEF2C transcriptional activity, and that MEF2C function in postnatal excitatory forebrain neurons is required for the biological events in response to sleep loss in C57BL/6J mice. These include altered gene expression, the increase and recovery of synaptic strength, and the rebound and resolution of SWS-SWA, which implicate MEF2C as an essential regulator of sleep function.

MEF2C Hypofunction in Neuronal and Neuroimmune Populations Produces MEF2C Haploinsufficiency Syndrome-like Behaviors in Mice.

BACKGROUND: Microdeletions of the MEF2C gene are linked to a syndromic form of autism termed MEF2C haploinsufficiency syndrome (MCHS). MEF2C hypofunction in neurons is presumed to underlie most of the symptoms of MCHS. However, it is unclear in which cell populations MEF2C functions to regulate neurotypical development. METHODS: Multiple biochemical, molecular, electrophysiological, behavioral, and transgenic mouse approaches were used to characterize MCHS-relevant synaptic, behavioral, and gene expression changes in mouse models of MCHS. RESULTS: We showed that MCHS-associated missense mutations cluster in the conserved DNA binding domain and disrupt MEF2C DNA binding. DNA binding-deficient global Mef2c heterozygous mice (Mef2c-Het) displayed numerous MCHS-related behaviors, including autism-related behaviors, changes in cortical gene expression, and deficits in cortical excitatory synaptic transmission. We detected hundreds of dysregulated genes in Mef2c-Het cortex, including significant enrichments of autism risk and excitatory neuron genes. In addition, we observed an enrichment of upregulated microglial genes, but this was not due to neuroinflammation in the Mef2c-Het cortex. Importantly, conditional Mef2c heterozygosity in forebrain excitatory neurons reproduced a subset of the Mef2c-Het phenotypes, while conditional Mef2c heterozygosity in microglia reproduced social deficits and repetitive behavior. CONCLUSIONS: Taken together, our findings show that mutations found in individuals with MCHS disrupt the DNA-binding function of MEF2C, and DNA binding-deficient Mef2c global heterozygous mice display numerous MCHS-related phenotypes, including excitatory neuron and microglia gene expression changes. Our findings suggest that MEF2C regulates typical brain development and function through multiple cell types, including excitatory neuronal and neuroimmune populations.

Emerging roles for MEF2 in brain development and mental disorders.

The MEF2 family of transcription factors regulate large programs of gene expression important for the development and maintenance of many tissues, including the brain. MEF2 proteins are regulated by neuronal synaptic activity, and they recruit several epigenetic enzymes to influence chromatin structure and gene expression during development and throughout adulthood. Here, we provide a brief review of the recent literature reporting important roles for MEF2 during early brain development and function, and we highlight emerging roles for MEF2 as a risk factor for multiple neurodevelopmental disorders and mental illnesses, such as autism, intellectual disability, and schizophrenia.

MEF2C regulates cortical inhibitory and excitatory synapses and behaviors relevant to neurodevelopmental disorders.

Numerous genetic variants associated with MEF2C are linked to autism, intellectual disability (ID) and schizophrenia (SCZ) - a heterogeneous collection of neurodevelopmental disorders with unclear pathophysiology. MEF2C is highly expressed in developing cortical excitatory neurons, but its role in their development remains unclear. We show here that conditional embryonic deletion of Mef2c in cortical and hippocampal excitatory neurons (Emx1-lineage) produces a dramatic reduction in cortical network activity in vivo, due in part to a dramatic increase in inhibitory and a decrease in excitatory synaptic transmission. In addition, we find that MEF2C regulates E/I synapse density predominantly as a cell-autonomous, transcriptional repressor. Analysis of differential gene expression in Mef2c mutant cortex identified a significant overlap with numerous synapse- and autism-linked genes, and the Mef2c mutant mice displayed numerous behaviors reminiscent of autism, ID and SCZ, suggesting that perturbing MEF2C function in neocortex can produce autistic- and ID-like behaviors in mice.

Modeling dopamine neuron degeneration in Caenorhabditis elegans.

Ongoing investigations into causes and cures for human movement disorders are important toward the elucidation of diseases, such as Parkinson's disease (PD). The use of animal model systems can provide links to susceptibility factors as well as therapeutic interventions. In this regard, the nematode roundworm, Caenorhabditis elegans, is ideal for age-dependent neurodegenerative disease studies. It is genetically tractable, has a short life span, and a well-defined nervous system. Fluorescent markers, like GFP, are readily visualized in C. elegans as it is a transparent organism; thus the nervous system, and factors that alter the viability of neurons, can be directly examined in vivo. Through expression of the human disease protein, alpha-synuclein, in the worm dopamine neurons, neurodegeneration is observed in an age-dependent manner. Furthermore, application of a dopamine neurotoxin, 6-hydroxy-dopamine, provides another independent model of PD. Described herein are techniques for C. elegans transformation to evaluate candidate neuroprotective gene targets, integration of the extrachromosomal arrays, genetic crosses, and methods for dopamine neuron analysis that are applicable to both types of neurotoxicity. These techniques can be exploited to assess both chemical and genetic modifiers of toxicity, providing additional avenues to advance PD-related discoveries.

Application of a C. elegans dopamine neuron degeneration assay for the validation of potential Parkinson's disease genes.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration. Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets. Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.