Overview on Research and Clinical Applications of Optogenetics.

Optogenetics is a method that uses light to control cells in living tissue, typically neurons, that have been modified to express light-sensitive ion channels and pumps. The approach facilitates neuromodulation in brain preparations and freely moving animals with unmatched spatial and temporal resolution. This optogenetics overview describes the vast array of light-sensitive proteins available and the methods used to deliver them to tissue and modulate them with light.

SOD1 silencing in motoneurons or glia rescues neuromuscular function in ALS mice.

Objective: Amyotrophic lateral sclerosis is an incurable disorder mainly characterized by motoneuron degeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for 20% of familial forms of the disease. Mutant SOD1 exerts multiple pathogenic effects through the gain of toxic properties in both neurons and glial cells.

Virally mediated optogenetic excitation and inhibition of pain in freely moving nontransgenic mice.

Primary nociceptors are the first neurons involved in the complex processing system that regulates normal and pathological pain. Because of constraints on pharmacological and electrical stimulation, noninvasive excitation and inhibition of these neurons in freely moving nontransgenic animals has not been possible. Here we use an optogenetic strategy to bidirectionally control nociceptors of nontransgenic mice.

Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening.

Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ∼1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ∼98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.

Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration.

Selective neuronal loss is the hallmark of neurodegenerative diseases. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons die but those innervating extraocular, pelvic sphincter, and slow limb muscles exhibit selective resistance. We identified 18 genes that show >10-fold differential expression between resistant and vulnerable motor neurons.

Intracerebroventricular injection of adeno-associated virus 6 and 9 vectors for cell type-specific transgene expression in the spinal cord.

In the context of motoneuron diseases, gene delivery as an experimental or therapeutic approach is hindered by the challenge to specifically target cell populations that are widely distributed along the spinal cord. Further complicating the task, transgenes often need to be delivered to motoneurons and/or glial cells to address the non-cell-autonomous mechanisms involved in disease pathogenesis. Intracerebroventricular (ICV) injection of recombinant adeno-associated viruses (AAVs) in newborn mice allows distributing viral vectors throughout the central nervous system while limiting undesired transduction of peripheral organs.

Optogenetic control of targeted peripheral axons in freely moving animals.

Optogenetic control of the peripheral nervous system (PNS) would enable novel studies of motor control, somatosensory transduction, and pain processing. Such control requires the development of methods to deliver opsins and light to targeted sub-populations of neurons within peripheral nerves. We report here methods to deliver opsins and light to targeted peripheral neurons and robust optogenetic modulation of motor neuron activity in freely moving, non-transgenic mammals.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown.

Next-generation carrier screening.

Purpose: Carrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing-based workflow to enable analysis of a more comprehensive set of disease-causing mutations.

Methods: We utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA).

Optical inhibition of motor nerve and muscle activity in vivo.

Introduction: There is no therapeutic approach that provides precise and rapidly reversible inhibition of motor nerve and muscle activity for treatment of spastic hypertonia.

Methods: We used optogenetics to demonstrate precise and rapidly reversible light-mediated inhibition of motor nerve and muscle activity in vivo in transgenic Thy1::eNpHR2.0 mice.

Distinctive genetic and clinical features of CMT4J: a severe neuropathy caused by mutations in the PI(3,5)P₂ phosphatase FIG4.

Charcot-Marie-Tooth disease is a genetically heterogeneous group of motor and sensory neuropathies associated with mutations in more than 30 genes. Charcot-Marie-Tooth disease type 4J (OMIM 611228) is a recessive, potentially severe form of the disease caused by mutations of the lipid phosphatase FIG4. We provide a more complete view of the features of this disorder by describing 11 previously unreported patients with Charcot-Marie-Tooth disease type 4J.

Alu-specific microhomology-mediated deletion of the final exon of SPAST in three unrelated subjects with hereditary spastic paraplegia.

Purpose: Autosomal dominant spastic paraplegia, type 4 (SPG4), a debilitating disorder of progressive spasticity and weakness of the lower limbs, results from heterozygous mutations in the SPAST gene. The full spectrum of SPAST mutations causing SPG4 and their mechanisms of formation remain to be determined.

Methods: We used multiplex ligation-dependent probe amplification, locus-specific array comparative genomic hybridization, and breakpoint DNA sequencing to identify and describe genomic rearrangements in three patients with a clinical presentation of hereditary spastic paraplegia.

Migration of breast cancer cells: understanding the roles of volume exclusion and cell-to-cell adhesion.

We study MCF-7 breast cancer cell movement in a transwell apparatus. Various experimental conditions lead to a variety of monotone and nonmonotone responses which are difficult to interpret. We anticipate that the experimental results could be caused by cell-to-cell adhesion or volume exclusion.

Neuroprotection by gene therapy targeting mutant SOD1 in individual pools of motor neurons does not translate into therapeutic benefit in fALS mice.

A major challenge in neurological gene therapy is delivery of the transgene to sufficient cell numbers in an atraumatic manner. This is particularly difficult for motor neuron (MN) diseases that have cells located across the entire spinal cord, brain stem, and cortex. We have used the familial mouse model of amyotrophic lateral sclerosis (ALS) to examine the feasibility of body-wide intramuscular injections of adeno-associated virus serotype 6 (AAV6), a vector capable of axonal retrograde transport, to deliver therapeutic genetic information across the lower MN axis.

GJB1/Connexin 32 whole gene deletions in patients with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is the second most common form of this genetically heterogeneous inherited peripheral neuropathy. CMT1X is caused by mutations in the GJB1 gene. Most of the mutations causative for CMT1X are missense mutations.

Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: rare CNVs as a cause for missing heritability.

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood.

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

Background: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression.

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6.

Retrograde transport of viral vectors in the rodent spinal cord provides a powerful means to administer a therapeutic transgene from the innervated musculature. With the aim of scaling up this approach to non-human primates, we have injected recombinant adeno-associated vectors (rAAV) serotype 6 expressing enhanced green fluorescent protein (eGFP) into the gastrocnemius muscle of African green monkeys to determine whether this results in efficient transgene delivery to lumbar motor neurons. Cells expressing eGFP were observed across more than 1 cm of the spinal cord 4 weeks after intramuscular injection, reaching more than half of motor neurons in some cross-sections.

The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans.

We recently proposed a DNA replication-based mechanism of fork stalling and template switching (FoSTeS) to explain the complex genomic rearrangements associated with a dysmyelinating central nervous system disorder in humans. The FoSTeS mechanism has been further generalized and molecular mechanistic details have been provided in the microhomology-mediated break-induced replication (MMBIR) model that may underlie many structural variations in genomes from all domains of life. Here we provide evidence that human genomic rearrangements ranging in size from several megabases to a few hundred base pairs can be generated by FoSTeS/MMBIR.

Lentiviral and adeno-associated vector-based therapy for motor neuron disease through RNAi.

RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. Lentivirus and adeno-associated virus have been used to knockdown levels of mutated superoxide dismutase 1 (SOD1) in the G93A SOD1 mouse model of familial amyotrophic lateral sclerosis (fALS) to result in beneficial therapeutic outcomes.

Regulation of episomal gene expression by KRAB/KAP1-mediated histone modifications.

KAP1 is an essential cofactor of KRAB zinc finger proteins, a family of vertebrate-specific epigenetic repressors of largely unknown functions encoded in the hundreds by the mouse and human genomes. So far, KRAB/KAP1-mediated gene regulation has been studied within the environment of chromosomal DNA. Here we demonstrate that KRAB/KAP1 regulation is fully functional within the context of episomal DNA, such as adeno-associated viral and nonintegrated lentiviral vectors, and is correlated with histone modifications typically associated with this epigenetic regulator.

Systemic AAV6 delivery mediating RNA interference against SOD1: neuromuscular transduction does not alter disease progression in fALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder arising from the selective death of motor neurons. Approximately 20% of familial ALS (fALS) cases are caused by toxic gain-of-function mutations in the superoxide dismutase 1 (SOD1) gene. We as well as others have provided proof-of-principle for the use of RNA interference (RNAi) against mutant SOD1 as a potential therapy for fALS.

Puromycin-sensitive aminopeptidase limits MHC class I presentation in dendritic cells but does not affect CD8 T cell responses during viral infections.

Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice.