Jason Arsenault

Reduced phenotypic severity following adeno-associated virus-mediated Fmr1 gene delivery in fragile X mice.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a trinucleotide repeat expansion in the FMR1 gene that codes for fragile X mental retardation protein (FMRP). To determine if FMRP expression in the central nervous system could reverse phenotypic deficits in the Fmr1 knockout (KO) mouse model of FXS, we used a single-stranded adeno-associated viral (AAV) vector with viral capsids from serotype 9 that contained a major isoform of FMRP. FMRP transgene expression was driven by the neuron-selective synapsin-1 promoter. The vector was delivered to the brain via a single bilateral intracerebroventricular injection into neonatal Fmr1 KO mice and transgene expression and behavioral assessments were conducted 22–26 or 50–56 days post injection. Western blotting and immunocytochemical analyses of AAV–FMRP-injected mice revealed FMRP expression in the striatum, hippocampus, retrosplenial cortex, and cingulate cortex. Cellular expression was selective for neurons and reached ∼50% of wild-type levels in the hippocampus and cortex at 56 days post injection. The pathologically elevated repetitive behavior and the deficit in social dominance behavior seen in phosphate-buffered saline-injected Fmr1 KO mice were reversed in AAV–FMRP-injected mice. These results provide the first proof of principle that gene therapy can correct specific behavioral abnormalities in the mouse model of FXS.

Synthetic self-assembling clostridial chimera for modulation of sensory functions.

Clostridial neurotoxins reversibly block neuronal communication for weeks and months. While these proteolytic neurotoxins hold great promise for clinical applications and the investigation of brain function, their paralytic activity at neuromuscular junctions is a stumbling block. To redirect the clostridial activity to neuronal populations other than motor neurons, we used a new self-assembling method to combine the botulinum type A protease with the tetanus binding domain, which natively targets central neurons. The two parts were produced separately and then assembled in a site-specific way using a newly introduced ‘protein stapling’ technology. Atomic force microscopy imaging revealed dumbbell shaped particles which measure ∼23 nm. The stapled chimera inhibited mechanical hypersensitivity in a rat model of inflammatory pain without causing either flaccid or spastic paralysis. Moreover, the synthetic clostridial molecule was able to block neuronal activity in a defined area of visual cortex. Overall, we provide the first evidence that the protein stapling technology allows assembly of distinct proteins yielding new biomedical properties.

Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces.

Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells

Precise cellular targeting of macromolecular cargos has important biotechnological and medical implications. Using a recently established ‘protein stapling’ method, we linked the proteolytic domain of botulinum neurotoxin type A (BoNT/A) to a selection of ligands to target neuroendocrine tumor cells. The botulinum proteolytic domain was chosen because of its well‐known potency to block the release of neurotransmitters and hormones. Among nine tested stapled ligands, the epidermal growth factor was able to deliver the botulinum enzyme into pheochromocytoma PC12 and insulinoma Min6 cells; ciliary neurotrophic factor was effective on neuroblastoma SH‐SY5Y and Neuro2A cells, whereas corticotropin‐releasing hormone was active on pituitary AtT‐20 cells and the two neuroblastoma cell lines. In neuronal cultures, the epidermal growth factor‐ and ciliary neurotrophic factor‐directed botulinum enzyme targeted distinct subsets of neurons whereas the whole native neurotoxin targeted the cortical neurons indiscriminately. At nanomolar concentrations, the retargeted botulinum molecules were able to inhibit stimulated release of hormones from tested cell lines suggesting their application for treatments of neuroendocrine disorders.

Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice

Silencing key neurons with botulinum toxin conjugates exerts long-lasting pain relief in mouse models of chronic pain. Relieving pain with botox Chronic pain affects more than 25 million Americans and is associated with reduced life span, anxiety, and depression. Opioid administration is often effective in relieving pain but, unfortunately, opioids have serious side effects, including risk of addiction and overdose. In a new study, Maiarù et al. have leveraged the inhibitory effects of botulinum toxin on neuronal activity. They developed two botulinum-conjugated molecules (SP-BOT and Derm-BOT) that were able to silence subpopulations of pain-related spinal neurons in several mouse models of chronic pain. Intrathecal administration of one dose of SP-BOT or Derm-BOT produced long-term pain relief in the mouse models that was comparable to the effects of opioid treatment. The results suggest that botulinum-conjugated molecules could be an opioid-free alternative for treating chronic pain. Chronic pain is a widespread debilitating condition affecting millions of people worldwide. Although several pharmacological treatments for relieving chronic pain have been developed, they require frequent chronic administration and are often associated with severe adverse events, including overdose and addiction. Persistent increased sensitization of neuronal subpopulations of the peripheral and central nervous system has been recognized as a central mechanism mediating chronic pain, suggesting that inhibition of specific neuronal subpopulations might produce antinociceptive effects. We leveraged the neurotoxic properties of the botulinum toxin to specifically silence key pain-processing neurons in the spinal cords of mice. We show that a single intrathecal injection of botulinum toxin conjugates produced long-lasting pain relief in mouse models of inflammatory and neuropathic pain without toxic side effects. Our results suggest that this strategy might be a safe and effective approach for relieving chronic pain while avoiding the adverse events associated with repeated chronic drug administration.

FMRP Expression Levels in Mouse CNS Neurons Determine Behavioral Phenotype.

Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector–based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lo...Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector–based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35–115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5–6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long-term curative gene therapy strategies for treating Fragile X Syndrome and other neurodevelopmental disorders.

Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP‐LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30–50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection. J. Cell. Biochem. 115: 2047–2054, 2014.

Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain

Principal neurons encode information by varying their firing rate and patterns precisely fine-tuned through GABAergic interneurons. Dysregulation of inhibition can lead to neuropsychiatric disorders, yet little is known about the molecular basis underlying inhibitory control. Here, we find that excessive GABA release from basket cells (BCs) attenuates the firing frequency of Purkinje neurons (PNs) in the cerebellum of Fragile X Mental Retardation 1 (Fmr1) knockout (KO) mice, a model of Fragile X Syndrome (FXS) with abrogated expression of the Fragile X Mental Retardation Protein (FMRP). This over-inhibition originates from increased excitability and Ca2+ transients in the presynaptic terminals, where Kv1.2 potassium channels are downregulated. By paired patch-clamp recordings, we further demonstrate that acutely introducing an N-terminal fragment of FMRP into BCs normalizes GABA release in the Fmr1-KO synapses. Conversely, direct injection of an inhibitory FMRP antibody into BCs, or membrane depolarization of BCs, enhances GABA release in the wild type synapses, leading to abnormal inhibitory transmission comparable to the Fmr1-KO neurons. We discover that the N-terminus of FMRP directly binds to a phosphorylated serine motif on the C-terminus of Kv1.2; and that loss of this interaction in BCs exaggerates GABA release, compromising the firing activity of PNs and thus the output from the cerebellar circuitry. An allosteric Kv1.2 agonist, docosahexaenoic acid, rectifies the dysregulated inhibition in vitro as well as acoustic startle reflex and social interaction in vivo of the Fmr1-KO mice. Our results unravel a novel molecular locus for targeted intervention of FXS and perhaps autism.

368. Advances in Fragile X Gene Therapy Using Adeno-Associated Viral Vectors Coding for the Fragile X Mental Retardation Protein

Fragile X syndrome (FXS) is a severe debilitating neurodevelopmental disorder characterized by a loss of proper translational control at the synapse. In FXS, an aberrant CGG trinucleotide expansion upstream of the Fragile X Mental Retardation Protein (FMRP) gene causes drastic downregulation of this translational modulator. The absence of FMRP impairs synaptic plasticity and leads to mental retardation and autistic spectrum-related phenotypes. To correct this neurological disorder we recently devised an adeno-associated viral (AAV) vector encoding FMRP where its cellular tropism was controlled by the neuron-specific synapsin promoter. Following intracerebroventricular (i.c.v.) injection in neonatal Fmr1 KO mice, transgene expression remained stable for over 7 months in terminally differentiated neurons. The FMRP transgene corrected PSD-95 protein hypo-expression in the cortex of Fmr1 KO animals, as well as lowered MeCP2 protein over-expression. Behavioral endophenotypes including hyperactivity, non-social anxiety, pre-pulse inhibition, repetitive stereotypies, and social dominance were fully or partially corrected using this viral construct. We combined i.c.v. injection with additional injections into the parenchyma of refractory brain regions to achieve wider transduction, while avoiding potentially pathological transgene over expression effects. This ongoing translational study enables us to determine the proper cellular tropism and the range of FMRP expression required for rescue, as well as the brain region dependent correlation of autistic behaviors implicated in FXS neuropathology. These findings are relevant to gene therapy strategies for treating human FXS, as well as other neurodevelopmental disorders.

17. Gene Therapy Strategies to Treat Fragile X Syndrome

Fragile X syndrome (FXS) is a severe debilitating neurodevelopmental disorder of the autism spectrum that results from an aberrant trinucleotide repeat extension in the 5’ region of the FMR1 gene. This extension pathologically reduces or eliminates the expression of the fragile X mental retardation protein (FMRP). FMRP is known to be a scrupulous translational modulator at the synapse and is also known to stabilize and traffic mRNAs important for proper neurological functions. We utilized C57/BL6 mice with a knock-out of the Fmr1 gene (FMRP-KO) in this study because this mouse model reproduces many of the behavioral phenotypes seen in human Fragile X patients. We used adeno-associated viral vectors (AAV) serotype 2/9, which contains the inverted terminal repeats of serotype 2, the human neuron specific synapsin promoter and the largest isoform of FMRP (isoform 1), as well as downstream woodchuck hepatitis virus post-transcriptional regulatory element and a poly A site to elevate mRNA stability, in order to re-introduce FMRP in the brain of neonatal FMRP KO mice. This vector, as well as a cognate null vector containing no transgene used as a control, were administered via bilateral intra-cerebroventricular injections at different early postnatal time points in wild type (WT) and FMRP-KO mice. AAVs were produced and purified by the University of Pennsylvania Vector Core facility. We also investigated whether FMRP overexpression in WT mice could induce behavioral changes. We tested the treated mice in an array of behavioral tests to monitor changes in autism-associated behaviors such as repetitive stereotypical motions, hyperactivity, deficits in social interaction, and general anxiety. Long term FMRP transgene expression was confirmed by immunocytochemistry and quantitative western immunoblotting where strong forebrain and neuron-specific expression was observed. Overall, we observed partial to full amelioration of certain fragile X phenotypes such as motor activity, sensorimotor gating and open field latency in FMRP-KO mice injected with the AAV-FMRP vectors. Also other observations points towards possible pathological effects due to FMRP overexpression in the forebrain of WT mice; an important consideration for future gene therapy of human FXS. These results show that the time of neonatal injection and the total number of AAV particles administered during intra-cerebroventricular injections can affect the efficacy, distribution of transgene expression, and ultimately, the successful reversal of abnormal behaviors.