Hematopoietic stem-cell gene therapy is associated with restored white matter microvascular function in cerebral adrenoleukodystrophy.

Blood-brain barrier disruption marks the onset of cerebral adrenoleukodystrophy (CALD), a devastating cerebral demyelinating disease caused by loss of ABCD1 gene function. The underlying mechanism are not well understood, but evidence suggests that microvascular dysfunction is involved. We analyzed cerebral perfusion imaging in boys with CALD treated with autologous hematopoietic stem-cells transduced with the Lenti-D lentiviral vector that contains ABCD1 cDNA as part of a single group, open-label phase 2-3 safety and efficacy study (NCT01896102) and patients treated with allogeneic hematopoietic stem cell transplantation. We found widespread and sustained normalization of white matter permeability and microvascular flow. We demonstrate that ABCD1 functional bone marrow-derived cells can engraft in the cerebral vascular and perivascular space. Inverse correlation between gene dosage and lesion growth suggests that corrected cells contribute long-term to remodeling of brain microvascular function. Further studies are needed to explore the longevity of these effects.

Peripheral Nerve Injury Induces Changes in the Activity of Inhibitory Interneurons as Visualized in Transgenic GAD1-GCaMP6s Rats.

Peripheral nerve injury induces cortical remapping that can lead to sensory complications. There is evidence that inhibitory interneurons play a role in this process, but the exact mechanism remains unclear. Glutamate decarboxylase-1 (GAD1) is a protein expressed exclusively in inhibitory interneurons. Transgenic rats encoding GAD1-GCaMP were generated to visualize the activity in GAD1 neurons through genetically encoded calcium indicators (GCaMP6s) in the somatosensory cortex. Forepaw denervation was performed in adult rats, and fluorescent Ca2+ imaging on cortical slices was obtained. Local, intrahemispheric stimulation (cortical layers 2/3 and 5) induced a significantly higher fluorescence change of GAD1-expressing neurons, and a significantly higher number of neurons were responsive to stimulation in the denervated rats compared to control rats. However, remote, interhemispheric stimulation of the corpus callosum induced a significantly lower fluorescence change of GAD1-expressing neurons, and significantly fewer neurons were deemed responsive to stimulation within layer 5 in denervated rats compared to control rats. These results suggest that injury impacts interhemispheric communication, leading to an overall decrease in the activity of inhibitory interneurons in layer 5. Overall, our results provide direct evidence that inhibitory interneuron activity in the deprived S1 is altered after injury, a phenomenon likely to affect sensory processing.

High-resolution MEMRI characterizes laminar specific ascending and descending spinal cord pathways in rats.

BACKGROUND: The spinal cord is composed of nine distinct cellular laminae that currently can only be visualized by histological methods. Developing imaging methods that can visualize laminar architecture in-vivo is of significant interest. Manganese enhanced magnetic resonance imaging (MEMRI) yields valuable architectural and functional information about the brain and has great potential in characterizing neural pathways in the spinal cord. Here we apply MEMRI to visualize laminae architecture in the thoracic region of the spinal cord with ultra-high resolution. NEW METHOD: Manganese chloride (MnCl2) was delivered systemically and imaging of the lumbar and thoracic spinal cord levels was acquired in high field, 11.7 T MRI scanner, 48 h following MnCl2 administration. RESULTS: Here we demonstrate laminar specific signal enhancement in the spinal cord of rats administered with MnCl2 with 69 µm in-plane resolution. We also report reduced T1 values over time in MnCl2 groups across laminae IIX. COMPARISONS WITH EXISTING METHODS: This is the first study to demonstrate that MEMRI is capable of identifying spinal laminae at a high resolution of 69 µm in a living animal. This would enable the visualization of architecture and function of distinct regions with improved resolution, in healthy and diseased animal models. CONCLUSIONS: The regions with the largest T1 enhancements were observed to correspond to laminae that contain either high cell density or large motor neurons, making MEMRI an excellent tool for studying spinal cord architecture, physiology and function in different animal models.

Regulation of Electromagnetic Perceptive Gene Using Ferromagnetic Particles for the External Control of Calcium Ion Transport.

Developing synthetic biological devices to allow the noninvasive control of cell fate and function, in vivo can potentially revolutionize the field of regenerative medicine. To address this unmet need, we designed an artificial biological "switch" that consists of two parts: (1) the electromagnetic perceptive gene (EPG) and (2) magnetic particles. Our group has recently cloned the EPG from the Kryptopterus bicirrhis (glass catfish). The EPG gene encodes a putative membrane-associated protein that responds to electromagnetic fields (EMFs). This gene's primary mechanism of action is to raise the intracellular calcium levels or change in flux through EMF stimulation. Here, we developed a system for the remote regulation of [Ca2+]i (i.e., intracellular calcium ion concentration) using streptavidin-coated ferromagnetic particles (FMPs) under a magnetic field. The results demonstrated that the EPG-FMPs can be used as a molecular calcium switch to express target proteins. This technology has the potential for controlled gene expression, drug delivery, and drug developments.

Multimodal Evaluation of TMS - Induced Somatosensory Plasticity and Behavioral Recovery in Rats With Contusion Spinal Cord Injury.

Introduction: Spinal cord injury (SCI) causes partial or complete damage to sensory and motor pathways and induces immediate changes in cortical function. Current rehabilitative strategies do not address this early alteration, therefore impacting the degree of neuroplasticity and subsequent recovery. The following study aims to test if a non-invasive brain stimulation technique such as repetitive transcranial magnetic stimulation (rTMS) is effective in promoting plasticity and rehabilitation, and can be used as an early intervention strategy in a rat model of SCI. Methods: A contusion SCI was induced at segment T9 in adult rats. An rTMS coil was positioned over the brain to deliver high frequency stimulation. Behavior, motor and sensory functions were tested in three groups: SCI rats that received high-frequency (20 Hz) rTMS within 10 min post-injury (acute-TMS; n = 7); SCI rats that received TMS starting 2 weeks post-injury (chronic-TMS; n = 5), and SCI rats that received sham TMS (no-TMS, n = 5). Locomotion was evaluated by the Basso, Beattie, and Bresnahan (BBB) and gridwalk tests. Motor evoked potentials (MEP) were recorded from the forepaw across all groups to measure integrity of motor pathways. Functional MRI (fMRI) responses to contralateral tactile hindlimb stimulation were measured in an 11.7T horizontal bore small-animal scanner. Results: The acute-TMS group demonstrated the fastest improvements in locomotor performance in both the BBB and gridwalk tests compared to chronic and no-TMS groups. MEP responses from forepaw showed significantly greater difference in the inter-peak latency between acute-TMS and no-TMS groups, suggesting increases in motor function. Finally, the acute-TMS group showed increased fMRI-evoked responses to hindlimb stimulation over the right and left hindlimb (LHL) primary somatosensory representations (S1), respectively; the chronic-TMS group showed moderate sensory responses in comparison, and the no-TMS group exhibited the lowest sensory responses to both hindlimbs. Conclusion: The results suggest that rTMS therapy beginning in the acute phase after SCI promotes neuroplasticity and is an effective rehabilitative approach in a rat model of SCI.

Transcranial magnetic stimulation and environmental enrichment enhances cortical excitability and functional outcomes after traumatic brain injury.

BACKGROUND: Therapeutic strategies for traumatic brain injury (TBI) in the last three decades have failed to show significant benefit in large scale studies. Given the multitude of pathological mechanisms involved in TBI, strategies focusing on multimodality regimen have gained interest as promising future interventions. HYPOTHESIS: We hypothesized that combining noninvasive transcranial magnetic stimulation (TMS) with rehabilitative training in an environmental enrichment (EE) can facilitate post-TBI recovery in rats via cortical excitability and reorganization. METHODS: We subjected rats to controlled cortical impact, and then assigned them to one of four groups: 1. No treatments (TBI), 2. EE after injury (TBI + EE), 3. TMS for one week (TBI + TMS), and 4. TMS for one week combined with EE (TBI + TMS/EE). For TMS, a 10 Hz repetitive TMS protocol was used. RESULTS: At 7 days, TBI + TMS and TBI + TMS/EE groups had significantly increased primary somatosensory cortex local field potential (LFP) compared to TBI and TBI + EE groups (P < 0.05). Also, TBI + TMS/EE group had significantly improved performance on beam walk test compared to TBI group (P < 0.005). At 6 weeks, there was significantly higher response in TBI + TMS/EE group compared to TBI + TMS for somatosensory cortex LFP (P < 0.05), bicep motor evoked potentials (MEP) (P < 0.05), challenge ladder test performance (P < 0.01), and fMRI responses to tactile forepaw stimulation. CONCLUSIONS: We demonstrate here for the first time the mechanism by which combined therapy using TMS and EE after TBI leads to functional improvement, possibly via cortical excitability and reorganization.

Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields.

The Kryptopterus bicirrhis (glass catfish) is known to respond to electromagnetic fields (EMF). Here we tested its avoidance behavior in response to static and alternating magnetic fields stimulation. Using expression cloning we identified an electromagnetic perceptive gene (EPG) from the K. bicirrhis encoding a protein that responds to EMF. This EPG gene was cloned and expressed in mammalian cells, neuronal cultures and in rat's brain. Immunohistochemistry showed that the expression of EPG is confined to the mammalian cell membrane. Calcium imaging in mammalian cells and cultured neurons expressing EPG demonstrated that remote activation by EMF significantly increases intracellular calcium concentrations, indicative of cellular excitability. Moreover, wireless magnetic activation of EPG in rat motor cortex induced motor evoked responses of the contralateral forelimb in vivo. Here we report on the development of a new technology for remote, non-invasive modulation of cell function.

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl- from acidic organelles in amacrine cells.

γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligand-gated ion channels support the influx of Cl- However, the electrochemical gradient for Cl- across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl- levels support inhibition, whereas higher levels of cytosolic Cl- can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl- from acidic organelles and transiently elevates cytosolic Cl-, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl- We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage-clamp recordings of currents through GABA-gated Cl- channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl- To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl- from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl- from acidic organelles.NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl- from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl- levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission.

Nitric oxide releases Cl(-) from acidic organelles in retinal amacrine cells.

Determining the factors regulating cytosolic Cl(-) in neurons is fundamental to our understanding of the function of GABA- and glycinergic synapses. This is because the Cl(-) distribution across the postsynaptic plasma membrane determines the sign and strength of postsynaptic voltage responses. We have previously demonstrated that nitric oxide (NO) releases Cl(-) into the cytosol from an internal compartment in both retinal amacrine cells and hippocampal neurons. Furthermore, we have shown that the increase in cytosolic Cl(-) is dependent upon a decrease in cytosolic pH. Here, our goals were to confirm the compartmental nature of the internal Cl(-) store and to test the hypothesis that Cl(-) is being released from acidic organelles (AO) such as the Golgi, endosomes or lysosomes. To achieve this, we made whole cell voltage clamp recordings from cultured chick retinal amacrine cells and used GABA-gated currents to track changes in cytosolic Cl(-). Our results demonstrate that intact internal proton gradients are required for the NO-dependent release of internal Cl(-). Furthermore, we demonstrate that increasing the pH of AO leads to release of Cl(-) into the cytosol. Intriguingly, the elevation of organellar pH results in a reversal in the effects of NO. These results demonstrate that cytosolic Cl(-) is closely linked to the regulation and maintenance of organellar pH and provide evidence that acidic compartments are the target of NO.

Expression and localization of CLC chloride transport proteins in the avian retina.

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl(-)/H(+) antiporters, ClCs 3-7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.