Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Effects of Perioperative Administration of Acetaminophen on Postoperative Shivering: A Randomized, Triple-Blind, Placebo-Controlled Trial.

BACKGROUND: In this randomized, triple-blind, placebo-controlled trial, we tested the hypothesis that perioperative acetaminophen administration has a prophylactic effect on postoperative shivering. METHODS: Forty-five women scheduled for gynecological laparotomy were randomized to either the acetaminophen or the placebo groups. After induction of general anesthesia, the test drug (acetaminophen 15 mg/kg) or placebo (0.9% saline) was intravenously administered over 15 minutes. The primary outcome measure was the incidence of severe postoperative shivering (ie, shivering score >2) in the postanesthesia care unit, where patients stayed for 30 minutes after their emergence from anesthesia. For the secondary outcomes, core body temperature (BT) was recorded at the forehead just before anesthesia induction (time 0 [T0]), at the start of surgery (time 1 [T1]), at the end of surgery (time 2 [T2]), at the initiation of postoperative observation in the postanesthesia care unit (time 3 [T3]), and 30 minutes after T3 (time 4 [T4]). At 1 hour after T4 (ie, time 5 [T5]), the BT was recorded from the axilla (BTA). Primary outcome was analyzed using a χ test. BT recorded at the forehead (BTF) and BTA were analyzed using a 2-way repeated-measures analysis of variance (ANOVA) and a 2-sample t test, respectively. For all comparisons, a P value <.05 was considered statistically significant. RESULTS: The study duration was 2 years. Of the 45 patients initially enrolled, 8 patients were excluded. The acetaminophen and placebo groups included 18 and 19 patients, respectively. The incidence of severe postoperative shivering in the postanesthesia care unit was significantly lower in the acetaminophen group (22.2%) than in the placebo group (73.7%) (relative risk, 0.302; 95% confidence interval, 0.122-0.746; P = .005). Two-way repeated-measures ANOVA showed a significant effect of time (F4,140 = 54.8; P < .001) and a significant time by treatment interaction (F4,140 = 9.61; P < .001) but did not show a main effect of the treatment (F1,35 = 1.83; P = .185) in BTF. Moreover, BTA at T5 was significantly lower in the acetaminophen group (mean [standard deviation {SD}], 37.2°C [0.48°C]) than in the placebo group (37.9°C [0.63°C]; P < .001). CONCLUSIONS: Our findings in patients undergoing gynecological laparotomy suggest that perioperative acetaminophen administration can prevent postoperative severe shivering. This prophylactic effect might be due to suppressing the postoperative increase in the BT set point, rather than lowering the threshold for shivering, as observed with clonidine.

The Effectiveness of Applying Soft Tissue Bonding Adhesive Composed of 2-Ethyl Cyanoacrylate to Epidural Catheter Fixations Using Film Dressings: An Open-Label, Randomized, Parallel-Group Comparative Study.

BACKGROUND: Insufficient fixation of an epidural catheter may result in migration of the catheter and eventual catheter failure. However, the best fixation method remains to be established. Aron Alpha A (2-ethyl cyanoacrylate) adhesive is approved for clinical use and can be used for surgical adhesion to both skin and blood vessels. We hypothesized that the addition of Aron Alpha A adhesive to film dressing would result in consistent and dependable catheter fixation. METHODS: In this study, 58 women who were scheduled for cesarean delivery under spinal and epidural anesthesia were recruited. Patients were randomly assigned to a control or treatment group. In the control group, the catheter was fixed solely by film dressing. In the treatment group, a small amount of Aron Alpha A was applied at 2 sites along the catheter. The fixation area was then covered by film dressing. The catheter insertion length was recorded after fixation (T0), immediately postoperatively (T1), on postoperative day 1 (T2), and when the catheter was removed (T3). The change in insertion length from T0 to T3 between the 2 groups was the primary outcome measure. The incidence of catheter failure was also recorded. For all comparisons, P < .05 was considered statistically significant. RESULTS: Initially, 58 women were enrolled; however, 3 patients were excluded. From the remaining 55 patients, 27 and 28 were assigned to the control and treatment groups, respectively, and were evaluated. The change in insertion length from T0 to T3 was significantly more in the control group compared with the treatment group (-1.9 ± 2.2 vs 0 ± 0 cm, respectively; P < .001). In the control group, 11 catheters (41%) failed; in the treatment group, all catheters provided effective analgesia throughout the study (P < .001). CONCLUSIONS: Epidural catheter fixation using film dressing combined with 2-ethyl cyanoacrylate adhesive application at 2 sites along the catheter resulted in secure fixation in patients receiving postoperative epidural analgesia for cesarean delivery.

Usefulness of stroke volume variation to assess blood volume during blood removal for autologous blood transfusion in pediatric patients.

BACKGROUND: Dynamic variables based on the heart-lung interaction induced by positive pressure ventilation have not been shown to be useful in assessing cardiac preload in pediatric patients. OBJECTIVE: To evaluate whether stroke volume variation (SVV) obtained from the FloTrac/Vigileo(TM) monitoring system can reflect a change in blood volume during the blood removal and fluid replacement protocol in acute normovolemic hemodilution (ANH). METHODS: Sixteen pediatric patients scheduled for elective cranioplasty were recruited. In the ANH protocol, 10 ml · kg(-1) blood removal and fluid replacement were performed. SVV, heart rate, mean blood pressure, and femoral venous pressure were recorded. Differences at four time points (T0: baseline, T1: 5 ml · kg(-1) blood loss, T2: 10 ml · kg(-1) blood loss, and T3: after fluid replacement) during ANH were compared. The blood volume (EBV) was estimated as 70 ml · kg(-1) at T0 and decreased to 60 ml · kg(-1) at T2. RESULTS: Of the 16 patients, four were excluded and 12 were analyzed. Significant differences in all of the parameters were observed between each time point. The SVV significantly increased after the blood removal and decreased after the fluid replacement (P < 0.01, Bonferroni adjustment). In addition, the increases in SVV during the blood removal, T0-T1 and T0-T2, were 70% ± 40% and 159% ± 91%, respectively. SVV showed a significant correlation with EBV during the blood removal in ANH (rs = -0.68, 95% confidence interval -0.73 to -0.63, P < 0.001). CONCLUSION: Stroke volume variation obtained from the FloTrac/Vigileo(TM) monitoring system revealed a strong correlation with EBV during ANH without surgical stimulation. The usefulness of this device as an indicator of cardiac preload under hypovolemic or normovolemic conditions in children during surgery remains to be determined.

The three-step method for ultrasound-guided pediatric internal jugular venous catheterization: a clinical trial.

Ultrasound guidance may be a valuable adjunct for pediatric internal jugular vein catheterization. We previously reported a long-axis in plane technique, called the "three-step method", resulting in high success and a low complication rate by novice operators in adult patients. This is the first report of ultrasound-guided internal jugular vein catheterization (US-IJV) using the three-step method in pediatric patients. Fourteen junior residents underwent simulation training, and then participated in a clinical trial. They performed US-IJV in 14 pediatric patients with congenital heart disease before undergoing cardiac surgery under supervision of an experienced clinician. The overall success rate was 93 %, and all catheterizations were performed within two venipunctures. There were no complications associated with the procedure. The three-step method may facilitate pediatric US-IJV even by a novice operator during their first experience.

[A Quantitative Verification for Operability of Three PCA Devices Attached to the Disposable Infusion Pumps].

BACKGROUND: In this study using 3 different PCA devices (Baxter infuser LVBB +PCM 2 ml: Pump B, Coopdech Balloonjector +PCA 3 ml: Pump C, Rakuraku fuser +PCA 3 ml: Pump S), we investigated how easily PCA devices could be handled. METHODS: In this study with 42 volunteers (14 elders and 28 nurses), we compared 3 PCA ejection volume and ejection rate among three PCA devices. PCA ejection rate was defined as the ratio of actual ejection volume to the maximum ejection volume (MEV) of each PCA device. RESULTS: Although not only elders but also nurses failed to produce accurate PCA ejection volume in the Pump B, Pump S could provide the MEV even by elders. In the Pump C, approximately 80% of MEV could be achieved by nurses, but 60% of MEV by elders (P < 0.05). CONCLUSIONS: Our data suggested that accuracy of PCA ejection volume might be dependent on PCA device.

[Anesthetic management of a patient with stiff-person syndrome undergoing thymectomy].

Stiff-person syndrome is an uncommon disease characterized by muscular rigidity and painful spasms in the axial and limb muscles. We report a 58-year-old woman with stiff-person syndrome undergoing thymectomy under general anesthesia. Before surgery, her medications were 25 mg of diazepam, 2 mg of clonazepam, and 15 mg of gabapentin per day. After epidural catheterization for the postoperative analgesia, general anesthesia was induced and maintained with continuous remifentanil infusion and propofol with target controlled infusion. With train-of-four ratio (TOFR) monitoring by stimulating the ulnar nerve, her trachea was intubated after 0.6mg x kg(-1) of rocuronium intravenous administration. Since then, additional rocuronium was not given for 4 hours. After surgery, she was fully awake and TOFR recovered to 100%, but tidal volume was too low to remove the tracheal tube, and mechanical ventilation was continued in ICU. On the next day, the tracheal tube was removed, and she was discharged from ICU. Because anesthetics may delay the recovery of respiratory function in a patient with stiff-person syndrome, careful assessment of respiratory function is needed at the emergence from general anesthesia.

Monitoring of Post-Brain Injuries By Measuring Plasma Levels of Neuron-Derived Extracellular Vesicles.

Background: Extracellular vesicles (EV) released from neurons into the blood can reflect the state of nervous tissue. Measurement of neuron derived EV (NDE) may serve as an indicator of brain injury. Methods: A sandwich immunoassay was established to measure plasma NDE using anti-neuron CD171 and anti-EV CD9 ([CD171 + CD9+]). Plasma samples were obtained from commercial sources, cross-country (n = 9), football (n = 22), soccer (n = 19), and rugby (n = 18) athletes over time. Plasma was also collected from patients undergoing total aortic arch replacement (TAR) with selective cerebral perfusion during cardiopulmonary bypass before and after surgery (n = 36). Results: The specificity, linearity, and reproducibility of NDE assay (measurement of [CD171 + CD9+]) were confirmed. By scanning electron microscopy and nanoparticle tracking, spherical vesicles ranging in size from 150 to 300 nm were confirmed. Plasma levels of NDE were widely spread over 2 to 3 logs in different individuals with a significant age-dependent decrease. However, NDE were very stable in each individual within a ± 50% change over time (cross-country, football, soccer), whereas rugby players were more variable over 4 years. In patients undergoing TAR, NDE increased rapidly in days post-surgery and were significantly (P = .0004) higher in those developing postoperative delirium (POD) (n = 13) than non-delirium patients (n = 23). Conclusions: The blood test to determine plasma levels of NDE was established by a sandwich immunoassay using 2 antibodies against neuron (CD171) and exosomes (CD9). NDE levels varied widely in different individuals and decreased with age, indicating that NDE levels should be considered as a normalizer of NDE biomarker studies. However, NDE levels were stable over time in each individual, and increased rapidly after TAR with greater increases associated with patients developing POD. This assay may serve as a surrogate for evaluating and monitoring brain injuries.

TERT promoter C228T mutation in neural progenitors confers growth advantage following telomere shortening in vivo.

BACKGROUND: Heterozygous TERT (telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis. However, no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. METHOD: We established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. The impact of the mutation was evaluated through the in vivo passage and in vitro experiment and analysis. RESULTS: Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with the TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type NPCs at initial in vivo passage presumably due to relatively long telomeres. However, the mutation recruited GA-Binding Protein and engendered low-level TERT expression resulting in enhanced tumorigenesis and maintenance of short telomeres upon secondary passage as observed in human GBM. These results provide the first insights regarding increased tumorigenesis upon introducing a TPM compared to isogenic controls without TPMs. CONCLUSION: Our novel GBM models presented the growth advantage of heterozygous TPMs for the first time in the context of GBM driver mutations relative to isogenic controls, thereby allowing for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice.

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1.

Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia. The sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.

Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats.

Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Subpial AAV Delivery for Spinal Parenchymal Gene Regulation in Adult Mammals.

The use of adeno-associated virus (AAV) vectors has become an attractive method for treatment of a variety of neurodegenerative disorders by permitting targeted gene upregulation or silencing in the CNS. Systemic and intrathecal infusion, while preferable routes of vector delivery, have shown encouraging but variable efficacy due to the poor permeability of AAV into spinal cord and brain parenchyma in adult mammals. Recently we have developed a novel and relatively noninvasive technique of spinal subpial vector delivery. This technique confers widespread transgene expression throughout the spinal parenchyma, including both white and gray matter. We have demonstrated that this technique can be performed safely, with a high level of accuracy, and is effective in both small (mouse or rat) and large preclinical (adult pig or nonhuman primate) animal models. In this chapter we provide a comprehensive description of the subpial vector delivery technique in adult rodents (mouse and rat) and large preclinical animals (adult pig and nonhuman primates).

The influence of preoperative mental health on clinical outcomes after laminectomy in patients with lumbar spinal stenosis.

OBJECTIVE: The influence of preoperative mental health on health-related quality of life (HRQOL) in patients with lumbar spinal stenosis (LSS) remains unclear. This study aims to investigate the influence of preoperative mental health HRQOL after laminectomy in patients with LSS. PATIENTS AND METHODS: We retrospectively reviewed 122 patients who had lumbar spinous process splitting laminectomy (LSPSL) for LSS. We assessed clinical information; Japanese Orthopedic Association (JOA) score; numerical rating scale (NRS) for low back pain, for leg pain, and for leg numbness; Zurich Claudication Questionnaire (ZCQ); JOA Back Pain Evaluation Questionnaire (JOABPEQ); Roland-Morris Disability Questionnaire (RMDQ); and Short Form 8 (SF-8) as patient reported outcomes. Patients were divided into two groups (Group L ≤ 36.2 points and Group NL > 36.2 points) based on the results of the preoperative mental health (MH) score in SF-8 to examine the influence of MH in LSS. We compared the HRQOL between the two groups postoperatively. RESULTS: The JOA score, NRS, and ZCQ score significantly improved after surgery. HRQOL outcomes including JOABPEQ, RMDQ, and SF-8 showed that the LSPSL improved not only the physical but also the mental function in patients with LSS. All HRQOL outcomes in Group L exhibited significantly worse scores preoperatively; however, no significant differences between two groups were found postoperatively. CONCLUSIONS: LSPSL greatly reduced low back pain, leg pain, and leg numbness. LSPSL resulted in a significant improvement based on HRQOL questionnaires even in patients with preoperative depressive mood. Not only the physical status but also the mental health may improve after LSPSL even in patients with LSS with a depressive mood preoperatively.

A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors.

BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells.

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation.

A First-in-Human, Phase I Study of Neural Stem Cell Transplantation for Chronic Spinal Cord Injury.

We tested the feasibility and safety of human-spinal-cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury (SCI). In this clinical trial, four subjects with T2-T12 SCI received treatment consisting of removal of spinal instrumentation, laminectomy, and durotomy, followed by six midline bilateral stereotactic injections of NSI-566 cells. All subjects tolerated the procedure well and there have been no serious adverse events to date (18-27 months post-grafting). In two subjects, one to two levels of neurological improvement were detected using ISNCSCI motor and sensory scores. Our results support the safety of NSI-566 transplantation into the SCI site and early signs of potential efficacy in three of the subjects warrant further exploration of NSI-566 cells in dose escalation studies. Despite these encouraging secondary data, we emphasize that this safety trial lacks statistical power or a control group needed to evaluate functional changes resulting from cell grafting.

ALS/FTD-Linked Mutation in FUS Suppresses Intra-axonal Protein Synthesis and Drives Disease Without Nuclear Loss-of-Function of FUS.

Through the generation of humanized FUS mice expressing full-length human FUS, we identify that when expressed at near endogenous murine FUS levels, both wild-type and ALS-causing and frontotemporal dementia (FTD)-causing mutations complement the essential function(s) of murine FUS. Replacement of murine FUS with mutant, but not wild-type, human FUS causes stress-mediated induction of chaperones, decreased expression of ion channels and transporters essential for synaptic function, and reduced synaptic activity without loss of nuclear FUS or its cytoplasmic aggregation. Most strikingly, accumulation of mutant human FUS is shown to activate an integrated stress response and to inhibit local, intra-axonal protein synthesis in hippocampal neurons and sciatic nerves. Collectively, our evidence demonstrates that human ALS/FTD-linked mutations in FUS induce a gain of toxicity that includes stress-mediated suppression in intra-axonal translation, synaptic dysfunction, and progressive age-dependent motor and cognitive disease without cytoplasmic aggregation, altered nuclear localization, or aberrant splicing of FUS-bound pre-mRNAs. VIDEO ABSTRACT.