Peripherally-derived LGI1-reactive monoclonal antibodies cause epileptic seizures in vivo.

One striking clinical hallmark in patients with autoantibodies to leucine-rich glioma inactivated 1 (LGI1) is the very frequent focal seizure semiologies, including faciobrachial dystonic seizures (FBDS), in addition to the amnesia. Polyclonal serum IgGs have successfully modelled the cognitive changes in vivo but not seizures. Hence, it remains unclear whether LGI1-autoantibodies are sufficient to cause seizures. We tested this with the molecularly precise monoclonal antibodies directed against LGI1 (LGI1-mAbs), derived from patient circulating B cells. These were directed towards both major domains of LGI1, LRR and EPTP and infused intracerebroventricularly over 7 days into juvenile male Wistar rats using osmotic pumps. Continuous wireless EEG was recorded from a depth electrode placed in hippocampal CA3 plus behavioural tests for memory and hyperexcitability were performed. Following infusion completion (Day 9), post-mortem brain slices were studied for antibody binding and effects on Kv1.1. The LGI1-mAbs bound most strongly in the hippocampal CA3 region and induced a significant reduction in Kv1.1 cluster number in this subfield. By comparison to control-Ab injected rats video-EEG analysis over 9 days revealed convulsive and non-convulsive seizure activity in rats infused with LGI1-mAbs, with a significant number of ictal events. Memory was not impaired in the novel object recognition test. Peripherally-derived human LGI1-mAbs infused into rodent CSF provide strong evidence of direct in vivo epileptogenesis with molecular correlations. These findings fulfill criteria for LGI1-antibodies in seizure causation.

Boosting neuregulin 1 type-III expression hastens SMA motor axon maturation.

Intercellular communication between axons and Schwann cells is critical for attaining the complex morphological steps necessary for axon maturation. In the early onset motor neuron disease spinal muscular atrophy (SMA), many motor axons are not ensheathed by Schwann cells nor grow sufficiently in radial diameter to become myelinated. These developmentally arrested motor axons are dysfunctional and vulnerable to rapid degeneration, limiting efficacy of current SMA therapeutics. We hypothesized that accelerating SMA motor axon maturation would improve their function and reduce disease features. A principle regulator of peripheral axon development is neuregulin 1 type III (NRG1-III). Expressed on axon surfaces, it interacts with Schwann cell receptors to mediate axon ensheathment and myelination. We examined NRG1 mRNA and protein expression levels in human and mouse SMA tissues and observed reduced expression in SMA spinal cord and in ventral, but not dorsal root axons. To determine the impact of neuronal NRG1-III overexpression on SMA motor axon development, we bred NRG1-III overexpressing mice to SMA∆7 mice. Neonatally, elevated NRG1-III expression increased SMA ventral root size as well as axon segregation, diameter, and myelination resulting in improved motor axon conduction velocities. NRG1-III was not able to prevent distal axonal degeneration nor improve axon electrophysiology, motor behavior, or survival of older mice. Together these findings demonstrate that early SMA motor axon developmental impairments can be ameliorated by a molecular strategy independent of SMN replacement providing hope for future SMA combinatorial therapeutic approaches.

p53-dependent c-Fos expression is a marker but not executor for motor neuron death in spinal muscular atrophy mouse models.

The activation of the p53 pathway has been associated with neuronal degeneration in different neurological disorders, including spinal muscular atrophy (SMA) where aberrant expression of p53 drives selective death of motor neurons destined to degenerate. Since direct p53 inhibition is an unsound therapeutic approach due carcinogenic effects, we investigated the expression of the cell death-associated p53 downstream targets c-fos, perp and fas in vulnerable motor neurons of SMA mice. Fluorescence in situ hybridization (FISH) of SMA motor neurons revealed c-fos RNA as a promising candidate. Accordingly, we identified p53-dependent nuclear upregulation of c-Fos protein in degenerating motor neurons from the severe SMNΔ7 and intermediate Smn2B/- SMA mouse models. Although motor neuron-specific c-fos genetic deletion in SMA mice did not improve motor neuron survival or motor behavior, p53-dependent c-Fos upregulation marks vulnerable motor neurons in different mouse models. Thus, nuclear c-Fos accumulation may serve as a readout for therapeutic approaches targeting neuronal death in SMA and possibly other p53-dependent neurodegenerative diseases.

SMN controls neuromuscular junction integrity through U7 snRNP.

The neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP)-which functions in the 3'-end processing of replication-dependent histone mRNAs-is required for NMJ integrity. Co-expression of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice-including NMJ denervation, decreased synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable muscles of SMA mice. These findings reveal a direct contribution of U7 snRNP dysfunction to neuromuscular pathology in SMA and suggest a role for histone gene regulation in maintaining functional synaptic connections between motor neurons and muscles.

Laser microscopy acquisition and analysis of premotor synapses in the murine spinal cord.

Loss of synapses on spinal motor neurons is a major feature of several neurodegenerative diseases; however, analyzing these premotor synapses is challenging because of their small size and high density. This protocol describes confocal and Stimulated Emission Depletion (STED) imaging of murine spinal premotor synapses and their segment-specific quantification by confocal microscopy. We detail the preparation of spinal cord segments, followed by image acquisition and analysis. This protocol enables in-depth analysis of pathological changes in spinal premotor synapses during neurodegeneration. For complete details on the use and execution of this protocol, please refer to Buettner et al. (2021).

Communicating With Diverse Patients About Participating in a Biobank: A Randomized Multisite Study Comparing Electronic and Face-to-Face Informed Consent Processes.

Some individuals' understanding of informed consent (IC) information may improve with electronic delivery, but others may benefit from face-to-face (F2F). This randomized, multisite study explores how individuals from diverse backgrounds understand electronic IC documents versus F2F, their confidence in understanding, and enrollment in research. A total of 501 patients at two U.S. biobanks with diverse populations participated. There were no overall differences between electronic and F2F understanding, but F2F predicted higher confidence in understanding and enrollment. Ethnicity and a higher educational level predicted higher understanding and confidence. Study findings suggest that electronic consent may lead to better understanding for non-Hispanic patients of higher socioeconomic status. F2F processes may lead to better understanding and higher enrollment of patients from Hispanic and lower socioeconomic levels. Researchers should carefully consider how they implement electronic IC processes and whether to maintain an F2F process to better address the needs and limitations of some populations.

Central synaptopathy is the most conserved feature of motor circuit pathology across spinal muscular atrophy mouse models.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by reduced survival motor neuron (SMN) protein. Recently, SMN dysfunction has been linked to individual aspects of motor circuit pathology in a severe SMA mouse model. To determine whether these disease mechanisms are conserved, we directly compared the motor circuit pathology of three SMA mouse models. The severe SMNΔ7 model exhibits vast motor circuit defects, including degeneration of motor neurons, spinal excitatory synapses, and neuromuscular junctions (NMJs). In contrast, the Taiwanese model shows very mild motor neuron pathology, but early central synaptic loss. In the intermediate Smn 2B/- model, strong pathology of central excitatory synapses and NMJs precedes the late onset of p53-dependent motor neuron death. These pathological events correlate with SMN-dependent splicing dysregulation of specific mRNAs. Our study provides a knowledge base for properly tailoring future studies and identifies central excitatory synaptopathy as a key feature of motor circuit pathology in SMA.

Impaired prenatal motor axon development necessitates early therapeutic intervention in severe SMA.

Gene replacement and pre-mRNA splicing modifier therapies represent breakthrough gene targeting treatments for the neuromuscular disease spinal muscular atrophy (SMA), but mechanisms underlying variable efficacy of treatment are incompletely understood. Our examination of severe infantile onset human SMA tissues obtained at expedited autopsy revealed persistence of developmentally immature motor neuron axons, many of which are actively degenerating. We identified similar features in a mouse model of severe SMA, in which impaired radial growth and Schwann cell ensheathment of motor axons began during embryogenesis and resulted in reduced acquisition of myelinated axons that impeded motor axon function neonatally. Axons that failed to ensheath degenerated rapidly postnatally, specifically releasing neurofilament light chain protein into the blood. Genetic restoration of survival motor neuron protein (SMN) expression in mouse motor neurons, but not in Schwann cells or muscle, improved SMA motor axon development and maintenance. Treatment with small-molecule SMN2 splice modifiers beginning immediately after birth in mice increased radial growth of the already myelinated axons, but in utero treatment was required to restore axonal growth and associated maturation, prevent subsequent neonatal axon degeneration, and enhance motor axon function. Together, these data reveal a cellular basis for the fulminant neonatal worsening of patients with infantile onset SMA and identify a temporal window for more effective treatment. These findings suggest that minimizing treatment delay is critical to achieve optimal therapeutic efficacy.

Chronic Pharmacological Increase of Neuronal Activity Improves Sensory-Motor Dysfunction in Spinal Muscular Atrophy Mice.

Dysfunction of neuronal circuits is an important determinant of neurodegenerative diseases. Synaptic dysfunction, death, and intrinsic activity of neurons are thought to contribute to the demise of normal behavior in the disease state. However, the interplay between these major pathogenic events during disease progression is poorly understood. Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the ubiquitously expressed protein SMN and is characterized by motor neuron death, skeletal muscle atrophy, as well as dysfunction and loss of both central and peripheral excitatory synapses. These disease hallmarks result in an overall reduction of neuronal activity in the spinal sensory-motor circuit. Here, we show that increasing neuronal activity by chronic treatment with the FDA-approved potassium channel blocker 4-aminopyridine (4-AP) improves motor behavior in both sexes of a severe mouse model of SMA. 4-AP restores neurotransmission and number of proprioceptive synapses and neuromuscular junctions (NMJs), while having no effects on motor neuron death. In addition, 4-AP treatment with pharmacological inhibition of p53-dependent motor neuron death results in additive effects, leading to full correction of sensory-motor circuit pathology and enhanced phenotypic benefit in SMA mice. Our in vivo study reveals that 4-AP-induced increase of neuronal activity restores synaptic connectivity and function in the sensory-motor circuit to improve the SMA motor phenotype.SIGNIFICANCE STATEMENT Spinal muscular atrophy (SMA) is a neurodegenerative disease, characterized by synaptic loss, motor neuron death, and reduced neuronal activity in spinal sensory-motor circuits. However, whether these are parallel or dependent events is unclear. We show here that long-term increase of neuronal activity by the FDA-approved drug 4-aminopyridine (4-AP) rescues the number and function of central and peripheral synapses in a SMA mouse model, resulting in an improvement of the sensory-motor circuit and motor behavior. Combinatorial treatment of pharmacological inhibition of p53, which is responsible for motor neuron death and 4-AP, results in additive beneficial effects on the sensory-motor circuit in SMA. Thus, neuronal activity restores synaptic connections and improves significantly the severe SMA phenotype.

Stasimon Contributes to the Loss of Sensory Synapses and Motor Neuron Death in a Mouse Model of Spinal Muscular Atrophy.

Reduced expression of the survival motor neuron (SMN) protein causes the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that adeno-associated virus serotype 9 (AAV9)-mediated delivery of Stasimon-a gene encoding an endoplasmic reticulum (ER)-resident transmembrane protein regulated by SMN-improves motor function in a mouse model of SMA through multiple mechanisms. In proprioceptive neurons, Stasimon overexpression prevents the loss of afferent synapses on motor neurons and enhances sensory-motor neurotransmission. In motor neurons, Stasimon suppresses neurodegeneration by reducing phosphorylation of the tumor suppressor p53. Moreover, Stasimon deficiency converges on SMA-related mechanisms of p53 upregulation to induce phosphorylation of p53 through activation of p38 mitogen-activated protein kinase (MAPK), and pharmacological inhibition of this kinase prevents motor neuron death in SMA mice. These findings identify Stasimon dysfunction induced by SMN deficiency as an upstream driver of distinct cellular cascades that lead to synaptic loss and motor neuron degeneration, revealing a dual contribution of Stasimon to motor circuit pathology in SMA.

Dysregulation of Mdm2 and Mdm4 alternative splicing underlies motor neuron death in spinal muscular atrophy.

Ubiquitous deficiency in the survival motor neuron (SMN) protein causes death of motor neurons-a hallmark of the neurodegenerative disease spinal muscular atrophy (SMA)-through poorly understood mechanisms. Here, we show that the function of SMN in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) regulates alternative splicing of Mdm2 and Mdm4, two nonredundant repressors of p53. Decreased inclusion of critical Mdm2 and Mdm4 exons is most prominent in SMA motor neurons and correlates with both snRNP reduction and p53 activation in vivo. Importantly, increased skipping of Mdm2 and Mdm4 exons regulated by SMN is necessary and sufficient to synergistically elicit robust p53 activation in wild-type mice. Conversely, restoration of full-length Mdm2 and Mdm4 suppresses p53 induction and motor neuron degeneration in SMA mice. These findings reveal that loss of SMN-dependent regulation of Mdm2 and Mdm4 alternative splicing underlies p53-mediated death of motor neurons in SMA, establishing a causal link between snRNP dysfunction and neurodegeneration.

Perspectives on Electronic Informed Consent From Patients Underrepresented in Research in the United States: A Focus Group Study.

Digital informed consent may better inform individuals about health research and increase participation. In the United States and elsewhere, minorities and rural populations are underrepresented in health research and may benefit from well-designed electronic informed consent (eIC). Seven focus groups were conducted with 50 Caucasian, African American, and rural patients in the United States. Participants were asked their preferences for a paper versus electronic informed consent document. Participants found the e-version easier to use, more interesting, and better for understanding. Minority participants emphasized limited access, computer literacy, and trust barriers to eIC. Rural participants were concerned about accessibility, connectivity, privacy, and confidentiality. People see value in electronic consenting. Researchers should consider barriers to eIC among underrepresented populations before recruitment.

Converging Mechanisms of p53 Activation Drive Motor Neuron Degeneration in Spinal Muscular Atrophy.

The hallmark of spinal muscular atrophy (SMA), an inherited disease caused by ubiquitous deficiency in the SMN protein, is the selective degeneration of subsets of spinal motor neurons. Here, we show that cell-autonomous activation of p53 occurs in vulnerable but not resistant motor neurons of SMA mice at pre-symptomatic stages. Moreover, pharmacological or genetic inhibition of p53 prevents motor neuron death, demonstrating that induction of p53 signaling drives neurodegeneration. At late disease stages, however, nuclear accumulation of p53 extends to resistant motor neurons and spinal interneurons but is not associated with cell death. Importantly, we identify phosphorylation of serine 18 as a specific post-translational modification of p53 that exclusively marks vulnerable SMA motor neurons and provide evidence that amino-terminal phosphorylation of p53 is required for the neurodegenerative process. Our findings indicate that distinct events induced by SMN deficiency converge on p53 to trigger selective death of vulnerable SMA motor neurons.

Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy.

Behavioral deficits in neurodegenerative diseases are often attributed to the selective dysfunction of vulnerable neurons via cell-autonomous mechanisms. Although vulnerable neurons are embedded in neuronal circuits, the contributions of their synaptic partners to disease process are largely unknown. Here we show that, in a mouse model of spinal muscular atrophy (SMA), a reduction in proprioceptive synaptic drive leads to motor neuron dysfunction and motor behavior impairments. In SMA mice or after the blockade of proprioceptive synaptic transmission, we observed a decrease in the motor neuron firing that could be explained by the reduction in the expression of the potassium channel Kv2.1 at the surface of motor neurons. Chronically increasing neuronal activity pharmacologically in vivo led to a normalization of Kv2.1 expression and an improvement in motor function. Our results demonstrate a key role of excitatory synaptic drive in shaping the function of motor neurons during development and the contribution of its disruption to a neurodegenerative disease.

A Stem Cell Model of the Motor Circuit Uncouples Motor Neuron Death from Hyperexcitability Induced by SMN Deficiency.

In spinal muscular atrophy, a neurodegenerative disease caused by ubiquitous deficiency in the survival motor neuron (SMN) protein, sensory-motor synaptic dysfunction and increased excitability precede motor neuron (MN) loss. Whether central synaptic dysfunction and MN hyperexcitability are cell-autonomous events or they contribute to MN death is unknown. We addressed these issues using a stem-cell-based model of the motor circuit consisting of MNs and both excitatory and inhibitory interneurons (INs) in which SMN protein levels are selectively depleted. We show that SMN deficiency induces selective MN death through cell-autonomous mechanisms, while hyperexcitability is a non-cell-autonomous response of MNs to defects in pre-motor INs, leading to loss of glutamatergic synapses and reduced excitation. Findings from our in vitro model suggest that dysfunction and loss of MNs result from differential effects of SMN deficiency in distinct neurons of the motor circuit and that hyperexcitability does not trigger MN death.

Interactive multimedia consent for biobanking: a randomized trial.

PURPOSE: The potential of interactive multimedia to improve biobank informed consent has yet to be investigated. The aim of this study was to test the separate effectiveness of interactivity and multimedia at improving participant understanding and confidence in understanding of informed consent compared with a standard, face-to-face (F2F) biobank consent process. METHODS: A 2 (face-to-face versus multimedia) × 2 (standard versus enhanced interactivity) experimental design was used with 200 patients randomly assigned to receive informed consent. All patients received the same information provided in the biobank's nine-page consent document. RESULTS: Interactivity (F(1,196) = 7.56, P = 0.007, partial η(2) = 0.037) and media (F(1,196) = 4.27, P = 0.04, partial η(2) = 0.021) independently improved participants' understanding of the biobank consent. Interactivity (F(1,196) = 6.793, P = 0.01, partial η(2) = 0.033), but not media (F(1,196) = 0.455, not significant), resulted in increased participant confidence in their understanding of the biobank's consent materials. Patients took more time to complete the multimedia condition (mean = 18.2 min) than the face-to-face condition (mean = 12.6 min). CONCLUSION: This study demonstrated that interactivity and multimedia each can be effective at promoting an individual's understanding and confidence in their understanding of a biobank consent, albeit with additional time investment. Researchers should not assume that multimedia is inherently interactive, but rather should separate the two constructs when studying electronic consent.

Activity Regulates the Incidence of Heteronymous Sensory-Motor Connections.

The construction of spinal sensory-motor circuits involves the selection of appropriate synaptic partners and the allocation of precise synaptic input densities. Many aspects of spinal sensory-motor selectivity appear to be preserved when peripheral sensory activation is blocked, which has led to a view that sensory-motor circuits are assembled in an activity-independent manner. Yet it remains unclear whether activity-dependent refinement has a role in the establishment of connections between sensory afferents and those motor pools that have synergistic biomechanical functions. We show here that genetically abolishing central sensory-motor neurotransmission leads to a selective enhancement in the number and density of such "heteronymous" connections, whereas other aspects of sensory-motor connectivity are preserved. Spike-timing-dependent synaptic refinement represents one possible mechanism for the changes in connectivity observed after activity blockade. Our findings therefore reveal that sensory activity does have a limited and selective role in the establishment of patterned monosynaptic sensory-motor connections.

Dysregulated IGFBP5 expression causes axon degeneration and motoneuron loss in diabetic neuropathy.

Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes. The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor (IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.

The PDZ-domain protein Whirlin facilitates mechanosensory signaling in mammalian proprioceptors.

Mechanoreception is an essential feature of many sensory modalities. Nevertheless, the mechanisms that govern the conversion of a mechanical force to distinct patterns of action potentials remain poorly understood. Proprioceptive mechanoreceptors reside in skeletal muscle and inform the nervous system of the position of body and limbs in space. We show here that Whirlin/Deafness autosomal recessive 31 (DFNB31), a PDZ-scaffold protein involved in vestibular and auditory hair cell transduction, is also expressed by proprioceptive sensory neurons (pSNs) in dorsal root ganglia in mice. Whirlin localizes to the peripheral sensory endings of pSNs and facilitates pSN afferent firing in response to muscle stretch. The requirement of Whirlin in both proprioceptors and hair cells suggests that accessory mechanosensory signaling molecules define common features of mechanoreceptive processing across sensory systems.