Cx43 hemichannels contribute to astrocyte-mediated toxicity in sporadic and familial ALS.

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here, we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In addition, Cx43 expression is up-regulated in human postmortem tissue and cerebrospinal fluid from ALS patients. Using human induced pluripotent stem cell­derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we establish that Cx43 is up-regulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We also demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes using GAP 19, a mimetic peptide blocker, and tonabersat, a clinically tested small molecule, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Extending the in vitro application of tonabersat with chronic administration to SOD1G93A mice results in MN protection with a reduction in reactive astrocytosis and microgliosis. Taking these data together, our studies identify Cx43 hemichannels as conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

COVID-19 and the peripheral nervous system. A 2-year review from the pandemic to the vaccine era.

Increasing literature has linked COVID-19 to peripheral nervous system (PNS) diseases. In addition, as we move from the pandemic to the vaccination era, literature interest is shifting towards the potential association between COVID-19 vaccines and PNS manifestations. We reviewed published literature on COVID-19, COVID-19 vaccines and PNS manifestations between 1 January 2020 and 1 December 2021. For Guillain-Barré syndrome (GBS), isolated cranial neuropathy (ICN) and myositis associated with COVID-19, the demographic, clinical, laboratory, electrophysiological and imaging features were included in a narrative synthesis. We identified 169 studies on COVID-19-associated complications, including 63 papers (92 patients) on GBS, 29 papers (37 patients) on ICN and 11 papers (18 patients) on myositis. Additional clinical phenotypes included chronic inflammatory demyelinating polyneuropathy, vasculitic neuropathies, neuralgic amyotrophy, critical care-related complications, and myasthenia gravis. PNS complications secondary to COVID-19 vaccines have been reported during randomized clinical trials, in real-world case reports, and during large-scale surveillance programs. These mainly include cases of GBS, Bell's palsy, and cases of neuralgic amyotrophy. Based on our extensive review of the literature, any conclusion about a pathophysiological correlation between COVID-19 and PNS disorders remains premature, and solely supported by their temporal association, while epidemiological and pathological data are insufficient. The occurrence of PNS complications after COVID-19 vaccines seems limited to a possible higher risk of facial nerve palsy and GBS, to a degree that widespread access to the ongoing vaccination campaign should not be discouraged, while awaiting for more definitive data from large-scale surveillance studies.

TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function.

OBJECTIVE: Distinct dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) typically cause nonoverlapping diseases of either the neuromuscular or skeletal systems. However, accumulating evidence suggests that some patients develop mixed phenotypes that include elements of both neuromuscular and skeletal disease. We sought to define the genetic and clinical features of these patients. METHODS: We report a 2-year-old with a novel R616G mutation in TRPV4 with a severe neuropathy phenotype and bilateral vocal cord paralysis. Interestingly, a different substitution at the same residue, R616Q, has been reported in families with isolated skeletal dysplasia. To gain insight into clinical features and potential genetic determinants of mixed phenotypes, we perform in-depth analysis of previously reported patients along with functional and structural assessment of selected mutations. RESULTS: We describe a wide range of neuromuscular and skeletal manifestations and highlight specific mutations that are more frequently associated with overlap syndromes. We find that mutations causing severe, mixed phenotypes have an earlier age of onset and result in more marked elevations of intracellular calcium, increased cytotoxicity, and reduced sensitivity to TRPV4 antagonism. Structural analysis of the two mutations with the most dramatic gain of ion channel function suggests that these mutants likely cause constitutive channel opening through disruption of the TRPV4 S5 transmembrane domain. INTERPRETATION: These findings demonstrate that the degree of baseline calcium elevation correlates with development of mixed phenotypes and sensitivity to pharmacologic channel inhibition, observations that will be critical for the design of future clinical trials for TRPV4 channelopathies.

Edaravone activates the GDNF/RET neurotrophic signaling pathway and protects mRNA-induced motor neurons from iPS cells.

BACKGROUND: Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. METHODS: We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. RESULTS: We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. CONCLUSIONS: This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

A novel HSPB1 mutation associated with a late onset CMT2 phenotype: Case presentation and systematic review of the literature.

Mutations in the HSPB1 gene are associated with Charcot-Marie-Tooth (CMT) disease type 2F (CMT2F) and distal hereditary motor neuropathy type 2 (dHMN2). More than 18 pathogenic mutations spanning across the whole HSPB1 gene have been reported. Three family members with a novel p.P57S (c.169C>T) HSPB1 mutation resulting in a late onset axonal neuropathy with heterogeneous clinical and electrophysiological features are detailed. We systematically reviewed published case reports and case series on HSPB1 mutations. While a genotype-phenotype correlation was not obvious, we identified a common phenotype, which included adult onset, male predominance, motor more frequently than sensory involvement, distal and symmetric distribution with preferential involvement of plantar flexors, and a motor and axonal electrophysiological picture.

Persistent Cyfip1 Expression Is Required to Maintain the Adult Subventricular Zone Neurogenic Niche.

Neural stem cells (NSCs) persist throughout life in the subventricular zone (SVZ) neurogenic niche of the lateral ventricles as Type B1 cells in adult mice. Maintaining this population of NSCs depends on the balance between quiescence and self-renewing or self-depleting cell divisions. Interactions between B1 cells and the surrounding niche are important in regulating this balance, but the mechanisms governing these processes have not been fully elucidated. The cytoplasmic FMRP-interacting protein (Cyfip1) regulates apical-basal polarity in the embryonic brain. Loss of Cyfip1 during embryonic development in mice disrupts the embryonic niche and affects cortical neurogenesis. However, a direct role for Cyfip1 in the regulation of adult NSCs has not been established. Here, we demonstrate that Cyfip1 expression is preferentially localized to B1 cells in the adult mouse SVZ. Loss of Cyfip1 in the embryonic mouse brain results in altered adult SVZ architecture and expansion of the adult B1 cell population at the ventricular surface. Furthermore, acute deletion of Cyfip1 in adult NSCs results in a rapid change in adherens junction proteins as well as increased proliferation and number of B1 cells at the ventricular surface. Together, these data indicate that Cyfip1 plays a critical role in the formation and maintenance of the adult SVZ niche; furthermore, deletion of Cyfip1 unleashes the capacity of adult B1 cells for symmetric renewal to increase the adult NSC pool.SIGNIFICANCE STATEMENT Neural stem cells (NSCs) persist in the subventricular zone of the lateral ventricles in adult mammals, and the size of this population is determined by the balance between quiescence and self-depleting or renewing cell division. The mechanisms regulating these processes are not fully understood. This study establishes that the cytoplasmic FMRP interacting protein 1 (Cyfip1) regulates NSC fate decisions in the adult subventricular zone and adult NSCs that are quiescent or typically undergo self-depleting divisions retain the ability to self-renew. These results contribute to our understanding of how adult NSCs are regulated throughout life and has potential implications for human brain disorders.

Role of Human-Induced Pluripotent Stem Cell-Derived Spinal Cord Astrocytes in the Functional Maturation of Motor Neurons in a Multielectrode Array System.

The ability to generate human-induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion. The sequence of plating, density, and age in which hiPSC-A are cocultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to coculture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron crosstalk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity, which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real-time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and coculture with neurons influences astrocyte maturation as well. Taken together, these data indicate that in a human in vitro spinal cord culture system, astrocytes support hiPSC-MN maturation in a time-dependent and species-specific manner and suggest a closer approximation of in vivo conditions. Stem Cells Translational Medicine 2019;8:1272&1285.

Pain at the onset of Amyotrophic Lateral Sclerosis: a cross-sectional study.

OBJECTIVE: We evaluated ALS patients reporting pain, either generalized or localized, at disease onset and determined whether this feature defined a specific ALS phenotype. PATIENTS AND METHODS: We considered all consecutive ALS patients referred to our Motor Neuron Diseases Center between 2006 and 2016 and included only patients who fulfilled the El Escorial revised criteria for probable and definite ALS diagnosis. We then identified those cases who reported pain at disease onset and compared them to all remaining cases. Secondary causes of pain have been excluded. RESULTS: Our initial sample consisted of 108 patients (55 men and 53 women). We identified 5 cases with generalized pain and 16 cases with localized pain at disease onset, corresponding to 4.6% and 14.8% of the initial sample, respectively. Cases with generalized pain were all female and had an earlier disease onset (49.6 ± 1.5 vs 66.6 ± 10.2 yrs, p = 0.002). Cases with localized pain showed a preponderance of upper motor neuron symptoms/signs at disease onset. Patients with pain, either localized or generalized, had a significantly higher involvement of the limbs (82.6% vs 100%, p = 0.022), while the bulbar district was spared at disease onset (17.4% vs 0%, p = 0.008). More specifically, the proximal upper and distal lower limbs were more frequently affected by ALS in patients with pain at disease onset. In two cases, the clinical presentation was notable for the resemblance with complex regional pain syndrome. CONCLUSION: The presence of pain at disease onset seems to relate to peculiar clinical features of ALS and may be pathophysiologically associated with neurodegeneration.

CSF biomarkers and amyloid PET: concordance and diagnostic accuracy in a MCI cohort.

Brain amyloid deposition is one of the main hallmarks of Alzheimer's disease (AD) and two approaches are available for assessing amyloid pathology in vivo: cerebrospinal fluid (CSF) biomarkers levels and amyloid load visualized by amyloid beta positron emission tomography imaging (Amy-PET) probes. We aimed to investigate the concordance between CSF biomarkers and Amy-PET in a memory clinic cohort. Moreover, using a proper clinical follow-up, we wanted to assess the diagnostic accuracy of CSF and PET biomarkers in predicting the progression of patients with mild cognitive impairment (MCI) to AD dementia. We included 31 MCI patients who underwent [18F]florbetaben PET and CSF sampling (Aß1-42, t-Tau, p-Tau). A semiquantitative visual scan assessment was used to quantify amyloid deposition in 5 brain regions, rating from 1 (negative), to 2 and 3 (positive). CSF biomarkers were considered abnormal if: Aß1-42 < 600 pg/ml, p-Tau/Aß1-42 > 0.08 and t-Tau/Aß1-42 > 0.52. We also applied less lenient cutoffs of 550 pg/ml and 450 pg/ml for Aß1-42. The concordance rate was 77% between Amy-PET and CSF Aß1-42 levels, and 89% between Amy-PET and p-Tau/Aß1-42 and t-Tau/Aß1-42. According to the clinical follow-up, Amy-PET (sensitivity [SE] 93.7%, specificity [SP] 80%) exhibited the best diagnostic accuracy in discriminating AD from non-AD, followed by p-Tau/Aß1-42 ratio and t-Tau/Aß1-42 ratio (SE 93.7%, SP 66.6%), and Aß1-42 levels (SE 81%, SP 60%). The regional uptake of [18F]florbetaben PET in the precuneus and the striatum showed the best SP (86.6%). In discordant cases, the clinical diagnosis was most often in agreement with PET results. In general, concordance between CSF biomarkers and Amy-PET was good, especially when the ratios between CSF amyloid and Tau biomarkers were used. However, Amy-PET proved to be superior to CSF Aß1-42 in terms of diagnostic accuracy for AD, with the possibility to further increase its specificity by focusing the analysis in specific areas such as the precuneus/posterior cingulate cortex and the striatum.

Cluster headache in relation to different age groups.

Cluster headache (CH) has always been considered a type of primary headache affecting predominantly male subjects in early and medium adulthood. However, recent studies carried out in large case series of patients with CH show that not infrequently it may set in also after age 50; by contrast, onset before adolescence is very rare. Additionally, when onset occurs before age 14 or from the sixth decade of life onward, male predominance decreases to the point that in chronic forms CH predominantly affects the female sex. This particular pattern of the gender ratio in relation to onset in different age groups suggests that hormonal factors may actually play a role in the genesis of CH. In particular, future studies should be aimed at investigating the possible protective role of estrogen.

Perfluorocarbon Labeling of Human Glial-Restricted Progenitors for 19 F Magnetic Resonance Imaging.

One of the fundamental limitations in assessing potential efficacy in Central Nervous System (CNS) transplantation of stem cells is the capacity for monitoring cell survival and migration noninvasively and longitudinally. Human glial-restricted progenitor (hGRP) cells (Q-Cells) have been investigated for their utility in providing neuroprotection following transplantation into models of amyotrophic lateral sclerosis (ALS) and have been granted a Food and Drug Administration (FDA) Investigational New Drug (IND) for intraspinal transplantation in ALS patients. Furthermore, clinical development of these cells for therapeutic use will rely on the ability to track the cells using noninvasive imaging methodologies as well as the verification that the transplanted GRPs have disease-relevant activity. As a first step in development, we investigated the use of a perfluorocarbon (PFC) dual-modal (19 F magnetic resonance imaging [MRI] and fluorescence) tracer agent to label Q-Cells in culture and following spinal cord transplantation. PFCs have a number of potential benefits that make them appealing for clinical use. They are quantitative, noninvasive, biologically inert, and highly specific. In this study, we developed optimized PFC labeling protocols for Q-Cells and demonstrate that PFCs do not significantly alter the glial identity of Q-Cells. We also show that PFCs do not interfere with the capacity for differentiation into astrocytes either in vitro or following transplantation into the ventral horn of the mouse spinal cord, and can be visualized in vivo by hot spot 19 F MRI. These studies provide a foundation for further preclinical development of PFCs within the context of evaluating Q-Cell transplantation in the brain and spinal cord of future ALS patients using 19 F MRI. Stem Cells Translational Medicine 2019;8:355-365.