LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons.

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) ß-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.

Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics.

The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aß1-40 levels could be observed in mice injected with anti-TfR/BACE1 bispecific antibodies when compared to vehicle-injected mice. In summary, we found two nanobodies that could bind both human and cynomolgus TfR with the potential to be used clinically to increase the brain permeability of therapeutic biologicals.

VHHs as tools for therapeutic protein delivery to the central nervous system.

BACKGROUND: The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to test its potential to shuttle a therapeutic relevant cargo. Since this VHH could not recognize the human TfR and hence its translational potential is limited, we also aimed to find and validate an anti-human transferrin VHH to deliver a therapeutic cargo into the CNS. METHODS: Alpaca immunizations with human TfR, and subsequent phage selection and screening for human TfR binding VHHs was performed to find a human TfR specific VHH (Nb188). Its ability to cross the BBB was determined by fusing it to neurotensin, a neuropeptide that reduces body temperature when present in the CNS but is not able to cross the BBB on its own. Next, the anti-ß-secretase 1 (BACE1) 1A11 Fab and Nb62 or Nb188 were fused to an Fc domain to generate heterodimeric antibodies (1A11AM-Nb62 and 1A11AM-Nb188). These were then administered intravenously in wild-type mice and in mice in which the murine apical domain of the TfR was replaced by the human apical domain (hAPI KI). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed to assess the concentration of the heterodimeric antibodies in the brain over time and the ability to inhibit brain-specific BACE1 by analysing the brain levels of Aß1-40. RESULTS: Selections and screening of a phage library resulted in the discovery of an anti-human TfR VHH (Nb188). Fusion of Nb188 to neurotensin induced hypothermia after intravenous injections in hAPI KI mice. In addition, systemic administration 1A11AM-Nb62 and 1A11AM-Nb188 fusions were able to reduce Aß1-40 levels in the brain whereas 1A11AM fused to an irrelevant VHH did not. A PK/PD experiment showed that this effect could last for 3 days. CONCLUSION: We have discovered an anti-human TfR specific VHH that is able to reach the CNS when administered systemically. In addition, both the currently discovered anti-human TfR VHH and the previously identified mouse-specific anti-TfR VHH, are both able to shuttle a therapeutically relevant cargo into the CNS. We suggest the mouse-specific VHH as a valuable research tool in mice and the human-specific VHH as a moiety to enhance the delivery efficiency of therapeutics into the CNS in human patients.

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease.

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer's disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes.

HDAC3 Inhibition Stimulates Myelination in a CMT1A Mouse Model.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, with currently no effective treatment or cure. CMT1A is caused by a duplication of the PMP22 gene, which leads to Schwann cell differentiation defects and dysmyelination of the peripheral nerves. The epigenetic regulator histone deacetylase 3 (HDAC3) has been shown to negatively regulate myelination as well as its associated signaling pathways, PI3K-AKT and MAPK-ERK. We showed that these signaling pathways are indeed downregulated in the C3-PMP22 mouse model, similar to what has been shown in the CMT1A rat model. We confirmed that early postnatal defects are present in the peripheral nerves of the C3-PMP22 mouse model, which led to a progressive reduction in axon caliber size and myelination. The aim of this study was to investigate whether pharmacological HDAC3 inhibition could be a valuable therapeutic approach for this CMT1A mouse model. We demonstrated that early treatment of CMT1A mice with the selective HDAC3 inhibitor RGFP966 increased myelination and myelin g-ratios, which was associated with improved electrophysiological recordings. However, a high dose of RGFP966 caused a decline in rotarod performance and a decline in overall grip strength. Additionally, macrophage presence in peripheral nerves was increased in RGFP966 treated CMT1A mice. We conclude that HDAC3 does not only play a role in regulating myelination but is also important in the neuroimmune modulation. Overall, our results indicate that correct dosing of HDAC3 inhibitors is of crucial importance if translated to a clinical setting for demyelinating forms of CMT or other neurological disorders.

Applicability of cerebral open flow microperfusion and microdialysis to quantify a brain-penetrating nanobody in mice.

The use of biologics in the therapeutic landscape has increased exponentially since the last 3 decades. Nevertheless, patients with central nervous system (CNS) related disorders could not yet benefit from this revolution because the blood-brain barrier (BBB) severely hampers biologics from entering the brain. Considerable effort has been put into generating methods to modulate or circumvent the BBB for delivery of therapeutics to the CNS. A promising strategy is receptor-mediated transcytosis (RMT). Recently, Wouters et al. (2020) discovered a mouse anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via RMT. The present study aims to sample a derivative of this brain-penetrating nanobody (Nb105) in the CNS. Therefore, we compared the applicability of cerebral open flow microperfusion (cOFM) and microdialysis as sampling techniques to directly obtain high molecular weight substances from the cerebral interstitial fluid. A custom AlphaScreen™ assay was validated to quantify nanobody concentrations in the samples. In vitro microdialysis probe (AtmosLM™, 1 MDa cut-off) recovery by gain and by loss for Nb105 was 18.3 ± 3.2% and 27.0 ± 2.5% respectively, whereas for cOFM it was 87.2 ± 4.0% and 97.3 ± 1.6%. Although a large difference in in vitro recovery is observed between cOFM and microdialysis, in vivo similar results were obtained. Immunohistochemical stainings showed an astrocytic and microglial reaction in the immediate vicinity along the implantation track for both probe types. Coronal sections showed higher fluorescein isothiocyanate-dextran and immunoglobulin G extravasation around the microdialysis probe track than after cOFM sampling experiments, however this leakage was clearly limited compared to a positive control where the BBB was disrupted. This is the first study that samples a bispecific nanobody in the brain's interstitial fluid in function of time, providing a pharmacokinetic profile of nanobodies in the CNS. Furthermore, this is the first time a cOFM study is performed in awake freely moving mice, providing data on inflammation and blood-brain barrier integrity in the mouse brain. Overall, this work demonstrates that, while taking into account the (bio)analytical considerations, both microdialysis and cOFM are suitable in vivo sampling techniques for quantification of nanobodies in the CNS.

Restoration of histone acetylation ameliorates disease and metabolic abnormalities in a FUS mouse model.

Dysregulation of epigenetic mechanisms is emerging as a central event in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). In many models of neurodegeneration, global histone acetylation is decreased in the affected neuronal tissues. Histone acetylation is controlled by the antagonistic actions of two protein families -the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). Drugs inhibiting HDAC activity are already used in the clinic as anti-cancer agents. The aim of this study was to explore the therapeutic potential of HDAC inhibition in the context of ALS. We discovered that transgenic mice overexpressing wild-type FUS ("Tg FUS+/+"), which recapitulate many aspects of human ALS, showed reduced global histone acetylation and alterations in metabolic gene expression, resulting in a dysregulated metabolic homeostasis. Chronic treatment of Tg FUS+/+ mice with ACY-738, a potent HDAC inhibitor that can cross the blood-brain barrier, ameliorated the motor phenotype and substantially extended the life span of the Tg FUS+/+ mice. At the molecular level, ACY-738 restored global histone acetylation and metabolic gene expression, thereby re-establishing metabolite levels in the spinal cord. Taken together, our findings link epigenetic alterations to metabolic dysregulation in ALS pathology, and highlight ACY-738 as a potential therapeutic strategy to treat this devastating disease.

Inhibition of histone deacetylase 6 (HDAC6) protects against vincristine-induced peripheral neuropathies and inhibits tumor growth.

As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the levels of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.

Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Genetic ablation of phospholipase C delta 1 increases survival in SOD1(G93A) mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, resulting in selective motor neuron degeneration and paralysis. Patients die approximately 3-5 years after diagnosis. Disease pathophysiology is multifactorial, including excitotoxicity, but is not yet fully understood. Genetic analysis has proven fruitful in the past to further understand genes modulating the disease and increase knowledge of disease mechanisms. Here, we revisit a previously performed microsatellite analysis in ALS and focus on another hit, PLCD1, encoding phospholipase C delta 1 (PLCδ1), to investigate its role in ALS. PLCδ1 may contribute to excitotoxicity as it increases inositol 1,4,5-trisphosphate (IP3) formation, which releases calcium from the endoplasmic reticulum through IP3 receptors. We find that expression of PLCδ1 is increased in ALS mouse spinal cord and in neurons from ALS mice. Furthermore, genetic ablation of this protein in ALS mice significantly increases survival, but does not affect astrogliosis, microgliosis, aggregation or the amount of motor neurons at end stage compared to ALS mice with PLCδ1. Interestingly, genetic ablation of PLCδ1 prevents nuclear shrinkage of motor neurons in ALS mice at end stage. These results indicate that PLCD1 contributes to ALS and that PLCδ1 may be a new target for future studies.

Modifying expression of EphA4 and its downstream targets improves functional recovery after stroke.

Functional recovery after stroke varies greatly between patients, potentially due to differences in gene expression. Several processes like angiogenesis, neurogenesis, axonal reorganization and synaptic plasticity act in concert to restore neurological functions. The ephrin family has known roles in all these processes. EphA4 is the most abundant ephrin receptor in the nervous system. Therefore, we investigated whether EphA4 affects functional recovery from stroke, and evaluated the potential of this receptor as a therapeutic target. Motor recovery after photothrombotic stroke was studied in transgenic mice in which expression of EphA4 was reduced. Furthermore, blocking a downstream target of EphA4, ROCK (Rho-associated kinase), by two different compounds was evaluated in the same model. Motor recovery after photothrombotic stroke was markedly enhanced in transgenic mice with reduced levels of EphA4, whereas infarct sizes were similar compared with non-transgenic controls. Pharmacological inhibition of the EphA4 signaling cascade using two ROCK inhibitors,Y-27632 and fasudil, improved motor function of mice after stroke. Infarct size was comparable in all groups studied, suggesting that the benefit obtained by EphA4 inhibition is not neuroprotective in nature but due to an effect on the mechanisms underlying recovery. Our findings show that reduction of EphA4 improves motor function after experimental stroke and demonstrate that ROCK inhibition is a promising therapeutic strategy to enhance recovery after ischemic stroke.

Neuronal overexpression of IP3 receptor 2 is detrimental in mutant SOD1 mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing progressive paralysis of the patient followed by death on average 3-5 years after diagnosis. Disease pathology is multi-factorial including the process of excitotoxicity that induces cell death by cytosolic Ca(2+) overload. In this study, we increased the neuronal expression of an endoplasmic reticulum (ER) Ca(2+) release channel, inositol 1,4,5-trisphosphate receptor 2 (IP(3)R2), to assess whether increased cytosolic Ca(2+) originating from the ER is detrimental for neurons. Overexpression of IP(3)R2 in N2a cells using a Thy1.2-IP(3)R2 construct increases cytosolic Ca(2+) concentrations evoked by bradykinin. In addition, mice generated from this construct have increased expression of IP(3)R2 in the spinal cord and brain. This overexpression of IP(3)R2 does not affect symptom onset, but decreases disease duration and shortens the lifespan of the ALS mice significantly. These data suggest that ER Ca(2+) released by IP(3) receptors may be detrimental in ALS and that motor neurons are vulnerable to impaired Ca(2+) metabolism.