Characterization of the bispecific VHH antibody gefurulimab (ALXN1720) targeting complement component 5, and designed for low volume subcutaneous administration.

The bispecific antibody gefurulimab (also known as ALXN1720) was developed to provide patients with a subcutaneous treatment option for chronic disorders involving activation of the terminal complement pathway. Gefurulimab blocks the enzymatic cleavage of complement component 5 (C5) into the biologically active C5a and C5b fragments, which triggers activation of the terminal complement cascade. Heavy-chain variable region antigen-binding fragment (VHH) antibodies targeting C5 and human serum albumin (HSA) were isolated from llama immune-based libraries and humanized. Gefurulimab comprises an N-terminal albumin-binding VHH connected to a C-terminal C5-binding VHH via a flexible linker. The purified bispecific VHH antibody has the expected exact size by mass spectrometry and can be formulated at greater than 100 mg/mL. Gefurulimab binds tightly to human C5 and HSA with dissociation rate constants at pH 7.4 of 54 pM and 0.9 nM, respectively, and cross-reacts with C5 and serum albumin from cynomolgus monkeys. Gefurulimab can associate with C5 and albumin simultaneously, and potently inhibits the terminal complement activity from human serum initiated by any of the three complement pathways in Wieslab assays. Electron microscopy and X-ray crystallography revealed that the isolated C5-binding VHH recognizes the macroglobulin (MG) 4 and MG5 domains of the antigen and thereby is suggested to sterically prevent C5 binding to its activating convertase. Gefurulimab also inhibits complement activity supported by the rare C5 allelic variant featuring an R885H substitution in the MG7 domain. Taken together, these data suggest that gefurulimab may be a promising candidate for the potential treatment of complement-mediated disorders.

Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism.

Regeneration failure after spinal cord injury (SCI) results in part from the lack of a pro-regenerative response in injured neurons, but the response to SCI has not been examined specifically in injured sensory neurons. Using RNA sequencing of dorsal root ganglion, we determined that thoracic SCI elicits a transcriptional response distinct from sciatic nerve injury (SNI). Both SNI and SCI induced upregulation of ATF3 and Jun, yet this response failed to promote growth in sensory neurons after SCI. RNA sequencing of purified sensory neurons one and three days after injury revealed that unlike SNI, the SCI response is not sustained. Both SCI and SNI elicited the expression of ATF3 target genes, with very little overlap between conditions. Pathway analysis of differentially expressed ATF3 target genes revealed that fatty acid biosynthesis and terpenoid backbone synthesis were downregulated after SCI but not SNI. Pharmacologic inhibition of fatty acid synthase, the enzyme generating palmitic acid, decreased axon growth and regeneration in vitro. These results support the notion that decreased expression of lipid metabolism-related genes after SCI, including fatty acid synthase, may restrict axon regenerative capacity after SCI.

Nociceptor Deletion of Tsc2 Enhances Axon Regeneration by Inducing a Conditioning Injury Response in Dorsal Root Ganglia.

Neurons of the PNS are able to regenerate injured axons, a process requiring significant cellular resources to establish and maintain long-distance growth. Genetic activation of mTORC1, a potent regulator of cellular metabolism and protein translation, improves axon regeneration of peripheral neurons by an unresolved mechanism. To gain insight into this process, we activated mTORC1 signaling in mouse nociceptors via genetic deletion of its negative regulator Tsc2. Perinatal deletion of Tsc2 in nociceptors enhanced initial axon growth after sciatic nerve crush, however by 3 d post-injury axon elongation rate became similar to controls. mTORC1 inhibition prior to nerve injury was required to suppress the enhanced axon growth. Gene expression analysis in purified nociceptors revealed that Tsc2-deficient nociceptors had increased activity of regeneration-associated transcription factors (RATFs), including cJun and Atf3, in the absence of injury. Additionally, nociceptor deletion of Tsc2 activated satellite glial cells and macrophages in the dorsal root ganglia (DRG) in a similar manner to nerve injury. Surprisingly, these changes improved axon length but not percentage of initiating axons in dissociated cultures. The pro-regenerative environment in naïve DRG was recapitulated by AAV8-mediated deletion of Tsc2 in adult mice, suggesting that this phenotype does not result from a developmental effect. Consistently, AAV8-mediated Tsc2 deletion did not improve behavioral recovery after a sciatic nerve crush injury despite initially enhanced axon growth. Together, these data show that neuronal mTORC1 activation induces an incomplete pro-regenerative environment in the DRG that improves initial but not later axon growth after nerve injury.

Voltage-Independent SK-Channel Dysfunction Causes Neuronal Hyperexcitability in the Hippocampus of Fmr1 Knock-Out Mice.

Neuronal hyperexcitability is one of the major characteristics of fragile X syndrome (FXS), yet the molecular mechanisms of this critical dysfunction remain poorly understood. Here we report a major role of voltage-independent potassium (K+)-channel dysfunction in hyperexcitability of CA3 pyramidal neurons in Fmr1 knock-out (KO) mice. We observed a reduction of voltage-independent small conductance calcium (Ca2+)-activated K+ (SK) currents in both male and female mice, leading to decreased action potential (AP) threshold and reduced medium afterhyperpolarization. These SK-channel-dependent deficits led to markedly increased AP firing and abnormal input-output signal transmission of CA3 pyramidal neurons. The SK-current defect was mediated, at least in part, by loss of FMRP interaction with the SK channels (specifically the SK2 isoform), without changes in channel expression. Intracellular application of selective SK-channel openers or a genetic reintroduction of an N-terminal FMRP fragment lacking the ability to associate with polyribosomes normalized all observed excitability defects in CA3 pyramidal neurons of Fmr1 KO mice. These results suggest that dysfunction of voltage-independent SK channels is the primary cause of CA3 neuronal hyperexcitability in Fmr1 KO mice and support the critical translation-independent role for the fragile X mental retardation protein as a regulator of neural excitability. Our findings may thus provide a new avenue to ameliorate hippocampal excitability defects in FXS.SIGNIFICANCE STATEMENT Despite two decades of research, no effective treatment is currently available for fragile X syndrome (FXS). Neuronal hyperexcitability is widely considered one of the hallmarks of FXS. Excitability research in the FXS field has thus far focused primarily on voltage-gated ion channels, while contributions from voltage-independent channels have been largely overlooked. Here we report that voltage-independent small conductance calcium-activated potassium (SK)-channel dysfunction causes hippocampal neuron hyperexcitability in the FXS mouse model. Our results support the idea that translation-independent function of fragile X mental retardation protein has a major role in regulating ion-channel activity, specifically the SK channels, in hyperexcitability defects in FXS. Our findings may thus open a new direction to ameliorate hippocampal excitability defects in FXS.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat.

The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior.

Gpr126/Adgrg6 Has Schwann Cell Autonomous and Nonautonomous Functions in Peripheral Nerve Injury and Repair.

Schwann cells (SCs) are essential for proper peripheral nerve development and repair, although the mechanisms regulating these processes are incompletely understood. We previously showed that the adhesion G protein-coupled receptor Gpr126/Adgrg6 is essential for SC development and myelination. Interestingly, the expression of Gpr126 is maintained in adult SCs, suggestive of a function in the mature nerve. We therefore investigated the role of Gpr126 in nerve repair by studying an inducible SC-specific Gpr126 knock-out mouse model. Here, we show that remyelination is severely delayed after nerve-crush injury. Moreover, we also observe noncell-autonomous defects in macrophage recruitment and axon regeneration in injured nerves following loss of Gpr126 in SCs. This work demonstrates that Gpr126 has critical SC-autonomous and SC-nonautonomous functions in remyelination and peripheral nerve repair. SIGNIFICANCE STATEMENT: Lack of robust remyelination represents one of the major barriers to recovery of neurological functions in disease or following injury in many disorders of the nervous system. Here we show that the adhesion class G protein-coupled receptor (GPCR) Gpr126/Adgrg6 is required for remyelination, macrophage recruitment, and axon regeneration following nerve injury. At least 30% of all approved drugs target GPCRs; thus, Gpr126 represents an attractive potential target to stimulate repair in myelin disease or following nerve injury.

Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.

Six3 cooperates with Hedgehog signaling to specify ventral telencephalon by promoting early expression of Foxg1a and repressing Wnt signaling.

Six3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/ß-catenin pathway.

Presenilin modulates Pen-2 levels posttranslationally by protecting it from proteasomal degradation.

The gamma-secretase complex functions to cleave several type I transmembrane proteins within their transmembrane domains. These include the amyloid precursor protein, which is central to Alzheimer's disease pathogenesis, as well as N-cadherin and Notch, which regulate transcription. This complex is composed of four requisite integral membrane proteins: presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin, Pen-2, and Aph-1. How these proteins coordinately regulate one another and assemble to form a functional complex is not well understood. In this report we demonstrate that PS1 selectively enhances the stability of Pen-2 protein but not that of nicastrin or Aph-1. In the absence of PS1, Pen-2 was rapidly degraded by the proteasome. As PS1 levels increased, so too did the half-life of Pen-2 and therefore its steady-state levels. In addition, Pen-2 protein levels correlated with PS1 levels not only in cell culture but in transgenic mouse models as well. The genetic absence of PS1 and PS2, and therefore of gamma-secretase-dependent mediation of transcriptional activity, did not affect Pen-2 mRNA levels. Rather, presenilin (PS) regulates Pen-2 levels posttranslationally by preventing its degradation by the proteasome. Thus, the amount of Pen-2 protein is effectively titrated by its PS binding partner, and the rapidity with which Pen-2 is degraded in the absence of PS interactions could provide a mechanism to tightly regulate gamma-secretase complex assembly.

The transmembrane domain region of nicastrin mediates direct interactions with APH-1 and the gamma-secretase complex.

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the gamma-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other gamma-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the gamma-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfide-bonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human gamma-secretase components, all of the ceNCT/hNCT chimeras interacted with gamma-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the gamma-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.

Membrane topology of gamma-secretase component PEN-2.

PEN-2 is an integral membrane protein that is a necessary component of the gamma-secretase complex, which is central in the pathogenesis of Alzheimer's disease and is also required for Notch signaling. In the absence of PEN-2, Notch signaling fails to guide normal development in Caenorhabditis elegans, and amyloid beta peptide is not generated from the amyloid precursor protein. Human PEN-2 is a 101-amino acid protein containing two putative transmembrane domains. To understand its interaction with other gamma-secretase components, it is important to know the membrane topology of each member of the complex. To characterize the membrane topology of PEN-2, we introduced single amino acid changes in each of the three hydrophilic regions of PEN-2 to generate N-linked glycosylation sites. We found that the N-linked glycosylation sites present in the N- and C-terminal domains of PEN-2 were utilized, whereas a site in the hydrophilic "loop" region connecting the two transmembrane domains was not. The addition of a carbohydrate structure in the N-terminal domain of PEN-2 prevented association with presenilin 1, whereas glycosylation in the C-terminal region of PEN-2 did not, suggesting that the N-terminal domain is important for interactions with presenilin 1. Immunofluorescence microscopy with selective permeabilization of the plasma membrane of cells expressing epitope-tagged forms of PEN-2 confirmed the lumenal location of both the N and C termini. A protease protection assay also demonstrated that the loop domain of PEN-2 is cytosolic. Thus, PEN-2 spans the membrane twice, with the N and C termini facing the lumen of the endoplasmic reticulum.

CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity.

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.

Beta-secretase processing in the trans-Golgi network preferentially generates truncated amyloid species that accumulate in Alzheimer's disease brain.

The amyloid beta (A beta) peptide that accumulates in Alzheimer's disease brain is derived from the proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase activities. The beta-secretase enzyme beta-site amyloid precursor protein-cleaving enzyme (BACE) generates the N terminus of A beta by cleavage at either Asp(1) (beta-site) or Glu(11) (beta'-site), ultimately leading to the production of full-length A beta 1-40/42 or truncated A beta 11-40/42. The functional significance of this variable cleavage site specificity as well as the relative pathological impact of full-length versus N-terminally truncated A beta remains largely unknown. In our analysis of BACE reactivity in cell culture, we found that the preference of the protease for either beta- or beta'-cleavage was strongly dependent on intracellular localization. Within the endoplasmic reticulum, beta-site proteolysis predominated, whereas in the trans-Golgi network, beta'-cleavage was favored. Furthermore, the contrasting cleavage site specificities of BACE were not simply due to differences in organelle pH or the oligosaccharide composition of the glycoproteins involved. Examination of post-mortem brain specimens revealed significant levels of A beta 11-40/42 within insoluble amyloid pools. Taken together, these data support an important role for beta'-cleavage in the process of cerebral amyloid deposition and localize the processing event to the trans-Golgi network.