Disruption of tubulin-alpha4a polyglutamylation prevents aggregation of hyper-phosphorylated tau and microglia activation in mice.

Dissociation of hyper-phosphorylated Tau from neuronal microtubules and its pathological aggregates, are hallmarks in the etiology of tauopathies. The Tau-microtubule interface is subject to polyglutamylation, a reversible posttranslational modification, increasing negative charge at tubulin C-terminal tails. Here, we asked whether tubulin polyglutamylation may contribute to Tau pathology in vivo. Since polyglutamylases modify various proteins other than tubulin, we generated a knock-in mouse carrying gene mutations to abolish Tuba4a polyglutamylation in a substrate-specific manner. We found that Tuba4a lacking C-terminal polyglutamylation prevents the binding of Tau and GSK3 kinase to neuronal microtubules, thereby strongly reducing phospho-Tau levels. Notably, crossbreeding of the Tuba4a knock-in mouse with the hTau tauopathy model, expressing a human Tau transgene, reversed hyper-phosphorylation and oligomerization of Tau and normalized microglia activation in brain. Our data highlight tubulin polyglutamylation as a potential therapeutic strategy in fighting tauopathies.

Muskelin regulates actin-dependent synaptic changes and intrinsic brain activity relevant to behavioral and cognitive processes.

Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation.

Alpha- and beta-tubulin isotypes are differentially expressed during brain development.

Alpha- and beta-tubulin dimers polymerize into protofilaments that associate laterally to constitute a hollow tube, the microtubule. A dynamic network of interlinking filaments forms the microtubule cytoskeleton, which maintains the structure of cells and is key to various cellular processes including cell division, cell migration, and intracellular transport. Individual microtubules have an identity that depends on the differential integration of specific alpha- and beta-tubulin isotypes and is further specified by a variety of posttranslational modifications (PTMs). It is barely understood to which extent neighboring microtubules differ in their tubulin composition or whether specific tubulin isotypes cluster along the polymer. Furthermore, our knowledge about the spatio-temporal expression patterns of tubulin isotypes is limited, not at least due to the lack of antibodies or antibody cross-reactivities. Here, we asked which alpha- and beta-tubulin mRNAs and proteins are expressed in developing hippocampal neuron cultures and ex vivo brain tissue lysates. Using heterologous expression of GFP-tubulin fusion proteins, we systematically tested antibody-specificities against various tubulin isotypes. Our data provide quantitative information about tubulin expression levels in the mouse brain and classify tubulin isotypes during pre- and postnatal development.

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits.

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory.

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs.

GSK3 and KIF5 regulate activity-dependent sorting of gephyrin between axons and dendrites.

The kinesin KIF5 transports neuronal cargoes into axons and dendrites. Isolated KIF5 motor domains preferentially move into axons, however KIF5 binding to GRIP1 or gephyrin drives the motor into dendrites, to deliver AMPA receptors (AMPARs) or glycine receptors (GlyRs), respectively. At postsynaptic sites, gephyrin forms a multimeric scaffold to anchor GlyRs and GABAA receptors (GABAARs) in apposition to inhibitory presynaptic terminals. Here, we report the unexpected observation that increased intracellular calcium through chronic activation of AMPARs, steers a newly synthesized gephyrin fusion protein (tomato-gephyrin) to axons and interferes with its normal delivery into dendrites of cultured neurons. Axonal gephyrin clusters were not apposed to presynaptic terminals, but colocalized with GlyRs and neuroligin-2 (NLG2). Notably, functional blockade of glycogen synthase kinase-3 (GSK3) and KIF5 normalized gephyrin missorting into the axonal compartment. In contrast, mutagenesis of gephyrin S270, a GSK3 target, did not contribute to axo-dendritic sorting. Our data are consistent with previous observations, which report regulation of kinesin motility through GSK3 activity. They suggest that GSK3 regulates the sorting of GlyR/gephyrin and NLG2 complexes in a KIF5-dependent manner.

Antigen-specific tolerance by autologous myelin peptide-coupled cells: a phase 1 trial in multiple sclerosis.

Multiple sclerosis (MS) is a devastating inflammatory disease of the brain and spinal cord that is thought to result from an autoimmune attack directed against antigens in the central nervous system. The aim of this first-in-man trial was to assess the feasibility, safety, and tolerability of a tolerization regimen in MS patients that uses a single infusion of autologous peripheral blood mononuclear cells chemically coupled with seven myelin peptides (MOG1-20, MOG35-55, MBP13-32, MBP83-99, MBP111-129, MBP146-170, and PLP139-154). An open-label, single-center, dose-escalation study was performed in seven relapsing-remitting and two secondary progressive MS patients who were off-treatment for standard therapies. All patients had to show T cell reactivity against at least one of the myelin peptides used in the trial. Neurological, magnetic resonance imaging, laboratory, and immunological examinations were performed to assess the safety, tolerability, and in vivo mechanisms of action of this regimen. Administration of antigen-coupled cells was feasible, had a favorable safety profile, and was well tolerated in MS patients. Patients receiving the higher doses (>1 × 10(9)) of peptide-coupled cells had a decrease in antigen-specific T cell responses after peptide-coupled cell therapy. In summary, this first-in-man clinical trial of autologous peptide-coupled cells in MS patients establishes the feasibility and indicates good tolerability and safety of this therapeutic approach.

T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses.

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of neutrophil functions and demonstrates a novel link between innate and adaptive immune responses.

Killer immunoglobulin-like receptor locus polymorphisms in multiple sclerosis.

OBJECTIVE: The objective of this study was to analyze whether inhibitory and activating killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I alleles defined by their KIR binding motifs are associated with multiple sclerosis (MS) susceptibility or severity. METHOD: We performed a population-based case-control study in 321 patients with clinically isolated syndrome (CIS) and clinically definite MS (CDMS) and 156 healthy blood donors (HD). Inhibitory and activating KIRs and HLA class I alleles were genotyped using polymerase chain reaction (PCR) sequence-specific primers. Allelic frequencies were correlated with prevalence, age of onset, disability and disease duration of CIS and CDMS. RESULTS: The frequency of the inhibitory KIR2DL3 gene was significantly reduced in patients with CIS and CDMS (p = 3.1 × 10(-5)). KIR2DL3-dependent risk reduction remained significant after elimination of patients carrying MS-associated DRB1*15, DRB1*03, DRB1*01 alleles. In addition, individuals carrying two copies for KIR2DL2/KIR2DS2 but lacking KIR2DL3 were overrepresented in the CIS/CDMS cohort. However, both genes did not affect disease risk in presence of KIR2DL3. We did not detect any association between the presence or absence of KIR genes with clinical disease parameters. CONCLUSION: Absence of the inhibitory KIR2DL3 gene is associated with the development of CIS/CDMS. These findings, if confirmed in larger cohorts, suggest that KIR-mediated recognition of HLA class I molecules should be further explored as potential disease mechanism in MS.

Central role of JC virus-specific CD4+ lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome.

Progressive multi-focal leucoencephalopathy and progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are caused by infection of the central nervous system with the JC polyoma virus. Both are complications of monoclonal antibody therapy in multiple sclerosis and other autoimmune diseases. Progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome can obscure the diagnosis of progressive multi-focal leucoencephalopathy and lead to severe clinical disability and possibly death. Different from progressive multi-focal leucoencephalopathy, in which demyelination results from oligodendrocyte lysis by JC virus in the absence of an immune response, tissue destruction in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome is caused by a vigorous immune response within the brain. The cells and mediators that are involved in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are as yet poorly understood. We examined two patients with multiple sclerosis, who developed progressive multi-focal leucoencephalopathy and later progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome under natalizumab therapy. Due to initially negative JC viral deoxyribonucleic acid testing in the cerebrospinal fluid, a diagnostic brain biopsy was performed in one patient. Histopathology revealed brain inflammation characterized by a prominent T cell infiltrate (CD4(+)> CD8(+) T cells), but also B/plasma cells and monocytes. Despite very low JC viral load, both patients showed high intrathecal anti-JC virus antibodies. Brain-infiltrating CD4(+) T cells were studied regarding antigen specificity and function. CD4(+) T cells were highly specific for peptides from several JC virus proteins, particularly the major capsid protein VP1. T cell phenotyping revealed CD4(+) Th1 and bifunctional Th1-2 cells. The latter secrete large amounts of interferon-γ and interleukin-4 explaining the strong brain inflammation, presence of plasma cells and secretion of intrathecal anti-VP1 antibodies. The functional phenotype of brain-infiltrating JC virus-specific CD4(+) T cells was confirmed and extended by examining brain-derived JC virus-specific CD4(+) T cell clones. Our data provide novel insight into the pathogenesis of progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome and indicate that JC virus-specific CD4(+) T cells play an important role in both eliminating JC virus from the brain, but also in causing the massive inflammation with often fatal outcome.