Benedikt Grünewald

Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

Human Stiff person syndrome IgG-containing high-titer anti-GAD65 autoantibodies induce motor dysfunction in rats.

Stiff person syndrome (SPS) is an autoimmune CNS disorder characterized by muscle rigidity, spasms and anxiety. The majority of patients have high-titer autoantibodies (ab) against glutamate decarboxylase (GAD65). A pathogenic role of SPS-associated IgG with ab against GAD65 has been shown for anxiety-like behavior but not for the core motor signs. We repetitively injected the purified IgG fraction of an SPS patient with severe motor impairment but without anxious comorbidity containing high titers of anti-GAD65 ab (SPS-IgG) into the lateral ventricle (i.c.v.) or intrathecally (i.th.) at the spinal level in experimental rats. We analyzed the effects on motor and anxiety-like behavior. Non-SPS human IgG fractions served as controls. Animals injected i.c.v. with SPS-IgG showed stiffness-like behavior with impaired walking ability and reduced grip strength of the upper limbs as well as postural and sensorimotor dysfunction. Testing for anxiety-like behavior revealed no significant differences between SPS and control IgG-treated rats. IgG deposits were found only in rats treated with SPS-IgG and were localized predominantly in CNS structures involved in motor control including globus pallidus, internal capsule, striatum and anterior thalamus. Double immunofluorescence staining revealed that predominantly GABAergic interneurons were positive for i.c.v. injected SPS-IgG. Rats injected i.th. with SPS-IgG did not present obvious motor symptoms and had a normal synaptic transmission at the spinal level. We conclude that SPS-like motor dysfunction can be induced in rats by passive transfer of IgG from an SPS-patient with high titer of anti-GAD65 ab. GABAergic dysfunction in supraspinal motor pathways rather than in the spinal cord may lead to motor deficits observed in the rats contrasting observations made in SPS with amphiphysin antibodies.

Human Stiff-Person Syndrome IgG Induces Anxious Behavior in Rats

Background Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. Methodology/Principal Findings We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patients amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. Conclusion/Significance The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.

Human IgG directed against amphiphysin induces anxiety behavior in a rat model after intrathecal passive transfer

Stiff person syndrome with auto-antibodies against amphiphysin is characterized by muscular stiffness, spasms, and anxiety which is a less appreciated core symptom. Here, we report that intrathecal application of purified immunoglobulin G-antibodies against amphiphysin from one patient induce anxiety behavior in rats. Immunostaining demonstrated binding of anti-amphiphysin antibodies to brain structures which are associated with anxiety disorders, such as the amygdala. We propose that antibody-mediated amphiphysin deficiency may account for anxiety behavior in stiff person syndrome via presynaptic dysregulation of GABAergic pathways.

Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition

Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex1-6) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.

Efficacy of Polyvalent Human Immunoglobulins in an Animal Model of Neuromyelitis Optica Evoked by Intrathecal Anti-Aquaporin 4 Antibodies

Neuromyelitis Optica Spectrum Disorders (NMOSD) are associated with autoantibodies (ABs) targeting the astrocytic aquaporin-4 water channels (AQP4-ABs). These ABs have a direct pathogenic role by initiating a variety of immunological and inflammatory processes in the course of disease. In a recently-established animal model, chronic intrathecal passive-transfer of immunoglobulin G from NMOSD patients (NMO-IgG), or of recombinant human AQP4-ABs (rAB-AQP4), provided evidence for complementary and immune-cell independent effects of AQP4-ABs. Utilizing this animal model, we here tested the effects of systemically and intrathecally applied pooled human immunoglobulins (IVIg) using a preventive and a therapeutic paradigm. In NMO-IgG animals, prophylactic application of systemic IVIg led to a reduced median disease score of 2.4 on a 0–10 scale, in comparison to 4.1 with sham treatment. Therapeutic IVIg, applied systemically after the 10th intrathecal NMO-IgG injection, significantly reduced the disease score by 0.8. Intrathecal IVIg application induced a beneficial effect in animals with NMO-IgG (median score IVIg 1.6 vs. sham 3.7) or with rAB-AQP4 (median score IVIg 2.0 vs. sham 3.7). We here provide evidence that treatment with IVIg ameliorates disease symptoms in this passive-transfer model, in analogy to former studies investigating passive-transfer animal models of other antibody-mediated disorders.

Measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

The lateral thoracic nerve and the cutaneous maximus muscle--a novel in vivo model system for nerve degeneration and regeneration studies.

We report a novel in vivo mouse model system to study regeneration of injured motor nerve and spatiotemporal pattern of denervation in experimental nerve diseases. The lateral thoracic nerve (LTN), as a pure motor nerve, innervates the cutaneous maximus muscle (CMM) by some of the shortest and the longest motor nerve fibers in the mouse body. Its branches and nerve terminals can be imaged in whole mount preparations. Here we describe the branching pattern of the LTN and its innervation of the CMM, and characterize degeneration and regeneration over time after a LTN crush by morphological and electrophysiological analyses. We demonstrate the utility of this model in a well-established neurotoxicity paradigm and in a genetic disease model of the peripheral neuropathy. Furthermore, this system enables punch biopsies that allow repeated and multi-location examinations for LTN regeneration and CMM reinnervation over time. The presence of the LTN and the CMM in a variety of species and its easy accessibility suggests that this in vivo model system offers considerable promise for future nerve degeneration and regeneration research.