Presynaptic Nerve Terminals Research Articles

P2X Receptors and Their Roles in Astroglia in the Central and Peripheral Nervous System

Astrocytes are a class of neural cells that control homeostasis at all levels of the central and peripheral nervous system. There is a bidirectional neuron–glia interaction via a number of extracellular signaling molecules, glutamate and ATP being the most widespread. ATP activates ionotropic P2X and metabotropic P2Y receptors, which operate in both neurons and astrocytes. Morphological, biochemical, and functional evidence indicates the expression of astroglial P2X1/5 heteromeric and P2X7 homomeric receptors, which mediate physiological and pathophysiological responses. Activation of P2X1/5 receptors triggers rapid increase of intracellular Na+ that initiates immediate cellular reactions, such as the depression of the glutamate transporter to keep high glutamate concentrations in the synaptic cleft, the activation of the local lactate shuttle to supply energy substrate to pre- and postsynaptic neuronal structures, and the reversal of the Na+/Ca2+ exchange resulting in additional Ca2+ entry. The consequences of P2X7 receptor activation are mostly but not exclusively mediated by the entry of Ca2+ and result in reorganization of the cytoskeleton, inflammation, apoptosis/necrosis, and proliferation, usually at a prolonged time scale. Thus, astroglia detect by P2X1/5 and P2X7 receptors both physiological concentrations of ATP secreted from presynaptic nerve terminals and also much higher concentrations of ATP attained under pathological conditions.

Ca2+-independent syntaxin binding to the C2B effector region of synaptotagmin

Although synaptotagmin I, which is a calcium (Ca2+)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca2+ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca2+ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca2+-independent manner, and the binding was abolished in the presence of 1M NaCl. Synaptotagmin contains 2 Ca2+-binding domains (C2A, C2B). Mutating the positively charged lysine residues in the putative effector-binding region of the C2B domain, which are critical for transmitter release, markedly inhibited synaptotagmin–syntaxin binding, while similar mutations in the C2A domain had no effect on binding. Synaptotagmin–syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C2B domain effector region in a Ca2+-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca2+ influx into presynaptic nerve terminals.

Synphilin-1-Binding Protein NUB1 is Colocalized With Nonfibrillar, Proteinase K-Resistant α-Synuclein in Presynapses in Lewy Body Disease

α-Synuclein is a major component of Lewy bodies in Parkinson disease (PD) and dementia with Lewy bodies (DLB). We recently showed that abnormal α-synuclein with resistance to proteinase K (PK) is deposited at presynapses of distinct brain anatomic regions from the early stages of PD and DLB. NUB1, a synphilin-1-binding protein, also accumulates in Lewy bodies, but it is not known whether abnormal α-synuclein is associated with NUB1. Here, we demonstrate that, in the brain of patients with PD and DLB, NUB1 accumulates in the presynapses in the hippocampus, cerebral neocortex, and substantia nigra in which PK-resistant α-synuclein is deposited. Endogenous NUB1 also accumulated with PK-resistant α-synuclein in the presynapses of transgenic mice that express human α-synuclein with an A53T mutation. Immunoelectron microscopy showed that NUB1 is localized to presynaptic nerve terminals where no abnormal filaments are seen. Biochemical analyses showed that NUB1 coexists with abnormal α-synuclein in the brain of DLB patients. These findings suggest that NUB1 along with abnormal α-synuclein is involved in the pathogenesis of Lewy body disease.

The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

Presynaptic nerve terminals contain between several hundred vesicles (for example in small CNS synapses) and several tens of thousands (as in neuromuscular junctions). Although it has long been assumed that such high numbers of vesicles are required to sustain neurotransmission during conditions of high demand, we found that activity in vivo requires the recycling of only a few percent of the vesicles. However, the maintenance of large amounts of reserve vesicles in many evolutionarily distinct species suggests that they are relevant for synaptic function. We suggest here that these vesicles constitute buffers for soluble accessory proteins involved in vesicle recycling, preventing their loss into the axon. Supporting this hypothesis, we found that vesicle clusters contain a large variety of proteins needed for vesicle recycling, but without an obvious function within the clusters. Disrupting the clusters by application of black widow spider venom resulted in the diffusion of numerous soluble proteins into the axons. Prolonged stimulation and ionomycin application had a similar effect, suggesting that calcium influx causes the unbinding of soluble proteins from vesicles. Confirming this hypothesis, we found that isolated synaptic vesicles in vitro sequestered soluble proteins from the cytosol in a process that was inhibited by calcium addition. We conclude that the reserve vesicles support neurotransmission indirectly, ensuring that soluble recycling proteins are delivered upon demand during synaptic activity.

The changes in GABA signaling after spinal cord injury

Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABAA receptors at postsynaptic sites. In this study, the role of gephyrin on GABAA receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABAA receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic γ2-subunit containing GABAA receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABAA receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABAA receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABAA receptor clusters in the right position but also for regulating both phasic and tonic inhibition.

An L-2 burst fracture and cauda equina syndrome due to tetanus

Thoracic vertebral compression fractures are a known complication of generalized tetanus. The authors report the first known case of an L-2 burst fracture leading to cauda equina syndrome, as a result of generalized tetanus. This 51-year-old man had generalized tetanus with a constellation of symptoms including compartment syndrome requiring fasciotomies, severe axial spasms and spasms of the extremities, autonomic dysreflexia, hypercarbic respiratory failure, and rhabdomyolysis. During the course of his illness, areflexic paraparesis developed in his lower extremities. He was found to have an L-2 burst fracture with retropulsion of a bone fragment resulting in cauda equina syndrome. Operative intervention was undertaken to decompress the cauda equina and stabilize the spine. The natural progression of tetanus can be complex, with a mixed picture ranging from spasms plus increased tone and reflexes to reduced tone and reflexes as presynaptic nerve terminals become damaged. The authors suggest that all sudden changes in the neurological examination should prompt consideration of diagnostic imaging before attributing such changes to natural progression of the disease.

AP180 and CALM

Communication between neurons largely occurs at chemical synapses by conversion of electric to chemical signals. Chemical neurotransmission involves the action potential-driven release of neurotransmitters from synaptic vesicles (SVs) at presynaptic nerve terminals. Fusion of SVs is driven by SNARE complex formation comprising synaptobrevin 2 on the SV membrane and syntaxin 1A and SNAP-25 on the plasma membrane. In order to maintain neurotransmission during repetitive stimulation and to prevent expansion of the presynaptic plasma membrane, exocytic SV fusion needs to be balanced by compensatory retrieval of SV components to regenerate functional vesicles. Our recent work has unraveled a mechanism by which the R-SNARE synaptobrevin 2, the most abundant SV protein and an essential player for exocytic fusion, is recycled from the presynaptic membrane. The SNARE motif of synaptobrevin 2 is directly recognized by the ANTH domains of AP180 and CALM, monomeric endocytic adaptors for clathrin-mediated endocytosis. Given that key residues involved in synaptobrevin 2-ANTH domain complex formation are also essential for SNARE assembly, we propose that disassembly of SNARE complexes is a prerequisite for synaptobrevin 2 retrieval, thereby preventing endocytic mis-sorting of the plasma membrane Q-SNAREs syntaxin 1A and SNAP-25. It is tempting to speculate that perturbed synaptobrevin 2 recycling caused by reduction of CALM or AP180 levels may lead to disease as suggested by the genetic association of ANTH domain proteins with neurodegenerative disorders.

Effects of propofol and pentobarbital on calcium concentration in presynaptic boutons on a rat hippocampal neuron

Numerous reports suggest that intravenously administered (IV) anesthetics affect postsynaptic events in the central nervous system. However, there is little evidence about how general anesthetics influence the presynaptic processes. The level of presynaptic calcium (Ca(2+)) concentration ([Ca(2+)](pre)) regulates neurotransmitter release. In this study, we investigated the effects of anesthetic propofol IV and the barbiturate pentobarbital on neurotransmitter release by measuring [Ca(2+)](pre) in the presynaptic nerve terminals (boutons) on a dissociated single hippocampal rat neuron. Sprague-Dawley rats 10-14days old were decapitated under pentobarbital anesthesia, and brain slices were prepared. The hippocampal CA1 area was touched with a fire-polished glass pipette, which vibrated horizontally, and neurons were dissociated, along with the attached presynaptic boutons. The presynaptic boutons were visualized under a confocal laser-scanning microscope after staining with FM1-43 dye, and [Ca(2+)](pre) was measured with acetoxymethyl ester of fluo-3 (fluo-3 AM). High potassium (K(+)) (15-90mM) increased the [Ca(2+)](pre) in the Ca(2+)-containing solution in a concentration-dependent manner. Whereas propofol (10μM) and pentobarbital (300μM) suppressed the high K(+) (60mM)-induced increase in [Ca(2+)](pre) in the boutons attached to the dendrite, they did not affect [Ca(2+)](pre) in the boutons attached to the soma or dendrite base. As a large majority of excitatory synapses are located on dendritic spines, these agents may affect Ca(2+) mobilization in the excitatory presynaptic boutons. Propofol and pentobarbital may affect neurotransmitter release from the excitatory presynaptic nerve terminals due to inhibition of increase in [Ca(2+)](pre).

Aging influences adaptations of the neuromuscular junction to endurance training

This investigation sought to determine if aging affected adaptations of the neuromuscular junction (NMJ) to exercise training. Twenty young adult (8 months) and 20 aged (24 months) rats were assigned to either a program of treadmill exercise, or sedentary conditions. Following the 10-week experimental period, rats were euthanized, and soleus and plantaris muscles were removed and frozen. Longitudinal sections of the muscles were fluorescently stained to visualize pre-synaptic nerve terminals and post-synaptic endplates on both slow- and fast-twitch fibers. Images were collected with confocal microscopy and quantified. Muscle cross-sections were histochemically stained to assess muscle fiber profiles (size and fiber type). Our analysis of NMJs revealed a high degree of specificity and sensitivity to aging, exercise training, and their interaction. In the soleus, slow-twitch NMJs demonstrated significant (P ≤ 0.05) training-induced adaptations in young adult, but not aged rats. In the fast-twitch NMJs of the soleus, aging, but not training, was associated with remodeling. In the plantaris, aging, but not training, remodeled the predominant fast-twitch NMJs, but only pre-synaptically. In contrast, the slow-twitch NMJs of the plantaris displayed morphologic adaptations to both aging and exercise in pre- and post-synaptic components. Muscle fiber profiles indicated that changes in NMJ size were unrelated to adaptations of their fibers. Our data show that aging interferes with the ability of NMJs to adapt to exercise training. Results also reveal complexity in the coordination of synaptic responses among different muscles, and different fiber types within muscles, in their adaptation to aging and exercise training.

Transsynaptic EphB/Ephrin-B Signaling Regulates Growth of Presynaptic Boutons Required for Classical Conditioning

Learning-related presynaptic remodeling has been documented in only a few systems, and its molecular mechanisms are largely unknown. Here we describe a role for the bidirectional EphB/ephrin-B signaling system in structural plasticity of presynaptic nerve terminals using an in vitro model of classical conditioning. Conditioning or BDNF application induced significant growth of auditory nerve presynaptic boutons that convey the conditioned stimulus to abducens motor neurons. Interestingly, bouton enlargement occurred only for those synapses apposed to motor neuron dendrites rather than to somata. Phosphorylation of ephrin-B1, but not EphB2, was induced by both conditioning and BDNF application and was inhibited by postsynaptic injections of ephrin-B antibody. Finally, suppression of postsynaptic ephrin-B function inhibited presynaptic bouton enlargement that was rescued by activation of EphB2 by ephrin-B1-Fc. These data provide evidence for ephrin-B-induced EphB2 forward signaling in presynaptic structural plasticity during classical conditioning. They also reveal a functional interaction between BDNF/TrkB and the Eph/ephrin signaling systems in the coordination of presynaptic and postsynaptic modifications during conditioning.

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Defective Neuromuscular Junction Organization and Postnatal Myogenesis in Mice With Severe Spinal Muscular Atrophy

A detailed pathologic analysis was performed on Smn(-/-);SMN2 mice as a mouse model for human type I spinal muscular atrophy (SMA). We provide new data concerning changes in the spinal cord, neuromuscular junctions and muscle cells, and in the organs of the immune system. The expression of 10 synaptic proteins was analyzed in 3-dimensionally reconstructed neuromuscular junctions by confocal microscopy. In addition to defects in postsynaptic occupancy, there was a marked reduction in calcitonin gene-related peptide and Rab3A in the presynaptic motor terminals of some, but not all, of the skeletal muscles analyzed. Defects in the organization of presynaptic nerve terminals were also detected by electron microscopy. Moreover, degenerative changes in muscle cells, defective postnatal muscle growth, and prominent muscle satellite cell apoptosis were also observed. All of these changes occurred in the absence of massive loss of spinal cord motoneurons. On the other hand, astroglia, but not microglia, increased in the ventral horn of newborn SMA mice. In skeletal muscles, the density of interstitial macrophages was significantly reduced, and monocyte chemotactic protein-1 was downregulated. These findings raise questions regarding the primary contribution of a muscle cell defect to the SMA phenotype.

The Sodium-Dependent Inorganic Phosphate Transporter SLC34A1 (NaPi-IIa) Is Not Localized in the Mouse Brain

The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.

Neuron–glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function

The NMJ (neuromuscular junction) serves as the ultimate output of the motor neurons. The NMJ is composed of a presynaptic nerve terminal, a postsynaptic muscle and perisynaptic glial cells. Emerging evidence has also demonstrated an existence of perisynaptic fibroblast-like cells at the NMJ. In this review, we discuss the importance of Schwann cells, the glial component of the NMJ, in the formation and function of the NMJ. During development, Schwann cells are closely associated with presynaptic nerve terminals and are required for the maintenance of the developing NMJ. After the establishment of the NMJ, Schwann cells actively modulate synaptic activity. Schwann cells also play critical roles in regeneration of the NMJ after nerve injury. Thus, Schwann cells are indispensable for formation and function of the NMJ. Further examination of the interplay among Schwann cells, the nerve and the muscle will provide insights into a better understanding of mechanisms underlying neuromuscular synapse formation and function.

Neurotoxic phospholipases directly affect synaptic vesicle function

Snake neurotoxic phospholipases (SPAN) exclusively affect pre-synaptic nerve terminals where they lead to a block of neurotransmission by not fully understood mechanisms. Here, we report that the SPANs, taipoxin and paradoxin, in nanomolar concentrations directly dissociate the synaptophysin/synaptobrevin (Syp/Syb) complex on isolated synaptic vesicles in the presence of synaptosomal cytosol. The phospholipase activity of SPANs depends on Ca(2+) but the dissociation of the Syp/Syb complex does not require Ca(2+). Ca(2+) (100 μM free) alone also dissociates the Syp/Syb complex in the presence of cytosol. Treatment with SPANs disturbs the lipid raft association of synaptophysin and synaptobrevin comparable to cholesterol depletion by β-methyl-cyclodextrin while Ca(2+) alone has no effect. SPANs but not Ca(2+) directly inhibit vesicular uptake of serotonin and glutamate. It is concluded that SPANs directly affect vesicular properties independent from their Ca(2+) -dependent phospholipase activity. SPANs and Ca(2+) dissociate the Syp/Syb complex as a prerequisite for exocytosis. SPANs also prevent the filling of synaptic vesicles thereby adding to the inhibition of neurotransmission.

Different effects of α-chloralose on spontaneous and evoked GABA release in rat hippocampal CA1 neurons

The effects of α-chloralose on presynaptic GABA A receptors were investigated with respect to spontaneous and evoked GABAergic transmission (sIPSCs and eIPSCs) in rat hippocampal CA1 pyramidal neurons. sIPSCs were recorded in mechanically dissociated CA1 neurons with intact GABAergic terminals, namely the “synaptic bouton preparation.” eIPSCs were elicited by focal electrical stimuli of a single GABAergic bouton on an isolated CA1 neuron using the whole-cell patch recording configurations under voltage-clamp condition. We found that α-chloralose potentiated the exogenous GABA-induced Cl − response in a concentration dependent manner, and the drug itself induced Cl − response at high concentrations (>100 μM). α-Chloralose at low concentrations (3–10 μM) increased sIPSC frequency without affecting the current amplitude and kinetics, but prolonged the slow current decay time constant ( τ s) at concentrations greater than 30 μM without changing either current amplitude or frequency. α-Chloralose at 10 μM enhanced amplitude of eIPSCs and decreased the failure rate (Rf), but at 30 μM decreased the amplitude and increased the Rf. Pretreatment with bumetanide, a blocker of NKCC-1, completely prevented the 30 μM α-chloralose-induced inhibition on eIPSC amplitude and Rf. These results suggest that α-chloralose activates GABA A receptors on GABAergic presynaptic nerve terminals and depolarizes the terminals, mediating presynaptic inhibition or autoregulation, in a concentration-dependent manner. In addition, α-chloralose at high concentrations activates not only extrasynaptic GABA A receptors on the postsynaptic soma membrane but also synaptic GABA A receptors resulting in prolongation of current decay phase. Thus α-chloralose induces complex and differential modulation of sIPSCs and eIPSCs in a concentration dependent manner.

Axonal Regeneration and Neuronal Function Are Preserved in Motor Neurons Lacking ß-Actin In Vivo

The proper localization of ß-actin mRNA and protein is essential for growth cone guidance and axon elongation in cultured neurons. In addition, decreased levels of ß-actin mRNA and protein have been identified in the growth cones of motor neurons cultured from a mouse model of Spinal Muscular Atrophy (SMA), suggesting that ß-actin loss-of-function at growth cones or pre-synaptic nerve terminals could contribute to the pathogenesis of this disease. However, the role of ß-actin in motor neurons in vivo and its potential relevance to disease has yet to be examined. We therefore generated motor neuron specific ß-actin knock-out mice (Actb-MNsKO) to investigate the function of ß-actin in motor neurons in vivo. Surprisingly, ß-actin was not required for motor neuron viability or neuromuscular junction maintenance. Skeletal muscle from Actb-MNsKO mice showed no histological indication of denervation and did not significantly differ from controls in several measurements of physiologic function. Finally, motor axon regeneration was unimpaired in Actb-MNsKO mice, suggesting that ß-actin is not required for motor neuron function or regeneration in vivo.

The norepinephrine transporter gene is a candidate gene for panic disorder

Panic disorder (PD) is an anxiety disorder characterized by recurrent panic attacks with a lifetime prevalence of 4.7%. Genetic factors are known to contribute to the development of the disorder. Several lines of evidence point towards a major role of the norepinephrine system in the pathogenesis of PD. The SLC6A2 gene is located on chromosome 16q12.2 and encodes the norepinephrine transporter (NET), responsible for the reuptake of norepinephrine into presynaptic nerve terminals. The aim of the present study was to analyze genetic variants located within the NET gene for association with PD. The case-control sample consisted of 449 patients with PD and 279 ethnically matched controls. All cases fulfilled the ICD-10 diagnostic criteria for PD. Genotyping was performed using the Sequenom platform (Sequenom, Inc, San Diego, USA). To test for allelic and haplotypic association, the PLINK software was used, and COMBASSOC was applied to test for gene-wise association. After quality control 29 single nucleotide polymorphisms (SNPs) spanning the gene-region were successfully analyzed. Seven SNPs located within the 5' end of the gene were significantly associated with PD. Furthermore, the NET gene showed overall evidence for association with the disease (P=0.000035). In conclusion, the present study indicates that NET could be a susceptibility gene for PD.

Follistatin-like 1 Suppresses Sensory Afferent Transmission by Activating Na+,K+-ATPase

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Microarray analysis of differentially regulated genes in human neuronal and epithelial cell lines upon exposure to type A botulinum neurotoxin

Among the seven serotypes (A–G), type A botulinum neurotoxin (BoNT/A) is the most prevalent etiologic agent and the most potent serotype to cause foodborne botulism, characterized by flaccid muscle paralysis. Upon ingestion, BoNT/A crosses epithelial cell barriers to reach lymphatic and circulatory systems and blocks acetylcholine release at the pre-synaptic cholinergic nerve terminals of neuromuscular junctions (NMJs) resulting in paralysis. One of the unique features of BoNT/A intoxication is its neuroparalytic longevity due to its persistent catalytic activity. The persistent presence of the toxin inside the cell can induce host cell responses. To understand the pathophysiology and host response at the cellular level, gene expression changes upon exposure of human HT-29 colon carcinoma (epithelial) and SH-SY5Y neuroblastoma cell lines to BoNT/A complex were investigated using microarray analysis. In HT-29 cells, 167 genes were up-regulated while 60 genes were down-regulated, whereas in SH-SY5Y cells about 223 genes were up-regulated and 18 genes were down-regulated. Modulation of genes and pathways involved in neuroinflammatory, ubiquitin–proteasome degradation, phosphatidylinositol, calcium signaling in SH-SY5Y cells, and genes relevant to focal adhesion, cell adhesion molecules, adherens and gap junction related pathways in HT-29 cells suggest a massive host response to BoNT/A. A clear differential response in epithelial and neuronal cells indicates that the genes affected may play a distinct role in BoNTs cellular mode of action, involving these two types of host cells.