Selective activation of microglia facilitates synaptic strength.

Synaptic plasticity is thought to be initiated by neurons only, with the prevailing view assigning glial cells mere specify supportive functions for synaptic transmission and plasticity. We now demonstrate that glial cells can control synaptic strength independent of neuronal activity. Here we show that selective activation of microglia in the rat is sufficient to rapidly facilitate synaptic strength between primary afferent C-fibers and lamina I neurons, the first synaptic relay in the nociceptive pathway. Specifically, the activation of the CX3CR1 receptor by fractalkine induces the release of interleukin-1ß from microglia, which modulates NMDA signaling in postsynaptic neurons, leading to the release of an eicosanoid messenger, which ultimately enhances presynaptic neurotransmitter release. In contrast to the conventional view, this form of plasticity does not require enhanced neuronal activity to trigger the events leading to synaptic facilitation. Augmentation of synaptic strength in nociceptive pathways represents a cellular model of pain amplification. The present data thus suggest that, under chronic pain states, CX3CR1-mediated activation of microglia drives the facilitation of excitatory synaptic transmission in the dorsal horn, which contributes to pain hypersensitivity in chronic pain states.

Induction of thermal hyperalgesia and synaptic long-term potentiation in the spinal cord lamina I by TNF-α and IL-1ß is mediated by glial cells.

Long-term potentiation (LTP) of synaptic strength in nociceptive pathways is a cellular model of hyperalgesia. The emerging literature suggests a role for cytokines released by spinal glial cells for both LTP and hyperalgesia. However, the underlying mechanisms are still not fully understood. In rat lumbar spinal cord slices, we now demonstrate that conditioning high-frequency stimulation of primary afferents activated spinal microglia within <30 min and spinal astrocytes within ~2 s. Activation of spinal glia was indispensible for LTP induction at C-fiber synapses with spinal lamina I neurons. The cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), which are both released by activated glial cells, were individually sufficient and necessary for LTP induction via redundant pathways. They differentially amplified 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)-propanoic acid receptor-mediated and N-methyl-D-aspartic acid receptor-mediated synaptic currents in lamina I neurons. Unexpectedly, the synaptic effects by IL-1ß and TNF-α were not mediated directly via activation of neuronal cytokine receptors, but rather, indirectly via IL-1 receptors and TNF receptors being expressed on glial cells in superficial spinal dorsal horn. Bath application of IL-1ß or TNF-α led to the release profiles of pro-inflammatory and anti-inflammatory cytokines, chemokines, and growth factors, which overlapped only partially. Heat hyperalgesia induced by spinal application of either IL-1ß or TNF-α in naive animals also required activation of spinal glial cells. These results reveal a novel, decisive role of spinal glial cells for the synaptic effects of IL-1ß and TNF-α and for some forms of hyperalgesia.

Converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system.

PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aß, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

Non-Hebbian plasticity at C-fiber synapses in rat spinal cord lamina I neurons.

Current concepts of memory storage are largely based on Hebbian-type synaptic long-term potentiation induced by concurrent activity of pre- and postsynaptic neurons. Little is known about non-Hebbian synaptic plasticity, which, if present in nociceptive pathways, could resolve a number of unexplained findings. We performed whole-cell patch-clamp recordings in rat spinal cord slices and found that a rise in postsynaptic [Ca(2+)]i due to postsynaptic depolarization was sufficient to induce synaptic long-term potentiation (LTP) in the absence of any presynaptic conditioning stimulation. LTP induction could be prevented by postsynaptic application of the Ca(2+) chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), the L-type voltage-gated calcium channel (VGCC) antagonist nifedipine, and by postsynaptic application of the NMDA receptor antagonist MK801. This indicates that synaptic potentiation was induced postsynaptically by Ca(2+) entry likely via L-type voltage-gated Ca(2+) channels (VGCC) and via NMDA receptor channels. The paired pulse ratio and the coefficient of variation remained unchanged in neurons expressing LTP, suggesting that this form of synaptic potentiation was not only induced, but also expressed postsynaptically. Postsynaptic depolarization had no influence on firing patterns, action potential shape, or neuronal excitability. An increase in [Ca(2+)]i in spinal lamina I neurons induces a non-Hebbian form of synaptic plasticity in spinal nociceptive pathways without affecting neuronal active and passive membrane properties.

Impaired excitatory drive to spinal GABAergic neurons of neuropathic mice.

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca(2+). Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca(2+)-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy.

Hyperalgesia by synaptic long-term potentiation (LTP): an update.

Long-term potentiation of synaptic strength (LTP) in nociceptive pathways shares principle features with hyperalgesia including induction protocols, pharmacological profile, neuronal and glial cell types involved and means for prevention. LTP at synapses of nociceptive nerve fibres constitutes a contemporary cellular model for pain amplification following trauma, inflammation, nerve injury or withdrawal from opioids. It provides a novel target for pain therapy. This review summarizes recent progress which has been made in unravelling the properties and functions of LTP in the nociceptive system and in identifying means for its prevention and reversal.