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Mitochondria are essential for proper cellular function through their critical roles in ATP synthesis, reactive oxygen species production, calcium (Ca 2+ ) buffering, and apoptotic signaling. In neurons, Ca 2+ buffering is particularly important as it helps to shape Ca 2+ signals and to regulate numerous Ca 2+ -dependent functions including neuronal excitability, synaptic transmission, gene expression, and neuronal toxicity. Over the past decade, identification of the mitochondrial Ca 2+ uniporter (MCU) and other molecular components of mitochondrial Ca 2+ transport has provided insight into the roles that mitochondrial Ca 2+ regulation plays in neuronal function in health and disease. In this review, we discuss the many roles of mitochondrial Ca 2+ uptake and release mechanisms in normal neuronal function and highlight new insights into the Ca 2+ -dependent mechanisms that drive mitochondrial dysfunction in neurologic diseases including epilepsy, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. We also consider how targeting Ca 2+ uptake and release mechanisms could facilitate the development of novel therapeutic strategies for neurological diseases.

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.

Voltage-gated Ca 2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α 2 δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α 2 δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α 2 δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α 2 δ-4 knockout (KO) mice. α 2 δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α 2 δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor coordination in the rotarod assay. α 2 δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α 2 δ-4 in sensorimotor gating and motor function and identify α 2 δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Voltage-gated Ca.sup.2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the [alpha].sub.2 [delta]-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. [alpha].sub.2 [delta]-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of [alpha].sub.2 [delta]-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of [alpha].sub.2 [delta]-4 knockout (KO) mice. [alpha].sub.2 [delta]-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas [alpha].sub.2 [delta]-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor coordination in the rotarod assay. [alpha].sub.2 [delta]-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for [alpha].sub.2 [delta]-4 in sensorimotor gating and motor function and identify [alpha].sub.2 [delta]-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7–12 but not 4‐week‐old mice. Inhibitors of PKA and of GluR2‐lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4‐week‐old WT mice. Inhibitors of GluR2‐lacking AMPA receptors also prevented LTP in 2 but not 3‐week‐old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2‐lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150‐anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8‐week‐old mice.

Peripheral nerve damage initiates a complex series of cellular and structural processes that culminate in chronic neuropathic pain. Our study defines local angiotensin signaling via activation of the Angiotensin II (Ang II) type-2 receptor (AT2R) on macrophages as the critical trigger of neuropathic pain. An AT2R-selective antagonist attenuates neuropathic, but not inflammatory pain hypersensitivity in mice, and requires the cell damage-sensing ion channel transient receptor potential family-A member-1 (TRPA1). Mechanical and cold pain hypersensitivity that are characteristic of neuropathic conditions can be attenuated by chemogenetic depletion of peripheral macrophages and AT2R-null hematopoietic cell transplantation. Our findings show no AT2R expression in mouse or human sensory neurons, rather AT2R expression and activation in macrophages triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on sensory neurons. Our study defines the precise neuro-immune crosstalk underlying nociceptor sensitization at the site of nerve injury. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic neuropathic pain, and therefore identifies multiple analgesic targets.

The fact that mitochondrial Ca 2+ signaling is key to controlling neuronal life and death makes the components of mitochondrial Ca 2+ transport attractive candidates for therapeutic targeting in the treatment of neurodegenerative and psychiatric disorders. This chapter reviews recent advancements in the molecular biology of mitochondrial Ca 2+ signaling in neurons and discusses how Ca 2+ transport into and out of neuronal mitochondria contributes to the control of neuron excitability, synaptic transmission, and gene expression. In neurons at rest, total Ca 2+ concentration in the matrix ([Ca] mt ) is low, usually <100 nM. During electrical or synaptic stimulation, [Ca] mt can increase to millimolar levels within seconds. Since, our knowledge of the expression and function of the MCU and MICU isoforms within various organ systems is limited, the chapter focuses on the data available for the nervous system.

Gene transfer into neuronal cells provides an important approach to study their function. Particle-mediated gene delivery was used to transfect rat dorsal root ganglion (DRG) and hippocampal neurons in primary culture with the genes for the enhanced blue and green fluorescent proteins (EBFP and EGFP) under control of the cytomegalovirus promoter. Quantitative analysis of marker protein fluorescence detected expression at 3 h that continued to increase for 48 h. For DRG neurons the optimal expression efficiency of 8+/-2% was obtained 24 h following transfection. In contrast, approximately 2+/-1% of hippocampal neurons in culture expressed EGFP at 3 h which subsequently declined. Co-transfection of DRG cultures with two plasmids produced reliable expression of both genes. Transfected DRG neurons exhibited normal electrophysiological properties, and resting and stimulated intracellular Ca2+ concentrations were unchanged. After transfection, 44% of hippocampal neurons remained in functional synaptic networks as indicated by glutamatergic Ca2+ spiking activity. Particle-mediated gene delivery provided a straightforward, reproducible and efficient method for transfection of neurons in primary culture. Transfected cells were easily identified by EGFP fluorescence, enabling subsequent physiological analysis. Biolistic particle bombardment was well tolerated by peripheral neurons, although caution was required when this method was applied to CNS cultures.

Voltage-gated Cav Ca2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α2δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α2δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α2δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α2δ-4 knockout (KO) mice. α2δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α2δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor learning/coordination in the rotarod assay. Female but not male α2δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α2δ-4 in cognitive and motor function and identify α2δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn. A tau-Fyn double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled Fyn KO in cognitive tasks and resembled tau KO mice in motor tasks and protection from pentylenetetrazole-induced seizures. In Ca2+ response, Fyn KO was decreased relative to WT and DKO had a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO resembled WT in its Ca2+ response, we investigated whether MAP2 served to compensate for tau, since its level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca2+ response. The downstream effect of the interaction between microtubule-associated protein tau and Src family non-receptor tyrosine kinase Fyn was investigated with a tau/Fyn double KO mouse. We demonstrate that tau has a Fyn-independent role in glutamate-induced calcium response and that MAP2 can compensate for tau in interacting with Fyn in dendrites.