Hong Z. Yin

AMPA/kainate receptor‐triggered Zn2+ entry into cortical neurons induces mitochondrial Zn2+ uptake and persistent mitochondrial dysfunction

Rapid Zn2+ influx through Ca2+‐permeable AMPA/kainate (Ca‐A/K) channels triggers reactive oxygen species (ROS) generation and is potently neurotoxic. The first aim of this study was to determine whether these effects might result from direct mitochondrial Zn2+ uptake. Adapting the mitochondrially sequestered divalent cation sensitive probe, rhod‐2, to visualize mitochondrial Zn2+, present studies indicate that Zn2+ is taken up into these organelles. The specificity of the signal for Zn2+ was indicated by its reversal by Zn2+ chelation, and its mitochondrial origin indicated by its speckled extranuclear appearance and by its elimination upon pretreatment with the mitochondrial protonophore, carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP). Consistent with inhibition of mitochondrial Zn2+ uptake, FCCP also slowed the recovery of cytosolic Zn2+ elevations in Ca‐A/K(+) neurons. Further studies sought clues to the high toxic potency of intracellular Zn2+. In experiments using the mitochondrial membrane polarization (ΔΨm)‐sensitive probe tetramethylrhodamine ethyl ester and the ROS‐sensitive probe hydroethidine, brief kainate exposures in the presence of 300 μm Zn2+ (with or without Ca2+) resulted in prolonged loss of ΔΨm and corresponding prolonged ROS generation in Ca‐A/K(+) neurons, in comparison to the more rapid recovery from loss of ΔΨm and transient ROS generation after kainate/1.8 mm Ca2+ exposures.

Zn2+ permeates Ca2+ permeable AMPA/kainate channels and triggers selective neural injury

BRIEF exposures of cortical cultures to kainate (100μM) plus Zn2+ (300μM) cause fluorescence of the Zn2+sensitive dye, TS-Q, to appear in virtually all neurons, probably reflecting depolarization and secondary Zn2+permeation through voltage-sensitive Ca2+ channels. However, if Na+ ions are removed from the media (to prevent depolarization), prominent TS-Q fluorescence is still observed in the small subset of neurons labeled by kainate stimulated Co2+ uptake (Co2+(+) neurons), a histochemical technique that identifies neurons expressing Ca2+ permeable AMPA/kainate receptor-gated channels. Kainate/Zn2+ exposures in Na+ containing media with lower (50–100 μM) Zn2+ concentrations resulted 24 h later in selective loss of the Co2+(+) neurons, suggesting that these channels may permit particularly high rates of Zn2+ passage. Thus, direct permeation of synaptically released Zn + through Ca2+permeable AMPA/kainate channels could contribute to selective degeneration of neurons in disease as well as subserving physiological signaling functions.

DENDRITIC LOCALIZATION OF CA2+-PERMEABLE AMPA/KAINATE CHANNELS IN HIPPOCAMPAL PYRAMIDAL NEURONS

Although it is well established that cortical and hippocampal γ‐aminobutyric acid (GABA)‐ergic neurons generally have large numbers of Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate channels (Ca‐A/K channels), their presence on pyramidal neurons is controversial. Ca2+ permeability of AMPA channels is regulated by expression of a particular glutamate receptor subunit (GluR2), which confers Ca2+ impermeability to heteromeric channels. Most electrophysiology studies, as well as in situ hybridization and immunolabeling studies demonstrating expression of GluR2 mRNA or peptide in pyramidal neurons, have provided evidence against the presence of Ca‐A/K channels on pyramidal neurons. However, observations that pyramidal neurons often appear to be labeled by kainate‐stimulated Co2+ influx (Co2+(+) cells), a histochemical stain that identifies cells possessing Ca‐A/K channels, suggests that they may have these channels. The present study futher examines cellular and subcellular distribution of Ca‐A/K channels on hippocampal pyramidal neurons in slice as well as in culture. To this end, techniques of kainate‐stimulated Co2+ influx labeling, supplemented by AMPA receptor subunit immunocytochemistry and fluorescent imaging of kainate‐stimulated intracellular Ca2+ ([Ca2+]i) rises are employed. Co2+ labeling is often seen in pyramidal neuronal dendrites in both slice and in culture. In addition, although GluR1 and 4 staining in these neurons is often seen in the soma and dendrites, GluR2 label, when evident, is generally more restricted to the soma. Finally, measurement of kainate‐stimulated [Ca2+]i rises in cultured neurons, assessed by using low affinity Ca2+ indicators in the presence of N‐methyl‐D‐aspartate (NMDA) receptor and voltage‐sensitive Ca2+ channel blockade, often shows dendritic rises to precede those in the somata. Thus, these data support the hypothesis that Ca‐A/K channels are present in dendritic domains of many pyramidal neurons, and may help to provide resolution of the apparently conflicting data regarding their distribution. J. Comp.Neurol. 409:250–260, 1999.

Glutamate triggers preferential Zn2+ flux through Ca2+ permeable AMPA channels and consequent ROS production.

ZN2+ co-released with glutamate at excitatory synaptic sites can enter and cause injury to postsynaptic neurons. While prior studies using the slowly desensitizing agonist kainate suggested preferential Zn2+ permeation through Ca2+ permeable AMPA/kainate (Ca-A/K) channels, the present study aims to assess relevance of those findings upon more physiological receptor activation. Microfluorimetric techniques were used to measure [Zn2+]i attained upon exposure to the rapidly desensitizing agonist AMPA or to the physiological agonist glutamate, in the presence of 300 microM Zn2+. Under these conditions, micromolar [Zn2+]i rises (delta[Zn2+]i) were still observed to occur selectively in the subset of neurons that express large numbers of Ca-A/ K channels. Further studies using the oxidation sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species generation that paralleled delta[Zn2+]i, with rapid oxidation only observed in the case of Zn2+ entry through Ca-A/K channels.

Slow development of ALS-like spinal cord pathology in mutant valosin-containing protein gene knock-in mice

Pathological features of amyotrophic lateral sclerosis (ALS) include, in addition to selective motor neuron (MN) degeneration, the occurrence of protein aggregates, mitochondrial dysfunction and astrogliosis. SOD1 mutations cause rare familial forms of ALS and have provided the most widely studied animal models. Relatively recent studies implicating another protein, TDP-43, in familial and sporadic forms of ALS have led to the development of new animal models. More recently, mutations in the valosin-containing protein (VCP) gene linked to the human genetic disease, Inclusion Body Myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD), were found also to be associated with ALS in some patients. A heterozygous knock-in VCP mouse model of IBMPFD (VCPR155H/+) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCPR155H/+ mice and find age-dependent degeneration of ventral horn MNs, TDP-43-positive cytosolic inclusions, mitochondrial aggregation and progressive astrogliosis. Aged animals (∼24–27 months) show electromyography evidence of denervation consistent with the observed MN loss. Although these animals do not develop rapidly progressive fatal ALS-like disease during their lifespans, they recapitulate key pathological features of both human disease and other animal models of ALS, and may provide a valuable new model for studying events preceding onset of catastrophic disease.

Intrathecal infusion of a Ca2+ permeable AMPA channel blocker slows loss of both motor neurons and of the astrocyte glutamate transporter, GLT-1 in a mutant SOD1 rat model of ALS

Elevated extracellular glutamate, resulting from a loss of astrocytic glutamate transport capacity, may contribute to excitotoxic motor neuron (MN) damage in ALS. Accounting for their high excitotoxic vulnerability, MNs possess large numbers of unusual Ca(2+)-permeable AMPA channels (Ca-AMPA channels), the activation of which triggers mitochondrial Ca(2+) overload and strong reactive oxygen species (ROS) generation. However, the causes of the astrocytic glutamate transport loss remain unexplained. To assess the role of Ca-AMPA channels on the evolution of pathology in vivo, we have examined effects of prolonged intrathecal infusion of the Ca-AMPA channel blocker, 1-naphthyl acetylspermine (NAS), in G93A transgenic rat models of ALS. In wild-type animals, immunoreactivity for the astrocytic glutamate transporter, GLT-1, was particularly strong around ventral horn MNs. However, a marked loss of ventral horn GLT-1 was observed, along with substantial MN damage, prior to onset of symptoms (90-100 days) in the G93A rats. Conversely, labeling with the oxidative marker, nitrotyrosine, was increased in the neuropil surrounding MNs in the transgenic animals. Compared to sham-treated G93A animals, 30-day NAS infusions (starting at 67+/-2 days of age) markedly diminished the loss of both MNs and of astrocytic GLT-1 labeling. These observations are compatible with the hypothesis that activation of Ca-AMPA channels on MNs contributes, likely in part through oxidative mechanisms, to loss of glutamate transporter in surrounding astrocytes.

Kainate-stimulated Zn2+ uptake labels cortical neurons with Ca2+-permeable AMPA/kainate channels

The endogenous cation, Zn2+, is synaptically released and may trigger neurodegeneration after permeating through NMDA channels, voltage sensitive Ca2+ channels (VSCC), or Ca2+ permeable AMPA/kainate channels (Ca-A/K). Neurons expressing Ca-A/K can be identified by a histochemical stain based upon kainate-stimulated Co2+ uptake (Co2+(+) neurons). The primary objective of this study was to determine whether a similar approach could be employed to visualize agonist-stimulated intracellular Zn2+ accumulation, and, thus, to test the hypothesis that Ca-A/K permit particularly rapid Zn2+ flux. Substituting Zn2+ for Co2+ during agonist-stimulated uptake, followed by Timms sulfide-silver staining to visualize intracellular Zn2+, resulted in distinct labeling of a subpopulation of cortical neurons (Zn2+(+) neurons) closely resembling Co2+(+) neurons, suggesting that, like Co2+, Zn2+ may permeate Ca-A/K with particular rapidity. Neither NMDA nor high K+ triggered comparable Zn2+ accumulation, indicating substantially greater permeation through Ca-A/K than through NMDA channels or VSCC. Both fluorescence studies of intracellular Zn2+ accumulation and double staining studies (using SMI-32 and anti-glutamate decarboxylase antibodies, both markers of cortical neuronal subsets), support the contention that Zn2+ and Co2+ labeling identify a common set of neurons characterized by expression of AMPA/kainate channels directly permeable to Zn2+ and Co2+ as well as Ca2+. Furthermore, the preferential destruction of Zn2+(+) neurons (like Co2+(+) neurons) after brief kainate exposures in the presence of lower, more physiologic concentrations of Zn2+ suggests that Zn2+ permeation through Ca-A/K could contribute to selective neurodegeneration in disease. Finally, the study provides a novel and potentially advantageous histochemical approach for kainate-stimulated Co2+ or Zn2+ uptake labeling, using a room temperature technique (Timms staining) rather than the usual hot AgNO3 development of the Co2+ stain.

A progressive translational mouse model of human valosin-containing protein disease: the VCP(R155H/+) mouse.

Mutations in the valosin‐containing protein (VCP) gene cause hereditary inclusion body myopathy (IBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently, these mutations have been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. A knock‐in mouse model offers the opportunity to study VCP‐associated pathogenesis.

The Homozygote VCPR155H/R155H Mouse Model Exhibits Accelerated Human VCP-Associated Disease Pathology

Valosin containing protein (VCP) mutations are the cause of hereditary inclusion body myopathy, Pagets disease of bone, frontotemporal dementia (IBMPFD). VCP gene mutations have also been linked to 2% of isolated familial amyotrophic lateral sclerosis (ALS). VCP is at the intersection of disrupted ubiquitin proteasome and autophagy pathways, mechanisms responsible for the intracellular protein degradation and abnormal pathology seen in muscle, brain and spinal cord. We have developed the homozygous knock-in VCP mouse (VCPR155H/R155H) model carrying the common R155H mutations, which develops many clinical features typical of the VCP-associated human diseases. Homozygote VCPR155H/R155H mice typically survive less than 21 days, exhibit weakness and myopathic changes on EMG. MicroCT imaging of the bones reveal non-symmetrical radiolucencies of the proximal tibiae and bone, highly suggestive of PDB. The VCPR155H/R155H mice manifest prominent muscle, heart, brain and spinal cord pathology, including striking mitochondrial abnormalities, in addition to disrupted autophagy and ubiquitin pathologies. The VCPR155H/R155H homozygous mouse thus represents an accelerated model of VCP disease and can be utilized to elucidate the intricate molecular mechanisms involved in the pathogenesis of VCP-associated neurodegenerative diseases and for the development of novel therapeutic strategies.

Marked synergism between mutant SOD1 and glutamate transport inhibition in the induction of motor neuronal degeneration in spinal cord slice cultures

Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1 week old pups, and after an additional week in vitro, some were exposed to either a low level (30 μM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3 weeks, or a higher level (50 μM) for 48 h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury.