Charles Krieger

Mitochondria, Ca2+ and neurodegenerative disease

Mitochondria play a central role in cell biology not only as producers of ATP, but also in the sequestration of Ca(2+) and the generation of free radicals. They are also repositories of several proteins which regulate apoptosis. Perturbations in the normal functions of mitochondria will inevitably disturb cell function, may sensitise cells to neurotoxic insults and may initiate cell death. Neuronal Ca(2+) overload, such as follows excessive stimulation of Ca(2+) permeant excitatory amino acid receptors, can cause cell death. Recent evidence suggests that the accumulation of Ca(2+) into mitochondria during episodes of cellular Ca(2+) overload initiates a cascade of events that culminate in cell death. Cell death appears to require not only mitochondrial Ca(2+) overload, but rather a combination of raised intramitochondrial Ca(2+) concentration with increased production of nitric oxide and possibly other oxyradical species. Cell death may proceed through either necrotic or apoptotic mechanisms, depending on the rate of consumption and depletion of ATP. Evidence is also accumulating to suggest that more subtle alterations in mitochondrial function may serve as predisposing factors in the pathogenesis of a number of neurodegenerative disorders.

Mislocalization of TDP-43 in the G93A mutant SOD1 transgenic mouse model of ALS.

Previous evidence demonstrates that TAR DNA binding protein (TDP-43) mislocalization is a key pathological feature of amyotrophic lateral sclerosis (ALS). TDP-43 normally shows nuclear localization, but in CNS tissue from patients who died with ALS this protein mislocalizes to the cytoplasm. Disease specific TDP-43 species have also been reported to include hyperphosphorylated TDP-43, as well as a C-terminal fragment. Whether these abnormal TDP-43 features are present in patients with SOD1-related familial ALS (fALS), or in mutant SOD1 over-expressing transgenic mouse models of ALS remains controversial. Here we investigate TDP-43 pathology in transgenic mice expressing the G93A mutant form of SOD1. In contrast to previous reports we observe redistribution of TDP-43 to the cytoplasm of motor neurons in mutant SOD1 transgenic mice, but this is seen only in mice having advanced disease. Furthermore, we also observe rounded TDP-43 immunoreactive inclusions associated with intense ubiquitin immunoreactivity in lumbar spinal cord at end stage disease in mSOD mice. These data indicate that TDP-43 mislocalization and ubiquitination are present in end stage mSOD mice. However, we do not observe C-terminal TDP-43 fragments nor TDP-43 hyperphosphorylated species in these end stage mSOD mice. Our findings indicate that G93A mutant SOD1 transgenic mice recapitulate some key pathological, but not all biochemical hallmarks, of TDP-43 pathology previously observed in human ALS. These studies suggest motor neuron degeneration in the mutant SOD1 transgenic mice is associated with TDP-43 histopathology.

Modulation of NMDA-mediated excitotoxicity by protein kinase C

Excessive activation of N‐methyl‐d‐aspartate (NMDA) receptors leads to cell death in human embryonic kidney‐293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA‐mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA‐mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down‐regulation. The region of NR2A that provides the PKC‐induced enhancement of cell death was localized to a discrete segment of the C‐terminus. Use of isoform‐specific PKC inhibitors showed that Ca2+‐ and lipid‐dependent PKC isoforms (cPKCs), specifically PKCβ1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by an in vitro kinase assay was also increased in NR1A/NR2A‐transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C‐terminus of NR2A to accentuate cell death in NR1/NR2A‐transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.

Bone marrow-derived cells in the central nervous system of a mouse model of amyotrophic lateral sclerosis are associated with blood vessels and express CX3CR1

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the CNS. However, it is unclear to what extent bone marrow (BM)‐derived cells contribute to this microgliosis. We have studied the adoptive transfer of green fluorescent protein (GFP)‐labeled whole BM cells and BM from mice that express GFP only in CX3CR1+ cells (CX3CR1+/GFP) into the CNS of a murine model of ALS having over‐expression of mutant superoxide dismutase (mSOD), and wt littermates. We find that most GFP+ and CX3CR1+/GFP cells are found adjacent to the microvasculature within the CNS, both in mSOD and wt mice. GFP+ and CX3CR1+/GFP cells within the CNS have a variety of morphologies, including cells with an elongated appearance, weak Iba‐1 immunoreactivity, and often mannose receptor immunoreactivity, indicating that these cells are perivascular microglia. Typically, less than 10% of BM‐derived cells had a stellate‐shape and expressed strong Iba‐1 immunoreactivity, as expected for parenchymal microglia, indicating that BM‐derived cells uncommonly generate parenchymal microglia. Adoptive transfer of BM‐derived cells from CX3CR1+/GFP mice revealed that many elongated cells are GFP+, demonstrating that some perivascular cells are derived from BM cells of the CX3CR1+ lineage. The significantly greater numbers of BM cells in mSOD than in control mice indicate that the presence of these BM cells in the spinal cord is regulated by conditioning stimuli that may include irradiation and inflammatory factors within the CNS.

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.

Neuromuscular dysfunction in the mutant superoxide dismutase mouse model of amyotrophic lateral sclerosis.

To better understand the interaction between motor neuron dysfunction and denervation in amyotrophic lateral sclerosis (ALS), we have evaluated motor neuron number and the retrograde uptake and transport of fluorogold by motor neurons in mice overexpressing mutant superoxide dismutase (mSOD), and wild‐type controls. N‐CAM immunoreactivity and protein kinase expression were determined in skeletal muscle during denervation. We found that in severely affected mSOD mice, motor neuron loss is moderate (approximate 40% reduction), whereas retrograde uptake/transport as assessed using fluorogold is profoundly impaired (approximately 90% reduction). The impairment in fluorogold uptake/transport corresponds to measures of progressive muscle denervation such as increased N‐CAM immunoreactivity of muscle and increased expression of protein kinase B (PKB) in denervated muscle. These data suggest that the debility in the mSOD mouse model of ALS is produced, in part, by impaired retrograde uptake/transport in motor neuron axons in spite of regenerative support from muscle such as elevated expression of PKB.

Reduced protein O-glycosylation in the nervous system of the mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis.

In the neurodegenerative disease amyotrophic lateral sclerosis (ALS), a number of proteins have been found to be hyperphosphorylated, including neurofilament proteins (NFs). In addition to protein phosphorylation, another important post-translational modification is O-glycosylation with β-N-acetylglucosamine residues (O-GlcNAc) and it has been found that O-GlcNAc can modify proteins competitively with protein phosphorylation, so that increased O-GlcNAc can reduce phosphorylation at specific sites. We evaluated a transgenic mouse model of ALS that overexpresses mutant superoxide dismutase (mSOD) and found that O-GlcNAc immunoreactivity levels are decreased in spinal cord tissue from mSOD mice, compared to controls. This reduction in O-GlcNAc levels is prominent in the motor neurons of spinal cord. We find that inhibition of O-GlcNAcase (OGA), the enzyme catalyzing removal of O-GlcNAc, using the inhibitor NButGT for 3 days, resulted in increased O-GlcNAc levels in spinal cord, both in mSOD and control mice. Furthermore, NButGT increased levels of O-GlcNAc modified NF-medium in spinal cords of control mice, but not in mSOD mice. These observations suggest that the neurodegeneration found in mSOD mice is associated with a reduction of O-GlcNAc levels in neurons, including motor neurons.

Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis

Myeloablative preconditioning using irradiation is the most commonly used technique to generate rodents having chimeric bone marrow, employed for the study of bone marrow-derived cell accumulation in the healthy and diseased central nervous system. However, irradiation has been shown to alter the blood-brain barrier, potentially creating confounding artefacts. To better study the potential of bone marrow-derived cells to function as treatment vehicles for neurodegenerative diseases alternative preconditioning regimens must be developed. We treated transgenic mice that over-express human mutant superoxide dismutase 1, a model of amyotrophic lateral sclerosis, with busulfan to determine whether this commonly used chemotherapeutic leads to stable chimerism and promotes the entry of bone marrow-derived cells into spinal cord. Intraperitoneal treatment with busulfan at 60 mg/kg or 80 mg/kg followed by intravenous injection of green fluorescent protein-expressing bone marrow resulted in sustained levels of chimerism (∼80%). Bone marrow-derived cells accumulated in the lumbar spinal cord of diseased mice at advanced stages of pathology at both doses, with limited numbers of bone marrow derived cells observed in the spinal cords of similarly treated, age-matched controls; the majority of bone marrow-derived cells in spinal cord immunolabelled for macrophage antigens. Comparatively, significantly greater numbers of bone marrow-derived cells were observed in lumbar spinal cord following irradiative myeloablation. These results demonstrate bone marrow-derived cell accumulation in diseased spinal cord is possible without irradiative preconditioning.

Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction.

ABSTRACT Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by protein kinase C (PKC). We have previously shown that the Drosophila adducin, Hu-li tai shao (Hts), plays a role in larval neuromuscular junction (NMJ) growth. Here, we find that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), we show that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.

Making the connection – shared molecular machinery and evolutionary links underlie the formation and plasticity of occluding junctions and synapses

ABSTRACT The pleated septate junction (pSJ), an ancient structure for cell–cell contact in invertebrate epithelia, has protein components that are found in three more-recent junctional structures, the neuronal synapse, the paranodal region of the myelinated axon and the vertebrate epithelial tight junction. These more-recent structures appear to have evolved through alterations of the ancestral septate junction. During its formation in the developing animal, the pSJ exhibits plasticity, although the final structure is extremely robust. Similar to the immature pSJ, the synapse and tight junctions both exhibit plasticity, and we consider evidence that this plasticity comes at least in part from the interaction of members of the immunoglobulin cell adhesion molecule superfamily with highly regulated membrane-associated guanylate kinases. This plasticity regulation probably arose in order to modulate the ancestral pSJ and is maintained in the derived structures; we suggest that it would be beneficial when studying plasticity of one of these structures to consider the literature on the others. Finally, looking beyond the junctions, we highlight parallels between epithelial and synaptic membranes, which both show a polarized distribution of many of the same proteins – evidence that determinants of apicobasal polarity in epithelia also participate in patterning of the synapse. Summary: Similarities between the regulation of structure and plasticity of occluding junctions and the synapse suggest a common derivation from an ancestral pleated septate junction. Thus, studies on one type of junction may be relevant to understand another.