Jochen Herms

The cellular prion protein binds copper in vivo

The normal cellular form of prion protein (PrPC) is a precursor to the pathogenic protease-resistant forms (PrPSc) believed to cause scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt–Jakob disease. Its amino terminus contains the octapeptide PHGGGWGQ, which is repeated four times and is among the best-preserved regions of mammalian PrPC. Here we show that the amino-terminal domain of PrPCexhibits five to six sites that bind copper (Cu(II)) presented as a glycine chelate. At neutral pH, binding occurs with positive cooperativity, with binding affinity compatible with estimates for extracellular, labile copper. Two lines of independently derived PrPCgene-ablated (Prnp0/0) mice exhibit severe reductions in the copper content of membrane-enriched brain extracts and similar reductions in synaptosomal and endosome-enriched subcellular fractions. Prnp0/0mice also have altered cellular phenotypes, including a reduction in the activity of copper/zinc superoxide dismutase and altered electrophysiological responses in the presence of excess copper. These findings indicate that PrPCcan exist in a Cu-metalloprotein form in vivo.

Real-time imaging reveals the single steps of brain metastasis formation

Brain metastasis frequently occurs in individuals with cancer and is often fatal. We used multiphoton laser scanning microscopy to image the single steps of metastasis formation in real time. Thus, it was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months. The essential steps in this model were arrest at vascular branch points, early extravasation, persistent close contacts to microvessels and perivascular growth by vessel cooption (melanoma) or early angiogenesis (lung cancer). Inefficient steps differed between the tumor types. Long-term dormancy was only observed for single perivascular cancer cells, some of which moved continuously. Vascular endothelial growth factor-A (VEGF-A) inhibition induced long-term dormancy of lung cancer micrometastases by preventing angiogenic growth to macrometastases. The ability to image the establishment of brain metastases in vivo provides new insights into their evolution and response to therapies.

Synapse Formation and Function Is Modulated by the Amyloid Precursor Protein

The amyloid precursor protein (APP) is critical in the pathogenesis of Alzheimers disease. The question of its normal biological function in neurons, in which it is predominantly located at synapses, is still unclear. Using autaptic cultures of hippocampal neurons, we demonstrate that hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor-mediated EPSCs. The size of the readily releasable synaptic vesicle pool was also increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP, whereas the amplitude of miniature synaptic currents was not found to be altered. Together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunohistochemical analysis revealing an increase of synaptophysin-positive puncta per cultured APP knock-out neuron. In conclusion, lack of APP affects synapse formation and transmission in cultured hippocampal neurons.

Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease

Microglia, the immune cells of the brain, can have a beneficial effect in Alzheimers disease by phagocytosing amyloid-β. Two-photon in vivo imaging of neuron loss in the intact brain of living Alzheimers disease mice revealed an involvement of microglia in neuron elimination, indicated by locally increased number and migration velocity of microglia around lost neurons. Knockout of the microglial chemokine receptor Cx3cr1, which is critical in neuron-microglia communication, prevented neuron loss.

The Secreted β-Amyloid Precursor Protein Ectodomain APPsα Is Sufficient to Rescue the Anatomical, Behavioral, and Electrophysiological Abnormalities of APP-Deficient Mice

It is well established that the proteolytic processing of the β-amyloid precursor protein (APP) generates β-amyloid (Aβ), which plays a central role in the pathogenesis of Alzheimers disease (AD). In contrast, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown. To address this question, we replaced the endogenous APP locus by gene-targeted alleles and generated two lines of knock-in mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPsα) or to a C-terminal (CT) truncation lacking the YENPTY interaction motif (APPΔCT15). Interestingly, the ΔCT15 deletion resulted in reduced turnover of holoAPP, increased cell surface expression, and strongly reduced Aβ levels in brain, likely because of reduced processing in the endocytic pathway. Most importantly, we demonstrate that in both APP knock-in lines the expression of APP N-terminal domains either grossly attenuated or completely rescued the prominent deficits of APP knock-out mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and long-term potentiation. Together, our data suggest that the APP C terminus is dispensable and that APPsα is sufficient to mediate the physiological functions of APP assessed by these tests.

Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members

The Alzheimers disease β‐amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor‐like proteins, termed APLP1 and APLP2. We previously documented that APLP2−/−APLP1−/− and APLP2−/−APP−/− mice die postnatally, while APLP1−/−APP−/− mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double‐mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.

hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations

Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.

Mouse cortical cells lacking cellular PrP survive in culture with a neurotoxic PrP fragment

To elucidate whether the neurotoxic effect of a prion protein fragment (PrP106–126) is in some way mediated by the cellular isoform of the prion protein (PrPC), dissociated cortical cell cultures were prepared from mice in which the PrP gene had been disrupted (PrPo/c mice). Cell survival after 10 days in culture was tested with an MTT assay. PrP106–126 applied every second day for 10 days in cultures from normal mice resulted in the death of 34% more cells than in untreated cells. When PrP106–126 was applied to cultures from mice lacking PrPC expression, survival was equal to or greater than that of untreated control cells. These results support the notion that expression of PrPC is required for the neurotoxic effect of PrP106–126.

Cellular prion protein function in copper homeostasis and redox signalling at the synapse

The fundamental physiological function of native cellular prion (PrPC) remains unknown. Herein, the most salient observations as regards prion physiology are critically evaluated. These include: (i) the role of PrPC in copper homeostasis, particularly at the pre‐synaptic membrane; (ii) involvement of PrPC in neuronal calcium disturbances; and (iii) the neuroprotective properties of PrPC in response to copper and oxidative stress. Ultimately, a tentative hypothesis of basic prion function is derived, namely that PrPC acts as a sensor for copper and/or free radical stimuli, thereby triggering intracellular calcium signals that finally translate into modulation of synaptic transmission and maintenance of neuronal integrity.

C-MYC expression in medulloblastoma and its prognostic value

To identify prognostic factors in medulloblastoma, a common malignant brain tumor of childhood, expression of the oncogene c‐myc was examined at the mRNA level by in situ hybridization. c‐myc mRNA expression was observed in 30 of 72 tumors (42%). The c‐myc gene copy number was determined by quantitative PCR from genomic DNA of paraffin‐embedded tumors. c‐myc gene amplification was present in 5 of 62 cases (8.3%). Therefore, c‐myc amplification was obviously not the cause of c‐myc mRNA expression in most samples. Kaplan‐Meier estimation revealed a significant correlation between c‐myc mRNA expression and survival (total mean follow‐up 4.6 ± 3.6 years, log‐rank p = 0.02). Multivariate logistic regression analysis including sex, age, histological type, degree of surgical resection and expression of synaptophysin, GFAP and c‐myc, was carried out on 54 patients who received both radiotherapy and chemotherapy. The analysis identified expression of c‐myc as an independent predictive factor of death from disease. Int. J. Cancer 89:395–402, 2000.