Neuron-specific translational control shift ensures proteostatic resilience during ER stress.

Proteostasis is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R-like endoplasmic reticulum (ER) kinase (PERK)-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK-deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK-deficient neurons. Haem-regulated inhibitor (HRI) mediates p-eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back-up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.

Enrichment of Glial Cells From Human Post-mortem Tissue for Transcriptome and Proteome Analysis Using Immunopanning.

Glia cells have a crucial role in the central nervous system and are involved in the majority of neurological diseases. While glia isolation techniques are well established for rodent brain, only recent advances in isolating glial cells from human brain enabled analyses of human-specific glial-cell profiles. Immunopanning that is the prospective purification of cells using cell type-specific antibodies, has been successfully established for isolating glial cells from human fetal brain or from tissue obtained during brain surgeries. Here, we describe an immunopanning protocol to acutely isolate glial cells from post-mortem human brain tissue for e.g. transcriptome and proteome analyses. We enriched for microglia, oligodendrocytes and astrocytes from cortical gray matter tissue from three donors. For each enrichment, we assessed the presence of known glia-specific markers at the RNA and protein levels. In this study we show that immunopanning can be employed for acute isolation of glial cells from human post-mortem brain, which allows characterization of glial phenotypes depending on age, disease and brain regions.

Humanized tau antibodies promote tau uptake by human microglia without any increase of inflammation.

Immunotherapies targeting pathological tau have recently emerged as a promising approach for treatment of neurodegenerative disorders. We have previously showed that the mouse antibody DC8E8 discriminates between healthy and pathological tau, reduces tau pathology in murine tauopathy models and inhibits neuronal internalization of AD tau species in vitro.Here we show, that DC8E8 and antibodies elicited against the first-in-man tau vaccine, AADvac1, which is based on the DC8E8 epitope peptide, both promote uptake of pathological tau by mouse primary microglia. IgG1 and IgG4 isotypes of AX004, the humanized versions of DC8E8, accelerate tau uptake by human primary microglia isolated from post-mortem aged and diseased brains. This promoting activity requires the presence of the Fc-domain of the antibodies.The IgG1 isotype of AX004 showed greater ability to promote tau uptake compared to the IgG4 isotype, while none of the antibody-tau complexes provoked increased pro-inflammatory activity of microglia. Our data suggest that IgG1 has better suitability for therapeutic development.

No evidence for cell-to-cell transmission of the unfolded protein response in cell culture.

The unfolded protein response (UPR) is one of the major cell-autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore actively investigated as therapeutic target. In this respect, cell non-autonomous effects of the UPR including the reported cell-to-cell transmission of UPR activity may be highly important. A pharmaca-based UPR induction was employed to generate conditioned media (CM) from CM-donating neuronal ('donor') cells (SK-N-SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal 'acceptor' cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry-over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co-culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry-over of pharmaca is a major confounding factor in pharmaca-based UPR transmission protocols that are therefore unsuitable to study cell-to-cell UPR transmission. In addition, the absence of UPR transmission in non-pharmaca-based models of UPR activation indicates that cell-to-cell UPR transmission does not occur in cell culture.

Granulovacuolar degeneration bodies are neuron-selective lysosomal structures induced by intracellular tau pathology.

Granulovacuolar degeneration bodies (GVBs) are membrane-bound vacuolar structures harboring a dense core that accumulate in the brains of patients with neurodegenerative disorders, including Alzheimer's disease and other tauopathies. Insight into the origin of GVBs and their connection to tau pathology has been limited by the lack of suitable experimental models for GVB formation. Here, we used confocal, automated, super-resolution and electron microscopy to demonstrate that the seeding of tau pathology triggers the formation of GVBs in different mouse models in vivo and in primary mouse neurons in vitro. Seeding-induced intracellular tau aggregation, but not seed exposure alone, causes GVB formation in cultured neurons, but not in astrocytes. The extent of tau pathology strongly correlates with the GVB load. Tau-induced GVBs are immunoreactive for the established GVB markers CK1δ, CK1ɛ, CHMP2B, pPERK, peIF2α and pIRE1α and contain a LAMP1- and LIMP2-positive single membrane that surrounds the dense core and vacuole. The proteolysis reporter DQ-BSA is detected in the majority of GVBs, demonstrating that GVBs contain degraded endocytic cargo. GFP-tagged CK1δ accumulates in the GVB core, whereas GFP-tagged tau or GFP alone does not, indicating selective targeting of cytosolic proteins to GVBs. Taken together, we established the first in vitro model for GVB formation by seeding tau pathology in primary neurons. The tau-induced GVBs have the marker signature and morphological characteristics of GVBs in the human brain. We show that GVBs are lysosomal structures distinguished by the accumulation of a characteristic subset of proteins in a dense core.

Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration.

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activation of alternative clearance pathways including Golgi membrane-associated and nucleophagy-based LaminB1 degradation and excretion. The combination of these alternative pathways and canonical autophagy blockade, results in dramatic nuclear pathology with disruption of the nuclear organization, bringing about terminal cell atrophy and degeneration. Thus, our findings identify a novel progressive mechanism for the terminal phases of neuronal cell degeneration and death in human neurodegenerative diseases and provide a link between autophagy block, activation of alternative pathways for degradation, and excretion of cellular components.

Differences in long-term memory stability and AmCREB level between forward and backward conditioned honeybees (Apis mellifera).

In classical conditioning a predictive relationship between a neutral stimulus (conditioned stimulus; CS) and a meaningful stimulus (unconditioned stimulus; US) is learned when the CS precedes the US. In backward conditioning the sequence of the stimuli is reversed. In this situation animals might learn that the CS signals the end or the absence of the US. In honeybees 30 min and 24 h following backward conditioning a memory for the excitatory and inhibitory properties of the CS could be retrieved, but it remains unclear whether a late long-term memory is formed that can be retrieved 72 h following backward conditioning. Here we examine this question by studying late long-term memory formation in forward and backward conditioning of the proboscis extension response (PER). We report a difference in the stability of memory formed upon forward and backward conditioning with the same number of conditioning trials. We demonstrate a transcription-dependent memory 72 h after forward conditioning but do not observe a 72 h memory after backward conditioning. Moreover we find that protein degradation is differentially involved in memory formation following these two conditioning protocols. We report differences in the level of a transcription factor, the cAMP response element binding protein (CREB) known to induce transcription underlying long-term memory formation, following forward and backward conditioning. Our results suggest that these alterations in CREB levels might be regulated by the proteasome. We propose that the differences observed are due to the sequence of stimulus presentation between forward and backward conditioning and not to differences in the strength of the association of both stimuli.

Ubiquilin 2 is not associated with tau pathology.

Accumulation of aberrant proteins in inclusion bodies is a hallmark of many neurodegenerative diseases. Impairment of proteolytic systems is a common event in these protein misfolding diseases. Recently, mutations in the UBQLN 2 gene encoding ubiquilin 2 have been identified in X-linked amyotrophic lateral sclerosis (ALS). Furthermore, ubiquilin 2 is associated with inclusions in familial and sporadic ALS/dementia, synucleinopathies and polyglutamine diseases. Ubiquilin 2 exerts a regulatory role in proteostasis and thus it has been suggested that ubiquilin 2 pathology may be a common event in neurodegenerative diseases. Tauopathies, a heterogenous group of neurodegenerative diseases accompanied with dementia, are characterized by inclusions of the microtubule-binding protein tau. In the present study, we investigate whether ubiquilin 2 is connected with tau pathology in Alzheimer's disease (AD), supranuclear palsy (PSP) and Pick's disease (PiD) and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show that ubiquilin 2 positive inclusions are absent in these tauopathies. Furthermore, we find decreased ubiquilin 2 protein levels in AD patients, but our results do not indicate a correlation with tau pathology. Our data show no evidence for involvement of ubiquilin 2 and indicate that other mechanisms underly the proteostatic disturbances in tauopathies.

Unfolded protein response activates glycogen synthase kinase-3 via selective lysosomal degradation.

The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser(21/9). We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer(21/9) GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer(21/9) GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.

The spinal muscular atrophy disease protein SMN is linked to the Rho-kinase pathway via profilin.

Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis.

Mice lacking basic fibroblast growth factor showed faster sensory recovery.

Neurotrophic factors have been shown to stimulate and support peripheral nerve repair. One of these factors is basic fibroblast growth factor (FGF-2), which is up-regulated after peripheral nerve injury and influences early sciatic nerve regeneration by regulating Schwann cell proliferation. Our previous study on FGF-2 deficient mice indicated that FGF-2 is important for axonal maturation and remyelination one week after sciatic nerve crush (Jungnickel, J., Claus, P., Gransalke, K., Timmer, M. and Grothe, C., 2004. Targeted disruption of the FGF-2 gene affects the response to peripheral nerve injury. Mol. Cell. Neurosci. 25, 444-452). However, the functional impact of these effects on sensory and motor fibers was not clear. After performing pinch test, walking track analysis and rotarod, we found faster recovery of mechanosensory but not of motor function in mutant mice. To elucidate the role of FGF-2 on structural recovery, we analyzed FGF-2 deficient mice and wild-type littermates 2 and 4 weeks after sciatic nerve crush. Two weeks after peripheral nerve injury, regenerating fibers of mutant mice showed both significantly increased axon and myelin size, but no difference in the number of myelinated and unmyelinated fibers. Molecular analysis indicated that the expression level of myelin protein zero was significantly enhanced in lesioned nerves in the absence of FGF-2. These results suggest that loss of FGF-2 could positively influence restoration of mechanosensory function by accelerating structural recovery transiently.

Fibroblast growth factor-2 regulates the stability of nuclear bodies.

Nuclear bodies are distinct subnuclear structures. The survival of motoneuron (SMN) gene is mutated or deleted in patients with the neurodegenerative disease spinal muscular atrophy (SMA). The gene product SMN is a marker protein for one class of nuclear bodies denoted as nuclear gems. SMN has also been found in Cajal bodies, which co-localize with gems in many cell types. Interestingly, SMA patients display a reduced number of gems. Little is known about the regulation of nuclear body formation and stabilization. We have previously shown that a nuclear isoform of the fibroblast growth factor-2 (FGF-2(23)) binds directly to SMN. In this study, we analyzed the consequences of FGF-2(23) binding to SMN with regard to nuclear body formation. On a molecular level, we showed that FGF-2(23) competed with Gemin2 (a component of the SMN complex that is necessary for gem stabilization) for binding to SMN. Down-regulation of Gemin2 by siRNA caused destabilization of SMN-positive nuclear bodies. This process is reflected in both cellular and in vivo systems by a negative regulatory function of FGF-2 in nuclear body formation: in HEK293 cells, FGF-2(23) decreased the number of SMN-positive nuclear bodies. The same effect could be observed in motoneurons of FGF-2 transgenic mice. This study demonstrates the functional role of a growth factor in the regulation of structural entities of the nucleus.